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Anaerobic oxidation of methane (AOM) mediated by anaerobic methanotrophic archaea (ANME) is the primary process that provides energy to cold seep ecosystems by converting methane into inorganic carbon. Notably, cold seep ecosystems are dominated by highly divergent heterotrophic microorganisms. The role of the AOM process in supporting heterotrophic population remains unknown. We investigate the acetogenic capacity of ANME-2a in a simulated cold seep ecosystem using high-pressure biotechnology, where both AOM activity and acetate production are detected. The production of acetate from methane is confirmed by isotope-labeling experiments. A complete archaeal acetogenesis pathway is identified in the ANME-2a genome, and apparent acetogenic activity of the key enzymes ADP-forming acetate-CoA ligase and acetyl-CoA synthetase is demonstrated. Here, we propose a modified model of carbon cycling in cold seeps: during AOM process, methane can be converted into organic carbon, such as acetate, which further fuels the heterotrophic community in the ecosystem.

Marine methylotrophs play a key role in the global carbon cycle by metabolizing reduced one-carbon compounds that are found in high concentrations in marine environments. Genome, physiology and diversity studies have been greatly facilitated by the numerous model organisms brought into culture. However, the availability of marine representatives remains poor. Here, we report the isolation of four novel species from North Sea sediment enrichments closely related to the Alphaproteobacterium Methyloceanibacter caenitepidi. Each of the newly isolated Methyloceanibacter species exhibited a clear genome sequence divergence which was reflected in physiological differences. Notably one strain R-67174 was capable of oxidizing methane as sole source of carbon and energy using solely a soluble methane monooxygenase and represents the first marine Alphaproteobacterial methanotroph brought into culture. Differences in maximum cell density of >1.5 orders of magnitude were observed. Furthermore, three strains were capable of producing nitrous oxide from nitrate. Together, these findings highlight the metabolic and physiologic variability within closely related Methyloceanibacter species and provide a new understanding of the physiological basis of marine methylotrophy., (© 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The methane removal capacity of mixed methane-oxidizing bacteria (MOB) culture in a biofilter setup using autoclaved aerated concrete (AAC) as a highly porous carrier material was tested. Batch experiment was performed to optimize MOB immobilization on AAC specimens where optimum methane removal was obtained when calcium chloride was not added during bacterial inoculation step and 10-mm-thick AAC specimens were used. The immobilized MOB could remove methane at low concentration (~1000 ppmv) in a biofilter setup for 127 days at average removal efficiency (RE) of 28.7 %. Unlike a plug flow reactor, increasing the total volume of the filter by adding a biofilter in series did not result in higher total RE. MOB also exhibited a higher abundance at the bottom of the filter, in proximity with the methane gas inlet where a high methane concentration was found. Overall, an efficient methane biofilter performance could be obtained using AAC as the carrier material.

Obtaining a reliable estimation of the methane potential of organic waste streams in anaerobic digestion, for which a biochemical methane potential (BMP) test is often used, is of high importance. Standardization of this BMP test is required to ensure inter-laboratory repeatability and accuracy of the BMP results. Therefore, guidelines were set out; yet, these do not provide sufficient information concerning origin of and the microbial community in the test inoculum. Here, the specific contribution of the methanogenic community on the BMP test results was evaluated. The biomethane potential of four different substrates (molasses, bio-refinery waste, liquid manure and high-rate activated sludge) was determined by means of four different inocula from full-scale anaerobic digestion plants. A significant effect of the selected inoculum on the BMP result was observed for two out of four substrates. This inoculum effect could be attributed to the abundance of methanogens and a potential inhibiting effect in the inoculum itself, demonstrating the importance of inoculum selection for BMP testing. We recommend the application of granular sludge as an inoculum, because of its higher methanogenic abundance and activity, and protection from bulk solutions, compared with other inocula., (© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Kitchen waste and molasses are organic waste streams with high organic content, and therefore are interesting substrates for renewable energy production by means of anaerobic digestion. Both substrates, however, often cause inhibition of the anaerobic digestion process, when treated separately, hence, co-digestion with other substrates is required to ensure stable methane production. In this research, A-sludge (sludge harvested from a high rate activated sludge system) was used to stabilize co-digestion with kitchen waste or molasses. Lab-scale digesters were fed with A-sludge and kitchen waste or molasses for a total period of 105 days. Increased methane production values revealed a stabilizing effect of concentrated A-sludge on kitchen waste digestion. Co-digestion of molasses with A-sludge also resulted in a higher methane production. Volumetric methane production rates up to 1.53 L L(-1) d(-1) for kitchen waste and 1.01 L L(-1) d(-1) for molasses were obtained by co-digestion with A-sludge. The stabilizing effect of A-sludge was attributed to its capacity to supplement various nutrients. Microbial community results demonstrated that both reactor conditions and substrate composition determined the nature of the bacterial community, although there was no direct influence of micro-organisms in the substrate itself, while the methanogenic community profile remained constant as long as optimal conditions were maintained., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Anaerobic digestion is considered a key technology for the future bio-based economy. The microbial consortium carrying out the anaerobic digestion process is quite complex, and its exact role in terms of "elasticity", i.e., the ability to rapidly adapt to changing conditions, is still unknown. In this study, the role of the initial microbial community in terms of operational stability and stress tolerance was evaluated during a 175-day experiment. Five different inocula from stable industrial anaerobic digesters were fed a mixture of waste activated sludge and glycerol. Increasing ammonium pulses were applied to evaluate stability and stress tolerance. A different response in terms of start-up and ammonium tolerance was observed among the different inocula. Methanosaetaceae were the dominant acetoclastic methanogens, yet, Methanosarcinaceae increased in abundance at elevated ammonium concentrations. A shift from a Firmicutes to a Proteobacteria dominated bacterial community was observed in failing digesters. Methane production was strongly positively correlated with Methanosaetaceae, but also with Bacteria related to Anaerolinaceae, Clostridiales, and Alphaproteobacteria. Volatile fatty acids were strongly positively correlated with Betaproteobacteria and Bacteroidetes, yet ammonium concentration only with Bacteroidetes. Overall, these results indicate the importance of inoculum selection to ensure stable operation and stress tolerance in anaerobic digestion.

