Biological Nitrogen Fixation in Sugar Cane: A Key to Energetically Viable Biofuel Production

The advantages of producing biofuels to replace fossil energy sources are derived from the fact that the energy accumulated in the biomass is captured directly from photosynthesis and is thus renewable, and that the cycle of carbon dioxide fixation by the crop, followed by burning of the fuel makes no overall contribution to atmospheric CO₂ or, consequently, to global warming. However, these advantages are negated if large quantities of fossil fuels need to be used to grow or process the biofuel crop. In this regard, the Brazilian bioethanol program, based on the fermentation/distillation of sugar cane juice, is particularly favorable, not only because the crop is principally hand harvested, but also because of the low nitrogen fertilizer use on sugar cane in Brazil. Recent ¹⁵N and N balance studies have shown that in some Brazilian cane varieties, high yields are possible without N fertilization because the plants are able to obtain large contributions of nitrogen from plant-associated biological N₂ fixation (BNF). The N₂-fixing acid-tolerant bacterium Acetobacter diazotrophicus was first found to occur within roots, stems, and leaves of sugar cane. Subsequently, two species of Herbaspirillum also have been found to occur within the interior of all sugar cane tissues. The discovery of these, and other N₂-fixing bacteria that survive poorly in soil but thrive within plant tissue (endophytic bacteria), may account for the high BNF contributions observed in sugar cane. Further study of this system should allow the gradual elimination of N fertilizer use on sugar cane, at least in Brazil, and opens up the possibility of the extension of this efficient N₂-fixing system to cereal and other crops with consequent immense potential benefits to tropical agriculture.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

Nitrogen fixation in Pseudomonas stutzeri

A recently developed oxygen gradient system and a complex medium were used to isolate a microaerobically N₂-fixing heterotrophic bacterium from the rhizosphere of a high fixing Sorghum nutans cultivar. The isolate was identified as nif(+) phenotype of Pseudomonas stutzeri on the basis of cultural, physiological and biochemical characteristics, including DNA/DNA hybridization. N₂ fixation was demonstrated by assimilation of ¹⁵N₂ into cellular protein; the physiology of nitrogen fixation was studied. The isolate contains one 30 MD plasmid and can be cured with associated loss of N₂ fixation capability.Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Nitrogen fixing cyanobacteria potential uses

The large scale production of nitrogen fixing cyanobacteria is discussed and the use of ammonia excreting mutants ofAnabaena siamensis is described.Gloeotrichia natans is considered for use as a biofertilizer and for the production of phycobiliproteins.     

The Improvement of Spinach Growth by Nano-anatase TiO<Subscript>2</Subscript> Treatment Is Related to Nitrogen Photoreduction

The improvement of spinach growth is proved to relate to N₂ fixation by nano-anatase TiO₂ in this study. The results show that all spinach leaves kept green by nano-anatase TiO₂ treatment and all old leaves of control turned yellow white under culture with N-deficient solution. And the fresh weight, dry weight, and contents of total nitrogen, , chlorophyll, and protein of spinach by nano-anatase TiO₂ treatment presented obvious enhancement compared with control. Whereas the improvements of yield of spinach were not as good as nano-anatase TiO₂ treatment under N-deficient condition, confirming that nano-anatase TiO₂ on exposure to sunlight could chemisorb N₂ directly or reduce N₂ to NH₃ in the spinach leaves, transforming into organic nitrogen and improving the growth of spinach. Bulk TiO₂ effect, however, was not as significant as nano-anatase TiO₂. A possible metabolism of the function of nano-anatase TiO₂ reducing N₂ to NH₃ was discussed.     

Nitrogen fixation in several grain legume species with contrasting sensitivities to copper nutrition

The nitrogen fixation response to copper nutrition in faba bean, yellow lupin and soybean was studied. Copper nutrition significantly increased the pod yields of all tested grain legumes but faba bean gave the greatest Cu-use efficiency for pod and grain production. The accumulation of dry matter in vegetative parts, nodules, N and leghemoglobin concentration in nodules and nitrogen accumulation in the whole plants were increased by copper supply in faba bean and yellow lupin in contrast with soybean. Cu nutrition significantly increased the Cu concentrations in nodules of all cultivated plants. The differential sensitivity of N₂ fixation in tested grain legume species to copper nutrition could be connected with the level of phenols in nodules and depended on both the host plants and strains of rhizobia, which differ in their ability to produce catechol-like siderophores. Copper requirements by symbiotic N₂ fixation could also depend on the nature of phenols in nodules (presence of o-dihydroxyphenols or number of hydroxyls in molecule).     

