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ABSTRACTWastewater is considered a reservoir of antimicrobial resistance genes (ARGs), where the abundant antimicrobial-resistant bacteria and mobile genetic elements facilitate horizontal gene transfer. However, the prevalence and extent of these phenomena in different taxonomic groups that inhabit wastewater are still not fully understood. Here, we determined the presence of ARGs in metagenome-assembled genomes (MAGs) and evaluated the risks of MAG-carrying ARGs in potential human pathogens. The potential of these ARGs to be transmitted horizontally or vertically was also determined. A total of 5,916 MAGs (completeness >50%, contamination 0.95). The dominant ARG classes detected were bacitracin, multi-drug, macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside, and 10.26% of them were located on plasmids. The main hosts of ARGs belonged to Escherichia, Klebsiella, Acinetobacter, Gresbergeria, Mycobacterium, and Thauera. Our data showed that 253 MAGs carried virulence factor genes (VFGs) divided into 44 gOTUs, of which 45 MAGs were carriers of ARGs, indicating that potential human pathogens carried ARGs. Alarmingly, the MAG assigned as Escherichia coli contained 159 VFGs, of which 95 were located on chromosomes and 10 on plasmids. In addition to shedding light on the prevalence of ARGs in individual genomes recovered from activated sludge and wastewater, our study demonstrates a workflow that can identify antimicrobial-resistant pathogens in complex microbial communities.IMPORTANCEAntimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.

Abstract Most recent advances for phosphorus (P) recovery using brewery yeast on laboratory scale were used to scale up to a pilot‐scale process (BioP‐Rec module) and applied in a full‐scale wastewater treatment plant (WWTP). A P balance was established for WWTP Markranstädt according to two thresholds: (1) the economic feasibility threshold for P recovery of 0.05 kg/m3 of free P, and (2) the German Sewage Sludge Ordinance (GSSO) threshold, which demands that all WWTPs with a P content in dry matter (DM) of biosolids of 20 gP/kgDM or higher in the coming years must perform mandatory P recovery. In terms of defined thresholds, return and excess sludges were identified as the most feasible WWTP process streams for P recovery. In a 1 m3 BioP‐Rec module a 3 stage process was established. From the P‐rich water‐phase of the return sludge produced in stage 1, which contained 0.051 kg/m3 of free P, 77.56% was taken up by P‐depleted brewer's yeast Saccharomyces pastorianus in 3 h in stage 2. In stage 3, the yeast was concentrated in 1 h to produce yeast sludge as a fertilizer product. We demonstrated a novel pilot‐scale process for the production of bio‐based P‐rich fertilizer.

Catalytic activity of microbial communities maintains the services and functions of soils. Microbial communities require energy and carbon for microbial growth, which they obtain by transforming organic matter (OM), oxidizing a fraction of it and transferring the electrons to various terminal acceptors. Quantifying the relations between matter and energy fluxes is possible when key parameters such as reaction enthalpy (∆rH), energy use efficiency (related to enthalpy) (EUE), carbon use efficiency (CUE), calorespirometric ratio (CR), carbon dioxide evolution rate (CER), and the apparent specific growth rate (μapp) are known. However, the determination of these parameters suffers from unsatisfying accuracy at the technical (sample size, instrument sensitivity), experimental (sample aeration) and data processing levels thus affecting the precise quantification of relationships between carbon and energy fluxes. To address these questions under controlled conditions, we analyzed microbial turnover processes in a model soil amended using a readily metabolizable substrate (glucose) and three commercial isothermal microcalorimeters (MC-Cal/100P, TAM Air and TAM III) with different sample sizes meaning varying volume-related thermal detection limits (LODv) (0.05−1mW L−1). We conducted aeration experiments (aerated and un-aerated calorimetric ampoules) to investigate the influence of oxygen limitation and thermal perturbation on the measurement signal. We monitored the CER by measuring the additional heat caused by CO2 absorption using a NaOH solution acting as a CO2 trap. The range of errors associated with the calorimetrically derived μapp, EUE, and CR was determined and compared with the requirements for quantifying CUE and the degree of anaerobicity (ηA). Calorimetrically derived μapp and EUE were independent of the instrument used. However, instruments with a low LODv yielded the most accurate results. Opening and closing the ampoules for oxygen and CO2 exchange did not significantly affect metabolic heats. However, regular opening during calorimetrically derived CER measurements caused significant measuring errors due to strong thermal perturbation of the measurement signal. Comparisons between experimentally determined CR, CUE,ηA, and modeling indicate that the evaluation of CR should be performed with caution.

With the increasing use of renewable energy resources for the power grid, the need for long-term storage technologies, such as power-to-gas systems, is growing. Biomethanation provides the opportunity to store energy in the form of the natural gas-equivalent biomethane. This study investigates a novel plug flow reactor that employs a helical static mixer for the biological methanation of hydrogen and carbon dioxide. In tests, the reactor achieved an average methane production rate of 2.5 LCH4LR∗d (methane production [LCH4] per liter of reactor volume [LR] per day [d]) with a maximum methane content of 94%. It demonstrated good flexibilization properties, as repeated 12 h downtimes did not negatively impact the process. The genera Methanothermobacter and Methanobacterium were predominant during the initial phase, along with volatile organic acid-producing, hydrogenotrophic, and proteolytic bacteria. The average ratio of volatile organic acid to total inorganic carbon increased to 0.52 ± 0.04, while the pH remained stable at an average of pH 8.1 ± 0.25 from day 32 to 98, spanning stable and flexible operation modes. This study contributes to the development of efficient flexible biological methanation systems for sustainable energy storage and management.

