Nitrogen-fixing methane-utilizing bacteria

Methane occurs abundantly in nature. In the presence of oxygen this gas may be metabolized by bacteria that are able to use it as carbon and energy source. Several types of bacteria involved in the oxidation of methane have been described in literature. Methane-utilizing bacteria have in common that they can only grow on methane or methanol and not on other carbon compounds. There is much confusion in the literature about the ability of these bacteria to fix atmospheric nitrogen. The object of the investigations presented in this thesis was to obtain more information about possible nitrogen fixation by methane-utilizing bacteria.Paper I is concerned with the isolation and description of the methaneoxidizing bacterium strain 41 of the Methylosinus type. This isolate is a curved rod, motile in young cultures, but on ageing of the culture motility is lost and exospores are formed. Only methane or methanol serves as growth substrate for this bacterium. Attempts to demonstrate nitrogen fixation by this organism were only successful after it was recognized that (i) the organism was sensitive to oxygen when dependent on N 2 as nitrogen source (ii) nitrogenase activity could not be assayed by acetylene reduction when the bacterium was growing on methane.Growth of strain 41 in nitrate-containing medium with methane was little influenced by varying the oxygen pressure. But increasing the oxygen pressure when growing the bacterium in nitrogen-free medium severely reduced growth. Similar phenomena were observed when the bacterium was grown on agar plates. Colony size on nitrate-containing plates was not affected by varying the oxygen pressure but on nitrogen-free plates the effect of oxygen became apparant. Incubation in an atmosphere containing 5% oxygen allowed normal growth on nitrogen-free medium while with 20% oxygen growth was only meagre. The nitrogen-fixing colonies that developed sporadically probably were a result of locally occurring clumps of the inoculated organism.Evidence of nitrogen fixation by strain 41 when growing with methane was obtained by using 15N 2 . Excess 15N percentages of the culture were measured after the bacterium was grown at reduced oxygen pressure in nitrogen-free medium with methane and 15N 2 .Although strain 41 fixed N 2 it did not reduce acetylene when growing on methane. In papers I and II two possible explanations were considered for this erratic behaviour. It was observed that nitrogenase activity could be assayed by acetylene reduction when the bacterium was growing on methanol in nitrogen-free medium and that ethylene was co-oxidized by methanegrown cells and not by methanol-grown cells. In spite of this observation, the hypothesis that failure of the acetylene-ethylene assay was due to co-oxidation of ethylene by the methane-oxidizing bacterium was incorrect because co- oxidation of ethylene by methane-grown cells was completely prevented by acetylene.Acetylene not only completely prevented the co-oxidation of ethylene, but it also inhibited very strongly the oxidation of methane. Thus, the supply of energy and reducing power to nitrogenase, needed for the reduction of acetylene, was impeded. This explanation for the failure of the acetyleneethylene assay with methane-grown bacteria was corroborated by the observation that methanol or other substrates oxidizable in the presence of acetylene supported acetylene reduction by methane-grown cells.In paper II, experiments are reported that were undertaken to study the mechanism of inhibition of methane oxidation by acetylene. Growth of the bacterium on methanol in a nitrate-containing medium was not affected by acetylene whereas growth on methane was completely prevented in the presence of acetylene. Apparently, only the first step in the degradation route of methane, the oxidation of methane to methanol, was blocked by acetylene. This was also shown by the course of the oxygen uptake of whole-cell suspensions of strain 41. Acetylene suppressed methane-dependent oxygen uptake, whereas it did not influence methanol-dependent oxygen uptake. Interaction of acetylene with methane hydroxylase, the enzyme involved in the oxidation of methane to methanol, was further demonstrated by showing that acetylene also inhibited co-oxidation of methane hydroxylase-dependent co-substrates. Acetylene itself was only co-oxidized by methane-grown cells. Methanolgrown cells, lacking methane hydroxylase, did not co-oxidize acetylene. Experiments with whole-cell suspensions were undertaken to study the mechanism of inhibition of methane hydroxylase by acetylene. The uptake by such suspensions of methane and of dissolved oxygen, both dependent on methane concentration, was measured. From these experiments it was tentatively concluded that acetylene inhibited methane oxidation competitively. Other strains of methane-oxidizing bacteria behaved similarly to strain 41 in that growth on methane was inhibited by acetylene. Furthermore, bacteria other than the methane-oxidizing bacteria that could grow on lower hydrocarbons were inhibited by acetylene as well when growing on the alkane but not when growing on non-hydrocarbon substrates. Thus the acetylene- ethylene assay likewise could not be employed for measuring nitrogenase activity in these bacteria when the alkane was the sole energy source.The study presented in paper III surveyed the nitrogen-fixing capacity among methane-oxidizing bacteria. Nitrogen-fixing methane-oxidizing bacteria grew readily in enrichment cultures that had been inoculated with material from various habitats. Methylosinus -type bacteria were most abundant in such enrichments but Methylomonas -type organisms occurred as well. The Methylosinus -type bacteria were isolated without difficulty from the enrichments. These strains all resembled strain 41. They were motile in young cultures and formed exospores upon ageing, but the cell size and shape varied. Both straight rods and vibrioid forms were identified. The Methylomonas -type bacteria were much more difficult to isolate and to cultivate than the Methylosinus -type strains. Growth of these bacteria was enhanced in mixed culture with a small motile rod that frequently appeared as a contaminant of pure cultures. The nitrogen-fixing Methylomonas -type strain 2 possessed the type I membrane system as opposed to the type II membrane system of strain 41. This result shows that nitrogen fixation is not restricted to type II methane-oxidizing bacteria.The occurrence in nature of methane-oxidizing bacteria with the capacity to fix nitrogen was investigated by incubating dilution series of samples in nitrate-containing and nitrogen-free media. Nitrate was not found to be a significantly better nitrogen source than N 2 , indicating that the capacity to fix N 2 is common among methane-oxidizing bacteria in nature. The higher dilutions of the samples contained coccoid bacteria that differed morphologically from the isolated Methylosinus and Methylomonas -type bacteria. Apparantly, these coccoid bacteria were more abundant in the mud and soil samples investigated than were the Methylosinus and Methylomonas -type bacteria. But in enrichments they were overgrown by these faster growing organisms. Isolation of the coccoid bacteria was difficult. The first transfer from the liquid enrichment cultures to plates with nitrogen-free medium yielded colonies, but subsequent transfers from these colonies only grew in a liquid medium. One isolated coccoid organism was found to possess the type II membrane system.Paper IV comprises a study of the hydrogenase activity of strain 41. This activity was assayed by measuring the uptake of H 2 by growing cultures. There was some hydrogenase activity when the bacterium was growing in nitrate-containing medium, but the enzymic activity increased markedly when bacterial growth was dependent on the fixation of N 2 . The function of the hydrogenase-nitrogenase association was not clear. Hydrogen gas supported the reduction of acetylene, indicating that its oxidation by hydrogenase supplied reductant and energy to the nitrogenase