In biotechnological processes, cell density and physiology are critical parameters for controlling the feed rate, harvest time, and process performance. We developed an automated flow cytometry approach that enables continuous, real-time (fully automated, hourly) monitoring of bacterial populations in continuous bioreactors. The method employed a double-staining protocol that combined DAPI to assess total DNA content and Alexa Fluor 488-EdU via Click-iT technology to identify the proportions of cells undergoing active DNA replication through EdU incorporation. The integrated workflow included fixation, permeabilization, staining, and measurement steps and was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. Automated analysis captured growth dynamics and cell cycle progression, providing insights into population behavior under different dilution rates. In this study, automated on-line sampling enabled hourly flow cytometry measurements of cell concentration and physiological indicators during continuous cultivation, supporting real-time monitoring and control in industrial biotechnology.

Soil, as the largest terrestrial carbon sink, plays a crucial role in carbon sequestration. Within soil systems, microorganisms decompose soil organic matter to generate energy and obtain carbon for growth, concomitantly release heat and CO2 as metabolic byproducts. The calorespirometric (CR) ratio-defined as the ratio of heat production to CO2 evolution, is a key indicator of carbon use efficiency and soil anaerobicity. However, conventional methodologies typically measure heat and CO2 separately, with CO2 often quantified by intermittent sampling. This discontinuous approach, compounded by the inherent heterogeneity of soil, introduces uncertainties in calorespirometric analysis. To address this limitation, an infrared CO2 sensor was mounted onto a stainless-steel calorimetric ampoule, containing soil-glucose mixtures, enabling simultaneous real-time measurements within an isothermal microcalorimeter. The novel configuration permits continuous monitoring of both parameters, validated through comparative analysis with traditional methods. The derived CR ratios aligned with theoretical predictions for carbohydrates metabolism. Furthermore, parallel oxygen measurements enabled quantification of CR ratio based on O2 (heat-to-O2), and the respiratory quotient (CO2-to-O2), offering deeper insight into microbial carbon-energy coupling and turnover in soil systems. This methodological advancement enhances the capacity to interrogate soil biogeochemical processes under dynamic environmental conditions.

Soil, as the largest terrestrial carbon sink, plays a pivotal role in the global carbon cycle. Soil microorganisms are fundamental to all biochemical processes in soil, ensuring its fertility and supporting a balanced ecosystem. Through their metabolic activities, these microorganisms drive energy and matter flows, mineralizing organic matter and releasing heat and CO2, which can be measured via calorespirometry. A key limitation of conventional calorimeters lies in their inability to combine high sample throughput with sufficiently large sample sizes while avoiding oxygen limitation during measurement. In order to overcome these weaknesses, we have developed a multi-channel macrocalorespirometer (CR-12) for soil analysis. To demonstrate its application, agricultural soil (Dikopshof, Luvisol) amended with 12C (unlabeled) and 13C (labeled) glucose was used in four experiments. Comparisons with commercial isothermal microcalorimeters confirmed the suitability of CR-12 for soil systems, providing reliable heat, CO2 measurements and calorespirometric ratios that align with known ranges for the aerobic turnover of carbohydrates. To further investigate the incorporation of carbon into the soil organic matter (SOM), a time series of soil samples amended with 13C-labeled glucose was subjected to mass spectrometric analysis (m/z 44 for 12C-CO2; m/z 45 for 13C-CO2) using thermogravimetry-differential scanning calorimetry-quadrupole mass spectrometry (TG-DSC-QMS). The integration of calorespirometric and mass spectrometric data demonstrated that combining these complementary techniques provides more detailed information on the fate of microbial carbon and energy turnover within SOM.

Lignocellulose is a promising renewable resource for anaerobic biochemical production, but its microbial conversion remains challenging. To elucidate metabolic networks in lignocellulose-degrading consortia, inocula of various origins were enriched on cellulose or xylan. Community composition and metabolic functions were revealed by amplicon sequencing, metagenomics, genome-scale metabolic modelling, and metabolic simulations. In cellulose-enriched communities, Fibrobacter and Lacrimispora consistently dominated as primary cellulose degraders, whereas Bacteroides likely functioned as secondary degraders. Acetic acid (up to 1.3 g l-1) and CO2 were the main fermentation products. Xylan enrichments produced C2-C6 fatty acids (up to 3.9 g l-1), lactic acid (up to 1.2 g l-1), ethanol (up to 1.2 g l-1), CO2, and H2. Clostridium dominated one xylan community and produced mainly butyric acid, while Bifidobacterium dominated another and produced mainly lactic acid. Caproic acid production was experimentally observed in one xylan enrichment. Metagenomic annotations and metabolic simulations suggest that Lacrimispora amygdalina degraded xylan and Robinsoniella peoriensis consumed xylobiose as a secondary consumer, both likely producing ethanol and lactic acid that supported caproic and butyric acid production by Caproicibacter fermentans. Integrated analysis identified functional guilds and clarified the roles of degraders and non-degraders, providing a blueprint for engineering synthetic consortia for sustainable biochemical production.

