Increased dependence on nitrogen-fixation of a native legume in competition with an invasive plant

Suppression of roots and/or their symbiotic microorganisms,such as mycorrhizal fungi and rhizobia,is an effective way for alien plants to outcompete native plants.However,little is known about how invasive and native plants interact with the quantity and activity of nutrient-acquisition agents.Here a pot experiment was conducted with monoculture and mixed plantings of an invasive plant,Xanthium strumarium,and a common native legume,Glycine max.We measured traits related to root and nodule quantity and activity and mycorrhizal colonization.Compared to the monoculture,fine root quantity(biomass,surface area)and activity(root nitrogen(N)concentration,acid phosphatase activity)of G.max decreased in mixed plantings;nodule quantity(biomass)decreased by 45%,while nodule activity in Nfixing via rhizobium increased by 106%;mycorrhizal colonization was unaffected.Contribution of N fixation to leaf N content in G.max increased in the mixed plantings,and this increase was attributed to a decrease in the rhizosphere soil N of G.max in the mixed plantings.Increased root quantity and activity,along with a higher mycorrhizal association was observed in X.strumarium in the mixed compared to monoculture.Together,the invasive plant did not directly scavenge N from nodule-fixed N,but rather depleted the rhizosphere soil N of the legume,thereby stimulating the activity of N-fixation and increasing the dependence of the native legume on this N source.The quantity-activity framework holds promise for future studies on how native legumes respond to alien plant invasions.

Microfluidic-oriented synthesis of enriched iridium nanodots/carbon architecture for robust electrocatalytic nitrogen fixation

Electrocatalytic nitrogen reduction reaction(NRR)is considered as a promising candidate to achieve ammonia synthesis because of clean electric energy,moderate reaction condition,safe operating process and harmless by-products.However,the chemical inertness of nitrogen and poor activated capacity on catalyst surface usually produce low ammonia yield and faradic efficiency.Herein,the microfluidic technology is proposed to efficiently fabricate enriched iridium nanodots/carbon architecture.Owing to in-situ co-precipitation reaction and microfluidic manipulation,the iridium nanodots/carbon nanomaterials possess small average size,uniform dispersion,high conductivity and abundant active sites,producing good proton activation and rapid electrons transmission and moderate adsorption/desorption capacity.As a result,the as-prepared iridium nanodots/carbon nanomaterials realize large ammonia yield of 28.73 μg h^(-1) cm^(-2) and faradic efficiency of 9.14%in KOH solution.Moreover,the high ammonia yield of 11.21 μg h^(-1) cm^(-2) and faradic efficiency of 24.30%are also achieved in H_(2)SO_(4) solution.The microfluidic method provides a reference for large-scale fabrication of nano-sized catalyst materials,which may accelerate the progress of electrocatalytic NRR in industrialization field.

Sustainable nitrogen fixation by bubble discharge plasma:Performance optimization and mechanism

Sustainable nitrogen fixation driven by renewable energy sources under mild conditions has been widely sought to replace the industrial Haber-Bosch process.The fixation of nitrogen in the form of NO_(x)^(-)and NH_4^(+)into aqueous solutions using electricity-driven gas-liquid discharge plasma is considered a promising prescription.In this paper,a scalable bubble discharge excited by nanosecond pulse power is employed for nitrogen fixation in the liquid phase.The nitrogen fixation performance and the mechanisms are analyzed by varying the power supply parameters,working gas flow rate and composition.The results show that an increase in voltage and frequency can result in an enhanced NO_(3)^(-)yield.Increases in the gas flow rate can result in inadequate activation of the working gas,which together with more inefficient mass transfer efficiencies can reduce the yield.The addition of O_(2) effectively elevates NO_(3)^(-)production while simultaneously inhibiting NH_4^(+) production.The addition of H_(2)O vapor increases the production of OH and H,thereby promoting the generation of reactive nitrogen and enhancing the yield of nitrogen fixation.However,the excessive addition of O_(2) and H_(2)O vapor results in negative effect on the yield of nitrogen fixation,due to the significant weakening of the discharge intensity.The optimal nitrogen fixation yield was up to 16.5 μmol/min,while the optimal energy consumption was approximately 21.3 MJ/mol in this study.Finally,the mechanism related to nitrogen fixation is discussed through the optical emission spectral(OES) information in conjunction with the simulation of energy loss paths in the plasma by BOLSIG+.The work advances knowledge of the effect of parameter variations on nitrogen fixation by gas-liquid discharge for higher yield and energy production.

