Free-living N2-fixing Bacteria Research Articles

EFFECT OF MAGNETITE AND CHITOSAN-MAGNETITE ADDITION ON BACTERIAL LEVELS AND NH3-N (FREE-LIVING N2-FIXING BACTERIA CULTURE)

Free-living N2-fixing bacteria are essential in the soil because they provide a source of nitrogen in the form of ammonium needed by plants to be used in building protein blocks. The fixation of free N2 needs to be increased by adding nanomaterials such as magnetite (Fe3O4), which has an active group and can be a source of nitrogenase enzyme cofactor. In this study, increased N2 fixation used N2-fixing bacteria in Burks medium. This increase was determined by measuring NH3 levels using the Phenate method formed after Burks medium was treated with Fe3O4 and chitosan-magnetite (CS-Fe3O4). The study found that the number of bacteria can be well decreased. This research showed that using Fe3O4 and CS-Fe3O4 increased NH3-N levels in cultures of free-living nitrogen-fixing bacteria by 15.40% and 75.54%. For future development, it can be in the form of optimization, the effect of adding the same material to plant secondary metabolites, and the mechanism of bacteria in using the material.

Analysis of Bacterial Microbiota of Aerated Compost Teas and Effect on Tomato Growth.

Mature composts and their water-based extracts, known as aerated compost teas (ACTs), are biofertilizers that share bioactive effects like soil restoration and plant health promotion, widely used for sustainable agriculture. Bioactive effects of compost and ACTs could be associated with their physicochemical and biological characteristics, like carbon/nitrogen (C/N) ratio and microbiota structure respectively. In our study, we elaborated ACTs using mature homemade compost, wheat bran, and grass clippings, following the C/N ratio criteria. Irrigation of tomato plantlets with ACT whose C/N ratio was close to the expected C/N ratio for mature compost evidenced plant growth promotion. Exploring the bacterial microbiota of elaborated ACTs and origin compost revealed significant structural differences, including phyla involved in N mineralization and free-living N-fixing bacteria. Therefore, ACTs harbor diverse bacterial microbiota involved in the N cycle, which would enrich plant and soil bacterial communities at the taxonomic and functional levels. Furthermore, ACTs are considered a part of agroecological and circular economy approaches.

Molecular Characterization of Free Living N2 Fixing Bacteria Isolated from Agricultural Soils of North Gujarat, India

Background: Molecular identification of a wide range of organisms capable of carrying out biological nitrogen fixation (BNF) are diverse in nature and significantly improves plant growth. Biological N2 fixation reflects the activity of a phylogenetically diverse list of microorganisms. Molecular characterization provides efficient means to identify organisms with the potential of N2 fixation. Applying these techniques in an array of environments has considerably broadened our understanding of the suite of organisms that can carry out BNF. Methods: Thirty-four strains of free living N2 fixing bacterial strains were isolated from diverse plants cultivated in North Gujarat, including wheat, cotton, castor and pearl millet, using a nitrogen-free selective medium. Acetylene reduction assay was used to check the ability of all bacteria to fix nitrogen. Hybridization with nifH probe derived from Azotobacter vinelandii with isolated free-living nitrogen-fixing bacteria showed a positive result. The selected strains were characterized by molecular analysis like; ARDRA and 16S rDNA sequencing. Result: Based on molecular characterization 17 strains to known groups of nitrogen-fixing bacteria, including organisms from the genus Azotobacter, Pseudomonas, Enterobacter, Arthrobacter, Bacillus, Variovorax, Nocardiodies, Rhodococcus, Mycobacterium, Planococcus, Microbacterium have been identified. One of the strains was identified as unknown bacteria. The potential strains were identified by 16srDNA analysis and also corroborated by morphological and biochemical characterization.

