abstract: Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene. / Dissertation/Thesis / Ph.D. Microbiology 2013

Microbial respiration can be highly diverse and adaptable, with many bacteria able to respond to changes in their environment promptly and efficiently. The regulation of respiratory enzymes by highly responsive and precise transcriptional regulators confers distinct advantage for survival in sometimes harsh and extreme conditions. The organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2 is able to utilise a wide range of electron acceptors and respiratory processes through tight regulation of respiratory machinery. An example of this tight regulation of respiratory machinery can been seen by biochemical analysis of the CRP-FNR-type transcriptional regulator family CprK, of which five are present in the strain. CprK1 is able to sense the presence of the physiological ligand, 3-chloro-4-hydroxyphenylacetic acid (CHPA), of reductive dehalogenase CprA1 with nM affinity. In this work we demonstrate that CprK1 is able to distinguish between the chlorinated CprA1 substrate CHPA and the non-chlorinated product 4-hydroxyphenylacetic acid (HPA) by ‘pKa interrogation’ of the 4-hydroxy moiety and by the atomic radius of the ortho-moiety. Through the use of in vitro biophysical and in vivo transcriptional response assays, we show that CprK1 is able to sense a number of halogenated phenols, including phenylacetic acids and nitrophenols. We also demonstrate that a 4-hydroxyl group is essential for CprK1 activation. In Chapter 4, an attempt to modify the effector sensitivity of CprK1 is performed by site-specific and random mutagenesis, and mutant selection assays are developed. We show that CprK1 is highly resistant to effector specificity modifications, with seemingly minor or conservative amino acid changes removing CprK1’s ability to initiate transcription. In Chapter 5, the CprK1 paralogue, CprK4 from D. hafniense DCB-2 is characterised by in vitro biophysical and in vivo transcriptional response assays in order to assess its potential as a biosensor. We show that CprK4 is able to bind cis-regulatory DNA elements dehaloboxes 7 and 10 in the absence of effector by Surface Plasmon Resonance (SPR) protein array; however, we were unable to identify its effectors reliably. Due to the unknown nature of CprK4’s effector, it is still unclear whether CprK4 could be a valuable biosensor.