Quantification Of Biological Nitrogen Fixation Research Articles

Probing Biological Nitrogen Fixation in Legumes Using Raman Spectroscopy.

Biological nitrogen fixation (BNF) by symbiotic bacteria plays a vital role in sustainable agriculture. However, current quantification methods are often expensive and impractical. This study explores the potential of Raman spectroscopy, a non-invasive technique, for rapid assessment of BNF activity in soybeans. Raman spectra were obtained from soybean plants grown with and without rhizobia bacteria to identify spectral signatures associated with BNF. δN15 isotope ratio mass spectrometry (IRMS) was used to determine actual BNF percentages. Partial least squares regression (PLSR) was employed to develop a model for BNF quantification based on Raman spectra. The model explained 80% of the variation in BNF activity. To enhance the model's specificity for BNF detection regardless of nitrogen availability, a subsequent elastic net (Enet) regularisation strategy was implemented. This approach provided insights into key wavenumbers and biochemicals associated with BNF in soybeans.

Quantification of biological nitrogen fixation by Mo-independent complementary nitrogenases in environmental samples with low nitrogen fixation activity

Biological nitrogen fixation (BNF) by canonical molybdenum and complementary vanadium and iron-only nitrogenase isoforms is the primary natural source of newly fixed nitrogen. Understanding controls on global nitrogen cycling requires knowledge of the isoform responsible for environmental BNF. The isotopic acetylene reduction assay (ISARA), which measures carbon stable isotope (13C/12C) fractionation between ethylene and acetylene in acetylene reduction assays, is one of the few methods that can quantify isoform-specific BNF fluxes. Application of classical ISARA has been challenging because environmental BNF activity is often too low to generate sufficient ethylene for isotopic analyses. Here we describe a high sensitivity method to measure ethylene δ13C by in-line coupling of ethylene preconcentration to gas chromatography-combustion-isotope ratio mass spectrometry (EPCon-GC-C-IRMS). Ethylene requirements in samples with 10% v/v acetylene are reduced from > 500 to ~ 20 ppmv (~ 2 ppmv with prior offline acetylene removal). To increase robustness by reducing calibration error, single nitrogenase-isoform Azotobacter vinelandii mutants and environmental sample assays rely on a common acetylene source for ethylene production. Application of the Low BNF activity ISARA (LISARA) method to low nitrogen-fixing activity soils, leaf litter, decayed wood, cryptogams, and termites indicates complementary BNF in most sample types, calling for additional studies of isoform-specific BNF.

Compound-specific amino acid 15N-stable isotope probing for the quantification of biological nitrogen fixation in soils

Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as plant nitrogen (N) is commonly a limiting nutrient for primary productivity. However, significant limitations have remained in our knowledge of the controls and rates of this process, due to technical difficulties in directly quantifying nitrogen (N2) fixation rates. To address this, we developed a novel compound-specific 15N-stable isotope probing method involving analysis of acid hydrolysable soil amino acids (AAs) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. By analysing 15N-enriched AAs (as N-acetyl, O-isopropyl derivatives), this new approach aimed to provide greater specificity compared to existing methods, and to contribute previously unobtainable quantitative information on the capture and flow of N2 fixed in soils. Laboratory incubations using 15N2 gas were carried out on surface peat over 15 days to obtain quantitative measures of N2 fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of 15N enrichment, i.e. N fixed. The enhanced detection limits of 15N-AAs by GC-C-IRMS, compared to bulk soil δ15N value determinations, was key to assessments of N2 fixation. Valuable insights into the assimilation pathway of the applied 15N2-substrate were revealed; for peat soils, 15N incorporation into glutamate dominated over other AAs.

