Isolation Of Nitrogen-fixing Bacteria Research Articles

Biofertilizer based on <i>Agrobacterium </i>as a key to food security

Background: The concept of food security is contingent upon the fulfillment of two fundamental requirements: physiological and socioeconomic. The former comprises providing sustenance that is both safeguarded and enhanced in line with people’s nutritional needs and priorities, whereas the latter includes the mechanisms by which these needs are supplied. The objective is to ensure a state of robust and healthy well-being. As a sustainable solution, biofertilizers play a pivotal role in addressing the challenges of food security. Biofertilizers represent a cost-effective and environmentally friendly solution, with the potential to enhance soil productivity through nitrogen fixation. They also enhance crop yield by increasing the concentration of nutrients. Access to functional foods derived from biofertilizer-enhanced yields may contribute to improved dietary diversification and overall well-being. Objective: The objective is to isolate and screen the most active nitrogen-fixing bacterium as a basis to a new biofertilizer. Methods: The isolation of nitrogen-fixing bacteria was conducted using the soil sowing technique. The selection of nitrogen-fixing bacteria was based on their morphophysiological characteristics. Bacterial growth was evaluated by viable cell count. Cultivation of the selected bacterium occurred in both flasks (on a shaker) and a bioreactor to compare growth conditions. The stimulatory effect of the selected bacterium on seed germination was assessed based on the final germination rate. The selected bacterium identified through molecular taxonomy. Results: A total of 100 pure cultures were isolated, from which nitrogen-fixing bacteria were selected. The cultivation conditions for these bacteria were optimized regarding pH, temperature and process duration in a flask on a shaker. Strain NB4 exhibited the most favorable development under optimal settings. Additionally, cultivation parameters for this strain were optimized in a laboratory bioreactor. Notably, a bacterial liquid culture water solution comprising 1.5% strain NB4 facilitated complete germination of wheat (Triticum aestivum L.), seeds and produced high-quality sprouts. The 16S rRNA gene sequence of strain NB4 was submitted to the GenBank database under accession number MT670424.1, identifying it as Agrobacterium Pusense RP 1. Conclusion: This research aimed to develop an effective biofertilizer to tackle the pressing issue of food security. The project’s cornerstone was Agrobacterium pusense RP 1, nitrogen-fixing strain isolated and identified through rigorous research. Keywords: Isolation and identification of nitrogen-fixing bacteria, cultivation in bioreactor, germination of wheat seeds, Agrobacterium pusense.

INDOLE ACETIC ACID AND EXOPOLYSACCHARIDE PRODUCTION OF NITROGEN FIXING BACTERIA ISOLATED FROM NUTMEG TREE

The constraint of plant cultivation in the tropics is low nitrogen (N) content in soil. The application of nitrogen fixing bacteria (NFB) is not only fulfill the N demand but also provide the phytohormones Indole acetic acid (IAA) and exopolysaccharides (EPS). The objective of the experiment was to was to isolate the NFB from the rhizosphere of nutmeg tree and characterized their IAA and EPS production in a liquid culture. The NFB was isolated by serial dilution plate method on Tryptic Soy Agar. In order to test the ability of various NFB isolates to produce IAA and EPS, all isolates were grown in Tryptic Soy Broth for 72 hours at room temperature. The results showed that at the end of experiment the media acidity was shift from neutral to alkaline. The population of five isolates of NFB was approximately 8-9 log10 (of CFU/mL). The IAA production in liquid culture of all isolates was similar but the EPS production was varied and depend on the isolates, The highest EPS content was in PL1 culture and the lowest was in the culture of PL5. This experiment verified that the NFB isolated from nutmeg rhizosphere enable to produce IAA and EPS.

Isolation and molecular identification of halotolerant diazotrophic bacteria from The Northern Coastal of Pemalang, Central Java, Indonesia

