Nitrogen-fixing Ability Research Articles

Complete genome sequence of a nitrogen-fixing bacterium, Klebsiella pasteurii (formerly Klebsiella oxytoca) NG13, a rice rhizosphere isolate.

Klebsiella oxytoca NG13 was isolated from the rice rhizosphere in the early 1980s, and its nitrogen-fixing ability has been investigated. For the further advanced research, we sequenced its genome. NG13 contains one chromosome of 5,897,538 bp and no plasmid. NG13 should be classified to Klebsiella pasteurii based on ANI.

C/N conversion and pigment production of Blastochloris sulfoviridis characterized by bacteriochlorophyll b under near-infrared single-wavelength laser

High carbon use efficiency (CUE) of photosynthetic bacteria (PSB) can concurrently increase biomass and reduce CO2 emissions. In the PSB study, light-oxygen conditions are crucial factors influencing the growth of these bacteria. Bacteriochlorophyll (BChl) b (absorption spectra: 970–1040 nm), due to its unique absorption spectrum, can be considered a crucial complement to BChl a (absorption spectra: 780–890 nm). We investigate the effects of four light-oxygen conditions on biomass, C/N conversion, and pigment production using five individual light sources in Blastochloris sulfoviridis (B. sulfoviridis), producing BChl b, isolated using 940±10 nm laser unexpectedly. Biomass, CUE, and pigment production follow the order under different light-oxygen conditions: light-anaerobic > light-aerobic > dark-aerobic > dark-anaerobic. The maximum biomass (1386.88 mg/L), highest CUE (0.95 g-biomass/g-CODremoved and 0.49 g-biomass carbon/g-CODremoved), and highest pigment production are observed under full spectrum (40 W)-anaerobic, 940-anaerobic, and 850-anaerobic conditions, respectively. Compared to light-aerobic, the nitrogen-fixing ability of B. sulfoviridis is enhanced under light-anaerobic conditions. This study contributes to research on resource recovery and greenhouse gas emissions reduction in PSB wastewater treatment.

IAA-producing bacteria from the rhizosphere of chickpea (Cicer arietinum L.): Isolation, characterization, and their effects on plant growth performance

Indole-3-acetic acid (IAA), a crucial plant hormone, regulates diverse physiological processes. This study aimed to isolate and characterize IAA-producing bacteria from the chickpea (Cicer arietinum L.) rhizosphere and evaluate their effects on plant growth. From 54 rhizosphere samples, 118 bacteria (designated as GAC) were isolated and screened for IAA production using a Salkowski colorimetric assay, and Bergey's manual was used for biochemical identification. Isolates were grown under various conditions and in vitro screened for their growth promotion traits. A PCR investigation was performed for IAA and nitrogen-fixing genes, and evaluated for greenhouse conditions. Among them, 27 isolates produced IAA, with eight high producers selected. Morphological and biochemical identification classified the six isolates as Pseudomonas and the other two as Bacillus. Optimal conditions for IAA production were observed at 500 μg/ml tryptophan, 35 °C, and pH 7.0. A 48-h incubation was ideal for IAA production, except for GAC-34 and GAC-73, which required 72 h. All the isolates achieved optimal IAA levels with tryptone and sucrose as nitrogen and carbon sources, respectively. Moreover, all isolates showed nitrogen fixation ability, and the six isolates exhibited phosphate solubilization. PCR confirmed the amplification of nifH (300 bp), nifK (360 bp), and ipdC (1170 bp) genes. Greenhouse experiments demonstrated that eight selected isolates significantly enhanced chickpea growth parameters (p < 0.001). These findings suggest that these IAA-producing bacteria have the potential to be used as biofertilizers to improve crop productivity, although further molecular identification and field studies are required.

Genome-wide association mapping identifies novel SNPs for root nodulation and agronomic traits in chickpea.

