<i>Methanospirillum</i> Respiratory mRNA Biomarkers Correlate with Hydrogenotrophic Methanogenesis Rate during Growth and Competition for Hydrogen in an Organochlorine-Respiring Mixed Culture

Molecular biomarkers hold promise for inferring rates of key metabolic activities in complex microbial systems. However, few studies have assessed biomarker levels for simultaneously occurring (and potentially competing) respirations. In this study, methanogenesis biomarkers for <i>Methanospirillum hungatei</i> were developed, tested, and compared to <i>Dehalococcoides mccartyi</i> biomarkers in a well-characterized mixed culture. Proteomic analyses of mixed culture samples (<i>n</i> = 4) confirmed expression of many <i>M. hungatei</i> methanogenesis enzymes. The mRNAs for two oxidoreductases detected were explored as quantitative biomarkers of hydrogenotrophic methanogenesis: a coenzyme F<sub>420</sub>-reducing hydrogenase (FrcA) and an iron sulfur protein (MvrD). As shown previously in <i>D. mccartyi</i>, <i>M. hungatei</i> transcript levels correlated linearly with measured (<i>R</i> = 0.97 for FrcA, <i>R</i> = 0.91 for MvrD; <i>n</i> = 7) or calculated respiration rate (<i>R</i> = 0.81 for FrcA, <i>R</i> = 0.62 for MvrD; <i>n</i> = 35) across two orders of magnitude on a log–log scale. The average abundance of MvrD transcripts was consistently two orders of magnitude lower than FrcA, regardless of experimental condition. In experiments where <i>M. hungatei</i> was competing for hydrogen with <i>D. mccartyi</i>, transcripts for the key respiratory hydrogenase HupL were generally less abundant per mL than FrcA and more abundant than MvrD. With no chlorinated electron acceptor added, HupL transcripts fell below both targets. These biomarkers hold promise for the prediction of in situ rates of respiration for these microbes, even when growing in mixed culture and utilizing a shared substrate which has important implications for both engineered and environmental systems. However, the differences in overall biomarker abundances suggest that the strength of any particular mRNA biomarker relies upon empirically established quantitative trends under a range of pertinent conditions.