Ecology and physiology of anaerobic microbial communities that degrade long chain fatty acids

Doutoramento em Engenharia Química e BiológicaWastewaters, particularly those from food processing industries, contain considerable amounts of long chain fatty acids (LCFA). These polluting compounds, resulting from the hydrolysis of oils and fats, can be used for the production of biogas, a renewable source of energy. Energetic valorization of LCFA-rich wastewaters in anaerobic bioreactors is, in its essence, a biological process, and therefore strongly linked to the performance and efficiency of the different microorganisms interacting in the process. Insight into the phylogenetic and functional diversity of microbial communities present in anaerobic bioreactors treating LCFA-containing wastewaters is necessary to understand and enable the effective performance of these bioreactors. Hence, this thesis describes the application of culture-dependent and culture-independent strategies to study microbiological aspects of the degradation of LCFA in anaerobic environments. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA, and palmitate, a saturated LCFA, both abundant in LCFA-rich wastewaters. The capability of anaerobic bioreactor sludge to degrade LCFA in cycles of continuous feeding (LCFA accumulation, loading) followed by batch incubation (LCFA degradation) was shown, first by incubating loaded sludge in batch assays and afterwards by simply interrupting the reactor’s feeding after sludge loading. The methanation kinetics of biomass-associated LCFA after continuous sludge loading with oleate was established in batch assays, according to an inhibition model based on Haldane’s enzymatic inhibition kinetics. An optimal value of about 1000 mgCOD-LCFA gVSS-1 was determined for the amount of accumulated LCFA that could be converted to methane at the maximal rate of 250 mgCOD-CH4 gVSS-1 day-1. The determination of this value is critical for the operation of a cyclic reactor treating LCFA-rich wastewaters. 16S rRNA gene analysis of sludge samples submitted to continuous oleate- and palmitate-feeding followed by batch degradation of the accumulated LCFA demonstrated that bacterial communities were dominated by members of the Clostridiaceae and Syntrophomonadaceae families. Archaeal communities, previously reported as very sensitive to contact with LCFA, could endure cycles of continuous/batch LCFA degradation, as shown by the relative high archaeal abundance measured by FISH and real-time PCR at the end of the cycle (38 to 75% of relative archaeal abundance). Archaeal populations were mainly comprised of hydrogen-consuming microorganisms belonging to the genus Methanobacterium, and acetate-utilizers from the genera Methanosaeta and Methanosarcina. Enrichment cultures growing on oleate and palmitate (as sole carbon and energy source) gave more insight into the major players involved in the degradation of unsaturated and saturated LCFA. Syntrophomonas-related species were identified as predominant microorganisms in different enrichment cultures, degrading both oleate and palmitate. Different microbial consortia were enriched with oleate and palmitate, evidencing a dependence on the saturation level of the LCFA. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture. Changes in the microbial composition of oleate- and palmitate-enrichment cultures submitted to a substrate swap were followed by denaturing gradient gel electrophoresis (DGGE), allowing the correlation between the appearance of a DGGE band corresponding to a Syntrophomonas-like microorganism (later on specified as related to S. zehnderi) with the capability to degrade unsaturated LCFA. Microorganisms clustering within the family Syntrophobacteraceae were also identified in the palmitate-enrichment cultures, though none of the thus far characterized bacteria from this family can utilize this compound. Competition between acetogenic and methanogenic microorganisms and sulfate-reducing bacteria (SRB) was also studied. Acetogenic bacteria were not out-competed by LCFA-degrading SRB and microorganisms belonging to the Syntrophomonadaceae and Syntrophobacteraceae families were present after successive culture transfers in the presence of sulfate. On the other hand, hydrogenotrophic archaea were rapidly out-competed by hydrogen-consuming SRB belonging to the Desulfovibrionales order. In conclusion, an increased understanding of the active microbial communities in LCFA-degrading bioreactors has been provided in this study. Further work on the genomics and proteomics of LCFA-degrading microorganisms might give more insight into the degradation of LCFA, including substrate-specific differences in the degradation of unsaturated and saturated LCFA.As águas residuais, particularmente as geradas pela indústria