Diversity of Anaerobic Microorganisms Involved in Long-Chain Fatty Acid Degradation in Methanogenic Sludges as Revealed by RNA-Based Stable Isotope Probing

ABSTRACT Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with 13C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect 13C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in 13C-labeled and unlabeled rRNA fractions. The bacterial populations in the 13C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.

[1]  Lutgarde Raskin,et al.  Microbial population dynamics during start‐up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge , 2004, Biotechnology and bioengineering.

[2]  B. Svensson,et al.  Mesophilic syntrophic acetate oxidation during methane formation in biogas reactors , 1999 .

[3]  T. Lueders,et al.  Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. , 2003, Environmental microbiology.

[4]  K. Kida,et al.  Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation. , 2005, Journal of bioscience and bioengineering.

[5]  B L Maidak,et al.  The RDP-II (Ribosomal Database Project) , 2001, Nucleic Acids Res..

[6]  Sang‐Hyoun Kim,et al.  Inhibitory effects of long-chain fatty acids on VFA degradation and β-oxidation , 2003 .

[7]  Amarjeet Bassi,et al.  Long-term performance of high-rate anaerobic reactors for the treatment of oily wastewater. , 2006, Environmental science & technology.

[8]  Philip Ineson,et al.  Stable-isotope probing as a tool in microbial ecology , 2000, Nature.

[9]  H. Harada,et al.  Pilot-plant study on anaerobic treatment of a lipid- and protein-rich food industrial wastewater by a thermophilic multi-staged UASB reactor. , 2002, Water science and technology : a journal of the International Association on Water Pollution Research.

[10]  C. Jeanthon,et al.  Characterization of long-chain fatty-acid-degrading syntrophic associations from a biodegraded oil reservoir. , 2005, Research in microbiology.

[11]  M. Wagner,et al.  Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community , 2004, Applied and Environmental Microbiology.

[12]  A. Whiteley,et al.  Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies. , 2006, Current opinion in biotechnology.

[13]  Y. Kamagata,et al.  Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium. , 2000, International journal of systematic and evolutionary microbiology.

[14]  H. Harada,et al.  Process Performance and Sludge Behaviors of Multi-staged UASB reactor for Treatment of Palm Oil Mill Effluent (POME) , 2003 .

[15]  P. Hugenholtz Exploring prokaryotic diversity in the genomic era , 2002, Genome Biology.

[16]  B. Schink Energetics of syntrophic cooperation in methanogenic degradation , 1997, Microbiology and molecular biology reviews : MMBR.

[17]  Alison M. Cupples,et al.  Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing , 2007 .

[18]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[19]  Y. Kamagata,et al.  Identification and Isolation of Anaerobic, Syntrophic Phthalate Isomer-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephthalate Manufacturing , 2004, Applied and Environmental Microbiology.

[20]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[21]  H. Harada,et al.  Identification and Cultivation of Anaerobic, Syntrophic Long-Chain Fatty Acid-Degrading Microbes from Mesophilic and Thermophilic Methanogenic Sludges , 2006, Applied and Environmental Microbiology.

[22]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[23]  Alfons J. M. Stams,et al.  Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids , 2006, Applied and Environmental Microbiology.

[24]  B. Patel,et al.  Anaerobaculum thermoterrenum gen. nov., sp. nov., a novel, thermophilic bacterium which ferments citrate. , 1997, International journal of systematic bacteriology.

[25]  M. Friedrich Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. , 2006, Current opinion in biotechnology.

[26]  L. Muxí,et al.  Physiological and Molecular Characterisation of an Anaerobic Thermophilic Oleate-degrading Enrichment Culture , 2001 .

[27]  Xiuzhu Dong,et al.  Syntrophomonas curvata sp. nov., an anaerobe that degrades fatty acids in co-culture with methanogens. , 2004, International journal of systematic and evolutionary microbiology.

[28]  I. Koster,et al.  Inhibition of Methanogenesis from Acetate in Granular Sludge by Long-Chain Fatty Acids , 1987, Applied and environmental microbiology.

[29]  T. Lueders,et al.  Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil , 2004, Applied and Environmental Microbiology.

[30]  Bo H. Svensson,et al.  Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration , 1994, Archives of Microbiology.