Synergism between feremycorrhizal symbiosis and free-living diazotrophs leads to improved growth and nutrition of wheat under nitrogen deficiency conditions

A controlled-environment study was conducted to explore possible synergistic interactions between the feremycorrhizal (FM) fungus Austroboletus occidentalis and soil free-living N2-fixing bacteria (diazotrophs). Wheat (Triticum aestivum) plants were grown under N deficiency conditions in a field soil without adding microbial inoculum (control: only containing soil indigenous microbes), or inoculated with a consortium containing four free-living diazotroph isolates (diazotrophs treatment), A. occidentalis inoculum (FM treatment), or both diazotrophs and A. occidentalis inoculums (dual treatment). After 7 weeks of growth, significantly greater shoot biomass was observed in plants inoculated with diazotrophs (by 25%), A. occidentalis (by 101%), and combined inoculums (by 106%), compared to the non-inoculated control treatment. All inoculated plants also had higher shoot nutrient contents (including N, P, K, Mg, Zn, Cu, and Mn) than the control treatment. Compared to the control and diazotrophs treatments, significantly greater shoot N content was observed in the FM treatment (i.e., synergism between the FM fungus and soil indigenous diazotrophs). Dually inoculated plants had the highest content of nutrients in shoots (e.g., N, P, K, S, Mg, Zn, Cu, and Mn) and soil total N (13–24% higher than the other treatments), i.e., synergism between the FM fungus and added diazotrophs. Root colonization by soil indigenous arbuscular mycorrhizal fungi declined in all inoculated plants compared to control. Non-metric multidimensional scaling (NMDS) analysis of the bacterial 16S rRNA gene amplicons revealed that the FM fungus modified the soil microbiome. Our in vitro study indicated that A. occidentalis could not grow on substrates containing lignocellulosic materials or sucrose, but grew on media supplemented with hexoses such as glucose and fructose, indicating that the FM fungus has limited saprotrophic capacity similar to ectomycorrhizal fungi. The results revealed synergistic interactions between A. occidentalis and soil free-living diazotrophs, indicating a potential to boost microbial N2 fixation for non-legume crops.

Effect of long-term nutrient management practices on soil health and paddy yield of rice-rice-fallow cropping system in tropic humid climate

Long-term repeated nutrient management practices may have impact on soil health and sustainable paddy (Oryza sativa) production. Therefore, a long-term (33 years) field experiment was conducted at Bangladesh Rice Research Institute in Gazipur with the objective of finding the impact of repeated inorganic and integrated nutrient management (INM) practices on soil biochemical properties, soil quality and its cumulative effect on paddy yield. Experiment was permanently laid out in a replicated complete block design with three replications and modern paddy varieties, BRRI dhan49 and BRRI dhan29, were grown in wet and dry seasons respectively. Treatments imposed were; balanced chemical fertilizer (BCF), K minus from BCF, P minus from BCF, N minus from BCF, Chemical fertilizer with 3 t ha−1 decomposed cowdung (CD), Chemical fertilizer with 2 t ha−1 decomposed poultry litter (PL) and Fertilizer control. The SOC decreased 8% due to 33 years BCF compared to initial soil and increased 18% in INM compared to BCF practice during last 10 years. Long term BCF practices did not bring significant changes in soil pH, however 0.09 units increased and 1.45% bulk density decreased in PL-INM practice. Urease and phosphatase activities, total bacteria, phosphate solubilizing bacteria, free-living N2 fixing bacteria, water stable aggregates, NH4+-N, available P, exchangeable K were increased in INM practices and between two INM treatments, PL was superior over CD. The highest SQI (0.93) and 10 years’ average paddy yield per season (4.99 t ha−1) was obtained in PL-INM followed by CD-INM (SQI, 0.80 and grain yield 4.12 t ha−1) and the lowest in the N minus from BCF practice (SQI, 0.16 and paddy yield 3.2 t ha−1) and control (SQI, 0.33 and paddy yield 2.9 t ha−1) treatment. SQI due BCF practice was 0.68 and average paddy yield was 4.36 t ha−1. The results of AreaSQI revealed that NH4–N, available P and enzyme activities are the most sensitive soil parameters that degraded due to different nutrient management practices. The study also proved that imbalance fertilizer application, especially removal of N nutrition, was the most detrimental for paddy soil quality and PL-INM treatment is the best nutrient management practice for maintenance of soil health in the hyperthermic vertic endoaquepts and tropical humid climate.

