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The effect of controlled pH upon the production of chemicals in several fermentations

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fHS EFFECT OF CONTROLLED pH UPON THE PRODUCTION OF
CHEMICALS IX m r n k h FERMENTATIONS
Richard James Hickey
A T hssia Subm itted to th e Graduate F aculty
f o r th e Degree o f
DOCTOR OF PHILOSOPHY
Major S u b ject B iophysical Chemistry
{L
Dean o f Graduate College M
Iowa S ta te C ollege
1941
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UMI N u m b er: D P 1 2 7 5 5
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Q ~ R \G 4 H 5 E £ e
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TABLE OF CONTENTS
Page
I. ACIOIOWLEDOFCTTS*........ ..... *...........
II*
III*
INTRODUCTION*..............
f
REVIM OF PREVIOUS INVESTIGATIONS
A*
B*
G.
IF*
8
......... *...........
H le to ric a l* * .
........
G lycerol Form ation by th e S a if it® Ferm entation
Methods*.
...........
1 . The Connatein aad IAidecke p r o c e s s * ....................
2. ft® Cocking s a l I l l l y p ro c e ss * ..•....................
3* O ther m e th o d s* .....« .* « . .* .. . .
..............
G lycerol by th e A lkaline P e rm u ta tio n M eth od s....*
1* The E off p ro c e a s * .... . . . . . . . . . . . . . . . . . . . . . . . . .
2.
O ther p r o c e s s e s * * . * . . . . . * . . . . . . . . . . . * . . . * . . . . .
U
14
22
22
26
2f
34
34
37
SFSIIKENTAL*.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4$
A#
45
45
45
4?
46
55
55
A
1.
p p a r a t u s . . . . . .
pH measuring in s tr u m e n ts ......
a* Cameron p i letfflp*.. . . . . . . . . . . . . *• ****»* **■
b* Coleman pH Ifetcr*
2* pH c o n tro l and record ing Instrum ent*. . . . . . . . . .
B# Methods o f P r o c e d u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1* A nalytical m eth o d s* ..* ..*
................
a* D etenainatioa o f s u g a r............... « . . . ..........
b . Dete®«dimti©s e tte s p lU ... . . . . . . . . . . . . . .
e* D eterm ination of s u lf ite * .
...............
d* Detert^Lnation o f a c e ta ld e h y d e * ............
©• D eterm ination o f g ly c e ro l* .
................
2 * Development e f am optimum a ^ s i-s y n th e tie
B edim s*..........« * * * * « .•* .* •* •...* * * •* ♦ .» * •* ♦
3* The a lk a lin e d is s im ila tio n o f d ex tro se f o r th e
p re p a ra tio n o f g ly cero l* * * .. . . . . . * . . * . . . . . . . . .
a . The us© o f aaaoniua hydroxide*. . . . . . . . . . . .
b* S tu d ies w ith ammonium s a l t b u f f e r s . . . . . . . .
c . The use o f sodium carbonate*
.
d« The use o f sodium h y d r o x i d e * ..............
4* The B U lfite-d Jistilfite d issim ila tio n s o f d«se»
tro s e fo r th e p rep aratio n o f glycerol*
jjau The use o f sisaaoaim s u lf ite * .* ..* * . * . . .. . .
v b* The use o f calcium and ssgneBitna s u lfite s *
T7 2 0 6
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56
66
6f
6?
68
68
77
77
W
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¥*
SWART A® COMGTtJSIOHS.........................................
Pag«
13?
U f M * CITED**...................
142
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I£S? OF TABLES
T&bl*
1 . GLYCEROL YIELDS USING ALKALINE SALTS IN THE FERMENTING
. ..............
*.
M EDIUM *.......*.............
2.
3.
4.
19
THE EFFECT OF INCREASING AMOUNTS OF SODIUM SULFITE IN
HAST FERMENTATIONS ON GLYCEROL Y X EID S.....
.........
20
REAGENTS CAUSING NEUBERG*S FIRST AND THIRD MECHANISMS
TO OCCUR........................................ ......... ............................... .
21
EFFECT ON GLZCK?GL YIELD OF USING HAST FROM ONE FER­
MENTATION AS SEED FOR THE NEXT*. . . . . . . . . . . . . . . . . . . . . . . .
24
5* YIELD OF GLYCHOL BY FERMENTATION WITH VARIOUS SALTS***
25
6 . AMMONIUM HYDROXIDE ADDITIONS WITH RESPECT TO TIMS IS
ORDER TO KEEP pH ABOUT SEVEN IN A JAVAN MOLASSES PER*
MENTATION OF THREE L IT E R S * * ........................... ............... .
43
f * SOMOGYI REAOSNT FOR THE CQPPEII~IGBOMmiC D^HMINA*
TIOS OF VERY SK&IL AMOUNTS OF SUGAR.................................... ..
5?
8*
STANDARDIZATION OF THE SOMOGYX SUGAR METHOD AGAINST
PORE DEXTROSE**
.......................................... ....................
§0
V* DEXTROSE ANALYSES BY THE MODIFIED SOMOGYI COPPERIOSOMSTRIC METHOD* .............................. .....................................
62
10*
COPPSK-IODOMETRIC REAGBET *G" FOR THE DETERMINATION OF
SUGAR ON A SEMI-MICRO S C jH S ..................................................
63
11*
THE BASAL M ED IU M ....*............ .................................................
69
12*
EFFECT OF YEAST EXTRACT CONCMmTION ON ETHANOL YIEID.
n
13* EFFECT OF AlSfONIUM CHLORIDE CONCENTRATION ON ETHANOL
YIELD.............................. ............................... ...................................
14*
EFFECT OF POTASSIUM ACID PHOSPHATE CONCENTRATION ON
ETHANOL YIELD......................................................... .......................
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n
73
Table
15* EFFECT OF MG1ISSIUM SULFATE CONCENTRATION 08 ETHANOL
.....................
16.
17.
18.
19.
20.
Page
.............14
EFFECT OF CALCIUM CHLORIDE GONCEimTION 08 ETHANOL
................................. . .......................*...............................
OPTIMUM S£kO>SBiTHmC MEDIUM FOE MAXIMUM EPH&80L PRO*
DUCTIOR UTILIZING A MINIMUM OF ADDED SOLIDS*...
75
7®
FBtffl»TATIOHS HAVING pH AUTOMATICALLY CONTROLLED BX
AMaOHIUM HYDROXIDE*.............................................
82
THE EFFECT OF A1I&8XUM CHLORIDE IN GREATER THAN
NUTRIMT QUANTITIES 08 THE ALCOHOLIC FERMENTATION OF
THE OPTIMUM SEMI-SYNTHETIC iffiDIHM..,.............................
THE EFFECT OF AMMONIUM CARBONATE OH PRODUCTS* EXTENT*
AND pH OF FERMENTATIONS WITH H A ST....................
83
85
2 1.
EFFECT m AMMONIUM CHIORID&^IKOHIUM CARBONATE RATIO ON
THE pH OF SOLUTIONS OF CONSTANT AMMONIUM 108 NOSKALlfX
{tmspemtooam * 25^ G*)*.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
22.
THE EFFECT OF SEVERAL AMJONIUM SALTS IN CONCENTRATIONS
APPROACHING 1 NORMAL ON THE ALCOHOLIC FERMENTATION. • **•
88
23* AMMONIUM SULFATB-GARBONATE BUFFERS 0*5 NORMAL 18
AMMONIUM ION CONCENTRATIONj THEIR EFFECT OS pH, GLYCMOL
FORMED, AND ON SUGAR CONSUSSPTIOM*.... . . . . . . . . . . . . . . . . . .
93
24*
SUGAR CONSUMPTION IS FERMENTATIONS AS RELATED TO pH AND ,
AMMONIUM CONCENTRATION*...
..............
300
25*
FERMENTATION AUTOMATICALLY CONTROLLED AT pH 7*5 BX SODIUM
CARBONATE SOLUTION IN THE ABSENCE OF APPRECIABLE
AMMONIUM S A L T S * . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 103
26* ACCLIMATIZATION OF INOCULUM WITH AMMONIUM SUIFITS*
27*
28.
OBSERVING pH.
108
SOLUBILITIES OF CALCIUM AND MAGNESIUM 3UIFITES AMD
BISULFITES...
..............
312
SOLUBILITIES OF HYDRATED AND ANHYDROUS CALCIUM SULFITES
AS FUNCTIONS OF pH*.
313
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Table
29. THE SOLUBILITY OF MAGNESIUM SUIFITB AS A FUNCTION OF
30*
Page
SOLUBILITY OF CALCIUM SULFITE AS A FUNCTION OF pH IN THE
m s s m m o f a co n stan t co n cen tratio n o f Asm um szoE**** 118
33U EFFECT OF pH ON GIYCEROL YIELD AND ON SUGAR CONSUMPTION
IN FERMENTATIONS IN THE PRESENCE OF ANHYDROUS CALCIUM
»* US)
SULFITE*
32*
EFFECT OF p i ON GLYCEROL YIELD AND ON SUGAR CONSUMPTION
IN FERMENTATIONS IS THE PRESENCE <F HYDRATED CAICIUH
121
SULFITE.
33% GLYCEROL YIELDS FROM YEAST FERMENTATIONS AT DIFFERENT
pH LEVELS USING CALCIUM SULFITE CASE FROM PREVIOUS PERMENTATIONS AT INOCULUM...
34*
THE EFFECT OF DEXTROSE CONCENTRATION ON THE GLYCEROL
H U B OF FERMENTATIONS IN THE PRESENCE OF CALCIUM
SULFHE. •» •
35*
PRELIMINARY FERMENTATIONS IN THE PRESENCE OF MAGNESIUM
SULFITE IITH pH UNADJUSTED* .........
* ..•• • 1 2 8
36.
THE RELATION OF pH TO THE GLYCEROL YIELD OF FEBUHSTA~
TIONS IN THE PRESMCE OF MAGNESIUM SULFITE.................... ..1 2 9
3 ?.
THE EFFECT OP DEXTROSE CONCENTRATION ON THE GLYCEROL
YIELD OF FERMENTATIONS IN THE PRESENCE OF MAGNESIUM
SU LFITE*.*.*.*..*...........
130
38* THE RELATIONSHIP BETWEEN K ® J S B ! 4 l AND CALCULATED
YIELDS GF GLYCEiOL FROM FOMENTATIONS INVOICING CALCIUM
SU IFIT 1...
..........
131
39.
THE RELATIONSHIP BETWEEN EXPERIMENTAL AND CALCULATED
YIELDS OF GLYCEROL FROM FERMENTATIONS INVOLVING
MAGNESIUM SUIFIT&...........................................
40.
GLYCEROL YIELDS FROM FERMENTATIONS WITH AUTOMATICALLY
CONT80LIED pH IN THE PRESENCE OF MAGNESIUM SU L F IT E ...... 133
41*
GLYCEROL HELD WHERE TWO SUCCESSIVE FERMENTATIONS ARE
RUN ON THE SAME SUBSTRATE IN THE PRESENCE OF CALCIUM
SULFITE*.
.........
........1 3 5
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•i-
LIST OF FIGURES
Figure
I* INITIAL pH CONTROL CIRCUIT*........................ . . . . . . . . . . . . . . .
Page
51
2*
SAFETY
51
3*
CIRCUIT ALLOWING BASE TO ALB ONLY IN THE RECORDING
PORTION OF THE C Y C L E .* ........ . . . ......... . . . . . . . . . . . . . . . . .
51
4* COMBINED SAFETY* PH CORTR0I* AND AUTOMATIC STIRRING
CIRCUIT.* .............
.* .* * .
53
5*
EFFECT OF THE RATIO OF AMMONIUM CHLORIDE TO AMMONIUM
CARBONATE ON pH OF SOLUTION OF CONSTANT AMMONIUM NORMAL*
m * . .............................
. * . . . 90
6*
EFFECT OF VARIOUS CONCENTRATIONS OF AlftlONIUH SALTS ON
TIE EXTENT OF THE ALCOHOLIC FERMENTATION***.***********
90
EFFECT OF pH OR YEAST FERMENTATIONS BUFFERED BI VARIOUS
MIXTURES OF AMMONIUM CARBONATE AND AMI,IONIUM SUIFAfE.*..
95
f*
8*
9.
20*
II*
22*
SUGAR CONSUMPTION IN FERMENTATIONS AS BELATED TO pH
AND AUfQMlUM CONCENTflAYIOII.......... . . . . . . . . . . . . . . . . . . .
101
RELATIONSHIP BETWEEN SULFITE’ 30LUBILITX AND p S . . . . . . . . . H 5
SOLUBILITY OF CALCIUM SUIFITE AS A FUNCTION OF pH IN
THE PRESENCE OF A CONSTANT CONCENTRATION OF
ACETALDEHYDB*•• ..........
H9
EFFECT OF INITIAL DEXTROSE CONCENTRATION ON GLYCEROL
HELD OF FERJOTATIOES IN THE PRESENCE OF CALCIUM
SULFITE*.............
127
EFFIGY OF INITIAL DEXTROSE CONCENTRATION ON RE5ULYIN0
GLYCEROL CONCENTRATION IN FSOffiHTATKH® DfFOLFBRJ
CALCIUM S U IF IT E ....**.. . . . . . .................
127
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I.
ACKHOWLEDQJMEHTS
Ihe au th o r wishes to tak e th is o p po rtun ity to express h is
s in e e re a t g ra titu d e to Dr. E U la 1* F u laer f o r suggesting th is
to p ic , and to him and to D r. Lel&nd A* U nderkofler f o r th e ir s o s t
v alu ab le sod generous a s s is ta n c e and advice during th e course o f
th is in v e s tig a tio n .
Mr. G. la rre n Buekaloo*s a ssista n c e in th e
developaent o f th e o p tim a media was most valuable* and Mr. Thoaas
& lees* o rig in a l id eas and a ssista n c e e sp e c ia lly along m echanical
lin e s and in a n a ly tic a l work were sin c e re ly a p p reciated .
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II*
INTRODUCTION
G lycerol has n o t boon produced on & commercial b a sis by ferm en tstlo n In normal tin e s w ith any g re a t degree o f economic success*
It
m s su c c e ssfu lly produced by ferm entation In Germany* however* during
th e World Iter o f 1914-1918 fo r use In th e m anufacture o f explosives*
Such a production m s necessary fo r a n a tio n a l emergency sho re th e
end ju s tif ie d th e means In s p ite of th e high c o st o f m anufacture.
The Connatein and tiidseke (1921) (1924) ferm entation process In Germany
produced g ly cero l from b e et sugar in th e presence o f sodium s u l f i te .
About tw enty-four fa c to rie s produced approxim ately one thousand ton s
o f g ly c e ro l p er month by th is r& thsr in e f f ic ie n t process*
Sm aller
amounts o f g ly ce ro l were produced In th e United S ta te s follow ing th e
success o f th e Germans* b u t sodium carbonate was used in p lace o f
sodium s u lfite *
Eoff ( ly l8 ) .
This a lk a lin e ferm entation method was p atented by
In England* th e Cooking and U lly (1922) process was
used to some extent*
I t i s sim ila r to th e method o f C oansteia and
Ludecke except th a t sodium b is u lf ite i s employed along w ith th e sodium
s u lf ite l a o rd er to in c re a se th e y ie ld o f g ly c e ro l.
I t i s claim ed
to be a means o f producing p ra c tic a lly a th e o re tic a l y ie ld o f g ly ce ro l
by ferm entation*
In most o f th e g ly c e ro l ferm entation processes sodium s a lts a re
added to in cre ase th e g ly ce ro l yield*
The re la tiv e ly high s o lu b ility
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o f th e se sodium s a lts causes c e rta in d if f ic u ltie s in th e recovery
o f to© g ly ce ro l from th e mash* The g ly c e ro l i s n o t e a s ily d is t il l e d
in to© presence o f such s a lts w ithout some decom position o r e th e ris a ­
tio n .
Here e f f ic ie n t recovery methods have been developed, however,
in re e a n t years*
G lycerol e x tra c tio n methods are u su a lly ra th e r
involved? expensive* o r im p ra c tic a l.
In o rd er f o r ferm entatio n g ly ce ro l to compete on an economic
b a sis w ith th a t produced as a by-product o f to e soap industry* th e
method o f form ation o f th e g ly ce ro l must be very e f f ic ie n t and a lso
th e procedure o f recovery must n o t re q u ire excessive expense to
o p e ra te .
The percentage recovery o f th e a v a ila b le g ly ce ro l should
be a t le a s t 90 percent*
The u t i l i t y and v alue o f the b y -p ro te c ts
must a lso be considered, as th ese m ight w ell be th e fa c to rs which
determ ine th e success o r f a ilu r e o f th e pro cess.
Recent methods fo r
tli© sy n th e sis o f g ly c e ro l from petroleum products appear to have
promise o f su ccess.
On th e o th e r hand, th e re a re very la rg e q u an ti­
t i e s o f b lac k strap m olasses a v a ila b le as a source o f sugar f o r fermen­
ta tio n a t very low c o s t.
Com o r com su g ar might a lso be p o te n tia l
carbohydrate sources*
G lycerol i s a v ery u se fu l commodity in an e x cep tio n ally wide
v a rie ty o f f ie ld s .
in d u stry a t present*
Xt i s produced alm ost ex clu siv ely by th e eoep
Since th is production i s lim ite d by th e q u a n tity
o f to e soap produced, g ly ce ro l i s not used as w idely in in d u stry a s
i t m ight be i f so re were a v a ila b le a t a more reasonable c o st to th e
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<■111
consumer.
If a consumer ware to use very large quantities of
glycerol, he wold no doubt desire practically an unlimited quantity
to be available at a stable price level*
He would net consider ex­
pansion of an industry or process which would be controlled by the
limited availability of one component*
Be could get no more than a
fixed amount of glycerol from the soap industry*
If he wanted more
than the soap industry could supply, be would, no doubt, have to
turn to either the fermentation processes or t© the synthetic pro­
cesses*
The price of his glycerol would then probably increase
to such an extent that his process would no laager be industrially
possible without economic difficulties*
Synthetic glycerol is still
in experimental stages, but according to Williams and associates
(1940)(1941) It can be made at approximately the market price*
Fermentation glycerol is as yet too expensive for widespread manu­
facturing uses*
Use as an antifreeze in automobiles would require
an inexpensive product*
The plastic industry could employ quantities
of glycerol if it were available at a low enough price*
number of other possible uses
are at hand*
A greet
The petroleum industry
is developing methods for the production of long chain fatty acids
from petroleum hydrocarbons and it is desired that these might be
converted Into glyceryl esters or fats to be used in the food Industry*
There has been a large amount of research done on the subject
of the production of glycerol by fermentation and it might appear
to sense that the glycerol fermentation has very little prospeet of
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becoming & th riv in g in d u stry .
th e fu tu re w ill prove*
That la a m atter o f co n jectu re which
I f th e r e m its o f th is work c o n trib u te some*
th in g o f value to e ith e r th e academic o r in d u s tria l fie ld s# th en
seme o f th e aims w ill have been re a lise d *
I t should be borne in
mind* however# th a t although a la rg e p a rt o f th e e sp e ria e n ta l work
has been done cm th e g ly c e ro l ferm entation# m ay o f th e p rin cip le s#
methods# and th e o rie s m ight w e ll be ap p lied to o th er ferm entatio ns
w ith q u ite s a tis fa c to ry re s u lts *
Some o f th e work i s concerned w ith
th e re la tio n s h ip o f pH to th e production o f g ly ce ro l by th e a lte ra *
tio n o f a y e ast ferm en tatio n from i t s normal course w herein th e pH
i s u su a lly about 3*
The pH i s a lte re d toward about ?# o r th e extreme
lim it o f endurance o f most s tr a in s o f yeast*
thereby g re a tly increased*
G lycerol production i s
There would be in te r e s t in determ ining
th e g ly c e ro l y ie ld as a m athem atical fu n ctio n o f th e pH o f th e medium*
Then l o d e e ffe c ts on th e course o f th e re a c tio n eould p o ssib ly be
stu d ied to more advantage*
Hie purposes o f th is th e s is a re to study c e rta in ferm entations
under abnormal pH conditions# to o b tain re la tio n s between pH and th e
y ie ld s o f th e products o f d issim ila tio n o f c e rta in carbohydrate*
in th e presence end in th e absence o f c e rta in fix in g agents# and to
o b tain methods fo r g ly c e ro l production by ferm entation such th a t
th e added s a lts o r o th e r compounds say be e a s ily removed by sim ple
methods such a s f i l t r a t i o n o r d i s t il l a t i o n o r by com binations o f 'the
two in o rd er to a s s is t in making th e g ly c e ro l ferm entation an
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in d u s tr ia l r e a lity .
T his th e s is i s concerned p rim arily w ith stu d ie s
on th e g ly c e ro l ferm en tatio n s.
S pecial a tte n tio n i s given to th e
r e la tio n between pH and th e y ie ld s o f v ario u s d issim ila tio n products
o f carbohydrates in th e presence o r absence o f c e rta in aldehyde
fix in g agents*
A tten tio n i s a ls o paid to th e development o f g ly c e ro l
ferm entation methods which should allow g re a tly sim p lifie d methods
o f g ly c e ro l recovery follow ing th e ferm entation*
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ra .
w n m o f p re w o ts in te s tig a tio k s
A* H isto ric a l
Glycerol* a ls o c a lle d glycerine* o r propan-1* 2*3 -trio l* i e s
th ic k liq u id which ie p ra c tic a lly c o lo rle ss and odorless*
ra th e r sweet ta s tin g and may be used as a food*
I t ie
lite r a tu r e on th e
su b je c t o f g ly cero l i s p ra c tic a lly a l l i t s a sp ec ts has been very
ably cowered up to 1928 in th e monograph by law rie (1928)*
Because
o f th e a v a ila b ility o f th is aonograph* to p ic s and refe ren c es up to
1928 w ill be mentioned v ery b rie fly o r om itted a lto g e th e r u n less th e
su b je c t m a teria l i s very p e rtin e n t to th e in v e stig a tio n s made fo r
th is th e sis*
The b r ie f h isto ry in th e follow ing paragraph was
taken d ir e c tly from I&wrie (1928)*
In 1779 Scheele prepared g ly ce ro l by h eating a m ixture o f o liv e
o i l and lith a rg e *
la te r* in 1784* he showed th a t th is m a te ria l could
be obtained from a v a rie ty o f o ils and f a ts both o f anim al and o f
v egetable o rig in *
The procedure o f Scheele m s about th e only method
o f eoarsercial production o f g ly cero l f o r many years*
In 1823 Chevreol
published work which le d to th e a lk a lin e h y d ro ly sis o f f a ts and o ils
as a method o f g ly ce ro l production*
la te r i t was found by Tilghaan*
in 1853» th a t i f f a te were mixed w ith w ater and heated under p ressu re
w ithout steam form ation, sa p o n ific a tio n occurred although a lkal i s were
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not present*
Xa 1856 W ilson improved th e procedure by meetxus o f a
steam d i s tilla tio n method* O ther improvements in th e production o f
g ly ce ro l from fa te and o i l s fo ilse e d la te r*
G lyceryl tr in itr a te *
o r "nitro g ly cerin e* * was discovered in 1846 by Sobrero*
was published in 1847*
th e work
Nobel dem onstrated i t s explosive value in
1863, and by absorbing i t in K leselguhr he developed dynamite in
1868*
In 1875 he developed b la s tin g g e la tin *
G lycerol has been prepared by a v a rie ty o f sy n th e tic organic
aethods*
One o f th e e a r l ie r methods was th a t o f Wurtss (1857) who
tre a te d l* 2 f 3*toibro»opropane w ith s ilv e r a c e ta te and o b tained
tr ia c e tin which gave g ly c e ro l on a lk a lin e hydrolysis*
Store re c e n t­
ly , methods have been proposed fo r th e production o f g ly ce ro l fro®
n a tu ra l o r cracked petroleum products such a s propane and propylene.
Although many o f th e o ld e r sy n th e tic methods had m ainly h is to r ic a l
and academic value and l i t t l e in d u s tria l p o s s ib ilitie s , some o f th e
newer methods a re considered a s p o ssib le g ly cero l sources*
W illiam s
and a sso c ia te s (1938>(1940)(1941) have d escrib ed a method involving
a high p ressu re c h lo rin a tio n o f propylene to form a lly ! chloride*
The a l l y l ch lo rid e i s converted to a ll y l alco h o l by a sodium
hydroxide treatm ent*
Qjrpcchloroua acid i s added to th e unsatur& ted
group to fo ra th e aonochlorohydrin which i s then hydrolysed to
g lycerol*
The above method has been operated re c e n tly on a sm all
sc a le a t a p ro fit*
Methods o f th is type have shown g re a t promise*
Thus w ith both th e g ly c e ro l and f a tty a c id s a v a ila b le from petroleum
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products* f a ts and o i ls sim ila r to th e n a tu ra l onee have been p re pared from petroleum products*
fb e f a s t th a t g ly c e ro l I s a product o f fo m e n ta tio n was f i r s t
noted by P asteu r (1858)*
He was studying pure y e ast c u ltu re s In
connection w ith th e production o f wines and beers* and o s q u ite
eo ap lete analyses o f th e fom ented mashes be faced th a t about 3*5
grams o f g ly cero l were norm ally formed from every 100 grams o f sugar*
For q u ite a w hile th is f a c t, though pu b lish ed , made very l i t t l e im­
p ressio n on s c ie n tis ts in general*
I t was n o t u n til about 1911 th a t
Reuberg and h is co-w orkers began to p u b lish some o f th e r e s u lts o f
th e ir in v e stig a tio n s on th e problem o f th e a lc o h o lic fo m e n ta tio n
mechanism (1913)(1915)(1918)(1919)*
T heir conclusions a s to th e
mechanism o f th e d issim ila tio n a re shown in th e follow ing equations!
(1 )
C H 0 - 23 0 ——» C I O (m ethyl glyoxal a ld d )
6 12 e
2
e ee
(2)
» 2CHa»C(OH)CHO o r 20^000® (m ethyl g ly o x al)
o ,
. ... . .
—
E^-C-COQH (pyruvid a d d s a d )
CH CHD
3
♦ H — » CH CH OH (e th y l alcoh o l)
a
3 a
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~17-
Masy Hechaniams f o r sugar d ise is& la tio n s have boon advanced, b u t
a t p re se n t th e most g e n erally accepted aech&nisa i s th a t o f Babden,
Meyerhof, and P&mas*
A review mid d iscu ssio n by Werkaan (1939)
e x c e lle n tly covered t h is su b je c t sad c e rta in re la te d to p ics*
A
comprehensive b ib liog raph y was i ncluded w ith th is work*
Weuberg*# stu d ie s le d h is to the id e a o f fix in g th e aeetaldehyde
fconed in th e fo m e n ta tio n by ag ents such a s a s u lf ite o r an org an ic
agent* diaedoa*
l a t e r , Xobel and Tychowski (1928) fix ed t in aldehyde
by iseana o f carbaBdnlc hydraside o r thiooarbaiainic hydraside during
fo m en tatio n *
Heuberg need sodiua s u lf ite fo r th e n e s t o f h is expert*
a e n is, b u t he a lso employed o th er s u lf ite s such a s those o f ca lo lu a ,
nagneaiua, and sine*
Since th e b is u lf ite ra d ic a l i s concerned w ith
th e fix a tio n o f ac»taldehyde, Heuberg suggested th e follow ing
th e o re tic a l reaction*
C H 0 4 a s SO 4 g 0
* ia 6
2 3 a
CH CHO«lfeBSO
3
3
4
JfaHGO 4 Q 8 0
3 3 8 1
Heuberg (1920) considered th a t th e sugar d issim ila tio n by y e a st
tak es p lace in th re e form s, namely*
(1 )
O S 0 —— » 2C H OH 4 200 (re g u la r alo o h o lic fe rn )
6 12 4
2$
2
(2 )
C S 0 , ——* CH OS) 4 00 4 0 HO
« W i
32
3 8 3
(3)
2 6 .3 0 * 1 0 ——►200 4 CH 000H 4 0 8 0 1 * 2 6 1 .0
ittt
2
2
3
25
383
(a lk a lin e mechanism)
( s u lf ite mechanism)
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R eaction ( l ) above le g e n e ra lly known a s tb s GeyLuesac reactio n *
For® ( 3 ) was considered a s a co n tin u atio n o f fo ra (2) w herein th e
a lk a li caused th e aeetaldehyde to undergo th e Cannizzaro re a c tio n
to fe rn th e eth an o l and a c e tic acid»
D iscussion and d a ta which
su b sta n tia te d Heuberg*s claim s axe adequately expounded in Laurie* a
monograph (1928)*
According to Anderson (1938)* Meyerhof considered th e follow ­
in g re a c tio n s to occur l a th e d issim ila tio n o f a hexose by y easts
m
U)
♦
caoH
C l 0 / ♦ 2Ha HFO
d 12 d
2 4
(B)
<*0H
——^ m m
CH OFO l a
COOS
3
COOH
|
2CH0H
— ^ 2Na HFO
eg OFO Ha
2 3 2
♦ 2 CO
*
*
CH
3
*
mmm
CH OFO Ha
CH OFO Ha
2 3 2
2 3 2
(«-glyo«r© (phosphophosphate) glyeer& te)
«
(glyceraldahyde
phosphate)
2
COOH
2.00
♦ 2
»
C®
2 gg
3
(pyruvic
a cid )
(C) C l 0
4 it A
2Sa HFO
2
4
CSO
I
2 CH cao
2CS0R ——
CH OFO Ha
2
3
2
_ ftty
GH 0 1
2 ktf*
3
COOH
|
♦ 2mm
CH OFO Ha
2
3
2
Meyerhof considered p a rts (A) and (B) to be th e " I n i t i a l phase",
and p a rt
(c)
was considered a s th e "S ta tio n a ry Condition" which was
supposed to be th e s itu a tio n a f t e r th e ferm entation had s ta rte d
through th e " I n i ti a l p h a se d and enough aeetaldehyde had formed to
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-19-
a c t a s th e hydrogen accep to r i s place o f th e glycer&ldehyde
phosphate which ie th e hydrogen accep to r I s th e absence &£ aeetaldehyde*
For exten sive d isc u ssio n s o s fers@ ntc.tirs and re s p ira to ry
aaechanisma, and o s th e ro la s o f v ario u s ag en ts i s netabollsat* one
should r e f e r to th e books by Stephenson (1959)* Oppenhelaer and
S tern (1939)# Anderson (1938)* and P re sc o tt and Dunn (l?40}«
C onaatein and iadaeke (1919)* a f te r eonsidering th e e a r lie r
mark o f Heuberg* stu d ied th e g ly ce ro l ferm en tation w ith th e view o f
producing g ly c e ro l on a commercial basis*
They decided* on a
th e o re tic a l basis* th a t a lk a lin e fe ra e n ta tio n s should r e s u lt in
in creased g ly cero l production*
This was found to be a fact* and
th e ir ob servations were in stru m en tal In th e d e riv a tio n o f Msuberg’ s
(3919) (1920) th ir d fer& en tatio n aschar&sa*
Som o f th e i r observa­
tio n s a re noted in Table 1* By p ercen t o f g ly ce ro l produced i s meant
TABLE 1
GLZCEBDL H U B S BSIHG ALKALINE SALTS IS THE
FSESBHTIKG MFDIBM
carbonate
00
30
46
to
34
30
300
100
300
300
100
200
fflyoerol Produced
I
So s a l ts (co n tro l)
Sedlua a c e ta te
riiw aA nt* (seC*5
^ l t t « tthttatthftta iS ^ e )
- B arts by Weight
Saga?
