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Enhancement of pathogen destruction and anaerobic digestibility using microwaves

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ENHANCEMENT OF PATHOGEN DESTRUCTION AND
ANAEROBIC DIGESTIBILITY USING MICROWAVES
by
Seung-Mo Hong
A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor o f Philosophy
(Civil and Environmental Engineering)
at the
UNIVERSITY OF WISCONSIN-MADISON
2002
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UMI Number: 3060492
Copyright 2002 by
Hong, Seung-Mo
All rights reserved.
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© Copyright by Seung-Mo Hong 2002
All Rights Reserved
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Committee’s Page. This page is not to be hand-written except for the
A dissertation entitled
ENHANCEMENT OF PATHOGEN DESTRUCTION AND
ANAEROBIC DIGESTIBILITY USING MICROWAVES
submitted to the Graduate School of the
University of Wisconsin-Madison
in partial fulfillment of the requirements for the
degree of Doctor of Philosophy
by
Seung-Mo Hong
Date of Final Oral Examination:
Committee's Page. This page is not to be hand-svritten except for the signatures
Month & Year Degree to be awarded:
May 28, 2002
December
May
August
Approval Signatures of Dissertation Committee
Signature, Dean of Graduate School
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2002
ABSTRACT
Enhancement of pathogen destruction and anaerobic digestibility using microwaves
Seung-Mo Hong
Under the Supervision o f Professor J. K. Park
At the University o f Wisconsin-Madison
Approximately 8 to 9 million dry tons o f biosolids are produced each year by
municipal wastewater treatment facilities in the United States (U.S.)- Sludge treatment
and disposal may account for up to 40 to 60% of the total wastewater treatment cost.
Beneficial use o f biosolids is expected to increase from 63% in 2000 to 70% in 2010. In
1992, the U.S. EPA promulgated a regulation (40 CFR part 503) to protect public health
and the environment from reasonably anticipated adverse effects of certain pollutants in
sewage sludges (58 FR 9248, 1993). For Class A sludges, fecal coliform densities must
be < 1,000 most probable number (MPN)/g-TS, or Salmonella sp. must be < 3 MPN/4
g-TS (US EPA, 1999).
Many wastewater treatment plants in the U.S. are currently
evaluating ways of increasing pathogen destruction in biosolids to make Class A sludge.
The processes approved by the US EPA (1999) are expensive and may not be applicable
for many wastewater treatment plants.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The objectives of this study were to evaluate the effects of irradiation of
microwaves to sludge on the destruction o f coliform as a pathogen indicator and
anaerobic biogas production, to compare the anaerobic performance after pretreatment
with microwaves and conventional heating, and to propose an economical, efficient way
o f generating Class A sludge.
The penetration depths o f a 2,450 MHz microwave unit were found to be 1.7 and
1.1 cm for primary and waste activated sludges, respectively. In batch tests, for the initial
5 days, the relative cumulative biogas productions of anaerobic digester sludge (ADS),
primary sludge (PS), and waste activated sludge (WAS) by microwave irradiation were
approximately 13, 20, and 5% higher than conventional heating, and 54, 52, and 3%
higher than the control, respectively.
Fecal coliforms were not detected at 65°C for PS and ADS and 85°C for WAS
when sludge was irradiated with microwaves. Pathogen die-away tests were performed
to evaluate the effect o f storage in a 4°C refrigerator on pathogen destruction. Despite
high fecal coliform destruction efficiencies ranging from 99.3 to 99.7%, the residual fecal
coliform counts were over 10,000 CFU/g TS even after 224 days of storage.
The variation o f toxicity appears to be caused by leaching o f materials from
sludge by microwave and thermal heating and evaporation of toxic volatile matter.
However, there is no significant inhibitory effect by microwave irradiation during
anaerobic digestion.
Cell membrane damage tests, electron transport system (ETS) activity assays, and
P-galactosidase enzyme activity tests were conducted to verify the microwave
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iii
inactivation efficiency.
Damages o f fecal coliform cell membranes by microwave
irradiation were observed using both SYTO 9 and propidium iodide stains. The ETS
activity was lineally related to (3-galactosidase activity, indicating both tests are suitable
for evaluating toxicity o f unknown organic compound leached from sludge after
pretreatment with microwaves or conventional heat. The genomic DNA bands from the
pure cultured fecal coliforms that had been irradiated with 1-kW microwaves were
increasingly faint with the increase in irradiation time. This phenomenon was not
observed in the externally heated samples. Therefore, it can be said that the microwaves
have synergistic effect on DNA disruption o f fecal coliforms.
During the bench scale anaerobic digester operation, the highest log reductions of
fecal coliforms were 2.66 for the anaerobic digester fed with microwave-pretreated
sludge. A statistical analysis was performed to evaluate the difference o f three benchscale anaerobic digesters. From the paired t-test results at the 95% confidence interval,
the results o f fecal coliform detection for pairs o f digesters were statistically significantly
different.
The digester fed with microwave-irradiated sludges was more efficient in
inactivation of fecal coliforms than the other two digesters fed with raw sludge and
heated sludge, respectively.
From the laboratory-scale experiments and economic
analyses, it was concluded that microwave technology is an effective, economical, and
clean method of generating environmentally safe sludge.
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ACKNOWLEDGMENTS
I greatly appreciate my advisor, Jae K. Park, for his encouragement and guidance
during my entire graduate study at the University of Wisconsin-Madison.
I would like to thank my committee members, Professors David Armstrong,
Tuncer Edil, Chang-Beom Eom, and Greg Harrington. Their constructive suggestions
have been invaluable and grateful.
Thanks are also extended to Emeritus Professors William Boyle, Mac Berthouex,
and Professors Daniel Noguera, and Jerry Eykholt.
I would like to thank the
Environmental Engineering program assistant, Nanette Kelsey, for her sincere help
during my study.
I would like to thank Nutthapong Teeradej, M.S. student, who worked together to
run anaerobic digesters for his hard work, and Robert Lisi, Eunkyu Lee, Changhoon, Ahn,
Min, Jang, and Soo-Hong, Min who spent many nights in the laboratory with me. I want
to express my gratitude to Daewoo construction and Engineering Company for the
financial support during my Ph.D. study.
I would like to extend my deepest gratitude to my wife, son, and daughter for
their love, support, patience, and understanding. I want to dedicate my thesis to my last
father, mother and father-in-law, both o f whom passed away during my Ph.D. study
Seung-Mo Hong
May 2002.
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TABLE OF CONTENTS
ABSTRACT................................................................................................................................I
A C K N O W L E D G M E N T S .....................................................................................................................................iv
T A B L E O F C O N T E N T S ..............................................................................................................................................V
L I S T O F F I G U R E S .................................................................................................................................................... V III
L I S T O F T A B L E S .........................................................................................................................................................X II
1. I N T R O D U C T I O N ........................................................................................................................................................ 1
1.1 B a c k g r o u n d ........................................................................................................................................................... 1
1.2 P r o b l e m s ....................................................................................................................... 2
1.3 O b j e c t i v e s ............................................................................................................................................................... 5
2. LITERATURE REVIEW .................................................................................................... 7
2 .1 S l u d g e G e n e r a t i o n s
2 .2 R e g u l a t i o n s
and
D is p o s a l
in t h e
U . S ............................................................................7
o n p a t h o g e n r e q u ir e m e n t s o f
B
i o s o l i d s ...................................................13
2.2.1 Regulations for Class A Pathogen Requirements............................................... 18
2.2.2 Regulations for Class B Pathogen Requirements.............................................. 22
2 .3 C u r r e n t S l u d g e T r e a t m e n t T e c h n o l o g i e s .............................................................................. 2 3
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2 .4 M
Physical Treatment................................................................................................24
Chemical Treatment..............................................................................................26
Biological Treatment............................................................................................27
Thermal Treatment................................................................................................42
Radiation................................................................................................................51
i c r o w a v e ...........................................................................................................................................................5 6
2.4.1
2.4.2
2.4.3
2.4.4
Electromagnetic Spectrum....................................................................................57
Microwave Generator...........................................................................................59
Microwave Heating Mechanism.......................................................................... 62
Penetration Depth..................................................................................................69
2 .5 B i o l o g i c a l E f f e c t
of
M
i c r o w a v e s .................................................................................................. 7 3
2.5.1 Temperature Limits for Microbial Growth......................................................... 74
2.5.2 Pathogen Reduction of Microwaves.................................................................... 76
2.5.3 Thermal and Nonthermal Effects on Microwaves............................................. 78
2.5.4 Microwave Effects on DNA and Chemical Bond...............................................81
2.6 G a p s in k n o w l e d g e .................................................................................................... 88
3. EXPERIMENTAL MATERIALS AND METHODS..................................................... 89
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vi
3 .1 S l u d g e S a m p l i n g
3 .2 M
ic r o w a v e
and
E x p e r i m e n t P r e p a r a t i o n s ................................................................ 8 9
P o w e r C a l i b r a t i o n .........................................................................................................91
3 .3 P e n e t r a t i o n D
epth
T e s t ............................................................................................................................9 2
3 .4 B a t c h T e s t s ......................................................................................................................................................... 9 4
3 .4 .1 F ir st B a tc h M ic r o w a v e T e s t .............................................................................................................. 9 5
3 . 4 . 2 S e c o n d B a tc h M i c r o w a v e T e s t ....................................................................................................... 9 6
3 .5 B e n c h - S c a l e A n a e r o b i c D
ig e s t e r
T e s t s .................................................................................1 0 2
3 .5 .1 E x p e r im e n ta l C o n d it io n s f o r B e n c h - S c a le T e s t s ............................................................... 1 0 2
3 . 5 .2 S ta tis tic a l A n a l y s is fo r D ig e s t e r E v a l u a t io n .........................................................................1 0 4
3 .6 T o x i c i t y S c r e e n i n g T e s t s
S M P S .......................................................................................1 0 6
u s in g
3 .6 .1 F u n d a m e n ta ls o f S M P ......................................................................................................................... 1 0 7
3 . 6 .2 F u n d a m e n ta ls o f R E T ( R e v e r s e E le c tr o n T r a n sfe r) A s s a y ..........................................1 0 8
3 .6 .3 R E T P r o t o c o l s .......................................................................................................................................... 1 0 9
3 .7 B i o a s s a y s
on
F e c a l C o l if o r m s
as an
In d i c a t o r .................................................................111
3 .7 .1 I s o la tio n o f F e c a l C o lif o r m s fr o m S l u d g e s ........................................................................... 1 1 2
3 .7 .2 L iv e /D e a d C e ll C o u n t s ........................................................................................................................1 1 5
3 .7 .3 E T S (E le c tr o n T r a n s p o r t S y s t e m ) A s s a y ................................................................................1 1 7
3 . 7 .4 P -G a la c t o s id a s e E n z y m e A s s a y ................................................................................................... 1 1 8
3 .7 .5 G e l E le c t r o p h o r e s is ............................................................................................................................... 1 2 0
3 .8 O t h e r A n a l y t i c a l M e t h o d s ............................................................................................................... 1 2 6
3 .8 .1 T e m p e r a t u r e ...............................................................................................................................................1 2 7
3 . 8 .2 p H ..................................................................................................................................................................... 1 2 8
3 .8 .3 S o l i d s .............................................................................................................................................................. 1 2 8
3 . 8 .4 C h e m ic a l O x y g e n D e m a n d ( C O D ) ............................................................................................ 1 2 8
3 .8 .5 V o la t ile F a tty A c id s ( V F A s ) .......................................................................................................... 1 2 9
3 . 8 . 6 A l k a l i n i t y .................................................................................................................................................... 1 2 9
3 . 8 .7 G a s P r o d u c tio n a n d C o m p o s i t i o n ............................................................................................... 1 3 0
3 .8 .8 T o ta l C o lif o r m s a n d E . c o l i A n a l y s i s ........................................................................................ 1 3 0
3 . 8 .9 F e c a l C o lif o r m A n a l y s i s ................................................................................................................... 1 3 2
3 .9 P r e s e r v a t i o n
4.
and
H o l d i n g T i m e s .................................................................................................. 1 3 3
R E S U L T S A N D D I S C U S S I O N .................................................................................................................. 1 3 4
4 .1 M
ic r o w a v e
P o w e r C a l i b r a t i o n ...................................................................................................... 1 3 4
4 . 2 P e n e t r a t i o n D e p t h E s t i m a t i o n ........................................................................................................1 3 8
4 .3 O r g a n i c R e l e a s e
4 .4 B a t c h M
by
ic r o w a v e
M
ic r o w a v e
Ir r a d i a t i o n ...................................................................... 1 4 5
T e s t s ........................................................................................................................1 4 7
4 .4 .1 F ir st B a tch M ic r o w a v e T e s t ............................................................................................................ 1 4 7
4 . 4 . 2 S e c o n d B a tc h M i c r o w a v e T e s t s ................................................................................................... 1 5 0
4 .5 P a t h o g e n D e s t r u c t i o n E f f i c i e n c y ............................................................................................... 1 6 2
4 .5 .1 C o lif o r m D e s t r u c t io n ...........................................................................................................................1 6 3
4 . 5 . 2 P a th o g e n D i e - A w a y T e s t s ................................................................................................................ 1 7 2
4 . 6 T o x i c i t y T e s t s ............................................................................................................................................... 1 7 4
4 .7 B io l o g ic a l E f f e c t s
4 .7 .1
of
M
ic r o w a v e s f o r
F e c a l C o l i f o r m s .....................................181
C e ll M e m b r a n e D a m a g e s b y M i c r o w a v e s .......................................................................... 181
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.7.2 Microbial Activity Tests.....................................................................................185
4.7.3 Gel Electrophoresis for Genomic DNA............................................................ 190
4 .8 B e n c h S c a l e A
n a e r o b ic
D
ig e s t e r
T e s t s .................................................................................. 1 9 7
4.8.1 Changes in Fecal Coliform during the Anaerobic Digester Operation........... 198
4.8.2 Statistical Analysis for Digester Evaluation.....................................................202
5. MICROWAVE SYSTEM DESIGN...............................................................................204
5.1 P r o p o s e d F u l l - S c a l e M
ic r o w a v e
S y s t e m ............................................................................. 2 0 4
5.1.1 Microwave Frequencies..................................................................................... 204
5.1.2 Proposed Microwave System Applications...................................................... 205
5.1.3 Description o f Full-Scale Microwave System..................................................208
5.2 E c o n o m i c A n a l y s i s .................................................................................................212
6. CONCLUSIONS AND RECOMMENDATIONS........................................................ 223
6.1 C o n c l u s i o n s .....................................................................................................................................................2 2 3
6.2 R e c o m m e n d a t i o n s ................................................................................................... 225
7. REFERENCES................................................................................................................ 227
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LIST OF FIGURES
F i g u r e 2 .1 : P r o j e c t i o n s
o f b i o s o l i d s g e n e r a t i o n s in t h e
U .S . ( S o u r c e : U .S . E P A ,
1 9 9 9 a ) ............................................................................................................................................................................12
F i g u r e 2 .2 : P r o j e c t i o n s
o f u s e a n d d i s p o s a l in t h e
U .S . ( S o u r c e : U .S . E P A 1 9 9 9 a ).
12
F i g u r e 2 .3 : S c h e m a t i c
F ig u r e 2 .4 : M e t h a n e
P a r k in
and
F i g u r e 2 .5 : T h e
o f g e n e r a t i o n o f s e w a g e s l u d g e s a n d b i o s o l i d s ....................... 14
f o r m a t i o n in a n a e r o b i c d i g e s t i o n
( S o u r c e : M c C a r t y , 1964;
O w e n , 1 9 8 6 ) ............................................................................................................................... 2 9
r e l a t io n s h ip a m o n g b ic a r b o n a t e a l k a l in it y , p e r c e n t a g e o f
C 02
in d i g e s t e r g a s , a n d p H in a n a e r o b i c t r e a t m e n t ................................................................... 3 5
F i g u r e 2 .6 : E f f e c t
COD
o f s o l i d s r e t e n t i o n t im e in a n a e r o b i c d i g e s t i o n o n
a n d v o l a t il e s o l id s r e m o v a l a n d g a s p r o d u c t io n ,
(b)
(a )
overall
p r o t e in ,
CELLULOSE, AND LIPID REMOVAL, AND (C ) VOLATILE ACID CONCENTRATIONS.............. 3 6
F i g u r e 2 .7 : D
SULZER
ia g r a m o f
pr e - p a s t e u r iz a t io n s y s t e m
F i g u r e 2 .8 : E n t e r o b a c t e r i a c e a e
..................................................4 6
c o u n t s in t h e p r e - p a s t e u r i z a t i o n s y s t e m in
S w i t z e r l a n d ...........................................................................................................................................................4 7
F i g u r e 2 .9 : S u b m e r g e d
c o m b u s t i o n h e a t in g e q u i p m e n t o f s e w a g e s l u d g e
(S o urce:
K i d s o n e t a l ., 1 9 8 2 ) .......................................................................................................................................... 4 8
F ig u r e 2 .1 0 : S c h e m a t i c
o f g a m m a r a y ir r a d i a t i o n f a c il i t y u s i n g c o b a l t - 6 0 a t
G e i s e l b u l l a c h , G e r m a n y ( S o u r c e : U .S . E P A , 1 9 7 9 ) ............................................................ 5 2
F i g u r e 2 .1 1 : S c h e m a t i c
o f beta r a y sc a n n e r a n d sl u d g e spr e a d e r
( S o u r c e : U .S .
E P A , 1 9 7 9 ) .................................................................................................................................................................5 4
F i g u r e 2 .1 2 : S c h e m a t i c
o f e l e c t r o m a g n e t ic s p e c t r u m
(A
llahyar a n d
Ro b i t a i l l e ,
2000 )............................................................................................................................................. 57
F i g u r e 2 .1 3 : S c h e m a t i c
o f a t y p ic a l m a g n e t r o n a s a m ic r o w a v e s o u r c e :
(a )
SCHEMATIC PLAN VIEW, (B ) SECTIONAL VIEW (SOURCE: GALLAWA.COM/MICROTECH).
60
F i g u r e 2 .1 4 : S c h e m a t i c
F i g u r e 2 .1 5 : V a r i a t i o n
M etaxas
and
M
o f t h e e l e c t r o n p a t h in a m a g n e t r o n ............................................. 61
of
e'
a n d s " a s a f u n c t io n o f f r e q u e n c y
e r e d it h ,
F i g u r e 2 .1 6 : C o n d u c t i o n
1 9 8 3 ) ................................................................................................................6 6
e f f e c t o f e l e c t r o n in e l e c t r i c f i e l d ..............................................6 7
F i g u r e 2 .1 7 : T h r e e - d i m e n s i o n a l
at
2 ,4 5 0 M H z ( l
kW
)
fo r
13
F i g u r e 2 .1 8 : T h r e e - d i m e n s i o n a l
at
9 1 5 M H z ( 1 .0 K W )
F i g u r e 2 .1 9 : ( a ) P L -1
externally to
(S o u r c e :
fo r
14
t e m p e r a t u r e g r a d i e n t s in a g a r c y l i n d e r h e a t e d
m in u t e s
(C o p s o n , 1 9 7 5 ) ............................................................ 7 0
t e m p e r a t u r e g r a d ie n t s in a g a r c y l in d e r h e a t e d
m in u t e s
p h a g e p a r t ic l e s
6 0 .4 ° C , ( b ) P L -1
96%
( S o u r c e : C o p s o n , 1 9 7 5 ) ......................................71
in a c t iv a t e d t h r o u g h h e a t in g
p h a g e p a r t ic l e s
98%
in a c t iv a t e d b y
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
IX
MICROWAVE IRRADIATION FOR 5 0 SECONDS TO 6 0 .4 ° C (SOURCE: KAKITA ET A L.,
1 9 9 5 ) ...............................................................................................................................................................................8 5
F i g u r e 2 .2 0 : T h e
R it t m a n n
a d e n o s i n m o n o p h o s p h a t e d e o x y n u c l e o t i d e in
and
M c C a r t y , 2 0 0 1 ) ............................................................................................................... 8 7
F i g u r e 3 .1 : S c h e m a t i c
M
a d is o n
D N A (S o u r c e :
of
N
in e
S p r in g s W W T P
M e t r o p o l it a n S e w e r a g e D
F i g u r e 3 .2 : S c h e m a t ic
M a d is o n , W is c o n s in ( S o u r c e :
in
is t r ic t ,
2 0 0 1 ) ..............................................................9 0
N
o f s a m p l i n g l o c a t i o n s in
in e
S p r in g s W W T P
in
M
a d is o n ,
W I .....................................................................................................................................................................................9 1
F i g u r e 3 .3 : S c h e m a t i c
o f d e v ic e s u s e d f o r t h e m e a s u r e m e n t o f p e n e t r a t io n d e p t h .
............................................................................................................................................................................................9 3
F i g u r e 3 .4 : D e t a i l s
of part
F i g u r e 3 .5 : P h o t o g r a p h s
A
in m i c r o w a v a b l e v e s s e l ................................................................9 3
o f e x p e r im e n t a l a p p a r a t u s e s
(a )
serum bo ttles fo r the
SECOND BATCH TEST AND (B ) WATER BATH SHAKER AND GAS VOLUME MEASURE
EQUIPMENT............................................................................................................................................................... 101
F i g u r e 3 .6 : S c h e m a t i c
of serum bo ttle
F i g u r e 3 .7 : S c h e m a t i c
F i g u r e 3 .8 : R E T
(8 0
m
L)
f o r b a t c h t e s t s .......................................101
o f a n a e r o b i c d i g e s t e r a p p a r a t u s e s .................................................. 1 0 3
a s s a y r e a c t i o n s c h e m e .................................................................................................1 0 8
F i g u r e 4 .1 : T e m p e r a t u r e
v a r ia t io n s o f t a p w a t e r a f t e r
60
s e c . o f m ic r o w a v e
IRRADIATION WITH DIFFERENT TAP WATER MASSES...................................................................... 1 3 5
F i g u r e 4 .2 : A
b so r b e d po w er v a r ia t io n s o f
1
k W m ic r o w a v e u n it fo r
60
se c . of
MICROWAVE IRRADIATION............................................................................................................................. 1 3 7
F i g u r e 4 .3 : S c h e m a t ic
o f t e m p e r a t u r e a t t e n u a t i o n in v e s s e l f o r d e t e r m i n a t i o n
o f p e n e t r a t i o n d e p t h .................................................................................................................................... 1 4 1
F i g u r e 4 .4 : T e m p e r a t u r e
v a r ia t io n s u s in g a c r y l - a ir b a f f l e d v e s s e l b y
MICROWAVES (AVERAGE 6 0 SEC. IRRADIATION)............................................................................. 1 4 2
F i g u r e 4 .5 : C h a n g e s
in s o l u b l e
COD
a t v a r io u s t e m p e r a t u r e s c a u s e d b y
MICROWAVE IRRADIATION............................................................................................................................. 1 4 5
F i g u r e 4 .6 : S C O D /T C O D
r a t io s o f
A D S, PS
W AS
and
a f t e r m ic r o w a v e
IRRADIATION......................................................................................................................................................... 1 4 7
F i g u r e 4 .7 : C u m u l a t i v e
g a s p r o d u c t io n o f
RADS
w it h m ic r o w a v e i r r a d i a t i o n
t i m e s ............................................................................................................................................................................1 4 9
F i g u r e 4 .8 : C u m u l a t i v e
g a s p r o d u c t i o n o f t h e s p e c i f i c m ix t u r e c o n d i t i o n s a l o n g
WITH MICROWAVE IRRADIATION TIME................................................................................................... 1 5 0
F i g u r e 4 .9 : V
a r ia t io n s o f
B M P /B M P COntrol r a t i o
A D S , PS,
fo r
and
W AS
at
35°C
..........................................................................................................................................................................................1 5 3
F i g u r e 4 .1 0 : C u m u l a t i v e
b io g a s p r o d u c t io n o f
ADS
at
3 5°C
a ft e r m ic r o w a v e
IRRADIATION........................................................................................................................................................... 1 5 7
F i g u r e 4 .1 1 : C u m u l a t i v e
b io g a s p r o d u c t io n o f
PS
at
35°C
aft e r m ic r o w a v e
IRRADIATION.......................................................................................................................................................... 1 5 8
F i g u r e 4 .1 2 : C u m u l a t i v e
a t
b io g a s p r o d u c t io n o f
W AS
ir r a d i a t e d b y m i c r o w a v e s
3 5 ° C ...................................................................................................................................................................... 1 5 9
F i g u r e 4 .1 3 : C u m u l a t i v e
b io g a s p r o d u c t io n r a t io s o f
A D S, PS,
and
W AS
for
INITIAL 5 DAYS (CONTROL = 1 , STANDARD ERROR BA R )............................................................. 1 6 0
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X
F i g u r e 4 .1 4 : C o l if o r m
F i g u r e 4 .1 5 : F e c a l
counts of
A D S, PS,
c o l if o r m r e d u c t i o n s o f
and
PS
W AS
at
N
in e
S p r i n g s W W T P . ... 1 6 4
w it h m i c r o w a v e s a n d t h e r m a l
HEATING......................................................................................................................................................................1 6 5
F i g u r e 4 .1 6 : F e c a l
c o l if o r m r e d u c t i o n s o f
ADS
b y m ic r o w a v e s a n d t h e r m a l
HEATING..................................................................................................................................................................... 1 6 9
F i g u r e 4 .1 7 : F e c a l
c o l if o r m r e d u c t i o n s o f
W AS
b y m ic r o w a v e s a n d
CONVENTIONAL HEATING................................................................................................................................. 171
F i g u r e 4 .1 8 : C h a n g e s
4°C .
in f e c a l c o l i f o r m c o u n t s o v e r t im e d u r i n g s t o r a g e a t
......................................................................................................................................................................................... 1 7 3
F i g u r e 4 .1 9 : % In h ib it io n
c h a n g e s in s o l u b l e l i q u i d s o f
ADS
m e d i u m w it h
m i c r o w a v e s a n d c o n v e n t i o n a l h e a t i n g ..................................................................................... 1 7 7
F i g u r e 4 .2 0 : T o x i c i t y
v a r i a t i o n s in s o l u b l e l i q u i d s o f
PS
m e d i u m w it h
m i c r o w a v e s a n d c o n v e n t i o n a l h e a t i n g ......................................................................................1 7 8
F i g u r e 4 .2 1 : T o x i c i t y
v a r i a t i o n s in s o l u b l e l i q u i d s o f
W AS
m e d i u m w it h
MICROWAVES AND CONVENTIONAL HEATING....................................................................................1 7 9
F i g u r e 4 .2 2 : C h a n g e s
in t o x i c i t y o f l i q u i d p r o d u c e d f r o m
A D S, PS,
and
W AS
at
VARIOUS STORAGE DURATIONS IN A 4 ° C REFRIGERATOR........................................................... 1 8 0
F i g u r e 4 .2 3 ( a ): F l u o r e s c e n t
m ic r o s c o p e
630*, 3
s e c . m ic r o w a v e ir r a d ia t io n t o
4 9 ± 3 ° C .......................................................................................................................................................................1 8 2
F i g u r e 4 .2 3 ( b ): F l u o r e s c e n t
m ic r o s c o p e
630*, 9
s e c . m ic r o w a v e ir r a d ia t io n t o
6 0 ± 3 ° C .......................................................................................................................................................................1 8 3
F i g u r e 4 .2 3 ( c ) : F l u o r e s c e n t
m ic r o s c o p e
6 3 0 * , 12
s e c . m ic r o w a v e ir r a d ia t io n t o
7 2 ± 3 ° C .......................................................................................................................................................................1 8 4
F i g u r e 4 .2 3 ( d ): F l u o r e s c e n t
m ic r o s c o p e
6 3 0 * , 15
s e c . m ic r o w a v e ir r a d ia t io n t o
1 0 0 ± 3 ° C .................................................................................................................................................................... 1 8 5
F i g u r e 4 .2 4 : E T S
a c t iv it ie s o f f e c a l c o l i f o r m s b y m i c r o w a v e s a n d c o n v e n t i o n a l
HEATING..................................................................................................................................................................... 1 8 7
F i g u r e 4 .2 5 :
b - g a l a c t o s i d a s e a c t i v i t i e s o f f e c a l c o l if o r m s b y m i c r o w a v e s a n d
CONVENTIONAL HEATING............................................................................................................................... 1 8 9
F ig u r e 4 .2 6 : R e l a t i o n s h i p
betw een
ETS
a c t i v i t y a n d b -g a l a c t o s i d a s e a c t i v i t y
..........................................................................................................................................................................................1 9 0
F i g u r e 4 .2 7 : G e l
e l e c t r o p h o r e s is o f g e n o m i c
DNA
is o l a t e d f r o m f e c a l
COLIFORMS PRETREATED BY MICROWAVES AND CONVENTIONAL HEATING................... 1 9 2
F i g u r e 4 .2 8 : O p t ic a l
d e n s it y o f g e n o m ic
DNA
is o l a t e d fro m f e c a l c o l if o r m s
PRETREATED BY CONVENTIONAL HEATING......................................................................................... 1 9 3
F i g u r e 4 .2 9 : O p t ic a l
d e n s it y o f g e n o m ic
DNA
is o l a t e d fr o m f e c a l c o l if o r m s
PRETREATED BY MICROWAVE IRRADIATION........................................................................................ 1 9 4
F i g u r e 4 .3 0 : F e c a l
F i g u r e 4 .3 1 : L o g
c o l if o r m v a r i a t i o n s d u r i n g b e n c h s c a l e a n a e r o b i c t e s t s .
. 199
r e d u c t io n s o f f e c a l c o l if o r m s d u r in g b e n c h s c a l e a n a e r o b ic
t e s t s ........................................................................................................................................................................... 2 0 0
F i g u r e 4 .3 2 : B o x
p l o t fo r f e c a l c o l if o r m d e t e c t io n s d u r in g t h e o p e r a t i o n
F i g u r e 5 .1 : E l e v a t i o n
201
v ie w o f t h e m i c r o w a v e d s l u d g e s / l i q u i d s t r e a t m e n t
a p p a r a t u s .............................................................................................................................................................. 2 0 9
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XI
F i g u r e 5 .2 : T o p
F i g u r e 5 .3 : M
v i e w o f t h e d i s t r i b u t i o n u n i t ................................................................................... 2 1 0
i c r o w a v e e x p o s u r e c h a m b e r ......................................................................................... 2 1 0
F i g u r e 5 .4 : P l a n
v ie w o f t h e m i c r o w a v e d s l u d g e s / l i q u i d s t r e a t m e n t a p p a r a t u s
(L I N E l-1 ) ...................................................................................................................................................................2 1 1
F i g u r e 5 .5 : P l a n
v ie w o f t h e m i c r o w a v e d s l u d g e s / l i q u i d s t r e a t m e n t a p p a r a t u s
( l i n e 2 - 2 )........................................................................................................................................ 211
F i g u r e 5 .6 : P r o p o s e d 3
a l t e r n a t i v e s f o r m i c r o w a v e u n i t a p p l i c a t i o n ....................... 2 1 3
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LIST OF TABLES
T a b l e 1.1 : P r o j e c t i o n s o f s l u d g e u s e a n d d i s p o s a l in t h e U S ...................................................2
T a b l e 2 .1 : T o t a l
b i o s o l id s g e n e r a t e d a n d m a n a g e m e n t p r a c t i c e s ..................................... 8
T a b l e 2 .2 : M a j o r
p a t h o g e n s a s s o c ia t e d w it h r a w d o m e s t ic s e w a g e a n d s e w a g e
SLUDGES..................................................................................................................................................................... 15
T a b l e 2 .3 : T y p i c a l
16
n u m b e r s o f m i c r o o r g a n i s m s in w a s t e w a t e r a n d s l u d g e
T a b l e 2 .4 : C o m m o n
p r o c e s s e s a n d b i o s o l i d s p a t h o g e n r e d u c t io n r e q u i r e m e n t s .
............................................................................................................................................................................................1 7
T a b l e 2 .5 : T i m e - t e m p e r a t u r e r e g i m e s f o r C l a s s A s l u d g e ....................................................... 1 9
T a b l e 2 .6 : M
e t h o d s t o a c c o m p l i s h v e c t o r a t t r a c t i o n r e d u c t i o n .................................... 2 1
p r o p e r t ie s o f s e w a g e s l u d g e ...............................................................................2 3
T a b l e 2 .7 : T y p i c a l
T a b l e 2 .8 : C l a s s i f i c a t i o n
T a b l e 2 .9 : T y p i c a l
o f s l u d g e t r e a t m e n t p r o c e s s e s .......................................................2 4
28
l o g r e m o v a l s o f p a t h o g e n s in s e w a g e s l u d g e t r e a t m e n t
T a b l e 2 .1 0 : C o e f f ic ie n t s
fo r s t o ic h io m e t r ic e q u a t io n s fo r a n a e r o b ic t r e a t m e n t .
........................................................................................................................................................................................... 3 2
T a b l e 2 .1 1 : T y p i c a l / ° ,
T a b l e 2 .1 2 : P e r m i t t e d
Y, a n d q v a l u e s f o r a n a e r o b i c p r o c e s s .......................................3 3
f r e q u e n c ie s fo r i n d u s t r ia l , m e d ic a l , a n d s c ie n t if ic u s e s .
........................................................................................................................................................................................... 5 9
T a b l e 2 .1 3 : U
p p e r t e m p e r a t u r e l im it s f o r g r o w t h o f v a r i o u s o r g a n i s m s
(B
rock,
2 0 0 1 ) ..............................................................................................................................................................................7 5
T a b l e 2 .1 4 : F r e q u e n c i e s
a n d q u a n t u m e n e r g i e s a s s o c i a t e d w it h v a r i o u s
PHENOMENA...............................................................................................................................................................8 3
T a b l e 3 .1 : S o l i d s
T a b l e 3 .2 : M
content
(% )
in t h r e e s a m p l i n g l o c a t i o n s .................................................. 9 0
i c r o w a v e p o w e r c a l i b r a t i o n t e s t .................................................................................. 9 2
T a b l e 3 .3 : E x p e r i m e n t a l
T a b l e 3 .4 : S l u d g e
c o n d i t i o n s f o r p e n e t r a t i o n d e p t h t e s t ....................................... 9 4
m i x in g c o n d i t i o n s f o r t h e f i r s t b a t c h t e s t .............................................9 6
T a b l e 3 .5 : E x p e r i m e n t a l
T a b l e 3 .6 : B e n c h - s c a l e
T a b l e 3 .7 : S a m p l e
c o n d i t i o n s f o r t h e s e c o n d b a t c h t e s t ........................................ 9 9
a n a e r o b i c d i g e s t e r e x p e r i m e n t a l c o n d i t i o n s ......................1 0 4
pretreatm ent for
T a b l e 3 .8 : P r e t r e a t m e n t
RET t e s t s .............................................................................. 1 1 0
c o n d i t i o n s o f i s o l a t e d f e c a l c o l if o r m s f o r b i o a s s a y s .
........................................................................................................................................................................................ 1 1 3
T a b l e 3 .9 : A g a r o s e
g e l c o n c e n t r a t i o n ...................................................................................................1 2 1
T a b l e 3 .1 0 : S u m m a r y
o f a n a l y t i c a l m e t h o d s .............................................................................. 1 2 7
T a b l e 3 .1 1 : R e c o m m e n d e d
T a b l e 4 .1 : D e t e r m i n a t i o n
T a b l e 4 .2 : In i t i a l
p r e s e r v a t i v e s a n d h o l d i n g t i m e s ........................................... 1 3 3
o f p e n e t r a t io n d e p t h a n d a t t e n u a t io n f a c t o r
144
a n d f in a l t e m p e r a t u r e s a f t e r a g i v e n d u r a t io n o f m i c r o w a v e
IRRADIATION......................................................................................................................................................... 151
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X lll
4.3: R e l a t i v e m e t h a n e p r o d u c t i o n r a t e s a t 35°C in t h e s e c o n d b a t c h t e s t .
......................................................................................................................................... 155
T a b l e 4.4: R e l a t i v e BMP v a l u e s a t 35°C c o m p a r e d w i t h BMP a t 25°C................ 162
T a b l e 4.5: D e t e c t i o n l i m i t s o f f e c a l c o l i f o r m s in ADS, PS, a n d WAS.................. 166
T a b l e 4.6: A v e r a g e c o l i f o r m r e d u c t i o n o f PS w i t h m i c r o w a v e s a n d t h e r m a l
t r e a t m e n t s ............................................................................................................................................................ 168
T a b l e 4.7: A v e r a g e c o l i f o r m r e d u c t i o n o f ADS w it h m i c r o w a v e s a n d t h e r m a l
TREA TM ENTS ................................................................................................................. 170
T a b l e 4.8: A v e r a g e c o l i f o r m n u m b e r r e d u c t i o n o f WAS w i t h m i c r o w a v e s a n d
T able
THERM AL TREATMENT...................................................................................................................................... 1 7 2
T a b l e 4 .9 : C h a n g e s
in f e c a l c o l i f o r m c o u n t s d u r i n g l o n g - t e r m s t o r a g e a t
4°C .
.........................................................................................................................................174
T a b l e 4 .1 0 : C h a n g e s
in
%
in h ib it io n a f t e r a s e r ie s o f m ic r o w a v e ir r a d ia t io n a n d
CONVENTIONAL HEATING................................................................................................................................ 1 7 6
T able
4.11: DNA c o n c e n t r a t i o n s
T a b l e 4 .1 2 : S t a t i s t i c a l
T a b l e 4 .1 3 : R e s u l t s
and
OD 260/280 r a t i o .................................................................196
v a l u e s o f f e c a l c o l i f o r m d e t e c t i o n i n e a c h r e a c t o r ..
