Biology subjects

Stouten, G. R.

Publications and source records attributed to Stouten, G. R..

5 recordsLinked to original sources

bioRxiv2026

Unraveling the metabolic interactions of a Dehalobacter-containinganaerobic mixed culture for bioremediation

Organohalide-respiring bacteria (OHRB), such as Dehalobacter, play key roles in the bioremediation of anoxic environments contaminated with chlorinated aromatic compounds. These obligate anaerobes rely on syntrophic interactions to obtain essential resources--hydrogen, acetate, and corrinoid cofactors--from acetogens and fermenters. However, the metabolic interactions enabling complete reductive dehalogenation of compounds like 1,2,4-trichlorobenzene (1,2,4-TCB) to benzene remain incompletely understood. In this study, we asked: (1) What are the key microbial taxa and their functional roles within a Dehalobacter-containing anaerobic microbial community detoxifying chlorinated benzenes? (2) How do syntrophic interactions enable complete dehalogenation of 1,2,4-TCB to benzene under anaerobic conditions? (3) Can genome-resolved metagenomics and genome-scale metabolic modeling elucidate the metabolic dependencies supporting organohalide respiration in complex consortia? To address these questions, we cultivated microbial communities in batch reactors using methanol as electron donor and either 1,2,4-TCB or monochlorobenzene (MCB) as electron acceptor. In active MCB-fed cultures, benzene increased from 0 to 62.3{micro}mol per bottle while MCB decreased from 88.3 to 22.0{micro}mol per bottle over 120 days, with this pattern repeating across multiple substrate additions. Using genome-resolved metagenomics to identify dominant taxa and select 12 high-quality metagenome-assembled genomes (MAGs) for modeling, we reconstructed genome-scale metabolic models (GEMs) to identify candidate metabolic interactions and predict syntrophic dependencies that may support organohalide respiration in these consortia. Community flux sampling predicted that methanol, H2, acetate, and CO2 formed the dominant exchange backbone of the modeled community, while also indicating competition for shared electron donors between the two Dehalobacter populations. Model-guided minimal-community analysis further identified a narrow dechlorinating core in which all feasible minimal consortia retained a Dehalobacter member together with Methanothrix. These results provide a modeling-informed framework for hypothesis generation and future experimental validation of anaerobic consortia relevant to bioremediation.

systems biology↗

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

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↗

bioRxiv2023

"Candidatus Siderophilus nitratireducens": a psychrophilic, nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-reducing iron-oxidizing (NRFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with NRFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens", carries central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). To our knowledge, this is the first report of nap-based lithoautotrophic growth, and we demonstrate that iron oxidation coupled to dissimilatory reduction of nitrate to nitrite is thermodynamically favourable under realistic Fe3+/Fe2+ and [Formula] concentration ratios. Ultimately, by bridging the gap between laboratory investigations and real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NRFOs taxonomic diversity and ecological role.

microbiology↗

bioRxiv2021

System characterization of dynamic biological systems through improved data analysis.

Determining the functional development and dominant competitive strategy in microbial community enrichments is complicated by the extensive measurement campaigns required for off-line system analysis. This study demonstrates that detailed system characterization of aerobic pulse fed enrichments can be established using on-line measurements combined with automated data analysis. By incorporating the physicochemical processes in on-line data processing with a Particle Filter and kinetic process model, an accurate reconstruction of the dominant biological rates can be made. We hereby can differentiate between storage compound production and biomass growth in sequencing batch bioreactors. The method proposed allows for close monitoring of changes in functional behavior of long-running enrichment cultures, without the need for off-line samples, therewith enabling the identification of new insights in process dynamics with a minimal experimental effort. Even though a specific example application of the method proposed is described here, the approach can readily be extended to a wide range of dynamic experimental systems that can be characterized based on on-line measurements.

microbiology↗