Microbial diversity of a full-scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing.

Tiago Palladino Delforno,Valéria M Oliveira,Isabel K Sakamoto,Gileno Vieira Lacerda Júnior,Maria Bernadete A Varesche,Melline F Noronha

doi:10.1002/mbo3.443

Tiago Palladino Delforno, Valéria M Oliveira + Show 4 more

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https://doi.org/10.1002/mbo3.443

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Abstract

The 16S rRNA gene amplicon and whole‐genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide‐spectrum profiles of microbial composition and metabolic diversity from a full‐scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one‐carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

Highlights

The anaerobic digestion (AD) corresponds to a consolidated technology that is successfully applied in different wastes and influents for the conversion of complex organic compounds to CH4 and CO2, nutrient recovery, pollutant removal and/or energy (CH4 and H2), and fermentative compound production (Batstone & Virdis, 2014; Battistoni et al, 1997).The large AD application is only possible due to the occurrence of complex interactions of microorganisms belonging to Bacteria and Archaea domains (Zinder, 1984)
The authors carried out a systematic comparison using 33 metagenomic datasets of human-associated bacterial communities and 71 datasets derived from 16S rRNA amplicon sequencing and observed significant differences between the community structures due to the distinct experimental approaches used, suggesting that sample replicates and dataset sizes had smaller influence in the microbial profile than the DNA extraction methods or sequencing protocols
The average size of the reads obtained in this study (454 bp for 16S amplicon sequencing and 233 bp for whole-genome shotgun metagenomic (WGSM) sequencing) and the hypervariable regions analyzed differed between both strategies, potentially leading to different accuracy levels in the phylogenetic affiliation and distinct microbial community profiles (Wang et al, 2007)

Summary

Introduction

The anaerobic digestion (AD) corresponds to a consolidated technology that is successfully applied in different wastes and influents for the conversion of complex organic compounds to CH4 and CO2, nutrient recovery (phosphorus and sulfur), pollutant removal and/or energy (CH4 and H2), and fermentative compound (butyrate, butanol) production (Batstone & Virdis, 2014; Battistoni et al, 1997).The large AD application is only possible due to the occurrence of complex interactions of microorganisms belonging to Bacteria and Archaea domains (Zinder, 1984). Previous studies have reported that microbial composition of anaerobic digestion sludge is highly diverse (Cabezas et al, 2015). Information on the key players in anaerobic conditions and their interactions is not comprehensive. These results indicate a high genetic potential that has been poorly explored and open promising perspectives for biotechnological applications. WGSM sequencing has been shown as an excellent approach to assess the genetic potential of a microbial community, unraveling the phylogenetic composition, metabolic capacity, and functional diversity (Streit & Schmitz, 2004) of community members. Comprehensive information on the phylogenetic composition is highly dependent on the sequence number and in general the shotgun metagenomic sequencing is not deep enough. WGSM sequencing does not involve the biased amplification of 16S rRNA genes, and more precise, the organism abundances are still dependent on the DNA extraction and sequencing protocols used (Kalyuzhnaya et al, 2008; Morgan, Darling, & Eisen, 2010)

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