S a lt
i
&
Allml-frvft
3*o
9 .9
i t .*>
25.6
12*7
13*4
(Adapted fro a Lawrie (1928))
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—20**
th e p ercen t o f th e w eight o f th e super found as glycerol* th a t 1%
a 10 g ra s y ie ld o f g ly c e ro l from 100 grans o f sugar i s considered
a s a 10 p ercen t y ield *
The th e o re tic a l y ie ld o f g ly ce ro l from e ith e r
th e a lk a lin e o r th e s u lf ite mechanism i s very clo se to $ t grams
from 100 p e n s o f heocose*
U nfortunately, th e pH v alues o f th ese media were n o t given*
The absence o f pH d a ta has been ty p ic a l of a Large, number o f p u b li­
c a tio n s .
Since c e rta in io n s, such a s th e phosphate io n , a re in ti*
n a te ly concerned w ith th e sugar breakdowns, i t i s ra th e r u n c e rta in
to what e x te n t th e e ff e c ts a re due to th e abnormal io n c o n cen tratio n s,
and to what e x ta n t th e e ffe c ts are pH functions*
I t was found th a t secondary in fe c tio n s caused c e rta in d i f f i ­
c u ltie s In th e a lk a lin e ferm entations* however, in s u lf ite ferm enta­
tio n s th e use o f high co n cen tratio n s o f sodium s u lf ite stopped most
TABLE 2
t m m rm t m isg k easih g amounts op sodium s u u t t b
I I IM S? FKBH2KXATX08S 08 GLXCEROL IIELDS
P a rts by W eight
Sodium S u lfite
Sugar
40
m
8o
100
120
ISO
200
100
100
100
100
100
100
100
Q lyoerol Produced
($ cm sugar)
23*1
24*8
27*3
3 0 .1
33*0
34*8
36*7
(Adapted from Iaw rie (1928))
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•214*
o f th e in fec tio n s*
liable 2 shows th e e ffe c t o f In creasin g amounts
o f sodium s u lf ite on th e y ie ld o f glyeerol*
For th e a lk a lin e ferm entation# Heuberg a ls o need o th e r rea g en ts
th an th o se mentioned above*
Sense o f h is reag en ts a re noted b a le rs
TABI£ 3
M km wm CAUSING NEOBERG’ S FIRST AiiU THUD MECHANISMS
TO OCCUR
F i r s t Mechanism
Atrwi^reypt RydrCQCide
F e rrie Hydroxide
Third Mechanism
Sodium Carbonate
Sodium B icarbonate
Potassium Carbonate
Potassium B icarbonate
iaignflBtww ftrtd f
Sodium Phosphate (norm al)
Sodium Phosphate (secondary)
Zinc Hydroxide
(Adapted from Laurie (1928))
In d iv id u al p rocesses and p a te n ts a re taken up in th e se c tio n s
which fo lio s*
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-22-
S* G lycerol Form ation by th e S u lfite Ferm entation
Stethoda
P relim inary work cm th e s u lf ite process of ferm entation was
mentioned in previous section s*
The a c tu a l processes* ©any o f which
were p a te n te d , a re nos presented in g re a te r d e ta il*
As was p rev io u sly m entioned, th e Connste in and Iiidecke (1519)
(1921) (1924) process was c a rrie d on in Germany during World War 1
in ahont tw enty-four f a c to r ie s , and about one thousand to n s o f g ly ce ro l
were produced each month in th is manner*
A fter th e war th e process
was p aten ted in many c o u n trie s and th e p a te n t referen ces a re noted
by Lawrle (1928)*
The follow ing fiv e claim s a re made in th e ir
B aited S ta te s p a te n t (1924) t
1*
The process f o r m anufacturing p ro p a n trlo l which c o n sists
in adding a lk a lin e s u lf ite s ( u n til a lk a lin e re a c tio n )
and y e a st to sugar and then allow ing th e m ixture to be
fermented*
2* She process f o r oaiaifacturing p ro p a n trlo l which c o n sists
in ending ciicfti j nA s u lf ite s ( u n til
re a c tio n )
and y e a st to su g ar, causing th e sugar to be ferm ented
in th e presenoe o f a lkal in e s u lf ite s , sep aratin g th e
y e a st sad aid in g th e separated y e a st and a lk a lin e r e ­
actin g substances to sug ar, whereupon th e process i s
repeated*
3*
The process fo r m anufacturing p ro p an trlo l which c o n sists
in adding a lk a lin e s u lf ite s ( u n til a lk a lin e re a c tio n ),
n e u tra l s a lts o f m agnesias in a h igher amount th an
necessary as y e a st n u trie n ts , and y e a st to su g ar, and
causing th e sugar to be fermented*
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-23**
4*
The process f o r raauufaeturing p ro p a n trlo l which con­
s i s t s In adding a lk a lin e s u lf ite s ( u n til a lk a lin e
re a c tio n ) and y e a st to sugar* causing a p o rtio n o f
th e sugar to be fermented* adding new p o rtio n s o f
sugar and causing the sugar to be fom ented*
5*
The process o f producing gly cero l* which c o n sists
in ferm enting & so lu tio n o f ferm entable sugar l a a a
a lk a lin e re a c tin g medics*
I t was claim ed th a t n e ith e r th e kind o f sugar nor th e y e a st
w ariety had any a p p reciab le e ffe c t on th e outcome o f th e ferm enta­
tio n *
This claim m e d isputed by o th er in v estig ato rs* such a s
Qeble (1922) and McDermott (1925)* e sp e c ia lly w ith regard to th e
s tr a in o r v a rie ty o f yeast*
fl*a*ih««MBW6ea
was employed
by m m in v e s tig a to rs in p referen ce to <3aac?y>rTT1TOf?ff ftereg islaa
stra in s* although th e £ •
ferm entation# were somewhat
slower*
Gormstein and Xudecke (1921)(1924) claim ed success using both
crude and refin e d sugars* end a lso aol&sses*
They employed th e
y e a st from one ferm entation to seed th e next fo r e ig h t ferm entatio ns
w ith apparent success*
sequently*
O bjections to th is method appeared sub­
Connstein and Iiideeke presented th e follow ing d a ta on
th e use o f th e y e a st o f one ferm entation a s th e inoculum f o r a
follow ing ferm entations
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*24*
nsm 4
EFFECT OS OZXCEROL HELD OF USING HAST FROM ONE FHUffimTHW
AS SEED FOE THE NEXT
Times l e a s t i s Used
G lycerol H e ld
on sugar)
18.8
21*4
22.9
22*8
22.3
20.9
19*9
21.2
1
2
3
4
5
6
I
8
(From law rie (l? 2 8 ))
D e ta ils o f th e process a re , f o r examples
To te a l i t e r s o f
were added one kilogram o f sugar and 100 grams o f fre s h yeast*
along w ith n u trie n t s a lts o f potassium , phosphorous, magnesium, and
nitrogen*
added*
F in a lly 400 g rass o f sodium s u lf ite (anhydrous) were a ls o
The medium was shaken q u ite w ell and was Incubated a t 30°
cen tig rad e f o r 48 to 60 hours*
I t was noted th a t th e re was not an
ap preciable in cre ase l a th e a lk a lin ity o f th e mash sin ce th e carbon
dioxide and aeetaldehyde re a c te d , supposedly, w ith th e sodium
s u lf ite to form some sodium bicarbonate and a lso some b is u lfite *
aldehyde a d d itio n product*
The v d a tl l e e were removed by d i s t i l l s *
t i e s , a f te r which calcium ch lo rid e and Haw were added to th e re*
m in in g medium to p re c ip ita te th e s u lfite *
The f i l t r a t e so obtained
m s tre a te d w ith sodium carbonate to p re c ip ita te th e excess o f so lu b le
calcium s a lts *
The re s u ltin g f i l t r a t e was concentrated by
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•*25*"
evap oratio n , and th e syrup -thus obtained was g ly ce ro l containing
sodtua c h lo rid e and eorae o th er so lu b le m aterials*
g ly c e ro l was procured by steam d is tilla tio n *
The p u rifie d
I t was re g u la rly
noted th a t a s th e amount o f s u lf ite was in creased , th e y ie ld o f
g ly o ero l and acetaldebyde in creased , and th a t o f th e a lco h o l de­
creased*
Heuberg*s f i r s t and second schemes both occur In th is
process*
Experim ental d a ta su b sta n tia te d Heuberg*s id ea s q u ite s e ll*
Ferm entations u sing o th er s a lts m e stu d ie d , but th e g ly c e ro l
y ie ld s e a rs lo s e r th an th o se obtained by th e s u lf ite ferm entations*
B esu lts a re shown in Sable 5*
TABLE 5
i ie i d m v m m o L m m m m T io n w ith v ario u s s a l ts
S a lt
Weight
o f sugar i t* )
G lycerol H e ld
& on sugar)
CaCl
40
8 .5
HH4C l
30
7*5
19
24
3 .0
M
48
3*0
34
5*5
11*8
8a SO
l ♦
8a SO
2 4
WfMrt
FeS04
FaSG4
FeSO^
60
90
120
1 2 .5
13*1
9 .4
Al (30 )
39
* 4 3
44
11.6
Al (SO )
a 43
Al (SO )
30
13*3
... *.
__________ - ........._ ............... .......... ........................._ .... ............
(F ree Iaw rie (1923) and Connatein and liidecke (1921)}
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- 26-
The above d a ta shoved th a t th e acid re a ctin g s a l ts (e*g*
fe rro u s s u lfa te and aluminum s u lfa te ) in in creasin g co n cen tratio n s
in creased th e y ie ld o f g ly c e ro l in c o n tra s t to th e id ea th a t in *
creased a lk a lin ity alone increased th e g ly ce ro l y ie ld s in th e absence
o f s u lfite s *
2* th e Cocking and U U y process
Cocking and L illy (1922) developed a process which was claim ed
to produce g ly cero l according to N euberg's second scheme in prac­
t ic a l l y th e o re tic a l q u a n titie s*
th e purpose o f th is cork was to
improve th e method o f Gonnetein and Ludecke in o rd er to in cre ase th e
g ly ce ro l yields*
I t was considered d e sira b le to in tro d u ce a c e rta in
co n cen tratio n o f b is u lf ite io n s in th e medium containing sodium
s u lf ite a t th e o nset o f th e ferm entation*
This was done in o rd er
to f ix th e acetaldehyde as i t formed a t th e s t a r t o f th e ferm enta­
tio n and not to w ait u n til a cid s formed by ferm entation reacted w ith
th e sodium s u lf ite to form some b is u lfite *
U rforfcunately, la rg e
amounts o f b is u lf ite io n s a re u n d esirab le during a su ccessfu l
g ly c e ro l ferm entation by c e rta in y e a sts because o f th e a n tis e p tic
action*
Cocking and L illy found th a t sodium s u lf ite and sodium
b is u lf ite could, be used in com binations in which th e re s u ltin g
so lu tio n m s n e arly n e u tra l to litm u s.
a n tis e p tic action*
There was then no se rio u s
Thus th e aldehyde was fix ed a t a much e a r lie r
stag e in th e ferm en tatio n than occurred in th e s u lf ite ferm en tatio n
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“27
o f Connateln and X&deake ( i f 21) (1924)*
I t was claim ed th a t th e tim e
o f ferm entation using th e m ixture o f acid and normal s u lf ite s was
about h a lf th e tim e o f ferm entatio n when th e normal s u l f i te alone
was used*
The t i e s o f th e ferm entation was u su a lly about s ix days*
and th e tem perature m s held a t about 35° to 37° centigrade*
The
g ly ce ro l y ie ld s u su a lly approximated 45 p ercen t o f the sugar weight*
I t appeared th a t th e h igh er y ie ld s o f g ly c e ro l were a sso c iated w ith
longer tim es o f ferm entation*
i t ....
The work o f Neuberg on sugar ferm entations by y e asts in th e
presence o f s u lf ite s was confirm ed by Gehle (1922)*
He noted th a t
w ith in creasin g s u lf ite co n cen tratio n s th e re was an in creasin g change
in th e products o f ferm entation* and th a t th e a lte r a tio n p ra c tic a lly
ceased when th e re were about 60 p a rts o f sodium s u lf ite to 100 p a rts
o f su g ar.
He a lso observed th a t d if f e r e n t ty p es o f y e a st d id not
ap p reciab ly a ffe c t th e q u a lita tiv e and q u a n tita tiv e d issim ilatio n *
although th e re was a ra th e r n o ticeab le d iffe re n c e In th e degree o f
re s ista n c e o f th e v ario u s y e a sts to th e s u lf ite .
Barb e t (1928) made use o f s u lfu r d io x id e fo r th e s u lf ite f e r ­
m entation.
To a m olasses mash was added some s u lfu r dio x id e previous
to in o c u la tio n to th e e x te n t o f about two grams p er l i t e r .
The
sash was then seeded and ferm entation proceeded w ith th e a d d itio n o f
s u lf lte d medium continuously o r in te rm itte n tly in q u a n titie s n o t
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-28*
g re a t enough to e x h ib it excessive to x ic e ffe c ts*
th e ferm entation
was allow ed to com plete i t s e l f , and a con sid erab le amount o f g ly ce ro l
m s found*
I t i s im portant to note th a t, in e f f e c t, an
add,
su lfu ro u s a c id , m s used a s an agent to in crease th e g ly c e ro l
y ie ld in a y e a st ferm entation a d n o t an a lk a lin e re a c tin g m aterial*
I t has been g en erally considered th a t in most any a cid medium a store
o r le s s ty p ic a l a lc o h o lic ferm entation must occur*
Thus B arbet
o btained in creased g ly c e ro l y ie ld s by th e use o f an a c id added to
a ferm enting mash* A ctual y ie ld s were n o t given by B arbet i s h is
patent*
th e re a re anmerous o th e r p a te n ts concerning th e use o f s a l ts ,
n e u tra l and o therw ise, along w ith s u lf ite s to produce glycerol*
According to Laurie (1928) ease p a te n ts claim ed th a t in
a d d itio n to
sodium s u lf ite , about 0*2 to 1 p ercent o f th e w eight o f
sugar should
be added in th e fo ra o f s a lts o f stro n g ly reducing s u lfu r acid s
such a s "hydrosulfates*1 and *su lfo x y lates* *
This a d d itio n was
claim ed to give g ly c e ro l y ie ld s g re a te r th an th o se obtained by th e
use o f sodium s u lf ite alone*
Ferrous and manganese s u lfa te s were
considered " c a ta ly s ts " to In crease g ly ce ro l form ation*
The addi­
tio n o f more sugar to th e mash a t i t s p o in t o f g re a te s t ferm entation
a c tiv ity w ith o r w ithout s a l t a d d itio n s was a lso described*
F u rther
d e ta ils a re found in th e monograph o f Lawrie (1928)*
In R ussia, Golovin (192?) d escrib ed a ferm entation using a
b is u lf ite .
The ferm en tatio n was complete in 48 hours, and on th e
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-29-
sugar w eight b a s is y ie ld s o f 20 p ercen t glycerol* 20 p ercen t
alcohol* and 8 p ercen t aoetaldehyde were G ained*
Uidecke and Iiidecke (1929) o b tain ed a p a te n t on a s u lf ite method
o f referm en tatio n follow ing a d i s t il l a t i o n o f th e f i r s t ferm entation
medium* The tem perature was h eld a t 30 ° to 35° c en tig ra d e fo r two
days*
Magnesium and n ie k e l s u lfa te s were added*
U g h t kilogram s
o f m olasses gave two kilogram s o f raw g ly ce ro l^ o r 960 grams o f pure
glycerol*
auger*
About 24 to 27 p ercen t o f g ly ce ro l was obtained from pure
fOaoda (1928) observed th a t in th e presence o f sodium s u lfite *
th e a lc o h o lic and g ly c e ro l ferm entations occurred p a r a lle l from
s t a r t to fin ish *
M athematical r e la tio n s were given*
Pankovaki (1929) stu d ied th e s u l f i te ferm entation o f sugar b e ets
and obtained 20 p ercen t glycerol* 17*5 p ercent ethanol* and ft p ercen t
aoetaldehyde*
I s th is year* Tomoda (1929) stu d ied fthe v e lo c ity o f
th e ferm entation in th e presence o f s u lfite *
Be a lso stu d ied th e
form ation o f 2 ,3 -b u ty len e g ly co l and a c e tic acid during a s u lf ite
g ly ce ro l ferm entation*
Im perial Chemical In d u stries* ltd * and Xdlly
(1930) improved on th e s u lf ite ferm entatio n o f carbohydrates and
molasses*
G iordani (1932) made a th e o re tic a l study o f th e s u lf ite f e r ­
m entation o f C onsteln and IAiaecke*
Be agreed th a t b d s u lflte s in ­
creased g ly ce ro l y ie ld s by combining w ith th e aeetaldehyde f i r s t form­
ed in such a way a s to prevent i t s fu rth e r conversion to acid and
alcohol*
A maximum y ie ld o f 25 p ercen t g ly ce ro l was found when ferm enta­
tio n was c a rrie d o u t in th e presence o f 20 p ercen t soditao b is u lfite *
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fakahaahi and As&i (1933) stu d ied both th e s u lf ite and a lk a lin e
d issim ila tio n s
of
sugar by means o f 23 v a rie tie s
o f |$ u sq e*
normally*
g ly ce ro l was produced to th e e x te n t o f 3*8 to 9«0 p ercen t f o r th e
a ssim ila te d sugar; production was approxim ately p a ra lle l to th a t o f
alcohol*
I t was found th a t th e a d d itio n o f sodium b is u lf ite o r
sodium carbonate g re a tly in creased th e g ly ce ro l yield*
The optimum
co n cen tratio n s o f th ese m alts were found to be* resp ectiv ely * 6
and 4 percent*
The corresponding y ie ld s o f g ly cero l were 21*5 and
23*5 p ercen t resp ectiv ely * fo r th e a ssim ila tio n o f glucose*
L illy (1935) described a method fo r a ferm entation w herein
so lu b le s u lf ite s and b is u lf ite s such a s th e sodium m alts were added
to a ferm entation medium in a manner sim ila r to th e o rig in a l Cocking
and L illy (1922) process* keeping th e medium about n eu tral*
However*
th e re were added to th e ferm enting mash a t in te rv a ls sm all quan ti»
t i e s o f regenerated s u lf ite - b is u lf ite so lu tio n con tain in g th e g ly cero l
o f a previous d issim ila tio n *
The Russian in v e stig a to rs* Kurbatova and Shakin (1936)* re*
ported th a t th ey used y e a st o f a s u lf ite ferm entation rep e ate d ly
w ithout lo s s o f ferm enting a c tiv ity * provided th a t a s u lf ite - f r e e
growth o f a c u ltu re i s in terp o sed between each s u lf ite ferm entation*
I t was recommended th a t th e y e a st c e lls be separated from th e s u lf ite
medium a s soon a s ferm entation i s complete*
Xwata (1936) rep o rted on a process fo r o b tain in g g ly c e ro l from
cane ju ic e by ferm entation*
He added em its and a q u a n tity o f sodium
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>31-
s u lf ite to th e ease Ju ic e and inoculated, w ith th e m olasses yeast*
ffaaahagaamraaf fo ra o n a n a fa nov.sp*
The h e a t y ie ld was 2 f *4f p ercen t
g ly c e ro l c alcu late d on th e ferm entable sugars*
With a m ixture o f
aodiua s u lf ite and sodium b is u lf ite he obtained a y ie ld o f 1?*?1
p ercen t o f th e sugar w eight a s gly cero l*
The pH was k ep t from 7
to 8*5 by means o f sodium carbonate o r sodium s u lfite *
For re ­
covery o f th e glycerol* Iw ata n e u tra lise d w ith s u lfu ric a d d * d ie*
t i l l e d v o la tile m aterials* evaporated to dryness* and ex tra cted
w ith fo u r voloses o f a b so lu te alco h o l to one volume o f carbon
te tra c h lo rid e *
The g ly c e ro l l e f t a f te r th e d is tilla tio n o f so lv e n ts
was 70 to 8© p ercen t pure*
liidecke (1938) obtained a p a te n t on a method whereby sugars o r
crude sugar so lu tio n s obtained by condensation o f formaldehyde
r*
p re fe ra b ly w ith th e a ssista n c e o f n a tu ra l o r a r t i f i c i a l lig h t were
ferm ented to form g ly cerol*
fi&o (1937) stu d ied th e w aste e&ne m olasses ferm en tatio n to forts
g lycerol*
fib ferm ented th e m olasses by means o f £• e e rc g isia a in
th e presence o f a lk a lin e carbonates* b icarb o n ates, and e sp e c ia lly
s u lf ite s .
He obtained a 10 to 25 p ercen t y ie ld o f g ly c e ro l calcu­
la te d on th e sugar*
The Jforddeutach© Hef© Industrie A*-Q* (1938) obtained a p a te n t
on th e g ly ce ro l ferm en tatio n in which about 3 percent sodium c h lo rid e
was considered im portant in a d d itio n to th e a lk a li s u lfite *
The ex­
ample described a medium containing su gar, sodium chloride* sodium
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b icarb o n ate, ammonium s u lf a te , tmgnesium s u lfa te and yeast*
th e
tem perature was m aintained a t 37° centigrade# end th e pH o f th e
medium m s 7*2 to 7*5*
Haehn (1938) (1940) developed a new method
f o r g ly c e ro l production*
His co n ten tio n was th a t a e ra tio n in th e
presence o f o x id atio n c a ta ly s ts , e* g*, iro n o r manganese sa lts #
m s advantageous fo r g ly c e ro l production*
Sodium b is u lf ite was
used# along w ith apgtfwttm phosphate and magnesium s u lfa te in th e
ferm entations*
M r was su pp lied through a porous cup# aad te e pH
was k ep t "w ithin U n its " *
A fter e ig h t hours# te e g ly c e ro l was r e ­
covered and found to be 25 to 30 p ercen t o f th e sugar weight*
to p y e a st was used which m s separated and reused*
A
In o th e r ex­
amples "aold y e asts" were employed such as th e genera
Ifo-coderma. Pjohia* and W illia..
An experim ent using a Myeodma^a
s tr a in y ield ed 30 to 34 percen t g ly c e ro l a f te r 23 hours a t 34°
centigrade*
Hesse (1935) in d ic ate d te a t tafiaciiwaa y ie ld s o f g ly c e ro l and
aeetaldehyd® were obtained when ferm entation occurred in th e presence
o f te e maximum amount o f sodium s u lf ite th a t te e y e a st could t o le r ­
ate*
When 33 grams o f sodium s u l f i te were used p er 100 g ram o f
sugar# th e re were o b tain ed 11*90 p ercen t o f aoetaldehyde and 22*37
p ercen t o f glycerol*
When 100 grams o f s u lf ite were used# th e y ie ld s
were 13*96 p ercen t aldehyde ana 36*99 p ercen t glycerol*
A sh o rt review on th e su b je c t o f b lac k strap m olasses a s a raw
m a teria l fo r th e production o f g ly ce ro l by ferm entation m s made by
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Gwen (1937) (1939)*
S ta tis tic a l and economic referen ces were made*
He considered th e p r e s e n t o f b io -c o llo id s end th e 111% to he o f
q u ite unsuspected im portance In a d issim ila tio n such as th e g ly cero l
ferm entation*
Gwen s ta te d (1937)(1939)*
I t has been found th a t th e a d d itio n o f minute amounts
o f th e fin e ly d iv id ed and h ig h ly d isp ersed absorbent
m a te ria ls l i i e c la y , bone-black, a c tiv a te d carbon, e tc * ,
a c t only a c c e le ra te th e r a te o f ferm entation o f sugars by
y e a s t, b u t a ls o provide co n dition s f o r th e e f f ic ie n t con­
v e rsio n o f su g ars in to alco h o l in so lu tio n s whose d e n s itie s
o r sugar co n cen tratio n s would ren d er i t im possible in th e
absence o f th e se b io -co llo id s*
While many exp lan atio ns have been advanced fo r th is
benign a c tio n o f th e se sub stances, a l l o f th e in v e s tig a to rs
have concurred in th e opinion th a t much o f i t i s to be
a ttrib u te d to th e a c tiv a tio n o f th e hydrogen lib e ra te d in
th e re a c tio n , and i t s consequently more e ffe c tiv e a c tio n
in reducing th e aoetaldehyde to alcohol* In so f a r a s
t h is i s tru e , i t re p re se n ts an e x ac tly opposite fu n ctio n
o f th ese substances to th a t o f a lk a li o r s u lf ite s in
ia n ^ b ills in g th e aoetaldehyde, mid thereb y preventing
i t s red u ctio n to a lc o h o l, and re s u ltin g in th e form ation
o f g ly c e rin from th e sugar molecule*
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0*
G lycerol by th e A lkaline Ferm entation Methods
th e production o f g ly cero l by means o f y e a st ferm en tation s o f
sugars In an a lk a lin e medium has n o t been considered a s a commercial
p o s s ib ility a s long a s has th e s u lf ite method*
The f a s t th a t th e
products a sso c ia te d w ith th e g ly c e ro l made by th e a lk a lin e methods
a re eth an o l and a c e tic a c id , as compered to aoetaldehyde by th e
s u lf ite method, m ight hare p e a t in flu en ce on th e value o f th e f e r ­
m entation*
O rd in a rily , ethan o l and a c e tic a d d a re more d e sira b le
than i s aoetaldehyde.
An a lk a lin e process fo r th e production o f
ferm entation g ly c e ro l was developed in th e United S ta te s about th e
tim e o f th e WorM War X.
1*
The &>f£ ProcessBearing th e World War X th e p ric e o f g ly ce ro l ro se q u ite rap id ­
ly to about s ix ty cen ts p er pound*
The United S ta te s Treasury
Department was n o tifie d by Dr* Alonso E. Taylor th a t g ly c e ro l was
being produced in Germany by a ferm entation method* Soon th re e
government la b o ra to rie s s ta rte d resea rch on th is problem .
S o ff,
L inder, and Beyer (1919) discovered th a t th e a d d itio n o f c e rta in
a lk a lin e reag en ts to a ferm entation medium caused a ra th e r la rg e
in crease in th e amount o f g ly cero l formed from a given amount o f
sugar*
Beagents used were sodium and potassium carb o n ates, b i-
carbonates, hydroxides, and some o th e r a lk a lin e compounds.
