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Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

While interest in underground gas storage (UGS) of hydrogen (H2) in salt caverns is increasing in a growing H2 economy, knowledge of the microbial communities inhabiting those hypersaline environments is still scarce. High salt concentrations and limited availability of carbon (C) sources in cavern environments reduce microbial growth rates and metabolic activities. Growth conditions potentially change once H2 is stored in salt caverns. H2 is a universal electron donor that can facilitate autotrophic growth and subsequently growth of heterotrophs. In this study, a mixed culture enriched from hypersaline UGS sites was investigated in microcosm experiments with H2 atmosphere, testing the effect of different salt concentrations and C sources on methanogenesis, sulfate reduction and homoacetogenesis. Sulfate, acetate and lactate concentrations were quantified throughout a time span of 125 days of incubation and correlations with the microbial community structure and function were explored through 16S rRNA gene-based amplicon sequencing at the end of the experiment. Many of the amplicon sequence variants (ASVs) were only assigned to family or order level, reflecting that a large number of ASVs belong to previously undescribed taxa. At 4.4 M NaCl, close to cavern brine salinity, members of the Desulfovibrionales were absent when no C source other than CO2 was offered as the sole C source. This is in line with the finding that no sulfate reduction occurred at these conditions. Acetogenic Halanaerobiia dominated in these high salinity levels. Based on metagenome sequencing of four selected samples, we found that acetogenesis at autotrophic conditions relies on the activity of a halophilic homoacetogen, Acetohalobium sp. and that sulfate reduction can most likely be associated with a so far undescribed member of the Desulfonatronovibrionales. We further discuss the discrepancy between sulfate reduction at heterotrophic conditions, while no sulfate reduction was observed under autotrophic conditions although acetate was produced through the homoacetogenic activity of Acetohalobium. For the application of UGS of H2, this means that the presence and activity of the aforementioned microorganisms must be investigated, as they can eventually lead to the formation of acetate and allow sulfate reduction at relevant concentrations.

In 2016 and 2018, Chung, Jansen and others described a new syndrome caused by haploinsufficiency of PHIP (pleckstrin homology domain interacting protein, OMIM *612,870) and mainly characterized by developmental delay (DD), learning difficulties/intellectual disability (ID), behavioral abnormalities, facial dysmorphism and obesity (CHUJANS, OMIM #617991). So far, PHIP alterations appear to be a rare cause of DD/ID. “Omics” technologies such as exome sequencing or array analyses have led to the identification of distinct types of alterations of PHIP, including, truncating variants, missense substitutions, splice variants and large deletions encompassing portions of the gene or the entire gene as well as adjacent genomic regions. We collected clinical and genetic data of 23 individuals with PHIP-associated Chung-Jansen syndrome (CHUJANS) from all over Europe. Follow-up investigations (e.g. Sanger sequencing, qPCR or Fluorescence-in-situ-Hybridization) and segregation analysis showed either de novo occurrence or inheritance from an also (mildly) affected parent. In accordance with previously described patients, almost all individuals reported here show developmental delay (22/23), learning disability or ID (22/23), behavioral abnormalities (20/23), weight problems (13/23) and characteristic craniofacial features (i.e. large ears/earlobes, prominent eyebrows, anteverted nares and long philtrum (23/23)). To further investigate the facial gestalt of individuals with CHUJANS, we performed facial analysis using the GestaltMatcher approach. By this, we could establish that PHIP patients are indistinguishable based on the type of PHIP alteration (e.g. missense, loss-of-function, splice site) but show a significant difference to the average face of healthy individuals as well as to individuals with Prader-Willi syndrome (PWS, OMIM #176270) or with a CUL4B-alteration (Intellectual developmental disorder, X-linked, syndromic, Cabezas type, OMIM #300354). Our findings expand the mutational and clinical spectrum of CHUJANS. We discuss the molecular and clinical features in comparison to the published individuals. The fact that some variants were inherited from a mildly affected parent further illustrates the variability of the associated phenotype and outlines the importance of a thorough clinical evaluation combined with genetic analyses for accurate diagnosis and counselling.