Novel digital assessment technique used to describe peanut nodulation life history

Biological nitrogen fixation (BNF) in legume root nodules is an important source of N globally. Typically, the quantification of BNF is an estimate of N fixation through techniques such as an acetylene reduction assay or isotopic signatures. However, these methods have large logistical limitations, and do not allow for assessments of the nodule morphology or the nodulation process itself, knowledge that is important to fully understand the BNF process. To provide an updated method of nodulation assessments, peanut (Arachis hypogaea L.) nodules were sampled from a two-year field trial and quantified using scanned images of excised nodules, allowing digital assessment of nodule number and size. Further, a digital color analysis method was developed to assess internal nodule color (a known proxy for nodule BNF activity and senescence). To apply and evaluate the new method, peanut nodulation trait data was used for an exploration of the relationship between nodulation traits, plant physiological processes, and nitrogen status in the plant, followed by a nodulation life history analysis. The nodulation assessment method was confirmed by strong correlation with measures of peanut tissue N, biomass, photosynthetic function, and yield. Nodulation life history data suggest novel findings regarding differences in the development of nodules between taproots and lateral roots, contrary to the previous understanding of peanut nodulation life history. This new nodulation assessment method can provide further insight into the BNF process by allowing the collection of detailed data in a relatively simplified manner in field conditions.

Contribution of a mixed inoculant containing strains of Burkholderia spp. and Herbaspirillum ssp. to the growth of three sorghum genotypes under increased nitrogen fertilization levels

Sorghum bicolor is a C4 plant that is normally associated with diazotrophs. Inoculation with selected bacteria can improve plant growth, biological nitrogen fixation (BNF) and nutrient uptake. Therefore, the objective of the current study was to evaluate the ability of twenty-two diazotroph strains to improve sorghum growth in a greenhouse. Then, a mixture of the best performing strains was applied in the field under different levels of N-fertilizer applied to three sorghum genotypes to evaluate crop productivity, N content, BNF contribution and the efficiency of the N fertilizer used under field conditions. Two Herbaspirillum and two Burkholderia species were selected under greenhouse conditions using a sterile substrate of three sorghum genotypes: grain sorghum lineage 9920044, grass BRS655 and sweet sorghum BR501. Four diazotrophs were selected for the field trial. Burkholderia kururiensis (16 and 109), B. tropica (PPe6) and Herbaspirillum seropedicae (ZMS176) were cultivated and mixed at planting in a Typic Hapladult soil using peat inoculant. Crop yield, N-content and BNF quantification were determined using the natural abundance of 15N, and the use efficiency of the applied N was assessed using urea labelled with 15N. The grain yield showed a 24% enhancement in the inoculated grain sorghum compared with the control without nitrogen. In addition, the aerial biomass was 13% higher in the grass genotype compared with the control. The sweet sorghum did not respond to any treatment. BNF quantification reached 31.4% in the grain, 22.7% in the grass and 19.4% in the sweet sorghum genotypes. Grain sorghum inoculation increased BNF by 4% (9.5kgha−1N fixed). Inoculation increased fertilizer efficiency recovery by 28.9%. Inoculation using a mixture containing the four diazotrophs improved the sorghum productivity by enhancing the dry mass, N-content, N from BNF and N use efficiency in the grain and grass genotypes. Under zero nitrogen fertilizer application, 22% of the N that accumulated in the biomass was derived from the air. New inoculants using a mixture of different bacterial species/strains can improve the knowledge and contribution of BNF in agricultural systems.