Plasma nitrogen fixation system with dual-loop enhancement for improved energy efficiency and its efficacy for lettuce cultivation

Plasma nitrogen fixation(PNF)has been emerging as a promising technology for greenhouse gasfree and renewable energy-based agriculture.Yet,most PNF studies seldom address practical application-specific issues.In this work,we present the development of a compact and automatic PNF system for on-site agricultural applications.The system utilized a gliding-arc discharge as the plasma source and employed a dual-loop design to generate NO_(x)from air and water under atmospheric conditions.Experimental results showed that the system with a dualloop design performs well in terms of energy costs and production rates.Optimal operational parameters for the system were determined through experimentation,resulting in an energy cost of 13.9 MJ mol^(-1)and an energy efficiency of 16 g kWh^(-1)for NO_(3)^(-)production,respectively.Moreover,the concentration of exhausted NO_(x)was below the emission standards.Soilless lettuce cultivation experiments demonstrated that NO_(x)^(-)produced by the PNF system could serve as liquid nitrate nitrogen fertilizer.Overall,our work demonstrates the potential of the developed PNF system for on-site application in the production of green-leaf vegetables.

Assessment of Nitrogen Fixation, Uptake, and Leaching in Maize/Soybean Intercropping System at Varied Soil Depths and under Phosphorus Application in Chinese Agricultural Settings

The study of Nitrogen fixation, uptake, and leaching at different soil depths in the co-cultivation of maize and soybean under phosphorus fertilization is important for sustainable agriculture. This study was conducted in Quzhou, Hebei Province, China, with MC812 maize and Jidou12 soybean varieties. Soil samples were taken from each plot to create a composite sample. The results show that nitrogen concentration varies at different depths and is higher in all treatments between 40 and 100 cm. Incorporating intercropping of maize and soybeans into farming practices can lead to more sustainable and environmentally friendly agriculture in China.

Estimation of Biological Nitrogen Fixation Capacity by Sugarcane Using 15N

[ Objective] The study aimed to reveal the biological nitrogen fixation capacity by sugarcane from Brazil under the ecological conditions of Guangxi, and to provide reference for study on the biological nitrogen fixation capacity by sugarcane and related generalization and application. [ Method] The ^15N isotopic fertilizer was solely applied on plants of three sugarcane cultivars planted in greenhouse with no other fertilizer forms applied, meanwhile virus-free stem seedling was regarded as control, to measure their biological nitrogen fixation capacity using ^15N isotope. [ Result ] The nitrogen fixation rate of B8 from Brazil reached 26.91%, while Guitang 11 and RIC16 presented no or poor nitrogen fixation capacity. [ Conclusion] The sugarcane eultivar B8 from Brazil showed some nitrogen fixation capacity under the ecological conditions of Guangxi.

