Influence of soil water potential on respiration and nitrogen fixation ofAzotobacter vinelandii

Summary Respiration and N₂-fixation (acetylene reduction) ofAzotobacter vinelandii have been studied at a variety of soil water potentials. Both processes were strictly linked and strongly reduced at water potentials between –0.6 and –1.3 MPa. Complete inhibition occurred below –2.1MPa. Osmotic potentials in soil compared to matric potentials of the same value were less inhibitory to respiration and acetylene reduction by Azotobacter. The N₂-fixing efficiency (mg N/g glucose) was not influenced by water potentials ranging from –0.1 to –2.1 MPa.     

Azotobacter numbers on broadbalk field,rothamsted

Summary Azotobacter chroococcum, an aerobic nitrogen-fixer, was counted during 1955–1958, and in 1962, in soil samples taken from nine plots from Broadbalk field at Rothamsted. In the fallow sections of the plots, and in sections carrying the fourth successive wheat crop, Azotobacter were few, especially in the plots given ammonium sulphate every year. Azotobacter numbers increased after fallowing. In cropped sections, Azotobacter were fewest in the plot with nitrogen only. With nitrogen and phosphate but no potash they were fewer than in the plots without nitrogen. The most Azotobacter were in the plots with nitrogen, phosphate and potash. These trends are correlated with yield, but Azotobacter were too few (ranging from <100 to a maximum of 9400 per gram soil) to fix enough nitrogen to affect crop growth.     

Counting viableAzotobacter chroococcum in vertisols

Methods for preparing soil suspensions for countingAzotobacter chroococcum in vertisols by soil dilution and plating were investigated. Mechanical methods to promote disaggregation of soil and Azotobacter microcolonies by shaking soil suspensions with glass beads (10% w/v) or coarse sand (1–2 mm fraction) increased soil dispersion and Azotobacter colony counts. Chemical methods for disaggregation were unsatis-factory. The non-ionic detergent Agral (0.004, 0.02, 0.1, 0.5 and 2.5% w/v) had no significant effect on soil dispersion and Azotobacter count. Both sodium pyrophosphate (0.03, 0.1, 0.3 and 0.9% w/v) and sodium metaphosphate as Calgon (0.022, 0.066, 0.2, 0.6 and 1.8% w/v) increased soil dispersion but were toxic to Azotobacter. Increasing time of shaking soil: distilled water suspensions increased deflocculation of the clay and Azotobacter counts to a maximum after 6–23 hours shaking. Comparable results were obtained within 30–60 minutes of shaking with coarse sand, but shaking with coarse sand beyond 2 hours reduced counts through mechanical damage to cells. Counts from suspensions in physiological saline (0.75% NaCl) and in distilled water were similar. Counts from suspensions in Jensen's mineral base shaken for <3 hrs were lower than from distilled water due to flocculation fo the soil by^Ca2+ ions, but were higher on extended shaking up to 23 hours due to better cell protection. Shaking soil suspensions in distilled water with 10% w/v coarse sand for 30 minutes is recommended when counting Azotobacter in vertisols.     

Nutritional and cultural conditions for production of poly-3-hydroxybutyric acid byAzotobacter chroococcum

Free-living nitrogen-fixing bacteria have been identified as a potential source of poly-3-hydroxybutyric acid (PHB). Systematic study of this ability of N₂-fixing organisms has lead to the isolation of an efficient strain, identified asAzotobacter chroococcum. Nutritional requirements and cultural conditions for optimal production of PHB by this strain under laboratory conditions were determined. In N-free liquid medium containing 2% glucose, the strain accumulated PHB up to 68% of its cell dry mass. Glucose and mannitol were found to be the best carbon sources, while organic nitrogen compounds were preferred as nitrogen source. Maximum yield (3.3 g/L) was obtained with 0.2% bactopeptone supplementation. Inorganic phosphate at a concentration suboptimal for growth had some growth-promoting effect. Under oxygen limiting conditions, biomass production was enhanced but a different response was obtained for PHB production.     

