Biology subjects

Molitor, B.

Publications and source records attributed to Molitor, B..

14 recordsLinked to original sources

bioRxiv2026

Tandem repeats in methanogenic archaea and their viruses reveal a shared potential for rapid genomic diversification

Methanogenic archaea drive global methane emissions, yet the molecular basis of their physiological flexibility is unclear. Archaeal adaptation is typically attributed to horizontal gene transfer, which is episodic and cannot explain rapid, reversible change. Here we show that direct tandem repeats (TRs) offer an intrinsic alternative. By screening 50,567 prokaryotic genomes, we find TR densities in Methanosarcina more than 30-fold above the prokaryotic average, paralleling Borg extrachromosomal elements. These TRs recur at conserved loci while sharing almost no sequence identity between strains, indicating independent origin, and evolve during routine cultivation. Methanogen TRs are transcribed, show ribosome occupancy, and lie near regulatory and cofactor-related genes, whilst being depleted around core energy-conserving and translational machinery. Genic TRs generate intrinsically disordered regions in proteins carrying nucleotide-binding, protein-protein interaction and cell-surface domains, and occur in structural and surface proteins of methanogen-infecting viruses. TRs are therefore an overlooked source of variability and adaptation potential in methanogens and their viruses.

microbiology↗

bioRxiv2026

Virus {Psi}M2 enters a non-lytic lifecycle alternative to lysogeny during infection of Methanothermobacter marburgensis

Methanogens, a diverse group of archaea that produce methane during energy metabolism, are prevalent in anoxic habitats where they are exposed to viral infections. The head-tailed virus {Psi}M2 is one of few virus isolates that infect a methanogen host and was described as a lytic virus that lyses its host, Methanothermobacter marburgensis. Here, we present evidence that {Psi}M2 does not exclusively follow the classic lytic lifecycle but can also enter an alternative, chronic infection-like state. Growth experiments showed that infected cultures in minimal medium, optimized for the methanogen host (MS medium) did not exhibit complete lysis but continuously released infective virions. In contrast, in a suboptimal complex medium (SAB medium), the {Psi}M2-infected culture released virions in the exponential growth phase and eventually lysed after 14 hours post infection. Although the genome of {Psi}M2 encodes a putative integrase, a prerequisite for lysogeny, whole genome sequencing of {Psi}M2-infected M. marburgensis cultures revealed that the viral DNA remained extrachromosomal rather than integrating into the host genome, likely due to a mutation in the integrase gene. These findings suggest that {Psi}M2 enters a non-lytic lifecycle alternative to lysogeny under suboptimal host growth conditions. Sequencing further identified a heterogeneous viral population including previously undescribed variants. A high density of non-synonymous mutations and frameshift-inducing insertions was observed within ORFs predicted to encode a capsid protein and a major spike protein, suggesting these viral variants to differ in host recognition and infectivity. Overall, our results show more complex viral behavior and greater genetic variability than previously described.

microbiology↗

bioRxiv2026

Establishing the fluorescence-activating and absorption-shifting tag as a fluorescent reporter protein in Methanothermobacter thermautotrophicus ΔH

The thermophilic methanogen Methanothermobacter thermautotrophicus {Delta}H is a model microbe for hydrogenotrophic methanogenesis and an emerging platform host for metabolic engineering. Despite recent advances in its genetic accessibility, the available molecular toolbox lacks fluorescent reporter proteins that are suitable for anaerobic and thermophilic conditions. Here, we established the fluorescence-activating and absorption-shifting tag (FAST) as a reporter protein in M. thermautotrophicus. We expressed codon-optimized variants of FAST by applying established genetic tools, and evaluated the performance for two temperatures and three fluorogens. We demonstrated that FAST is functional in M. thermautotrophicus but exhibits temperature-dependent instability, which is more pronounced at 60{degrees}C compared to 50{degrees}C. Among the tested fluorogens, TFLime and TFAmber yielded comparable fluorescence intensities, while TFCoral resulted in significantly lower fluorescence intensity. Exploiting the partial thermolability of FAST, we characterized the dynamic expression profiles of several promoters, which revealed growth phase-dependent regulation patterns. Our findings challenge previous assumptions of constitutive expression for several promoters. Notably, we identified distinct expression patterns for promoters that are associated with methanogenesis and energy-converting hydrogenases. Our results establish FAST as a versatile fluorescent reporter for thermophilic methanogens and provide new insights into promoter regulation in M. thermautotrophicus. This work expands the genetic toolbox for this microbe and lays the foundation for advanced studies in archaeal cell biology and biotechnology.

