Graduate Thesis Or Dissertation
 

Factors controlling halogenated and nonhalogenated alkene growth substrate range of vinyl chloride-utilizing bacteria

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  • This thesis explores the factors limiting the alkene substrate range of the vinyl chloride (VC)-utilizing bacteria, and describes a method for measuring VC transformation in situ. Vinyl fluoride (VF) was evaluated as a surrogate for monitoring aerobic VC-transformation utilizing three isolates, Mycobacterium EE13a, Mycobacterium JS60 and Nocardioides JS614. JS614 grew on VF in addition to VC, making it the first bacterium reported to use VF as a sole carbon and energy source. There was little difference among the three strains in Ks or kmax for VC or VF. Rates of VF transformation and F- accumulation were correlated with the rate of VC transformation through a competitive inhibition model, and showed promise for estimating VC rates in situ. Addition of supplemental ethene oxide (EtO) extended the growth substrate range of JS614 to propene, butene, and vinyl bromide (VB), whereas propene oxide (PrO) and butene oxide had no effect. Despite EtO and PrO being both inducers of AkMO and EaCoMT and intermediates in the alkene metabolic pathway, they exerted reversible inhibition on growth. Poor growth on propene was likely caused by the low rate of PrO consumption compared to PrO production which caused PrO accumulation to inhibitory levels; and reduced net reductant gain caused by a combination of PrO consumption "bottleneck" and the requirement of a NADPH-dependent CoM-carboxylase for metabolism of ≥C₃ alkenes. EtO consumption was inhibited by PrO, but overall the combined rate of EtO plus PrO consumption was better than that of PrO alone, perhaps explaining the beneficial effect of EtO as generating reductant to support growth on propene. EtO-stimulated growth on VB was significantly slower than growth rates on VF and VC, but the maximum rate of VB consumption by Eth-grown cells was actually ~50% greater. Since 30% of the maximum rate of EtO consumption accompanied the maximum rate of VB consumption, it indicated that the "epoxide consumption bottle neck" must also apply to VB-epoxide, and is likely a factor limiting effective VB metabolism. VB was more inhibitory to growth of JS614 both on acetate and during active turnover than either VC and VF.
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file details THESIS_10_9_08_final.pdf 2017-08-08 Público Baixar