Solubilization of rock phosphates byRhizobium andBradyrhizobium

The ability ofRhizobium andBradyrhizobium strains to solubilize phosphate from hydroxyapatite was determined in a medium containing NH₄Cl or KNO₃. The presence of NH₄ ⁺ in the medium resulted in higher solubilization of phosphate as compared to the presence of KNO₃, with the exception ofR. leguminosarium bv. viceae strain TAL 1236 and 1402 which solubilized comparable amounts of phosphate in a medium containing either KNO₃ or NH₄Cl. These results suggest that the strains employ two different mechanisms for phosphate solubilization, one depending on the presence of NH₄ ⁺, the other not requiring its presence. Temperature and aeration (O₂ demand) optima were 30°C and 4.2 Hz (shaking frequency), respectively. In nonsterile soil the tested strain (R. meliloti TAL 1236) was very effective in solubilizing rock phosphate.     

Plant Growth-Promoting Effect of the Chitosanolytic Phosphate-Solubilizing Bacterium Burkholderia gladioli MEL01 After Fermentation with Chitosan and Fertilization with Rock Phosphate

Phosphate-solubilizing bacteria (PSB) are important plant growth-promoting rhizobacteria that can increase soil fertility through the solubilization of insoluble inorganic phosphate and organophosphorus. In this study, a PSB, Burkholderia gladioli MEL01, was isolated and identified from rice–wheat rotation rhizosphere soil. MEL01 had an excellent phosphate-solubilizing capacity (reaching 107.69 mg/L) toward insoluble inorganic phosphate rock phosphate. HPLC analysis revealed that the mechanism of phosphate solubilization of MEL01 was probably due to secreted oxalic acid and gluconic acid transformation of phosphate from insoluble to soluble. MEL01 also exhibited 4030 U/L specific chitosanase activity when cultured with chitosan fermentation medium. Interestingly, the chitosan hydrolysis product chitooligosaccharide could significantly enhance the MEL01 phosphate-solubilizing capacity. Pot experiments showed that MEL01 chitosan medium fermentation liquor (MCMFL) could promote improvement of soil available phosphorus and pakchoi growth when supplemented with phosphate rock phosphate as the phosphate fertilizer. In addition, pot experiments demonstrated that MCMFL could also promote the growth of wheat, which could decrease the amount of compound fertilizer used. Microbial diversity analysis showed that the genera Pseudomonas, Burkholderia, Mycoplana, and Cellvibrio were enriched, which might participate in synergetic phosphate solubilization. Therefore, after fermentation with chitosan and fertilization with rock phosphates, MEL01 has potential as a phosphate biofertilizer in ecological agricultural production.

     

Characterization of the mineral phosphate solubilizing activity of <Emphasis Type="Italic">Serratia marcescens</Emphasis> CTM 50650 isolated from the phosphate mine of Gafsa

The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO₄), tri-calcium phosphate (Ca₃(PO₄)₂) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO₄, Ca₃(PO₄)₂, hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP.     

Effect of phosphate-solubilizing bacteria and vesicular-arbuscular mycorrhizal fungi on the utilization of Bayovar rock phosphate by alfalfa plants using a sand-vermiculite medium

Phosphate-solubilizing bacteria (PSB) exhibited a high efficiency to improve plant growth and nutrition in the presence of Bayovar rock phosphate when sand-vermiculive was used as a culture medium. Treatments with dual inoculum (PSB plus mycorrhiza) significantly (P≤0.05) increased alfalfa growth. Bacteria-microbial fungi interactions resulted in a greater utilization of the rock phosphate added to the rooting medium. Although Bayovar rock phosphateper se can be considered an inert substrate because it did not stimulate plant growth, metabolites released by PSB were able to transform the rock into available forms which could be utilized by alfalfa plants.Glomus fasciculatum was the most efficient mycorrhizal endophyte under the experimental conditions employed.     