Nitrogen fixation in grasses - gluconacetobacter activates genes in sugarcane

Nitrogen-fixing bacteria have been isolated from sugarcane (Saccharum spp.) and other grasses in an endophytic and beneficial interaction that promotes plant growth. In this interaction, bacteria colonize the intercellular spaces and vascular tissues of most plant organs without causing disease symptoms. The best characterized sugarcane endophytic diazotrophs are Gluconacetobacter diazotrophicus, clustered in the alpha subclass of Proteobacteria, and Herbaspirillum seropedicae, Herbaspirullum rubrisubalbicans and Burkholderia sp, clustered in the beta subclass of the Proteobacteria (Reis et al. 2000). These particular types of endophytic plant growth promoting bacteria (PGPB) can offer several benefits to host plants such as to provide nitrogen trough Biological Nitrogen Fixation (BNF) and to produce plant growth hormones (eg. auxins and gibberellins), promoting plant development, increase in biomass, defense against pathogens and tolerance to abiotic stresses [1,2]. In Brazil, BNF plays a fundamental role in sugarcane cultivation by reduction of the use of nitrogen fertilizers, making Brazilian sugarcane culture more competitive in global markets. It has been suggested that the relatively low mineral N-inputs used for Brazilian sugarcane production over the last 100 years has historically selected for varieties with a low response to applied mineral N and a high N2-fixing ability [3]. Studies on the quantification of BNF to the Brazilian sugarcane varieties, using 15N isotope dilution and 15N natural abundance, have demonstrated that the amount of fixed N2 can be highly variable. Very large BNF-inputs were observed in several sugarcane varieties, especially the wild noncommercial species Krakatau (Saccharum spontaneum) used in plant breeding in Brazil, as well as the commercial varieties SP 70-1143 and CB 45-3, that exhibit high yields in lowfertility soils. SP70-1143 could obtain 72% of N-requirement from BNF, while Chunee (Saccharum barberi), a wild noncommercial species, obtained only 14% [3,4]. An important biotechnological challenge of this century is to develop tools to apply for a sustainable agriculture, that would increase productivity using less fertilizers, pesticides, water and cultivated area. The associations that occur between sugarcane and other grasses with nitrogenfixing endophytic bacteria have raised a large interest in their use in agriculture, in view of the positive effects on root development, and the increase in biomass and productivity. Experiments carried out at EMBRAPA Agrobiologia have shown significant results in biomass increase and grain yield due the use of diazotrophic endophytic bacteria inoculation in sugarcane, maize and rice inoculants. However, studies have also shown that the plant genotype and the environment where the association is established can influence the degree of beneficial results obtained by the plant caused by the association with endophytic bacteria. This plant / nitrogen-fixing endophytic bacteria interaction represents a novel system of beneficial plantmicroorganism association, which has unique features that remain to be characterized. The studies on BNF quantification have indicated that the selection of the best combination of endophytic diazotroph strains as well as sugarcane varieties needs to be exploited to obtain the maximum benefit from this association in agriculture. The signaling pathways by which sugarcane plants can decipher bacterial signals and respond properly for a successful association, by controlling endophyte recognition, colonization and nitrogen fixation rates, are still not clearly understood. Therefore, the major goal of our research is to develop biotechnological tools to help to obtain novel plant varieties more responsive to bacteria Laboratorio de Biologia Molecular de Plantas, Instituto de Bioquimica Medica, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro, 21.941-590, Rio de Janeiro, RJ, Brazil Full list of author information is available at the end of the article Carvalho et al. BMC Proceedings 2014, 8(Suppl 4):O20 http://www.biomedcentral.com/1753-6561/8/S4/O20

Heterotrophic and phototrophic 15N2 fixation and distribution of fixed 15N in a flooded rice–soil system

A range of different N2 fixing systems can contribute to the nitrogen (N) economy of flooded rice fields, but the direct evidence of N2 fixation and the proportional contribution of heterotrophic and phototrophic N2 fixation are difficult to assess. Here we report on the development and application of a field-based 15N2 labelling technique in which a flooded rice–soil system was exposed to a 15N2-enriched atmosphere to assess the biological nitrogen fixation (BNF) in paddy fields. One month old rice seedlings (Oryza sativa L.) were transplanted to non N-fertilized pots of flooded soil and exposed to a 15N2-enriched atmosphere (approx. 7 atom% 15N) in a gas-tight growth chamber for 70 days. The highest 15N-enrichment 3.7551 atom% was observed in blue green algae in unplanted pots. After 70 days incubation, 49% of the total 15N fixed in flooded rice–soil system was found in the plants (rice and weeds), while 51% was found in the flooded soils. Rice planting enhanced both phototrophic and heterotrophic BNF and increased the proportion of heterotrophic BNF to phototrophic BNF from 0.50 in treatment without rice (fallow) to 0.99 in treatment with rice. The findings suggested that rice benefits greatly from BNF processes and a major improvement for quantification of BNF in flooded rice fields based on field 15N2 labelling technique.

