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Optimization of metagenomic DNA extraction from activated sludge samples.

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ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING
Asia-Pac. J. Chem. Eng. 2009; 4: 780–786
Published online 31 July 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI:10.1002/apj.338
Special Theme Research Article
Optimization of metagenomic DNA extraction
from activated sludge samples
Yuan-Yuan Qu,1 Qiang Zhang,1 Li Wei,2 Fang Ma,2 * Ji-Ti Zhou,1 Wen-Qing Pi1 and Min Gou1
1
School of Environmental and Biological Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of
Education, China), Dalian University of Technology, Dalian 116024, China
2
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
Received 7 October 2008; Revised 10 March 2009; Accepted 18 March 2009
ABSTRACT: Metagenomic DNA extraction is essential for metagenomic technology. Therefore, optimization of a
conventional total DNA extraction from activated sludge was investigated in detail in this study. Throughout two
distinct orthogonal experiments, it was shown that the highest yield for metagenomic DNA could be obtained using
TENP buffer, lysozyme of 1 mg ml−1 (1 h), protease K (200 µg ml−1 ), SDS (1%, 1 h). Furthermore, the quality of the
differentially extracted DNA was subsequently assessed by the molecular fingerprint technology, such as denaturing
gradient gel electrophoresis (DGGE) and ribosomal intergenic spacer analysis (RISA). The results indicated that the
microbial diversity was dramatically different by different combined methods, and the DNA template quality for
RISA was much better than that for polymerase chain reaction (PCR)-DGGE. This study provides detail process for
metagenomic DNA extraction of activated sludge, and it would be useful for metagenomic DNA extraction of other
environment samples.  2009 Curtin University of Technology and John Wiley & Sons, Ltd.
KEYWORDS: metagenome; RISA; activated sludge; DNA extraction; DGGE
INTRODUCTION
In numerous environments, only less than 1% of
microorganisms can be readily cultivated using
cultivation-dependent approaches,[1,2] and the genetic
information of over 99% uncultured microorganisms
cannot be obtained. Moreover, conventional culture
methods have limitations in both quantitative and qualitative studies on the gene levels. With the emergence of
metagenome technique, this problem can be solved. The
immense and unknown world of microorganisms can be
investigated in different ways, such as polymerase chain
reaction (PCR) amplification and molecular phylogenetic techniques.[3 – 6] Metagenome is the environmental
DNA comprising the genetic blueprints of entire microbial consortia.[7] Metagenomic technology is a cultureindependent method, in which metagenomic library
is constructed from environment samples for further
screening and isolating the target gene.[8] Metagenomic
process includes extraction of DNA, construction of
metagenomic libraries, screening of object materials
such as biocatalyst, gene and active materials.[9]
*Correspondence to: Fang Ma, State Key Laboratory of Urban
Water Resource and Environment, Harbin Institute of Technology,
Harbin 150090, China. E-mail: dlutbest@yahoo.cn
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
During the process, DNA extraction is a critical
step of metagenome technology.[10,11] Because in these
modern molecular PCR-based studies, purified DNA is
required for many applications such as gene cloning,
PCR and microarrays, etc.[5] Therefore, the availability of effective DNA extraction methods is essential
for environmental microbiology studies. Many methods
of DNA extraction have been studied during the last
20 years, including total microbial community DNA
from soils and sediments,[12 – 15] water samples,[16] activated sludge[17,18] and other samples.[19 – 21] However,
protocols of DNA extracted from activated sludge samples have only been published recently. Although some
comparative studies of different methods for DNA
extraction and purification have been developed, the
effects of main factors on the yield and purity of
DNA have not been analyzed. Previous studies mainly
focused on single factor investigation, and effects of
factors in each step within one method have not been
fully explored. Therefore, it is necessary to investigate the main factors for DNA extraction from activated sludge samples. In this study, we chose the
method called “lysozyme (L)-SDS-proteinase K” of
DNA extraction from activated sludge described by Qu
et al .[22] This method was mostly based on five steps:
retreatment, lysis of the cells, extraction of nucleic
acids, removal of protein, polysaccharides and humic
Asia-Pacific Journal of Chemical Engineering
METAGENOMIC DNA EXTRACTION FROM ACTIVATED SLUDGE SAMPLES
acid, DNA deposition. Orthogonal tests were designed
for six factors in the DNA extraction and purification,
and ultraviolet (UV) detection was used to measure
the yield and purity of extracted DNA. PCR-denaturing
gradient gel electrophoresis (DGGE) were used to estimate the community diversity while ribosomal intergenic spacer analysis (RISA) was conducted in parallel.