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27^T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.     

Production of poly(β-hydroxybutyrate-β-hydroxyvalerate) copolymer from sugars by Azotobacter salinestris

Azotobacter salinestris, a sodium-dependent, microaerophilic N₂-fixing soil bacterium, formed polyhydroxyalkanoate copolymers comprised of β-hydroxybutyric acid and 9–12 mol% β-hydroxyvaleric acid (HV) during growth on sugars. Increased HV content was achieved by feeding valeric acid to the culture growing on glucose, but propionic acid could be directed to HV formation only when it served as the sole C source. Polymer production in nitrogen-fixing cells was increased at higher aeration, provided that a complex organic nitrogen source was also present, but there was no HV in the polymer. HV production was increased to 28 mol% in nitrogen-fixing cells when aeration was lower and acetate was provided with glucose in the medium. Enzymes leading to the production of polyhydroxyalkanoate copolymers were found to be similar in A. salinestris and Azotobacter vinelandii, but A. vinelandii is unable to form HV from propionate or from sugars without valeric acid addition. A biochemical scheme is proposed for the production of HV in A. salinestris, whereby the glyoxylate bypass assimilates acetate to generate succinate, which may be converted into propionyl-CoA for HV synthesis. The results suggest that it may be possible to control the molar yield of HV formed from sugars by A. salinestris. Received: 21 January 1997 / Received revision: 7 April 1997 / Accepted: 13 April 1997     

Ecology of soil fungi of a sal forest with emphasis on fungistasis

Summary An ecological study of the soil micro-fungi in three forest regions of different age was undertaken. The pH of the soil varied from 6.6 to 6.9. Soil samples were collected from three different depthsviz 0–6, 7–12, and 13–18 inches with aseptic precautions and were studied by a dilution-plate method using peptone-dextrose agar with rose bengal for the fungal analysis. Determinations were made of physico-chemical characters of soil such as organic matter, total nitrogen, phosphorus, water-holding capacity and pH of the soil. The organic matter and nitrogen favoured the growth of fungi in the soil. The upper horizon contained more of the organic matter, nitrogen, phosphorus, and perhaps other elements, which in turn favoured the growth of fungi in soil. The number of fungi decreased according to increase in depth of the soil samples. The frequency and abundance of some dominant fungi have been calculated and the fungal population has been represented both qualitatively and quantitatively. The pattern of colonization of nutrient agar by fungi from soil inocula changed with the increase of concentration of staling products produced by earlier established fungal colonies. At the highest concentration of staling the fungi colonizing the agar disks were species ofTrichoderma, Aspergillus, Penicillium, Curvularia, Alternaria andFusarium. The capacity of species to colonize the staled nutrient medium was due partly to tolerance of the metabolic products diffused in agar disks and partly to density of the population in the inoculated soil. The comparative account of germinated spores on unsterilized and sterilized soil and in controlled condition shows that fungistatic activity is higher in unsterilized soil. In Table 4 the F₃, an oldest soil sample has more fungistatic activity than F₁ and F₂, due to more litter deposition on the floor, which increases the population of micro-organisms and fungistatic activity.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Parasitism ofAzotobacter andRhizobium species byBdellovibrio bacteriovorus

Attempts to isolate, from soil, strains ofBdellovibrio bacteriovorus parasitic on variousAzotobacter andRhizobium species were not successful. However, a strain ofBdellovibrio bacteriovorus isolated from soil as a parasite ofEscherichia coli was shown to produce plaques onAzotobacter chroococcum as well but not onAzotobacter vinelandii or variousRhizobium species.The nutritive conditions required for thisBdellovibrio strain to yield discrete countable plaques onAzotobacter chroococcum plates were examined, and it was proposed thatAzotobacter slime be used as a preservative agent for the maintenance ofBdellovibrio bacteriovorus strains in frozen culture.This research was authorized for publication as paper No. 3309 in the journal series of the Pennsylvania Agricultural Experiment Station, on October 3, 1967.     

Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms

A range of pasture grass (Danthonia richardsonii and Phalaris aquatica) and legume (Medicago polymorpha, M. sativa, Trifolium repens and T. subterraneum) species showed limited capacity to obtain phosphorus (P) from inositol hexaphosphate (IHP), when grown in either sterile agar (pH 5.0 or 5.5) or sand-vermiculite media (pH 5.0). The total P content of shoots from IHP-supplied plants grown in agar was between 20% and 34% of that for seedlings supplied with an equivalent amount of P as inorganic phosphate (P_i), while in sand-vermiculite, the total P content of IHP-grown plants was between 5 and 10% of control plants. The poor ability of plants to utilize P from IHP resulted in significantly lower tissue P concentrations and, in general, reduced plant dry weight accumulation. In contrast, the P nutrition of plants supplied with IHP was significantly improved by inoculating media with either a cultured population of total soil micro-organisms or with a specific isolate of Pseudomonas sp., selected for its ability to release phosphate from IHP (strain CCAR59; Richardson and Hadobas, 1997 Can. J. Micro. 43, 509-516). In agar and sand-vermiculite media, respectively, the P content of IHP-grown plants increased with inoculation by up to 3.9- and 6.8-fold, such that the dry weight and P content of the plant material were equivalent to those observed for control plants supplied with P_i. However, the response to inoculation was dependent on the growth medium and the source of micro-organisms used. In sand-vermiculite, the cultured population of soil micro-organisms was effective when IHP was supplied at an equivalent level of P_i required for maximum plant growth. By comparison, inoculation of plants with the Pseudomonas strain was only effective at very high levels of IHP supply (×36), whereas in agar a response to inoculation occurred at all levels of IHP. The ability of pasture plants to acquire P from phytate was, therefore, influenced by the availability of IHP substrate, which was further affected by the presence of soil micro-organisms. Our results show that in addition to having an effect on the sorption characteristics of the growth media, soil micro-organisms also provided a source of phytase for the dephosphorylation of phytate for subsequent utilization of P_i by plants.     

Increased root exudation of14C-compounds by sorghum seedlings inoculated with nitrogen-fixing bacteria

Summary Organic components leaked fromSorghum bicolor seedlings (‘root exudates’) were examined by recovering¹⁴C labelled compounds from root solutions of seedlings inoculated withAzospirillum brasilense, Azotobacter vinelandii orKlebsiella pneumoniae nif-. Up to 3.5% of the total¹⁴C recovered from shoots, roots, and nutrient solutions was found in the root solutions. Inoculation with Azospirillum and Azotobacter increased the amounts of¹⁴C and decreased the amounts of carbohydrates in the root solutions. When sucrose was added as a carbon source for the bacteria, the increase of¹⁴C in the solutions did not occur. Quantities of¹⁴C found in the root solutions were proportional to amounts of mineral nitrogen supplied to the plants. Bacterial growth also was proportional to nitrogen levels. When sorghum plants were grown in soil and labelled with¹⁴CO₂, about 15% of the total¹⁴C recovered within 48 hours exposure was found in soil leachates.     

Efficacy of brain heart infusion-egg albumen agar,yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.     

Transconjugation studies in <Emphasis Type="Italic">Azospirillum</Emphasis> sp. negative to mineral phosphate solubilization

It has been previously shown that certain gram-negative bacteria do not have the ability to solubilize insoluble phosphates due to the lack of pyrroloquinoline-quinone synthesis genes (pqq). PQQ is required as cofactor for the assembly of the glucose dehydrogenase (GDH) holoenzyme, which acts in the oxidation of glucose to gluconic acid. In this context the transconjugation and expression of pqq genes in Azospirillum sp. was studied using the construct pMCG 898. pMCG 898 containing pqq gene/s was mobilized into an Azospirillum strain negative to mineral phosphate solubilization by biparental mating. The presence of the construct was also confirmed by minipreps of the transconjugants. The transconjugants were able to solubilize dicalcium phosphate while the wild type was not able to do so. The nitrogen-fixing ability of the transconjugants was also examined and they retained the ability to fix nitrogen. Further detailed studies are required to confirm the utility of such strains in releasing inorganic P from fixed phosphates in soil.     

