Free-living Nitrogen Fixing Research Articles

The Hypoxia-Associated Localization of Chemotaxis Protein CheZ in Azorhizorbium caulinodans.

Spatial organization of chemotactic proteins is important for cooperative response to external stimuli. However, factors affecting the localization dynamics of chemotaxis proteins are less studied. According to some reports, the polar localization of chemotaxis system I is induced by hypoxia and starvation in Vibrio cholerae. However, in V. cholerae, the chemotaxis system I is not involved in flagellum-mediated chemotaxis, and it may play other alternative cellular functions. In this study, we found that the polar localization of CheZ, a phosphatase regulating chemotactic movement in Azorhizobium caulinodans ORS571, can also be affected by hypoxia and cellular energy-status. The conserved phosphatase active site D165 and the C-terminus of CheZ are essential for the energy-related localization, indicating a cross link between hypoxia-related localization changes and phosphatase activity of CheZ. Furthermore, three of five Aer-like chemoreceptors containing PAS domains participate in the cellular localization of CheZ. In contrast to carbon starvation, free-living nitrogen fixation can alleviate the role of nitrogen limitation and hypoxia on polar localization of CheZ. These results showed that the localization changes induced by hypoxia might be a strategy for bacteria to adapt to complex environment.

Biosorption of Malachite Green by Dry Cells of Isolated Free Living Nitrogen Fixing Bacteria

Contamination of water with Malachite green (MG) may threaten aquatic and human life. Nitrogen-fixing Azotobacter sp. is an efficient adsorbent for the removal of MG from dye solutions. The optimum pH for the biosorption process was determined. The maximum adsorption capacity and the effect of different adsorbate concentrations were detected. The kinetics and isotherm models for biosorption were constructed. Optimum adsorption of MG by Azotobacter sp. was obtained at pH 6.0, biomass concentration was 0.05%, initial dye concentration was 50 mg.L-1, and contact time was 600 mins. Dye adsorption exhibited an increase with contact time and initial malachite green concentration. The kinetics of the adsorption process was best followed by the pseudo-second-order kinetic model which confirms the chemisorption process. The adsorption equilibrium data fit well to the Langmuir model indicating a monolayer adsorption behavior onto a surface of Azotobacter sp. with a finite number of active sites. Maximum biosorption capacity was found to be 142.8 mg.g-1 of bacterial biomass. The dry biomass of Azotobacter sp. has proved to be an efficient biosorbent for the removal of synthetic dyes from actual industrial effluent that is contaminated with up to 400 mg.L-1 dye concentration.

Jeevamrut and Panchagavya’s Consequences on Growth, Quality and Productivity of Organically Grown Crops: A Review

Organic farming and organically produced food products are gaining popularity very rapidly in India and world. To trounce the reliance on chemical fertilizers for crop production liquid organic bio-fertilizers and manures are very excellent and cheap sources on the earth because of easy availability and good source of nutrients. Among these Jeevamrut, Beejamrut and panchagavya are the one of the best, which contains almost essential plant nutrients (nitrogen, phosphorus, potassium, zinc, copper, manganese etc.), enzymes (acid phosphatise, alkaline phosphatise, dehydrogenase etc.) and microbes (bacteria, fungi, actinomycetes, free living nitrogen fixers and phosphorus solubilising organisms), which directly enhances plant metabolism resulting better growth and development. Jeevamrut is well thought-out to be a brilliant source of natural carbon, nitrogen, phosphorous, potassium and lot of other micro nutrients required for the crops and popularly used as means of organic farming. Panchagavya was used in conventional Hindu rituals which is prepared by mixing five ingredients viz., cow dung, urine, milk, curd and ghee. Jeevamrut and Panchagavya have potential to play the role of promoting growth and provide immunity in plant system. In this manuscript various effects of applications of Jeevamrut and Panchagavya on crops grown in organic farming and their consequences will be discussed.

