Oxidation of gaseous hydrocarbons by alkene-utilizing bacteria

Gaseous alkenes are widespread in the environment due to the emission of these hydrocarbons by industry and due to their production from natural sources as for instance ethene by plants, fungi and bacteria. Micro-organisms have developed the potential to oxidize these hydrocarbons. Alkenes can either be co-oxidized by alkane-utilizing bacteria and/or used as carbon and energy source by specific alkene-utilizing bacteria. Alkane-utilizing micro-organisms have extensively been investigated and described. Potential applications of alkane-utilizing micro-organisms e.g. the epoxidation of alkenes have been recorded. in numerous patents. The scope of the research presented was to gain, in a concerted action between microbiologists and process engineers, more fundamental knowledge on the behaviour of alkene-utilizing bacteria in either a gas/solid bioreactor or in a multiphase bioreactor. This thesis mainly deals with microbiological aspects.Chapters 2, 3 and 4 subsequently deal with the isolation and characterization of propene- and 1-butene-utilizing Xanthobacter spp., with Nocardia spp. utilizing both 1,3-butadiene and isoprene as sole source of carbon and energy and with a Nocardia sp. capable of growth on trans -2-butene. The initial oxidation of propene, 2-butene, 1,3-butadiene and isoprene by these bacteria is mediated by a mono- oxygenase. The mono-oxygenase present in propene-grown Xanthobacter spp. is different from hydrocarbon mono-oxygenases described until now in view of substrate specificity towards hydrocarbons and in view of activities measured in the presence of potential inhibitors of the mono-oxygenase. Both Xanthobacter spp. and alkadiene-utilizing Nocardia spp. possess a mono-oxygenase which catalyses an epoxidation reaction. On the other hand, the trans -2-butene-grown Nocardia sp. which is also able to grow on gaseous n-alkanes, carries out a hydroxylation reaction instead of an epoxidation reaction. A degradation route of trans -2-butene via crotonic acid was proposed on basis of inhibitor experiments, simultaneous adaptation studies and enzyme activities.Possible applications of alkene-grown bacteria are the production of (chiral) epoxyalkanes and the removal of alkenes from gas phases and therefore an optimal production of the bacteria is essential. Microbial growth on either ethene or propene in chemostat cultures is dealt with in chapter 5. By using a simple growth model and experimentally derived growth parameters with a Xanthobacter sp. and a Mycobacterium sp. the dilution rate resulting in the optimal biomass production could be calculated. Measured and mathematically derived production rates agreed well.The potential to produce epoxyalkanes from alkenes was investigated using washed cell suspensions of alkene-grown Xanthobacter spp. The results are given in chapter 6. Chapter 7 represents a extended survey of gaseous hydrocarbon utilization and oxidation by alkene-grown bacteria. Some selected alkene-utilizing micro-organisms were investigated in more detail to provide a better understanding of the ability to grow on hydrocarbons and the oxidation of gaseous and volatile hydrocarbons. From such observations it was obvious that a 1-hexene-utilizing Pseudomonas and a trans -2-butene-utilizing Nocardia TB1 resemble alkane-utilizing bacteria. Other alkene-utilizing micro-organisms consist of a specific group, which are not able to hydroxylate alkanes. Alkene-grown bacteria were capable to excrete epoxides and using an appropriate combination of bacterium and alkene almost every epoxide could be produced. Finally, it was shown that most epoxides formed were either oxidized to CO 2 and H 2 O, or hydrolysed by alkene-grown bacteria.Chapter 8, 9 and 10 deal with some aspects of the behaviour of alkene-utilizing bacteria in gas/solid bioreactors. 1,2-Epoxyethane formation by propene-grown Mycobacterium Py1 cells was studied in such a reactor because no accumulation of the toxic epoxide occurs in the vicinity of the bacteria. Prolonged 1,2-epoxyethane formation was dependent on co-factor regeneration. In a subsequent experiment, it was demonstrated that the presence of a metabolizable co-substrate enhanced the epoxide production.Ethene is a plant hormone and has already detrimental effects on stored fruits and vegetables at concentrations of 1 vpm in the gas phase. Therefore, ethene has to be removed from the vicinity of stored agricultural products. alkene-grown bacteria capable of oxidizing ethene may be an alternative of known chemical/physical ethene-removal systems from storage facilities. Ethene-grown Mycobacterium E3 oxidizes ethene to the desired low concentrations even when immobilized on carriers like lava, perlite or in alginate. Chapter 9 describes the characteristics of ethene-grown Mycobacterium E3 immobilized on various supports. However, the operational stability of Mycobacterium E3 immobilized on the supports tested was insufficient. In a subsequent investigation the operational stability of Mycobacterium E3 on compost was tested, and surprisingly, a good operational stability was found while possibly even cellgrowth or induction of mono-oxygenase enzyme was obtained also at very low ethene concentrations. From the efficiency of conversion of ethene and the rate of ethene production by fruits and vegetables, it was calculated that bioscrubbers can be of relatively small dimensions in relation to storage facilities.