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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.

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.

A stable anoxic enrichment culture was obtained that degraded benzene with chlorate as an electron acceptor. The benzene degradation rate was 1.65 mM benzene per day, which is similar to reported aerobic benzene degradation rates but 20-1650 times higher than reported for anaerobic benzene degradation. Denaturing gradient gel electrophoresis of part of the 16S rRNA gene, cloning and sequencing showed that the culture had a stable composition after the seventh transfer. Five bacterial clones were further analyzed. Two clones corresponded to bacteria closely related to Alicycliphilus denitrificans K601. The three other clones corresponded to bacteria closely related to Zoogloea resiniphila PIV-3A2w, Mesorhizobium sp. WG and Stenotrophomonas acidaminiphila. DGGE analysis of cultures grown with different electron donors and acceptors indicated that the bacterium related to Alicycliphilus denitrificans K601 is able to degrade benzene coupled to chlorate reduction. The role of the other bacteria could not be conclusively determined. The bacterium related to Mesorhizobium sp. WG can be enriched with benzene and oxygen, but not with acetate and chlorate, while the bacterium related to Stenotrophomonas acidaminophila grows with acetate and chlorate, but not with benzene and oxygen. As oxygen is produced during chlorate reduction, an aerobic pathway of benzene degradation is most likely. © 2007 Federation of European Microbiological Societies.

Nitric oxide production was measured during nitrification in a laboratory-scale bioreactor, operated at conditions relevant to municipal nitrifying wastewater treatment plants. This study aims to determine which type of microorganism and which metabolic pathway is responsible for nitric oxide emission during nitrification. Simulation studies were used to identify which pathway is the main source of nitric oxide emission, based on the following three hypothetical pathways for nitric oxide emission: (a) nitrification, (b) denitrification by ammonia-oxidizing bacteria with ammonium as electron donor, and (c) heterotrophic denitrification. The results of the study suggest that, in a nitrifying reactor treating wastewater containing solely ammonium and nutrients, denitrification by ammonia- oxidizing bacteria is the main nitric-oxide-producing pathway. During the experiments, 0.025% of the treated ammonium is emitted as nitric oxide, independent of the aeration rate imposed. Nitrite presence and oxygen limitation were found to increase the nitric oxide emission. Water Environ. Res., 79, 2499 (2007).

Perchlorate and chlorate are electron acceptors that during reduction result in the formation of molecular oxygen. The produced oxygen can be used for activation of anaerobic persistent pollutants, like benzene. In this study chlorate was tested as potential electron acceptor to stimulate benzene degradation in anoxic polluted soil column. A chlorate amended benzene polluted soil column was operated over a period of 500 days. Benzene was immediately degraded in the column after start up, and benzene removal recovered completely after omission of chlorate or a too high influent chlorate concentration (22 mM). Mass balance calculations showed that per mole of benzene five mole of chlorate were reduced. At the end of the experiment higher loading rates were applied to measure the maximal benzene degradation rate in this system; a breakthrough of benzene was not observed. The average benzene degradation rate over this period was 31 micromol l(-1) h(-1) with a maximal of 78 micromol l(-1) h(-1). The high degradation rate and the necessity of chlorate indicate that oxygen produced during chlorate reduction indeed is used for the activation of benzene. This is the first column study where benzene biodegradation at a high rate coupled with anaerobic chlorate reduction is observed.

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l(-1)) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l(-1)) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge.

Complete biodegradation of azo dyes requires an anaerobic and aerobic step, in the anaerobic step sulfonated azo dyes (SADs) are reduced, yielding (sulfonated) aromatic amines ((S)AAs) which can be degraded aerobically. The complete biodegradation of the SAD Mordant Yellow 10 (MY10) was studied in a sequential anaerobic and aerobic bioreactor. Anaerobically, MY10 was reductively cleaved and the resulting aromatic amines, 5-aminosalicylic acid (5-ASA) and sulfanilic acid (SA), were both recovered in high stoichiometric yields. One of the AAs, 5-ASA, was readily degraded under aerobic conditions. However, SA was not degraded aerobically in the continuous experiment because no SA-degrading bacterial activity was present in the system. Therefore, a SA-degrading enrichment culture derived from Rhine sediment was used as an inoculum source. This enrichment culture was bioaugmented into the aerobic reactor by increasing the hydraulic retention time (HRT), thus enabling SA-degrading activity to develop and maintain in the aerobic reactor. After decreasing the HRT, the SA-degrading activity remained in the bioreactor and the stoichiometric recovery of sulfate (a SA biodegradation product) indicated the mineralization of SA after bioaugmentation. Batch experiments with aerobic reactor sludge confirmed the biodegradation of SA and 5-ASA. The sequential anaerobic and aerobic bioreactor was able to completely remove the sulfonated azo dye MY10 at a maximum loading rate of 210 mg MY10 (lreactor d)-1 after the appropriate microorganisms for aerobic degradation of SA were bioaugmented into the aerobic bioreactor.