Altering Nitrogen Sources Affects Growth Carbon Costs in Vachellia nilotica Growing in Nutrient-Deficient Grassland Soils.

Vachellianilotica (L.) Willd. Ex Del. is a multipurpose leguminous tree that is common in grassland and savanna ecosystems in southern and eastern Africa. These ecosystem soils are reported to be acidic and nutrient-limited, specifically with regards to nitrogen (N) and phosphorus (P). The presence of this plant in these terrestrial ecosystems improves soil fertility benefiting the surrounding vegetation due to its ability to fix atmospheric N. This study seeks to understand the N-fixing bacteria symbiosis and physiological adaptations of V. nilotica in these acidic and nutrient-deficient KwaZulu-Natal soils. The soils used for this study were collected from the Ukulinga Grassland Nutrient Experiment located at the Ukulinga research farm of the University of KwaZulu-Natal, Pietermaritzburg, South Africa. Due to long-term soil nutrient addition treatments, these soils offered a diverse nutrient variation for better understanding the effects of acidity and nutrient variation on microbial symbiosis, plant nutrition, and biomass accumulation of V. nilotica. V. nilotica was able to maintain growth by relying on both atmospheric and soil-derived N across all treatments decreasing carbon (C) growth costs. There was an increased reliance on atmospheric-derived N of un-nodulated high N-treated plants. The plants grown in high N + P soils were able to nodulate with various species from the Mesorhizobium genus, which resulted in increased biomass compared to other plants. The results of this study show that V. nilotica can alter N sources to reduce C growth costs. In addition, both nodulating and free-living soil N2 fixing bacteria such as Caulobacter rhizosphaerae, Sphingomonas sp. and Burkholderia contaminans identified in the experimental soils may play an important role under P-deficient conditions.

Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability.

Biological nitrogen fixation (BNF) refers to a microbial mediated process based upon an enzymatic “Nitrogenase” conversion of atmospheric nitrogen (N2) into ammonium readily absorbable by roots. N2-fixing microorganisms collectively termed as “diazotrophs” are able to fix biologically N2 in association with plant roots. Specifically, the symbiotic rhizobacteria induce structural and physiological modifications of bacterial cells and plant roots into specialized structures called nodules. Other N2-fixing bacteria are free-living fixers that are highly diverse and globally widespread in cropland. They represent key natural source of nitrogen (N) in natural and agricultural ecosystems lacking symbiotic N fixation (SNF). In this review, the importance of Azotobacter species was highlighted as both important free-living N2-fixing bacteria and potential bacterial biofertilizer with proven efficacy for plant nutrition and biological soil fertility. In addition, we described Azotobacter beneficial plant promoting traits (e.g., nutrient use efficiency, protection against phytopathogens, phytohormone biosynthesis, etc.). We shed light also on the agronomic features of Azotobacter that are likely an effective component of integrated plant nutrition strategy, which contributes positively to sustainable agricultural production. We pointed out Azotobacter based-biofertilizers, which possess unique characteristics such as cyst formation conferring resistance to environmental stresses. Such beneficial traits can be explored profoundly for the utmost aim to research and develop specific formulations based on inoculant Azotobacter cysts. Furthermore, Azotobacter species still need to be wisely exploited in order to address specific agricultural challenges (e.g., nutrient deficiencies, biotic and abiotic constraints) taking into consideration several variables including their biological functions, synergies and multi-trophic interactions, and biogeography and abundance distribution.

