Biology subjects

Angenent, L. T.

Publications and source records attributed to Angenent, L. T..

At least 19 recordsLinked to original sources

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

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↗

bioRxiv2026

Development of a continuous bioreactor to maintain stable nasal microbiomes from swab specimens and synthetic communities

BackgroundThe nasal microbiome is a collection of diverse microbial populations that inhabit the nose. Staphylococcus aureus is the most common opportunistic pathogen that colonizes the nasal mucosa, increasing the risk of invasive infections in immunocompromised and hospitalized patients. Clinicians usually prescribe antibiotics to decolonize the nasal cavities of at-risk patients from S. aureus. However, their broad antimicrobial activity can damage the resident nasal microbiome. Instead, naturally occurring compounds or resident bacteria in nasal microbiomes can effectively and safely exclude S. aureus from the nose. Cell culture and animal models have been used for nasal microbiome studies. However, their unstable microbiomes reduce the accuracy and reliability of the results. Recently, continuous bioreactors have been proposed as alternatives to these models. ResultsWe designed and operated a continuous bioreactor system to maintain stable nasal microbiomes. Next, we inoculated the bioreactor with nasal-swab specimens that we had collected from healthy volunteers. We operated the bioreactors under varying conditions (i.e., operating mode, dilution rate, temperature, pH, and medium composition), and determined the optimal conditions (continuous mode, 1 d-1, 30xlink:href=" pH 6.5, and synthetic nasal medium 3), resulting in stable microbiomes consisting of the main nasal bacterial species. The nasal microbiomes in the optimized bioreactors showed high reproducibility and resilience during a pH perturbation. Moreover, all microbiomes in the bioreactor, which were inoculated with six different nasal-swab specimens, maintained stable bacterial and metabolite compositions. In addition, we applied a synthetic microbial community (SynCom), which was derived from one of the volunteers, to demonstrate a S. aureus decolonization strategy. The bioreactor, inoculated with this SynCom, maintained a stable nasal microbiome for more than one month. Finally, different S. aureus strains that we inoculated in the SynCom showed distinct growth patterns within the otherwise stable community. ConclusionsThe continuous bioreactor enables the cultivation of stable nasal microbiomes for longer than one month by mimicking the environmental conditions of the human nose. The bioreactor is a valuable model for understanding the functions of the nasal microbiome and devising new decolonization strategies against S. aureus.

bioengineering↗

bioRxiv2026

Community composition and strain identity drive metabolic competition and Staphylococcus aureus colonization resistance in Synthetic Nasal Communities

The human nasal microbiome is a low-diversity ecosystem whose assembly principles and mechanisms of colonization resistance remain poorly understood. Staphylococcus aureus is a member of the nasal microbiome of some individuals with variable abundance. We hypothesized that nutritional competition, strain-level diversity, and nutrient availability shape community stability and the ability of commensal species to inhibit S. aureus. To test this, we constructed 50 defined synthetic communities composed of representative human nasal bacteria differing in strain and species composition and tracked their temporal dynamics, S. aureus growth, metabolic profiles, and nutritional interactions. The composition of synthetic communities with 5-10 species showed robust and reproducible dynamics and converged in one of three stable states. Synthetic communities dominated by a specific strain of Corynebacterium propinquum were highly stable and consistently excluded S. aureus. Growth curves and coculture assays showed that C. propinquum outcompetes S. aureus under poor nutritional conditions resembling the nasal environment, whereas S. aureus dominates in nutrient-rich conditions. Metabolomics analyses revealed that nutritional competition, including siderophores utilization and amino acid limitation, likely underlies this colonization resistance. These results establish a tractable synthetic community model for the human nasal microbiome and identify nutrient-dependent competition and microbial metabolite production as key drivers of community structure and pathogen exclusion.

microbiology↗

bioRxiv2025

Dried-bakery waste as a substrate for n-caproate and n-caprylate production via chain elongation: bakeroate

