CHARACTERIZATION OF BACTERIAL COMMUNITIES IN BIOGAS

PRODUCTION STEPS
Lia Vania Dewi, Rika Fithri Nurani, Andriessa Prameswara, and Elizabeth Caroline Situmorang
Plant Production and Biotechnology Division, PT. SMART, Tbk, Sinarmas Land Plaza, 2 nd Tower, 10th Floor, Jl. MH Thamrin
no. 51, Jakarta 10350, Indonesia,
Tel: +62 21 392 5720, E-mail: biotechnology@sinarmas-agri.com
Abstract:

Fossil fuel crisis, air pollution, and global warming have urged human society to seek environmentally
friendly, clean, and renewable energy. Therefore, biogas production by utilizing biomass has gained more
attention not only in developed, but also developing countries. Biogas production involves complex
microbial communities, mainly bacterial and fungal, and metabolic pathways that are poorly understood.
The aim of this study is to determine the possibility of applying Denaturing Gradient Gel Electrophoresis
(DGGE) to characterize bacterial communities in biogas production. In the future, information regarding
dominant microbial communities in biogas production can help to boost production efficiency, increase
yield, and generate improved microbial additives. Nine points of sampling were done in different stages of
biogas production in West Kalimantan, including POME Feed Pump (1), Incoming POME Equalization
Concrete Sump (IPECS) (2), Biodigester tank 1 (3, 4, 5), Biodigester tank 2 (6, 7, 8), and Treated POME
Collection Concrete Sump (TPPCS) (9), followed by total community DNA isolation and purification.
Amplification of 16S rRNA genes and DGGE was conducted to characterize dominant communities in each
point. Then, the dominant bands were sliced, sequenced, and analyzed. The results indicated that
Pseudomonas stutzeri was dominant in point 1-5, Pseudomonas fluorescens (6), Clostridum sp. (7),
Escherichia coli (8), and Clostridium sp. (9) as prominent bacterial species in respective points. As for the
next step, further research and trial are required to know the specific role of each species in the process.

Keywords:

Biogas, Bacterial communities, DGGE, POME

1. Introduction
Biogas production by utilizing biomass has
gained more attention in many developed and
developing countries these days. Fossil fuel
crisis, air pollution, and global warming have
urged human society to seek environmentfriendly, clean, and renewable energy sources
(Li et al., 2013).
Palm Oil Mill Effluent (POME) is the liquid
waste produced from oil extraction process of
Fresh Fruit Bunches (FFB) in palm oil mills.
POME contains high organic carbon content.
Thus, it is a promising source for biogas
production
and
may potentially boost up the renewable energy
sector. Biogas produced from anaerobic
digestion of POME are also able to substitute
palm kernel shell and mesocarp fiber as boiler
fuel (Chin, Poh, Tey, Chan, & Chin, 2013)
Microorganisms play important roles in
biogas production. However, only a little
knowledge was found regarding the interaction
of microorganisms inside a biogas reactor.
Therefore, it is important to understand and
describe the microbial diversity and growth
dynamics inside the bioreactor in order to
further optimize the conditions (Weiland, 2010).

DGGE
(Denaturing
Gradient
Gel
Electrophoresis) is a popular technique for
analyzing microbial diversity based on the
sequence differences that results in differential
denaturing characteristics of the DNA (Muyzer,
de Waal, & Uitterlinden, 1993; Shimano,
Sambe, & Kasahara, 2012). Melting pattern of
double strand DNA fragments is based on their
hydrogen
bond
content.
DNA
fragments, which are GC rich, melts at
higher denaturant area of the gradient
(Cetecioglu, Ince, & Ince, 2012)
The aims of this study are to determine the
possibility of applying DGGE to characterize
bacterial consortium that develops in biogas
production. In the future, the information
regarding dominant microbial communities in
biogas production can help to boost production
efficiency, increase yield, and generate
improved microbial additives.

2. MaterialsandMethod

Sampling
POME was retrieved from nine (9) points of
biogas production stages in West Kalimantan i.e.
Pome Feed Pump point 1; Incoming POME
Equalization Concrete Sump (IPECS) point 2;
Biodigester tank 1 point 3, 4, 5; Biodigester tank 2
point 6, 7, 8; and Treated POME Collection
Concrete Sump (TPPCS) point 9.