Plasmid Profiles of Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on a medium with Fe²⁺, as well as adapted to such oxidation substrates as S⁰, FeS₂, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on a medium with Fe²⁺. One plasmid was found in strain TFL-2; two plasmids, in strains TFO, TFBk, and TFV-1; and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S⁰, FeS₂, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS₂, four after growth on S⁰, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Kinetics of the Removal of Iron Pyrite from Coal by Microbial Catalysis

Different strains of Thiobacillus ferrooxidans and Thiobacillus thiooxidans were used to catalyze the oxidative dissolution of iron pyrite, FeS₂, in nine different coal samples. Kinetic variables and parametric factors that were determined to have a pronounced effect on the rate and extent of oxidative dissolution at a fixed Po₂ were: the bacterial strain, the nitrogen/phosphorus molar ratio, the partial pressure of CO₂, the coal source, and the total reactive surface area of FeS₂. The overall rate of leaching, which exhibited a first-order dependence on the total surface area of FeS₂, was analyzed mathematically in terms of the sum of a biochemical rate, ν₁, and a chemical rate, ν₂. Results of this study show that bacterial desulfurization (90 to 98%) of coal samples which are relatively high in pyritic sulfur can be achieved within a time-frame of 8 to 12 days when pulp densities are ≤20% and particle sizes are ≤74 μm. The most effective strains of T. ferrooxidans were those that were isolated from natural systems, and T. ferrooxidans ATCC 19859 was the most effective pure strain. The most effective nutrient media contained relatively low phosphate concentrations, with an optimal N/P molar ratio of 90:1. These results suggest that minimal nutrient additions may be required for a commercial desulfurization process.     

Solubilization of inorganic phosphate byRhizobium

A large number of strains ofRhizobium were able to solubilize the insoluble phosphate compound, hydroxy-apatite, in liquid culture. Solubilization of hydroxyapatite byRhizobium was not mediated by an enzyme but acidity developed in the cultures was involved in the process. An inverse relationship between the level of soluble phosphate and medium pH was evident. The ability to solubilize hydroxyapatite varied among the strains. In a medium without NH ₄ ⁺ , some of the strains showed better activity than when NH ₄ ⁺ was present, suggesting involvement of different mechanisms for phosphate solubilization.R. meliloti SU 47 produced 2-ketogluconic acid along with an unidentified acid in the medium containing NH ₄ ⁺ . 2-Ketogluconic acid was identified as the major factor in inorganic phosphate solubilization. Initial presence of soluble phosphate in the medium had no discernible influence on the extent of hydroxyapatite solubilization. Initial presence of calcium reduced solubilization of phosphate and addition of EDTA to stationary phase cultures caused an increase in the level of soluble phosphate.     

Continuous microbial leaching of a pyritic concentrate by Leptospirillum-like bacteria

Summary Continuous leaching of a pyritic flotation concentrate by mixed cultures of acidophilic bacteria was studied in a laboratory scale airlift reactor. Enrichment cultures adapted to the flotation concentrate contained Thiobacillus ferrooxidans and Thiobacillus thiooxidans. During the late stationary growth phase of these thiobacilli growth of Leptospirillum-like bacteria was observed, too. In discontinuous cultivation no significant influence of Leptospirillum-like bacteria on leaching rates could be detected. During continuous leaching at pH 1.5 Leptospirillum-like bacteria displaced Thiobacillus ferrooxidans. The iron leaching rate achieved by Leptospirillum-rich cultures was found to be up to 3.9 times higher than that by Leptospirillum-free cultures.     

Effects of organic compounds on growth of chemostat cultures of Thiomicrospira pelophila,Thiobacillus thioparus and Thiobacillus neapolitanus

Summary Cultures of Thiomicrospira pelophila, Thiobacillus thioparus and Thiobacillus neapolitanus were grown in thiosulfate-limited chemostats in a mineralsthiosulfate medium with and without organic supplements. Acetate, succinate and mixtures of amino acids increased the dry weight by 12–24% and the protein by 11–38%. Addition of both acetate and succinate had a cumulative effect. Saccharose, glucose, fructose, ribose, glycerol, glycerate, pyruvate, lactate or malate were without effect. The increase in dry weight of T. neapolitanus by ¹⁴C-acetate was directly related to the relative contribution of this compound to the total cell carbon.In CO₂-limited cultures of T. neapolitanus the effects of acetate on dry weight and protein were similar to those found in thiosulfate-limited cultures. In CO₂-limited cultures of T. pelophila a combination of acetate and succinate caused an increase in dry weight of 27% and of 50% in protein, the increase in protein being twice as high as in thiosulfate-limited cultures.There were no measurable differences in the activities of ribulosediphosphate carboxylase (RudPcase) in cell free extracts obtained from thiosulfate- or CO₂-limited cultures of T. pelophila or T. neapolitanus grown in the presence or absence of organic compounds. In T. pelophila the RudPcase activity was almost constant at all growth rates tested, and independent of the type of growth-limitation. For T. neapolitanus the specific RudPcase activity varied slightly with the growth rate. In CO₂-limited cultures the activity was three times that found in thiosulfate-limited cultures, thus showing that the RudPcase activity can be influenced by nutritional conditions.     