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Hoff
-35-
(1918) obtained a p a te n t on g ly c e ro l production by a y e a st ferm enta­
tio n in an a lk a lin e medium*
He claim ed th a t about 20 to 23 p ercent
o f th e ferm entable sugar was converted in to g ly cero l*
a s o th e r products a o e tio acid* ethanol? and acetone*
■that he used were pure c u ltu re s o f yeasts*
both
and £*,
He lis te d
The organisms
He stu d ied s tra in s o f
He claim ed b e st r e s u lts
by th e u se o f 3& «rtunpn<a*m«r v a rie ty "Steinberg"? known as a
C a lifo rn ia wine yeast*
37° centigrade*
Optimum tem perature was s ta te d to be about
la rg e sc a le ferm entatio ns were run ty b u ild in g up
in ocu la from sm aller ones, causing each successive c u ltu re to be
grown in a medium made s lig h tly a lk a lin e by th e reag en t to be used
f o r a lk a liz in g th e la rg e ferm entation*
This procedure amounted to
an a c c lim a tisa tio n o f th e organism to th e a lk a lin e ferm en tatio n
conditions*
Use a lk a li was added from tim e to time*
I t was re *
commended th a t th e degree o f a lk a lin ity be m aintained a t a p o in t
ju s t sh o rt o f th e p o in t o f in h ib itio n o f th e c u ltu re growth*
It
was mentioned th a t sodium? potassium? and c& leiua carbonates? phos­
phates? and hydroxides may be used} sodium carbonate was p refe rred
because o f i t s cheapness and a v a ila b ility *
A ty p ic a l medium was
made from m olasses and contained about 11 p ercen t to ta l sugar*
About 20 p ercen t o f th e sugar was converted to glycerol*
S off (l$19) s ta te d th a t the g ly ce ro l y ie ld was p ro p o rtio n al
to th e amount o f a lk a li used up to th e endurance lim it o f the
organism*
He did n o t mention pH a s being th e c o n tro llin g facto r*
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•3^»
He showed th a t th e o p tim a tem perature was 3©® to 32° cen tig rad e
in c o n tra st to 37° a s he had given p rev io u sly in h is patent*
The
b e st sugar co n cen tratio n was s ta te d to be 17*5 to 20 grams p e r 100
ce* o f medium* In h is m olasses ferm entations the sugar was calcu­
la te d by th e Fehling re d u c in g sugar method*
I t m s shown th a t th e
a c tu a l ferm entable sugar was only 72 p ercen t o f to e F ehling value*
The co rrected g ly ce ro l y ie ld s were th en 19*6 to 27*1 p ercen t on th e
b a sis o f ferm entable sugar*
For each 100 p a rts o f Cuban m olasses
used (47*8 percent a s in v e rt sugar) th e re would be produced*
.................
G ly c e r o l....
E th a n o l.... . . . . . . . . . *.........
A cetic a c id , e tc * .....................
A s h * ..... . . . . . . . . . . . . . . . . . . . . . . .
Sodium c arb o n a te .
..........
Organic n o n -su g ars. .................
10*04 p a rts
17*21 (removed by
3*3*
d is tilla tio n )
8.00
15*94
22.20
76*73 p a rts
I t m y be no ticed th a t in th e re s u ltin g medium a f te r alco h o l
removal* fo r every te n p a rts o f g ly ce ro l th e re were 49*5 p a rts o f
non*glyeerol s o lid s .
O riginally * only about $0 percent o f th e g ly ce ro l
p resen t was a c tu a lly recovered by d ir e c t vacuum d i s t il l a t i o n .
However*
treatm en t w ith fe rro u s s u lfa te follow ed by the use o f hydrated lim e
powder caused a r a th e r la rg e p a rt o f th e organic non-cugars to be
p re c ip ita te d .
E vaporation to 30 p ercen t g ly c e ro l, follow ed by steam
d i s t il l a t i o n under vacuum, brought th e g ly c e ro l recovery up to about
85 to 90 percent*
Such & c la r if ic a tio n method i s ra th e r c o s tly ,
as a re some o th er methods*
The procedure h a s, however, emergency
v a lu e .
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C oanstein and liideck© (1924) d escrib ed c e rta in e th e r ty p es o f
a lk a lin e fe ra e n te tio n s .
Most o f th ese d a ta m y be found in th e
monograph o f Iaw rle (192S)*
Inform ation on pH was not included*
unfortim ately*
McDermott , in & note fo r Lawrle’ s monograph (1928)* explained
h is v is e s on th e m olasses ferm entation in th e presence o f a lk a li to
for® glycerol*
He s ta te d th a t o rd in a rily fro® pH 3*5 to 6*8 th e
(kty-Dussae reaction* o r Heuberg*s f i r s t equation! predoainated*
When th e pH ranged fro® 7 to 8*8* a g re a t in crease in g ly c e ro l
form ation was noted in accordance w ith Neuberg*s th ird equation*
Be claim ed th a t th e change o f th e hydrogen-ion co n cen tratio n on th e
a d d itio n o f th e a lk a li caused a change in th e re a c tio n o f th e y e a st
enzymes on th e carbohydrate being ferm ented.
Be noted th a t th e re
m s an in d ic a tio n in th e lite r a tu r e th a t high s a l t o r sugar concen­
tr a tio n s alone enhance th e production o f glycerol*
McDermott th en
mentioned th a t th e main advantage o f th e us© o f a m olasses medium
over o th e r media f o r a lk a lin e ferm entations i s th a t th e m olasses
a c ts a s a b u ffer fo r th e added a lk a li.
The im p licatio n was th a t
th e b e tte r th e b u ffe r system* th e b e tte r th e g ly ce ro l yield *
Maintenance o f co n stan t pH was stressed *
Experim ental d a ta were
given to show th a t m olasses mash b u ffered th e pH o f th e medium when
sodium carbonate was added In te rm itte n tly much b e tte r than did
•sy n th e tic * mash* The pH o f both media averaged about e ig h t,
though th e pH o f th e m olasses medium was much more co n stan t in i t s
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*
v a lu e .
33*
The g ly ce ro l y ie ld s fo r th e m olasses and Hsy n th etic* media
were 18*54 p ercen t and 15*24 p ercent resp ectiv ely *
The q u estio n n ig h t a ris e a s to whether th e in cre ase in th e
g ly ce ro l y ie ld was caused by th e constancy o f th e pH o r by th e
higher co n cen tratio n o f s o lid s in th e m olasses mash* The e f f e c t o f
b io * co llo id s a s mentioned by Owen (1937) m ight come in to e f f e c t in
th e m olasses mash*
Heuberg and Kobel (1930) made stu d ie s on th e decom position o f
nosFphosphOTylated sugar by y e ast w ith th e form ation o f g ly c e ro l and
pyruvic a c id .
Kobel {1931} made s tu d ie s on th e form ation o f e q u i-
m olecular amounts o f g ly c e ro l and o f pyruvic acid in e e ll* fre e
ferm entations o f glu co se.
The e ffe c ts o f phosphates and o th er io n s
were studied*
C arothera, H tll, and Van N atta {1934} o f S. I . du Pont de
Honours and Company, obtained a United S ta te s p a te n t on ferm en tatio n
g ly c e ro l wherein th e sugar was ferm ented in an a lk a lin e medium; th e
g ly cero l formed was d i s t il l e d from th e ferm ented product, and th is
g ly ce ro l was made a lk a lin e w ith lin e*
The re s u ltin g m ixture was
blown w ith a ir in o rd er to d estro y phenols which were present*
Hesse {1935) noted th a t th e maximum r a te o f ferm entation
to aldehyde o f an 8*55 p ercen t sugar so lu tio n in th e presence o f
4*27 p ercen t sodium b icarb o n ate, was reached in 2*5 h ou rs, which
was 40 to ?0 tim es g re a te r than th e u su a l ferm entation rate *
aoetaldehyde produced was changed to alco h o l and a c e tic a c id .
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The
-39-
Equal part® o f g ly c e ro l and a c e tic acid were formed*
The Norddeutsehe H efein d u strie, A*«*G* (1938) described a method
wherein th e acid s formed during & ferm entation were n e u tra liz e d by
magnesium carbonate.
A p a te n t o f p o ssib le im portance was granted to Krug and McDermott
(1935)*
I t was concerned w ith th e use o f ammonium hydroxide to con­
t r o l th e a lk a lin ity o f ferm entation mashes in o rd er to produce
glycerol*
An im portant f a c t i s th a t th e ammonium hydroxide i s n o t
a "fixed* reag en t, in th a t i t i s d is tilla b le its e lf * and th e ammonium
carbonate o r bicarbonate th a t no doubt form a re a lso n o t "fixed*
and may be removed on d i s t il l a t i o n , as th e ammonium carbonate I s
q u ite therm ol& bile, even in so lu tion *
The purposes o f th is p a te n t o f Krug and McDenaott a re very
c lo se ly aligned w ith some o f th e purposes o f th is th e sis*
They
s ta te d w ith regard to th e Eoff (1918) method th a t "w hile such soda
ash process produces a v ery good y ie ld o f g ly c e ro l, — * th e a d d itio n
o f la rg e amounts o f soda ash and th e lik e introd u ces d i f f ic u lt
problems in th e recovery and p u rific a tio n o f glycerol* p a rtic u la rly
when b lac k stra p m olasses c o n s titu te s th e source o f th e sugar**
An extended q u o tatio n from th e p a te n t o f Krug and McDermott
(1935), who a re a ssig n o rs to E* I* du Pont de Nemours and Company,
should len d co n sid erab le w eight to th e im portance o f th e work con­
cerning g ly c e ro l production by means o f ferm entation*
The qu o tatio n follow s*
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
•*40«
A b la c k stra p m olasses m y have approxim ately th e follow ing
com positions
Percent
Ferm entable sugars*.............................. • •
P ro te in s (8 x 6 * 2 5 ) . . .. . .. ...........
Qms and organic m atters o th e r than
su g a r, p ro te in , o r a c id s * * * .....* .*
O rganic a c id s (as la c tic } * . . . . . . . . . . .
A sh * *............ ..................... .........................
W a te r * .....................# .........
a co leu ses o f th is typo* such fo r in stan ce a s ona
containing 45 p ercen t o f ferm entable su g ar, 5 p ercen t o f u nfarsw m table sugar and 6 p ercen t o f a sh , i s d ilu te d w ith
about th re e tim es i t s volume o f w ater and ferm ented i s th e
presence o f aodii® carbonate, in accordance w ith th e d is c lo su re o f E o ff, in an amount o f about 4*5 percent by
w eight o f th e sa s h o r 28 p ercen t o f th e w eight o f to ta l sugar
in th e mash, each g a llo n o f m olasses would y ie ld about 1*25
pounds o f gly cerol* th is g ly c e ro l would be admixed w ith
about 0*72 pounds o f a sh , o rig in a lly p resen t in th e m olasses,
and about 1*67 pounds of th e soda ash added in th e process
which would give ■tee f in a l r a tio o f g ly ce ro l to ash o f about
1*1*91* Thame ash c o n stitu e n ts in te r f e r e w ith th e recovery
o f th e g ly c e ro l by ra is in g th e b o ilin g p o in t o f th e solution*
th e se s a l ts fu rth e r tend to a c c e le ra te polym erisation and
decom position o f th e g ly c e ro l during th e d i s t il l a t i o n th e re ­
o f, thereby decreasing th e yield * This i s illu s tr a te d in
th® a r t i c l e by A* C. Iangrauir in lad* and log* Chew* o f A p ril,
1932, pages 378-380 in clu siv e* These s a lts fu rth e r tend to
cause decom position o f th e non-glycerol organic m atter p resen t
in th e wash producing d if f ic u ltly removable im p u ritie s in th e
g ly c e ro l, which r e s u lt i s favored by stro n g ly a lk a lin e re a c tio n
o f th e m a te ria l from which th e g ly ce ro l most be recovered*
An o b je c t o f th is in v en tio n io to provide a new process
fo r th e production o f gly cero l* A fu rth e r o b je c t i s to pro­
vide such a' process which w ill n o t have th e obvious abovementioned disadvantages o f th e p rio r processes* A s t i l l
fu rth e r o b je c t i s to provide a a cre economic process fo r th e
production o f glycerol* Another o b je c t i s to provide a
process o f producing g ly c e ro l in such a manner th a t th e g ly ce ro l
may be more re a d ily and com pletely recovered and p u rified *
O ther o b je c ts w ill appear from a co n sid eratio n o f th e follow ­
ing d e sc rip tio n o f our invention*
In th e ir p a te n t v ario u s sugar so lu tio n s, and e sp e c ia lly those
made from m olasses, were ferm ented w ith th e a lk a lin ity c o n tro lle d
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-41-
by th e a d d itio n o f assaoniua hydroxide*
secured*
Good g ly cero l y ie ld s were
I t was mentioned th a t tip to 1932 i t was thought by those
s k ille d in th e a r t th a t “fre e ammonia* could not be used in p lace
o f soda ash w ith "com m ercially s a tis fa c to ry r e s u lts ” •
A wain mash m s prepared and in ocu lated w ith a s tr a in o f yeast*
In about fiv e to e ig h t hours th e ferm en tatio n became a c tiv e , a t
which tim e mtmofam hydroxide was added in such a q u a n tity a s to
make th e ferm enting medium s lig h tly alk alin e*
Ferm entation was th en
allow ed to proceed u n t il th e m sh had become n e u tra l o r s lig h tly
acid whereupon more ammonium hydroxide was again added to s lig h t
a lk a lin ity *
This procedure was repeated a s o ften m was necessary
to keep th e medium a t th e d e sired a lk a lin ity *
Various y e a sts were
used, b u t y e a st “Ho* 16” o f McDermott was preferred*
E ith e r ammonia
gas o r ammonium hydroxide so lu tio n s could be employed*
In th e
examples c ite d , commercial «sffi»nium hydroxide so lu tio n co ntaining
26 to 28 p ercen t ammonia was used*
The incu b atio n tem perature m s
about 30° centigrade*
About 0*75 gram of ammonium s u lfa te was added p er l i t e r o f
m olasses medium con tain in g about 16 to 17 grams o f in v e rt sugar
(c a lc u la te d ) per 3i50 ec» o f medium* About 0*3 grams o f secondary
ammonium phosphate was added p er l i t e r o f medium in some eases*
The tim e o f ferm entation m s about 72 to 84 hours*
The pH v alues
d escribed u su a lly o s c illa te d between about 6*8 to about 7*3*
It
was mentioned in an example th a t a pH o f 7*2 to 7*4 was produced
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-42*"
follow ing an ammonium hydroxide a d d itio n a f te r which "ferjiien tation
then ceases fear a sh o rt tim e .” When th e ferm entation “rev iv e®
in about two to th re e hours th e pH m s found to have dropped to
about 6*8 to 7*0*
The pH was measured c o lo rim e trie ally *
The
sugar ferm ented m s mentioned to have been 90*09 p ercen t in one
case and 92*16 p ercent in another*
I t m e sta te d th a t " a f te r f e r -
m entation th e sash w ill co n tain from about 6*0»6*5 p ercen t by
volume o f alcohol and about 2*70*3*15 grams o f g ly cero l p er 100 ee*
This In d ic a te s a y ie ld on th e to ta l sugar (3950 pounds) in th e m olasses
o f 15*8~18*5 p ercen t g ly c e ro l and 27 *9*30*5 p ercent alcohol*"
The alco h o l m s removed in th e u su al manner and from 20 to 40
p ercen t o f th e ammonia was recovered by a su ita b le scrubber in th e
alco h o l vapor lin e *
Vacuum evaporation concentrated th e d ealcoh o lised
medium to a heavy syrup*
The g ly ce ro l was recovered fro® th is syrup
by th e u se o f a spray tow er such as th a t in d ic ate d by fcawric £1928)*
However* such a tow er i s n o t e n tire ly necessary sin ce th e low s a l t
co n cen tratio n allow s th e use o f th e more u su a l types o f d is tillin g
equipment*
A cetic a cid was formed during th e ferm entation and was recover­
ed by th e u su al methods*
In th e examples o f th is p a te n t, i t was shown th a t th e r a tio o f
g ly c e ro l to ash m s about 1*0 to 0*7 a s compared to th e correspond­
ing r a tio o f 1*1*91 f o r th e lo f f method* This was "a red u ctio n o f
th e ash w ith re sp e c t to th e g ly cero l to about 40 p ercent o f th a t
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
•43*
which would he obtained w ith th e soda ash process** The handling
o f ammonia was sim pler th an th e handling o f stro n g c au stics* sad th e
amount o f ammonia req u ired c o n stitu te d a n o tic ea b le econotqy* Be*
eovery was much sim pler and more fea sib le*
I t i s w ell to note th a t in th is p a te n t Krug and McDermott
claim ed m aintenance o f pH frost 7 to 8 although th e d e sc rip tio n s
h ard ly mention any pH v alu e more a lk a lin e than about 7*3 o r 7*4*
Such a v alue was not alw ays m aintained h u t was reached in te rm itte n t*
ly upon th e a d d itio n o f th e ammonium hydroxide* a f te r which th e pH
sometimes f e l l to 7 o r below*
I t appeared th a t perhaps tits© in *
te rv a ls o f ferm entation in th e acid range may have been necessary
f o r a su ccessfu l ferm en tation when ammonium hydroxide was used to
c o n tro l th e pH* Vokorny (1913) noted a to x ic a ctio n o f ammonia on
yeast*
fh e use o f ammonium hydroxide req u ired th e form ation o f ammonium
carbonate o r ammonium b icarbonate o r both during th e ferm entation*
TABLE 6
AMMONIUM HXDRQXIBE ADDITIONS WITH RESPECT TO TIME IS ORDER TO
KEEP pH ABOUT SEVEN IN A JAVAN MOLASSES FERMENTATION OF
THREE LITERS
Dose
number
1
2
3
4
Volume
cc«
Hours a f te r
seeding
20
15
10
5
5 *1©
10*55
14*05
16*40
(From Krug am McDermott (1935))
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
Both s a l ts were deriv ed fro a a weak base and a very weak acid*
th e
presence o f such s a l ts had th e advantage o f b u ffer e f f e c t in th e
medium*
The volumes o f ammonium hydroxide so lu tio n (co n taining 28
p ercen t ammonia) added to th re e l i t e r s o f a Javan cans m olasses
medium containing 10 grams o f in v e rt sugar (ca lcu late d ) p e r 100 oc«
o f medium a re noted in ta b le 6 .
A fu rth e r d isc u ssio n o f th e ammonium hydroxide ferm en tatio n
method w ill be found in a l a t e r m o tio n o f th is th e sis*
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-45-
Tim MPffiBiEHfAl,
A* A pparatus
%
Cameron pH M ater,
She pH m eter used m s a Cameron Meter a f th e
double sc a le ty p e which w ill give d ir e c t read in g s in e ith e r m illi­
v o lts o r pH* I t was made by th e E isendrath Memorial la b o ra to rie s
o f R acine, Wisconsin* and was obtained by th is lab o ra to ry fro® th e
Wilkens-An&ereon Company o f Chicago, Illin o is *
I t i s a g la ss
e le ctro d e m eter which o p erates e n tire ly from b a tte r ie s and i s m a*
p o rtab le inasmuch as a sto rag e b a tte ry i s necessary*
I t s o p eratio n
m s most s a tis fa c to ry when op eratin g d ire c tio n s were follow ed
c a re fu lly .
I t was ad v isab le to keep a l l so lu tio n s used i n connection
w ith th e pH measurements a t th e same tem perature a s n early a s
p o ssib le to e lim in ate any e rro rs a ris in g from tem perature d if f e r ­
ences o f th e opposing h a lf-c e lls *
Inasmuch a s th e instrum ent i s
w idely used in in d u stry and o th e r s c ie n tif ic f ie ld s , i t w ill not
to described fu rth e r as d esired inform ation may to obtained from
th e m anufacturers o r from the d istrib u to rs *
V*
PflJfe&SS*
a e te r i s sim ila r to th e Cameron
Meter in some re s p e c ts , b u t I t i s d iffe re n t in th a t i t i s portable*
I t has th e sample cup contained in th e m eter beat, w hile th e
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
Cameron m eter has & se p ara te d e te c to r u n it allow ing e a s ie r access to
th e sample cup and sim pler m anipulation*
However, e ith e r instrum ent
I s q u ite s a tis fa c to ry f o r pH measurements where a g la ss e le ctro d e
measurement i s desired*
th is instrum ent i s made by th e Coleman
H e e tr ic Company o f Maywood, Illin o is *
I t i s a b a llls tie a lly oper­
a te d Instrum ent, w hile th e Cameron Meter i s n o t, th e balance p o in t
o f the l a t t e r being a n u ll p o in t on a galvanometer*
A pH measurement
may be made w ith e ith e r instrum ent in a m atter o f seconds*
2*
p H C ontrol
and Recording Instrum ent
Inasmuch a s i t i s d i f f ic u lt to c o n tro l pH by means o f b u ffe rs
a t a l l d e sired le v e ls , a means o f very a ccu rate c o n tro l was d e sire d .
A permanent record o f th e pH a g a in st tim e was a lso very d e sirab le*
B uffers may beep th e pH o f a medium a t approxim ately th e pH sought,
perhaps w ith in 0*5 o r 1 pH u n it*
Sometimes ra th e r high s a l t concen­
tr a tio n s a re necessary fo r b u ffe rs to be s a tis fa c to ry , b u t th is s a l t
may a l t e r th e process being stu d ied to such an e x te n t a s to be un­
sa tisfa c to ry *
I t was d e sire d in some cases to operate ferm entations
w ith c e rta in reag en ts th a t could be e a s ily removed a t th e com pletion
o f th e ferm entation*
Some reag en ts a re n o t s a tis fa c to ry fo r b u ffe rs
i f th e ease o f s a lt removal is to be m aintained*
Such pH c o n tro l
agents a re calcium hydroxide and ammonium hydroxide*
C ontrol o f
pH by s u lfu r dio x id e In th e presence o f ammonium s u l f i te , calcium
s u l f i te , o r magnesium s u lf ite were a lso considered o r used*
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited without perm ission.
-47-
C onsiderable correspondence was made w ith v ario u s i» !» ifaetu rere
and d e a le rs o f pH c o n tro l instrum ents o p eratin g from a g la s s electrode*
I t was found th a t such Instrum ents a re q u ite new* com plicated, and
expensive*
The p ric e o f such an In stru a s n t was found to be in th e
neighborhood o f $1000# With th is in mind some experim ents were made
w ith c e rta in vacuum tube c ir c u its to see i f a c o n tro l instrum ent might
be b u ilt which would be sim pler th an th e commercial in stru m en ts, a s
w ell a s »uch cheaper*
S h o rtly a f te r th is work had been begun* a
commercial pH r e c o r d s was obtained which was adapted to pH control*
Because o f th is a c q u is itio n , fu rth e r work on th e development o f such
an instrum ent was discontinued*
The equipment secured was a Cameron pH Heeorder, obtained from
th e W ilkens-Andersoa Company in th e sp rin g o f 1939*
and te s te d as a reco rd er o f pH a t f ir s t*
I t was s e t up
Beoorda were made o f pH
a g a in st tim e f o r y e a st ferm entations a id f o r th e b u ty laeeto n le f e r ­
m entation*
The curves obtained wore ex cellen t*
The machine record­
ed th e pH continuously fo r 24 hours w ith l i t t l e a tte n tio n except to
o ccasio n ally check p o te n tio a e tric balance*
The record from 10 P*H^
to 8 A.M. was e sp e c ia lly convenient*
A fter i t was found th a t th e instrum ent operated, s a tis f a c to r ily ,
th e c ir c u it was adapted to co n tro l operation*
A m agnetically con­
tr o lle d Marooid sw itch m s in s ta lle d on th e instrum ent in such a way
th a t & magnet nsoved in conjunction w ith th e recording pen connected
to th e reco rd in g potentiom eter screw*
For th e d e ta ils o f th e opera­
tio n o f th e re c o rd e r, one should see th e o p eratin g d ire c tio n s th a t
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
*48-
came w ith th e instrum ent? th e c ir c u its a re a lso Included*
The de*»
t a i l s o f th e added c ir c u its o f th e c o n tro l mechanisms a re shown be*
low? th ey were developed to a la rg e e x te n t in those la b o ra to rie s*
No
attem pt i s node to re to d escrib e th e commercial Instrum ent a s i t I s
exceedingly complex and i s described by th e m anufacturers*
tony o f
th e refe ren c es to p a rts and co n tact numbers and trip -w h eel p o sitio n s
r e f e r to th e code used in th e m anufacturers diagrams*
The follow ing diagram s o f c ir c u its and p a rts a re concerned only
w ith th e c o n tro l mechanism*
The f i r s t c ir c u it involves th e sim ple
m agnetically operated Mereoid sw itch which came w ith th e instrum ent*
I t i s norm ally clo sed , b u t opens under m agnetic influence*
The magnet
i s u su a lly behind th e sw itch keeping i t open, thus keeping th e solenoid
valve in o p era tiv e w hile th e pH i s a t th e d e sired lev el*
The c o n tro l
c ir c u it i s shown in F igure 1*
I t was found th a t th is type o f c ir c u it was not e n tir e ly s a t is fa c to ry sin ce c o n tro l was m aintained only w hile the pH o f th e medium
tended to decrease* i*e*» w hile acid s formed ae tim e proceeded*
How­
e v e r, toward th e end o f some ferm entations* th e medium became le s s
a c id , th a t i s , th e pH increased*
The c ir c u it was designed to add
base a s th e pH decreased w ith th e magnet proceeding to th e r ig h t
p a st Sa o f Figure 1*
When th e magnet goes to th e rig h t o f S2 a sh o rt
d ista n c e , S2 closes* th u s o p eratin g th e valve which allow s th e base
to How u n til th e pH increases*
The magnet th en moves to th e le ft*
th u s opening S2 again and sh u ttin g o f f th e solenoid valve*
But when
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-
49-
th e pH in creases from th e c o n tro lle d pH value* then th e sw itch S2
opens ag ain causing th e base to add a s before* and the magnet goes
f a rth e r and fa rth e r to th e le f t*
The base adds u n til th e base r e ­
se rv o ir i s empty* C ontrol o f pH i s lo st* and th e experim ent i s
spoiled*
A sa fe ty c ir c u it was designed to c o rre c t fo r t h is d if f ic u lty so
th a t c o n tro l o f pH would be m aintained a s u su al when th e pH o f th e
medium ten d s to decrease* b u t when i t ten d s to increase* th e base
cannot be added? m erely a pH record re s u lts *
This m odified c ir c u it
i s shami in F igure 2*
The magnet o p erates norm ally behind Sa which cans© w ith th e in ­
strum ent! in th is p o sitio n th e sw itch i s "o ff* as noted p rev io u sly
in F igure 1* As th e magnet proceeds to th e r ig h t w ith d ecreasing pH*
th e sw itch Sa clo ses th u s op eratin g re la y C which th en opens th e
solenoid valve*
again*
Base i s added* and pH in cre ases u n tH S2 opens
But should th e occasion a ris e th a t th e medium becomes so re
b asic th an th e value f o r which th e c o n tro l i s s e t, th e magnet w ill
proceed to th e l e f t o f S2, th u s causing th e a d d itio n o f some excess
base f o r an in sta n t*
quickly c lo ses
However, a s Sf i s a d ja ce n t to Sg* th e magnet
which o p erates re la y s A and B* Having been oper­
ated* th e se re la y s do no t allow any more base to add sin c e re la y G
i s no® open*
Belays A* B* and C w ill now remain in th is p o sitio n
re g a rd le ss o f what happens u n til s u ffic ie n t a c id ity forms in th e
medium to b ring th e magnet back to th e r ig h t to %* o r u n t il th e
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
o p erato r can observe th e situ a tio n *
I f a c id fo rm causing th e magnet
to move behind S2 th us opening th a t c o n ta c t, then a l l re la y s f a l l
back to normal p o sitio n , and normal o p eratio n w ill proceed*
Whan
th e pH In creases from th e pH c o n tro l value again in stea d o f the
re v e rse , then th e procedure would be repeated*
th is sa fe ty c ir c u it was q u ite successful*
The o p eratio n o f
The c lo se r St and Sf a re
to g e th e r, th e g re a te r i s th e accuracy o f control*
f o r a ferm entation
th a t c o n tin u a lly becomes more a lk a lin e th a rc i s no c o n tro l w ithout
re v is in g th e above c ir c u it by rev ersin g th e p o sitio n s o f St and S2
and by p lacin g a d d in th e re se rv o ir In stead o f base*
Since re la y s
A and B keep them selves closed a f te r th ey a re o p erated , i t i s necessary
n o t to j a r them, a s c o n tro l w ill be l o s t i f th ey a re opened by any
means o th e r th an by S2*
I t was found a f te r se v e ra l o p eratio n s th a t sometimes th e b a l­
ancing mechanism o f th e R ecorder, which balances th e potentiom eters
once every fo u r m inutes, commenced o p e ra tio n a t th e tim e th a t bass
was being added* When th is i s th e case, b ass i s added again when th e
tim er trlp -w h ee l i s in p o sitio n 1-4*
This i s because o f previous
design o f th e instrum ent in co rp o ratin g an autom atic sampling period
during th e fo u r minute tim e cycle*
Through th e advice o f Dr* Cameron,
ano th er re la y was In s ta lle d a s shown in F igure 3 80 th a t th e solenoid
valve can n o t o perate when the trip -w h eel i s in p o sitio n 1-4*
The
valve should be a b le to o p erate only during th e recording c y cle, o r
when th e trip -w h eel i s in p o sitio n 4*
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
IIOV.
feo~
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7 -1r *
IIov.
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timer
090090
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25V.
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PATH
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VALVE
c
T( - T r a n s f o r m e r
S 2 - M e r c o i d S w i t c ■\j Magnetically Opened
C - 2.5V. Valve Corit rol Relay
I N I TI AL
II OV
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pH
CONTROL
F ig ure I .
CIRCUIT
IIOV.
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VALVE
A , B and C — R e l a y s , 25V .C oils
S , - Mercoid S w it c h , Mag. Closed
S 2 ~ M ereo id S w it c h , Mag.Opened
T, -- T r a n s f o rm er
SA F E T Y CIR C UIT
Figure 2 .
