Nitrogen transformations in stratified aquatic microbial ecosystems

New analytical methods such as advanced molecular techniques and microsensors have resulted in new insights about how nitrogen transformations in stratified microbial systems such as sediments and biofilms are regulated at a μm–mm scale. A large and ever-expanding knowledge base about nitrogen fixation, nitrification, denitrification, and dissimilatory reduction of nitrate to ammonium, and about the microorganisms performing the processes, has been produced by use of these techniques. During the last decade the discovery of anammmox bacteria and migrating, nitrate accumulating bacteria performing dissimilatory reduction of nitrate to ammonium have given new dimensions to the understanding of nitrogen cycling in nature, and the occurrence of these organisms and processes in stratified microbial communities will be described in detail.     

Distribution and fate of anthropogenic nitrogen in the Calamagrostis angustifolia wetland ecosystem of Sanjiang Plain, Northeast China

Wetlands are important for the protection of water quality of rivers and lakes, especially those adjacent to agricultural landscapes, by intercepting and removing nutrients in runoff. In this study, the 15N tracer technique was applied to study the distribution and fate of anthropogenic nitrogen (15N-fertilizer) in Calamagrostis angustifolia Kom wetland plant-soil microcosms to identify the main ecological effects of it. 15NH415NO3 solution (14.93 mg N/L, 20.28 at.% 15N) was added to each microcosm of the first group, which was approximate to the current nitrogen concentration (CNC) of farm drainage, and 29.86 mg NIL 15NH415NO3 solution was added to another group, which was approximate to the double nitrogen concentration (DNC) of farm drainage, while no nitrogen (NN) was added to the third group. The results suggest that the Input of anthropogenic nitrogen has positive effects on the biomass and total nitrogen content of plant, and the positive effects will be elevated as the increase of its Input amount. The increase of 15N-fartilizer can also elevate its amounts and proportions in plant nitrogen. Soil nitrogen is still the main source of plant nitrogen, but its proportion will be reduced as the increase of 15N-fertilizer. The study of the fate of 15N-fartilizar indicates that, in CNC treatment, only a small proportion is water-dissolved (0.13±0.20%), a considerable proportion is soil-immobilized (17.02±8.62%), or plant-assimilated (23.70±0.92%), and most is lost by gaseous forms (59.15±8.35%). While in DNC treatment, about 0.09±0.15% is water-dissolved, 15.33±7.46% is soil-immobilized, 23.55±2.86% is plant-assimilated, and 61.01±5.59% is lost by gaseous forms. The double input of anthropogenic nitrogen can not elevate the proportions of plant-assimilation, soil-immobilization and water-dissolution,but it can enhance the gaseous losses.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulation of gymnosperm Podocarpus macro-phyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

Structural and functional analysis of denitrification genes in Pseudomonas stutzeri A1501

Denitrification is the process by which nitrates are converted to nitrogen gas under the action of microor-ganism, and in a bioenergetics viewpoint, a kind of respiration of bacteria in anoxia condition. In such a process, nitrogen in oxidation state replaces oxygen as the terminal electron acceptor in cell membrane, gen-erates potential gradient with the action of a series of oxidoreductase, and finally converts nitrate into nitro-gen[1]. Denitrification is widely present in nature, and resea…     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

The development of policy approaches for reducing nitro-gen pollution to coastal waters of the USA

