Graduate Thesis Or Dissertation
 

Kinetic and molecular effects of sulfate reduction on a dechlorinating culture under chemostat growth conditions

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  • Chlorinated ethenes are common groundwater contaminants that may be treated through in-situ bioremediation. Relationships between the reducing environment, available electron donors and acceptors, reaction kinetics, and microbial community composition must be further understood to successfully engineer remediation schemes in the complex subsurface environment. This thesis work investigated the effect of sulfate reduction on a dehalogenating culture grown under very controlled conditions. Two chemostats containing the Point Mugu (PM) culture were maintained using an influent containing tetrachloroethene (PCE) as an electron acceptor and lactate as a fermenting electron donor. One of these chemostats, PM-5L, was used as a control, while the influent to the PM-2L chemostat was amended with sulfate on an equal electron-equivalent basis to PCE. The effluent composition of these two chemostats was monitored over time, and periodic batch rate tests and molecular analyses were performed with cells harvested from the chemostat to elucidate the changes in performance and microbial composition within the chemostat culture. A numerical model based on Monod kinetics with competitive inhibition was developed to fit data from batch PCE-to-ethene rate tests by simultaneously solving for the k[subscript m]X parameters of each CAH dechlorination step given a standard set of Ks values. Non-linear regression of multi-equilibrium VC Monod test data provided the Monod parameters (k[subscript m]X and K[subscript s]) for VC dechlorination. These parameters were used to quantify changes in dechlorinating performance of each chemostat over time and compare the performance of the two chemostats. The effluent chemical composition of the PM-5L chemostat appeared to be steady after approximately six residence times, with 1120 μM PCE being transformed to 98% ethene and 2% VC, H₂ tensions remaining between 2-3 nM, acetate around 4.3 mM, and biomass around 23 mg protein/L. Batch rate tests during this time showed rapid rates of transformation for all CAHs, agreeing well with chemostat performance. The k[subscript m]X parameters derived from the PCE-to-ethene data and the pseudo-mixed order rate coefficient of VC dechlorination determined through multi-equilibrium VC Monod tests also remained essentially constant over the one-year period of study. Changes in the PM-2L chemostat performance following the initiation of sulfate reduction were observed. Sulfate reduction began almost immediately after its addition to the chemostat, and total sulfide concentrations rose to 100-300 μM. Chemostat performance with respect to CAH and H₂ concentrations was roughly steady over approximately 250 days, with PCE being dechlorinated to 9 μM cis-DCE, 230 μM VC, and 860 μM ethene under H₂ tensions around 4 nM. Total protein levels nearly doubled during this period, increasing from 25 to 47 mg protein/L. Sulfate reduction then rapidly increased to completion, resulting in 620-720 μM dissolved sulfide and a decrease in the H₂ concentration to 2 nM. At this time, the extent of PCE dechlorination also decreased to 280, 760, and 80 μM cis-DCE, VC, and ethene, respectively. Batch rate tests showed a decrease in all chlorinated ethene reduction rates; however, VC dechlorination was the most effected by sulfate reduction, showing a 97% reduction in rate following sulfate addition. Multi-equilibrium VC rate tests were impossible to conduct following the sharp increase in sulfate reduction in the chemostat due to lack of measurable dechlorination over a days' time. A simple chemostat model employed Monod kinetics for the series of CAH reactions to determine the steady-state extent of dechlorination in the chemostat predicted by the best-fit kinetic parameters of each PCE-to-ethene rate test. The extent of dechlorination was well-modeled for the PM-5L chemostat when a H₂ limitation factor of 0.3 was applied to the rate of each CAH. Using a dual Monod kinetic model with H₂ as the electron donor, the limitation factor corresponded with a half-velocity coefficient (K[subscript H]) of 4.6 nM. When this same K[subscript H] was used to model the PM-2L chemostat, a greater extent of dechlorination was predicted than what was observed in the chemostat, possibly suggesting other inhibitory factors of dechlorination were present in the PM-2L chemostat. DNA and RNA analyses of cells periodically harvested from the chemostat were performed by Ian P.G. Marshall at Stanford University. His work revealed shifts in the chemostats' Dehalococcoides population over time. Analysis of the PM-5L culture using the H2ase chip he developed and a clone library of hupL genes showed that the Dehalococcoides population was predominately related to strains BAV-1 or CBDB1/GT and did not undergo a significant shift over time. Clone libraries constructed for cells harvested from the PM-2L chemostat revealed two shifts in the chemostat Dehalococcoides population. A genetically homogenous strain relative of BAV-1 was eliminated following a decline in chemostat H2 tensions from around 27 nM to 2 nM and a corresponding increase in dehalogenation efficiency. In a second shift, a strain 195 relative outcompeted the CBDB1/GT relatives following enhanced sulfate reduction. A general decrease in the Dehalococcoides concentration within the chemostat culture was also suggested by qPCR analysis of Dehalococcoides 16S genes. These molecular results correlated well with the decline in VC reduction rates reported in batch kinetic tests given the characteristic co-metabolic VC reduction of the dominant strain 195 relative and overall lower concentrations of Dehalococcoides. Our work suggests that sulfate reduction in the anaerobic chemostat environment caused a shift in the dechlorinating microbial population to a strain with less efficient VC reduction. This shift was also accompanied by a decline in Dehalococcoides concentration within the culture. Both of these factors contributed to the decline in chlorinated ethene transformation rates observed through batch rate tests. Competition for H₂ was not expected to be the primary cause for the changes observed in the PM-2L chemostat. Long-term batch tests involving the control culture are proposed to elucidate whether sulfide or other factors of sulfate reduction are responsible for this shift, and to confirm the suspected role of H₂ competition between dechlorination and sulfate reduction in the chemostat-grown PM culture.
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