1
0
I
3 4
1 1? 0
s
9
0
^--
5 6 O
7 8 4 10
0
0
*— •
D
0
0
0
Time Relay B o x o f
C o n t r o l C a bi n et
I n s e r t in S e r i e s w it h t h e
S o l e n o i d Valve C i r c u i t
D - Relay , IIOV, 6 0 ^ Coil
C IR C U I T ALLOWING BASE TO ADD ONLY IN T H E RE CO RD IN G
PORTION OF THE CYCLE
Figure 3 .
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
I t was found a f te r th e I n s ta lla tio n o f th e re la y i> a s in d icated
in fig u re 3 th a t o p eratio n o f th e e n tire c o n tro l mechanism m s s a t i s ­
factory#
The only d if f ic u ltie s encountered were d if f ic u ltie s such as
were caused hy an open condenser in th e p h o to e le c tric a m p lifie r c irc u it*
an open c ir c u it in a d ry c e ll* a i r bubbles in th e s a l t bridge* an
e le c tr ic a l leak in a g la ss electrode* o ccasio n al low charge in th e
sto rag e b attery * and a few o th e r situ a tio n s*
on one occasion*
A re la y sp rin g broke
In an instrum ent a s complex a s i s th is autom atic
pH B ecorder-C ontroller* th e re a re many p o s s ib ilitie s fo r a m echanical
d e fe c t to a rise *
O peration i s successful* b u t the machine m a t be
watched ra th e r c a re fu lly in o rd er to d e te c t rapidly* i f possible*
th e source o f tro u b le i f i t a rises*
I t was decided to fu rth e r com plicate th e alread y complex c ir c u it
by adding th e necessary c ir c u it fo r au to m atically s tir r in g th e f e r ­
menting medium f o r 35 seconds o f every fo u r minutes*
The tim e con­
t r o l re la y operated on th e cycle which would allow th is c o n tro l to
be adapted*
4*
The re s u ltin g e n tire c o n tro l c ir c u it i s shown in Figure
The medium i s s tir r e d 15 seconds out o f fo u r minutes* and a ls o
during th e tim e th a t th e so lenoid valve c o n tro l i s allow ing th e base
to add to th e medium*
Bach op eratio n i s independent o f th e o th e r
and th e re i s no In te rfe re n c e between c irc u its *
The f in a l c ir c u it
made use o f th e advantageous p a rts o f previous c ir c u its w ith an
in te rm itte n t s t i r r e r c o n tro l added a s shown in Figure 4*
The c ir c u it diagramed in F igure 4 operated q u ite successfully *
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
IIOV.
TO
STIRRER
MSUMJ
ttm f
25V.
TO
S OLENOI D
o
VALVE
T O POINTS 2 AND 3
OF TIME RELAY BOX
AS IN FIGURE 3 .
o
o
§
A, B, and C- Relays, 25V. Coils.
E
R e l a y , IIOV. C
oil.
Relay
E r e p la c e s R e l a y D
o f Figure 3 .
S , — Mercoid S w i t c h , M ag . Closed.
S>2l “ Mercoid S w i t c h , Mag. Opened.
T , —T r a n s f o r m e r .
C O M B I N E D S A F E T Y , pH C O N T R O L , A N D A U T O M A T I C
Figure 4-.
STIRRING C IR C U IT
►54-
There were occasion al m echanical d if f ic u ltie s such as th e breaking
o f a sp rin g o f a re la y and o th er each actions* h at a f te r a few con­
tr o ls th e d if f ic u ltie s were minimized*
The method seamed q u ite pro­
m ising and se v e ra l c o n tro l o p eratio n s have been run fo r s ix days
CKjntinuously w ithout any tro u b le w hatsoever e ith e r w ith th e machine
o r i t s adjuncts*
C ontrol was m aintained w ith ease to w ith in 0*1 pH
u n it o f th e deaired value*
O ccasional cheeks by th e pH m eters v er­
if ie d th e record o f th e control*
The place a t which c o n tro l o f th e
pH ceased was where th e pH began to in cre ase toward th e end o f some
ferm entations*
However* th is in cre ase was u su a lly v e ry s lig h t and
gradual*
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B* Methods o f Procedure
th e a n a ly sis o f th e ferm entation was designed to he c a rrie d o a t
a s eeoasiB leally a s p o ss ib le , and a lso w ith th e g re a te s t p ra c tic a l
degree o f speed and accuracy*
The accuracy i s , o f course, o f prim ary
iaportanoef however, where la rg e numbers o f d eteraL m tio n s a re to be
made, i t i s most d e sira b le to be ab le to run th ese determ inations
M gfeB f « ith accu rate c o n tro l o f a n a ly tic a l conditions*
One o f th e
most common methods fo r th e determ ination o f reducing sugar i s th a t
o f S h affer aM Hartmann (1921)*
th e amounts o f th e necessary reag en ts
f o r a sin g le sugar d etcm d n atio n a re la rg e thu s making a sin g le d e te rm iratio n r e la tiv e ly expensive*
The method a lso has th e
disadvant­
age o f being ra th e r inconvenient to use f o r la rg e numbers o f an aly ses
sim ultaneously*
A djustm ents o f copper reducing con d itio n s w ist be
v ery accu rate in o rd er to o b tain rep ro d u cib le re su lts*
d o st g ly ce ro l
a n a ly sis methods a re very complex and te d io u s! th ey u su a lly depend
o s th e complete removal o f sugars p rio r to a c tu a l a n a ly s is .
In
some methods th e re a re days between th e s t a r t and th e fin is h o f a
sin g le an aly sis*
Even th en , th e accuracy o f th e determ ination i s
sometimes doubtful*
D eterm inations o f g ly o ero l
masse in th e pre­
sence o f sugar by some o f th e methods would su rely tr y th e p atien ce
o f even th e most p a tie n t analyst*
In many c ase s, i t i s very advanta-
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geous to have th e g ly c e ro l r e s u lt w ith in a few hours a t moat o f th e
tim e th e sample i s tak en , th u s rendering th e long a n a ly sis q u ite
u n d esirab le i f another more rap id and a c c u ra te method i s av ailab le*
at,
^ tf ll4 a a 3 to jtf ..a a g a g »
T hsre a re numerous sugar a n a ly sis methods
th a t may be found in most o f the standard textbooks on food a n a ly sis
and re la te d subjects* b u t most o f th e isethods a re m od ification s o f
th e fe rric y a n id e red u ctio n procedure o f Hagedora and Jensen (1923)
o r o f th e o rig in a l g rav im etric copper red u ctio n method o f Bunsen and
Walker (1906)*
The most commonly used m odification o f th e Munsen and
Walker method in th is country i s th e volum etric method o f L a f f e r
and Hartmann (1921)*
The fe rric y a n id e method depends on th e redu ctio n o f potassium
fe rric y a n id e to ferrocyanide*
M odifications o f the Hagedom and
Jensen method have been mad© by B lish and S&odstedt (1933) «ad by
Gore and S te ele (1935)*
O bjections to th is method have been pointed
o u t by S h affer and Somogyi (1933) and by Soaogyi (1937)* A main
o b jec tio n was th e la c k o f s p e c ific ity to sugar in e o a te a si to tb s
s p e c ific ity shown by copper red u ctio n methods*
The method o f S h affer
and Hartmann (1921) i s a m acso-determ inatlon* being run in 3^0 ml*
ISrlenaeyer flask s*
I t was q u ite inconvenient to run la rg e numbers
o f determ inations in a sh o rt tim e by th is method because o f th e ex act
c o n tro l necessary during th e copper red u ctio n by th e sugar*
Improve­
ments in th e a n a ly tic a l methods were made by S tiles* P eterso n , and
Fred (1926) and l a t e r , as was mentioned p rev io u sly , by S h affer and
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-5 7 -
Sosogyi (1933), and a t i l l l a t e r by Somogyi (1937)*
The aethod o f
Soaogyi (1937) was employed as th e te a ls f o r development of th e
a n a ly tic a l methods need during th is research*
A m odified method
d escribed in th e th e s is o f Guyaon (19-39) was a lso used*
The o p p o s itio n o f th e reducing sugar reagent recommended by
Sosaogyi (1937) fo r d eterm in ation s o f a ic ro q u a n titie s o f sugar in
blood i s given in Table 7*
TABUS f
SGMGGII USAGE® FOR THE COPPER*IGDGMEmiC BETERMIHATION
OF VEHX m i l AMOUNTS OF SUGAR
Reagent
Q uantify
NazCOj (anhydrous)
R ochelle s a l t
CuSo**5H-.G
Ra»303
8a 2S04. {anhydrous* a n a ly tic a l grade)
KI
Water
25 g»*
25 ga*
4 g»*
20 gm*
200 g».
1*5 pi*
6*0 cd . o f 1 H. so lu tio n
To asks 1 l i t e r o f so lu tio n
According to Soaogyi (1937) th e sodium s u lfa te has th e e ff e c t o f
s ta b ilis in g th e so lu tio n * elim in atin g s e lf •red u c tio n o f tb s reagent*
I t a lso depresses th e carbonate io n isatio n * and thu s th e a lk a lin ity
o f the so lu tion *
The method o f preparing o f th e reag en t i s quoted
f r e e th e paper o f Somogyil
P rep aratio n - The carbonate and R ochelle s a l t a re disso lv ed
in about SOO ec* o f water* th en 40 ec* o f a 10 p ercen t GsSSi
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-58-
so lu tio n a re introduced w ith s tirrin g * This i s follow ed
by t i n a d d itio n o f th e b icarb o n ate, s u lf a te , and iodide*
th e d ilu tio n i s heated to b o ilin g , k ep t b o ilin g f o r about
th ir t y seconds, eooled» and a f te r th e a d d itio n o f th e
UQ% d ilu te d to 1 l i t e r .
Since th e chemica l s , seen though o f a n a ly tic a l grade,
co n tain so lid im p u ritie s, th e reag en t re q u ire s f iltr a tio n *
Upon standing fo r a day o r two b efo re f i l t r a t i o n , th e in p u r itie s s e t tl e o u t a s flo e o u le n t p a r tic le s and ean be
removed so re com pletely than i f th e so lu tio n i s f ilte r e d
fresh*
I t should be noted th a t f i l t r a t i o n o f th e above reag en t should
n ot be attem pted through f i l t e r paper o r o th er e e llu lo s ic m a te ria ls
sin ee th e re i s m m h y d ro ly sis to reducing sugars which w ill n atu r­
a lly a ff e c t subsequent sugar d eterm in atio n s by means o f th e rea g en t.
Cuprous oxide w ill g rad u a lly s e ttle o u t on prolonged stan d in g .
The
presence o f fin e ly d iv id ed p a rtic le s in th e reag ent induces reoxida­
tio n o f cuprous o xid e.
These e ffe c ts a re discussed by Soaogyi (193?)
and by B enedict (1931)*
^ro ced n rf* The reag en ts used f o r th e m icro sugar d eterm in atio n s were*
(A)
The oopper-iodom etrie rea g en t.
(B)
reag en t which contains 2*5 percent o f each in
w ater.
(C) 5 Bonsai HgSQ* so lu tio n (Keep away from ru b b e rit).
(B)
BasSfGi stan d ard so lu tio n approxim ately 0 .0 0 5 norm al.
The re a c tio n was run in 22 x 250 «n. Fyrex t e s t tu b es a t f ir s t*
The procedure employed m s a s follow s*
The sugar sample to be d e te r­
mined i s c la r if ie d w ith b asic lead a c e ta te and an a liq u o t i s added
to the t e s t tube such th a t th e so lu tio n added s h a ll co n tain from
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-59-
0*05 to 0*5 mg* o f d ex tro se o r i t s e q u iv alen t in a volume o f 5*0
ml*
fo th e 5*0 a l . o f sugar so lu tio n in th e t e s t tube i s added
5*0 &U o f reag en t (&)•
A fter m ixing, th e tube is placed in a b o il*
is g w ater bath fo r e x a c tly 20 m inutes as reeoEm sded by Smog? i
(1937), a f te r which i t i s placed in w ater to cool th e co n ten ts to
30° centigrade*
so lu tio n (C ).
Then 2 ml* o f (S) i s added* follow ed by 1*0 ml* o f
The re s u ltin g so lu tio n i s thoroughly mixed and allow *
ed to stand fo r about fiv e m inutes a t 30° w ith o ccasional shaking#
The m ixture i s th en t it r a t e d w ith standard sodium th io s u lfa te so lu tio n
(B) w ith about s ix drops o f sta rc h so lu tio n used a s an in d icato r*
A 5 o r 10 a l* a io ro -b u re tte i s ad v isab le f o r th e th io su lfa te *
The
volume o f th io s u lfa te should be estim ated to th e n e a re st 0*01 ml*
A blank should be ru n w ith a l l sugar determ inat ions*
P referab ly two
blanks should be run a t th e same tim e th a t th e sugar i s determ ined,
and th e sugar d eterm inatio ns should be run in duplicate*
I t was
found a f te r a number o f determ inations th a t a lin e a r equation could
be used fo r c a lc u la tin g sugar from th e th io s u lfa te t i t r a t i o n value*
Equation ( l ) was f a i r l y s a tis fa c to ry fo r th e d a ta obtained*
& • 0*116 % * O *01*
In th e equation, £ i s expreseed a s m illigram s o f dextro se and %
i s th e volume o f th io s u lfa te in m il l il i te r s obtained by su b tra ctin g
th e experim ental t it r a t i o n value from th e blank t i t r a t i o n v alu e, and
converting th is volume to m illilite r s o f 0*005 normal tu lo su lfa t© i f
th e reag en t i s n o t o f th a t norm ality*
R ep ro d u ced with p erm ission o f the copyright ow ner. Further reproduction prohibited without perm ission.
-6g -
I t m y be noted ■teat £ I s d ir e c tly p ro p o rtio n al to th e quantity*
X * 0*01, and i t i s d ir e c tly p ro p o rtio n al to th e w eight o f cuprous
oxide p re c ip ita te d which i s p ro p o rtio n al to th e sugar*
X I s n o t th e
th io s u lfa te t it r a t i o n v a lu e .
ty p ic a l r e s u lts a re given in Table 8 .
th e dextro se used fo r th e
sta n d a rd isa tio n was P fa n stie h l’ s C.P. anhydrous dextrose*
I t was
d rie d one hour a t H 0 ° cen tig rad e and th e ro ta tio n was measured*
sugar was sa tisfa c to ry *
th e
A standard so lu tio n was made to co n tain
TABLE 8
STAMMRBIZATIGM OF THE SOHDGZI SUGAR METHOD ASAIHST
PURE DEXTROSE
M 3H graas Volume, £ M illigram s M illigram s Volume, £ M illigram s
D extrose o f 0*005 M* D extrose
D extrose o f 0*005 ^.D extrose
P resen t th io s u lfa te Pound
P resen t th io s u lfa te Pound
0*050
0.050
0*050
0*100
0*18
0.08
0.34
0.82
0*02
0*01
0.049
0.305
0*300
1*74
2*5?
2*28
2*44
0.212
0.308
0*274
0.293
0*200
0*300
0*30 ©
0.100
0.100
0*300
0*200
o*n
0*82
o*T3
urn
0*101
0.305
0.095
0*206
0*400
0*400
0*400
0*500
3.38
3.36
3.50
4.32
0.402
0*400
0.416
0*510
0.200
0.200
0*200
1.14
1*53
1.50
0*212
0*188
0*184
0*500
0.500
4*16
4.24
m
0*492
0.502
m
0*1 mg* o f dextrose p er ml* o f so lution*
a 5 ml* b u rette*
Volumes were measured from
l e so lu tio n was used which was over 24 hours o ld ,
sin c e th e growth o f molds g en erally a lte r s th e so lu tio n s by about
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-
61-
th a t time*
The an aly ses, as in d ic ate d in Table 8* v ara n o t a l l th a t could
be desired*
There appeared to be an evaporation during th e heating*
th u s changing th e co n cen tratio n o f reagents* and a le e th e presence
o f oxygen o f th e a i r s ig h t cause reascidation o f th e cuprous oxide*
Hence* i t was decided to modify th e above procedure by covering th e
t e s t tu b es w ith g la ss bulbs during h eatin g and previous to titr a tio n *
I t was thought th a t lengthening th e tim e o f h eatin g s ig h t a lso be
advantageous.
A fter some p relim inary te s ts i t was decided to sake
th e follow ing two m od ificatio n s to th e procedure described abovet
Cl} The tim e o f h eatin g in th e w ater bath i s increased from
20 m inutes to 35 m inutes.
(2)
The re a c tio n tubes a re covered w ith g la ss bulbs a t a l l
tim es up to th e tim e o f t i t r a t i o n w ith sodium th io s u lfa te solution*
I t was necessary to determ ine a new equation f o r sugar d e te ra ia *
a tio n s by th e titr a tio n s w ith th e m odified method*
i s th e new eq u atio n .
Equation 1*
Equation (2)
The d e fin itio n o f term s i s th e same a s fo r
I t any be noted th a t th e co n stan t 0*001 i s n e a re r se re
than was th e sim ila r c o n stan t o f Equation 1*
<2) £ « 0.2071 X ♦ 0*001
The reag en t *8* (KI~K2C20}, should be added so th a t i t rem ains
on th e su rface o f th e m ixture in o rd er th a t io d in e as i t i s formed
w ill have to go through potassium Iodide in o rd er to escape and
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<-62*
w ill be disso lved th u s preventing escape w hile effervescence pro­
ceeds*
T opical r e s u lts appear in Thble 9*
TABLE 9
dextbqse m s s s s s by th e m odified s o ie o n oow m riom m m iG
method
M illigram s Helune, £ M illigram s
D extrose o f 0*005 X* D esires*
th io su lfftt* Found
0 .0 3 0
0 .0 5 0
0 .1 0 0
0*200
0*200
0*300
0 .2 8
0*4?
0*93
1*84
M illigram s f d s N | £ M illigram s
D extrose o f 0*(X)5 X* D extrose
P resen t th io s u lfa te Found
0*030
0*051
0*101
0*193
1*88
0*202
2*?9
0*300
0*300
0*400
0.400
0*400
0*500
0.500
2*?9
M 2
M2
3*73
4.5?
4*53
0*300
0*400
0*400
0.401
0*492
0.491
The r e s u lts o f Table 9 in d ic a te th a t th e m odified sugar iaethod
I s q u ite s a tis fa c to ry provided th a t te e q u a n tity o f sugar in th e
sample does n o t g re a tly exceed 0*4 mg.
I t nay be noted th a t th e
low v alu es se re found f a i r l y accurately* however te e percentage
e rro r s i t e th ese sm all amounts i s great*
The main o b jec tio n to th is
method i s th a t a high d ila tio n i s necessary fo r many ferm en tatio n
analyses*
The determ ination o f sugar has a ls o been considered by o th e r
in v e s tig a to rs o f th e se la b o ra to ries*
Zn o rd er to elim in ate te e high
d ilu tio n s req u ired f o r sugar determ inations such as mere necessary
in th e previous methods described* and to rend er te e reag en t and
r e s u lts more reproducible* Gtsymon (1939) developed what i s c a lle d
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout p erm ission .
»63»
reag en t •G" fo r sugar determ inations where d ilu tio n requirem ents
a re n o t so high*
Reproduction o f r e s u lts m s q u ite s a tis fa c to ry when
subsequent reag en ts s e re made up in th e same way*
I t was found to
be a d v isab le to sake th e pH o f th e reag en t th e im portant fa c to r
ra th e r th an th e a c tu a l amounts o f b a sis reag en ts employed uinoe th e
m e o f d if f e re n t batches o f chem icals m y produce a d if f e r e n t pH
in th e f in a l reagent*
Reagent *0* o f Gammon i s given in Table 10*
TABLE 10
CGPPER-IOBGMETRIG REAGBKf *G*» FOR THE RSTERMINiTIOH
OF SGGAR 08 A SME-MICRO SGAUE
Q uantity
He^CGj (anhydrous)
RaOH
53 g»*
About 11*4 ga* sa tu ra te d solu­
tio n la added to pH 5*4$) th is
re q u ire s about 7*5 ml«
(R ochelle s a l t )
125 ga*
OuSO4-*5Ha0
(add a s HaO so lu tio n )
37*5 go*
KX
1*0 gn*
SIO3
3.567 ga* (exact am ount).
HajSO* (anhydrous)
J®*0 ga*
Water
to 1 l i t e r f in a l volume*
The procedure o f making tqj th e rea g en t i s as follow s*
The sodium
carbonate and R ochelle s a l t a re d issolv ed in approxim ately 300 ml*
o f d is tille d w ater a f te r which th e copper s u lfa te i s added a s an
aqueous so lu tio n w ith s tir r in g so th a t carbon dioxide w ill s o t be
evolved*
The use o f a fu n n el w ith i t s e x it under th e su rfa c e o f th e
carbonate so lu tio n i s q u ite h e lp fu l.
A fter th is th e potassium
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io d id e and sodium s u lfa te a re added*
The so lu tio n w ill now have a
pH o f about 9*1 as measured by a g la ss e le c tro d e pH meter*
The
sodium e rro r i s s o t considered end would not be im portant so long
a s th e re i s a co n stan t deviation*
volume to about 950 m l.
W ater i s added to b rin g th e to ta l
The sodium hydroxide so lu tio n i s now added
l i t t l e by l i t t l e w ith s tir r in g u n til a pH o f 9*48 i s ob tained as
measured by a g la ss e le c tro d e system*
I f th e pH i s fro® 9*4 to
9*5 th e r e s u lts w ill be reasonably sa tisfa c to ry *
The so lu tio n i s heated to b o ilin g and th e b o ilin g i s continued
f o r about th re e to fiv e minutes*
The so lu tio n i s then cooled* th e
lod&te i s added* and w ater i s added to make th e f in a l volume one
lite r *
C onsiderable cuprous oxide u su a lly forma*
The so lu tio n should
be allow ed to stand fo r se v e ra l days* o r perhaps a week* and i t should
be kept in a Pyrew b o ttle o r flask *
i t th e end of th is time* th e
reag en t m y be f ilte r e d through asb esto s o r o th e r in e r t m aterial*
F ilte r paper should n o t be used as i t hydrolyses m rm /t& t under th e
in flu en ce o f th e a lk a lin e solution*
As an a lte rn a tiv e , th e d e a r
su p ern atan t liq u id m y be siphoned fro® th e p re c ip ita te i f s u ita b le
p recau tio n s a re taken a g a in st a g ita tio n o r th e p re c ip ita te -
la y
suspended p re c ip ita te in th e reag en t i s very u n d esirab le, and e rro rs
w ill be noted in th e re s u lts * e sp e c ia lly i f th e p re c ip ita te i s
cuprous oxide*
The reag en ts f o r th e auger d eterm ination by th is method a re
s im ila r in some ways to th e reag en ts o f Soaogyi (1937) bu t they
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
*
45-
r e a u lt i» about te n tim es th e o x id isin g capacity*
The rea g en ts to
he used are*
(1)
Besgent *G"*
(2)
S olution co n tain in g 12*5 g»* £1 cod 25 ga* K-jC^O^HaO
p er 100 ml* of d i s t il l e d water*
(3 )
S o lu tio n T*5 normal in H2S0*. (Keep away from ru b b e r!!)
(4 )
Sodium th io s u lfa te so lu tio n about 0*05 normal*
(5 )
S tarch so lu tio n in sa tu ra te d aqueous BaGl solu tio n*
The method o f procedure i s sim ila r to th e method p rev io u sly
described*
The t e s t tu b es w ed a re sm aller (25 x 150 a s u ), although
th e a c tu a l e ls e i s n o t c r itic a l*
To 5 ml* o f reag en t •0* i s added
5 ml* o f c la r if ie d sugar sample con taining n o t over 10 stg* o f sugar*
Mix thoroughly and h e at covered in a b o ilin g w ater bath fo r 30 m inutes)
then cool to JG° centigrade*
Add 2 ml* o f th e iodide-ox& late reag en t
( 2 ) , and then add th e s u lfu ric acid w ry c au tio u sly u n til a l l th e
io d in e th a t w ill fo ra i s lib e ra te d .
S lig h tly over 1 ml* i s necessary*
F in a lly t i t r a t e w ith th e th io s u lfa te so lu tio n using th e sta rc h in d i­
c a to r fo r th e endpoint*
Analyses a re m e t s a tis fa c to ry when th e sugar in th e sample i s
between 1 and 10 eg*
In some ferm ented media p ra c tic a lly no d ila ­
tio n i s necessary f o r a d eterm in atio n .
however.
C la rific a tio n i s recommended*
B asie lead a c e ta te o r alumina cream a re u su a lly used* b u t
o th e r agents a re a p p lica b le depending on th e mediun*
Dock (1939)
described a new method u tilis in g cadmium hydroxide fo r p la n t e x tra c t
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
66-
—
treatm ents*
In analyses* Equation (3) was found to hold q u ite w ell
f o r th e reag en t and technique used*
(3)
In th is equ atio n , £ i s expressed a s m illigram s o f dex tro se and £
i s th e volume in si* o f 0*05 normal sodium th io s u lfa te so lu tio n which
i s obtained by su b tra c tin g th e experim ental t it r a t i o n v alu e from tits
blank v alu e a s d escribed on page 59*
I t i s ad v isab le to d eriv e a
new eq uation each week o r so in o rder to be sure o f the equ atio n con­
sta n t*
I t changes w ith decreasing ra p id ity as th e so lu tio n ag es, b u t
th e r a te o f change c lo se ly approaches aero a f te r about a month o f
standing*
The co n stan t v a rie s fo r d if f e r e n t sugars*
i s considered e n tir e ly s a tis fa c to ry .
The method
One must he very c a re fu l w ith
th e s u lfu ric acid reag en t to keep i t fre e o f o x id isab le m a te ria ls such
a s become dissolved in th e aeld on long exposure to rubber.
The o n -
sa tu ra te d m aterial e x tra c te d fro a rubber caused tro u b le u n til th e
source o f th e d if f ic u lty m s discovered*
Tbs m odified method o f Soao&ri was used in th e a x p eriae n ta l work
u n til th e method o f Quynon (1939) became av ailab le*
Both methods
were s a tis fa c to ry ! however, th e method o f Quymon was mors convenient
f o r sugar determ inations in th e co n cen tratio n s g en erally encountered
in t h is work.
h
fo tfw ajim t10*1 o f »th& nol.
The ethan o l determ ination was based on
th e standard d i s t il l a t i o n - d e n sity method o f th e A sso ciatio n o f
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-67-
O ffic ia l A g ric u ltu ra l G heaists (1925)*
U sually about 300 ral* ©f
aediua wore d is tille d In th e presence o f calcium o arto n ate in o rd er
to keep v o la tile a cid s from d is tillin g *
K Jeldahl d is tilla tio n appara­
tu s was used# and 100 ml* o f d i s t i l l a t e were c o lle c te d in a 100 ml*
volum etric flask *
The d e n sity o f th is so lu tio n was measured a t 25®
cen tig rad e ty means o f a chainom atlc Westpfcal balance*
Standard
conversion ta b le s were employed to o b ta in th e gratae o f eth an o l p er
100 ml* o f d i s t i l l a t e .
In cases where ammonia might d i s t i l w ith the ethan ol and water*
a second d i s t il l a t i o n was necessary* s u lfu ric acid being added before
th e d i s t il l a t i o n u n til an acid re a c tio n to a non-alcoholic in d ic a to r
was noted*
F a rth e r a n a ly tic a l d ire c tio n s have been given by Gehle
(1922)*
g»
flSQ&UfaB« S u lfite was determ ined by t i t r a t i o n
w ith io d in e so lu tio n as d escribed by K olthoff and S andell (1937)* and
K olthoff (192^.
Free s u lf ite m s f i r s t titr a te d in a c id so lu tio n *
Then on a d d itio n o f sodium b icarb o n ate, th e b isu lfite -a ld e h y d e com­
p lex decomposed lib e ra tin g th e combined s u lf ite which m s titr a te d
w ith io d in e solution*
This determ ination i s c lo sely re la te d to th e
aeetaldehyde determ ination*
d* b e te m lm tio n o f aoetaldahvde*
The aeetaldehyde m y be estim -ted
by m io d im e tric determ ination o f th e d iffe re n c e between t o ta l su l­
f i t e and fix ed s u lf ite in th e medium*
The aeetaldehyde fix e s th e
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b is u lf ite in a q u a n tity p ro p o rtio n al to th e q u a n tity o f aoet&ldehyde
present*
so lu tio n .
The fix e d s u l f i te i s lib e ra te d by a lk a lis a tio n o f th e
D etailed d ire c tio n s may be found in th e papers of Tosoda
(1929) and Jsulm es and E speael (1915)*
monograph o f Iaw rie (1928)*
ftti
1 note i s a lso given in th e
O ther methods se re d escribed by Gehle (1922).
tOjCgcal*
The g ly c e ro l was determ ined by th e
method o f Fulmer* Hickey and U nderkofler (1940).
This method
c o n sists o f a d eterm ination by a eopper red u ctio n method follow ed
by an o x id atio n o f both th e sugar and th e g ly ce ro l by 0 .1 normal
e e rie s u lfa te solution*
fo r th ese stu d ie s .
tlo a e d .
