Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing microorganisms from the tea rhizosphere. The fungus developed a phosphate solubilization zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya broth showed high solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization of inorganic phosphates by the fungus was also observed under high stress levels of aluminium, iron, and desiccation, though the significant decline in phosphate solubilization was marked in the presence of aluminium than iron. The fungal isolate showed 100 % identity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence; and 28S rRNA gene, partial sequence.     

Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium

Solubilization of rock phosphate by Aspergillus niger was studied in solid-state fermentation on sugar-beet waste. This combination was selected after testing three agroindustrial waste materials, namely rice hulls, sugar-beet waste and alperujo. Sugar-beet waste was the best substrate for fungal growth with 69% mineralization, followed by rice hulls and alperujo. The fungus was successfully cultivated on sugar-beet waste supplemented with 3.0 g/l rock phosphate, acidifying the medium and thus decreasing the pH to 3–3.5. Solubilization of insoluble phosphate increased during the first half of the process, reaching a maximum of 292 g phosphate/ml, although a part of it was probably consumed by the mycelium.     

Utilization of phosphate from different sources by six plant species

Summary Six plant species, wheat, paspalum grass (Paspalum plicatulum), maize, molasses grass (Melinis minutiflora), soybean and buckwheat (Fagopyrum esculentum) were compared for their abilities to utilize phosphate from superphosphate, a calcined aluminum phosphate and four rock phosphates.Buckwheat showed an exceptional behaviour in that it could utilize all phosphates. For the other plants, only the calcined aluminum phosphate and one rock phosphate (hyperphosphate) had significant fertilizing values. Their efficiencies, relative to superphosphate, were 0.45 and 0.11 for wheat, 0.73 and 0.43 for paspalum grass, 0.50 and 0.37 for maize, 0.46 and 0.42 for molasses grass, 0.28 and 0.38 for soybean, and 0.72 and 1.08 for buckwheat, respectively.For three P sources, superphosphate, calcined aluminum phosphate and hyperphosphate, a relationship between soil acidity and P uptake was found. Soil pH in its turn was negatively related to the ratio of total equivalents of cations and those of anions absorbed. Consequenly, P uptake was positively related to the ratio of total equivalents of cations to those of anions absorbed. The same effect of plant species on soil pH could also explain the difference in uptake of P from sparingly soluble phosphates. The relative efficiencies of calcined aluminum phosphate and hyperphosphate for the various plant species were closely related to the ratio of total cations and total anions absorbed by these plants.on leave at the Agricultural University during 1977.     

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.     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).     

Solubilization of low-grade Indian rock phosphates and inorganic phosphates byBacillus licheniformis

Bacillus licheniformis solubilized a range of inorganic phosphates and five different Indian rock phosphates (low grade) to varying extent in broth culture and in soil. Statistical evaluation indicated that broth cultures alone were inadequate indicators of the potential for microbial solubilization of rock phosphates.     

Endophytic bacteria of <Emphasis Type="Italic">Mammillaria fraileana</Emphasis>, an endemic rock-colonizing cactus of the southern Sonoran Desert

The small cactus Mammillaria fraileana is a pioneer rock-colonizing plant harboring endophytic bacteria with the potential for nitrogen fixation and rock weathering (phosphate solubilization and rock degradation). In seeds, only a combination of culture-independent methods, such as fluorescence in situ hybridization, scanning electron microscopy, and fluorescence vital staining, detected significant amounts of non-culturable, but living, endophytic bacteria distributed underneath the membrane covering the embryo, in the undifferentiated tissue of the embryo, and in the vascular tissue. Large populations of culturable endophytic bacteria were detected in stems and roots of wild plants colonizing rocks in the southern Sonoran Desert, but not in seeds. Among 14 endophytic bacterial isolates found in roots, four isolates were identified by full sequencing of their 16S rRNA gene. In vitro tests indicated that Azotobacter vinelandii M2Per is a potent nitrogen fixer. Solubilization of inorganic phosphate was exhibited by Pseudomonas putida M5TSA, Enterobacter sakazakii M2PFe, and Bacillus megaterium M1PCa, while A. vinelandii M2Per, P. putida M5TSA, and B. megaterium M1PCa weathered rock by reducing the size of rock particles, probably by changing the pH of the liquid media. Cultivated seedlings of M. fraileana, derived from disinfected seeds and inoculated with endophytic bacteria, showed re-colonization 105 days after inoculation. Their densities decreased from the root toward the stem and apical zones. Functional traits in planta of culturable and non-culturable endophytic bacteria in seeds remain unknown.     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Phosphate supplying power of rock phosphates in an oxisol

