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Environmental Biotechnology |
Electronic Journal of Biotechnology ISSN: 0717-3458 |
Vol.
12 No. 3, Issue of July 15, 2009 |
© 2009 by Pontificia Universidad Católica
de Valparaíso -- Chile |
Received September 23, 2008
/ Accepted April 13, 2009 |
DOI: 10.2225/vol12-issue3-fulltext-4 |
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PCR-based DGGE and FISH analysis of methanogens
in an anaerobic closed digester tank for treating palm oil mill effluent
Meisam Tabatabaei*
Department of Bioprocess Technology
Faculty of Biotechnology and Biomolecular
Sciences
University Putra Malaysia 43400, Serdang
Selangor, Malaysia
Tel: 603
89467590
Fax: 603 89467593
E-mail: meisam_tab@yahoo.com
Mohd Rafein Zakaria
Department of Bioprocess Technology
Faculty of Biotechnology and Biomolecular
Sciences
University Putra Malaysia 43400, Serdang
Selangor, Malaysia
Raha Abdul Rahim
Department of Cell and Molecular Biology
Faculty of Biotechnology and Biomolecular
Sciences
Universitiy Putra Malaysia 43400, Serdang
Selangor, Malaysia
André-Denis G. Wright
CSIRO Livestock Industries
Queensland Bioscience Precinct
St. Lucia, Queensland 4067, Australia
Yoshihito Shirai
Graduate School of Life Science and
Systems Engineering
Kyushu Institute of Technology
Wakamatsu-ku, Kitakyushu 808-0196, Japan
Norhani Abdullah
Department of Microbiology
Faculty of Biotechnology and Biomolecular
Sciences
University Putra Malaysia 43400, Serdang
Selangor, Malaysia
Kenji Sakai
Laboratory of Soil Microorganisms
Department of Plant Resources
Graduate School of Bioresources and Bioenvironmental
Sciences
Kyushu University
Higashi-ku, Fukuoka, Japan
Shinya Ikeno
Graduate School of Life Science and
Systems Engineering
Kyushu Institute of Technology
Wakamatsu-ku, Kitakyushu 808-0196, Japan
Masatsugu Mori
Laboratory of Soil Microorganisms
Department of Plant Resources
Graduate School of Bioresources and Bioenvironmental
Sciences
Kyushu University
Higashi-ku, Fukuoka, Japan
Nakamura Kazunori
Laboratory of Soil Microorganisms
Department of Plant Resources
Graduate School of Bioresources and Bioenvironmental
Sciences
Kyushu University
6-10-10Hakozaki, Higashi-ku
Fukuoka, Japan
Alawi Sulaiman
Department of Food and Process Engineering
Faculty of Engineering
University Putra Malaysia 43400, Serdang
Selangor, Malaysia
Mohd
Ali Hassan
Department of Food and Process Engineering
Faculty of Engineering and
Department of Bioprocess Technology
Faculty of Biotechnology and Biomolecular
Sciences
University Putra Malaysia 43400, Serdang
Selangor, Malaysia
*Corresponding author
Financial
support: FELDA (Federal
Land Development Authority), MOSTI (Ministry of Science, Technology and
Innovation, Malaysia) and JSPS (Japan Society for the Promotion of Science).
Keywords: anaerobic digestion, DGGE, FISH, methanogens, POME.
Abbreviations: |
CCD: charge-coupled
device
CDM:
clean development mechanism
CDT:
closed digester tank
COD:
chemical oxygen demand
DGGE:
denatured gradient gel electrophoresis
FISH : fluorescent in situ hybridization
NCBI: National
Center for Biotechnology Information
PBS:
phosphate buffer saline
PCR:
polymerase chain reaction
POME:
palm oil mill effluent
RFLP:
restriction fragment length polymorphism
rRNA:
ribosomal RNA
SEM:
scanning electron microscopy
UASB:
upflow anaerobic sludge blanket
VFA: volatile fatty acids |
16S
ribosomal RNA (rRNA)-targeted fluorescent in situ hybridization
combined with polymerase chain reaction (PCR)-cloning,
light microscopy using Gram stains, scanning electron microscopy
and denatured gradient gel electrophoresis were used to reveal
the distribution of methanogens within an anaerobic closed digester
tank fed with palm oil mill effluent. For specific detection
of methanogens, 16S rRNA-cloning analysis was conducted followed
by restriction fragment length polymorphism (RFLP) for presumptive
identification of methanogens. To cover the drawbacks of the
PCR-cloning study, the organization of the microorganisms was
visualized in the activated sludge sample by using fluorescent
oligonucleotide probes specific to several different methanogens,
and a probe for bacteria. In situ hybridization
with methanogens and bacterial probes and denatured gradient gel
electrophoresis within activated sludge clearly confirmed the presence of Methanosaeta sp.
and Methanosarcina sp. cells. Methanosaeta concilii was
found to be the dominant species in the bioreactor. These results revealed the
presence of possibly new strain of Methanosaeta in the bioreactor
for treating palm oil mill effluent called Methanosaeta concilii SamaliEB
(Gene bank accession number: EU580025). In addition, fluorescent hybridization
pictured the close association between the methanogens and bacteria and that
the number of methanogens was greater than the number of bacteria.
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