Abstract Background The ability to quantitatively predict ecophysiological functions of microbial communities provides an important step to engineer microbiota for desired functions related to specific biochemical conversions. Here, we present the quantitative prediction of medium-chain carboxylate production in two continuous anaerobic bioreactors from 16S rRNA gene dynamics in enriched communities. Results By progressively shortening the hydraulic retention time (HRT) from 8 to 2 days with different temporal schemes in two bioreactors operated for 211 days, we achieved higher productivities and yields of the target products n-caproate and n-caprylate. The datasets generated from each bioreactor were applied independently for training and testing machine learning algorithms using 16S rRNA genes to predict n-caproate and n-caprylate productivities. Our dataset consisted of 14 and 40 samples from HRT of 8 and 2 days, respectively. Because of the size and balance of our dataset, we compared linear regression, support vector machine and random forest regression algorithms using the original and balanced datasets generated using synthetic minority oversampling. Further, we performed cross-validation to estimate model stability. The random forest regression was the best algorithm producing more consistent results with median of error rates below 8%. More than 90% accuracy in the prediction of n-caproate and n-caprylate productivities was achieved. Four inferred bioindicators belonging to the genera Olsenella, Lactobacillus, Syntrophococcus and Clostridium IV suggest their relevance to the higher carboxylate productivity at shorter HRT. The recovery of metagenome-assembled genomes of these bioindicators confirmed their genetic potential to perform key steps of medium-chain carboxylate production. Conclusions Shortening the hydraulic retention time of the continuous bioreactor systems allows to shape the communities with desired chain elongation functions. Using machine learning, we demonstrated that 16S rRNA amplicon sequencing data can be used to predict bioreactor process performance quantitatively and accurately. Characterizing and harnessing bioindicators holds promise to manage reactor microbiota towards selection of the target processes. Our mathematical framework is transferrable to other ecosystem processes and microbial systems where community dynamics is linked to key functions. The general methodology used here can be adapted to data types of other functional categories such as genes, transcripts, proteins or metabolites. Video Abstract

Abstract The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine, a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation.

The increasing share of renewable electricity in the grid drives the need for sufficient storage capacity. Especially for seasonal storage, power-to-gas can be a promising approach. Biologically produced methane from hydrogen produced from surplus electricity can be used to substitute natural gas in the existing infrastructure. Current reactor types are not or are poorly optimized for flexible methanation. Therefore, this work proposes a new reactor type with a plug flow reactor (PFR) design. Simulations in COMSOL Multiphysics ® showed promising properties for operation in laminar flow. An experiment was conducted to support the simulation results and to determine the gas fraction of the novel reactor, which was measured to be 29%. Based on these simulations and experimental results, the reactor was constructed as a 14 m long, 50 mm diameter tube with a meandering orientation. Data processing was established, and a step experiment was performed. In addition, a kLa of 1 h−1 was determined. The results revealed that the experimental outcomes of the type of flow and gas fractions are in line with the theoretical simulation. The new design shows promising properties for flexible methanation and will be tested.

Cell density is an important factor in all microbiome research, where interactions are of interest. It is also the most important parameter for the operation and control of most biotechnological processes. In the past, cell density determination was often performed offline and manually, resulting in a delay between sampling and immediate data processing, preventing quick action. While there are now some online methods for rapid and automated cell density determination, they are unable to distinguish between the different cell types in bacterial communities. To address this gap, an online automated flow cytometry procedure is proposed for real-time high-resolution analysis of bacterial communities. On the one hand, it allows for the online automated calculation of cell concentrations and, on the other, for the differentiation between different cell subsets of a bacterial community. To achieve this, the OC-300 automation device (onCyt Microbiology, Zürich, Switzerland) was coupled with the flow cytometer CytoFLEX (Beckman Coulter, Brea, USA). The OC-300 performs the automatic sampling, dilution, fixation and 4′,6-diamidino-2-phenylindole (DAPI) staining of a bacterial sample before sending it to the CytoFLEX for measurement. It is demonstrated that this method can reproducibly measure both cell density and fingerprint-like patterns of bacterial communities, generating suitable data for powerful automated data analysis and interpretation pipelines. In particular, the automated, high-resolution partitioning of clustered data into cell subsets opens up the possibility of correlation analysis to identify the operational or abiotic/biotic causes of community disturbances or state changes, which can influence the interaction potential of organisms in microbiomes or even affect the performance of individual organisms.