Optimized nitrogen application methods to improve nitrogen use efficiency and nodule nitrogen fixation in a maize-soybean relay intercropping system

In China, the abuse of chemical nitrogen （N） fertilizer results in decreasing N use efficiency （NUE）, wasting resources and causing serious environmental problems. Cereal-legume intercropping is widely used to enhance crop yield and improve resource use efficiency, especially in Southwest China. To optimize N utilization and increase grain yield, we conducted a two-year field experiment with single-factor randomized block designs of a maize-soybean intercropping system （IMS）. Three N rates, NN （no nitrogen application）, LN （lower N application： 270 kg N ha-1）, and CN （conventional N application： 330 kg N ha-1）, and three topdressing distances of LN （LND）, e.g., 15 cm （LND1）, 30 cm （LND2） and 45 cm （LND3） from maize rows were evaluated. At the beginning seed stage （R5）, the leghemoglobin content and nitrogenase activity of LND3 were 1.86 mg plant-1 and 0.14 mL h-1 plant-1, and those of LND1 and LND2 were increased by 31.4 and 24.5%, 6.4 and 32.9% compared with LND3, respectively. The ureide content and N accumulation of soybean organs in LND1 and LND2 were higher than those of LND3. The N uptake, NUE and N agronomy efficiency （NAE） of IMS under CN were 308.3 kg ha-1, 28.5%, and 5.7 kg grain kg-1 N, respectively; however, those of LN were significantly increased by 12.4, 72.5, and 51.6% compared with CN, respectively. The total yield in LND1 and LND2 was increased by 12.3 and 8.3% compared with CN, respectively. Those results suggested that LN with distances of 15-30 cm from the topdressing strip to the maize row was optimal in maize-soybean intercropping. Lower N input with an optimized fertilization location for IMS increased N fixation and N use efficiency without decreasing grain yield.

Effect of nitrogen fertilization on yield, N content, and nitrogen fixation of alfalfa and smooth bromegrass grown alone or in mixture in greenhouse pots

Planting grass and legume mixtures on improved grasslands has the potential advantage of realizing both higher yields and lower environmental pollution by optimizing the balance between applied N fertilizer and the natural process of legume biological nitrogen fixation. However, the optimal level of N fertilization for grass-legume mixtures, to obtain the highest yield, quality, and contribution of N2 fixation, varies with species. A greenhouse pot experiment was conducted to study the temporal dynamics of N2 fixation of alfalfa （Medicago sativa L.） grown alone and in mixture with smooth bromegrass （Bromus inermis Leyss.） in response to the addition of fertilizer N. Three levels of N （0, 75, and 150 kg ha-1） were examined using 15N-labeled urea to evaluate N2 fixation via the 15N isotope dilution method. Treatments were designated NO （0.001 g per pot）, N75 （1.07 g per pot） and N150 （2.14 g per pot）. Alfalfa grown alone did not benefit from the addition of fertilizer N; dry matter was not significantly increased. In contrast, dry weight and N content of smooth bromegrass grown alone was increased significantly by N application. When grown as a mixture, smooth bromegrass biomass was increased significantly by N application, resulted in a decrease in alfalfa biomass. In addition, individual alfalfa plant dry weight （shoots＋roots） was significantly lower in the mixture than when grown alone at all N levels. Smooth bromegrass shoot and root dry weight were significantly higher when grown with alfalfa than when grown alone, regardless of N application level. When grown alone, alfalfa＇s N2 fixation was reduced with N fertilization （R2=0.9376,P=0.0057）. When grown in a mixture with smooth bromegrass, with 75 kg ha-1 of N fertilizer, the percentage of atmospheric N2 fixation contribution to total N in alfalfa （%Ndfa） had a maximum of 84.07 and 83.05% in the 2nd and 3rd harvests, respectively. Total 3-harvest %Ndfa was higher when alfalfa was grown in a mixture than when grown alone （shoots： ｜t｜=3.39, P=0.0096; root： ｜t｜=3.57, P=0.0073）. We believe this was due to smooth bromegrass being better able to absorb available soil N （due to its fibrous root system）, resulting inlower soil N availability and allowing alfalfa to develop an effective N2 fixing symbiosis prior to the 1st harvest. Once soil N levels were depleted, alfalfa was able to fix N2, resulting in the majority of its tissue N being derived from biological nitrogen fixation （BNF） in the 2nd and 3rd harvests. When grown in a mixture, with added N, alfalfa established an effective symbiosis earlier than when grown alone; in monoculture BNF did not contribute a significant portion of plant N in the N75 and N150 treatments, whereas in the mixture, BNF contributed 17.90 and 16.28% for these treatments respectively. Alfalfa has a higher BNF efficiency when grown in a mixture, initiating BNF earlier, and having higher N2 fixation due to less inhibition by soil-available N. For the greatest N-use-efficiency and sustainable production, grass-legume mixtures are recommended for imDrovino orasslands, usino a moderate amount of N fertilizer （75 kq N ha-l） to provide optimum benefits.

