Abstract
Integration of anaerobic and aerobic biodegradation is a perspective approach for remediating multiple volatile organic compounds (VOCs). The objective of this study was to elucidate aerobically degrading microorganisms of coexisting VOCs; toluene, benzene, and dichloromethane (DCM) in a microbial consortium with DNA-stable isotope probing, after anaerobically degradation of chlorinated ethylenes. The consortium was added with a 13C-labeled VOC and the other two unlabeled VOCs at a concentration of 30 mg/L each. DNA was extracted from the periodically sampled consortia according to degradation degree of the VOCs for 32 days. The DNA extracts were applied to next generation sequencing (NGS) to evaluate bacterial communities in the consortia, and density gradient fractionation was performed to clarify the bacteria that degraded the VOCs. Bacterial 16S rRNA gene amplicons in each fraction were analyzed with terminal restriction fragment length polymorphism (T-RFLP) using HhaI. The NGS confirmed the existence of Pseudomonas and Hyphomicrobium in the consortium. As for the T-RFLP, peaks of normalized DNA (558 bp with HhaI) in the treatment added with 13C-toluene shifted toward heavier fractions. The T-RF was derived from relatives of Pseudomonas stutzeri. In the treatment added with 13C-benzene, peaks of normalized DNA (201 bp with HhaI) shifted toward heavier fractions. The T-RF was originated from relatives of Pseudomonas alcaligenes. Such peak shifts did not occur in the treatment added with 13C-DCM, though Hyphomicrobium similar to a known DCM degrader existed. The results suggested that the bacteria involved in degrading toluene and benzene were inferred to be related species to P. stutzeri and P. alcaligenes, respectively. In sequential anaerobic and aerobic biodegradation, maintaining viability of nitrate-reducing bacteria such as Pseudomonas would be significantly useful for enhancing aerobic degradation of the aromatic compounds.