Phages are ubiquitous in soil, shaping microbial diversity and nutrient cycling. Phage replication requires maintaining infectivity and finding the right host. Yet, there are limited data on phage persistence and transport in soil under field conditions. The potential presence of hosts enabling phage replication impedes the assessment of the mobility of autochthonous phages in soils. In lysimeters installed in forest and pasture topsoil, we elucidated the transport of the tailed marine Pseudoalteromonas phage HS2 in comparison to deuterium. Transport of infectious phages as well as numbers of tracer phage genomes and tracer capsid-bound genomes were quantified to account for phage retention and inactivation. Phages were transported up to 4 times faster than the simultaneously applied deuterium tracer, which was attributed to pore size exclusion. Retention in immobile regions and remobilization during precipitation caused pronounced tailing in tracer breakthroughs. High phage survival in pasture soil resulted in mass recoveries of infectious phages that were up to 6 times higher than those in forest soil. However, long-term observations showed that the infectivity was also preserved in forest soil, enabling event-driven remobilization. This remobilization underscores the importance of distinguishing between phage retention and inactivation, which is crucial for accurately predicting phage transport dynamics and their ecological impact in terrestrial environments.

Soil, as the largest terrestrial carbon sink, plays a crucial role in carbon sequestration. Within soil systems, microorganisms decompose soil organic matter to generate energy and obtain carbon for growth, concomitantly release heat and CO2 as metabolic byproducts. The calorespirometric (CR) ratio – defined as the ratio of heat production to CO2 evolution, is a key indicator of carbon use efficiency and soil anaerobicity. However, conventional methodologies typically measure heat and CO2 separately, with CO2 often quantified by intermittent sampling. This discontinuous approach, compounded by the inherent heterogeneity of soil, introduces uncertainties in calorespirometric analysis. To address this limitation, an infrared CO2 sensor was mounted onto a stainless-steel calorimetric ampoule, containing soil-glucose mixtures, enabling simultaneous real-time measurements within an isothermal microcalorimeter. The novel configuration permits continuous monitoring of both parameters, validated through comparative analysis with traditional methods. The derived CR ratio aligned with theoretical predictions for carbohydrates metabolism. Furthermore, parallel oxygen measurements enabled quantification of CR ratio based on O2 (heat-to-O2), and the respiratory quotient (CO2-to-O2)), offering deeper insight into microbial carbon-energy coupling and turnover in soil systems. This methodological advancement enhances the capacity to interrogate soil biogeochemical processes under dynamic environmental conditions.

Soil is one of the most important natural carbon sinks, alongside its many other critical functions. The ratio of carbon incorporated into the soil system for instance as biomass as a proportion of carbon consumption from a given substrate is defined as carbon use efficiency (CUE). CUE is assumed to depend on environmental conditions. However, the conservation of energy within the soil system, quantified as energy use efficiency (EUE), has not yet been thoroughly studied related to CUE. To study the effects of environmental conditions on cellulose degradation in the absence of soil organic matter background, we utilized artificial soil to analyse their impact on CUE and EUE. We quantified CO2 evolution rate, heat production rate, and cellulose degradation, at three water contents (10%, 14.4% and 19%), two C/N ratios (9 and 18), and two temperatures (7 degrees C and 20 degrees C). Environmental conditions significantly influenced the primary parameters such as CO2 evolution, heat production, and cellulose degradation rate while the derived parameters CUE and in particular EUE were less sensitive. Among the studied factors, water content had the least influence on ultimate CUE and EUE. Conversely, decreases in nitrogen supply and temperature tended to increase CUE without significantly affecting EUE, which is generally higher than CUE. Over the degradation process, CUE often started from high values > 90% and then decreased to lower ones around 50%, whereas the EUE remained more constant on a higher level. The variations of CUE and EUE suggest alterations in the stoichiometry of the microbial growth reaction and thus add additional factors (e. g. time lapse of the degradation process) to be considered for their assessment.

