Soils,a sink for N2O? A review

Soils are the main sources of the greenhouse gas nitrous oxide (N₂O). The N₂O emission at the soil surface is the result of production and consumption processes. So far, research has concentrated on net N₂O production. However, in the literature, there are numerous reports of net negative fluxes of N₂O, (i.e. fluxes from the atmosphere to the soil). Such fluxes are frequent and substantial and cannot simply be dismissed as experimental noise.
Net N₂O consumption has been measured under various conditions from the tropics to temperate areas, in natural and agricultural systems. Low mineral N and large moisture contents have sometimes been found to favour N₂O consumption. This fits in with denitrification as the responsible process, reducing N₂O to N₂. However, it has also been reported that nitrifiers consume N₂O in nitrifier denitrification. A contribution of various processes could explain the wide range of conditions found to allow N₂O consumption, ranging from low to high temperatures, wet to dry soils, and fertilized to unfertilized plots. Generally, conditions interfering with N₂O diffusion in the soil seem to enhance N₂O consumption. However, the factors regulating N₂O consumption are not yet well understood and merit further study.
Frequent literature reports of net N₂O consumption suggest that a soil sink could help account for the current imbalance in estimated global budgets of N₂O. Therefore, a systematic investigation into N₂O consumption is necessary. This should concentrate on the organisms, reactions, and environmental factors involved.     

Sources of nitrous oxide production following wetting of dry soil

Abstract Production of N₂O was detected within 30 min of adding water to very dry soil (matric water potential < −9 MPa) sampled at the end of the dry season from an annual grassland of California, U.S.A. Using C₂H₂ to inhibit nitrification, we demonstrate that nitrification was a modest source of N₂O in sieved soil wetted to a water content below field capacity, but that denitrification was the major source of N₂O in sieved soils wetted to a water content above field capacity and in intact cores wetted either below or above field capacity. Significant abiological sources of N₂O were not detected. De novo enzyme synthesis began within 4–8 h of wetting, and denitrifying enzyme activity doubled within 26 h, indicating that denitrifying bacteria can quickly transform their metabolic state from adaptation to severe drought stress to rapid exploitation of changing resources.     

Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Abstract: Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, ¹⁵N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N₂ and N₂O were both produced from NH₄⁺, NO₃⁻ and NO₂⁻. Periplasmic nitrate reductase was present in aerobically grown cells.     

真菌对土壤N2O释放的贡献及其研究方法

N₂O是一种重要的温室气体,而土壤作为N₂O的重要来源之一,其N₂O主要产生于硝化和反硝化作用的生物过程.研究表明细菌和古菌是这些生物过程的主要参与者,然而在特定土壤生态系统中,真菌在N循环过程中起主要作用.但真菌对土壤N₂O释放贡献的研究报道甚少.本文阐述了土壤真菌N₂O产生机制的研究进展,介绍了自养硝化、异养硝化和反硝化过程的发生机理、关键微生物和功能基因.详细介绍了与真菌有关的N₂O产生过程,真菌的异养硝化作用和反硝化作用,并且比较了真菌和细菌反硝化系统的差异.此外,本文重点总结了研究土壤真菌N₂O产生的主要方法,包括选择抑制剂法、^15N标记、分离和纯培养以及免培养的分子生态学方法,对各种方法的优势和弊端进行了探讨,并对今后的研究工作提出了展望.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Field determination of denitrification in water-logged forest soils

Abstract Denitrification rates were measured as N₂O production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibition and the addition of nitrate, glucose, acetate and celloboise in field incubations was examined.
N₂O release from the two soils was very low, 26 mg N₂m⁻²y⁻¹ in ash and 178 mg N₂ in alder. In acetylene inhibited incubations N₂O production was higher, 296 and 486 mg N₂m⁻² y⁻¹ in ash and alder respectively. After addition of nitrate and C-sources to a 10 mM concentration, denitrification rates increased to 5–15 times higher values.
The denitrification rates below 4°C were low and most N₂O was produced in late spring and summer.
The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

Silicon promotes nodule formation and nodule function in symbiotic cowpea (Vigna unguiculata)