Pyrite oxidation in acid sulphate soils: The role of miroorganisms

Summary A study has been made of microbial processes in the oxidation of pyrite in aicd sulphate soil material. Such soils are formed during aeration of marine muds rich in pyrite (FeS₂). Bacteria of the type ofThiobacillus ferrooxidans are mainly responsible for the oxidation of pyrite, causing a pronounced acidification of the soil. However, becauseThiobacillus ferrooxidans functions optimally at pH values bellow 4.0, its activity cannot explain the initial pH drop from approximately neutral to about 4. This was shown to be a non-biological process, in which bacteria play an insignificant part. AlthoughThiobacillus thioparus andThiobacillus thiooxidans were isolated from the acidifying soil, they did not stimulate oxidation of FeS₂, but utilized reduced sulphur compounds, which are formed during the non-biological oxidation of FeS₂.Ethylene-oxide-sterilized and dry-sterilized soil inoculated with pure cultures of mixtures of various thiobacilli or with freshly sampled acid sulphate soil soil did not acidify faster than sterile blanks.Thiobacillus thiooxians. Thiobacillus thioparus. Thiobacillus intermedius andThiobacillus perometabolis increased from about 10⁴ to 10⁵ cells/ml in media with FeS₂ as energy source. However, FeS₂ oxidation in the inoculated media was not faster than in sterile blanks.Attempts to isolate microorganisms other thanThiobacillus ferrooxidans, like metallogenium orLeptospirillum ferrooxidans, which might also be involved in the oxidation of FeS₂ were not successful.Addition of CaCO₃ to the soil prevented acidification but did not stop non-biological oxidation of FeS₂.     

Isolation and screening of microorganisms dissolving low-grade rock phosphate

Several yeasts, fungi and bacteria isolated from the rhizosphere of leguminous crops and soils of rock phosphate deposit area were found to solubilize low-grade Mussorie rock phosphate. Of the several yeasts and fungi,Schawanniomyces occidentalis, Aspergillus awamori andPenicillium digitatum were better than others in rock phosphate solubilization. Among bacterial isolates from soils of rock phosphate deposits, Gram-negative motile rods were more effective than Gram-negative non-motile rods in dissolving rock phoshates. The most efficient bacteria were identified as strains ofPseudomonas striata. All the microorganisms acidified the liquid medium but there was no relationship between the rock phosphate dissolved and the decrease in pH of the culture broth.     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of ¹³C into metabolic intermediates and end products when cells were labelled with [1‐¹³C], [2‐¹³C], or [¹³C₆]glucose, in combination with ¹⁴C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.     

Dissolution of Fluorapatite by Pseudomonas fluorescens P35 Resulting in Fluorine Release

Chemical weathering of fluorine-bearing minerals is widely accepted as the main mechanism for the release of fluorine (F) to groundwater. Here, we propose a potential mechanism of F release via microbial dissolution of fluorapatite (Ca₅(PO₄)₃F), which has been neglected previously. Batch culture experiments were conducted at 30°C with a phosphate-solubilizing bacteria strain, Pseudomonas fluorescens P35, and rock phosphates as the sole source of phosphate for microbial growth in parallel with abiotic controls. Rock phosphates consisted of 55–91% of fluorapatite and 5–10% of dolomite before microbial dissolution as indicated by X-ray diffraction (XRD). Mineral composition and morphology changed after microbial dissolution characterized by the disappearance of dolomite and the development of etched cavities on rock phosphate surfaces. The pH of media used was approximately 7.4 at the beginning and increased gradually to 7.7 in abiotic controls; with the inoculum, the pH decreased to acidic values of 3.7–3.8 after 27 h. Phosphate, calcium, and fluoride were released from the rock phosphate to the acidified medium. At 42 h, the concentration of F reached 8.1–10.3 mg L^?1. The elevated F concentration was two times higher than the F levels in groundwater in regions diagnosed with fluorosis, and was toxic to the bacteria, as demonstrated by a precipitous decrease in live cells. Geochemical modeling demonstrated that the oxidation of glucose (the carbon source for microbial growth in the medium) to gluconic acid could decrease the pH to 3.7–3.8 and result in the dissolution of fluorapatite and dolomite. Dolomite and fluorapatite remained unsaturated, while concentrations of dissolved phosphorus (P), calcium (Ca), and F increased throughout the time course Fluorite reached saturation [saturation index (SI) 0.22–0.42] after 42 h in rock phosphate–amended biotic systems. However, fluorite was not detected in XRD patterns of the final residue from microcosms. Given that phosphate-solubilizing bacteria are ubiquitous in soil and groundwater ecosystems, they could play an important role in fluorapatite dissolution and the release of F to groundwater.     