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world's oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30-55 cm below the sea floor (bsf); the second is at 165-205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry.

Although microorganisms coexist in the same environment, it is still unclear how their interaction regulates ecosystem functioning. Using a methanotroph as a model microorganism, we determined how methane oxidation responds to heterotroph diversity. Artificial communities comprising of a methanotroph and increasing heterotroph richness, while holding equal starting cell numbers were assembled. We considered methane oxidation rate as a functional response variable. Our results showed a significant increase of methane oxidation with increasing heterotroph richness, suggesting a complex interaction in the cocultures leading to a stimulation of methanotrophic activity. Therefore, not only is the methanotroph diversity directly correlated to methanotrophic activity for some methanotroph groups as shown before, but also the richness of heterotroph interacting partners is relevant to enhance methane oxidation too. In this unprecedented study, we provide direct evidence showing how heterotroph richness exerts a response in methanotroph-heterotroph interaction, resulting in increased methanotrophic activity. Our study has broad implications in how methanotroph and heterotroph interact to regulate methane oxidation, and is particularly relevant in methane-driven ecosystems.

Anaerobic oxidation of methane (AOM) is a crucial process limiting the flux of methane from marine environments to the atmosphere. The process is thought to be mediated by three groups of uncultivated methane-oxidizing archaea (ANME-1, 2 and 3). Although the responsible microbes have been intensively studied for more than a decade, central mechanistic details remain unresolved. On the basis of an integrated analysis of both environmental metatranscriptome and single-aggregate genome of a highly active AOM enrichment dominated by ANME-2a, we provide evidence for a complete and functioning AOM pathway in ANME-2a. All genes required for performing the seven steps of methanogenesis from CO2 were found present and actively expressed. Meanwhile, genes for energy conservation and electron transportation including those encoding F420H2 dehydrogenase (Fpo), the cytoplasmic and membrane-associated Coenzyme B-Coenzyme M heterodisulfide (CoB-S-SCoM) reductase (HdrABC, HdrDE), cytochrome C and the Rhodobacter nitrogen fixation (Rnf) complex were identified and expressed, whereas genes encoding for hydrogenases were absent. Thus, ANME-2a is likely performing AOM through a complete reversal of methanogenesis from CO2 reduction without involvement of canonical hydrogenase. ANME-2a is demonstrated to possess versatile electron transfer pathways that would provide the organism with more flexibility in substrate utilization and capacity for rapid adjustment to fluctuating environments. This work lays the foundation for understanding the environmental niche differentiation, physiology and evolution of different ANME subgroups.

Background: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments., Results: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features., Conclusions: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

Methane (CH4) release from wetlands is an important source of greenhouse gas emissions. Gas exchange occurs mainly through the aerenchyma of plants, and production of greenhouse gases is heavily dependent on rhizosphere biogeochemical conditions (i.e. substrate availability and redox potential). It is hypothesized that by introducing a biocatalyzed anode electrode in the rhizosphere of wetland plants, a competition for carbon and electrons can be invoked between electrical current-generating bacteria and methanogenic Archaea. The anode electrode is part of a bioelectrochemical system (BES) capable of harvesting electrical current from microbial metabolism. In this work, the anode of a BES was introduced in the rhizosphere of rice plants (Oryza sativa), and the impact on methane emissions was monitored. Microbial current generation was able to outcompete methanogenic processes when the bulk matrix contained low concentrations of organic carbon, provided that the electrical circuit with the effective electroactive microorganisms was in place. When interrupting the electrical circuit or supplying an excess of organic carbon, methanogenic metabolism was able to outcompete current generating metabolism. The qPCR results showed hydrogenotrophic methanogens were the most abundant methanogenic group present, while mixotrophic or acetoclastic methanogens were hardly detected in the bulk rhizosphere or on the electrodes. Competition for electron donor and acceptor were likely the main drivers to lower methane emissions. Overall, electrical current generation with BESs is an interesting option to control CH4 emissions from wetlands but needs to be applied in combination with other mitigation strategies to be successful and feasible in practice.

