The Anaerobic Biotransformation Of Hexachlorocyclohexane Isomers,Chlorinated Benzenes And Benzene In Contaminated Groundwater

Groundwater is an important resource in China and has been used widely for drinking,irrigation and industry.However,with the rapid social economic development,groundwater resources stress and water pollution loads have gradually increased.As China is a vast agricultural country,the hexachlorocyclohexane(HCH)isomers have been used as pesticide extensively since 1950s,which resulted in heavily contaminated soil and groundwater.Chlorinated benzenes(CBs)are also typical contaminants in groundwater in China,as a result of their intensive use in the manufacture of dyes,herbicides,pesticides and other chemicals,as solvents or reactants.Benzene is a toxic petroleum-derived compound that is a widespread and problematic groundwater contaminant owing to accidental spills and releases at industrial facili-ties,oil refineries or from pipelines and mining operations.The HCH isomers,chlorinated benzenes and benzene are persistent anaerobically,accumulate in the food chain and are(potential)carcinogens.They are all included in the priority pollutant list by Chinese govemnet.Among the groundwater remedy alternatives,in situ bioremediation has the advantages of cost-effectiveness,less disruption,no re-contamination and has draw more and more attention.The availability of dissolved oxygen is often limiting due to low water solubility and rapid consumption by aerobic microorganisms,and the difficulty and expense associated with injecting sufficient oxygen underground.The anaerobic transformation processes are important determinants of the fate of these compounds in situ.In this study,we obtained four anaerobic enrichment cultures,derived from HCHcontaminated soil,capable of sustainably dechlorinating α-,β-,γ-,and δ-HCH to benzene and monochlorobenzene(MCB).The dechlorination rates of the four isomers increased from 3.3±0.7 to 11.9±2.1 μM/day averagely over 595 days of incubation.The ratios of benzene to MCB varied by isomer,i.e.,β(0.8±0.16)>α(0.57±0.13)>γ(0.13±0.02)>δ(0.06±0.01);this was consistent with the theoretical preferential dechlorination pathways based on the stereochemistry of the HCH isomers.The microbial community of the enrichment cultures was surveyed using the 16S rRNA gene amplicon sequencing.We observed significant increases in the relative abundance of Geobacter and Dehalobacter spp.,which were possibly involved in the dechlorination of HCH isomers.The α-and β-HCH enrichment cultures had similar microbial communities,yet different from that of the γ-or the δ-HCH dechlorinating cultures.This study provides the first direct comparison of shifts in anaerobic microbial communities induced by the dechlorination of distinct HCH isomers and uncovers candidate microorganisms responsible for the dechlorination of α-,β-,γ-,and δ-HCH.We set up a laboratory microcosm study using contaminated soil and groundwater from an industrial field site in Nanjing,China.The microcosm study demonstrated that 1,2,4-trichlorobenzene(TCB)was dechlorinated to 1,2-,1,3-,and 1,4-dichlorobenzene(DCB)in approximately 2%/10%/88%molar proportions.The DCB isomers were dechlorinated via MCB to benzene,and finally benzene was degraded under prevailing sulfate-reducing conditions.To identify the microbes responsible for the observed chlorobenzene dechlorination,the microbial community was surveyed using 16S rRNA gene amplicon sequencing.Members of the Dehalobacter and Dehalococcoides are reported to respire chlorobenzenes,however neither were abundant in our sediment microcosms.Instead,we observed a significant increase in the relative abundance of Dehalogenimonas from<1%to 16～30%during dechlorination of 1,2,4-TCB,1,2-DCB and 1,3-DCB over 19 months.Quantitative PCR(qPCR)confirmed that Dehalogenimonas gene copies increased by 2 orders of magnitude with an average yield of 3.9 ± 2.1 g cells per mol Cl-released(N=12).In transfer cultures derived from sediment microcosms,dechlorination of 1,4-DCB and MCB was carried out by Dehalobacter spp.with a growth yield of 3.0 ± 2.1 g cells per mol Cl~- released(N=5).This is the first time that any Dehalogenimonas has been shown to respire 1,2,4-TCB and DCB isomers.While anaerobic benzene degradation was mediated by microbes affiliated to a Deltaproteobacterium ORM2,previously associated with this activity.The relative abundance of ORM2 increased from less than 1%to 10～20%in the sediment microcosms after 379 days of incubation and to even over 50%in the enrichment cultures.The qPCR confirmed that the 16S rRNA gene copies increased from 104 copies/mL to 107 copies/mL sediment microcosms and to 108 copies/mL enrichment culture with a growth yield of 10~5～10~7 copies/μmol benzene consumed.The contaminated site in Nanjing was examined the natural attenuation using the technical protocol for evaluating natural attenuation of chlorinated solvents in groundwater proposed by United States Environmental Protection Agency(USEPA).The main contaminants were 1,2,4-TCB reaching concentrations up to 7300μg/L,DCB isomers,MCB and benzene.Over time,these contaminants naturally attenuated to below regulatory levels under anaerobic conditions.The trend of the concentrations of the contaminants revealed a pattern corresponding to reductive dechlorination,where parent compound 1,2,4-TCB concentrations decreased,while concentrations of daughter products(DCBs,MCB and benzene)increased.The site hydrogeochemical parameters including the low dissolved oxgen(0.5mg/L)and low concentration of nitrate,as well as the detection of nitrite and methane,suggested that the site was suitable for natural attenuation.The concentration of chloride in most contaminated zone was twice higher than that in the background,suggesting the dechlorination reactions might happen.The values of oxidation-reduction potential(ORP)in part of the monitoring wells were lower than -100mV,which would benefit the reductive dechlorination reactions.The hydrogeochemical parameters at the site revealed that the site has the potential of natural attenuation.Coupling the laboratory microcosm study results explained above,the chlorinated benzenes and benzene can be attenuated at the site.Moreover,unidentified organic compounds(measured as TOC),possibly aromatic compounds related to past on-site production processes,were fueling the dechlorination reactions in situ.The higher concentration of sulfate in the groundwater leaked during the used of sulfuric acid served as the electron acceptor for anaerobic benzene degradation.No exogenous compounds are needed to stimulate the microbial activities.In other words,the contaminants ate the site in Nanjing can be naturally attenuated by indigenous microbes without human intervention.