| Trichloroethylene(TCE) was widely used in dry cleaning, medicine anesthesia, pesticide and degreasing industries as a solvent. However,it has classified into the list of priority pollutants and suspected carcinogen by many countries due to its toxic essence to creatures, it has already become the most detected chlorinated organic compounds in subsurface water attribute to artificial improper disposal and leak. It is significant to remediate TCE-contaminated groudwater effectively. Microbial anaerobic reductive dechlorination was a promising technique among all the remediation technologies owned to its superiority of inexpensive and environmentally friendly properties.At present, microbial resources for remediation were deficient, researches on mix clutures were somewhat less than isolates. Related studies were hardly reported in China. Thus this thesis regards an enrichment that dechlorination TCE to ETH as the research object and firstly systematically analyzed its degradation characteristics and molecular biological properties. Based on research results above we further focus on its adaptability under disadvantage conditions. Then, the carbon isotope fractionation when dechlorination of chlorinated ethene by this culture was studied. Finally, remediation effects by in-situ biostimulation and bioaugmentation in laboratory-scale study were also investigated.Firstly, researches on degradation characteristics and molecular biological properties of this enrichment culture showed that this enrichment culture was capabale of dechlorinating TCE stepwise to ETH with methanol, sodium lactate and hydrogen(sodium acetate) as electron donors, methanogenesis was coexisted with dechlorination when all these substracts served as electron donors, in addition, six chlorinated ethylene were all available to serve as electron acceptor. All the results obtained above indicated that this enrichment culture was composed of many kinds of microorganisms besides D.mccartyi,especially rather than one types of D.mccartyi.Dechlorinating efficiency of this enrichment culture was greatly suppressed under the unfavourable conditions such as a temperature of 10 OC or a acidity of p H=6. However, it’s thrilled that TCE eventually was enabled to slowly translated to ETH with adequate time.The dynamic experiments based on Monod equation revealed that the mix culture have higher maximum specific degradation rate in dechlorinating of TCE and cis-1,2-DCE than that of VC, while the half rate constants of these three chlorinated ethylene obtained no obvious difference. Microbial community structure achieved by high throughput sequencing analysis showed that besides D.mccartyi, the non-dechlorinating organisms within mixed cultures were abundant, the representative including acetobacter, methanogens, et. al. And these organisms were dominant organisms with a higher ratios than the functional dechlorinator. The PCR with specific primers target D.mccartyi 16 S r RNA and functional genes received positive results also consolidating the conclusion that the culture was capabale of dechlorinating TCE to ETH.Functional dechlorinators and non-dechlorinators were coexisted in the mix culture, we further explored their roles and respons under some disadvantage conditions. The results showed thatdechlorinating ability of this mix culture was hardly influenced in the absense of cobalamin in the medium, an essential substance for D.mccartyi. Further addition of archaeal inhibitor 2-BES and bacterial inhibitor ampicillin to the cobalamin-absent medium resulted in distinct results. The 2-BES treatment was capable of compeletely dechlorinating TCE to ETH with consecutive transfer although with a lower rate, while the ampicillin-amended treatment can only unaffected by exogenous supplementing of cobalamin after a consecutive transfer. high throughput sequencing analysis indicated that the unclassified Peptococcaceae and unclassified Spirochaetaceae were the most potential candidates denovo synthesis of this substance in this mix culture.Dechlorinating rate of TCE by this enrichment culture was greatly suppressed when exposure the consortium to oxygen, the reaction was reinitialized with the consumption of oxygen and ethene was eventually observed. The q PCR results indicated that D.mccartyi strainscontaining tce A gene were less sensitive than D. mccartyi containing vcr A gene to the presence of oxygen. Multiple non-dechlorinators within the enrichment culture were likely to be pivotal in scavenging the oxygen present in the medium to recreate an anaerobic environment which is suitable for the D.mccartyi to survive and reinitialize the reaction. Reversibility experiment demonstrated that the oxygen exposed culture could retrieve when inoculated it back to the anaerobic medium. Dechlorinator within the enrichment cluture were responsible for dechlorinating TCE to ETH, the non-dechlorinators, also play a crucial role in maintaining stability of the mix culture and offer better resistance to the harsh condition than the pure strain.Then, evaluation of in-situ bioremediation effectsby concentration measurement was always affected by physical factors such as adsorption, dilution, volatilization, dissolution, et.al. C-CSIA technique was thus emerged and developed, appied this technique to complex enrichment cultures, however, was not adequately studied. Therfore, the carbon isotope fractionation when dechlorination of chlorinated ethene by this culture was studied. The results showed thatthere are obvious carbon isotopic fractionation effects when TCE and cis-1,2-DCE were degraded by the enrichment culture, and the 13 C enrichment effects in the residual cis-1,2-DCE was stronger than TCE, ?C of-7.24±0.59‰ and-14.60 ±1.71‰ for TCE and cis-1,2-DCE degradation by this enrichment culture were obtained under the standard conditions. Variation of cultivate factors, microbial community structure composition, et.al.,obtained no statistical difference ?Cwithin the 95% confidence interval, which indicated the specificity and stability of ?C of this culture. The tce A and the vcr A gene-containing D.mccartyi are coexisted in the enrichment culture, the carbon stable isotope analysis conducted in this study indicated that two of the functinal D.mccartyi intrinsic in this enrichment culture are responsible for different steps of TCE reduction.CSIA proved to be an effective tool to calculate bioremediation efficiency due to its superiority of excluding the physicalinterference and solely concentrating on targetcompound.Finally, remediation effects by in-situ biostimulation and bioaugmentation in laboratory-scale study were investigated. The results showed that advection with groudwater and moving downstream due to gravity effect were the main reasons led to TCE distribution in simulation tanks, the remediation effects in the course of 33 weeks by these two strategies, however, obtained opposite results. Biostimulationobtained no obvious results, whilecis-1,2-DCE as well as ETH were detected the 19 th weeks in the bioaugmentation tank. It was found out that the composition of indigenous microorganism in simulation tanks was complex, the addition of electron donors stimulated some special fermentative microorganism. However, the absence of D. mccartyi that completely reductive dechlorination of TCE to ETH compeled the bioaugmentation way to obtained prospective results.In the AU simulation tank, initiation of TCE reduction was not monitored until the 19 th weeks likely due to the low temperature condition and the low level of functional microorganisms. Carbon-CSIA result indicated that physical factors impacted TCE concentration and distribution in the simulation tanks didn’t result in carbon isotope fractionation effect. The steady δ13C values of TCE before dechlorination initiationsimultaneously excluded other approaches that probably transformation of TCE. Further calculation of degradation rate by various ways obtained different results after dechlorination, the resultscalculated by carbon isotope composition datas, however,reflecting the intrinsic biodegradation. |
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