Nitrogen cycling in low input legume-based agriculture,with emphasis on legume/grass pastures

Low input legume-based agriculture exists in a continuum between subsistence farming and intensive arable and pastoral systems. This review covers this range, but with most emphasis on temperate legume/grass pastures under grazing by livestock. Key determinants of nitrogen (N) flows in grazed legume/grass pastures are: inputs of N from symbiotic N₂ fixation which are constrained through self-regulation via grass/legume interactions; large quantities of N cycling through grazing animals with localised return in excreta; low direct conversion of pasture N into produce (typically 5–20%) but with N recycling under intensive grazing the farm efficiency of product N: fixed N can be up to 50%; and regulation of N flows by mineralisation/immobilisation reactions. Pastoral systems reliant solely on fixed N are capable of moderate-high production with modest N losses e.g. average denitrification and leaching losses from grazed pastures of 6 and 23 kg N ha^–1 yr^–1. Methods for improving efficiency of N cycling in legume-based cropping and legume/grass pasture systems are discussed. In legume/arable rotations, the utilisation of fixed N by crops is influenced greatly by the timing of management practices for synchrony of N supply via mineralisation and crop N uptake. In legume/grass pastures, the spatial return of excreta and the uptake of excreta N by pastures can potentially be improved through dietary manipulation and management strategies. Plant species selection and plant constituent modification also offer the potential to increase N efficiency through greater conversion into animal produce, improved N uptake from soil and manipulation of mineralisation/immobilisation/nitrification reactions.     

现场模拟添加磷、铁及胶体对北部湾2007年春季生物固氮的影响

生物固氮是海洋氮循环的重要过程。固氮作用能够促进海洋初级生产力的提高,增强海洋吸收CO₂能力,对于降低大气CO₂浓度,减缓温室效应具有重要意义。2007年春季对北部湾海区进行了现场模拟添加P,Fe和胶体实验,并应用乙炔还原法（ARA,Acetylene Reducing Activity）分析其固氮速率,研究其主要影响因素。结果表明,温盐与固氮速率相关性不显著（P > 0.05）。对固氮速率来说,外加P其变化范围为-97%-545%,外加Fe其变化范围为-86%-146%,外加胶体其变化范围为-96%-1456%。对叶绿素含量而言,外加P其变化范围为-24%-50%,外加Fe其变化范围为-32%-36%,外加胶体其变化范围为-53%-41%。N可能是春季北部湾海区浮游植物生长的限制因子,固氮作用主要受到P限制,而外加胶体对固氮生物和固氮速率表现出较大的促进作用。     

Nitrogen fixation by non-legumes in tropical agriculture with special reference to wetland rice

Summary Of the 143 million hectares of cultivated rice land in the world, 75% are planted to wetland rice. Wet or flooded conditions favour biological nitrogen fixation by providing (1) photic-oxic floodwater and surface soil for phototrophic, free-living or symbiotic blue-green algae (BGA), and (2) aphotic-anoxic soil for anaerobic or microaerobic, heterotrophic bacteria. TheAzolla-Anabaena symbiosis can accumulate as much as 200 kg N ha^–1 in biomass. In tropical flooded fields, biomass production from a singleAzolla crop is about 15 t fresh weight ha^–1 or 35 kg N ha^–1. Low tolerance for high temperature, insect damage, phosphorus requirement, and maintenance of inoculum, limit application in the tropics. Basic work on taxonomy, sporulation, and breeding ofAzolla is needed. Although there are many reports of the positive effect of BGA inoculation on rice yield, the mechanisms of yield increase are not known. Efficient ways to increase N₂-fixation by field-grown BGA are not well exploited. Studies on the ecology of floodwater communities are needed to understand the principles of manipulating BGA. Bacteria associated with rice roots and the basal portion of the shoot also fix nitrogen. The system is known as a rhizocoenosis. N₂-fixation in rhizocoenosis in wetland rice is lower than that ofAzolla or BGA. Ways of manipulating this process are not known. Screening rice varieties that greatly stimulate N₂-fixation may be the most efficient way of manipulating the rhizocoenosis. Stimulation of N₂-fixation by bacterial inoculation needs to be quantified.     

