Role of syntrophic microbial communities in high-rate methanogenic bioreactors

Anaerobic purification is a cost-effective way to treat high strength industrial wastewater. Through anaerobic treatment of wastewaters energy is conserved as methane, and less sludge is produced. For high-rate methanogenesis compact syntrophic communities of fatty acid-degrading bacteria and methanogenic archaea are essential. Here, we describe the microbiology of syntrophic communities in methanogenic reactor sludges and provide information on which microbiological factors are essential to obtain high volumetric methane production rates. Fatty-acid degrading bacteria have been isolated from bioreactor sludges, but also from other sources such as freshwater sediments. Despite the important role that fatty acid-degrading bacteria play in high-rate methanogenic bioreactors, their relative numbers are generally low. This finding indicates that the microbial community composition can be further optimized to achieve even higher rates.Our research is funded by grants from the division of Chemical Sciences (CW) and Earth and Life Sciences (ALW) of The Netherlands Organisation for Scientific Research (NWO) and by the Technology Foundation (STW), the applied science division of NWO

Syntrophic LCFA-degraders: “bacteria that can clean soap”

Wastewaters contain substantial amounts of long-chain fatty acids (LCFA) which, when in the form of sodium salts, are what we normally call soaps. These compounds, resulting from fats' hydrolysis, can be converted to high amounts of methane. Developing new technological solutions for LCFA methanation, but also understanding the physiology and microbiology of LCFA degradation is fundamental for the bioenergy valorization of fatty wastewaters. In this work we present an overview of our results on anaerobic LCFA microbial degradation. Molecular techniques were used to investigate the structure of microbial communities present in different LCFA-degrading communities, such as continuous oleate- and palmitate-fed bioreactors and several enrichment cultures degrading these two LCFA as well. Choice of oleate and palmitate as model substrates was due to their predominance in wastewaters and to the fact that they represent mono-unsaturated and saturated LCFA, respectively. DGGE fingerprinting and sequencing evidenced the importance of syntrophic bacteria, affiliated with the Syntrophomonas genus, in the degradation of these compounds. Enrichment on oleate or palmitate resulted in distinct bacterial communities, which might be related to LCFA chain-saturation differences. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture. The fact that S. zehnderi can degrade a wide range of fatty-acids with different chain length (C4-C18) and is also able to use unsaturated LCFA (e.g. oleate) makes it a destined and dedicated key for the anaerobic treatment of wastewaters, in which an assembly of different fatty-acids is normally present. Genome sequencing of S. zehnderi is currently undergoing

The microbiology of conversion of long-chain fatty acids (LCFA) to biogas

Wastewaters, mainly the ones from food processing industries, contain considerable amounts of long-chain fatty acids (LCFA). These pollutant compounds, resulting from the hydrolysis of lipids, can be used as energetic resources for the production of biogas. A large amount of methane can be produced from LCFA; theoretically 1g of oleate, one of the most common LCFA found in wastewaters, can be converted to 1.01 L of methane (at standard temperature and pressure), while 1 g of glucose yields only 0.37 L methane. In its core this is a biological process, thus strongly linked to the performance and efficiency of the different microorganisms interacting in the process. Insight into the phylogenetic and functional communities involved in LCFA degradation is necessary to understand and enable the effective performance of bioreactors treating these compounds. In this work we describe the application of culture-dependent and culture-independent strategies to study microbiological and physiological aspects of the degradation of LCFA in anaerobic environments. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA (C18:0), and palmitate, a saturated LCFA (C16:0), both abundant in LCFA-rich wastewaters. LCFA-degrading communities were developed by selective enrichments growing on oleate and palmitate. Changes in the microbial composition during enrichment were analyzed by DGGE profiling of PCR-amplified 16S rRNA gene fragments. Predominant DGGE-bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. 16S rRNA gene sequences clustering within the Syntrophomonadaceae family were identified as corresponding to predominant DGGE-bands in the oleate- and palmitateenrichment cultures. In stable palmitate-enrichment cultures members of the Syntrophobacteraceae family were also present. Further on, a new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture. This mesophilic, syntrophic, fatty acid oxidizing bacterium degrades straight-chain fatty acids with 4 to 18 carbon atoms but, also, unsaturated LCFA, such as oleate. The presence of Syntrophomonas zehnderi related bacteria in several sludges after contact with oleate was, subsequently, verified by DGGE-fingerprinting analysis and suggests its important role in anaerobic oleate degradation in bioreactor sludge. Future work on the performance of bioaugmented reactors with this versatile LCFA-degrading bacterium promise new results on the efficient conversion of LCFA to methane