Two-thirds of the coastal rivers and bays in the United States are degraded from nutrient pollution, and nitrogen inputs these waters continue to increase. The nitrogen comes from a variety of sources, including runoff from agricultural fields, concentrated animal feeding operations, atmospheric deposition from fossil fuel combustion, and sewage and septic wastes. Technical solutions for nitrogen pollution exist at reasonable cost. That most of these solutions have not yet been implemented to any significant extent across the United States suggests that new policy approaches are necessary. The best solution may involve a combination of voluntary and mandatory approaches, applying different approaches to different sources of nitrogen pollution. A watershed-based approach that relies heavily on voluntary mechanisms (such as crop-yield insurance to reduce over-fertilization) is likely to be the most effective for some sources of nitrogen (such as runoff from agricultural fields), while a uniform national regulatory approach may be better for others (such as NOx emissions from fossil fuel combustion). Implementation of management strategies should be carefully coupled to monitoring programs to assess the effectiveness of these strategies. While both nitrogen and phosphorus are important to control, the focus should be on nitrogen management, in part because nitrogen is more generally the causal agent of coastal eutrophication. Also, while nitrogen-control practices tend to also reduce phosphorus pollution, phosphorus-control practices often have little effect on nitrogen. Although current scientific and technical knowledge is sufficient to begin to make substantial progress toward solving coastal nitrogen pollution, progress will be made more quickly and more cost effectively with increased investment in appropriate scientific research.     

混合培养真菌和细菌对废水的生物去氮作用

Denitrificationis a biological processin which nitrateand/or nitrite is reduced to gaseous nitrogen,dinitrogen(N2)or nitrous oxide(N2O)while carbon dioxide is thesecond gaseous product of the process.This is one of themain mechanisms of the global nitrogen cycle,and playsanimportant role as the reverse reaction of nitrogen fixa-tion in maintaining global environmental homeostasis[1].Denitrification has beenlongthought to be a unique char-acteristic of prokaryotes[2,3].Anumber of bacteria(such…     

Soil nitrogen balance assessment and its application for sustainable agriculture and environment

Soil nitrogen balance assessment (SNBA) serves as an effective tool for estimating the magnitude of nitrogen loss/gain of the agro-eco systems and to appraise their sustainability. SNBA brings forth awareness of soil fertility problems, besides providing information relating to the resultant release of nitrogen into the environment consequent to agricultural practices. Quantitative information relating to nitrogen escape into the environment through such exercises can be gainfully utilized for identification of causative factors, enhancing fertilizer use efficiency and formulating programmes aimed at plugging N leakages. An overview of nitrogen balance approaches and methodologies is presented. A deeper understanding and insight into the agro-eco systems provided by the SNBA exercises can lay the basis for the formulation of effective agronomic interventions and policies aimed at promoting sustainable agriculture and a benign environment.     

Evaluation of complexed NO reduction mechanism in a chemical absorption–biological reduction integrated NO<Subscript><Emphasis Type="BoldItalic">x</Emphasis></Subscript> removal system

Biological reduction of nitric oxide (NO) from Fe(II) ethylenediaminetetraacetic acid (EDTA)-NO to dinitrogen (N(2)) is a core process for the continual nitrogen oxides (NO(x)) removal in the chemical absorption-biological reduction integrated approach. To explore the biological reduction of Fe(II)EDTA-NO, the stoichiometry and mechanism of Fe(II)EDTA-NO reduction with glucose or Fe(II)EDTA as electron donor were investigated. The experimental results indicate that the main product of complexed NO reduction is N(2), as there was no accumulation of nitrous oxide, ammonia, nitrite, or nitrate after the complete depletion of Fe(II)EDTA-NO. A transient accumulation of nitrous oxide (N(2)O) suggests reduction of complexed NO proceeds with N(2)O as an intermediate. Some quantitative data on the stoichiometry of the reaction are experimental support that reduction of complexed NO to N(2) actually works. In addition, glucose is the preferred and primary electron donor for complexed NO reduction. Fe(II)EDTA served as electron donor for the reduction of Fe(II)EDTA-NO even in the glucose excessive condition. A maximum reduction capacity as measured by NO (0.818 mM h(-1)) is obtained at 4 mM of Fe(II)EDTA-NO using 5.6 mM of glucose as primary electron donor. These findings impact on the understanding of the mechanism of bacterial anaerobic Fe(II)EDTA-NO reduction and have implication for improving treatment methods of this integrated approach.     

Effects of nitrate and nitrite on dissimilatory iron reduction by Shewanella putrefaciens 200.