MATERIALS AND METHODS
Activated sludge samples
The activated sludge samples were taken from a local
sewage farm (Chunliu River Wastewater Treatment
Plant). The sludge samples without pretreatment were
cultured in mineral salt medium for 48 h at 30 ◦ C, then
were collected by centrifuge (8000 ×g, 10 min) and
stored in plastic containers at −80 ◦ C until use.
DNA extraction buffers
Lysis of the cells was performed after suspending
sludge samples (1.0 g wet wt) by 5 ml extraction buffer
in 10-ml centrifuge tubes at 37 ◦ C water bath. Three
extraction buffers were used: TEN (100 mM Tris-HCl,
pH 8.0; 100 mM Na-EDTA, pH 8.0; 1.5 M NaCl);
TENC (100 mM Tris-HCl, pH 8.0; 100 mM Na-EDTA,
pH 8.0; 1.5 M NaCl; 1% CTAB); TENP (100 mM TrisHCl, pH 8.0; 100 mM Na-EDTA, pH 8.0; 1.5 M NaCl;
1% PVP). The phenol/chloroform solution (pH 8.0)
was prepared by mixing 100 ml of Tris-EDTA buffer
saturated phenol with 96 ml of chloroform and 4 ml
of isoamyl alcohol. All the reagents were purchased
from TaKaRa Biotechnology (Dalian) Co. Ltd. (TaKaRa
Dalian).
Distinct orthogonal experiments
Lysis treatments and removal of protein and polysaccharides were two key steps for DNA extraction. In
the lysis procedure, extraction buffer, concentration of
lysozyme and 37 ◦ C water bath time for lysozyme were
tested (Table 1). Concentration of SDS, concentration of
proteinase K and 55 ◦ C water bath time for proteinase K
were designed for the removal of protein and polysaccharides (Table 2).
According to the orthogonal experiment design
method, we designed nine groups of parallel experiment to investigate the influence of the three factors
and three levels on the extraction process. For the first
nine parallel groups, removal of protein and polysaccharides was performed under the following conditions:
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Table 1. Influencing factors and their levels for lysis
treatment.
No.
Extraction buffer
Concentration of
lysozyme (mg ml−1 )
Water bath
time (h)
TENP
TEN
TENC
TENP
TEN
TENC
TENP
TEN
TENC
1
1
1
2
2
2
3
3
3
1
2
0.5
2
0.5
1
0.5
1
2
1
2
3
4
5
6
7
8
9
Table 2. Influencing factors and their levels for removal
of protein and polysaccharides treatments.
No.
Concentration of
protease K (µg ml−1 )
Water bath
time (h)
Concentration
of SDS (%)
300
100
200
300
100
200
300
100
200
1
1
1
0.5
0.5
0.5
2
2
2
1
2
0.5
2
0.5
1
0.5
1
2
1
2
3
4
5
6
7
8
9
SDS 1%, Proteinase K 200 µg ml−1 and 55 ◦ C water
bath 1 h.
Purity and yield of DNA
A gross assessment of metagenomic DNA extracted
was generated by agarose gel electrophoresis using
λ-Hind III digested DNA marker (TaKaRa Biotechnology Co. Ltd., China). The purity of extracted DNA was
confirmed by measuring the ratio of OD260 /OD280 . Yield
of DNA was calculated as follows:
OD260 × 500
× dilutated multiple × V
Yield of DNA (µg g−1 ) =
1000
Primers
To test possible selectivity of lysis and purification
treatments, DGGE and RISA of DNA were used.
DNA extracts were used as the template for PCR
amplification. The primers used for DGGE and RISA
analysis were:
338FGC: 5 ATTACCGCGGCTGCTGG 3 ; 518R:
Asia-Pac. J. Chem. Eng. 2009; 4: 780–786
DOI: 10.1002/apj
781
782
Y.-Y. QU ET AL.
Asia-Pacific Journal of Chemical Engineering
5 CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGG
CACGGGGGGCCCCA ACGGG AGG CAG CAG 3
and 926 F: 5 CTY AAA KGA ATT GAC GG 3 ; 189
R: 5 TACTGAGATGYTTMARTTC 3 , respectively.