microbiology↗

bioRxiv2026

A host-directed virulence factor of Clostridium perfringens is modulated by gut commensal strains

In a healthy host, the residential microbes help regulate the growth of pathobionts, which are common members of the human gut microbiome, preventing them from causing diseases, including infections, under certain conditions. In cases of dysbiosis, this protection may be compromised. Targeted microbiome modulation offers a promising approach to restore healthy conditions in a disrupted community and consequently prevent infections using the natural colonization resistance of the microbiome. Elucidating the interaction mechanisms between microbial species within a microbiome is crucial for understanding how a microbiome can be modulated precisely and effectively to benefit the hosts well-being. Here, we investigated the interactions between the pathobiont C. perfringens and human gut commensals on physiological and molecular levels. We found that commensal strains affect C. perfringens growth by competing for substrates such as amino acids or a carbon source other than glucose. We further observed that Bacteroidaceae strains altered the levels of C. perfringens proteins, among others, the host-directed {theta}-toxin. Our findings reinforce the notion that modulating the composition of the gut microbiome is an effective strategy to prevent infections.

microbiology↗

bioRxiv2026

Towards complete carbon utilization: Improved methane yield from formate and hydrogen co-feeding through constitutive formate dehydrogenase-gene expression in Methanothermobacter thermautotrophicus ΔH

Formate is emerging as an important molecule in carbon capture and utilization technologies. However, its low electron density makes formate less attractive for energy storage. Some hydrogenotrophic methanogens can reduce formate to methane, thereby upgrading it into an established energy carrier. The bottleneck in this process is that 75% of the carbon is lost as carbon dioxide, and achieving a complete formate-to-methane conversion requires co-feeding hydrogen. However, hydrogen-dependent genetic regulation of formate metabolism inhibits simultaneous formate and hydrogen utilization in hydrogenotrophic methanogens. Here, we compared the catalytic performance of the genetically modified strain Methanothermobacter thermautotrophicus {Delta}H (pFdh) with M. thermautotrophicus Z-245 by conducting continuous cultivation at different hydrogen concentrations. While M. thermautotrophicus Z-245 is a natural formatotroph, M. thermautotrophicus {Delta}H (pFdh) was engineered to enable formate utilization via episomal expression of a formate dehydrogenase-gene cassette. We found that M. thermautotrophicus {Delta}H (pFdh) can simultaneously utilize formate and hydrogen. It continuously consumed formate at {approx} 0.1 mM dissolved hydrogen, enabling a 75.6% formate-to-methane conversion efficiency. M. thermautotrophicus Z-245 showed a declining formate consumption at {approx} 0.016 mM and only reached a maximum stable efficiency of 36.3%. These results suggest that M. thermautotrophicus {Delta}H (pFdh) is largely insensitive to hydrogen-induced genetic regulation; however, it still faces redox-related metabolic limitations at dissolved hydrogen concentrations above 0.4 mM. Overall, the findings reveal a potential strategy to circumvent hydrogen-induced regulation of formate metabolism and identify M. thermautotrophicus {Delta}H (pFdh) as a promising biocatalyst for formate-to-methane conversion.

microbiology↗

bioRxiv2025

Metabolic engineering of Methanothermobacter thermautotrophicus ΔH for recombinant acetoin production