201
o f p a ir e d t -t e s t o f f e c a l c o l if o r m s f o r e a c h p r e t r e a t m e n t
..........................................................................................................................................................................................2 0 3
T able
T able
5.1:
5.2:
S um m ary
Sum m ary
S p r in g s
T able
5.3:
o f c a p i t a l a n d t o t a l p r e s e n t w o r t h c o s t s .....................................
o f c o s t e s t im a t io n s a t t h e s o u t h c o m p l e x o f t h e
N
215
in e
WWTP................................................................................................................................................... 2 1 6
c o m p a r i s o n b e t w e e n TP AD a n d m i c r o w a v e p r e / p o s t
C ost
PASTEURIZATION...................................................................................................................................................2 1 7
T a b l e 5 .4 : E s t i m a t i o n
o f e l e c t r i c i t y c o s t s f o r p o s t / p r e p a s t e u r i z a t i o n ..................2 2 0
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1
1. INTRODUCTION
1.1 Background
The volume o f wastewater produced around the world has been significantly
increased due to urbanization and industrialization.
Thousands of American cities
disposed their raw sewage directly into lands, rivers, lakes, and bays thirty years ago.
Current wastewater treatment plants use a combination o f physical, chemical, and
biological processes to treat wastewater. As a result, biosolids, the nutrient-rich organic
materials resulting from the treatment of sewage sludge, are produced and increased with
increase in wastewater.
The United States currently has about 16,000 wastewater treatment facilities
(Pincince et al., 1997). Approximately 8 to 9 million dry tons o f biosolids are produced
each year by municipal wastewater treatment facilities in the United States (U.S.). In
total, these facilities generate about 20,000 dry tons o f biosolids per day. Furthermore,
their management cost is approximately one half of the total annual cost for wastewater
treatment (Barrett, 1996; WERF, 1996). The U.S. Environmental Protection Agency
statistics indicate that nearly 50% o f the biosolids are applied to the land for beneficial
use. Table 1.1 shows the projections on the percentage o f biosolids that will be used or
disposed o f in 2000, 2005, and 2010. Beneficial use of biosolids is expected to increase
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2
from 60% in 1998 to 66% in 2005 and 70% in 2010. Thus, by 2010, disposal might only
account for about 30% of all biosolids generated.
Table 1.1: Projections o f sludge use and disposal in the US.
Beneficial use (%)
Year
Land
Advanced
application
treatment
Disposal (%)
Other
Surface
beneficial
disposal
use
landfill
Incineration
Other
1998
41
12
7
17
22
1
2000
43
12.5
7.5
14
22
1
2005
45
13
8
13
20
1
2010
48
13.5
8.5
10
19
1
Source: U.S. EPA (1999a).
1.2 Problems
Wastewater treatment systems have been developed to remove both soluble and
suspended organic matter from sewage wastewater, resulting in a liquid sludge containing
about 2-3% by weight of dry solids.
The biological wastewater treatment process
concentrates various pollutants in wastewater into sludge. The sludge contains not only
organic and inorganic matter, but also hazardous contaminants such as bacteria and
viruses as pathogens and vector attractions, oil and grease, nutrients (nitrogen and
phosphorus), heavy metals, and synthetic organic compounds.
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3
Particularly, pathogens and vector attractions are major concerns during the final
disposal of sludges. There are four major types o f human pathogenic organisms found in
biosolids: (1) bacteria, (2) viruses, (3) protozoa, and (4) helminthes. All contaminants
and pathogens accumulated in sludge must be removed when disposed o f for beneficial
use, such as land application as a fertilizer or soil conditioner. Domestic wastewater also
contains many pathogenic microorganisms that can cause harmful diseases to human
beings (U.S. EPA, 1999).
Sludge treatment and disposal are worldwide problems. Nowadays, ocean
disposal is forbidden in most countries, and incineration ash o f sludges should be treated
like a hazardous waste.
It is undoubtedly considered that land application of sludge
would be the most sustainable method.
Recently, the New York Times and other prominent newspapers have reported
accidents due to infection from biosolids applied to farmlands. In addition, on July 31,
2000, the National Whistleblower Center in Washington, D.C. requested the U.S. EPA to
immediately suspend regulations that allow the dumping of sewage sludge onto farmland.
According to the Hazard Identification Report by the Centers for Disease Control's
National Institute o f Occupational Safety and Health (NIOSH), approximately 220,000
colony forming unit (CFU) fecal coliforms per gram o f sample were found at collected
bulk samples from different locations within the biosolids storage site.
Furthermore,
enteric bacteria were detected in the collected air samples at the Class B biosolids land
application site. This indicates that there remains a significant exposure risk (NIOSH,
1999).
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4
On November 25, 1992, pursuant to Section 405 o f the Clean Water Act (CWA),
the U.S. EPA promulgated a regulation (40 CFR part 503) to protect public health and the
environment from reasonably anticipated adverse effects of certain pollutants in sewage
sludges (58 FR 9248, February 19, 1993). The final rule was issued effective on October
25, 1995. For Class A sludges, fecal coliform densities must be less than 1,000 most
probable number (MPN) per gram o f total dry solids, or Salmonella sp. bacteria must be
less than 3 MPN per 4 grams o f total dry solids. Class A sludge is allowed for general
distribution while Class B sludge is allowed only for application to agricultural land and
some fruit trees (U.S. EPA, 1999b). The hazard that is associated with Class B biosolids
is a function o f the number and type o f pathogens in the treated sludge relative to the
minimum infective dose and the exposure level. To protect public health, the U.S. EPA
rule prescribes a restricted period o f up to one year to limit public access to lands where
Class B biosolids have been applied.
Class B biosolids may contain pathogens in
sufficient quantity to warrant restricted public access and special precautions for exposed
workers (U.S. EPA, 1999a). Class A biosolids are sewage sludge that has undergone
treatment by processes that further reduce pathogen concentrations resulting in an end
product that is virtually pathogen-free. These processes include irradiation, composting,
heat drying, heat treatment, pasteurization, thermophilic aerobic digestion, and alkaline
stabilization.
Many wastewater treatment plants in the U.S. are currently evaluating ways of
increasing pathogen destruction in biosolids to make Class A sludge. Many technologies
have been developed to reuse biosolids for safe land application. However, they require
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5
the energy-intensive pre-drying and pre-heating o f biosolids to satisfy the regulation of
70~80% moisture content (U.S. EPA, 1999).
1.3 Objectives
Several technologies have so far been developed for generation o f Class A
sludges.
Technologies were designed to simultaneously increase the efficiency of
pathogen reduction and enhance digestibility for anaerobic digestion.
Microwave
irradiation has been assumed to be an attractive method because of the synergistic
microwave effect on pathogen destruction and thermal heating for anaerobic digestion at
35°C. Since substances in sludges from wastewater treatment plants can be easily heated
with microwave power, and microwave energy has a strong ability to penetrate dielectric
materials to produce thermal or synergistic microwave effects on microbes, it has been
postulated that microwave irradiation technology could be applied to biosolids treatment.
The objective of this study was to develop a new technology of generating Class
A sludge from wastewater treatment plant (WWTP) sludge using microwaves. There has
been extensive research on the use of microwaves for treatment of wastewater treatment
plant sludge.
No study has so far been reported in this area except on the use of
microwaves for sludge drying. The specific objectives of this study are as follows:
1. Compare the effect o f microwave irradiation on pathogen destruction with
conventional heating;
2. Evaluate pathogen destruction mechanisms by microwaves;
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6
3. Assess anaerobic digestibility and organic reduction after pretreatment o f sludge
with microwaves; and
4. Conduct an economic analysis of microwave technology in biosolids treatment.
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7
2. LITERATURE REVIEW
2.1 Sludge Generations and Disposal in the U.S.
The population in the United States has increased dramatically over the past 20
years.
These increases have contributed to an increase in the volume of biosolids
produced since 1972 (Bastian, 1977).
In recent years, the emphasis on biosolids
management has changed from disposal to the beneficial use. According to BioCycle's
1997 survey, land application and surface disposal account for almost 60 percent of
biosolids use. Over the year, land application has been increasingly managed to protect
human health and environment from various potential harmful constituents typically
found in sewage sludge, such as bacteria, viruses, and other pathogens; metals (e.g.,
cadmium and lead); toxic organic chemicals; and nutrients (U.S. EPA, 1995).
As shown in Table 2.1, there are over 16,000 wastewater treatment plants and
approximately 7 million dry tons o f biosolids are produced as o f 1999 in the United
States (BioCycle, 1999). The estimated biosolids generation amounts in 2000, 2005, and
2010 in the United States estimated by EPA are shown in Figure 2.1. The production of
biosolids is approximately over 1 pound per person per day, and that is roughly
equivalent to the weight of solid waste produced daily (U.S. EPA, 1999a).
Future
biosolids production is expected to increase from 6.9 million dry tons in 1998 to 7.1
million dry tons in 2000, 7.6 million dry tons in 2005, and 8.2 million dry tons in 2010.
These increases are mostly due to anticipated increases in population, the increase in
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8
POTWs (Publicly Owned Treatment Works) using secondary treatment, and the
subsequent increases in quantities o f biosolids (U.S. EPA, 1999a).
Table 2.1: Total biosolids generated and management practices.
State
Total
WWTP
Total
Land
Landfill Incineration Surface Composting Other*
generated
application
disposal
biosolids
Dry tons/yr
%
%
%
%
%
%
Alabama
268
47,000
59
25
1
0
1
14
Alaska
70
n/a
n/a
n/a
n/a
n/a
n/a
n/a
Arizona
150
50,000
n/a
n/a
n/a
n/a
n/a
n/a
Arkansas
350
n/a
n/a
n/a
n/a
n/a
n/a
n/a
California
238
750,000
68
9
6
0
11
6
Colorado
450
76,800
80
17
0
3
n/a
0
Connecticut
84
23,700
0
10
82
0
8
0
Delaware
29
21,000
16
1
0
0
3
80
Florida
2798
270,000
66
17
8
1
3
6
Georgia
507
n/a
12
26
2
0
3
57
Hawaii
34
20,000
0
84
0
1
15
0
Idaho
n/a
n/a
65
0
0
20
10
5
Illinois
538
391,400
68
30
0
1
0
I
Indiana
500
60,000
95
0
0
0
5
0
Iowa
76
50,000
95
0
3
0
2
0
Kansas
175
n/a
60
25
5
0
5
5
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9
State
Total
WWTP
Total
Land
generated
Landfill Incineration Surface Composting Other*
application
disposal
biosolids
Dry tons/yr
%
%
%
%
%
%
Louisiana
250
n/a
27
38
15
10
5
5
Maine
101
31,800
49
8
0
0
43
0
Maryland
308
194,000
56
8
5
0
26
5
Massachusetts
130
174,000
1
30
15
0
13
41
Michigan
n/a
84,000
n/a
n/a
n/a
n/a
n/a
n/a
Minnesota
210
265,000
29
20
51
0
0
0
Mississippi
335
n/a
n/a
n/a
n/a
n/a
n/a
n/a
Missouri
277
227,000
31
2
60
7
0
0
Montana
300
9,100
56
35
0
3
1
5
Nebraska
350
n/a
95
0
0
0
3
5
Nevada
20
38,500
15
80
0
0
5
0
New
Hampshire
76
18,000
11
47
22
0
20
0
New Jersey
342
262,700
4
4
25
0
7
60
New Mexico
48
20,000
5
40
n/a
10
25
n/a
New York
584
360,000
10
17
31
0
15
27
North
Carolina
342
150,000
n/a
n/a
n/a
n/a
n/a
n/a
North Dakota
290
4,160
25
75
0
0
0
0
Ohio
1200
392,000
58
8
31
1
n/a
2
Oklahoma
400
70,000
85
15
0
0
0
0
Oregon
217
50,000
99
1
0
0
3
2
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10
State
Total
WWTP
Total
Land
generated
Landfill Incineration Surface Composting Other*
application
disposal
biosolids
Dry tons/yr
%
%
%
%
%
%
South
Carolina
102
120,000
30
54
I
1
5
9
South Dakota
287
5,900
73
3
0
24
0
0
Tennessee
240
n/a
66
34
0
0
0
0
Texas
2500
450,000
50
35
0
3
9
3
Utah
25
45,000
53
4
0
16
27
0
Vermont
92
7,000
20
26
4
0
50
0
Virginia
300
225,000
54
10
30
0
6
0
Washington
30
75,000
85
5
10
n/a
n/a
0
-
n/a
-
Wisconsin
485
n/a
98.4
Wyoming
10
3,600
95
West Virginia
-
-
-
-
1.2
0.4
0
0
0
0
0
5
0
0
-
♦Other: Includes lime stabilization, heat drying/pelletization, and other methods.
Source:
BioCycle (1999).
Disposal o f biosolids is expected to decrease because o f regulatory influences,
voluntary improvements in biosolids quality, and the resulting increase in biosolids use.
Regulatory influences include the increased restrictions on incineration, surface disposal,
and landfilling in the Part 503 Biosolids Rule, the Part 258 Landfill Rule, and various
state requirements, which also have driven up the costs of these disposal methods (U.S.
EPA, 1994). Figure 2.2 provides projections on the percentages of biosolids that will be
beneficial use or disposal in 2000, 2005, and 2010. Beneficial use (e.g., land application,
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11
composting, and landfill cover) o f biosolids is expected to increase from 60% in 1998 to
63% in 2000,6 6 % in 2005, and 70% in 2010.
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12
8.2
l/tsC.'.-.
6.9
£
c
o
■>f.««!
7
w.c~
g%:'.
-4:6m
1972
2000
1998
2005
2010
Year
Figure 2.1: Projections of biosolids generations in the U.S. (Source: U.S. EPA, 1999a).
IV W
22
22
17
14
20
80
60
E
o
<u
a.
40
20
13
19
□ O ther disposal
10
□ Incineration
8.5
7.5
—
12
□ Surface d isposal
13
125
135
mam
□ O ther beneficial u se
II I
1996
2000
2005
□ A dvanced treatm ent
■ Land application
2010
Year
Figure 2.2: Projections of use and disposal in the U.S. (Source: U.S. EPA 1999a).
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13
Thus, disposal might only account for about 30% of all biosolids generated in
2010. The amount o f biosolids used beneficially is expected to increase to 4.5, 5.0, and
5.7 million dry tons in 2000, 2005, and 2010, respectively.
The beneficial use of
biosolids is expected to continue to increase in the future, while the trend toward disposal
decreases slightly.
2.2 Regulations on pathogen requirements of Biosolids
Sewage sludge, the residue generated during treatment of domestic sewage, is
often used as an organic soil conditioner and particle fertilizer in the United States and
many other countries (U.S. EPA, 1999b). The US EPA 503 rule defines sewage sludge
biosolids as a solid, semi-solid, or liquid residue generated during the treatment of
domestic sewage in a treatment works (U.S. EPA, 1999b). The term “sewage sludge” has
largely been replaced by the term “biosolids.” “Biosolids” specially refers to sewage
sludge that has been undergone treatment and meets federal and state standards for
beneficial use (U.S. EPA, 1999b). Figure 2.3 shows the typical flows of generation of
sewage sludges and biosolids. When treated properly, sewage sludge becomes biosolids,
which can be safely recycled and applied as fertilizer.
As mentioned in Chapter 1, the use and disposal of treated sewage sludge
(biosolids), including domestic septage, are regulated under 40 CFR Part 503 (Title 40 of
the Code o f Federal Regulation, Part 503). This regulation, promulgated on February 19,
1993, was issued under the authority o f Clean Water Act (CWA) as amended in 1977 and
the 1976 Resource Conservation and Recovery Act (RCRA). The regulations are based
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14
on how the residual solids are handled and processed at the treatment facility and/or at
subsequent processing facilities (U.S. EPA, 1999b).
Domestic Sewage ~|—|—| Industry (wastewater)
WWTPs
Effluent
V
Class A
Sewage Sludges
h
i
Sewage Sludge Treatment |im ^> | Biosolids
i D igestion, Drying, C om posting
| Lim e stabilization, Irradiation
' H eat treatm ent, Etc
Class B
Figure 2.3: Schematic o f generation o f sewage sludges and biosolids.
The Part 503 regulation mostly discusses pathogens, which are organisms or
substances capable of causing disease.
The four major types o f human pathogenic
organisms are bacteria, viruses, protozoa, and helminthes.
All may be present in
domestic sewage (U.S. EPA, 1999b). Major pathogens o f concern that may be present in
sewage sludges are listed in Table 2.2. These pathogens can cause infection or disease if
humans and animals are exposed to sufficient levels.
Typical levels of pathogens in various stages of wastewater and sludge are shown
in Table 2.3. It can be clearly seen that significant level o f pathogens exist in biosolids
after sludge digestion. The U.S. EPA classified sewage sludges into Class A and Class B
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15
according to pathogen reduction requirements (U.S. EPA, 1999b). The goal o f the Class
A requirements is to reduce the pathogens in sewage sludge including enteric viruses,
pathogenic bacteria, and viable helminth ova to below detectable level at which they are
infectious.
Table 2.2: Major pathogens associated with raw domestic sewage and sewage sludges.
Pathogen
class
Bacteria
Examples
Disease
Shigella sp.
Salmonella sp.
Salmonella typhi
Vibrio cholerae
Enteropathogenic-Escherichia coli
Yersinia sp.
Campylobacter jejuni
Campylobacteriosis (gastroenteritis)
Viruses
Hepatitis A virus
Norwalk viruses
Rotaviruses
Polioviruses
Coxsackie viruses
Echoviruses
Infectious hepatitis
Acute gastroenteritis
Acute gastroenteritis
Poliomyelitis
"flu-like" symptoms
"flu-like" symptoms
Protozoa
Entamoeba histolytica
Giardia lamblia
Cryptosporidium sp.
Balantidium coli
Amebiasis (amoebic dysentery)
Giardiasis (gastroenteritis)
Cryptosporidiosis (gastroenteritis)
Balantidiasis (gastroenteritis)
Ascaris sp.
Taenia sp.
Necator americanus sp.
Trichuris trichuria sp.
Ascariasis (roundworm infection)
Taeniasis (tapeworm infection)
Ancylostomiasis (hookworm
infection)
Trichuriasis (whipworm infection)
Helminths
Bacillary dysentery
Salmonellosis (gastroenteritis)
Typhoid fever
Cholera
A variety of gastroenteric diseases
Yersiniosis (gastroenteritis)
Source: National Research Council (1996).
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16
Table 2.4 shows common processes and biosolids pathogen
reduction
requirements for Class A and Class B. The processes approved by the U.S. EPA for class
A sludge require in general higher temperatures than those for class B sludge because
most pathogens will be killed at a higher temperature for a certain duration.
Table 2.3: Typical numbers o f microorganisms in wastewater and sludge.
Raw sewage
Raw sludge
Digested sludge3
(No./100 mL)
(No./g sludge)
(No./g sludge)
109
107
106
8 ,0 0 0
1,800
18
Enteric viruses
(PFUC)
50,000
1,400
210
Helminth ova
800
30
10
1 0 ,0 0 0
140
43
Organism
Fecal coliforms
(MPNb)
Salmonella (MPN)
Giardia lamblia
cysts
a Mesophilic anaerobic digestion,
MPN = Most Probable Number, c PFU = Plaque-forming
units. Source: Dean and Smith (1973); Engineering Science Inc. (1987); Feachem et al. (1980);
Gerba et al. (1983); Logsdon et al. (1985); National Research Council (1996); U.S. EPA (1991);
U.S. EPA (1992).
Fecal coliforms are used as an indicator organism, indicating that they were
selected and monitored with other indicator organisms. The correlations between fecal
coliforms and Salmonella sp. were developed by Farrell in 1993. It was reported that
fecal coliform correlate with very low level of Salmonella sp. in composted sewage
sludge (Farrell, 1993; U.S. EPA, 1992a).
In addition, lime effectively destroyed Salmonella sp. in sludges, but leaves
surviving fecal coliforms (Farrell et al., 1974). The fecal coliforms re-grew higher than
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17
1,000 MPN per gram. Fecal coliform densities may be high compared to other pathogen
densities and may be overly conservative. For this reason, all o f the Part 503 Class A
alternatives allow the direct measurement of Salmonella sp. or fecal coliform analysis,
but do not require both (U.S. EPA, 1999b).
Table 2.4: Common processes and biosolids pathogen reduction requirements.
Class A
Process
Class B
Composting1
Composting 2
Heat drying (pasteurization)
Air drying
Lime sterilization
Lime stabilization
Thermophilic aerobic digestion
Anaerobic digestion
Beta, gamma ray irradiation
Aerobic digestion
AND
OR
Fecal coliforms <1000 MPN/g TS
(averaging 7 samples over 2
(averaging 7 samples over 2 weeks.)
weeks.)
Pathogen
Fecal coliforms < 2 million MPN/g TS
OR
OR
Salmonella sp. < 3 MPN/4 g TS
Enteric viruses < 1 PFU/4 g TS
Salmonella sp. < 2 million MPN/g TS
Helminth ova < 1/4 g TS
composting material is maintained at 55°C for 3 days.
Using windrow composting, the
temperature is maintained at 55°C for 15 days or longer, during which time the windrow piles are
turned a minimum o f five times.
2Using either in-vessel, aerated static piles, or windrow
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composting, the temperature is maintained at 40°C for 5 days. For 4 hours within the five-day
period, the temperature is to exceed 55°C. Source: U.S. EPA (1999b).
2.2.1 Regulations for Class A Pathogen Requirements
Alternatives for meeting Class "A" pathogen requirements regulated by the US
EPA are summarized below:
Alternative 1: The temperature o f sludges that is used or disposed shall be
maintained a specific value for a period o f time. Biosolids must be subject to one
o f the following time-temperature regimes (Table 2.5).
Alternative 2: Biosolids treated in a high pH-high temperature process with specific
pH, temperature, and air-drying requirements as follows:
o
pH must be elevated greater than 12 for 72 hours or longer,
o
Temperature must be maintained above 52 °C for at least 12 hours during
the period that the pH is greater than 12.
o
Biosolids must be air dried to over 50 % solids after the 72-hour period of
elevated pH.
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19
Table 2.5: Time-temperature regimes for Class A sludge.
Temperature-Time
Heating
Percent solids
Time
temperature
period equation
TS > 7 %
Shall be >
50°C
20
Shall be >
min.
D = 131,700,000/10°141
TS ^ 7 %, heated by either
warmed gases or immiscible
liquid
Shall be >
50°C
Shall be >
15 sec.
D = 131,700,000/ 10°
TS < 7 %
-
TS < 7 %
Shall be >
50°C
15 sec. <
30 min.
Shall be >
30 min.
141
D = 131,700,000/10°141
D = 50,070,000/10 ° ,4t
t: °C, D: days
Alternative 3: Biosolids treated in other known processes with comprehensive
monitoring o f the biosolids for enteric viruses and viable helminth ova until the
process has shown adequate reduction o f pathogens.
Alternative 4 : Biosolids treated in other unknown processes.
All pathogen
requirements must be met.
Alternative 5: Biosolids treated in processes to further reduce pathogens (PFRP,
Appendix B to Part 503) such as:
o
Composting: In-vessel or aerated pile composting must maintain biosolids
at 55°C or higher for 3 days, or windrow compost biosolids must maintain
55°C for 20 days or longer and must be turned at least 5 times,
o
Heat Drying: Biosolids are heated with hot gases to raise the percent of
solids to 90 % or higher. Temperatures must exceed 80°C.
o
Heat Treatment: Liquids biosolids are heated above 180°C for 30 minutes.
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20
o
Thermophilic Aerobic Digestion: Aerobic conditions must be maintained
while the holding time o f 10 days is maintained at 55-60°C.
o
Beta Ray Irradiation: Beta rays are used from an accelerator at a minimum
of 1.0M radat20°C.
o
Gamma Ray Irradiation: Certain isotope gamma rays at 20°C used for
irradiation.
o
Pasteurization: Biosolids are maintained at 70°C or above for 30 minutes
or longer.
Alternative 6 : Biosolids treated in a process equivalent to a PFRP process approved
by the authority issuing permits and meeting all pathogen requirements.
In addition, methods to reduce vector attraction outlined and defined in Part 503
are summarized in Table 2.6.
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21
Table 2.6: Methods to accomplish vector attraction reduction.
Option 1
Option 2
Meet 38% reduction in volatile solids content.
Demonstrate VAR with additional anaerobic digestion in a bench-scale
unit.
Option 3
Demonstrate VAR with additional aerobic digestion in a bench-scale
unit.
Option 4
Meet specific oxygen uptake rate for aerobically digested biosolids.
Option 5
Use aerobic processes at greater than 40 °C for 14 days or longer.
Option
Alkali addition under specific conditions.
6
Option 7
Dry biosolids with no unstabilized solids to at least 75% solids.
Option
Dry biosolids with unstabilized solids to at least 90% solids.
8
Option 9
Option 10
Inject biosolids beneath the soil surface.
Incorporate biosolids into the soil within 6 hours o f application to or
placement on the land.
Option 11
Cover biosolids placed on a surface disposal site with soil or other
material at the end o f each operating day.
Option 12
Alkaline treatment o f domestic septage to pH 12 or above for 30
minutes without adding more alkaline material.
Source: U.S. EPA (1999b).
Vector attraction reduction (VAR) is an attempt by the U.S. EPA to address
stability.
“Exceptional Quality” (EQ) biosolids must satisfy one o f the first eight
methods (Options 1-8 in Table 2.6) for vector attraction reduction, whereas “NonExceptional Quality” (Non-EQ) biosolids are required to meet any one (Options 1-12)
reduction in vector attraction.
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22
EPA has used the term "Exceptional Quality" (EQ) to refer to sludge that meets
specified low pollutant and pathogen limits and that has been treated to reduce the level
o f degradable compounds that attract vectors. EQ sludge is a product that required no
further regulation (U.S. EPA, 1999b). Biosolids that are disposed o f in a landfill are not
required to meet any method of VAR.
2.2.2 Regulations for Class B Pathogen Requirements
Class B pathogen requirements can be met in three different ways. The objective
o f all three alternatives is to ensure that pathogenic bacteria and enteric viruses are
reduced in density. It should be demonstrated that a fecal coliform density is less than 2
million MPN or CFU per gram total solids biosolids in the treated sewage sludge.
However, viable helminth ova are not necessarily reduced in Class B biosolids (U.S. EPA,
1999b). Three alternatives for Class B sludges are as follows:
Alternative 1: Fecal coliforms as an indicator organism are used prior to the biosolids
use or disposal.
Fecal coliforms should be less than 2 million MPN or CFU
(colony forming units) per gram o f dry solids.
Alternative 2: Biosolids must be treated by one o f the five Processes to Significantly
Reduce Pathogens (PSRP) such as: 1) aerobic digestion, 2) air drying, 3)
anaerobic digestion, 4) composting, and 5) lime stabilization.
Alternative 3: Biosolids should be treated in a process equivalent to a PSRP
permitted by U.S. EPA.
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23
2.3 Current Sludge Treatment Technologies
All sludges are not produced equally. The physical, chemical, and biological
characteristics of sludges (biosolids) are dramatically affected by the treatment processes.
The first source of biosolids is the primary sludge. The secondary treatment produces
activated sludge containing 0.8~2.5% solids, which are harder to thicken and dewater
than the primary sludge. Both primary and secondary sludges are further treated through
stabilization in an anaerobic digestion, an aerobic digestion, or a lime treatment system.
After stabilization, biosolids are usually dewatered through centrifugation, a
vacuum filter, a filter press, bed drying, or heat drying with varying degrees of efficiency.
Typical characteristics o f biosolids are presented in Table 2.7. The general processes
including PSRPs (processes to significantly reduce pathogens) and PFRPs (processes to
further reduce pathogens) that can be applied to sludge treatments are classified in Table
2.8.
Table 2.7: Typical properties of sewage sludge.
Primary sludge (%) Secondary sludge (%) Digested sludge (%)
Total solids
4.0-10.0
0.8-2.5
2.5-7.0
Volatile solids
60-80
59-88
30-60
Source: Metcalf and Eddy (1994).
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24
Table 2.8: Classification of sludge treatment processes.
Treatments
Sludge treatment processes
Physical treatment
Thickening, dewatering, air drying
Chemical treatment
Lime stabilization
Biological treatment
Anaerobic digestion 1, thermophilic anaerobic digestion (TP AD),
aerobic digestion, composting
Thermal treatment
Radiation treatment
Pasteurization, heat treatment, heat-drying (pelletization),
Zimpro®-process, wet air oxidation, incineration, pyrolysis
P-ray, y-ray, electron beam
2.3.1 Physical Treatment
2.3.1.1 Thickening and Dewatering
Thickening and dewatering are necessary before sludge treatment or use. such as
composting, heat drying, or biosolids preparation for land application.
Thickening
processes include those that partially decrease the water content of liquid sludge to 1 - 2 %
solids content, while dewatering occurs generally greater than 5% solids. The purpose of
thickening ahead of dewatering is to improve the solids content of dewatered material
and/or to increase the capacity o f the dewatering units (WERF, 1996). The typical sludge
dewatering utilizes gravity thickening, flotation thickening, centrifugation, vacuum
filtration, and pressure filtration. Prior to dewatering, biosolids are usually conditioned
and thickened.
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25
In conditioning, chemicals, such as ferric chloride, lime, or polymers, are added to
facilitate the separation of solids by aggregating small particles into larger masses (U.S.
EPA, 1995). It was reported that the activated sludge floes are held together to form a 3dimensional matrix by means o f exocellular polymers and divalent cations (Higgins and
Novak, 1997).
In addition, potassium ions appear to play a more important role in
determining the nature o f activated sludge floes compared to sodium.
An optimal
potassium concentration exists (0.25-0.50 meq/L) to achieve optimal dewatering
properties and to minimize supernatant turbidity (Murthy and Novak, 1998). More exotic
technologies, which have been applied to the dewatering of sewage sludge, are electro­
dewatering (Lockhart, 1986), acoustic dewatering (Ensminger, 1986), and vacuum
combined with electrical and ultrasonic fields (Muralidhara, 1986).
Centrifugation is used both to thicken and to dewater sludges. Thickening by
centrifugation involves the settling o f sludges particles under the influence o f centrifugal
forces (Metcalf and Eddy, 1994). Historically, centrifuges have been largely applied to
dilute sludges with solids content in the range o f
1
to 2% dry solids and have produced
sludges in the range of 7 to 13% dry solids.
Vacuum and pressure filtration are also well-developed technologies, which are
widely used to produce dewatered sludge cake normally in the range of 25 to 40% dry
solids. New technical developments continue to improve the performance o f pressure
dewatering equipment (Gildemeister, 1988).
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26
23.1.2 Air drying
A conventional sludge treatment is to digest the sludge followed by evaporation
on a drying bed. Air drying involves placing biosolids on a sand bed and allowing them
to dry through evaporation and drainage. This process can produce solids content in
primary biosolids o f as high as 45 to 90%. Air drying systems are relatively simple in
terms o f operation but require large land areas and relatively long periods of time and,
therefore, tend to be used by small POTWs that generate small amounts o f biosolids.
Larger POTWs rely on mechanical dewatering systems such as vacuum filters, plate-andframe filter presses, centrifuges, and belt filter presses (U.S. EPA, 1995).
According to the PSRP (processes to significantly reduce pathogens) in the U.S.
EPA regulation, sewage sludges dries for a minimum 3 months during air-drying.
However, traditional distribution o f this type of sludge is dependent on die-away of
Ascaris lumbricoids eggs for at least six months. This limitation was established based
on bacteriological and helminthological data. However, enteric viruses are known to
accumulate in sludge, and being capable to survive for a long period (Vasl et al., 1983).
Such air drying can achieve Class B pathogen control. Extensive and long term drying
may be able to produce Class A biosolids, but the specific requirements have not yet been
defined for this level of treatment (Schafer, 2001).
2.3.2 Chemical Treatment
Historically, alkaline stabilization has been implemented using either quicklime
(CaO) or hydrated lime [Ca(OH)2 ], which is added to either liquid biosolids before
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27
dewatering or dewatered biosolids in a contained mechanical mixer. The addition of lime
(CaO) to dewatered sludge results in a heating of the sludge to temperatures between 55
and 70°C. Traditional lime stabilization processes are capable o f producing biosolids
meeting the minimum pathogen and vector attraction reduction requirements found in the
40 CFR Part 503 rules governing land application of biosolids; sufficient lime is added so
that the pH of the biosolids/lime mixture is raised to 12 or above for a period of 2 hours.
The elevated pH helps to reduce biological action and odors (U.S. EPA, 1995).
As a pretreatment step, accelerated hydrolysis o f long chain polymeric material
under acidic or alkaline conditions or by extracellular enzymes and heat treatment of the
sludge were studied.
While alkaline hydrolysis has been reported to significantly
increase organic acid yield from acidogenesis (Hashimoto et al., 1991).
23.3 Biological Treatment
23.3.1 Anaerobic Digestion
Anaerobic digestion is one of the oldest biological wastewater treatment processes,
having first been used more than a century ago (McCarty, 1982). Conventional anaerobic
digestion is widely used in sewage treatment plants, its main purpose being to stabilize
organic sludges prior to disposal on land or water. During anaerobic digestion, anaerobic
bacteria carry out wide range o f reactions to break down complex organic molecules.
Anaerobic digestion involves biologically stabilizing biosolids in a closed tank to reduce
the organic content, mass, odor, and pathogen content o f biosolids. Typical log removals
o f pathogens in sewage sludge treatment are summarized in Table 2.9.
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28
Anaerobic bacteria that thrive in the oxygen-free environment convert organic
solids to carbon dioxide, methane, and ammonia.
Anaerobic digestion is typically
operated at about 35°C, but also can be operated at higher temperatures (> 55°C) to
further reduce solids and pathogen content o f the stabilized biosolids (U.S. EPA, 1995).
Table 2.9: Typical log removals o f pathogens in sewage sludge treatment.
PSRP 1 treatment
Bacteria
Viruses
Parasites
Anaerobic digestion
0.5-4.0
0.5-2.0
0.5
Aerobic digestion
0.5-4.0
0.5-2.0
0.5~4.0
Composting
2.0-4.0
2.0-4.0
2.0-4.0
Air drying
0.5-4.0
0.5~4.0
0.5~4.0
Lime stabilization
0.5~4.0
4.0
0.5
: PSRP (processes to significantly reduce pathogens), Source: U.S.EPA (1999b).
i.
Microbiology and Stoichiometry
In
the
anaerobic
chemoheterotrophic,
digestion,
mixed
culture
of
anaerobic
organisms,
non-methanogenic and methanogenic microorganisms,
work
together to bring about the conversion of organic sludges and waste. Three theoretical
stages are thought to occur during the anaerobic digestion process, ( 1 ) hydrolysis and
fermentation, (2) acetogenesis and dehydrogenation, and (3) methane fermentation.
As shown in Figure 2.4, hydrolysis is the first process in which organism can
breakdown complex organic matter, such as carbohydrates and proteins, into monomers
and oligomers such as carbohydrates, proteins, and lipids, to simple carbohydrates, amino
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29
acids, glycerol, fatty acids, and hydrogen plus carbon dioxide (Metcalf and Eddy, 1994;
Rittmann and McCarty, 2001), and hydrolytic products, then, are fermented into some
short chain fatty acids including acetate, alcohols, carbon dioxide, and hydrogen.
Complex Organics
• Carbohydrates
• Proteins
• Lipids________
100%
Hydrolysis &
Fermentation
jq
60 %,
o/o
15%
Other intermediates
(Long-chain fatty acids)
Acetogenesis &
Dehydrogenation
5%
Propionate
13%
50 V
2%
10%
Acetate
28%
72%
Methane fermentation
CH4. CO,
Bacteria Group:
I: Fermentative bacteria
2: Hydrogen-producing, Acetogenic bacteria
3: Hydrogen-consuming. Acetogenic bacteria
4: CO,-reducing Methanogens
5: Aceticlastic Methanogens
Figure 2.4: Methane formation in anaerobic digestion (Source: McCarty, 1964; Parkin
and Owen, 1986).
In the second stage, acidogens or acid formers ferment the breakdown products
from the first stage to simple organic acids (acetogenesis stage). These microorganisms
are nonmethenogen and consist o f facultative and obligate anaerobic bacteria (Metcalf
and Eddy, 1994). The final stage involves the conversion of the hydrogen and acetic acid
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30
formed by the acid formers to simpler end products, principally methane and carbon
dioxide by methanogenic bacteria. In this stage, hydrogen is used as an electron donor,
with carbon dioxide as an electron acceptor to form methane, while acetate is cleaved to
form methane from the methyl group and carbon dioxide from the carboxyl group in
fermentation reaction.
Waste stabilization is accomplished when methane and carbon dioxide are
produced during the anaerobic digestion process. Methanogenic bacteria can only use a
limited number o f substrates for the formation o f methane, such as CO 2 + H2, formate,
acetate, methanol, methylamines, and carbon monoxide. The specifics o f each reaction
are shown as the follows (Speece, 1996):
4H 2 + H + + HCO 3"
CH 4 + 3 H ,0
4HCOOH + H ,0 -» C H 4 + 3H C 03~ + 3I-T
CHjCOO" + H ,0 -►CH 4 + H C O j'
kJ/mole = -136 (2.1)
kJ/mole =-130 (2.2)
kJ/mole - -30 (2.3)
CH 3 C H 2COO + 2 H ,0 + H+ ->1.75CH 4 +1.25CO, + 1.5H ,0
kJ/mole = -53 (2.4)
4CH3OH
3CH 4 + HCO3' + H+ + H 20
kJ/mole = -314 (2.5)
As shown in all biological processes, an electron balance should be maintained,
because most electron equivalents or BODl entering the anaerobic process are conserved
in CH4. Therefore, the removal of BODl, or waste stabilization, depends totally on the
formation o f methane.