Summary The P-supplying power of triple superphosphate, three apatitic rock phospates and a calcined aluminum rock phosphate were tested by measuring the quantities of fertilizer P recovered in soybean and in four chemical extractants, after 3-day and 75-day periods of contact between soil and fertilizer.The triple superphosphate supplied the highest amounts of P, but it lost efficiency during the longer incubation period. The rock phosphates maintained their original efficiencies, probably as a result of a balance between P released from the fertilizer and P converted into non-labile forms.The following coefficients of correlation between P uptake by soybean from an acid oxisol and P extracted by chemical extractants, after the two incubation periods, were found: 0.902^** for 0.01M CaCl₂; 0.823^** for anion-exchange resin; 0.720^** for 0.03N NH₄F+0.025N HCl; –0.037 (n.s.) for 0.025N H₂SO₄+0.050N HCl.The acid NH₄F solubilized residual calcined aluminum phosphate particles, and double acid extracted P from residual apatite particles, thus accounting for their poorer performances in predicting availability of fertilizer P.The relative efficiencies of the rock phosphates could largely be predicted after an incubation period of only three days. This finding attests to the presence in these rock phosphates of an easily soluble fraction of P which is not indicative of the degree of reactiveness of the phosphate as a whole.on leave at the Agricultural University during 1977.     

Microbial acceleration of aerobic pyrite oxidation at circumneutral pH

Pyrite (FeS₂) is the most abundant sulfide mineral on Earth and represents a significant reservoir of reduced iron and sulfur both today and in the geologic past. In modern environments, oxidative transformations of pyrite and other metal sulfides play a key role in terrestrial element partitioning with broad impacts to contaminant mobility and the formation of acid mine drainage systems. Although the role of aerobic micro‐organisms in pyrite oxidation under acidic‐pH conditions is well known, to date there is very little known about the capacity for aerobic micro‐organisms to oxidize pyrite at circumneutral pH. Here, we describe two enrichment cultures, obtained from pyrite‐bearing subsurface sediments, that were capable of sustained cell growth linked to pyrite oxidation and sulfate generation at neutral pH. The cultures were dominated by two Rhizobiales species (Bradyrhizobium sp. and Mesorhizobium sp.) and a Ralstonia species. Shotgun metagenomic sequencing and genome reconstruction indicated the presence of Fe and S oxidation pathways in these organisms, and the presence of a complete Calvin–Benson–Bassham CO₂ fixation system in the Bradyrhizobium sp. Oxidation of pyrite resulted in thin (30–50 nm) coatings of amorphous Fe(III) oxide on the pyrite surface, with no other secondary Fe or S phases detected by electron microscopy or X‐ray absorption spectroscopy. Rates of microbial pyrite oxidation were approximately one order of magnitude higher than abiotic rates. These results demonstrate the ability of aerobic microbial activity to accelerate pyrite oxidation and expand the potential contribution of micro‐organisms to continental sulfide mineral weathering around the time of the Great Oxidation Event to include neutral‐pH environments. In addition, our findings have direct implications for the geochemistry of modern sedimentary environments, including stimulation of the early stages of acid mine drainage formation and mobilization of pyrite‐associated metals.     