Continuous culture of Thiobacillus ferrooxidans on a zinc sulfide concentrate

A zinc sulfide concentrate was leached microbiologically by Thiobacillus ferrooxidans in a continuous stirred tank reactor. A model was developed to predict, the leaching kinetics when the bacterial growth rate was not limited by any substrate other than the zinc concentrate, and it was modified to explain the observed results. Stable steady sates were obtained over a range of dilution rates from 0.0171 to 0.1038 hr^?1. Because a solid substrate was used, the specific growth rate of the bacaeria was not a unique function of the subastrate concentration, and conventional contnuous culture theory based on the Monod equation did not apply to this system. The leaching rates and bacterial growth rates were first order in mineral surface area cocentration.     

Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans

From cell yields of Thiomicrospira denitrificans grown in the chemostat at different growth rates under anaerobic conditions a value of 1.4mm S₂O _inf3 ^sup= per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 g dry wt per mole S₂O _inf3 ^sup= for the true growth yield.Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans. Though in Thiobacillus denitrificans at D=0.03 h^-1 under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans. Under aerobic conditions at D=0.03 h^-1 values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate.As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate.Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans. This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase.     

Use of partially acidulated rock phosphate as a possible means of minimising phosphate fixation in acid soils

Summary To examine the possibility of minimising phosphate fixation the lateritic soil at various levels of liming was incubated with phosphate rock from U.A.R. acidulated to different degree viz. 0, 10, 20, 50, and 100 per cent both with phosphoric and nitric acid. The soil was incubated for 90 days on addition of different phosphate carriers at the rate of 100 ppm total P₂O₅ containing different proportion of water-soluble, citrate-soluble and insoluble phosphorus. Samples were drawn at an interval of 30 days. Bray's p₁ and pH of the soil samples were measured. The dry-matter yield and uptake of phosphorus by two successive crops of maize grown in pots, the treatments being same as in incubation study, were well correlated with the Bray's p₁. Ground rock phosphate and 10 per cent acidulated material were effective in minimising the fixation in soil of pH 4.0 whereas 50 per cent acidulation was suitable for soils of higher pHi.e. 5.6 and 6.5. H₃PO₄ acidulated material was proved superior to HNO₃ acidulated product. The use of partially acidulated rock phosphate for acid soils may be recommended to receive economic return. Associate Professor Senior Research Assistant.     

Effect of phosphate concentration on the production of dextransucrase by<Emphasis Type="Italic"> Leuconostoc mesenteroides</Emphasis> NRRL B512F

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K₂HPO₄. In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.     

STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

Combined degradation of covellite by Thiobacillus thiooxidans and Thiobacillus ferrooxidans

Summary In the presence of iron, which is always associated with natural sulphide ores, the percentages of copper dissolution in the bioleaching of covellite were 34 and 45 % when Thiobacillus thiooxidans and Thiobacillus ferrooxidans were used together and when an indirect bioleaching with attached bacteria was performed respectively. In the latter, the percentage of copper dissolution was still higher than the percentages obtained with pure cultures (36 % with a T. thiooxidans culture and 40 % with a T. ferrooxidans culture).     

Studies on the growth of Thiobacillus ferrooxidans

Summary A method for enumeration of viable numbers of Thiobacillus ferrooxidans using membrane filters on ferrous-iron agar is presented. Factors affecting colony production were the concentration and brand of agar, pH of the medium, and type of membrane filter. The results suggest that inhibition of T. ferrooxidans by agar is a result of the acid hydrolysis of agar, the main product of which is d-galactose. Colony development was suppressed by aged medium, by acid-hydrolysed agar and by 0.1% galactose. Sartorius and Millipore membrane filters were suitable for the experiments, whereas Oxoid MF-50 membranes virtually suppressed the production of colonies. The method was employed to follow growth of T. ferrooxidans in pH 1.3 medium. The viable cell numbers were correlated with ¹⁴CO₂-fixation and ferrous iron oxidation. Generation time was 6 h 22 min with a yield of 2.2×10¹² organisms/g atom Fe²⁺ oxidized. Growth of T. neapolitanus on thiosulphate medium was not affected by agar-type or membrane filters and yield of the organism was 1.5×10¹³ organisms/g molecule Na₂S₂O₃ oxidized.