The empirical molecular formula for the organic matter (and
electron donor, or BOD l) can be C nHaObNc (Rittmann and McCarty, 2001). A certain
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31
portion o f its electron equivalents, fs, is net synthesized into biomass. Equations 2.6 to
2 .8
show the conversion o f acetate to methane, which take place through the acetoclastic
reaction. Assume that acetate reacts as an electron donor and CO2 as electron acceptor
for the reaction, then using half reaction (Rittmann and McCarty, 2001):
-Rd
CHjCO" + - H 20 - > - C 0 2 + -H C O J + H+ + e‘
8
8
8
R. : - C 0 7 + H + + e " - » - C H 4 + —H 20
a g
8
4
(2.7)
R :-C H ,C O O ‘ + - H , 0 -►- C H 4 + -H C 0 7
8
3
8
‘
8
(2.6)
8
4
8
(2.8)
3
In addition, balanced overall stochiometric equation for generalized organic
wastes is:
-
9df
df
df
C„H O N + (2n + c - b
s .)H ,0 -> —*-CH4 +
n a b c
20
4
*
8
4
df df
df
df
df
(n-c
5 .- ^ - ) C O , + ^ - C 5 H 70 2N + ( c
5.)NH: + ( c - —^ )H C 0 3
5
8
2
20 5
2
20
4
20
3
where d =4n + a - 2b - 3c , fs = fraction o f waste organicmatter
convertedto
(29)
synthesized or
cells, fe = portion converted for energy, such that fs + fe = 1. At steady
state, fs can be estimated:
‘fs
"l + ( l - f d)b©,
= ‘f su
l+ b 0 x
(2 . 10)
where fd = fraction of biodegradable biomass, 0 x = solid retention time.
Typical values for fs° and b (decay rate, time'1) for methane fermentation of
common organic compounds are summarized in Table 2.10. The fs° values include the
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32
methanogens and all bacteria needed to convert the original organic matter to acetate and
H2.
Table 2.10: Coefficients for stoichiometric equations for anaerobic treatment.
Typical chemical
Waste components
Carbohydrates
Proteins
Fatty acids
Municipal sludge
/,°
YfgVSSa/gCODcons.,
b (d ay 1)
C 6H 10O 5
0.28
0 .2 0
0.05
C 16H24O5N4
0.08
0.056
0 .0 2
C 16H32O2
0.06
0.042
0.03
C 10H 19O3N
0 .1 1
0.077
0.05
formula
CODcons.: COD consumed. Source: Rittmann and McCarty (2001).
Typically, when microorganism uses an electron donor for synthesis, a fraction of
electrons ( fe° ) is transferred to the electron accepter to provide energy for conversion of
the other fraction o f electrons ( f ° ) into microbial cells. The fraction f ° can be converted
into biomass (Y = VSS production/CODremoved = yield for cell synthesis). In the microbial
kinetics, the maximum specific growth rate is the function of maximum substrate
utilization rate (q ) and biomass yield. Table 2.11 shows the typical f ° , Y, and q values
for anaerobic process.
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33
Table 2.11: Typical / “ , Y, and q values for anaerobic process.
Acetate
H 2 oxidizer
Sulfate reducers
fermenters
H2 and formate
h2
e* acceptors
Acetate
C 02
s o 42-
(Z>
C-source
Acetate
C0 2
Acetate
/"
0.05
0.08
0.05
0.08
0.04
0.45
0.28
0.057
g VSS/g Ac
g VSSa/g H2
g VSS/g h 2
g VSS/g BODl
7
3
1.05
8.7
Y
O
1
Acetate BOD
O
Acetate BOD
e‘donor
hO
J
Parameters
11
g BOD[/VSS-d
g H2/g VSSad
g H2/g VSS-d
g Ac/g VSS*d
Y was computed using a cellular VSSa (C5H7O2N), q is computed using q =le'eq/g VSSa-d.
Source: Rittmann and McCarty (2001).
ii. Major Requirements for Anaerobic Digestion
pH and Alkalinity Requirements
The desired pH for anaerobic treatment is between
6 .6
and 7.6.
The main
chemical species controlling pH in anaerobic treatment are those related to the carbonic
acid system, as governed by following reactions (Rittmann and McCarty, 2001):
CO, (aq) = C 0 2 (gas)
(2 . 1 1 )
C 0 2 (aq) + H 20 = H 2C 0 3
(2 . 1 2 )
h 2c o
(2.13)
hco3
-
3
= h + + h c o 3_
=
h
+ + c o 32-
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(2.14)
34
H 20 = H+ + 0 H '
(2.15)
Alkalinity is defined as the acid-neutralizing capacity of water. Alkalinity can be
quantified from proton condition on the species o f interest (Sawyer et al., 1994):
[H+]+ [Alkalinity] = [h C O ' j+ 2[C0*' ]+ [OH'J (mol/L)
(2.16)
With respect to the usual pH and conditions of anaerobic treatment, the
concentrations of H+, CO 3", and O H ' are relatively low compared with H C O j. In
addition, alkalinity can be expressed in the units o f mg/L as CaC0 3 . Equation 2.16 can
be converted to following approximation:
^ ! “ £ = [h c o ;]
50,000
1
1
(2.17)
Equation 2.17 shows that the total alkalinity in an anaerobic process is effectively
equal to the bicarbonate concentration (bicarbonate alkalinity).
Figure 2.5 shows the
limits o f normal anaerobic treatment regarding bicarbonate alkalinity, CO2 percentage in
digester gas, and pH in anaerobic treatment (Rittmann and McCarty, 2001).
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35
50
40
/Limits of / s
normal anaerobic ^
/ treatment
^
$
uT
co
? 30
3</5
60
•S
O
u
xv
20
10
0
250
500
1000
2500
5000
10,000
Bicarbonate alkalinity - mg/1 as CaC03
25,000
Figure 2.5: The relationship among bicarbonate alkalinity, percentage o f CO 2 in digester
gas, and pH in anaerobic treatment.
Solids Retention Time (SRT) and Complex Substrates
Most sludges produced in municipal wastewater treatment plants contain protein,
carbohydrates, and fats.
Because o f this, it was impossible to measure biomass
production. O’Rourke evaluated the effect o f solids retention time (cell residence time)
on overall performance (Rittmann and McCarty, 2001). Figure 2.6 shows the effect of
SRT on overall performance by COD, VS (volatile solids), and methane production for
25°C. Also shown are details for the variations o f proteins, carbohydrates, lipids, and the
individual volatile acids.
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36
320
a
ou
CH.
E
16
160
COD
-SO
U
Volatile solids
0
16
U
S'.
L ipids
8
\
E
=o
P rotein
Cellulose
0
4
Total
.3 °
2 <
3
2 =
= J?
3“
"E
> S
SL
A cetic
/P r o p i o n ic
B utyric
/V a le r ic
0
10
20
30
40
50
SRT (day)
Figure 2.6: Effect of solids retention time in anaerobic digestion on (a) overall COD and
volatile solids removal and gas production, (b) protein, cellulose, and lipid removal, and
(c) volatile acid concentrations.
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37
Temperature
Another effect o f organism growth rate in anaerobic digestion process is
temperature. In anaerobic treatment, microbial growth rate increases in general roughly
two times for each 10°C rise in temperature within the usual mesophilic operation
(10~35°C). The mesophilic microorganisms can function effectively at the optimum
temperature around 40 to 45°C, and thermophilic microorganisms at the range o f 55 to
65°C (Rittmann and McCarty, 2001).
The advantage o f high temperature is faster
reactions and smaller required tank volume (Rittmann and McCarty, 2001). The change
in rate o f chemical reaction with temperature is generally expressed with the following
Arrhenius equation:
k,
RT2 T,
(2.18,
where Ti andT 2 = temperature, Ea = activation energy, but generally unknown, and R =
gas constant. The ratio o f E /R is taken to be a constant. The term 'T | T2’will not vary
greatly at mesophilic and thermophilic range, and this may be taken to be a constant.
Then, Equation 2.18 can be simplified to the following form (Rittmann and McCarty,
2001):
k 2 = k,e*(T2' T,)
(2.19)
Therefore, the change in rate of reaction with different temperature can be
quantified by <j> values, which has been experimentally obtained by several researchers.
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38
iii. Pretreatment of Sludge for Increase in Digestibility
Thermophilic Aerobic Pretreatment
In Japan, the study o f using thermophilic aerobic digestion as a pretreatment for
anaerobic digestion to solubilize organic sludge was investigated. The bacterium type
SPT2-1 isolated from the thermophilic aerobic digestion reactor was selected, which
secreted protease and solubilized the sludge most efficiently (Hasegawa et al., 2000).
Hasegawa et al. reported that sludge was rapidly solubilized by the thermophilic aerobic
bacteria, approximately 40% o f the organic sludge removed after the treatment for 1-2
days.
Production of biogas during anaerobic digestion of the pretreated sludge was
increased by 50% when compared with the sludge without pretreatment.
Ultrasonic Pretreatment
Ultrasonic lysis is a cell disruption technique that has been used in the
biochemical field. The bacteria cells can be disintegrated by pressure waves and shear
forces generated by high-power ultrasonic generator leading to release intracellular
organic substances (Wang et al., 1999). As a result, methane generation during anaerobic
digestion increased by over 64% after ultrasonic pretreatment. Optimum pretreatment
time was about 30 minutes.
Neis et al., (2000) demonstrated a decrease in sludge residence time from 16 to 4
days without any decrease in degradation efficiency.
The organic substances in the
ultrasonic-pretreated sludges showed higher methane production than untreated
substances. It appears that complex matter is solubilized into biodegradable substances
that are more easily converted into methane gas.
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39
Ozonation Pretreatment
The study of an oxidative pre-treatment with ozone to disintegrate the sludge cells
and increase their biodegradability was presented in recent years (Weemaes et al., 2000).
The results showed that an ozone dose o f 0.1 g
0 3 /g
biosolids could enhance the
anaerobic digestion of WAS by a factor 1.35, and the overall COD removal efficiency of
the biosolids increased to approximate 72%.
Mechanical Pretreatment
The study of using mechanical pretreatment process with high-pressure jet was
performed with WAS by jetting and colliding to a collision-plate at 30 bar pump pressure
and an anaerobic digestion with pretreated WAS in a bench scale (Choi et al., 1997).
Choi et al. reported that the range o f volatile solids removal efficiencies increased from
2-35 % to 13-50 % with WAS pretreated under 30 bar, and the digestion rate
coefficients increased from 0.01 day'1 to 0.04 day'1.
Nah et al. (2000) stated that mechanical pretreatment with WAS decreased the
required SRT in anaerobic digester from 13 to 6 days without major adverse effects on
process efficiency and effluent quality, and enhanced volatile mass reduction and unit gas
production.
2.3.3.2 Aerobic Digestion
Aerobic digestion involves biologically stabilizing biosolids in an open or closed
vessel or lagoon using aerobic bacteria to convert the organic solids content to carbon
dioxide, water, and nitrogen. Pathogens and odors (and the potential to generate odors)
are reduced in the process. Aerobic digestion is commonly used by smaller POTWs (U.S.
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40
EPA, 1995). The reduction o f fecal coliforms and fecal streptococci was increased as the
temperature was raised from 20°C to 40°C (Kuchenrither and Benefield, 1983). The
high-temperature operation (i.e., higher than 55°C) of aerobic digestion is becoming more
popular because it can produce biosolids with lower pathogen levels and higher solids
content.
23.3.3 Temperature Phased Anaerobic Digestion (TPAD)
The objective o f the TPAD (Temperature phased anaerobic digestion) is to
increase volatile solids destruction and pathogen reduction (Class A) during anaerobic
digestion. The system combines the advantages of thermophilic (40~80°C) digestion
with advantages of mesospheric digestion (less-odorous digested sludge). In the lab tests,
TPAD achieved more destruction o f volatile solids and fecal coliform than conventional
single-stage digestion, with solids retention time of 28 days versus 40 days (Han and
Daegue, 1996). Fecal coliforms were less than 100 MPN/g solids and Salmonella sp. was
not detected at the TPAD sludge treatment facility operated with 55°C (Kelly et al.,
1993).
2.3.3.4 Composting
Composting is the decay o f organic matter by microorganisms in an environment
that controls the size and porosity o f the pile, thereby facilitating an increase in
temperature (typically to about 55 to 60°C) to destroy most pathogens.
Composting
involves mixing dewatered biosolids with a bulking agent (such as wood chips, municipal
yard trimmings, bark, rice hulls, straw, or previously composted material) and allowing
the biosolids mixture to decompose aerobically for a period of time. The biosolids mass
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41
is initially increased due to the addition o f the bulking agent. The bulking agent is used
to lower the moisture content o f the biosolids mixture, increase porosity, and add a source
o f carbon. Depending on the method used, the biosolids compost can be ready in about 3
to 4 weeks o f active composting followed by about one month of less-active composting.
Three different composting processes are typically used: windrow composting, aerated
static piles, and in-vessel composting (U.S. EPA, 1995).
Composting is a proven method for pathogen reduction, provides considerable
volume reduction, and produces a valuable product. The 1997 survey indicated there are
338 biosolids composting facilities in the U.S., representing a small increase from the
number o f facilities reported (Pollution Engineering, 1997).
The safety o f biosolids
composting programs and biosolids use are governed by federal and state regulations
(U.S. EPA, 1994). Applicable options in Part 503 for vector attraction reduction include
either reduction of VS (volatile solids) mass by a minimum of 38% or use o f aerobic
processes at greater than 40°C (average temperature 45°C) for 14 days or longer.
Biosolids compost that achieves pathogen reduction, vector attraction reduction, and
pollutant concentration limits has the designation o f EQ (exceptional quality).
If the finished compost product meets 40 CFR Part 503 Biosolids Rule Class A
specifications for the highest level o f pathogen and vector control and specific metals
limits, the compost product can be widely used, like any other fertilizer or soilconditioning product. Additionally, biosolids compost is often used for soil blending,
landfill cover, application to golf courses, mine reclamation, degradation o f toxics,
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42
pollution prevention, erosion control, and wetlands restoration.
It also is used for
agricultural purposes, such as application to citrus crops (BioCycle, 1997).
A number o f studies have been performed on pathogen levels at composting
facilities. Burge and Cramer (1978) compared the pathogen survival during windrow
composting with stockpiling. Survival o f the pathogens in a windrow was significantly
higher than in static pile. Clark et al. (1984) reported the health risks associated with the
exposure to composted sewage sludge.
Clark et al. found that the workers showed
frequently positive for Aspergillus fumigatus.
Yanko (1988) expressed the concern o f
pathogen regrowth in compost products. Hu et al. (1996) found that composting reduced
Giardia cyst concentrations to approximately the same levels as sludge storage, but over
a much shorter time period.
2.3.4 Thermal Treatment
Thermal treatment is normally used as a conditioning process for raw or digested
sludges. As a result, thermal treatment greatly improves the dewatering properties of
waste activated and primary sludges (Everett, 1972; Marshall, 1974).
Thermal
pretreatment of activated sludge resulted in an increase of 60% in methane production
and a 36% decrease in effluent volatile suspended solids (VSS) from an anaerobic
digester (Haug et al., 1978). While primary sludge did not increase methane production,
but appeared to increase dewaterability. The optimum temperature for activated sludge
pretreatment was approximately 175°C and above 175°C gas production decreased
because o f inhibitory substances.
On the other hand, odorous compounds normally
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43
associated with heat treatment are significantly reduced during digestion o f thermally
pretreated sludge. Thermal pretreatment o f mixture of WAS and PS in a 1:1 ratio lead to
14% increase in methane gas production, 16% VSS reduction in effluent, and increased
sludge dewaterability. No toxicity problems were reported (Haug et al., 1978).
2.3.4.1 Heat Treatment
Liquid sewage sludge is heated to a temperature of 180°C or higher with or
without pressure for over 30 minutes. Heat treatment is for stabilization and conditioning.
It is used to coagulate solids, break down the gel structure, and reduce the water affinity
o f sludge solids. As a result, the sludge is sterilized and dewatered readily. A typical
heat treatment process is the low pressure Zimpro
(fb
system.
However, in general,
supernatant from the heat treatment process shows high BOD concentrations, and thus
special side stream treatment is required before it is transferred to the mainstream
wastewater treatment process.
Several types of heat treatment processes have been
developed, but many are no longer in operation (Metcalf and Eddy, 1994).
2.3.4.2 Wet Air Oxidation
The process is the same as the heat treatment, except higher pressure and
temperature are required to oxidize the volatile solids more completely. The Zimpro*
process involves wet oxidation o f untreated sludge at elevated temperature and pressure.
Untreated sludge is ground and mixed with a compressed air. The mixture is pumped
through a heat exchanger and then enters a reactor, which is pressurized to keep the water
in liquid phase at the reactor operating temperature of 175 to 315°C with up to 20 MN/m 2
(Metcalf and Eddy, 1994).
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44
Organic matter by wet oxidation is oxidized to carbon dioxide and water, with the
ash produced remaining in the water. However, a major disadvantage associated with
this process is the high-strength recycled liquor. For that reason, wet air oxidation has
been implemented in only a limited number of installations since its introduction in the
early 1960s (Metcalf and Eddy, 1994).
2 J .4 J Heat Drying and Pelletizing
Sewage sludge is dried by direct or indirect contact with the hot gases to reduce
the moisture content of the sludge to 10% or lower. Either the temperature o f the sewage
sludge particles exceeds 80°C or the wet bulk temperature of the gas leaves the dryer
exceeds 80°C (U.S. EPA, 1999a). Heat drying involves using active or passive dryers to
remove water from biosolids. It is used to destroy pathogens and eliminate most o f the
water content, which greatly reduces the volume of biosolids. Several highly successful
products are prepared using this process (e.g., Milorganite produced and sold by the city
of Milwaukee since the 1920s) (U.S. EPA, 1995). In some cases, the heat-dried biosolids
are formed into pellets. These products are very dry and, therefore, can save significantly
on transportation costs over compost or other forms of biosolids with higher moisture
contents.
Thus, heat drying and pelletizing might be process of choice for urban
communities where distances to agricultural land can be substantial.
Boston,
Massachusetts, and New York City, for example, pelletize and transport their biosolids
out o f state (U.S. EPA, 1995).
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45
23.4.4 Sterilization or Pasteurization
The temperature o f the sewage sludge should be maintained at 70°C or higher for
30 minutes or longer for pasteurization (U.S. EPA, 1999a).
Based on European
experience, heat pasteurization is a proven technology, but requires skills such as boiler
operation and the understanding o f high temperature and pressure processes (Metcalf and
Eddy, 1994).
Sterilization can be achieved by a number of techniques that employ various
combinations of high temperature, extended time, and high pH (Strauch, 1988). The
following simple temperature/time combinations can be employed: 1) 65°C for at least 30
minutes; 2) 70°C for at least 25 minutes; 3) 75°C for at least 20 minutes; 4) 80°C for at
least 10 minutes. Batch-oriented pre-pasteurization (prior to anaerobic digestion) has
been conducted in Europe. Most o f these installations were designed to meet pathogen
requirements, which typically mandated 65 to 70°C for about 30 minutes, or 55 to 60°C
for several hours (Schafer, 2001).
In 1972, the first pre-pasteurization plant (MTS) was put into service in Dissen in
Germany. In 1979, the first commercial pre-pasteurization plant (SULZER) unit was
installed in Switzerland. The pasteurization system was designed to handle between 5
and 20 m3 of sludge per day.
Figure 2.7 shows the diagram of SULZER pre­
pasteurization system. The temperature range was 65 to 70°C for 30 minutes to achieve
disinfection of sludges (Clements, 1982). As a result, the enterobacteriaceae was well
below 100 enterobacteriaceae per gram of sludge (Figure 2.8).
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46
Pasteurization is a heat treatment process where the sludge is pretreated at
elevated temperature for a certain period. The pasteurization process is always carried
out in combination with a stabilization process. Sludge pasteurization after digestion has
been abandoned because on subsequent storage massive regrowth of Salmonella took
place, sometimes to levels even higher than original raw sludge.
from
T hickener
to Digester
1.
2.
3.
4.
Feed pump (fresh sludge)
Sludge/sludge heat exchanger
Feed pump (preheated sludge)
Pasteurization tank
5.
6.
7
8.
9.
Circulation pump
Heat exchanger (hot water/sludge)
Discharge pump (past, sludge)
Control cabinet
Boiler
Figure 2.7: Diagram o f SULZER pre-pasteurization system.
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47
03
LL
Figure 2.8: Enterobacteriaceae counts in the pre-pasteurization system in Switzerland.
For this reason, post-pasteurization has been prohibited in Switzerland since May
1981 (Berger, 1982).
This phenomenon is not fully understood but it may be
hypothesized that the elimination o f competitive flora is an important factor.
If the
pasteurization process is carried out prior to digestion, the final product is no longer
susceptible to regrowth of Salmonella (Bruce, 1982).
2.3.4.5 Submerged Combustion
Most pasteurization processes has always been in the second stage operation. In
1973, the Niersverband (France) tried submerged combustion heating in pilot tests to
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48
evaluate whether this process was effective for a sewage sludge operation. The unit
consists o f a heat exchanger and a burner with a down-flow tube. The burner with the
incinerator chamber is arranged above the heat exchanger (Figure 2.9).
The
pasteurization and digestion chamber work as one unit, and thus, is considered as
simultaneous pasteurization digestion (SPD).
flue gas cnincev
(30* - W C )
ignition burner
gas or oil burner
cascade ore-heating
combustion
chanber
ore-heated sludge
(30* - «5*C1
combustion
air
mnersion
tube
sludge overflow oioe
discharge oioe
Figure 2.9: Submerged combustion heating equipment of sewage sludge (Source: Kidson
el al., 1982).
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49
The SPD unit is operated continuously for 24 hours, when the raw sludge is
temporarily heated up to 75~85°C. As a result, the original content of enterobacteria in
loaded sludge o f 10s/mL in the digestion chambers decreased below the demanded
100/mL in roughly 70 days (Braun et al., 1982).
KJdson et al. (1982) investigated submerged combustion for pasteurization of
sludge before digestion. The conception, design, and early operation of an anaerobic
mesophilic digester modified to enable investigation of the conditions necessary for
pasteurization o f sewage sludge were discussed. Retention o f the sludge at temperatures
up to 55°C for a minimum period o f 3 hours is followed by digestion for a period of
12~30 days.
2.3.4.6 Incineration
Incineration of biosolids involves burning volatile matters in biosolids at high
temperatures in the presence o f oxygen.
Incineration o f biosolids produces ash as
byproduct, which is approximately 20 % o f the original volume. The process destroys
nearly all o f the volatile solids and pathogens, although compounds such as dioxin may
be formed (U.S. EPA, 1999b).
The main types of equipment for incineration are multiple-hearth and fluidized
bed. Fluidized-bed furnaces are known to have fewer problems than multiple hearth units
because o f more uniform combustion o f biosolids. The main product o f incineration, ash,
generally is disposed of to landfill. Incinerated ash shows that cadmium range from 70 to
900 mg/kg (Theis et al., 1984) and mercury range from 2 to 9 mg/kg. Dewling et al.
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50
(1980) found that 97.6% of the mercury in the sludge was in the exhaust gases even after
a water scrubbing system.
In the near future, incinerators will be subjected to regulation under provisions of
the Clear Air Act that are more stringent (U.S. EPA, 1999b). Numerous facilities that
incinerated biosolids have closed over the past decade because other biosolids
management options became more publicly acceptable or less expensive, even when the
facilities recovered energy (U.S. EPA, 1999a).
2J.4.7 Pyrolysis
In contrast to the combustion process such as incineration, the other technique
that can be applied to dewatered sewage sludge is pyrolysis. This technique generally
results in the production of a liquid fuel and an ash in absence of oxygen that can then be
burnt to provide heat energy for drying the sludge. The liquid tar and/or oil components
normally obtained are represented by the molecular structure of C6HgO. The following
equation has been suggested as a pyrolysis reaction (Metcalf and Eddy, 1994):
3(C6 H 10O 5) -►8H ,0 + C 6 HgO + 2CO + 2CO, + CH 4 + H 2 + 7C
(2.20)
Digested or dried sewage sludge has been pyrolyzed in an indirectly heated
fluidized bed reactor at temperatures ranging from 620 to 750°C.
The main gas
constituents by pyrolysis are hydrogen, methane, ethane, ethene, propene, carbon
monoxide and carbon dioxide. The oil fraction contains up to 30% aromatic compounds
(Kaminsky and Kummer, 1989).
The feasibility of biomass can be an alternative to
traditional fossil fuels for heat and power generation, which are equivalent to the existing
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51
combined reserves of oil, gas and coal, but future use will depend on technological
developments and environmental concerns.
2J.S Radiation
Radiation can be used to disinfect sewage sludge.
Radiation destroys certain
organisms by altering the colloidal nature o f cell contents. Gamma rays and beta rays are
representatives o f the two potential energy sources for use in sewage sludge disinfection.
However, there are important differences between beta and gamma rays. Gamma rays
can penetrate substantial thickness o f sewage sludges, but beta rays have limited
penetration ability and therefore are introduced by passing a thin layer o f sewage sludge
under the radiation source (U.S. EPA, 1999b).
23.5.1 Gamma Ray Irradiation
Gamma irradiation is one o f processes to further reduce pathogens (PFRPs) with
beta ray irradiation. Sewage sludge is irradiated with gamma rays from certain isotopes,
such as Cobalt 60 and Cesium 137, at dosages of at least 1 megarad at room temperature
(Figure 2.10).
Gamma irradiation technologies have already been investigated in the
disinfection of sludge and approved by the U.S. EPA to generate Class A sludge.
Irradiation o f dried sludges has been developed at the Sandia National Laboratory using
Cs-137, and the construction o f a commercial plant is planned in Albuquerque, New
Mexico. The construction of a gamma irradiation plant is in the planning stage in Canada
for disinfection of virus-contaminated wastewater effluents (U.S. EPA; U.S. EPA, 1979
& 1999a).
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52
Sludge
Inlet
Ground
Level
w.
Cobalt
Rods
Concrete,
Shielding
Sludge
Outlet
Figure 2.10: Schematic of gamma ray irradiation facility using cobalt-60 at
Geiselbullach, Germany (Source: U.S. EPA, 1979).
Vasl et al. (1983) compared gamma radiation with microwaves for the
disinfection o f bacteria and viruses. According to their results, gram-negative bacteria
are the most sensitive, followed by fecal streptococci. They suggested that both gamma
and microwave radiation of sludge are effective as possible solutions for the disinfection
o f sludge for reuse.
In addition, Watanabe et al. reviewed for the disinfection of municipal sewage
sludge cake by gamma-irradiation. Total bacterial count in the sludge cake in Japan was
in the range o f
1 .6
x 108 to 4.1 x 109/g. Coliform count in aerobically activated sludge
was from 1.8 x 107 to 4.8x 108/g, while in anaerobically digested sludge it was less than
8.3 x 107/g. The dosage to reduce the coliforms to undetectable levels ranged from 0.3 to
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53
0.5 Mrad, depending on the season.
In addition, it was observed that no coliforms
reappeared in 0.5 Mrad irradiated sludge cake during storage at 6~16°C and at 30°C. The
adequate disinfection dose is therefore considered to be 0.5 Mrad (Watanabe, 1984;
Watanabe et al., 1985).
Gamma and electron radiation have already been investigated in the disinfection
o f sludge and approved by the U.S. EPA as a means of generating Class A sludge. One
type o f disinfection, which has been less studied, is microwave radiation.
2.3.5.2 Beta Ray (electron beam) Irradiation
Sewage sludge is irradiated with beta rays from an electron accelerator at dosage
o f at least 1 Mrad at room temperature (20°C). Beta rays are electrons accelerated in
velocity by electrical potentials in the vicinity o f 1 millions volts (Figure 2.11).
The effectiveness of beta radiation in reducing pathogens depends on the radiation
dose. A dose of 1 Mrad or more will reduce pathogenic viruses, bacteria, and helminths
to below detectable levels. Lower doses may successfully reduce bacteria and helminth
ova but not viruses. Since organic matter has not been destroyed, sewage sludge must be
properly stored after processing to prevent contamination (U.S. EPA, 1999a).
The Japan Atomic Energy Research Institute (JAERI) has developed this
technology to increase the rate o f composting sludges by radiation for use in agriculture.
The technique relies on irradiating a thin layer o f sludge with an electron beam by a
Cockcroft-Walton accelerator. The penetration o f the electron beam achieved to a depth
of
6
mm (Hashimoto et al., 1991).
Although this type of process is proven, the
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54
application o f electron beams has been more limited than the application o f Cobalt due to
the limited penetration (U.S. EPA, 1979).
Input
Electron
Beam
Electron Beam
Scanner
Constant
Head Tank .
Underflow
Weir
Inclined X
Feed Ramp
High Energy
Disinfection
Zone
Sludge
Receiving
TanR
•*. Output
(Disinfected
Sludge)
Figure 2.11: Schematic o f beta ray scanner and sludge spreader (Source: U.S. EPA, 1979).
23.5.3 Infrared and Microwaves
The usage of microwave/infrared irradiation has risen over the last ten years.
Microwaves have been applied to many areas such as organic decomposition, medical
waste sterilization, killing o f pathogen in food, animal manures, and soil. There have
been studies on heating mechanisms, disinfection o f food, and industrial applications.
However, little research has been conducted on sludge disinfection using microwaves. In
fact, microwave efficiency for sewage sludge medium depends on the sludge
characteristics, water content, and composition (Reimers et al., 1986b).
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55
During the 1980s, various biophysicists identified a minimum o f six different
DNA denaturing frequencies within the lower microwave region. Each frequency had a
different effects on DNA, such as a helix zipper frequency, individual cation site parting,
coil tightening, and complete chemical bond disassociation (Reimers et al., 1986b).
Harris et al. (1989) evaluated the effectiveness and convenience o f microwave
irradiation as a method of disinfecting soft contact lenses.
Significant reductions in
bacteria colony counts were found after 30 seconds of microwave irradiation. Few of the
bacteria survived after 60 seconds o f microwave exposure and none survived after 90
seconds.
Najdovski et al. (1991) studied on the killing activity o f microwaves on some
non-sporogenic and sporogenic medically important bacterial strains.
Microwave
disinfection o f medical waste was studied by Cusack (1991). The energy efficient soil
disinfections by microwaves were studied by Mavrogianopoulos et al. (2000). The effect
o f initial soil temperature and soil moisture on energy consumption by microwave
radiation used for soil disinfection was studied. It is concluded that humidity o f the soil
and initial soil temperature are critical for a low-cost use of microwaves for soil
disinfections (Mavrogianopoulos et al., 2000). Cwiklinski et al. (1998) studied on the
use o f microwave energy and other thermal methods for eradicating fungi on agricultural
seeds. Improvements in seed quality were achieved by controlled application o f heat at
defined seed moisture contents.
The microwave effect on sterilizing bacteria (or virus) by high-power microwaves
was studied by Wu in 1996.
A series o f sterilizing experiments on several typical
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56
bacteria or viruses, such as Bacillus subtilis var.
nigar, Bacillus stearothermophilus,
Bacillus pumilus, Staphylococcus aureas, and Bacillus cereus, were performed. Under
the conditions of different sterilization duration and unequal intensity o f microwave
power irradiation onto the bacteria or virus, the sterilization efficiency was evaluated, i.e.,
the Bacillus subtilis can be considered as an optimum indicated bacterium for microwave
sterilization (Wu, 1996).
Since a purpose of this research is the evaluation of pathogen reduction by
microwaves, more discussion will be presented in Section 2.4 and Chapter 4.
2.4 Microwave
Microwave application as an industrial process has been originally conceived
since 1950s. “The advent of magnetron during World War II presented engineers and
scientists in industry, universities, and government establishments with a unique
challenge to put such a device for generating microwaves into peaceful and profitable use.
During the late 1940s and early 1950s, an intensive effort was made to obtain reliable
data on material properties, led by von Hippel (1954) and his co-workers at
Massachusetts Institute of Technology (MIT). Their pioneering work on the properties o f
many organic and inorganic materials in the frequency region between 100 and 10 10 Hz
has since formed, and still remains, a solid basis for the establishment o f radio frequency
and microwave energy techniques in industry” (Metaxas and Meredith, 1983).
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57
2.4.1 Electromagnetic Spectrum
Visible light is a type o f electromagnetic radiation. Other types include radio
waves, microwaves, infrared radiation, ultraviolet rays (UV), X-rays, and gamma rays.
All of these are basically similar in that they move at 299,792,458 meter/second (the
speed of light). The only difference between them is their wavelength, which is inversely
related to the amount o f energy. As shown in Figure 2.12 (Allahyar and Robitaille, 2000),
if the wavelength o f the radiation is short, the energy will be high (Metaxas, 1996).
Wavalangth
(inmalar*)
10* 10* 10’ 1
<
IQ-' Iff* 10•* Iff4 Iff* Iff* Iff* 10* 10* Iff'* 1Q-" Iff’*
to n u w
Sizaola
a
^
id
b o u se
b a s n h a tt
*
*
th is d o c
c e ll
-
/
bedena
I
v iru s
p ro te m
w a t e r m o le c u le
Common
__
fla m * O f w a v *
w*v®5
infrared
s
uaraviolel
iru c n > w a v e s
Sourcas
^
A M r a d io
Frequency
(w iv ts p i r u c )
" e o ft" x - r a y s
MR1
F M ra d io
■
*hanf i-n y j
tem j
ra d a r
s
p e o p le lig h t b u f t
a
l\^
, fd y
w f w a c tiv e
m a c ah i n e s e l e m■ e n t s
________
JQ*
g a m m a ra y s
._____ ,_________
10a
10«
10io
1 0 11
1 0 13
1013
jQis
fQic
io iT 101#
101*
1(H°
Energyof photon _____________________________________ ________________ ___
(alactronvolts) 10« 1(p 10r 10-» 10» io-« «•* io* 10-’ 1 10’ 10* 10* 104 10* 10*
Figure 2.12: Schematic o f electromagnetic spectrum (Allahyar and Robitaille, 2000).
Each frequency may be used in devices according to special characteristics. It has
been well known that low frequencies as well as sound are useful in communications,
microwaves and infrared in heating, visual light in vision and photosynthesis, X-rays in
the visualization of internal structures, lasers in holography, and UV in the activation of
the provitamin or the sterilization o f microorganisms (Copson, 1975).
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58
Microwaves lie in the region o f the electromagnetic spectrum between millimeter
waves and radio waves. Particularly, they are defined as those waves with wavelengths
between 0.01 and 1 m, corresponding to frequencies between 30 and 0.3 GHz (Figure
2.12). In a microwave oven, the radio waves generated are tuned to frequencies that can
be absorbed by the polar materials. The polar material simply absorbs the energy and
gets warmer. Microwaves are also emitted from the earth, cars, planes, and from the
atmosphere (Kingston and Haswell, 1997).
Microwave radar equipment operates at the lower wavelengths (0.01-0.25 m), and
much o f this band is used for telecommunications. In order to avoid interference with
these uses, the wavelengths at which industrial and domestic microwave apparatus may
operate isregulated at both national andinternationallevels (Copson,
majority
1975).
o f countries, 2,450±0.050 MHz is the majoroperatingfrequency
In the
for this
purpose, although other frequency allocations exist (Table 2.12).
Where an apparatus is built to operate outside these bands, efficient shielding
must be used to prevent radiation leakage. The frequencies of microwave heating come
under the rules of the Federal Communications Commission (FCC), which provide
certain frequencies for industrial, scientific, and medical (ISM) uses. The communication
has assigned four ISM frequencies, which conformed to the international radio
regulations adopted at Geneva in 1959 (Copson, 1975). These frequencies are:
o
915±25 MHz
o
2,450±50 MHz
o
5,800±75 MHz
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59
o
22,125±125 MHz
O f these frequencies, 2,450 MHz has been most widely used in the U.S. and other
countries.
Table 2.12: Permitted frequencies for industrial, medical, and scientific uses.
Area permitted
Frequency (MHz) Tolerance
434
0 .2 %
Austria, Netherlands, Portugal, Germany, Switzerland
896
10 MHz
United Kingdom
915
13 MHz
North and South America
Albania, Bulgaria, CIS, Hungary, Romania,
50 MHz
2,375
Czech /Slovak Republics
World-wide, except where 2.375 is used
2,450
50 MHz
3,390
0 .6 %
Netherlands
5,800
5 MHz
World-wide
6,780
0 .6 %
Netherlands
24,150
25 MHz
World-wide
40,680
25 MHz
United Kingdom
Source: Copson (1975).
2.4.2 Microwave Generator
Most industrial microwave heating systems demand power in excess o f 10 kW,
often extending into the range 100 kW to 1 MW.
The magnetron, a source o f
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60
microwaves, was developed intensively during the Second World War to cover a very
wide frequency range (500-100,000 MHz) in a wide range of power outputs for radar
and microwave heating (Roger, 1998).
The assembly o f the typical high power
magnetrons at 915 MHz (33 cm) and 2,450 MHz (12.2 cm) is illustrated in Figure 2.13.
O f the many parts of a microwave device, the magnetron is the most important
component since it is not only the source o f microwaves but also the determinant of
microwave intensity.
W AVEGUIDE
O U T PU T A N TEN N A
C E R A M IC
MAGNET
C O C L JN G
F IN S
\
•VA N ES
ANODE
BLOCK—"
STRA P,
CATHODE
RING S
filament
MACNETRON
H O U S IN G
FILAM ENT AND
C A T h C O E LEADS
(a)
T E R M IN A L S W»TH
R F C A P A C IT O R S
(b)
Figure 2.13: Schematic o f a typical magnetron as a microwave source: (a) schematic
plan view, (b) sectional view (Source: gallawa.com/microtech).
For the design of a magnetron, Figure 2.14 shows the schematic o f an electron
path in a magnetron shown in Figure 2.13. Assume that the radius o f the cathode is ‘a’
and the radius of the anode from center o f the magnetron is ‘b \ As soon as DC power is
supplied, electrons will be released from the cathode to the anode with a constant path
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based on given frequency. One important mechanism is that electrons cannot reach the
anode due to magnetic fields located outside of magnetron.
Correspondingly, a
continuously moving electron path inside of the magnetron allows for the production of
microwaves.
R=b
Cathode
m
R=a
Anode
Figure 2.14: Schematic of the electron path in a magnetron.
The following two equations with respect to the electric field and magnetic field
can be obtained:
(2.21)
V = b ! ( & n ~ F )!