PCR amplification and DGGE analysis
The PCR mixture (50 µl) contained 10× buffer (Mg2+
contained) 5 µl, dNTP 8 µl, 0.5 µl primer forward and
0.5 µl primer reverse, 2 µl template DNA, TaKaRa
rTaq enzyme 0.5 µl and ddH2 O 33.5 µl. Cycling was
designed with a pre-cycle (94 ◦ C for 4 min), 20 cycles
for Touch Down (94 ◦ C for 30 s, 65 ◦ C for 30 s, 72 ◦ C
for 1 min) and 10 cycles for constant temperature (94 ◦ C
for 30 s, 55 ◦ C for 30 s, 72 ◦ C for 1 min). After a
final extension step of 6 min at 72 ◦ C samples were
maintained at 4 ◦ C.
The PCR products of 16S rDNA V3 region were performed using a DGGE apparatus. Polyacrylamide gels
were composed of 8% (w/v) polyacrylamide, 30–60%
denaturant from top to bottom (100% denaturant contained 7 mol L−1 carbamide and 40% deionized formamide). DGGE was operated in 1 × TE buffer
(10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) 5 h at
60 ◦ C. Then, polyacrylamide gels were dyed with normal temperature for 30 min in the 1 × TE buffer 120 ml
(1/10 000 v/v gene finder contained).
RISA fingerprint technique
The RISA PCR mixture (50 µl) included 10 × buffer
5 µl, dNTP 8 µl, 0.5 µl primer forward and 0.5 µl
M 1 2 3 4 5 M 6
7 8
primer reverse, 2 µl template DNA, TaKaRa rTaq
enzyme 0.5 µl and ddH2 O 33.5 µl. Cycling conditions
contained initial denaturation (94 ◦ C for 2 min) followed by 35 cycles for amplification (94 ◦ C for 1 min,
48 ◦ C for 1 min, 72 ◦ C for 1.5 min). After a final extension step of 5 min at 72 ◦ C, samples were maintained
at 4 ◦ C.
RESULTS AND DISCUSSION
Extraction of metagenomic DNA from sludge
samples
DNA extracted from all the samples were shown in
Fig. 1, which was suggested that most DNA recovered
was of high molecular weight. From these results, it was
obvious that all the DNA fragments were larger than
23 kb, which could meet the demand of constructing
a small metagenome library. Therefore, the libraries
would be analyzed for novel genes and pathways
with sequence-based techniques or through screening
proteins and drugs.[23]
The yield and purity of DNA extracted from the first
nine groups were shown in Table 3. Crude DNA yields
from the nine samples ranged from 81.6 to 1812 µg g−1
(sludge wet wt). The highest yield was 22 times higher
than the lowest one. DNA extracted from all the groups
was pure enough because the value of OD260 /OD280 was
more than 1.5.[24,25] Because the values of OD260 and
OD260 /OD280 could be used to evaluate the yield and
purity of DNA, the optimum conditions were obtained
by the model calculation as follows: extracted buffer
9
M 1
2 3 4
M 5 6 7 8 9
23.1kb
23.1kb
(a)
(b)
Figure 1. Agarose gel electrophoresis of DNA fragments. anes: M, λ-Hind III DNA marker;
1–9, DNA samples. (a) samples of lysis treatment; (b) samples of protein and polysaccharides
removal treatments.
Table 3. Yields and purity of DNA for lysis treatment.
Samples
OD260 /OD280
Yield (µg g−1 )
1
2
3
4
5
6
7
8
9
1.891
1812
1.967
677
1.855
81.6
2.042
545.6
1.789
406
1.842
243.2
1.889
299.2
1.798
876
1.795
766
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2009; 4: 780–786
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
METAGENOMIC DNA EXTRACTION FROM ACTIVATED SLUDGE SAMPLES
Table 4. Orthogonal analysis for lysis treatment.
No.
1
2
3
4
5
6
7
8
9
Mean One
Mean Two
Mean Three
Range
Extracted buffer
Lysozyme (mg ml−1 )
Water bath time (h)
Yield of DNA [µg g−1 (sludge wet wt)]
TENP
TEN
TENC
TENP
TEN
TENC
TENP
TEN
TENC
885.600
653.000
363.600
522.000
1
1
1
2
2
2
3
3
3
856.876
396.267
647.057
458.600
1
2
0.5
2
0.5
1
0.5
1
2
262.267
977.067
662.867
714.800
1812
677
81.6
545.6
406
243.2
299.2
876
766
Table 5. Yields and purity of DNA for removal of protein and polysaccharides.
