On-line monitoring of the denitrification process by measurement of NADH fluorescence

On-line monitoring method of the denitrification process was developed by NADH fluorescence measurements using the facultative microorganism, Ochrobactrum anthropi SY509. The NADH fluorescence signals showed a rapid drop and a rise at the initiation and termination points of the denitrification, respectively. This NADH fluorescence method could be applied successfully to the monitoring of the denitrification process in sequential batch and continuous operations while these rapid changes of fluorescence were not observed in a batch operation due to accumulation of some metabolites secreted from the microorganism.     

Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria,Ochrobactrum anthropi SY509, which was isolated from soil samples.O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to 70°C. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membrane-bound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.     

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.     

Permeabilization of<Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> SY509 cells with organic solvents for whole cell biocatalyst

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Failure of denitrifying bacteria to utilize benzoic acid under anaerobic conditions with nitrate as the only terminal electron acceptor

Summary (Ring-U)-¹⁴C-benzoate was not utilized by various denitrifying bacteria (pure cultures of Pseudomonas aeruginosa, Acinetobacter sp. and Moraxella sp. or a mixed population) in the presence of nitrate as the only electron acceptor (completely anaerobic conditions). In the presence of only traces of molecular oxygen (introduced by porous tubing), denitrification did occur under inappropriate experimental conditions. This indicates that an apparent anaerobic fission of the benzene nucleus may occur.     

Denitrification under various aeration conditions in Comamonas sp., strain SGLY2

Abstract A number of bacteria were isolated from different anoxic reactors. Those having denitrifying potential were tested for their ability to denitrify under aerobic conditions. The activity of their denitrifying enzymes varied from partial inactivation by oxygen (strains NO2B9 and TCET1) to oxygen-independent activity in a strain named SGLY2 which was tentatively identified as Comamonas sp. The effect of different aeration conditions on growth and on denitrification of SGLY2 was studied more extensively. This strain was able to consume oxygen and nitrate simultaneously with the production of nitrogen and without build-up of nitrite. The dissimilatory nitrate-reductase of nitrate-adapted cells was found to be more active in the presence of oxygen than in micro-aerobic or strictly anaerobic conditions.     

微生物驱动硝酸盐还原耦合亚铁氧化成矿过程的锌胁迫

【目的】探究中性厌氧条件下,金属锌影响下硝酸盐依赖型铁氧化菌Pseudomonas stutzeri LS-2驱动的硝酸盐还原耦合亚铁氧化成矿过程机制,对深入理解中性厌氧环境中微生物亚铁氧化驱动的反硝化作用及重金属固定机制具有重要意义。【方法】以不同Zn(Ⅱ)浓度构建LS-2驱动的亚铁氧化成矿体系,分析不同体系中亚铁氧化速率、硝酸盐还原速率以及形成矿物的结构变化规律。【结果】LS-2驱动的硝酸盐还原耦合亚铁氧化成矿过程中,共存Zn(Ⅱ)降低该过程中硝酸盐的还原速率和亚铁氧化速率。同时,随着Zn(Ⅱ)浓度提高,抑制作用增强。微生物亚铁氧化形成的矿物通过吸附、共沉淀和离子置换等过程固定Zn(Ⅱ),降低Zn(Ⅱ)活性。Zn(Ⅱ)浓度对形成的矿物结构有较大的影响:低浓度Zn(Ⅱ)体系中,形成的矿物为纤铁矿;随着Zn(Ⅱ)浓度的提高,矿物结构与结晶度都有一定程度的变化,当Zn(Ⅱ)达到4 mmol/L时,形成的矿物主要为铁锌尖晶石。【结论】明确了重金属锌对LS-2菌株反硝化及亚铁氧化过程的抑制规律,同时阐明了Zn(Ⅱ)浓度对形成矿物结构的影响。研究结果有助于深入认识中性厌氧环境中重金属与微生物驱动的铁循环和反硝化过程的耦合作用,为土壤重金属污染防治提供理论支撑。     