B=
y 8 V m /q
b(1 - F >
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(2 .22 )
62
where V= voltage, B = magnetic intensity (kg/m2), b = radius of anode (m), a = radius of
cathode (m), q = charge of electron (1.602 x I O' 19 coulomb), m = mass o f electron (9.107
x
1 0 ’31
kg).
Using Equations 2.21 and 2.22, the critical path of the electron should be
determined by B and V. When the magnetron is designed, the combination between B
and V will determine the frequency (to, Hz). Since B (magnetic intensity), a, and b
(cylinder radiuses o f the anode and cathode) would typically be fixed, and the microwave
intensity will be determined by V (voltage).
In other words, the magnetron design
parameters such as power, density, frequency, and even efficiency should be determined
by the radius o f the anode and cathode, voltage, and magnetic intensity.
2.43 Microwave Heating Mechanism
The conventional methods of applying heat to material by the combustion o f fuels
such as wood, coal, oil, and gas can be classified as: ( 1) conduction by thermal
conductivity, (2) convection by circulation, and (3) radiation by heat waves (Copson,
1975).
The theory o f microwave heating has been developed by many scientists such as
Debye (1929), Frohlich (1958), Hill et al. (1969), and Hasted (1973). It has been known
that materials may be heated with the use o f high frequency electromagnetic waves. The
heating effect arises from the interaction of the electric field component of the wave with
charged particles in the material. Two major effects are responsible for the heating that
results from this interaction.
If the charged particles are free to travel through the
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63
material, a current will be induced which will travel in phase with the electric field. If, on
the other hand, the charged particles are bound within regions of the material, the electric
field component will cause them to move until opposing forces balance the electric force.
The result is a dipolar polarization in the material. Conduction and dipolar polarization
may give rise to heating under microwave irradiation. Therefore, two principal heating
methods exist; dielectric polarization and conduction. A third mechanism is interfacial
polarization, which is of limited importance.
2.4J.1 Dielectric Polarization
The dielectric effect on polar molecules has been known since 1912 (Debye,
1929). The heating of a material with microwave power is the result o f the complex
interaction between the electromagnetic field and the dielectric.
Particles within the
dielectric are displaced from their equilibrium positions due to the alternating
electromagnetic field. This is mainly the result of induced polarization and orientation
polarization (Curtis, 1999).
Dielectric polarization (dipolar polarization) is the phenomenon responsible for
the majority o f microwave heating effects observed in solvent systems. In substances
such as water, the different electro-negativities o f individual atoms results in the
existence o f a permanent electric dipole on the molecule. The dipole is sensitive to
external electric fields, and will attempt to align with them by rotation (“orientation” or
“pearl effect”), the energy for this rotation (dipole moment) being provided by the field
(Copson, 1975).
This realignment is rapid for a free molecule, but in liquids,
instantaneous alignment is prohibited by the presence o f other molecules.
A limit is
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64
therefore placed on the ability o f the dipole to respond to a field, which affects the
behavior o f the molecule with different frequencies of electric field (Kingston and
Haswell, 1977).
Under low frequency irradiation, the dipole may react by aligning itself in phase
with the electric field. The overall heating effect is small, because the molecule gains
some energy by this behavior and some is also lost in collisions. Under the influence o f a
high frequency electric field, on the other hand, the dipoles do not have sufficient time
(over relaxation time) to respond to the field, and so do not rotate. Since no motion is
induced in the molecules, no energy transfer and no heating take place in material
(Metaxas and Meredith, 1983).
Between these two extremes, at frequencies that are approximately those o f the
response times o f the dipoles, is the microwave region (Figure 2.15). The microwave
frequency is low enough that the dipoles have time to respond to the alternating field, and
therefore to rotate, but high enough that the rotation does not precisely follow the field.
This phase difference causes energy to be lost from the dipole in random collisions and
gives rise to dielectric heating. For any material, both the real and complex dielectric
constants will vary with frequency.
Debye formed the basis for current understanding of dielectrics (Debye, 1929 &
1935). The dielectric constant ( e f ) represents the ability of the material to store the
electric energy. While the loss factor (e*) represents the loss of the electric field energy
in the material (Stuchly, 1978).
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65
The complex permittivity (e*) defines the material behavior in the electric field,
indicating the relative permittivity o f the dielectric material. The complex permittivity is
expressed in terms o f the dielectric constant and loss factor as:
e - = e '- je *
(2.23)
Or the real complex dielectric constant attains a complex form as follows:
e* = e '- j s l ff
(2.24)
where j = V -T , s' = relative dielectric constant, e" = loss factor, and s'W = effective
loss factor. The complex permittivity of materials when normalized to the permittivity of
vacuum is referred to as the relative permittivity (e*r):
8 *r = £ ! = £ z i f L
e0
s0
(2 .2 5 )
where e0= 8.854 *10' 12 (Farads/meter). The relative dielectric constant and loss factor
are dimensionless quantities. The relative dielectric constant (real part) describes how
well the material can store electromagnetic energy and the dielectric loss factor
(imaginary part) represents the material's ability to convert this energy to heat (Curtis,
1999).
The term s" (loss factor) quantifies the power dissipation in the capacitor having a
dielectric filling with relative permittivity. The loss angle
8
is usually given in the form
of its tangent. It is related to the complex dielectric constant by:
Tan5 = —
e'
(2.26)
where 5 = phase difference between the electric field and the polarization o f the material.
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66
The variation o f the dielectric constants, s' (relative dielectric constant) and s"
(relative dielectric loss) with frequency for water is shown in Figure 2.15 (Metaxas and
Meredith, 1983). The term “loss” implies the conversion of electrical energy into heat,
and the term “relative” means relative to free space (Singh and Heldman, 1993).
Dielectric
lo sses d u e to
dipolar polarisation
Radio and
microwave
Dielectric losses
due to a to m ic a n d
electronic p o larisatio ns
In f r a - r e d
; 20GHz
o
2.45GHz
Frequency (f)
Figure 2.15: Variation o f e' and e" as a function of frequency (Source: Metaxas and
Meredith, 1983)
The maximum dielectric loss (e") for water would be at a frequency of
approximately 20 GHz (water), the same point at which the dielectric constant s' goes
through a point of inflection as it decreases with increasing frequency. The 2.45 GHz
operating frequency o f domestic ovens is selected to be distant from this maximum in
order to limit the efficiency of the absorption (Metaxas and Meredith, 1983). The heating
mechanism by microwaves, in fact, is associated with the frequency and penetration
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67
depth. For that reason, too efficient absorption by the outer layers would inevitably lead
to poor heating of the internal volume in large samples.
2.43.2 Conduction
If the irradiated sample is an electrical conductor, the charge carriers (electrons,
ions, etc.) are moved through the material under the influence o f the electric field,
resulting in a polarization (Figure 2.16). These induced currents will cause heating in the
sample due to any electrical resistance. For a very good conductor, complete polarization
may be achieved in approximately
18
10‘
seconds, indicating that under the influence o f a
2.45 GHz microwave, the conducting electrons move precisely in phase with the field.
,
1
Resistance
Microwave
Electric Field !
Sample
Figure 2.16: Conduction effect of electron in electric field.
If the sample is too conductive, such as a metal, most of the microwave energy
does not penetrate the surface of the material, but is reflected. However, the enormous
surface voltages that may still be induced are responsible for the arcing that is observed
from metals under microwave radiation.
2.4J.5 Theoretical Aspects of Electromagnetic Volumetric Heating
The microwave power will be attenuated as the electromagnetic fields penetrate
the dielectric, an effect depending upon the dielectric properties (Metaxas and Meredith,
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68
1983). Microwave heating involves the conversion of electromagnetic energy into heat.
Energy is transported through space or medium by means of electromagnetic waves. The
average power of microwave energy conversion to heat can be derived from Metaxas and
Meredith (1983) as follows:
Pw =cne 0 B i E 2V
(2.27)
where E = V/m and V = the volume o f the material (m3). Inserting the eo (permittivity of
free space) value of 8 .8 x 10‘12 (F/m) and m = 2 n f into Equation 2.27, the average power
o f conversion to heat is given by Metaxas and Meredith (1983):
Pav = 0 .556xlO 'lofe'E 2V
(2.28)
where Pm = power o f the fraction o f the electromagnetic fieldthatis converted to heat
(watts) and f =frequency (Hz).
Rearranging Equation 2.28, the dielectric heating
equation becomes:
P(power/volume) = 27if£0el,E2
(2.29)
where the volume o f dielectric material between the plate (the electromagnetic field) is
A(area)*d (distance) (m3), E = V/d (volt/meter), P (power/m3) = Pav/Ad (watts/m3), and to
= 2ttf (radians/second). This means that the power density dissipation is proportional to
frequency where the other parameters are constant. The volume of workload in the
microwave oven may be reduced as the frequency rises.
In addition, the heating conversion o f microwave energy was approximately
quantified with the following equation (Decarau and Peterson, 1986):
Pd =55.61 xlO ",4 E 2fe'tan 6
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(2.30)
69
where Pd = power dissipation (W/cm3) and E = electrical field strength (V/cm). The
dielectric constant (s') and the loss tangent (tan 5) depend on the properties o f the
material.
2.4.4 Penetration Depth
The electric field strength (E) and the frequency (f) represent the energy source of
microwaves. It is noted that the increasing E has a dramatic effect on the power density.
The energy transfer is influenced by the electrical properties o f the material.
The
distribution of energy within a material is determined by the attenuation factor a ' (Singh
and Heldman, 1993):
(2.31)
With the attenuation factor, the penetration depth of an electric field can be
calculated. The penetration depth is defined as the distance from the surface o f the
material at which the power drops to 1/e from its value at the surface (Copson, 1975).
von Hippel (1954) found that the depth Z below the surface of the material is the inverse
o f the attenuation factor when the electric field strength is 1/e of the electric field in the
space (Singh and Heldman, 1993). Thus,
(2.32)
From Equation 2.32, the penetration depth of microwave at 915 MHz is estimated
to be deeper than the penetration depth at 2,450 MHz. Figures 2.17 and 2.18 show the
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70
relationship between microwave frequency and penetration depth using two frequencies
(2,450 MHz and 915 MHz) applied to a cylinder of agar.
The temperature o f bactonutrient agar medium in one liter o f water was
determined by thermocouple probes after heating for 13 minutes for 2,450 MHz and 14
minutes for 915 MHz, respectively. The temperature gradient at 2,450 MHz is peripheral,
while that at 915 MHz may be described as “core” heating. This experiments shows
clearly the differences between frequency and penetration depth in microwaves heating
(Copson, 1975).
HEAT P E N E T R A T IO N O P A CAR C r i ' N O f R
AT 2 4 5 0 M e IRAQARA n GC HGIA)
Di m e n s i o n s 9 * h t x i o ‘ d i a
a l i g h t 2 6 LBS
PER CFNT AGAR 2 J
POWFR AT 2 4 SO Me I 0 * *
TIME OF HFATING lA M lN
INITIAL T FM P£A A TuftC
/
75 *T
/
I3O0
I2O0
IIQ 0
3
H E IG H T C IN C H E S }
F 100 0
90 0
BO V
70*O sA M LtL R
{INCHES!
Figure 2.17: Three-dimensional temperature gradients in agar cylinder heated at 2,450
M H z(l kW) for 13 minutes (Copson, 1975).
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71
In addition, the depth o f penetration for microwave power is defined in two
different ways. First, Lambert’s expression for power absorption gives:
P = P0e"2otd
(2.33)
where P = power at the penetration depth, Po = initial incident power; d = penetration
depth, and a ' = attenuation factor. If the power is reduced to 1/e o f the incident power at
depth d, P/Po = 1/e, then, from Equation 2.33,2a'd = 1 or d = l/2a'.
H U T DtM eTftATlO* O f AGAR
C r u N O E R AT
DIMENSIONS » V,
* HT
X lO* D<*
NEI6NT 2 9 l 6 S
PER CENT AGAR 2 3
PO RE*» AT 9 1 D M e
I0
tm
tim e O f h e a t i n g ia m i n u t e s
I N TIAL T E M P E R A T U R E
*
7I * HEIGHT UNCmESI
d ia m e t e r
(in c h e si
Figure 2.18: Three-dimensional temperature gradients in agar cylinder heated at 915
MHz (1.0 KW) for 14 minutes (Source: Copson, 1975).
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72
Secondly, the penetration depth is defined as half-power depth. Therefore, at half
power, depth P/Po = 0.5. From Equation 2.33, e'2ad = 0.5 and d = 0.347/a' (Singh and
Heldman, 1993).
As mentioned above, the dielectric constants (s') o f materials in EM
(electromagnetic) field and loss tangent (tan 5) will be needed to determine the
attenuation factor. Then, the penetration depth and half power depth can be calculated.
In fact, those factors will be most important in design and operation of the microwave
application. Several measuring techniques have been used to measure the dielectric
constant such as Roberts and Hippel method, X-band techniques, and cavity perturbation
techniques (Metaxas and Meredith, 1983).
In other words, as the microwave energy is absorbed in the material, its
temperature increases at a rate depending upon a number of distinct parameters. The
power required to raise the temperature of a mass of material from To to T°C in t seconds
is given by extending Equation 2.33:
(2.34)
P = M aC p( T - T 0) /t
where P = required power (J/sec), Ma = mass (kg), To = initial temperature, T = final
temperature, and Cp = specific heat (J/kg°C) for a given material heated by high
frequency energy at a given f (frequency). Combining Equations 2.30 and 2.34 yields:
[°C/ second]
(2.35)
where p = density o f the material (kg/m3). The rate of rise of T depends on e"fE 2 (Singh
and Heldman, 1993).
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73
2.4.4 Advantages of Microwave Heating
When microwave heating is used properly, more materials can be heated in
shorter time at a lower cost. Historically, conventional heating has been the only way to
heat materials; i.e., apply heat to its surface. About 50 years ago, industrial engineers
began developing microwave-heating techniques that avoid some limitations of
conventional heating (Copson, 1975).
O f many applications, microwave energy can also be used for disinfection of
wastewater and wastes.
In fact, the time required for destruction of bacteria by
microwaves was reduced over that o f conventional heating (Iskandar et al., 1980).
Compared to conventional heating, microwave heating has the following advantages:
o
Rapid and uniform heating: Heating occurs instantly and uniformly throughout
the material, thus reducing heat loss as a result o f highly localized heating,
o
Instant control: Microwave heating can be controlled instantly and the power
applied can be accurately regulated,
o
Selective heating: As with radio frequency heating, the power will selectively
concentrate on the material that has the highest dielectric loss factor.
2.5 Biological Effect of Microwaves
While nonionizing electromagnetic wave effects on biological materials have
been discussed for over
100
years, it has been noted that the maximum recommended
safe power density for human exposure varies from 10 mW/cm 2 in the US to 0.01
mW/cm2 in the USSR (Johnson, 1972). Johnson claimed that electromagnetic fields in
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74
the spectrum between 1 MHz and 100,000 MHz have special biological significance
since they can readily be transmitted through, absorbed by, and reflected at biological
tissue boundaries in varying degrees, depending on material size, tissue properties, and
frequency.” However, it appears that biological effect is not clearly known and has been
arguing on this point although the Bioelectromagnetics Society has indexed 10,000
papers on this subject. A specific review relating to interaction between electromagnetic
energy with microorganisms was made by Fung and Cunningham in 1980 (Johnson,
1972). However, as mentioned above, electromagnetic levels to induce harmful effects
are still not clear; furthermore, many opposite opinions exist.
2.5.1 Temperature Limits for Microbial Growth
Temperature is generally one of the most important environmental factors in
living species o f microorganisms. Microorganisms differ remarkably in their ability to
adapt to high temperatures.
Biologists recognize three major categories of living
organisms: eucarya, archaea, and bacteria.
Archaea and bacteria are much simpler
organisms, seldom occurring as multicellular forms, and lacking true nuclei and mitosis.
They are called prokaryotic. Table 2.13 shows the upper temperature limits for growth of
various types o f living organisms, which is useful in determining the survivability o f
various microorganisms during microwave irradiation.
It should be noted that the eucaryotes are unable to adapt to high temperatures
because their upper limit is about 60~62°C. The only organisms capable o f surviving at
temperatures above 60~62°C are prokaryotes. The upper limit o f photosynthetic bacteria
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75
is about 70~73°C. At higher temperatures, only nonphotosynthetic bacteria are able to
grow.
At the highest temperatures, over 100°C, the only bacteria found are a few
unusually heat-adapted Archaea called hyperthermophiles (Brock, 2001).
Table 2.13: Upper temperature limits for growth of various organisms (Brock, 2001).
Microorganisms
Upper limits
Group
(°C)
56
Protozoa
Eucaryotic
Algae
55-60
Fungi
60-62
Cyanobacteria
70-73
(oxygen-producing photosynthetic bacteria)
Prokaryotes (Bacteria) Other photosynthetic bacteria
70-73
(do not produce oxygen)
Heterotrophic bacteria (use organic nutrients)
90
Methane-producing bacteria
110
Sulfur-dependent bacteria
115
Prokaryotes (Archaea)
Methane-producing bacteria can survive up to 110°C.
Therefore, if the
temperature raised by microwave irradiation is lower than 110°C, while the pathogens
such as coliforms, Salmonella sp., enteric viruses, and helminth ova are killed because of
increased temperature, the activity o f methane-producing bacteria, which are active in
anaerobic digester sludge, will be less affected relatively by heating.
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76
2.5.2 Pathogen Reduction of Microwaves
2.5.2.1 Inactivation and Pasteurization
Microwave energy has a strong ability to penetrate dielectric materials, and
produces thermal or non-thermal effects on microbes (Wu, 1996). Microwave radiation
differs from electron or gamma radiation in the manner by which microorganisms are
killed. The gamma waves or electrons directly hit molecules of the microorganisms or
create radicals in the medium, which in turn destroy the microorganisms. Microwave
radiation causes friction in the molecules by reversing their polarity with a magnetron
10 9
times per second, hence causing a great increase in the temperature of the medium, which
in turn leads to killing of the microorganisms.
For microwave and gamma radiation, Vasl et al. (1983) reported that microwave
irradiating sludge showed a greater die-away of seeded viruses in sludges for four types
o f viruses (Poliovirus 1, Echovirus 7, Coxsachie virus Bl, Coxsachie virus B5). In other
words, experiments with microwave radiation showed that more viruses were inactivated
with an increase in the medium thickness compared with gamma radiation.
This
indicates that an entirely different mechanism is involved in the inactivation of the
viruses by microwaves.
The thermal and specific effects o f microwaves on cell systems were investigated.
It was shown that cell damage in Escherichia coli and Saccharomyces cerevisiae were
induced by microwave heating more effectively than by equivalent thermal heat
(Morozov II et al., 1995). A microwave oven operating at 2,450 MHz resulted in a
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77
satisfactory sterilization process, that is, the destruction of heat-resisting spore forms (B.
stearothermophilus), provided that the materials undergoing sterilization were placed in
sealed containers with sufficient water present to provide steam during the heating
process (Fitzpatrick et al., 1978).
2.S.2.2 Kinetics of Pathogen Destruction
For the application of disinfection using microwaves and conventional heating,
the following factors should be considered: (1) contact time, (2) radiation intensity, (3)
temperature, (4) number of organisms, (5) types of organism, and ( 6 ) nature of
suspending liquids. One of the most important variables in the disinfection process will
be contact time.
The longer the contact time, the greater the disinfection.
The
destruction o f microorganisms by chemical addition is generally based on first order rate
kinetics. This observation was first formalized by Chick in 1908 (Metcalf and Eddy,
1994). Chick’s law is
dt
(2.36)
where Nt = number of organisms at time t, t = time, and k = constant (tim e1).
(2.37)
where No is the number of organism when t = 0 .
To formulate a valid relationship for the kill of organisms under a variety of
conditions, the following assumption was made:
N
In-—5- = - k t m
N0
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(2.38)
78
where m = constant. If m <l, then the rate of kill decreases with time. Equation 2.38 can
be obtained by plotting -In(N/No) versus the contact time t on Iog-log paper. Therefore,
the straight-line form o f the equation is
N
log(—In—-) = log k + m log t
No
(2.39)
In addition, the effect o f temperature on the rate o f kill can be represented by
van’t Hoff-Arrhenius relationship.
In terms of the time t, required to effect a given
percentage kill, the relationship is:
In— = JKT2 ~~T,)
t2
r t,t2
(2.40)
where t|, t2 = time for given percentage kill at temperature T| and T 2, respectively, E =
activation energy (J/mol or cal/mol), and R = gas constant (8.314 J/mol*K or 1.99 cal/Kmol).
2.5.3 Thermal and Nonthermal Effects on Microwaves
The effectiveness o f microwaves for sterilization has been well established by
numerous studies over the past few decades (Braun et al., 1978; Goldblith, 1967; Latimer
and Matsen, 1977; Sanborn et al., 1982). The exact nature o f the sterilization effect and
whether it is due solely to thermal effects or to the 'microwave effect' have continued to
be a matter of controversy.
Duringthe 1930s, the effects of low frequency electromagnetic
biologicalmaterials were studied in depth by physicists, engineers,
waves on
and biologists.
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79
Studies of the effects o f microwaves on bacteria, viruses, and DNA were performed in
the 1960s and included research on heating, biocidal effects, dielectric dispersion,
mutagenic effects, and induced sonic resonance.
Some o f the early biophysicists
investigating microwave absorption claimed evidence o f a 'microwave effect' which was
distinct in its biocidal effect from the effect of external heating (Barnes and Hu, 1977;
Cope, 1976; Furia et al., 1986; Hu et al., 1996). Many biologists in turn claimed there
was no evidence of a microwave effect and that the biocidal effects o f microwaves were
either due entirely to heating or were indistinguishable from external heating (Fujikawa et
al., 1992; Goldblith, 1967; Jeng et al., 1987; Lechowich et al., 1969; Vela and Wu, 1979;
Welt etal., 1994).
These experiments typically fell into two categories; controlled temperature
experiments and dry experiments.
In the controlled temperature experiments, the
researchers controlled the temperature of the irradiated specimen through various timing,
pulsing, or cooling techniques (Lechowich et al., 1969; Welt et a l, 1994). For example,
Welt et al. investigated the effects o f microwave irradiation on Clostridium spores and
found no additional lethality caused by microwaves that could not be accounted for by
conventional heating.
However, spores may not be representative of microwave
irradiation effects on active growing bacterial cells. However, the assumption that the
microwave effect is independent of, and separable from, temperature was always implicit
in these studies, but was never acknowledged.
The
second
type
of
unacknowledged assumptions.
experiment,
the
dry
experiment,
also
contains
Studies have shown that in the absence of water or
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80
moisture, biocidal effects o f microwaves are severely diminished, or require considerably
longer exposures (Jeng et al., 1987; Vela and Wu, 1979). This was typically taken as
evidence that non-thermal microwave effects did not exist; however, since water is the
primary medium by which microwaves are converted to heat, the absence o f biocidal
effects in the absence o f water would only indicate that water is necessary for sterilization
whether or not heating is the cause.
As mentioned above, thermal and non-thermal effects by electromagnetic energy
have been debated for many years. The term “non-thermal effect” generally relates to an
effect that is not associated with an increase temperature (Johnson, 1972).
Schwan
(1965) has indicated that the effect occurs in biological tissue at fields where damaging
thermal effect will occur. On the other hand, microwave fields can cause polarized side
chains o f the macromolecules to line up with the direction of the electric field (pearl
chain), leading to a possible breakage o f hydrogen bonds and to alteration of the
hydration zone (Teixeira-Pinto, 1960; Wilderbank, 1959).
Such effects can cause
denaturation or coagulation of molecules, which was confirmed experimentally by
Fleming (1961). Lately, Stuerga and Gaillard (1996) reported that electromagnetic fields
induce structuring and orienting effect within the irradiated medium.
The magnetic
energy converted to heating under thermal conversion (Brownian movements), while
allowing in induced organization o f the irradiated medium under athermal condition.
These hypothetical effects are called “non-thermal effects,” “athermal effects,”
“microwave effect,” or “specific effects” o f electromagnetic irradiation.
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81
On the other hand, the early studies showed that DNA tended to absorb
microwave radiation (Grandolfo et al., 1983; Grant et al., 1978; Takashima, 1963 &
1966), but no biocidal effects in the range o f 1 MHz to 60 MHz were observed. One
notable exception, however, was an early experiment, which found that frequencies
between 11 and 350 MHz had lethal effects on bacteria, with a peak at 60 MHz (Fleming,
1944). In addition, Takashima et al. (1984) reported that DNA has a dielectric dispersion,
where microwaves are readily absorbed, at much lower frequencies than water.
2.5.4 Microwave Effects on DNA and Chemical Bond
A question arises regarding whether microwaves can disrupt the covalent or other
chemical bonds of DNA. Numerous studies have concluded that there is no evidence to
support the existence of the 'microwave effect', and yet, some recent studies to be
discussed below have demonstrated that microwaves are capable of breaking the covalent
bonds of DNA. The exact nature o f this phenomenon is not well understood and no
theory currently exists to explain it.
Hydrogen bonds, as present in DNA, have a binding energy of only 0.1 eV.
Concentrated wave photons even below that for ultraviolet, that is visible, and infrared,
will affect bonds through the phenomenon of coincident photons if the bonds are very
weak (Copson, 1975). Since the activation energy of microwaves is approximately 10*5
electron volts, it appears that microwaves cannot disrupt the hydrogen bonds (0.04-0.44
eV) or covalent bonds (5 eV) in DNA (Table 2.14).
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82
Microwaves are not sufficiently energetic to be called ionizing radiation; however,
they may affect chemical bonds under certain circumstances. The water o f hydration o f
molecules such as proteins exerts a pronounced effect on the dielectric properties.
It
appears that the principal factors affecting dielectric materials are strength (frequency),
radiation time, concentration, particle size, viscosity, and penetration depth.
Tissues as protein solutions or electrolyte solutions contain lipid-protein and
protein molecules.
These would then have to be dielectrically special proteins or
lipoproteins to have non-thermal responses to microwaves. The bound water on proteins
may account for the dielectric properties, at least to a significant extent. In other words,
the water content o f the hydrated protein is a principal influence on the dielectric
response (Schwan, 1958).
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83
Table 2.14: Frequencies and quantum energies associated with various phenomena.
Phenomenon
Ionization
Energy (eV)
7.6-10
Frequency (GHz)
2.4 x 106
x
Covalent bond disruption
5
Reversible conformational
0.4
9.7 x 104
0.04-0.44
1.9 x 104~4.8 x 104
0.026
6.3 x 103
y radiation
12.3 x 106
3 x 10 12
X rays
12.3 x 103
3
1 .2
106
changes in protein
Hydrogen bond disruption
Thermal or brownian motion
x
109
Visible light
1.6-3 .2
3.8 x 1 0 5—7.5 x 105
Microwave
0.4 x 10'5~ 1.2 x 10' 3
1-300
RF wave
0.4 x 10'7~0.4 x 10' 5
0 .0 1 - 1
Photons
1 0 '5
Source: Stuchly (1978); Stuerga and Gai lard (1996).
An experiment performed by Kakita et al. (1995) demonstrated that a microwave
effect is distinguishable from external heating. The experiment involved irradiating PL-1
bacteriophage cultures with a 2,450 MHz microwave oven and comparing this with PL-1
bacteriophage cultures heated externally to the same temperature. The results showed
that the microwave radiation was capable of extensively fragmenting the viral DNA,
while external heating to the same temperature left the viral DNA intact.
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Analysis
84
through electrophoresis and electron microscopy showed that the DNA o f the microwave
irradiated samples had mostly disappeared (Figure 2.19).
The electron micrograph reveals that most of the phage particles in the microwave
irradiated sample were found to have empty heads. One possible explanation is that the
genomic DNA of the phages is susceptible to microwave irradiation. Furthermore, DNA
was extracted from the phage particles and analyzed by agarose gel electrophoresis. The
gel revealed that “microwave irradiation breaks the DNA located deep in the core of
phages, whereas heating the phage particles from the outside does not” (Kakita et al.,
1995).
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85
Figure 2.19: (a) PL-1 phage particles 96% inactivated through heating externally to
60.4°C, (b) PL-1 phage particles 98% inactivated by microwave irradiation for 50
seconds to 60.4°C (Source: Kakita et al., 1995).
There is currently no theory to explain the DNA fragmentation by microwaves,
but given the results of these researchers, it is not too far fetched to believe that the
microwave effect does indeed exist, even if it cannot be explained now.
Although Jeng et al. (1987) and Fujikawa et al. (1992) claimed that microwaves
were incapable of breaking the covalent bonds of DNA", non-thermal effect has
apparently occurred in the study by Kakita et al. (1995), despite the fact that this may
show only an indirect effect o f the microwaves. There is, in fact, sufficient of evidence
to indicate that there are alternate mechanisms for causing DNA covalent bond breakage
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86
without invoking the energy levels of ionizing radiation (Ishibashi et al., 1982; Kakita et
al., 1995; Kashige et al., 1990; Kashige et al., 1994; Watanabe et al., 1989; Watanabe et
al., 1985). However, no conclusive theory currently exists to explain the phenomenon of
DNA fragmentation by microwaves although research is ongoing which may elucidate
the mechanism (Kakita et al., 1995).
Based on the various referenced experiments, two reasonable hypotheses would
be considered. First, it may simply indicate that a threshold temperature to accumulate
sufficient energy has been reached. Second, the microwave radiation process may create
oxygen radicals, which will certainly dissociate the covalent bonds o f DNA. That is,
oxygen radicals can be generated by the disruption of a hydrogen bond on a water
molecule that exist alongside DNA molecules as "bound" water two or three layers thick.
These water molecules share a hydrogen bond with component atoms of the DNA
backbone including carbon, nitrogen, and other oxygen atoms (Figure 2.20).
O f hydrogen atoms, one hydrogen atom may be primarily bonded to either an
oxygen atom on the water molecule or to other oxygen atom on the DNA backbone. The
fluctuating character o f these shared or exchanged bonds is enhanced by microwave
heating temperature. Most of the oxygen radicals would immediately bond to the nearest
available site. If this site is an oxygen atom on the DNA backbone, a covalent bond will
be broken (www.em fauru.com).
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87
Base
rAdenine (A)
Ester bond forme
with release of H
\
o — P—O — (
II
O
4
H—
Glycosidic bond formed
with release of H-jO
Deoxyribose unit
OH
H
DNA Backbone
Figure 2.20: The adenosin monophosphate deoxynucleotide in DNA (Source: Rittmann
and McCarty, 2001).
Although DNA tends to repair itself naturally, the simultaneous breakage of a
sufficient number of covalent bonds would lead to the failure o f the entire DNA molecule
(emfguru.com/EMF/microwave-dna.htmiy This is the theory, and it awaits experimental
verification.
A similar experiment was performed in this study to compare the effects of
conventional heating and microwave irradiation on bacterial genomic DNA. Particularly,
fecal coliform bacteria cultures were used and were subjected to either microwave
irradiation or external heat from a water bath.
Then, the DNA o f the bacteria was
analyzed with gel electrophoresis. Such an analysis was meant to help elucidate the
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88
mechanism o f destruction by microwaves on bacteria and give credence to any one side
o f the argument over the existence of the microwave non-thermal effect.
2.6 Gaps in knowledge
Microwave irradiation has been used in inactivation of microorganisms in food,
soil, and other medium. Although there have been numerous studies on the destruction of
pathogens in sludge with various techniques, there was no study on the use of
microwaves for pathogen destruction to make class A sludge regulated by U.S. EPA. It
will be beneficial to understand the mechanism of pathogen destruction by microwaves.
New technical approaches such as gel electrophoresis for DNA quantification,
live/dead cell tests, P-galactosidase tests for enzyme activity, etc., can be used for this
study. Engineering design and application of microwave technology is a viable option
for generating class A sludge. Optimum exposure time or temperature, and penetration
depth o f the microwave unit in wastewater treatment plants must be determined based on
the destruction efficiency of pathogens such as fecal coliforms, Salmonella, and helminth
ova.
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89
3. EXPERIMENTAL MATERIALS AND METHODS
3.1 Sludge Sampling and Experiment Preparations
In wastewater treatment processes, two types of sludge are generally produced:
sludge from the primary sedimentation tank and waste activated sludge (WAS) from
aeration basins or secondary clarifiers. These sludges are further treated to reduce their
organic content and offensive odors. Sludges tested for this study were obtained from the
Nine Springs wastewater treatment plant (WWTP) in Madison, Wisconsin (Figure 3.1).
The three sampling locations are shown in Figure 3.2.
The primary sludge (PS) was taken from a gravity thickener underflow pipe. The
thickened WAS was taken from the dissolved air flotation (DAF) thickened sludge pipe.
The anaerobic digester sludge (ADS) was obtained from the recirculation line for heating.
All samples were stored at a 4°C refrigerator separately until they were tested. The solids
content in three sampling locations and typical ranges (Metcalf and Eddy, 1994) are
summarized in Table 3.1.
Glass bottles were used to collect the sludge samples. They were cleaned by
washing with deionized (DI) water and phosphorus-free soap, rinsing, washing with 20%
hydrochloric acid, rinsing again with DI water, washing with acetone, and finally rinsing
with sterilized water.
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90
Table 3.1: Solids content (%) in three sampling locations.
Anaerobic digester
Primary sludge
Sludges
from thickener
WAS from thickener
sludge
Nine Springs
5.0-7.0
3.5-4.5
2.7-3.3
Typical range
4.0-10.0
0.8-2.5
2.5-7.0
Moorland Road
Figure 3.1: Schematic of Nine Springs WWTP in Madison, Wisconsin (Source: Madison
Metropolitan Sewerage District, 2001)
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91
Primary Settling
Gravity Thickener
Aeration Basin
Thickener (DAF)
Secondary
Clarifier
I
wastewater
Sludge
- f
S3
Anaerobic Digester
Heat
Exchanger
Figure 3.2: Schematic o f sampling locations in Nine Springs WWTP in Madison, WI.
3.2 Microwave Power Calibration
Based on the U.S. EPA method 3052 (1985 Annual Book of ASTM Standards),
the microwave unit was calibrated for the normalization. The calibration was
accomplished by measuring the temperature rise in water samples exposed to microwave
radiation. Normalized microwave power was used for the incident and absorbed power
calculations o f all microwave-irradiated sludges in this research.
100, 200, 300, 400, 500, and 1000 g of DI water were weighed into a suitable
glass beaker (1,000 mL). The initial temperature o f the water was 24±2°C measured to
±0.2°C.
The beaker was circulated continuously through the microwave field for 1
minute at the maximum power setting with the system exhaust fan on maximum. The
beaker was removed and the water vigorously stirred. A magnetic stirring bar was used
immediately after microwave irradiation and the maximum temperature was recorded
within the first 30 seconds to ±0.2°C (Table 3.2).
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92
Table 3.2: Microwave power calibration test.
DI water
Microwave irradiation
Initial
Final
volume (mL)
time (seconds)
temperature (°C)
temperature (°C)
60
24±0.2
Measured (±0.2)
100,
200,300,
500, and 1000
3 3 Penetration Depth Test
To obtain the penetration depth, an acryl-air baffled vessel was developed and
placed in a 2,450 MHz microwave unit (Figures 3.3 and 3.4). The purpose o f the acrylair baffled vessel was to protect both heat transfer through each vessel and mixing of
samples. The total thickness o f the acryl-air baffle was total 7 mm including two 1-mm
thick acryl walls and 5 mm o f air spaces (Figure 3.4). The right side of the vessel wall
was tightly attached at the hole o f the microwave exit (wave guider) to reduce the wave
loss before testing. The temperature was measured using a thermo-coupled thermometer
(Cole Parmer p-92900-20, T-type) within 30 seconds as soon as microwave power
stopped. Figure 3.4 shows the details o f Part A (first reservoir) in the microwavable
vessel.
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93
Thermo coupled
Thermometer
Transparent Acryl-Air baffle
Sample 5
130 mL
Sample 3
110 mL
Sample 4
l l OmL
Attenuation o f power
Sample 2
50 mL
Sample 1
50 mL
50 MHz microwave
< ■
Part A
Figure 3.3: Schematic of devices used for the measurement o f penetration depth.
Acryl baffle (1 mm)
Air space (5 mm)
1st reservoir
Sample, 50 mL
2,450 MHz microwave
Sample depth (12 mm)
Figure 3.4: Details o f part A in microwavable vessel.
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94
Into each reservoir section (sections 1 to 5), 50, 50, 110, 110, and 130 mL of
samples (DI, tap, 10% NaCl water, PS, WAS) were added. Initial temperatures of three
different water and sludge samples were 20 and 10±0.2°C, respectively (Table 3.3).
Table 3.3: Experimental conditions for penetration depth test.
Volume, mL
MW irradiation time, sec
DI water
50, 50, 110, 110, 130
30, 60, 90
Tap water
50, 50, 110, 110, 130
30, 60, 90
10% NaCl solution
50, 50, 110, 110, 130
30, 60,90
Primary sludge
50, 50, 110, 110, 130
30, 60,90
Waste activated sludge
50, 50, 110, 110,130
30, 60,90
Samples
3.4 Batch Tests
The objective of the batch tests was to evaluate both the anaerobic digestibility
using the BMP (biochemical methane potential) test and the pathogen reduction (fecal
coliforms as an indicator) after microwave irradiation of sewage sludge.
The first batch test (1st BMP) was performed only to evaluate the effect of
microwave irradiation for digestibility o f anaerobic digester sludge. The second batch
(2nd BMP) test was used to investigate the difference of anaerobic digestibility at 25 and
35°C between microwave irradiation and conventional heating with different types of
sludge. Furthermore, the coliform destruction rate was evaluated to determine optimal
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95
irradiation time and temperature conditions. From the two batch tests, the applicability o f
microwave irradiation was evaluated.
3.4.1 First Batch Microwave Test
Batch tests were conducted with only ADS (anaerobic digester sludge) to evaluate
the effect of microwave irradiation of ADS (anaerobic digested sludge) on the total gas
production. Approximately 100 mL of ADS was irradiated with microwaves in a 500mL beaker for 15, 30, 60, and 120 seconds.