Cartap hydrochloride induced stress response in Anabaena variabilis ARM 441

Cartap hydrochloride is a moderately hazardous nereistoxin analogue insecticide that is predominantly applied in paddy fields of India, at a recommended dose of 10 μg ml−1 to kill chewing and sucking insect pests of rice crop. Toxicity of cartap hydrochloride was studied on non-target free-living nitrogen fixing cyanobacterium Anabaena variabilis ARM 441 commonly used as algal biofertilizer in rice cultivation. Anabaena sp. could tolerate commercial grade insecticide up to 30 μg ml−1. However, at the recommended dose of 10 μg ml−1, it caused reduction in algal growth, total nitrogen and heterocyst frequency by 47.28, 24.29 and 17.72% respectively, as well as photosynthetic pigments under pure culture conditions. Scanning electron micrographs revealed cell rupture and breakage in filaments due to cartap exposure with the formation of akinetes. Cartap hydrochloride induced stress, since level of superoxide dismutase, peroxidase and catalase were increased by 108.57, 187.5 and 117% respectively. Generation of superoxide radicals and hydrogen peroxide were also increased by 152.48 and 34% respectively. Lipid peroxidation was increased by 31.03%, whereas there was decline in ascorbate content by 48.45%, however the glutathione content was increased by 128.57%. Increase in osmolytes such as proline from 8.6 to 32.8% and sucrose from 61.22 to 90.13% indicates their possible role in overcoming cartap induced oxidative stress and can be helpful in assessing its detrimental effect on Anabaena variabilis ARM 441, since cyanobacterial biofertilizers are purposely used in paddy fields as nitrogen contributors.

Morphological study of nitrogen fixers

Biological diversity, the common term of biodiversity, in the simplest word means the variation and variety of life forms. The biodiversity being the amalgamation of dual word i.e. ‘Bios’ meaning “Life” and ‘Diversitas’ meaning “Variety” or “Difference”. Nitrogen fixation on one hand and Photosynthesis on the other hand is the foundation of all the living system in this planet. Nitrogen fixation can be defined as a chemical process involving the conversion of atmospheric nitrogen into the nitrogenous compound, either biologically or physically. Biological Nitrogen Fixation (BNF) contributes about 90% of the process and rest 10% is carried out by the physical activities which include lightening, thunder, etc. Each and every year, approximately 17.2x10^7 tons of nitrogen are biologically fixed worldwide. The main contributors in BNF are the microorganisms which falls under two categories i.e. may be aerobic or anaerobic. Majority of the microbes do not have the potentiality to reduce nitrogen. The microorganisms having the potentiality of reducing nitrogen play a vital role in the nitrogen fixation along with nitrogen cycle in the nature. The microbes involved in BNF are usually the prokaryotes which make use of a biocatalyst or enzyme nitrogenise to bring about the catalysis of atmospheric nitrogen (N2) to form ammonia (NH3). These prokaryotes consists of aquatic organisms like blue green algae, free living nitrogen fixers like Azotobacter, Clostridium, symbiotic nitrogen fixers like Rhizobium and associative symbiotic includes Azospirillium. The current review paper delineates with reference to the morphological study of nitrogen fixers.

Impact of nutrient additions on free-living nitrogen fixation in litter and soil of two French-Guianese lowland tropical forests

Impact of nutrient additions on free-living nitrogen fixation in litter and soil of two French-Guianese lowland tropical forests

Yield and Rhizosphere - microflora of Cowpea in Response to Magnesium Fertilization in Lateritic Soils of Kerala

Background: Magnesium deficiency has become a major nutritional disorder in lateritic soils of Kerala. Appropriate magnesium fertilization is the best strategy to combat deficiency issues. Apart from correcting nutritional deficiency, magnesium fertilization has an influence on the growth of beneficial microbes such as nitrogen fixing bacterias and arbuscular mycorrhizal fungi. The experiment aimed to investigate the effect of magnesium fertilization on crop yield and population rhizosphere micoflora of cowpea in lateritic soils of Kerala.Methods: A pot culture experiment was conducted with a gradient of magnesium additions ranging from 5 mg kg-1 to 80 mg kg-1 of soil along with recommended dose of fertilizers. Population of rhizobium, free living nitrogen fixing bacteria, spore count of arbuscular mycorrhizal fungi and per cent root colonization of arbuscular mycorrhizal fungi were studied during flowering. The available magnesium and magnesium uptake were also worked out during harvest. Yield and yield contributing characteristics of cowpea were measured during harvest stage.Result: Magnesium addition produced significant variations in population of rhizobium and free- living nitrogen fixing bacteria whereas spore count of AMF and per cent root colonization of AMF did not vary according to the added doses of magnesium. A higher population of rhizobium, free living nitrogen fixers, root nodules, magnesium uptake, plant height and yield were obtained in the treatment where magnesium was applied @ 10 mg kg-1 soil.

