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Ex situ biomethanation allows the conversion of hydrogen produced from surplus electricity to methane. The flexibility of the process was recently demonstrated, yet it is unknown how intermittent hydrogen feeding impacts the functionality of the microbial communities. We investigated the effect of starvation events on the hydrogen consumption and methane production rates (MPRs) of two different methanogenic communities that were fed with hydrogen and carbon dioxide. Both communities showed functional resilience in terms of hydrogen consumption and MPRs upon starvation periods of up to 14 days. The origin of the inoculum, community structure and dominant methanogens were decisive for high gas conversion rates. Thus, pre-screening a well performing inoculum is essential to ensure the efficiency of biomethanation systems operating under flexible gas feeding regimes. Our results suggest that the type of the predominant hydrogenotrophic methanogen (here: Methanobacterium ) is important for an efficient process. We also show that flexible biomethanation of hydrogen and carbon dioxide with complex microbiota is possible while avoiding the accumulation of acetate, which is relevant for practical implementation. In our study, the inoculum from an upflow anaerobic sludge blanket reactor treating wastewater from paper industry performed better compared to the inoculum from a plug flow reactor treating cow manure and corn silage. Therefore, the implementation of the power-to-gas concept in wastewater treatment plants of the paper industry, where biocatalytic biomass is readily available, may be a viable option to reduce the carbon footprint of the paper industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Logroño, Popp, Nikolausz, Kluge, Harms and Kleinsteuber.)

Hitherto, few species have been reported to convert lactate to n -caproate. Here, we report the high-quality draft genomes of three Clostridia strains isolated on lactate as the sole carbon source. The genomes were assembled using a hybrid short- and long-read sequencing approach. The genes involved in lactate-based chain elongation were identified., (Copyright © 2020 Liu et al.)

Diverse intestinal microbiota is frequently used in in vitro bioreactor models to study the effects of diet, chemical contaminations, or medication. However, the reproducible cultivation of fecal microbiota is challenging and the resultant communities behave highly dynamic. To approach the issue of reproducibility in in vitro models, we established an intestinal microbiota model community of reduced complexity, SIHUMIx, as a valuable model for in vitro use. The development of the SIHUMIx community was monitored over time with methods covering the cellular and the molecular level. We used microbial flow cytometry, intact protein profiling and terminal restriction fragment length polymorphism analysis to assess community structure. In parallel, we analyzed the functional level by targeted analysis of short-chain fatty acids and untargeted metabolomics. The stability properties constancy, resistance, and resilience were approached both on the structural and functional level of the community. We show that the SIHUMIx community is highly reproducible and constant since day 5 of cultivation. Furthermore, SIHUMIx has the ability to resist and recover from a pulsed perturbation, with changes in community structure recovered earlier than functional changes. Since community structure and function changed divergently, both levels need to be monitored at the same time to gain a full overview of the community development. All five methods are highly suitable to follow the community dynamics of SIHUMIx and indicated stability on day five. This makes SIHUMIx a suitable in vitro model to investigate the effects of e.g. medical, chemical, or dietary interventions.

Medium-chain carboxylates such as n -caproate and n -caprylate are valuable chemicals, which can be produced from renewable feedstock by anaerobic fermentation and lactate-based microbial chain elongation. Acidogenic microbiota involved in lactate-based chain elongation and their interplay with lactic acid bacteria have not been characterized in detail yet. Here, the metabolic and community dynamics were studied in a continuous bioreactor with xylan and lactate as sole carbon sources. Four succession stages were observed during 148 days of operation. After an adaptation period of 36 days, a relatively stable period of 28 days (stage I) was reached with n- butyrate, n- caproate and n- caprylate productivities of 7.2, 8.2 and 1.8 gCOD L -1 d -1 , respectively. After a transition period, the process changed to another period (stage II), during which 46% more n- butyrate, 51% less n- caproate and 67% less n- caprylate were produced. Co-occurrence networks of species based on 16S rRNA amplicon sequences and correlations with process parameters were analyzed to infer ecological interactions and potential metabolic functions. Diverse functions including hydrolysis of xylan, primary fermentation of xylose to acids (e.g., to acetate by Syntrophococcus , to n- butyrate by Lachnospiraceae , and to lactate by Lactobacillus ) and chain-elongation with lactate (by Ruminiclostridium 5 and Pseudoramibacter ) were inferred from the metabolic network. In stage I, the sub-network characterized by strongest positive correlations was mainly related to the production of n- caproate and n- caprylate. Lactic acid bacteria of the genus Olsenella co-occurred with potentially chain-elongating bacteria of the genus Pseudoramibacter , and their abundance was positively correlated with n- caproate and n- caprylate concentrations. A new sub-network appeared in stage II, which was mainly related to n- butyrate production and revealed a network of different lactic acid bacteria ( Bifidobacterium ) and potential n- butyrate producers ( Clostridium sensu stricto 12). The synergy effects between lactate-producing and lactate-consuming bacteria constitute a division of labor cooperation of mutual benefit. Besides cooperation, competition between different taxa determined the bacterial community assembly over the four succession stages in this resource-limited system. During long-term reactor operation under constant conditions, chain-elongating bacteria were outcompeted by butyrate-producing bacteria, leading to the increase of n- butyrate yield at the cost of medium-chain carboxylate yields in this closed model system., (Copyright © 2020 Liu, Kleinsteuber, Centler, Harms and Sträuber.)

