A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments

Abstract Newly developed low capacity columns were used in suppressed ion chromatography for rapid and highly reproducible determination of SO₄²⁻ in porewater samples from freshwater sediments without preconcentration of samples. With a 50 μl injection the detection limit for SO₄²⁻ was ca. 50 pmol (= 1 μ M) with a precision of 1–3% at the 10–200 μM level and <1% at concentrations above 200 μM. SO₄²⁻ could be measured in 4–5 min with the routinely used eluent (3.0 mM NaHCO₃/0.8 mM Na₂CO₃). When the strength of the eluent was increased to 3.0 mM NaHCO₃/2.0 mM Na₂CO₃, sulfate analysis was possible in less than 3 min, provided that samples were nitrate-free. Under these conditions S₂O₃²⁻ could also be sensitively determined in about 6 min. Examples of application of the method are given for measurements of sulfate reduction rates in freshwater sediment samples from Lake Constance.     

Potential rates and pathways of microbial nitrate reduction in coastal sediments

Nitrate reduction plays a key role in the biogeochemical dynamics and microbial ecology of coastal sediments. Potential rates of nitrate reduction were measured on undisturbed sediment slices from two eutrophic coastal environments using flow-through reactors (FTR). Maximum potential nitrate reduction rates ranged over an order of magnitude, with values of up to 933 nmol cm(-3) h(-1), whereas affinity constants for NO(3) (-) fell mostly between 200 and 600 microM. Homogenized sediment slurries systematically yielded higher rates of nitrate reduction than the FTR experiments. Dentrification was the major nitrate removal pathway in the sediments, although excess ammonium production indicated a contribution of dissimilatory nitrate reduction to ammonium under nitrate-limiting conditions.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of nitrate.     

微生物异化硝酸盐和亚硝酸盐产铵研究进展

异化硝酸盐和亚硝酸盐还原产铵是氮转化附属途径,为生态系统中氮的重复利用提供了依据,已成为近年来的研究热点。据报道,氮源的种类及浓度不同异化还原产铵的发生机制及强度具有差异性,决定着微生物产铵的效率,因此,有必要明确不同氮源异化还原产铵的代谢机制。本文详细论述了参与硝酸盐和亚硝酸盐异化还原产铵过程的相关微生物种类、产铵途径及其机理;系统分析了单一氮源和混合氮源对不同微生物产铵的影响和差异,比较了放线菌与其他微生物产铵的优势,并对未来的研究方向进行了展望,旨在为微生物异化硝酸盐和亚硝酸盐还原产铵提供理论基础。     

Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H₂ and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO₂ fixation. The following enzymes were found [values in parentheses = μmol min^–1 (mg protein)^–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F₄₂₀-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F₄₂₀ (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO₂ fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N₂O with NO as intermediate (0.1 μmol N₂O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity. Received: 23 August 1996 / Accepted: 6 November 1996     

Nitrite reduction to nitrous oxide by propionibacteria: Detoxication mechanism

Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N₂O. Recovery of added nitrite-N as N₂O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N₂O production were 3 to 6 orders of magnitude lower than specific rates of N₂ production by common denitrifiers. Oxygen but not acetylene inhibited N₂O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N₂O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.     

Denitrification,dissimilatory nitrate reduction to ammonium,and nitrogen fixation along a nitrate concentration gradient in a created freshwater wetland

Wetlands are often highly effective nitrogen (N) sinks. In the Lake Waco Wetland (LWW), near Waco, Texas, USA, nitrate (NO₃⁻) concentrations are reduced by more than 90% in the first 500 m downstream of the inflow, creating a distinct gradient in NO₃⁻ concentration along the flow path of water. The relative importance of sediment denitrification (DNF), dissimilatory NO₃⁻ reduction to ammonium (DNRA), and N₂ fixation were examined along the NO₃⁻ concentration gradient in the LWW. “Potential DNF” (hereafter potDNF) was observed in all months and ranged from 54 to 278 μmol N m⁻² h⁻¹. “Potential DNRA” (hereafter potDNRA) was observed only in summer months and ranged from 1.3 to 33 μmol N m⁻² h⁻¹. Net N₂ flux ranged from 184 (net denitrification) to −270 (net N₂ fixation) μmol N m⁻² h⁻¹. Nitrogen fixation was variable, ranging from 0 to 426 μmol N m⁻² h⁻¹, but high rates ranked among the highest reported for aquatic sediments. On average, summer potDNRA comprised only 5% (±2% SE) of total NO₃⁻ loss through dissimilatory pathways, but was as high as 36% at one site where potDNF was consistently low. Potential DNRA was higher in sediments with higher sediment oxygen demand (r ² = 0.84), and was related to NO₃⁻ concentration in overlying water in one summer (r ² = 0.81). Sediments were a NO₃⁻ sink and accounted for 50% of wetland NO₃⁻ removal (r ² = 0.90). Sediments were an NH₄⁺ source, but the wetland was often a net NH₄⁺ sink. Although DNRA rates in freshwater wetlands may rival those observed in estuarine systems, the importance of DNRA in freshwater sediments appears to be minor relative to DNF. Furthermore, sediment N₂ fixation can be extremely high when NO₃⁻ in overlying water is consistently low. The data suggest that newly fixed N can support sustained N transformation processes such as DNF and DNRA when surface water inorganic N supply rates are low.     