This a n a ly tic a l method was developed e sp e c ia lly
F u rth er refe ren c es may be found in th e paper sen*
Numerous o th e r a n a ly tic a l methods a re g iv es in th e monograph
o f Iaw rie (1928) and in th e paper o f Gehle (1922)*
Development o f an optimum seml-fG rathetie medium.
I t m e d e sired to have a aediua w ith which to study th e g ly c e ro l
ferm en tatio n which would be as n early sy n th e tic a s possible* and
a lso would have a minimum amount o f “fix e d sa lts" *
The purpose o f
using such a medium was to make th e a n a ly sis of d e sire d products*
such as glycerol* sim pler and more expedient* in o rd er to follow
th e d esired in v e stig a tio n s w ith more ease*
Such a medium would be
used only to develop o r improve methods f o r g ly ce ro l pro d uctio n .
D extrose was used f o r th e p relim inary medium in o rd er to have as
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-
69*“
few a n a ly tic a l com plications as p o ssib le , such as in v ersio n o f sucrose
b efo re reducing sugar determ inations can be made* le a s t e x tra c t
(Difco anhydrous) m s se le c te d as th e only req u ired m a te ria l which
was n o t a pure chem ical o f known com position a s i t la one o f th e most
concentrated y e a st n u trie n t sources; thus* very l i t t l e should be
required*
The work o f Fulmer* Nelson and Sherwood (1521} and inform ation
given by Buchanan and Fulm er (1930b) were considered preceding th e
experim ental work,
th e term* "optimum medium"* i s intended to in ­
d ic a te optimum w ith re sp e c t to alco h o l yield * minimum s a lts * and
speed o f re a c tio n ra th e r th an to y e ast growth*
The optimum m ed ia
was prepared fo r th e normal a lc o h o lic ferm entation*
The com position o f Table 11 was assumed fo r an i n i t i a l b a sal
medium*
TABLE 11
THE BASAL MEDIUM
Reagent
Dextrose (anhydrous)
le a s t e x tra c t (D ifco an h .)
EfefG*
JEtHFQr 3%0
MgStVTHaG
Fe(RS*)2<S0*)2*6Ba0
CaCl*
Weight p er 100 ml*
o f Medium
15
0*5
0.188
0 .05
0 .0 5
0.01
0.01
0*01
m*
gm.
ga­
g a.
ga.
ga.
m*
gm.
The o rig in a l medium employed f o r carry ing th e y e ast c u ltu re
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-70-
contained 5 grama o f d ex tro se and 0*5 grama o f y e ast e x tra c t p er
100 ral* o f Hsedltstu
Tap w ater was used*
lbs* steam p ressu re f o r o n e -h a lf hour*
S te r ilis a tio n was a t 10
C ulture fla s k s w a s 1 0 el*
E rlem ey er fla s k s co n tain in g 10 s i* o f th e d ex tro se-y east e x tra c t
medium*
The pH o f th e medium was ad ju sted to $ b efore s te riliz a tio n *
A medium con taining 5 grams o f sugar in 100 ml* o f so lu tio n i s
c a lle d a $ p ercen t medium* O ther media a re described sim ila rly 1 fo r
convenience*
The b a sa l medium was th u s a 15 percent medium*
Xsoeula
f o r th e fla s k s e rie s were 10 p ercent media* a s se e r optimum w ith
regard to m ateria ls o th e r than sugar as were found from tim e to
tim e so th a t th e in ocu la would a l t e r th e com position o f th e media
being stu d ied a s l i t t l e a s possible*
A 5 p ercen t in o c u la tio n means
th a t 5 ml* o f inoculum i s added p er 100 ml* o f medium*
A s e rie s o f media was ferm ented f i r s t keeping a l l in g re d ie n ts
co n stan t except th e y e a st e x tra c t which m s v aried in o rd er to fixed
th e le a s t amount o f th is agent which could be used and s t i l l o b ta in
a mcudsBB o f alcohol*
The s e rie s i s d escribed in Table 12*
By o b serv atio n o f Table 12* i t i s evid en t th a t th e o p tim a con­
c e n tra tio n o f y e ast e x tra c t i s about 0*7 grams in 200 ml* o f aeditaa,
o r 0*35 grams p er 100 ml*
Accompanying analyses were made fo r re s id u a l
sugar by th e method o f Somogyi (1937)*
le a s t
e x tra c t was found to have
n e g lig ib le reducing e ff e c t on th e copper complex*
The fla s k s con­
ta in in g 0*? grams and over o f y e a st e x tra c t showed about 1 p ercen t
o r le s s o f re sid u a l dextrose*
The o th e r fla s k s contained more th an
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1 p ercen t o f re sid u a l sugar*
I t was estim ated th a t th e d e sira b le
amount o f y east e x tra c t should be about 0*375 grains per 100 a t* o f
medium* th is valu e was used f o r subsequent in v estig atio n s*
’CABLE 12
EFFECT* OF IEAST BCTRACT COHCBSmTIOH OK 1HUB0L HELD
flask s*
Medium!
200 al* medium in 500 m l. S rlen sey er fla s k s .
lg £ dioctrose s e d ita shown in Table 21* exempt fo r y e a st
e x tra c t. pH « 5*
S te riliz a tio n * 10 lbs* fo r 30 a iru
Inoculum* 10$ d ex tro se medium* 10 m l. to each flask * (5& in o c u latio n )
Incubation* 5 days a t 30 C*
Grams o f
le a s t 1st*
in 200 ml*
D ensity o f
d is tilla te
0 ||
Grams o f
Ethanol*
T o tal
0*0
0*0
0*1
0 .1
0*2
0.9925
0*9924
0.9900
0*9900
0*9863
4.13
4.19
5*60
5*60
7*89
0*2
0*3
0*3
0*4
0*4
0.9868
0*9846
0*9843
0*9817
0*9824
7*58
8*95
9*25
10.85
10*39
0*5
0*5
0 .6
0*6
0*7
0.9798
0*9796
0*9796
0*9791
0.9786
12*12
12*26
12*26
12*60
12*94
©*T
0 .8
0*8
1*0
0.9789
0.9793
0.9791
0.9792
12.73
12*46
12*60
12*53
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-72-
Having determ ined th e optimum ye&st e x tra c t concentration* tb s
nex t m a te ria l taken fo r study was ammonium chloride*
A s e rie s o f
fla s k s was prepared in a manner analogous to th e s e rie s o f Table 12*
Hie d a ta obtained a re given in fa b le 13*
TABLE 1 3 .
EFFECT Of AMMONIUM C W fflm CONCENTRATION ON ETHtNOX. HELD
F lask et
Medium!
200 ml* in $00 m l. E rleaaeyer flask s*
1$% dextrose? 0.37JP » y e a st e x tra c tj o ilie r m ateria ls a s in
fa b le 119 except NS*C1. pH » 5*
S te r ilis a tio n ! 1 3 lb s . f o r 20 m inutes.
Inoculum* 10$ d ex tro se medium? $$ in o c u la tio n .
Incubations 4 days a t 30 C.
Grams o f
NHfCl
i s 200 ml*
D ensity o f
____ _____ 4 i f
Grams o f
Ethanol?
f e ta l
.........................__
0 .0
0 .0
0 .1
0 .1
0*2
0.9801
0.9152
0.9802
0.9799
0.9809
11.91
12.53
11.85
32.05
11.39
0 .2
0.9805
0.9806
0.9198
0.9803
0.9811
11.64
11.57
12.12
11.78
11.25
0.9807
0.9821
0.9810
0.9809
0.9805
11.51
30.59
11.31
11.37
11.64
0 .3
0 .3
0.35
0.35
0 .4
0 .4
0 .5
0 .5
0 .?
An o bservation o f fa b le 13 in d ic a te s no decided, optimum fear
ammonium chloride*
Some ammonlun s a l ts a re p resen t in th e y e a st
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-73-
ex traet*
The ferm en tativ e a o tiv ity m s g reater* however* where an
ap p reciab le amount o f ammonium ch lo rid e was present*
A co n cen tratio n
o f 8*25 grans p er 100 nl* o f medium m s used in subsequent media*
th e next n u trie n t stu d ied m s th e phosphate*
Potassium a cid
phosphates were used w herein h a lf o f th e s a l t was th e prim ary and
th e o th e r h a lf m s th e secondary sa lt*
R esu lts a re shown in Table
TABLE 14
w m m OP POTASSIUM ACID PHOSPHATE CONCESTMTIOH
OH ETHANOL YIELD
Flasks*
Mediums
200 s i* in 500 ml* arlenm eyer flask s*
25$ dextrose* 0*375$ y e a st e x tra ct* 0*15$ NH*C1* aud o th e r
m a te ria ls a s in Table 11* except fo r th e phosphate where
th e w eight in each fla s k was made up o f 50$ prim ary and
50$ secondary potassium phosphates* as designated below*
p i * 5*
S te rilis a tio n * 13 lbs* f o r 20 minutes*
Inoculum! 10$ d ex tro se medium* 5$ inoculation*
Incubations 4 days a t 30° C*
D ensity o f
d is tilla te *
Grams o f
ethanol*
T o tal
0 .0
0 .0
0 .1
0 .1
0 .2
0.7757
0.9774
0*9775
0.9775
0.9795
12.19
12.39
12*32
12*32
12*32
0 .2
0*4
0*4
0 .6
0 .6
0.9794
0.9794
0.9795
0*9799
0*9801
12*39
12.39
12*32
12.05
21*91
Gres® o f
phosphates
in 200 m l.
m
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•>^4*
Shese s a lts do s o t seem to be c r i t ic a l in requirem ent*
Some
phosphates e re no doubt p resen t in th e y e a st e x tra c t allow ing fe r~
a e n ta tlo n w ith no o th e r phosphate added*
O bservation o f th e d a ta
and re a c tio n v ig o r in d ic a te d th a t about #*25 gram p ar 100 al* o f th e
nixed phosphates i s advantageous*
The e ff e c t o f varying co n cen tratio n s o f magnesium s u lfa te
(MgSOe+fiJatO) was stu d ied n&ct*
I t van noted by Connstein and Iiideeke
TABLE 15
w n c t 0f 1A0HESIUM SUIFAfR CGHCSimTICH OS
ETHANOL YIELD
F lask s:
Medina:
200 a l* in 500 ml* Srlecaaeyer flask s*
1 0 d e x tro se, 0*31 0 y e a st e x tra c t, 0*15$ HH*€1# 0 * 1 ^
phosphate m ixture# and o th er m aterials m in fe b le 11
except fo r % S04*f% 0 which i s as shown below* pH ® 5*
S te rilis a tio n s 13 lbs* fo r 20 minutes*
InOCUlusn* 3f3$ d ex tro se wetHtHB, 0 Iw w ulw tf nn.
fcw ubattoai 4 days a t
C*
drams o f
% so4*ts*0
in 200 ml*
D ensity o f
d is tilla te ,
4?
G rass o f
eth an o l
f e ta l
0
0
0.02
0*02
0*04
0*9793#
0*97921
0*97930
0.97920
0.97920
12*46
12*52
12.46
12.53
12*53
0*04
0.06
0*06
0*08
0*08
0*97930
0.97930
0*97922
0.97925
0.97929
12*46
12.46
12*51
12.50
12*54
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-75-
(1924) l a th e ir g ly c e ro l ferm entation work th a t m g n e siia s u lfa te
was Im portant as a ferm entatio n a c c e le ra to r.
Work was done by
o th e r in v e stg a to rs, hut a s th e ir purpose was s o t to o b ta in a medium
w ith m iniiaat added s o lid s , th e in v e s tig a tio n described in fa b le 15
was made*
Hie r e s u lts o f fa b le 25 obviously a re inconclusive in s o fa r as
TABLE 16
EFFECT OF G4ICXUM CHIDRIDE G0SCSHTR4T30B
OS ETH&.NOL YIELD
F lack st
Mediun*
200 ml* in 500 si* B rletm eyer fla s k s .
1$$ d ex tro se, 0*375!^ y e a st e x tra c t, 0.15$ BB*Q2« 0*25$
phosphate m ix tu re, 0*02^ MgSQ4 *7 %Q and o th e r m a te ria ls
a s in Table 11 except fo r Cafifewhich i# shown below*
pH * 5*
Steriliaatiaat 13 lbs. for 20 minutes,
inoculums 10$ dextrose medium, 5$ inoculation*
Incubation*
4 days a t 30° C*
(brass o f
G a d , in
200 ml*
O snsity o f
d is tilla te
__ J S L _
Grams o f
eth an o l
to ta l
O
0
0*02
0.02
0.04
0*97825
0*97800
0*97770
0*97745
0.9777©
12*97
13*18
13*35
13*18
0.04
0.06
0,06
0*06
0.08
0*97780
0*97820
0,97850
0*97905
0.97780
13*11
12*83
12.63
12*33
13*11
1 2 .8 0
optim m co n cen tratio n o f magnesium s u lfa te i s concerned.
However*
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-7 6 -
© bservatlona on th e r a te o f ferm entation in d icated ^fcat th e presence
o f some o f th e s a l t i s to be d e sire d .
She fla s k s o f h ig h er m agnesias
s u lfa te co ncentratio ns were q u iescen t sooner than were th e fla s k s o f
low er concentration*
A co n cen tratio n o f *02 grams p er 100 ml* o f
medium was used fo r subsequent media*
The e ff e c t o f th e co n cen tratio n o f calcium ch lo rid e on th e ethan ol
y ie ld from a y east ferm en tatio n o f d ex tro se was stu d ied next*
a re found in Table 16*
Data
From th e r e s u lts o f Table 16 i t m s decided
to use a co n cen tratio n o f 0*01 g rass o f calcium ch lo rid e p e r 100 ml*
Of
raoriiuw u
Mo p a rtic u la r advantage was found in th e ad d itio n o f an iro n
s a l t , ao no iro n s a lts were included in th e f in a l b asal medium*
TABLE 1?
OmWBM SUdl-SIHTHETIC MEDIUM FOR MAXIMUM ETHANOL PRODUCTION
UTILIZING A MINIMUM OF ADDED SOLIDS
Neagent
Y east E x tra c t (D ifeo anh*}
NH*G1
K2HF0**3H*0
KHaFOe
«gSQ**7%©
CftClj
D extrose f o r carry in g c u ltu re
D extrose fo r inoculum
D extrose fo r experim ental media
Weight p er
100 n l. o f Medium
0*375
0 .1 5
0*075
0*075
0.02
0*01
5*0
10*0
15*0
ga*
gm.
go*
gm*
gat*
m*
gm*
gs*
ga*
The optimum medium was taken to be a s described in Table 17*
The sugar content v aried according to th e purpose o f th e medium*
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-7T-
fap w ater was used i s making up th e media.
The q u a n titie s o f th e s a lts employed i s th e medium were n o t
found to be c r i t ic a l in th e experim ental s e rie s ! hence* th e b a sa l
medium d escribed in fa b le IT was chosen fo r th e sake o f co nsisten cy
in 'toe work.
3*
The a lk a lin e d issim ila tio n o f d ex tro se fo r to e p re p a ratio n o f g lv c a ro l.
a.
The use o f ammonium hydroxide.
T his reag en t m s used by Krug end
McDermott (1935) in th e ir p a te n t on th e ferm entation o f sugar to
glycerol*
They obtained 15*8 to 18*5 p ercen t g ly ce ro l based on p ercen t
o f sugar weight* o r 31 to 36*3 p ercen t o f th e th e o re tic a l yield*
Three
l i t e r s o f a m olasses medium req u ired a t o ta l o f about 50 ml* o f aiaaonturn hydroxide (28 p ercen t HHj) which m s added in portions*
The d ilu *
tio n e ff e c t m s n o t very g re a t w ith th a t sm all an added volume.
ammonia was added a t in te rv a ls to keep th e pH m a r n eu tral*
The
U sually
th e pH was about f*2 o r ?«3 im m ediately a f te r th e ammonia addition*
but i t soon approached n e u tra lity o r s lig h t a c id ity (pH * 8*8)*
ammonia was th en added*
More
In using ammonia* no "fixed* s u its were add-
ed* and th e g ly ce ro l was more e a s ily recovered*
Some c o n tro lle d ferm en tatio n s were d e sired a t v ario u s pH lev els*
I t would be advantageous from a th e o re tic a l p o in t o f view to be a b le
to express g ly ce ro l y ie ld a s a fu n ctio n o f pH.
The Gameron pH
Recorde5Mk>ntroUer I s to c o n tro l ferm entations a t a d e sired pH le v e l
by au to m atically adding th e base as required*
The equipment was
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-?8-
prepared fo r th e autoia&tie a d d itio n o f liq u id s so th e a d d itio n o f
asmonifna hydroxide so lu tio n s ana possible*
- Three l i t e r s o f optlm aa medium were prepared In th e u su al way in
« jW L lter, 3-neeked flask *
th e experim ent was known as t r i a l *L"*
AaaonltaB hydroxide so lu tio n (14 p ercen t BJH^) was p w p n d and placed
in th e re s e rv o ir fo r a u to a a tie addition*
In o cu latio n mm made w ith
300 nl* o f y e a st c u ltu re Ho* 43 and c o n tro l m s attem pted a t pit * $
to tr y o u t th e machine so th a t any d if f ic u ltie s a ris in g in th e process
would be due to something o th e r th an th e c u ltu re growth i t s e l f .
Qper-
a tio n m s f a i r l y good fo r th e f i r s t th re e days* but a t th e end o f th a t
tin e th e aediua became s lig h tly le s s acid w ithout th e a d d itio n o f
more b ase, and as soon as th e pH reached 5*3# c o n tro l was lo s t and
th e whole o f th e amrnoniiim hydroxide so lu tio n emptied in to th e medium
asking th e pH * 9* The c o n tro l had n o t y e t been adapted fo r th e
co n d itio n in which th e pH Increased* ra th e r than decreased tr m th e
v alu e s e t by th e instrum ent*
A sa fe ty c ir c u it m s devised and has
hem d escribed in th e se c tio n above*
The fo m e n ta tio n c o n tro l m s
th u s unauecessful in th is experim ent except th a t i t led to develop*
s e n t o f th e sa fe ty c irc u it*
A second batch o f medium was prepared a s before*
f o r 20 m inutes.
This was known as t r i a l % •#
a t 7*5 by ammonium hydroxide add itio ns*
w ith t r i a l *W except f o r th e pH.
I t m s s te r ilis e d
The pH was c o n tro lle d
The method m s th e same as
The sa fe ty c ir c u it m s used to pro­
te c t a g a in st lo sin g pH c o n tro l by a pH in c re a se .
The medium was
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au to m atically s tir r e d fo r 30 seconds o f every fo u r m inutes, a id e ls e
when th e has# was au to m atically added*
The pH was fo u r a f te r in ocu la­
tio n , b u t i t was brought to about 7*5 g rad u ally over a p eriod o f fiv e
hours*
The machine worked ex cellen tly * b u t th e ferm entation was found
to have ceased a f te r about one day a t th is pH* Sugar was n o t being
consumed, and no base was added*
Among th e reasons considered fo r the death o f th e c u ltu re wares
(1)
lack o f a c c lim a tisa tio n o f th e c u ltu re to th e abnormal
conditions*
(2)
T sadelty re la te d in some manner to th e ammonites ion*
(3 )
Unfavorable pH*
(4)
Too rap id a pH change in th e beginning*
(5)
C ulture too week a t the s t a r t o f th e co n tro l period*
A b atch o f medium was prepared a s above which m s to be co n tro lle d
a t pH * 6*3 by emaonlm hydroxide (14 p ercen t MH^}* T his was known
a s t r i a l •» » .
in o c u la tio n .
The pH was c o n tro lle d a t fiv e f o r 12 hours follow ing
Vigorous growth resu lted *
The pH was g rad u ally brought
to 6*9, th e process re q u irin g a period o f about nine hours*
A fter th e
c u ltu re m s 90 hours o ld , c o n tro l was found to be unnecessary and m s
sh a t o ff w hile allow ing th e recording to proceed*
about 7*3 a f te r 118 h o u rs.
The pH had r is e n to
This seem to be a ty p ic a l phenomenon*
At f t hours and a t U S hours about 0*5 p ercen t o f th e o rig in a l sugar
m s found remaining*
The pB was checked a t in te rv a ls w ith a Cameron
pH M eisrf th e record was sa tisfa c to ry *
Analyses showed 13*8 grams o f
g ly c e ro l and 3&«6 grams o f eth an o l were obtained from 100 grams o f
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d ex tro se, o r 27*1 p ercen t o f the th e o re tic a l g ly c e ro l, o r 13*8 p ercen t
©a th e eager basis*
About 99*5 p ercen t o f th e sugar m * consumed*
About 92 percen t o f th e d ex tro se was accounted fo r, taking in to consid­
e ra tio n th e f a c t th a t some eth an ol i s fonsed by th e th ird equation o f
Beuberg, and th e r e s t by th e f i r s t equation (a lc o h o lic ).
The g ly c e ro l
y ie ld o f th is fe re se n ta tio n n ig h t be expected to be a b i t lo n e r th an
those rep o rted by Krug and McDermott (1935) a s th is was ra n a t pH
8 .8 to give 13*8 p ercen t g ly ce ro l on th e su g ar, while they v aried th e
pH from 6 .8 to about 7*3 and obtained 15.8 to 18*5 percen t o f th e
sugar a s glycerol*
Another d ifferer.e e was th a t they used m olasses
in stea d o f d ex tro se.
The next t r i a l , known a s *0*, m s ru n a s b efo re, except th a t some
so lid calcium carbon:-;te was added to th e medium to keep th e pH n earer
n e u tra l before the c o n tro l was ap p lied .
About 10 grams o f calcium
carbonate were added to th e th re e l i t e r s o f sodium.
A fter in o c u la tio n ,
th e c u ltu re grew lu x u ria n tly fo r 24 hours b efore c o n tro l was applied*
Some in stru m en tal d if f ic u ltie s toad a ris e n .
The pH o f th e medium a t
24 hours w ith calcium carbonate p resen t was about 5*8.
Ammonia w ater
was added as u su al fo r about s ix hours to b rin g th e pH to 7*7 fo r th e
com pletion o f th e ferm en tatio n .
tio n should have gone w e ll.
With such a stro n g s t a r t th e ferm enta­
However, ferm en tation ceased w ith 33 p e r­
cen t o f th© sugar untouched*
The n ex t t r i a l , known a s *P*, was run in th e u su al way*
The
pH m e g rad u ally brought to 7*5 very c a re fu lly over a p erio d ©f nine
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-81-
hours a f te r itx x m latio n .
But th is run was in h ib ite d a s ware th e
r e s t o f th e unsuccessful experim ents a t high pH m ines#
With such c o n siste n t unsuccessful ferm entations a t co n tro lle d
pH m in e s over pH - 7, i t was thought th a t perhaps th e d if f ic u lty could
be a sso c iated w ith th e u se o f ammonium hydroxide ra th e r th an pH i t s e l f .
Also* Krug and McDermott (1935) did n o t d e scrib e ferm entations w ith
average pH such more th an seven which were successful* although
they claim ed th e pH range from seven to e ig h t to be d esirab le#
th e re
have been numerous re fe re n c e s to a lk a lin e ferm entations l a th e lite m *
tu re , so th e pH alone i s n o t th e m use o f th e lack o f com pletion o f
th e ferm entations d escribed above#
th e c o n tro lle d ferm entations
"I* through *P* a re compared in fa b le 18*
They a re arranged in th e
o rd er o f th e c o n tro lle d pH valves*
b»
S tu d ies w ith ammonium s a l t b u ffers*
Because o f th e r e s u lts o f
th e above experiments* and because o f th e f a c t th a t only one experim ent
can be ru n a t a tin e on th e instrum ent* i t mas decided to study th e
e ffe c ts o f o th e r ammonium s a lts on th e ferm entation in o rd er to o b tain
system s o f ammonium s a l t b u ffe rs which could be used to study th e f e r ­
m entation a t v ario u s pH le v e ls in numerous fla s k s sim ultaneously.
An acid re a c tin g ammonium s a l t such as ammonium c h lo rid e m ight be
used w ith a s a lk a lin e re a c tin g s a l t such a s ammonium carbonate i s
v ario u s proportions to ask s a b u ffe r s e r ie s .
The ammonium carbonate
would ten d to hydrolyze somewhat fowning some ammonium hydras ids*
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•82**
The presence o f HS4G1 would tend to suppress th e d is so c ia tio n o f th e
ajsaoniua hydroxide th u s aaking th e pH in e ff e c t more a c id .
TABLE 18
FURS®TATIOHS HAVIHG pH ABTOmTICALIY C0STSOLI® BT AMMONIUM
HTBROXIBE
Flask* 5 lite r * >*necke&, co ntain ing 3 l i t e r s o f asdiiiB .
Mediant Optimum s e a l-s y n th e tic w ith 1 0 d ex tro se; 3 lite r* } pH * 5
a t s ta r t.
S te rilis a tio n * 30 n lm te s a t 19 lb s . steam p ressu re .
Znoealont 300 ml* o f c u ltu re Ho. 43 In 1 0 d ex tro se m edian.
Ineubation* 4 to 8 days a t 39 to 3* 0 .
Instrum ent* Cameron pH (k»ntroller—Beccrdar*
Baslo reagent* Ammonium hydrcnd.de (14 p e rc en t H8j)
S tirrin g * Automatic* 30 sec* o a t o f 4 &ln*9 and on a d d itio n o f
base*
T ria l
ps
&
5 .0
N
6 ,8
7.4
*• M
X
P
? .$
0
T .f
E thanol G lycerol B esidual
Yield*
Yield*
Sugar*
% on sugar % on sugar % o a f arm.
-
36.6^
m
-
m
li#
0 .4 ^
Kssstrics
Mechanical d if f ic u ltie s .
C ontrol l o s t . Excess
N1L0H added a a to a a tic S u ccessful.
m
Ower 50
Mechanical o p eratio n
e x c e lle n t, B issiaila**
•
Over 50
Mechanical o p eratio n
ex cellen t* t u t growth
ceased.
p re s e n t.
Mechanical o p eratio n
bad th e 1 s t day* so
th a t medium had a 24
how s t a r t o f grow th.
But a f te r pH was con­
tr o lle d a t 7*7* growth
ceased.
33
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-83-
8
# «* «*
N*1
I
KUTRISKT
QHMTITHS
OS
I”;
<£A S«U
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S0# 0-S*0S*0*l0*i 0R* 0*H0#0B* . .
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GBEEORIDE
XH
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ill.
THE EFFECT OF
MMMim
5 1
SB.
1 h 4
sn**
J•H? f0*. 0i- l0 l0*s0 • SisSSS
OOOOOO
i j l 'i
i#&§
OOOOO
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r^r4<!¥
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-84»
Xt was f i r s t d e sired to a s c e rta in What maximal co n cen tratio n o f
ammonium c h lo rid e could be used in a b u ffer w ithout suppressing o r
in h ib it i n g th e ferm entation*
Aa experim ent mas designed to shoe th e
e ff e c t o f ammonium ch lo rid e on ferm entations o f media which a re optimum
except fo r th e ammonium c h lo rid e concentration*
th e r e s u lts and d a ta
a re shown in Table 19*
O bservation o f fa b le 19 stows th a t th e sugar wm consumed s a tis ­
f a c to r ily when th e ammonim c h lo rid e co n cen tratio n was below 1*5 grams
p e r XOO al*
At h ig h er co n cen tratio n s o f th e sa lt* th e usferm entod sugar
con ten t ro se rap id ly#
There seemed to be a gradual in crease in th e
g ly ce ro l formed a s th e s a lt co n ten t increased*
in g g ly cero l a re given a lso in Table 29*
The d a ta on result**
Xt was suspected a t th is
p o in t th a t perhaps th e ch lo rid e io n s ig h t have been th e in h ib itin g
facto r# ra th e r th an both th e ammonium and ch lo rid e ions*
A s e rie s o f
ferm entations was designed to study th e e ffe c t o f ammonium carbonate
on th e ferm entatio ns by adding varying amounts to th e medium*
The
r e m its a re given in Table 20#
Xt may be noted ag ain th a t when th e pH was ap p reciab ly g re a te r
th an 7# no growth occurred#
The y ie ld o f g ly ce ro l in creased a s th e
f in a l pH in creased up to th e p o in t where growth, did n o t occur#
F lask
*0% having th e sane ammonium norm ality a s fla s k "7" o f fa b le 19 t o t
le s s chloride* had le s s re s id u a l sugar than fla s k *?*•
This was to
have been expected i f ch lo rid e io n s ware th e in h ib ito rs*
Although i t was considered th a t th e ch lo rid e io n co n cen tratio n
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
•8«£»
THE EFF1X3T OF 1MUQXXB8 CARBONA®
0» PRODUCTS, Wm%
AND p i
I
@9 ^ 0
S * ■&■&;§§
< lo S 3 3 2
4 4 * * 4
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• * I §
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«
36—
was th e in h ib itin g fa c to r in ferm en tatio n s o f a c id pH and o f r e la tiv e ­
ly high ammonias ch lo rid e concentrations# i t waa d e sired to knew th e
type and th e e x te n t o f e ffe c tiv e n e ss o f v ario u s m ixtures o f ammonium
ch lo rid e and ammonium carbonate a s b u ffers fo r p o ssib le use in f a r m entations*
The ammonium earbonate used in a l l eases was th e mono-
hydrate b u t w eights were c alcu late d on th e anhydrous basis*
Normal
TABLE 21
EFFECT OF AMMONIUM CHUORILl-AMaOHIBM CARBONATE RATIO
OS THE pH OF SOLUTIONS Of CONSTANT
AMUOHIUM W8 NORMALITI
(fo a p e n ttu re • 25® C .)