Dependence of Wheat and Rice Respiration on Tissue Nitrogen and the Corresponding Net Carbon Fixation Efficiency Under Different Rates of Nitrogen Application

To quantitatively address the role of tissue N in crop respiration under various agricultural practices, and to consequently evaluate the impact of synthetic fertilizer N application on biomass production and respiration, and hence net carbon fixation efficiency （Encf）, pot and field experiments were carried out for an annual rotation of a rice-wheat cropping system from 2001 to 2003. The treatments of the pot experiments included fertilizer N application, sowing date and planting density. Different rates of N application were tested in the field experiments. Static opaque chambers were used for sampling the gas. The respiration as CO2 emission was detected by a gas chromatograph. A successive biomass clipping method was employed to determine the crop autotrophic respiration coefficient （Ra）. Results from the pot experiments revealed a linear relationship between Ra and tissue N content as Ra = 4.74N-1.45 （R^2= 0.85, P 〈 0.001）. Measurements and calculations from the field experiments indicated that fertilizer N application promoted not only biomass production but also increased the respiration of crops. A further investigation showed that the increase of carbon loss in terms of respiration owing to fertilizer N application exceeded that of net carbon gain in terms of aboveground biomass when fertilizer N was applied over a certain rate. Consequently, the Encf declined as the N application rate increased.

Photocatalytic nitrogen fixation: An attractive approach for artificial photocatalysis

Ammonia synthesis via the Haber-Bosch process, which has been heralded as the most important invention of the 20 th century, consumes massive amounts of energy, around 1%–2% of the world’s annual energy consumption. Developing green and sustainable strategies for NH3 synthesis under ambient conditions, using renewable energy, is strongly desired, by both industrial and sci-entific researchers. Artificial photosynthesis for ammonia synthesis, which has recently attracted significant attention, directly produces NH3 from sunlight, and N2 and H2O via photocatalysis. This has been regarded as an ideal, energy-saving and environmentally-benign process for NH3 produc-tion because it can be performed under normal temperature and atmospheric pressure using re-newable solar energy. Although sustainable developments have been achieved since the pioneering work in 1977, many challenging issues（e.g., adsorption and activation of nitrogen molecules on the surface of photocatalysts under mild conditions） have still not been well solved and the photocata-lytic activities are generally low. In this miniature review, I summarize the most recent progress of photocatalytic N2 fixation for ammonia synthesis, focusing specifically on two attractive aspects for adsorption and activation of nitrogen molecules： one is engineering of oxygen vacancies, and the other is mimicking natural nitrogenase for constructing artificial systems for N2 fixation. Several representative works focusing on these aspects in artificial systems have been reported recently, and it has been demonstrated that both factors play more significant roles in photocatalytic N2 re-duction and fixation under ambient conditions. At the end of the review, I also give some remarks and perspective on the existing challenges and future directions in this field.