Cyanobacteria are highly promising microorganisms in forthcoming biotechnologies. Besides the systematic development of molecular tools for genetic engineering, the design of chassis strains and novel reactor concepts are in focus. The latter includes capillary biofilm reactors (CBR), which offer a high surface area-to-volume ratio and very high cell densities. In this context, Tolypothrix sp. PCC 7712 was found to be highly suited for this reactor system due to maximal surface coverage, extraordinarily strong biofilm attachment, and high biomass formation. Here, we provide the genome sequence of Tolypothrix sp. PCC 7712 to potentially allow targeted strain engineering. Surprisingly, it was almost identical to an available incomplete genome draft of Tolypothrix sp. PCC 7601. Thus, we completely sequenced this strain as well and compared it in detail to strain PCC 7712. Comparative genome analysis revealed 257 and 80 unique protein-coding sequences for strains PCC 7601 and PCC 7712, respectively. Clustering genomes based on average nucleotide identity (ANI) and 16S rRNA homology showed 99.98% similarity and only minor distance, respectively, between the two strains in contrast to 21 other cyanobacterial genomes. Despite these high similarities, both strains differ in the ability to fix atmospheric nitrogen and show specific sequence variations, which are discussed in the paper.

ABSTRACT The present study was conducted under the hypothesis that, in field peas, type of plant material, stage of maturity, ensiling, silage additive, and aerobic stress affect the composition and diversity of epiphytic microbial communities. Epiphytic microbial composition and diversity of pea seeds, partial crop peas, and whole crop peas was analyzed at different stages of late maturity, before and after ensiling, and with or without the use of lactic acid bacteria (LAB) as inoculant. Suitable combinations among pea crop variants, maturity stages, and inoculant use for the production of stable silages with sufficient aerobic stability after opening and during feed-out were identified. Genomic DNA was extracted, and 16S and 18S rRNA gene amplicons were sequenced. To assess the quality of the various silages, nutrient concentration, pH value, concentration of lactic acid, short chain fatty acids, and alcohols, and aerobic stability were determined. Pea seeds were barely colonized by epiphytic microorganisms. In partial and whole crop peas, composition and α-diversity (Shannon index) of bacterial communities did not differ between crop variants but differed among maturity stages. Epiphytic eukaryotes were rarely found on partial and whole crop peas. Bacterial composition and α-diversity were affected by ensiling and subsequent aerobic storage. In partial and whole crop peas, plant maturation caused an increase of the relative abundance of naturally occurring LAB (Weissella, Pediococcus, and Lactobacillus spp.). As a possible result, natural LAB support stable ensiling conditions even without the use of inoculants beginning with a maturity of 78 on the BBCH scale. This corresponded with a dry matter (DM) concentration of 341 and 363 g/kg in partial and whole crop peas, respectively. Addition of LAB inoculants, however, reduced ammonia, acetic acid, and butanol concentrations, and supported aerobic stability. Earlier stages of plant maturity (BBCH 76 and 77, 300 g DM/kg or less) were more prone to microbial spoilage. Stable pea seed silages can be produced at a maturity between BBCH 78 (427 g DM/kg) and 79 (549 g DM/kg), but they undoubtedly require LAB inoculation or application of other ensiling agents. IMPORTANCE Field peas are important protein suppliers for human and animal nutrition. They can be grown in many areas of the world, which may reduce imports of protein plants and has beneficial economic and ecological effects. Ensiling is a method of preserving feed that can be implemented easily and cost-effectively at the farm. Peas harvested as seeds, partial crop, or whole crop at different maturities enable a wide range of applications. The study characterized epiphytic microbial communities on peas in terms of composition and diversity depending on the maturity of the plants and feed conservation by ensiling as they play an essential role for the production of silages. Even if this study did not consider year, site, or cultivar effects, the results would show which part of the plant is probably well suited for the production of stable and high-quality silages and at which stage of maturity.

Analyzing microbial communities using metagenomes is a powerful approach to understand compositional structures and functional connections in anaerobic digestion (AD) microbiomes. Whereas short-read sequencing approaches based on the Illumina platform result in highly fragmented metagenomes, long-read sequencing leads to more contiguous assemblies. To evaluate the performance of a hybrid approach of these two sequencing approaches we compared the metagenome-assembled genomes (MAGs) resulting from five AD microbiome samples. The samples were taken from reactors fed with short-chain fatty acids at different feeding regimes (continuous and discontinuous) and organic loading rates (OLR). Methanothrix showed a high relative abundance at all feeding regimes but was strongly reduced in abundance at higher OLR, when Methanosarcina took over. The bacterial community composition differed strongly between reactors of different feeding regimes and OLRs. However, the functional potential was similar regardless of feeding regime and OLR. The hybrid sequencing approach using Nanopore long-reads and Illumina MiSeq reads improved assembly statistics, including an increase of the N50 value (on average from 32 to 1740 kbp) and an increased length of the longest contig (on average from 94 to 1898 kbp). The hybrid approach also led to a higher share of high-quality MAGs and generated five potentially circular genomes while none were generated using MiSeq-based contigs only. Finally, 27 hybrid MAGs were reconstructed of which 18 represent potentially new species—15 of them bacterial species. During pathway analysis, selected MAGs revealed similar gene patterns of butyrate degradation and might represent new butyrate-degrading bacteria. The demonstrated advantages of adding long reads to metagenomic analyses make the hybrid approach the preferable option when dealing with complex microbiomes.

