Free-living Nitrogen Fixing Research Articles

Improved method for isotopic measurement of free‐living nitrogen fixation

AbstractThe use of 15N‐labeled dinitrogen (15N2) affords the only direct means of measuring free‐living nitrogen fixation (FLNF); however, progress in utilizing this approach has been impeded by methodological limitations arising from the presence of nitrogenous contaminants, a lack of atmospheric uniformity, and incomplete description of procedural details. Such constraints are eliminated with an ex situ technique comprehensively described herein, which involves circulating 15N2 generated by hypobromite oxidation through a closed system that includes chemical (sulfuric acid–potassium permanganate) and cryogenic (isopentane–liquid N2) traps for atmospheric purification and an incubation chamber consisting of a desiccator equipped with a pressure gauge. Studies to evaluate the circulation system described showed that a uniform atmosphere was readily achieved with a 10‐L desiccator by pumping for 30 min, and that both chemical and cryogenic traps were necessary to ensure complete (98.8%–99.6%) removal of gaseous contaminants subject to physicochemical retention by sterilized soil samples. The method proposed was successfully demonstrated in detecting the stimulatory effect of organic carbon (C) on FLNF in active soils, and can be further utilized to improve the reliability of ex situ assessment of FLNF in relation to soil processing and storage, climatic conditions, microbial dynamics, and land management practices.

The rhizosphere contributes disproportionately to free-living nitrogen fixation in subalpine forest soils

Root activity creates a unique microbial hotspot in the rhizosphere, profoundly regulating free-living nitrogen fixation (FLNF). However, empirical assessments of rhizosphere FLNF and its ecological consequences for nitrogen (N) budgets remain lacking, particularly for different root functional modules. Here, we separately collected rhizosphere soils attached to two root functional modules, absorptive roots and transport roots, and investigated the rates (15N2 incorporation) and regulators of rhizosphere FLNF in a N-limited subalpine coniferous forest. The measured rates were further extrapolated to annual fluxes based on the volume of the rhizosphere and the relationship between FLNF rate and temperature. We found that absorptive roots drove significantly higher rhizosphere FLNF rates than did transport roots (12.2 vs. 7.5 ng N g−1 d−1), with both rhizosphere compartments exhibiting rates well exceeding those in the bulk soil (0.9 ng N g−1 d−1). The FLNF rates were positively correlated with soil organic carbon content but showed no significant relationship with the abundance or composition of the diazotrophic community. Moreover, when extrapolated to the ecosystem level the FLNF fluxes were 2-fold greater in the rhizosphere of absorptive roots than in that of transport roots (0.13 vs. 0.04 kg N ha−1 yr−1). Taken together, despite representing only 6.0% of the soil volume, the two rhizosphere compartments contributed as much as 47.2% to the total soil FLNF fluxes. Overall, we provide empirical evidence that despite its limited volume, the rhizosphere contributes disproportionately to the FLNF in subalpine forest soils. Our findings also underscore the critical role of root functional differentiation in regulating rhizosphere FLNF, which is essential for integrating this process into the ecosystem-level N cycle.

Plant and Microbial Carbon Are Important Drivers of Free‐Living Nitrogen Fixation in Tropical Forest Soils: A New Discovery of Carbon‐Driven Nitrogen Input

AbstractNitrogen (N) availability limits plant growth and soil carbon (C) sequestration in N‐limited ecosystems, however, plant and soil C feedback on the free‐living nitrogen fixation (FLNF) process is poorly understood. Moreover, whether this feedback is influenced by initial N availability in leguminous and non‐leguminous forest soils has not been clarified. Here, we found that the addition of plant‐ and microbial‐derived C significantly enhanced soil nitrogenase activity (13∼28%) and that microbial‐derived C had a more positive impact. These positive effects were attributed to the direct C‐energy supply (0.49∼0.84) rather than variations in soil microbial activity (−0.01∼0.21) and substrate resources (−0.45∼0.27). Long‐term N addition did not inhibit FLNF. C addition promoted FLNF in soils of the two forests, but the response rate was higher in the leguminous forest soils. Our study reveals that increased soil C availability can drive FLNF in tropical forests, enhancing our understanding of the soil C‐N coupling mechanism.

