N.C.G. Tan

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

ABSTRACT A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.

N2O emission hotspots at different spatial scales and governing factors for small scale hotspots

Chronically nitrate-loaded riparian buffer zones show high N(2)O emissions. Often, a large part of the N(2)O is emitted from small surface areas, resulting in high spatial variability in these buffer zones. These small surface areas with high N(2)O emissions (hotspots) need to be investigated to generate knowledge on the factors governing N(2)O emissions. In this study the N(2)O emission variability was investigated at different spatial scales. Therefore N(2)O emissions from three 32 m(2) grids were determined in summer and winter. Spatial variation and total emission were determined on three different scales (0.3 m(2), 0.018 m(2) and 0.0013 m(2)) at plots with different levels of N(2)O emissions. Spatial variation was high at all scales determined and highest at the smallest scale. To test possible factors inducing small scale hotspots, soil samples were collected for slurry incubation to determine responses to increased electron donor/acceptor availability. Acetate addition did increase N(2)O production, but nitrate addition failed to increase total denitrification or net N(2)O production. N(2)O production was similar in all soil slurries, independent of their origin from high or low emission soils, indicating that environmental conditions (including physical factors like gas diffusion) rather than microbial community composition governed N(2)O emission rates.

Effect of Nitric Oxide on Anammox Bacteria

ABSTRACT The effects of nitrogen oxides on anammox bacteria are not well known. Therefore, anammox bacteria were exposed to 3,500 ppm nitric oxide (NO) in the gas phase. The anammox bacteria were not inhibited by the high NO concentration but rather used it to oxidize additional ammonium to dinitrogen gas under conditions relevant to wastewater treatment.

Physiological and phylogenetic study of an ammonium-oxidizing culture at high nitrite concentrations

Oxidation of high-strength ammonium wastewater can lead to exceptionally high nitrite concentrations; therefore, the effect of high nitrite concentration (> 400 mM) was studied using an ammonium-oxidizing enrichment culture in a batch reactor. Ammonium was fed to the reactor in portions of 40-150 mM until ammonium oxidation rates decreased and finally stopped. Activity was restored by replacing half of the medium, while biomass was retained by a membrane. The ammonium-oxidizing population obtained was able to oxidize ammonium at nitrite concentrations of up to 500 mM. The maximum specific oxidation activity of the culture in batch test was about 0.040 mmol O(2)g(-1)proteinmin(-1) and the K(s) value was 1.5 mM ammonium. In these tests, half of the maximum oxidation activity was still present at a concentration of 600 mM nitrite and approximately 10% residual activity could still be measured at 1200 mM nitrite (pH 7.4), or as a free nitrous acid (FNA) concentration of 6.6 mg l(-1). Additional experiments showed that the inhibition was caused by nitrite and not by the high sodium chloride concentration of the medium. The added ammonium was mainly converted into nitrite and no nitrite oxidation was observed. In addition, gaseous nitrogen compounds were detected and mass balance calculations revealed a nitrogen loss of approximately 20% using this system. Phylogenetic analyses of 16S rRNA and ammonium monooxygenase (amoA) genes of the obtained enrichment culture showed that ammonium-oxidizing bacteria of the Nitrosomonas europaea/Nitrosococcus mobilis cluster dominated the two clone libraries. Approximately 25% of the 16S rRNA clones showed a similarity of 92% to Deinococcus-like organisms. Specific fluorescence in situ hybridization (FISH) probes confirmed that these microbes comprised 10-20% of the microbial community in the enrichment. The Deinococcus-like organisms were located around the Nitrosomonas clusters, but their role in the community is currently unresolved.

Role of nitrogen oxides in the metabolism of ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) can use oxygen and nitrite as electron acceptors. Nitrite reduction by Nitrosomonas is observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with nitrogen dioxide (NO(2)) and (iii) NO(x)-induced aerobic ammonia oxidation. NO(x) molecules play an important role in the conversion of ammonia and nitrite by AOB. Absence of nitric oxide (NO), which is generally detectable during ammonia oxidation, severely impairs ammonia oxidation by AOB. The lag phase of recovery of aerobic ammonia oxidation was significantly reduced by NO(2) addition. Acetylene inhibition tests showed that NO(2)-dependent and oxygen-dependent ammonia oxidation can be distinguished. Addition of NO(x) increased specific activity of ammonia oxidation, growth rate and denitrification capacity. Together, these findings resulted in a hypothetical model on the role of NO(x) in ammonia oxidation: the NO(x) cycle.