Abstract. Purwanto, Oktaviani E, Leana NWA. 2022. Isolation and molecular identification of halotolerant diazotrophic bacteria from The Northern Coastal of Pemalang, Central Java, Indonesia. Biodiversitas 23: 5814-5821. Plant Growth Promoting Bacteria can fix nitrogen, a very important macronutrient for plant growth. However, the application of urea as a source of this macronutrient has a negative impact on the environment. Therefore, developing biofertilizers using N-fixing bacteria is an environmentally friendly technology. Therefore, this research aimed to isolate and analyze the diversity of N2-fixing bacteria from saline rice fields. The soil samples were taken from the rhizosphere of rice plants in Nyamplung Sari Village, Petarukan District, Pemalang Regency. Meanwhile, 9 (nine) isolates of nitrogen-fixing bacteria have been isolated, which can fix N2. The isolates can bind to N2, but only a few can produce IAA. The nitrogen-fixing ability of diazotrophic bacteria ranged from 17.85 ppm to 29.05 ppm. Isolate Jn3 has the highest ability to fix nitrogen, reaching 29.05 ppm, while Jn3, J, J12, J5, Kn1, and A3 can produce IAA with values of 2.00, 2.69, 2.22, 1.76, 3.47, and 1.79 ppm, respectively. Based on the 16S rRNA analysis and phylogeny construction, the isolated bacteria were identified in 3 (three) clusters. The first cluster was identified as Pseudomonas stutzeri and Acinetobacter junii (Kn1 and Jn3), while the second was identified as Bacillus cereus, Bacillus tropicus, Bacillus altitudinis, and Bacillus subtilis (Jn, Jn1, A3, and K3 isolates). The third cluster was identified as Bacillus pumilus, Acinetobacter baumanii, and Acinetobacter schindleri (J12, J5, and J). In addition, our study reported the findings of Acinetobacter baumannii and Acinetobacter schindleri species that can fix nitrogen.

SELEKSI Seleksi Dan Identifikasi Bakteri Penambat Nitrogen Pada Perakaran Tanaman Kacang Hijau (Vigna radiata L) Dan Tomat (Solanum lycopersicum L) Di Kabupaten Belu

Nitrogen fixing bacteria are often called diazotroph bacteria which are able to use air nitrogen as a nitrogen source for their growth. Nitrogen fixing bacteria have the ability to increase the efficiency of N- available in the soil. The purpose of this study was to determine the morphological and biochemical characters of nitrogen-fixing bacteria from the roots of mung bean and tomato plants in Belu Regency. Isolation of nitrogen fixing bacteria by scratch method and spread on NA media. While the selection of nitrogen fixing bacteria using Jensen agar media. The results of the isolation obtained 12 isolates. Six isolates from roots of tomato plants and six isolates from roots of mung bean plants. The morphological characters of the 12 isolates were generally round in shape, small in size, flat in elevation and milky white in color. The selection results showed that 3 isolates grew on Jansen agar media, namely RKHB05, RKHB06, and RTB06. The result of gram staining showed that RKHB05, RKHB06 isolates were gram negative and had cocci cell form, while RTB06 isolates were gram positive with bacillus cell forms. The 3 isolates were positive for motility test, Citrate test, TSIA, and Catalase test

Exploration non-symbiotic nitrogen-fixing bacteria from several lakes in East Java, Indonesia

Abstract. Nafisah W, Prabaningtyas S, Witjoro A, Saptawati RT, Rodiansyah A. 2022. Exploration non-symbiotic nitrogen-fixing bacteria from several lakes in East Java, Indonesia. Biodiversitas 23: 1752-1758. Potential microorganisms, mainly nitrogen-fixing bacteria spread in almost lake areas in East Java. The biogeochemical processes that occur between biotic-biotic or biotic-abiotic factors can accelerate the growth of autotrophic microorganisms in waters as like microalgae. Non-symbiotic nitrogen-fixing bacteria in the water can supply nitrogen to support the growth of microalgae. This study is aimed to explore and calculate the ammonium concentration produced by non-symbiotic nitrogen-fixing bacteria that isolated from several lakes in East Java. The highest ammonium producing bacteria were characterized based on morphology, physiology, and genetic with 16S rRNA gene sequencing. The bacterial isolates were screened with a nitrogen-free bromothymol blue medium to select bacterial isolates capable of fixing nitrogen. The content of NH3/NH4 was produced by isolates of nitrogen-fixing bacteria was measured by using the prodac test kit NH3/NH4. The ammonium concentration was calculated with Nessler’s reagent by using the spectrophotometry method at a wavelength of 425 nm. The result was obtained 31 isolates of non-symbiotic nitrogen-fixing bacteria. The bacterial isolate B2 exhibited highest activity of fixing nitrogen that produced ammonium (14.46 mg/L) among the other screened bacteria. The morphological and physiological characterization from that isolate was identified as genus Bacillus, whereas the genetic characterization similar to Bacillus paramycoides, that included in the Bacillus cereus group.