Nitrogen acquisition,fixation and transfer in maize/alfalfa intercrops are increased through root contact and morphological responses to interspecies competition

Nitrogen(N)fixation by legumes and nitrogen transfer to cereals have been considered as important pathways for overyielding and higher N use efficiency in cereal/legume intercropping systems.However,the extent to which root morphology contributes to N fixation and transfer is unclear.A two-factorial greenhouse experiment was conducted to quantify the N fixation,transfer and root morphology characteristics of the maize/alfalfa intercropping system in two consecutive years using the 15N-urea leaf labeling method,and combining two N levels with three root separation techniques.N application could inhibit N fixation and transfer in a maize/alfalfa intercropping system.Irrespective of the N application level,compared with plastic sheet separation(PSS),no separation(NS)and nylon mesh separation(NNS)significantly increased the total biomass(36%)and total N content(28%),while the N fixation rate also sharply increased by 75 to 134%,and the amount of N transferred with no root barrier was 1.24–1.42 times greater than that with a mesh barrier.Redundancy analysis(RDA)showed that the crown root dry weight(CRDW)of maize and lateral root number(LRN)of alfalfa showed the strongest associations with N fixation and transfer.Our results highlight the importance of root contact for the enhancement of N fixation and transfer via changes in root morphology in maize/alfalfa intercropping systems,and the overyielding system was achieved via increases in maize growth,at the cost of smaller decreases in alfalfa biomass production.

SWAT Modeling of Nitrogen Dynamics Considering Atmospheric Deposition and Nitrogen Fixation in a Watershed Scale

The Soil and Water Assessment Tool (SWAT) nitrogen (N) water quality model considers the artificial inputs associated with human activities, including point and nonpoint source pollution loads. Although SWAT has the ability to simulate atmospheric N deposition and fixation, they were not considered in the modeling research. N deposition from the air is an important and considerable pathway for the input of N species into watersheds and water bodies, causing soil and water body acidification and the leaching of N into surface and groundwater, resulting in eutrophication and degraded water quality. The goal of this study is to assess the effects of atmospheric and agricultural N loads on stream water quality at the watershed scale. For a 6642 km2 Chungju dam watershed, SWAT was calibrated for 4 years (2003-2006) and validated for another 4 years (2007-2010) using daily anthropogenic N data (sewage discharge pollutants and fertilizer) and monthly measured atmospheric deposition data for NO3ˉ, NH4+, and dissolved organic N (DON). At the watershed outlet, the Nash-Sutcliffe (1970) efficiency (NSE) of daily streamflow during the validation period was 0.74. The coefficient of determination (R2) of total N was 0.69 considering atmospheric deposition, whereas it was 0.33 when removing the deposition effect. The results of this study demonstrate the potential for using the N dynamics between the atmosphere and land for SWAT assessments of nonpoint source pollution and for modeling stream water quality.

Influences of frequency on nitrogen fixation of dielectric barrier discharge in air

The influences of frequency on nitrogen fixation of dielectric barrier discharge in air were studied by electrical diagnostics, gas detection and infrared detection methods. The system power, nitrogen oxide concentration, voltage-current waveform, dielectric surface temperature distribution and filamentous discharge pictures were measured, and then the energy yield was calculated; paper studied their changing tendencies in the presence of frequency. Results show that frequency has strong influences on nitrogen fixation. When the parameters of reaction chamber and amplitude of applied voltage is fixed, with the increasing of frequency, the system power increases; in 5-10 kHz, nitrogen oxide gas concentration up to 1113.7 mg m-3, and 7 kHz is the optimal nitrogen fixation frequency whose energy yield is 20.5 mR （m3 W）-1.

Comparative genome analysis on intraspecific evolution and nitrogen fixation of Leptospirillum ferriphilum isolates

To reveal the intraspecific evolution of Leptospirillum ferriphilum isolates which thrived in industrial bioleaching ecosystems and acid mine drainages,genome sequences of L.ferriphilum YSK,L.ferriphilum DX and L.ferriphilum ZJ were determined to compare with complete genome of L.ferriphilum ML-04.The genome comparisons reveal that extensive intraspecific variation occurs in their genomes,and that the loss and insertion of novel gene blocks of probable phage origin may mostly contribute to heterogeneity of gene content among L.ferriphilum genomes.Surprisingly,a nif gene cluster is identified in L.ferriphilum YSK and L.ferriphilum ZJ genomes.Intensive analysis and further experiments indicate that the nif gene cluster in L.ferriphilum YSK inherits from ancestor rather than lateral gene transfer.Overall,results suggest that the population of L.ferriphilum undergoes frequent genetic recombination,resulting in many closely related genome types in recent evolution.The combinatorial processes profoundly shape their physiologies and provide the basis for adaptation to different niches.

