Degradation of di (2-ethylhexyl) phthalate by marine sediment bacteria and identification of key degraders and degradative genes

Abstract

Di (2-ethylhexyl) phthalate (DEHP) is a toxic phthalate ester (PAE) plasticizer that is predominantly detected in marine sediment and biota. DEHP degradation by bacteria from several environments has been studied, but very little is known about marine sediment bacteria that can degrade DEHP and other PAEs. Therefore, in this study, we enriched a bacterial consortium C10 that can degrade four PAEs of varying alkyl chain lengths (DEHP, dibutyl phthalate, diethyl phthalate, and dimethyl phthalate; separately and as a mixture) from marine sediment. The major bacterial genera in C10 during the degradation of the PAEs were Glutamicibacter, Ochrobactrum, Pseudomonas, Bacillus, Stenotrophomonas, and Methylophaga. Meanwhile, Brevibacterium, Ochrobactrum, Achromobacter, Bacillus, Sporosarcina, and Microbacterium populations were enhanced by DEHP intermediates (monoethylhexyl phthalate, 2-ethylhexanol, phthalic acid, and protocatechuic acid). Through network analyses, it was predicted that Bacillus, Stenotrophomonas, and Microbacterium were the key phthalate-degraders in C10. Twenty-one isolates were obtained from C10 through selective isolation and the best DEHP-degraders were Microbacterium sp. OR21, Stenotrophomonas acidaminiphila OR13, Microbacterium sp. OR16, Sporosarcina sp. OR19, and Cytobacillus firmus OR20 (84.5, 83.7, 59.1, 43.4, and 40.6% degradation of 100 mg/L DEHP in 8 d), thus lending support to the prediction of key degraders. This is the first report of DEHP degradation by S. acidaminiphila, Sporosarcina sp., and Cytobacillus firmus. Furthermore, several isolates of Bacillus, Microbacterium, Stenotrophomonas, and Sporosarcina could utilize DEHP intermediates as sole carbon source. Isolates Microbacterium sp. OR21, Microbacterium sp. OR16, and Sporosarcina sp. OR05 were selected based on DEHP degradation efficiency, ability to utilize DEHP intermediates, and predicted interactions. A defined consortium of these three isolates (A02) could degrade multiple PAEs more efficiently than the individual strains, which could be attributable to synergistic interactions among the bacterial strains in A02. Furthermore, Consortium A02 could degrade DEHP intermediates (monoethylhexyl phthalate, phthalic acid, and protocatechuic acid). Bioaugmentation with A02 could enhance DEHP degradation (80% in 26 d) by indigenous microbes in microcosms of saline sediment from a shrimp farm. Genomic analyses of the three strains revealed the presence of several phthalate-degradation genes. Based on this information and experimental degradation results, the pathway of PAE degradation by Consortium A02 was predicted. Thus, this study reveals as yet unknown insights into the PAE-degrading potential of marine sediment bacteria and demonstrates a simple approach for the prediction and isolation of key pollutant-degraders from complex bacterial communities. Furthermore, a defined consortium with potential applicability for DEHP/PAE degradation in saline environments was developed.