Autochthonous Bioaugmentation-Modified Bacterial Diversity of Phenanthrene Degraders in PAH-Contaminated Wastewater as Revealed by DNA-Stable Isotope Probing

Abstract

To reveal the mechanisms of autochthonous bioaugmentation (ABA) in wastewater contaminated with polycyclic aromatic hydrocarbons (PAHs), DNA-stable-isotope-probing (SIP) was used in the present study with the addition of an autochthonous microorganism Acinetobacter tandoii LJ-5. We found LJ-5 inoculum produced a significant increase in phenanthrene (PHE) mineralization, but LJ-5 surprisingly did not participate in indigenous PHE degradation from the SIP results. The improvement of PHE biodegradation was not explained by the engagement of LJ-5 but attributed to the remarkably altered diversity of PHE degraders. Of the major PHE degraders present in ambient wastewater ( Rhodoplanes sp., Mycobacterium sp., Xanthomonadaceae sp. and Enterobacteriaceae sp.), only Mycobacterium sp. and Enterobacteriaceae sp. remained functional in the presence of strain LJ-5, but five new taxa Bacillus, Paenibacillus, Ammoniphilus, Sporosarcina, and Hyphomicrobium were favored. Rhodoplanes, Ammoniphilus, Sporosarcina, and Hyphomicrobium were directly linked to, for the first time, indigenous PHE biodegradation. Sequences of functional PAH-RHD genes from heavy fractions further proved the change in PHE degraders by identifying distinct PAH-ring hydroxylating dioxygenases between ambient degradation and ABA. Our findings indicate a new mechanism of ABA, provide new insights into the diversity of PHE-degrading communities, and suggest ABA as a promising in situ bioremediation strategy for PAH-contaminated wastewater.

Stable-Isotope Probing-Enabled Cultivation of the Indigenous Bacterium Ralstonia sp. Strain M1, Capable of Degrading Phenanthrene and Biphenyl in Industrial Wastewater

“…Conventional biological treatment is considered to be a cost-effective and ecofriendly approach for eliminating PAHs and PCBs from industrial wastewater (12,34,40). However, a major drawback of biological treatment is the fact that the majority of indigenous microbes responsible for degradation are not cultivable (16,41), and the active indigenous PAH or PCB degraders are difficult to isolate and study. DNA-SIP is a cultivation-independent technique that provides the opportunity to link the identities of microorganisms to their in situ functions (31,42).…”

“…Although various cultivable PAH and PCB degraders have been isolated from contaminated sites (23,24,62), their application for bioaugmentation in soil remediation and wastewater treatment is subject to certain restrictions, because their survival and degradation ability are highly dependent on environmental conditions (16). Autochthonous bioaugmentation has been proposed as an alternative approach to overcome these difficulties but suffers from the fact that the active indigenous degraders are scarcely cultivable (15,41).…”

“…DNA was extracted from the cell pellets by centrifuging 50 ml of each sample from all treatments and was quantified using an ND-2000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). DNA ultracentrifugation was conducted as described previously (16). The hypervariable V4 region of bacterial 16S rRNA gene fragments was amplified for DNA extracted from each fraction derived from the [ 12 C]PHE, [ 12 C]BP, [ 13 C]PHE, and [ 13 C]BP microcosms using primer set 515f/806r (Table 1), as described previously (12).…”

“…The bioremediation or microbial degradation of PAHs and PCBs involves the use of microorganisms to remove the pollutants from wastewater or to neutralize them by conversion to carbon dioxide, water, and other by-products (13,14). This is considered to be a cost-effective and ecofriendly remediation option and is therefore used as the dominant treatment strategy in industrial WWTPs (15,16). Microbial degradation or mineralization depends on the metabolic potential of microorganisms to detoxify or transform these pollutants.…”

“…Phenanthrene (PHE) and BP have been used as model compounds for the study of PAH and PCB degradation (32,33). Stable-isotope probing studies have successfully explored the biodegradation of PAHs or PCBs in real-world habitats, such as industrial wastewater (16,34), seawater (35), and soils or sediments (36,37). However, previous studies have only identified indigenous degraders of PAHs or PCBs individually, whereas degraders of both PAHs and PCBs have not been reported.…”