Characterisation of anaerobic digester microbiome in various operating conditions using next-generation and third-generation sequencing technologies

Abstract

Anaerobic digestion is a common wastewater treatment technology that alleviates pressure on sludge disposal and generates energy via combustion of biogas. Diverse groups of microorganisms are responsible for the biodegradation, To manipulate the process better, we study the anaerobic digestion process through the lens of genomic analysis. In the past decade, the characterisation of the microbial community in anaerobic digestion was primarily achieved by using the next-generation sequencing technologies. However, the next-generation short-read approach has inherent primer bias and low phylogenetic resolution. A more accurate phylogenetic identification is desired. In this study, we employed the next generation sequencing offered by Illumina and the third-generation sequencing technology offered by Pacific Bioscience to characterise microbiome in anaerobic digesters operated under different conditions. From sample preparation to data analysis, this work serves as a reference to utilise third-generation sequencing for 16S survey. The high-quality circular consensus sequences provided a superior resolution for diversity profiling. Unexpectedly, Methanothrix concilii and Smithella were found to have universal presence in different operating conditions. Unique methanogenic and syntrophic species (Methanosarcina horonobensis, Methanosarcina flavescens, Thermanaerovibrio acidaminovorans, Ca. Cloacimonas acidaminovorans, etc.) were observed in divergent operating conditions. The species-level information facilitated our understanding of their survival niches and how they become dominant in respect of the operating conditions. We also compared the two sequencing technologies in terms of taxonomic classification and diversity indices. The results reflected the use of different technology might lead to inconsistent interpretation of the microbiome. Last but not least, shotgun metagenomics sequencing and mcrA gene amplicon sequencing were performed on lab-scale microaerated fermenters. The facultative bacteria promote hydrolysis by switching to more thermodynamic favourable pathways under microaerated condition, ultimately facilitating methane production.