Carbon type and quantity regulate soil free-living nitrogen fixation through restructuring diazotrophic community

Free-living nitrogen fixation (FLNF) is a ubiquitous and significant source of new nitrogen in ecosystems. This process requires a substantial amount of energy, and as a result, the input of labile carbon to soils may enhance FLNF. Here, we aim to explore the precise influence of different types and quantities of carbon on FLNF. We conducted experiments where sugars, carboxylic acids, and amino acids (common labile carbon inputs via root exudates) were added to cropland and forest soils. We monitored the changes in FLNF rate and diazotrophic community in response to these additions. Our findings revealed that sugars and carboxylic acids significantly stimulated FLNF rate, with an increase of up to three orders of magnitude, while amino acids either restricted FLNF or had no effect. When a mixture of sugars and carboxylic acids was added, the stimulation of FLNF was not observed until a threshold quantity of carbon was reached, indicating competition between diazotrophs and other microbes. Furthermore, the enhanced FLNF rate following carbon addition was attributable to shifts in diazotrophic community composition (e.g., increased abundance of Paenibacillus, a genus frequently used as biofertilizers), rather than to changes in the overall abundance of diazotrophs (reflected by nifH gene copy number). Our findings highlight the significant role of carbon type and quantity in regulating soil FLNF, partly through restructuring the diazotrophic community. The results have implications for predicting the response of rhizosphere FLNF to shifts in root exudation under environmental changes and can contribute to the application of diazotrophic biofertilizers in sustainable agriculture.

Impact of two acquisitive plants on N cycle on different soils: The invasive Fallopia japonica does it and so does the native Dactylis glomerata!

Invasive plants may alter ecological and ecosystem processes, including the N-cycle. The Fallopia species complex is a well-studied invasive species whose N-resource acquisition traits define it as an acquisitive species. However, the study of the impacts of invasive plants on the N-cycle never considers the N-acquisition strategy as a reference for choosing another suitable plant control. The purpose of this study is to assess the impacts of an invasive species (Fallopia japonicaFJ) on the N-cycle and to compare with those caused by a native acquisitive species (Dactylis glomerataDG), all compared to unplanted soils. A four-months mesocosm experiment was conducted by growing FJ and DG on nine different soils and measuring their impacts on N-cycle microbial activities (free-living nitrogen fixation FLNF, denitrification DEA, nitrification NEA), on N-mineral forms and on functional N-cycle gene abundance (nifH, AOA, AOB, nirS, nirK) as well as the total bacterial community gene (rRNA 16S). The nine soils differ in microbial enzymatic activities, N-mineral form concentrations, physico-chemical factors, texture, and gene abundances. Plant effects on FLNF, NEA and DEA are only soil dependent and no effect of invasive status was found. In addition, the native plant DG generally affected microbial parameters over a wider range of soils than the invasive plant. Stronger impacts of the native DG on microbial gene abundances were found compared to the invasive FJ. A stronger effect of the invasive plant was found for the soil NO3− concentration, with a significant decrease under the FJ than under the DG. Under both FJ and DG, NH4+ concentrations were not significantly affected. In conclusion, the invasive status in the ecosystem of the two plants studied cannot be explained through their impacts on microbial enzymatic activities and gene abundances of the soil N-cycle and the soil N mineral pools.

Estuarine mangrove niches select cultivable heterotrophic diazotrophs with diverse metabolic potentials-a prospective cross-dialog for functional diazotrophy.

