13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation

The influence of microbial degradation on the ¹³C/¹²C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors αC for toluene degradation by Pseudomonas putida (1.0026 ± 0.00017), Thauera aromatica (1.0017 ± 0.00015), Geobacter metallireducens (1.0018 ± 0.00029) and the sulphate-reducing strain TRM1 (1.0017 ± 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar ¹³C/¹²C isotope fractionation factor (αC = 1.0015 ± 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil–hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a ¹³C/¹²C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms

Nitrogen isotope fractionation in the assimilation of inorganic nitrogenous compounds was studied using marine diatoms (Phaeodactylum tricornutum and Chaetoceros sp.). The isotopic composition (δ¹⁵N) of the diatoms ranged from 7 to ‐18‰ relative to that of the nitrogen source, i.e., ammonium, nitrite, or nitrate. When the growth was light‐limited, the isotope fractionation in nitrate assimilation was inversely correlated with the growth rate. The highest fractionation factor of 1.016 was obtained when the growth rate was as low as 0.025 day^‐1. Fractionation was negligible when the growth, rate was higher than 1 day^‐1. A steady‐state kinetic model was applied to explain the isotope fractionation in nitrate assimilation. The nitrogen isotope fractionation primarily takes place at the step of N‐O bond breaking in nitrate reduction to nitrite. The extent of the isotope fractionation associated with the nitrate uptake is very small, and barely exceeds the limit of detection.     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

Autotrophic nitrite removal in the cathode of microbial fuel cells

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.     

Simultaneous nitrification,denitrification, and phosphorus removal in a lab-scale sequencing batch reactor

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic-enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis showed that the final denitrification product was mainly nitrous oxide (N(2)O), not N(2). Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen-accumulating organisms (DGAOs), rather than denitrifying polyphosphate-accumulating organisms (DPAOs), were responsible for the denitrification activity.     

Nitrogen‐isotope fractionation in the nitrate respiration by the marine bacterium Serratia marinorubra

Abstract

The isotopic composition (δ¹⁵N) of nitrite produced during nitrate respiration by both growing and washed cells of the marine bacterium, Serratia marinorubra, was determined. In both the growing and washed cells, δ¹⁵N of the nitrite changed considerably with time. At least two reaction steps, producing different isotope effects (active transport of nitrate across membranes and reduction of nitrate to nitrite), appear to be involved. With washed cells, a fractionation factor as high as 1.039 was obtained—the highest ever reported for biologicalnitrate reduction. The physiological state of nitrate‐respiring bacteria in oxygen‐depleted subsurface waters of the sea is discussed from the viewpoint of isotope fractionation.     

Stoichiometry and kinetics of microbial toluene degradation under denitrifying conditions

Batch experiments were carried out to investigate the stoichiometry and kinetics of microbial degradation of toluene under denitrifying conditions. The inoculum originated from a mixture of sludges from sewage treatment plants with alternating nitrification and denitrification. The culture was able to degrade toluene under anaerobic conditions in the presence of nitrate, nitrite, nitric oxide, or nitrous oxide. No degradation occurred in the absence of Noxides. The culture was also able to use oxygen, but ferric iron could not be used as an electron acceptor. In experiments with¹⁴C-labeled toluene, 34%±8% of the carbon was incorporated into the biomass, while 53%±10% was recovered as¹⁴CO₂, and 6%±2% remained in the medium as nonvolatile water soluble products. The average consumption of nitrate in experiments, where all the reduced nitrate was recovered as nitrite, was 1.3±0.2 mg of nitrate-N per mg of toluene. This nitrate reduction accounted for 70% of the electrons donated during the oxidation of toluene. When nitrate was reduced to nitrogen gas, the consumption was 0.7±0.2 mg per mg of toluene, accounting for 97% of the donated electrons. Since the ammonia concentration decreased during degradation, dissimilatory reduction of nitrate to ammonia was not the reductive process. The degradation of toluene was modelled by classical Monod kinetics. The maximum specific rate of degradation, k, was estimated to be 0.71 mg toluene per mg of protein per hour, and the Monod saturation constant, K _s , to be 0.2 mg toluene/l. The maximum specific growth rate, _max , was estimated to be 0.1 per hour, and the yield coefficient, Y, was 0.14 mg protein per mg toluene.Abbreviations NVWP Non Volatile Water-soluble Products     