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.

Microbial communities are pervasive in the natural environment, associated with many hosts, and of increasing importance in biotechnological applications. The complexity of these microbial systems makes the underlying mechanisms driving their dynamics difficult to identify. While experimental meta-OMICS techniques are routinely applied to record the inventory and activity of microbiomes over time, it remains difficult to obtain quantitative predictions based on such data. Mechanistic, quantitative mathematical modeling approaches hold the promise to both provide predictive power and shed light on cause-effect relationships driving these dynamic systems. We introduce μbialSim (pronounced “microbial sim”), a dynamic Flux-Balance-Analysis-based (dFBA) numerical simulator which is able to predict the time course in terms of composition and activity of microbiomes containing 100s of species in batch or chemostat mode. Activity of individual species is simulated by using separate FBA models which have access to a common pool of compounds, allowing for metabolite exchange. A novel augmented forward Euler method ensures numerical accuracy by temporarily reducing the time step size when compound concentrations decrease rapidly due to high compound affinities and/or the presence of many consuming species. We present three exemplary applications of μbialSim: a batch culture of a hydrogenotrophic archaeon, a syntrophic methanogenic biculture, and a 773-species human gut microbiome which exhibits a complex and dynamic pattern of metabolite exchange. Focusing on metabolite exchange as the main interaction type, μbialSim allows for the mechanistic simulation of microbiomes at their natural complexity. Simulated trajectories can be used to contextualize experimental meta-OMICS data and to derive hypotheses on cause-effect relationships driving community dynamics based on scenario simulations. μbialSim is implemented in Matlab and relies on the COBRA Toolbox or CellNetAnalyzer for FBA calculations. The source code is available under the GNU General Public License v3.0 at https://git.ufz.de/UMBSysBio/microbialsim.

Abstract Background Demand-driven biogas production could play an important role for future sustainable energy supply. However, feeding a biogas reactor according to energy demand may lead to organic overloading and, thus, to process failures. To minimize this risk, digesters need to be actively steered towards containing more robust microbial communities. This study focuses on acetogenesis and methanogenesis as crucial process steps for avoiding acidification. We fed lab-scale anaerobic digesters with volatile fatty acids under various feeding regimes and disturbances. The resulting microbial communities were analyzed on DNA and RNA level by terminal restriction fragment length polymorphism of the mcrA gene, 16S rRNA gene amplicon sequencing, and a [2-13C]-acetate assay. A modified Anaerobic Digestion Model 1 (ADM1) that distinguishes between the acetoclastic methanogens Methanosaeta and Methanosarcina was developed and fitted using experimental abiotic and biotic process parameters. Results Discontinuous feeding led to more functional resilience than continuous feeding, without loss in process efficiency. This was attributed to a different microbial community composition. Methanosaeta dominated the continuously fed reactors, while its competitor Methanosarcina was washed out. With discontinuous feeding, however, the fluctuating acetic acid concentrations provided niches to grow and co-exist for both organisms as shown by transcription analysis of the mcrA gene. Our model confirmed the higher functional resilience due to the higher abundance of Methanosarcina based on its higher substrate uptake rate and higher resistance to low pH values. Finally, we applied our model to maize silage as a more complex and practically relevant substrate and showed that our model is likely transferable to the complete AD process. Conclusions The composition of the microbial community determined the AD functional resilience against organic overloading in our experiments. In particular, communities with higher share of Methanosarcina showed higher process stability. The share of these microorganisms can be purposefully increased by discontinuous feeding. A model was developed that enables derivation of the necessary feeding regime for a more robust community with higher share of Methanosarcina.