Bakery waste is a promising feedstock for the circular economy; however, its use for producing medium-chain carboxylates (MCCs) via microbial chain elongation remains unexplored. This study investigated pretreatment, pertraction, and chain elongation strategies to convert bakery waste into n-caproate and n-caprylate. Bakery waste was mechanically and enzymatically processed, and different inocula were tested to optimize the conversion of the resulting glucose-rich solution into lactate and ethanol (intermediates). These intermediates were fed into continuous chain elongation systems, which operated for over 386 days at 37{degrees}C and pH 5.5. Results showed that 30-35% of bakery waste carbon was converted into n-caproate and 10-15% into n-caprylate. Enzymatic starch hydrolysis proved essential, and lactate was a superior intermediate for chain elongation compared to ethanol. A maximum volumetric MCC production rate of 131 mM C L-1 d-1 (0.1 g L-1 h-1) was achieved. This integrated approach, named "bakeraote," demonstrates an efficient pathway for valorizing bakery waste.

bioengineering↗

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↗

bioRxiv2025

D- and L-lactate consumers in the human gut are taxonomically, biochemically, and energetically different

The human gut microbiota routinely produces D- and L-lactate during fermentation; however, the microbial fate of these stereoisomers, particularly the neurotoxic D-lactate, remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion, understanding its microbial turnover is essential for linking gut metabolism to host health. Here, we used chemostat bioreactors (pH 7.0, 37{degrees}C, and a solids retention time [SRT] of 4 d) to simulate gut-relevant, transient nutrient conditions and to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched from a human-derived microbiota and then inoculated into duplicate bioreactors, which were fed exclusively D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed this divergence at the pathway level. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization in the gut, with implications for understanding the ecology and clinical impact of lactate metabolism.

bioengineering↗

bioRxiv2025

MMonitor: Software for Real-Time Monitoring of Microbial Communities Using Long Reads

Real-time monitoring of microbial communities offers valuable insights into microbial dynamics across diverse environments. However, many existing metagenome analysis tools require advanced computational expertise and are not designed for monitoring. We present MMonitor, an open source software platform for the real-time analysis and visualization of metagenomic Oxford Nanopore Technologies (ONT) sequencing data. MMonitor includes two components: a desktop application for running bioinformatics pipelines through a graphical user interface (GUI) or command-line interface (CLI), and a web-based dashboard for interactive result inspection. The dashboard provides taxonomic composition over time, quality scores, diversity indices, and taxonomy-metadata correlations. Integrated pipelines enable automated de-novo assembly and reconstruction of metagenome-assembled genomes (MAGs). To validate MMonitor, we tracked human gut microbial populations in three bioreactors using 16S rRNA gene sequencing, and applied it to whole-genome sequencing (WGS) data to generate high-quality annotated MAGs and reveal functional insights. We also compare MMonitor to other software for real-time metagenomic analysis, highlighting the strengths and limitations of each tool for this use case. By performing automated time-series analyzes, sample management, and updating of reference databases, MMonitor addresses current limitations and supports dynamic microbiome research in various fields. IMPORTANCEMetagenome monitoring is essential for understanding and managing microbial communities in dynamic environments. Rapid and accurate analysis of these communities allows for timely interventions in biologically active environments, for example, in industrial biotechnology, environmental surveillance, and medical diagnostics. Metagenome monitoring tools should have real-time capabilities, intuitive interfaces, convenient setup and distribution, and suitable visual representations for effective monitoring. MMonitor addresses these challenges by offering a platform that combines real-time analysis of nanopore sequencing data with customizable pipelines and advanced visualization tools. It is designed to be accessible to researchers of all levels of computational skill. It supports efficient taxonomic and functional analyses based on popular metagenome software and automates taxonomic profiling, genome assembly, binning, and annotation. The dashboard helps to interpret data and present results. MMonitor is designed to track metagenomes and we hope that it can impact microbiome research and applications in health, biotechnology, agricultural, and environmental sciences.

bioinformatics↗

bioRxiv2025

Genetic modification of Clostridium kluyveri for heterologous n-butanol and n-hexanol production