Performance of Centrocema grown on mercury-contaminated tailing inoculated with beneficial bacteria: Preliminary study

Low levels of major essential nutrients and high mercury (Hg) content in gold mine tailings can inhibit plant growth. An inexpensive and effective strategy to overcome these obstacles was inoculation of plant-growth promoting bacteria and planting legume cover crops (LCC). A greenhouse experiment was conducted to evaluate the effect of isolates of Nitrogen Fixing Bacteria (NFB) and Phosphate Solubilizing Bacteria (PSB) on some growth characteristics of LCC Censtrosema pubescens (butterfly pea) grown in Hg-contaminated tailing of gold mine tailing. Moreover, the research was aimed to verify the viability of two bacterial groups in tailing after inoculation. The experiment was set up in randomized block design to test single and mixed inoculation of NFB and PSB. The experiment verified that the single inoculation and mixture of two bacteria did not change plant properties compared to the control treatment. Although the statistical analysis was not significant, there were slightly increase in root length as well as NFB and PSB populations of inoculated plants.

Eksplorasi Bakteri Penambat Nitrogen dari Tanah Hutan Mangrove Sungai Peniti, Kabupaten Mempawah

Peniti Mangrove Forest is one of the forests in West Kalimantan that has high forest productivity, thus supporting the presence of nitrogen-fixing bacteria. Nitrogen fixing bacteria is one of the nitrogen-fixing microorganisms that can bind the free nitrogen needed for the nitrogen cycle process. This study aimed to determine the types of nitrogen-fixing bacteria that can be found in the soil of Peniti mangrove forest, Mempawah Regency, West Kalimantan. Taking soil samples used a purposive sampling method. Bacterial isolation used dilution techniques and pour plate method on the Nitrogen Free Bromthymol Blue (NFB) media. The nitrogen fixing was obtained by 3 isolates of nitrogen fixing bacteria. After identification, 3 genera were obtained, namely members of the genera Azotobacter, Azospirillum, and Pseudomonas.

Nitrogen Fixation in Cereals.

Cereals such as maize, rice, wheat and sorghum are the most important crops for human nutrition. Like other plants, cereals associate with diverse bacteria (including nitrogen-fixing bacteria called diazotrophs) and fungi. As large amounts of chemical fertilizers are used in cereals, it has always been desirable to promote biological nitrogen fixation in such crops. The quest for nitrogen fixation in cereals started long ago with the isolation of nitrogen-fixing bacteria from different plants. The sources of diazotrophs in cereals may be seeds, soils, and even irrigation water and diazotrophs have been found on roots or as endophytes. Recently, culture-independent molecular approaches have revealed that some rhizobia are found in cereal plants and that bacterial nitrogenase genes are expressed in plants. Since the levels of nitrogen-fixation attained with nitrogen-fixing bacteria in cereals are not high enough to support the plant’s needs and never as good as those obtained with chemical fertilizers or with rhizobium in symbiosis with legumes, it has been the aim of different studies to increase nitrogen-fixation in cereals. In many cases, these efforts have not been successful. However, new diazotroph mutants with enhanced capabilities to excrete ammonium are being successfully used to promote plant growth as commensal bacteria. In addition, there are ambitious projects supported by different funding agencies that are trying to genetically modify maize and other cereals to enhance diazotroph colonization or to fix nitrogen or to form nodules with nitrogen-fixing symbiotic rhizobia.

Endophytic Azospirillum for enhancement of growth and yield of wheat

The present study was aimed at the isolation of nitrogen fixing bacteria belonging to genus Azospirillum from wild wheat and their growth promoting effect on the bread wheat, under pot culture and filed conditions in semi-arid environment. Two bacterial strains were obtained on the NFb (N-free malate medium) medium with ability to produce phytohormone (indoleacetic acid; IAA) and fix atmospheric nitrogen. Based on 16S rRNA sequence analysis, one isolate (Sp1) showed 99% homology with Azospirillum brasilense and the second isolate (Sp2) showed 99% homology with Azospirillum zeae. Effect of these two isolated strains on bread wheat was examined under pot experiment. Both bacterial strains were found to be effective in enhancing wheat growth. However, best results were obtained with isolate Sp2. Therefore, only this strain was introduced as inoculum in field experiment, to investigate its effect on wheat yield under semi arid conditions during winter growing season 2016–2017. Based on the obtained field results, wheat inoculated with isolate Sp2 displayed an increased total grain yield by up to 18% as compared to un-inoculated plants. These results show the potential of these bacteria to be used as biofertilizer.