Biological treatment using methane-oxidizing bacteria (MOB) immobilized on six porous carrier materials have been used to mitigate methane emission. Experiments were performed with different MOB inoculated in building materials at high (~20 % (v/v)) and low (~100 ppmv) methane mixing ratios. Methylocystis parvus in autoclaved aerated concrete (AAC) exhibited the highest methane removal rate at high (28.5 ± 3.8 μg CH₄ g⁻¹ building material h⁻¹) and low (1.7 ± 0.4 μg CH₄ g⁻¹ building material h⁻¹) methane mixing ratio. Due to the higher volume of pores with diameter >5 μm compared to other materials tested, AAC was able to adsorb more bacteria which might explain for the higher methane removal observed. The total methane and carbon dioxide-carbon in the headspace was decreased for 65.2 ± 10.9 % when M. parvus in Ytong was incubated for 100 h. This study showed that immobilized MOB on building materials could be used to remove methane from the air and also act as carbon sink.

Termite-derived methane contributes 3 to 4% to the total methane budget globally. Termites are not known to harbor methane-oxidizing microorganisms (methanotrophs). However, a considerable fraction of the methane produced can be consumed by methanotrophs that inhabit the mound material, yet the methanotroph ecology in these environments is virtually unknown. The potential for methane oxidation was determined using slurry incubations under conditions with high (12%) and in situ (∼0.004%) methane concentrations through a vertical profile of a termite (Macrotermes falciger) mound and a reference soil. Interestingly, the mound material showed higher methanotrophic activity. The methanotroph community structure was determined by means of a pmoA-based diagnostic microarray. Although the methanotrophs in the mound were derived from populations in the reference soil, it appears that termite activity selected for a distinct community. Applying an indicator species analysis revealed that putative atmospheric methane oxidizers (high-indicator-value probes specific for the JR3 cluster) were indicative of the active nest area, whereas methanotrophs belonging to both type I and type II were indicative of the reference soil. We conclude that termites modify their environment, resulting in higher methane oxidation and selecting and/or enriching for a distinct methanotroph population.

Methanotrophs can form the basis of a methane-driven food web on which heterotrophic microorganisms can feed. In return, these heterotrophs can stimulate growth of methanotrophs in co-culture by providing growth additives. However, only a few specific interactions are currently known. We incubated nine methanotrophs with 25 heterotrophic strains in a pairwise miniaturized co-cultivation setup. Through principal component analysis and k-means clustering, methanotrophs and heterotrophs could be grouped according to their interaction behaviour, suggesting strain-dependent methanotroph-heterotroph complementarity. Co-cultivation significantly enhanced the growth parameters of three methanotrophs. This was most pronounced for Methylomonas sp. M5, with a threefold increase in maximum density and a fourfold increase in maximum increase in density in co-culture with Cupriavidus taiwanensis LMG 19424. In contrast, co-cultivation with Methylobacterium radiotolerans LMG 2269 and Pseudomonas aeruginosa LMG 12228 inhibited growth of most methanotrophs. Functional genomic analysis suggested the importance of vitamin metabolism for co-cultivation success. The generated data set was then successfully exploited as a proof-of-principle for predictive modelling of co-culture responses based on other interactions of the same heterotrophs and methanotrophs, yielding values of the area under the receiver operating characteristic curve of 0.73 upon 50% missing values for the maximum increase in density parameter. As such, these modelling-based tools were shown to hold great promise in reducing the amount of data that needs to be generated when conducting large co-cultivation studies., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Methane-oxidizing bacteria (MOB) possess the ability to use methane for energy generation and growth, thereby, providing a key ecosystem service that is highly relevant to the regulation of the global climate. MOB subgroups have different responses to key environmental controls, reflecting on their functional traits. Their unique features (C1-metabolism, unique lipids and congruence between the 16S rRNA and pmoA gene phylogeny) have facilitated numerous environmental studies, which in combination with the availability of cultured representatives, yield the most comprehensive ecological picture of any known microbial functional guild. Here, we focus on the broad MOB subgroups (type I and type II MOB), and aim to conceptualize MOB functional traits and observational characteristics derived primarily from these environmental studies to be interpreted as microbial life strategies. We focus on the functional traits, and the conditions under which these traits will render different MOB subgroups a selective advantage. We hypothesize that type I and type II MOB generally have distinct life strategies, enabling them to predominate under different conditions and maintain functionality. The ecological characteristics implicated in their adopted life strategies are discussed, and incorporated into the Competitor-Stress tolerator-Ruderal functional classification framework as put forward for plant communities. In this context, type I MOB can broadly be classified as competitor-ruderal while type II MOB fit more within the stress tolerator categories. Finally, we provide an outlook on MOB applications by exemplifying two approaches where their inferred life strategies could be exploited thereby, putting MOB into the context of microbial resource management., (© 2012 John Wiley & Sons Ltd and Society for Applied Microbiology.)