The microwave oven (Emerson, MW
8107WA, 1 K.W) was operated at a frequency o f 2,450 megacycles that rotates molecules
180°, 2.45 x 109 times per second. The temperature was measured as soon as the beaker
was removed from the microwave oven. A 160-mL serum bottle was used for the batch
test with microwave-irradiated samples. Experimental conditions are shown in Table 3.4.
Typically, when BMP tests are performed, inoculums (anaerobic digester sludge)
are inoculated in the serum bottles, and then substances are fed in to the bottles (Speece,
1996). In this chapter, the terms “seed sludge” and “mixing sludge” will be used to
represent the first inoculums, and the sludges that were fed with microwave irradiated
sludge, or heated sludge, respectively.
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96
Table 3.4: Sludge mixing conditions for the first batch test.
Mixing condition (mL)
Microwave
Microwave
Seed
Mixing
Irradiation time
Irradiation time for
sludge
sludge
for seed sludge
mixing sludge
(ADS)
(ADS)
Sec.
Temp.
Sec.
Temp.
0
100
0
-
0
10±2°C
2
0
100
0
-
15
36±2°C
3
0
100
0
-
30
48±2°C
4
0
100
0
-
60
84±2°C
5
0
100
0
-
120
100±2°C
6
50
50
0
10±2°C
15
36±2°C
7
50
50
0
10±2°C
30
48±2°C
8
50
50
0
10±2°C
60
84±2°C
so
50
0
10±2°C
120
100±2°C
Run
1
(control)
9
r
T e st tem perature - 35°C (m e so p h ilic )
The batch test was performed in a temperature-controlled water bath shaker
(Gyratory, New Brunswick Scientific Co. Inc, G76D) at 120 rpm and 35°C.
The
cumulative gas production was measured using a water displacement method for 69 days.
3.4.2 Second Batch Microwave Test
The purpose o f the second batch test was to investigate the difference between
microwave irradiation and conventional heating with different types o f sludge and to
determine the best microwave irradiation point. Coliform reduction was assayed for
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97
optimal irradiation time and temperature conditions. Simultaneously, anaerobic
digestibility was evaluated at 25 and 35°C during the second batch test.
The tests were performed to determine the degree o f degradability for three
different types (PS, WAS, and ADS) o f sludge and mixed sludges. An 80-mL serum
bottle was used for the test. Sludge samples were irradiated with microwaves at 0.70,
1.40, 2.80, 4.19, and 5.59 wattshr/g total dried solids (TS) for ADS, 0.34, 0.67, 1.35,
2.02, and 2.69 watts hr/g TS for PS, and 0.53, 1.06, 2.11, 3.17, and 4.23 watts-hr/g total
dried solids (TS) for WAS, corresponding to 15, 30, 60, 90, and 120 seconds of
irradiation time per 200 mL sludge samples, respectively, and the temperature raised
accordingly to 25, 45, 65, 85, and I00±2°C. The experimental conditions o f the second
batch test are summarized in Table 3.5. Photographs of the experimental apparatus are
shown in Figure 3.5.
First, 200 mL o f PS, WAS, and ADS were irradiated with
microwaves in a 500 mL beaker, respectively for 0 (control), 15, 30, 60, 90 and 120
seconds and the temperature was measured as soon as microwave irradiation stopped.
Second, 200 mL o f PS, WAS, and ADS were heated in a water bath to
temperatures of 10 (control), 45, 65, and 100°C. An aliquot o f 10 mL o f pretreated
samples were added to serum bottles with 30 mL of seed sludge.
Serum bottles were sealed with butyl rubber stoppers and cramped with an
aluminum cap (Figure 3.6). All serum bottles were placed in a water bath shaker at 120
rpm. The tests were performed at 25 and 35°C. The gas volume was measured at 1, 3, 5,
10, 15, 20, 25, 35, and 50 days. The volume of gas generated from each bottle was
measured by
10 %
saturated sodium chloride sulfuric acid solution so that carbon dioxide
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98
gas was not dissolved into the solution. After releasing excess pressure from the bottles,
a 200 pL gas sample was collected with a gas-tight syringe. The bottle was shaken a few
times before taking a gas sample. Care was taken to avoid contact between the content
and the rubber stopper. The syringe was flushed three times wflh at least 5 pL o f gas
from the bottle, the plunger was set to the 200 pL mark, and then the gas was allowed to
equilibrate at 35°C for at least 30 seconds. The valve on the syringe was closed and the
needle was removed from the stopper. The gas sample (200 pL) was injected to a gas
chromatograph (Varian Model 3300) equipped with a TCD (thermal conductivity
detector) to measure carbon dioxide, oxygen, nitrogen, and methane. The results were
expressed as cumulative gas volume multiplied by the methane composition and divided
by the volume of sludge (vol. methane/vol. o f sludge in serum bottle).
In addition, the initial samples pretreated by microwaves and conventional
heating were analyzed for fecal coliforms and E. coli prior to sludge mixing. These
analyses were conducted in accordance with standard methods (APHA et al., 1995).
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99
Table 3.5: Experimental conditions for the second batch test.
Run conditions
Mixing condition
Microwave
(mL)
Run
Mixing sludge
Seed Sludge
Seed
Mixing
sludge
sludge
Irradiation time
for mixing sludge
(sec)
1
RADS1
-
40
0
0
2
RADS
RPS"
30
10
0
3
RADS
RWASJ
30
10
0
4
RADS
ADS (15S-MWI)4
30
10
15
5
RADS
ADS (30S-MWI)
30
10
30
6
RADS
ADS (60S-MWI)
30
10
60
7
RADS
ADS (90S-MWI)
30
10
90
8
RADS
ADS (120S-MWI)
30
10
120
9
RADS
PS (15S-MWI)
30
10
15
10
RADS
PS (30S-MWI)
30
10
30
11
RADS
PS (60S-MWI)
30
10
60
12
RADS
PS (90S-MWI)
30
10
90
13
RADS
PS (120S-MWI)
30
10
120
14
RADS
WAS (15S-MWI)
30
10
15
15
RADS
WAS (30S-MWI)
30
10
30
16
RADS
WAS (60S-MWI)
30
10
60
17
RADS
WAS (90S-MWI)
30
10
90
18
RADS
WAS (120S-MWI)
30
10
120
19
RADS
ADS (45C-CH)
30
10
0
20
RADS
ADS (65C-CH)
30
10
0
21
RADS
ADS (100C-CH)
30
10
0
22
RADS
PS (45C-CH)i
30
10
0
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100
Mixing condition
Run conditions
Microwave
(mL)
Irradiation time
Run
Seed Sludge
Mixing sludge
Seed
Mixing
sludge
sludge
for mixing sludge
(sec)
23
RADS
PS (65C-CH)
30
10
0
24
RADS
PS (100C-CH)
30
10
0
25
RADS
WAS (45C-CH)
30
10
0
26
RADS
WAS (65C-CH)
30
10
0
27
RADS
WAS (100C-CH)
30
10
0
dotations: RADS1 - anaerobic digester sludge without pretreatment, RPS2 - primary sludge
without pretreatment, RWAS3 - waste activated sludge w/o pretreatment, ADS (15S-MWI)4 anaerobic digester sludge treated by microwave for 15 seconds), and PS (45C-CH)5 - primary
sludge treated at a temperature o f 45°C with conventional heating (water bath). Test temperature
- 2 5 and 35°C.
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101
Figure 3.5: Photographs o f experimental apparatuses (a) serum bottles for the second
batch test and (b) water bath shaker and gas volume measure equipment.
Aluminum Cap
Rubber Stopper
Head Space
Mixing Sludge
1— ^ Seed Sludge (ADS)
Serum Bottle
Figure 3.6: Schematic o f serum bottle (80 mL) for batch tests
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102
3.5 Bench-Scale Anaerobic Digester Tests
Based on the first and second batch tests, bench scale anaerobic digester tests
were performed to evaluate both anaerobic digestibility and fecal coliform destruction
during continuous operation of digesters.
3.5.1 Experimental Conditions for Bench-Scale Tests
Three 6-L anaerobic digesters were operated in a semi-batch
mode.
Approximately 4 L o f anaerobic digester sludge was inoculated as seed sludge in each
reactor.
The first three months were spent in set-up, sludge seeding and biomass
acclimation with sludge obtained at the Nine Springs WWTP. The schematic o f the
anaerobic digester apparatus is shown in Figure 3.7.
Each anaerobic digester was equipped with a mechanical mixer that operated at a
speed o f 20 rpm. The first reactor was a control simulating a conventional anaerobic
digester. The second reactor received the feed irradiated with microwaves. The third
reactor received the feed heated to a temperature achieved by corresponding microwave
irradiation to simulate conventional heating by a water bath. The feed contained the
same volumes of PS and WAS (1:1). A 1-kW microwave oven designed for household
use was used for the study.
The feed was manually added once or twice a day to
anaerobic digesters based on the SRT (solids retention time). The SRT was controlled by
removing or adding the same volume of sludge.
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103
Temperature was controlled by winding the outer wall o f the anaerobic digester
with a Vi-inch Tygon® tube and circulating water at 35°C from a water bath (Haake Bath
and Circulator, P-12203-00). The amount o f gas production was measured with a 4.2 L
gas-sampling bag (standard Teflon® bags with on/off valves). The total gas volume was
determined using a wet gas meter. The temperature and gas measurement devices were
calibrated at least once every two weeks.
1: Sludge Input
4: Gas Port
2: Sludge Output
3: Thermometer
5: Wet G as Meter
6: Agitator (120 rpm)
Figure 3.7: Schematic of anaerobic digester apparatuses.
At least once a week, fecal coliforms were counted for the influent and effluent
samples from the three reactors. The following operational parameters were measured
regularly: pH, temperature, CODcr, solids (TS, VS, TSS, and VSS), total volatile fatty
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104
acids (TVFA) including acetic acid, propionic acid, and butyric acid, total alkalinity,
bicarbonate alkalinity, gas production, and gas composition.
The experimental
conditions are summarized in Table 3.6.
Table 3.6: Bench-scale anaerobic digester experimental conditions.
Description
Run
Seed
Feed
1
RADS
RPS and RWAS
(Control)
4L
(1:1)
Sludge/Reactor
SRT (day)
volume (L)
4/6
20, 15,10 and 5
4/6
20, 15, 10 and 5
4/6
20, 15,10 and 5
Microwave
RADS
2
4L
pretreated PS and
WAS (1:1)
Conventional
RADS
3
4L
pretreated PS and
WAS (1:1)
Operation temperature: 35°C
3.S.2 Statistical Analysis for Digester Evaluation
A statistical analysis was performed to evaluate the difference in the two average
results.
Three alternatives such as raw sludge, microwave-irradiated sludge, and
conventional heated sludge were compared each other to evaluate the level of difference
in each pair.
The standard procedure for comparing two methods is to construct a null
hypothesis, which is tested statistically using a paired t-test (Berthouex and Brown, 1994).
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105
In statistical analysis, the classical null hypothesis represents no difference between the
mean o f population from two methods as follows:
H0 :p , = p 2, H, :p, * p 2
(3.1)
where pi = average fecal coliform numbers by a pretreatment method and \n = average
fecal coliform numbers by the other pretreatment method.
An alternative method to construct a null hypothesis is to calculate average
difference anduseconfidence interval in which thedifference isexpected to fall. This
paired t-test method was used toassess the significance o f theaveragedifferences
from
two digesters. Box et al. (1978) stated that a statistically significant difference occurs if
the null hypothesis is rejected. To apply this statistical method, Berthouex and Brown
(1994) suggested the equations below.
The true mean of differences between random variables digester 1 and digester 2
will be zero value when the results from which digester 1 and 2 are drawn and equal.
d = - Y .d ,
n
(3.2)
where d = average of difference between paired observations, d, = difference between
two digesters in each pair o f values, and n = number of pairs o f values. The variance of
the average difference of samples ( S] ) is calculated using the following equation:
Sj =
n -l
(3.3)
The standard error o f the average difference ( Sj ) is calculated using the
following equation:
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The significant differences would be accepted if the confident interval has not
included zero. The confident intervals were defined as follows:
( 3 -5 >
where to/2 =
value from a t-distribution table, a = significant level (95% = 0.05), and n-
1 = the number of degrees of freedom.
Accordingly, three result groups o f fecal coliform numbers were run in the paired
t-test.
3.6 Toxicity Screening Tests using SMPS
Thermal pretreatment before anaerobic digestion shows promise for increasing
biodegradability of sludges although thermal pretreatment at 200~225°C was found to
cause the production of inhibitory materials that adversely affected digester performance
(Haug et al., 1978). Since both microwave irradiation and conventional heating were the
thermal pretreatment for sludges, toxicity screening tests should be required. Although
the temperature of sludges by microwaves and conventional heating varied from 10 to
100°C, toxicity results will be useful to evaluate the effect of byproducts created by
pretreatment on anaerobic digestibility. Consequently, the objective o f the toxicity assay
was to determine whether sludges pretreated with microwaves or conventional heating
have greater toxicity after pretreatment.
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107
3.6.1 Fundamentals of SMP
SMP (submitochondrial particles) tests are becoming an efficient method for rapid
determination of toxicity in water samples or aqueous extracts. The SMPs contain the full
complement of oxidative enzymes.
SMP membranes are inside out relative to
mitochondria, which place the important enzymes on the outside rather than inside,
making them easier to manipulate and poison (Blondin et al., 1987).
The MitoScan® assay is used to utilize SMPs and is based on the membranelinked enzymes associated with cellular electron transport and oxidative phosphorylation
(Argese et al., 1995). Mitochondria are the energy centers of the cell where oxidative
metabolism takes place. MitoScan tests provide information about toxicant disruption
that impacts higher levels of biological organization such as are usually only available
from whole organism tests. Two such bioassay (ETr and RET) protocols using MitoScan
SMP are widely used.
The Electron Transfer (ETr) assay replicates mitochondrial
biochemistry as it occurs in normally functioning cells. The Reverse Electron Transfer
(RET) assay uses only a part o f the enzyme system by adding an alternative energy
source to force the electron flow in the reverse direction of normal living systems. In this
study, RET assay was used to evaluate the toxicity of pretreated sludge samples.
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108
3.6.2 Fundamentals of RET (Reverse Electron Transfer) Assay
The RET protocol is more complex biochemically, but once set up it is as simple
to run as the ETr protocol. As shown in Figure 3.8, the RET assay monitors the rate o f
appearance of NADH as NAD+ is reduced.
Complex
Succinate
Furmarate
NAD+
NADH
Complex I
jCoO
■^Complex III
ATPase
ADP
Complex IV
Antimycin Block
ATP
S\1
Figure 3.8: RET assay reaction scheme.
To run this test, three components must be provided: a source o f electrons, a
source o f energy, and NAD+.
Normally, succinate is used to provide a source o f
electrons, while ATP is used as an activator to provide a source o f energy. Upon addition
o f ATP, the ATPase enzyme (Complex V) couples energy from the oxidation of ATP to
ADP and uses it to drive the reduction o f NAD+ to NADH (Knobeloch et al., 1990).
Antimycin is used to block the pathway to Complex III, Cytochrome c, and
Complex IV, thus preventing the NADH produced from being oxidizing back to NAD+
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109
(Bolndin et al., 1987).
Accordingly, the rate of reaction is measured spectrophoto-
metrically by taking absorbance readings at 340 nm at timed intervals (Knobeloch et al.,
1990).
3.63 RET Protocols
SMPs (Biorenewal Technologies, Madison, WI) were prepared from bovine heart
mitochondria. The reverse electron transfer (RET) of SMP assay was conducted in 96well microplates using 300 pL per well following the procedures below (Gustavson et al.,
1998):
1. Prepare the assay medium containing 0.25 M sucrose, 50 mM HEPES
buffer (pH 7.5), 6.0 mM MgCh, 5.0 mM potassium succinate, 0.2 pg/mL
antimycin A, 1 mM NAD+ (nicotinamide adenine dinucleotide) and 0.05
mg SMP protein.
2. Adjust pH to 7.5 and warm to 25°C.
3.
Dispensed mixed assay medium into pipette basin (45 pL * number of
samples).
4. 235 pL volumes o f filtered sludge samples (Table 3.7) were added in 96well microplates.
5. Mix filtered sludge sample with RET assay medium (235 pL + 45 pL = 280
pL).
6. The reaction was started by adding 0.5 pmol ATP (adenosine 5'triphosphate) solution.
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110
7. The production o f NADH overtime was monitored at 340 nm using a
Dynatech® (Chantilly, VA, model MRX microplate reader). Toxicity is
indicated by a reduction in the rate of NADH production.
Table 3.7: Sample pretreatment for RET tests.
Sludge
Treated mixing sludge
Temp. (±2°C)
Raw
RADS1, RPS2, RWASJ
10
ADS (15S-MW1)4, ADS (30S-MWI),
ADS (60S-MWI), ADS (90S-MWI),
Microwave
irradiated
sludge
25,45, 65, 85,
and 100
ADS (120S-MWI)
PS (15S-MWI), PS (30S-MW1), PS (60S-MWI), PS
(90S-MWI), PS (120S-MWI)
WAS (15S-MWI), WAS (30S-MWI), WAS (60SMWI), WAS (90S-MWI), WAS (120S-MWI)
25,45, 65, 85,
and 100
25,45,65, 85,
and 100
4 5 ,6 5 ,and
ADS (45C-CH), ADS (65C-CH), ADS (100C-CH)
Conventional
heated sludge
100
45,65, and
PS (45C-CH)5, PS (65C-CH), PS (100C-CH)
100
4 5 ,6 5 ,and
WAS (45C-CH), WAS (65C-CH), WAS (100C-CH)
100
dotations: RADS ; anaerobic digester sludge w/o pretreatment, RPS2; primary sludge w/o
pretreatment, RWAS3; waste activated sludge w/o pretreatment, ADS (I5S-MW I)4; anaerobic
digester sludge treated by microwave for 15 seconds), PS (45C-CH)5; primary sludge treated at a
temperature o f 45°C with conventional heating (water bath). All samples were filtered with 0.45
pm filter.
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I ll
In RET assays, the Microtox assay was performed according to the established
procedure using 15 min toxicant exposure period. A least-squares fit was conducted to
determine the rate o f absorbance change with time.
3.7 Bioassays on Fecal Coliforms as an Indicator
Microwave irradiation can be an appropriate method for destruction o f pathogens
in sludge. Sludge with reduced pathogens may be recycled through land application as a
soil conditioner or fertilizer.
However, the mechanism of microwave sterilization of
pathogens has not been understood clearly.
The existence o f “non-thermal” effects
(athermal effects or specific effects) o f electromagnetic irradiation when irradiating
biological specimen with microwaves is currently in debate (Kingston and Haswell,
1997).
Destruction o f an indicator organism, the fecal coliforms, by microwave
irradiation may be achieved at temperatures lower than by conventional heating methods
(e.g., boiler). In this research, three possible reactions were thought to destruct coliform
bacteria. Cell wall damages were expected to occur first and then the reduction of fecal
coliform bacteria’s activity.
The last reaction may be that DNA isolated from fecal
coliforms would be denatured more by a microwave effect.
Live/Dead Cell Counts bioassay was used to investigate the cell wall damages of
fecal coliforms by microwaves compared with that by conventional heating. An ETS
(electron transport system) assay was applied using 2-(4-iodophenyl)-3-(4-nitrophenyl)5-phenyltetrazolium chloride (INT, C 19H 13CIIN5O2, Sigma® 18377) for the activity of
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112
fecal coliforms by microwave irradiation and conventional heating. The other assay for
the reduction o f fecal coliforms’ activity, (3-Galactosidase assay, was used as a reporter
molecule. The |3-Galactosidase Enzyme Assay System with Reporter Lysis Buffer is a
method for assaying P-galactosidase activity in lysates. As a third investigation, gel
electrophoresis was used to quantitatively compare the effects of conventional heating
and microwave irradiation on fecal coliforms. Genomic DNA measurement of bacterial
death at specific temperatures was also simply used to quantitatively compare the effects
of conventional heating and microwave irradiation.
3.7.1 Isolation of Fecal Coliforms from Sludges
To obtain a pure culture o f fecal coliform bacteria, which were isolated from a
primary or WAS sludge sample using the membrane filtration (MF, Standard Method
9222 D) method.
An appropriate volume o f a diluted sludge sample (106 fold) was
passed through a sterile, gridded, 0.45 pm membrane that retains the bacteria present in
the sample.
This filter containing microorganisms was placed on an absorbent pad
saturated with m-FC (Cole Parmer, ATCC 25922, PN# 14020-01) broth ampoules in a
sterile 50 * 9 mm petri dish. The dish was then inverted and incubated at 44.5±0.5°C for
24±2 hours.
After incubation, the fecal coliform colonies were blue while other coliforms were
gray. A sterile loop was touched to one o f the blue fecal coliform colonies and an agar
plate with Luria broth (LB) was inoculated with the bacteria. This plate was incubated at
35±0.5°C for 24±2 hours.
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113
Next, 400 mL o f liquid LB broth base was prepared using sterilized water and 8 g
o f the nutrient broth base in an Erlenmeyer flask. A sterile wooden probe was used to
inoculate the broth with a fecal coliform colony from the final petri dish to ensure the
purity of the culture. The flask was then placed in the cooling room at 4°C until the
analysis of the coliforms.
3.7.I.1 Pretreatment of Cultured Fecal Coliforms
After isolation and pure culture o f fecal coliforms, 20 mL portions o f the infected
liquid broth were transferred by a pipette into a 50-mL graduated cylinder for microwave
irradiation and a 25-mL test tube for conventional heating, respectively (Table 3.8).
Table 3.8: Pretreatment conditions of isolated fecal coliforms for bioassays.
Heating
Microwave irradiation
Conventional heating
Microwave oven, 1 kW
Water bath
type
Heating
equipment
Heating time,
Temp, °C
sec
Time
&
temperature
Heating time,
Temp, °C
sec
0
20.4±0.2°C
0
20.4±0.2°C
7
38.0±0.2°C
58
38.0±0.2°C
12
56.8±0.2°C
185
56.8±0.2°C
13
61.9±0.2°C
270
61.9±0.2°C
15
70.5±0.2°C
392
70.5±0.2°C
19
78.0±0.2°C
598
78.0±0.2°C
21
82.1±0.2°C
706
82.1±0.2°C
23
86.9±0.2°C
866
86.9±0.2°C
Autoclaved sample: 121 °C, IS min
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These samples were then heated using either microwave radiation at 2,450 MHz
with a 1 kW Emerson microwave oven, or a hot water bath on a hot plate. The control
samples were not heated. The pretreatment conditions of isolated fecal coliforms for
following bioassays such as Live/Dead cell count, ETS assay, (i-galactosidase enzyme
assay, and gel electrophoresis are summarized in Table 3.8.
3.7.1.2 Determination of the Survival Fraction of Fecal Coliforms
The membrane filter (MF) test was used to obtain the survival fraction o f fecal
coliforms in the heated and irradiated samples. An appropriate volume o f sample or its
dilution (* 102, x 104, or * 106) was passed through a sterile, gridded, 0.45 pm membrane
filter that retains all bacteria present in the sample. This filter was placed on an absorbent
pad saturated with m-FC (Standard Method 9222 D) in a petri dish.
The dish was
incubated at 44.5±0.5°C for 24±2 hours.
After incubation, the blue fecal coliform colonies were counted under low
magnification and the number was reported per 100 mL of initial sample or per g-TS.
These numbers were then divided by the number o f colonies present in the control sample
to determine the survival fraction. The procedure to obtain the survival fraction o f fecal
coliforms was as follows:
1. Take 1 mL o f samples (refer to Table 3.8). A 102x, 104x, or 106x dilution o f the
original sample in sterilized water was often necessary to reduce the number of
bacteria to measurable levels.
The recommended colony count was 20~80
colonies and less than 200 total bacterial colonies per filter after dilution.
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2. The sample bottle was shaken vigorously to ensure even distribution of the
bacteria, taking care to secure the screw cap and prevent leakage during shaking.
3. A sterile membrane was placed on the filter base using sterile forceps and the top
funnel of the filter unit was attached.
4. The sample was shaken vigorously and poured into the funnel with the vacuum
off.
5. The vacuum was turned on and the walls of the funnel were rinsed with sterile
water.
6. The vacuum was turned off and the top funnel of the filter unit was removed.
7. The membrane was taken from the filter base using sterile forceps and placed,
grid side up, in a petri dish on an absorbent pad saturated with m-FC broth in
such a way as to prevent air bubbles from being trapped between the pad and
membrane. At least two plates were prepared for each volume.
8. The petri dishes were placed, inverted, in the incubator at 44.5±0.5°C for 24±2
hours.
9. After incubation, using a binocular microscope with a magnification of 10 or 15 *,
the blue colonies were counted and the total number of colony forming units per
1mL was recorded.
3.7.2 Live/Dead Cell Counts
Live/Dead Cell
Counts
bioassay (Catalog No.:
L-7012,
LIVE/DEAD®
fiacLight™ Bacterial Viability Kit) was used to determine the number of viable and dead
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cells in pure cultured fecal coliforms that are irradiated with microwaves and heated in a
water bath. Using this protocol, call wall damages by microwaves were investigated.
The chemicals (Catalog No.: L-7012, LIVE/DEAD® 5acLight™ Bacterial Viability Kit)
used in this bioassay are as follows:
1.
Component A (SYTO 9 dye, 3.34 mM): 300 pL solution in DMSO
(dimethylsulfoxide)
2.
Component B (Propidium iodide, 20 mM)
3.
Component C: BacLight™ mounting oil, 10 mL for bacteria immobilized on
membrane
4.
The protocols o f the live/dead cell counts are as follows:
5.
Combine an equal volume o f components A and B in a micro centrifuge tube
and mix thoroughly (0.3% DMSO solution).
6.
Add 3 (j.L of the dye mixture for each mL of sample (refer to Table 3.8)
suspension.
7.
Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
8.
Trap 5 pL of bacterial suspension between a slide and an 18-mm square cover
slip.
9.
Observe in a fluorescence microscope with the filters (FL for live cells, Cy3
for dead cells). Otherwise, the procedure above will be processed with a 0.2pm membrane and mount oil to reduce the errors and bias (Haug et al., 1977).
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3.73 ETS (Electron Transport System) Assay
The tetrazolium salts INT has been used as an indicator o f bacterial respiration
through their reduction to insoluble, intracellular formazan crystals (Zimmermann et al.,
1978; Trevors and Starodub, 1983; Swannell and Williamson, 1988; Kang et al., 1998).
Respiring bacteria deposit accumulated INT-formazan intracellularly as dark red spots.
Corresponding to electron transport system activity, these deposits attain a size
and a degree o f optical density, which allows them to be examined by light microscopy
(Zimmermann et al., 1978).
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium
chloride (INT, C 19H 13CIIN 5O 2 , Sigma® 18377) was used to measure the electron transport
system (ETS) activity o f fecal coliforms by microwave irradiation and conventional
heating. Since INT-formazan production is sensitive for dehydrogenase activity and the
formazan is a stable compound (Trevors and Starodub, 1982), a spectrophotometric assay
was conducted to determine the ETS activity at 480 nm. The chemicals and protocols
used in this bioassay are as follows:
1. Dissolve 0.4 g INT in 100 mL sterilized Milli-Q water (0.4% INT solution)
2. Dissolve 0.0270 g INT-formazan in 500 mL MeOH (54 mg/L INT-formazan
solution). Dilute 2, 5, 10, 50, and 100 times with MeOH as a solvent for standard
curve.
3. Prepare the pure cultured fecal coliform samples (refer to Table 3.8), which were
pretreated by microwave, conventional heating, and autoclave.
4. An aliquot of 100 pL each sample was added to a 1.5-mL micro tube.
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5. Add 10 pL o f 0.4 % INT solution to micro tubes for each o f sample.
6. Mix thoroughly and incubate at room temperature (20°C) in the dark for 24 hours.
7. Add 1.5 mL of methanol and vortex vigorously for 30 seconds.
8. Centrifuge the micro tubes at 10,000 rpm for 5 minutes.
9. Measure the absorbance o f the supernatant and calibration by spectrophotometer
at 480 nm.
10. Alternatively, a sterilized control sample can be prepared either by autoclaving or
adding 10 pL of formalin (40%) one hour prior to the assay.
3.7.4 0-Galactosidase Enzyme Assay
P-Galactosidase is a commonly used reporter molecule.
The P-Galactosidase
Enzyme Assay System with Reporter Lysis Buffer (Part No. TB097, Promega®) is a
suitable method for assaying (3-galactosidase activity in lysates prepared from cells
transfected with P-galactosidase reporter vectors. The standard assay is performed by
adding a sample to an equal volume o f Assay 2* Buffer, which contains the substrate
ONPG (o-nitrophenyl-P-D-galactopyranoside). Samples are incubated for 30 minutes,
during which time the P-Galactosidase hydrolyzes the colorless substrate to o-nitrophenol,
which is yellow. The reaction is terminated by addition o f sodium carbonate, and the
absorbance is read at 420nm with a spectrophotometer (Rosenthal, 1987).
In this study, p-galactosidase enzyme assay was conducted to measure the enzyme
activity o f fecal coliforms pretreated by microwaves and conventional heating.
results were compared with ETS assays. The analytical protocols are as follows:
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119
1. As mentioned at ETS protocol, the same pure cultured fecal coliform samples
(refer to Table 3.8), which were pretreated by microwave, conventional heating,
and autoclaved were prepared.
2. An aliquot of 1 mL of each sample was added to 1.5 mL micro tube.
3. Centrifuge at 1,300* g for 2 minutes.
4. Discard supernatants, and dry on an absorbed paper.
5. Wash the centrifuged cells in micro tube with PBS 1x (phosphate buffered saline
without Mg2+ and Ca2+) buffer. Remove as much of the final wash as possible
using a pipet tip.
6. Centrifuge at 1,300* g for 2 minutes
7. Discard supernatants carefully, and dry on an absorbed paper again.
8. Add 4 volumes o f sterilized DI water to 1 volume of 5* RLB (reporter lysis
buffer) to produce a 1*stock solution (1.6 mL of sterilized DI water + 0.4 mL of
5X RLB = 2 mL o f 1x RLB).
9. Add a sufficient volume o f 1* RLB to cover the cells (200 pL for 1 mL initial
sample). Pipetting slowly several times to ensure complete coverage of the cells.
10. Incubate at room temperature for 15 minutes
11. Place the micro tube (sample) on ice for 2 minutes.
12. Vortex the tube for 10-15 seconds, then centrifuge at top speed in a micro
centrifuge for 2 minutes at 4°C. Transfer the supernatant (150 pL to a fresh tube).
13. The lysates may be assayed directly or stored at -70°C for at least 2 months.
14. Mix each component well before use. Place the Assay 2* Buffer on ice.
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15. As a negative control, prepare the same dilution of a cell lysate made from cells
that have not been transfected with the P-galactosidase gene.
16. Add 150 pL o f Assay 2* Buffer to each o f the tubes.
17. Mix all samples by vortexing briefly.
18. Incubate the reactions at 35°C for 30 minutes or until a faint yellow color has
developed. Color development continues for approximately 3 hours. If enzyme
activity is low, samples may be incubated overnight if the reaction tubes are
tightly capped.
19. Stop the reactions by adding 500 pL o f 1 M sodium carbonate. Mix by vortexing
briefly.
20. Read the absorbance at 420 nm.
21. One unit o f P-Galactosidase hydrolyzes 1 pmole of o-nitrophenyl-P-Dgalactopyranoside (ONPG) to o-nitrophenol and galactose per minute at pH 7.5
and 35°C.
3.7.5 Gel Electrophoresis
Gel electrophoresis is a technique, which allows for the separation o f DNA
(single-stranded, double-stranded, and super coiled) and RNA molecules based on their size.
The DNA on the gel is stained with ethidium bromide, and then visualized with UV light
(Roser, 1980). That is, more negatively charged molecules will migrate in an electric field
toward the positively charged cathode. Since DNA is negatively charged, it migrates in an
electric field toward the positively charged cathode.
The agarose matrix retards DNA
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migration roughly proportionally to DNA length when the DNA being separated is small.
Longer oligos have a harder time traveling through the matrix, while shorter oligos breeze
right through it.
3.7.5.1 Determination of Gel Concentration
The agarose gel concentration should be determined to achieve better separation
for a specific range of sizes. Table 3.9 shows the optimal relationship between gel
concentration and specific DNA size (Maniatis et al., 1987). Based on the Lambda
DNA/Hind HI Markers and table 3.9, the size of base pairs of the purified genomic DNA
from fecal coliforms was above 20 kbp. Thus 0.5% agarose solution was prepared using
the agarose (LE, analytical) and l*TAE (40 mM Tris base, 40 mM Acetic acid, 1 mM
EDTA) buffer.
Table 3.9: Agarose gel concentration.
Gel concentration (%W/V)
DNA size range (base pair)
0.3
5,000-60,000
0.6
1,000-20,000
0.7
800-10,000
0.9
500-7,000
1.2
400-6,000
1.5
200-3,000
2.0
100-2,000
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3.7.S.2 Isolation of Genomic DNA from Fecal Coliforms
To isolate the genomic DNA, the Promega Wizard Genomic DNA Purification
Kit was used. The procedure for the DNA purification occurred as follows (Promega
#TM050):
1. Pipet an aliquot of lmL of each sample (refer to Table 3.8) that pretreated by
microwave or water bath into a sterile 1.5 mL micro centrifuge tube.
2. Centrifuge the tubes at 14,000* g for 2 minutes to pellet the cells.
3. Remove the supernatant with a pipet.
4. Add 600 pL o f Nuclei Lysis Solution to each tube and carefully pipet the mixture
until the cells were re-suspended in the solution.
5. Place the tubes in an 80°C water bath for 5 minutes to lyse the cells; then cool
back to room temperature.
6. Add an aliquot of 3 pL o f RNase solution to the cell lysate. Invert the tubes 3~5
times to mix.
7. Place the tubes in the incubator at 35°C for 60 minutes; then cool back to room
temperature.
8. Add an aliquot of 200 pL o f Protein Precipitation Solution to the cell lysate
solution in each tube.
9. Votex each tube vigorously at high speed for 20 seconds to thoroughly mix the
protein Precipitation Solution with the cell lysate.
10. Place each tube in an ice bath for 5 minutes.
11. Centrifuge tubes at 14,000* g for 3 minutes to pellet the precipitate proteins.
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12. Transfer the supernatant containing the DNA in each tube to a clean 1.5-mL
micro centrifuge tube containing 600 pL o f room temperature isopropanol
(+99%) very carefully to minimize the amount of precipitated proteins
transferred with the supernatant.
13. Mix the new tubes gently by inversion to precipitate the DNA and then
centrifuged at 14,000* g for 2 minutes to pellet the DNA.
14. Carefully pipet the supernatant and add 600 pL of room temperature 70% ethanol
to each tube.
15. Invert the tubes several times to wash the DNA pellet.
16. Centrifuge the tubes at 14,000* g for 2 minutes to pellet the DNA.
17. Carefully pipet the ethanol supernatant and air-dry the DNA containing tubes for
15 minutes.
18. Add an aliquot of 100 pL o f DNA Rehydration Solution to each tube.
19. Incubate the samples to rehydrate at 65°C for one hour.
20. Store the DNA samples at 2~8°C until assay.
3.7.5.3 Electrophoresis of Genomic DNA Purified from Fecal Coliforms
Following heating or microwave irradiation and genomic DNA purification, an
agarose gel electrophoresis of the fecal coliform genomic DNA was performed on each
sample. The procedure to perform this technique was as follows (Sharp et al., 1973):
1. Based on DNA size range (20-25 kbp), prepare 50 mL of a 0.5% agarose
solution by measuring 0.25 g o f agarose (LE, analytical) into flask containing 50
mL o f 1* TAE (40 mM Tris base, 40 mM acetic acid, 1 mM EDTA) buffer.
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2. Irradiate the solution with microwaves- for 90 seconds.
3. Allowing solution to cool to about 50°C, pour the solution into gel tray, which is
sealed and contains a gel comb.
4. Leave the solution for 20 minutes to cool and solidify.
5. Carefully remove the comb and bring down the walls o f the gel box and place in
the electrophoresis chamber.
6. Submerge the gel completely with 1* TAE buffer in the chamber.
7. To prepare samples for electrophoresis, add 2 pL of 6* gel loading dye
(blue/orange) to each 2.5 pL of DNA solution. Mix the DNA solution and dye
through gentle pipetting on Para film.
8. Load an aliquot of 4.5 pL of dyed DNA solution into the wells. In addition, load
1.5 pL o f marker (Lambda DNA/Hind HI Markers) solution with 2pL into the
first well.Electrophorese the samples at 50 volts for 35 minutes (The length of
the electrophoresis box was such that the voltage was approximately 5V/cm).
10. After the run, the gel was stained in 0.5 pg/mL ethidium bromide (EtBr) by
removing the gel from the electrophoresis chamber and submerging it in
ethidium bromide for 15 minutes.
11. Finally, submerge the gel in de-ionized water to remove the excess ethidium
bromide and examined using UV Transilluminator.
12. Take a photograph of the gel using a digital camera.
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3.7.5.4 Determination of Genomic DNA Concentration
In order to isolate the genomic DNA from the fecal coliforms, the Promega®
Wizard Genomic DNA Purification Kit® and protocol were used. This protocol enables
to purify approximately 5-100 pg of DNA base on the various material.
The
concentration o f the DNA was determined using the UV-spectrophotometer (Spectral
Instruments, Inc., Tucson, Arizona, 400 series, Model No. 420).
After agarose gel
electrophoresis o f the fecal coliform genomic DNA, the following protocol was
employed:
1. Transfer an aliquot o f 1 mL o f sterilized DI water to a 1.5-mL micro centrifuge
tube.
2. Pipet an aliquot of 50 pL o f each sample that purified by DNA isolation
protocols (the Promega Wizard Genomic DNA Purification Kit®) into a sterile
1.5-mL micro centrifuge tube. This sample contains DNA rehydration solution
with isolated genomic DNA.
