Effect of oxygen on denitrification in continuous chemostat culture withComamonas sp SGLY2

Continuous cultures ofComamonas sp SGLY2 were grown anaerobically prior to establishing steady states at different oxygen flow rates. At a low oxygen transfer rate, no dissolved oxygen accumulated in the medium and all nitrate was reduced to dinitrogen. Concurrently with the increase of dissolved oxygen concentration in the liquid phase, the rate of denitrification decreased. However, at a dissolved oxygen concentration near saturation (33 mg L^–1), a part of the electron flow always diverted to nitrate with production of dinitrogen: the aerobic denitrification rate was equivalent to 35% of that calculated under anaerobic conditions. These experiments reflected the co-utilization of oxygen and N-oxides and the production of dinitrogen, up to saturated conditions, which implied synthesis and activity of the four denitrifying enzymes under various aeration conditions.     

Influence of reactive media composition and chemical oxygen demand as methanol on autotrophic sulfur denitrification

Sulfur-utilizing autotrophic denitrification relies on an inorganic carbon source to reduce the nitrate by producing sulfuric acid as an end product and can be used for the treatment of wastewaters containing high levels of nitrates. In this study, sulfur-denitrifying bacteria were used in anoxic batch tests with sulfur as the electron donor and nitrate as the electron acceptor. Various medium components were tested under different conditions. Sulfur denitrification can drop the medium pH by producing acid, thus stopping the process half way. To control this mechanism, a 2:1 ratio of sulfur to oyster shell powder was used. Oyster shell powder addition to a sulfurdenitrifying reactor completely removed the nitrate. Using 50, 100, and 200 g of sulfur particles, reaction rate constants of 5.33, 6.29, and 7.96 mg(1/2)/l(1/2)·h were obtained, respectively; and using 200 g of sulfur particles showed the highest nitrate removal rates. For different sulfur particle sizes ranging from small (0.85-2.0 mm), medium (2.0-4.0 mm), and large (4.0-4.75 mm), reaction rate constants of 31.56, 10.88, and 6.23 mg(1/2)/l(1/2)·h were calculated. The fastest nitrate removal rate was observed for the smallest particle size. Addition of chemical oxygen demand (COD), methanol as the external carbon source, with the autotrophic denitrification in sufficiently alkaline conditions, created a balance between heterotrophic denitrification (which raises the pH) and sulfur-utilizing autotrophic denitrification, which lowers the pH.     

Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Effect of Chloramphenicol on Denitrification in Flexibacter canadensis and "Pseudomonas denitrificans"

It was recently reported that chloramphenicol inhibits existing denitrification enzyme activity in sediments and carbon-starved cultures of "Pseudomonas denitrificans." Therefore, we studied the effect of chloramphenicol on denitrification by Flexibacter canadensis and "P. denitrificans." Production of N(inf2)O from nitrate by F. canadensis cells decreased as the concentration of chloramphenicol was increased, and 10.0 mM chloramphenicol completely inhibited N(inf2)O production. "P. denitrificans" was less sensitive to chloramphenicol, and production of N(inf2)O from nitrate was inhibited by only about 50% even in the presence of 10.0 mM chloramphenicol. These results suggested that inhibition of denitrification enzyme activity depended on the concentration of chloramphenicol. Increasing the concentration of chloramphenicol decreased the rate of production of nitrite from nitrate by F. canadensis cells, and the concentration of chloramphenicol which resulted in 50% inhibition of production of nitrite from nitrate was 2.5 mM. In contrast, the rates of production of nitrite from nitrate by intact cells and cell extracts of "P. denitrificans" were inhibited by only 58 and 54%, respectively, at a chloramphenicol concentration of 10.0 mM. Chloramphenicol caused accumulation of NO from nitrite but not from nitrate and inhibited NO consumption in F. canadensis; however, it had neither effect in "P. denitrificans." Chloramphenicol did not affect N(inf2)O consumption by these organisms. We concluded that chloramphenicol inhibits denitrification at the level of nitrate reduction and, in F. canadensis, also at the level of NO reduction.     

Quantitative selection of denitrifying bacteria in continuous cultures and requirement for organic carbon. I. Starch

A mixed population of bacteria from bottom sludge of nitrogen wastewater reservoir was incubated in continuous culture in medium containing 1000 mg nitrate nitrogen/l and starch. Maximal efficiency of denitrification was 5 mg N/l/h. Marked changes in participation of denitrifying bacteria (16-76%) among total number of bacteria was observed, this being dependent on the ratio of starch concentration (CS) to nitrogen concentration (CN) in the medium. The optimal CS/CN ratio ensuring highest participation of denitrifying bacteria was 3.2. The amount of starch required for the denitrification of a defined quantity of nitrogen is negatively correlated (r = -0.98) with the frequency of the occurrence of denitrifying bacteria (XD) and is: CS = (5.53-0.028XD) CN. The denitrifying bacteria in continuous culture were dominated, depending on CS/CN ratio and flow rate of medium, by Alcaligenes faecalis, Paracoccus denitrificans or Pseudomonas mendocina, that is species unable to hydrolyse starch.     

