A systems-level understanding of electron flow in TCE-dechlorinating microbial communities using modeling and molecular biology tools

Groundwater and soils have been frequently contaminated by trichloroethene (TCE), perchloroethene (PCE) and other chlorinated compounds in the U.S. and worldwide, in spite of their established toxicity and mutagenicity towards many organisms, including humans. In order to protect public health, bioremediation using Dehalococcoides-containing microbial communities is a promising approach to reach ecotoxicological-safety endpoints. The overall goal of this research is to understand electron flows in complex dechlorinating microbial communities, and to develop mathematical models to predict the performance of the microbial communities in different environmental conditions. To accomplish these goals, we first studied the electron flow and material exchange of constructed TCE-dechlorinating consortia. We also applied emerging molecular techniques to study TCE-dechlorinating microbial communities under different remediation conditions. Furthermore, we developed integrated thermodynamic and kinetic models to predict the dechlorination performance and microbial growth of syntrophic consortia under batch and continuous-flow conditions, and the suite of models were further validated using enrichment cultures./nThe first objective of this research was to understand the material and energy exchange between Dehalococcoides and its supporting syntrophic bacteria. We investigated dechlorination activity, cell growth, cell aggregate formation, and global gene expression of D. mccartyi strain 195 (strain 195) grown with Syntrophomonas wolfei in co-cultures amended with butyrate and TCE. By applying thermodynamically consistent rate laws to study the electrons flows in the co-culture, we found that the growth rates of the two species were strictly coupled by H2 transfer, and that the growth yield of syntrophic bacteria and the ratio maintained in the co-cultures were mainly controlled by thermodynamics. We demonstrated, for the first time, that D. mccartyi could form cell aggregates with its supporting fermenter S. wolfei on butyrate. Furthermore, we found carbon monoxide (CO) may serve as a supplemental energy source for S. wolfei during syntrophic fermentation with strain 195, and that the observed increased cell yields of strain 195 is likely due to the continuous removal of CO in the co-culture./nIn order to understand the microbial community structure shift from "feast-and-famine" condition (semi-batch) to the continuous feeding of low nutrients condition (completely-mixed flow reactor (CMFR)), molecular techniques based on 16S I-tags and metagenomic sequencing were applied to investigate the dechlorinating community structural shift after transition from semi-batch to a long-term steady-state CMFR condition. A Dehalococcoides genus-wide microarray was also applied to study the transcriptional dynamics of D. mccartyi strains within the CMFR community that was grown in the continuous-flowing diluted, nutrient poor environment. I-tags and metagenomic sequencing analysis revealed that dominant species in the CMFR shifted significantly from the semi-batch culture condition while the ratio of D. mccartyi was maintained at relatively stable levels. Transcriptional analysis identified tceA and vcrA to be among the most expressed genes in the CMFR, hup and vhu were more critical hydrogenases utilized by Dehalococcoides sp. in the continuous-flowing system. In contrast, corrinoid-related uptake and modification genes were expressed at lower levels in the CMFR than in the semi-batch culture during active dechlorination./nA systems-level approach was applied to determine accurate kinetic parameters involved in reductive dechlorination from simple constructed syntrophic consoria to complex microbial communities. The results demonstrated that the kinetic parameters involved in reductive dechlorination were in similar ranges for simple and complex Dehalococcoides-containing cultures. Cell growth calculations showed H2 was the most sensitive factor limiting the growth of H2-utilizing microorganisms involved in dechlorinating communities. High concentrations of acetate resulted in slower dechlorination rates by inhibiting the growth of specific fermenting bacteria. High sulfate concentrations also hindered dechlorination performance due to either sulfide inhibition or competition with sulfate reduction. The mechanism for observed slower dechlorination rates with lower bicarbonate concentrations was not clear and further experiments need to be conducted to evaluate the role of bicarbonate in reductive dechlorination communities./nBased on the knowledge obtained in the previous studies, an integrated thermodynamic and kinetic model was developed to predict reductive dechlorination and cell growth under batch growth conditions. The model parameters calculated to fit the experimental data were at the same levels as those determined experimentally. The resultant model accurately captured the dechlorination kinetics in two Dehalococcoides-containing syntrphic co-cultures using different fermenting substrates. The model was validated at different donor to acceptor ratios in syntrophic co-cultures and in syntrophic tri-cultures and enrichment cultures performing hydrogenotrophic methanogenesis. The sensitivity of kinetic parameters on model stability was tested. Half velocity constants and inhibition coefficients were found to be the most sensitive factors affecting model predictions./nThe significance of this research is to provide a more fundamental understanding of the metabolic exchange and energy transfer among the key players of TCE-dechlorinating communities, as well as the physiology of dechlorinating microbial communities experiencing different environmental stresses. The integrated thermodynamic and kinetic models developed in this study could be used as a platform to incorporate more biological processes under different experimental conditions. The knowledge developed in this research will aid practitioners to better design, monitor and optimize future in situ bioremediation systems.

Advisor: Alvarez-Cohen, Lisa