Advances of structure, function, and catalytic mechanism of methyl-coenzyme M reductase / 生物工程学报

Methanogens are unique microorganisms for methane production and the main contributor of the biogenic methane in atmosphere. Methyl-coenzyme M reductase (Mcr) catalyzes the last step of methane production in methanogenesis and the first step of methane activation in anaerobic oxidation of methane. The genes encoding this enzyme are highly conserved and are widely used as a marker in the identification and phylogenetic study of archaea. There has been a longstanding interest in its unique cofactor F430 and the underpinning mechanisms of enzymatic cleavage of alkane C-H bond. The recent breakthroughs of high-resolution protein and catalytic-transition-state structures further advanced the structure-function study of Mcr. In particular, the recent discovery of methyl-coenzyme M reductase-like (Mcr-like) enzymes that activates the anaerobic degradation of non-methane alkanes has attracted much interest in the molecular mechanisms of C-H activation without oxygen. This review summarized the advances on function-structure-mechanism study of Mcr/Mcr-like enzymes. Additionally, future directions in anaerobic oxidation of alkanes and greenhouse-gas control using Mcr/Mcr-like enzymes were proposed.

Insights into the microbial community structure of anaerobic digestion of municipal solid waste landfill leachate for methane production by adaptive thermophilic granular sludge

BACKGROUND: The amount of municipal solid waste (MSW) gradually increased along with the rapid development of modern cities. A large amount of landfill leachate are generated with excessive chemical oxygen demand (COD), which create a great deal of pressure on the environment-friendly treatment process. Anaerobic digestion is an ideal technique to solve the above problem. RESULTS: A thermophilic granular sludge was successfully adapted for anaerobic digestion of MSW leachate (from an aging large-scale landfill) for methane production. The COD degradation efficiency improved by 81.8%, while the methane production rate reached 117.3 mL CH4/(g VS d), which was 2.34-fold more than the control condition. The bacterial and archaeal communities involved in the process were revealed by 16S rRNA gene high-throughput pyrosequencing. The richness of the bacterial community decreased in the process of thermophilic granular sludge, while the archaeal community structure presented a reverse phenomenon. The bacterial genus, Methanosaeta was the most abundant during the mesophilic process, while Methanobacterium, Methanoculleus, Methanosaeta and Methanosarcina were more evenly distributed. The more balanced community distribution between hydrogenotrophic and acetotrophic methanogens implied a closer interaction between the microbes, which further contributed to higher methane productivity. The detailed relationship between the key functional communities and anaerobic digestion performances were demonstrated via the multivariate canonical correspondence analysis. Conclusions: With the assistance of adaptive thermophilic granular sludge, microbial community structure was more evenly distributed, while both of COD degradation rate and methane production was improved during anaerobic digestion of MSW landfill leachate.

Effect of Para Substituted Anilines Chemical Structure on Methane Biosynthesis by the Methanogens.

The present paper aims to study the effect of aromatic structure on the inhibition of biogas production and more specifically the effect of para substituted anilines functional groups (chemical structure) on methane biosynthesis by the digested pig manure methanogens. The objective of this study was also to examine the structure-toxicity relationships of aromatic compounds to acetoclastic methanogens. Study Design: Anaerobic digestion of pig manure, anaerobic toxicity essay, The effects of functional group nature on inhibition of methane production by acetoclastic methanogens. Correlation of the methanogenic toxicity (IC50) with aromatic compounds hydrophobicity (logPoct). Place and Duration of Study: Department of Chemistry, University of Kinshasa (DR Congo), between August 2011 and May 2012. Methodology: The toxicity to acetoclastic methanogenic bacteria was performed with the standard method of serum bottles, digested pig manure was utilized as inoculums, acetate as substrate and the methane gas volume produced was measured by serum bottles liquid displacement systems (Mariotte flask system). Results: The obtained results indicate that relationships exist between para substituted anilines functional groups nature (chemical structure) and their inhibitory effects on methanogens. The toxicity of para bisubstituted anilines increases in the following order: SO3 < OH < H < CH3 < Cl < NO2 From this sequence of increasing toxicity, it can be seen that the methanogenic toxicity varies with the functional group nature which is in the para position of the main function. Indeed, p-Nitroaniline and benzene with 45.76 and 208.78 mg/l as IC50 values respectively were the most toxic compounds, while p-Aminophenol and p-Aminosulfonic acid (Sulfanilic acid) with 1800.39 and 2777.82 mg/l IC50 values were the less toxic. A very significant negative linear correlation between the toxicity of para substituted anilines compounds and their hydrophobicity was found. Conclusion: The results of this study indicate that relationships exist between para substituted anilines functional groups nature and their inhibitory effects in methane biosynthesis by the methanogens.

Inhibitory Effects of Phenolic Monomers on Methanogenesis in Anaerobic Digestion.