In industrial biotechnology, cell density is a critical parameter that influences key process control variables such as feed rate, harvest timing, and product recovery. Traditional biomass measurements are indirect, incapable of quantifying population heterogeneity, and often prone to inaccuracies due to manual handling and interference from culture media compounds. This study introduces an automated flow cytometry approach to enable continuous, real-time monitoring of bacterial cultures in a continuous bioreactor. Our method employs a double-staining protocol that combines DAPI for assessing total DNA content with Alexa Fluor 488 via Click-iT technology to detect the percentage of cells with active DNA replication through EdU incorporation. This streamlined protocol, which integrates cell fixation, permeabilization, staining, and measurement, was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. The approach successfully captured both growth dynamics and cell cycle progression, providing rapid and quantitative insights into culture composition. The enhanced temporal resolution achieved by this method facilitates timely adjustments of process parameters, ultimately improving bioprocess efficiency and product quality. These results underscore the potential of automated flow cytometry as a powerful tool for real-time monitoring and control in industrial biotechnology. ### Competing Interest Statement The authors have declared no competing interest.

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 <10%) were recovered, covering 68 phyla and 279 genera. MAGs were dereplicated into 1,204 genome operational taxonomic units (gOTUs) as a proxy for species ( average nucleotide identity >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.

Biofiltration is a simple and low-cost method for the cleanup of contaminated water. However, the reduced availability of dissolved chemicals to surface-attached degrader bacteria may limit its efficient use at certain hydraulic loadings. When a direct current (DC) electric field is applied to an immersed packed bed, it invokes electrokinetic processes, such as electroosmotic water flow (EOF). EOF is a surface-charge-induced plug-flow-shaped movement of pore fluids. It acts at a nanometer distance above surfaces and allows the change of microscale pressure-driven flow profiles and, hence, the availability of dissolved contaminants to microbial degraders. In laboratory percolation columns, we assessed the effects of a weak DC electric field (E = 0.5 Vcm(-1)) on the biodegradation of waterborne naphthalene (NAH) by surface-attached Pseudomonas fluorescens LP6a. To vary NAH bioavailability, we used different NAH concentrations (C-0 = 2.7, 5.1, or 7.8 x 10(-5) molL-1) and Darcy velocities typical for biofiltration (U = 0.2-1.2 x 10(-4) ms(-1)). In DC-free controls, we observed higher specific degradation rates (q(c)) at higher NAH concentrations. The q(c) depended on U, suggesting bioavailability restrictions depending on the hydraulic residence times. DC fields consistently increased q(c) and resulted in linearly increasing benefits up to 55% with rising hydraulic loadings relative to controls. We explain these biodegradation benefits by EOF-altered microscale flow profiles allowing for better NAH provision to bacteria attached to the collectors even though the EOF was calculated to be 100-800 times smaller than bulk water flow. Our data suggest that electrokinetic approaches may give rise to future technical applications that allow regulating biodegradation, for example, in response to fluctuating hydraulic loadings.

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.

This study aimed to examine the bacterial, methanogenic archaeal, and eukaryotic community structure in both the midgut and hindgut of Pachnoda marginata larvae using an amplicon sequencing approach. The goal was to investigate how various diets and the soil affect the composition of these three-domain microbial communities within the gut of insect larvae. The results indicated a notable variation in the microbial community composition among the gut compartments. The majority of the bacterial community in the hindgut was composed of Ruminococcaceae and Christensenellaceae. Nocardiaceae, Microbacteriaceae, and Lachnospiraceae were detected in midgut samples from larvae feeding on the leaf diet, whereas Sphingomonadaceae, Rhodobacteraceae, and Promicromonasporaceae dominated the bacterial community of midgut of larvae feeding on the straw diet. The diet was a significant factor that influenced the methanogenic archaeal and eukaryotic community patterns. The methanogenic communities in the two gut compartments significantly differed from each other, with the midgut communities being more similar to those in the soil. A higher diversity of methanogens was observed in the midgut samples of both diets compared to the hindgut. Overall, the microbiota of the hindgut was more host-specific, while the assembly of the midgut was more influenced by the environmental microorganisms.