Enrichment of carbon monoxide utilising microorganisms from methanogenic bioreactor sludge

Conversion of CO is the rate limiting step during anaerobic conversion of syngas (a gaseous mixture mainly composed of CO, CO2 and H2). In this work we study the microbial diversity in anaerobic sludge submitted to extended contact to syngas in a multi-orifice baffled bioreactor (MOBB). Methane was the main product resulting from syngas conversion in the MOBB. Enrichment cultures started with this sludge produced methane as final product, but also acetate. 16S rRNA gene analysis revealed a predominance of Acetobacterium and Sporomusa species in the enrichments. These are homoacetogenic bacteria that might be involved in CO conversion to acetate. Hydrogen was formed as intermediary from CO conversion and likely used by hydrogenotrophs with the formation of methane. Pasteurisation and serial dilutions of stable CO-converting enrichments resulted in a microbial culture dominated by two Sporomusa species that are able to use CO as sole substrate

Anaerobic LCFA degradation: a role for non-syntrophic conversions?

For many years the focus of lipids/long-chain fatty-acids (LCFA) wastewater treatment was on technological and process developments. More recently, promising results on the anaerobic treatment of LCFA-containing wastewaters[1] widened the attention to the microbiology aspects as well. In anaerobic bioreactors, LCFA can be β-oxidized to acetate and H2 by acetogenic bacteria, in obligatory syntrophy with methanogens. Presently, 14 species have been described that grow on fatty-acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae[2]. Among these, only 4 species are able to degrade mono- and/or polyunsaturated LCFA. The reason why the degradation of unsaturated LCFA is not more widespread remains unknown. Early studies suggested that degradation of unsaturated LCFA requires complete chain saturation prior to β-oxidation[2]. Unsaturated LCFA, such as linoleate (C18:2) and oleate (C18:1), would be metabolized through a hydrogenation step yielding stearate (C18:0), then entering the β-oxidation cycle. However, this theory is inconsistent with the observed accumulation of palmitate (C16:0) in continuous bioreactors fed with oleate[1]. We hypothesize that LCFA chain saturation might be a non-syntrophic process, i.e. unsaturated LCFA can function as electron donors and acceptors, as protons released in a first β-oxidation step can be used to hydrogenate the unsaturated hydrocarbon. To test this, linoleate (C18:2), oleate (C18:1) and a mixture of stearate (C18:0) and palmitate (C16:0) were continuously fed to bioreactors with methanogenesis-active or -inhibited anaerobic sludge. In the reactors fed with linoleate and oleate, palmitate accumulated in methanogenesis-active and -inhibited bioreactors up to concentrations of approximately 2 mM and 8 mM, respectively. In methanogenesis-inhibited bioreactors fed with a mixture of saturated LCFA (stearate and palmitate) no biological activity occurred. These results suggest the occurrence of a non-syntrophic step during the degradation of unsaturated LCFA in anaerobic bioreactors. The identification of microbial communities involved in non-syntrophic linoleate/oleate to palmitate conversion will give more insights into this novel biochemical mechanism

Characterization of an anaerobic thermophilic glycerol-degrading enrichment culture

