Biodegradation of chlorinated and unsaturated hydrocarbons in relation to biological waste-gas treatment.

The original goal of the research described in this thesis was to develop a biological process for the removal of vinyl chloride from waste gases. The gaseous and carcinogenic vinyl chloride is used to produce the plastic polyvinyl chloride (PVC). During this production process waste gases containing vinyl chloride are generated. As a microorganism capable of growth on vinyl chloride as the sole carbon and energy source had been isolated it was envisaged that it might be possible to remove vinyl chloride from waste gases with a biological process.Besides the original strain, Mycobacterium L1, three additional vinyl chlorideutilizing strains were isolated subsequently. All strains were tentatively identified as Mycobacterium aurum.The first step in vinyl chloride metabolism in strain L1, was shown to be the oxidation of vinyl chloride to the corresponding epoxide, chlorooxirane, by alkene monooxygenase. Chlorooxirane is also the product of vinyl chloride oxidation in the human liver and is responsible for the carcinogenic properties of vinyl chloride. Alkene monooxygenase is also present in Mycobacterium E3 after growth on ethene. Extracts from strain E3 could be fractionated yielding two fractions which upon combination exhibited alkene monooxygenase activity, indicating that the enzyme consists of at least two components. One fraction was inhibited by acetylene, indicating it contained the oxygenase component of alkene monooxygenase, whereas the other fraction contained significant reductase activity. The corresponding fractions could also be obtained from extracts of vinyl chloride- grown cells of strain L1, Alkene monooxygenase appeared to be similar to the soluble three-component methane monooxygenases. These enzymes also oxidize alkenes to the corresponding epoxides.The capacity of alkene monooxygenase to oxidize vinyl chloride to the mutagenic and toxic chlorooxirane was exploited to generate and select monooxygenase mutants of the ethene-utilizing strain E3. As long as cells exhibit monooxygenase activity they produce chlorooxirane from vinyl chloride and are hampered in their growth. However, when monooxygenase activity is lost, due to a mutation, these cells are no longer inhibited by the presence of vinyl chloride. Using this technique a mutant of strain E3 no longer capable of growth on ethene was isolated. Subsequently, it was shown that this mutant lacks the reductase component of alkene monooxygenase. Growth of the mutant on epoxyethane (oxirane) resulted in synthesis of the other alkene monooxygenase component(s). Extracts of such cells could be used to detect and subsequently purify the reductase component of alkene monooxygenase.During growth of strain L1, on vinyl chloride in chemostat cultures it became evident that the original mineral salts medium was not optimal. The addition of extra iron to the medium resulted in an enhanced vinyl chloride consumption. Chemostat cultures were also used to determine to what levels vinyl chloride could be removed. This type of experiment was also done with the Xanthobacter autotrophicus GJ10 isolated by D.B. Janssen (University of Groningen) to determine 1,2-dichloroethane removal from air. 1,2-Dichloroethane is the precursor in the major production process of vinyl chloride. The concentrations of both compounds in the air that had passed through the cultures were significantly higher than the maximal allowable concentrations in waste gases according to the German legislation (TA-Luft). Therefore the affinity of both strains towards the respective substrates is too low to apply them in waste-gas treatment.The enzyme transforming chlorooxirane appeared to be very unstable. After a short interruption in the supply of vinyl chloride to a culture of strain L1, inactivation took place upon restoring the vinyl chloride supply to the culture. This is probably caused by chlorooxirane accumulation due to an insufficient activity of the chlorooxirane transforming enzyme after such an interruption in the supply of vinyl chloride. The chlorooxirane subsequently inactivates cell components including alkene monooxygenase. Based on the observed inactivation and the relatively low affinity for vinyl chloride it was concluded that development of a process to remove vinyl chloride based on the application of Mycobacterium aurum L1, was not feasible.As the formation of chlorooxirane is one of the major drawbacks of strain L1, microorganisms were isolated on compounds structurally related to vinyl chloride. Using this approach we hoped to isolate strains which add a water molecule to the double bond of vinyl chloride, resulting in formation 2-chloroethanol or acetaldehyde. 3-Chloroacrylic acid and styrene were used as vinyl chloride analogues in enrichment cultures. The enrichment cultures with 3-chloroacrylic acid did indeed result in the isolation of bacteria which hydrated the double bond of 3-chloroacrylic as the initial step in the degradation pathway of this compound. Unfortunately these enzymes did not exhibit any activity with vinyl chloride as substrate. With styrene as carbon source a number of microorganisms were isolated. Almost all of these isolates oxidized the unsaturated alifatic moiety of styrene yielding styrene oxide (phenyloxirane). The flavine adenine dinucleotide- dependent styrene monooxygenase has a high substrate specificity, only oxidizing phenyl substituted alkenes. In contrast to alkene monooxygenase the enzyme appears to require only one component for activity.Besides vinyl chloride, biodegradation of methyl chloride, the simplest chlorinated hydrocarbon was studied. Methyl chloride-grown cells of the isolated Hyphomicrobium strain MC1 could dechlorinate methyl chloride only under aerobic conditions. Simple hydrolytic dehalogenation was therefore not taking place. No methane monooxygenase activity could be detected in methyl chloride-grown cells.To be able to study a bioreactor for the removal of a chlorinated hydrocarbon from air, dichloromethane was selected as model contaminant. Dichloromethane is applied on a large scale as a solvent and consequently is present in numerous industrial waste gases. The strains isolated and characterized by the group of Th. Leisinger (ETH Zürich), as well as a new isolate, were shown to have a much better affinity towards dichloromethane than the value published for the purified dehalogenase. Due to this high affinity these microorganisms can be applied to remove dichloromethane to concentrations well below the maximal allowable levels in waste gases.A 0.066 m <sup>3</SUP>trickle-bed bioreactor was studied for the elimination of dichloromethane from waste gases. The reactor was filled with a polypropylene packing material on which a biofilm developed. The air containing dichloromethane was forced through the reactor counter-current to a circulating aqueous phase. The aqueous phase is used to neutralize and remove the hydrochloric acid formed during dichloromethane degradation. The biological system was very stable and not sensitive to fluctuations in the dichloromethane supply.Dichloromethane elimination with the trickle-bed bioreactor was determined at various gas and liquid flows and dichloromethane concentrations. It was possible to simulate the observed dichloromethane elimination efficiencies surprisingly well with a model incorporating Michaelis-Menten kinetics, diffusion in the biofilm and mass-transfer resistance in the gaseous as well as the liquid phase.On the basis of the experimental results it was concluded that removal of dichloromethane is technically feasible with the trickle-bed reactor