Thermophilic methanogens of the genus Methanothermobacter are established biocatalysts in power-to-gas applications, converting H2 and CO2 into CH4 through the process of methanogenesis. Further expanding this platform for the bioproduction of value-added compounds (power-to-x) has the potential to increase the economic viability of such processes. This requires a genetic toolset that enables the controlled expression of recombinant pathways. Here, we report the fully autotrophic inducible recombinant bioproduction of acetoin from H2 and CO2 in Methanothermobacter thermautotrophicus {Delta}H. To facilitate inducible gene expression, we implemented an anhydrotetracycline (aTc)-inducible promoter system, expanding our available set of promoters. The aTc-inducible system enabled controlled expression of a codon-optimized acetoin-production operon comprising the acetolactate synthase- and acetolactate decarboxylase-encoding genes from Streptococcus thermophilus. Batch cultivation at 42{degrees}C demonstrated aTc-dependent acetoin formation, yielding up to 0.45 {+/-} 0.08 mM acetoin. Fed-batch bioreactor experiments confirmed growth-coupled, recombinant acetoin production, while eliminating the non-specific acetoin accumulation that we observed during non-growth phases in batch cultivation. Continuous cultivation in a chemostat resulted in stable acetoin production rates of 1.28 {+/-} 0.07 {micro}mol L-{superscript 1} h-{superscript 1} at 42{degrees}C. Elevated temperatures led to reduced acetoin production, suggesting diminished activity or thermal instability of the heterologous enzymes. This study demonstrates the feasibility of value-added bioproduction in Methanothermobacter and establishes an inducible expression system suitable for pathway engineering in thermophilic methanogens. Together with genome-scale modeling and emerging enzyme engineering strategies, these results lay the foundation for developing robust, CH4-co-producing power-to-x bioprocesses with Methanothermobacter species.

microbiology↗

bioRxiv2024

The deletion of aldehyde:ferredoxin oxidoreductase-encoding genes in Clostridium ljungdahlii results in changes in the product spectrum with various carbon sources

Biofuels, such as ethanol, can be produced by the microbial fermentation of waste gases that contain carbon dioxide (CO2) and carbon monoxide (CO). The acetogenic model microbe Clostridium ljungdahlii converts those substrates into acetyl-CoA with the Wood-Ljungdahl pathway. During autotrophic conditions, acetyl-CoA can be reduced further to ethanol via acetic acid by the enzymes aldehyde:ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase. Here, the genes encoding both tungsten-dependent AORs (aor1, CLJU_c20110 and aor2, CLJU_c20210) were deleted from the genome of C. ljungdahlii. Ethanol formation was enhanced for C. ljungdahlii {Delta}aor1 with different carbon sources, that is, fructose, a mixture of hydrogen (H2) and CO2, and CO. The highest and lowest ethanol:acetic acid ratio was detected during growth with H2/CO2 and CO, respectively. Oscillating patterns were observed during growth with CO, underpinning the importance of a balanced redox metabolism.

microbiology↗

bioRxiv2024

Proton-pump inhibitors increase C. difficile infection risk by altering pH rather than by affecting the gut microbiome based on a bioreactor model

Clostridioides difficile infections often occur after antibiotic use, but they have also been linked to proton-pump inhibitor (PPI) therapy. The underlying mechanism--whether infection risk is due to a direct effect of PPIs on the gut microbiome or changes in gastrointestinal pH--has remained unclear. To disentangle both possibilities, we studied the impact of the proton-pump inhibitor omeprazole and pH changes on key members of the human gut microbiome and stool-derived microbial communities from different donors in vitro. We then developed a custom multiple-bioreactor system to grow a model human microbiome community in chemostat mode and tested the effects of omeprazole exposure, pH changes, and their combination on C. difficile growth within this community. Our findings show that changes in pH significantly affect the gut microbial communitys biomass and the abundances of different strains, leading to increased C. difficile growth within the community. However, omeprazole treatment alone did not result in such effects. These findings imply that the higher risk of C. difficile infection following proton-pump inhibitor therapy is probably because of alterations in gastrointestinal pH rather than a direct interaction between the drug and the microbiome. This understanding paves the way for reducing infection risks in proton-pump inhibitor therapy.

