A novel experimental method to determine substrate uptake kinetics of gaseous substrates applied to the carbon monoxide-fermenting Clostridium autoethanogenum.

Syngas fermentation has gained momentum over the last decades. The cost-efficient design of industrial-scale bioprocesses is highly dependent on quantitative microbial growth data. Kinetic and stoichiometric models for syngas-converting microbes exist, but accurate experimental validation of the derived parameters is lacking. Here, we describe a novel experimental approach for measuring substrate uptake kinetics of gas-fermenting microbes using the model microorganism Clostridium autoethanogenum. One-hour disturbances of a steady-state chemostat bioreactor with increased CO partial pressures (up to 1.2 bar) allowed for measurement of biomass-specific CO uptake- and CO2 production rates ( q CO ${q}_{{CO}}$ , q CO 2 ${q}_{{{CO}}_{2}}$ ) using off-gas analysis. At a pCO of 1.2 bar, a q CO ${q}_{{CO}}$ of -119 ± 1 mmol g-1 X h-1 was measured. This value is 1.8-3.5-fold higher than previously reported experimental and kinetic modeling results for syngas fermenters. Analysis of the catabolic flux distribution reveals a metabolic shift towards ethanol production at the expense of acetate at pCO ≥ $\ge $ 0.6 atm, likely to be mediated by acetate availability and cellular redox state. We characterized this metabolic shift as acetogenic overflow metabolism. These results provide key mechanistic understanding of the factors steering the product spectrum of CO fermentation in C. autoethanogenum and emphasize the importance of dedicated experimental validation of kinetic parameters.

NovoLign: metaproteomics by sequence alignment.

Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here, we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. We assessed the NovoLign pipeline for taxonomic coverage and false positive annotations using a wide range of in silico and experimental data, including pure reference strains, laboratory enrichment cultures, synthetic communities, and environmental microbial communities. In summary, we present NovoLign, a de novo metaproteomics pipeline that employs large-scale sequence alignment to enable rapid taxonomic profiling, evaluation of database searching outcomes, and the creation of reference sequence databases. The NovoLign pipeline is publicly available via: https://github.com/hbckleikamp/NovoLign.

Overflow metabolism at the thermodynamic limit of life: How carboxydotrophic acetogens mitigate carbon monoxide toxicity.

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.