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。     

Vertical distribution of denitrification in an estuarine sediment: integrating sediment flowthrough reactor experiments and microprofiling via reactive transport modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N(2)O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N(2)O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm(-3) h(-1). The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (+/- 10) nmol cm(-2) h(-1), which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

帽儿山地区不同类型河岸带土壤的反硝化效率

以帽儿山地区森林背景下的森林、皆伐、草地河岸带和农田背景下的森林、裸地河岸带土壤为研究对象,采用硝态氮消失法,研究了不同背景下各类型河岸带的反硝化强度及其影响因素．结果表明：各类型河岸带中,农田背景下的森林河岸带土壤反硝化强度最大,其硝态氮消失率的变化范围为46．79％～91．13％,农田背景下的裸地河岸带土壤反硝化强度最小,其硝态氮消失率的变化范围为15．64％～81．84％;森林背景下土壤反硝化强度的大小顺序为皆伐河岸带〉森林河岸带〉草地河岸带,其硝态氮消失率的变化范围依次为42．06％～90．39％、28．24％～85．73％、21．44％～83．11％．研究区河岸带表层土壤的反硝化强度大于底层．河岸带土壤反硝化强度均受可利用碳、硝态氮的限制,各类型河岸带以农田背景下森林河岸带土壤反硝化潜力最大．     

Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.     

Denitrification in lucerne nodules and bacteroids supplied with nitrate

Nodulated lucerne plants ( Medicago sativa L. cv. Aragón) were supplied with 20 m M nitrate. Anaerobically isolated bacteroids of Rhizobium meliloti from these plants were able to denitrify after 48 h treatment. R. meliloti bacteroids behave as total denitrifiers, reducing nitrate to dinitrogen: when acetylene was omitted from the assay medium very little nitrous oxide was recovered. The onset of denitrification activity was coincident with the induction of nitrite reductase activity (EC 1.7.99.3) whereas nitrate reductase activity (EC 1.7.99.4) was constitutive. Whole nodules from plants receiving several doses of nitrate were assayed, in a nitrate-free medium, to monitor denitrification activity dependent on nitrate within the nodules. Denitrification activity was detected after 2 days of 20 m M nitrate supply or after 3 days in the presence of 10 or 5 m M nitrate. These results are discussed in relation to current controversy about nitrate entry into the infection region of nodules. It is concluded that this process occurs more rapidly than suggested in recent research.     

Interactions between Benthic Copepods,Bacteria and Diatoms Promote Nitrogen Retention in Intertidal Marine Sediments

The present study aims at evaluating the impact of diatoms and copepods on microbial processes mediating nitrate removal in fine-grained intertidal sediments. More specifically, we studied the interactions between copepods, diatoms and bacteria in relation to their effects on nitrate reduction and denitrification. Microcosms containing defaunated marine sediments were subjected to different treatments: an excess of nitrate, copepods, diatoms (Navicula sp.), a combination of copepods and diatoms, and spent medium from copepods. The microcosms were incubated for seven and a half days, after which nutrient concentrations and denitrification potential were measured. Ammonium concentrations were highest in the treatments with copepods or their spent medium, whilst denitrification potential was lowest in these treatments, suggesting that copepods enhance dissimilatory nitrate reduction to ammonium over denitrification. We hypothesize that this is an indirect effect, by providing extra carbon for the bacterial community through the copepods'' excretion products, thus changing the C/N ratio in favour of dissimilatory nitrate reduction. Diatoms alone had no effect on the nitrogen fluxes, but they did enhance the effect of copepods, possibly by influencing the quantity and quality of the copepods'' excretion products. Our results show that small-scale biological interactions between bacteria, copepods and diatoms can have an important impact on denitrification and hence sediment nitrogen fluxes.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Denitrification in Marl and Peat Sediments in the Florida Everglades

The potential for denitrification in marl and peat sediments in the Shark River Slough in the Everglades National Park was determined by the acetylene blockage assay. The influence of nitrate concentration on denitrification rate and N₂O yield from added nitrate was examined. The effects of added glucose and phosphate and of temperature on the denitrification potential were determined. The sediments readily denitrified added nitrate. N₂O was released from the sediments both with and without added acetylene. The marl sediments had higher rates than the peat on every date sampled. Denitrification was nitrate limited; however, the yields of N₂O amounted to only 10 to 34% of the added nitrate when 100 μM nitrate was added. On the basis of measured increases in ammonium concentration, it appears that the balance of added nitrate may be converted to ammonium in the marl sediment. The sediment temperature at the time of sampling greatly influenced the denitrification potential (15-fold rate change) at the marl site, indicating that either the number or the specific activity of the denitrifiers changed in response to temperature fluctuations (9 to 25°C) in the sediment. It is apparent from this study that denitrification in Everglades sediments is not an effective means of removing excess nitrogen which may be introduced as nitrate into the ecosystem with supply water from the South Florida watershed and that sporadic addition of nitrate-rich water may lead to nitrous oxide release from these wetlands.     