Comparative genomic analysis of diverse rhizobia and effective nitrogen-fixing clover-nodulating Rhizobium strains adapted to Egyptian dry ecosystems

Rhizobium strains that nodulate Egyptian winter clover belong to two symbiotic species, Rhizobium aegyptiacum sv. trifolii and Rhizobium bangladeshense sv. trifolii. Rhizobia from Egyptian clover are often tolerant of extreme environmental conditions and are generally poorly characterized, despite the importance of this crop species. Here, we report on the comparative genomic analysis of four stress-tolerant, highly effective nitrogen-fixing, Egyptian-clover-nodulating rhizobia, R. aegyptiacum sv. trifolii strain Rhiz950 and Rhizobium bangladeshense sv. trifolii strains Rhiz1002, Rhiz1017 and Rhiz1024, as well as other diverse Rhizobium strains. While Rhizobium bangladeshense sv. trifolii strains Rhiz1002, Rhiz1017, and Rhiz1024 had similar genome sizes of around 6.6 Mb, the salt tolerant strain Rhiz950 had a slightly larger genome size of 7.4 Mb. All four strains lacked genes responsible for methylation and sulfation of nod factors (nodS and nodHPQ). They also lacked nodZ, and instead had the functionally-equivalent nod factor fucosylation gene nolK. No strains contained homocitrate synthase (encoded by nifV) that is essential for free-living nitrogen fixing by diazotrophs. A number of genes unique to the Egyptian clover strains and to other rhizobia are potential candidates for variation in stress tolerance and host-range.

Predicting the influence of fertilization regimes on potential N fixation through their effect on free-living diazotrophic community structure in double rice cropping systems

Free-living nitrogen fixation (FLNF) by diazotrophs is a ubiquitous renewable resource and may constitute an attractive viable solution to the problem of environmental degradation caused by nitrogen over-fertilization. However, the biotic and abiotic control mechanisms of free-living nitrogen fixation are poorly understood. Moreover, there is rare information to link the composition and functions of diazotrophic communities; additionally, there are insufficient or inadequate predictors of the potential N fixation rate (PNFR) to help improve fertilization strategies. Here, through high-throughput sequencing, network analysis, and 15N2 labelling methods, we investigated the long-term effects of partial substitution of mineral N by organic fertilizers on diazotroph abundance and composition in double rice cropping systems. The field trial was conducted for 34 years and included five treatments: control without fertilizer (CK); only mineral NPK fertilizer (NPK); 70% inorganic N + 30% organic N + PK (NPKM1); 50% inorganic N + 50% organic N + PK (NPKM2); 30% inorganic N + 70% organic N + PK (NPKM3). Our results revealed that at the same nutrient input level, increasing the percentage of organic N substitution reduced diazotrophic abundance. Compared with the CK and NPK treatments, the organic substitution regimes reduced the fluctuation range of the PNFR between the early and late rice growing seasons. NH4+-N was the primary factor that negatively correlated (p < 0.01) with diazotrophic abundance, while the potential N fixation rate was positively correlated (p < 0.01) with soil C/N. Among abiotic factors, the models incorporating soil C/N and Fe2+/Mo could accurately predict the potential N fixation rate. High-throughput sequencing and network analyses revealed that Bradyrhizobium and Geobacter were the dominant genera in diazotrophic co-occurrence and keystone taxa patterns under all fertilization regimes. Soil pH and NH4+-N were the most important factors in shaping the diazotrophic co-occurrence and keystone patterns in paddy soils, respectively. Diazotrophic keystone composition was more suitable for predicting and characterising the potential N fixation rate than the co-occurrence pattern of diazotrophs. Moreover, compared with the CK and NPK regimes, partial substitution of mineral N with organic N fertilizer improved the prediction accuracy of potential N fixation rate more effectively. Overall, we highlight the benefits of organic substitution fertilizer regime in stabilizing and improving the potential N fixation rate. Our findings provide relevant insights into the potential of key microbial taxa for predictive N fixation for optimising chemical N fertilizer application strategies in paddy soils.