Nitrogen Transfer Between Plants: A 15N Natural Abundance Study with Crop and Weed Species

An increasing amount of evidence indicates that N can be transferred between plants. Nonetheless, a number of fundamental questions remain. A series of experiments was initiated in the field to examine N transfer between N₂-fixing soybean (Glycine max [L.] Merr.) varieties and a non-nodulating soybean, and between N₂-fixing peanut (Arachis hypogaea L.) or soybean and neighboring weed species. The experiments were conducted in soils with low N fertilities and used differences in N accumulation and/or ¹⁵N natural abundance to estimate N transfer. Mixtures of N₂-fixing and non-nod soybean indicated that substantial inter-plant N transfer occurred. Amounts were variable, ranging from negligible levels to 48% of the N found in the non-nod at maturity. Transfer did not appear to strongly penalize the N₂-fixing donor plants. But, in cases where high amounts of N were transferred, N content of donors was noticeably lowered. Differences were evident in the amount of N transferred from different N₂-fixing donor genotypes. Results of experiments with N₂-fixing crops and the weed species prickly sida (Sida spinosa L.) and sicklepod (Senna obtusifolia [L.] Irwin & Barneby) also indicated substantial N transfer occurred over a 60-day period, with amounts accounting for 30–80% of the N present in the weeds. Transfer of N, however, was generally very low in weed species that are known to be non-hosts for arbuscular mycorrhizae (yellow nutsedge, Cyperus esculentus L. and Palmer amaranth, Amaranthus palmeri [S.] Watson). The results are consistent with the view that N transfer occurs primarily through mycorrhizal hyphal networks, and they reveal that N transfer may be a contributing factor to weed problems in N₂-fixing crops in low N fertility conditions.     

Biological nitrogen fixation: Investments,expectations and actual contributions to agriculture

Inputs of biologically fixed N into agricultural systems may be derived from symbiotic relationships involving legumes and Rhizobium spp., partnerships between plants and Frankia spp. or cyanobacteria, or from non-symbiotic associations between free-living diazotrophs and plant roots. It is assumed that these N₂-fixing systems will satisfy a large portion of their own N requirements from atmospheric N₂, and that additional fixed N will be contributed to soil reserves for the benefit of other crops or forage species. This paper reviews the actual levels of N₂ fixation attained by legume and non-legume associations and assesses their role as a source of N in tropical and sub-tropical agriculture. We discuss factors influencing N₂ fixation and identify possible strategies for improving the amount of N₂ fixed.     

土壤磷含量与有效性对N2O排放量的影响元分析

土壤氧化亚氮(N₂O)主要产生于土壤微生物参与的氮循环过程,其排放量受磷含量及有效性影响。添加磷肥有助于缓解陆地生态系统磷限制,提升土壤有效磷含量,进一步影响土壤微生物对氮的利用,同时控制N₂O排放。然而不同独立实验中N₂O对外源磷添加的响应差异较大。研究从发表的中、英文文献中收集了54份关于施用磷肥与N₂O排放量的观测结果,采用元分析方法确定添加外源磷后N₂O排放量的响应差异及潜在影响因素。结果表明：(1)外源磷添加对土壤N₂O排放量影响不显著;但在磷肥施用量> 50 kg P/hm²的室外实验中土壤N₂O排放量显著降低了32.5%;施用NaH₂PO₄的室内实验中N₂O排放量显著降低了18.4%。(2)土壤N₂O对外源磷添加响应的高变异性是磷肥施用量和土壤含水量、土壤pH、土地利用类型、磷肥种类、纬度和实验时间多种影响...     