The chickpea (Cicer arietinum L.) is well-known for having climate resilience and atmospheric nitrogen fixation ability. Global demand for nitrogenous fertilizer is predicted to increase by 1.4% annually, and the loss of billions of dollars in farm profit has drawn attention to the need for alternative sources of nitrogen. The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in determining the growth and production of chickpea. To support findings on nodule formation in chickpea and to map the genomic regions for nodulation, an association panel consisting of 271 genotypes, selected from the global chickpea germplasm including four checks at four locations, was evaluated, and data were recorded for nodulation and 12 yield-related traits. A genome-wide association study (GWAS) was conducted using phenotypic data and genotypic data was extracted from whole-genome resequencing data of chickpea by creating a hap map file consisting of 602,344 single-nucleotide polymorphisms (SNPs) in the working set with best-fit models of association mapping. The GWAS panel was found to be structured with sufficient diversity among the genotypes. Linkage disequilibrium (LD) analysis showed an LD decay value of 37.3 MB, indicating that SNPs within this distance behave as inheritance blocks. A total of 450 and 632 stringent marker-trait associations (MTAs) were identified from the BLINK and FarmCPU models, respectively, for all the traits under study. The 75 novel MTAs identified for nodulation traits were found to be stable. SNP annotations of associated markers were found to be related to various genes including a few auxins encoding as well as nod factor transporter genes. The identified significant MTAs, candidate genes, and associated markers have the potential for use in marker-assisted selection for developing high-nodulation cultivars after validation in the breeding populations.

Glucose addition in natural forest soils has higher biological nitrogen fixation capacity than other types of soils

Land use changes soil microbial and chemical properties, but the mechanism of biological nitrogen fixation under different land use patterns is rarely reported, so we used four types of soil: Natural forest soil (NS), healthy banana soil (HS), diseased banana soil (DS) and paddy soil (PS). Treatments included the control (CK), addition of glucose (G), addition of glucose and ammonium nitrate (GN), addition of banana straw (BS), addition of banana straw and ammonium nitrate (BSN), addition of banana root (BR), and addition of banana root and ammonium nitrate (BRN). The study found that the change of soil utilization types, glucose addition increased carbon dioxide emissions (Compared with the control, increased by 963.11%, 508.39%, 794.77% and 511.34%, respectively) and enhanced the ability of soil microbial nitrogen fixation. Importantly, natural forest soil microorganisms have a higher biological nitrogen fixation capacity compared to other types of soils. Glucose addition caused the accumulation of ammonium nitrogen (Compared with the control, increased by 426.08%, 934.21%, 420% and 1065.95%, respectively), indicating that microorganisms had higher utilization efficiency of soluble carbon and enhanced the biological nitrogen fixation capacity, and nitrogen addition caused the accumulation of ammonium nitrogen, thereby weakening the biological nitrogen fixation capacity. At the same time, glucose significantly increased the Fimicutes phylum (83.73%, 66.38%, 67.18% and 70.36%) and lowered the level of other bacterial phylums, thereby reducing the bacterial network structure, and the stability of the soil environment has decreased. Forest analysis showed that CO2 was an important factor in predicting the bacterial community structure of different soil types, an increase in CO2 content can predict drastic changes in the bacterial community. Bacteria at the Fimicutes phylum level preferred glucose, which may also have a negative effect on bacteria at the level of other phylums.

Nitrogen Fertilization in Snap Bean Following a Legume in the Sandy Soils of North Florida

Despite being a legume, snap bean (Phaseolus vulgaris L.) lacks nodulation genes, restricting its ability for nitrogen (N) fixation through rhizobia, making N fertilization essential for maximizing yields. However, conventional fertilizer application may lead to N losses due to a lack of synchronization between plant uptake and N availability. A promising option could be controlled-release fertilizers (CRFs), which can be custom-formulated to synchronize nutrient release with plant growth needs, promoting efficient resource use. Field studies were conducted at the North Florida Research and Education Center-Suwannee Valley (NFREC-SV) in Live Oak, FL, during the spring of 2021 and 2022. The experiments also investigated broadcasting and banding application methods of the University of Florida’s recommended N rate (112 kg·ha−1). The experimental design comprised 10 treatments, including five N rates (0, 56, 112, 168, and 224 kg·ha−1 N) and two fertilizer sources (ammonium nitrate and CRF), applied to ‘Caprice’ in a randomized complete block design with four replications. Results showed a significant yield improvement with N fertilization vs. the zero-N control. However, no yield increase was observed beyond 56 kg·ha−1. This finding could be due to the residual N from a preceding peanut crop rotation in both years. No notable variation in quality metrics (pod length and width) occurred above the 56 kg·ha−1 threshold. The results also revealed that the choice between conventional or CRF did not exert any statistically significant differences in snap bean yield. In addition, fertilizer broadcast and banding treatments yielded the same results.