The aim of this review is to give a summary of natural lignocellulose-degrading systems focusing mainly on animal digestive tracts of wood-feeding insects and ruminants in order to find effective strategies that can be applied to improve anaerobic digestion processes in engineered systems. Wood-feeding animals co-evolved with symbiotic microorganisms to digest lignocellulose-rich biomass in a very successful way. Considering the similarities between these animal gut systems and the lignocellulose-based biotechnological processes, the gut with its microbial consortium can be a perfect model for an advanced lignocellulose-degrading biorefinery. The physicochemical properties and structure of the gut may provide a scheme for the process design, and the microbial consortium may be applied as genetic resource for the up-scaled bioreactor communities. Manipulation of the gut microbiota is also discussed in relation to the management of the reactor communities.

The widely used artificial sweetener acesulfame K has long been considered recalcitrant in biological wastewater treatment. Due to its persistence and mobility in the aquatic environment, acesulfame has been used as marker substance for wastewater input in surface water and groundwater. However, recent studies indicated that the potential to remove this xenobiotic compound is emerging in wastewater treatment plants worldwide, leading to decreasing mass loads in receiving waters despite unchanged human consumption patterns. Here we show evidence that acesulfame can be mineralized in a catabolic process and used as sole carbon source by bacterial pure strains isolated from activated sludge and identified as Bosea sp. and Chelatococcus sp. The strains mineralize 1 g/L acesulfame K within 8-9 days. We discuss the potential degradation pathway and how this novel catabolic trait confirms the "principle of microbial infallibility." Once the enzymes involved in acesulfame degradation and their genes are identified, it will be possible to survey diverse environments and trace back the evolutionary origin as well as the mechanisms of global distribution and establishment of such a new catabolic trait., (Copyright © 2019 Kleinsteuber, Rohwerder, Lohse, Seiwert and Reemtsma.)

Syngas fermentation has been successfully implemented in commercial-scale plants and can enable the biochemical conversion of the driest fractions of biomass through synthesis gas (H 2 , CO 2 , and CO). The process relies on optimized acetogenic strains able to reach and maintain high productivity of ethanol and acetate. In parallel, microbial communities have shown to be the best choice for the production of valuable medium-chain carboxylates through anaerobic fermentation of biomass, demanding low technical complexity and being able to realize simultaneous hydrolysis of the substrate. Each of the two technologies benefits from different strong points and has different challenges to overcome. This review discusses the rationales for merging these two seemingly disparate technologies by analyzing previous studies and drawing opinions based on the lessons learned from such studies. For keeping the technical demands of the resulting process low, a case is built for using microbial communities instead of pure strains. For that to occur, a shift from conventional syngas-based to "syngas-aided" anaerobic fermentation is suggested. Strategies for tackling the intricacies of working simultaneously with communities and syngas, such as competing pathways, and thermodynamic aspects are discussed as well as the stoichiometry and economic feasibility of the concept. Overall, syngas-aided anaerobic fermentation seems to be a promising concept for the biorefinery of the future. However, the effects of process parameters on microbial interactions have to be understood in greater detail, in order to achieve and sustain feasible medium-chain carboxylate and alcohol productivity.