microbiology↗

bioRxiv2022

An integrated systems-biology platform for power-to-gas technology

Methanogenesis allows methanogenic archaea (methanogens) to generate cellular energy for their growth while producing methane. Hydrogenotrophic methanogens thrive on carbon dioxide and molecular hydrogen as sole carbon and energy sources. Thermophilic and hydrogenotrophic Methanothermobacter spp. have been recognized as robust biocatalysts for a circular carbon economy and are now applied in power-to-gas technology. Here, we generated the first manually curated genome-scale metabolic reconstruction for three Methanothermobacter spp.. We investigated differences in the growth performance of three wild-type strains and one genetically engineered strain in two independent chemostat bioreactor experiments. In the first experiment, with molecular hydrogen and carbon dioxide, we found the highest methane production rate for Methanothermobacter thermautotrophicus {Delta}H, while Methanothermobacter marburgensis Marburg reached the highest biomass growth rate. Systems biology investigations, including implementing a pan-model that contains combined reactions from all three microbes, allowed us to perform an interspecies comparison. This comparison enabled us to identify crucial differences in formate anabolism. In the second experiment, with sodium formate, we found stable growth with an M. thermautotrophicus {Delta}H plasmid-carrying strain with similar performance parameters compared to wild-type Methanothermobacter thermautotrophicus Z-245. Our findings reveal that formate anabolism influences the diversion of carbon to biomass and methane with implications for biotechnological applications of Methanothermobacter spp. in power-to-gas technology and for chemical production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/522236v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d468aeorg.highwire.dtl.DTLVardef@24a595org.highwire.dtl.DTLVardef@1f2dd60org.highwire.dtl.DTLVardef@1a3d8fc_HPS_FORMAT_FIGEXP M_FIG C_FIG Broader contextRenewable energy sources (e.g., wind and solar) provide carbon-free electric power. However, their intermittency and offset between peak production and demand generate the need to store this electric power. Furthermore, these technologies alone do not satisfy the demand for carbon-based commodities. Power-to-gas technology provides a means to store intermittent renewable electric power with concomitant carbon dioxide recycling into a chemical energy carrier, such as methane, on a centralized and decentralized scale. This is particularly important to establish equitable energy strategies for all countries, as is highlighted by the United Nations Sustainable Development Goals. With this work, we provide an integrated systems-biology platform for Methanothermobacter spp. to optimize biological power-to-gas technology and formulate strategies to produce other value-added products besides methane.

microbiology↗

bioRxiv2022

The targeted deletion of genes responsible for expression of the Mth60 fimbriae leads to loss of cell-cell connections in M. thermautotrophicus ΔH

This study was continued by the Environmental Biotechnology Group of the University of Tubingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is important to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus {Delta}H are one of only few known archaeal cell appendages that do not assemble via the type-IV assembly mechanism. Here, we report the constitutive expression of Mth60 fimbriae-encoding genes from a shuttle-vector construct, as well as the deletion of the Mth60 fimbriae-encoding genes from the genomic DNA of M. thermautotrophicus {Delta}H. We expanded our system for genetic modification of M. thermautotrophicus {Delta}H by an allelic-exchange method. While overexpression of the respective genes resulted in an increase of the Mth60 fimbriae, deletion of the Mth60 fimbriae-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus {Delta}H. This either increased or decreased number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus {Delta}H strains compared to the wild-type strain. Originality-Significance StatementMethanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, due to the lack of genetic tools, the detailed investigation of certain aspects, such as regulatory processes, was not possible. Here, we amend our genetic toolbox for M. thermautotrophicus {Delta}H with an allelic exchange method. We report the deletion of genes that encode for the Mth60 fimbriae. Our findings provide a first insight into the regulation of the expression of these genes and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus {Delta}H.