alimentar, contêm quantidades consideráveis de ácidos gordos de cadeia longa (AGCL). Estes compostos poluentes, resultantes da hidrólise de óleos e gorduras, podem ser utilizados na produção de biogás, uma fonte de energia renovável. A valorização energética de águas residuais ricas em AGCL em bioreactores anaeróbios é, na sua essência, um processo biológico e, por isso, fortemente relacionado com o desempenho e eficiência dos diferentes microrganismos que intervêm no processo. Nesse sentido, para o tratamento de águas residuais com elevados teores de AGCL em bioreactores anaeróbios, é importante ter conhecimento sobre a diversidade filogenética e funcional das comunidades microbianas presentes nesses bioreactores. Esta tese descreve a aplicação de métodos moleculares e de cultura para estudar aspectos microbiológicos da degradação de AGCL em ambientes anaeróbios. Foram usados dois AGCL como substratos modelo: oleato, um AGCL mono-insaturado, e palmitato, um AGCL saturado, ambos abundantes em águas residuais ricas em AGCL. A capacidade da biomassa anaeróbia degradar AGCL em ciclos sequenciais de alimentação contínua (promoção da acumulação de AGCL) seguida de incubação em “batch” (degradação dos AGCL acumulados) foi demonstrada, primeiro através da incubação em ensaios “batch” de biomassa contendo AGCL associados e, depois, simplesmente através da interrupção da alimentação contínua de um bioreactor laboratorial. Estabeleceu-se a cinética de mineralização dos AGCL associados à biomassa, de acordo com um modelo de inibição baseado na cinética de inibição enzimática de Haldane. Determinou-se um valor óptimo de aproximadamente 1000 mgCQO-AGCL gSSV-1 como a quantidade de AGCL que podem ser acumulados e posteriormente degradados a uma taxa máxima de 250 mgCQO-CH4 gSSV-1 dia-1. Este valor é crítico para a operação de um reactor cíclico para o tratamento de águas residuais ricas em AGCL. A análise do gene 16S rRNA presente em amostras de biomassa submetidas a alimentação contínua de oleato e palmitato, seguida de degradação “batch” dos AGCL acumulados, demonstrou que as comunidades bacterianas eram dominadas por membros das famílias Clostridiaceae e Syntrophomonadaceae. As comunidades arquea, consideradas como muito sensíveis ao contacto com AGCL, demonstraram suportar ciclos de alimentação contínua e degradação batch, tal como indicado pela elevada abundância relativa de arquea medidas através de FISH e PCR em tempo real. As populações arquea eram compostas essencialmente por microrganismos utilizadores de hidrogénio pertencentes ao género Methanobacterium, e utilizadores de acetato dos géneros Methanosaeta e Methanosarcina. Culturas enriquecidas em oleato e palmitato (fornecidos como única fonte de carbono e energia) permitiram um maior conhecimento dos microrganismos envolvidos na degradação de AGCL saturados e insaturados. Foram identificadas espécies relacionadas com Syntrophomonas como predominantes em vários enriquecimentos diferentes, degradando ambos oleato e palmitato. Os consórcios microbianos desenvolvidos durante o enriquecimento com oleato ou com palmitato eram diferentes, evidenciando uma especialização dependente do nível de saturação da cadeia do AGCL. Foi isolada uma nova bactéria sintrófica, Syntrophomonas zehnderi, de uma cultura que degradava oleato. Foram monitorizadas alterações na composição microbiana de culturas enriquecidas com oleato e palmitato submetidas a troca de substrato através da utilização de electroforese em gel desnaturante, permitindo a correlação do aparecimento de uma banda, correspondente a um microrganismo relacionado com Syntrophomonas (posteriormente especificado como relacionado com S. zehnderi), com a capacidade de degradar AGCL insaturados. Foram também identificados microrganismos relacionados com a família Syntrophobacteraceae, nas culturas enriquecidas com palmitato, embora nenhuma das bactérias caracterizadas pertencentes a esta família possam utilizar este composto. Foi também estudada a competição de microrganismos acetogénicos e metanogénicos por bactérias redutoras de sulfato (BRS). Verificou-se a presença de microrganismos pertencentes às famílias Syntrophomonadaceae e Syntrophobacteraceae, depois de transferências sucessivas em meio contendo sulfato, indicando que as bactérias acetogénicas não foram suprimidas por BRS capazes de degradar AGCL. Contrariamente, os microrganismos hidrogenotróficos foram rapidamente substituídos por BRS pertencentes à ordem Desulfovibrionales. Em conclusão, este estudo permitiu uma melhor compreensão das comunidades microbianas activas em bioreactores a degradar AGCL. Estudos adicionais ao nível do genoma e proteoma de microrganismos que degradam AGCL permitirão um melhor entendimento da degradação dos AGCL, especificamente das diferenças na degradação de AGCL saturados e insaturados