The mesophilic microbe Clostridium kluyveri serves as the most commonly used model microbe to elucidate the physiology and biochemistry of ethanol-based chain elongation via reverse {beta}-oxidation. In this pathway, ethanol and acetate are converted into short- and medium-chain carboxylates. However, to date, no genetic system has been published in a peer-reviewed publication. Here, we report the development of versatile genetic tools for C. kluyveri, utilizing the pMTL Clostridia shuttle vector system and thiamphenicol as a selective marker. We identified the native restriction-modification system of C. kluyveri as a critical barrier to DNA transfer and overcame it by identifying and characterizing the crucial methyltransferase. To mimic the native DNA methylation pattern of C. kluyveri, we performed in-vivo methylation of the shuttle vector plasmid by expressing the methyltransferase in Escherichia coli, followed by DNA transfer via conjugation. After validating the genetic system, we demonstrated heterologous expression of different combinations of both NADH and NADPH-dependent alcohol dehydrogenases from Clostridium acetobutylicum. The expression of these genes was controlled by the Pthl promoter, which is commonly used in Clostridia, and the PadhE2 promoter, leading to n-butanol and n-hexanol production of the mutant strains. This genetic system for C. kluyveri will not only enable further research on the metabolism of this microbe but also enable more profound insights into ethanol-based chain elongation in general. IMPORTANCEMedium-chain carboxylates are required in various everyday products, including cosmetics, pharmaceuticals, and fragrances, and show a natural antimicrobial property. Further, they represent food additives and serve as chemical building blocks for several other compounds. Traditionally, these carboxylates are produced from fossil resources, contributing to increased greenhouse gas emissions. Alternatively, they are derived from animal- or plant-based fat (e.g., coconut oil), which competes with agricultural land that is needed for food production. However, microbial chain elongation, which is a biotechnological approach relying on microbes, such as Clostridium kluyveri, is sustainable and a promising alternative to the conventional production of medium-chain carboxylates. Notably, it enables the use of industrial waste streams (e.g., off-gases, carbohydrate-rich industrial waste) as substrates, making the process more environmentally friendly. By applying our genetic system for C. kluyveri, a better understanding of microbial chain elongation can be achieved, and potentially even an extension of its product portfolio.

microbiology↗

bioRxiv2025

Tyrosine availability shapes Staphylococcus aureus nasal colonization and interactions with commensal communities

Only certain individuals are nasally colonized by Staphylococcus aureus, making them more susceptible to developing endogenous infections. The role of the resident microbiota in modulating S. aureus nasal colonization remains largely unclear. Using a newly assembled nasal strain collection, we demonstrate that nasal commensals can either promote or inhibit S. aureus proliferation through strain- and community-specific effects in vitro and within gnotobiotic animals. We identified nutrient-dependent interactions as a key driver of these dynamics and found that S. aureus strains auxotrophic to tyrosine rely on amino acid-providing nasal communities to thrive in the nutrient-limited environment of the nose. Large-scale screenings revealed tyrosine dependency to affect 8% of nasal S. aureus strains and to impair S. aureus colonization. Our findings uncover a crucial factor in S. aureus physiology and its interactions as a commensal nasal species, paving the way for new decolonization strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/651429v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@17b6289org.highwire.dtl.DTLVardef@182249forg.highwire.dtl.DTLVardef@1324dc1org.highwire.dtl.DTLVardef@18ec3bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Steering the chain-elongating microbiome to specific medium-chain carboxylates with ethanol and lactate as co-electron donors: maximizing C8 or C6

Microbial chain elongation is a sustainable process to convert organic residues into valuable biochemicals via anaerobic fermentation. The operating conditions for the bioreactor and the ecological interactions among functional populations are crucial in controlling this process, but they have not been completely ascertained. Here, we unraveled two operating conditions (i.e., environmental factors): (1) the substrate ratio of ethanol and lactate as co-electron donors, and (2) the temperature, which affected both the product specificity (i.e., function) and microbial dynamics of chain-elongating microbiomes in a continuously fed bioreactor with product extraction. Specifically, we found that the increase in the substrate ratio of ethanol to lactate shifted the microbiomes toward n-caprylate (C8) production, while the slightly higher operating temperatures of 37{degrees}C or 42{degrees}C were advantageous to n-caproate (C6) production. We detected a core microbiome that was similar for all environmental conditions and the two bioreactors, consisting of populations from Sphaerochaeta spp., Caproiciproducens spp., and Oscillospiraceae. Besides the core microbiome, we observed positive correlations between Erysipelaclostridiaceae UCG-004, Bacteroides spp., Oscillospiraceae NK4A214, Rikenellaceae RC9, and Pseudoclavibacter spp. with n-caprylate production. Similar populations compared to the core microbiome were positively correlated with n-caproate production. We showed that we can steer microbiomes toward a high specificity of certain medium-chain carboxylates. Abstract Graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/637006v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@70557eorg.highwire.dtl.DTLVardef@fcee81org.highwire.dtl.DTLVardef@18e64feorg.highwire.dtl.DTLVardef@1888a8d_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISGenerating medium-chain carboxylates is a promising open-culture biotechnology production platform for converting organic waste streams into biofuels and chemicals.

