Syntrophomonas zehnderi sp. nov., an anaerobe that degrades long-chain fatty acids in co-culture with Methanobacterium formicicum

An anaerobic, mesophilic, syntrophic fatty-acid-oxidizing bacterium, designated strain OL-4T, was isolated as a co-culture with Methanobacterium formicicum DSM 1535NT from an anaerobic expanded granular sludge bed reactor used to treat an oleate-based effluent. Strain OL-4T degraded oleate, a mono-unsaturated fatty acid, and straight-chain fatty acids C4 : 0–C18 : 0 in syntrophic association with Methanobacterium formicicum DSM 1535NT. Even-numbered fatty acids were degraded to acetate and methane whereas odd-numbered fatty acids were degraded to acetate, propionate and methane. Branched-chain fatty acids were not degraded. The bacterium could not grow axenically with any other substrate tested and therefore is considered to be obligately syntrophic. Fumarate, sulfate, thiosulfate, sulfur and nitrate could not serve as electron acceptors for strain OL-4T to degrade oleate or butyrate. Cells of strain OL-4T were curved rods, formed spores and showed a variable response to Gram staining. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain OL-4T was most closely related to the fatty-acid-oxidizing, syntrophic bacterium Syntrophomonas sp. TB-6 (95% similarity), Syntrophomonas wolfei subsp. wolfei DSM 2245T (94% similarity) and Syntrophomonas erecta DSM 16215T (93% similarity). In addition to this moderate similarity, phenotypic and physiological characteristics, such as obligate syntrophy, spore formation and utilization of a broader substrate range, differentiated strain OL-4T from these Syntrophomonas species. Therefore strain OL-4T represents a novel species, for which the name yntrophomonas zehnderi sp. nov. is proposed. The type strain is OL-4T (=DSM 17840T=JCM 13948T).Fundação para a Ciência e a Tecnologia (FCT)( Fundo Social Europeu (FSE); Wageningen Institute for Environmental and Climate Research (WIMEK)

Effect of sulfate on methanogenic communities that degrade unsaturated and saturated long-chain fatty acids (LCFA)

Anaerobic bacteria involved in the degradation of long-chain fatty acids (LCFA), in the presence of sulfate as electron acceptor, were studied by combined cultivation-dependent and molecular techniques. The bacterial diversity in four mesophilic sulfate-reducing enrichment cultures, growing on oleate (C18:1, unsaturated LCFA) or palmitate (C16:0, saturated LCFA), was studied by denaturing gradient gel electrophoresis (DGGE) profiling of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. These enrichment cultures were started using methanogenic inocula in order to assess the competition between methanogenic communities and sulfate-reducing bacteria. Phylogenetic affiliation of rRNA gene sequences corresponding to predominant DGGE bands demonstrated that members of the Syntrophomonadaceae, together with sulfate reducers mainly belonging to the Desulfovibrionales and Syntrophobacteraceae groups, were present in the sulfate-reducing enrichment cultures. Subculturing of LCFA-degrading methanogenic cultures in the presence of sulfate resulted in the inhibition of methanogenesis and, after several transfers, archaea could no longer be detected by real-time PCR. Competition for hydrogen and acetate was therefore won by sulfate reducers, but acetogenic syntrophic bacteria were the only known LCFA-degrading organisms present after subculturing with sulfate. Principal component analysis of the DGGE profiles from methanogenic and sulfate-reducing oleate- and palmitate-enrichment cultures showed a greater influence of the substrate than the presence or absence of sulfate, indicating that the bacterial communities degrading LCFA in the absence/presence of sulfate are rather stable.This work was possible through grants attributed to D. Z. Sousa by Fundacao para a Ciencia e Tecnologia (FCT) and Fundo Social Europeu (FSE) (SFRH/BD/8726/2002), and by the Wageningen Institute for Environmental and Climate Research (WIMEK)

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFAaccumulation/ degradation cycles.Fundação para a Ciência e a Tecnologia (FCT); Fundo Social Europeu (FSE)

Waste lipids to energy: how to optimize methane production from long-chain fatty acids (LCFA)

The position of high-rate anaerobic technology (HR-AnWT) in the wastewater treatment and bioenergy market can be enhanced if the range of suitable substrates is expanded. Analyzing existing technologies, applications and problems, it is clear that, until now, wastewaters with high lipids content are not effectively treated by HR-AnWT. Nevertheless, waste lipids are ideal potential substrates for biogas production, since theoretically more methane can be produced, when compared with proteins or carbohydrates. In this minireview, the classical problems of lipids methanization in anaerobic processes are discussed and new concepts to enhance lipids degradation are presented. Reactors operation, feeding strategies and prospects of technological developments for wastewater treatment are discussed. Long-chain fatty acids (LCFA) degradation is accomplished by syntrophic communities of anaerobic bacteria and methanogenic archaea. For optimal performance these syntrophic communities need to be clustered in compact aggregates, which is often difficult to achieve with wastewaters that contain fats and lipids. Driving the methane production from lipids/LCFA at industrial scale without risk of overloading and inhibition is still a challenge that has the potential for filling a gap in the existing processes and technologies for biological methane production associated to waste and wastewater treatment.Fundação para a Ciência e a Tecnologia (FCT) - project FAT-METHANE (POCTI/CTA/46328/2002), grants PRAXIS XXI/BD/20326/99, SFRH/BPD/14591/2003, SFRH/BD/24256/2005Instituto Nacional da Propriedade Industrial (INPI)Netherlands Science FoundationLettinga Associates Foundatio

Ecophysiology of syntrophic communities that degrade saturated and unsaturated long-chain fatty acids

Syntrophic relationships are the key for biodegradation in methanogenic environments. We review the ecological and physiological features of syntrophic communities involved in the degradation of saturated and unsaturated long-chain fatty acids (LCFA), as well as their potential application to convert lipids/fats containing waste to biogas. Presently, about 14 species have been described with the ability to grow on fatty acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae. The principle pathway of LCFA degradation is through β-oxidation, but the initial steps in the conversion of unsaturated LCFA are unclear. Communities enriched on unsaturated LCFA also degrade saturated LCFA, but the opposite generally is not the case. For efficient methane formation, the physical and inhibitory effects of LCFA on methanogenesis need to be considered. LCFA adsorbs strongly to biomass, which causes encapsulation of active syntrophic communities and hampers diffusion of substrate and products in and out of the biomass. Quantification of archaea by real-time PCR analysis suggests that potential LCFA inhibitory effect towards methanogens might be reversible. Rather, the conversion of adsorbed LCFA in batch assays was shown to result in a significant increase of archaeal cell numbers in anaerobic sludge samples.The authors thank J. Prosser for the invitation to write this minireview. We appreciated the critical reading of I.M. Head and of the anonymous reviewers, and we thank them for their constructive comments and suggestions. This work was possible through the financial support provided by the Portuguese Science Foundation (FCT) and European Social Fund (ESF) (grant SFRH/BD/8726/2002), and by the Wageningen Institute for Environmental and Climate Research (WIMEK)

Crystallization and preliminary X-ray characterization of an NAD(P)-dependent butanol dehydrogenase A from Geobacillus thermodenitrificans

Job file for the creation/design of stained glass from either the Charles J. Connick Studio (1912-1945) or the Charles J. Connick Associates studio (1945-1986). The job file contains a job number, location information, date of completion, size, contact information, price, and a description of the project. This particular job file contains information on a job located at: Glendale, California. Forest Lawn Memorial Park