ABSTRACT Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes. Here, we analyzed whether and to which degree hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of the obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy, and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches, allowing for spore germination, growth, and fermentative activity of the obligate anaerobe in an otherwise inhabitable environment. Hypoxic liquid films containing 80% ± 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Hyphae hence may serve as good networks for the activity and spatial organization of obligate anaerobic bacteria in oxygenated heterogeneous environments such as soil. IMPORTANCE Although a few studies have reported on the presence of anoxic microniches in fungal biofilms, knowledge of the effects of fungal oxygen consumption on bacterial-fungal interactions is limited. Here, we demonstrate the existence and persistence of oxygen-free zones in air-exposed mycelia enabling spore germination, growth, fermentative activity, and dispersal of the obligate anaerobe. Our study points out a previously overlooked role of aerobic fungi in creating and bridging anoxic microniches in ambient oxic habitats. Air-exposed hyphae hence may act as a scaffold for activity and dispersal of strictly anaerobic microbes. Given the short-term tolerance of strict anaerobes to oxygen and reduced oxygen content in the mycosphere, hyphae can promote spatial organization of both obligate anaerobic and aerobic bacteria. Such finding may be important for a better understanding of previously observed co-occurrences of aerobes and anaerobes in well-aerated habitats such as upland soils.

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.

The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting.

In order to partially mimic the efficient lignocellulose pretreatment process performed naturally in the gut system of Pachnoda marginata larvae, two wheat straw pretreatments were evaluated: a mechanical pretreatment via cutting the straw into two different sizes and an alkaline pretreatment with calcium hydroxide. After pretreatment, gut enrichment cultures on wheat straw at alkaline pH were inoculated and kept at mesophilic conditions over 45 days. The methanogenic community was composed mainly of the Methanomicrobiaceae and Methanosarcinaceae families. The combined pretreatment, size reduction and alkaline pretreatment, was the best condition for methane production. The positive effect of the straw pretreatment was higher in the midgut cultures, increasing the methane production by 192%, while for hindgut cultures the methane production increased only by 149% when compared to non-pretreated straw. Scanning electron microscopy (SEM) showed that the alkaline pretreatment modified the surface of the wheat straw fibers, which promoted biofilm formation and microbial growth. The enrichment cultures derived from larva gut microbiome were able to degrade larger 1 mm alkaline treated and smaller 250 µm but non-pretreated straw at the same efficiency. The combination of mechanical and alkaline pretreatments resulted in increased, yet not superimposed, methane yield.

Summary The detection and quantification of Legionella pneumophila (responsible for legionnaire's disease) in water samples can be achieved by various methods. However, the culture‐based ISO 11731:2017, which is based on counts of colony‐forming units per ml (CFU·ml‐1) is still the gold standard for quantification of Legionella species (spp.). As a powerful alternative, we propose real‐time monitoring of the growth of L. pneumophila using an isothermal microcalorimeter (IMC). Our results demonstrate that, depending on the initial concentration of L. pneumophila, detection times of 24–48 h can be reliably achieved. IMC may, therefore, be used as an early warning system for L. pneumophila contamination. By replacing only visual detection of growth by a thermal sensor, but otherwise maintaining the standardized protocol of the ISO 11731:2017, the new procedure could easily be incorporated into existing standards. The exact determination of the beginning of metabolic heat is often very difficult because at the beginning of the calorimetric signal the thermal stabilization and the metabolic heat development overlap. Here, we propose a new data evaluation based on the first derivation of the heat flow signal. The improved evaluation method can further reduce detection times and significantly increase the reliability of the IMC approach.

The effects of the inoculum origin, temperature or operational changes on ex situ biomethanation by complex microbial communities have been investigated; however, it remains unclear how the diversity of the inoculum influences the process and its stability. We explored the effect of microbial diversity of four inocula (coded as PF, WW, S37 and Nrich) on methane production, process stability and the formation of volatile fatty acids as by-products. The highest methane amounts produced were 3.38 ± 0.37 mmol, 3.20 ± 0.07 mmol, 3.07 ± 0.27 mmol and 3.14 ± 0.06 mmol for PF, WW, S37 and Nrich, respectively. The highest acetate concentration was found in less diverse cultures (1679 mg L−1 and 1397 mg L−1 for S37 and Nrich, respectively), whereas the acetate concentrations remained below 30 mg L−1 in the more diverse cultures. The maximum concentration of propionate was observed in less diverse cultures (240 mg L−1 and 37 mg L−1 for S37 and Nrich cultures, respectively). The highly diverse cultures outperformed the medium and low diversity cultures in the long-term operation. Methanogenic communities were mainly composed of hydrogenotrophic methanogens in all cultures. Aceticlastic methanogenesis was only active in the highly diverse sludge community throughout the experiment. The more diverse the inocula, the more methane was produced and the less volatile fatty acids accumulated, which could be attributed to the high number of microbial functions working together to keep a stable and balanced process. It is concluded that the inoculum origin and its diversity are very important factors to consider when the biomethanation process is performed with complex microbial communities.

In the present study, we investigated whether a non-invasive metabolic heat flux analysis could serve the determination of the functional traits in free-living saprotrophic decomposer fungi and aid the prediction of fungal influences on ecosystem processes. For this, seven fungi, including ascomycete, basidiomycete, and zygomycete species, were investigated in a standardised laboratory environment, employing wheat straw as a globally relevant lignocellulosic substrate. Our study demonstrates that biocalorimetry can be employed successfully to determine growth-related fungal activity parameters, such as apparent maximum growth rates (AMGR), cultivation times until the observable onset of fungal growth at AMGR (tAMGR), quotients formed from the AMGR and tAMGR (herein referred to as competitive growth potential, CGP), and heat yield coefficients (YQ/X), the latter indicating the degree of resource investment into fungal biomass versus other functional attributes. These parameters seem suitable to link fungal potentials for biomass production to corresponding ecological strategies employed during resource utilisation, and therefore may be considered as fungal life history traits. A close connection exists between the CGP and YQ/X values, which suggests an interpretation that relates to fungal life history strategies.

