Application of Pseudomonas sp. strain DCA1 for the removal of chlorinated hydrocarbons

The large-scale application of chlorinated aliphatic hydrocarbons (CAHs) has resulted in many cases of groundwater contamination. Contaminated groundwater can be remediated by pump-and-treat: the groundwater is pumped to the surface and treated. The groundwater can be treated in bioreactors, in which the contaminants are biodegraded. Since extracted groundwater is often anaerobic, it has to be oxygenated in order to allow aerobic biodegradation of the contaminants. However, direct oxygenation of (anaerobic) groundwater may result in two undesired effects: stripping of volatile contaminants and coagulation of iron oxides. The use of a membrane-aerated biofilm reactor (MABR) can solve these problems. At one side of the membrane a gas phase is present for the supply of oxygen. A biofilm is located at the other side of the membrane, where the contaminant is present in the liquid phase. The contaminant diffuses into the biofilm, where biodegradation takes place. The combination of a membrane with a biofilm allows oxygenation and biodegradation without stripping the contaminant and coagulation of iron oxides.The main contaminant studied in this thesis is 1,2 dichloroethane (DCA), a synthetic chemical that is mainly used as a feedstock for the production of vinyl chloride. Known bacterial strains that are able to aerobically degrade DCA, Xanthobacter autotrophicus GJ10 and Ancylobacter autotrophicus AD25, do not have the desired characteristics for treatment of DCA-contaminated groundwater. Both strains require additional organic nutrients, such as vitamins, for optimal growth. Moreover, strain GJ10 has a very low affinity for DCA ( K m value of 570 µM).Two major objectives were proposed at the start of this research. The first objective was to isolate DCA-degrading microorganisms with a high affinity for DCA and without requirements for additional organic nutrients for optimal growth. The second objective was to apply the isolated microorganisms in a MABR that allows aerobic biodegradation of DCA, without direct oxygenation of the contaminated groundwater.In Chapter 2, the isolation and characterization of a bacterial strain, designated Pseudomonas sp. strain DCA1, is described. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients for optimal growth. The maximum specific growth rate of strain DCA1 on DCA is 0.14 h -1 . The affinity of strain DCA1 for DCA is very high, with a K m value below the detection limit of 0.5 µM. Instead of a hydrolytic dehalogenation, as was shown in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells, and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.Strain DCA1 was applied in a MABR for the removal of DCA from (ground)water (Chapter 3). A hydrophobic membrane was used to create a barrier between the liquid and the gas phase. Inoculation of the MBR with cells of strain DCA1 grown in a continuous culture resulted in the formation of a stable and active DCA-degrading biofilm on the membrane. The maximum DCA removal rate in the MABR was 410 g m -3 h -1 . This maximum removal rate was reached at a DCA concentration of approximately 80 µM. Simulation of the DCA fluxes into the biofilm showed that the MABR performance at lower concentrations was limited by the DCA diffusion rate rather than by kinetic constraints of strain DCA1. To determine the effect of bacterial kinetics on reactor performance, we also simulated the performance of strain GJ10 in our MABR. At 80 µM the simulated DCA flux into a strain GJ10 biofilm is more than 8 times lower. At a concentration of 4 µM (the intervention value for DCA in The Netherlands), the simulated flux into a biofilm of strain GJ10 is less than 5% of the flux generated into a biofilm of strain DCA1. Aerobic biodegradation of DCA present in anoxic water could be achieved by supplying oxygen solely from the gas phase to the biofilm grown on the liquid side of the membrane.Since the degradation of DCA by strain DCA1 is mediated by a monooxygenase, and these enzymes generally have a broad substrate spectrum, strain DCA1 was used to co-metabolically oxidize chlorinated methanes, ethanes, propanes and ethenes (Chapter 4).Chloroaceticacid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCA1 and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethene were oxidized by cells expressing DCA monooxygenase. Strain DCA1 could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was tested with 1,2-dichloropropane, since this compound shows structure analogy to DCA but is recalcitrant to aerobic biodegradation. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCA1. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2-dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.An alternative to pump-and-treat is in situ bioremediation. This technology is based on microorganisms biodegrading the contaminants in the subsurface. Besides addition of electron donors, electron acceptors and/or nutrients to stimulate the biodegradation ('biostimulation'), also non-indigenous microorganisms can be injected into the subsurface, a process called 'bioaugmentation' (Chapter 5). Bioaugmentation can be applied in case the indigenous microorganisms are:- degrading the contaminant at rates that are too low;- inhibited by the presence of multiple contaminants;- killed as a result of drastic (abiotic) remediation techniques;- not capable to carry out the desired reactions.The latter case seems to be the most promising field for application of bioaugmentation. A good example is the incomplete reduction of tetrachloroethene, which often stalls at cis- 1,2-dichloroethene. Bioaugmentation with members of the Dehalococcoides group has proven its success, resulting in complete reduction of tetrachloroethene to the harmless end product ethene. To efficiently bioaugmentate a contaminated site, the introduced microorganisms have to be distributed in the subsurface and come into close contact with the contaminant(s). Transport of cells in the subsurface is often limited because injected cells are filtered by soil particles, but can be enhanced by using solutions of low ionic strength, surfactants and bacteria with limited adhesive properties. The survival of injected bacteria depends on factors such as competition for electron donors and acceptors, contaminant toxicity and the availability of inorganic nutrients. The use of genetically engineered microorganisms (GMOs) for bioaugmentation has not made much progress over the past decades due to regulatory constraints and public adversity. To justify the cost of bioaugmentation it is important to accurately assess the effect of bioaugmentation on the biodegradation. Molecular techniques have evolved rapidly, allowing the determination of both numbers and types of bacteria present. Future research on bioaugmentation should focus on large-scale field studies and more attention has to be paid to proper control plots and assessment of bioaugmentation efficacy.