microbiology↗

bioRxiv2021

Genetic evidence reveals the indispensable role of the rseC gene for autotrophy and the importance of a functional electron balance for nitrate reduction in Clostridium ljungdahlii

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: 1) the RNF complex-encoding gene cluster rnfCDGEAB; 2) the putative RNF regulator gene rseC; and 3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy and contribute to understand the connection of nitrate reduction to energy metabolism. Significance StatementAcetogenic bacteria are widely known for their ability to convert gaseous substrates, such as hydrogen, carbon dioxide, and carbon monoxide, into short-chain fatty acids and alcohols, which can be utilized as sustainable platform chemicals and fuels. However, acetogenic bacteria conserve energy at the thermodynamic limit of life during autotrophy, and thus the production of more complex and energy-dense chemicals is limited due to low ATP yields. Therefore, it is key to decipher the interplay of the electron balancing reactions to understand and optimize the acetogenic metabolism. Recent findings with alternative electron acceptors that accelerated the cellular growth and ATP yield during autotrophy, such as nitrate, provide an opportunity to overcome energetic barriers in the acetogenic metabolism. The interrogation of the nitrate metabolism and the interplay between nitrate reduction and energy conservation in C. ljungdahlii, will contribute to fine-tuning of the acetogenic metabolism for biotechnological applications.

microbiology↗

bioRxiv2021

A shuttle-vector system allows heterologous gene expression in the thermophilic methanogen Methanothermobacter thermautotrophicus ΔH

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis), because of their short doubling times and robust growth with high growth yields. Yet, a genetic system for these model microbes was missing despite intense work for four decades. Here, we report the establishment of tools for genetic modification of M. thermautotrophicus. We developed the modular Methanothermobacter vector system, which provided shuttle-vector plasmids (pMVS) with exchangeable selectable markers and replicons for both Escherichia coli and M. thermautotrophicus. For M. thermautotrophicus, a thermostable neomycin-resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The pMVS-plasmid DNA was transferred from E. coli into M. thermautotrophicus via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus, we demonstrated heterologous gene expression of a thermostable {beta}-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assays, we found significantly different {beta}-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus on formate as the sole growth substrate, while this was not possible for the empty vector control. These genetic tools provide the basis to investigate hypotheses from four decades of research on the physiology and biochemistry of Methanothermobacter spp. on a genetic level. Significance StatementThe world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production, but also for production of high-value platform chemicals in power-to-x processes.

microbiology↗

bioRxiv2020

Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

synthetic biology↗

bioRxiv2019

An open-source multiple-bioreactor system for replicable gas-fermentation experiments: Nitrate feed results in stochastic inhibition events, but improves ethanol production of Clostridium ljungdahlii with CO2 and H2

The pH-value in fermentation broth has a large impact on the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiment. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be easily achieved in commercially available bioreactors. However, their acquisition is costly and their operation is time consuming, and therefore the experiment is often limited to a single bioreactor run. Here, we present a self-built, relatively cheap, and easy to handle open-source multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. The functionality of the MBS was tested in three experiments by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments were addressing the questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; (2) does feeding acetate alter the production rate of ethanol; and (3) does feeding nitrate influence the product spectrum under controlled pH conditions with CO2 and H2? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. Our data show high reproducibility for gas-fermentation experiments with C. ljungdahlii, using the MBS. We found that feeding acetate did not improve ethanol production, but rather impaired growth and reduced acetate production. Using nitrate as sole N-source, on the other hand, enhanced biomass production even at a low pH. However, we observed differences in growth, acetate, and ethanol production rates between triplicate bioreactors (n=3). We explained the different performances because of stochastic inhibition events, which we observed through the accumulation of nitrite, and which led to complete crashes at different operating times. One of these bioreactors recovered after the crash and showed enhanced ethanol production rates while simultaneously producing less acetate. The MBS offers a great opportunity to perform bench-scale bioreactor experiments at steady-state conditions with replicates, which is especially attractive for academia.

microbiology↗