Biological nitrogen fixation (BNF), an unparalleled metabolic novelty among living microorganisms on earth, globally contributes ~88-101 Tg N year-1 to natural ecosystems, ~56% sourced from symbiotic BNF while ~22-45% derived from free-living nitrogen fixers (FLNF). The success of symbiotic BNF is largely dependent on its interaction with host-plant, however ubiquitous environmental heterotrophic FLNFs face many limitations in their immediate ecological niches to sustain unhindered BNF. The autotrophic FLNFs like cyanobacteria and oceanic heterotrophic diazotrophs have been well studied about their contrivances acclimated/adapted by these organisms to outwit the environmental constraints for functional diazotrophy. However, FLNF heterotrophs face more adversity in executing BNF under stressful estuarine/marine/aquatic habitats. In this study a large-scale cultivation-dependent investigation was accomplished with 190 NCBI accessioned and 45 non-accessioned heterotrophic FLNF cultivable bacterial isolates (total 235) from halophilic estuarine intertidal mangrove niches of Indian Sundarbans, a Ramsar site and UNESCO proclaimed World Heritage Site. Assuming ~1% culturability of the microbial community, the respective niches were also studied for representing actual bacterial diversity via cultivation-independent next-generation sequencing of V3-V4 rRNA regions. Both the studies revealed a higher abundance of culturable Gammaproteobacteria followed by Firmicutes, the majority of 235 FLNFs studied belonging to these two classes. The FLNFs displayed comparable selection potential in media for free nitrogen fixers and iron-oxidizing bacteria, linking diazotrophy with iron oxidation, siderophore production, phosphorus solubilization, phosphorus uptake and accumulation as well as denitrification. This observation validated the hypothesis that under extreme estuarine mangrove niches, diazotrophs are naturally selected as a specialized multidimensional entity, to expedite BNF and survive. Earlier metagenome data from mangrove niches demonstrated a microbial metabolic coupling among C, N, P, S, and Fe cycling in mangrove sediments, as an adaptive trait, evident with the co-abundant respective functional genes, which corroborates our findings in cultivation mode for multiple interrelated metabolic potential facilitating BNF in a challenging intertidal mangrove environment.

Low temperature-mediated changes in soil free-living nitrogen fixation functional groups in tree species at different successional stages in subtropical forests

Low temperature-mediated changes in soil free-living nitrogen fixation functional groups in tree species at different successional stages in subtropical forests

Tripartite interactions among free‐living, N‐fixing bacteria, arbuscular mycorrhizal fungi, and plants: Mutualistic benefits and community response to co‐inoculation

AbstractInteractions between arbuscular mycorrhizal (AM) fungi and free‐living nitrogen fixers (FLNF) occur in the rhizosphere where they can enhance plant nutrient acquisition, impact plant growth, and affect soil processes. Tripartite mutualism commonly occurs between nodule‐forming plants, symbiotic diazotrophs, and AM fungi, and can occur between non‐nodulating plants, FLNF, and AM fungi. However, information on the extent of, and controls on, tripartite mutualism in non‐nodulating plant systems is limited to a small number of crop plants and culturable microbial inoculum, mostly in greenhouse growing conditions. We conducted a systematic literature review to synthesize the current understanding of the responses of plants, AM fungi, and FLNF to co‐inoculation, as well as the conditions affecting tripartite mutualism and the magnitude and range of benefits conferred. Our review shows that plants generally benefit from co‐inoculation with AM fungi and FLNF taxa, but benefits are highly variable and context dependent, ranging from 94% reduction in plant shoot biomass to 255% increase in total plant biomass. Additionally, the presence of AM fungi can increase abundance of FLNF and the presence of FLNF can increase AM fungal root colonization, but these responses also vary widely. Major factors influencing variation in response to co‐inoculation by all organisms include plant phenology/age, soil type and nutrient availability, and partner pairing. There is potential for leveraging these tripartite mutualisms to improve plant productivity and soil microbial function, but successful application is more likely with a thorough understanding of the environmental and mechanistic controls on these relationships and testing of field‐scale implementation.

The Attributes of Biofertilizer as an Alternative to Chemical Fertilizer: A Mini Review

Traditional soil management relies heavily on inorganic fertilizers, raising environmental and health concerns. A fertile soil requires a precise ratio of inorganic and organic components, with topsoil crucial for plant growth. Essential plant elements include macronutrients (nitrogen, phosphorus, potassium) and micronutrients (zinc, iron). Soil composition must balance minerals, air, water, and living matter. Macronutrients (nitrogen, phosphorus, potassium) are pivotal in plant growth. Potassium influences water regulation, root development, and crop resilience. Phosphorus, crucial for seed development, is essential for legume development. Nitrogen from nitrates, ammonium, and urea is indispensable for protein synthesis and overall plant growth. Biofertilizers, containing active microorganisms, offer an alternative to inorganic fertilizers. They enhance soil fertility, water and nutrient uptake, and plant tolerance to environmental variables. Nitrogen-fixing bacteria, phosphate solubilizing microorganisms (PSM), silicate solubilizing bacteria (SSB), plant growth-promoting rhizobacteria (PGPR), and arbuscular mycorrhizal fungi (AM fungi) are common groups of bacteria that play different specific roles in defining biofertilizer. Bacterial genera such as Rhizobium (a symbiotic nitrogen fixer known for forming nodules on legumes), Azotobacter (a free-living nitrogen fixer known for enhancing sugar content in crops), Azospirillum (a bacterium known for enhancing nitrogen content in non-leguminous plants) and Anabaena-Azollae (a symbiotic relationship between a cyanobacterium and lower plants known in fixing nitrogen and promotes growth in various crops). As agriculture continues to evolve, embracing biofertilizers represents a promising step toward a more sustainable and resilient future.