The role of biological nitrogen fixation by non-legumes in the sustainable production of food and biofuels

The 12th International Symposium on biological nitrogen fixation (BNF) with Non-Legumes took place in Buzios, Rio de Janeiro, Brazil from October 3 to 8, 2010. It was the latest in a series of meetings that, fittingly, first began in Brazil, in 1977 in the city of Piracicaba. This Special Issue of Plant and Soil comprises 21 papers from the Buzios Symposium, as well as a commentary and three obituaries. Given the volatility (and general upward trend) in oil prices and global attempts to alleviate greenhouse gas (GHG) emissions associated with the agricultural use of Ncontaining mineral fertilizers produced by the energyintensive Haber-Bosch process, it was inevitable that the main focus of the 2010 Symposium would be very much on the substitution of fertilizers in favour of the increased use of BNF in non-legume cropping systems. In the intervening years since the last time the symposium was held in Brazil (August 1987 in Rio de Janeiro), knowledge about non-legume BNF has increased enormously, encompassing everything from more accurate N-based quantification studies in the field through to the molecular identification of their associated diazotrophs (both culturable and unculturable) and the localization of these bacteria via high resolution microscopy. The completely sequenced genomes of many diazotrophic bacteria are also now available, and more are deposited in databases every year. Indeed, such technologies, which only a few years ago were considered remarkable, are now becoming standard tools in this field. In the case of sugarcane and other biofuel crops, the location of the 2010 meeting in Brazil was especially pertinent, as the highly advanced Brazilian bioethanol programme, which produces over 27 billion litres of ethanol per year, is based upon the cultivation of sugarcane, and Brazilian cane has long been known to be able to derive much of its N-requirements via BNF. The consequent low fertilizer use by Brazilian cane producers not only makes for enormous economic savings, but also mitigates against GHG emissions from the production of N-containing fertilizers, as well as their transport and application and nitrous oxide emissions. However, in spite of the strong likelihood that cane can benefit significantly from BNF, little is known about how much N it can really fix, and as field-based BNF quantification studies may take several years to complete, we are thus fortunate to include two such studies in this Special Issue (both utilizing the N natural abundance technique): one on sugarcane by Urquiaga et al., and one on the C-4 “energy crop” Plant Soil (2012) 356:1–3 DOI 10.1007/s11104-012-1317-1

Effect of 15n-labeled hairy vetch and nitrogen fertilization on maize nutrition and yield under no-tillage¹

This study evaluated the effect of hairy vetch (Vicia villosa Roth) as cover crop on maize nutrition and yield under no tillage using isotope techniques. For this purpose, three experiments were carried out: 1) quantification of biological nitrogen fixation (BNF) in hairy vetch; 2) estimation of the N release rate from hairy vetch residues on the soil surface; 3) quantification of 15N recovery by maize from labeled hairy vetch under three rates of mineral N fertilization. This two-year field experiment was conducted on a sandy Acrisol (FAO soil classification) or Argissolo Vermelho distrófico arênico (Brazilian Soil Classification), at a mean annual temperature of 18 ºC and mean annual rainfall of 1686 mm. The experiment was arranged in a double split-plot factorial design with three replications. Two levels of hairy vetch residue (50 and 100 % of the aboveground biomass production) were distributed on the surface of the main plots (5 x 12 m). Maize in the sub-plots (5 x 4 m) was fertilized with three N rates (0, 60, and 120 kg ha-1 N), with urea as N source. The hairy vetch-derived N recovered by maize was evaluated in microplots (1.8 x 2.2 m). The BFN of hairy vetch was on average 72.4 %, which represents an annual input of 130 kg ha-1 of atmospheric N. The N release from hairy vetch residues was fast, with a release of about 90 % of total N within the first four weeks after cover crop management and soil residue application. The recovery of hairy vetch 15N by maize was low, with an average of 12.3 % at harvest. Although hairy vetch was not directly the main source of maize N nutrition, the crop yield reached 8.2 Mg ha-1, without mineral fertilization. There was an apparent synergism between hairy vetch residue application and the mineral N fertilization rate of 60 kg ha-1, confirming the benefits of the combination of organic and inorganic N sources for maize under no tillage.

