Biology subjects

Lawson, C. E.

Publications and source records attributed to Lawson, C. E..

12 recordsLinked to original sources

bioRxiv2026

Automated anaerobic cultivation and phenotyping of diverse chain-elongating bacteria

Anaerobic bacteria perform diverse metabolic functions with considerable biotechnological potential, yet their physiology remains poorly characterized because conventional anaerobic cultivation is low throughput. Here, we developed a Platform for Automated Cultivation of Microbial Anaerobes (PACMAN) and applied it to systematically characterize the physiology of diverse chain-elongating bacteria (CEB) that produce medium-chain carboxylic acids (MCCAs). High-throughput screening across nine CEB and 20 potential electron donors revealed extensive metabolic diversity, including previously unreported conversion of glycerol to octanoate by Pseudoramibacter alactolyticus and xylose to hexanoate by Caproiciproducens sp. 7D4C2. Comparison with genome-derived metabolic pathways further revealed widespread discrepancies between predicted and observed substrate utilization. Across diverse CEB and electron donors, decreasing the acetate-to-electron-donor ratio consistently shifted product distributions toward longer-chain carboxylates, revealing a general principle governing chain-length specificity, although maximum attainable chain length remained strain dependent. Finally, screening CEB across increasing carboxylate concentrations revealed substantial variation in MCCA tolerance, with Gram-positive MCCA producers exhibiting greater resilience than Gram-negative and butyrate-producing bacteria. Together, these findings reveal organizing principles underlying the metabolic versatility and product specificity of CEB and establish automated anaerobic phenotyping as a scalable approach for discovering microbial functions relevant to biomanufacturing.

microbiology↗

bioRxiv2026

Coupling Stickland fermentation with reverse β-oxidation represents a new mode of medium-chain carboxylate production

Biotechnologies for producing medium-chain carboxylic acids (MCCAs) by chain elongating bacteria (CEB) promise to shift the oleochemical industry away from existing land use practices, vulnerable supply chains, and significant environmental costs. Realizing this potential requires a broader understanding of CEB physiology, which is currently based on lactate and ethanol utilization, despite these bacteria thriving in protein-rich digestive tracts and organic waste bioreactors. Through comparative genomic analysis of known MCCA producers and physiological examination of the recently isolated species Peptonella octanoica and other members of its genus as a model system, we show that amino acid utilization is a broadly distributed yet largely overlooked trait of many CEB. Among several amino acid-fermenting lineages, a conserved metabolic architecture couples Stickland fermentation with reverse beta-oxidation (R{beta}O), enabling MCCA production when acetate or electron-accepting amino acids are limiting. We show how this coupling manages thermodynamic constraints on lactate and amino acid oxidation by driving longer-chain product formation as hydrogen partial pressure increases. We further reveal that amino acid-fermenting CEB are abundant not only in gut microbiomes but also engineered bioreactors, where their novel physiology provides a mechanistic basis for high titer n-octanoate production from proteinaceous organic wastes. Beyond biomanufacturing, these findings broaden our understanding of CEB across anaerobic microbiomes in which amino acid fermentation and chain elongation are intersecting ecological strategies.

microbiology↗

bioRxiv2026

Peptonella octanoica gen. nov., sp. nov., a new medium-chain carboxylate-producing bacterium, and the reclassification of Eubacterium pyruvativorans as Peptonella pyruvativorans comb. nov.

A strictly anaerobic bacterial strain, F2T, was isolated from an anaerobic bioreactor fermenting source-separated organic waste. Cells of strain F2T are non-spore-forming, rod-shaped (1.5-2.5 x 0.27-0.33 m), and Gram-negative, although they possess a monoderm cell wall architecture. The strain grew at 37 degrees C within a pH range of 5 to 8 and produced short-, branched-, and medium-chain carboxylates as well as ammonium, H2 and CO2, with acetate and propanoate produced or consumed depending on fermentation conditions. The genome consists of a single 2.4 Mbp chromosome with a G+C content of 50.2% and 2,131 predicted genes. Phylogenetic analysis of the 16S rRNA gene against other isolates revealed that strain F2T is most similar to Eubacterium pyruvativorans I-6T (92.06% 16S rRNA identity). Based on further phenotypic, genomic, and phylogenetic analysis, strain F2T represents a novel genus and species within the family Anaerovoracaceae with the proposed name Peptonella octanoica gen. nov. sp. nov. The type strain is F2T (strain accession pending). As a member of this same genus-level clade, we propose reclassifying Eubacterium pyruvativorans as Peptonella pyruvativorans comb. nov. These findings disambiguate Peptonella spp. from the phylogenetically distant and phenotypically distinct Eubacterium limosum ATCC 8486T.

