Enrichment and specific quantification of Methanocalculus in anaerobic digestion

• For the first time Methanocalculus was enriched from anaerobic digestion processes.
• Methanocalculus was enriched under hydrogenotrophic methanogenesis condition only.
• We subsequently developed the first Methanocalculus-specific qPCR assay.
• The qPCR assay was shown to have high coverage, specificity, and efficiency.

Members of the genus Methanocalculus are characterized as hydrogenotrophic methanogens and present in diverse natural and engineered environments. Methanocalculus populations were enriched from anaerobic digesters treating dairy waste using formate as the substrate. Methanocalculus sequences retrieved from the enrichment cultures were subsequently used to develop a Methanocalculus-specific TaqMan qPCR assay to determine the abundance of Methanocalculus populations in the environment, representing the first quantitative tool specifically targeting Methanocalculus. The Methanocalculus-specific primer/probe set was shown to have high coverage with perfect match to >80% of all Methanocalculus 16S rRNA gene sequences in the Ribosomal Database Project (RDP). High specificity of the qPCR assay was also validated by both in silico and experimental analyses. Amplification efficiency of the qPCR assay was determined to be 91.9%, which is satisfactory for quantitative applications. Results from the Methanocalculus-specific qPCR analysis of formate-enriched methanogenic cultures were consistent with those from clone library analysis of the same cultures, validating the accuracy of the qPCR assay. Subsequent field application of the qPCR assay found low relative abundance of Methanocalculus in anaerobic digesters treating dairy waste, accounting for 0.01% of the archaeal populations. The qPCR results were consistent with the lack of detection of Methanocalculus in previous studies of the same anaerobic digesters with clone library analyses, which are less sensitive than qPCR. Thus, the Methanocalculus-specific qPCR assay developed in this study is a highly sensitive tool for the rapid and efficient quantification of Methanocalculus populations in methanogenic environments and understanding of the ecological functions of these methanogens.