The present study aimed to investigate whether and how non-invasive biocalorimetric measurements could serve for process monitoring of fungal pretreatment during solid-state fermentation (SSF) of lignocellulosic agricultural residues such as wheat straw. Seven filamentous fungi representing different lignocellulose decay types were employed. Water-soluble sugars being immediately available after fungal pretreatment and those becoming water-extractable after enzymatic digestion of pretreated wheat straw with hydrolysing (hemi)cellulases were considered to constitute the total bioaccessible sugar fraction. The latter was used to indicate the success of pretreatments and linked to corresponding species-specific metabolic heat yield coefficients (YQ/X) derived from metabolic heat flux measurements during fungal wheat straw colonisation. An YQ/X range of about 120 to 140 kJ/g was seemingly optimal for pretreatment upon consideration of all investigated fungi and application of a non-linear Gaussian fitting model. Upon exclusion from analysis of the brown-rot basidiomycete Gloeophyllum trabeum, which differs from all other here investigated fungi in employing extracellular Fenton chemistry for lignocellulose decomposition, a linear relationship where amounts of total bioaccessible sugars were suggested to increase with increasing YQ/X values was obtained. It remains to be elucidated whether an YQ/X range being optimal for fungal pretreatment could firmly be established, or if the sugar accessibility for post-treatment generally increases with increasing YQ/X values as long as “conventional” enzymatic, i.e. (hemi)cellulase-based, lignocellulose decomposition mechanisms are operative. In any case, metabolic heat measurement–derived parameters such as YQ/X values may become very valuable tools supporting the assessment of the suitability of different fungal species for pretreatment of lignocellulosic substrates. • Biocalorimetry was used to monitor wheat straw pretreatment with seven filamentous fungi. • Metabolic heat yield coefficients (YQ/X) seem to indicate pretreatment success. • YQ/X values may support the selection of suitable fungal strains for pretreatment.

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.

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.

Wastewater-based monitoring of SARS-CoV-2 has become a promising and useful tool in tracking the potential spread or dynamics of the virus. Its recording can be used to predict how the potential number of infections in a population will develop. Recent studies have shown that the use of passive samplers is also suitable for the detection of SARS-CoV-2 genome copies (GC) in wastewater. They can be used at any site, provide timely data and may collect SARS-CoV-2 GC missed by traditional sampling methods. Therefore, the aim of this study was to evaluate the suitability of passive samplers for the detection of SARS-CoV-2 GC in wastewater in the long-term at two different scales. Polyethylene-based plastic passive samplers were deployed at the city-scale level of Leipzig at 13 different locations, with samples being taken from March 2021 to August 2022. At the smaller city district level, three types of passive samplers (cotton-cloth, unravelled polypropylene plastic rope and polyethylene-based plastic strips) were used and sampled on a weekly basis from March to August 2022. The results are discussed in relation to wastewater samples taken at the individual passive sampling point. Our results show that passive samplers can indicate at a city-scale level an accurate level of positive infections in the population (positiverate: 86 %). On a small-scale level, the use of passive samplers was also feasible and effective to detect SARS-CoV-2 GC easily and cost-effectively, mirroring a similar trend to that at a city-scale level. Thus, this study demonstrated that passive samplers provide reproducible SARS-CoV-2 GC signals from wastewater and a time-integrated measurement of the sampled matrix with greater sensitivity compared to wastewater. We thus recommend the use of passive samplers as an alternative method for wastewater-based epidemiology. Passive samplers can in particular be considered for a better estimation of infections compared to incidence levels.

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.

For anaerobic mixed cultures performing microbial chain elongation, it is unclear how pH alterations affect the abundance of key players, microbial interactions, and community functioning in terms of medium-chain carboxylate yields. We explored pH effects on mixed cultures enriched in continuous anaerobic bioreactors representing closed model ecosystems. Gradual pH increase from 5.5 to 6.5 induced dramatic shifts in community composition, whereas product range and yields returned to previous states after transient fluctuations. To understand community responses to pH perturbations over long-term reactor operation, we applied Aitchison PCA clustering, linear mixed-effects models, and random forest classification on 16S rRNA gene amplicon sequencing and process data. Different pH preferences of two key chain elongation species─one Clostridium IV species related to Ruminococcaceae bacterium CPB6 and one Clostridium sensu stricto species related to Clostridium luticellarii─were determined. Network analysis revealed positive correlations of Clostridium IV with lactic acid bacteria, which switched from Olsenella to Lactobacillus along the pH increase, illustrating the plasticity of the food web in chain elongation communities. Despite long-term cultivation in closed systems over the pH shift experiment, the communities retained functional redundancy in fermentation pathways, reflected by the emergence of rare species and concomitant recovery of chain elongation functions.

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.