Non-targeted effects of nitrification inhibitors on soil free-living nitrogen fixation modified with weed management

Biological nitrogen fixation and nitrification inhibitor applications contribute to improving soil nitrogen (N) availability, however, free-living N fixation affected by nitrification inhibitors has not been effectively evaluated in soils under different weed management methods. In this study, the effects of the nitrification inhibitors dicyandiamide (DCD) and 3, 4-dimethylpyrazole phosphate (DMPP) on the nitrogenase, nifH gene，and diazotrophic communities in soils under different weed management methods (AMB, weeds growth without mowing or glyphosate spraying; GS, glyphosate spraying; MSG, mowing and removing weeds and glyphosate spraying; and WM, mowing aboveground weeds) were investigated. Compared to the control counterparts, the DCD application decreased soil nitrogenase activity and nifH gene abundance by 4.5 % and 37.9 %, respectively, under the GS management method, and the DMPP application reduced soil nitrogenase activity by 20.4 % and reduced the nifH gene abundance by 83.4 % under the MSG management method. The application of nitrification inhibitors significantly elevated soil NH4+-N contents but decreased NO3−-N contents, which had adverse impacts on soil nifH gene abundance and nitrogenase activity. The nifH gene abundances were also negatively impacted by dissolved organic N and Geobacter but were positively affected by available phosphorus and diazotrophic community structures. Nitrification inhibitors significantly inhibited Methylocella but stimulated Rhizobiales and affected soil diazotrophic communities. The nitrification inhibitors DCD and DMPP significantly altered soil diazotrophic community structures, but weed management outweighed nitrification inhibitors in reshaping soil diazotrophic community structures. The non-targeted effects of the nitrification inhibitors DMPP and DCD on soil free-living N fixation were substantially influenced by the weed management methods.

Biological nitrogen fixation in barren soils of a high-vanadium region: Roles of carbon and vanadium

Free-living nitrogen fixation (FLNF) is a vital source of nitrogen for the initiation and development of ecosystems on bare lands. This process is catalyzed by the enzyme nitrogenase and is energy intensive. Of the three nitrogenase isoforms, molybdenum nitrogenase (Mo-Nase) is more efficient than its vanadium (V) and iron (Fe) counterparts at room temperature. However, the acquisition of Mo, one of the scarcest biometals in soils, represents a major resource investment for soil diazotrophs. In a vegetation restoration chronosequence developed on barren (low carbon and nitrogen) soils of a high-V region, we tested the following two hypotheses. First, the FLNF rate would be limited by the supply of energy. Second, high V availability would cut the cost of V acquisition, and it would be beneficial for diazotrophs to use V-Nase as a complement of Mo-Nase. For the soils collected along the chronosequence, the addition of a carbon cocktail remarkably stimulated the FLNF rate by relieving the energy constraint and by enriching the diazotrophic genus Paenibacillus. Despite the high V content in the soils, we failed to detect the vnf genes of V-Nase by metagenomic sequencing. Meanwhile, we observed a limited contribution of V-Nase activity to FLNF by checking the R ratio (ratio of FLNF rates measured by acetylene reduction and 15N2 incorporation) and the production of ethane during acetylene reduction. The reason might be that the Mo availability and temperature in our study area did not reach the thresholds for the onset of V-Nase activity. Overall, our results highlight the importance of carbon supply for FLNF in barren soils, indicating that plants may effectively regulate rhizosphere diazotrophs by secreting root exudates. This may be an important mechanism by which nonnitrogen-fixing plants survive on nitrogen-poor soils at the beginning of primary succession.

