Biological removal of phosphorus and nitrogen from wastewater : new insights from metagenomic and metatranscriptomic approaches

Abstract

The study was conducted to reveal the insights of microbial diversity, functional profile and gene expression of microorganisms responsible for enhanced biological phosphorus removal (EBPR) and hydrogen-oxidizing autotrophic denitrification mainly by using metagenomic and metatranscriptomic analysis based on high-throughput sequencing.

Two sequencing batch reactors (SBRs) were operated to remove phosphorus (15 mg/L) from synthetic wastewater. The integrated metagenomic and metatranscriptomic approaches were adopted to retrieve a nearly complete draft genome of Candidatus Accumulibacter phosphatis (referred to Accumulibacter) in Clade IB (CAP IB HKU-1) from one of the SBRs treating saline wastewater. The CAP IB HKU-1 draft genome, being different from CAP IIA UW-1, does not possess the phosphotransferase in polyphosphate metabolism and V-ATPase for orthophosphate transport. Additionally, unlike CAP IA UW-2, CAP IB HKU-1 carries the genes for carbon fixation and nitrogen fixation. The metatranscriptomic results revealed that the most significantly up-regulated genes in CAP IB HKU-1 from the anaerobic to the aerobic phase were responsible for assimilatory sulfate reduction, genetic information processing and phosphorous absorption, while the down-regulated genes were involved in N2O reduction, PHA synthesis and acetyl-CoA formation. From another SBR, a draft genome affiliated to Accumulibacter Clade IIC (CAP IIC HKU-2) was reconstructed using two metagenomic sequence data sets. Comparative genomic analysis demonstrated that Accumulibacter of Clades IA, IB, IIA and IIC conserved the genes encoding for enzymes in glycolysis, the TCA cycle, acetate uptake, PHA synthesis and polyP metabolism, but differed in the abilities of nitrate reduction, nitrogen fixation and carbon fixation.

The abundances of the Accumulibacter clades in 18 activated sludge (AS) samples from the globally distributed sewage treatment plants (STPs) were quantified by the qPCR-ppk1 assay. Clades IIC and IID were found to be dominant among the five Accumulibacter clades in 11 AS samples. And two novel Accumulibacter Clades IIH and II-I were identified. The results indicated that the wastewater characteristics could be more important to determine the proliferation of Accumulibacter clades in STP AS systems rather than the geographic location.

Geographical distribution of putative polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) among 14 globally located STPs and their monthly microbial variation in AS of Sha-Tin STP over five years were studied by using 16S rRNA gene pyrosequencing. The structure of EBPR-related microbial community should be the result of interaction of multiple operational variables and wastewater characteristics. Thauera-dominated (with relative abundances of 47% - 62%) microbial communities carrying out hydrogenotrophic denitrification were successfully enriched from various seed sludges and characterized by 16S rRNA gene Illumina high-throughput sequencing. Nitrogen removal rates of the enriched culture were comparable to that of the model organism Paracoccus denitrificans.

A genome binning pipeline was proposed to retrieve the dominant genome from an enriched hydrogenotrophic denitrifying consortium using metagenomic sequence data. A draft genome (Thauera R4) affiliated with a novel Thauera species was assembled by employing the strategies of coverage clustering, GC content filtering and paired-end tracking. Genes encoding enzymes for hydrogen oxidation, carbon metabolism and denitrification were intensively analyzed to provide new insights of this microbe.