The inhibitory effects of nitrate (NO3-) and nitrite (NO2-) on dissimilatory iron (FE3+) reduction were examined in a series of electron acceptor competition experiments using Shewanella putrefaciens 200 as a model iron-reducing microorganism. S. putrefaciens 200 was found to express low-rate nitrate reductase, nitrite reductase, and ferrireductase activity after growth under highly aerobic conditions and greatly elevated rates of each reductase activity after growth under microaerobic conditions. The effects of NO3- and NO2- on the Fe3+ reduction activity of both aerobically and microaerobically grown cells appeared to follow a consistent pattern; in the presence of Fe3+ and either NO3- or NO2-, dissimilatory Fe3+ and nitrogen oxide reduction occurred simultaneously. Nitrogen oxide reduction was not affected by the presence of Fe3+, suggesting that S. putrefaciens 200 expressed a set of at least three physiologically distinct terminal reductases that served as electron donors to NO3-, NO2-, and Fe3+. However, Fe3+ reduction was partially inhibited by the presence of either NO3- or NO2-. An in situ ferrozine assay was used to distinguish the biological and chemical components of the observed inhibitory effects. Rate data indicated that neither NO3- nor NO2- acted as a chemical oxidant of bacterially produced Fe2+. In addition, the decrease in Fe3+ reduction activity observed in the presence of both NO3- and NO2- was identical to the decrease observed in the presence of NO2- alone. These results suggest that bacterially produced NO2- is responsible for inhibiting electron transport to Fe3+.     

Effects of electron donor and acceptor conditions on reductive dehalogenation of tetrachloromethane by Shewanella putrefaciens 200.

Shewanella putrefaciens 200 is a nonfermentative bacterium that is capable of dehalogenating tetrachloromethane to chloroform and other, unidentified products under anaerobic conditions. Since S. putrefaciens 200 can respire anaerobically by using a variety of terminal electron acceptors, including NO3-, NO2-, and Fe(III), it provides a unique opportunity to study the competitive effects of different electron acceptors on dehalogenation in a single organism. The results of batch studies showed that dehalogenation of CT by S. putrefaciens 200 was inhibited by O2, 10 mM NO3-, and 3 mM NO2-, but not by 15 mM Fe(III), 15 mM fumarate, or 15 mM trimethylamine oxide. Using measured O2, Fe(III), NO2-, and NO3- reduction rates, we developed a speculative model of electron transport to explain inhibition patterns on the basis of (i) the kinetics of electron transfer at branch points in the electron transport chain, and (ii) possible direct inhibition by nitrogen oxides. In additional experiments in which we used 20 mM lactate, 20 mM glucose, 20 mM glycerol, 20 mM pyruvate, or 20 mM formate as the electron donor, dehalogenation rates were independent of the electron donor used. The results of other experiments suggested that sufficient quantities of endogenous substrates were present to support transformation of tetrachloromethane even in the absence of an exogenous electron donor. Our results should be significant for evaluating (i) the bioremediation potential at sites contaminated with both halogenated organic compounds and nitrogen oxides, and (ii) the bioremediation potential of iron-reducing bacteria at contaminated locations containing significant amounts of iron-bearing minerals.     

Production of nitrous oxide from nitrite in Klebsiella pneumoniae: mutants altered in nitrogen metabolism.

Under anaerobic conditions, Klebsiella pneumoniae reduced nitrite (NO2-), yielding nitrous oxide (N2O) and ammonium ions (NH4+) as products. Nitrous oxide formation accounted for about 5% of the total NO2- reduced, and NH4+ production accounted for the remainder. Glucose and pyruvate were the electron donors for NO2- reduction to N2O by whole cells, whereas glucose, NADH, and NADPH were found to be the electron donors when cell extracts were used. On the one hand, formate failed to serve as an electron donor for NO2- reduction to N2O and NH4+, whereas on the other hand, formate was the best electron donor for nitrate reduction in either whole cells or cell extracts. Mutants that are defective in the reduction of NO2- to NH4+ were isolated, and these strains were found to produce N2O at rates comparable to that of the parent strain. These results suggest that the nitrite reductase producing N2O is distinct from that producing NH4+. Nitrous oxide production from nitric oxide (NO) occurred in all mutants tested, at rates comparable to that of the parent strain. This result suggests that NO reduction to N2O, which also uses NADH as the electron donor, is independent of the protein(s) catalyzing the reduction of NO2- to N2O.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

Nitrous oxide as end product of denitrification by strains of fluorescent pseudomonads.