3. Make the total volume o f each sample 1,050 pL, which contains 50 pL o f DNA
rehydration solution and 1 mL o f sterilized DI water without DNA.
4. Place the DNA sample in a 2-mL vial to read the optical density (OD).
5. Measure the concentration of DNA using a spectrophotometer at the absorbance
o f A260.
The A260 of DNA with the concentration of 50 pg/mL is 1 OD unit.
A260
readings should be between 0.1 and 1 to be accurate. Since DNA sample was diluted 50
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126
|iL into 1 mL sterilized DI water, dilution factor was 21. Thus, the concentration of
original DNA solution was calculated as follows (Equation 3.6):
DNA concentration ((ig/mL) = Reading value o f OD units
x (50 pg/ml)/l OD unit) * dilution factor
In addition, the DNA was quantified with a UV-spectrophotometer at 260 nm and
280 nm. The absorbance of DNA sample at A280 indicates the concentration o f both
proteins and nucleic acids. The ratio o f A260/A280 represents the purity o f DNA sample.
Typically, A260/A280 should range between 1.75 and 2 for good quality DNA without
protein and other nucleic acids. However, it is noted that estimates of purity o f nucleic
acids based on OD260/OD280 ratios are unreliable and that estimates o f concentration
are inaccurate if the sample contains significant amounts of phenol.
Nucleic acid
preparations free of phenol should have OD260:OD280 ratios of -1.2 (Sambrook and
Russel, 2001).
3.8 Other Analytical Methods
The samples were analyzed for several properties: temperature, pH, solids [total
solids (TS), volatile solids (VS), total suspended solids (TSS), CODcr, and volatile
suspended solids (VSS)], total volatile fatty acids (TVFA) including acetic acid,
propionic acid, and butyric acid, total alkalinity, bicarbonate alkalinity, gas production,
gas composition, and total coliforms (fecal coliforms and E. coli). The analyses were
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conducted in accordance with Standard Methods (APHA et al., 1995).
Analytical
methods used in this study are summarized in Table 3.10.
Table 3.10: Summary o f analytical methods
Measurement
Measurement
Method
Period
pH
Daily
Orion digital pH/mV meter equipped with
the glass electrode
Alkalinity
3 times per week
Titration from Standard Method, Part 2320B
Temperature
Daily
Thermometer or thermocouples (T type)
Gas Production
Daily
Collected with gas sampling bay, and
measured volume with wet gas meter
Gas chromatography (GC), Varian 3300 GC
Gas Composition
1 time per week
equipped with column Hayesep and
Molesieve
COD
3 times per week
High range Hach’s Kit (Cat.21259-15),
0-1500 ppm range
Solids, Total
3 times per week
Standard Method, Part 2540B and 2540E
3 times per week
Standard Method, Part 2540D and 2540G
Solids,
Suspended
Gas chromatography (GC), Varian Star 3600
VFA
3 times per week
Cx equipped with column EC™-1060
3.8.1 Temperature
Water resistant thermocouple thermometer (Cole Parmer p-92900-20) and flexible
insulated wire probes (Cole-Parmer p-08506-75, T-type, -250°C~150°C) were used to
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accurately measure temperature. Response time was 0.5 seconds. Grounding the sheath
o f the thermocouple prevented the wire o f the thermocouple itself from coming into
contact with the microwave field (Kingston and Haswell, 1997).
3.8.2 pH
The pH of the samples was measured with an Orion digital pH/mV meter
equipped with a glass electrode. The pH measurement was reported as the average of
three readings for each sample after calibration with reference buffers following the
electrometric method in Standard Methods 4500-H+ B (APHA et al., 1995).
3.8.3 Solids
TS and VS concentrations were obtained from samples in accordance with Parts
2540B and 2540E, respectively, o f Standard Methods (APHA et al., 1995). TSS and
VSS concentrations were measured by following the procedure outlined in Parts 2540D
and 2540G o f Standard Methods (APHA et al., 1995).
3.8.4 Chemical Oxygen Demand (COD)
Chemical oxygen demand (COD) was determined by colorimetric Hach® methods
(reagent vial No. 21259-25,15, USEPA-approved, 40CFR). The samples were diluted
with DI water by volume to achieve the most reproducible result. Membrane filter (0.45
pm) was used for the filtering of samples. An aliquot o f 2 mL of the samples was added
to a Hach® COD vial and digested for 2 hours at 150°C. A well-defined standard COD
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129
curve was obtained using the same COD procedure outlined above without any dilution
for
various
concentrations
of
potassium
hydrogen
phthalate
(KHP).
The
spectrophotometer used was a Spectronic® Genesys™ 20 (Thermo spectronic, Rochester,
New York). Each sample’s absorbance was measured at a wavelength o f 620 nm.
3.8.5 Volatile Fatty Acids (VFAs)
Since all complex organic compounds are hydrolyzed and fermented to produce
hydrogen and short chain fatty acid, especially acetic acid, before converting to methane
gas by methanogenesis, the measurement o f VFA concentration in this batch operation
was desired in order to assess the relation of VFAs consumed in reactors. Volatile fatty
acid compounds were measured by GC Varian Star 3600-Cx equipped with an EC™1060 column. This column was set up to detect four organic acids - acetic, propionic,
butyric, and n-valeric acid.
All samples were pre-filtered by 0.45-pm filter syringes
before inoculating into GC. VFA concentrations were measured by following the
procedure outlined in Parts 5560 o f Standard Methods (APHA et al., 1995).
3.8.6 Alkalinity
Since the concentration o f alkalinity has an effect on pH, the measurement was
done in order to evaluate the reactor operation. The alkalinity was measured as described
in Standard Method 2320-B. The alkalinity results were obtained by titration samples
with 0.2 N sulfuric acid, and the results were computed using the following equation:
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130
aii, r
n r\ //rL = -----------------A xN x50000
Alkalinity,
mg nCaC03
mL sample
^
where A = mL standard acid used, and N = normality o f standard acid
3.8.7 Gas Production and Composition
Gas production from each reactor was collected daily in a 4.2-L gas-sampling bag
(standard Teflon® bags with on/off valves). Hereafter, the amount of gas production was
measured with a wet gas meter. The gas production rate can indicate roughly the amount
of methane gas. The amount o f methane gas in the total gas production was calculated
from multiplying the percentage of CH4 by the quantities of total gas production.
The composition o f biogas produced from each digester was analyzed with gas
chromatography (GC), model Varian 3300 GC equipped with column Hayesep and
Molesieve. In this study, these two columns were set up to detect three gas compounds,
methane, carbon dioxide, and nitrogen gas. The biogas concentrations were measured by
following the procedure outlined in Parts 6211 of Standard Methods (APHA et al., 1995).
3.8.8 Total Coliforms and E. coli Analysis
The membrane filter (MF, standard method 9222B) test was used for total
coliform count.
The MF test is a primary indicator o f the bacteriological quality of
potable water, distribution system waters, and public water supplies. The MF results are
obtained in 24 hours. An appropriate volume of sludge sample or its dilution (x 102, x 103
or *106) is passed through a MF that retains the bacteria present in the sample. The filter
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131
containing microorganisms is placed on an absorbent pad saturated with m-ColiBlue 24®
(U.S. EPA approved method 10029) in a petri dish. The dish is incubated at 35±0.5°C for
24±2 hours. After incubation, the typical red (coliforms) and blue (E. coli) colonies are
counted under low magnification and the number o f total coliforms, total number of blue
and red colonies, will be reported per 100 mL of the original sample. The procedure of
the total coliform and E. coli analysis is as follows (U.S. EPA, 1987):
1. Take 100 mL of samples. One thousand or one million dilutions of the original
sample are often necessary to reduce the number of bacteria to measurable levels.
These volumes usually provide the recommended colony count o f 20-80
colonies and less than 200 total bacterial colonies per filter after dilution.
2. Transfer 1 mL o f sample through a series of known volumes (99 mL) of
sterilized water. Repeated this procedure until the desired range o f bacterial
densities is reached.
3. Shake sample bottle vigorously (25 times) to evenly distribute the bacteria,
taking care to secure the screw cap and prevent leakage during shaking.
4. Place a sterile membrane on the filter base using a sterile forceps, grid side up.
Attach the top of the filter unit.
5. Shake the sample vigorously 25 times. Measure and pour the sample into the
funnel with the vacuum off.
6. Filter the sample and rinse the sides o f the funnel walls at least twice with 20-30
mL of sterile dilution water.
7. Turn off the vacuum and remove the funnel from the filter base.
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8. Using sterile forceps, aseptically remove the membrane from the filter base.
Place the filter, grid side up, on the absorbent pad saturated with m-ColiBlue
24® broth using a rolling motion to prevent air bubbles from being trapped
between the pad and membrane. Mark the dish and bench forms with sample
identity and sample volume.
9. Prepare at least two replicate plates for each sample volume.
10. Invert the dishes and incubate for 24±2 hours at 35±0.5°C in an atmosphere
with the humidity near saturation.
11. After incubation, using a binocular (dissection) microscope with a magnification
of 10x or 15*, count the red and blue colonies on those membrane filters
containing 20-80 colonies and less than 200 total bacterial colonies.
12. Run duplicate analyses on all sample volumes filtered. Tentative limits are set
until 20 data points are collected.
3.8.9 Fecal Coliform Analysis
The fecal coliform analysis was also adopted from the MF test (standard method
9222D) for the analysis. The membrane filter method provides direct enumeration of the
fecal coliform group without enrichment or subsequent testing.
An appropriate volume o f sludge sample or its dilution (*102, *103 or *106) is
passed through a membrane filter that retains the bacteria present in the sample. The
filter containing the microorganisms is placed on an absorbent pad saturated with m-FC
broth in a petri dish. The dish is incubated at 44.5±0.2°C for 24 hours. After incubation,
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the typical blue colonies are counted under low magnification and the number of fecal
coliforms is reported per 100 mL o f original sample. For the detection o f fecal coliforms,
m-FC broth was used. Incubation was performed with the petri dishes for 24±2 hours at
44.5±0.2°C.
Those plates with 20-60 blue (sometimes greenish-blue) colonies were
counted. Non-fecal colonies were gray, buff, or colorless and were not counted. The
total coliform detection protocol (refer to Section 3.6.8) was also used for observation
and determination.
3.9 Preservation and Holding Times
Sample preservation methods and recommended holding times by the U.S. EPA
shown in Table 3.11 were adopted for the study.
Table 3.11: Recommended preservatives and holding times.
Parameter
Preservative
Maximum holding time
Coliforms (fecal and total)
Cool, 4°C; 0.008% Na2S20 3
6 hours
Alkalinity
Cool, 4°C
14 days
COD (TCOD, SCOD)
1 mL conc. H2S0 4 to pH < 2;
28 days
Room temp.
pH
Solids (TS, VS, TSS,
VSS), VFAs
Cool, 4°C
24 hours
Cool, 4°C
7 days
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134
4. RESULTS AND DISCUSSION
4.1 Microwave Power Calibration
Based on the U.S. EPA method 3052 (1985 Annual Book of ASTM Standards),
the microwave unit was calibrated for the normalization. The microwave unit, 1 kW of
total output power, used in this research was comprised of an exhausting fan, inside lamp,
rotating equipment, and a microwave source (magnetron). For this reason, the power of
the microwave unit must be calibrated before the performed test.
The calibration was accomplished by measuring the temperature rise in water
samples exposed to microwave radiation. 100, 200, 300, 400, 500, and 1000 g of water
were weighed into a suitable glass beaker (1,000 mL). The initial temperature of the
water was 24±2°C measured to ±0.2°C. The beaker was circulated continuously through
the microwave field for 1 minute at the maximum power setting with the system exhaust
fan on maximum. The beaker was removed and the water vigorously stirred. A magnetic
stirring bar was used immediately after microwave irradiation and the maximum
temperature was recorded within the first 30 seconds to ±0.2°C. As shown in Figure 4.1,
the temperature decreased nonlinearly from 74.5 to 34.8±0.2°C for 1-minute microwave
irradiation, corresponding to 100, 200, 300, 500, and lOOOg of tap water, respectively.
The regression line was well fitted with second order power regression (y = 429.57 x-036,
R2 = 0.99).
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135
100
80 -
u
23
2u
o
Q.
S
4>
f-
60 Y = 3 3 .4 4 + 6 4 .4 1 e x p (-0 .0 0 3 3 x )
R 2 = 0 .9 9 7
40 -
20
-
0
200
400
600
800
1000
1200
Df water, g
Figure 4.1: Temperature variations o f tap water after 60 sec. of microwave irradiation
with different tap water masses.
The absorbed power was determined by the following relationship (from equation
2.34):
P = KMCp( T - T 0) / t
(4.1)
where P = power absorbed by the sample in watts (W, W = joule/sec ), K. = 4.184
(conversion factor from cal/sec to watts), Cp = heat capacity, thermal capacity, or specific
heat (cal/g °C ) o f water, M = mass o f the water sample (g), T-T0 = final temperature initial temperature (°C), and t = irradiation time in seconds (s).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
136
Figure 4.2 shows the absorbed power (watts) with microwave irradiation for 1
minute. Although the microwave unit used for the calibration was 1 kW, approximately
750 watts were absorbed into 1,000 g of water, indicating that approximately 18% of
energy loss will be associated with the exhausting fan, circulating plate, and small light
inside of the microwave unit. In addition, the moisture content in the microwave cavity
will be a possible reason for loss o f energy during microwave irradiation.
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137
1000
800 ts
C3
£
S
£o
a.
J
l*
O
(fl
X
<
y = 812.23(1-exp(-0.0065x))
R2 = 0.983
600 -
400 -
200
-
0
200
400
600
800
1000
1200
Water, g
Figure 4.2: Absorbed power variations o f 1 kW microwave unit for 60 sec. of microwave
irradiation.
When a small volume of water (< 500 g) was applied in the microwave unit,
absorbed power was below 700 watts. Thus, it can be said that the microwave power
absorption is a function of sample volume (area and size), penetration depth of a material,
and peripherals inside of the microwave unit. The maximum absorbed power o f the
microwave unit used in this research was determined to be approximately 812 watts
(joule/second) for the experiments.
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138
4.2 Penetration Depth Estimation
The microwave energy in itself is not thermal energy but the interactions between
microwave and dielectric material (Decarau and Peterson, 1986). The heating conversion
(power absorption) o f microwave energy can be approximately quantified with electrical
field strength (E) and the relative dielectric loss (e"). As discussed in Chapter 2, the
dielectric constant (s') and the loss tangent (tan 8) are the properties of the material. The
electrical field strength and the frequency (f) represent the energy source. It is noted that
the increasing electric field strength has a remarkable effect on the power density. The
energy transfer is influenced by the electrical properties of the material.
Two different definitions have been proposed to assess the penetration depth of
microwave power. The first definition is Lambert’s expression for power absorption,
which is defined as the distance from the surface o f a dielectric material where the
incident power is decreased to 1/e o f the incident power. Second, the penetration depth is
defined as half-power depth. If the power is reduced to half of the incident power, P/Po at
half power depth = 1/2. Therefore, e‘2ad = Vi, and d = 0.347/a' (Singh and Heldman,
1993).
According to Equations 2.34 and 2.35, if frequency (f) is constant, and the
variations of material density (p) and specific heat (Cp) are relatively small, the
temperature increase by microwave irradiation will be the function of s".
The penetration depth can be estimated in terms of half-power depth (P o/2), that is,
the distance from the surface of the material where the power is one half o f the incident
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139
power. The following four assumptions were made. First, electric field strength (E) and
frequency (f) in the microwave unit are constant at the moment of measurement. Second,
no heat transfers through the samples occur. Third, microwave power is absorbed to only
the sample medium (total incident power = total dissipated heating energy in sample
medium) in the microwave cavity. Last, the specific heat (Cp) is constant (1 cal/g°C) for
water, tap water, 10% NaCl water, and sludges. Then, the equation for determination of
the penetration depth can be simply derived using second definition o f penetration depth
as follows:
K C ,X M ,( T ,-T 0)
d --------------------- = I X-------- s=1-------------------
t
2
(4.1)
t
where Mj = mass o f sample where the power is one half of the incident power penetration
depth (penetration depth * width * height, g), Mj = total mass o f sample in reservoir
(depth x width * height, g), Po = experimental total power absorbed by the samples in
watts (W ), Tj-To = final temperature at j 1*1reservoir - initial temperature (°C ), Tj-To = final
temperature at i,h reservoir - initial temperature (°C), and t = microwave irradiation time
in seconds (s).
In other words, the temperature of samples in each reservoir will be attenuated as
Figure 4.3. If Mi is the first volume of medium in the first reservoir from microwave
source, Mi will have the highest temperature because most microwave energy will
dissipate in the first medium. Then, transmitted wave energy from the first medium will
be absorbed in the second medium (M2). Likewise, M3, M4, and M5 will be absorbed
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140
energies from transmitted energy o f the previous medium. To satisfy this assumption,
other microwave energy loss should be ignored.
On the other hand, microwave power dissipation depends on the material, wave
frequency, water content, and so on.
If a 915-MHz microwave system is used, the
temperature trends can be shown conversely. That is, as shown in Figure 2.17 and 2.18
(Copson, 1975), the temperature o f the first medium will show lower than the second
medium, indicating that microwave energy absorption (heating) is inversely proportional
to penetration depth.
However, the 2,450-MHz microwave unit used in this study
showed typical temperature trends as shown in Figure 4.3.
The left term o f Equation 4.3 indicates the absorbed microwave heating energy
(w) from the surface o f the material to penetration depth. The right term represents half
o f the total absorbed microwave energy. Since the equation involves the penetration
depth term (Dp) and P = 0.5Po, the penetration depth of the sample by 2,450-MHz
microwaves can be calculated.
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141
width
T em perature, T,
Mj = mass o f sam ple up to penetration depth (g)
Ml
Penetration depth, D.
M2
M3
2.7
M4
M5
2.7
2.8
-► Distance from
material surface
(cm )
Figure 4.3: Schematic of temperature attenuation in vessel for determination o f
penetration depth.
As shown in Figure 4.4, the temperature varied with each reservoir along with the
distance from the microwave source.
Initial temperatures of DI, tab, and 10% NaCl
solution were controlled at 20±0.2°C and primary and waste activated sludge at 10±0.2°C.
Since penetration depth was expected to be 0 to 2.4 cm (first or second reservoir), the
depth o f first and second reservoirs was 1.2 cm. Metaxas and Meredith (1983) and von
Hippel (1954) reported that the relative dielectric loss factor of 3% NaCl solution was
over three times higher (41.87) than DI water (12.0) at 25°C. However, 10% NaCl
solution showed slightly high power dissipations of 1.5% and 6.4% at first reservoir
compared with DI water and tap water, respectively.
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142
100
□ DI water
■ Tap water
■ 10% NaCl
BPS
□ WAS
90
80
p
70
2J
60
1
50
|
40
£
30
Contro
20
10
0
0
1.2
2.4
5.1
7.8
10.6
Distance, cm
Figure 4.4: Temperature variations using acryl-air baffled vessel by microwaves (average
60 sec. irradiation).
Although the initial temperature o f PS and WAS was 10±0.2°C, primary sludge
and WAS absorbed 254.14±35.1 and 241.74±40.5 watts at the first reservoir, respectively,
indicating that primary sludge absorbed the highest microwave energy among the five
samples. It appears that microwave energy absorption depends on the characteristics of
materials such as water content, ionic strength, percentage of protein and fat, viscosity,
and so on (Metaxas, 1996).
It is noted that the higher the power absorption, the less the penetration depth.
Table 4.1 shows the average o f the results o f three runs on penetration depth tests. The
penetration depths of PS and WAS were 1.73 and 1.11 cm, respectively. These values
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143
were relatively small compared with the water sample. NaCl solution had the highest
penetration depth (2.56 cm), followed by DI water (2.21 cm).
As discussed above,
penetration depth of materials is determined by many complicated factors. One o f the
factors is solid contents (water contents) because total solid concentrations o f WAS and
PS used in the test was 29,800 and 41,300 mg/L, respectively.
Lin et al. (1978) investigated the effect o f microwave radiation on human tissue
media and biological materials.
To perform the study, biological materials may be
classified into three major groups according to their water content (90, 80, and 50%).
The dielectric constant and conductivity o f tissues with low water content are an order of
magnitude lower than those with higher water content characterized by polar properties
o f water and macromolecules (proteins) in electrolytes (Lin and Dept o f Physical
Medicine and Rehabilitation, 1978). Water molecules play several roles in all systems.
Conventionally, water is divided in free water and bound water (Oliveira and Oliveira,
1999). The bond between water molecules and the solid matrix can have very diverse
intensities such as physical adsorption, hydrogen bond, polar bonds, ionic bonds, and so
on. Some o f the bound water can actually be made available for use by endogenous
metabolism or microbial activity (Karel, 1975).
Another possible reason considered was viscosity. WAS taken from the thickener
(DAF) showed a high viscosity, although it was not tested. Since the heating mechanism
o f the microwave is regarded as the interaction o f dielectric materials (dipole moments of
polar materials like water molecules), heating is affected by viscosity (Metaxas and
Meredith, 1983).
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144
The results of penetration depth (Dp) are also associated with the attenuation
factor (a'), which are shown in Table 4.1. Attenuation factor can be calculated using a ' =
0.347/penetration depth (Lambert’s expression). Those values will be useful for design
o f a microwave unit for municipal sludge application.
Table 4.1: Determination o f penetration depth and attenuation factor.
10% NaCl
PS
WAS
-
29,800
41,300
60
60
60
60
20
20
20
10
10
Ml
78.3
74.7
79.5
78.7
76.0
Temperature
M2
46.0
42.0
41.8
41.7
35.3
(±0.2°C)
M3
31.7
29.0
30.7
20.0
16.8
M4
29.7
27.0
30.3
19.0
13.5
M5
29.7
27.3
31.0
19.0
14.5
Total absorbed (P, watts)
559.11
503.86
586.38
576.62
425.18
Standard error of P
24.88
25.47
27.36
35.10
40.48
Penetration depth (cm)
2.21
2.16
2.56
1.73
1.11
8.53
8.15
8.60
10.40
10.33
0.157
0.160
0.136
0.200
0.313
Sample
DI water
Tap water
Total solids (mg/L)
-
-
Microwave, sec
60
Initial
Standard error of
sol.
temperature
Attenuation factor (a', cm)
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145
4.3 Organic Release by Microwave Irradiation
It is expected that suspended solids or some colloidal nonsoluble materials in
sludge can be solubilized when sludge is irradiated with microwaves, leading to leaching
of soluble COD (SCOD) from sludge into bulk solution. A series o f experiments was
performed with 20 mL o f sludge samples in a 50 mL container at the microwave
irradiation times of 0 (control), 3, 6, 9, 12, 15 and 18 seconds, corresponding to 12, 21,
30, 42, 51, 63, and 70±3°C, to evaluate the degree of solubilization of organics by
microwave irradiation. The sludge samples tested were anaerobic digester sludge (ADS),
primary sludge (PS), and waste activated sludge (WAS). Figure 4.5 shows the changes in
soluble COD at various temperatures caused by microwave irradiation.
10000
8000
>
16% increase.
6000
125%jj
4000
:ase
WAS &
ADS
2000
0
[
AY'
45% increase
20
40
60
80
Temperature, °Z
Figure 4.5: Changes in soluble COD at various temperatures caused by microwave
irradiation.
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146
PS had the greatest initial soluble COD (SCOD) concentration (6,220 mg/L),
followed by WAS (3,510 mg/L), and ADS (2,340 mg/L). Since ADS has been stabilized
in anaerobic digesters for over 20 to 30 days, it is natural to have lower soluble COD
values than PS and WAS. While, the soluble COD value of WAS showed the greatest
increase (125%) compared with that of PS. It is expected that WAS basically contains
more non-biodegradable suspended materials, which can be easily solubilized by heating,
than PS or ADS. This expectation was proved at BMP test. In other words, the gas
production of WAS was not linearly proportional to the increase o f SCOD. It was
discussed at Section 4.4.2 in detail. In the case of ADS, the soluble COD value reached a
maximum at 51.5°C and then decreased slightly. Soluble COD values o f PS and WAS
increased when the sludge sample temperature rose to over 30°C by microwave
irradiation. The temperatures o f initial samples ranged from 10 to 15°C (the first data
point of each curve in Figure 4.5). Temperatures increased from 21 to 72.5°C, depending
on the microwave irradiation time and type of sludge samples.
WAS had a greater
temperature increase than ADS and PS. The soluble COD values o f PS, WAS, and ADS
increased from 6,220 to 7,200 mg/L, 3,510 to 7,890 mg/L, and 2,340 to 3,400 mg/L,
leading to a soluble COD increase o f 16, 125, and 45%, respectively. The variations of
the SCOD/total COD (TCOD) ratio are shown in Figure 4.6.
With the increase in temperature, the SCOD/TCOD ratio increased in the case of
WAS. However, PS and ADS had a slight increase in the SCOD/TCOD ratio at all
temperatures.
As discussed above, SCOD values after 12-18 seconds o f irradiation
increased by 124.7, 45.4, and 16.4 % for WAS, ADS, and PS, leading to an increase of
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147
the SCOD/TCOD ratio to 9.0,4.6, and 1.5% for WAS, ADS, and PS, respectively. These
increases in SCOD are thought to increase the gas production rate and the efficiency of
anaerobic digestion.
20.0
N°
; i5.o
_o
§ ADS
■ PS
■ WAS
2
Q
O 10.0
U
§
O
oC/3
5.0
0.0
3
6
9
12
15
18
Microwave irradiation time, sec
Figure 4.6: SCOD/TCOD ratios o f ADS, PS and WAS after microwave irradiation.
4.4 Batch Microwave Tests
4.4.1 First Batch Microwave Test
The first batch microwave tests were conducted to investigate the effect of
microwave irradiation o f ADS on the total gas production. An aliquot o f a 100 mL ADS
sample was irradiated in a 500-mL beaker for 15, 30, 60, and 120 seconds in a
microwave oven. The temperatures rose to 36, 48, 84 and 100±3°C accordingly from the
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148
initial temperature o f 10°C. After microwave irradiation, a 100 mL ADS sample was
added to a 150-mL serum bottle and the gas volume was monitored following incubation
at 35°C.
The cumulative total gas production of raw anaerobic digester sludge (RADS)
after a series o f microwave irradiation procedures is shown in Figure 4.7. It appears that
at 15 seconds (MI-ADS), anaerobic bacteria such as acetogenic and methanogenic
bacteria were not completely killed, but other organics were broken down so that gas
production was slightly higher than the control where the seed ADS was not irradiated
with microwaves. From 30-seconds o f microwave irradiation, anaerobic bacteria started
to be inactivated.
When ADS was irradiated with microwaves for 60 seconds, no gas was produced
for 16 days. It appears that methanogenic, sulfate reducing, and acid forming bacteria
were inactivated for 16 days but were capable of surviving in a dormancy state for that
period.
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149
400
Control
C
o
-a
oh »
CL
3so
u>
Stopped at
16.4 day
E
Gas producti/n
started
15 M I-ADS 100 mL
►
30 M I-ADS 100 mL
3
u
RADS 100 mL
6 0 M I-ADS 100 mL
Dormancy
120 M I-ADS 100 mL
►
80
Time, days
Figure 4.7: Cumulative gas production of RADS with microwave irradiation times.
A mixture of 50 mL o f RADS and 50 mL of microwave-irradiated ADS were
added in a 150-mL serum bottle and the cumulative gas production was evaluated. The
results are shown in Figure 4.8. The cumulative gas productions of the control and the
15-second microwave-irradiated ADS were almost the same. The relative cumulative gas
productions for 16.4 days were 0.97, 1.25, 1.02, and 1.22 when the control was set at 1,
corresponding to 36, 48, 84, and 100°C, respectively. Based on this result, it can be said
that the gas production will increase by 22-25% when a portion o f ADS is irradiated by
microwaves compared to a conventional mesophilic operation.
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150
400
300
200
100
0
5
RADS
RADS
RADS
RADS
RADS
100 mL
50 mL + 15 MI-ADS 50 mL
50 mL + 30 MI-ADS 50 mL
50 mL + 60 MI-ADS 50 mL
50 mL + 120 MI-ADS 50 mL
10
15
20
Time, days
Figure 4.8: Cumulative gas production o f the specific mixture conditions along with
microwave irradiation time.
4.4.2 Second Batch Microwave Tests
The objective o f the second batch microwave test was to evaluate the gas
production between microwave-irradiated and conventionally heated sludges using the
BMP test at 25 and 35°C. Initial and final temperatures after microwave irradiation or
conventional heating and times to reach the final temperatures are summarized in Table
4.2.
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151
Table 4.2: Initial and final temperatures after a given duration of microwave irradiation.
Run no.
Sample
Initial temp.
±3°C
Final temp.
±3°C
AT
±3°C
0
Raw ADS for seeding
10
-
_
-
1,4
Raw ADS (control)
10
.
_
-
2 ,5
Raw PS (control)
10
_
.
_
3 ,6
Raw WAS (control)
10
.
_
_
7
ADS (MW 15s)
10
25
15
15
8
ADS (MW 30s)
10
45
35
30
9
ADS (MW 60s)
10
65
55
60
10
ADS (MW 90s)
10
85
75
90
11
ADS (MW 120s)
10
100
90
120
12
PS (MW 15s)
10
25
15
15
13
PS (MW 30s)
10
45
35
30
14
PS (MW 60s)
10
65
55
60
15
PS (MW 90s)
10
85
75
90
16
PS (MW 120s)
10
100
90
120
17
WAS (MW 15s)
10
25
15
15
18
WAS (MW 30s)
10
45
35
30
19
WAS (MW 60s)
10
65
55
60
20
WAS (MW 90s)
10
85
75
90
21
WAS (MW 120s)
10
100
90
120
22
ADS (CH 45°C)
10
45
35
120
23
ADS (CH 65°C)
10
65
55
190
24
ADS (CH 100°C)
10
100
90
302
25
PS (CH 45°C)
10
45
35
118
26
PS (CH 65°C)
10
65
55
175
27
PS (CH I00°C)
10
100
90
288
28
WAS (CH 45°C)
10
45
35
125
29
WAS (CH 65°C)
10
65
55
210
30
WAS (CH I00°C)
10
100
90
320
Time for target
temp., sec
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152
BMP test at 3S°C
After a series of microwave irradiation, batch tests were performed to evaluate the
gas production and pathogen destruction efficiency.
Experimental conditions are
described in detail in Section 3.4.2. An aliquot of 10 mL of a sludge sample treated
under various conditions was mixed with 30 mL of anaerobic digester seed and the
volume of gas produced was measured. The composition of gas in the headspace was
analyzed with a gas chromatograph (GC). The volume of methane produced after various
treatments was divided by that of the control to obtain the relative methane production.
As shown in Figure 4.9, the volume o f methane produced by microwaves or
conventional heat pretreatments was divided by that of the control, respectively, to obtain
the
ratio (relative
B M P /B M P co n tro i
B M P /B M P c o n tro i
ratios o f
ADS
Conversely,
methane
ratio of the control for A D S ,
and
W AS
PS
PS,
and
W AS
Assuming that the
should be 1 , the relative
showed a relatively greater BMP value at BMP 10. indicating that
was greater than
BM P
were over 1.5 times greater than that of the control at BMP5 .
contains more non-biodegradable materials than
PS
production).
A D S.
ADS
or
PS.
WAS
After 10 days, the ratio of
As a whole, the relative BMP ratios (BMP/BMPcontroi) of
microwave-irradiated sludges were approximately
10-15% higher than that of
conventional heated sludges. However, almost the same trend was shown at the ratio of
W AS.
Therefore, it can be said that the effect o f microwave irradiation on methane
production is significant for ADS and PS but less for WAS. The effect of microwave
irradiation on methane production was most pronounced at 5 days but less apparent at 15
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153
and 30 days, indicating that it is possible to shorten the solids retention time (SRT) in the
anaerobic digester.
2.00
o
03
*1
1.50
o
c
o
CU
2 1.00
Controf
CQ
a:
s
ca 0.50
o
o
■RA D S
RADS
RADS
• RADS
RADS
RA D S
30
30
30
30
30
30
+
+
+
+
+
+
ADS 10 (M W )avg.
PS 10 (M W )avg.
W AS 10 (M W )avg.
AD S 10 (C H )avg.
PS 10 (C H )avg.
W AS 10 (C H )avg.
0.00
5
10
15
20
25
30
35
BMP, days
Figure 4.9: Variations o f B M P /B M P c o n tro i ratio for A D S ,
PS,
and
W AS
at 35°C
Table 4.3 shows the summary of methane contents and relative BMP values at 2nd
BMP tests. Methane contents of the controls, ADS, PS, and WAS at 35°C showed 54.1,
57.6, and 62.5%, respectively. The average methane contents of ADS, PS, and WAS
varied from 62.8 to 69.8% during the mesophilic BMP tests. PS had approximately 70%
o f the average methane content, which was the highest in the second batch test.
In the case o f microwave irradiation, the methane volume increase, when using
WAS, was only significant when the microwave irradiation time was 30 seconds or
greater. From 60 seconds of irradiation, the methane volume increased by 0 to 25% at 45,
65, and 100°C of final temperatures for the 30 day BMP test (BMP30) compared with the
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154
control WAS. For PS, the methane volume increased by 70 to 87% after 5 days (BMP5),
54 to 74% after 15 days (BMP 15), and 52 to 67% after 30 days (BMP 30) at 45, 65, and
100°C of final temperature. Also, the methane volume increased by 49 to 113% after 5
days, 34 to 72% after 15 days, and 32 to 6 6 % after 30 days for ADS.
In the case o f conventional heating with a water bath, the methane volume of
WAS increased by 0 to 27% for the 30 day BMP test (BMP30) compared with the control
WAS at 45, 65, and 100°C of final temperatures. This result showed nearly the same
trend of microwave irradiation. For PS, the methane volume increased by 53 to 64%
after 5 days (BMP5), 48 to 61% after 15 days (BMP 15), and 51 to 59% after 30 days
(BMP 30). In addition, the methane volume increased by 43 to 77% after 5 days, 35 to
45% after 15 days, and 34 to 40% after 30 days for ADS. In general, the average of
microwave-irradiated sludges (ADS, PS, and WAS) resulted in
8%
and 48% more
methane production (BMP) than conventional heating and the control, respectively.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
155
Table 4.3: Relative methane production rates at 35°C in the second batch test.
Mixing conditions
Sample Methane
No.
(%)
Measurement time
5 days
10 days 15 days 30 days
Relative methane production
1.00
1.00
1.00
1.00
RADS 30 + RADS 10 (control)
1,4
54.1
RADS 30 + RPS 10 (control)
2 ,5
57.6
1.00
1.00
1.00
1.00
RADS 30 + RWAS 10 (control)
3 ,6
62.5
1.00
1.00
1.00
1.00
58.1
1
1
I
1
Average
RADS 30 + ADS (15S MW) 10
7
66.0
1.58
1.60
1.46
1.44
RADS 30 + ADS (30S MW) 10
8
64.6
1.49
1.48
1.34
1.32
RADS 30 + ADS (60S MW) 10
9
65.4
1.92
1.74
1.54
1.48
RADS 30 + ADS (90S MW) 10
10
65.3
2.16
1.95
1.72
1.66
RADS 30 + ADS (120S MW) 10
11
65.2
2.13
1.93
1.72
1.65
65.3
1.86
1.74
1.56
1.51
Average
RADS 30 + PS (15S MW) 10
12
68.0
1.03
0.97
1.03
1.10
RADS 30 + PS (30S MW) 10
13
70.2
1.74
1.61
1.60
1.60
RADS 30 + PS (60S MW) 10
14
68.1
1.70
1.57
1.54
1.52
RADS 30 + PS (90S MW) 10
15
65.4
1.77
1.64
1.61
1.56
RADS 30 + PS (120S MW) 10
16
63.7
1.87
1.75
1.74
1.67
67.1
1.62
1.61
1.47
1.44
Average
RADS 30 + WAS (15S MW) 10
17
69.7
0.59
0.85
0.89
0.93
RADS 30 + WAS (30S MW) 10
18
64.3
0.98
1.20
1.21
1.21
RADS 30 + WAS (60S MW) 10
19
64.9
1.08
1.25
1.22
1.20
RADS 30 + WAS (90S MW) 10
20
61.2
1.00
1.21
1.18
1.15
RADS 30 + WAS (120S MW) 10
21
59.3
1.04
1.20
1.17
1.15
63.9
0.94
1.14
1.13
1.13
Average
RADS 30 + ADS (45°C CH) 10
22
64.3
1.43
1.48
1.35
1.34
RADS 30 + ADS (65°C CH) 10
23
62.8
1.64
1.58
1.40
1.35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
156
Measurement time
Mixing conditions
RADS 30 + ADS (100°C CH) 10
Sample Methane
No.
(%)
24
Average
10 days 15 days 30 days
5 days
Relative methane production
59.5
1.77
1.63
1.45
1.40
62.2
1.61
1.56
1.40
1.36
RADS 30 + PS (45°C CH) 10
25
70.4
1.53
1.45
1.48
1.51
RADS 30 + PS (65°C CH) 10
26
69.3
1.64
1.53
1.52
1.53
RADS 30 + PS (100°C CH) 10
27
69.8
1.63
1.60
1.61
1.59
69.8
1.60
1.53
1.54
1.55
Average
RADS 30 + WAS (45°C CH) 10
28
75.2
1.18
1.26
1.24
1.22
RADS 30 + WAS (65°C CH) 10
29
65.4
1.01
1.18
1.15
1.13
RADS 30 + WAS (100°C CH) 10
30
67.6
1.07
1.27
1.25
1.22
69.4
1.09
1.23
1.21
1.19
Average
MW: microwave irradiation, CH: conventiona heating
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157
Cumulative total gas productions with microwaves for ADS are shown in Figure
4.10. After microwave irradiation, total gas production increased by 3 to 26% after 50
days.