bioengineering↗

bioRxiv2024

A comparison study between liquid- and vapor-fed anode zero-gap bioelectrolysis cells

Improving microbial electrosynthesis could be one solution for transitioning towards sustainable chemical production, offering a pathway to convert CO2 into valuable commodities from renewable energy sources. Therefore, we examined the performance differences between liquid- and vapor-fed anode zero-gap bioelectrochemical cells for electromethanogenesis, utilizing a membrane electrode assembly to enhance mass and ohmic transport. Focusing on CH4 and H2 production, we compared two ion-exchange membranes with the liquid-fed anode system and selected the best performing ion-exchange membrane for the vapor-fed anode system. Liquid-fed anode systems did not show significant differences in volumetric CH production rates compared to vapor-fed anode systems, although the latter demonstrated advantages in reducing electrocatalyst degradation and maintaining stable cell voltages. The research underscores the need for further optimization to address performance losses and suggests potential for industrial applications of microbial electrosynthesis, highlighting the importance of catalyst protection.

bioengineering↗

bioRxiv2024

Toxicity and Biodegradation of Two Different Hydrothermal Liquefaction Process Waters to Anaerobic Digestion and the Effect of Microaeration

Hydrothermal liquefaction (HTL) can convert a considerable portion of the carbon in complex feedstocks into renewable bio-oil, but it also generates a liquid byproduct (i.e., HTL process water) that retains 15 - 55% of the carbon from the HTL feedstock. Feeding HTL process water to anaerobic digestion (AD) is a promising approach for maximizing resource recovery, enabling the conversion of the retained carbon into biogas. However, various toxic and poorly biodegradable compounds in HTL process water make its treatment with AD challenging. Presently, the underlying mechanisms remain often unclear. We investigated the impact of HTL process water from two different feedstocks - a food-waste proxy (i.e., dog food, rich in proteins) and wheat straw (rich in lignocellulose) - on the different trophic groups in the food web of AD. We found that methanogens rather than acidogens were inhibited by HTL process water. Comparative toxicity and biodegradability analyses showed that wheat-straw process water had a higher biodegradability regardless of its higher toxicity to acetoclastic methanogens than food-waste process water, due to its higher content in toxic but easily degradable aromatic compounds. Microaeration enhanced the biodegradation and methane yields of food-waste process water, particularly under anoxic conditions. However, microaeration was ineffective for wheat-straw process water. These findings highlight the importance of feedstock-specific strategies to optimize AD for biogas production from HTL process water.

bioengineering↗

bioRxiv2024

Lactate production from lactose-rich wastewater: A comparative study on reactor configurations to maximize conversion rates and efficiencies