E quivalent %
of
0^
1
2
4
10
20
40
60
80
100
E quivalent %
©f N]f*Cl
pH o f Solution*
X 8# in
pH o f S olutions
0*5 n. in nhJ .
%
90
4*89
6*94
7*15
7*40
7*74
5*03
7*09
7*30
7*52
7*91
80
60
40
20
O
8*00
8*27
8*43
8*58
8*72
8.20
8.48
8*67
8*79
8.92
100^
n
n
o f both
p a y in d is t il l e d water*
c h lo rid e
carbonate were p re ­
Various m ixtures o f th e two so lu tio n s were
made up which re s u lte d in a co n stan t ammonium io n concentration# b u t
w ith th e anions o f varying eq u iv alen t proportions*
The pH o f th ese
so lu tio n s was measured a t 25° cen tig rad e by means o f th e Cameron
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-87-
g lasa e le c tro d e pH meter*
A fter th e se measurement# mere made, th e
so lu tio n s mate d ilu te d w ith an equal volume o f d is t il l e d meter*
re s u ltin g so lu tio n s were 0*5 normal in ammonium ions*
ments were ag ain node a s before*
She
th e pH measure­
The d a ta a re given in ta b le 21 and
Figure 5*
The d a ta in Table 21 in d ic a te th a t such m ixtures o f asmaoniua
s a lts s ig h t be s a tis fa c to ry fo r bu ffered pH ferm entations i f o th er
fa c to rs do not make th e ir u se im practical*
Other ammonium s a lts
such a# th e s u lfa te should a c t i s a manner sim ila r to th e a c tio n o f
ammonium ch lo rid e i n such b u ffe rs .
A f a ir ly high co n cen tratio n o f
ammonium carbonate should be used so th a t th e a c e tic aeid th a t forms
during th e ferm entation would convert only a s n a il fra c tio n o f th e
carbonate to acetate*
In o rd er to hare some d a ta regarding th e in h ib itin g e ffe c ts
o f various anions o f ammonium s a lts on a lc o h o lic ferm entatio ns o f
th e optimum medium being studied* where th e s a l t co n cen tratio n i s
g re a te r than n u trie n t requirem ents, th e ferm entations described in
Table 22 and in F igure 6 atom what s a l t o r s a lts may be used in
co ncen tratio n s approaching 1 normal*
I t was shown in Table 19 th a t
ammonium ch lo rid e eould n o t be used in co n centratio ns above about
0*25 noxm l w ithout leavin g more than 0*5 percen t o f th e sugar u n ferraented*
The c h lo rid e , s u lfa te , and n itr a te were stu d ied se p a ra te ­
ly and as m ixtures*
The r e s u lts proved th a t ammonium s u lfa te was by f a r the most
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*68**
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- 90-
9
8
E ffect of th e R atio of
Ammonium Chloride to Ammonium
C arb o n ate on pH of Solutions of
C onstant Ammonium Normality.
pH
7
(Temperature =25°C.)
6 1
% ( n h 4 )2 co3 0
% n h 4 ci
10
90
100
20
30
40
50
60
70
80
70
60
50
40
30
Mole Equivalent Percent of the Salt
Figure
80
20
90
10
100
0
5
100
Effect
80
voa>
)
£ 60
0)
on
of Vari< >us Concentration:
of Amrrionium S alts
the Ejwent ot the A lc o r lOllC
F ermenl lotion
Age: Nl-l4 CI Sfsries, 7 Days
- All Others , 6 Da ys
Nrh4 ci
/% ■
"r
—
a
/ r ' XNH4 N03
/r
|
a:
N)h4 no 3
Nr■r. c i
1
(N H ^SO *
NH. NO.
•4- -3 --Nr14 CI
>>
40
cc
o
w
a>
a 20
V
✓
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.~ w
r
^ *
4t
S
-a '
*
'
\X
A
s
_
4
^
___ (NH 4)aso4
•
N h4 no 3
r—1
^ - • - ( nh
0
0.3
Ammonium
0 .5
Ion Normality
0 .7
in Medium
0 .9
Figure 6
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aI S04
1.1
s a tis fa c to ry s& lt o f those studied* in so fa r as re s id u a l sugar was
concerned*
I t was a lso shown th a t th e ammonium io n was n o t th e
in h ib itin g fa c to r a s much a s was th e ch lo rid e ion*
Both ch lo rid e
and n itr a te were u n sa tisfa c to ry in con cen tratio n s up to about 0*5
normal* C hile th e s u lfa te m s sa tisfa c to ry *
M ixtures o f th e m rlo u e
s a lts were no t a s s a tis fa c to ry as m s aaoonina s u lfa te alone*
It
i s w ell to note th a t a l l th ese ferm entations were o f th e s tra ig h t
a lc o h o lic ty p e , i*e** th e pH was about th re e and s u l f i te was absent*
B uffer m ixtures o f ammonium carbonate and ammonium s u lfa te should
be s a tis fa c to ry fo r studying th e pH e ff e c t on g ly ce ro l form ation
during sugar breakdowns by y e a st, w ith th e ammonium io n present*
It
should be remembered th a t ammonium s a lts a re d e sira b le ra th e r than
sodium o r potassium s a lts because th e c a tio n may be removed by
v o la tiliz a tio n a f te r a lk a liz a tio n .
A complete s e rie s o f ferm entations was d esired where pH i s con*
tr o lle d by ammonium s a l t b u ffe rs where th e tren d o f a number o f
ferm entations m ight be stu d ied a t one time*
Such stu d ie s would
ta k e much too long a tim e w ith th e autom atic c o n tro lle r which can
c o n tro l but one ferm entation a t a time*
Since i t was shown in fa b le
22 th a t ammonium s u lfa te could be used s a tis f a c to r ily in concentre*
tio n s somewhat g re a te r th an 0*5 normal* i t was decided to prepare a
s e rie s of ferm en tatio n s containing v ario u s m ixtures of ammonium
s u lfa te and ammonium carbonate along w ith th e sm all amount o f
ammonium c h lo rid e added a s a n u trie n t to th e optimum medium* The
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-92-
to ta l ammonium ion co ncentratio n was made to be 0*5 normal In a l l
eases*
The experim ent I s d escribed in fa b le 23) th e r e s u lts and
im p licatio n s nay be more e a s ily observed in Figure 7*
Once ag ain i t i s obvious th a t th e ferm entations v en t to
com pletion q u ite w all a s long as th e ferm enting medium teed a pH
b ales 7* approxim ately*
As soon a s th e pH exceeded 7* th e ra te
o f sugar consumption f e l l o ff q u ite ra p id ly and th e growth was
sluggish*
Although th e media were n o t buffered a t e x a c tly fix e d pH
v alu es, th e v alu es fo r most o f th e media were n ear enough to a fix ed
v alu e so th a t tre n d s could be noted and conclusions may be draws
w ith in reasonable lim its o f erro r*
The technique used was n o t fo r th e purpose o f g e ttin g maximum
p o ssib le g ly c e ro l y ie ld s , but ra th e r to study th e ferm entative trends*
The g ly ce ro l y ie ld increased w ith th e pH a s the alco ho l decreased,
up to approxim ately pH - 7* th e more a lk a lin e media contained h ig h er
re s id u a l sugar th an th e media having pH values below 7*
E vidently something happened a t about pH » 7» when ammonium
s a lts were used in r e la tiv e ly la rg e q u a n titie s , which caused in h ib itio n
o f d issim ilatio n *
This e ff e c t was n o t shown in th e a lk a lin e ferm enta­
tio n s using soda ash*
The change th a t seemed obvious a s th e pH
approached and th en passed 7 was th e very rap id form ation o f m olecular
ammonium hydroxide which i s in equilibrium w ith m olecular ammonia*
The e n tire s e t o f e q u ilib ria i s as follow s*
♦ OB'
» BH|0H ---- - H*0 ♦ HHj{sol*a) ------
8H3 (gaa)
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100
90
80
— - - o __ Ethanol
o
0)
-C 7 0
.....................................“
%
\
60
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E ffe c t
o f pH
on
Y east
F e rm e n t a t i o n s
-----•
B uffered by V arious M ixtures o f
Ammonium C arbonate and Ammonium
S u lp h a te
/
__________________________________________
Ammonium
Ion
•
%
Normality * 0.5
•
TZL
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Dextros
5
6
pH
of
Figure
Medium
7
-O"
J
}
V
-96-
Tb» emxxolmi io n teas not a t fa u lt* near m s th e s u lf a te , as m s
shorn in Table 22.
Carbonate ions were not a t f a u lt, as soda ash
ferm entations were successful*
th e sodium its e lf *
u la r
Ammonia gas m s , o f course, out o f
The compounds l e f t f o r su sp icio n were th e molec­
hydroxide, and m olecular
in solution*
The con*
centratlon® o f th e se agents were probably no t as Im portant a s were
th e chem ical a c tiv itie s a s defined by Lewis and R andall (1923), o r
th e chem ical p o te n tia ls , a s they a re a lso known* The re la tio n o f
otwwrfwil pn-fcatrMni
b acterio lo g y h*»« been discu ssed by
and Fulmer (1930a)*
The a c tiv ity o f m olecular ammonia* fo r example, i s p ra c tic a lly
n e g lig ib le a t pH v alu es more a cid than about pH - 6*8, w hile th e
a c tiv ity begins to appear and r is e very ra p id ly as th e pH p asses ?
and goes on to 8 o r sore*
As pH « f I s reached and passed, th e
hydroxide io n co n cen tratio n in c re a se s very rap idly *
This allow s th e
form ation o f so re and so re as®oniua hydroxide m olecules which, being
i n eq uilibrium w ith ammonia, in cre ases th e a c tiv ity o f m olecular
ammonia*
The presence of ap p reciab le co n centratio ns o f a m a n ita
s a lts a t a given pH w ill have th e e ff e c t o f in creasin g th e a c tiv ity
o f Use ammonia over th a t a c tiv ity o r chem ical p o te n tia l in so lu tio n s
o f low ammonium s a l t co n cen tratio n s.
This m ight be considered in
some re sp e c ts analogous to th e in cre ase in b a c te ric id a l a c tiv ity o f
phenol so lu tio n s brought about by added sodium c h lo rid e .
The added
s a l t decreases th e s o lu b ility o f th e phenol, thu s bring ing th e r e ­
su ltin g so lu tio n n earer to satu ratio n *
The phenol th en has a
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-9 f-
g re a te r "escaping tendency®*
Such an e ffe c t s ig n ifie s g re a te r
chem ical a c tiv ity o r p o te n tia l*
The phenol Is* i s e ff e c t, d riv en
more fo rc ib ly in to th e organism*
Likew ise, afimtiia. would be le s s
so lu b le in a so lu tio n o f high m m i m s a l t co n cen tratio n th an in
one o f 2m concentration* and th e ammonia a c tiv ity would thus be
g reater*
A fix e d concentration, o f ammonia would have a g re a te r
a c tiv ity in a 2 normal so lu tio n o f sodium carbonate th an i t would
have in a 1 normal so lu tio n o f th e same s a l t - o th e r fa c to rs r e sa in in g th e same*
The a c tiv ity would, a ls o be in creased w ith an
in cre ase in pH*
This th eo ry ex p lain s every su ccessfu l and every unsu ccessfu l
fo m e n ta tio n mentioned thu s f a r where ammonium co n cen tratio n s over
n u trie n t q u a n titie s were used*
Removal o f most o f th e ammonium
s a lts should allow ferm entations a t g re a te r a lk a lin ity th an those
where ammonium s a l ts a re present*
I t w ill be noted l a t e r th a t th e
f i r s t ferm entatio n c o n tro lle d by soda ash was su ccessfu l a t pH ?*5»
Krug and McDermott (3935) obtained g ly ce ro l y ie ld s from th e f e r ­
m entation o f m olasses media which were 15*8 to 18*5 p ercen t o f th e
sugar weight* whereas th e maximum obtained a s shown in Table 23 was
about 10 p ercen t o f th e t o ta l sugar w eight, o r about 11 to 12*3 per­
cen t on th e sugar ferm ented in fla s k s *P" and «Q**
However, th e
a v a ila b le ammonium io n co n cen tratio n in th e experim ents o f Krug and
McDermott was about 0*28 to 0*28 norm al, w hile th e experim ents o f
Table 23 were ru n in media which were 0*5 normal in a v a ila b le asBsohiua
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io n co n cen tratio n .
Seer pH « 7 such o f th e erataonium io n s would be
converted to m olecular am oaiw a hydreadd* and ajmaonia* th e a c tiv ity *
©r chem ical p o te n tia l, o f which would in crease as a fu n ctio n o f in *
creasin g pH and o f in creasin g awaonlu® o r ammonia concentrations*
Thus th e ammonia a c tiv ity would be g re a te r a t a given pH i s tb s
experim ents o f Table 23 th an i s th e ferm entations o f Krug and McDermott
Since more ammonium io n s were a v a ila b le fo r ammonia form ation near
th e n e u tra l pH.
Then a lower p i would be req u ired fo r ferm entations
o f high a v a ila b le aaaaonia in o rd er to d ecrease th e a c tiv ity o f th e
ammonia enough so th a t in h ib itio n o f growth ia not predominant*
Thus
th e higher y ie ld s would n o t be expected in Table 23* A lso Krug and
McDermott used about 16 to 18 grams o f m olasses sugars per 100 cc* o f
medium* w hile 15 g rass p e r 100 ml* were employed in Table 23*
The
b io -c o llo id s o f th e m olasses may have bad some e ff e c t also# accord­
in g to Owen (1737}*
though not- Id e n tic a l*
The r e s u lts th u s f a r a re perhaps reasonable*
Higher sugar con centrations a re conducive to
g re a te r g ly c e ro l form ation according to McDermott (1929)*
He a lso
noted th a t m olasses media in the presence o f r e la tiv e ly la rg e amounts
o f sodium earbonate gave a s lig h t p re c ip ita tio n along w ith th e develop­
ment o f a s lig h t ammoniacal odor*
The ammonia a c tiv ity i s th e m olasses
s ig h t lim it th e maximum pH a t which ferm entation would be com plete.
The odor o f ammonia would be obvious evidence of i t s presence and
a c tiv ity *
An experim ent, described below in th e se c tio n d ealing w ith th e
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-9 9 -
ammonium sulfite fermentation* further showed the difficulties of
using very high concentrations of ammonium wits in fomentations
near
f o r over*
It m s desired to obtain evidence of a more quantitative nature
concerning the toxic effects of free ammonia or ammonium hydroxide
in fermentation media*
Several series of fermentations were designed
to show that at constant pH the toxicity increases as a function of
the initial a s m i t m Ion concentration*
The series were also design­
ed to show that at constant initial aaeaoHtwa Ion concentration the
toxicity increases with increasing pH and that pH » f is the critical
value*
A semi-synthetic medium was prepared which was optimum except
that a nitrogen equivalent of urea was substituted for ammonium
chloride In order to have the ammonium concentration equal to sereIt was prepared in 300-ml* Erlemeyer flasks such that 100 ml* on
dilation to 200 ml* would result in a 15 percent dextrose medium*
Ammonium sulfate was added to each flask in 20 ml* of aqueous solution
to give the media the desired ammonium ion concentrations*
sation was at 15 pounds for 15 minutes*
Sterili­
th e media of the various
series were inoculated with 20 ml* of inoculum grown 1m 20 percent
dextrose medium*
The organism was £• ceravlalae Ho* 43*
cultures were incubated for 200 hoars at 30° centigrade*
The
The pH
adjustments were made at frequent intervals by additions of solid
sodium earbonate.
Bata are given in Table 24 and in Figure 8*
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•JjQO-
TABLE 24
SUGAR COHSUMPTI0H W FER^JTATIONS AS RILAfSB
TO pH AMD AMMOHUBI CONCBRBATI0B
( ® g asQ4
pH * 8*0
H araality
Grams ©f MaaQD. R esidual
Added p er F la w D extrose,
ga./lL0Oral*
0 .0
0*1
0 .2
0 .3
0 .4
o*5
( k% )2so4
H om aH ty
1 4 .5 m »
13*0
8 .4
5*4
4*f
4 .2
pH * ?*2
Grams o f IfeuGO. R«»fi-trinc1
Added p er F lask D extrose,
p u /tO Q a l.
0*0
0 .1
0*2
5*3 gm*
4 .7
0*3
4*4
4*1
3*8
0*4
0*5
(H%)2SD*
H o rm llty
0*0
0*1
0 .2
0*3
0*4
0*5
2.34 p*.
3*50
?-78
11.36
12*36
13.00
4*5
0*50 g * .
0*05
0*f6
5*44
9*24
10.70
pH * 6*8
Grams o f HeuOO. R esidual
Added p er F la w D extrose,
gffi*/LO0al»
2*1 pi*
2*4
2 .3
2*3
2 .2
2*2
0*40 gm.
0*18
0.19
0*30
0*61
2*89
pH * 7 .5
Grass o f Hb£@0« R esidual
Added p er F lask Deartaroae,
gffi./LOGral*
11*3 8®*
20*3
3*0
4.8
3*4
3*2
1*25 s®*
1*044
f.4 6
11.92
14*26
14*50
pH - 7 .0
Grams o f 8&J3CL
Added p e r F le a s Dextrose*
gsa./iOOal.
3*5 g®*
3*4
3*4
3 .3
3*3
3*2
0.74 gn.
0*16
0.41
0*32
3-34
3*22
pH * 6*5
Grams o f Sft*00* R esidual
A44_a ---- W1**
J*JUB>
U&
88*
gm./LQCtal.
0*98 gs*
1*03
3*05
1.16
1*16
1.21
0.12 gB*
0*31
0*13
0.11
0*14
0*16
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•a o i-
S u g a r Consumption i n ___
F erm e n ta tio n s a s
Related to pH and
Ammonium C oncentration
Initial Dextrose=l5 gm. per 100 ml.
a. 8
cn
x6
-'Y
I
0.2
N orm ality of
0.3
(NH4 )2 S 0 4
Figure
pH =6.5
0.4
in M
edium
8
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0.5
-202-
ffem th e r e s u lts in d icated in Table 24 and in F igure % I t i s
obvious th a t a t co n stan t pH near o r above 7# in creasin g th e ammonium
co n cen tratio n decreases d issim ila tio n o f th e dextrose*
One aay a lso
conclude th a t a t co n stan t ammonium co n cen tratio n , in creasin g pH de­
c reases th e sugar d issim ila tio n *
T his evidence su b sta n tia te s th e
previous evidence th a t ammonia o r ammonium hydroxide can e x h ib it
to x ic e ffe c ts ©a y e a st cu ltu res*
Zt i s undoubtedly tru e th a t th e
to x ic ity i s a fu n ctio n o f th e a c tiv ity o f th e ammonia o r ammonium
hydroxide*
The ammonium io n co n cen tratio n s up to 6*5 normal were
in h ib ltiv e in a cid so lu tio n as shown by th e f a i r l y complete sugar
consumption a t pH * 6*5*
M athem atical re la tio n sh ip s could be derived*
no doubt* between th e to x ic ity and ammonia a c tiv ity *
However* sin ce
th e main purpose o f th e to x ic ity in v e stig a tio n s was attain ed * no
fu rth e r work in tho se lin e s was considered necessary*
Zt may be noted in Table 24 th a t th e sugar was g en erally eonsumed more e x ten siv ely when th e ammonium io n co n cen tratio n was
0*1 normal than when i t was zero*
I t should be remembered th a t
u rea was su b s titu te d fo r ammonium ch lo rid e in th e i n i t i a l optimum
medium* A d isc u ssio n concerning th e u se o f ammonia and u rea in th e
n itro g en m etabolism o f y e a st was given by S chu ltz, A tkin and Frey
(1940).
Thus* sin ce i t was proven beyond doubt th a t th e use o f ammonium
hydroxide and o th e r ammonium ag en ts f o r a lk a lin e ferm entations was
im practical* a tte n tio n was focused on o th er types o f ferm entations
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4S>
f o r g ly c e ro l production*
jt.„ .H a , M»,,gC .8aa4m,,aMr)aflaft1a»
Sodium carbonate has been consider­
ed very e x te n siv e ly , a f te r th e work o f Boff (1918) and o f E off,
U nder and Beyer (1919)* a s an ag en t to taring about th e form ation o f
in creased amounts o f g ly cero l from th e a lte re d a lc o h o lic ferm entation*
The sodium carbonate process baa been g e n erally im p ra c tic a l because
o f th e recovery d if f ic u ltie s p rev io u sly mentioned which were caused
TABLE 25
FERMENTATION AUTOMATICALLT CONTROLLED AT pH 7*5 BX SODIUM CARBONATE
SOLUTION IN THE ABSENCE Of APPRECIABLE AMMONIUM SALTS
Flask*
5 l it e r * 3»Beckedv co ntaining 3 l i t e r s o f lf $ medium a t
s ta r t*
Medium* 15$ dextrose* optimum except th a t HH^Cl was replaced by
a n itro g e n eq u iv alen t o f urea* to remove ammonia effe ct*
pH » 5 in itia lly *
S te riliz a tio n * 30 m inutes a t 10 lbs*
I nocaliaa* 300 ml* a t W$> d extrose cu ltu re* le a s t No* 43*
Incubation* $ l/2 days a t 32 0*
Aeratlon* m ild and in te rm itte n t fo r the f i r s t 7 hours*
Instrum ent* Cameron pH R eoorder-C ontrdler*
R esidual Dextrose* 0 .35^ o f th a t o rig in a lly present*
G lycerol found* 70> o f th eo ry , o r
o f the sugar weight*
Sodium carbonate* added by so lu tio n (150 gm* Ha2CQ, p e r l i t e r o f
so lu tio n )
by th e high s a l t co ncen tratio n s necessary*
As wee in d ic a te d under th e ammonium hydroxide se c tio n , ap p reci­
a b le q u a n titie s o f ammonium s a lts appear to cause in h ib itio n o r
suppression o f ferm entation when th e pH allow s a c tiv e ammanl* to be
present*
A t r i a l ferm entation a t an au to m atically c o n tro lle d pH
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—
204—
value o f 7*5 was d e sire d , b a t so ferm entation had been completed
su c ce ssfu lly w ith an ap p reciab le ammonium io n co n cen tratio n p resen t
a t th is degree o f a lk a lin ity *
Three l i t e r s o f o p tio n median were s te r ilis e d in a 5 * ltts %
3-necked flask *
The experim ental d a ta a re given In liable 25*
The ferm en tatio n p a r tia lly describ ed in Table 25 was autom atic
e a lly c o n tro lle d a t pH * 7*5 by a sodium carbonate so lu tio n contain*
le g 150 grans o f th e
anhydrous s a l t
p er l i t e r o f so lu tio n *
The f e r -
m entation was f a i r l y
rapid* The pHwas co n tro lled a t 6*8 f o r about
th e f i r s t 20 hours, to allow a good growth o f y e a st to g e t under
way; then th e pH was
held a t th a t value*
changedto 7*5
over a period o f two hours and
fifty -o n e hours a f te r in o c u la tio n , 1660 ml* o f
th e a lk a lin e so lu tio n had been added, corresponding to 249 grams o f
sodium carbonate*
The t o ta l volume in th e ferm entation fla s k was
ra p id ly approaching th e 5 l i t e r fla s k cap acity by th is time*
fo re , 500 nl* o f th e m ixture was removed a t th is point*
There­
T his pro­
cedure caused com plications in th e a n a ly tic a l computations*
In 96
hours a t o ta l o f 1945 ml* o f th e carbonate had been added and th e
ferm entation m s complete*
Keeping th e sodium carbonate so lu tio n
in a graduated c o n tain er allowed a v is ib le record o f th e r a te o f
consumption o f th e s a l t so lu tio n to be kept*
The r a te o f consumption
corresponded to th e r a te o f ferm entation*
The low y ie ld o f g ly c e ro l i s n o t su rp risin g f o r th is ferm enta­
tio n as th e pH was 6*8 fo r about 20 h o urs.
A lso, th e a d d itio n o f
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-205-
aodiua carbonate by d ilu te so lu tio n i s not conducive to high g ly c e ro l
y ie ld s according to McDermott (1929) a s decreases were g eneral when
d ilu te so lu tio n s were used*
S olid sodium carbonate was g en erally
used when h igher y ie ld s were obtained*
However, i t was th e f i r s t
su ccessfu l au to m atically c o n tro lle d ferm entation a t a pH a s a lk a lin e
e s 7*5* toward th e com pletion o f th e ferm entation a f te r 90 hours
o f incubation and c o n tro l had occurred* i t m s found th a t th e con­
tr o ll e r m s so lo n g er needed*
Ho more soda ash m s added a f te r th a t,
and th e pH g rad u a lly in creased to 7*9*
The fo m e n ta tio n may h are
been complete in 90 hours*
fhe use o f sodium carbonate w ith anraonium s u lf a te i s ferm enta­
tio n s m s described in th e d isc u ssio n concerning th e d a ta o f Ifebls
24*
d*
th e u se o f sodium hydroxide.
Since a s extrem ely concentrated
so lu tio n o f sodium carbonate could n o t be prepared s a tis fa c to rily *
th e d ilu tio n o f th e pH c o n tro lle d medium described p rev io u sly was
an un d esirab le re s u lt*
The low g ly ce ro l y ie ld was presumably due to
th e d ilu tio n o f th e ferm enting medium
However, a much so re concentrated
so lu tio n may be made employing sodium hydroxide in p lace of aodlua
carbonate*
P
The volume o f so lu tio n req u ired fo r autom atic pH c o n tro l
would be much le e s using concentrated sodium hydroxide both because
o f th e h ig h er co n cen tratio n p o ssib le and bem use o f th e g re a te r
a lk a lis in g cap acity o f th e pure base*
An autom atic pH c o n tro l was attem pted on 3 l i t e r s o f optimum
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-106-
medium where th e sodium hydroxide so lu tio n employed f o r pB c o n tro l
had a co n cen tratio n o f 0*5 gt»* P*r ®1* th e f i r s t attem p t ended l a
f a ilu r e when a re la y broke and a l l o f th e base in th e re s e rv o ir
emptied la te th e ferm enting medium* Ferm entation eeased*
The second attem p t mas successful*
There ware prepared 1*5
l i t e r s o f optimum sen d -ey n th etic medics containing 15 p e rc en t dextrose*
S te r ilis a tio n was fo r 20 m inutes a t 15 pounds ste e a pressure*
In ­
o cu latio n m s w ith 200 ml* o f a 48 hour c u ltu re o f y e a st Mo* 43*
The inoculum medium was i n i t i a l l y 10 percen t in dextrose*
cubation tem perature m s 30° centigrade*
The in ­
Over a p erio d o f s ix to u rs
a f te r inoculation* th e pH was g rad u ally increased to 8 by toad*
The
pH c o n tro l was th en s e t a t pH * 8* At in te rv a ls th e c o n tro l was
c u t o u t and th e decrease in pH a s shown on th e record m s an in d ic a ­
tio n o f ferm en tativ e a c tiv ity *
encountered*
Very l i t t l e m echanical d if f ic u lty was
A fte r about 80 hours o f ferm entation* th e r a te o f bam
consumption f e l l o ff In d ica tin g th a t th e ferm entation was n early
completed*
analysed*
At 218 hours th e c o n tro l was stopped and th e medium m s
The volume o f base added au to m atically m s 175 ml*
The
t o ta l volume o f re s u ltin g medium was 1875 ml** accounting fo r both
th e inoculum and added b a sic solu tio n*
The dextro se co n cen tratio n
I n i ti a l l y , c a lc u la te d on th e b a sis o f th e f in a l volume, m s 12*3 gm*
p e r 100 ml* o f medium* A nalysis showed 2*5 p ercen t re s id u a l d ex tro se
and a g ly cero l y ie ld o f 22*8 p ercen t on th e sugar weight*
This m s
p ra c tic a lly tw ice th e g ly ce ro l y ie ld found in th e pH c o n tro lle d f a r m entation using a sodium carbonate so lu tio n fo r control*
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-3© f-
U Q A -flT & te ra U
at, ,Thft gflft ffiC B8«»Bfo« guAfrUfl*
The u se o f m m aSim s u lf ite and o f
aaraoniuBi b is u lf ite had n o t Iwm aentioned in lite ra tu re * previous to
a note in th e a n a ly tic a l paper o f F a ls e r, Hickey, and U nderkofler
(1940)*
Water so lu tio n s o f ajBoonium s u lf ite a re m t a s a lk a lin e as
a re e a te r so lu tio n s o f sodium s u lfite * though they a re alk alin e*
The u se o f amraonluHi s u lf ite and b is u lf ite was considered a s a
p o s s ib ility f o r th e s u lf ite ferm en tatio n as th e added s a lts would
be ra th e r e a s ily renewed* presumably#
The oonteapl& ted s a l t removal
method would o o n sist o f th e a d d itio n o f e a le iu a oxide o r calcium
hydroxide to th e fom ented medium th u s p re c ip ita tin g calcium s u lfite *
F iltr a tio n would reaeve th e p re c ip ita te * and carbon dioxide could be
used to remove th e excess lim e.
D is tilla tio n o f th e f i l t r a t e would
remove v o la tile m a te ria l including ammonia which might be recovered
and reused*
The resid u e should be a g ly ce ro l syrup containing
m ainly n u trie n t s a lts u n less a ssc c h a rin lc m a te ria l such a s m olasses
m s used.