Model based evaluation of a contaminant plume development under aerobic and anaerobic conditions in 2D bench-scale tank experiments

The influence of transverse mixing on competitive aerobic and anaerobic biodegradation of a hydrocarbon plume was investigated using a two-dimensional, bench-scale flow-through laboratory tank experiment. In the first part of the experiment aerobic degradation of increasing toluene concentrations was carried out by the aerobic strain Pseudomonas putida F1. Successively, ethylbenzene (injected as a mixture of unlabeled and fully deuterium-labeled isotopologues) substituted toluene; nitrate was added as additional electron acceptor and the anaerobic denitrifying strain Aromatoleum aromaticum EbN1 was inoculated to study competitive degradation under aerobic / anaerobic conditions. The spatial distribution of anaerobic degradation was resolved by measurements of compound-specific stable isotope fractionation induced by the anaerobic strain as well as compound concentrations. A fully transient numerical reactive transport model was employed and calibrated using measurements of electron donors, acceptors and isotope fractionation. The aerobic phases of the experiment were successfully reproduced using a double Monod kinetic growth model and assuming an initial homogeneous distribution of P. putida F1. Investigation of the competitive degradation phase shows that the observed isotopic pattern cannot be explained by transverse mixing driven biodegradation only, but also depends on the inoculation process of the anaerobic strain. Transient concentrations of electron acceptors and donors are well reproduced by the model, showing its ability to simulate transient competitive biodegradation.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.     

Effect of Medium Composition on the Denitrification of Nitrate by Paracoccus denitrificans

The course of denitrification of nitrate in static cultures of Paracoccus denitrificans was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by P. denitrificans is not stable and depends on the nutritional composition of the culture medium.     

Denitrification by a soil bacterium with phthalate and other aromatic compounds as substrates.

A soil bacterium, Pseudomonas sp. strain P136, was isolated by selective enrichment for anaerobic utilization of o-phthalate through nitrate respiration. o-Phthalate, m-phthalate, p-phthalate, benzoate, cyclohex-1-ene-carboxylate, and cyclohex-3-ene-carboxylate were utilized by this strain under both aerobic and anaerobic conditions. m-Hydroxybenzoate and p-hydroxybenzoate were utilized only under anaerobic conditions. Protocatechuate and catechol were neither utilized nor detected as metabolic intermediates during the metabolism of these aromatic compounds under both aerobic and anaerobic conditions. Cells grown anaerobically on one of these aromatic compounds also utilized all other aromatic compounds as substrates for denitrification without a lag period. On the other hand, cells grown on succinate utilized aromatic compounds after a lag period. Anaerobic growth on these substrates was dependent on the presence of nitrate and accompanied by the production of molecular nitrogen. The reduction of nitrite to nitrous oxide and the reduction of nitrous oxide to molecular nitrogen were also supported by anaerobic utilization of these aromatic compounds in this strain. Aerobically grown cells showed a lag period in denitrification with all substrates tested. Cells grown anaerobically on aromatic compounds also consumed oxygen. No lag period was observed for oxygen consumption during the transition period from anaerobic to aerobic conditions. Cells grown aerobically on one of these aromatic compounds were also adapted to utilize other aromatic compounds as substrates for respiration. However, cells grown on succinate showed a lag period during respiration with aromatic compounds. Some other characteristic properties on metabolism and regulation of this strain are also discussed for their physiological aspects.     

Factors promoting emissions of nitrous oxide and nitric oxide from denitrifying sequencing batch reactors operated with methanol and ethanol as electron donors

The emissions of nitrous oxide (N₂O) and nitric oxide (NO) from biological nitrogen removal (BNR) operations via nitrification and denitrification is gaining increased prominence. While many factors relevant to the operation of denitrifying reactors can influence N₂O and NO emissions from them, the role of different organic carbon sources on these emissions has not been systematically addressed or interpreted. The overall goal of this study was to evaluate the impact of three factors, organic carbon limitation, nitrite concentrations, and dissolved oxygen concentrations on gaseous N₂O and NO emissions from two sequencing batch reactors (SBRs), operated, respectively, with methanol and ethanol as electron donors. During undisturbed ultimate‐state operation, emissions of both N₂O and NO from either reactor were minimal and in the range of <0.2% of influent nitrate‐N load. Subsequently, the two reactors were challenged with transient organic carbon limitation and nitrite pulses, both of which had little impact on N₂O or NO emissions for either electron donor. In contrast, transient exposure to oxygen led to increased production of N₂O (up to 7.1% of influent nitrate‐N load) from ethanol grown cultures, owing to their higher kinetics and potentially lower susceptibility to oxygen inhibition. A similar increase in N₂O production was not observed from methanol grown cultures. These results suggest that for dissolved oxygen, but not for carbon limitation or nitrite exposure, N₂O emission from heterotrophic denitrification reactors can vary as a function of the electron donor used. Biotechnol. Bioeng. 2010; 106: 390–398. © 2010 Wiley Periodicals, Inc.     