Integration of anaerobic and aerobic conditions in a single bioreactor is a good strategy for the complete mineralization of azo dyes. In order for this strategy to work, azo dye reduction should occur in biofilms exposed to oxygen. Therefore, the effect of oxygen on the azo dye reduction by methanogenic granular sludge was studied using Mordant Orange 1 (MO1) as a model. Azo dye-reduction rates by two different granular sludges were determined in batch assays with various concentrations of oxygen in the headspace. Azo dye reduction occurred in the presence of oxygen if co-substrates, either ethanol or acetate, were added. The rate of dye reduction was highly positively correlated with the oxygen-consuming activity of the sludge. The results suggest that co-substrates stimulate oxygen respiration, which lowers oxygen penetration into the biofilm and thereby creates anaerobic microniches where azo dye reduction can occur.

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.

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.

The lateral etch rate of the epitaxial lift-off (ELO) process was determined as a function of the total HF concentration Cup and the O 2 partial pressure Po 2 . For this purpose samples were grown by metallorganic chemical vapor deposition and etched using a weight-induced ELO process. It was found that the etch rate increases linearly with C HF , which is in accordance with the model on the ELO process presented in a previous paper. This result and composition calculations of HF solutions show that the first step in the etch process of AlAs with an HF solution most probably takes place by chemical attack of undissociated HF on AlAs surface bonds. Furthermore, it is shown that the ELO rate increases slightly over a Po 2 range varying from 0.046 to 0.98 atm and that for Po 2 = 0.003 atm, a significantly lower etch rate is found. We suggest that the observed decrease is the result of surface passivation by elemental arsenic, which is formed by the reaction of AlAs with H + . An oxygen-poor atmosphere may allow the build-up of elemental arsenic on the surface, thus slowing down the AlAs reaction with HF. Oxygen, by removing arsenic as As 2 O 3 , keeps the surface active.

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 protein min −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.

Nitric oxide (NO) and nitrous oxide (N2O) emissions from nitrifying ecosystems are a serious threat to the environment. The factors influencing the emission and the responsible microorganisms and pathways were studied using a laboratory-scale nitrifying reactor system. The nitrifying culture was established at growth rates relevant to wastewater treatment plants (WWTPs). During stable ammonia oxidation, 0.03% of ammonium was emitted as NO and 2.8% was emitted as N2O. Although mixed cultures were used, clear responses in emission of ammonia oxidizing bacteria (AOB) could be detected and it was concluded that the denitrification pathway of AOB was the main source of the emissions. Emissions of nitrogen oxides in the system were strongly influenced by oxygen, nitrite, and ammonium concentrations. Steady state emission levels greatly underestimate the total emission, because changes in oxygen, nitrite, and ammonium concentrations induced a dramatic rise in NO and N2O emission. The data presented can be used as an indication for NO and N2O emission by AOB in plug-flow activated sludge systems, which is highly relevant because of the atmospheric impact and potential health risk of these compounds.