microbiology↗

bioRxiv2026

Long-term Production and Recovery of Medium-Chain Carboxylates from Source-Separated Organics

Source-separated organics (SSO) are widely processed via anaerobic digestion to produce biogas, yet alternative conversion pathways could generate higher-value products. Here, we demonstrate long-term continuous production and recovery of medium-chain carboxylic acids (MCCAs) from SSO via microbial chain elongation using a bench-scale anaerobic bioreactor operated for 911 days. The bioreactor was fed with SSO samples collected from two full-scale municipal organics processing facilities in Toronto, Canada, capturing facility-specific and seasonal variability in SSO composition. MCCA production was closely associated with the availability of lactate as an electron donor, which varied with SSO preprocessing operations and outdoor collection temperatures. To mitigate product inhibition, an in-line extraction system using hollow-fiber polydimethylsiloxane (PDMS, also known as silicone polymer) membranes was integrated with the anaerobic membrane bioreactor, providing a robust and solvent-free alternative to solvent-based extraction methods. Selective extraction reduced MCCA product inhibition when lactate availability was not limiting, resulting in maximum MCCA yields and production rates of 0.31 g MCCA g-1 VSfeed and 0.84 g L-1 day-1, respectively, with notable octanoic acid production (up to 20% of total MCCA). Acidification of the alkaline extract produced a phase-separated MCCA-rich oil (0.88 g MCCA g-1 oil) without the addition of downstream separation steps. Microbial community analysis of the bioreactor revealed enrichment of putative chain-elongating bacteria, including Eubacterium and Pseudoramibacter species, while shifts in SSO feedstock microbiomes influenced substrate availability and product spectra. These results demonstrate the feasibility of sustained MCCA production from municipal organic waste streams and highlight opportunities to integrate chain elongation with existing anaerobic digestion infrastructure.

bioengineering↗

bioRxiv2026

CAZyme domain architectures suggest fine-scale functional differentiation among anaerobic fungi and bacteria during lignocellulose conversion to volatile fatty acids

Anaerobic fermentation with microbial communities (microbiomes) is an emerging platform for conversion of lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), which can be achieved by arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow faeces, goat faeces, and anaerobic digester sludge, selecting for fungal or bacterial dominated communities that convert sorghum biomass into VFAs. Subsequently we reconstructed metabolic networks across these enrichments based on recovered bacterial metagenome-assembled genomes (MAGs) and fungal isolate genomes and profiled their metabolic activity using metatranscriptomics to identify potential functional niches. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix-dominated fungal enrichments converted lignocellulose to lactate, acetate and formate. Analysis of carbohydrate-active enzymes (CAZymes) revealed fine-scale differences between microbes that expressed unique multi-functional enzymes linking two or more CAZymes together with distinct carbohydrate binding motifs, implicating lignocellulose structure as a key driver of selection and niche differentiation. Most of these multi-functional enzymes localized complementary degradation functions together, likely conferring synergistic degradation effects within and between microbiome members. We anticipate that these findings will help inform efforts to develop synthetic microbiomes with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals.

microbiology↗

bioRxiv2025

Engineering anaerobic fungal-bacterial consortia for medium-chain fatty acid production from lignocellulosic biomass

