Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing

In the past decade, the characterisation of the microbial community in anaerobic digestion was primarily done by using high-throughput short-read amplicon sequencing. However, the short-read approach has inherent primer bias and low phylogenetic resolution. Our previous study using Illumina MiSeq suggested that the heterogeneity of AD microbiome was operation-driven. To advance our knowledge towards the complexity of the AD microbiome, we performed full-length 16S rRNA gene amplicon sequencing using PacBio Sequel for a more accurate phylogenetic identification. To this end, purified DNA samples from 19 global anaerobic digesters were sequenced. Sixteen methanogenic archaea were identified at the species level. Among them, Methanosarcina horonobensis and Methanosarcina flavescens had significant presence under specific operating conditions. Methanothrix concilii presented in all digesters sequenced. Unexpectedly, over 90% of the Smithella detected were closely related to alkane-degrading Smithella strains D17 and M82, not Smithella propionica. Using LEfSe and network analysis, the interspecies relationship between the fermentative and syntrophic bacteria was addressed. Comparison of the short- and long-read sequencing results were performed and discussed. From sample preparation to data analysis, this work characterised the digester microbiomes in a superior resolution.