Strong cooperations among diazotroph and arbuscular mycorrhizal fungi taxa promote free-living nitrogen fixation at soil-rock mixing layer

Soil nitrogen (N) is a renewable resource of N fixed by free-living N fixation (FLNF) diazotrophs. Thus, understanding the microbial driving mechanism of FLNF activity can aid in the optimization of N input. However, the role of co-symbiosis between diazotrophs and arbuscular mycorrhizal fungi (AMF) in the FLNF activity at different soil depths has been largely overlooked, particularly in karst ecosystems. Thus, we investigated soil properties and the characteristics of diazotroph and AMF across soil depths, from topsoil to soil-rock mixing layer, based on the soil profile. Soil properties such as soil organic matter and ammonium N decreased with increasing soil depth, whereas pH showed the opposite trend. Similarly, diazotroph abundance and diversity and AMF abundance were higher by 8–73% in 0–20 cm soil than in 20–40 cm and soil-rock mixing layer. Despite high diazotroph abundance in the topsoil, the FLNF activity was higher by 30% at soil-rock mixing layer than in 0–20 cm. The co-occurrence network analysis revealed a strengthening of the cooperative relationship between the diazotroph and AMF taxa at the soil-rock mixing layer via an increase in the number of unique AMF taxa. A structural equation model indicated that increasing soil depth improves the FLNF activity due to increasing soil pH and mutualistic cooperations between diazotrophs and AMF taxa, such as Bradyrhizobium to AMF unclassified taxa and Bradyrhizobium to Racocetra. This study provides novel insights into the interspecific interactions between diazotrophs and AMF, rather than their abundance and diversity, which were found to be the most important driving factors of FLNF activity at the soil-rock mixing layer. Consequently, the roles of biotic factors in influencing mutually beneficial microbial taxa regulating FLNF activity should be considered during vegetation recovery in the fragile karst region.

Codon usage behavior distinguishes pathogenic Clostridium species from the non-pathogenic species

Genus Clostridium is of the largest genus in class Clostridia. It is comprised of spore-forming, anaerobic, gram-positive organisms. The members of this genus include human pathogens to free-living nitrogen fixing bacteria. In the present study, we have performed a comparison of the choice of preferred codons, codon usage patterns, dinucleotide and amino acid usage pattern of 76 species of Genus Clostridium. We found the pathogenic clostridium species to have smaller AT-rich genomes as compared to opportunistic and non-pathogenic clostridium species. The choice of preferred and optimal codons was also influenced by genomic GC/AT content of the respective clostridium species. The pathogenic clostridium species displayed a strict bias in the codon usage, employing 35 of the 61 codons encoding for 20 amino acids. Comparison of amino acid usage revealed an increased usage of amino acids with lower biosynthetic cost by pathogenic clostridium species as compared to opportunistic and non-pathogenic clostridium species. Smaller genome, strict codon usage bias and amino acid usage lead to lower protein energetic cost for the clostridial pathogens. Overall, we found the pathogenic members of genus Clostridium to prefer small, AT-rich codons to reduce biosynthetic costs and match the cellular environment of its AT-rich human host.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

Diazotrophic bacterium Azotobacter vinelandii as a mutualistic growth promoter of an aquatic plant: Lemna minor

Lemnaceae plants, commonly referred to as duckweeds, are small planktonic terrestrial freshwater plants that live in symbiosis with various microbial communities. Azotobacter vinelandii are typical free-living nitrogen fixing soil bacteria that indirectly benefit plants by providing nitrogen compounds. In this study, Lemna minor RDSC 5512 and A. vinelandii ATCC 12837 = NBRC 13581 were co-cultured under gnotobiotic conditions. The growth of L. minor colonized by A. vinelandii was accelerated in both nitrogen-containing and nitrogen-free water conditions. The growth promotion effect is attributed to several plant growth promotion factors produced by the bacterium as well as biological nitrogen fixation in nitrogen-free condition. Moreover, L. minor elevated the nitrogen fixing activity of A. vinelandii and the cell number of A. vinelandii on L. minor increased continuously over 30 d. These observations indicated that L. minor provides a favorable environment for A. vinelandii colonization, allowing them to mutually benefit and flourish through syntrophism.