Endophytic Bacterial Biofilm-Formers Associated with Antarctic Vascular Plants.

Deschampsia antarctica and Colobantus quitensis are the only two vascular plants colonized on the Antarctic continent, which is usually exposed to extreme environments. Endophytic bacteria residing within plant tissues can exhibit diverse adaptations that contribute to their ecological success and potential benefits for their plant hosts. This study aimed to characterize 12 endophytic bacterial strains isolated from these plants, focusing on their ecological adaptations and functional roles like plant growth promotion, antifungal activities, tolerance to salt and low-carbon environments, wide temperature range, and biofilm formation. Using 16S rRNA sequencing, we identified several strains, including novel species like Hafnia and Agreia. Many strains exhibited nitrogen-fixing ability, phosphate solubilization, ammonia, and IAA production, potentially benefiting their hosts. Additionally, halotolerance and carbon oligotrophy were also shown by studied bacteria. While some Antarctic bacteria remain strictly psychrophilic, others demonstrate a remarkable ability to tolerate a wider range of temperatures, suggesting that they have acquired mechanisms to cope with fluctuations in environmental temperature and developed adaptations to survive in intermediate hosts like mammals and/or birds. Such adaptations and high plasticity of metabolism of Antarctic endophytic bacteria provide a foundation for research and development of new promising products or mechanisms for use in agriculture and technology.

Utilization of Aquatic Plants and Microalgae for Sustainable Aquaculture Production and Potential Biotechnological Applications

Locally available feedstuffs, such as grain seed cakes, oilseeds, and vegetable waste, hold immense potential as alternative feed sources in fish farming. However, these plant-based ingredients have low crude protein content and lack essential fatty acids, which lowers palatability and feed conversion efficiencies, leading to suboptimal fish growth. Traditional feedstuffs like fishmeal and soybean meal face sustainability challenges such as local unavailability, the presence of anti-nutritional factors, and energy-intensive processing. The rising costs of commercial aqua-feeds and feed scarcity necessitate exploring alternative fish feed options. Aquatic plants like Ipomoea aquatica, Lemna minor, and Azolla pinnata, along with green and blue-green microalgae such as Chlorella spp. and Arthrospira spp. (Spirulina), are promising alternatives due to their high protein content, availability of essential omega-3 fatty acids such as EPA (Eicosapentaenoic acid) and DHA (Docosahexaenoic acid), and beneficial bioactive compounds. These plants and microalgae, with crude protein content ranging from 25% to 65%, can significantly enhance fish growth, health, and product quality by partially or entirely replacing fishmeal. Their nitrogen-fixing abilities contribute to their high protein levels. Additionally, these organisms have various biotechnological applications, including phytoremediation, Integrated Multi-trophic Aquaculture (IMTA), aquaponics, biofloc technology, and constructed wetlands. Despite their potential, challenges in scaling up and integrating these alternatives into existing systems remain. Collaborative efforts and advocacy among farmer groups are crucial for knowledge sharing and fostering sustainable biotechnological solutions. Long-term strategies should focus on upscaling local feed production and research and development to achieve self-sufficiency and cost-effective natural feed production systems in fish farming.

Role of Wilting Time on the Chemical Composition, Biological Profile, and Fermentative Quality of Cereal and Legume Intercropping Silage

Agricultural production in the Azores primarily focuses on the livestock sector, notably, dairy production, where cows graze year-round in a rotational system. To maintain pasture productivity, farmers often rely on synthetic nitrogen fertilizers, which have adverse environmental impacts like ammonia emissions and nitrate leaching. Alternatively, nitrogen-fixing crops like legumes are explored as green manures to enhance soil quality and reduce dependence on chemical fertilizers. The traditional practice of using mixed forages of legumes and grasses, known as “outonos” or intercrops, has been crucial but is declining over time. These mixtures include plants such as lupins, Vicia faba, oats, and vetch, noted for their adaptability and nitrogen-fixing ability. Due to the high perishability of these crops, effective conservation strategies like ensiling are essential to preserve forage nutritional quality through controlled fermentation. This study evaluates the productivity and quality of intercrop forages in the Azores, focusing on fresh samples and silage prepared with wilting times of 0, 24, 48, and 96 h, followed by comprehensive chemical analyses. Results showed significant changes in fiber components (neutral detergent fiber, acid detergent fiber, and acid detergent lignin) with increased wilting time, leading to reduced digestibility. However, wilting improved dry matter content.