Chicken feather waste-derived protein hydrolysate as a potential biostimulant for cultivation of mung beans

Feathers, a waste product of poultry industries have highly recalcitrant structure and are a rich source of organic nitrogen. The use of keratinolytic microbes for bioconversion of feather waste to a value added hydrolysate is gaining the interest of researchers. Bacillus aerius NSMk2 has been reported as a potential keratinolytic strain that converts chicken feather waste into a hydrolysate rich in proteins, small peptides and amino acids. In the present study, biostimulating potential of chicken feather waste-derived protein hydrolysate (PH) generated by biocatalytic action of Bacillus aerius NSMk2 has been evaluated for the growth of mung beans (Vigna radiata), under potted conditions. Soil treated with PH, unhydrolyzed feathers (UF) or diammonium phosphate (DAP) was transferred to different pots, and seeds of mung beans were transplanted. PH treated groups showed superior germination frequency and early growth of pods. The highest number of pods with highest weight of seeds was observed in test groups as compared to control. Higher weight of roots was observed in plants treated with PH, and content of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and total protein was found to be high. The content of phosphate, potassium and carbon:nitrogen ratio (C:N) was substantially high in soil after application of hydrolysate. Appreciable count of free living nitrogen fixers and phosphate solubilizers was also observed in PH treated groups. The present study shows improved plant performance, yield and soil fertility after application of Bacillus aerius NSMk2 derived protein hydrolysate, and therefore indicates its application as a potential biostimulant in agroindustry.

Low-pyrolysis-temperature biochar promoted free-living N2-fixation in calcareous purple soil by affecting diazotrophic composition

Free-living nitrogen fixation (FNF) is an important input of nitrogen to minimise the use of inorganic nitrogen fertiliser in agricultural ecosystems, but knowledge regarding response of FNF to biochar amendment is still limited. A microcosm study was performed to investigate changes in soil nitrogen content and FNF efficiency for calcareous purple soil applied with biochars prepared at 300 °C (CB300) and 600 °C (CB600). After 56 d of incubation, both CB300 and CB600 applied at any rates significantly (P < 0.01) increased soil total nitrogen (TN) concentrations relative to the controls. However, the soil TN is higher in the CB300 treatment group than in the CB600 group, especially for biochar applied at a 2% rate. Moreover, the concentrations of different N forms (NH4+, NO3−, microbial biomass nitrogen, and dissolved organic nitrogen) increased with biochar addition during the incubation, among which the peak value of NO3− was lagging behind NH4+. The acetylene reduction activity (ARA) as well as nifH abundance significantly increased in soil applied with 2% of biochars and peaked at day 56. Notably, the maximum values of ARA and nifH abundance were ~50% greater in soil applied with CB300 than CB600. Further analysis on the soil diazotrophic community composition showed that Azotobacter, Bradyrhizobium, and Skermanella were the most dominant genera. The proportion of Azotobacter, reported as free-living diazotroph, reached the maximum in soil amended with 2% of CB300 at day 56. The redundancy analysis indicated that electrical conductivity was the main factor leading to the difference in diazotrophic community among treatments. Besides, the rapid consumption of dissolved organic carbon (DOC) in CB300 treatment group facilitated soil diazotrophic proliferation, and finally led to the increase of ARA. Increased pH and abundant sulfur in soil applied with biochar promoted the concentrations of available molybdenum, which was positively related with the abundance of Azotobacter. Overall, our study provided clear evidence that application of low-pyrolysis-temperature biochars in calcareous soils greatly contributes to FNF and increased soil N accumulation.