Nitrogen inputs and losses from New Zealand dairy farmlets, as affected by nitrogen fertilizer application: year one

Inputs and losses of nitrogen (N) were determined in dairy cow farmlets receiving 0, 225 or 360 kg N ha^-1 (in split applications as urea) in the first year of a large grazing experiment near Hamilton, New Zealand. Cows grazed perennial ryegrass/white clover pastures all year round on a free-draining soil. N₂ fixation was estimated (using ¹⁵N dilution) to be 212, 165 and 74 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. The intermediate N rate had little effect on clover growth during spring but favoured more total pasture cover in summer and autumn, thereby reducing overgrazing and resulting in 140% more clover growth during the latter period.Removal of N in milk was 76,89 and 92 kg N ha^-1 in the 0, 225 and 360 N treatments, respectively. Denitrification losses were low (7–14 kg N ha^-1 yr^-1), increased with N application, and occurred predominantly during winter. Ammonia volatilization was estimated by micrometeorological mass balance at 15, 45 and 63 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. Most of the increase in ammonia loss was attributed to direct loss after application of the urea fertilizer.Leaching of nitrate was estimated (using ceramic cup samplers at 1 m soil depth, in conjunction with lysimeters) to be 13, 18 and 31 kg N ha^-1 yr^-1 in a year of relatively low rainfall (990 mm yr^-1) and drainage (170–210 mm yr^-1). Drainage was lower in the N fertilized treatments and this was attributed to enhanced evapotranspiration associated with increased grass growth.Nitrate-N concentrations in leachates increased gradually over time to 30 mg L^-1 in the 360 N treatment whereas there was little temporal variation evident in the 0 (mean 6.4 mg L^-1) and 225 (mean 10.1 mg L^-1) N treatments. Thus, the 360 N treatment had a major effect by greatly reducing N₂ fixation and increasing N losses, whereas the 225 N treatment had little effect on N₂ fixation or on nitrate leaching. However, these results refer to the first year of the experiment and further measurements over time will determine the longer-term effects of these treatments on N inputs, transformations and losses.     

Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

Production of NO and N2O by the Heterotrophic Nitrifier Alcaligenes faecalis parafaecalis under Varying Conditions of Oxygen Saturation

Production of NO and N ₂ O by the heterotrophic nitrifier Alcaligenes faecalis subsp. parafaecalis was studied during growth in batch and continuous culture on peptone-meat extract medium. Depending on oxygen saturation level, medium redox status and amount of substrate supplied, the microorganisms produced 0.002–0.25 mg NO-N h^{- 1} (g protein)^{- 1} and 0.16–2.4 mg N ₂ O-N h^{- 1} (g protein)^{- 1} . Maximum rates of nitrogen oxides production were observed during peak events initiated by sudden changes of oxygen supply in the medium and were due to combined nitrification/denitrification taking place simultaneously within the cells. Based on model simulations of enzymatic kinetics of denitrification, possible mechanisms of increased nitrogen oxides production during periods of changes in oxygen supply are suggested.     

Short term effect of ploughing a permanent pasture on N2O production from nitrification and denitrification

Soils are an important source of N₂O, which can be produced both in the nitrification and the denitrification processes. Grassland soils in particular have a high potential for mineralization and subsequent nitrification and denitrification. When ploughing long term grassland soils, the resulting high supply of mineral N may provide a high potential for N₂O losses. In this work, the short-term effect of ploughing a permanent grassland soil on gaseous N production was studied at different soil depths. Fertiliser and irrigation were applied in order to observe the effect of ploughing under a range of conditions. The relative proportions of N₂O produced from nitrification and denitrification and the proportion of N₂ gas produced from denitrification were determined using the methyl fluoride and acetylene specific inhibitors. Irrespectively to ploughing, fertiliser application increased the rates of N₂O production, N₂O production from nitrification, N₂O production from denitrification and total denitrification (N₂O + N₂). Application of fertiliser also increased the denitrification N₂O/N₂ ratio both in the denitrification potential and in the gaseous N productions by denitrification. Ploughing promoted soil organic N mineralization which led to an increase in the rates of N₂O production, N₂O production from nitrification, N₂O production from denitrification and total denitrification (N₂O + N₂). In both the ploughed and unploughed treatments the 0–10 cm soil layer was the major contributing layer to gaseous N production by all the above processes. However, the contribution of this layer decreased by ploughing, gaseous N productions from the 10 to 30 cm layer being significantly increased with respect to the unploughed treatment. Ploughing promoted both nitrification and denitrification derived N₂O production, although a higher proportion of N₂O lost by denitrification was observed as WFPS increased. Recently ploughed plots showed lower denitrification derived N₂O percentages than those ploughed before as a result of the lower soil water content in the former plots. Similarly, a lower mean nitrification derived N₂O percentage was found in the 10–30 cm layer compared with the 0–10 cm.     