Engineering a Sinorhizobium meliloti Chassis with Monopartite, Single Replicon Genome Configuration.

Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.

Mapping the molecular landscape of Lotus japonicus nodule organogenesis through spatiotemporal transcriptomics

Legumes acquire nitrogen-fixing ability by forming root nodules. Transferring this capability to more crops could reduce our reliance on nitrogen fertilizers, thereby decreasing environmental pollution and agricultural production costs. Nodule organogenesis is complex, and a comprehensive transcriptomic atlas is crucial for understanding the underlying molecular events. Here, we utilized spatial transcriptomics to investigate the development of nodules in the model legume, Lotus japonicus. Our investigation has identified the developmental trajectories of two critical regions within the nodule: the infection zone and peripheral tissues. We reveal the underlying biological processes and provide gene sets to achieve symbiosis and material exchange, two essential aspects of nodulation. Among the candidate regulatory genes, we illustrate that LjNLP3, a transcription factor belonging to the NIN-LIKE PROTEIN family, orchestrates the transition of nodules from the differentiation to maturation. In summary, our research advances our understanding of nodule organogenesis and provides valuable data for developing symbiotic nitrogen-fixing crops.

Bioremediation of soils contaminated with nicosulfuron by the bacterial complex ES58

Nicosulfuron residues in soil can be harmful to the environment. In this study, Klebsiella oxytoca ES5 and Klebsiella grimontii ES8 were isolated to construct the ES58 bacterial complex. The culture medium was optimized using response surface methodology, nicosulfuron degradation by ES58 reached 97.21 % in 96 h. The optimal degradation conditions were: temperature: 25℃, pH 6, initial nicosulfuron concentration: 50 mg L−1 and inoculum volume: 2 %. The two individual strains and the bacterial complex were tested for the ability of nitrogen fixation, phosphorus solubilization, potassium decomposition and iron carrier production. The results revealed that both strains and the bacterial complex had the ability to promote the growth, with bacterial complex being more capable than the two individual bacterial strains. After 28 d of bioremediation of nicosulfuron-contaminated soil by ES58, degradation rate of nicosulfuron was 90.84 % in the unsterilized soil. Furthermore, the remediation process restored the activities of catalase, invertase, dehydrogenase and urease in the soil. The ES5-specific gene budA and ES8-specific gene ldh were detected in the soil at the 28th day. ES58 exhibited different effects on the growth indexes of nicosulfuron-sensitive crops. The study provides a novel strategy for nicosulfuron degradation effective by constructing and utilizing bacterial complex.

Power of seeds: exploring the growth boosting potential of endophytes

Insect pests on agricultural crops cause biotic stress, resulting in enormous yield losses. Nowadays, microbial insecticides for insect pests have been explored to overcome insect resistance and environmental hazards caused by chemical insecticides. Bacillus thuringiensis (Bt) is known as the most successful microbial insecticide globally, but its commercial application is limited due to its vulnerability to abiotic factors. To overcome these factors, the establishment of Bt as an endophyte in maize plants by seed treatment has been proposed. So, for that reason, the present study aimed to investigate the effect of seed endophytic bacteria in maize on Bt isolates and vice versa. Furthermore, the study was also carried out to assess the antibiotic resistance of these bacteria as well as their role in plant growth promotion. A total of 12 maize seed endophytic bacterial isolates were used for this study. In that, seven isolates belong to the Bacillus genus, two to the Staphylococcus genus, and one each from Paenibacillus, Pantoea, and Enterobacter. No antagonistic effect of seed endophytes was observed against Bt strains. Five isolates had the ability to solubilize phosphate and potassium, seven showed zinc solubilization, and all the isolates showed nitrogen fixation ability. Thus, these seed endophytic bacteria can be explored as potential bio-inputs for improving plant growth, and their harmonizing effect on Bt has been found to be promising for the establishment of Bt as an endophyte in maize plants.