Abstract Background The carboxylate platform is a promising technology for substituting petrochemicals in the provision of specific platform chemicals and liquid fuels. It includes the chain elongation process that exploits reverse β–oxidation to elongate short-chain fatty acids and forms the more valuable medium-chain variants. The pH value influences this process through multiple mechanisms and is central to effective product formation. Its influence on the microbiome dynamics was investigated during anaerobic fermentation of maize silage by combining flow cytometric short interval monitoring, cell sorting and 16S rRNA gene amplicon sequencing. Results Caproate and caprylate titres of up to 6.12 g L−1 and 1.83 g L−1, respectively, were achieved in a continuous stirred-tank reactor operated for 241 days. Caproate production was optimal at pH 5.5 and connected to lactate-based chain elongation, while caprylate production was optimal at pH 6.25 and linked to ethanol utilisation. Flow cytometry recorded 31 sub-communities with cell abundances varying over 89 time points. It revealed a highly dynamic community, whereas the sequencing analysis displayed a mostly unchanged core community. Eight key sub-communities were linked to caproate or caprylate production (rS > | ± 0.7|). Amongst other insights, sorting and subsequently sequencing these sub-communities revealed the central role of Bifidobacterium and Olsenella, two genera of lactic acid bacteria that drove chain elongation by providing additional lactate, serving as electron donor. Conclusions High-titre medium-chain fatty acid production in a well-established reactor design is possible using complex substrate without the addition of external electron donors. This will greatly ease scaling and profitable implementation of the process. The pH value influenced the substrate utilisation and product spectrum by shaping the microbial community. Flow cytometric single cell analysis enabled fast, short interval analysis of this community and was coupled with 16S rRNA gene amplicon sequencing to reveal the major role of lactate-producing bacteria.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

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.

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.

The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter, Methanoculleus, and Methanosarcina. Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut.

Mixed or pure cultures can be used for biomethanation of hydrogen. Sodium 2-bromoethanesulfonate (BES) is an inhibitor of methanogenesis used to investigate competing reactions like homoacetogenesis in mixed cultures. To understand the effect of BES on the hydrogenotrophic metabolism in a biomethanation process, anaerobic granules from a wastewater treatment plant, a hydrogenotrophic enrichment culture, and pure cultures of Methanococcus maripaludis and Methanobacterium formicicum were incubated under H2/CO2 headspace in the presence or absence of BES, and the turnover of H2, CO2, CH4, formate and acetate was analyzed. Anaerobic granules produced the highest amount of formate after 24 h of incubation in the presence of BES. Treating the enrichment culture with BES led to the accumulation of formate. M. maripaludis produced more formate than M. formicicum when treated with BES. The non-inhibited methanogenic communities produced small amounts of formate whereas the pure cultures did not. The highest amount of acetate was produced by the anaerobic granules concomitantly with formate consumption. These results indicate that formate is an important intermediate of hydrogenotrophic metabolism accumulating upon methanogenesis inhibition.

Recent studies have demonstrated that phages can be co-transported with motile non-host bacteria, thereby enabling their invasion of biofilms and control of biofilm composition. Here, we developed a novel approach to isolate non-host bacteria able to co-transport phages from soil. It is based on the capability of phage-carrying non-host bacteria to move along mycelia out of soil and form colonies in plaques of their co-transported phages. The approach was tested using two model phages of differing surface hydrophobicity, i.e., hydrophobic Escherichia virus T4 (T4) and hydrophilic Pseudoalteromonas phage HS2 (HS2). The phages were mixed into soil and allowed to be transported by soil bacteria along the mycelia of Pythium ultimum. Five phage-carrying bacterial species were isolated (Viridibacillus sp., Enterobacter sp., Serratia sp., Bacillus sp., Janthinobacterium sp.). These bacteria exhibited phage adsorption efficiencies of ≈90–95% for hydrophobic T4 and 30–95% for hydrophilic HS2. The phage adsorption efficiency of Viridibacillus sp. was ≈95% for both phages and twofold higher than T4-or HS2-adsorption to their respective hosts, qualifying Viridibacillus sp. as a potential super carrier for phages. Our approach offers an effective and target-specific way to identify and isolate phage-carrying bacteria in natural and man-made environments.

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.