真菌异化硝酸盐还原机理的研究进展

真菌异化硝酸盐还原途径的发现打破了反硝化仅存在于原核细胞这一传统观念。真菌异化硝酸盐还原途径是在环境中氧供给受限的情况下发生的, 包括反硝化和氨的发酵。硝酸盐能诱导产生反硝化作用的酶, 其中, 硝酸盐还原酶与亚硝酸还原酶位于线粒体中, 它们所催化的酶促反应能偶联呼吸链ATP合成酶合成ATP, 同时产生NO。与参与反硝化作用前两个酶不同, 真菌NO还原酶能以NADH为直接电子供体将NO还原为N2O, 在NAD+的再生和自由基NO的脱毒中起着重要作用。氨发酵则将硝酸盐还原成NH4+, 同时偶联乙酸的生成和底物水平磷酸化。此文从参与该过程的关键酶、关键酶的表达调节、真菌与细菌异化硝酸盐还原的比较等角度综述了真菌异化硝酸盐还原的最新研究进展。     

Influence of bioturbation on denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in freshwater sediments

Intensive agriculture leads to increased nitrogen fluxes (mostly as nitrate, NO₃ ^?) to aquatic ecosystems, which in turn creates ecological problems, including eutrophication and associated harmful algal blooms. These problems have focused scientific attention on understanding the controls on nitrate reduction processes such as denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Our objective was to determine the effects of nutrient-tolerant bioturbating invertebrates (tubificid oligochaetes) on nitrogen cycling processes, specifically coupled nitrification–denitrification, net denitrification, DNRA, and biogeochemical fluxes (O₂, NO₃ ^?, NH₄ ⁺, CO₂, N₂O, and CH₄) in freshwater sediments. A mesocosm experiment determined how tubificid density and increasing NO₃ ^? concentrations (using N¹⁵ isotope tracing) interact to affect N cycling processes. At the lowest NO₃ ^? concentration and in the absence of bioturbation, the relative importance of denitrification to DNRA was similar (i.e., 49.6 and 50.4 ± 8.1 %, respectively). Increasing NO₃ ^? concentrations in the control cores (without fauna) stimulated denitrification, but did not enhance DNRA, which significantly altered the relative importance of denitrification compared to DNRA (94.6 vs. 5.4 ± 0.9 %, respectively). The presence of tubificid oligochaetes enhanced O₂, NO₃ ^?, NH₄ ⁺ fluxes, greenhouse gas production, and N cycling processes. The relative importance of denitrification to DNRA shifted towards favoring denitrification with both the increase in NO₃ ^? concentrations and the increase of bioturbation activity. Our study highlights that understanding the interactions between nutrient-tolerant bioturbating species and nitrate contamination is important for determining the nitrogen removal capacity of eutrophic freshwater ecosystems.     

Effects of pH,phosphate and ammonia on the rate of uptake of nitrate and ammonia by freshwater phytoplankton

The effect of phosphate (PO₄ ⁺³) and pH in regulating nitrate (NO₃) and ammonia (NH₃ ⁺) uptake by phytoplankton was investigated in two Oklahoma lakes using ¹⁵N tracers. Addition of PO₄ ⁺³ above ambient concentrations had a negligible effect on the rate of uptake of NO₃ ^– or NH₃ ⁺. Manipulation of pH of lake water had little effect on uptake of either NO₃ ^– or NH₃ ⁺. A correlation analysis suggested that NO₃ ^– is not used by phytoplankton when NH₃ ⁺ concentrations exceed about 210 µg NH₃ ⁺-N(1)^–1.     

Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp. (Lupinus)

Induction, energy gain, effect on growth, and interaction of nitrate and nitrite reduction of Bradyrhizobium sp. (Lupinus) USDA 3045 were characterized. Both nitrate and nitrite were reduced in air, although nitrite reduction was insensitive to ammonium inhibition. Anaerobic reduction of both ions was shown to be linked with energy conservation. A dissimilatory ammonification process was detected, which has not been reported in rhizobia so far. Nevertheless, anaerobic conversion of nitrate to ammonium was lower than 40%, which suggests the presence of an additional, nitrite reductase of denitrifying type. Nitrite toxicity caused a non-linear relationship between biomass produced and >2 mM concentrations of each N oxyanion consumed. At > or =5 mM initial concentrations of nitrate, a stoichiometric nitrite accumulation occurred and nitrite remained in the medium. This suggests an inhibition of nitrite reductase activity by nitrate, presumably due to competition with nitrate reductase for electron donors. Lowering of growth temperature almost completely diminished nitrite accumulation and enabled consumption as high as 10 mM nitrate, which confirms such a conclusion.     