Microbial Protein Production Using Lignocellulosic Biomass (Switchgrass) and Klebsiella oxytoca M5A1—A Nitrogen Fixer

The expanding global population has increased the demand for sustainable protein sources, and microbial protein (MP) has emerged as a promising alternative. However, conventional carbon (glucose) and nitrogen (ammonia, urea) sources needed for MP production pose environmental and economic issues. This study aims to produce protein using lignocellulosic biomass (LCB) as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The study investigates the pretreatment of LCB (switchgrass), enzymatic hydrolysis, protein quantification, nitrogen fixation, glucose utilization and organic acids production. K. oxytoca M5A1 harnessed free nitrogen from the atmosphere and used abundant, cheap glucose from LCB to produce MP and organic acids as by-products. Protein production occurred in two phases: first within the initial 8 h and secondly, within the last 16 h. The highest protein concentration was at 40 h, with approximately 683.46 µg/mL protein content. High-performance liquid chromatography system (HPLC) analysis revealed a dynamic profile of glucose utilization and organic acids (Lactic acid, Propionic acid, Acetic acid, and Succinic acid) production. K. oxytoca M5A1 exhibited an early high rate of glucose consumption, and conversion to organic acids, that were later used for second-phase protein production. The acids profile revealed intra-conversion from one acid to another via metabolic pathways (glycolysis and tricarboxylic acid cycle). Overall, leveraging LCB and the nitrogen-fixing ability of K. oxytoca M5A1 for MP production offers an eco-friendly and cost-effective alternative to traditional protein sources, contributing to a sustainable circular economy.

Effects of Semi‐Film and Full‐Film Mulching on Soybean Growth, Biological Nitrogen Fixation and Grain Yield

ABSTRACTSoybean film mulching has shown promise in maintaining consistent and high yields in semi‐arid regions. However, the specific impacts of full‐film and semi‐film mulching on soybean growth, root nodule traits and grain yield are poorly understood. This 2‐year study (2021–2022) investigates the effects of full‐film and semi‐film mulching on soil moisture, soybean growth and yield. Our findings revealed that semi‐film mulching increased soybean yield by 18.12% compared to full‐film mulching, averaged across 2 years. Furthermore, the semi‐film treatment significantly enhanced biological nitrogen fixation ability, increasing root nodule numbers by 24.81%–33.43%, compared to full‐film mulching. This improvement also positively affected soybean quality, with crude protein content increasing by 5.89% in 2021 and 4.14% in 2022 compared to full‐filming. Moreover, semi‐film mulching helped maintain soil moisture and temperature during later soybean growth stages. There findings suggest that semi‐film mulching is a viable agricultural strategy for soybean cultivation in semi‐arid regions, improving soybean quality and efficiency while promoting environmental sustainability.

Inter-species interaction of bradyrhizobia affects their colonization and plant growth promotion in Arachis hypogaea.

Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

Combined dynamic transcriptome and flavonoid metabolome reveal the role of Mo nanoparticles in the nodulation process in soybean

Symbiotic nitrogen fixation can reduce the impact of agriculture on the environment by reducing fertilizer input. The rapid development of nanomaterials in agriculture provides a new prospect for us to improve the biological nitrogen fixation ability of leguminous crops. Molybdenum is an important component of nitrogenase, and the potential application of MoO3NPs in agriculture is largely unexplored. In this study, on the basis of verifying that MoO3NPs can improve the nitrogen fixation ability of soybean, the effects of MoO3NPs on the symbiotic nitrogen fixation process of soybean were investigated by using dynamic transcriptome and targeted metabolome techniques. Here we showed that compared with conventional molybdenum fertilizer, minute concentrations of MoO3NPs (0.01–0.1 mg kg−1) could promote soybean growth and nitrogen fixation efficiency. The nodules number, fresh nodule weight and nitrogenase activity of 0.1 mg kg−1 were increased by 17 %, 14 % and 27 %, and plant nitrogen accumulation increased by 17 %. Compared with conventional molybdenum fertilizer, MoO3NPs had a greater effect on apigenin, kaempferol and other flavonoid, and the expression of nodulation related genes such as ENOD93, F3'H. Based on WGCNA analysis, we identified a core gene GmCHS9 that was positively responsive to molybdenum and was highly expressed during MoO3NPs induced nodulation. MoO3NPs could improve the nitrogen fixation ability of soybean by promoting the secretion of flavonoids and the expression of key genes. This study provided a new perspective for the nano-strengthening strategy of nodules development and flavonoid biosynthesis by molybdenum.