Applying silicon in the form of metasilicic acid (H₄SiO₃) or silicic acid (H₄SiO₃) to Bradyrhizobium -infected, hydroponically grown cowpea seedlings resulted in a significant ( P 0.05) increase in the number of nodules, nodule dry matter, and nitrogen fixed on a per plant basis. Total dry matter of plants increased with silicon supply, and the differences were significant ( P 0.05) at the higher silicon concentrations. Cowpea plants cultured in sand were also assessed for their response to silicic acid. Nodule number and nodule mass increased with silicon supply to sand cultured plants, though nitrogen fixation was unaltered. Although silicon is not essential for growth of cowpea, it is important for nodule formation and nodule functioning in hydroponically grown plants. Consequently, data collected and conclusions drawn from earlier glasshouse experiments, which have excluded silicon from nutrient solutions, may be flawed. Future studies on nodulation and nitrogen fixation using legumes in liquid culture must therefore include silicon as a nutrient element.     

Denitrification in sediment determined from nitrogen isotope pairing

Abstract A new method for accurate and easy measurement of denitrification in sediments is presented. The water overlying intact sediment cores was enriched with ¹⁵NO₃⁻ which mixed with the ¹⁴NO₃⁻ of the natural sources of NO₃⁻. The formation by denitrification of single-labeled (¹⁴N¹⁵N) and double-labeled (¹⁵N¹⁵N) dinitrogen pairs was measured by mass spectrometry after a few hours incubation. Total denitrification including the formation of unlabeled (¹⁴N¹⁴N) dinitrogen could be calculated assuming random isotope pairing by denitrification of the uniformly mixed NO₃⁻ species. In contrast to previous approaches, by this method it is possible to measure denitrification of both NO₃⁻ diffusing from the overlying water and NO₃⁻ from nitrification within the sediment.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

Abstract: Different reduced sulfur compounds (H₂S, FeS, S₂O₃²⁻) were tested as electron donors for dissimilatory nitrate reduction in nitrate-amended sediment slurries. Only in the free sulfide-enriched slurries was nitrate appreciably reduced to ammonia (     ), with concomitant oxidation of sulfide to S⁰ (     ). The initial concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of free S²⁻ (metal sulfides) nitrate was reduced via denitrification whereas at higher S²⁻ concentrations, dissimilatory nitrate reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of NO- and N₂O- reductases is proposed as being responsible for the driving part of the electron flow from S²⁻ to NH₄⁺.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

Abstract A mesophilic cellulolytic bacterium ( Clostridium strain C7) capable of N₂ fixation and a non-cellulolytic bacterium ( Klebsiella strain W1), both isolated from freshwater environments rich in decaying plant material, were co-cultured in a chemically defined, vitamin-deficient medium containing cellulose as the carbon and energy source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as fermentable substrates for the Klebsiella . In turn, the Klebsiella excreted growth factors, identified as biotin and p -aminobenzoic acid, which were required by the Clostridium . Furthermore, demonstration of NH₄⁺-repressible acetylene reduction by co-cultures growing in medium lacking combined nitrogen showed that the Clostridium fixed N₂, thus allowing growth of the Klebsiella , which was not a nitrogen fixer. The mutualistic relationships observed in the co-cultures may be representative of interactions that take place in natural environments in which cellulose-containing plant materials are biodegraded.     

Exploitation of N2-fixation capacity for enrichment of anoxygenic photosynthetic bacteria in ecological studies

Abstract A procedure is described for the selective enrichment of anoxygenic photosynthetic bacteria from diverse natural sources containing numerous types of microorganisms. The enrichment medium contains a mixture of organic acid carbon sources, and the conditions used favor the growth of organisms that can multiply relatively rapidly anaerobically with N₂ as the nitrogen source and light as the source of growth energy; the development of oxygenic cyanobacteria is effectively excluded.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

CO_2浓度升高对水体硝化、反硝化作用的影响研究进展

大气CO_2浓度升高潜移默化地影响着水体生态系统的碳循环过程.然而,该过程如何影响与其耦合的氮循环过程仍不明确.水体硝化、反硝化过程作为水体氮循环的重要环节,必然会对大气CO_2浓度升高产生一系列的响应.本文总结了国内外关于大气CO_2浓度升高对水体理化性质、硝化作用、反硝化作用及N形态转化影响方面的研究工作,发现大气CO_2浓度升高会降低水体的p H,增加水中CO_2和HCO_3^-含量,但对富营养化与寡营养化水体中硝化、反硝化作用的影响具有明显差异.大气CO_2浓度升高抑制寡营养化水体的硝化作用和反硝化作用,降低N2_O的释放通量,抑制富营养化水体的硝化作用,但当水体pH在7～9时,可能促进反硝化作用,增加N2_O的释放通量,最终可能导致水体中NH_4^+的积累及NO_3^-浓度的降低,影响水体中微生物的多样性.在此基础上提出目前相关研究存在的瓶颈问题及值得深入探讨的科学问题,为进一步深入理解温室效应背景下全球CO_2浓度升高对水体生态系统N循环的影响提供参考.     