Lignocellulosic biomass is a renewable feedstock for sustainable fuels and chemicals, yet industrial conversion remains constrained by carbohydrate solubilization. Inspired by herbivore rumen microbiomes, we engineered an anaerobic fungal-bacterial consortium converting native lignocellulose into medium-chain fatty acids (MCFAs) without pretreatment. Systematic screening identified a newly isolated anaerobic fungus, Neocallimastix sp. FC1, in co-culture with Megasphaera hexanoica as a top-performing pair, achieving a lignocellulose-to-MCFA yield of 21.0 % (carbon-to-carbon basis) through tight lactate cross-feeding without competition for soluble sugars. Because fungal lactate production rate constrained the growth of M. hexanoica, the bacterium reallocated protein from growth toward chain elongation, resulting in increased MCFAs production over butyrate. These results demonstrate that high lignocellulose-to-MCFA conversion by the consortium requires high lactate-producing capability and operating regimes sustaining low lactate concentrations at high flux. Technoeconomic analysis further identifies the cost and yield thresholds required for economically viable deployment, establishing quantitative design targets for pretreatment-free fungal-bacterial lignocellulose upgrading.

bioengineering↗

bioRxiv2025

Enabling Plasmid-based Expression in Clostridium kluyveri using a Biparental Methylation-Conjugation System

Clostridium kluyveri is a promising biocatalyst for producing medium-chain fatty acids (MCFAs) from waste-derived carbon via chain elongation. MCFAs are platform chemicals with diverse applications across agriculture, food, cosmetics, and fuels, and could support efforts towards tandem resource recovery and sustainable chemical production. However, genetic intractability has hindered efforts to engineer C. kluyveri for improved product yields, control over chain length and selectivity, and production of non-native oleochemicals. Here, we report a streamlined, biparental methylation-conjugation system developed for C. kluyveri DSM555T to bypass the organisms restriction-modification barriers and enable stable plasmid delivery. We use this system to demonstrate heterologous expression of the Fluorescence-Activated absorption-Shifting Tag (FAST), an anaerobic fluorescent reporter. This system supports advances in metabolic engineering of C. kluyveri and the broader adoption of genetic tools in chain elongating bacteria to expand the applications of anaerobic chain elongation in industrial biomanufacturing. Lay SummaryWe developed a streamlined method for genetically modifying Clostridium kluyveri, a promising strain for industrial anaerobic biomanufacturing, and used it to express a fluorescent protein useful for downstream applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/657078v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11e2a58org.highwire.dtl.DTLVardef@16e1099org.highwire.dtl.DTLVardef@103c72forg.highwire.dtl.DTLVardef@10a1fce_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Distinct Acetate Utilization Strategies Differentiate Butyrate and Octanoate Producing Chain-Elongating Bacteria

Chain elongating bacteria (CEB) are a unique guild of anaerobes that upcycle organic waste into valuable short- and medium-chain carboxylic acids (MCCAs), enabling a circular bioeconomy. However, the metabolic rules that determine product chain length have remained elusive. Here, we combine 13C isotope tracing, proteomics, enzyme assays, and metabolic modelling to show that distinct acetate utilization strategies underlie the divergence between butyrate- and MCCA-producing CEB. MCCA-producing strains recycle acetate to maximize lactate use under acetate limitation, but at the cost of slower growth. In contrast, butyrate-producing strains grow faster by favoring acetate assimilation, at the cost of restricted lactate utilization when acetate is scarce. These physiological trade-offs are encoded in the substrate specificity of coenzyme A transferase, the terminal enzyme in reverse {beta}-oxidation. Our findings uncover a fundamental constraint shaping chain-length selectivity in CEB and offer new strategies to optimize MCCA production from organic waste streams.

microbiology↗

bioRxiv2024

Evaluating the feasibility of medium chain oleochemical synthesis using microbial chain elongation

