The effect of ammonia on methanogenic biomass from a full-scale agricultural digester treating nitrogen-rich materials was characterized in batch activity assays subjected to increasing concentrations of total ammonia N. Acetotrophic and methanogenic profiles displayed prolonged lag phases and reduced specific activity rates at 6.0 gN-TAN L-1, though identical methane yields were ultimately reached. These results agreed with the expression levels of selected genes from bacteria and methanogenic archaea (qPCR of 16S rRNA and mrcA cDNA transcripts). Compound-specific isotope analysis of biogas indicated that ammonia exposure was associated to a transition in methanogenic activity from acetotrophy at 1.0 gN-TAN L-1 to intermediate and complete hydrogenotrophy at 3.5 and 6.0 gN-TAN L-1. Such pattern matched the results of 16S-Illumina sequencing of genes and transcripts in that predominant methanogens shifted, along with increasing ammonia, from the obligate acetotroph Methanosaeta to the hydrogenotrophic Methanoculleus and the poorly understood methylotrophic Methanomassiliicoccus. The underlying bacterial community structure remained rather stable but, at 6.0 gN-TAN L-1, the expression level increased considerably for a number of ribotypes that are related to potentially syntrophic genera (e.g. Clostridium, Bellilinea, Longilinea, and Bacteroides). The predominance of hydrogenotrophy at high ammonia levels clearly points to the occurrence of the syntrophic acetate oxidation (SAO), but known SAO bacteria were only found in very low numbers. The potential role of the identified bacterial and archaeal taxa with a view on SAO and on stability of the anaerobic digestion process under ammonia stress has been discussed.
Keywords: Active microbiome; Ammonia; Anaerobic-digestion; C-isotopic biogas fractionation; Syntrophic-acetate-oxidation (SAO).
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