Soil is populated by highly diverse microbial communities mediating important processes and functions. The distribution of microbes, however, is neither uniform nor random. Instead, it is dictated by physical, chemical, and biological processes and conditions and varying over small spatial and temporal scales. The feedbacks between these processes make the soil-microbe complex a self-organizing system capable of adapting to the continuously changing conditions mainly driven by the highly fluctuating water content. For making meaningful predictions on the spatiotemporal dynamics of soil microbes and their functions, we need to integrate knowledge from physics, chemistry, and biology in our modeling approaches. Here, we review modeling studies with a focus on spatiotemporal dynamics of bacteria and bacterial functions in soil microhabitats. We compare these studies along four dimensions: specific aim, model type (individual-based, population-based), scale, and considered physical, chemical, biological processes and aspects. A special emphasis is laid on modeling approaches considering processes and aspects influencing the spatial distribution of bacteria such as motility, vector-based dispersal and biofilm formation. This includes factors like soil structure, carbon and oxygen gradients, temporal variations in hydration conditions or anthropogenic disturbance events. By assessing the importance of different microscale bacterial processes, this review should contribute to the ongoing discussion on challenges related to the upscaling from the microscopic via the profile to the landscape scale. Recent technical advances to observe bacteria in soils or soil-like environments combined with multidisciplinary collaborations will help to shed light on currently understudied physical, chemical and biological interactions in the soil-microbe complex.

Abstract Background Dimethylphenols (DMP) are toxic compounds with high environmental mobility in water and one of the main constituents of effluents from petro- and carbochemical industry. Over the last few decades, the use of constructed wetlands (CW) has been extended from domestic to industrial wastewater treatments, including petro-carbochemical effluents. In these systems, the main role during the transformation and mineralization of organic pollutants is played by microorganisms. Therefore, understanding the bacterial degradation processes of isolated strains from CWs is an important approach to further improvements of biodegradation processes in these treatment systems. Results In this study, bacterial isolation from a pilot scale constructed wetland fed with phenols led to the identification of Delftia sp. LCW as a DMP degrading strain. The strain was able to use the o-xylenols 3,4-DMP and 2,3-DMP as sole carbon and energy sources. In addition, 3,4-DMP provided as a co-substrate had an effect on the transformation of other four DMP isomers. Based on the detection of the genes, proteins, and the inferred phylogenetic relationships of the detected genes with other reported functional proteins, we found that the phenol hydroxylase of Delftia sp. LCW is induced by 3,4-DMP and it is responsible for the first oxidation of the aromatic ring of 3,4-, 2,3-, 2,4-, 2,5- and 3,5-DMP. The enzyme may also catalyze both monooxygenation reactions during the degradation of benzene. Proteome data led to the identification of catechol meta cleavage pathway enzymes during the growth on ortho DMP, and validated that cleavage of the aromatic rings of 2,5- and 3,5-DMPs does not result in mineralization. In addition, the tolerance of the strain to high concentrations of DMP, especially to 3,4-DMP was higher than that of other reported microorganisms from activated sludge treating phenols. Conclusions LCW strain was able to degraded complex aromatics compounds. DMPs and benzene are reported for the first time to be degraded by a member of Delftia genus. In addition, LCW degraded DMPs with a first oxidation of the aromatic rings by a phenol hydroxylase, followed by a further meta cleavage pathway. The higher resistance to DMP toxicity, the ability to degrade and transform DMP isomers and the origin as a rhizosphere bacterium from wastewater systems, make LCW a suitable candidate to be used in bioremediation of complex DMP mixtures in CWs systems.

Modern isothermal microcalorimeters (IMC) are able to detect the metabolic heat of bacteria with an accuracy sufficient to recognize even the smallest traces of bacterial contamination of water, food, and medical samples. The modern IMC techniques are often superior to conventional detection methods in terms of the detection time, reliability, labor, and technical effort. What is missing is a systematic analysis of the influence of the cultivation conditions on calorimetric detection. For the acceptance of IMC techniques, it is advantageous if already standardized cultivation techniques can be combined with calorimetry. Here we performed such a systematic analysis using Lactobacillus plantarum as a model bacterium. Independent of the cultivation techniques, IMC detections were much faster for high bacterial concentrations (>102 CFU⋅mL–1) than visual detections. At low bacterial concentrations (

Abstract Ecological disturbances are important drivers of biodiversity patterns. Many biodiversity studies rely on endpoint measurements instead of following the dynamics that lead to those outcomes and testing ecological drivers individually, often considering only a single trophic level. Manipulating multiple factors (biotic and abiotic) in controlled settings and measuring multiple descriptors of multi-trophic communities could enlighten our understanding of the context dependency of ecological disturbances. Using model microbial communities, we experimentally tested the effects of imposed disturbances (i.e. increased dilution simulating density-independent mortality as press or pulse disturbances coupled with resource deprivation) on bacterial abundance, diversity and community structure in the absence or presence of a protist predator. We monitored the communities immediately before and after imposing the disturbance and four days after resuming the pre-disturbance dilution regime to infer resistance and recovery properties. The results highlight that bacterial abundance, diversity and community composition were more affected by predation than by disturbance type, resource loss or the interaction of these factors. Predator abundance was strongly affected by the type of disturbance imposed, causing temporary relief of predation pressure. Importantly, prey community composition differed significantly at different phases, emphasizing that endpoint measurements are insufficient for understanding the recovery of communities.

Abstract Background The widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious. Results In this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL−1 and 1.8 x 109 (± 1.1 × 108) cells mL−1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results. Conclusions The presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.

The maintenance of bacterial and fungal activity is essential for ecosystem functioning, particularly in dry soils where the two phyla co-exist. Here, Worrich and colleagues show experimentally that mycelia traffic water and nutrients and thereby stimulate bacterial activity in stressful conditions.