Use of biochar with immobilized free-living N-fixers as mineral fertilizer

It is known that biochar application as soil fertilizer lead to reduce N loss. At the same time because of its porosity biochar is high quality substrate for immobilization microorganisms. The practice of using free-living N fixing bacteria (FLNFB) in organic farming is widely used to increase the nitrogen content in the soil. In this study immobilisation FLNFB on two types of biochar (made of chicken manure and sewage sludge) was applied to increase soil quality and crop productivity. The effectiveness of immobilization has been proven using SEM method: biofilms of FLNFB on the porous surface of both types of biochars are visible in the SEM images. The effectiveness of application 1% by mass biochar with FLNFB was estimated during 30 days vegetation experiment in greenhouse conditions by estimating the chemical composition of the soil (pH, CEC, OM, Corg, Ntot), by estimating change the functional activity of the soil microbial community (soil respiration activity, leucine aminopeptidase enzyme activity), by estimating change the structure of the microbial community (qPCR were used to analyze nifH and 16S rRNA genes in order to study the total count of bacterial and fungi), by estimating change the plants characteristics (chlorophyll content, germination, root and shoot length, biomass). It was found that application biochar with FLNFB lead to higher content of Ntot (0.11-0.19%) in soil in the end of the experiment as comparing than that for soil samples with mineral fertilizer ( 0.09%) and control soil (0.08%). The application biochar with FLNFB lead to significantly increase nifH-genes in soil. The soil leucine aminopeptidase and respiration activity have a similar trend for samples with and without biochar. The use of biochar as fertilizer led to increase chlorophyll content (13-22%) and barley biomass (10-84%) and decrease the length of the root (3-33%) in comparison with the control soil.

Rare microbial taxa rather than phoD gene abundance determine hotspots of alkaline phosphomonoesterase activity in the karst rhizosphere soil

The spatial distribution of alkaline phosphomonoesterase (ALP) activity in the rhizosphere and bulk soil of three plants (Zea mays, Medicago sativa, and Cyperus rotundus) grown in a karst soil (pH 8.27) was visualized using in situ soil zymography. According to the zymogram images, we identified and precisely collected soil samples from hotspots and non-hotspots of ALP activity, and then analyzed the phoD genes that encoded alkaline phosphomonoesterase and assessed the microbial community. The results showed that (1) the phoD abundance in the plant types varied and was highest in the alfalfa and lowest in the maize; (2) Proteobacteria dominated the phoD-harboring microbes in the rhizosphere and Actinobacteria dominated the phoD-harboring microbes in the bulk soil, and (3) the ALP activity was positively correlated with the relative abundance of the diazotrophic Azotobacter, but negatively correlated with the phoD gene abundance, microbial community richness, and diversity. By coupling zymography and microbial molecular approaches, we identified hotspots of enzymatic and microbial activity in rhizosphere soil and evaluated the relative contributions from potential active microorganisms. We found that the function of specific phoD-harboring microorganisms in these hotspots differed depending on the plant in the soil, which had implications for phosphorus (P) management in P-limited karst soils. The results also suggested that free-living N2-fixing bacteria (Azotobacter) might promote ALP activity, thereby emphasizing vital linkages between, and coupling of, the soil nutrient cycles.

Enrichment of Nitrogen-Fixing Bacteria in a Nitrogen-Deficient Wastewater Treatment System.

Anthropogenic nitrogen fixation is essential to sustain a global population of 7.7 billion. However, there has been a long-standing desire to find cheaper and more environmentally friendly alternatives to the Haber-Bosch process. In this study, we developed a new strategy of nitrogen fixation by enriching free-living N2-fixing bacteria (NFB) in reactors fed with low nitrogen wastewater, analogous to those usually found in certain industrial effluents such as paper mills. Our reactors fixed appreciable quantities of nitrogen with a rate of 11.8 mg N L-1 day-1. This rate is comparable to recent "breakthrough" nitrogen-fixing technologies and far higher than observed in low C/N reactors (fed with organic matter and nitrogen). NFB were quantified using quantitative polymerase chain reaction (qPCR) of the nifH (marker gene used to identify biological nitrogen fixation) and 16S rRNA genes. The nifH gene was enriched by a factor of 10 in the nitrogen-fixing reactors (compared to controls) attaining 13% of the bacterial population (1:4.2 copies of nifH to 16S rRNA). The Illumina MiSeq 16S rRNA gene amplicon sequencing of reactors showed that the microbial community was dominated (19%) by Clostridium pasteurianum. We envisage that nitrogen-enriched biomass could potentially be used as a biofertilizer and that the treated wastewater could be released to the environment with very little post-treatment.