Dissimilatory nitrate reduction by Pseudomonas alcaliphila with an electrode as the sole electron donor

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were considered two alternative pathways of dissimilatory nitrate reduction. In this study, we firstly reported that both denitrification and DNRA occurred in Pseudomonas alcaliphila strain MBR with an electrode as the sole electron donor in a double chamber bio‐electrochemical system (BES). The initial concentration of nitrate appeared as a factor determining the type of nitrate reduction with electrode as the sole electron donor at the same potential (?500 mV). As the initial concentration of nitrate increased, the fraction of nitrate reduced through denitrification also increased. While nitrite (1.38 ± 0.04 mM) was used as electron acceptor instead of nitrate, the electrons recovery via DNRA and denitrification were 43.06 ± 1.02% and 50.51 ± 1.37%, respectively. The electrochemical activities and surface topography of the working electrode catalyzed by strain MBR were evaluated by cyclic voltammetry and scanning electron microscopy. The results suggested that cells of strain MBR were adhered to the electrode, playing the role of electron transfer media for nitrate and nitrite reduction. Thus, for the first time, the results that DNRA and denitrification occurred simultaneously were confirmed by powering the strain with electricity. The study further expanded the range of metabolic reactions and had potential value for the recognization of dissimilatory nitrate reduction in various ecosystems. Biotechnol. Bioeng. 2012; 109: 2904–2910. © 2012 Wiley Periodicals, Inc.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Light-activated,-inhibited and-independent denitrification by a denitrifying phototrophic bacterium

Effects of illumination on denitrification by a freshly isolated denitrifying phototrophic bacterium were investigated. Denitrification activity was induced when cells were grown in either light or darkness in the presence of nitrate without oxygen. Denitrification of nitrate with malate as the electron donor by cells at a phase of exponential growth occurred independently of illumination while that by cells in a stationary phase was activated. Effects of illumination on denitrification varied with electron donors. Using malate or succinate, denitrification by cells in a stationary phase was accelerated by illumination, inhibited when glucose or lactate was used, and independent of illumination when pyruvate was used. Denitrification by cells in an exponential phase was independent of illumination when succinate, malate or pyruvate was used and inhibited by it when glucose or lactate was used. Effects of illumination on the denitrification of nitrite were similar to those involving nitrate. Effects of various inhibitors on denitrification were examined in light-succinate and dark-lactate systems. Differences between the two systems are discussed.Abbreviations KCN potassium cyanide - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TTFA 2-thenoyltrifluoroacetone - CCCP carbonyl cyanide-m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide     

Effect of sulfide concentration on autotrophic denitrification from nitrate and nitrite in vertical fixed-bed reactors

Autotrophic denitrification coupled with sulfide oxidation represents an interesting alternative for the simultaneous removal of nitrate/nitrite and sulfide from wastewaters. The applicability of such bioprocess is especially advantageous for the post treatment of effluents from anaerobic reactors, since they usually produce sulfides, which can be used as endogenous electron donor for autotrophic denitrification. This study evaluated the effect of sulfide concentration on this bioprocess using nitrate and nitrite as electron acceptors in vertical fixed-bed reactors. The results showed that intermediary sulfur compounds were mainly produced when excess of electron donor was applied, which was more evident when nitrate was used. Visual evidences suggested that elemental sulfur was the intermediary compound produced. There was also evidence that the elemental sulfur previously formed was being used when sulfide was applied in stoichiometric concentration relative to nitrate/nitrite. Nitrite was more readily consumed than nitrate. For both electron acceptors and sulfide concentrations tested, autotrophic denitrification was not affected by residual heterotrophic denitrification via endogenic activity, occurring as a minor additional nitrogen removal process.