For biogas-producing continuous stirred tank reactors, an increase in dilution rate increases the methane production rate as long as substrate input can be converted fully. However, higher dilution rates necessitate higher specific microbial growth rates, which are assumed to have a strong impact on the apparent microbial biomass yield due to cellular maintenance. To test this, we operated two reactors at 37°C in parallel at dilution rates of 0.18 and 0.07 days -1 (hydraulic retention times of 5.5 and 14 days, doubling times of 3.9 and 9.9 days in steady state) with identical inoculum and a mixture of volatile fatty acids as sole carbon sources. We evaluated the performance of the Anaerobic Digestion Model No. 1 (ADM1), a thermodynamic black box approach (TBA), and dynamic flux balance analysis (dFBA), to describe the experimental observations. All models overestimated the impact of dilution rate on the apparent microbial biomass yield when using default parameter values. Based on our analysis, a maintenance coefficient value below 0.2 kJ per carbon mole of microbial biomass per hour should be used for the TBA, corresponding to 0.12 mmol ATP per gram dry weight per hour for dFBA, which strongly deviates from the value of 9.8 kJ Cmol h -1 that has been suggested to apply to all anaerobic microorganisms at 37°C. We hypothesized that a decrease in dilution rate might select taxa with minimized maintenance expenditure. However, no major differences in the dominating taxa between the reactors were observed based on amplicon sequencing of 16S rRNA genes and terminal restriction fragment length polymorphism analysis of mcrA genes. Surprisingly, Methanosaeta dominated over Methanosarcina even at a dilution rate of 0.18 days -1 , which contradicts previous model expectations. Furthermore, only 23-49% of the bacterial reads could be assigned to known syntrophic fatty acid oxidizers, indicating that unknown members of this functional group remain to be discovered. In conclusion, microbial maintenance was found to be much lower for acetogenesis and methanogenesis than previously assumed, likely due to the exceptionally low growth rates in anaerobic digestion. This finding might also be relevant for other microbial systems operating at similarly low growth rates.

Benzene mineralization under nitrate-reducing conditions was successfully established in an on-site reactor continuously fed with nitrate and sulfidic, benzene-containing groundwater extracted from a contaminated aquifer. Filling material from the reactor columns was used to set up anoxic enrichment cultures in mineral medium with benzene as electron donor and sole organic carbon source and nitrate as electron acceptor. Benzene degradation characteristics and community composition under nitrate-reducing conditions were monitored and compared to those of a well-investigated benzene-mineralizing consortium enriched from the same column system under sulfate-reducing conditions. The nitrate-reducing cultures degraded benzene at a rate of 10.1 ± 1.7 μM d -1 , accompanied by simultaneous reduction of nitrate to nitrite. The previously studied sulfate-reducing culture degraded benzene at similar rates. Carbon and hydrogen stable isotope enrichment factors determined for nitrate-dependent benzene degradation differed significantly from those of the sulfate-reducing culture (Λ H/C nitrate  = 12 ± 3 compared to Λ H/C sulfate  = 28 ± 3), indicating different benzene activation mechanisms under the two conditions. The nitrate-reducing community was mainly composed of Betaproteobacteria, Ignavibacteria, and Anaerolineae. Azoarcus and a phylotype related to clone Dok59 (Rhodocyclaceae) were the dominant genera, indicating an involvement in nitrate-dependent benzene degradation. The primary benzene degrader of the sulfate-reducing consortium, a phylotype belonging to the Peptococcaceae, was absent in the nitrate-reducing consortium.

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mL N CH 4 g VS -1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Natural microbial communities generally have an unknown structure and composition because of their still not yet cultivable members. Therefore, understanding the relationships among the bacterial members, prediction of their behaviour, and controlling their functions are difficult and often only partly successful endeavours to date. This study aims to test a new idea that allows to follow community dynamics on the basis of a simple concept. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S ribosomal RNA genes was used to describe a community profile that we define as composition of a community. Flow cytometry and analysis of DNA contents and forward scatter characteristics of the single cells were used to describe a community profile, which we define as structure of a community. Both approaches were brought together by a non-metric multidimensional scaling (n-MDS) for trend interpretation of changes in the complex community data sets. This was done on the basis of a graphical evaluation of the cytometric data, leading to the newly developed Dalmatian plot tool, which gave an unexpected insight into the dynamics of the unknown bacterial members of the investigated natural microbial community. The approach presented here was compared with other techniques described in the literature. The microbial community investigated in this study was obtained from a BTEX contaminated anoxic aquifer. The indigenous bacteria were allowed to colonise in situ microcosms consisting of activated carbon. These microcosms were amended with benzene and one of the electron acceptors nitrate, sulphate or ferric iron to stimulate microbial growth. The data obtained in this study indicated that the composition (via T-RFLP) and structure (via flow cytometry) of the natural bacterial community were influenced by the hydro-geochemical conditions in the test site, but also by the supplied electron acceptors, which led to distinct shifts in relative abundances of specific community members. It was concluded that engineered environments can be successfully monitored by single cell analytics in combination with established molecular tools and sophisticated statistical analyses, a combination that holds great promise for studying and monitoring natural microbial community behaviour.