Responses of soil diazotrophs to legume species and density in a karst grassland, southwest China

Diazotrophs have potential for fixing atmospheric nitrogen (N) in terrestrial ecosystems. The effects of legumes on free-living N-fixing diazotrophs in the karst grassland were determined to provide a basis for ecosystem restoration. In a field experiment, diazotroph responses were examined in five treatments: control, low, and high densities of Amorpha fruticosa and Indigofera atropurpurea. The abundance and community composition of diazotrophs were significantly affected by sampling time and the interaction between different leguminous shrubs and planting density. Legume treatments significantly increased diazotroph abundance compared with the control in December (non-growing season). Greater N input with I. atropurpurea at high density in July (growing season) may suppress soil free-living N-fixing bacteria by decreasing diazotroph abundance. Diazotroph diversity was highest under I. atropurpurea in July. Phyllobacteriaceae and Beijerinckiaceae were abundant in the A. fruticosa plots, while Burkholderiaceae, Comamonadaceae, and Geobacteraceae were abundant in the I. atropurpurea plots. Regardless of the treatments, dissolved organic carbon was the key factor driving seasonal changes in diazotroph abundance and community composition. It suggests seasonal changes in temperature and soil moisture that drive nutrient availability (e.g., ammonium N and nitrate N) under planting legumes may explain the variation in diazotroph communities. Legumes reduced negative interactions among the species in network analyses. These findings suggest that increasing N availability by legume N fixation reduced inter-specific competition among the diazotrophs, with essential roles of rare taxa during the restoration of degraded lands in the karst region.

Nutrient Mineralization and Soil Biology as Influenced by Temperature and Fertilizer Management Practices

High soil temperature due to climate change may influence nutrient mineralization and soil biology. An incubation study was conducted at Bangladesh Rice Research Institute to determine the effect of temperature (28°C and 45°C) on nutrient mineralization and soil microbial population of two different soils (terrace and saline soil) having different nutrient management practices (chemical fertilizer and integrated nutrient management). Terrace soil was clay loam and saline (6 ds m-1) soil was sandy loam in texture. Total N and organic C content was significantly high in terrace soil compared to saline soil. High temperature (45°C) enhanced C mineralization by 33% in integrated nutrient management (INM) of terrace soil and 41% in chemical fertilizer treatment in saline soil. The NH4+-N mineralization was increased by 3 fold in saline soil at 45°C as compared to the same at normal temperature of 28°C. Temperature and nutrient management options also significantly influenced phosphorus (P) and potassium (K) mineralization. High temperature significantly enhanced P mineralization in INM compared to chemical fertilizer amendment. In terrace soil, at 28°C temperature K mineralization was high in chemical fertilizer amended soil as compared to INM treatment. Temperature and nutrient sources affected soil bacterial population significantly compared to fungi, and actinomycetes. Phosphate solubilizing bacteria (PSB) were more resistant to high temperature compared to free-living N2 fixing bacteria. In general, high temperature and nutrient management practices affected C, N, P, K mineralization and soil biology; although mode of action varied and depending on soil types and nutrient management practices.

Microbiological Indicators of Soil Quality Under Native Forests are Influenced by Topographic Factors.

Several microbiological indicators of soil quality present high sensitivity, but little is known about the influence of topographic factors on them. This work aimed to evaluate variability of biological indicators of soil quality across a hillslope under native forest and the influence of topographic factors on them. Four positions on a hillslope were evaluated. Activity of the enzymes β-glucosidase, acid phosphatase, urease and fluorescein diacetate (FDA) hydrolysis were determined, as well as basal and substrate-induced respiration, and density of microorganisms: total bacteria, total fungi, actinobacteria, phosphate solubilizers, ammonifiers, native rhizobia, free-living N2-fixing bacteria, spores of arbuscular mycorrhizal fungi and percentage of root colonization by arbuscular mycorrhizal fungi. Activity and density of microorganisms were correlated with topographic factors. The relation of these factors to the variations of the evaluated indicators was determined using the random forest algorithm. Microbiological indicators varied according to the hillslope positions. The indicators urease, basal respiration, spore density, mycorrhizal colonization, total bacteria and fungi, phosphate solubilizers, and free-living N2-fixing bacteria detected in JNFB and FAM culture medium did not vary with terrain attributes and were therefore more indicated in cases of topographic variations. This and future studies can help to select the best microbiological indicators for different conditions.

