Analysis of a σ⁵⁴ Transcription Factor L420P Mutation in Context of Increased Organic Nitrogen Tolerance of Photofermentative Hydrogen Production in Cereibacter sphaeroides Strain 2.4.1 Substrain H2

Photofermentative hydrogen production with non-sulfur purple bacteria like Cereibacter sphaeroides (formerly Rhodobacter sphaeroides) is a promising and sustainable process to convert organic waste into the energy carrier hydrogen gas. However, this conversion is inhibited by elevated organic nitrogen concentrations in the substrate, which limits its applicability to nitrogen-poor organic waste. We present genomic and transcriptomic insights into a substrain of Cereibacter sphaeroides strain 2.4.1 that shows unexpected high levels of photofermentative hydrogen evolution when fed with glutamate. Genome sequencing revealed 222 single nucleotide variances (SNVs) between the reference genome of C. sphaeroides strain 2.4.1 and the analyzed substrain H2. These affect 61 protein coding genes. A leucine-proline exchange is present in the σ⁵⁴ factor (rpoN2 gene), a global hydrogen and nitrogen metabolism regulator. We propose a model how this mutation alters DNA-binding properties that explain the unexpected organic nitrogen tolerance of hydrogen production. Transcriptomic analyses under varying glutamate concentrations support this finding. Thus, we present the first thorough genomic and transcriptomic analysis of a Cereibacter strain that shows promising metabolic characteristics for biotechnological hydrogen gas production from organic waste. These results suggest a potential target for strain optimization. Possibly, our key finding can be transferred to other hydrogen producing microorganisms.