Hippuric acid and benzoic acid inhibition of urine derived N2O emissions from soil

Atmospheric concentrations of the greenhouse gas nitrous oxide (N₂O) have continued to rise since the advent of the industrial era, largely because of the increase in agricultural land use. The urine deposited by grazing ruminant animals is a major global source of agricultural N₂O. With the first commitment period for reducing greenhouse gas emissions under the Kyoto Protocol now underway, mitigation options for ruminant urine N₂O emissions are urgently needed. Recent studies showed that increasing the urinary concentration of the minor urine constituent hippuric acid resulted in reduced emissions of N₂O from a sandy soil treated with synthetic bovine urine, due to a reduction in denitrification. A similar effect was seen when benzoic acid, a product of hippuric acid hydrolysis, was used. This current laboratory experiment aimed to investigate these effects using real cow urine for the first time. Increased concentrations of hippuric acid or benzoic acid in the urine led to reduction of N₂O emissions by 65% (from 17% to <6% N applied), with no difference between the two acid treatments. Ammonia volatilization did not increase significantly with increased hippuric acid or benzoic acid concentrations in the urine applied. Therefore, there was a net reduction in gaseous N loss from the soil with higher urinary concentrations of both hippuric acid and benzoic acid. The results show that elevating hippuric acid in the urine had a marked negative effect on both nitrification and denitrification rates and on subsequent N₂O fluxes. This study indicates the potential for developing a novel mitigation strategy based on manipulation of urine composition through ruminant diet.     

Elevated atmospheric CO2 lowers leaf litter nutritional quality for stream ecosystem food webs

Up to 99% of the carbon fuelling the food webs of temperate woodland streams is derived from inputs of terrestrial leaf litter. Aquatic bacteria, fungi, and detritivore invertebrates directly utilize these inputs, transferring this energy to other components of the food web. Increases in atmospheric CO₂ could indirectly impact woodland stream food webs by chemically altering leaf litter. This study evaluated CO₂-induced chemical changes in aspen ( Populus tremuloides ) leaf litter, and the corresponding effects on stream bacteria, fungi and leaf-shredding cranefly larvae ( Tipula abdominalis : Diptera). Leaf litter from plants grown under elevated CO₂ had decreased nutritional value to aquatic decomposers and detritivores because of higher levels of structural compounds and lower nitrogen content. Consequently, elevated CO₂-grown leaf litter supported 59% lower bacterial production in a stream than litter grown at ambient CO₂ levels, while not affecting fungal biomass. Larval craneflies fed elevated CO₂-grown microbially colonized leaves consumed less, assimilated less, and grew 12 times slower than their ambient fed counterparts.     

CO2浓度升高对水体硝化、反硝化作用的影响研究进展

大气CO₂浓度升高潜移默化地影响着水体生态系统的碳循环过程.然而,该过程如何影响与其耦合的氮循环过程仍不明确.水体硝化、反硝化过程作为水体氮循环的重要环节,必然会对大气CO₂浓度升高产生一系列的响应.本文总结了国内外关于大气CO₂浓度升高对水体理化性质、硝化作用、反硝化作用及N形态转化影响方面的研究工作,发现大气CO₂浓度升高会降低水体的pH,增加水中CO₂和HCO₃^-含量,但对富营养化与寡营养化水体中硝化、反硝化作用的影响具有明显差异.大气CO₂浓度升高抑制寡营养化水体的硝化作用和反硝化作用,降低N₂O的释放通量,抑制富营养化水体的硝化作用,但当水体pH在7~9时,可能促进反硝化作用,增加N₂O的释放通量,最终可能导致水体中NH₄⁺的积累及NO₃^-浓度的降低,影响水体中微生物的多样性.在此基础上提出目前相关研究存在的瓶颈问题及值得深入探讨的科学问题,为进一步深入理解温室效应背景下全球CO₂浓度升高对水体生态系统N循环的影响提供参考.