Towards a comprehensive understanding of free-living nitrogen fixation

Free-living nitrogen fixation (FNF) is a ubiquitous phenomenon that plays a modest role in the N economy of an ecosystem. However, sampling difficulties, methodological constraints and environmental controls have presented challenges for predicting the actual rate of FNF. Therefore, the understanding of the accuracy to design models that consider dynamics, heterogeneity, influences and other limitations is needed. This review presents an overview of the biology and diversity of microorganisms related to FNF as well as various ecological controls that influence these microorganisms. We also discussed the contributions of FNF to the N input of various ecosystems. Overall, previous research shows that a considerable spatiotemporal variability exists in microbial types at both biome and microbiome scales, that results significant variation in FNF. Beyond this, rate of FNF is controlled by certain factors such oxygen and metal ions availability, source of energy and soil nutrients, temperature and pH. Empirically evidences are increasingly available showing significant contribution of FNF to N inputs of natural, agricultural and aquatic ecosystems. It is inferred from this review that for the expanded exploitation of biological nitrogen fixation (BNF), we must give attention to FNF because it occupies a central role within the process. Finally, we propose a framework for the quantification of FNF and a set of recommendations that would increase our understanding of FNF.

Nitrogen Fixation Using Symbiotic and Non-Symbiotic Microbes: A Review Article

Nitrogen (N) is one of the key drivers of global agricultural production and needs 150-200 million tons each year by plants in agricultural systems to produce the world’s food, animal feed and industrial products. Hence, to minimize this problem industrially producing nitrogen fertilizer is necessary. However, this industrially produced nitrogen fertilizer affect the world ecosystem through different mechanisms and effective exploitation and utilization of biologically fixed nitrogen in agricultural systems is necessary. This naturally fixed nitrogen minimizes the rate of cost of crop production, urea volatilization and is the sustainable soil fertility maintenance. Despite the importance of biological nitrogen fixation is sustainable and environmentally friend approach, some researches were done on legume crops through exploring variety specific rhizobia species for legume crops and shortage of information on free living nitrogen fixer of bacteria species for cereal crops which will be the future concerned research. This paper review discusses biological nitrogen fixation mechanism symbiotically and non-symbiotically either through free living bacteria or associative with host plant. It also focused on types of bacteria in which fix atmospheric nitrogen in cereal crops and factors affecting biological nitrogen fixation in lesser amount.

Comparable canopy and soil free-living nitrogen fixation rates in a lowland tropical forest

Biological nitrogen fixation (BNF) is a fundamental part of nitrogen cycling in tropical forests, yet little is known about the contribution made by free-living nitrogen fixers inhabiting the often-extensive forest canopy.We used the acetylene reduction assay, calibrated with 15N2, to measure free-living BNF on forest canopy leaves, vascular epiphytes, bryophytes and canopy soil, as well as on the forest floor in leaf litter and soil. We used a combination of calculated and published component densities to upscale free-living BNF rates to the forest level.We found that bryophytes and leaves situated in the canopy in particular displayed high mass-based rates of free-living BNF. Additionally, we calculated that nearly 2 kg of nitrogen enters the forest ecosystem through free-living BNF every year, 40% of which was fixed by the various canopy components.Our results reveal that in the studied tropical lowland forest a large part of the nitrogen input through free-living BNF stems from the canopy, but also that the total nitrogen inputs by free-living BNF are lower than previously thought and comparable to the inputs of reactive nitrogen by atmospheric deposition.

Effect of Free-Living Nitrogen Fixing and Phosphate Solubilizing Bacteria on Growth of Gossypium hirsutum L

Effect of Free-Living Nitrogen Fixing and Phosphate Solubilizing Bacteria on Growth of Gossypium hirsutum L

Potential yield challenges to scale-up of zero budget natural farming.

