Physiology, ecology and biochemistry of anaerobic, phototrophic oxidation of nitrite

This thesis describes the novel process of anaerobic oxidation of nitrite to nitrate performed by phototrophic bacteria and its qualitative and, in parts, its quantitative distribution in the environment. Bicarbonate-buffered enrichment cultures which had 1 mM nitrite as sole electron donor, were obtaioned from many freshwater and some saltwater sites. In these cultures, nitrite was almost stoichiometrically oxidized to nitrate with concomitant increase in optical density in the light. Quantitative measurements of three sampling sites via the MPN-method revealed cell densities of 104 cells per ml in activated sewage sludge whereas sediments of Lake Constance and sediments of the slightly acidic lake Dingelsdorfer Ried contained substantially less cells per ml. Also in nitrite oxidation, enrichment cultures from activated sewage sludge were the most active ones, from which two morphological different bacterial strains could be isolated: strain KS1 and strain LQ17.Both strains oxidized nitrite to nitrate anaerobically in the light with concomitant biomass formation. Without light, no growth or nitrite oxidation was detectable.While strain LQ17 oxidized 1 mM nitrite incompletely to 0.6 mM nitrate within three months, strain KS1 oxidized nitrite stoichiometrically to nitrate within few days. If these strains were fed with nitrite at concentrations higher than 1.5 mM, the lag phase increased and growth was slowed down, and at concentrations above 4 mM no nitrite oxidation was observed and the OD of the cultured decreased permanently. Cultivation of strain KS1 in molybdenum-free medium with nitrite as sole electron donor revealed no nitrite oxidation or growth unless molybdenum (300 nM) was added. With organic electron donors in darkness, no anaerobic growth was observed with both strains, neither with nitrate nor with sulfate as alternative electron acceptor, whereas both strains were able to utilize organic substrates under air. When grown phototrophically, both strains utilized many organic and some inorganic substrates, and further physiological experiments such as, e.g., utilized nitrogen or sulfur sources or the in-vivo absorption spectra together with 16S rRNA gene analyses allowed to assign strain LQ17 to the genus Rhodopseudomonas and of strain KS1 to the genus Thiocapsa. Of already isolated strains, the two Thiocapsa roseopersicina strains DSM221 and DSM217 were also able to oxidize nitrite stoichiometrically to nitrate.When grown with nitrite as sole electron donor, cell-free extracts of strain KS1 exhibited no nitrite oxidase but a specific nitrate reductase activity of more than 1 U per mg protein. Comparison with cell-free extracts of strain KS1 grown with fructose as e-donor and nitrate as N-source exhibited only few mU per mg protein. Subsequent SDS-PAGE analysis revealed two protein bands of 130-150 kDa and 55-60 kDa, which were strongly expressed specifically after growth with nitrite, and resembled the α- and β-subunit of the membrane-bound nitrate reductase.