Syntrophic LCFA degradation: generating a high-energy carrier from low-energy metabolism

For many years the interest in anaerobic degradation of lipids and long-chain fatty acids (LCFA) focused on technology and process developments. Attention to the microbiology of LCFA conversion was boosted by results showing that high methane yields could be obtained during anaerobic degradation of LCFA. The principle pathway of LCFA degradation is through -oxidation, producing acetate and hydrogen. For this conversion to be thermodynamically feasible, in methanogenic environments, acetogenic LCFA-degrading bacteria have to cooperate in syntrophy with hydrogen-consuming archaea. DGGE fingerprinting and 16S rRNA gene sequencing showed the importance of Syntrophomonas-like bacteria during batch and continuous degradation of unsaturated and saturated LCFA. The 7 species described thus far with the ability to grow on LCFA (with more than 12 carbon atoms), all belong to the families Syntrophomonadaceae and Syntrophaceae and, among these, only 4 species have the capability of utilizing mono- and/or polyunsaturated LCFA. Syntrophomonas zehnderi is able to degrade a wide range of saturated and unsaturated LCFA (C4 to C18) and could be detected in sludge samples from fed-batch and continuous reactors degrading oleate (monounsaturated C18). Batch bioaugmentation assays showed that addition of S. zenhderi could improve methane recovery from LCFA. The molecular mechanisms of anaerobic-LCFA degradation by S. zehnderi are being further studied by proteomics and whole genome sequencing. Identifying the components involved in the conversion of unsaturated LCFA-conversion is of particular interest. Related to this, the microbiology of oleate to palmitate (saturated C16) conversion by mixed cultures is also being studied using stable isotope probing and metaproteomics

Innovative start-up strategies for optimal methane production from lipids in anaerobic bioreactors

Lipids and long-chain fatty acids (LCFA) are energy-rich compounds that can be used as carbon and energy source by anaerobic microbial communities. Theoretically, large amounts of methane, a valuable energy carrier, may be generated during this process. However, operational problems, mainly associated with LCFA accumulation onto the sludge, have limited the use of anaerobic technology to produce methane from LCFA. In this work, two novel start-up strategies were tested for optimal methane production from LCFA: (i) bioreactor start-up using a intermittent feeding strategy, and (ii) bioreactor bioaugmentation with a LCFA-degrading bacterium. Intermittent feeding start-up resulted in efficient continuous methane production from high LCFA loads (up to 21 kgCOD m-3 day-1, 50% COD as oleate (unsaturated LCFA, C18:1)). Alternating continuous bioreactor feeding and batch degradation periods, during bioreactor start-up, was crucial for sludge acclimation and contributed to the development of a metabolically specialized anaerobic microbial community that was able to efficiently convert oleate to methane. After intermittent feeding bioreactor start-up, methane yields higher than 70% were achieved, and neither LCFA nor VFA accumulated in the system. Bioaugmentation experiments were performed using Syntrophomonas zehnderi, a bacterium able to degrade saturated and unsaturated LCFA [1]. Anaerobic sludge amended with active and inactive S. zehnderi was incubated with 1 mM oleate as sole carbon and energy source. Methane production from oleate in bioaugmented batches was faster and high methane yields (89±5%) were achieved. This work highlights the importance of the start-up strategy for the development of balanced syntrophic communities specialized in methane production from LCFA. Intermittent feeding and bioaugmentation with LCFA-degrading bacteria may be applied as alternative or complementary strategies.Fundação para a Ciência e a Tecnologia (FCT) - FCOMP-01-0124-FEDER-014784, SFRH/BD/24256/2005.FEDER - COMPETE programEuropean Social Fund (ESF

Syntrophic LCFA-degrading microbial ecosystems

Long-chain fatty acids (LCFA) are energy-rich compounds, which are abundantly present in raw and waste materials. Thus, wastewaters that contain LCFA may yield high levels of methane in an anaerobic digestion process. Biogas formation from LCFAcontaining wastewater is a sustainable technology that warrants further investigation, specifically in terms of more fundamental microbiological aspects. The aim of this work is to get more insights into the syntrophic microbial communities that degrade LCFA anaerobically. Bacterial shifts of a mesophilic sludge incubated in the presence of palmitic, stearic or oleic acid was estimated by means of automated ribosomal intergenic spacer analysis (ARISA). Slightly differences were observed between the communities incubated with saturated LCFA (palmitic and stearic acids) and the ones of the blank assay. On the other hand, evident changes were found between ARISA profiles of the communities that were incubated with oleic acid and the ones obtained for the blank assay. These results suggest that the microbial communities that degrade saturated fatty acids are very close to each other and different from the ones that degrade unsaturated fatty acids

Microbial syngas conversion by mesophilic and thermophilic anaerobic mixed-cultures