The methanotrophic potential in sewage treatment sludge was investigated. We detected a diverse aerobic methanotrophic community that potentially plays a significant role in mitigating methane emission in this environment. The results suggest that community structure was determined by conditions specific to the processes in a sewage treatment plant.

Life in hypersaline environments is typically limited by bioenergetic constraints. Microbial activity at the thermodynamic edge, such as the anaerobic oxidation of methane (AOM) coupled to sulphate reduction (SR), is thus unlikely to thrive in these environments. In this study, carbon and sulphur cycling was investigated in the extremely hypersaline cold seep sediments of Mercator mud volcano. AOM activity was partially inhibited but still present at salinity levels of 292 g L(-1) (c. eightfold sea water concentration) with rates of 2.3 nmol cm(-3) day(-1) and was even detectable under saturated conditions. Methane and evaporite-derived sulphate comigrated in the ascending geofluids, which, in combination with a partial activity inhibition, resulted in AOM activity being spread over unusually wide depth intervals. Up to 79% of total cells in the AOM zone were identified by fluorescence in situ hybridization (FISH) as anaerobic methanotrophs of the ANME-1. Most ANME-1 cells formed monospecific chains without any attached partner. At all sites, AOM activity co-occurred with SR activity and sometimes significantly exceeded it. Possible causes of these unexpected results are discussed. This study demonstrates that in spite of a very low energy yield of AOM, microorganisms carrying this reaction can thrive in salinity up to halite saturation., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Methane-oxidizing bacteria (MOB) have a large potential as a microbial sink for the greenhouse gas methane as well as for biotechnological purposes. However, their application in biotechnology has so far been hampered, in part due to the relative slow growth rate of the available strains. To enable the availability of novel strains, this study compares the isolation of MOB by conventional dilution plating with miniaturized extinction culturing, both performed after an initial enrichment step. The extinction approach rendered 22 MOB isolates from four environmental samples, while no MOB could be isolated by plating. In most cases, extinction culturing immediately yielded MOB monocultures making laborious purification redundant. Both type I (Methylomonas spp.) and type II (Methylosinus sp.) MOB were isolated. The isolated methanotrophic diversity represented at least 11 different strains and several novel species based on 16S rRNA gene sequence dissimilarity. These strains possessed the particulate (100%) and soluble (64%) methane monooxygenase gene. Also, 73% of the strains could be linked to a highly active fast-growing mixed MOB community. In conclusion, miniaturized extinction culturing was more efficient in rapidly isolating numerous MOB requiring little effort and fewer materials, compared with the more widely applied plating procedure. This miniaturized approach allowed straightforward isolation and could be very useful for subsequent screening of desired characteristics, in view of their future biotechnological potential., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Anaerobic digestion is an important technology in the framework of renewable energy production. The anaerobic digestion system is susceptible to perturbations due to the sensitivity of the methanogens towards environmental factors. Currently, technology is evolving from conventional waste treatment, i.e. the removal of pollutants, to very intensive biogas production from concentrated wastes, in the framework of bio-energy production. In the latter configuration Methanosarcina species appear to be of crucial importance. Methanosarcina sp. are, compared to other methanogens, quite robust towards different impairments. They are reported to be tolerant to total ammonium concentrations up to 7000 mg L(-1), salt concentrations up to 18,000 mg Na(+)L(-1), a pH shock of 0.8-1.0 units and acetate concentrations up to 15,000 mg CODL(-1). The possibilities of Methanosarcina sp. as key organisms in specific types of anaerobic digestion systems are demonstrated in this review., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The biomass of industrially grown Phaeodactylum tricornutum was subjected in a novel way to bio-methanation at 33°C, i.e., in an anaerobic membrane bioreactor (AnMBR) at a hydraulic retention time of 2.5 days, at solid retention times of 20 to 10 days and at loading rates in the range of 2.6-5.9 g biomass-COD L(-1) day(-1) with membrane fluxes ranging from 1 to 0.8 L m(-2) h(-1). The total COD recovered as biogas was in the order of 52%. The input suspension was converted to a clear effluent rich in total ammonium nitrogen (546 mg TAN L(-1)) and phosphate (141 mg PO(4)-P L(-1)) usable as liquid fertilizer. The microbial community richness, dynamics, and organization in the reactor were interpreted using the microbial resource management approach. The AnMBR communities were found to be moderate in species richness and low in dynamics and community organization relative to UASB and conventional CSTR sludges. Quantitative polymerase chain reaction analysis revealed that Methanosaeta sp. was the dominant acetoclastic methanogen species followed by Methanosarcina sp. This work demonstrated that the use of AnMBR for the digestion of algal biomass is possible. The fact that some 50% of the organic matter is not liquefied means that the algal particulates in the digestate constitute a considerable fraction which should be valorized properly, for instance as slow release organic fertilizer. Overall, 1 kg of algae dry matter (DM) could be valorized in the form of biogas ( euro 2.07), N and P in the effluent (euro 0.02) and N and P in the digestate (euro 0.04), thus totaling about euro 2.13 per kilogram algae DM.