Nitrogen fixation of different seasons and parts of seagrass Enhalus acoroides in Xincun Bay, northern South China Sea

In coastal ecosystems, the nitrogen fixation of seagrasses is crucial. However, the nitrogen fixation rates of seagrasses can vary spatially and temporally, especially in tropical regions. To investigate the nitrogen fixation rates of Enhalus acoroides, we used principal component analysis to examine the rates of nitrogen fixation in the roots, rhizome, and leaves of E. acoroides during the southwest monsoon (July) and the northeast season (October and January), respectively. The results showed that the nitrogen fixation rates in all three parts of E. acoroides were significantly higher during the southwest monsoon period compared to the northeast period. The rates of nitrogen fixation in the leaves of E. acoroides were found to be higher than in the roots and rhizomes. The difference in nitrogen fixation between the various parts of E. acoroides was highly significant (p<0.01). The nitrogen fixation activity of E. acoroides was significantly affected by temperature, as determined by principal component analysis. The spatial variations observed in different regions of E. acoroides were influenced by environmental factors in addition to the temperature-driven seasonal changes. The results suggest that temperature plays a vital role in the nitrogen fixation ability of E. acoroides.

Adaptations to nitrogen availability drive ecological divergence of chemosynthetic symbionts.

Bacterial symbionts, with their shorter generation times and capacity for horizontal gene transfer (HGT), play a critical role in allowing marine organisms to cope with environmental change. The closure of the Isthmus of Panama created distinct environmental conditions in the Tropical Eastern Pacific (TEP) and Caribbean, offering a "natural experiment" for studying how closely related animals evolve and adapt under environmental change. However, the role of bacterial symbionts in this process is often overlooked. We sequenced the genomes of endosymbiotic bacteria in two sets of sister species of chemosymbiotic bivalves from the genera Codakia and Ctena (family Lucinidae) collected on either side of the Isthmus, to investigate how differing environmental conditions have influenced the selection of symbionts and their metabolic capabilities. The lucinid sister species hosted different Candidatus Thiodiazotropha symbionts and only those from the Caribbean had the genetic potential for nitrogen fixation, while those from the TEP did not. Interestingly, this nitrogen-fixing ability did not correspond to symbiont phylogeny, suggesting convergent evolution of nitrogen fixation potential under nutrient-poor conditions. Reconstructing the evolutionary history of the nifHDKT operon by including other lucinid symbiont genomes from around the world further revealed that the last common ancestor (LCA) of Ca. Thiodiazotropha lacked nif genes, and populations in oligotrophic habitats later re-acquired the nif operon through HGT from the Sedimenticola symbiont lineage. Our study suggests that HGT of the nif operon has facilitated niche diversification of the globally distributed Ca. Thiodiazotropha endolucinida species clade. It highlights the importance of nitrogen availability in driving the ecological diversification of chemosynthetic symbiont species and the role that bacterial symbionts may play in the adaptation of marine organisms to changing environmental conditions.

Exploration of nyamplung rizosphere nitrogen fixing bacteria in mine reclamation

Nitrogen fixing bacteria have the ability to utilize free nitrogen as a nitrogen source for their growth. These bacteria are able to make efficient availability of N- in the soil, so that it can be utilized by plants. The aim of this study was to obtain nitrogen fixing bacteria isolates from the rhizosphere of nyamplung plants in limestone reclamation lands. Isolation and testing of nitrogen fixation ability by nitrogen fixing bacteria was carried out using selective Ashby's Mannitol Agar media. The isolation results obtained 5 isolates on Ashby's Mannitol Agar media. The morphological characters of the colonies of the five isolates showed round and irregular shapes, flat and convex elevations with flat edges, white, cloudy white and clear. Microscopic observation showed that the isolates consisted of gram-positive and negative bacteria with bacilli cell shape. The catalase test showed 4 positive isolates and 1 negative. The conclusion of this study was that 5 nitrogen fixing bacteria isolates were obtained from the rhizosphere of the nyamplung limestone land plant. Isolates 1, 3, and 5 showed the character of the genus Clostridium, isolate 2 led to the genus Bacillus, and isolate 4 had the same character as the genus Klabsiella.