Phosphate potential and phosphate capacity of soils

Summary The relationship between the phosphate potential (I) and the amount of phosphate (Q), added to the soil has been examined by equilibrating soil samples with 0.001M or 0.01M CaCl₂ solutions containing various amounts of phosphate. For one neutral and two alkaline soils the Q/I relationship depends on the CaCl₂ concentration and the pH in such a way that the apparent values of I decrease when the CaCl₂ concentration increases from 0.001 M to 0.01M. The difference between the two values increases when the pH increases. When correction is made for the formation of the soluble calcium phosphate complex, CaHPO₄, the Q/I relationship becomes independent of the CaCl₂ concentration. The initial phosphate potential (I₀) determined by interpolation, is also found to be independent of the CaCl₂ concentration. The necessary amount of phosphate to be added or removed per gram of soil in order to obtain a certain alteration of the phosphate potential is designated the differential phosphate potential buffering capacity, DPBC. For ten soils DPBC-values are determined on the basis of the Q/I relationships, (ΔQ/ΔI)_Io, and found to be independent of the CaCl₂ concentration. The content of colloids and of inorganic phosphate accounts for a significant part of the variation in the DPBC for different soils. The importance of the DPBC for characterization of the phosphate status of soils in respect to phosphate supply to plants is briefly discussed. The author is indebted to professor, Dr. H. C. Aslyng, head of the department for his suggestions and helpful criticism during the progress of this work.     

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

Organic Acid Secretion and Phosphate Solubilizing Efficiency of Pseudomonas sp. PSB12: Effects of Phosphorus Forms and Carbon Sources

Secretion of organic acids is an important mechanism for phosphate solubilizing bacteria (PSB) to dissolve insoluble phosphorus in soil. However, the composition of organic acids produced by PSB in the presence of different phosphorus compounds is poorly known, and little is known about the ability of PSB to degrade pollutants in sediment. In this study, we isolated a strain Pseudomonas sp. PSB12 from the sediment of the Qihe River. PSB12 had maximum phosphate solubilization index (SI) of 3.86 on Pikovskaya's agar medium. The phosphate solubilizing activity was associated with the release of organic acids produced from glucose, while the composition of organic acids produced by PSB12 was dependent on the phosphorus forms. When initial soluble phosphorus was insufficient (in MP1 and MP2 media), gluconic acid was the predominant organic acid. In contrast, formic acid, butyrate, and propanedioic acid were the main organic acids produced when only soluble phosphorus (MP3) was supplied. RT-PCR indicated that the expression of glucose dehydrogenase gene (gcd) of PSB12 was two- to four-fold higher in MP1 than in MP3. PSB12 also possessed the phenol hydroxylase gene (phe) suggesting that phenol could be used as the carbon source to dissolve insoluble phosphorus. PSB12 is a potential candidate for in situ bioremediation and for promoting plant growth in soil contaminated by phenol with low levels of soluble phosphorus.     

Development of a New Biofertilizer with a High Capacity for N2 Fixation,Phosphate and Potassium Solubilization and Auxin Production

Biofertilizers that possess a high capacity for N₂ fixation (Azotobacter tropicalis), and consist of phosphate solubilizing bacteria (Burkhoderia unamae), and potassium solubilizing bacteria (Bacillus subtilis) and produce auxin (KJB9/2 strain), have a high potential for growth and yield enhancement of corn and vegetables (Chinese kale). For vegetables, the addition of biofertilizer alone enhanced growth 4 times. Moreover, an enhancement of growth by 7 times was observed due to the addition of rock phosphate and K-feldspar, natural mineral fertilizers, in combination with the biofertilizer.