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.

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.