About 90% of global lactate production is derived from bacterial fermentation of sugars via pure cultures of homofermentative bacteria in batch mode. Acid whey, which is a wastewater from the yogurt industry with lactose and galactose as the main sugars, can be used as an alternative substrate for the commercial production of lactate. Operating open cultures of microbial consortia (i.e., reactor microbiomes) reduces the costs of lactate production by circumventing sterilization, while continuous operation achieves higher productivity at shorter production times. Homofermentation can be achieved by maintaining acidic and thermophilic conditions, while product formation in continuous systems can be increased with biomass retention strategies. To find the best reactor configuration for lactate production from acid whey, we operated three different reactor configurations: (1) an upflow anaerobic sludge blanket (UASB) reactor; (2) an anaerobic filter reactor (AFR); and (3) an anaerobic continuously stirred tank reactor (CSTR) with a hollow-fiber membrane module. We operated at different hydraulic retention times (HRTs) to find the optimum parameters to maximize the lactose and galactose-into-lactate (LG-into-LA) conversion efficiency. We did not use an inoculum but enriched the endogenous D-lactate-producing Lactobacillus spp. that later dominated the reactor microbiomes (> 90% relative abundance). Undissociated lactic acid concentrations of more than 60 mmol C L-1 inhibited the microbiomes. We alleviated the inhibition effect by shortening the HRT to 0.6 days and using diluted acid-whey substrate (1.67-fold dilution) to achieve almost complete conversion of the acid-whey sugars to lactate. At the 0.6-day HRT commencement, the AFR and CSTR performed better than the UASB reactor due to their better cell retention abilities. During the period between Day 365-384, we experienced an error in the pH control of the CSTR system during which the pH value dropped to 4.3. After this pH-error period, the LG-into-LA conversion efficiency for the CSTR considerably improved and surpassed the AFR. We achieved the highest lactate conversion rate of 1256 {+/-} 46.3 mmol C L-1 d-1 (1.57 {+/-} 0.06 g L-1 h-1) at a LG-into-LA conversion efficiency of 82.2 {+/-} 3.4% (in mmol C), with a yield of 0.85 {+/-} 0.02 mmol C mmol C-1 (product per consumed substrate) for the CSTR.

bioengineering↗

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

Toward industrial C8 production: Oxygen intrusion drives renewable n caprylate production from ethanol and acetate via intermediate metabolite production

Previous bioreactor studies achieved high volumetric n-caprylate (i.e., n-octanoate) production rates and selectivities from ethanol and acetate with chain-elongating microbiomes. However, the metabolic pathways from the substrates to n-caprylate synthesis were unclear. We operated two n-caprylate-producing upflow bioreactors with a synthetic medium to study the underlying metabolic pathways. The operating period exceeded 2.5 years, with a peak volumetric n-caprylate production rate of 190 {+/-} 8.4 mmol C L-1 d-1 (0.14 g L-1 h-1). We identified oxygen availability as a critical performance parameter, facilitating intermediate metabolite production from ethanol. Bottle experiments in the presence and absence of oxygen with 13C-labeled ethanol suggest acetyl-coenzyme A-based derived production of n-butyrate (i.e., n-butanoate), n-caproate (i.e., n-hexanoate), and n-caprylate. Here, we postulate a trophic hierarchy within the bioreactor microbiomes based on metagenomics, metaproteomics, and metabolomics data, as well as experiments with a Clostridium kluyveri isolate. First, the aerobic bacterium Pseudoclavibacter caeni and the facultative anaerobic fungus Cyberlindnera jadinii converted part of the ethanol pool into the intermediate metabolites succinate, lactate, and pyroglutamate. Second, the strict anaerobic C. kluyveri elongated acetate with the residual ethanol to n-butyrate. Third, Caproicibacter fermentans and Oscillibacter valericigenes elongated n-butyrate with the intermediate metabolites to n-caproate and then to n-caprylate. Among the carbon chain-elongating pathways of carboxylates, the tricarboxylic acid cycle and the reverse {beta}-oxidation pathways showed a positive correlation with n-caprylate production. The results of this study inspire the realization of a chain-elongating production platform with separately controlled aerobic and anaerobic stages to produce n-caprylate renewably as an attractive chemical from ethanol and acetate as substrates. Broader contextNext to renewable electric energy, carbon-based chemicals have to be produced sustainably and independently from fossil sources. To meet this goal, we must expand the portfolio of bio-based conversion technologies on an industrial scale to cover as many target chemicals as possible. We explore the bioprocess of chain elongation to provide medium-chain carboxylates that can function as future platform chemicals in the circular economy. The most valuable medium-chain carboxylate produced with chain elongation is n-caprylate (i.e., n-octanoate). This molecule with eight carbon atoms in a row (C8) is challenging to produce renewably for the chemical industry. Previous reports elucidated that elevated ethanol-to-acetate ratios, which are found in syngas-fermentation effluent, stimulated n-caprylate production. Until now, studies have suggested that chain elongation from high concentrations of ethanol and acetate is a fully anaerobic process. We refine this view by showing a trophic hierarchy of aerobic and anaerobic microbes capable of facilitating this process. Appropriate oxygen supplementation enables the synthesis of succinate, lactate, and pyroglutamate that permit high-rate chain elongation to n-caprylate under anaerobic conditions. Given these results, future research should focus on the segregated study of aerobic and anaerobic microbes to further enhance the process performance to produce n-caprylate renewably at an industrial scale.