Enrichment
Enrichment was done prior to total community
DNA isolation stage with the addition of 2 ml
homogenized sample into 98 ml Nutrient Broth and
incubated at 37C, overnight.

Total Community
Purification

DNA

Isolation

and

Total community DNA isolation and purification

was conducted using GeneJET Genomic DNA
Purification
Kit
(Fermentas,
USA).
DNA
concentration was measured using NanoDrop
instrument (Thermo Scientific, USA) and qualitative
analysis was done using agarose gel electrophoresis.

Amplification of 16S rRNA gene

The isolated total community DNA was used as
template. Bacterial 16S rRNA gene fragments were
amplified using 63F1-GC Clamp and 518R primer
set. The PCR amplification conditions were: Predenaturation was done at 95C for 5 min, followed

by 35 cycles of denaturation at 94C for 45 s,

annealing at 55C for 1 min, extension at 72C for
1.5 min, and a final step of 72C for 7 min.

DGGE
DGGE was performed according to (Muyzer et
al., 1993) using the D-code system (Bio-Rad, USA)
in 8% (w/v) polyacrylamide gels containing
denaturing gradients of 4060%. One-hundred
percent denaturing conditions corresponded to 7 M
urea and 40% formamide. Then, 50l sample and
15l loading dye were loaded into the well.
Electrophoresis was undertaken at 150 V and 60C
for 3 h. After electrophoresis, gels were stained with
Ethidium Bromide (Sigma Aldrich, USA) and
visualized under Gel Doc (Bio-Rad, USA).
Dominant bands from each well were excised from
acrylamide gels under UV light using a sterile blade
and subsequently incubated in 50l of sterile distilled
water at 4C for 24 h, followed by amplification
using 63F2 and 518R primer set to release the GC
Clamp, then prepared for sequencing.

Sequencing and Bioinformatics Analysis

Sequencing analysis was performed by First
BASE Laboratories, Singapore. Bioinformatics
analysis was undertaken to identify the dominant
species. Sequence alignment was performed using
BioEdit and identification was done using BlastN
(NCBI).

3. ResultsandDiscussion
Separation of DGGE bands is important to
distinguish single base-pair changes of DGGE
fragments (Shimano et al., 2012) that indicate
the different bacterial community. DGGE
analysis provides a picture composed of an array
of bands with different intensities. Band
intensities indicate the frequency of individual
PCR products in the reaction mixture
(Valkov & Baldrian, 2009). Therefore, it can
be concluded that dominant bands signified as
dominant populations.

The community profiles in Figure 1

revealed fewer strong bands on each well
(around 2 to 4) and more numbers of weaker
bands. Although the bands in Figure 1 were not
clearly separated, especially BLNM 3-6, we had
adjusted the lights for better visualization and
decided bands with the red arrows were the
dominant ones. In addition, the imperfect
separation might be caused by primer dimer or
other PCR impurities.

Figure 1. Negative image of EtBr-stained separation pattern using DGGE of 9 BLNM samples

Those dominant bands were sliced, prepared and sequenced. Then, bioinformatics analysis was
conducted for sequence alignment and identification. The results are provided in Table 1.
Table 1. Identified dominant bacterial species found in the biogas production steps collected at West Kalimantan

Sampl
e
B1
B2

Primer

Identification

F
R

Pseudomonas stutzeri
Pseudomonas stutzeri
Bacillus cereus, Bacillus
thuringiensis
Bacillus thuringiensis, Bacillus
anthracis, Bacillus cereus

F
R

B3
B4
B5
B6
B7
B8
B9

F
R
F
R
F
R
F
R
F
R
F
R
F
R

Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas stutzeri
Pseudomonas fluorescens
Clostridium sp.
Clostridium bifermentans
Escherichia coli
Escherichia coli
Clostridium sp.
Clostridium bifermentans

Query
(%)
100
100

Identity
(%)
100
100

74

97

100

99

100
100
98
98
100
100
95
100
100
100
100
100
100
100

100
100
94
97
99
99
85
96
95
95
99
99
99
99

Conclusion
Pseudomonas
stutzeri
Bacillus sp.
Pseudomonas
stutzeri
Pseudomonas
stutzeri
Pseudomonas
stutzeri
Pseudomonas
fluorescens
Clostridium sp.
Escherichia coli
Clostridium sp.

