The 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.

Efficient and low-emission diesel engine combustion processes require an optimal fuel-air mixing and combustion process. Therefore, already in an early engine design phase there is a strong interest among injector and engine developers for a simple and reliable method to predict fuel spray penetration characteristics at engine-like conditions. At the Institute of Piston Machines and Internal Combustion Engines of Rostock University a new application-oriented spray penetration model considering injector type individual behaviour was developed.

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.

This study was continued by the Environmental Biotechnology Group of the University of Tübingen 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 ΔH 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 ΔH. We expanded our system for genetic modification of M. thermautotrophicus ΔH 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 ΔH. 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 ΔH 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 ΔH 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 ΔH.

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