- The cometabolic ability of Burkholderia vietnamiensis G4 grown on aromatic substrates was first assessed by resting suspended cells, and subsequently for cells encapsulated with and without slow release compounds (SRCs). In Chapter 3, benzyl alcohol was assessed as a food-grade growth substrate for promoting cometabolism of trichloroethene (TCE), via the toluene-2-monooxygenase (T2MO) of Burkholderia vietnamiensis G4. Benzyl alcohol was evaluated by comparing the level of induction of the T2MO to that seen in toluene-grown cells. The level of T2MO expression was evaluated by two methods: 1) Activity based labeling (ABL); a gel assay method used to identify catalytically active monooxygenases (e.g. T2MO), and 2) resting suspended cell kinetic tests. Other substrates were also tested for their ability to support TCE cometabolism including phenol, benzyl acetate, benzyl butyrate, acetate, butyrate, and lactate. Both the ABL assay and resting cell kinetic tests demonstrated the level of T2MO expression in cells grown on benzyl alcohol was comparable to that observed with toluene, with similar levels of fluorescence detected and similar TCE transformation rates and capacities. The benzyl esters induced T2MO expression, with benzyl butyrate having the highest TCE transformation rate and capacity of all substrates tested. Acetate, butyrate, and lactate results showed minimal levels of induction, verifying benzyl alcohol was responsible for T2MO expression in benzyl ester grown cells, and growth on lactate could not support cometabolism. ABL assay and resting cell kinetic tests largely agreed, demonstrating that ABL is a reliable method of determining suitable substrates to support cometabolism. Additionally, kinetic tests with either benzyl alcohol or toluene and TCE were performed to determine whether, like toluene, benzyl alcohol competitively inhibits contaminant transformation by the T2MO. Results suggest that benzyl alcohol inhibition occurs, but at concentrations greater than 10 mg/L and to a lesser degree than toluene at the same concentration. Finally, kinetics for 1,2-cis-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC) were determined for cells grown on benzyl alcohol and compared to toluene-grown cells. Toluene-grown cells had higher initial rates for VC, cis-DCE, and 1,1-DCE, however, benzyl alcohol-grown cells had approximately twice the transformation capacity for the same compounds.
In Chapter 4, the cometabolic activity of encapsulated cells of Burkholderia vietnamiensis G4 was first determined through kinetic tests in batch systems over time. A singlet bottle of encapsulated Burkholderia vietnamiensis G4 saw transformation of TCE over 400 days with two external additions of toluene to re-stimulate cometabolic activity after the transformation capacity of resting cells was reached. Encapsulated cells at a higher biomass loading of 8 mgTSS/gbead verified that long-term transformation of a mixture of TCE and 1,1-DCE could be achieved over 100 days through external additions of toluene to recommence cometabolism. Given these results, possible SRCs were investigated for a co-encapsulated system with both cells and SRC present in the hydrogel bead. The two SRCs assessed were benzyl butyrate and tetraphenoxysilane (TPhOS), which hydrolyze at the ester bonds to produce benzyl alcohol and phenol, respectively. Though it was shown in the resting cell kinetic tests that benzyl butyrate is a promising substrate for supporting cometabolism, when co-encapsulated, it hydrolyzed too rapidly indicated by a high oxygen uptake rate, making it an unsuitable SRC for in situ use. TPhOS, which hydrolyzes to produce phenol, was co-encapsulated at 1.9% (w/w) and 0.5 mgTSS/gbead. TCE transformation was achieved for 180 days by the co-encapsulated cells with a minimal oxygen uptake rate for the majority of transformation. This setup was repeated (1.9% TPhOS and 0.5 mgTSS/gbead) in a microcosm system with a TCE and cis-DCE mixture, and while TCE rates were lower and the oxygen uptake rate was higher than in media, TCE and cis-DCE were transformed for 100 and 175 days, respectively. TPhOS as a SRC provided proof of concept that a co-encapsulated system with Burkholderia vietnamiensis G4 could promote long-term transformation of chlorinated ethenes in both batch and microcosm systems.