Comparison of genomic DNA extraction methods for the next-generation sequencinganalysis of bacterial community in the activated sludge after exposing to 1,3-butadiene

Wipoo Prasitwuttisak, Kanyapak Sapsanyakorn, Vasimon Ruanglek, Thunyarat Pongtharangkul


The effective DNA extraction method is essential for an investigation of environmental microbial communitiesusing next-generation sequencing (NGS).In this study, two DNA extracting protocols were evaluated based on yield and purity of the DNA. While an Al-precipitation method gave higher DNA yield, a commercial soil DNA extraction kit gave higher DNA purity and therefore selected as a suitable extracting protocol. This study also investigated effects of 1,3-butadiene (1,3-BD) exposure on bacterial community in activated sludge samples (designated as SL2 and SL4) collected from 2 different wastewater treatment facilities of petrochemical industry. While SL2 represented a bacterial community with no prior exposure to 1,3-BDand/or toluene, SL4 represented a bacterial community with prior exposure to both compounds. The sludge sampleswere enriched with100 ppm of 1,3-BD (contributing 8,600 ppm of toluene as a solvent) for 6 weeks.The sludge samples exposed with only toluene were also conducted in parallel. Changes in microbial community profile was monitored based on 16s rDNA using next-generation sequencing platform. The sequences were assigned to the phylum as well as lower levels to represent the relative microbial abundance in each sample. Before an enrichment (Week 0), Proteobacteria(Alpha- and Beta-proteobacteria)were the dominant bacterial populations in both sampleswhereas Firmicutes was found only in SL2.After an enrichment withtoluene,Firmicutes (Bacilli) was found to be the dominated taxa in SL2 while Proteobacteria (Alpha- and Beta-proteobacteria) decreased over time. In case of 1,3-BD exposure, Proteobacteriaincreased in both samples. Taxonomic classification into class level indicated that Beta-proteobacteria (mainly Burkholderiales) was the highest dominated bacterial populations in both samples exposed with 1,3-BD suggested that Burkholderialesmay involve with degradation of 1,3-BD or its metabolites. An understanding on the bacterial community in toxic waste as well as how they evolve after an exposure can be applied further for selection of remediation solutions and environmental management.

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