Since most decomposable organics attributed to anaerobic stabilization during
anaerobic digestion, it was expected that the gas production may be relatively lower than
Ps and WAS. When sludge was irradiated with microwaves for over 90 seconds, the
cumulative total gas production was almost the same, indicating that organic
concentration s was lower than PS and WAS when sampled from Nine Springs WWTP.
200.0
E
o 150.0
o
3
T3
e
*CQ loo.o
00
0>
1
E
RADS 30 + ADS (15S MWI) 10
- O - RADS 30 + ADS (30S MWI) 10
- O - RADS 30 + ADS (60S MWI) 10
- £ r - RADS 30 + ADS (90S MWI) 10
RADS 30 + ADS (120S MWI) 10
— RADS 30 + RADS 10 (control)
50.0
3
u
0.0
0.0
10.0
20.0
30.0
40.0
50.0
Time, days
Figure 4.10: Cumulative biogas production o f ADS at 35°C after microwave irradiation.
Cumulative total gas productions with microwaves for PS are shown in Figure
4.11. After microwave irradiation, total gas production increased by 25 to 52% after 50
days.
With the increase in the microwave irradiation time, the cumulative total gas
production also increased. The cumulative gas production was almost 10% greater at the
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
158
microwave irradiation time of 120 seconds corresponding to 100°C than at 90 seconds.
On the other hand, primary sludge irradiated with microwaves for 15 seconds had
approximately 20% less gas production than the control, indicating potential leaching of
inhibitory compounds as discussed by Haug et al. (1978).
500
-------------------------------------------------------------------------------
E
c- 400
_o
c5
|
300
Q.
C/5
«
“ 200
£
-2
E 100
<3
RADS
RADS
RADS
RADS
RADS
RADS
RADS
RADS
- RADS
—
i
0
0
30 +
30 +
30 +
30 +
30 +
30 +
PS (15S MWI) 10
PS (30S MWI) 10
PS (60S MWI) 10
PS (90S MWI) 10
PS (120S MWI) 10
RPS 10 (control)
------------------------------------
10
20
30
40
50
Time, days
Figure 4.11: Cumulative biogas production o f PS at 35°C after microwave irradiation.
Cumulative total gas productions with microwaves for WAS are shown in Figure
4.12.
Surprisingly, the cumulative total gas production was lower at the microwave
irradiation time of 15 seconds than the control. Unknown inhibitory compounds appear
to be released at low temperatures (25°C). There was little difference in the cumulative
total gas production when sludge was irradiated with microwaves for 30 to 120 seconds.
The microwave irradiation time for WAS must be determined by pathogen destruction
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159
rather than gas production. After microwave irradiation, total gas production increased
by 8 to 14% after 50 days, indicating less effectiveness of microwave irradiation than
ADS and PS. WAS had approximately 16—18% lower gas production per volume than
PS. It appears that the microwave penetration depth and viscosity play significant roles
in the efficiency of microwaves on WAS.
300
-j
E
c
.o
|
200
T3
O
w
Q.
e/l
CO
00
u
>
RADS
RADS
- O - RADS
A RADS
—O— RA D S
RADS
100
Jo
3
E
3
o
0
0
10
20
30
30
30
30
30
30
+
+
+
+
+
+
30
WAS (1 5S M W I) 10
WAS (30S M W I) 10
WAS (60S M W I) 10
WAS (90S M W I) 10
WAS (120S M W I) 10
RWAS 10 (control)
40
50
Time, days
Figure 4.12: Cumulative biogas production o f WAS irradiated by microwaves at 35°C.
As shown in Figure 4.13, the cumulative biogas productions (mL) o f ADS, PS,
and WAS for the initial 5 days were divided by that of each control. In other words,
relative biogas productions o f ADS, PS, and WAS for 5 days were obtained to compare
them with the control (control = 1 ).
Much higher biogas productions were shown in
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160
proportion to the pretreated temperature increase, although BMP tests were performed at
35°C.
For the initial 5 days, the relative cumulative biogas productions o f ADS, PS, and
WAS by microwave irradiation were approximately 13, 20, and 5% higher than
conventional heating, and 54, 52, and 3% higher than the control.
As a result, it is
expected that microwave irradiation as a pretreatment for ADS and PS will be effective to
obtain higher biogas production.
2.5
■£o
<S2 2.0
U
rn
□ ADS 30 +
□ ADS 30 +
BADS 30 +
BADS 30 +
QUADS 30 +
BADS 30 +
ADS 10 (MW)
ADS 10 (CH)
PS 10 (MW)
PS 10 (CH)
WAS 10 (MW)
WAS 10 (CH)
TO
3
S 1.5
o.
A
00
o
IE
<
u 1.0
>
I
5
0.5
45
Control = I
65
100
Temperature, °C
Figure 4.13: Cumulative biogas production ratios of ADS, PS, and WAS for initial 5 days
(control =1, standard error bar).
In summary, PS was most sensitive to microwave irradiation in terms of
cumulative gas production followed by ADS and WAS. PS was readily biodegradable
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
161
within 5 days. Although WAS had greater soluble COD values after microwave
irradiation than ADS or PS (Figure 4.6), the non-biodegradable soluble material content
o f WAS may be high.
In general, when microwave-irradiated sludge is mixed with
anaerobic digester seed sludge, the gas production will be higher than conventionally
heated sludge.
BMP test at 35°C vs. 25°C
Second batch tests were also performed to evaluate the methane production at 35
and 25 °C. Experimental conditions and sample preparations were the same as performed
at 35°C. The results are summarized in Table 4.4.
The average methane content o f ADS, PS, and WAS at 25°C was 54.4%, which
was approximately 9% lower than at 35°C. To compare the BMP values at 25°C with
35°C, the volume of methane was divided by BMP values at 25°C, respectively.
Assuming that the BMP/BMP ratio for ADS, PS, and WAS at 25°C is 1, the relative
BMP ratios o f ADS, PS, and WAS at 35°C were evaluated.
The average BMP at 35°C increased by 87% after 5 days (BMPs), 60 to 69% after
10, 15, 20, and 30 days (BMPio, BMPis, BM P2o,and BMP30), compared with 25°C. In
general, the average BMP values at 35°C were approximately 69% higher than at 25°C.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
162
Table 4.4: Relative BMP values at 35°C compared with BMP at 25°C.
Mixing conditions
B M Ps
B M Pio
B M P ,s
BM P20
BMP30
ADS
30 + A D S 10 (control)
1.69
1.64
1.78
1.70
1.76
ADS
30 + PS 10 (control)
1.46
1.28
1.31
1.26
1.37
ADS
30 + W A S 10 (control)
1.61
1.32
1.38
1.35
1.46
ADS
30 + A D S 10 (M W )
1.89
1.75
1.76
1.66
1.72
ADS
30 + PS 10 (M W )
2.13
1.63
1.60
1.50
1.58
ADS
30 + W A S 10 (M W )
1.79
1.69
1.74
1.68
1.81
ADS
30 + A D S 10 (CH)
2.26
2.09
2.07
1.96
2.04
ADS
30 + PS 10 (CH)
2.15
1.70
1.70
1.62
1.72
ADS
30 + W A S 10 (CH)
1.83
1.66
1.71
1.65
1.77
4.5 Pathogen Destruction Efficiency
Thermal treatments such as heat drying, composting, lime sterilization, and
thermophilic anaerobic digestion are recognized by the U.S. EPA as proven technologies
for generating Class A sludge for unlimited beneficial land applications. Total coliform,
fecal coliform, and E. coli have been used as an indicator to verify the reduction of
pathogen and vector attraction.
Typically, the MPN (most probable number) method is used in the EPA
regulations.
However, the CFU (colony forming unit) by the MF technique was used in
this study since the MF method was also approved by the U.S. EPA and coliforms are
detected simply and concisely in 24 hours.
Coliform number reductions between
microwave irradiation and conventional heating were compared.
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163
4.5.1 Coliform Destruction
Coliform bacteria were used as an indicator in evaluating Class A sludge in this
experiment.
The samples were taken from the second batch tests performed for
determining gas production.
An aliquot o f 200 mL o f sludge in a 500-mL beaker was irradiated with
microwaves in a 1-kW household microwave oven at 0.021, 0.042, 0.083, 0.125, and
0.167 watts-hr/g samples. Total solid concentrations depend on sludge characteristics,
which are 0.7, 1.4, 2.8, 4.19, and 5.59 watts-hr/g total solids (TS) for ADS, 0.34, 0.67,
1.35, 2.02, and 2.69 watts-hr/g TS for PS, and 0.53, 1.06, 2.11, 3.17, and 4.23 watts-hr/g
total solids (TS) for WAS, corresponding to 15, 30,60,90, and 120 seconds of irradiation
time in a 500-mL beaker, and a respective temperature increase to 25, 45, 65, 85 and
100±3°C. Sludges were also heated to 25, 45, 65, 85 and 100±3°C in a water bath to
simulate conventional heating. These are the same temperatures obtained by microwave
irradiation times o f 15, 30, 60, 90, and 120 seconds, respectively. When a water bath was
used to achieve the same fecal coliform destruction, there was a serious odor problem
during heating. During microwave irradiation, very little odor was produced.
The initial total and fecal coliforms and E. coli count numbers are shown in
Figure 4.14. PS had the greatest count numbers followed by WAS and ADS.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
164
le+8
le+7 -
_]
E
o
o
le+ 6 -
lc+5 -
■ i E-coli
-HU Fecal coll
IB
Total coli
Ie+4
ADS (control)
WAS (control) PS (control)
Figure 4.14: Coliform counts o f ADS, PS, and WAS at Nine Springs WWTP.
In order to compare the effectiveness of microwaves with conventional heating,
the same volume of sludge was heated in a water bath to the corresponding temperature
achieved by microwave irradiation and total coliform, fecal coliform, and E. coli were
measured. The first test for pathogen reduction was the irradiation o f primary sludge
taken from the gravity thickener underflow pipe at the Nine Springs wastewater treatment
plant (WWTP) in Madison, Wisconsin. The fecal coliform counts for various treatments
o f primary sludge are shown in Figure 4.15. The x-axis is the temperature increase
caused by microwave irradiation and conventional heating in a water bath.
Initially,
microwave-irradiated samples had much lower coliform counts than conventional heating.
When temperature reached 65°C for microwave irradiation, the fecal coliform count was
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165
not detected while conventional heating had to reach 85°C to achieve the same efficiency,
indicating that fecal coliforms were lower than detection limits. The detection limits of
fecal coliforms were determined by dilution factors and total solid concentration (Table
4.5). Based on the standard methods, the dilution factors were determined that the colony
numbers on the membrane filter should be 20-80.
Microwave
Conventional heating
Detection limit (MW)
Detection limit (CH)
on
~2?
3
u,
U
CO
'I
c2
c
_o
o
o3
Q
(. Ia>s A l i mi l
0
20
40
60
80
100
120
Temperature, °C
Figure 4.15: Fecal coliform reductions of PS with microwaves and thermal heating.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
166
Table 4.5: Detection limits o f fecal coliforms in ADS, PS, and WAS.
Total solids
Dilution factors
Detection limits
(mg/L)
00
(CFU/gTS)
RADS (Control)
29808
10000
3354.8
RPS (Control)
61909
10000
1615.3
RWAS (Control)
39440
10000
2535.5
ADS (MW 15 sec)
29463
1000
339.4
ADS (MW 30 sec)
29836
1000
335.2
ADS (MW 60 sec)
29514
100
33.9
ADS (MW 90 sec)
31210
100
32.0
ADS (MW 120 sec)
34764
100
28.8
PS (MW 15 sec)
60844
1000
164.4
PS (MW 30 sec)
61419
1000
162.8
PS (MW 60 sec)
62057
100
16.1
PS (MW 90 sec)
64812
100
15.4
PS (MW 120 sec)
70004
100
14.3
WAS (MW 15 sec)
37757
1000
264.9
WAS (MW 30 sec)
38424
1000
260.3
WAS (MW 60 sec)
37266
1000
268.3
WAS (MW 90 sec)
39053
100
25.6
WAS (MW 120 sec)
42335
100
23.6
ADS (CH 25°C)
31640
1000
316.1
ADS (CH 45°C)
30679
1000
326.0
ADS (CH 65°C)
29671
100
33.7
ADS (CH 85°C)
29990
100
33.3
ADS (CH 100°C)
29958
100
33.4
PS (CH 25°C)
48860
1000
204.7
PS (CH 45°C)
49360
1000
202.6
PS (CH 65°C)
64602
100
15.5
PS (CH 85°C)
67750
100
14.8
Sludges
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167
Total solids
Dilution factors
Detection limits
(mg/L)
(x)
(CFU/gTS)
PS (CH 100°C)
71667
100
14.0
WAS (CH 25°C)
37200
1000
268.8
WAS (CH 45°C)
37528
1000
266.5
WAS (CH 65°C)
38488
1000
259.8
WAS (CH 85°C)
39880
100
25.1
Sludges
WAS (CH 100°C)
24.6
40586
100
RADS, RPS, and RWAS: anaerobic digester sludge, primary sludge, and waste activated sludge
without pretreatment by microwave or external heating, MW: microwave irradiation, CH:
conventional heating.
The total coliform, fecal coliform, and E. coli counts along with final
temperatures for primary sludge are summarized in Table 4.6. Total coliform counts
were always greatest followed by fecal coliform and E. coli as expected. At > 1.35
watts-hr/g TS (65°C), total coliform, fecal coliform, and E. coli were not detected. In the
case o f conventional heating, all three were not detected only at 85°C. For conventional
heating, typically it took 0.9, 1.9,2.9,3.8, and 4.8 minutes to raise the temperatures to 25,
45,65, 85, and I00°C, respectively, in a water bath. In general, pathogen destruction was
insignificant up to 45°C.
The irradiation of PS with microwaves will result in pre-pasteurization (or
destruction o f pathogens) and breakdown of particulate organics into more readily
biodegradable organics. This step will significantly destruct pathogens, sufficient for
meeting Class A sludge requirements.
Furthermore, the detention time in sludge
stabilization processes will be shortened significantly. In order to reduce the volume for
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168
microwave irradiation, it is better to thicken the primary sludge with a thickener. The
microwave irradiation will increase the influent sludge temperature to approximately
65 °C or more. Therefore, if anaerobic digestion is used as a stabilization process, it may
need significantly less heating or virtually no additional heating for maintaining 35°C
inside the anaerobic digester.
Table 4.6: Average coliform reduction o f PS with microwaves and thermal treatments.
Treatment
MW - no radiation (control)
MW - 0.34 watts-hr/g TS
M W -0 .6 7 watts-hr/g TS
M W -1 .3 5 watts-hr/g TS
MW - 2.02 watts-hr/g TS
MW - 2.69 watts-hr/g TS
Heating to 25°C
Heating to 45 °C
Heating to 65°C
Heating to 85°C
Heating to 100°C
Temp.
°C
10 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
E. coli
Fecal coliform Total coliform
CFU/g TS
CFU'/g TS
CFU/g TS
7.11E+04
6.99E+04
6.11E+04
N.D.
N.D.
N.D.
7.08E+04
5.67E+04
2.32E+03
N.D.
N.D.
6.27E+05
3.26E+05
2.76E+04
N.D.
N.D.
N.D.
5.94E+05
3.55E+05
1.09E+04
N.D.
N.D.
4.00E+06
4.07E+06
4.48E+06
N.D.
N.D.
N.D.
4.18E+06
4.00E+06
2.22E+06
N.D.
N.D.
Colony forming unit, N.D.: not detected
Typically, sludge treatments for the pathogen reduction are classified with (1)
pretreatment, (2) post treatment, and (3) submerged treatment using ADS recycling. The
second trial was to test the feasibility o f irradiating a recycle stream from a sludge
stabilization process with microwaves. This irradiation will lead to pathogen destruction
and heating. This will ensure the complete exposure o f the sludges in a stabilization
process to microwaves for generation of Class A sludge. For heating only or partial
pathogen destruction, the exposure volume may be reduced. The test results for ADS are
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
169
shown in Figure 4.16. The results were very similar to those for primary sludge. ADS by
microwave irradiation had not detected any fecal coliform above 65°C. Conventional
heating had to reach 85°C to have no detection of fecal coliform count.
106
106
m
io 5
u
104
Microwave
Conventional heating
Detection limit (MW)
Detection limit (CH)
10s
c/5
H
'S?
104 U
£
<2
=3
103
c
103 _o
ow
u
Clas> A limit
"3
CJ
102 ■
102
10'
10'
0
20
40
60
80
100
Q
120
Temperature, °C
Figure 4.16: Fecal coliform reductions o f ADS by microwaves and thermal heating.
The total coliform, fecal coliform, and E. coli counts along with final
temperatures for ADS are summarized in Table 4.7. At > 2.8 watts-hr/g TS (65°C) by
microwaves, total coliform, fecal coliform, and E. coli were all not detected. In the case
of conventional heating, all three were not detected only above 85°C.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
170
Table 4.7: Average coliform reduction o f ADS with microwaves and thermal treatments.
Treatment
MW - no radiation (control)
MW - 0.7 watts-hr/g TS
MW - 1.4 watts-hr/g TS
M W - 2 .8 watts-hr/g TS
M W -4 .1 9 watts-hr/g TS
MW - 5.59 watts-hr/g TS
Heating to 25°C
Heating to 45°C
Heating to 65°C
Heating to 85°C
Heating to 100°C
Temp.
°C
E. coli
CFU'/gTS
10 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
1.36E+04
4.24E+03
4.69E+03
N.D.
N.D.
N.D.
1.27E+04
8.76E+03
8.43E+02
N.D.
N.D.
Fecal coliform Total coliform
CFU/g TS
CFU/g TS
7.46E+04
3.76E+04
1.75E+04
N.D.
N.D.
N.D.
6.86E+04
4.88E+04
2.58E+04
N.D.
N.D.
7.15E+06
7.73E+06
2.59E+06
N.D.
N.D.
N.D.
9.75E+05
9.98E+05
1.10E+05
N.D.
N.D.
C o lo n y form ing unit, N .D .: not d etec te d
The third test was the irradiation of thickened WAS taken from a dissolved air
flotation (DAF) system in the Nine Springs WWTP with microwaves.
Microwave
irradiation of WAS is thought to destruct pathogens and hydrolyze biomass. This is a
pre-pasteurization step before disposal or further treatment in sludge stabilization
processes. Due to hydrolysis of biomass or breakdown o f biomass into smaller molecular
weight organics, the detention time in the sludge stabilization process will be shortened.
The test results for WAS are shown in Figure 4.17. Conventional heating showed
almost the same trends as microwave irradiation up to 45°C. Above 65°C, microwave
irradiation had not much better fecal coliform destruction than conventional heating. The
results were relatively different from PS and ADS discussed above. In the case o f PS and
ADS by microwave irradiation, fecal coliforms were not detected at 65°C. The fecal
coliforms were not detected until the temperature reached 85°C for WAS.
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171
The reason may be due to the limitation in microwave penetration depth due to a
high-suspended solids (> 0.45 pm) concentration. However, it appears that the efficiency
o f microwaves depends not on the water content (solids content) but viscosity. When
WAS was sampled from the Nine Springs WWTP, it was slimy and sticky with a particle
size smaller than PS and ADS. Note that total solids contents of WAS varied from
37,000 to 42,000 mg/L, while that of PS from 37,000 to 70,000 mg/L. Lower pathogen
destruction efficiency of WAS is supported by the shallower microwave penetration
depth than PS as described in Section 4.2.
Microwave
Conventional heating
Detection limit (MW)
Detection limit (CH)
Hso
tn
E—
■
D
u.
U
U.
U
Io
c
_o
ou
<L>
Q
o
"a
ou
u-
10 '
0
20
40
60
80
100
10 '
120
Temperature, °C
Figure 4.17: Fecal coliform reductions of WAS by microwaves and conventional heating.
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172
Further studies should be needed to determine important factors controlling the
pathogen destruction in sludges such as mixing, solids content (water content), and
viscosity.
The total coliform, fecal coliform, and E coli counts along with final
temperatures for WAS are summarized in Table 4.8.
Table 4.8: Average coliform number reduction o f WAS with microwaves and thermal tre
atment.
°C
E. coli
CFU'/gTS
Fecal
coliform
CFU/g TS
Total coliform
CFU/g TS
10 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
25 ± 3
45 ± 3
65 ± 3
85 ± 3
100 ± 3
1.14E+05
6.89E+04
5.47E+04
1.48E+03
N.D.
N.D.
4.88E+04
9.33 E+03
6.75E+03
N.D.
N.D.
4.16E+05
2.37E+05
7.15E+04
4.05 E+03
N.D.
N.D.
3.14E+05
9.35E+04
1.36E+04
N.D.
N.D.
1.81E+06
8.00E+05
3.31E+05
3.43E+05
N.D.
N.D.
5.89E+06
7.54E+05
9.06E+05
N.D.
N.D.
Temp.
Treatment
MW - no radiation (control)
MW - 0.53 watts-hr/g TS
MW - 1.06 watts-hr/g TS
MW -2 .1 1 watts-hr/g TS
MW -3 .1 7 watts-hr/g TS
MW - 4.23 watts-hr/g TS
Heating to 25°C
Heating to 45 °C
Heating to 65°C
Heating to 85°C
Heating to 100°C
C o lo n y form in g unit, N .D .: not d etected
4.5.2 Pathogen Die-Away Tests
In many wastewater treatment plants including the Nine Springs WWTP,
stabilized sludge (biosolids) is stored in storage tanks for over 3 to 6 months, although the
temperature o f storage is not constant. In this research, pathogen die-away tests were
performed to evaluate the effect of storage in a 4°C refrigerator on pathogen destruction.
Figure 4.18 shows fecal coliform changes over storage time. Despite high fecal coliform
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173
destruction efficiencies ranging from 99.3 to 99.7%, the residual fecal coliform counts
were over 10,000 CFU/g TS which is well above the regulatory limit o f Class A sludge,
1,000 MPN/g TS even after 224 days o f storage at 4°C. Therefore, if the sludges are
stored at 4°C refrigerator, another process will be required for compliance with this limit.
1.0E+07
£
O Digester sludge
■O—primary sludge
DAF sludge
X GBT sludge
-+- Detection limit
1.0E+06
Z)
1.0E+05
a
oo
I.0E+04
Detection limit
I.0E+03
[•.PA limit for Class A
1.0E+02
0
50
100
150
200
250
Storage Time, days
Figure 4.18: Changes in fecal coliform counts over time during storage at 4°C.
The total coliform, fecal coliform, and E. coli counts over time for ADS, PS, and
WAS samples stored at 4°C are summarized in Table 4.9. Total coliform and E. coli
counts were still well above 100,000 CFU/g TS.
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174
Table 4.9: Changes in fecal coliform counts during long-term storage at 4°C.
Storage time
E. coli
Fecal coliforms
Total coliforms
(Days)
CFU/g TS
CFU/g TS
CFU/g TS
ADS1
224
9.83E+04
1.06E+04
9.83E+04
ADS2
91
1.98E+05
5.03E+04
1.98E+05
ADS3
18
1.49E+06
1.75E+05
1.49E+06
ADS4
3
2.01E+06
9.34E+05
2.01E+06
PS1
224
3.66E+05
4.72E+04
3.66E+05
PS2
91
1.25E+05
1.78E+04
1.25E+05
PS3
18
4.16E+05
7.99E+05
4.16E+05
PS4
3
8.10E+06
5.28E+06
8.10E+06
WAS1
224
7.09E+04
1.69E+04
7.09E+04
WAS2
91
2.21E+05
1.47E+05
2.21E+05
WAS3
18
1.70E+06
1.25E+06
1.70E+06
WAS4
3
3.72E+06
3.35E+06
3.72E+06
GBT4
3
7.89E+05
1.15E+05
7.89E+05
Sludge
GBT: gravity belt thickener - thic cened anaerobic digester sludge
4.6 Toxicity Tests
The objective o f this study was to determine whether ADS, PS, and WAS treated
with microwaves or conventional heating produce toxic compounds which inhibit the
microbial growth during aerobic or anaerobic stabilization of sludge.
The potential
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175
toxicity increase after thermal treatment must also be evaluated since the supernatant of
sludge is typically recycled back to the head o f a wastewater treatment plant.
The toxicity screening tests were performed using RET assay (MitoScan Co.,
Madison, WI) with three sludge samples taken from the Nine Springs WWTP.
The
results o f the toxicity screening tests are summarized in Table 4 10. The slope was
determined from the NADH concentrations at 340 nm. The higher NADH concentration
represents a lower toxicity, which results in a higher slope from 5 to 30 minutes. ADS,
PS, and WAS were filtered with a 0.45-pm filter before and after various treatments, and
the filtrates were used for RET toxicity assays. In general, under the same treatment
conditions, WAS had the greatest toxicity followed by ADS and PS since the slope was
the smallest among the three sludge samples. The increase in % inhibition may be due to
the leaching of organic compounds inhibiting microbial activities. The reduction in %
inhibition may be caused by decomposition o f inhibitory organics by thermal treatment.
The % inhibition was estimated using the slopes of the samples that were not irradiated
with microwaves as a control.
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176
Table 4.10: Changes in % inhibition after a series o f microwave irradiation and conventio
nal heating.
PS
ADS
Treatment
Temp.
°C
Slope
WAS
%
%
%
Slope
Slope
Inhibition
Inhibition
Inhibition
No irradiation (control)
10 + 3
8.66
0
10.27
0
7.41
0
MW - 30 sec. irradiation
45 ±3
9.26
-6.88
12.17
-18.55
6.20
16.33
MW - 60 sec. irradiation
65 ±3
8.37
3.33
11.26
-9.70
5.68
23.36
MW - 120 sec. irradiation 100 ±3
11.10
-28.22
9.48
7.71
4.67
36.96
Heating to 45°C
45 ±3
7.60
12.23
11.92
-16.05
7.55
-1.92
Heating to 65°C
65 ±3
8.81
-1.73
13.14
-27.99
7.09
4.25
Heating to 100°C
100 ±3
8.92
-2.97
11.57
-12.63
7.53
-1.66
The % inhibition values for ADS samples treated with microwaves or
conventional heating are shown in Figure 4.19. The % inhibition remained unchanged
but decreased sharply when the temperature was above 65°C.
It is expected that a
toxicity variation can affect the digestibility in an anaerobic digester. In fact, the
cumulative gas production also increased as the temperature rose (Figure 4.10).
Conventional heating to 45°C led to a slight increase in % inhibition and then remained
similar to the control when the temperature was raised further.
It can be said that
microwave irradiation o f ADS does not produce inhibitory compounds to biological
treatment processes.
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177
Conventional heating
. O SD: 7.01, SE: 3.50
- Control
RADS = 0
Microwave
SD: 14.17
SE: 7.09
-30 -40
0
20
40
60
80
100
120
Temperature, °C
Figure 4.19: % Inhibition changes in soluble liquids of ADS medium with microwaves
and conventional heating.
The % inhibition values for PS samples treated with microwaves or conventional
heating are shown in Figure 4.20. The % inhibition values of microwave irradiation and
conventional heating were low until 65°C compared with the control. However, when the
temperature increased to 85°C, microwave irradiation might have produced inhibitory
compounds. The % inhibition value greater than the control at 100°C did not affect the
gas production (Figure 4.11). This indicates that if the microwave-irradiated sludge is
mixed with anaerobic digester seed at less than 1 to 3, then there will be no significant
inhibitory effect by microwave irradiation during anaerobic digestion.
The effect of
recycling liquid generated during sludge pretreatment on activated sludge processes
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178
needs to be confirmed further from respirometer tests or RET assays with a series of
dilutions.
Control
RPS = 0
Microwave
SD: 11.43
SE: 5.72 ,
£
'3
‘x
2
u>
-20
-
Conventional heating SD: 11.51, SE: 5.76
-25 -30
0
20
40
60
80
100
120
Temperature, °C
Figure 4.20: Toxicity variations in soluble liquids o f PS medium with microwaves and
conventional heating.
The % inhibition values for WAS samples treated with microwaves or
conventional heating are shown in Figure 4.21. The % inhibition values o f WAS after
microwave irradiation increased gradually while the % inhibition after conventional
heating remained almost the same as the control. After 15 seconds o f microwave
irradiation, the cumulative gas volume was almost 20% lower than the control (Figure
4.12) despite a slight increase in the % inhibition.
However, after 30 seconds of
microwave irradiation, the cumulative gas production was 8% greater than the control.
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179
The variation of toxicity appears to be caused by leaching of materials from sludge by
microwave and thermal heating and evaporation o f toxic volatile matter. It appears that if
the microwave-irradiated sludge is mixed with anaerobic digester seed at the ratio less
than 1 to 3, then there will be no significant inhibitory effect by microwave irradiation
during anaerobic digestion. Further study is needed to evaluate the effect of the liquid
produced from sludge during pretreatment on anaerobic biological processes.
30 20
Microwave
SD: 15.38
SE: 7.69
-
o>>
Control
RWAS 50 ----Conventional heating ■
SD: 2.85. SE: 1.43
-10
0
20
40
60
80
100
120
Temperature, °C
Figure 4.21: Toxicity variations in soluble liquids of WAS medium with microwaves and
conventional heating.
Changes in toxicity of liquids generated from ADS, PS, and WAS at different
storage durations were investigated.
The highest initial toxicity was shown in WAS
followed by ADS, and PS. Soluble samples from sludge medium were analyzed. Since
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180
the sludge samples were stored in a 4°C refrigerator, the result could be used to evaluate
the effect of sludge storage in a sludge storage tank in a 4°C. Figure 4.22 shows the
effect o f long-term storage on % inhibition. The three sludge samples before storage
were used as the controls. The % inhibition values increased over time for WAS and
ADS while the value for PS decreased significantly.
30 -i
■---------1--------- 1---------1---------'-------- 1---------1---------r
Control
RADS = 0
RPS = 0
RW AS = 0
y = 0.376-0.055x
Storage time at 4°C, days
Figure 4.22: Changes in toxicity o f liquid produced from ADS, PS, and WAS at various
storage durations in a 4°C refrigerator.
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181
4.7 Biological Effects of Microwaves for Fecal Coliforms
4.7.1 Cell Membrane Damages by Microwaves
Pathogen destruction efficiency by microwaves was
evaluated using a
LIVE/DEAD® ZtocLight™ Bacterial Viability Kit developed by Molecular Probes Co.
(Eugene, Oregon). LIVE/DEAD® ZtacLight™ Kit has been used for determination of
total viable counts as a rapid detection method (Duffy and Sheridan, 1988). The twocolor fluorescent assay kit, which contains a mixture o f nucleic acid stains, is typically
used to verify bacterial membrane integrity. These stains differ both in the spectral
characteristics and in their ability to penetrate healthy bacterial cell membranes. The
green stain contains a small molecule (SYTO 9) capable of penetrating intact bacteria,
while the red stain contains a larger molecule (propidium iodide) which penetrates only
through damaged membranes (Braux et al., 1997).
Pure-cultured fecal coliform bacteria were either irradiated with microwaves or
heated in a water bath. Then, an aliquot of 10 mL of fecal coliform in an autoclaved test
tube was irradiated with microwaves for 3 ,6 ,9 , 12, and 15 seconds, which resulted in the
final temperatures o f 49, 60, 70, 72, and 100±3°C, respectively. The protocols were
described in Section 3.7 in detail.
As described in Section 3.7, green spots represent live fecal coliform bacteria
cells, and red spots are dead cells.
Based on the experimental protocols, a red spot
indicates cell membrane damage by microwaves.
Most cell membranes of fecal
coliforms were not damaged at 49±3°C (Figure 4.23 (a)).
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182
Figure 4.23 (a): Fluorescent microscope 630x , 3 sec. microwave irradiation to 49±3°C
However, when the temperature increased above 60±3°C after microwave
irradiation, the number of viable cells was rapidly reduced. Woo et al. (2000) reported
that the viable E. coli counts in cell suspensions were found to noticeably diminish
relative to an increase in the microwave heating temperatures. The highest reduction ratio
in the viable counts was observed when the temperature was increased from 50 to 60°C,
which was a 3-log reduction in E. coli organisms. When the microwave heating
temperature exceeded 60°C, viable fecal coliforms decreased rapidly.
In addition, Woo et al. (2000) found that untreated E. coli cells had a smooth
surface, while most of the microwave-radiated cells exhibited severe destruction. The
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183
surfaces o f the microwave-irradiated cells were damaged and had become rough and
swollen.
Similar results were found in the tests using the LIVE/DEAD ZtacLight™ Kit.
The red spots, which represent damage o f the cell membrane, were rapidly increased after
irradiation to 60±3°C (Figure 4.23 (b)), and all spots were red at 100±3°C (Figure 4. 23
(d)).
Figure 4.23 (b): Fluorescent microscope 630X, 9 sec. microwave irradiation to 60±3°C
It is believed that microwave heating for fecal coliform inactivation is highly
efficient when the temperature exceeds 60±3°C, and the cell membrane damages of fecal
coliforms by microwave irradiation were proved using both SYTO 9 and propidium
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184
iodide stains. In this bioassay, live and dead cell counts were not performed, because the
purpose o f this assay was confirm whether fecal coliform cell membranes were damaged
or not by microwaves.
Figure 4.23 (c): Fluorescent microscope 630X, 12 sec. microwave irradiation to 72±3°C
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185
Figure 4.23 (d): Fluorescent microscope 630X, 15 sec. microwave irradiation to 100±3°C
4.7.2 Microbial Activity Tests
There have been many debates on the existence of “non-thermal” effects of
electromagnetic irradiation. As described in Chapter 3, three possibilities were thought to
prove the destruction of coliform bacteria such as cell membrane damages, reduction of
biological activity, and DNA disruption.
For the microbial activity, the ETS (electron transport system) assay using INT
(C19H13CIIN5O2, Sigma® 18377) and P-Galactosidase assay were chosen to verify
destruction of fecal coliforms.
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186
4.7.2.1 ETS (Electron Transfer System) Activity
Tetrazolium salts are used as indicators o f reducing systems such as the electron
transport system (ETS). Bacteria with an active ETS reduce these vital redox dyes from
water soluble tetrazolium salts to a colored formazan product (Roslev and King, 1993).
The INT assay depends on the detection o f respiration by determining dehydrogenase
activity o f the electron transport chain. The formazan produced by the reduction of INT
is insoluble in water. It is therefore accumulated in actively respiring cells, and can be
detected by its red color under bright field illumination (Swannell and Williamson, 1988).
INT salts were used to measure the electron transport system (ETS) activity o f
fecal
coliforms
by
microwave
irradiation
and
conventional
heating.
A
spectrophotometric assay was conducted to determine the ETS activity at 480 nm. The
chemicals and protocols used in this bioassay were described in Section 3.7.3 in detail.
The absorbance of INT-Formazan at 480nm for fecal coliforms that have been
treated by microwave and conventional heating is shown in Figure 4.24. INT-formazan
concentrations were expressed as ETS activity (INTF/g).
The control sample had approximately 1.78 mg INTF/g sample.
With a
temperature increase, the ETS variation o f fecal coliforms by microwaves can be
classified with 3 phases. In the first phase, the activity was slightly low at 20 to 57°C
compared with the control, while ETS activity o f fecal coliform by conventional heating
increased slightly from 20 to 57°C. It appears that the inactivation of fecal coliform as an
indicator o f pathogens may be due to not only conventional heating, but also another
thermal effect. That is, fecal coliforms were possibly stressed more during rapid increase
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187
o f temperature (temperature rate) or stressed by molecule collisions o f 2.45 x 109 times
by microwaves, although the temperature was lower than 60°C.
2.5
|
<b
-SP
u,
|
|
—
o-
Microwave
Conventional heating
E-
Z
00
E
£
’>
ocs
CZ)
E—
W
Autoclave = 0
flhasejl
Phase 1
0
20
40
60
Phase 3
80
100
120
140
Temperature, °C
Figure 4.24: ETS activities o f fecal coliforms by microwaves and conventional heating.
In the second phase, the ETS activity rapidly decreased by conventional heating
and microwaves. Furthermore, the ETS activity was very close to 0.1 (Autoclave sample
= 0.158) over 62°C in the third phase.
4.7.2.2 p-Galactosidase Enzyme Activity
The (3-galactosidase enzyme assay was used for isolated fecal coliforms. The
assay was performed by adding pretreated fecal coliform samples to assay 2* Buffer,
which contains the substrate ONPG (o-nitrophenyl-P-D-galactopyranoside).
The P-
galactosidase hydrolyzed the colorless substrate to o-nitrophenol (yellow) for 30 minutes
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188
o f incubation. One unit o f P-Galactosidase hydrolyzes 1 micromole o f ONPG to onitrophenol and galactose per minute at pH 7.5 and 37°C. The absorbance was read at
420 nm with a spectrophotometer. The analytical protocols were described in Section
3.7.4 in detail.
The trend o f results was somewhat similar to the ETS activity test (Figure 4.25).
In the first phase, the enzyme activity o f fecal coliforms irradiated by microwaves
decreased at 38°C compared with the control and conventional heating, while the samples
exposed to conventional heating rather increased. The biological activities in the
temperature range (20 to 40°C) showed reverse trends between microwaves and
conventional heating.
In the second phase, the P-Galactosidase enzyme activity rapidly decreased as
temperature increased. In this phase, the difference between conventional heating and
microwaves was not found. In the third phase, the activity was also relatively close to the
autoclaved sample when the temperature was over 78°C as shown in the ETS activity test
results. As a whole, it is expected that microwave irradiation has more efficiency with
respect to the inactivation o f coliforms in sludges.
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189
CO
□
C
3
'g
1.0 -
1 08
«
<D
0.6
Microwave
Coventional heating
Control
'
-
CO
tj
0.4 -
CO
O
2 0-2
-
Autoclave = 0 .
co
f
0.0 Phase I
CO.
0
20
P h a se 2
40
60
P hase 3
80
100
120
14 0
Temperature, °C
Figure 4.25: P-galactosidase activities o f fecal coliforms by microwaves and conventional
heating
Since ETS and P-galactosidase activities for isolated fecal coliforms were shown
to be similar in their activity trends, the relationship between the two tests was considered.