Samples
OD260 /OD280
Yield (µg g−1 )
1
2
3
4
5
6
7
8
9
1.81
282
1.92
153.2
1.90
1446.8
1.87
113.6
1.90
154.4
1.81
460
2.04
38.4
1.81
1030.8
1.86
351.6
Table 6. Orthogonal analysis for removal of protein and polysaccharides treatments.
No.
1
2
3
4
5
6
7
8
9
Mean One
Mean Two
Mean Three
Range
Protease K (mg ml−1 )
Water bath time (h)
SDS (%)
Yield of DNA [µg g−1 (sludge wet wt)]
300
100
200
300
100
200
300
100
200
466.133
752.800
144.667
608.133
1
1
1
0.5
0.5
0.5
2
2
2
242.667
627.333
473.600
384.666
1
2
0.5
2
0.5
1
0.5
1
2
206.133
590.933
546.533
384.800
282
253.2
1446.8
113.6
154.4
460
38.4
1030.8
351.6
TENP, lysozyme 1 mg ml−1 , 37 ◦ C water bath 1 h
(Table 4).
As for removal of protein and polysaccharides
(Table 5), there was great difference among the yields
of DNA extracted from the nine groups, and Group 3
had the highest yield [1446.8 µg g−1 (sludge wet wt)].
But the purity of DNA for all samples was more than
1.8. For the review of quality and yield, the optimum
conditions were obtained as follows: SDS 1%, Proteinase K 200 µg ml−1 , 55 ◦ C water bath 1 h (Table 6).
It was suggested that although the optimal value was not
included in the design of these nine groups, it was just
consistent with the conditions of purification in the first
nine groups’ experiments. Therefore, such conditions
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
should be taken into account for further application. The
test under the optimal conditions was shown as Group 1
in Table 4, and the crude DNA yield was 1812 µg g−1
(sludge wet wt) with DNA purity of 1.81.
Helmut Bürgmann et al .[26] provided a bead beating
method for optimizing quality and quantity of DNA
extracted from soil. They extracted DNA from six
soils of different texture and chemical characteristics
with selected bead beating protocols and the maximum
DNA yield obtained was 136 ± 17 µg g−1 (soil dry
wt), which was greatly lower than the yield of this
study. And there are some other reports revealing that
the quality of the extracted DNA depended on the
characteristics of the tested samples.[15,16,18,26]
Asia-Pac. J. Chem. Eng. 2009; 4: 780–786
DOI: 10.1002/apj
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Y.-Y. QU ET AL.
Asia-Pacific Journal of Chemical Engineering
Metagenomic DNA for RISA fingerprint
analysis
Generally, RISA targets length and sequence heterogeneities of the intergenic transcribed spacer (ITS)
regions between SSU and LSU rRNA genes.[27] RISA
can distinguish closely related microbial species and
requires PCR amplification without subsequent enzymatic restriction.[28] Therefore, RISA fingerprint technique has been widely used in environment pollutants
treatment systems.
The polyacrylamide gel electrophoresis of the RISA
products for 1–9 samples from lysis treatment part
was shown in Fig. 2(a). It was exhibited that sample
6 and sample 7 had no obvious band, and band 3 was
relatively weak. No significant differences in the other
samples. As reported, RISA was based on intergenic
spacers, which were between the 16S and 23S rRNA
genes.[29] Although only a few specific bands were
obtained from the nine templates, all of them were
located between 1200 bp and 1400 bp. The results
were the same with agarose gel electrophoresis of PCR
amplified products, which indicated that the amount
of DNA template had significant effects on amplified
results.
For the removal of protein and polysaccharides, it
could be seen that sample 7 had no obvious band, and
band 3 and 9 were relatively weak in the polyacrylamide
gel electrophoresis of the RISA products [Fig. 2(b)].
All the other bands were concentrated in 800–1000
bp and their specific products were basically identical.
The results demonstrated that water bath time, the
concentration of SDS and proteinase K also had effects
on the yield of DNA, but little effects on its size of
fragment and species.