Vertical Distribution of Denitrification in an Estuarine Sediment: Integrating Sediment Flowthrough Reactor Experiments and Microprofiling via Reactive Transport Modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N₂O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N₂O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm⁻³ h⁻¹. The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (± 10) nmol cm⁻² h⁻¹, which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.     

Dissimilatory ammonia production vs. denitrification in vitro and in inoculated agricultural soil samples

A dissimilatory ammonia-producing isolate identified as Enterobacter amnigenus and a denitrifier identified as Agrobacterium radiobacter isolated from the same soil were studied. The products of nitrate reduction in a minimal medium, enriched with glucose and containing nitrate N as the sole nitrogen source, were quantified when each of these isolates was cultured anaerobically, alone or mixed together in the presence or absence of C(2)H(2). When they were cultured together, ammonia was the principal product of nitrate reduction. The distribution between denitrification and dissimilatory ammonia production (DAP) for nonsterilised soil samples inoculated with E. amnigenus or A. radiobacter, or a mixture of these two isolates, was also investigated. Production of NH(4)(+) was increased under these conditions (strict anaerobiosis and much available fermentable carbon), but the inoculation of soil samples with 1.2?×?10(7) cells of E. amnigenus·g dried soil(-1) was not sufficient to shift nitrate reduction from nitrous oxide (denitrification) to ammonia production, suggesting that inoculation with a greater number of DAP bacteria than introduced would probably be required to enable ammonia production to exceed nitrous oxide release. Key words: dissimilatory ammonia production, denitrification, Enterobacter amnigenus, Agrobacterium radiobacter.     

Denitrification of wastewater containing high nitrate and calcium concentrations

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO(3)(-)-N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO(3)(-)-N/gVSSh and very low residual COD in the effluent.     

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     

Study of a combined sulfur autotrophic with proton-exchange membrane electrodialytic denitrification technology: sulfate control and pH balance

A novel combined system established for nitrate removal from aqueous solution consisted of two parts: sulfur autotrophic denitrification and bio-electrochemical denitrification based on proton-exchange membrane electrodialysis (PEMED). The system was operated at various hydraulic retention times (HRT) and current intensities. Its optimum operation condition was also determined. The combined process had pH adjustment thus generating less nitrite than PEMED process. The denitrification rate of sulfur autotrophic part was dependent on HRT, while shorter HRT could reduce the sulfate generated by the sulfur autotrophic process. The denitrification rate of PEMED process depended on the applied current. For 32 ± 1 mg-N/L nitrate in influent, the optimum operation parameters of combined process were: HRT 2 h; applied current 350 mA. The combined reactor could achieve 95.8% nitrate removal without nitrite accumulation, the pH of effluent kept neutral and the sulfate of effluent was 202.1 mg/L, lower than the drinking water standard in China.     

Selection of organic substrates as potential reactive materials for use in a denitrification permeable reactive barrier (PRB)

The aim of the present study was to select a suitable natural organic substrate as a potential carbon source for use in a denitrification permeable reactive barrier (PRB). A number of seven organic substrates were first tested in batch tests. The materials attained varying degrees of success at promoting denitrification. Some of the organic substrates performed very well, achieving complete nitrate removal (>98%), while others were considered unsuitable for a variety of reasons, including: insufficient nitrate or nitrogen removal, excessive release of leachable nitrogen from the substrate or excessive reduction of nitrate to ammonium rather than removing it as gaseous N2. The top performing substrate in terms of denitrification extent (>98%) and rate (0.067 mgNO3(-)-N dm(-3)d(-1)g(sub)(-1)) was then selected for two bench-scale column experiments in an attempt to simulate the PRB. The inlet concentration was 50 mg dm(-3) NO3(-)-N and the columns operated at two different flow rates: 0.3 cm3 min(-1) (Column 1) and 1.1cm3 min(-1) (Column 2). The two columns showed different general patterns, making it clear that the flow rate was a key factor at the nitrate removal. Nitrate was completely removed (>96%) by the passage through Column 1, while only partially removed in Column 2 (66%). The results indicated that the selected organic substrate (Softwood) was applicable for further use as a filling material for a PRB.