Under current trends, 60% of India's population (>10% of people on Earth) will experience severe food deficiencies by 2050. Increased production is urgently needed, but high costs and volatile prices are driving farmers into debt. Zero budget natural farming (ZBNF) is a grassroots movement that aims to improve farm viability by reducing costs. In Andhra Pradesh alone, 523,000 farmers have converted 13% of productive agricultural area to ZBNF. However, sustainability of ZBNF is questioned because external nutrient inputs are limited, which could cause a crash in food production. Here, we show that ZBNF is likely to reduce soil degradation and could provide yield benefits for low-input farmers. Nitrogen fixation, either by free-living nitrogen fixers in soil or symbiotic nitrogen fixers in legumes, is likely to provide the major portion of nitrogen available to crops. However, even with maximum potential nitrogen fixation and release, only 52-80% of the national average nitrogen applied as fertilizer is expected to be supplied. Therefore, in higher-input systems, yield penalties are likely. Since biological fixation from the atmosphere is possible only with nitrogen, ZBNF could limit the supply of other nutrients. Further research is needed in higher-input systems to ensure that mass conversion to ZBNF does not limit India's capacity to feed itself.

Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2. FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g−1 day−1. We present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.

Study on the efficiency of free-living nitrogen fixing bacteria isolated from rice rhizospheresoil on auxin and giberellin production

Study on the efficiency of free-living nitrogen fixing bacteria isolated from rice rhizospheresoil on auxin and giberellin production

To Fix or Not To Fix: Controls on Free-Living Nitrogen Fixation in the Rhizosphere

Free-living nitrogen fixation (FLNF) in the rhizosphere, or N fixation by heterotrophic bacteria living on/near root surfaces, is ubiquitous and a significant source of N in some terrestrial systems. FLNF is also of interest in crop production as an alternative to chemical fertilizer, potentially reducing production costs and ameliorating negative environmental impacts of fertilizer N additions. Despite this interest, a mechanistic understanding of controls (e.g., carbon, oxygen, nitrogen, and nutrient availability) on FLNF in the rhizosphere is lacking but necessary. FLNF is distinct from and occurs under more diverse and dynamic conditions than symbiotic N fixation; therefore, predicting FLNF rates and understanding controls on FLNF has proven difficult. This has led to large gaps in our understanding of FLNF, and studies aimed at identifying controls on FLNF are needed. Here, we provide a mechanistic overview of FLNF, including how various controls may influence FLNF in the rhizosphere in comparison with symbiotic N fixation occurring in plant nodules where environmental conditions are moderated by the plant. We apply this knowledge to a real-world example, the bioenergy crop switchgrass (Panicum virgatum), to provide context of how FLNF may function in a managed system. We also highlight future challenges to assessing FLNF and understanding how FLNF functions in the environment and significantly contributes to plant N availability and productivity.

Isolation, Identification and Screening for Nitrogen Fixing Activities by &amp;lt;i&amp;gt;Azotobacter chroococcum&amp;lt;/i&amp;gt; Isolated from Soil of Keffi, Nigeria as Agent for Bio-fertilizer Production

Free living nitrogen fixing bacteria are those that lives freely on rhizosphere of a young growing plant or those living in a close association with root of plants (Rhizobacteria) but not symbiotically. In most tropical soil, Nitrogen and other essential nutrients element are the most limiting nutrients which deficiencies could lead to slow growth and reduced crop production. Free living nitrogen fixing bacteria has the potential of secreting nitrogenase enzymes and produces organic acids such as glycolic, acetic, malic, succunic acids which fix atmospheric nitrogen directly into the soil for plants growth and development. To this end, the study on isolation, identification and screening of Azotobacter chroococcum from soil of Keffi, Nasarawa State, Nigeria was carried out from May to September 2018. Soil samples were collected from eight different locations and Azotobacter strains were isolated and identified using standard microbiological methods. The 16SrRNA gene sequence analysis of the strain showed maximum similarity of 96% with Azotobacter chroococcum of the reference type strain deposited in RDP Gen Bank database. Azotobacter strains isolated from four different locations showed coloured zone ranging between 16-10mm. Similarly, Percentage amount of nitrogen released by each Azotobacter strain in the culture broth ranging between 1.19% - 5.11% in an increasing order.