Background: Anaerobic oxidation of methane coupled to sulphate reduction (SR-AOM) prevents more than 90% of the oceanic methane emission to the atmosphere. In a previous study, we demonstrated that the high methane pressure (1, 4.5, and 8 MPa) stimulated in vitro SR-AOM activity. However, the information on the effect of high-pressure on the microbial community structure and architecture was still lacking., Results: In this study we analysed the long-term enrichment (286 days) of this microbial community, which was mediating SR-AOM in a continuous high-pressure bioreactor. 99.7% of the total biovolume represented cells in the form of small aggregates (diameter less then 15 μm). An increase of the total biovolume was observed (2.5 times). After 286 days, the ANME-2 (anaerobic methanotrophic archaea subgroup 2) and SRB (sulphate reducing bacteria) increased with a factor 12.5 and 8.4, respectively., Conclusion: This paper reports a net biomass growth of communities involved in SR-AOM, incubated at high-pressure.

Effluents of anaerobic wastewater treatment plants are saturated with methane, an effective greenhouse gas. We propose a novel approach to treat such effluents using a coculture of methane oxidizing communities and microalgae, further indicated as methalgae, which would allow microbial methane oxidation with minimal CO(2) emissions. Coculturing a methane oxidizing community with microalgae in sequence batch reactors under continuous lightning yielded a factor of about 1.6 more biomass relative to the control without microalgae. Moreover, 55% less external oxygen supply was needed to maintain the methane oxidation, as oxygen was produced in situ by the microalgae. An overall methane oxidation rate of 171±27 mg CH(4) L(-1) liquid phase d(-1) was accomplished in a semi-batch setup, while the excess CO(2) production was lower than 1mg CO(2) L(-1) d(-1). Both nitrate and ammonium were feasible nitrogen sources for the methalgae. These results show that a coculture of microalgae and methane oxidizing communities can be used to oxidize dissolved methane under O(2)-limiting conditions, which could lead to a novel treatment for dissolved methane in anaerobic effluents., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Effluents of anaerobic digesters are an underestimated source of greenhouse gases, as they are often saturated with methane. A post-treatment with methane-oxidizing bacterial consortia could mitigate diffuse emissions at such sites. Semi-continuously fed stirred reactors were used as model systems to characterize the influence of the key parameters on the activity of these mixed methanotrophic communities. The addition of 140 mg L(-1) NH (4) (+) -N had no significant influence on the activity nor did a temperature increase from 28 degrees C to 35 degrees C. On the other hand, addition of 0.64 mg L(-1) of copper(II) increased the methane removal rate by a factor of 1.5 to 1.7 since the activity of particulate methane monooxygenase was enhanced. The influence of different concentrations of NaCl was also tested, as effluents of anaerobic digesters often contain salt levels up to 10 g NaCl L(-1). At a concentration of 11 g NaCl L(-1), almost no methane-oxidizing activity was observed in the reactors without copper addition. Yet, reactors with copper addition exhibited a sustained activity in the presence of NaCl. A colorimetric test based on naphthalene oxidation showed that soluble methane monooxygenase was inhibited by copper, suggesting that the particulate methane monooxygenase was the active enzyme and thus more salt resistant. The results obtained demonstrate that the treatment of methane-saturated effluents, even those with increased ammonium (up to 140 mg L(-1) NH (4) (+) -N) and salt levels, can be mitigated by implementation of methane-oxidizing microbial consortia.