ANALYSIS OF GENETIC VARIABILITY IN SOURSOP (Annona muricata L.) POPULATIONS FROM CENTRAL JAVA AND EAST JAVA BASED ON RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKER

Nitrogen fixing bacteria (Azospirillum and Azotobacter) were isolated from coastal mangrove in Pulau seribu. The aims of this experiment was to find out isolates of nitrogen fixing bacteria which were tolerant to high salinity. The isolates can be used as a biofertilizer to support coastal agriculture. A total of 28 isolates (14 isolates of Azospirillum and 14 isolates of Azotobacter) were tested their tolerance to salt by growing them in their respective media containing 1% 2% and 3 % NaCl,. Salt tolerant isolates obtained, then used as inoculants in paddy. The experiment laid out factorial based randomized complete block design which was comprised of 7 fertilizer treatments and 5 watering treatments with 5 replicates for each treatment at green house of Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences. The first factor was fertilizer treatments 1. Compost + mixed bacteria (K1), 2. Control without inoculant, 3. Compost, 4. NPK, 5. Compost + NPK + mixed microbial, 6. Azotobacter isolates; 7. Azospirillum isolates. The second factor was watering treatments, plant was watered by : 1. freshwater, 2. freshwater + sea water at mixture ratio 1:1, 3. Sea water, 4. sea water + 2% NaCl (20 g NaCl / l), 5. freshwater + 5% NaCl (50 g NaCl / l). The result showed that there were 9 isolates of Azospirillum and 4 isolates of Azotobacter which were tolerant to grow at media with 3% NaCl. The green house experimental result revealed that the plants were treated with bacteria can survive up to the level of salinity 12.43 dS-1m.

THE EFFECT OF SALT TOLERANT NITROGEN FIXING BACTERIA ON THE GROWTH OF PADDY RICE (Oryza sativa. L)

Nitrogen fixing bacteria (Azospirillum and Azotobacter) were isolated from coastal mangrove in Pulau seribu. The aims of this experiment was to find out isolates of nitrogen fixing bacteria which were tolerant to high salinity. The isolates can be used as a biofertilizer to support coastal agriculture. A total of 28 isolates (14 isolates of Azospirillum and 14 isolates of Azotobacter) were tested their tolerance to salt by growing them in their respective media containing 1% 2% and 3 % NaCl,. Salt tolerant isolates obtained, then used as inoculants in paddy. The experiment laid out factorial based randomized complete block design which was comprised of 7 fertilizer treatments and 5 watering treatments with 5 replicates for each treatment at green house of Microbiology Division, Research Center of Biology, Indonesian Institute of Sciences. The first factor was fertilizer treatments 1. Compost + mixed bacteria (K1), 2. Control without inoculant, 3. Compost, 4. NPK, 5. Compost + NPK + mixed microbial, 6. Azotobacter isolates; 7. Azospirillum isolates. The second factor was watering treatments, plant was watered by : 1. freshwater, 2. freshwater + sea water at mixture ratio 1:1, 3. Sea water, 4. sea water + 2% NaCl (20 g NaCl / l), 5. freshwater + 5% NaCl (50 g NaCl / l). The result showed that there were 9 isolates of Azospirillum and 4 isolates of Azotobacter which were tolerant to grow at media with 3% NaCl. The green house experimental result revealed that the plants were treated with bacteria can survive up to the level of salinity 12.43 dS-1m.

16s rRNA Sequence Analysis and Ammonium Excretion Ability of Nitrogen Fixing Bacteria Isolated from Mineral Acid Soil