Background: The glycerol market was totally changed by the biodiesel industry, which resulted in the production of an excess of this compound as an industrial by-product. As a consequence, the price of glycerol dropped and a huge interest in alternatives for its valorisation emerged since then. In the field of Biotechnology research, glycerol is an attractive compound for the microbial production of chemical building blocks. Objectives: The aim of this work was to investigate thermophilic anaerobic communities capable of conversion of glycerol. Methods: Thermophilic sludge from a lab-scale anaerobic reactor fed with skim milk and sodium oleate (50:50% chemical oxygen demand) was incubated at 55°C in closed bottles containing bicarbonate-buffered medium supplemented with 10mM glycerol. Periodic successive transfers of the glycerol-converting enrichment culture, combined with serial dilutions were performed. After eight generations a highly enriched, low diversity (microscopic observations and 16s rRNA DGGE profiling) microbial culture was obtained. Conclusions: The enriched culture converted glycerol mainly to methane (6mM) and acetate (7mM) within 6 days of incubation. A yet unknown organic compound was also produced. Sequencing results obtained on the Illumina platform showed the bacterial predominance of an uncultured Thermotoga species (75 % of the retrieved sequences), an uncultured Anaerobaculum species (13 %) and a close relative to Thermoanaerobacter pseudethanolicus (5 %). Isolation of the new uncultured Thermotoga and Anaerobaculum species is ongoing and their role in glycerol degradation will be assessed

Microbial communities involved in anaerobic degradation of unsaturated or saturated long-chain fatty acids

Anaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria within Syntrophomonadaceae and Syntrophobacteraceae families. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate- specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with the Syntrophomonas genus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.Fundação para a Ciência e a Tecnologia (FCT)Fundo Social Europeu (FSE) Wageningen Institute for Environmental and Climate Research (WIMEK)

Microbial communities involved in anaerobic degradation of unsaturated-and saturated-LCFA in methanogenic bioreactors

Long chain fatty acids (LCFA) are frequently found in wastewaters as the main product of lipid hydrolysis. These compounds hold a high energetic potential and thus are attractive substrates for methane production. Insight into the microbial populations involved in anaerobic LCFA-degradation is important for the development and improvement of technologies for lipids/LCFA-rich wastewater valorisation. This study identifies putative LCFA-degrading bacteria by combining selective enrichments with molecular techniques. Two distinct enrichment series of anaerobic cultures growing on unsaturatedand saturated-LCFA were obtained by successive transfers in medium containing oleate (C18:1) and palmitate (C16:0), respectively, as the sole carbon and energy source. This procedure resulted in two stable and highly enriched cultures that could convert oleate and palmitate to acetate and methane. Changes in the microbial composition during the enrichment were analyzed by 16S rRNA gene PCRDGGE profiling. Upon enrichment a decrease in microbial diversity was observed. Prominent bands in the DGGE profiles of stable enriched cultures were identified by 16S rRNA gene sequencing, and nearly full sequences were compared using ARB software. A major part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Organisms corresponding to dominant DGGE bands in oleate- and palmitate-enrichment cultures clustered with fatty-acid oxidizing syntrophic bacteria within Syntrophomonadaceae and Syntrophaceae families. Despite the absence of sulphate in the medium, a Desulfovibrio-like organism was detected as a dominant band in the DGGE profile of the oleate-enrichment culture. In other studies Desulfovibrio species have been detected in methanogenic reactors without added sulphate. They were proposed to grow acetogenically. The role of such bacteria in the oleate-enrichment culture is not clear yet, and this needs further investigation. A low methane yield (12%) was observed in the oleate-enrichment and acetate, produced according to the expected stoichiometry, was no further converted to methane. In the palmitate-enrichment culture, the acetate produced was completely mineralised and a total methane yield of about 83% was achieved from palmitate degradation. Furthermore, the oleate-enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialised consortia degraded oleate only after a lag phase of three months, after which the DGGE profile was changed. A dominant band appeared and sequence analysis showed affiliation with the Syntrophomonas genus. This band was also present in the oleate-enrichment culture, suggesting that this bacterium is important for oleate degradation, emphasizing possible differences between the degradation of unsaturated- and saturated-LCFA