Models of biological nitrogen fixation of legumes. A review

Leguminous crops have the ability to fix nitrogen (N) biologically from the atmosphere. This can benefit not only the legumes themselves but also any intercropped or subsequent crops, thus reducing or removing the need to apply N fertilizers. Improved quantification of legume biological nitrogen fixation (BNF) will provide better guidance for farmers on managing N to optimise productivity and reduce harmful losses to the environment. There are many techniques available for the direct quantitative measurement of legume BNF in the field and in controlled environments. However, these are time-consuming and therefore expensive, and generate data relevant only to the time and place of measurement. Alternatively, legume BNF can be estimated by either empirical models or dynamic mechanistic simulation models. Comparatively, simulation by a dynamic model is preferable for quantifying legume BNF, because of its capability to simulate the response of N fixation to a wide range of environmental variables and legume growth status. Currently there is no published review of the approaches used to simulate, rather than measure, legume BNF. This review of peer-reviewed literature shows that most simulation models estimate the N fixation rate from a pre-defined potential N fixation rate, adjusted by the response functions of soil temperature, soil/plant water status, soil/plant N concentration, plant carbon (C) supply and crop growth stage. Here, we highlight and compare the methods used to estimate the potential N fixation rate, and the response functions to simulate legume BNF, in nine widely-cited models over the last 30 years.We then assess their relative strengths in simulating legume BNF with varying biotic and abiotic factors, and identify the discrepancies between experimental findings and simulations. After this comparison, we identify the areas where there is the potential to improve legume BNF simulation in the future. These include; (1) consideration of photosynthetic C supply, (2) refining the various effects of soil mineral N concentration, (3) characterization and incorporation of excess soil water stress and other factors into models, and (4) incorporation of the effects of grazing, coexistence and competition with intercrops and weeds into models to improve their practical relevance to sustainable agricultural systems. This review clarifies, for the first time, the current progress in legume BNF quantification in simulation models, and provides guidance for their further development, combining fundamental experimental and modelling work.

A preliminary study on quantification of biological nitrogen fixation in sugarcane grown in Sevanagala in Sri Lanka

The extent of nitrogen(N) fixation in sugarcane grown in reddish brown earth soils in Sevanagala was determined using the difference in 15 N abundance in the top visible dewlap leaves of sugarcane (variety CO 775, SL 8306) compared to those of neighbouring weeds. Results indicate that biological N 2 fixation contributed to an average of 18% of the total N in sugarcane in the study site.

Non-isotopic method for the quantification of biological nitrogen fixation and wheat production under field conditions

A field experiment was conducted in the Inshas area (Sharkeia governorate) to study the potential of biofertilizers, when the quantities of commercial fertilizers were reduced, for optimal wheat production. The different treatments were arranged in a completely randomized block design with seven replicates. N fertilizer was applied in three treatments with one control, i.e. zero, full, half and one-fourth rates, in the presence or absence of inocula. Azospirillum brasilense strain Sp245 was used as a biofertilizer. Generally, inoculation increased the accumulation of shoot dry matter and grain yield by about 35%, relative to the control treatment. Similar trends were observed in the case of N and P uptake by shoots and grains, as well as the efficient use of both, where inoculation increased the acquisition of the two elements as compared with the uninoculated plants. The obtained data showed that N2 fixed by shoots and grains ranged from 2 to 10 kg N ha–1 and from 8 to 19 kg N ha–1, respectively. With respect to fixed N2, the best treatment was inoculation combined with the one-fourth dose of N, followed by inoculation combined with half of the recommended N dose. Most of the fixed N was utilized by grains and the results clearly reflected the negative effect of high N fertilizer rates on biological N fixation. It is obvious that inoculation, in general, enhanced the N fertilizer utilized by both shoots and grains of wheat plants. In conclusion, the application of biofertilization technology to a light-textured soil with low fertility had a positive effect on plant growth, N gained from the air and enhancement of fertilizer N uptake (apparent recovery fraction).