Potential Breeding for High Nitrogen Fixation in <i>Pisum sativum</i>L.: Germplasm Phenotypic Characterization and Genetic Investigation

Nitrogen (N) is the most yield-limiting crop nutrient worldwide. Industrially produced N has increased in cost over the past years, and is unavailable in many regions around the globe. Biological N fixation by rhizobial bacteria is a great underutilized resource that this project aims to maximize. Grain legumes fix approximately 20 to 100 kg·N·ha-1·yr-1. The amount of N supplied by fixation is affected by genes and traits of both the bacterial and plant partners. The objectives of this study are to identify Pisum sativum varieties with high nitrogen fixation efficiency. This is achieved by germplasm screening and phenotypic evaluation of nodule formation, total plant nitrogen, and residual nitrogen in soil. Significant differences in plant total nitrogen among the various cultivated genotypes were found, with heritability of 0.57. These pea varieties left in the soil a residual N that varies between 11.21 to 65.018 kg.N.ha-1. Our findings reveal a unique opportunity for improving N fixation through genetic crossing and selection.

Construction of 2D/2D Z-scheme MnO_(2-x)/g-C_(3)N_(4) photocatalyst for efficient nitrogen fixation to ammonia

Reducing nitrogen to ammonia with solar energy has become a wide concern when it comes to photocatalysis research.It is considered to be one of the more promising alternate options for the conventional Haber-Bosch cycle.Herein,2D g-C_(3)N_(4)composites with modifying ultrathin sheet MnO_(2-x)were prepared and used as nitrogen fixation photocatalyst.With the assistance of the nature of MnO_(2-x),the generation rate of NH_(3)reached 225 mmol g^(-1)h^(-1),which is more than twice over the rate of pristine 2D g-C_(3)N_(4)(107 mmol g^(-1)h^(-1)).The presence of ultrathin sheet MnO_(2-x)shortens the gap of the carriers to the surface of photocatalyst.Thus the speed of electron transfer gets increased.Besides,the construction of Z-scheme heterojunction boosts the separation and migration of photogenerated carriers.As a result,the nitrogen reduction reaction(NRR)performance gets enhanced.The work may provide an example of promoting the NRR performance of non-metallic compound.

Nitrogen Fixation into HNO_3 and HNO_2 by Pulsed High Voltage Discharge

Plasma processing induced by discharge offers a unique way to activate nitrogen molecules. Direct nitrogen fixation into water can be realized through this approach. In this study, air or pure nitrogen gas was used as the major nitrogen source bubbled into the discharge reactor. When a discharge occurred, nitrogen was dissociated to active species to take part in the aqueous chemical process. HNO3 and HNO2 were produced. The nitrogen fixation process was influenced distinctly by the presence of hydroxyl radicals. During a discharge of 21 min, HNO3 was the main product and occupied 95% of the total nitrogen content in water. Its concentration was 1.36 × 10^-3 mol/L^-1 with bubbling air and was 1.53 × 10^-3 mol L^-1 with bubbling nitrogen, while the yield was 2.32 × 10^-3 mol J^-1S^-1 and 2.06 × 10^-8 mol J^-1S^-1, respectively.