Anaerobic oxidation of methane coupled to sulphate reduction (SR-AOM) prevents oceanic methane emissions and is considered as a major environmental process in the deep-sea sediments. To stimulate in vitro SR-AOM activity, we designed a (continuous) high-pressure bioreactor system. Fed-batch mode incubations showed that the elevated methane pressure stimulated SR-AOM activity: when the methane pressure increased from 1 to 4.5 to 8 MPa, the initial SR-AOM activity increased from 3.46 to 8.64 to 9.22 micromol sulphide production/g(dw)/day; the apparent affinity (K(m)) for methane was 37 mM. However, in each fed-batch mode incubation, there was an inhibitory effect observed after 2 days, probably due to the accumulation of sulphide and the increase of pH. When the reactor was operated in a continuous mode, the SR-AOM activity was constantly increasing within 18 days at both 1 and 8 MPa pressures., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In the past decades, large amounts of non-insecticidal hexachlorocyclohexane (HCH) isomers (alpha-, beta-, delta- and epsilon-HCH) have been dumped as side-products of the insecticide gamma-HCH (lindane). This study investigates the effect of HCH isomers on methane oxidation, an important soil function performed by methanotrophic bacteria. Both activity and structure of the methanotrophic community were assessed, using methane oxidation assays and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) respectively. Methane oxidation assays with historically polluted soils revealed that on the long-term methane oxidation was inhibited by HCH pollution. PCR-DGGE and diversity analysis based on Lorenz curves showed that the type I methanotrophic community was less evenly distributed in historically HCH-polluted soils compared with less polluted reference soils. Short-term experiments with methane-enriched consortia further demonstrated that only gamma- and delta-isomers inhibited methane oxidation. Type I methanotrophs of methane-enriched microbial consortia that received gamma- or delta-HCH evolved towards higher species richness. Apparently, for historically HCH-polluted soils, a narrow community remained after long-term exposure while in case of short-term exposures, methane-enriched consortia were converted into less active, but richer communities when they were stressed by the presence of gamma- or delta-HCH. This work demonstrates the importance of incorporating all isomers and possible other side-products in risk assessment studies of persistent organic pollutants and the use of structural analysis of type I methanotrophic communities as evaluating tool.

Background: Molasses is a dense and saline by-product of the sugar agroindustry. Its high organic content potentially fuels a myriad of renewable products of industrial interest. However, the biotechnological exploitation of molasses is mainly hampered by the high concentration of salts, an issue that is nowadays tackled through dilution. In the present study, the performance of microbial communities derived from marine sediment was compared to that of communities from a terrestrial environment (anaerobic digester sludge). The aim was to test whether adaptation to salinity represented an advantage for fermenting molasses into renewable chemicals such as volatile fatty acids (VFAs) although high sugar concentrations are uncommon to marine sediment, contrary to anaerobic digesters. Results: Terrestrial and marine microbial communities were enriched in consecutive batches at different initial pH values (pHi; either 6 or 7) and molasses dilutions (equivalent to organic loading rates (OLRs) of 1 or 5 gCOD L-1 d-1) to determine the best VFA production conditions. Marine communities were supplied with NaCl to maintain their native salinity. Due to molasses inherent salinity, terrestrial communities experienced conditions comparable to brackish or saline waters (20-47 mS cm-1), while marine conditions resembled brine waters (>47 mS cm-1). Enrichments at optimal conditions of OLR 5 gCOD L-1 d-1 and pHi7 were transferred into packed-bed biofilm reactors operated continuously. The reactors were first operated at 5 gCOD L-1 d-1, which was later increased to OLR 10 gCOD L-1 d-1. Terrestrial and marine reactors had different gas production and community structures but identical, remarkably high VFA bioconversion yields (above 85%) which were obtained with conductivities up to 90 mS cm-1. COD-to-VFA conversion rates were comparable to the highest reported in literature while processing other organic leftovers at much lower salinities. Conclusions: Although salinity represents a major driver for microbial community structure, proper acclimation yielded highly efficient systems treating molasses, irrespective of the inoculum origin. Selection of equivalent pathways in communities derived from different environments suggests that culture conditions select for specific functionalities rather than microbial representatives. Mass balances, microbial community composition, and biochemical analysis indicate that biomass turnover rather than methanogenesis represents the main limitation to further increasing VFA production with molasses. This information is relevant to moving towards molasses fermentation to industrial application. [ABSTRACT FROM AUTHOR]

Microbial interaction is an integral component of microbial ecology studies, yet the role, extent, and relevance of microbial interaction in community functioning remains unclear, particularly in the context of global biogeochemical cycles. While many studies have shed light on the physico-chemical cues affecting specific processes, (micro)biotic controls and interactions potentially steering microbial communities leading to altered functioning are less known. Yet, recent accumulating evidence suggests that the concerted actions of a community can be significantly different from the combined effects of individual microorganisms, giving rise to emergent properties. Here, we exemplify the importance of microbial interaction for ecosystem processes by analysis of a reasonably well-understood microbial guild, namely, aerobic methane-oxidizing bacteria (MOB). We reviewed the literature which provided compelling evidence for the relevance of microbial interaction in modulating methane oxidation. Support for microbial associations within methane-fed communities is sought by a re-analysis of literature data derived from stable isotope probing studies of various complex environmental settings. Putative positive interactions between active MOB and other microbes were assessed by a correlation network-based analysis with datasets covering diverse environments where closely interacting members of a consortium can potentially alter the methane oxidation activity. Although, methanotrophy is used as a model system, the fundamentals of our postulations may be applicable to other microbial guilds mediating other biogeochemical processes. [ABSTRACT FROM AUTHOR]