B1: POME Feed Pump; B2: Incoming POME Equalization Concrete Sump (IPECS); B3, B4, B5: Biodigester tank 1; B6,
B7, B8: Biodigester tank 2; B9: Treated POME Collection Concrete Sump (TPPCS).

The results on Table 1 show that POME Feed Pump and Biodigester tank 1 were dominated by
Pseudomonas stutzeri. However, biodigester tank 2 was more diverse. It was dominated by
Pseudomonas fluorescens, Clostridium sp., and Escherichia coli. While Incoming POME Equalization
Concrete Sump (IPECS) was dominated by Bacillus sp., Treated POME Collection Concrete Sump
(TPPCS) was dominated by Clostridium sp. That bacterial profile was similar to microbial
communities established in another biogas system in Sweden using agar plate technique (Erdgas,
2007).

4. Conclusion
It has been demonstrated that DGGE
technique was able to characterize bacterial
consortium that develops in biogas production.
While biodigester tank 1 was dominated by
Pseudomonas stutzeri, biodigester tank 2 was
more diverse with dominant populations of
Pseudomonas fluorescens, Clostridium sp., and
Escherichia coli. However, further research in
other communities, including fungal and
archaeal, are highy required constructing a
complete profile. In the future, the information
regarding dominant microbial communities in
biogas production can help to boost production
efficiency, increase yield, and generate
improved microbial additives.

5. Acknowledgement
We would like to acknowledge the
management of PT SMART Tbk. We would
also like to express our gratitude to Mr. Zulfikar
A. Tanjung for providing the bioinformatics
analysis.

6. References
Cetecioglu, Z., Ince, O., & Ince, B. (2012). Gel
Electrophoresis Based Genetic Fingerprinting
Techniques on Environmental Ecology. In S.

Magdeldin (Ed.), Gel Electrophoresis - Advanced

Techniques (pp. 5166). InTech. doi:10.5772/2688
Chin, M. J., Poh, P. E., Tey, B. T., Chan, E. S., & Chin, K.
L. (2013). Biogas from palm oil mill effluent
(POME): Opportunities and challenges from
Malaysias perspective. Renewable and Sustainable
Energy
Reviews,
26,
717726.
doi:10.1016/j.rser.2013.06.008
Erdgas. (2007). Microbiological community in biogas
systems and evaluation of microbial risks. Retrieved
September
14,
2015,
from
http://www.dvgw.de/uploads/media/ewp2007_12_g
as-usage.pdf
Li, A., Chu, Y., Wang, X., Ren, L., Yu, J., Liu, X., Li, S.
(2013). A pyrosequencing-based metagenomic
study of methane-producing microbial community
in solid-state biogas reactor. Biotechnology for
Biofuels, 6(1), 3. doi:10.1186/1754-6834-6-3
Muyzer, G., de Waal, E. C., & Uitterlinden, a G. (1993).
Profiling of complex microbial populations by
denaturing gradient gel electrophoresis analysis of
polymerase chain reaction-amplified genes coding
for 16S rRNA. Applied and Environmental
Microbiology,
59(3),
695700.
doi:00992240/93/030695-06$02.00/0
Shimano, S., Sambe, M., & Kasahara, Y. (2012).
Application of Nested PCR-DGGE (Denaturing
Gradient Gel Electrophoresis) for the Analysis of
Ciliate Communities in Soils. Microbes and
Environments,
27(2),
136141.
doi:10.1264/jsme2.ME11287
Valkov, V., & Baldrian, P. (2009). Denaturing gradient
gel electrophoresis as a fingerprinting method for
the analysis of soil microbial communities. Plant
and Soil, 2009(526), 413423. Retrieved from
http://www.journals.uzpi.cz/publicFiles/11516.pdf
Weiland, P. (2010). Biogas production: current state and
perspectives.
Applied
Microbiology
and
Biotechnology, 85(4), 84960. doi:10.1007/s00253009-2246-7