硝态氮异化还原机制及其主导因素研究进展

硝态氮(NO_3~-)异化还原过程通常包含反硝化和异化还原为铵(DNRA)两个方面,是土壤氮素转化的重要途径,其强度大小直接影响着硝态氮的利用和环境效应(如淋溶和氮氧化物气体排放)。反硝化和DNRA过程在反应条件、产物和影响因素等方面常会呈现出协同与竞争的交互作用机制。综述了反硝化和DNRA过程的研究进展及其二者协同竞争的作用机理,并阐述了在NO_3~-、pH、有效C、氧化还原电位(Eh)等环境条件和土壤微生物对其发生强度和产物的影响,提出了今后应在产生机理、土壤环境因素、微生物学过程以及与其他氮素转化过程耦联作用等方面亟需深入研究,以期增进对氮素循环过程的认识以及为加强氮素管理利用提供依据。     

细菌硝酸盐异化还原成铵过程及其在河口生态系统中的潜在地位与影响

细菌硝酸盐异化还原成铵(DNRA)过程能够将河口沉积物中的硝氮转化为氨氮,是河口生态系统中潜在的重要氮循环过程之一。本文介绍DNRA机理与分类,综述河口生态系统中DNRA的地位与影响,并总结河口生态系统中几种重要生态因子对DNRA过程的调控与影响。目前DNRA的机理还有待完善。深入研究各类河口生态系统中环境因子对DNRA的调控与影响机制,并研发新的研究方法,将为我国河口地区的水资源保护和生态治理提供科学依据。     

Nitrogen fixation and nitrate reduction in the root nodules of legumes

Published data on, and hypotheses regarding the effect of NO⁻₃ on functioning of legume root nodules are reviewed. It is concluded that a short-term reversible effect of NO⁻₃ may act via an increased resistance to O₂ diffusion in nodules; this is coupled to decreased bacteroid respiration. For longer exposures to NO⁻₃ nodule activity is irreversibly lost, but how this relates to carbohydrate deprivation or NO-₂ accumulation is unclear. Complicating factors include denitrification reactions and the interaction of NO⁻₂ with leghaemoglobin.     

Effect of sulfide to nitrate ratios on the simultaneous anaerobic sulfide and nitrate removal

Present investigation deals with the effect of sulfide to nitrate (S/N) molar ratio on the simultaneous anaerobic sulfide and nitrate removal on capacity, stability and selectivity of the process. The volumetric sulfide-sulfur and nitrate-nitrogen removal rates at molar S/N ratio of 5:2 were 4.86 kg (m³ d)⁻¹ and 0.99 kg (m³ d)⁻¹, respectively, which were higher than those at S/N molar ratios of 5:5 and 5:8. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios. During the operation, the ratio of converted sulfide to converted nitrate tended to approach 5:2. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. The process became unstable if the influent sulfide surpassed its critical concentration. The electron balance between reactants was also analyzed for different S/N molar ratios.     

Microbes involved in dissimilatory nitrate reduction in the human large intestine

Nitrate-limited batch cultures, incorporating 20 different fermentation substrates and inoculated with human faeces, mainly selected for the growth of enterobacteria. The microbial diversity involved was determined by a combination of phenotypic and genotypic procedures. Continuous culture with lactate as the sole electron donor selected for similar micro-organisms, but when antibiotics were incorporated to inhibit Escherichia coli and lactate was replaced with choline, there was a wider microbial diversity recovered. Clostridium ramosum and Bacteroides vulgatus were then isolated as well as enterobacteriaceae.     

Simultaneous sulfide and nitrate removal in anaerobic reactor under shock loading

The performance of anaerobic reactor for simultaneous sulfide and nitrate removal under substrate shock loading was studied. The response to the shock loading could be divided into three stages i.e. disturbance, inertial and recovery periods. The effect of the shock loading was directly proportional to the intensity of the shock loads. The reactor performance was stable at a relatively lower intensity (1.5 times shock load), while it was considerably affected by higher intensity (higher than 2.0 times shock load). Nevertheless, the reactor performance recovered from disturbances at all the tested shock loads. The effluent sulfide-sulfur concentration was found as sensitive parameter, which increased up to 18 times of that at steady state; it could be used as an indicator of the reactor’s performance.