The process of anaerobic digestion in which waste biomass is transformed to methane by complex microbial communities has been modeled for more than 16 years by parametric gray box approaches that simplify process biology and do not resolve intracellular microbial activity. Information on such activity, however, has become available in unprecedented detail by recent experimental advances in metatranscriptomics and metaproteomics. The inclusion of such data could lead to more powerful process models of anaerobic digestion that more faithfully represent the activity of microbial communities. We augmented the Anaerobic Digestion Model No. 1 (ADM1) as the standard kinetic model of anaerobic digestion by coupling it to Flux-Balance-Analysis (FBA) models of methanogenic species. Steady-state results of coupled models are comparable to standard ADM1 simulations if the energy demand for non-growth associated maintenance (NGAM) is chosen adequately. When changing a constant feed of maize silage from continuous to pulsed feeding, the final average methane production remains very similar for both standard and coupled models, while both the initial response of the methanogenic population at the onset of pulsed feeding as well as its dynamics between pulses deviates considerably. In contrast to ADM1, the coupled models deliver predictions of up to 1,000s of intracellular metabolic fluxes per species, describing intracellular metabolic pathway activity in much higher detail. Furthermore, yield coefficients which need to be specified in ADM1 are no longer required as they are implicitly encoded in the topology of the species’ metabolic network. We show the feasibility of augmenting ADM1, an ordinary differential equation-based model for simulating biogas production, by FBA models implementing individual steps of anaerobic digestion. While cellular maintenance is introduced as a new parameter, the total number of parameters is reduced as yield coefficients no longer need to be specified. The coupled models provide detailed predictions on intracellular activity of microbial species which are compatible with experimental data on enzyme synthesis activity or abundance as obtained by metatranscriptomics or metaproteomics. By providing predictions of intracellular fluxes of individual community members, the presented approach advances the simulation of microbial community driven processes and provides a direct link to validation by state-of-the-art experimental techniques.

The platform chemicals n-caproate and iso-butyrate can be produced by anaerobic fermentation from agro-industrial residues in a process known as microbial chain elongation. Few lactate-consuming chain-elongating species have been isolated and knowledge on their shared genetic features is still limited. Recently we isolated three novel clostridial strains (BL-3, BL-4, and BL-6) that convert lactate to n-caproate and iso-butyrate. Here, we analyzed the genetic background of lactate-based chain elongation in these isolates and other chain-elongating species by comparative genomics. The three strains produced n-caproate, n-butyrate, iso-butyrate, and acetate from lactate, with the highest proportions of n-caproate (18%) for BL-6 and of iso-butyrate (23%) for BL-4 in batch cultivation at pH 5.5. They show high genomic heterogeneity and a relatively small core-genome size. The genomes contain highly conserved genes involved in lactate oxidation, reverse β-oxidation, hydrogen formation and either of two types of energy conservation systems (Rnf and Ech). Including genomes of another eleven experimentally validated chain-elongating strains, we found that the chain elongation-specific core-genome encodes the pathways for reverse β-oxidation, hydrogen formation and energy conservation, while displaying substantial genome heterogeneity. Metabolic features of these isolates are important for biotechnological applications in n-caproate and iso-butyrate production.

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.

Metagenomics analysis revealing the composition and functional repertoire of complex microbial communities typically relies on large amounts of sequence data. Numerous analysis strategies and computational tools are available for their analysis. Fully integrated automated analysis pipelines such as MG-RAST or MEGAN6 are user-friendly but not designed for integrating specific knowledge on the biological system under study. In order to facilitate the consideration of such knowledge, we introduce a modular, adaptable analysis pipeline combining existing tools. We applied the novel pipeline to simulated mock data sets focusing on anaerobic digestion microbiomes and compare results to those obtained with established automated analysis pipelines. We find that the analysis strategy and choice of tools and parameters have a strong effect on the inferred taxonomic community composition, but not on the inferred functional profile. By including prior knowledge, computational costs can be decreased while improving result accuracy. While automated off-the-shelf analysis pipelines are easy to apply and require no knowledge on the microbial system under study, custom-made pipelines require more preparation time and bioinformatics expertise. This extra effort is minimized by our modular, flexible, custom-made pipeline, which can be adapted to different scenarios and can take available knowledge on the microbial system under study into account.

Biomethanation is a promising solution to convert H2 (produced from surplus electricity) and CO2 to CH4 by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H2/CO2. We enriched a hydrogenotrophic microbiota on CO2 and H2 as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH4. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH4 (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH4 from H2/CO2 by adapting the media composition, the reducing agent and the stirring intensity.