Saw m a te ria ls m ight be m olasses, pure sugars* o r hydrols*
ammonia* s u lfu r dioxide* and e a le iu a oxide o r hydroxide*
The ammonia
and s u lfu r dioxide m ight be used e y c lid y i f e ff ic ie n t enough re*
covery methods were obtained*
Automatic pH e o n tro l using s u lfu r
dioxide a s th e c o n tro llin g c h ea ic al would be d esirab le*
The ferm ent*
ing mediua becomes wore a lk a lin e a s fo m e n ta tio n proceeds sin ce th e
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-3S8-
b is u lf ite becomes tie d to th e aldehyde and ammonium carbonate o r
bicarbonate r e s u lt, thus in creasin g th e pi*
Hence, s u lfu r dio xid e
would be used l a g re a te r amounts than would ammonia*
For prelim in ary experim ents anmonitna s u lf ite m nohydrate was
prepared by running su lfu r dioxide gas in to concen tra te d ammonium
hydroxide so lu tio n u n til th e ammonia odor disappeared*
Then enough
ammonium hydroxide was added to ag ain bring about th e odor o f ammonia*
This p recau tio n was to remove th e p o s s ib ility o f b is u lf ite form ation*
TABUS 26
A0CUSATIZATIOM OF XK0CUUJ11 WITH AMMONIUM S&lttTB, GBSS8FIH3 pH
Medium* One 250 ml* fla s k containing 1$) ml* o f optimum medium,
1 0 In dextrose*
Inoculum* W ml* o f c u ltu re No* 43 in $% d ex tro se medium*
Incubation* 75 hours a t 30 C*
A®aoniua s u lf ite added*
added a s & e o lid j 5 gm* added
over a period o f 28 1/fe hours In 1 gram portions*
Time
pH
Growth
vwiw aww*
27 hr*
# 6 1/2 hr*
74 hr*
6.12
6*18
6*4?
cood
aatrf
mood
^^eneee
The a d d itio n o f e th y l alco h o l to th e c h ille d m ixture re su lte d in an
e x c e lle n t y ie ld o f pure w hite c ry s ta ls which were f ilte r e d and dried*
T his s a l t was prepared sin ce non® was a t hand and th e p rep aratio n was
simple*
In o rd er to a cc lim atise th e y e a st c u ltu re Ho* 43 to th e so lu b le
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- 109-
anaonlum s u lfite * a s was recommended by Muazey (1932)* a fla s k o f
optimum medium was prepared and tre a te d a s described in Table 26*
Z t m s found to be necessary to add th e s u lf ite i s m a ll q u a n titie s*
a s th e a d d itio n o f th e e n tir e amount in one a d d itio n g e n erally stepped
a l l growth*
Z t nay be noted th a t pS in creased w ith tim e according
to th e experim ent o f Sable 26* A very a c tiv e growth was noted
throughout*
The above sodium was used as an inoculum fo r fo u r fla s k s which
were to co n tain varying amounts o f ammonium s u lfite *
To each fla s k
containing 300 ml* o f 1$ p ercent d ex tro se madias was added 10 g ra ss
o f ammonium s u lfite *
The pH o f each fla s k m s measured before In ­
o cu la tio n and was found to be 7*44*
Then 30 ml* o f th e inoculum
ware added to each fla a k j th e pH m s then found by g la s s e le c tro d e
to be f*38*
At th e end o f fo u r days o f in cubation a t 2$* cen tig rad e
th e re was found to be p ra c tic a lly no ferm entation*
Growth had
ceased*
This experim ent was attem pted b efo re th e in h ib itin g e ffe c ts o f
ammonia under a lk a lin e pH co n dition s were noted a s described in th e
amffioniura hydroxide se c tio n .
However* i t i s u se fu l aa fu rth e r proof
o f th e in h ib itin g c h a ra c te ris tic s o f m olecular ammonia o r ammonium
hydroxide in so lu tion *
F u rth er work rem ains to be done on ferm enta­
tio n s w ith ammonium s u lf ite to prove th e m in e o f such a ferm entation*
However* I t was shown th a t yeast- w ill ferm ent d extro se in th e p re ­
sence o f th is s u lfite *
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-010*
It, „IfrmML
.miZUm* ®» »»• °t ©aids**
and magnesia* s u lf ite s fo r acetaldehyde fix a tio n in ferm entations
had been noted fcy Neuberg and R ein fu rth (1919)*
They a ls o mad© a
few stu d ie s using sin o s u l f i te , b u t th e r e s u lts u sin g t h is reag en t
s e re n o t promising*
They found th a t a g ita tio n induced Increased
acetaldehyde fix a tio n during ferm en tation s in th e presence o f s lig h t­
ly so lu b le s u lfite s *
They a ls o founi th a t th e use ©f fre s h ly pre­
c ip ita te d calcium s u lf ite in ferm entations re s u lte d in h igher a e e ta ld e byde fix a tio n than d id th e use o f th e commercial anhydrous sa lt*
The stu d ie s o f Meuberg and R einfurth were d e ig n e d to g iv e fu rth e r
evidence concerning th e proof o f th e sugar d issim ila tio n mscftetti -ffW*
Acetaldehyde was ap p aren tly th e main product beiz^ studied*
Nothing
was in d icated a s to p o ssib le In d u s tria l a p p lic a tio n fo r th e production
o f glycerol*
Two experim ents wore run hy Meuberg and R einfu rth where attem pts
were made to a d ju s t the a c id ity o f th e ferm enting media i s th e pre­
sence o f o a le im s u lfite *
In one experim ent, th e a d d reag en t used
was prim ary potassium phosphate? in th e o th er experim ent th e acid re ­
agent was phosphoric acid*
a top yeast*
The organism used fo r ferm en tatio n w&s
The ferm entations were completed in about 26 and 42
days resp ectiv ely *
The acetaldehyde found was 14*86 percen t and
15*22 percen t o f th e hexose w eight, re sp e c tiv e ly .
A purpose o f th e se
experim ents was to show th a t acetaldehyde fix a tio n occurred in a d d
media*
Such methods would not be in d u s tria lly p ra c tic a l because o f
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lo ng tim e involved.*
The ex ten siv e employment o f reag en te such as calcium end magnesium
s u lf ite s fo r ferm entations producing g ly ce ro l has n o t bees reported*
The use o f such reag en ts would have th e advantage o f easy removal
fro® ferm en tatio n media In contrast- to th e d if f ic u ltie s Involved
when sodium s u lf ite and b is u lf ite a re used.
I t I s g en erally acknow­
ledged th a t th e b is u lf ite Ion i s necessary fo r acetaldehyde fix a tio n
in th ese ferm entations* and i t I s ra th e r u n c ertain what th e b is u lf ite
io n co n cen tratio n in so lu tio n would be i f an access o f e a le iu a o r
magnesium s u l f i te were added to a fo m e n ta tio n mediwa.
The s o lu b ility
o f th e s u lf ite would be an im portant fa c to r to consider* a s w ell a s
th e e ffe c t o f a c id s formed by ferm entation on th e s o lu b ility o f th e
s u lfite *
The e ff e c t o f carbon dioxide on th e s a l t would be expect*
ed to be ra th e r sm all in s lig h tly acid so lu tio n s sin c e su lfu ro u s
a cid i s a stro n g e r acid than i s earbonle acid*
I t i s obvious th a t
decreasing th e pH o f the m ixture would in crease th e b is u lf ite Ion
c o n cen tratio n .
The id e a l s itu a tio n would be such th a t th e re would let
a v a ila b le s u ffic ie n t b is u lf ite io ns to f ix a l l o f th e acetaldehyde
and s t i l l not enough to e x h ib it to x ic c h a ra c te ris tic s *
An approach
toward th e id e a l n ig h t be made by pH c o n tro l u sing s u lfu r dioxide
as th e pH c o n tro llin g agent in th e presence o f calcium o r magnesium
s u lfite *
An a cid pH should be m aintained*
The id e a l pH v alu es would
be expected to be somewhat d if f e r e n t fo r calcium and magnesium
s u lf ite s because o f the e ffe c t o f th e ir d iffe re n t s o lu b ilitie s on
th e b is u lf ite io n co n cen tratio n .
The s o lu b ilitie s o f calcium and
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-322-
aagnesiaa s u lf ite s a re given in Table 2?*
m m 27
SQLSBILITIES OF CALCIUM AND MAGNESIUM SCUTIES A® BISULFXTES
S o la b ility in P arte
p er 100 P arte HgO
.
CaSG3*2H a0....................
......0 * 0 0 4 3 a t 18° C.
CfeS03* W
0.0027 a t 90° C*
Hi®®3
**♦*.
. . . . 1*25 eold
MgS03.6% 0 *..................
.....0 * 8 3 3 hot
GaCaSOj)*
.....s o l u b l e ; e x is ts in so lu tio n o n ly .
%(HS03)2 *................................ ...s o lu b le ; e x is ts in so lu tio n only*
S ato teU ltx J jl
,jg aM la aa
GaS0|
................. 0*0625 gs* In 1 l i t e r 3 0 sugar sola*
CeSO,
........................ .. 0*800 ga* In 2 l i t e r 30$ sugar sola*
3
(18 0*)
0.533 g®* GaSOj d ieso lv ee in 200 ee* % 0 eo ntaining 9 gn* S02*
(Adapted from H e lle r (1930))
A aethod f o r g ly e e ro l production u tiliz in g s u lfu r d io x id e In
connection w ith calcium o r magnesium s u lf ite s would be re la te d in
a way to th e aethod of B arbet (192$) who used on ly s u lfu r dioacida
(o r sulfarouB a c id ) in fern en tatio n s*
Free eu lfu ro u s a d d would be
r a th e r d i f f ic u lt to use* probably* because o f th e to x ic ity o f high
b is u lf ite c o n cen tratio n .
I f th e co n cen tratio n were kept low enough
to be non-toxie* then th e re would a o a t probably be low acetaldehyde
fix a tio n .
The la c k o f ex tensiv e in d u s tria l u tilis a tio n o f th e method
o f B arbet s ig h t be a. p o in t o f evidence supporting th is co n ten tio n .
The v o la tility o f s u lfu r dioxide would be an advantage in g ly ce ro l
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-113-
recovery compared w ith th e methods necessary when sodium s a l ts a re
used*
The s o lu b ilitie s o f calcium and magnesium s u lf ite s In d icated in
Table 2? were n o t given m functio n * o f pS .
Zt was deemed d e sira b le
to determ ine th e s o lu b ilitie s o f th e se s a l ts as fu n ctio n s o f pH*
sin ce they a re to be used subsequently under c a rio u s pH conditions*
The s o lu b ility o f both Merck’ s anhydrous calcium s u l f i te and
fre s h ly p re c ip ita te d ealclum s u l f i te were stu died a* pH fu n ctio n s
a t 25° centigrade*
S atorated aqueous so lu tio n * were prepared where
to e pH values were ad ju sted by means o f s u lfu ric acid*
The m ixtures
TABLE 28
SOLDBZLITIES OP HIBMTED AMD AHHBDBOUS CALCIUM
SULFITES AS FUKCTIONS OF pH
Anhydrous CaSOj
pa
Ml* o f 0*1 M l*
S olu tio n Consumed
by 10 ml* a t Sul­
f i t e Solution*
Hydrated GaSOj
Grams p er 106
Ml. o f 0*1 H I 2 drams
ml* o f D isso lv S o lu tio n Consumed p e r 166
pH by 10 ml* o f
m l. o f
ed CftS6|
(c a lc u la te d }
S u lfite S o lu tio n B lssolw ed CaS03
(calcu lated }
3.55
3.T8
4.00
4*12
4.29
16*6
12*44
8*n
3*81
3*95
4*31
4.81
5.45
16.71
9*71
5 .04
2.56
1.08
1.002
0.583
7 .4 4
5*86
0.996
0*746
0.538
0.446
0.352
4.49
4*62
4*91
5.42
6.42
7-52
4 * |6
3*89
2*20
6*98
0 .1 5
0*05
0.270
0*233
0.132
0.059
0.O09
0*003
5*81
6.44
7.68
0.48
0.28
0*04
'4*
0.029
0 .0 1 7
0.0 0 2
-
0.354
0.065
*
m
m
0 .3 0 2
•as
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■m
- 214-
were shaken a t In te rv a le over a p erio d o f teal hours*
were determ ined by means o f th e Cameron pH Meter*
The pH v alues
th e d isso lv ed s u l-
f i t s was determ ined v o lm e trlc a lly by t itr a tin g 20 ml* samples o f
th e d e a r so lu tio n s w ith a 0*1 normal io d in e solution*
so lu tio n was used a s an in d icato r*
S tandi
On m il l il i te r o f th e standard
io d in e so lu tio n i s eq u iv alen t to 0*0060 gram o f Q&leium s u lf ite
according to th e equation*
®&S®3 ♦ *2 ♦ % 0 —
►CaSO* * 2HX
The d a ta f o r th e s o lu b ilitie s o f e a le iu a s u lf ite a re given in
Table 28 and in F igu re 9*
The s o lu b ility o f magnesium s u l f i te was measured a s a fu n ctio n
o f pH in a manner analogous to th e methods used f o r ealdUas s u lf ite
determ inations*
S u lfu ric acid was used fo r pH adjustm ents, and th e
pH was measured a t 25° cen tig rad e a s b efo re.
One m i l l i l i t e r o f 0*1
normal io d in e so lu tio n i s eq u iv alen t to 0*0052 gram o f magnesium
s u lf ite (MgSOj}* E&jwrimental s o lu b ility d a ta a re given in fa b le
29 and in F igure 9.
The r e s u lts shown in Tables 28 and 29 and in Figure 9 show a
sharp in crease in th e s o lu b ility o f calcium s u lf ite as th e pH
decreases below s ix , and in a s im ila r in cre ase in th e s o lu b ility o f
magnesium s u lf ite was noted a t about pH « 6*5*
I t was considered
th a t as th e pH went below th ese v a lu e s, in creasin g acetaldehyde
fix a tio n would occur along w ith in creased to x ic ity *
P ate e re given
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
R elatio nsh ip B etw een
S u lf ite S o lub ility an d
0.8
Q.
• Anhydrous Ca S 0 3
o H ydrated Ca S 0 3
0.6
CJ>
= 0.4
Ca S O 3
tn
Q 0.2
Figure 9
«2JUs*»
subsequently re la te d to th is consideration*
I t m s thought th a t perhaps th e presence o f acetaldehyde would
in cre ase th e s o lu b ility o f calcium s u lf ite bem use o f th e form ation
o f t i e a c e ta ld e h y d e -b isu lfite complex*
Some q n a n tita tim d a ta oca-
oerning such a s o lu b ility re la tio n s h ip to pH m s desired*
Such r e ­
a c tio n s occur in ferm entation media* and i t m e hoped th a t soa©
TABLE 2f
TW 80LUBILITX OF MGHESI0M SOU'ITE AS A FOTCTION OF pH
pH
Ml. o f 0 .1 N. I 2
S o lu tio n Consumed
by 10 m l. o f S u lfite
S o lu tio n
Oram p e r ICO m l.
o f D issolved J&SO**
(c a lc u la te d )
6.16
6*f0
21*60
19.08
10*83
1 .3 ?
i.to »
0.??2
0*564
0*384
T.2T
T.81
8*32
7*36
7.2?
7.28
0 .3 J3
0*380
0.37?
5.35
%n
inform ation m ight be obtained which would be o f v alu e in ferm enta­
tio n s fo r g ly ce ro l production*
S tudies were made w ith sa tu ra te d hydrated calcium s u l f i te solu ­
tio n s in th e presence o f a f ir e d con cen tration o f acetaldehyde but
w ith varying pH le v e ls*
Aqueous so lu tio n s co ntaining an excess o f
so lid calcium s u l f i te were prepared*
These so lu tio n s were ad lu sted
to v ario u s pH le v e ls by means o f s u lfu ric a d d solution*
Acetaldehyde
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added to th ese so lu tio n s a s an aqueous so lu tio n so th a t th e re
was m f in a l co n cen tratio n 0*7? gm» o f acetaldehyde p er 100 ml* o f
solution*
On th e a d d itio n o f th e acetaldehyde th e pH o f th e solu­
tio n s in creased markedly*
The pH ro se in one so lu tio n from 3*6 to
8*5* and from 7*68 to 10*5 in another so lu tion *
th e acetaldehyde un­
doubtedly combined w ith th e a v a ila b le b is u lf ite io n s th u s causing an
in crease in th e pH*
The fre e s u l f i te was determ ined by d ire c t io d in e titr a tio n * and
th e combined s u l f i te m s determ ined by fu rth e r io d in e t i t r a t i o n upon
th e a d d itio n o f excess s o lid sodium bicarbonate to th e solution*
Data
a re given in Table 30 and in fig u re 30*
The r e s u lts o f fa b le 30 and F igure 10 in d ic a te a g re e t in crease
in th e s o lu b ility o f calcium s u lf ite In th e presence o f acetaldehyde
a s compared to i t s s o lu b ility in w ater (see Table 28)«
I t may be
noted* e sp e c ia lly in F igure 1% th a t th e a e e ta ld e h y d e -b ia u lfite complex
commences to decompose w ith in creasin g ra p id ity as th e pH in cre ases
p a st 4*5*
This f a c t i s in d icated by th e decrease in fix e d s u lf ite
w ith an in cre ase in pH p o st 4*5*
A p relim in ary pH recording was made on 1*585 l i t e r s o f 15
p ercen t dex trose medium w ith 160 go* o f M erck's anhydrous calcium
s u lf ite added*
o f y e a st Ho* 43*
Inoculum w&s 15$ ml* o f 10 percent d ex tro se c u ltu re
The I n i t i a l pH was 6*6* At 24 hours th e pH was 4*5
and a t 48 hours th e pH was 5* At about 57 hours th e re was & sharp
ra te o f pH change and pH * 6*5 was sh o rtly reached*
At % hours
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•*H8*>
th e pH was 6.9*
A nalysis showed 0*8 p ercent re sid u a l dextro se and
a y ie ld o f 8*81 p ercen t of th e dextro se w eight as g ly cero l*
When
th e pH was ? o r store* th e odor o f acetaldehyde was q u ite apparent*
TABUS 30
smmmn
op calcium s u if it e a s a pusctdoh op pH in the
PRESENCE OF A CONSTANT CONCENTRATION OF ACETAILEHHJE
f Ml* o f 0*1 H. Ml. o f 0*1 S.
la f o r T etal
S u lfite in
S u lfite in
10 tCU o f
10 ffll* o f
S olution
S olu tio n
pH 12 f o r F ree
HI* o f 0*1 «•"
I 2 fo r Gem*
bined S u lfite
in 10 m l. o f
se ln tlo fi
F re e m m *
ae CaSOj*
Qa* p e r 100
m l.
Coabined'
S u lfite a s
OaSOy*
Qs. p er 100
sal*
1*21
3*22
3*64
4.19
1*52
1*88
1.87
1*72
27*93
28.05
23*02
28*72
28.41
26.19
26*39
27*00
0.091
0*112
0.108
0*303
1*59
1*57
1.58
1.62
4.33
4*85
5*38
8*46
1*75
0.62
0 .21
0 .21
28*43
27*05
26*52
24.87
26*88
26*43
26*31
24*66
0.105
0.037
0*013
0*013
1*60
1.59
1.58
1*48
8.71
7.18
f.8 0
8*80
0*19
0.15
0.12
0*08
22*83
21*27
13*21
8*48
22.64
21.12
13*09
8.42
0.011
0.009
0*007
0*004
1*36
1.27
0.79
0*51
S sp erisen ts were run to check th e statem ents of Neuberg and
R einfurth (1919) th a t fre s h ly prepared e a le iu a s u l f i te was b e tte r
fo r acetaldehyde fix a tio n than was th e commercial anhydrous s a lt*
The fo m e n ta tio n s were conducted a t v ario u s pH v alu es to see i f
th e re were v a ria tio n s in g ly ce ro l production a t d iffe re n t pH le v e ls
fo r th e d iffe re n t typ es o f e a le iu a s u lfite *
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.0
!
Total
Sulfite
Combined
Sulfite
CL
O'
Solubility
of
Calcium
S u lfite
ro
CO
O
0.8
T3
As a Function of pH in the P resen ce
of a C onstant C oncentration of
A cetaldehyde.
CH3 C H 0 = 0 . 7 7 gm. per 100 ml.
0.4
Free
Sulphite
Figure 10
•220-
Optimum sem i-sy n th etic medium was prepared con taining 25 p e rcen t dextrose#
Two hundred ml* o f th is medium were placed in each
o f 20 Erlenisieyer fla s k s o f 300 ml# cap acity and s te r ilis e d in th e
u su al way* Twenty m l. o f 10 p ercen t dex trose Inoculum o f y e a st So#
43 were added to each flask *
in Tables 31 and 32*
Calcium s u lf ite m s added a s shown
The in creased so lu tio n volumes in Table 32
over th e volumes in Table 31 were due to th e fe e t th a t th e added
e ale iu a s u lf ite o f Table 32 was damp#
Incubation was fo r fo u r
TABUS 31
EFFECT OF pH OS GLTCIRQL HELD AND QM SUGAR CQMUMFflOH IS
FERMSlimONS ZH THE PRESENCE OF AHHHJR0OS CAESIUM SUIFITS
T o tal D extrose Weight o f F in a l Volume Percent G lgeerol Found
CaS03
E xclusive o f B esidual % oa Em* % on
oculum,
p er F lask , S o lid ,
D extrose tro s e
T o tal
Crams
Grams
Ml*
Consumed Dextrose
pH Including In -
4*0
4 .5
5*0
5*5
6*0
33
33
33
33
33
20
20
20
20
20
days a t 30° centigrade*
220
220
220
22Q
220
80.6
3*06
0*94
0*54
0*54
24,7
11*00
10*31
10*68
10*39
4.79
10*66
10.20
10.68
10*31
The fla s k s were freq u e n tly shaken by hand*
The pH was a d ju ste d a t In te rv a ls by s u lfu ric acid ad d itio n s*
The r e s u lts in d ic a te th a t anhydrous calcium s u lf ite i s not a s
e ffe c tiv e as i s th e hydrated s a lt in acetaldehyde fix atio n *
These
observation* v e rify th e observ ation s o f Beuberg and R ein furth (1919)*
I t m s a lso observed th a t decreasing pH in the range stu d ied using
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anhydrous e a le iu a s u lf ite did not appreciably a ffe c t th e g ly ce ro l
y ie ld o f experim ents in which th e sugar m s consumed f a i r l y complete­
ly*
However, when th e hydrated s a l t m s used, an in cre ase in g ly cero l
y ie ld m s noted w ith decreasing pH*
TABIE 32
EFFECT OF pH OH GIXCIROL IIELD ASP OH SUQ&R CQKSDMFTIOH IN
flWfflraJtTIOKS IN THE PRESENCE OF BXDMTS3 0&1GXHJS SfJIFITE
S c ta l D extrose
pH TamlwtHwg Tf%»
oculu%
Grams
4*0
4*5
5*0
5 .5
6*0
33
33
33
33
33
Weight o f F in a l fa^ at% P ercent ( ^ c c r o lF o w r a ...
OaSOa p e r
italia* o f Rma-fdmal
pn Dw» ^ ~pa
F lask , An* S o lid ,
D extrose tro s e Oon* T o tal
hydrous
MOL*
smasd
Be®*
B asis,
tre e s
Grass
20
20
20
20
20
245
245
245
245
245
34*0
36*0
0.89
0*86
0*594
27.8
26*4
14*23
12*38
11*39
4*45
3* TO
14.1
12*2?
11*31
I t i s very probable th a t continuous a g ita tio n and th e use o f
la rg e r q u a n titie s of e a le iu a s u lf ite would have Increased th e
g ly c e ro l y ield s*
In th e experim ents described in Tables 31 &ad 32, i t i s noteworthy
th a t a sharp decrease in th e sugar consumption occurred as th e pH went
from 5 to **5 and below* B is u lf ite io n to x ic ity was no doubt th e
cause o f th ese re su lts*
A m u ltip le s t i r r e r m s acquired which was capable o f s tir r in g
th e media o f s ix fla s k s sim ultaneously and continuously*
A die*
advantage o f th e s t i r r e r was th e f a c t th a t i t could not be placed
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- 122-
in an in cub ator very re a d ily , so th e ferm entations were ru n a t room
tem perature which m s u su a lly about 26° cen tig rad e in ste a d o f 30 ®
which was th e incub ator tem perature*
A ferm en tatio n s e rie s m s prepared in which 118 p u o f m oist
hydrated e a le iu a s u lf ite (eq u iv alen t to 60 gs» o f anhydrous s a l t)
were added to th e ferm en tatio n fla s k s each o f which contained 200 ml*
o f 15 p ercen t optimum medium* The inoculum was 20 ml* o f a 48 hour
c u ltu re o f y e a st Bo* 43 grown in 10 p ercen t d ex tro se medium) th e pH
v alu es were 4*7, 4*8, 4*8, 5*0, and 5*5*
These v alu es ware chosen
sin ce they seemed to be d o s s to th e c r i t i c a l pH range*
The most
acid ferm entatio n p o ssib le was considered d e sira b le in o rd er to have
maximum b is u lf ite co n cen tratio n f a r maximal aeetaldehyds fix a tio n *
The media were continuously s tir r e d a t about 26® centigrade*
m entations were u n su ccessfu l where th e pH was below 5*
F er­
Lack o f a c e li-
a a lis a tio n o f th e c u ltu re to th e above co n d itio n s was considered
resp o n sib le fo r th e lac k o f growth in th e media more a d d th an pH * 5*
However th e ferm entations a t pH * 5*0 and 5*5 re s u lte d in g ly ce ro l
y ie ld s o f 15*1 and 12*2 p ercen t on th e sugar w eight, resp ectiv ely *
R esidual sugar was approxim ately 0*5 p ercen t o f th a t i n i t i a l l y pre­
se n t.
Since so many members o f th e s e rie s d id not ferm ent, th e re ­
s u lts were n o t e sp e c ia lly co n clu siv e.
I t was observed th a t th e
medium a t pH * 5*0 did n o t ferm ent w ell fo r about th e f i r s t 48 hours
a f te r in o c u la tio n a s compared to th e immediate ferm entation when th e
5*5*
Howfivsr$ once th e ferm en tatio n s ta rte d a t p i * 5# i t
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-123-
proceeded to com pletion smoothly*
This o bserv ation s ig h t be consider*
ed evidence th s t unacclim atized y e a st w ill grow in such a medium
i t given s u ffic ie n t tim e to g e t used to th e conditions*
ad ju sted when necessary w ith 18 normal s u lfu ric acid*
The pH was
The volume
req u ired p er fla s k was n eg lig ib le*
I t was considered a s p o ssib ly d e sira b le to f i l t e r th e s o lid c a ld m s u lf ite and y east c u ltu re s from th e previou sly ferm ented media
and to u se th e re s u ltin g cake a s inoculum fo r a subsequent s e rie s
o f ferm entations*
Such a c u ltu re in d ir e c t co n tact w ith e a le iu a
s u lf ite would su re ly be a c c lia a tls e d to th e s u l f i te to some extent*
The cake containing th e c u ltu re was always k e p t damp*
A s e rie s o f fiv e fla s k s was prepared containing 200 ml* o f
optimum medium*
necessary*
S te r ilis a tio n o f th e medium was n o t considered
Inch fla s k was in o cu lated w ith cm e-fifth o f th e cake from
th e two su c ce ssfu lly ferm ented fla s k s describ ed above*
A d ditio n al
hydrated calcium s u l f i te m s added such th a t the t o ta l m ig h t o f th e
s a l t p er fla s k was eq u iv alen t to 60 ge* o f the anhydrous s a lt*
The
pH v alues stu d ied were near th e c r i t ic a l pH range where th e b is u lf ite
Ion con cen tratio n begins to r is e ra p id ly w ith decreasing pH.
The
r e s u lts o f th e experim ent a re given in Table 33*
From th e d a ta o f Table 33 one may conclude th a t th e g ly ce ro l
y ie ld approaches a co n stan t value o f about 10 p ercent o f th e d ex tro se
w eight p ra c tic a lly independent o f pH v a ria tio n s under th e co n d itio n s
described above*
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-124*
The follow ing eq u ilib riu m was considered*
The r a te o f decom position o f th e acetaldehyde complex would be in ­
dependent o f th e co n cen tratio n s o f c&lciura b is u lf ite and acetaldehyde.
The r a te o f form ation, however, would be dependent upon th e concentra­
tio n s o f th e se compounds. As th e co n cen tratio n o f th e complex b u ild s
TABLE 33
GXXCSROI. XIELBS FB0M XEAST FERMBITATIOKS AT DIFFMSST pH LEVELS
8SIWG GkWJM SULFITE CASE FROM PREVIOUS FEHtOKTATIOHS
AS IHQCUIAJM
pH
F ilte re d
V dusie,
&L«
5 .3
5*1
4 .?
4*7
4 .5
20®
206
266
200
200
I n itia l
D extrose,
g s. p e r
100 m l.
H esidaal
D extrose
%
H .2 2
18.95
18.95
18.95
18.95
0.42
0 .5 0
0.52
0*52
High?