As shown in Figure 4.26, the linear regression was well fitted to two data populations (y
= 0 .5 7 8 x - 0.96, R2 = 0.91).
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190
1.50
-------------r
-
1-------------- 1
1
1
------------- --------------------1------------------T --------------r
1
c
3
£
1.00
-
y = 0.578x - 0.96, R2
=091
S
s '
T
m
/
cs
"
/
0.50
¥
8
u
c/5
\J
’>
95% prediction intervals
rs
"55
%
a
co
l
0.00
^9
S
S '
95% confidence intervals
s
co.
-0.50 ___ 1
<
0.0
0.5
1.0
1.5
2.0
2.5
ETS activity, mg INTF/g
Figure 4.26: Relationship between ETS activity and P-galactosidase activity
4.7.3 Gel Electrophoresis for Genomic DNA
It is not clear whether a non-thermal effect of microwaves exists or not, as many
scientists have been debating on this issue for over 10 years. Gel electrophoresis was
performed with fecal coliforms to evaluate whether a non-thermal effect exists.
Genomic DNA was extracted from fecal coliforms cultured for 1 day that had
been subjected to microwave irradiation or to external heating and analyzed by agarose
gel (0.5%, IX TAE buffer) electrophoresis (Sambrook and Russel, 2001). The optical
density o f cultured fecal coliforms was approximately 1.3, indicating that the number of
cells was over 109 cells/mL (OD600 = 1 0 ,0.8><109 cells/mL).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The temperatures o f pretreated fecal coliform samples were 20.4 (control, lane A),
38.0 (lane B), 56.8 (lane C), 61.9 (lane D), 70.5 (lane E), 78.0 (lane F), 82.1 (lane G), and
86.9±0.2°C (lane H) using microwaves and external heating (conventional heating), plus
the autoclaved sample (121°C, lane I). As shown in Figure 4.27, the genomic DNA
bands from the pure cultured fecal coliforms that had been irradiated with 1 kW
microwaves were increasingly faint with the increase in irradiation time. In particular,
the DNA band from lane G (82.1±0.2°C) faded away. This phenomenon is not observed
in the externally heated samples. In the case o f external heating, the DNA bands were
relatively clear.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4.27: Gel electrophoresis o f genomic DNA isolated from fecal coliforms
pretreated by microwaves and conventional heating
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193
In addition, after agarose gel electrophoresis o f the fecal coliform genomic DNA,
quantitative analysis o f DNA in each sample was performed with an ultraviolet
spectrophotometer. Figures 4.28 and 4.30 show the optical density (absorbance) of
genomic DNA isolated from fecal coliforms pretreated by microwaves and conventional
heating. As expected from electrophoresis results, the optical densities o f microwave
irradiated samples decreased more at 260 nm compared with that o f conventional heated
samples. This result indicates that microwaves can disrupt DNA in the fecal coliform
cells more than external heating.
D l w ater
Control. 20.4°C
CH. 38°C
CH. 56.8°C
CH. 70.5°C
CH. 78°C
CH. 82.1°C
Autoclave
CH. 87°C
Autoclave. I21°C
Dl water
150
200
250
3 00
350
400
Wave length, nm
Figure 4.28: Optical density o f genomic DNA isolated from fecal coliforms pretreated by
conventional heating
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194
D l w ater
0.6
C o n tro l, 20.4°C
M W , 38°C
Q
2,
M W , 6 1 .9°C
0.4
M W , 70.5°C
&
55
cu
2
M W , 78°C
M W ,8 2 .1 °C
0.2
M W , 87°C
Autoclave. V
a
a
A utoclave, I21°C
o
0.0
Dl water
150
200
250
300
350
400
Wave length, nm
Figure 4.29: Optical density o f genomic DNA isolated from fecal coliforms pretreated by
microwave irradiation
DNA concentration was calculated using optical density obtained by UV
spectrophotometer. Since the A260 of DNA with the concentration o f 50 pg/mL is 1 OD
unit, the concentration o f DNA solution can be calculated using equation 3.1. Table 4.11
shows the DNA concentrations and OD260/280 ratio obtained from DNA absorbance values.
The DNA concentration o f the control was approximately 22.67 pg/mL.
concentrations decreased as temperature increased.
The
The DNA concentration of
microwave-irradiated samples showed over a 74% decrease above 82.1±0.2°C compared
with the control.
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195
Additionally, since the absorbance o f DNA samples at A280 indicates the
concentration of both proteins and nucleic acids, the ratio of A260/A280 represents the
purity o f DNA samples. Typically, A260/A280 should range between 1.75 and 2 for
good quality DNA. On the other hand, nucleic acid preparations free of phenol should
have OD260:OD280 ratios of -1 .2 (Sambrook and Russel, 2001). Most A260/A280
ratios in Table 4.11 varied from 0.9 to 1.8, which were lower than 1.75. It is expected
that a portion of proteins or RNAs were not perfectly removed during DNA extraction.
In summary, the gel electrophoresis results revealed that the DNA quantity (DNA
band on agarose gel) from bacteria decreased when the microwave irradiation time
increased while the extent o f DNA quantity decrease was less pronounced for the
conventionally heated bacteria. However, it was not clear whether genomic DNA bands
and concentrations were related to the death of the bacteria achieved at a specific
temperature when microwave irradiated and water bath heated samples were compared.
In any case, it appears that microwave irradiation, at least, will be a better choice in
destructing pathogens in sludges from sewage treatment plants. More study is needed to
investigate “non-thermal’ microwave effects.
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196
Table 4.11: DNA concentrations and OD 260/280 ratio
Samples
Temperature
DNA conc.
OD260
OD 280
-
0.022
0.018
-
Autoclave
121°C
0.077
0.062
3.87
Control
20.4±0.2°C
0.453
0.359
22.67
MW1
38.0±0.2°C
0.562
0.446
28.09
MW2
56.8±0.2°C
0.396
0.290
19.82
MW3
61.9±0.2°C
0.367
0.230
18.37
MW4
70.5±0.2°C
0.292
0.316
14.61
MW5
78.0±0.2°C
0.248
0.132
12.40
MW6
82.1±0.2°C
0.169
0.194
8.46
MW7
86.9±0.2°C
0.065
0.053
3.27
CHI
38.0±0.2°C
0.467
0.368
23.36
CH2
56.8±0.2°C
0.600
0.478
30.02
CH3
61.9±0.2°C
0.461
0.365
23.07
CH4
70.5±0.2°C
0.452
0.358
22.60
CH5
78.0±0.2°C
0.400
0.315
20.02
CH6
82.1±0.2°C
0.343
0.274
17.15
CH7
86.9±0.2°C
0.293
0.239
14.65
Dl water
(ug/mL)
MW: fecal coliform sample treated by microwave, CH: fecal coliform sample treated by
conventional heating.
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197
4.8 Bench Scale Anaerobic Digester Tests
Bench scale anaerobic digester tests were performed to evaluate both anaerobic
digestibility and fecal coliform destruction during continuous operation o f digesters.
Approximately 4 L o f anaerobic digester sludge was inoculated as seed sludge in each 6L reactor. The first three months were spent in set-up, biomass acclimation with seeding
sludges obtained from the anaerobic digester at Nine Springs WWTP. The other one and
half months were spent in fixing minor problems such as gas-leaking, temperature control,
and so on. As a result, the experimental data were collected between July 15, 2001 and
December 17,2001.
The first reactor was a control simulating a conventional anaerobic digester. The
second reactor received the feed irradiated with 1-kW microwaves. The third reactor
received the feed heated using a water bath to a temperature achieved by corresponding
microwave irradiation. For the second and third reactors, the temperature after heating
with microwaves and water bath was controlled at 60±3°C. The feed contained the same
volumes of PS and WAS (1:1). The feed was manually added to anaerobic digesters
based on the SRT (solids retention time) of 20, 15, 10, 7.5, and 5 days. At least once a
week, fecal coliforms were counted for the influent and effluent samples ffom the three
reactors. The experimental conditions are summarized in Table 3.6 in detail.
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198
4.8.1 Changes in Fecal Coliform during the Anaerobic Digester Operation
The changes in fecal coliforms during the operation periods are shown in Figure
4.30. Total operation periods were about 160 days for fecal coliform detection. The first
two months were operated with 20 day SRT. The SRT was changed to observe the
changes in fecal coliform count at 20, 15,10, 7.5 and 5 days.
The initial fecal coliform values of raw sludges (WAS + PS = 1:1) ranged from
104 to 105 CFU/g TS. During the 20-day SRT, fecal coliform count fluctuated from 103
to 104 CFU/g TS. Anaerobic digestion is known to have ability to reduce the pathogen
due to microbial competition (U.S. EPA, 1999b). When SRT was changed to 10 days,
fecal coliform count decreased gradually, indicating that the sludge input volume affects
fecal coliform count.
As sown in Figure 4.30, for initial 2 months, the fecal coliforms by microwave
pretreatment showed nearly the same reduction as the control (R l) and conventionally
preheated sludges (R3). The reason may be why the initial values of fecal coliforms may
be almost same in three reactors when ADS inoculated as seed sludges. However, after 2
months, the fecal coliforms were rather decreased compared with the control (Rl) and
conventionally preheated sludges (R3). Conventional digester sludges still contained over
104 CFU/gTS, and preheated sludges varied fromlO2 to 104 CFU/gTS, while fecal
coliforms were sometimes not detected in microwave-pretreated sludges. In addition,
Figure 4.31 shows the log reduction for R l, R2, and R3, which were calculated by log
(N/No). The numbers of fecal coliforms not detected in this experiments were used
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199
detection limit of each sample. The detection limits o f fecal coliforms in this performance
varied from 1.3 to 1.6 log CFU/ gTS (102 CFU/lOOmL) for 3 output sludges, 3.0 to 3.6
log CFU/ gTS (104 CFU/lOOmL) for input sludges. The detection limits were determined
by dilution factors and total concentrations of sludges. Therefore, fecal coliforms can be
survived at zero detection, but this value should be lower than detection limit.
6.0
Class B limit
SRT
£
-S? 5.0
D
u.
U
Input sludge
C/J
CO
-o
00
4.0
c2
Class A limit
ou 3.0
"3
o<u
2.0
C/5
(CH)
R2 (MW)
Detection limits
1.0
5
0
25
50
75
100
125
150
Operation, days
Figure 4.30: Fecal coliform variations during bench scale anaerobic tests.
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200
0
25
50
75
100
125
150
0 .0
-0.5
1.0
oo 1.5
c
-2 .0
-2.5
"3
(j -3.0
w
<u
Lu
-3.5
R l(?ontrol), Log( N /N 0)1
R2 (M W ), Log(N/NO)
R3 (CH ), Log(N/NO)
-4.0
Operation, days
Figure 4.31: Log reductions of fecal coliforms during bench scale anaerobic tests.
Raw sludge containing PS and WAS was Class B based on U.S. EPA regulation
limit. Digesters receiving raw sludge and preheated sludge could not generate Class A
sludge consistently.
However, the pretreatment using microwaves provided Class A
sludge consistently compared with conventional heating.
Figure 4.32 shows the box plot for fecal coliform detections during the operation.
The average values (log unit) o f fecal coliform detection during the operation were
approximately 4.8 for raw sludges, 3.5 for conventional digester sludges, 2.1 for
microwave-heated sludges, and 2.7 for external heated sludges. The statistical analysis
results are summarized in Table 4.12. During the operation, the average log reductions of
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201
fecal coliforms were approximately 1.27, 2.66, and 2.08 for R l, R2, and R3 reactors,
respectively.
C/D
E- 5
-2?
A
^
U 4
00
jD
I 3
<2
oo 2
Class A limit
"5
u<U I
u,
1
Input
R l: output
R2: output
R3: output
Figure 4.32: Box plot for fecal coliform detections during the operation
Table 4.12: Statistical values of fecal coliform detection in each reactor
Input
R l out
R2 out
R3 out
22
22
22
22
Max
5.140
4.033
3.210
3.637
Min
4.037
2.853
1.500
1.520
Mean
4.806
3.537
2.147
2.729
-
1.269
2.659
2.077
S.D.
0.323
0.372
0.586
0.502
S.E
0.068
0.079
0.125
0.107
95% conf. int.
0.143
0.165
0.259
0.222
Statistic
Number
Log reduction
Unit: log CFU/gTS
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202
There has recently been a growing concern on the regrowth problems of fecal
contaminants or salmonella in water or sludges.
Some data in conventional heated
sludges or conventionally digested sludges appeared to increase slightly after 75-day
operation. However, the number o f fecal coliforms was not greater than that of raw
sludges. Further research is needed on this issue.
4.8.2 Statistical Analysis for Digester Evaluation
The analyzed paired t-test results from the sludge obtained by the control (Rl),
microwave (R2), and conventional heating (R3) are shown in Tables 4.13. Based on the
paired t-test results, the 95% confidence intervals for the average o f the differences in the
fecal coliform number (log CFU/gTS) for most pairs did not include the value zero.
Therefore, it is believed that the fecal coliform results for pairs of digesters were different.
It can be said that the results o f fecal coliform detection for pairs o f digesters were
significantly different and it is concluded that the second digester receiving microwave
irradiated sludge (R2) is more efficient in inactivation of fecal coliforms than the other
two digesters (Rl and R3).
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203
Table 4.13: Results o f paired t-test o f fecal coliforms for each pretreatment
Statistically
Digesters
N
d—
Better digester
’ ^a/2,n-l
Significant?
Rl (control) vs. R2
(microwaves)
Rl (control) vs. R3
22
(1.037,1.743)
Yes
R2
22
(0.517, 1.099)
Yes
R3
22
(-0.815, -0.350)
Yes
R2
(Conventional heating)
R2 (microwave) vs. R3
(conventional heating)
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204
5. MICROWAVE SYSTEM DESIGN
5.1 Proposed Full-Scale Microwave System
When sludges and liquids are exposed to microwaves at an effective level of
irradiation, pathogens are significantly destroyed and the performance o f sludge
stabilization processes is improved, leading to significant organic and volume reduction.
When this microwave system is applied, the sludge retention time in stabilization
processes could be reduced significantly, leading to significant cost savings.
Compared to conventional heating, microwave heating has the following
advantages (Copson, 1975): (1) rapid and uniform heating, (2) instant control, and (3)
selective heating.
5.1.1 Microwave Frequencies
The proposed microwave system pertains to the destruction o f pathogens and
reduction o f vector attraction by irradiating sludges with microwaves at an effective level
o f irradiation at the frequencies for heating required under the International Radio
Regulations (IRR) adopted at Geneva in 1959 or frequencies assigned by the FCC for
industrial, scientific, and medical use. The adopted frequencies for microwave heating
under the IRR are 0.915, 2.45, 5, 8, and 22.125 GHz.
In addition, 0.43392 GHz is
available in West Germany, Switzerland, Austria, Portugal, and Yugoslavia and 17.85 to
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205
18.15 GHz is legal in limited areas.
Although the proposed full-scale system was
developed at 2.45 GHz, the principal concept of the system does not change. Because of
the difference in penetration depth depending on microwave frequency, it may be better
to use the combination o f 2.45 and 0.915 GHz microwaves in the system as well as single
frequency microwaves.
An effective level o f irradiation, as used herein, means an amount of irradiation
that is sufficient to materially and substantially increase the pathogen destruction and
vector attraction reduction. An example irradiation requirement for anaerobic digester
sludge ranges from 2.8 to 5.6 watt hr/g total solids at 2,450 MHz. The medium subject to
microwave irradiation in this proposed microwave system includes sludges and liquids
generated during wastewater treatment and industrial operation.
Example media are
sludges from primary settling tanks, waste activated sludges, digested sludges, and
sludges contaminated with organic compounds.
5.1.2 Proposed Microwave System Applications
The first application o f this proposed microwave system is the irradiation of
primary sludge (generated from primary sedimentation tanks) with microwaves. This
will result in pre-pasteurization o f primary sludge before disposal or further treatment in
sludge stabilization processes.
The second application is the irradiation o f waste
activated sludge (generated from excess growth of microorganisms participating in
wastewater treatment) with microwaves. This will result in pre-pasteurization o f waste
activated sludge before disposal or further treatment in sludge stabilization processes.
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206
The third application of the microwave system is the irradiation of a recycle stream from
sludge stabilization processes with microwaves.
destroying o f pathogens before disposal.
This will result in heating and
During all three applications, besides the
benefits of pathogen destruction and vector attraction reduction, increased organic
reduction and volume reduction will be achieved. If anaerobic digestion is used as a
sludge stabilization process, increased methane production will be accomplished as well.
The fourth application is the irradiation o f stabilized sludge in a dewatered or
undewatered stage with microwaves. This will result in destruction o f pathogens before
final disposal.
microwaves.
The fifth application is the irradiation of side-stream liquids with
This will result in pathogen destruction prior to recycling back to the
liquids treatment processes.
Microwave frequency has an effect on heating. For example, at 915 MHz, the
temperature rises from the center, while at 2,450 MHz the temperature rises from the
outside. This phenomenon is due to the penetration depth of the microwave system.
Therefore, for homogeneous heating, it is better to use different microwave frequencies in
series.
Here are three examples of microwave frequency combinations: (1) all 2,450
MHz, (2) 2,450 and 915 MHz in series, and (3) 915 and 2,450 MHz in series.
The use o f microwave technology for the destruction of pathogen and complex
organics has the following advantages:
i.
Ease of operation: Unlike conventional heating, microwave heating can be turned
on or off with a switch, and thus, it is easier to automate the system.
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207
ii. Simplicity of the apparatus: The microwave sludge treatment apparatus is more
compact and simpler than conventional heating, alkaline chemical addition, or other
technologies approved by the U.S. EPA for Class A sludge.
iii. Effectiveness: Since microwaves have synergistic effects on pathogens, the
pathogen destruction efficiency will be greater than conventional heating;
iv. Lower cost: From cost estimation (Section 5.2) for a 108,000 gallon-per-day
wastewater treatment facility, the microwave sludge treatment system was 10 to
20% cheaper than pre-pasteurization or post-pasteurization methods.
v. Safe and clean system: The microwave sludges/liquids treatment system does not
need high pressure or high temperature.
After irradiation o f sludges with
microwaves, the sludges lose viscosity so there should be no sludges accumulated
in the system.
vi. No obnoxious odor release: During conventional heating, obnoxious odor is
released.
However, sludges do not produce any serious odor when exposed to
microwaves. In addition, there is no opening in the system and thus, there is no
danger o f releasing odorous gas during operation.
Microwave systems have been used for baking bread, increasing the rate of
chemical reactions, blanching vegetables, ashification, fat extraction, liquification of
crude oil, mineral extraction, rendering bacon, soil sterilization, defrosting frozen meat,
etc.
A full-scale microwave system used for bread baking could be modified for
application to sludge treatment.
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208
5.13 Description of Full-Scale Microwave System
A microwave irradiated sludge/liquid treatment apparatus o f the proposed fullscale microwave system is shown in Figures 5.1 through 5.5. Figure 5.1 is an elevation
view of the microwave irradiated sludges/liquids treatment apparatus o f the present
invention, comprising the preheating unit, distribution unit, microwave exposure chamber,
and collection unit along with inlet and outlet pipes. Sludges or liquids enter into the
microwave system via a distribution system 6 from pipe 2 and are then evenly dispersed
throughout the entire width of the system.
When liquids are irradiated with the
microwave system, valve 11 is partially closed to create internal pressure inside the
microwave exposure chamber and to control the detention time. The system is generally
comprised of a preheating unit 1, inlet pipe 2, inlet valve 3, backwash outlet pipe 4,
backwash outlet valve 5, distribution unit 6, microwave exposure chamber 7, magnetrons
8, microwave cut-off walls 9, collection unit 10, outlet valve 11, backwash inlet pipe 12,
backwash inlet valve 13, and outlet pipe 14. The preheating unit 1 is a typical heat
exchanger. Inlet and outlet valves 3, 12 isolate the microwave sludge treatment apparatus
for maintenance and clean up. In addition, the outlet valve 11 is used to control the
pressure and thus the detention time inside the microwave exposure chamber.
Microwave cut-off walls 9 are installed surrounding the microwave exposure chamber
and the top where magnetrons 8 are located to prevent any leakage o f microwaves outside
the system.
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209
Linel-l
L m e l-j/
Figure 5.1: Elevation view of the microwaved sludges/liquids treatment apparatus.
Figure 5.2 is a top view o f the distribution unit 6 before entering the microwave
sludge treatment apparatus o f the present invention. The distribution unit 6 is made of
either plastic or steel and is designed to distribute sludge/liquid from a pipe to the
microwave exposure chamber. The distribution unit is connected to the pipe 2 and the
microwave exposure chamber 7 with flange type joints 15, 16.
Figure 5.3 is a microwave exposure chamber 7 made of microwavable plastics.
The microwave exposure chamber has wedge-type distributors 17 in a zigzag pattern to
ensure good mixing and no accumulation o f sludges inside the chamber.
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210
Line 2 -2
16 3
4
12
C IO
14
: i»<
15
18
\
6
13
'7
10
Line 2 -2
Figure 5.2: Top view o f the distribution unit.
16
17
17
14
18
10
Figure 5.3: Microwave exposure chamber.
Figure 5.4 is a plan view o f the microwave irradiated sludges/liquids treatment
apparatus taken along the line 1-1 o f Figure 5.1. Figure 5.5 is a front view of the
microwave irradiated sludges/liquids distribution 6 and collection 10 units.
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211
Figure 5.4: Plan view o f the microwaved sludges/liquids treatment apparatus (line 1-1).
Part B
Part A
Figure 5.5: Plan view o f the microwaved sludges/liquids treatment apparatus (line 2-2).
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212
5.2 Economic Analysis
As mentioned in the literature review on microwave irradiation techniques,
Reimers et al., (1986a) discussed the use o f microwave radiation in industry and
development projects along with guidelines for the economic feasibility o f utilizing
microwave radiation in several areas including (1) the heating of thick sections of
material, (2) the heating o f temperature sensitive material, and (3) the use of minimum
microwave energy (Reimers et al., 1986b). Meanwhile, Graham and Domingue (1996)
indicated potential benefits, but the cost drops as the organic content increases. Similarly,
infrared drying has been applied for biosolids drying and obtained effective results. This
infrared drying process reduces side-stream effects and can produce up to 85% solids.
The problems are related to high energy costs and a very thorough maintenance o f the
process. The cost of the process for a disinfected biosolids was $540 per dry ton or five
times the cost of existing technologies (Graham and Domingue, 1996).
According to these literature reviews, it appears that microwave-heating systems
for water (not slurry conditions) and sludge drying (85% solid content) will not be
competitive with conventional methods. However, the proposed method in this research
is to substitute a microwave heating/pasteurization system for a conventional
heating/drying system. As shown in Figure 5.6, three alternatives were considered for the
application o f microwave units. Among them, microwave-irradiated sludge will be fed to
an existing mesophilic (35°C) anaerobic digester (pre-pasteurization) and then to a
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213
thickener for dewatering (post-pasteurization).
If Class A sludge is obtained using a
microwave system, existing sludge thickener or drying facilities may not be necessary.
(Option^)
(Option3j
MW|
[Option2)
Figure 5.6: Proposed 3 alternatives for microwave unit application.
Based on the report by Donohue and Associates (Gerbitz and Marten, 2000) for
the Madison Metropolitan Sewage District (MMSD) Nine Springs wastewater treatment
plant (WWTP), a similar cost estimation was performed with the microwave technology
(Table 5.1). According to Tables 5.2, 5.3, and 5.4, it can be seen that microwave
technology will be approximately 14% for post-pasteurization and 6% for prepasteurization cheaper than pre-pasteurization using a temperature-phased anaerobic
digester (TPAD), although energy savings (methane production increase) is not
considered (). The Nine Springs WWTP technical memorandum No.9.E indicated that
total sludge flow would increase from 309,458 to 432,814 gallons/day (1,171 m3/day in
2000 to 1,638 m3/day in 2020). The capacity expansion of sludge treatment processes
will be needed. Total 15 processes for sludge treatment were reviewed using total 15
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214
weighted criteria evaluations; present worth cost, reliability, operability, constructibility,
and so on by Donohue and Associates. As a result, four processes were first screened by
non-economic evaluations such as the construction of an additional primary digester, prepasteurization, aerobic thermophilic pre-treatment, and TPAD (Table 5.3).
Finally,
TP AD at a total digestion hydraulic retention time (HRT) of 15 days was recommended
for the following reasons:
i.
It has the lowest capital and total present worth cost, representing an estimated
savings of almost $6 million over the next closest alternative.
ii.
It will provide reliable, effective, and enhanced stabilization of biosolids and
production o f biogas, based on the MMSD’s bench scale testing and industry’s
reported experiences, for the planning periods.
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215
Table 5.1: Summary o f capital and total present worth costs.
Process
No.
Alternatives
Construct additional primary
digester
9.E.l.b
Capital
Annual
Total present
worth
$8,752,000
$146,000
$10,285,000
9.E.3.c(l)
Pre-pasteurization
$16,885,000
$206,000
$19,048,000
9.E.3.c(2)
Aerobic thermophilic pre­
treatment
$18,610,000
$232,000
$21,043,000
9.E.3.c(3)
Pre-pasteurization, 25% flow
option
$12,797,000
$174,000
$14,618,000
9.E.4.b(l)
Temperature phased anaerobic
digestion
$10,034,000
$334,000
$13,534,000
9.E.4.b(2)
Temperature phased anaerobic
digestion, 25% flow option
$11,952,000
$233,000
$14,294,000
Temperature phased anaerobic
9.E.4.b(3) digestion, 15 day total HRT
option
$3,839,000
$284,000
$6,816,000
Source: Gerbitz and Marten (2000).
It is difficult to compare 9.E.4.b.(3) in Table 5.1 (15-day HRT TP AD) with the
microwave system because three existing east digesters would serve as thermophilic
digesters. Thus, 9.E.4.b.(2) has been chosen to compare the estimation o f microwave
pasteurization with TP AD at the same condition. The general description o f 9.E.4.b.(2)
and the comparison condition between the microwave system and TP AD are shown
below (general description, at south complex only, 25% flow, 108,000gpd):
i.
Add a new south digester complex to produce class A biosolids on 25% of the
raw sludge flow.
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216
ii.
Overall digestion HRT = 20 days.
iii.
The east and west complexes would produce class B.
Table 5.2: Summary o f cost estimations at the south complex of the Nine Springs WWTP.
TP AD
MW: post­
pasteurization
MW: pre­
pasteurization
25%
25%
25%
(108,000gpd)
(108,000gpd)
(108,000gpd)
342,500
342,500
342,500
2. Concrete
2,252,125
2,252,125
2,252,125
3. Buildings
644,000
322,000
322,000
2,411,400
1,961,900
2,361,900
8,071,464
6,969,321
7,540,750
1,614,293
1,393,864
1,508,150
2,421,439
2,090,796
2,262,225
1,816,079
1,568,097
1,696,669
9. Electrical
485,245
362,111
544,050
10. Chemical (Ferric chloride1)
101,822
101,822
101,822
Total annual cost (No.9+No.l0)
587,067
463,933
645,872
14,510,343
12,486,012
13,653,666
240 KW x 4
240 KW x 6
Applications
Sludge flow (%, gpd)
1. Earthwork
4. Process mechanical equip. &
Piping
5. Initial cost
(No.l+2+3+0.3initial = initial)
6. Contingency (20% of initial)
7.Contract overhead & profit
makeup (25% o f No.6+No.7)
8. Engineering (15% of No.5+6+7)
Total
Microwaves and boilers
Ferric chloride1: for struvite control. Total cost was estimatet except for interest & present worth
factor.
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217
Table 5.3: Cost comparison between TP AD and microwave pre/post pasteurization.
TPAD
Works
Work detail
MW-post
MW-pre
Operation
Initial cost Initial cost Initial cost
$
$
$
Dewatering
5,000
5,000
5,000
Excavation
300,000
300,000
300,000
Structural fill
37,500
37,500
37,500
Total
342,500
342,500
342,500
967,875
967,875
967,875
%
Location
Earth work
Walls
Concrete
Structural slab
Total
1,284,250 1,284,250 1,284,250
2,252,125 2,252,125 2,252,125
644,000
322,000
322,000
644,000
322,000
322,000
Recirculation pump
3,216
0
0
25
east
Hot water pump
3,216
0
0
25
east
Mixing system
13,723
13,723
13,723
100
east
Mixing system
20,585
20,585
20,585
100
west
Recirculation pump
15,439
0
0
100
south
Hot water pump
1,287
0
0
25
south
Hot water pump
7,719
0
0
100
south
Mixing system
42,886
42,886
42,886
100
south
Digested sludge
pump
5,146
5,146
5,146
100
south
Two story
Building
Total
Electrical
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218
Recirculation pump
643
0
0
25
east
Hot water pump
643
0
0
25
east
6,862
6,862
6,862
100
east
Heating with electric
boiler
363,878
272,909
454,848
Total
485,245
362,111
544,050
Ferric chloride
(struvite)
69,350
69,350
69,350
Polymer (GBT)
32,472
32,472
32,472
101,822
101,822
101,822
594,000
594,000
594,000
445,500
445,500
445,500
Digester
recirculation pumps
75,000
0
0
Heat exchangers
(east)
45,500
0
0
Heat exchangers
(south)
208,000
0
0
Heat exchangers
(south)
85,000
0
0
Hot water pumps
36,000
0
0
Digested sludge
pumps
117,000
117,000
117,000
Mixing system
Chemical
Total
Mechanica Mixing system
l&
(meso)
equipment
Mixing system
(thermo)
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219
Digester grit pumps
Electric boiler
(240KW)
Total
5,400
800,000
5,400
5,400
800,000 1,200,000
2,411,400 1,961,900 2,361,900
Sludge flow: 25%, 108,000 gpd
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220
Table 5.4: Estimation o f electricity costs for post/pre pasteurization.
Post Pasteurization with microwave
$/kW
$/day
742
0.042
747.70
0.8
1484
0.042
1495.39
0.8
2967
0.042
2990.77
$/kW
$/day
n (efficiency) P(kW )
M3/d
gal/d
To
Tf
409
108000
35
65
0.8
818
216001
35
65
1637
432000
35
65
Pre Pasteurization with microwave
n (efficiency) P (kW)
M3/d
gal/d
To
Tf
409
108000
15
65
0.8
1236
0.042
1246.16
818
216001
15
65
0.8
2473
0.042
2492.32
1637
432000
15
65
0.8
4945
0.042
4984.62
Standard mesophilic digestion with boiler and heat exchanger
$/kW
$/day
495
0.042
498.46
0.8
989
0.042
996.93
0.8
1978
0.042
1993.85
$/kW
$/day
M3/d
gal/d
To
Tf
409
108000
15
35
0.8
818
216001
15
35
1637
432000
15
35
n (efficiency) P (kW)
TP AD with boiler and heat exchanger
M3/d
gal/day
To
Tf
409
108000
15
55
0.8
989
0.042
996.93
818
216001
15
55
0.8
1978
0.042
1993.85
1637
432000
15
55
0.8
3956
0.042
3987.70
n (efficiency) P (kW)
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221
1 cal = 4.2 W-sec = 1.16 * I O'3 W-hr, 1 kW = 862 kcal; AT = H/Cp-M (A T = temp, increment, H
= cal, M = g, Cp = cal/g °C); P (W) = 4.2 Cp-M-AT/n t; n = Pen/Pin efficiency, t = seconds, n
(efficiency) = 0.8 (Roger, 1998); P (kW) = M Cp A T (1.16 x 10'3)/n (M = kg/hrs).
Construct:
o
one 1,000,000 gal. digester at east
o
three 225.000 gal. thermo digester at south
o
two 820,000 gal. meso digesters at south
o
Fixed concrete covers on all digesters
o
Mechanical mixing systems
o
Re-circulation pumps
o
Hot water pumps
o
Heat exchangers to provide digester heating
o
Sludge transfer pumps to transfer digested sludge to secondary digesters or
Install:
gravity belt thickeners (GBTs)
o
Sludge-sludge heat exchanger for thermo digester
o
Assumptions for comparison:
o
Cost of heating energy (cost o f electricity, gas, or fuel) for digesterswill
be the same based on 80% efficiencies for all of the heating facilities,
o
Cost of electricity will be $0.042/kW.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
222
o
The heat energy cost for a TPAD and the microwave system were
calculated with total input volume of sludges (25%, 108,000 gpd) and
target temperature (TPAD: 55°C, MW:65°C).
o
Cost benefits o f biogas increase with a TPAD and microwave radiated
digester were ignored.
o
In case of the TPAD estimation, an electric
boiler (240 kW,
$200,000/each) was included, and a conveyer belt type microwave system
(240 kW, $200,000/each) was considered for the microwave heating
system.
o
Life cycle analysis (interest and number of years) was ignored.
The above cost comparison between conventional and microwave technologies is
an example. More detailed cost estimation must be made to compare the economics of a
15-day TPAD and microwave system. Despite the lower cost, the microwave technology
must be further evaluated for destruction o f Salmonella sp., enteric viruses, and helminth
ova since they are also regulated indicator organisms for Class A sludge although it is
anticipated that these pathogens are destructed completely. In addition, the best location
for the microwave irradiation must be selected in a wastewater treatment plant based on
pilot testing. Since microwave irradiation is a new technology, the approval must be
obtained from the U.S. EPA.
It is believed that the microwave technology can be
approved if a more thorough study is performed on all four pathogens (fecal coliform,
Salmonella sp., enteric viruses, and helminth ova).
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223
6. CONCLUSIONS AND RECOMMENDATIONS
6.1 Conclusions
Several
technologies
have
so
environmentally save Class A sludge.
far
been
developed
for
generation
of
Microwave irradiation could be an attractive
method because o f the synergistic microwave effect on pathogen destruction and thermal
heating for anaerobic digestion at 35°C. Since substances in sludge from wastewater
treatment plants can be easily heated with microwave power and microwave energy has a
strong ability to penetrate dielectric materials to produce thermal or synergistic-thermal
effects on microbes, it has been postulated that microwave irradiation technology could
be applied to biosolids treatment.
The objective o f this study was to develop a new technology of generating Class
A sludge from wastewater treatment plant (WWTP) sludge using microwaves. There has
been extensive research conducted on the use of microwaves for treatment of wastewater
treatment plant sludge. No study has so far been reported in this area except on the use of
microwaves for sludge drying. The specific objectives of this study were as follows: 1)
Compare the effect o f microwave irradiation on pathogen destruction with conventional
heating; 2) Evaluate pathogen destruction mechanisms by microwaves; 3) Assess
anaerobic digestibility and organic reduction after pretreatment of sludge with
microwaves; and 4) Conduct an economic analysis of microwave technology in biosolids
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224
treatment.
In order to accomplish the specific objectives above, several tests were
performed, and the following conclusions were drawn.
1.
The penetration depths o f a 2,450 MHz microwave unit were 1.7 and 1.1 cm for
primary and waste activated sludges, respectively. This value can be used as a
design parameter for 2,450 MHz microwave units to apply on sewage sludge.
2.
In the batch BMP tests, the relative cumulative biogas productions o f ADS, PS, and
WAS pretreated with microwaves after 5 days were approximately 13, 20, and 5%
higher than conventional heating, and 54, 52, and 3% higher than the control.
3.
Pathogen die-away tests performed at 4°C demonstrated that despite high fecal
coliform destruction efficiencies ranging from 99.3 to 99.7%, the residual fecal
coliform counts could not meet the Class A sludge requirement o f < 1,000 CFU/g
TS even after 224 days of storage at 4°C. On the other hand, fecal coliforms were
not detected at 65°C for PS and ADS and at 85°C for WAS when sludge was
irradiated with microwaves.
4.
When sludge was heated to < 65°C by conventional heating or microwaves, it
appeared that unknown inhibitory organic compounds leached from sludge leading
to a decrease in methane production or higher toxicity values than the control.
However, there was no inhibitory effect on anaerobic digestion when sludge was
irradiated with microwaves > 65°C.
5.
Damages of fecal coliform cell membranes by microwave irradiation were observed
using both SYTO 9 and propidium iodide stains. Cell membrane damages of fecal
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225
coliform were thought to be caused by microwave heating. The ETS activity was
lineally fitted to P-galactosidase activities. In addition, the genomic DNA bands
extracted from the pure cultured fecal coliforms that had been irradiated with 1 kW
microwaves were increasingly faint with the increase in irradiation time. This
phenomenon was not observed in the externally heated samples. The non-thermal
effect should be studied as further research.
6.
During the bench scale anaerobic digester operation, the highest log reductions of
fecal coliforms were approximately 2.66 for the digester fed with microwavepretreated sludge. Based on the paired t-test results at the 95% confidence interval,
fecal coliform detection results for pairs of digesters were statistically significantly
different. The digester fed with microwave-irradiated sludge was more efficient in
inactivation of fecal coliforms than the other two digesters receiving raw sludge and
preheated sludge.
7.
From the economic analyses and laboratory-scale experiments, microwave
technology is an economical, effective, method of producing environmentally safe
sludge.
6.2 Recommendations
From the study performed so far, the following recommendations were made:
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226
1. Based on the U.S. EPA regulation, Salmonella, helminth ova, and enteric viruses
should be tested along with fecal coliforms to confirm the similar pathogen
destruction efficiency.
2. The comparison between microwave heating and external heating will be needed at
the same temperature increase rate and at the same energy levels.
3. Long-term batch tests should be performed at various SRTs such as 35, 30, 25, 20, 15,
10, and 5, because the kinetic relationship between pathogen reduction and anaerobic
digestibility will be required.
4. Performing a scale-up test (pilot-scale test) will be needed to obtain scale up factors
o f microwave unit design.
5. Pathogen regrowth problems should be further investigated.
However, sludge
irradiated with microwaves appeared to be more effective in preventing regrowth.
6. During the research, microorganisms appear to be affected differently by magnetic
electro field waves. It will be not the intensity of waves but frequency. If so, it will
be possible to inactivate selectively. In addition, the pathogen destruction tests should
be evaluated with various ranges in microwaves for sludges. It will be worth studying
this area in the near future.
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227
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