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1 - 7 of 7 for "Mike S. M. Jetten"
Physiological role of the respiratory quinol oxidase in the anaerobic nitrite-reducing methanotroph ‘Candidatus Methylomirabilis oxyfera’
The anaerobic nitrite-reducing methanotroph ‘Candidatus Methylomirabilis oxyfera’ (‘Ca. M. oxyfera’) produces oxygen from nitrite by a novel pathway. The major part of the O2 is used for methane activation and oxidation which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes such as respiration. We have found that the genome of ‘Ca. M. oxyfera’ harbours four sets of genes encoding terminal respiratory oxidases: two cytochrome c oxidases a third putative bo-type ubiquinol oxidase and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed albeit at low levels. Oxygen-uptake and inhibition experiments UV–visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by ‘Ca. M. oxyfera’ notably the membrane-bound bo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems and are an additional indication of the flexibility of terminal oxidases of which the distribution among anaerobic micro-organisms may be largely underestimated.
Nitrogen fixation by the verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ SolV
The ability to utilize atmospheric nitrogen (N2) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N2 fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ strain SolV was investigated for its ability to fix N2. Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes encoding the subunits of the nitrogenase. ‘M. fumariolicum’ SolV was able to fix N2 at low oxygen (O2) concentration (0.5 % v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h−1 (mg cell dry weight)−1]. Based on acetylene reduction assay and growth experiments the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N2 fixation within the phylum Verrucomicrobia.
Denitrification and ammonia oxidation by Nitrosomonas europaea wild-type, and NirK- and NorB-deficient mutants
The phenotypes of three different Nitrosomonas europaea strains – wild-type nitrite reductase (NirK)-deficient and nitric oxide reductase (NorB)-deficient strains – were characterized in chemostat cell cultures and the effect of nitric oxide (NO) on metabolic activities was evaluated. All strains revealed similar aerobic ammonia oxidation activities but the growth rates and yields of the knock-out mutants were significantly reduced. Dinitrogen (N2) was the main gaseous product of the wild-type produced via its denitrification activity. The mutants were unable to reduce nitrite to N2 but excreted more hydroxylamine leading to the formation of almost equal amounts of NO nitrous oxide (N2O) and N2 by chemical auto-oxidation and chemodenitrification of hydroxylamine. Under anoxic conditions Nsm. europaea wild-type gains energy for growth via nitrogen dioxide (NO2)-dependent ammonia oxidation or hydrogen-dependent denitrification using nitrite as electron acceptor. The mutant strains were restricted to NO and/or N2O as electron acceptor and consequently their growth rates and yields were much lower compared with the wild-type. When cells were transferred from anoxic (denitrification) to oxic conditions the wild-type strain endogenously produced NO and recovered ammonia oxidation within 8 h. In contrast the mutant strains remained inactive. For recovery of ammonia oxidation activity the NO concentration had to be adjusted to about 10 p.p.m. in the aeration gas.
Ammonium and hydroxylamine uptake and accumulation in Nitrosomonas
Starved cells of Nitrosomonas europaea and further ammonia oxidizers were able to rapidly accumulate ammonium and hydroxylamine to an internal concentration of about 1 and 0·8 M respectively. In kinetic studies the uptake/accumulation rates for ammonium [3·1 mmol (g protein)−1 min−1] and hydroxylamine [4·39 mmol (g protein)−1 min−1] were determined. The uptake and accumulation process of ammonium and hydroxylamine was not coupled to ammonia or hydroxylamine oxidation and nitrite was not produced. In the presence of uncouplers the ammonium accumulation was completely inhibited indicating an active membrane-potential-driven transport mechanism. When the external ammonium or hydroxylamine pool was depleted the internal ammonium and hydroxylamine was consumed within 12 h or 20 min respectively. The binding of ammonium/ammonia was correlated with an energized membrane system and hydroxylamine may bind to the hydroxylamine oxidoredutase.
Ammonia oxidation by Nitrosomonas eutropha with NO2 as oxidant is not inhibited by acetylene
The effect of acetylene (14C2H2) on aerobic and anaerobic ammonia oxidation by Nitrosomonas eutropha was investigated. Ammonia monooxygenase (AMO) was inhibited and a 27 kDa polypeptide (AmoA) was labelled during aerobic ammonia oxidation. In contrast anaerobic NO2-dependent ammonia oxidation (NO2/N2O4 as oxidant) was not affected by acetylene. Further studies gave evidence that the inhibition as well as the labelling reaction were O2-dependent. Cells pretreated with acetylene under oxic conditions were unable to oxidize ammonia with O2 as oxidant. After these cell suspensions were supplemented with gaseous NO2 ammonia oxidation activity of about 140 μmol NH4 + (g protein)−1 h−1 was detectable under both oxic and anoxic conditions. A significantly reduced acetylene inhibition of the ammonia oxidation activity was observed for cells incubated in the presence of NO. This suggests that NO and acetylene compete for the same binding site on AMO. On the basis of these results a new hypothetical model of ammonia oxidation by N. eutropha was developed.
Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor
Summary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I−1 h−1) and a decrease in the nitrate production rate (0.5 mmol I−1 h−1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.
Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor
An autotrophic synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed. This medium contained ammonium and nitrite as the only electron donor and electron acceptor respectively while carbonate was the only carbon source provided. Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity. The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m−3 d−1 when fed with 30 mM NH4 +. This is equivalent to a specific anaerobic ammonium oxidation rate of 1000–1100 nmol NH4 +h−1 (mg volatile solids)−1. The maximum specific oxidation rate obtained was 1500 nmol NH4 +h−1 (mg volatile solids)−1. Per mol NH4 + oxidized 0.041mol CO2 were incorporated resulting in a estimated growth rate of 0.001 h−1. The main product of the Anammox reaction is N2 but about 10% of the N-feed is converted to NO3 −. The overall nitrogen balance gave a ratio of NH4 −-conversion to NO2 −-conversion and NO3 −-production of 1:1·31±0·06:2·02±0·02. During the conversion of NH4 + with NO2 − no other intermediates or end-products such as hydroxylamine NO and N2O could be detected. Acetylene phosphate and oxygen were shown to be strong inhibitors of the Anammox activity. The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology. During the enrichment for Anammox micro-organisms on synthetic medium an increase in ether lipids was observed. The colour of the biomass changed from brownish to red which was accompanied by an increase in the cytochrome content. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.