Denitrification in Lake Kinneret in the presence of oxygen*

SUMMARY. Denitrification experiments under anaerobic and aerated conditions were carried out in the laboratory with Lake Kinneret water and with pure cultures of the denitrifying bacteria Pseudomonas aeruginosa 2 Kin isolated from the lake. Although losses of nitrogen in Lake Kinneret due to denitrification have been found to occur during periods when dissolved oxygen exceeded 5 mg l^?1 it was found that under aerated conditions glucose as a carbon source must be added in order to get denitrification in the laboratory. Disappearance of nitrogen during the experiments was due to denitrification as shown by the nitrogen balance calculated for each sampling. The ATP content showed that no proliferation of cells took place during the experiment. The rate of denitrification was strongly influenced by and was directly proportional to nitrate concentrations. Temperature has a very slight effect on the denitrification rate. Q₁₀ for the range 15–30°C was 1.35. The role of denitrification in the nitrogen balance of Lake Kinneret is discussed.     

Effect of agitation and aeration on the citric acid production by Yarrowia lipolytica grown on glycerol

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24-0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.     

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium

A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO₂. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Received: 6 November 1998 / Accepted: 19 January 1999     

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.     

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.     

Nitrate-dependent anaerobic carbon monoxide oxidation by aerobic CO-oxidizing bacteria

Two dissimilatory nitrate-reducing (Burkholderia xenovorans LB400 and Xanthobacter sp. str. COX) and two denitrifying isolates (Stappia aggregata IAM 12614 and Bradyrhizobium sp. str. CPP), previously characterized as aerobic CO oxidizers, consumed CO at ecologically relevant levels (<100 ppm) under anaerobic conditions in the presence, but not absence, of nitrate. None of the isolates were able to grow anaerobically with CO as a carbon or energy source, however, and nitrate-dependent anaerobic CO oxidation was inhibited by headspace concentrations >100-1000 ppm. Surface soils collected from temperate, subtropical and tropical forests also oxidized CO under anaerobic conditions with no lag. The observed activity was 25-60% less than aerobic CO oxidation rates, and did not appear to depend on nitrate. Chloroform inhibited anaerobic but not aerobic activity, which suggested that acetogenic bacteria may have played a significant role in forest soil anaerobic CO uptake.     

Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus

Lavendamycin methyl ester (LME) is a derivative of a highly functionalized aminoquinone alkaloid lavendamycin and could be used as a scaffold for novel anticancer agent development. This work demonstrated LME production by cultivation of an engineered strain of Streptomyces flocculus CGMCC4.1223 ΔstnB1, while the wild-type strain did not produce. To enhance its production, the effect of shear stress and oxygen supply on ΔstnB1 strain cultivation was investigated in detail. In flask culture, when the shaking speed increased from 150 to 220 rpm, the mycelium was altered from a large pellet to a filamentous hypha, and the LME production was almost doubled, while no significant differences were observed among varied filling volumes, which implied a crucial role of shear stress in the morphology and LME production. To confirm this suggestion, experiments with agitation speed ranging from 400 to 1,000 rpm at a fixed aeration rate of 1.0 vvm were conducted in a stirred tank bioreactor. It was found that the morphology became more hairy with reduced pellet size, and the LME production was enhanced threefolds when the agitation speed increased from 400 to 800 rpm. Further experiments by varying initial k _L a value at the same agitation speed indicated that oxygen supply only slightly affected the physiological status of ΔstnB1 strain. Altogether, shear stress was identified as a major factor affecting the cell morphology and LME production. The work would be helpful to the production of LME and other secondary metabolites by filamentous microorganism cultivation.     

Novel 3-dimensional bioelectrode for mediatorless bioelectrochemical denitrification

A novel bioelectrochemical method for denitrification was developed using electricity as the electron donor. The novel electrode contained both Ochrobactrum anthropi SY509, which was permeabilized as a biocatalyst, and copper powder as a conducting material. Using this electrode, a high denitrification efficiency of 1 mmol N-NO (3) (-) /g dry cell.h was achieved via direct electron transfer without using mediator.     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996