Showing papers by "J.G. Kuenen published in 1997"

Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (Anammox) sludge

Abstract: The anaerobic ammonium oxidation (Anammox) process is a promising novel option for removing nitrogen from wastewater. In this study it was shown that the Anammox process was inhibited reversibly by the presence of oxygen. Furthermore, aerobic nitrifiers were shown not to play an important role in the Anammox process.

Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor.

Abstract: 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.

Novel principles in the microbial conversion of nitrogen compounds.

Abstract: Some aspects of inorganic nitrogen conversion by microorganisms like N2O emission and hydroxylamine metabolism studied by Beijerinck and Kluyver, founders of the Delft School of Microbiology, are still actual today. In the Kluyver Laboratory for Biotechnology, microbial conversion of nitrogen compounds is still a central research theme. In recent years a range of new microbial processes and process technological applications have been studied. This paper gives a review of these developments including, aerobic denitrification, anaerobic ammonium oxidation, heterotrophic nitrification, and formation of intermediates (NO2-, NO, N2O), as well as the way these processes are controlled at the genetic and enzyme level.

Sulfur production by obligately chemolithoautotrophic Thiobacillus species

Abstract: Transient-state experiments with the obligately autotrophic Thiobacillus sp. strain W5 revealed that sulfide oxidation proceeds in two physiological phases, (i) the sulfate-producing phase and (ii) the sulfur- and sulfate-producing phase, after which sulfide toxicity occurs. Specific sulfur-producing characteristics were independent of the growth rate. Sulfur formation was shown to occur when the maximum oxidative capacity of the culture was approached. In order to be able to oxidize increasing amounts of sulfide, the organism has to convert part of the sulfide to sulfur (HS(sup-)(symbl)S(sup0) + H(sup+) + 2e(sup-)) instead of sulfate (HS(sup-) + 4H(inf2)O(symbl)SO(inf4)(sup2-) + 9 H(sup+) + 8e(sup-)), thereby keeping the electron flux constant. Measurements of the in vivo degree of reduction of the cytochrome pool as a function of increasing sulfide supply suggested a redox-related down-regulation of the sulfur oxidation rate. Comparison of the sulfur-producing properties of Thiobacillus sp. strain W5 and Thiobacillus neapolitanus showed that the former has twice the maximum specific sulfide-oxidizing capacity of the latter (3.6 versus 1.9 (mu)mol/mg of protein/min). Their maximum specific oxygen uptake rates were very similar. Significant mechanistic differences in sulfur production between the high-sulfur-producing Thiobacillus sp. strain W5 and the moderate-sulfur-producing species T. neapolitanus were not observed. The limited sulfide-oxidizing capacity of T. neapolitanus appears to be the reason that it can convert only 50% of the incoming sulfide to elemental sulfur.

Polythionate degradation by tetrathionate hydrolase of Thiobacillus ferrooxidans

Abstract: Cell-free extracts of Thiobacillus ferrooxidans grown with thiosulfate as energy source and prepared at high ammonium sulfate concentrations and at low pH are capable of polythionate hydrolysis. The enzyme responsible for the hydrolysis of tetrathionate (S4O2- 6) and pentathionate (S4O2- 6) was purified to homogeneity. Enzyme activity during the purification procedure was based on a continuous spectrophotometric method that detects soluble intermediates that absorb in the UV region. The end products of hydrolysis of both polythionates by the pure enzyme were thiosulfate, sulfur and sulfate. The purified enzyme has a pH optimum of around 4 and a temperature optimum of 65 °. The activity is strongly influenced by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of molecular mass 52 kDa. During purification of tetrathionate hydrolase, fractions able to hydrolyse trithionate and tetrathionate were separated, indicating that the two substrates are hydrolysed by different enzymes.

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

Abstract: A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the Vmax was 2.1 ± 0.1 μmol cytochrome c (mg protein)–1 min–1, and the Km values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product.

Isolation of the tetrathionate hydrolase from Thiobacillus acidophilus

Abstract: An enzyme capable of hydrolysing tetrathionate was purified from cell-free extracts of Thiobacillus acidophilus. The purified enzyme converts tetrathionate into thiosulfate, sulfur and sulfate. In addition, pentathionate could also be converted by the same enzyme. Measurement of the enzyme activity during purification is based on the absorbance of the initial intermediates formed from tetrathionate in the ultraviolet region, which have not been identified. Enzyme activity could also be measured by the scattering of insoluble sulfur in the visible region. The purified enzyme has a pH optimum of 2.5 and a temperature optimum of 65°C. Enzyme activity is strongly stimulated by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of 48kDa. The ultraviolet–visible absorption spectra and denaturation experiments indicate the presence of an organic cofactor.

Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase.

Abstract: Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield. In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite. In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification. The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 µm. Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase. The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases fou nd in autotrophic bacteria.

Thiobacillus sp. W5, the dominant autotroph oxidizing sulfide to sulfur in a reactor for aerobic treatment of sulfidic wastes

Abstract: The floating filter technique was successfully adapted for the isolation of the dominant, chemolithoautotrophic, sulfide-oxidizing bacterium from a sulfur-producing reactor after conventional isolation techniques had failed. The inoculated polycarbonate filters, floating on mineral medium, were incubated under gaseous hydrogen sulfide at non-toxic levels. This technique gave 200-fold higher recoveries than conventional isolation techniques. Viable counts on the filters, making up 15% of the total count, appeared to be all of the same species. Chemostat cultures of the new isolate had a very high sulfur-forming capacity, converting almost all hydrogen sulfide in the medium to elemental sulfur under high sulfide loads (27.5 mmol l-1 h-1) and fully aerobic conditions. This behaviour closely resembled that of the microbial community in the sulfur-producing reactor. Moreover, similar protein patterns were obtained by electrophoresis of cell-free extracts from the isolate and the mixed culture. It has therefore been concluded that this isolate represents the dominant sulfide-oxidizing population in the reactor. The isolate has been shown to be a new Thiobacillus species, related to Thiobacillus neapolitanus. In view of the general confusion currently surrounding the taxonomy of the thiobacilli, a new species has not been formally created. Instead, the isolate has been given the working name Thiobacillus sp. W5.