In situ identification of polyhydroxyalkanoate (PHA)-accumulating microorganisms in mixed microbial cultures under feast/famine conditions

Donhatai Sruamsiri,Benjaporn Boonchayaanant Suwannasilp,Parinda Thayanukul

doi:10.1038/s41598-020-60727-7

Abstract

The accumulation of plastic waste in the environment has become a serious environmental problem worldwide. Biodegradable plastics, such as polyhydroxyalkanoate (PHA), could serve as green alternatives to petroleum-based plastics. In this study, a mixed microbial culture was enriched under feast/famine conditions using a sequencing batch reactor (SBR) with acetate as a carbon source. The enrichment could accumulate a maximum PHA concentration of 32.3% gPHA/g mixed liquor suspended solids (MLSS) in the 12th cycle of SBR operation. The microbial community in this sludge sample was analyzed using 16 S rRNA gene amplicon sequencing (MiSeq). The results showed the dominance of Proteobacteria, represented by Alphaproteobacteria (13.26% of total sequences), Betaproteobacteria (51.37% of total sequences), and Gammaproteobacteria (23.44% of total sequences). Thauera (Betaproteobacteria) had the highest relative abundance, accounting for 48.88% of the total sequences. PHA-accumulating microorganisms in the enrichment were detected using fluorescence in situ hybridization (FISH) and a fluorescent dye, Nile blue A. Alphaproteobacteria and Betaproteobacteria were capable of accumulating PHA, while no Gammaproteobacteria were detected. Thauera spp. from Betaproteobacteria constituted 80.3% of the total PHA accumulating cells.

Highlights

Massive generation of plastic waste has long been considered a worldwide environmental problem
The mixed liquor suspended solids (MLSS) decreased at the end of the cycles, www.nature.com/scientificreports suggesting biomass decay during the second half of the cycles, consistent with the famine conditions observed based on the chemical oxygen demand (COD) levels
The average COD removal efficiency for all of the sequencing batch reactor (SBR) cycles was 92.0 ± 1.9%, which was comparable to typical aerobic treatment processes, suggesting that this system can still effectively serve as a wastewater treatment system

Summary

Introduction

Massive generation of plastic waste has long been considered a worldwide environmental problem. Previous research has investigated microbial communities in PHA production systems with mixed microbial cultures under feast/famine conditions. PHA can be stained using fluorescent dyes such as Nile blue A This technique, supported by FISH, enables us to identify the groups of microorganisms that accumulate PHA. With the current advances in molecular techniques, more complete information on microbial communities can be achieved via next-generation sequencing, which, in combination with FISH and the fluorescence staining technique, can greatly assist in identifying PHA-accumulating microorganisms. This study aimed to analyze the microbial community in a mixed-culture PHA-accumulating system under feast/famine feeding conditions using 16 S rRNA gene amplicon sequencing (MiSeq) and FISH in conjunction with the fluorescence-based PHA staining technique. This study expands our knowledge of the microorganisms capable of accumulating PHA in situ in mixed microbial cultures, certain key microorganisms in which may not be individually cultivable

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