Chain elongating bacteria are a unique guild of strictly anaerobic bacteria that have garnered interest for sustainable chemical manufacturing from carbon-rich wet and gaseous waste streams. They produce C6-C8 medium-chain fatty acids which are valuable platform chemicals that can be used directly, or derivatized to service a wide range of chemical industries. However, the application of chain elongating bacteria for synthesizing products beyond C6-C8 medium-chain fatty acids has not been evaluated. In this study, we assess the feasibility of expanding the product spectrum of chain elongating bacteria to C9-C12 fatty acids, along with the synthesis of C6 fatty alcohols, dicarboxylic acids, diols, and methyl ketones. We propose several metabolic engineering strategies to accomplish these conversions in chain elongating bacteria and utilize constraint-based metabolic modelling to predict pathway stoichiometries, assess thermodynamic feasibility, and estimate ATP and product yields. We also evaluate how producing alternative products impacts the growth rate of chain elongating bacteria via resource allocation modelling, revealing a trade-off between product carbon length and class versus cell growth rate. Together, these results highlight the potential for using chain elongating bacteria as a platform for diverse oleochemical biomanufacturing and offer a starting point for guiding future metabolic engineering efforts aimed at expanding their product range. Graphical AbstractIn this work, the authors use constraint-based metabolic modelling and enzyme cost minimization to assess the feasibility of using metabolic engineering to expand the product spectrum of anaerobic chain elongating bacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/592817v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@725c90org.highwire.dtl.DTLVardef@1b2575org.highwire.dtl.DTLVardef@382c95org.highwire.dtl.DTLVardef@1f71ea0_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryIn this work, the authors use constraint-based metabolic modelling and enzyme cost minimization to assess the feasibility of using metabolic engineering to expand the product spectrum of anaerobic chain elongating bacteria.

bioengineering↗

bioRxiv2021

Investigating the chemolithoautotrophic and formate metabolism of Nitrospira moscoviensis by constraint-based metabolic modeling and 13C-tracer analysis

Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used constraint-based analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and 13C-tracer experiments with bicarbonate and formate coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings support that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation. We also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospiras in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. ImportanceNitrospira are globally abundant nitrifying bacteria in soil and aquatic ecosystems and wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of N. moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite- oxidizing bacteria.

microbiology↗

bioRxiv2019

Autotrophic and mixotrophic metabolic network fluxes suggest versatile lifestyle for the anammox bacterium Candidatus Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus Kuenenia stuttgartiensis using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactors side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

microbiology↗

bioRxiv2018

Metatranscriptomic and thermodynamic insights into medium-chain fatty acid production using an anaerobic microbiome

Biomanufacturing from renewable feedstocks represents a sustainable strategy to offset fossil fuel-based chemical production. One potential biomanufacturing strategy is production of medium chain fatty acids (MCFA) from organic feedstocks using either pure cultures or microbiomes. While the set of microbes in a microbiome can often metabolize more diverse organic materials than a single species, and the role of specific species may be known, knowledge of the carbon and energy flow within and between organisms in MCFA producing microbiomes is only starting to emerge. Here, we integrate metagenomic, metatranscriptomic, and thermodynamic analyses to predict and characterize the metabolic network of an anaerobic microbiome producing MCFA from organic matter derived from lignocellulosic ethanol fermentation conversion residue. A total of 37 high quality (>80% complete, < 10% contamination) metagenome-assembled genomes (MAGs) were recovered from the microbiome and metabolic reconstruction of the 10 most abundant MAGs was performed. Metabolic reconstruction combined with metatranscriptomic analysis predicted that organisms affiliated with Lactobacillus and Coriobacteriaceae degraded carbohydrates and fermented sugars to lactate and acetate. Lachnospiraceae and Eubacteriaceae affiliated organisms were predicted to transform these fermentation products to MCFA. Thermodynamic analyses identified conditions in which H2 is expected to be either produced or consumed, suggesting a potential role of H2 partial pressure on MCFA production. From an integrated systems analysis perspective, we propose that MCFA production could be improved if microbiomes are engineered to use homofermentative instead of heterofermentative Lactobacillus, and if MCFA-producing organisms are engineered to preferentially use a thioesterase instead of a CoA transferase as the terminal enzyme in reverse {beta}-oxidation.\n\nImportanceMixed communities of microbes play important roles in health, the environment, agriculture, and biotechnology. While tapping the combined activity of organisms within microbiomes may allow the utilization of a wider range of substrate over the use of pure cultures for biomanufacturing, harnessing the metabolism of these mixed cultures remains a major challenge. Here, we predict metabolic functions of bacteria in a microbiome that produces medium-chain fatty acids from a renewable feedstock. Our findings lay the foundation to begin addressing how to engineer and control microbiomes for improved biomanufacturing; to build synthetic mixtures of microbes that produce valuable chemicals from renewable resources; and to better understand microbial communities that contribute to health, agriculture, and the environment.

systems biology↗