English

Piliostigma reticulatum shrub is a native legume found in fallow areas in dry and semi-dry savanna soil and is used in intercropping systems. The aim was to understand the functioning of the rhizosphere, particularly the involvement of symbiotic and free living-N fixing bacteria. Soil extracts collected from P. reticulatum roots were sampled in two contrasting areas and endophytic bacterial communities were isolated using three trap host species (F. albida, A. bivenosa and V. seyal). Potential endophytic bacteria (PEB) were characterized by RFLP, nifH PCR and by 16S rRNA gene sequencing. The subsequent behavior of P. reticulatum was monitored in vitro by measuring leaf weight, biomass and chlorophyll content, after inoculation with PEB. This approach enabled isolation of 59 bacteria belonging to different genotypes. The most abundant genera were Cohnella (27.65%) among which 11 isolates clustered together and could represent a new species closely related to C. plantaginis. The other dominant genera were Paenibacillus (21.27%), Bradyrhizobium (14.89%) and Ensifer (8.5%). The nitrogen fixing gene (nifH) was detected in 21 strains and in particular, detected in a single isolate (PZS_S04) close to Cohnella xylanilytica. The strains PZS_S05 (Ensifer) and PZG_A18 (Cohnella) significantly increased certain parameters including shoot dry weight, shrub height at 90 days and photosynthetic activity (SPAD), compared to  non-inoculated controls.The result obtained showed that soil under the influence of P. reticulatum roots harbored a specific diversity of endophytic bacteria including two free living-N fixing bacteria with the potential to improve the growth of P. reticulatum in natural conditions. Key words: Piliostigma reticulatum, microbiology, phylogeny, Potential endophytic bacteria (PEB), nitrogen-fixing bacteria. &nbsp

Influence of Microbial Composition on the Carbon Storage Capacity of the Mangrove Soil at the Land-ocean Boundary in the Sundarban Mangrove Ecosystem, India

ABSTRACTSpatiotemporal assessment of the mangrove soil of Indian Sundarban revealed that decomposition rate of the organic matter was significantly lower in the anoxic condition than that of the oxic condition. Higher degree of enzyme activity in the oxic soil than the anoxic condition suggested that slower biomineralization in anoxic condition would facilitate long-term storage of organic matter in that particular ecosystem. Microbial population of nitrifying bacteria, phosphate solubilizing bacteria, cellulose degrading bacteria and fungi showed significant reduction in anoxic incubation than that in oxic incubation. In contrary, sulfate reducing bacteria and free living N2 fixing bacteria showed higher population in anoxic incubation indicating their preference for anaerobic condition. Soil CO2 emission rate decreased with the increase in anoxicity and was largely dependent on the soil redox potential, organic carbon and microbial population of the mangrove soil.

Brassicaceae cover crops reduce Aphanomyces pea root rot without suppressing genetic potential of microbial nitrogen cycling