Nitrogen retention versus methane emission: Environmental benefits and risks of large-scale wetland creation

Coastal eutrophication by nutrient fluxes from agricultural land to marine recipients is presently combated by measures such as the implementation of watershed-scale wetland creation programs aimed at nitrogen removal. Such created agricultural wetlands - termed ‘nitrogen farming wetlands’ (NFWs) - receive nitrogen (N) loads predominantly as nitrate, facilitating N removal by denitrification. However, the conversion of agricultural soils into waterlogged wetland area is likely to increase climate gas emissions, particularly methane (CH₄). There is thus a need to evaluate the benefits and risks of wetland creation at a large, watershed-scale.Here we investigate N retention and CH₄ emission originating from watershed-scale wetland creation in South Sweden, the relation between both processes, and how CH₄ emission depends on individual wetland parameters. We combine data from intensively studied reference wetlands with an extensive wetland survey to predict N retention and CH₄ emission with simple models, to estimate the overall process rates (large-scale effects) as well as spatial variation among individual NFWs.We show that watershed-scale wetland creation serves targeted environmental objectives (N retention), and that CH₄ emission is comparably low. Environmental benefit and risk of individual wetlands were not correlated, and may thus be managed independently. High cover of aquatic plants was the most important wetland property that suppressed CH₄ net production, potentially facilitating N retention simultaneously. Further, differences between wetlands in water temperature and wetland age seemed to contribute to differences in CH₄ net production. The nationally planned wetland creation (12,000 ha) could make a significant contribution to the targeted reduction of N fluxes (up to 27% of the Swedish environmental objective), at an environmental risk equaling 0.04% of the national anthropogenic climate gas emission.     

Outdoor mass culture ofAzolla spp.: yields and efficiencies of nitrogen fixation

Summary The productivity of three species of Azolla (A. pinnata, A. filiculoides andA. caroliniana) in outdoor culture has been evaluated at different planting densities. The highest yields were obtained with biomass concentration ranging from 40 to 70g d.w. m^–2. The mean productivity over a 90 days period (from May 10th to August 10th) ranged from 10g d.w. m^–2 day^–1 forA. filiculoides up to 11.5 g d.w. m^–2 day^–1 forA. caroliniana. The nitrogen content of the dried biomasses was 48.3 mg (g d.w.)^–1 forA. pinnata, 51.5mg (g d.w.)^–1 forA. filiculoides and 52.3 mg (g d.w.)^–1 forA. caroliniana. Very little variations of the nitrogen content of the ferns during the experimental period were observed.The nitrogen-fixing efficiency of the Azolla-Anabaena azollae symbiosis grown in outdoor conditions was evaluated both by direct measurement of the amount of N₂ fixed by the culture and by the C₂H₂-reduction and H₂-evolution tests in an air atmosphere. These tests were performed outdoor under the same environmental conditions as the growing cultures. For all the species the ratios of C₂H₂-reduced to N₂-fixed were unexpectedly low, ranging from 2.04 (A. pinnata) to 1.50 (A. caroliniana).The results suggest that the reliability of the C₂H₂-reduction assay, particularly when applied to complex biological N₂-fixing systems, must be re-examined.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Nitrogen fixation in a large shallow lake: rates and initiation conditions

The fixation of molecular nitrogen (N₂fix) by cyanobacteria in situ and in PO₄-P enrichment experiments was investigated in large shallow Lake Võrtsjärv in 1998–2000. In this lake, N₂fix started when TN/TP mass ratio was about 20, which is much higher than Redfield mass ratio 7. The rate of N₂fix varied between 0.81 and 2.61 gN l^–1 d^–1 and maximum rate (2.61 gN l^–1 d^–1) was measured in 15.08.2000. In L. Võrtsjärv a lag period of a couple of weaks occurred between the set-up of favourable conditions for N₂fix as the appearance of N₂-fixing species and depletion of mineral nitrogen, and the real N₂fix itself. However, if the favorable conditions for N₂fix occurred in the lake, N₂fix started after enrichment with PO₄-P in mesocosms even then when no N₂fix was detected in the lake. N₂fix in mesocosms was also more intensive than in lake water. In our experiments PO₄-P concentrations higher than 100 gP l^–1started to inhibit N₂fix.