Aims: The purpose of this study was to evaluate the inhibitory effects of phenolic monomers on methanogenesis in anaerobic digestion and to assess the effect of hydroxyl groups’ number of phenolic monomers (aromatic structure) on inhibition of methane production by acetoclastic methanogens (archaea). Study Design: Anaerobic digestion of pig manure, anaerobic toxicity essay, The effect of the hydroxyl group’s number on the methanogenic toxicity as exhibited by monomeric tannins, Correlation of the methanogenic toxicity (IC50) with aromatic compounds hydrophobicity (logPoct), Correlation of the methanogenic toxicity (IC50) with Cresols boiling point (bp). Place and Duration of Study: Department of Chemistry, University of Kinshasa (DR Congo), between September 2011 and May 2012. Methodology: The toxicity to acetoclastic methanogenic bacteria was performed with the standard method of serum bottles; digested pig manure was utilized as inoculums and acetate as substrate. The methane gas volume produced was measured by serum bottles liquid displacement systems (Mariotte flask system). Results: The results of this study indicate that an increase in the number of hydroxyl groups on the aromatic compound was associated with a decrease in the compound’s toxicity to methanogens (archea). The toxicity of various phenolic monomers are decreasing in the following order: pyrogallol < hydroquinone < resorcinol < phenol < benzene with 3172, 2745, 1725, 1249 and 209 mg/l IC50 values respectively. A significant negative linear correlation between the toxicity of phenolic monomers together with the reference compound (benzene) and their hydrophobicity was found. Moreover, a high positive linear correlation has been found between the IC50 values of phenolic monomers and their boiling temperatures. Conclusion: The obtained results indicate that relationships exist between the phenolic monomers structure and their inhibitory effects in methane biosynthesis. The analysis of experimental results suggests that an increase in the number of hydroxyl groups on the aromatic compounds was associated with a decrease in the phenolic monomers toxicity.

Effect of sediment composition on methane concentration and production in the transition zone of a mangrove (Sepetiba Bay, Rio de Janeiro, Brazil) / Efeito da composição do sedimento sobre a concentração e a produção do metano na zona de transição de um mangue tropical, na Baía de Sepetiba-RJ, Brasil

The aim of this research was to evaluate the effect of sediment composition on methane (CH4) dynamics in sediments of different areas in the transition zone between a mangrove and the sea. This research was conducted in a mangrove at Coroa Grande, on the southern coast of Rio de Janeiro. Samples were collected at three stations: (1) region colonised by Rhizophora mangle L. on the edge of the mangrove, (2) region colonised by seagrasses and (3) infra-littoral region without vegetation. Samples were collected from the surface layer of the sediment to determine the concentrations of nutrients (C, N and P) and CH4 concentration and production. We observed that concentrations of CH4 and carbon (C) were significantly higher (p < 0.05) in station 1 than station 3. The molar ratios (C:N, C:P and N:P) suggest that the origin of the substrate is mainly autochthonous. Methanogenesis was initially low, possibly due to competition between methanogens and sulfate reducers, and increased significantly (p < 0.05) on the twenty-sixth day in the sediment of station 1, probably due to higher organic matter (OM) availability in this region. Results indicate that methanogenic activity observed herein is not regulated by the amount or quality of OM, but by other factors. The concentration of CH4 in the sea-land ecotone at Mangrove Coroa Grande is a function of available OM suggesting a possible inhibition of methanotrophy by intense oxygen consumption in the soil surface covered by detritus of Rhizophora mangle vegetation.

O objetivo desta pesquisa foi avaliar a dinâmica do metano (CH4) no sedimento em diferentes áreas na região de transição entre a borda do manguezal e o mar, em função da composição do sedimento. A pesquisa foi realizada no Manguezal de Coroa Grande, no litoral sul do Estado do Rio de janeiro. Foram coletadas amostras em três estações: (1) região colonizada por Rhizophora mangle L. na borda do mangue, (2) região colonizada por macrófitas, e (3) região sem vegetação, permanentemente inundada pelo mar. Foram coletadas amostras da fração superficial do sedimento para determinação da concentração de nutrientes (C, N e P), e da concentração e da produção de CH4. Foi observado que as concentrações de CH4 e carbono (C) foram significativamente maiores (p < 0,05) na estação 1, em relação à estação 3. Com relação às razões molares (C:N, C:P e N:P), estas indicam que a origem da composição do substrato é principalmente autóctone. Quanto à metanogênese, foram observados, inicialmente, baixos valores de CH4 produzido, indicando provável competição entre metanogênicos e sulfatorredutores. Seguiu-se, então, um significativo aumento (p < 0,05) da metanogênese no 26º dia, no sedimento da estação 1, provavelmente em função da maior disponibilidade de matéria orgânica (MO) nessa região. Os resultados mostram que a atividade metanogênica observada pela presente pesquisa não é regulada pela quantidade e a qualidade da MO e sim por outros fatores. A concentração de CH4 no ecótono mar-terra no manguezal de Coroa Grande, ocorre em função da MO disponível, sugerindo possível inibição da metanotrofia, em razão de intenso consumo de oxigênio na superfície do solo coberto pelo detrito da vegetação de Rhizophora mangle.

PCR-based DGGE and FISH analysis of methanogens in an anaerobic closed digester tank for treating palm oil mill effluent

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.

Contributions of available substrates and activities of trophic microbial community to methanogenesis in vegetative and reproductive rice rhizospheric soil.

Potential of methane production and trophic microbial activities at rhizospheric soil during rice cv. Supanburi 1 cultivation were determined by laboratory anaerobic diluents vials. The methane production was higher from rhizospheric than non-rhizospheric soil, with the noticeable peaks during reproductive phase (RP) than vegetative phase (VP). Glucose, ethanol and acetate were the dominant available substrates found in rhizospheric soil during methane production at both phases. The predominance activities of trophic microbial consortium in methanogenesis, namely fermentative bacteria (FB), acetogenic bacteria (AGB), acetate utilizing bacteria (AB) and acetoclastic methanogens (AM) were also determined. At RP, these microbial groups were enhanced in the higher of methane production than VP. This correlates with our finding that methane production was greater at the rhizospheric soil with the noticeable peaks during RP (1,150 ± 60 nmol g dw-1 d-1) compared with VP (510 ± 30 nmol g dw-1 d-1). The high number of AM showed the abundant (1.1x104 cell g dw-1) with its high activity at RP, compared to the less activity with AM number at VP (9.8x102 cell g dw-1). Levels of AM are low in the total microbial population, being less than 1% of AB. These evidences revealed that the microbial consortium of these two phases were different.