Preliminary study of an open-air water-contacting discharge for direct nitrogen fixation

Efficient nitrogen fixation through a reactive plasma process attracts intense interest due to the environmental issues induced by the conventional Haber–Bosch method. In this work, we present a direct and simple fixation routine without any catalysts for nitrogen in open air using an atmospheric-pressure pin-to-solution plasma electrolytic system. Nitrate, nitrite, and ammonia as the nitrogen-derived chemicals in solution were analyzed as indicators under various discharge conditions to estimate the energy efficiency of this process. The results show that the nitrogen fixation process was much more efficient by the pin-positive discharge compared to the negative one. N chemicals preferred to be formed when the solution was of negative polarity. It was also found that, with the help of solution circulation, the energy efficiency was enhanced compared to that of static liquid. However, an inverse trend was observed with the increase of the discharge current. Further study by optical emission spectroscopy indicates the important roles of active N2* and water vapour and their derived species near the plasma–water interface in the fixation process.

Oxygen defect modulating the charge behavior in titanium dioxide for boosting photocatalytic nitrogen fixation performance

Extremely high-temperature and high-pressure requirement of Haber-Bosch process motivates the search for a sustainable ammonia synthesis approach under mild conditions.Photocatalytic technology is a potential solution to convert N2 to ammonia.However,the poor light absorption and low charge carrier separation efficiency in conventional semiconductors are bottlenecks for the application of this technology.Herein,a facile synthesis of anatase TiO_(2)nanosheets with an abundance of surface oxygen vacancies(TiO_(2)-OV)via the calcination treatment was reported.Photocatalytic experiments of the prepared anatase TiO_(2)samples showed that TiO_(2)-OV nanosheets exhibited remarkably increased ammonia yield for solar-driven N2 fixation in pure water,without adding any sacrificial agents.EPR,XPS,XRD,UV-Vis DRS,TEM,Raman,and PL techniques were employed to systematically explore the possible enhanced mechanism.Studies revealed that the introduced surface oxygen vacancies significantly extended the light absorption capability in the visible region,decreased the adsorption and activation barriers of inert N2,and improved the separation and transfer efficiency of the photogenerated electronhole pairs.Thus,a high rate of ammonia evolution in TiO_(2)-OV was realized.This work offers a promising and sustainable approach for the efficient artificial photosynthesis of ammonia.

Low-Density Co-Inoculation with Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 Promotes Plant Growth and Nitrogen Fixation in Soybean Cultivars

Inoculation density has a marked effect on nodulation and N fixation in soybean (Glycine max L.). Therefore, we conducted this study to determine the optimal inoculation density of Bradyhizobium japonicum SAY3-7 (SAY3-7) and Streptomyces griseoflavus P4 (P4) for plant growth, nodulation, and N fixation, and to investigate the effect of co-inoculation on selected soybean cultivars, using the optimal inoculation density. Nitrogen fixation, in terms of an acetylene reduction activity value, was measured using a flame ionization gas chromatograph. In this study, low-density single inoculation with P4 (105 or 106 cells mL-1) was associated with the highest plant biomass, compared with normal- and high-density single inoculation with P4 (107 or 108 cells mL-1). Moreover, low-density single or co-inoculations with SAY3-7 and/or P4 produced the highest nodule biomass and highest nitrogenase activity, compared with single or dual inoculation at other inoculation densities. Therefore, we evaluated low-density co-inoculation with P4 and SAY3-7, at the rate of 105 cells mL-1, on selected soybean cultivars. Low-density co-inoculation increased the plant biomass, compared with un-inoculated plants. The effects of single and co-inoculation on nodulation did not differ significantly for any of the cultivars, except “Yezin-9” in the first experiment and “Shan Seine” in the second experiment. Low-density inoculation with both bacteria increased N fixation by 15% - 75% for seven of the cultivars in the first experiment and by 15% - 39% for three of the cultivars in the second experiment, compared with single inoculation with SAY3-7. Based on the overall results, we concluded that low-density co-inoculation with P4 and SAY3-7 gave improved plant growth and N fixation.