Microbial inoculants with inorganic fertilizers slacken the chlorosis impact on Kalmegh [Andrographis paniculata (Burm. F.) Wall ex., Nees] improve yields and quality traits

A common problem witnessed in Kalmegh [Andrographis paniculata (Burm. F.) Wall ex., Nees] plants are yellowing of leaves, when cultivated during rainy season which drastically affects the yield and quality of andrographolide. In the present study, four strains of bioinoculants (Pseudomonas monteilii, Cedecea devisae, Cronobacter dublinensis, Advenella species) were scrutinized along with combinations of inorganic fertilizers to identify the best bioinoculant-fertilizer combination which can curb the effect of yellowing in Kalmegh plants most efficiently as well as enhance its biomass and secondary metabolite content. Minimum percentage of yellowing of leaves was recorded in the plantlets inoculated with bioinoculants (Cedecea devisae) and N:P:K applied at the ratio of 80:60:40 kg ha−1. Among all the treatments, Cedecea devisae (T5) gave maximum fresh and dry herb yields. The results showed a significant variation in photosynthetic pigments along with the treatments. Maximum andrographolide yield was recorded in the plantlets inoculated with Cedecea devisae which was 63.07% more compared to control. Moreover, the concentration of andrographolide in different parts of Kalmegh plants followed the trend-leaves>stem>inflorescence>flowers>seeds>seed husk>root. Except soil pH, other analysed soil parameters of the post harvest soil were significantly improved. Significant enhancement of the soil dehydrogenase, alkaline and acid phosphatase enzymes activity was 14.8% to 54.0%, 57.0% to 234%, and 51.6% to 276% greater, respectively over control. Principal component analysis revealed a strong positive interrelationship between vegetative attributes, soil enzyme activity, biomass yield and andrographolide content. We can suggest from our study that, bioinoculants especially free living nitrogen fixers (Cedecea davisae) along with recommended doses of NPK can effectively control the problem of yellowing (chlorosis) in Kalmegh plants during rainy season.

Bulk and Spatially Resolved Extracellular Metabolome of Free-Living Nitrogen Fixation

ABSTRACTSoil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is an important soil N transformation and key N input to terrestrial systems, but the forms of N contributed to soil by FLNF are poorly understood. To address this knowledge gap, a focus on microorganisms and microbial scale processes is needed that links N-fixing bacteria and their contributed N sources to FLNF process rates. However, studying the activity of soil microorganisms in situ poses inherent challenges, including differences in sampling scale between microorganism and process rates, which can be addressed with culture-based studies and an emphasis on microbial-scale measurements. Culture conditions can differ significantly from soil conditions, so it also important that such studies include multiple culture conditions like liquid and solid media as proxies for soil environments like soil pore water and soil aggregate surfaces. Here we characterized extracellular N-containing metabolites produced by two common, diazotrophic soil bacteria in liquid and solid media, with or without N, across two sampling scales (bulk via GC-MS and spatially resolved via MALDI mass spec imaging). We found extracellular production of inorganic and organic N during FLNF, indicating terrestrial N contributions from FLNF occur in multiple forms not only as ammonium as previously thought. Extracellular metabolite profiles differed between liquid and solid media supporting previous work indicating environmental structure influences microbial function. Metabolite profiles also differed between sampling scales underscoring the need to quantify microbial scale conditions to accurately interpret microbial function.IMPORTANCE Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms in situ, such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity). Thus, upscaling important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, requires microbial-scale measurements in controlled systems. Our work generated bulk and spatially resolved measurements of nitrogen released during free-living nitrogen fixation under two contrasting growth conditions analogous to soil pores and aggregates. This work allowed us to determine that diverse forms of nitrogen are likely contributed to terrestrial systems by free-living nitrogen bacteria. We also demonstrated that microbial habitat (e.g., liquid versus solid media) alters microbial activity and that measurement of microbial activity is altered by sampling scale (e.g., bulk versus spatially resolved) highlighting the critical importance of quantifying microbial-scale processes to upscaling of ecosystem function.

Microbiological properties of Beejamrit, an ancient Indian traditional knowledge, uncover a dynamic plant beneficial microbial network.