Nitrogen fixing bacteria defined as bacteria which is capable to transform free nitrogen molecules into ammonium v (PCR). Nitrogenase activity of these selected isolates was measured using Acetylene Reduction Assay (ARA). The ability of these selected isolates in ammonium excretion was qualitatively and quantitavely measured using Nessler reagent and spectrophotometry method respectively. Taxonomic position of the selected bacteria were determined based on their 16S rRNA sequence analysis. Genetic diversity analysis of these 15 isolates of nitrogen fixing bacteria yield eight selected bacteria for subsequent analysis. Sequence of nifH gene from all of these selected bacteria were successfully amplified. Nitrogenase assay of these selected bacteria revealed 6 isolates with high nitrogen fixation capasity namely GMA3, GMA5, GMA6, GMA9, GMA12 AND GMA 13. Ammonium excretion analysis revealed 4 isolates which have remarkable ability of producing high level of ammonium namely GMA1, GMA3, GMA6, and GMA9. The 16S rRNA sequence analysis shown that isolates GMA3, GMA5, GMA11 and GMA12 had a close relationship with Brevibacillus formosus strain DSM 9885T, Flexibacter canadensis strain ISSDS-428, Rhizobium tropici strain rif 200849, and Azotobacter tropicalis strain RBS. Respectively, isolate GMA1 and GMA13 had a close relationship with Sthenotropphomonas sp. Strain MFC-C, while isolate GMA6 and GMA9 had a close relationship to Azotobacter vinelandii strain ISSDS-428. ', 'string'), (105, 'en_US', 'subject', 'nitrogen fixing bacteria, ammonium excretion, identification', 'string'), (105, 'en_US', 'sponsor', '', 'string'), (107, 'en_US', 'title', 'Effect of Probiotic Lactobacillus sp. Dad13 on Humoral Immune Response of Balb/C Mice Infected with Salmonella typhimurium', 'string'), (107, 'en_US', 'abstract', 'An indigenous strain of lactic acid bacterium (LAB) identified as Lactobacillus spp. Dad13 (Dad13), isolated from traditional fermented buffalo milk, was found to be potential as probiotic. The aim of this research was to study the effect of probiotic Dad13 on humoral immune response of Balb/C mice infected with Salmonella typhimurium. Thespecific objective was to find out the effect of different Dad13 consumption time (before and along with infection of S. typhimurium) on the humoral immune response of Balb/C mice. The experiment was conducted by in vivo trial on 20 males of Balb/C mice, age of 6-8 weeks, fed with AIN-93 standard diet. The mice were assigned into 4 groups. Each group received the following treatments, ie. Dad13 only, Dad13 before infection, Dad13 along with infection and Salmonella infection only. A volume of 100 ml Dad13 cell suspensions (1010 CFU/ml) were given by oral forced feeding daily for a week, at week 3 for group before infection and at week 4 for group of Dad13 only and Dad13 along with infection. Salmonella infection (109 CFU/ml) was given once orally at week 4 to all groups except group treated with Dad13 only. The humoral immune response of Balb/C mice was detected 2 weeks after infection by measuring the titers of IgG and IgA specific from serum and mucosal intestinal liquid samples using Enzyme-linked Immunosorbent Assay (ELISA) method. The result indicated that humoral immune response of Balb/C mice consuming Dad13 before and along with Salmonella infection were significantly different (pl0.05). Dad13 consumption along with Salmonella infection increased circulated IgG and IgA as well as secretory IgA. It can be concluded that Dad13 probiotic feeding along with infection increased humoral immune response more significantly compared to that before infection.

Producción de un biofertilizante a partir de un aislamiento de Azotobacter nigricans obtenido en un cultivo de Stevia rebaudiana Bert