Methane-oxidizing cultures from five different inocula were enriched to be used for co-metabolic degradation of micropollutants. In a first screening, 18 different compounds were tested for degradation with the cultures as well as with four pure methane-oxidizing bacterial (MOB) strains. The tested compounds included pharmaceuticals, chemical additives, pesticides, and their degradation products. All enriched cultures were successful in the degradation of at least four different pollutants, but the compounds degraded most often were sulfamethoxazole (SMX) and benzotriazole (BTZ). Addition of acetylene, a specific methane monooxygenase (MMO) inhibitor, revealed that SMX and BTZ were mainly degraded co-metabolically by the present MOB. The pure MOB cultures exhibited less degradation potential, while SMX and BTZ were also degraded by three of the four tested pure strains. For MOB, copper (Cu) concentration is often an important factor, as several species have the ability to express a soluble MMO (sMMO) if the Cu concentration is low. In literature, this enzyme is often described to have a broader compound range for co-metabolic degradation of pollutants, in particular when it comes to aromatic structures. However, this study indicated that co-metabolic degradation of the aromatic compounds SMX and BTZ was possible at high Cu concentration, most probably catalyzed by pMMO. [ABSTRACT FROM AUTHOR]

Abstract: Two types of non-axenic algal cultures, one dominated by the freshwater microalgae Scenedesmus obliquus and the other by the marine microalgae Phaeodactylum tricornutum, were cultivated in two types of simple photobioreactor systems. The production rates, expressed on dry matter (DM) basis, were in the order of 0.12 and 0.18g DML−1 d−1 for S. obliquus and P. tricornutum respectively. The biogas potential of algal biomass was assessed by performing standardized batch digestion as well as digestion in a hybrid flow-through reactor (combining a sludge blanket and a carrier bed), the latter under mesophilic and thermophilic conditions. Biomethane potential assays revealed the ultimate methane yield (B 0) of P. tricornutum biomass to be about a factor of 1.5 higher than that of S. obliquus biomass, i.e. 0.36 and 0.24LCH4 g−1 volatile solids (VS) added respectively. For S. obliquus biomass, the hybrid flow-through reactor tests operated at volumetric organic loading rate (Bv) of 2.8gVSL−1 d−1 indicated low conversion efficiencies ranging between 26–31% at a hydraulic retention time (HRT) of 2.2days for mesophilic and thermophilic conditions respectively. When digesting P. tricornutum at a Bv of 1.9gVSL−1 d−1 at either mesophilic or thermophilic conditions and at an HRT of 2.2days, an overall conversion efficiency of about 50% was obtained. This work indicated that the hydrolysis of the algae cells is limiting the anaerobic processing of intensively grown S. obliquus and P. tricornutum biomass. [Copyright &y& Elsevier]

Anaerobic digestion is a well-established microbial-based technology for the treatment of organic waste streams and subsequent biogas recovery. A robust and versatile microbial community to ensure overall stability of the process is essential. Four full-scale anaerobic digestion plants were followed for one year to link operational characteristics with microbial community composition and structure. Similarities between digesters, community dynamics and co-occurrence between bacteria and archaea within each digester were analysed. Free ammonia concentration (>200 mg N L −1 ) and conductivity (>30 mS cm −1 ) hindered acetoclastic methanogenesis by Methanosaetaceae. Thus, methanogenesis was pushed to the hydrogenotrophic pathway carried out by Methanobacteriales and Methanomicrobiales. Firmicutes dominated the overall bacterial community in each of the digesters (>50%), however, principal coordinate analysis of Bray-Curtis indices showed that each of the four digesters hosted a unique microbial community. The uniqueness of this community was related to two phylotypes belonging to the Syntrophomonas genus (Phy32 and Phy34) and to one unclassified bacterium (Phy2), which could both be considered marker populations in the community. A clear differentiation in co-occurrence of methanogens with several bacteria was observed between the different digesters. Our results demonstrated that full-scale anaerobic digestion plants show constant dynamics and co-occurrence patterns in function of time, but are unique in terms of composition, related to the presence of marker populations. [ABSTRACT FROM AUTHOR]

Anaerobic digestion is regarded as a key environmental technology in the present and future bio-based economy. The microbial community completing the anaerobic digestion process is considered complex, and several attempts already have been carried out to determine the key microbial populations. However, the key differences in the anaerobic digestion microbiomes, and the environmental/process parameters that drive these differences, remain poorly understood. In this research, we hypothesized that differences in operational parameters lead to a particular composition and organization of microbial communities in full-scale installations. A total of 38 samples were collected from 29 different full-scale anaerobic digestion installations, showing constant biogas production in function of time. Microbial community analysis was carried out by means of amplicon sequencing and real-time PCR. The bacterial community in all samples was dominated by representatives of the Firmicutes, Bacteroidetes and Proteobacteria, covering 86.1 ± 10.7% of the total bacterial community. Acetoclastic methanogenesis was dominated by Methanosaetaceae, yet, only the hydrogenotrophic Methanobacteriales correlated with biogas production, confirming their importance in high-rate anaerobic digestion systems. In-depth analysis of operational and environmental parameters and bacterial community structure indicated the presence of three potential clusters in anaerobic digestion. These clusters were determined by total ammonia concentration, free ammonia concentration and temperature, and characterized by an increased relative abundance of Bacteroidales, Clostridiales and Lactobacillales, respectively. None of the methanogenic populations, however, could be significantly attributed to any of the three clusters. Nonetheless, further experimental research will be required to validate the existence of these different clusters, and to which extent the presence of these clusters relates to stable or sub-optimal anaerobic digestion. [ABSTRACT FROM AUTHOR]