Ten sulfonated aromatic amines were tested for their aerobic and anaerobic biodegradability and toxicity potential in a variety of environmental inocula. Of all the compounds tested, only two aminobenzenesulfonic acid (ABS) isomers, 2- and 4-ABS, were degraded. The observed degradation occurred only under aerobic conditions with inocula sources that were historically polluted with sulfonated aromatic amines. Bioreactor experiments, with non-sterile synthetic wastewater, confirmed the results from the aerobic batch degradation experiments. Both ABS isomers were degraded in long-term continuous experiment by a␣bioaugmented enrichment culture. The maximum degradation rate in the aerobic bioreactor was 1.6¿1.8 gl-1 d-1 for 2-ABS and a somewhat lower value for 4-ABS at hydraulic retention times (HRT) of 2.8¿3.3h. Evidence for extensive mineralization of 2- and 4-ABS was based on oxygen uptake and carbon dioxide production during the batch experiments and the high levels of chemical oxygen demand (COD) removal in the bioreactor. Furthermore, mineralization of the sulfonate group was demonstrated by high recovery of sulfate. The sulfonated aromatic amines did not show any toxic effects on the aerobic and anaerobic bacterial populations tested. The poor biodegradability of sulfonated aromatic amines indicated under the laboratory conditions of this study suggests that these compounds may not be adequately removed during biological wastewater treatment.

Anaerobic wastewater treatment (AnWT) is considered as the most cost-effective solution for organically polluted industrial waste streams. Particularly the development of high-rate systems, in which hydraulic retention times are uncoupled from solids retention times, has led to a world-wide acceptance of AnWT. In the last decade up to the present, the application potentials of AnWT are further explored. Research shows the feasibility of anaerobic reactors under extreme conditions, such as low and high temperatures. Also toxic and/or recalcitrant wastewaters, that were previously believed not to be suitable for anaerobic processes, are now effectively treated. The recent advances are made possible by adapting the conventional anaerobic high-rate concept to the more extreme conditions. Staged anaerobic reactor concepts show advantages under non-optimal temperature conditions as well as during the treatment of chemical wastewater. In other situations, a staged anaerobic - aerobic approach is required for biodegradation of specific pollutants, e.g. the removal of dyes from textile processing wastewaters. The current paper illustrates the benefits of reactor staging and the yet un-exploited potentials of high-rate AnWT.

Integration of anaerobic and aerobic conditions is a good strategy for the complete mineralization of azo dyes. The success of the strategy relies on anaerobic azo dye reduction occurring inside biofilms exposed to oxygen and subsequent aerobic mineralization of the formed aromatic amines. First, the effect of oxygen on the azo dye reduction by methanogenic granular sludge was studied using Mordant Orange 1 (MOI, Color Index no. 14030) as a model azo dye and ethanol as a cosubstrate. Cosubstrate is required for the formation of anaerobic microniches and supply of reducing equivalents for the reduction. Aromatic amines released from MOI reduction were not mineralized due to the absence of appropriate aromatic amine degrading bacteria in the methanogenic sludge utilized. Second, the mineralization of 4-phenylazophenol (4-PAP, Color Index no. 11800) was studied under integrated anaerobic/aerobic conditions. Aniline and 4-aminophenol (4-AP) were formed during the anaerobic reduction of 4-PAP. It was examined whether aerobic microorganisms from aerobic sludge and sediment from the Rhine were capable to degrade these aromatic amines. Aniline was readily degraded but 4-AP autoxidized easily under aerobic conditions. No anaerobic degradation could be found for either aromatic amines, indicating the necessity of an aerobic step for removal of these aromatic amines. The bacterial cocultures, anaerobic granular sludge together with the aerobic enrichment culture, were able to degrade the azo dye under integrated anaerobic/aerobic conditions.

A bacterial culture (LET-13) was enriched, which uses toluene as sole carbon and energy source, and manganese oxide as terminal electron acceptor. The culture is able to degrade a variety of substituted monoaromatic compounds like p-hydroxy-benzylalcohol, p-hydroxy-benzaldehyde, p-hydroxy-benzoate, phenol and the three isomers of cresol. Benzene, ethylbenzene, all xylenes and naphthalene were not degraded under the experimental conditions used. Based on the results of growth experiments and the detection of intermediates, it is concluded that toluene is degraded via a methyl hydroxylation. A possible side reaction can lead to the formation of cresol. The organisms in the culture look similar; motile rods, which are Gram-negative, oxidase-negative and catalase-negative. The culture was partly identified by phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria shows 70–80% similarity (based on 16S rRNA gene sequence data) with the Bacteroides-Cytophaga group, and one group consists of members of the β-subclass of the Proteobacteria.