bioengineering↗

bioRxiv2024

Performance effects from different shutdown methods of three electrode materials for the power-to-gas application with electromethanogenesis

Industrial applications of microbial electrochemical systems will require regular maintenance shutdowns, involving inspections and component replacements to extend the lifespan of the system. Here, we examined the impact of such shutdowns on the performance of three electrode materials (i.e., platinized titanium, graphite, and nickel) as cathodes in a microbial electrochemical system that would be used for electromethanogenesis in power-to-gas applications. We focused on methane (CH4) production from hydrogen (H2) and carbon dioxide (CO2) using Methanothermobacter thermautotrophicus. We showed that the platinized titanium cathode resulted in high volumetric CH4 production rates and Coulombic efficiencies. Using a graphite cathode would be more cost-effective than using the platinized titanium cathode in microbial electrochemical systems but showed an inferior performance. The microbial electrochemical system with the nickel cathode showed improvements compared to the graphite cathode. Additionally, this system with a nickel cathode demonstrated the fastest recovery during a shutdown experiment compared to the other two cathodes. Fluctuations in pH and nickel concentrations in the catholyte during power interruptions affected CH4 production recovery in the system with the nickel cathode. This research enhances understanding of the integration of biological and electrochemical processes in microbial electrochemical systems, providing insights into electrode selection and operating strategies for effective and sustainable CH4 production.

bioengineering↗

bioRxiv2024

The gases H2 and O2 in open-culture reactors influence the performance and microbiota of chain elongation into n-caproate and n-caprylate.

Medium-chain carboxylates are used in various industrial applications. These chemicals are typically extracted from palm oil, which is deemed not sustainable. Recent research has focused on microbial chain elongation using reactors to produce medium-chain carboxylates, such as n-caproate (C6) and n-caprylate (C8), from organic substrates such as wastes. Even though the production of n-caproate is relatively well-characterized, bacteria and metabolic pathways that are responsible for n-caprylate production are not. Here, three 5-L reactors with continuous membrane-based liquid-liquid extraction (i.e., pertraction) were fed ethanol and acetate and operated for an operating period of 234 days with different operating conditions. Metagenomic and metaproteomic analyses were employed. n-Caprylate production rates and reactor microbiomes differed between reactors even when operated similarly due to differences in H2 and O2 between the reactors. The complete reverse {beta}-oxidation pathway was present and expressed by several bacterial species in the Clostridia class. Several Oscillibacter spp., including Oscillibacter valericigenes, were positively correlated with n-caprylate production rates, while Clostridium kluyveri was positively correlated with n-caproate production. Pseudoclavibacter caeni, which is a strictly aerobic bacterium, was abundant across all the operating periods, regardless of n-caprylate production rates. This study provides insight into microbiota that are associated with n-caprylate production in open-culture reactors and provides ideas for further work. ImportanceMicrobial chain elongation pathways in open-culture biotechnology systems can be utilized to convert organic waste and industrial side streams into valuable industrial chemicals. Here, we investigated the microbiota and metabolic pathways that produce medium-chain carboxylates, including n-caproate (C6) and n-caprylate (C8), in reactors with in-line product extraction. Although the reactors in this study were operated similarly, different microbial communities dominated and were responsible for chain elongation. We found that different microbiota were responsible for n-caproate or n-caprylate production, and this can inform engineers on how to operate the systems better. We also observed which changes in operating conditions steered the production toward and away from n-caprylate, but more work is necessary to ascertain a mechanistic understanding that could be predictive. This study provides pertinent research questions for future work.

microbiology↗