Beejamrit is an ancient organic formulation commonly used as a seed treatment in organic and natural farming in India. This low-cost formulation is primarily a product of dairy excreta (e.g., cow dung and cow urine) and forest soil, often supplemented with limestone. Growing data suggest that dairy excreta are the potential sources of enriched microbial niche, including several plant growth-promoting bacteria capable of synthesizing plant growth regulators. However, the microbiological properties of Beejamrit and their temporal changes after different incubation periods, delineating its application in seed treatment, remain largely unexplored. Here, we aimed to analyze the decomposition rate of Beejamrit over 7-consecutive days of incubation. This study further elucidates the microbial niche and their dynamics in Beejamrit, including the plant beneficial bacteria. We have shown that the population of plant beneficial bacteria, such as the free-living nitrogen fixers (FNFs) and the phosphate solubilizers (PSBs), proliferates progressively up to 4- and 5-days of incubation, respectively (p < 0.0001). This study also reports the total indolic content of Beejamrit, including indole 3-acetic acid (IAA), which further tends to oscillateinconcentration based on the incubationperiods incurred during the Beejamrit preparation. Our analyses, together, establish that Beejamrit provides a dynamic, microbe-based metabolic network and may, therefore, act as a plant biostimulant to crop plants. A plant-based bioassay finally demonstrates the role of Beejamrit in the seed treatment to improve seed germination, seedling survival rate, and shoot length trait in French beans (p < 0.01). In conclusion, this study highlights, for the first time, the scientific insights of Beejamrit as a potential seed priming agent in agriculture.

IMPORTANCE OF BIO FERTILIZERS AS ALTERNATIVE SOIL FERTILITY AMENDMENTS

There is a current interest in agrarian sustainability with soil microorganisms instead of agro-chemicals. Key constraints in the use of bio fertilizers are; inadequate awareness about bio inoculants and lack of promotion network and publicity among the end users. This review discusses current technical information a way of creating awareness in order to promote the use of bio fertilizers. Publon, Google Scholar, Science Direct and Microsoft Academic data bases were used for the review 2021. Research and review articles published from 2019 onwards were considered as current information for the review. The findings of the review are that; both primary and secondary macronutrients can be provided by bio fertilizers. Potential microbes are; free-living nitrogen fixing bacteria and cyanobacteria, symbiotic nitrogen-fixing bacteria and fungi such as mycorrhiza. Other important functions of bacteria are; conferring to plants the ability for salt tolerance, lignin degradation and remediation of heavy metals from the soil. Bio Compost, vermicompost and termite soil with their rich microorganism content can be used as bio fertilizers for soil nutrient increase. In order to reap maximum benefit from bio fertilizers there is need to formulate them in appropriate materials. Apart from addition of nutrients to the soil, bio fertilizers play an important role in plant health, conclusion. This paper has brought to the fore the need to improve rhizosphere management in a sustainable way particularly at this point in time when there are strong indications that it has deteriorated in the face of continued use of chemical fertilizers.

Low Frequency of Plants Associated with Symbiotic Nitrogen-Fixers Exhibits High Frequency of Free-Living Nitrogen Fixing Bacteria: A Study in Karst Shrub Ecosystems of Southwest China

Plants associated with symbiotic nitrogen-fixers and soil free-living nitrogen-fixing bacteria are good indicators for detecting the source of nitrogen in natural ecosystems. However, the community composition and diversity of plants associated with symbiotic nitrogen-fixers and soil free-living nitrogen-fixing bacteria in karst shrub ecosystems remain poorly known. The community composition and diversity of soil free-living nitrogen-fixing bacteria and plants, as well as the soil physical–chemical properties were investigated in 21 shrub plots (including different topographies and plant types). The frequency of plants associated with symbiotic nitrogen-fixers was found to be low in the 21 shrub plots. The soil free-living nitrogen-fixing bacterial community structure varied among the 21 shrub soils. Based on a variance partitioning analysis, topography, plant type, and soil pH explained 48.5% of the observed variation in bacterial community structure. Plant type had a predominant effect on community structure, and topography (aspect and ascent) and soil pH had minor effects. A negative correlation between the abundance of the soil free-living nitrogen-fixing bacterial community and the richness index for plants associated with symbiotic nitrogen-fixers was observed. The result of the low frequency of plants associated with symbiotic nitrogen-fixers highlights the importance of sources of fixed nitrogen by soil free-living nitrogen-fixing bacteria in the nitrogen limitation shrub ecosystem of the karst regions.