Microbiomes of High-Rate Anaerobic Digestors Reveal ‘Study’-Specific Factors and Limitations of Synthetic Wastewater

Anaerobic digestion (AD) is a key technology for the treatment of organic wastes and the production of renewable energy. The stability of the process hinges on the underlying microbial populations. Amplicon sequencing is increasingly used to characterise AD microbiomes, yet sequencing efforts have not translated to process engineering of the microbiome or prediction of failure using microbial tools. Using high-rate biofilm wastewater bioreactors as a study system, we aimed to i) discern trends in archaeal and bacterial diversity, ii) identify a core AD microbiome, iii) determine the functional stability of AD microbiomes, and iv) correlate taxa to experimental conditions. We analysed amplicon sequencing data from 32 high-rate anaerobic digestor studies (> 1,258 samples) at various operational conditions and applied a suite of statistical microbiome tools. We found that taxonomic archaeal diversity was highly study dependent, while functional diversity was highly shared across studies. A core AD microbiome was identified with > 100 bacterial genera and 6 archaeal genera which were present at > 1 % relative abundance in at least 50% of samples. Interestingly, we observed that microbiome stability was significantly impacted by the choice of real or synthetic wastewater, with synthetic wastewaters yielding a more stable and less complex microbiome. This was correlated to the abundances of 37 taxa in the synthetic wastewater, including 3 key methanogens (Methanothrix, Methanobacterium, and Methanosphaerula). This suggests that when synthetic wastewater is used in experimental studies, it may not result in an AD microbiome representative of real wastewater treatment systems.