Bio-refineries produce large volumes of waste streams with high organic content, which are potentially interesting for further processing. Anaerobic digestion (AD) can be a key technology for treatment of these sidestreams, such as molasses. However, the high concentration of salts in molasses can cause inhibition of methanogenesis. In this research, concentrated and diluted molasses were subjected to biomethanation in two types of submerged anaerobic membrane bioreactors (AnMBRs): one with biogas recirculation and one with a vibrating membrane. Both reactors were compared in terms of methane production and membrane fouling. Biogas recirculation seemed to be a good way to avoid membrane fouling, while the trans membrane pressures in the vibrating MBR increased over time, due to cake layer formation and the absence of a mixing system. Stable methane production, up to 2.05 L L−1 d−1 and a concomitant COD removal of 94.4%, was obtained only when diluted molasses were used, since concentrated molasses caused a decrease in methane production and an increase in volatile fatty acids (VFA), indicating an inhibiting effect of concentrated molasses on AD. Real-time PCR results revealed a clear dominance of Methanosaetaceae over Methanosarcinaceae as the main acetoclastic methanogens in both AnMBRs. [ABSTRACT FROM AUTHOR]

Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ± 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ± 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ± 0.03 g of CaCO3 g of Ca(CHOOH)2-1 calcium carbonate precipitate yield was obtained when a culture of 109 cells ml-1 and 5 g of calcium formate liter-1 were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry. [ABSTRACT FROM AUTHOR]

Abstract: Anaerobic digestion (AD) is a well-established technology for energy recovery from organic waste streams. Several studies noted that inserting a bioelectrochemical system (BES) inside an anaerobic digester can increase biogas output, however the mechanism behind this was not explored and primary controls were not executed. Here, we evaluated whether a BES could stabilize AD of molasses. Lab-scale digesters were operated in the presence or absence of electrodes, in open (no applied potential) and closed circuit conditions. In the control reactors without electrodes methane production decreased to 50% of the initial rate, while it remained stable in the reactors with electrodes, indicating a stabilizing effect. After 91 days of operation, the now colonized electrodes were introduced in the failing AD reactors to evaluate their remediating capacity. This resulted in an immediate increase in CH4 production and VFA removal. Although a current was generated in the BES operated in closed circuit, no direct effect of applied potential nor current was observed. A high abundance of Methanosaeta was detected on the electrodes, however irrespective of the applied cell potential. This study demonstrated that, in addition to other studies reporting only an increase in methane production, a BES can also remediate AD systems that exhibited process failure. However, the lack of difference between current driven and open circuit systems indicates that the key impact is through biomass retention, rather than electrochemical interaction with the electrodes. [Copyright &y& Elsevier]

Abstract: Anaerobic digestion is a key technology in the bio-based economy and can be applied to convert a wide range of organic substrates into CH4 and CO2. Kitchen waste is a valuable substrate for anaerobic digestion, since it is an abundant source of organic matter. Yet, digestion of single kitchen waste often results in process failure. High-rate activated sludge or A-sludge is produced during the highly loaded first stage of the two-phase ‘Adsorptions-Belebungsverfahren’ or A/B activated sludge system for municipal wastewater treatment. In this specific case, the A-sludge was amended with FeSO4 to enhance phosphorous removal and coagulation during the water treatment step. This study therefore evaluated whether this Fe-rich A-sludge could be used to obtain stable methanation and higher methane production values during co-digestion with kitchen waste. It was revealed that Fe-rich A-sludge can be a suitable co-substrate for kitchen waste; i.e. methane production rate values of 1.15 ± 0.22 and 1.12 ± 0.28 L L−1 d−1 were obtained during mesophilic and thermophilic co-digestion respectively of a feed-mixture consisting of 15% KW and 85% A-sludge. The thermophilic process led to higher residual VFA concentrations, up to 2070 mg COD L−1, and can therefore be considered less stable. Addition of micro- and macronutrients provided a more stable digestion of single kitchen waste, i.e. a methane production of 0.45 L L−1 d−1 was obtained in the micronutrient treatment compared to 0.30 L L−1 d−1 in the control treatment on day 61. Yet, methane production during single kitchen waste digestion still decreased toward the end of the experiment, despite the addition of micronutrients. Methane production rates were clearly influenced by the total numbers of archaea in the different reactors. This study showed that Fe-rich A-sludge and kitchen waste are suitable for co-digestion. [Copyright &y& Elsevier]