[31]  B. Patel,et al.  Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil-producing well. , 1995, International journal of systematic bacteriology.

[32]  C. Kitts,et al.  Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. , 2001, Current issues in intestinal microbiology.

[33]  R. Menes,et al.  Detection of fatty acid beta-oxidizing syntrophic bacteria by fluorescence in situ hybridization. , 2006, Water science and technology : a journal of the International Association on Water Pollution Research.

[34]  C. Etchebehere,et al.  Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge. , 1998, International journal of systematic bacteriology.

[35]  H. Harada,et al.  Syntrophomonas palmitatica sp. nov., an anaerobic, syntrophic, long-chain fatty-acid-oxidizing bacterium isolated from methanogenic sludge. , 2007, International journal of systematic and evolutionary microbiology.

[36]  E. Madsen The use of stable isotope probing techniques in bioreactor and field studies on bioremediation. , 2006, Current opinion in biotechnology.

[37]  F. Rainey,et al.  Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum. , 1996, International journal of systematic bacteriology.

[38]  Lutgarde Raskin,et al.  Quantification of Syntrophic Fatty Acid-β-Oxidizing Bacteria in a Mesophilic Biogas Reactor by Oligonucleotide Probe Hybridization , 1999, Applied and Environmental Microbiology.

[39]  Hauke Smidt,et al.  Syntrophomonas zehnderi sp. nov., an anaerobe that degrades long-chain fatty acids in co-culture with Methanobacterium formicicum. , 2007, International journal of systematic and evolutionary microbiology.

[40]  M. P. Bryant,et al.  Syntrophomonas wolfei subsp. saponavida subsp. nov., a Long-Chain Fatty-Acid-Degrading, Anaerobic, Syntrophic Bacterium; Syntrophomonas wolfei subsp. wolfei subsp. nov.; and Emended Descriptions of the Genus and Species , 1989 .

[41]  B. Schink,et al.  Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. , 1996, International journal of systematic bacteriology.

[42]  A. Ogram,et al.  Phylogeny of Acetate-Utilizing Microorganisms in Soils along a Nutrient Gradient in the Florida Everglades , 2006, Applied and Environmental Microbiology.

[43]  E. Samain,et al.  Synthrophomonas sapovorans sp. nov., a new obligately proton reducing anaerobe oxidizing saturated and unsaturated long chain fatty acids , 1986, Archives of Microbiology.

[44]  Mark J. Bailey,et al.  RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny , 2002, Applied and Environmental Microbiology.

[45]  P A Vanrolleghem,et al.  Coupling the SHARON process with anammox: model-based scenario analysis with focus on operating costs. , 2005, Water science and technology : a journal of the International Association on Water Pollution Research.

[46]  M. P. Bryant,et al.  Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses. , 1993, International journal of systematic bacteriology.

[47]  Richard E. Speece,et al.  Anaerobic Biotechnology for Industrial Wastewaters , 1996 .

[48]  Y. Kamagata,et al.  Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors. , 2005, Water science and technology : a journal of the International Association on Water Pollution Research.

[49]  A. Ogram,et al.  Fatty Acid-Oxidizing Consortia along a Nutrient Gradient in the Florida Everglades , 2006, Applied and Environmental Microbiology.

[50]  S. Wuertz,et al.  Three-dimensional distribution of GFP-labeled Pseudomonas putida during biofilm formation on solid PAHs assessed by confocal laser scanning microscopy. , 2003, Water science and technology : a journal of the International Association on Water Pollution Research.

[51]  Y. Kamagata,et al.  Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate. , 2000, International journal of systematic and evolutionary microbiology.

[52]  K. Kida,et al.  Microbial diversity of mesophilic methanogenic consortium that can degrade long-chain fatty acids in chemostat cultivation. , 2006, Journal of bioscience and bioengineering.

[53]  A. Stams,et al.  Molecular monitoring of microbial diversity in expanded granular sludge bed (EGSB) reactors treating oleic acid. , 2002, FEMS microbiology ecology.

[54]  Y Y Li,et al.  High-rate methane fermentation of lipid-rich food wastes by a high-solids co-digestion process. , 2002, Water science and technology : a journal of the International Association on Water Pollution Research.

[55]  C. Woese,et al.  Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms , 1999, Archives of Microbiology.