Objetivo. Realizar un aislamiento de bacterias fijadoras de nitrógeno para emplearlas en un programa de fertilización bajo un esquema de agricultura orgánica. Materiales y métodos. El aislamiento de bacterias fijadoras de Nitrógeno se realizó en medio Ashby-benzoato a partir del suelo de un cultivo de Stevia rebaudiana Bert. Los aislamientos identificados como Azotobacter nigricans fueron evaluados mediante una cinética de crecimiento y la cepa con mayor velocidad se utilizó para la elaboración de un biofertilizante por fermentación discontinua. La evaluación preliminar del biofertilizante se realizó mediante su inoculación en 3 eras de un cultivo de S. rebaudiana Bert. y el rendimiento se determinó con base en la producción de biomasa y concentración de glucósidos. Resultados. Dos aislamientos (A5 y A6) fueron identificados como A. nigricans con base en la caracterización fenotípica y genotípica. El aislamiento A5 se seleccionó para la elaboración del biofertilizante debido a que presentó mejor estabilidad, pigmentación, mayor velocidad de crecimiento 0,1405 h-1 fase exponencial de 18 horas y una producción de AIA promedio de 38,4 mg/ml a las 150 horas. El biofertilizante se obtuvo en medio leche con una concentración celular de 4x1012 UFC/ml. Conclusiones. La evaluación preliminar en campo mostró una correlación positiva entre el aumento de la concentración de glucósidos en las hojas de S. rebaudiana y una mayor producción de biomasa en respuesta a la aplicación del biofertilizante.Palabras clave: AIA, Azotobacter nigricans, glucósidos, Stevia rebaudiana Bert.AbstractBio-fertilizer production from an isolate of Azotobacter nigricans obtained from a plantation of Stevia rebaudiana Bert. Objective. To isolate nitrogen fixing bacteria to be used in a fertilization regime of an organic agriculture program. Materials and methods. The isolation of nitrogen fixing bacteria was done in an Ashby-benzoate medium from soil of a Stevia rebaudiana plantation. Isolates identified as Azotobacter nigricans were evaluated by their growth kinetics and the strain with the fastest growth was used for the production of a biofertilizer by discontinuous fermentation. The preliminary evaluation of the biofertilizer was done by its inoculation into three ridges of a plantation of S. rebaudiana and yield determination was based upon biomass production and glycoside concentration. Results. Two isolates (A5 and A6) were identified as A. nigricans based on their phenotypic and genotypic characterization. Isolate A5 was selected for preparing the biofertilizer because it showed a better stability, pigmentation, a faster growth rate (0.1405 h-1 exponential phase of 18 hours) and an average IAA production of 38.4 g/ml after 150 hours. The bio-fertilizer was obtained in milk medium with a cell concentration of 4x1012 CFU/ml. Conclusions. The preliminary field evaluation showed a positive correlation between the increase of the glycoside concentration in the leaves of S. rebaudiana and a higher production of biomass in response to the bio-fertilizer application.Key words: IAA, Azotobacter nigricans, glycosides, Stevia rebaudiana Bert.ResumoProdução de um bio-fertilizante a partir do isolamento de Azotobacter nigricans obtido num cultivo de Stevia rebaudiana Bert. Objetivo: Realizar um isolamento de bactérias fixadoras de nitrogênio para empregá-las num programa de fertilização sob um esquema de agricultura orgânica. Materiais e métodos: O isolamento de bactérias fixadoras de nitrogênio realizou-se no meio Ashby-benzoato a partir do solo de um cultivo de Stevia rebaudiana Bert. Os isolamentos identificados como Azotobacter nigricans foram avaliados mediante uma cinética de crescimento e a cepa com maior velocidade utilizou-se para a elaboração de um bio-fertilizante por fermentação descontínua. A avaliação preliminar do bio-fertilizante realizou-se mediante sua inoculação em 3 eras de um cultivo de S. rebaudiana Bert., e o rendimento determinou-se com base na produção de biomassa e concentração de glucosideos. Resultados: Dois isolamentos (A5 e A6) foram identificados como A. nigricans com base na caracterização fenotípica e genotípica. O isolamento A5 selecionou-se para a elaboração do bio-fertilizante por apresentar melhor estabilidade, pigmentação, maior velocidade de crescimento (0,1405 h-1 fase exponencial de 18 horas) e uma produção de AIA media de 38,4 µg/ml as 150 horas. O bio-fertilizante obteve-se no meio leite com uma concentração celular de 4x1012 UFC/ml. Conclusões. A avaliação preliminar no campo apresentou uma correlação positiva entre o aumento da concentração de glucosideos nas folhas de S. rebaudiana e uma maior produção de biomassa como resposta à aplicação do bio-fertilizante.Palavras chave: AIA, Azotobacter nigricans, glucosideos, Stevia rebaudiana Bert.

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

ABSTRACTBiological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 104 CFU mL‐1) and Beijerinckia (13.2 x 104 CFU mL‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 104 CFU mL‐1) and Clostridium pasteurianum 39.1 x 104 CFU mL‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.

Isolation of nitrogen-fixing bacteria containing molybdenum-independent nitrogenases from natural environments.

Seven diazotrophs that grow well under Mo-deficient, N(2)-fixing conditions were isolated from a variety of environments. These isolates fall in the gamma subdivision of the class Proteobacteria and have genes that encode the Mo nitrogenase (nitrogenase 1) and the V nitrogenase (nitrogenase 2). Four of the isolates also harbor genes that encode the iron-only nitrogenase (nitrogenase 3).

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10(2) to 4.6×10(2)/ml for seawater, 4.0×10(4) to 4.3×10(5)/g wet wt for eelgrass-bed sediment, and 2.1 × 10(5) to 1.2 × 10(7)/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.