Ammonia inhibition is an important reason for reactor failures and economic losses in anaerobic digestion. Its impact on acetic acid degradation is well-studied, while its effect on propionic and butyric acid degradation has received little attention and is consequently not considered in the Anaerobic Digestion Model No. 1 (ADM1). To compare ammonia inhibition of the degradation of these three volatile fatty acids (VFAs), we fed a mixture of them as sole carbon source to three continuous stirred tank reactors (CSTRs) and increased ammonium bicarbonate concentrations in the influent from 52 to 277 mM. The use of this synthetic substrate allowed for the determination of degradation efficiencies for the individual acids. While butyric acid degradation was hardly affected by the increase of ammonia concentration, propionic acid degradation turned out to be even more inhibited than acetic acid degradation with degradation efficiencies dropping to 31 and 65% for propionic and acetic acid, respectively. The inhibited reactors acclimatized and approximated pre-disturbance degradation efficiencies toward the end of the experiment, which was accompanied by strong microbial community shifts, as observed by amplicon sequencing of 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of mcrA genes. The acetoclastic methanogen Methanosaeta was completely replaced by Methanosarcina. The propionic acid degrading genus Syntrophobacter was replaced by yet unknown propionic acid degraders. The butyric acid degrading genus Syntrophomonas and hydrogenotrophic Methanomicrobiaceae were hardly affected. We hypothesized that the ammonia sensitivity of the initially dominating taxa Methanosaeta and Syntrophobacter led to a stronger inhibition of the acetic and propionic acid degradation compared to butyric acid degradation and hydrogenotrophic methanogenesis, which were facilitated by the ammonia tolerant taxa Syntrophomonas and Methanomicrobiaceae. We implemented this hypothesis into a multi-taxa extension of ADM1, which was able to simulate the dynamics of both microbial community composition and VFA concentration in the experiment. It is thus plausible that the effect of ammonia on VFA degradation strongly depends on the ammonia sensitivity of the dominating taxa, for syntrophic propionate degraders as much as for acetoclastic methanogens.

Anaerobic digestion (AD) is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The lower diversity of methanogenic archaea compared to the bacterial groups involved in AD and the corresponding lack of functional redundancy cause a stronger susceptibility of methanogenesis to unfavorable process conditions such as trace element (TE) deprivation, thus controlling the stability of the overall process. Here, we investigated the effects of a slowly increasing TE deficit on the methanogenic community function in a semi-continuous biogas process. The aim of the study was to understand how methanogens in digester communities cope with TE limitation and sustain their growth and metabolic activity. Two lab-scale biogas reactors fed with distillers grains and supplemented with TEs were operated in parallel for 76 weeks before one of the reactors was subjected to TE deprivation, leading to a decline of cobalt and molybdenum concentrations from 0.9 to 0.2 mg/L, nickel concentrations from 2.9 to 0.8 mg/L, manganese concentrations from 38 to 18 mg/L, and tungsten concentrations from 1.4 to 0.2 mg/L. Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing TE limitation, the relative abundance of Methanosarcina dropped to 67% and a slight decrease of acetoclastic methanogenic activity was observed in batch tests with 13C-methyl-labeled acetate, suggesting a shift toward syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways. Proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase from Methanosarcinaceae increased, confirming our hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. Both Methanosarcina and Methanoculleus increased the abundance of N5-methyltetrahydromethanopterin-coenzyme M methyltransferase and methyl-coenzyme M reductase. However, these efforts to preserve the ion motive force for energy conservation were seemingly more successful in Methanoculleus. We conclude that both methanogenic genera use different strategies to stabilize their energy balance under TE limitation. Methanosarcina switched from TE expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis. Methanoculleus showed a higher robustness and was favored over the more fastidious Methanosarcina, thus stabilizing reactor performance under TE limitation.

Helicases are motor proteins that play a central role in the metabolism of DNA and RNA in biological cells. Using the energy of ATP molecules, they are able to translocate along the nucleic acids and unwind their duplex structure. They have been extensively characterized in the past and grouped into superfamilies based on structural similarities and sequential motifs. However, their functional aspects and the mechanism of their operation are not yet well understood. Here, we consider three helicases from the major superfamily 2--Hef, Hel308 and XPD--and study their conformational dynamics by using coarse-grained relaxational elastic network models. Specifically, their responses to mechanical perturbations are analyzed. This enables us to identify robust and ordered conformational motions which may underlie the functional activity of these proteins. As we show, such motions are well-organized and have large amplitudes. Their possible roles in the processing of nucleic substrate are discussed.