Growing cultures of several strains of Pseudomonas fluorescens and Pseudomonas chlororaphis produced N2O as the only detectable gaseous product of denitrification, and other strains produced N2 as the gaseous end product of denitrification. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2O-producing strains P. fluorescens PJ 185 and P. chlororaphis B-560 was recovered as N2O. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2-producing strain P. fluorescens PJ70 was converted to N2. Cell extracts of P. fluorescens PJ 70, PJ 185, and P. chlororaphis B-560 exhibited NO3- reductase activity when sodium succinate was the electron donor. Reduced nicotinamide adenine dinucleotide and flavine adenine dinucleotide were required to demonstrate NO2- reductase activity in cell extracts.     

Inorganic Carbon-Stimulated O2 Photoreduction Is Suppressed by NO2- Assimilation in Air-Grown Cells of Synechococcus UTEX 625

The effect of NO2- assimilation on O2 exchange and CO2 fixation of the cyanobacterium, Synechococcus UTEX 625, was studied mass spectrometrically. Upon addition of 1 mM inorganic carbon to the medium, inorganic carbon pools developed and accelerated O2 photoreduction 5-fold when CO2 fixation was inhibited. During steady-state photosynthesis at saturating light, O2 uptake represented 32% of O2 evolution and balanced that portion of O2 evolution that could not be accounted for by CO2 fixation. Under these conditions, NO2- assimilation reduced O2 uptake by 59% but had no influence on CO2 fixation. NO2- assimilation decreased both CO2 fixation and O2 photoreduction at low light and and increased net O2 evolution at all light intensities. The increase in net O2 evolution observed during simultaneous assimilation of carbon and nitrogen over carbon alone was due to a suppression of O2 photoreduction by NO2- assimilation. When CO2 fixation was precluded, NO2- assimilation inhibited O2 photoreduction and stimulated O2 evolution. When the electron supply was limiting (low light), competition among O2, CO2, and NO2- for electrons could be observed, but when the electron supply was not limiting (saturating light), O2 photoreduction and/or NO2- reduction caused electron transport that was additive to that for maximum CO2 fixation.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Nitric oxide reduction in BioDeNOx reactors: kinetics and mechanism

Biological reduction of nitric oxide (NO) to di-nitrogen (N(2)) gas in aqueous Fe(II)EDTA(2-) solutions is a key reaction in BioDeNOx, a novel process for NOx removal from flue gases. The mechanism and kinetics of the first step of NO reduction, that is, the conversion of NO to N(2)O, was determined in batch experiments using various types of inocula. Experiments were performed in Fe(II)EDTA(2-) medium (5-25 mM) under BioDeNOx reactor conditions (55 degrees C, pH 7.2 +/- 0.2) with ethanol as external electron donor. BioDeNOx reactor mixed liquor gave the highest NO reduction rates (+/-0.34 nmol s(-1) mg(prot)(-1)) with an estimated K(m) value for NO lower than 10 nM. The specific NO (to N(2)O) reduction rate depended on the NO (aq) and Fe(II)EDTA(2-) concentration as well as the temperature. The experimental results, complemented with kinetic and thermodynamic considerations, show that Fe(II)EDTA(2-), and not ethanol, is the primary electron donor for NO reduction, that is, the BioDeNOx reactor medium (the redox system Fe(II)EDTA(2-)/Fe(III)EDTA(-)) interferes with the NO reduction electron transfer chain and thus enhances the NO denitrification rate.