Aims Brassicaceae cover crops can be used to suppress soil-borne pathogens. The aim was to investigate the effect of different brassicas with different glucosinolate profiles on the development of Aphanomyces pea root rot in subsequent pea plants, and the genetic potential of free-living N2-fixing bacteria and ammonia oxidising bacteria (AOB) and archaea (AOA) performing key soil ecosystem services. Methods The Brassicaceae species Brassica juncea and Sinapis alba and non-Brassicaceae species Secale cereale were grown for 11-weeks in Aphanomyces euteiches infested soil at low and high nitrogen (N) fertiliser doses. After removing both shoots and roots of the cover crops, peas were grown as a bioassay to evaluate Aphanomyces pea root rot development. Soil was sampled before harvesting the cover crops and at the end of the bioassay. Volatile compounds were collected in the root-soil environment before harvesting the Brassicaceae cover crops to determine the concentration of isothiocyanates. The abundance of genes involved in N2-fixing bacteria and ammonia oxidation in AOA and AOB were assessed. Results Pea root rot disease severity was reduced in Brassicaceae grown soil at the high N fertiliser dose. This was associated with increased growth of the cover crops. The growth of Brassicaceae did not suppress the abundance of N-cycling microbial communities, but rather increased the AOB at the end of the bioassay, most likely due to increased N availability. The disease suppressive effect was higher with S. alba than with B. juncea, and this coincided with a more diverse composition and higher concentration of aliphatic ITCs released from S. alba roots. Fewer nodules were formed after the Brassicaceae crops, especially Sinapis alba. Conclusions Brassicaceae cover crops, particularly S. alba, can be used to control soil-borne pathogens without major side effects on the genetic potential of beneficial soil microorganisms involved in N cycling. However, less nodule formation after brassicas indicates an effect on rhizobium activity.

Recent Trends in Microbial Inoculants in Agriculture

Recent Trends in Microbial Inoculants in Agriculture

Reversible oxygen‐tolerant hydrogenase carried by free‐living N2‐fixing bacteria isolated from the rhizospheres of rice, maize, and wheat

Hydrogen production by microorganisms is often described as a promising sustainable and clean energy source, but still faces several obstacles, which prevent practical application. Among them, oxygen sensitivity of hydrogenases represents one of the major limitations hampering the biotechnological implementation of photobiological production processes. Here, we describe a hierarchical biodiversity-based approach, including a chemochromic screening of hydrogenase activity of hundreds of bacterial strains collected from several ecosystems, followed by mass spectrometry measurements of hydrogenase activity of a selection of the H2-oxidizing bacterial strains identified during the screen. In all, 131 of 1266 strains, isolated from cereal rhizospheres and basins containing irradiating waste, were scored as H2-oxidizing bacteria, including Pseudomonas sp., Serratia sp., Stenotrophomonas sp., Enterobacter sp., Rahnella sp., Burkholderia sp., and Ralstonia sp. isolates. Four free-living N2-fixing bacteria harbored a high and oxygen-tolerant hydrogenase activity, which was not fully inhibited within entire cells up to 150–250 μmol/L O2 concentration or within soluble protein extracts up to 25–30 μmol/L. The only hydrogenase-related genes that we could reveal in these strains were of the hyc type (subunits of formate hydrogenlyase complex). The four free-living N2-fixing bacteria were closely related to Enterobacter radicincitans based on the sequences of four genes (16S rRNA, rpoB, hsp60, and hycE genes). These results should bring interesting prospects for microbial biohydrogen production and might have ecophysiological significance for bacterial adaptation to the oxic–anoxic interfaces in the rhizosphere.

Diversity of free-living nitrogen-fixing bacteria associated with Korean paddy fields

Nitrogen (N)-fixing microorganisms play a major role in maintaining soil fertility and are thereby important for sustainable rice production. Among a total of 165 bacterial isolates recovered from seven paddy field soils through an isolation process on four N-free media, 32 were found to be positive for PCR amplification of nifH gene. On screening, the BOX-PCR fingerprint technique grouped the nifH gene positive isolates into seven clusters. Clustering of bacteria revealed a very low level of similarity (20%), indicating the existence of a high degree of genetic diversity among the N-fixing isolates. Further characterization based on fatty acid methyl ester (FAME) showed that the isolates were members of 16 different genera, with maximum number belonged to the genus Burkholderia followed by Sphingomonas. Our results provide evidence for wide diversity of free-living N-fixing bacteria that can be used in future as a feasible alternative to N fertilizers in rice-paddy ecosystems.

High Rate of N 2 Fixation by East Siberian Cryophilic Soil Bacteria as Determined by Measuring Acetylene Reduction in Nitrogen-Poor Medium Solidified with Gellan Gum

For evaluating N(2) fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N(2)-fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N(2) fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N(2)-fixing soil bacteria isolated from the microbiota exhibited 10- to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N(2) fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.