Ocorrência de bactérias diazotróficas em diferentes genótipos de cana-de-açúcar

O objetivo deste trabalho foi avaliar a localização e o número de bactérias endofíticas em quatro genótipos de cana-de-açúcar e investigar sobre a possível existência de correlação com os resultados apresentados em trabalhos de quantificação da fixação biológica de nitrogênio (FBN). Fez-se um levantamento das bactérias diazotróficas presentes, e quantificou-se a população de Herbaspirillum spp. e Acetobacter diazotrophicus, em genótipos de cana-de-açúcar contrastantes quanto à capacidade de obter N da FBN. De acordo com o levantamento realizado neste trabalho, as bactérias estudadas (Azospirillum lipoferum, A. brasilense, A. amazonense, Herbaspirillum spp. e Acetobacter diazotrophicus) estavam presentes nos quatro genótipos avaliados e em todas as partes da planta, exceto A. amazonense, que não foi isolado de amostras de folhas. A quantificação das bactérias Herbaspirillum spp. e A. diazotrophicus mostrou não haver diferenças significativas entre os genótipos, e que, geralmente, elas estão presentes em maior número nas raízes. Enquanto Herbaspirillum spp. mantém-se mais estável ao longo do ciclo da cultura, a população de A. diazotrophicus decresce com a aproximação do final do ciclo comercial. Pode-se sugerir que as diferenças entre as taxas de FBN encontradas nos diversos genótipos não é causada por diferenças na presença ou no número das bactérias aqui estudadas.

Quantification of biological nitrogen fixation associated with sugar cane using a 15N aided nitrogen balance

A nitrogen balance study was performed on four commercial sugar cane cultivars grown in pots containing 64 kg of soil from a sugar cane growing area, with the objective of quantifying possible contributions of biological nitrogen fixation (BNF) to the plants. Distillery (stillage) waste from an alcohol distillery was added to half of the pots, and all treatments were replicated 10 times. The pots were maintained in the field and amended with the equivalent of 80 kg N ha −1 of 15N-labelled urea fertilizer. After 12 months of growth the plants accumulated the equivalent of between 10 and 24% of the total N in the soil and added fertilizer. In the next 9 months of growth, with no more N additions, the plants further accumulated the equivalent of between 8.5 and 19% of the original soil + fertilizer N. The cultivar CB 47–89 accumulated significantly more N than the other cultivars. This difference was greatest in the absence of distillery waste, and in this treatment the 15N enrichment of this cultivar was almost half that of the others, which suggests that there was a contribution of unlabelled N from the air to this cultivar via BNF. After 21 months of growth the N content of the soil and roots was determined on 5 replicates. These analyses revealed that the planted plots lost between 7.5 and 12.5 g of N from soil + fertilizer, but all accumulated more than this in plant tissue. In view of the fact that unplanted plots lost between 10 and 14 g of N during this period, the data suggest that all of the cultivars received some N from associated BNF. In the case of the cultivar CB 47–89, in the absence of distillary waste the plants removed approx. 10 g of N from the soil and fertilizer but accumulated almost 35 g of N. These data constitute the first direct evidence of very significant contributions of plant-associated BNF to a sugar cane cultivar although further experiments must be performed to determine whether such large contributions would be encountered under field conditions.

Methods for quantification of nitrogen fixation associated with gramineae

In recent years, the question of whether grasses or cereals are able to obtain significant quantities of nitrogen from plant‐associated heterotrophic nitrogen‐fixing bacteria has raised a great deal of controversy. This controversy is principally due to the difficulties involved in the reliable quantification of plant‐associated biological nitrogen fixation (BNF). The acetylene reduction assay has contributed immensely to the understanding of diazotroph/ gramineae associations, but in its use for definitive quantification of BNF, it suffers from many disadvantages, mainly due to the fact that it is a short‐term assay of enzyme activity and such activity is drastically reduced when plants are disturbed. Few nitrogen‐balance studies have been performed (due no doubt to their laborious nature), but recent data on rice and sugar cane have proved most valuable. Isotope techniques (15N) have been more popular. Incorporation of 15N‐labeled N2 provides definitive proof of incorporation of biologically fixed N, bu...