Synthesis gas (or syngas) can be produced from the gasification of a variety of recalcitrant or biodegradable wastes. Syngas is a mixture composed of mainly H2, CO and CO2 that can be used in a biological process for the production of fuels or usable chemicals. The main goal of this work was to study the physiology and microbial composition of anaerobic cultures able to utilize syngas. The results indicated that the thermophilic sludge inoculum presents a promising carboxydotrophic potential comparing to the mesophilic sludge inoculum. Monitoring of microbial structure of thermophilic enriched cultures by using PCR-DGGE and cloning techniques showed that bacterial community profiles clustered in three different groups

Methane production from fat : assessing the bioaugmentation potential of syntrophomonas zehnderi

Fundação para a Ciência e a Tecnologia (FCT

Biological fermentation of syngas

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

New perspectives for methane production from oleate : bioaugmentation of anaerobic sludge with Syntrophomonas zehnderi

Biogas production from waste lipids is a promising technology for sustainable energy production. In anaerobic bioreactors, lipids and long-chain fatty acids (LCFA) are easily removed from the liquid medium, mainly by adsorption. However, further LCFA degradation is rate-limiting and possible dependent on the development of syntrophic communities. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes was used to follow the changes in bacterial communities during continuous and fed-batch reactors operation with oleate, an unsaturated LCFA. A specific dominant DGGE-band corresponding to bacteria deeply clustering with Syntrophomonas zehnderi (99% identity) was found in all the sludges that could degrade oleate, thus suggesting the involvement of this bacterium in unsaturated LCFA catabolism. Therefore, the potential of S. zehnderi as bioaugmenting strain for improving methane production from oleate was further studied in batch assays. Oleate was added to the medium at a final concentration of 1 mM and the assays were performed with and without the solid microcarrier sepiolite. Methane production was faster in the bioaugmented assays, and this effect was more pronounced in the presence of sepiolite. The positive effects of sepiolite can be related to a decrease in oleate toxicity towards the acetoclastic methanogens, or to an improvement of the syntrophic relationships. Bioaugmentation with S. zehnderi might be a suitable strategy for accelerating LCFA conversion to methane in anaerobic bioreactors, shortening the start-up period of high rate continuous processes or recover LCFA-inhibited sludge

Enhancing methane production from fat by bioaugmenting Syntrophomonas zehnderi to anaerobic sludge

Fundação para a Ciência e a Tecnologia (FCT) - bolsa SFRH/BD/24256/200

Anaerobic treatment of LCFA-rich wastewater: assessing the bioaugmentation potential of Syntrophomonas zehnderi

Long-chain fatty acids (LCFA) are commonly present in fatty-wastewaters. Complete LCFA degradation depends on the coordinated activity of syntrophic bacteria, which convert LCFA to acetate and hydrogen, and methanogenic archaea, that utilize these substrates, making the overall conversion energetically possible. LCFA-degrading bacteria are fastidious microorganisms with low predominance in bioreactors. Thus, addition of LCFA-degrading bacteria to anaerobic sludge can possibly improve LCFA biodegradation and enhance methane production. In this work, a co-culture of Syntrophomonas zehnderi and Methanobacterium formicicum was added to non-acclimated granular sludge. Two sets of bottles were prepared, with and without sepiolite, a solid microcarrier. Sludge was bioaugmented with co-culture and supplemented with 1 mM oleate. Blanks (without oleate) and controls (with inactivated co-culture) were also prepared. Methane, VFA and LCFA were quantified. Addition of S. zehnderi enhanced LCFA degradation, both in the assays prepared with and without microcarrier. In the bottles containing bioaugmented sludge and no microcarrier, acetate accumulated in the medium indicating a fast LCFA β-oxidation: after 15 days of incubation, maximum acetate concentrations (approx. 5 mM) were attained and 77% of the added oleate could be accounted for the acetate and methane measured. In non-bioaugmented sludge, acetate accumulation started later and, after 15 days of incubation, was not higher than 1.5 mM. In bottles containing microcarrier methane was produced at a higher rate. In this case only residual acetate concentrations were measured, indicating balanced syntrophic relations, maybe due to stimulation of bacteria-archaea relation by the microcarrier. Methane production from oleate was most favored in bottles supplemented with the syntrophic co-culture and containing microcarrier: 71% of the added oleate was recovered as methane after 12 days of incubation and a maximum methane production rate of 1.12 mMCH4day-1 was observed. Bioaugmentation with S. zehnderi enhances oleate biodegradation and can be potentially useful for a faster reactor start-up