Abstract Background Species recovery after disturbances depends on the strength and duration of disturbance, on the species traits and on the biotic interactions with other species. In order to understand these complex relationships, it is essential to understand mechanistically the transient dynamics of interacting species during and after disturbances. We combined microcosm experiments with simulation modelling and studied the transient recovery dynamics of a simple microbial food web under pulse and press disturbances and under different predator couplings to an alternative resource. Results Our results reveal that although the disturbances affected predator and prey populations by the same mortality, predator populations suffered for a longer time. The resulting diminished predation stress caused a temporary phase of high prey population sizes (i.e. prey release) during and even after disturbances. Increasing duration and strength of disturbances significantly slowed down the recovery time of the predator prolonging the phase of prey release. However, the additional coupling of the predator to an alternative resource allowed the predator to recover faster after the disturbances thus shortening the phase of prey release. Conclusions Our findings are not limited to the studied system and can be used to understand the dynamic response and recovery potential of many natural predator–prey or host–pathogen systems. They can be applied, for instance, in epidemiological and conservational contexts to regulate prey release or to avoid extinction risk of the top trophic levels under different types of disturbances.

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.

Due to their strong antimicrobial activity, silver nanoparticles (AgNPs) are massively produced, applied, consumed and, as a negative consequence, released into wastewater treatment plants. Most AgNPs are assumed to be bound by sludge, and thus bear potential risk for microbial performance and stability. In this lab-scale study, flow cytometry as a high-throughput method and 16S rRNA gene amplicon Illumina MiSeq sequencing were used to track microbial community structure changes when being exposed to AgNPs. Both methods allowed deeper investigation of the toxic impact of chemicals on microbial communities than classical EC50 determination. In addition, ecological metrics were used to quantify microbial community variations depending on AgNP types (10 and 30 nm) and concentrations. Only low changes in α- and intra-community β-diversity values were found both in successive negative and positive control batches and batches that were run with AgNPs below the EC50 value. Instead, AgNPs at EC50 concentrations caused upcoming of certain and disappearance of formerly dominant subcommunities. Flavobacteriia were among those that almost disappeared, while phylotypes affiliated with Gammaproteobacteria (3.6-fold) and Bacilli (8.4-fold) increased in cell abundance in comparison to the negative control. Thus, silver amounts at the EC50 value affected community structure suggesting a potential negative impact on functions in wastewater treatment systems.

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.

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.

In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

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.

Bacterial degradation of organic compounds is an important ecosystem function with relevance to, e.g., the cycling of elements or the degradation of organic contaminants. It remains an open question, however, to which extent ecosystems are able to maintain such biodegradation function under recurrent disturbances (functional resistance) and how this is related to the bacterial biomass abundance. In this paper, we use a numerical simulation approach to systematically analyze the dynamic response of a microbial population to recurrent disturbances of different spatial distribution. The spatially explicit model considers microbial degradation, growth, dispersal, and spatial networks that facilitate bacterial dispersal mimicking effects of mycelial networks in nature. We find: (i) There is a certain capacity for high resistance of biodegradation performance to recurrent disturbances. (ii) If this resistance capacity is exceeded, spatial zones of different biodegradation performance develop, ranging from no or reduced to even increased performance. (iii) Bacterial biomass and biodegradation dynamics respond inversely to the spatial fragmentation of disturbances: overall biodegradation performance improves with increasing fragmentation, but bacterial biomass declines. (iv) Bacterial dispersal networks can enhance functional resistance against recurrent disturbances, mainly by reactivating zones in the core of disturbed areas, even though this leads to an overall reduction of bacterial biomass.

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.

ABSTRACT Natural microbial communities affect human life in countless ways, ranging from global biogeochemical cycles to the treatment of wastewater and health via the human microbiome. In order to probe, monitor, and eventually control these communities, fast detection and evaluation methods are required. In order to facilitate rapid community analysis and monitor a community’s dynamic behavior with high resolution, we here apply community flow cytometry, which provides single-cell-based high-dimensional data characterizing communities with high acuity over time. To interpret time series data, we draw inspiration from macroecology, in which a rich set of concepts has been developed for describing population dynamics. We focus on the stability paradigm as a promising candidate to interpret such data in an intuitive and actionable way and present a rapid workflow to monitor stability properties of complex microbial ecosystems. Based on single-cell data, we compute the stability properties resistance, resilience, displacement speed, and elasticity. For resilience, we also introduce a method which can be implemented for continuous online community monitoring. The proposed workflow was tested in a long-term continuous reactor experiment employing both an artificial and a complex microbial community, which were exposed to identical short-term disturbances. The computed stability properties uncovered the superior stability of the complex community and demonstrated the global applicability of the protocol to any microbiome. The workflow is able to support high temporal sample densities below bacterial generation times. This may provide new opportunities to unravel unknown ecological paradigms of natural microbial communities, with applications to environmental, biotechnological, and health-related microbiomes. IMPORTANCE Microbial communities drive many processes which affect human well-being directly, as in the human microbiome, or indirectly, as in natural environments or in biotechnological applications. Due to their complexity, their dynamics over time is difficult to monitor, and current sequence-based approaches are limited with respect to the temporal resolution. However, in order to eventually control microbial community dynamics, monitoring schemes of high temporal resolution are required. Flow cytometry provides single-cell-based data in the required temporal resolution, and we here use such data to compute stability properties as easy to interpret univariate indicators of microbial community dynamics. Such monitoring tools will allow for a fast, continuous, and cost-effective screening of stability states of microbiomes. Applicable to various environments, including bioreactors, surface water, and the human body, it will contribute to the development of control schemes to manipulate microbial community structures and performances.

Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n = 135, ascites n = 92, duodenal fluid n = 54) from 135 patients with liver cirrhosis and 52 samples (blood n = 26, duodenal fluid n = 26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x10(1) copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p = 0.123) and the median level of Candida DNA was 3.8x10(5) copies ml-1 (2.3x10(2)-6.3x10(9)). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation.

Complex ecosystems harbor multiple predators and prey species whose direct and indirect interactions are under study. In particular, the combined effects of predator diversity and resource preference on prey removal are not known. To understand the effect of interspecies interactions, combinations of micro-predators—i.e., protists (generalists), predatory bacteria (semi-specialists), and phages (specialists)—and bacterial prey were tracked over a 72-h period in miniature membrane bioreactors. While specialist predators alone drove their preferred prey to extinction, the inclusion of a generalist resulted in uniform losses among prey species. Most importantly, presence of a generalist predator enabled coexistence of all predators and prey. As the generalist predator also negatively affected the other predators, we suggest that resource partitioning between predators and the constant availability of resources for bacterial growth due to protist predation stabilizes the system and keeps its diversity high. The appearance of resistant prey strains and subsequent evolution of specialist predators unable to infect the ancestral prey implies that multitrophic communities are able to persist and stabilize themselves. Interestingly, the appearance of BALOs and phages unable to infect their prey was only observed for the BALO or phage in the absence of additional predators or prey species indicating that competition between predators might influence coevolutionary dynamics.

Environmental surveys on soil viruses are still rare and mostly anecdotal, i. e., they mostly report on viruses at one location or for only a few sampling dates. Detailed time-series analysis with multiple samples can reveal the spatio-temporal dynamics of viral communities and provide important input as to how viruses interact with their potential hosts and the environment. Such surveys, however, require fast, easy-to-apply and reliable methods. In the present study we surveyed monthly across 13 months the abundance of virus-like particles (VLP) and the structure of the viral communities in soils along a land use transect (i.e., forest, pasture, and cropland). We evaluated 32 procedures to extract VLP from soil using different buffers and mechanical methods. The most efficient extraction was achieved with 1× saline magnesium buffer in combination with 20 min vortexing. For community structure analysis we developed an optimized fingerprinting approach (fluorescent RAPD-PCR; fRAPD) by combining RAPD-PCR with fluorescently labeled primers in order to size the obtained fragments on a capillary sequencing machine. With the concomitantly collected data of soil specific factors and weather data, we were able to find correlations of viral abundance and community structure with environmental variables and sampling site. More specifically, we found that soil specific factors such as pH and total nitrogen content played a significant role in shaping both soil viral abundance and community structure. The fRAPD analysis revealed high temporal changes and clustered the viral communities according to sampling sites. In particular we observed that temperature and rainfall shaped soil viral communities in non-forest sites. In summary our findings suggest that sampling site was a key factor for shaping the abundance and community structure of soil viruses, and when site vegetation was reduced, temperature and rainfall were also important factors.

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs·L−1·day−1 indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance.

Background: Mass cytometry (Cytometry by Time of Flight, CyTOF) allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry protocol was developed for the detection and quantification of silver in single bacterial cells of different physiological states. The silver quantities were generally heterogeneously distributed among cells in a population, the degree of which was dependent on micro-environmental conditions and on silver applied either in ion or nanoparticle-aggregated form.

Previously, Planted Fixed-Bed Reactors (PFRs) have been used to investigate microbial toluene removal in the rhizosphere of constructed wetlands. Aerobic toluene degradation was predominant in these model systems although bulk redox conditions were hypoxic to anoxic. However, culture-independent approaches indicated also that microbes capable of anaerobic toluene degradation were abundant. Therefore, we aimed at isolating anaerobic-toluene degraders from one of these PFRs. From the obtained colonies which consisted of spirilli-shaped bacteria, a strain designated 15-1 was selected for further investigations. Analysis of its 16S rRNA gene revealed greatest similarity (99%) with toluene-degrading Magnetospirillum sp. TS-6. Isolate 15-1 grew with up to 0.5 mM of toluene under nitrate-reducing conditions. Cells reacted to higher concentrations of toluene by an increase in the degree of saturation of their membrane fatty acids. Strain 15-1 contained key genes for the anaerobic degradation of toluene via benzylsuccinate and subsequently the benzoyl-CoA pathway, namely bssA, encoding for the alpha subunit of benzylsuccinate synthase, bcrC for subunit C of benzoyl-CoA reductase and bamA for 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase. Finally, most members of a clone library of bssA generated from the PFR had highest similarity to bssA from strain 15-1. Our study provides insights about the physiological capacities of a strain of Magnetospirillum isolated from a planted system where active rhizoremediation of toluene is taking place.