Metagenomic DNA for PCR-DGGE analysis
PCR-DGGE has been successfully used to differentiate
bacterial isolates[30,31] and to characterize complex bacterial communities in soil since it was first introduced
bp
into environmental studies in 1993.[32,33] In recent years,
separation of DNA fragments by DGGE analysis has
been widely used in the microbial ecology studies.[25]
First, we used DGGE analysis of template with the
same volume, but results showed that only a few
samples could be observed. Therefore, we diluted the
template into the same concentration in the later study.
After PCR amplification of the 16S rDNA V3 region
of DNA extracts from the first nine groups, the PCR
products were then analyzed by DGGE [Fig. 3(a)].
DGGE band pattern represented the dominant bacterial
species in samples. According to Fig. 3(a), no obvious
differences could be seen among the DGGE bands
except that the intensity of band 7 was relatively weak.
It showed that the nine DGGE lanes had the similar
bands and distribution. Therefore, it suggested that all
the samples had enough DNA template for molecular
studies, and all the factors had almost no effects on the
biological diversity.
We used the same procedure to treat the nine samples
obtained from the parallel experiments for the removal
of protein and polysaccharides. From Fig. 3(b), the
nine bands had no differences in their intensity and all
of them remained the same level, then we could get
the same conclusions as mentioned above. The results
showed that the microbial diversity was dramatically
different by different combined methods, some bands
had differences in their intensity, and there were also
some bands even totally missed. According to Figs. 2
and 3, it was indicated that the DNA template quality
for RISA was much better than PCR-DGGE analysis.
In both Figs. 3(a) and (b), DGGE analysis for each
sample presented a single band, which could be due
to the sludge samples we used.
The sludge samples we used here were without
pretreatment and only cultured in mineral salt medium
for 48 h at 30 ◦ C. Thus, the diversity was not obvious.
But the objective of our study was to compare the
effects of factors on the DNA extraction, characteristics
of materials were not of our concern. Therefore, it had
no obvious effects on our conclusions. In this study,
the SDS-based method presented by Zhou et al .[15]
M1 9 8 7 6 5 4 3 2 1 M 2
M1 9 8 7 6 5 4 3 2 1 M2
4000
bp
3000
1400
1200
2000
1000
800
2000
500
1000
250
750
Figure 2. Polyacrylamide gel electrophoresis of the RISA products. Lanes: M1 , 200bp DNA
Ladder Marker; M2 , DL2000; 1–9, RISA profiles. (a) RISA products of lysis treatment; (b) RISA
products of protein and polysaccharides removal treatment.
 2009 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2009; 4: 780–786
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
1
2
3
4
5
METAGENOMIC DNA EXTRACTION FROM ACTIVATED SLUDGE SAMPLES
6
7
8
9
(a)
1
2
3
4
5
6
7
8
9
(b)
Figure 3. PCR products of 16S rDNA V3 region. Lanes: M, 100 bp Ladder
Marker; 1–9, PCR products of the 16S rDNA V3 region. (a) DGGE detection
of lysis treatment; (b) DGGE detection of protein and polysaccharides
removal treatment.
was optimized and modified in detail. A simple and
rapid DNA extraction method with higher obtainable
DNA recovery and inhibitor removal was confirmed and
justified in this study.
CONCLUSIONS
By the orthogonal tests, the optimization of metagenomic DNA extraction was as follows: best cell lysis
could be obtained using TENP buffer, lysozyme 1 mg
ml−1 , 37 ◦ C water bath 1 h; protein and DNA could
separate more completely with SDS 1%, proteinase
K 200 µg ml−1 , 55 ◦ C water bath 1 h. Under the optimal operation conditions, the yield of DNA extracted
could be up to 1812 µg g−1 sludge and OD260 /OD280
was 1.891, which could be sufficient for construction of
metagenomic library and analysis of RISA and DGGE
fingerprint. In addition, by DGGE and RISA analysis, we proved that operation parameters had significant
effects on quantity of DNA without affecting the size
of DNA segments and biological diversity.
Acknowledgements
We gratefully acknowledge the financial supports from
the National Natural Science Foundation of China
(NSFC) (No. 50608011) and State Key Lab of Urban
Water Resource and Environment (HIT) (QA200811).
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metagenomic, sludge, extraction, dna, samples, optimization, activated
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