*■
—
Ok* p e r
% an
100 ml*
D extrose
________________
10*06
1.94
10.01
1.92
1.95
30,03
2*00
10,05
fom en tation ceased.
u p , th e r a te o f decom position would in cre ase th u s allow ing a g re a te r
co n cen tratio n o f acetaldehyde to be p resen t th u s allow ing i t s g re a t­
e r enzym atic red u ctio n to ethan ol and a corresponding red u ctio n in
th e g ly c e ro l yield*
I f th e se su p po sitio n s a re tru e , then decreasing
th e i n i t i a l sugar co n cen tratio n s in th e media should r e s u lt i s in ­
creased percentage g ly c e ro l y ie ld s .
The low er sugar co n cen tratio n s
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•125-
ahould g iv e le e s p o ssib le acotaldehyde, th e o re tic a lly * and th u s th e
co n cen tratio n o f the a M e h y d e -b isu lfite oonplex would be low er in
e o la tio n th u s allow ing a g re a te r percentage o f th e acetaldehyde to be
fixed*
This would r e s u lt in h igher g ly c e ro l y ield s*
A s e rie s o f media was prepared to study th e re la tio n s h ip between
th e i n i t i a l dextrose co n cen tratio n of th e medium and th e g ly ce ro l
y ie ld in th e presence o f hydrated calcium s u lfite *
The inoculum
was th e f ilte r e d s o lid e d c iu a s u lf ite cake containing th e y east No*
43 o f previous ferm entations*
The calcium s u lf ite preeent p e r fla a k
m s eq u iv alen t to 60 ga* o f th e anhydrous s a lt*
tin u o u sly s tir r e d a t 26° centigrade*
The media were con­
The ferm entation tin s decreased
w ith decreasing i n i t i a l d extro se concentration? however* a l l media
were allow ed to stand fo r fo u r days before analyse# were run*
The
pH was not co n tro lled a c c u ra te ly in th e media* but a l l members o f th e
s e rie s were tre a te d alik e*
given in Table 34*
The re s u lt# o f th is in v e s tig a tio n a re
The im p licatio n# may be more c le a rly observed
in Figure# 11 and 12*
The d a ta o f Tablo 34 prove co n clu sively th a t th e percentage
g ly c e ro l y ie ld in cre ases w ith decreasing i n i t i a l dextro se eo n een tratio n under th e p rev io u sly described conditions*
Jk c o ro lla ry to th is
conclusion i s th a t th e p ercen t o f aeetaldehyde fix a tio n decreases
w ith In creasin g I n i t i a l d extrose concentration*
This evidence
supports ra th e r stro n g ly th e contentions described above concerning
th e a d e ta ld e iy d e -b is u lfite eq u ilib riu m .
The a c e ta ld e h y d e -b isu lfite
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• 226**
co ncen tratio n very lik e ly approaches a «i«3clamK under a
sp e c ifie d s e t o f fo m e n ta tio n c o n d itio n s.
A m th eraatieal r e la tio n ­
ship between th e i n i t i a l dextro se co n cen tratio n and th e g ly c e ro l y ie ld
i s presented follow ing 'fable 3?*
Following th e in v e stig a tio n s on fo m e n ta tio n s in th e presence
o f calcium s u lf ite , i t was decided to run sane y e ast fo m en tatio n # In
TABLE 34
THE EFFECT CT DEXTROSE COHCEHTHATIOS OS THE GLYCEROL
x iw > m fm m m m m s m th e p re s b k » m m a x m
SULFITE
Ty.4-f.4«l
D extrose,
pa
g** p er
10C a l .
5. 5- 5.1
5.5~5.0
5 .5 -5 .1
5*5-5*0
4
3
12
16
CaSOl
P re se n t,
Cfe* Dry
B asis
30
30
30
30
F U tered
Voluiaa,
ml*
200
200
200
200
cayeeroi Y ield
E esldnal
D extrose, "Gs* p e r % on
100 a l* D extrose
g mrn per
100 al*
0 .0 6 4
0*83
0*084
0.053
0.093
1*32
20.8
13*5
12*8
9*8
1*51
1*56
th e presence o f magnesium s u l f i te (%SGj*6%©) w ith sp e c ia l a tte n tio n
being paid to pH, g ly ce ro l y ie ld s and i n i t i a l sugar concentration*
Two prelim inary ferm en tation s were ran using magnesium s u lf ite
in p lace o f o a ld u st s u lfite *
The technique employed was th e same a s
was used in th e fo m e n ta tio n s involving calcium s u lfite *
Zt i s o f
im portance th a t magnesium s u l f i te i s somewhat taore so lu b le than i s
c a le iu a s u lfite *
S o lu b ility d a ta a re given in Table 27* The g re a te r
s o lu b ility o f magnesium s u lf ite should r e s u lt in a h igher acetaldehyde
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20
1.4
Medium
1.6
a>
tn
ml. of
o
S 15
Q
c
1.2
TJ
T
E ffe c t o f Initial
Dextrose i C oncentra tion
on Giyr erol Yield
of Ferm entations in
the Pre sence of
Calcium Sulphite.
ao>
>•.
o
4
8
12
Gm. Dextrose per 100 ml
Figure II
Glycerol
per
100
o
Gm.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25
16
/
1.0
/
/
0.8
E ffe c t of Initial
Dextro s e Concent ration
on R«ssulting Gly cerol
Conce ritration in
Fermer itations Invo Iving
Calciur n Sulphite.
0.6
8
Gm.
12
Dextrose
Figure
per 100 ml
12
16
•128**
fix a tio n than occurred when calcium s u lf ite was used; hence, & g re a te r
g ly ce ro l y ie ld should be obtained*
The inoculum used was a f ilte r e d
c a le iu a s u lf ite eake a s d escrib ed p rev io u sly ,
th e i n i t i a l unadjusted
pH o f both media a f te r magnesium s u lf ite was added was about seven*
On ferm enting about 24 hours th e pK dropped to about 6*3* At about
TABLE 35
p relim in arx w m m m km im in th e presence o f magnesium
SDUTTE WITH pH UHADJOSSED
pH
Range
I n itia l
D extrose,
ga* per
100 ml*
J&SQq.6SaO Volume o f iteelduftX M m m tA J m tik ....
iknctroM* i n . p e r % on
P resen t,
Medium
gnu
nil#
gm. p er IjOO s i* Deoctrowe
100 ml*
7 .1 -6 .3
8
50
200
0.048
1.765
22.05
7 .1 -6 .2
8
50
200
0.076
1*777
22.2
50 hours tb s pH had in creased to 7*1 and th e media were q u ie sc e n t.
Analyses were ru n fo u r days a f te r inoculation*
to c o n tro l th e pH in th e m edia.
No attem pt was made
R esu lts a re given in Table 35*
The r e s u lts o f Table 35 in d ic a te th a t taagnesiu® s u lf ite i s an
e f f ic ie n t aeetaldehyfie fix in g agent sin ce about 22 p ercen t g ly ce ro l
was found*
This value i s about 45 p ercen t o f th e th e o re tic a l yield*
The ferm entation tim e m s b u t a fra c tio n o f th e tim e req u ired in th e
experim ents o f Seuberg and R einfu rth (1919).
These d ata prove th a t
magnesium s u lf ite i s & more e ff ic ie n t aeet&ldehyde fix in g agent then
i s calcium s u lfite *
G reater g ly ce ro l y ie ld s resu lted * th us v e rify ­
in g th e previous pred iction *
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-129-
I t wui d e sired to study th e e ffe c t o f pH on tb s y ie ld o f g ly ce ro l
l a ferm entations in th e presence o f magnesites s u lfite *
The inoculum
was th e f ilte r e d magnesium s u l f i te cake containing y e a st Ho* 43 from
th e ferm en tatio ns o f Table 35*
The media were not s te r iliz e d .
The
a n tis e p tic a c tio n o f th e magnesit&s s u l f i te in so lu tio n m a s u ffic ie n t
to keep contam ination a t a low lev el*
The pH o f each medium was
ad ju sted by means o f freq u e n t a d d itio n s o f se v e ra l drops o f 7*5 normal
s u lfu ric acid*
The media were s tir r e d continuously*
was about 26 ° centigrade*
The tem perature
Vigorous ferm entations were noted*
ferm entation tim e was fo u r days*
The
B esu lts a re given in Table 36 *
TABLE 3d
THE RELATION Of pH TO THE QUGEROL HELD OF FERMEHTATIOIS
m THE PBESEHC1 OF MAQHESIBH SiflSITt
pH
Range
5 .5
5*8
6.0
6*3
6*5
7*0
f ilte r e d
VolnaOf
a l.
T o tal
I n itia l f in a l
H eight
D extrose, Dextrose*
%3®3*6H20 go* p er gn» peer
p i*
100 a l*
100 m l.
204*4
207.4
205*6
204*5
203*0
2Q1.Q
60
60
60
66
60
6@
9.68
9*66
9*75
9*78
9*86
9*95
0.14
0.14
0*12
0 .1 3
0*13
0*13
g ly ce ro l Found
Ga* p e r % m
Dextrose
200 m l.
H Few w
2.25
2.235
2.185
2*20
2*24
2.37
23*3
23*1
22*4
22*5
22*7
23*8
The r e s u lts o f Table 36 in d ic a te th a t pH i s n o t a c r i t i c a l
fu n ctio n in th e range stu d ied and under th e co n d itio n s employed.
These r e s u lts a re sim ila r to th e corresponding r e s u lts in th e stu d ie s
w ith calcium s u lf ite .
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-1 3 0 -
The next investigation#
concerned with the effect of inital
dextrose eonmntration on the glycerol yield of fermentations la the
presence of magnesium sulfite*
The theoretical considerations described
previously for the use of calcium sulfite in ferment&tiona apply queli*
tatively in the investigations with magnesium sulfite*
The optimum
TABLE 3f
Tim EFFECT OF DEXTROSE CONCENTRATION OK THE QXIGWQh YIELD
m m m m k T i Q w s m the presence of mghesihm s h ifx te
a :r s ra r r :\r a r ':r z r r :" M r ”u ::g iK ;^ r;,:r:';,^ := n r:::;;iil: r : ^ ,a ri^ :r a ! J ir i:iri,,:?.;i;;';.i; ’s a .: a '.v'.x j
Final
Weight of
Filtered HgS03*&ia©
Vduse, per FlaSkf
Initifil Time to
Final
,..jlyegp^ F^jd,,,
Dextrose,
Quiescence, Dextrose,
Qst* per % m
ga* peer hours
gm* per 100 ml* Dextrose
ml*
g»*
300 a l*
220
22®
220
m
220
40
40
40
40
40
4*54
9.08
13*62
17.94
22*70
100 a l .
35
&
48
>72
>72
0*127
O*08C
0.080
0.121
0*126
1*079
2.032
2.780
3.320
3*985
23*8
22.4
20.4
18*5
17*6
seal-synthetic medium was used as a hasia for these fermentations as
in previous experiments.
The inoculm m e an acclimatised culture of
yeast So* 43 grown in the presence of magnesium sulfite without pH
adjustments*
Inoculation was 20 ml* of a 48 hour culture of this
yeast grown in a 10 percent dextrose medium*
2?° centigrade*
The temperature m s about
Continuous stirring was maintained*
of the media were uncontrolled*
The pH values
In all flasks the pH m s initially
about seven; at 48 hours the pH was about 6*3* and at four days the
pB had again returned to seven*
The experimental data are given in
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-131-
Tahle 37 .
The r e s u lts o f Table 37 in d ic a te th a t in creasin g th e I n i t i a l
dex trose co n cen tratio n causes an In crease in th e f in a l g ly ce ro l eon*
c e n tra tio n and a decrease in th e percentage y ie ld o f glycerol*
This
T&B1E 38
THE RELATIONSHIP SSTWEgS IXPERIBMTAL AHB CALCULATED
TIELDS OP GLXCBRQL FROM FERMEHTATIOHS IHVOLSflKG
CALCIUM S lim H
I n itia l
Dsactrose*
8®. p e r
G lycerol X ield, P ercent on Dextrose
........ "■"u"
.....
-I—
—
£K perlaentel
C alculated
300
4
8
20.8
14.5
20.4
14.0
12
12.6
12.5
26
9*8
9*8
tren d i s q u ite sim ila r to th a t found f o r ferm entations in th e
o f c a l c i c s u l f i te .
A nathesaetical co n sid era tio n o f th e d a ta o f Tnblae 34 and 37
shoes th e p ercen t y ie ld o f g ly c e ro l, (g, to be an exponential fu n ctio n
o f th e i n i t i a l co n cen tratio n o f d ex tro se, £» th a t i s ,
lo g Q * a J» ♦ log k
th e equations fo r th e calcium s u lf ite and magnesium s u lf ite a re ,
resp ectiv ely *
lo g G| « -0.0267 £
♦ 1.417
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-132-
Sk
* - 0 * 0 0 7 3 0 & ♦ i,4 i7
An e x tra p o la tio n in d ic a te s th e mexlsua y ie ld , a t & d extrose
© oseentration o f se re gram per 100 al** to be about 26 p ercen t under
th e co n d itio n s o f th e experim ents*
However, whether such an extern*
p o latio n i s e n tire ly J u s tif ie d wouM need to be st^porbsd by a d d itio n ­
a l d a te In th e lower ran g es o f co n cen tratio n o f dextrose*
The agreem ent between th e experim ental values and th o se c a lc u la te d
by mmm o f th e above equatio ns i s shown in T ables 3& and 39*
TABLE 39
THE H1UTI0HSHIP BSSHOK EXPERIMENTAL ABD CALCULATED XILLDS Of
GI^CEROL FROM FMMBNTATIOHS HimVlMG UOHESXOI StJIFITl
Tw4f.ini
D extrose,
rfrfl, p er
100 a l .
4*5a
9*s 8
13*62
17*94
22.70
G lycerol fie ld s Percent on D extrose
Ix p eriiaen tal
C alculated
23*8
22.4
20.4
18*5
17*6
24*2
22*4
20*8
19*3
17*8
Data were d esired concerning th e success o f autom atic p H -^ cn tro lled fo m e n ta tio n s in th e presenoe o f s o lid m gneeiust s u lfite *
Media
were prepared and th e pH m s au to m atically co n tro lled by an a c e tic
acid so lu tio n (SO p ercen t by volume)#
H* c e re v la la f Ho* 43*
The ln o cu la were c u ltu re s o f
The s ta r tin g media were 1*5 l i t e r s o f ©ptiawa
aed ia co ntaining 15 p ercen t d e x tro se.
Ferm entation tem perature was
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•133*
30° centigrade*
each b a tc h ,
About 280 gm. o f magnesium s u lf ite m re added to
Experim ental d a ta a re given in Table 40.
TABLE 40
QLSCEROL 11EIS5 FROM FERHE8TATIQHS WITH AUTOMATICAL!# CONTROLLED
pa IH THE PRESENCE € f MAGNESIUM SUIFITB
35S3S5SSSBS&5S5398MB9nSRSE09HM3K8SSMM^^
IQ,* ^
ffoffjjdfcwil
Glycerol Foira^
A cetic Volume
D extrose, Dextrose* t a T i e r a on
'
Acid
Excluding go. p er ga* p e r
100 a l* DeocAdded S o lid s
100 a l .
100 m l.
tro s e
___________ _________ a l *
'__________________________________
pH
6*5
195 ®1*
60
1T55
12*82
0*124
3*04
23*4
65
1565
14*3®
O*0?O
3*325
23*2
13f
d ex tro se
6*2 %S03
cake
The r e s u lts o f th e in v e s tig a tio n s o f Table 40 su b s ta n tia te th o se
o f previous ferm entations in th e presence o f magnesias s u lf ite is s o f a r as th e g ly c e ro l y ie ld i s concerned.
The y ie ld s o f g ly c e ro l u sin g
pH c o n tro l a t an a cid le v e l appear s lig h tly h ig h er than th o se o f un­
co n tro lled ferm entations o f corresponding i n i t i a l dextrose concentra­
tio n s d escribed in Table 37*
standing* however*
The d iffe re n c e s in y ie ld s a re n o t o u t­
The use o f a c e tic acid fo r pH c o n tro l i s n o t id e a l
by any means sin ce th e magnesium a c e ta te formed mould cause d i f f i ­
c u ltie s in th e g ly ce ro l recovery*
The u se o f s u lfu r d io x id e fo r
autom atic pH c o n tro l would have th e advantage o f sim ple removal on
cp ap letio n o f th e ferm entation*
One o f th e proposed advantages o f th e method o f form ation o f
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-134*
g ly ce ro l by th e use o f s lig h tly so lu b le s u lf ite s i s th e f a s t th a t
such confounds may be e a s ily p re c ip ita te d by s u ita b le pH adjustm ents
and subsequently be e a s ily f ilte r e d from th e fom ented liquor*
The
re s u ltin g medium aay then be d is t il l e d fre e from so lv en ts and any
a d d itio n a l p re c ip ita te filte re d *
With sugar and perhaps n u trie n ts
added, th e re s u ltin g medium could be refe rae n ted to b u ild up th e
g ly ce ro l co n ten t in o rd er to make i t s recovery more fe a s ib le eeonosdL#*
a lly *
Such ferm entations a re described below*
The so lv e n ts were d i s t il l e d from prev io u sly ferm ented media in
th e presence o f enough calcium hydroxide to keep th e so lu tio n s lig h t*
ly alk alin e*
Hie re s u ltin g m ixtures ware then filte re d * d ex tro se mas
added to th e f i l t r a t e s which were then d ilu te d to th e ir i n i t i a l v o l­
umes before d is tilla tio n o f th e so lv e n ts.
In o cu latio n was by means
o f a calcium s u lf ite cake containing y e ast No. 43 from a previous
ferm entation*
Ferm entation occurred a t about 2?° to 30® cen tig rad e
w ith in te rm itte n t s tir r in g fo r about fo u r days*
R esu lts a re shown
in Table 41*
The r e la tiv e ly low y ie ld s in d icated in Table 41 were probably
p a r tia lly due to nan-continuous s tir r in g and p o ssib ly to th e r e la ­
tiv e ly wide pH range found sin c e pH was n o t c o n tro lle d .
However,
the d a ta show th a t more th an one ferm entation can be run on th e same
b asic su b stra te w ith com parative ease*
The g ly ce ro l co n ten t o f th e
medium was increased* more e f f ic ie n t methods should in crease the
g ly ce ro l y ie ld s ig n ific a n tly .
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-135*
42
l
a
oo•
M i
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9>
*
t-
g
Jf;
*8
4
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OS
CVl 04*
it
*n
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os
8s
I:
VO
ON
4
A
1*
m
•
3
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is
t*y
•
3
SO
g*H g«"l
S|
si
m
i
% s
3
3
t“1
•
rl
W**k
\r\
u\
SI
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136-
I t m s d eaired to fin d whether a second ferm entation oould a lso
he run using magnesium s u lf ite in place o f calcium s u lfite *
The
i n i t i a l g ly c e ro l co n cen tratio n o f th e medium employed m s 3*04 gm*
p er 100 a l* o f so lu tio n *
Two hundred al* were taken and v o la tile *
were removed a s describ ed above where c a lc iu a s u lf ite was used*
D extrose was added so th a t th e re s u ltin g medium d ilu te d back to i t s
i n i t i a l volume would co n tain 1$ ga* p er DO ml* T h irty a l . o f y e a st
c u ltu re No. 43 grown in 10 p ercent dextrose medium were added a s
inoculum.
The n e t d ex tro se co n cen tratio n was th en 13*04 gm* per
100 ml* assuming a l l o f th e d ex tro se m s gone from th e inoculum .
The inoculum contained 0*12 ga* o f glycerol*
co n cen tratio n was 2*69 gm. p er DO a l .
The to ta l I n i t i a l g ly ce ro l
The f in a l g ly c e ro l co n ten t
m s found to have been 5*05 gm. p er 100 ml* Tims* th e in crease in
g ly ce ro l con tent due to th e second ferm entation was 2.36 ga* p er 100
m l.
This corresponds to a y ie ld o f 18*1 p ercen t on th e d ex tro se w eight.
Though th e co n d itio n s were n o t n e c e ssa rily ideal* th is experim ent
showed th a t more than one ferm entation could be ru n on th e sane b a sic
su b stra te using magnesium s u lf ite to b u ild up th e g ly cero l concentra­
tio n in th e medium toward a d e sira b le value*
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v*
sm m i Am
conclusions
JU The pa o f th e ferm enting media m s follow ed by means o f a
Cameron pa Recorder employing & g la s s electrode*
A d ditio nal c ir c u its
end equipment were devised to a u to m tie a lly c o n tro l th e pH a t th e d e sire d lev e ls*
£•
Improvements were made in th e reducing-sugar determ ination
method o f Semogyi which re s u lte d in more ex act and rep rod u cible re ­
su lts*
J*
An optimum sem i-sy n th etic medium m s developed to serve a s
a b asal median fo r stu d ie s on th e g ly c e ro l ferm entations*
The medium
contained a minimum o f fix ed s o lid s , th a t i s , s o lid s such a s n u trie n t
s a lts which a re d if f ic u ltly removeable from th e ferm ented medium* A
minimum o f fix ed s o lid s was an im portant requirem ent inasmuch as la rg e
amounts o f such m a te ria ls cause con siderable d if f ic u lty i n subsequent
g ly c e ro l recovery*
4,* S tu d ies were made on th e a lk a lin e ferm entatio n o f dextro se
by means o f y e a st using ammonium hydroxide a s th e a lk a lia in g agent*
I t was found th a t ferm en tatio n s were unsuccessful when an ap p reciab le
ammonium co n cen tratio n e x iste d in media in which th e pH v alu e was
above seven*
I t was concluded th a t m olecular ammonia o r ammonium
hydroxide in so lu tio n was a to x ic agent*
The to x ic ity o f th e m olecular
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-138*
ammonia o r ammonium hydroxide i s a fu n ctio n o f th e a c tiv ity o f th e
m olecular compound* Ammonium io ns a re r e la tiv e ly n o n -to x ic.
The
a c tiv ity and th u s th© to x ic ity o f ammonia o r ammonium hydroxide i s
a fu n ctio n o f both pH and th e a v a ila b le ammonium concentration#
Consequently, sin c e th e to x ic ity in c re a se s sh arp ly as pH in c re a se s
p a st seven, th e a lk a lin e ferm en tatio n to prepare g ly ce ro l i s n o t very
s a tis fa c to ry , sinew g re a te r a lk a lin ity i s necessary fo r high g ly ce ro l
yield s*
The advantage o f th© u se o f ammonium hydroxide f o r pH
c o n tro l o f ferm entations would, have been i t s s ta p le removal follow ­
ing ferm entation*
£•
f e a s t w ill ferm ent d ex tro se in th e presence o f ammonium
s u lf ite provided th e pH o f th e medium i s s u ffic ie n tly below seven
to ob v iate th e to x ic e ff e c ts o f ammonia* A cclim atisatio n o f th e
c u ltu re to aMcsoniua s u lf ite i s necessary*
Ammonium s u lf ite i s not
a * fixed s a lt* such a s i s sodium s u lf ite sin ce i t may be removed
from ferm ented media r e la tiv e ly easily*
The use o f th is s a l t in
ferm entations fo r g ly ce ro l production should be fea sib le*
I t is
probable th a t pH c o n tro l o f th e ferm enting medium by means o f s u lfu r
fHrfvHHo would be ad v isab le.
J»« I t was shown th a t autom atic a lk a lin e pH -controlled ferm enta­
tio n s could be run using aqueous so lu tio n s o f sodiwa carbonate and
sodium hydroxide a s th e c o n tro llin g agents*
The u se o f th e sodium
hydroxide so lu tio n fo r c o n tro l mas more s a tis fa c to ry , however*
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*339*
X* Calcium s u lf ite can be used in ferm entations in o rd er to in *
crease th e g ly c e ro l y ield *
in Im portant advantage to th e use o f
calcium s u lf ite fo r th© production o f g ly ce ro l by ferm entation i s
t i e f s e t th a t i t may be removed from th e medium by f iltr a tio n *
justm ent o f pH before f i l t r a t i o n i s ad v isab le.
Ad­
The s o lu b ility o f
calcium s u lf ite i s a fu n ctio n o f pH* In creasing th e i n i t i a l d«e»
tro se co n cen tratio n In d ie ferm en tatio n medium decreases th e p er­
centage g ly c e ro l y ield *
The percentage y ie ld o f g ly ce ro l i s an
exponential fu n ctio n o f th e i n i t i a l dextro se concentration*
The
fo m e n ta tio n tin e was found to be re la tiv e ly sh o rt, from one to about
fo u r days, depending on th e i n i t i a l d ex tro se concentration*
F iltr a ­
tio n o f a ferm ented medium con taining calcium s u lf ite r e s u lts in a
f ilte r e d cake o f so lid calcium s u lf ite con taining th e c u ltu re o f
yeast*
This cake may be used §s a very a c tiv e inoculum fo r subse­
quent ferm entations*
Contam ination was very r a r e .
The maximum
g ly ce ro l y ie ld approached 26 p ercen t o f th e sugar w eight a s th©
in i t i a l sugar co n cen tratio n approached zero*
8*
Magnesium s u lf ite was found to a c t in a manner s im ila r to
e a le im s u lf ite in ferm en tatio n s fo r th e production o f gly cero l*
It
was observed th a t th e g ly c e ro l y ie ld was g re a te r in ferm entations
involving magnesium s u lf ite than in those employing calcium s u lf ite
fo r th e sugar co n cen tratio n s studied*
However, th e maximum g ly c e ro l
y ie ld approached 26 p ercen t of th e sugar w eight a s th e i n i t i a l sugar
co n cen tratio n approached aero j th is i s th e same lim itin g v alu e a s
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- 140-
th a t calculated, f o r calcium s u lfite *
£♦
I t was shown th a t g ly c e ro l was formed to to# presence o f
s lig h tly eoiufcle s u lf ite s under acid conditions*
The tim e f o r c o s-
p le tio n o f to e ferm en tatio n s stu d ied was r e la tiv e ly short*
2&* The s o lu b ilitie s o f calcium and magnesium s u lf ite s a re
fu n ctio n s o f pH* The b to u lfit© io n co n cen tratio n i s a fu n ctio n o f
to e s o lu b ility o f to e s u lf ite and o f pH* T oxicity is a fu ao tlo n o f
b is u lf ite io n co n cen tratio n! hence* i t i s a fu n ctio n o f pH* A cetaldehyde fix a tio n i s a fu n c tio n o f to e b is u lf ite to n co n cen tratio n )
hence* i t i s a lso a fu n ctio n o f pH* Tbs g ly c e ro l y ie ld i s a fu n ctio n
o f to e degree o f aeetaldehyde fix atio n *
The v a ria tio n o f g ly c e ro l
y ie ld v ith p i was n o t outstanding* however* Aeetaldehyde in cre ases
tb s s o lu b ility o f calcium s u lf ite a t & given pH* With a given con­
c e n tra tio n o f aeetaldehyde, to e s o lu b ility o f calcium s u lf ite decreases
w ith in cre asin g pH* Tbs a c e ta ld e h y d e -b isu lfite complex to s ta b le
o nly under acid co n d itio n s and to e s t a b il i ty decreases w ith in c re a s­
ing pH*
J£*
Two o r more successiv e ferm entations involving calcium o r
magnesium s u lf ite may be run on th e same su b stra te provided so lv e n ts
a re removed between ferm entations*
Such a procedure in cre ases th e
g ly c e ro l co n cen tratio n in th e medium th u s making g ly ce ro l recovery
methods g en erally more e ffic ie n t*
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-141*
12.
I t was shown th a t th e a c e ta ld e h y d e -b isu lflte complex
approaches a maximum co n cen tratio n in fo m e n ta tio n media u tiliz in g
calcium o r magnesium s u lf ite fo r fix a tio n o f acetaldehyde#
As th e
co n cen tratio n o f th e complex in c re a se s, th e co n cen tratio n o f
acetaldehyde, o r i t s e q u iv a le n t, a ls o in c re a se s, th e o re tic a lly #
The in creased co n cen tratio n o f aeetaldebyde r e s u lts in a g re a te r
p ro b a b ility o f I t s red u c tio n to eth an o l by means o f glyceraldehyds
phosphate: consequently a decreased y ie ld o f g ly ce ro l would be
expected*
The e ffic ie n c y o f acetaldehyde fix a tio n should be g re a t­
e s t a t th e begin lo g o f th e ferm en tatio n before th e acetaldehydeb ls u lf ite complex co n cen tratio n i s appreciable#
A method o f co a-
tin u cu aly removing th e acetaldehyde from a s u lf ite ferm entation
medium should r e s u lt in a g re a te r g ly ce ro l y ield #
I t may be p o ssib le
to continuously remove th e acetaldehyde from a c irc u la te d p o rtio n o f
the ferm enting medium by a d i s t il l a t i o n , fear example, under low
pressure#
An in crease in pH would more e a s ily allow d i s t il l a t i o n
o f th e acetaldehyde from th e medium* Following d i s t il l a t i o n , th e
acetaldehyde—fre e medium could be read ju sted to th e a d d pH o f th e
ferm enting medium fo r fu rth e r aldehyde fix a tio n and g ly ce ro l forma­
tio n by fu rth e r ferm en tation s and d is tilla tio n s *
R ep ro d u ced with p erm ission o f th e copyright ow ner. Further reproduction prohibited w ithout perm ission.
-
142*
LITERATURE CITED
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