Trace elements play an essential role in all organisms due to their functions in enzyme complexes. In anaerobic digesters, control and supplementation of trace elements lead to stable and more efficient methane production processes while trace element deficits cause process imbalances. However, the underlying metabolic mechanisms and the adaptation of the affected microbial communities to such deficits are not yet fully understood. Here, we investigated the microbial community dynamics and resulting process changes induced by trace element deprivation. Two identical lab-scale continuous stirred tank reactors fed with distiller’s grains and supplemented with trace elements (cobalt, molybdenum, nickel, tungsten) and a commercial iron additive were operated in parallel. After 72 weeks of identical operation, the feeding regime of one reactor was changed by omitting trace element supplements and reducing the amount of iron additive. Both reactors were operated for further 21 weeks. Various process parameters (biogas production and composition, total solids and volatile solids, trace element concentration, volatile fatty acids, total ammonium nitrogen, total organic acids/alkalinity ratio, and pH) and the composition and activity of the microbial communities were monitored over the total experimental time. While the methane yield remained stable, the concentrations of hydrogen sulfide, total ammonia nitrogen, and acetate increased in the trace element-depleted reactor compared to the well-supplied control reactor. Methanosarcina and Methanoculleus dominated the methanogenic communities in both reactors. However, the activity ratio of these two genera was shown to depend on trace element supplementation explainable by different trace element requirements of their energy conservation systems. Methanosarcina dominated the well-supplied anaerobic digester, pointing to acetoclastic methanogenesis as the dominant methanogenic pathway. Under trace element deprivation, Methanoculleus and thus hydrogenotrophic methanogenesis was favored although Methanosarcina was not overgrown by Methanoculleus. Multivariate statistics revealed that the decline of nickel, cobalt, molybdenum, tungsten, and manganese most strongly influenced the balance of mcrA transcripts from both genera. Hydrogenotrophic methanogens seem to be favored under nickel- and cobalt-deficient conditions as their metabolism requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways. Thus, trace element supply is critical to sustain the activity of the versatile high-performance methanogen Methanosarcina.

Contaminant biodegradation in soils is hampered by the heterogeneous distribution of degrading communities colonizing isolated microenvironments as a result of the soil architecture. Over the last years, soil salinization was recognized as an additional problem especially in arid and semiarid ecosystems as it drastically reduces the activity and motility of bacteria. Here, we studied the importance of different spatial processes for benzoate biodegradation at an environmentally relevant range of osmotic potentials (ΔΨo) using model ecosystems exhibiting a heterogeneous distribution of the soil borne bacterium Pseudomonas putida KT2440. Three systematically manipulated scenarios allowed us to cover the effects of i) substrate diffusion ii) substrate diffusion and autonomous bacterial dispersal, and iii) substrate diffusion and autonomous as well as mediated bacterial dispersal along glass fiber networks mimicking fungal hyphae. To quantify the relative importance of the different spatial processes, we compared these heterogeneous scenarios to a reference value obtained for each ΔΨo by means of a quasi optimal scenario in which degraders were ab initio homogenously distributed. Substrate diffusion as the sole spatial process was insufficient to counteract the disadvantage due to spatial degrader heterogeneity at ΔΨo ranging from 0 to 1 MPa. In this scenario, only 13.8 to 21.3 % of the quasi-optimal biodegradation performance could be achieved. In the same range of ΔΨo values, substrate diffusion in combination with bacterial dispersal allowed between 68.6 % and 36.2 % of the performance showing a clear downwards trend with decreasing ΔΨo. At ¬ 1.5 MPa, however, this scenario performed worse than the diffusion scenario, possibly as a result of energetic disadvantages associated with flagellum synthesis and emerging requirements to exceed a critical population density to resist osmotic stress. Network mediated bacterial dispersal kept biodegradation almost consistently high with an average of 70.7 ± 7.8 %, regardless of the strength of the osmotic stress. We propose that especially fungal network mediated bacterial dispersal is a key process to achieve high functionality of heterogeneous microbial ecosystems also at reduced osmotic potentials. Thus, mechanical stress by, for example, soil homogenization should be kept low in order to preserve fungal network integrity.

Cattle manure is frequently used as an inoculum for the start-up of agricultural biogas plants or as a co-substrate in the anaerobic digestion of lignocellulosic feedstock. Ruminal microbiota are considered to be effective plant fiber degraders, but the microbes contained in manure do not necessarily reflect the rumen microbiome. The aim of this study was to compare the microbial community composition of cow rumen and manure with respect to plant fiber-digesting microbes. Bacterial and methanogenic communities of rumen and manure samples were examined by 454 amplicon sequencing of bacterial 16S rRNA genes and mcrA genes, respectively. Rumen fluid samples were dominated by Prevotellaceae (29%), whereas Ruminococcaceae was the most abundant family in the manure samples (31%). Fibrobacteraceae (12%) and Bacteroidaceae (13%) were the second most abundant families in rumen fluid and manure, respectively. The high abundances of fiber-degrading bacteria belonging to Prevotellaceae and Fibrobacteraceae might explain the better performance of anaerobic digesters inoculated with rumen fluid. Members of the genus Methanobrevibacter were the predominant methanogens in the rumen fluid, whereas methanogenic communities of the manure samples were dominated by the candidate genus Methanoplasma. Our results suggest that inoculation or bioaugmentation with fiber-digesting rumen microbiota can enhance the anaerobic digestion of lignocellulosic biomass.