A critical stage in the formation of acid mine drainage: Colonization of pyrite by Acidithiobacillus ferrooxidans under pH-neutral conditions

A dominant Acidithiobacillus ferrooxidans ssp. was isolated from the supergene copper deposit in Morenci, Arizona, USA. Washed bacterial suspensions (10⁸ MPN per treatment), in pH‐neutral buffer, were inoculated onto pyrite cubes for 24 h. Heterogeneous bacterial absorption onto the pyrite removed approximately 90% of the viable bacteria from the inoculum. At T = 0, the bacteria were observed primarily in regions enriched in phosphorus. Over 30 days, the bacterial population on the pyrite cubes increased from 1.3 × 10⁷ to 2.9 × 10⁸ bacteria cm^?2. During this growth stage, low levels of thiobacilli (228 ± 167 MPN mL^?1) were also recovered from the fluid phase; however, this population decreased to zero within 30 days. Growth on pyrite occurred as micrometre‐scale planar microcolonies, a biofilm, coating the mineral surfaces. These microcolonies possessed viable thiobacilli, even after 4 months at ‘circumneutral pH’. Imaging the pyrite cubes using SEM‐EDS and scanning force microscopy demonstrated that the thiobacilli grew as iron oxy‐hydroxide‐cemented cells, leading to the formation of mineralized microcolonies. Removing the iron oxy‐hydroxides with oxalic acid did not dislodge the bacteria, demonstrating that the secondary minerals were not responsible for ‘gluing’ the bacteria to the pyrite surface. Removing organic material, i.e. the cells, by an oxygen plasma treatment revealed the presence of corrosion pits the size and shape of bacteria. Because of the inherent geochemical constraints on pyrite oxidation at neutral pH, the colonization of pyrite under circumneutral pH conditions must be facilitated by the development of an acidic nanoenvironment between the bacteria and the pyrite mineral surface.     

Morphology and ethology of the Late Devonian trace fossil Rhizocorallium from the Xichuan section of Central China

The trace fossil Rhizocorallium is abundant in the shallow-marine settings of the Upper Devonian Wangguangou Formation in Xichuan, Henan Province, northern margin of the South China block. Based on its morphological characteristics, the studied Rhizocorallium is attributed to Rhizocorallium commune var. auriforme. There is a significant compositional difference between the fillings of the marginal tube and the spreite. The fillings in the marginal tube are similar to the surrounding rock, with abundant bioclasts. The fillings in the spreite are relatively fine-grained and obviously affected by biological activities. Four types of pyrite framboids (pyritohedral, octahedron, cube and spherical) have been found within the Rhizocorallium. Moreover, the ratio of pyrite framboids diameter (D) to submicron crystal diameter (d) is <10, suggesting that they were formed by microbial activities. Relatively large pyrite framboids with smooth surface mainly occur in the marginal tube, while small pyrite framboids with sheath are abundant in the spreite. Energy spectrometer (EDS) analysis showed that the S/Fe ratio in the pyrite increased with decreasing crystal diameter, indicating different redox conditions in the marginal tube and the spreite. These features indicate that Rhizocorallium from the Xichuan section is not simply a foraging trace, but is probably a multi-functional, complex burrow system utilized for living, caching and farming microorganisms.     

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.     

Rock-phosphate mobilization induced by the alkaline uptake pattern of legumes utilizing symbiotically fixed nitrogen

Summary Soybean and alfalfa were grown on sand and soil to which P was added in the form of finely ground rock phosphates. When the legumes depended on NO₃ as N source, more anionic than cationic nutrients were absorbed. This resulted in a pH increase in the growth medium and in very low availability of P added as rock phosphate. When, however, the legumes made use of symbiotically fixed N, more cationic than anionic nutrients were absorbed leading to an acidification of the growth medium and an ensuing mobilization and higher availability of the rock phosphates.Symbiotic N fixation which initiates the chain of reactions leading to an increased availability of rock phosphate-P is dependent on photosynthate supply and on the availability of phosphate. Therefore, in a separate experiment it was investigated whether a priming effect exerted by a small quantity of added easily soluble phosphate, could enhance the availability of rock phosphate-P to legumes. Results obtained indicated that easily soluble phosphate might indeed be effective in this respect.     

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.     

Evidence For In Vitro and In Situ Pyrite Weathering By Microbial Communities Inhabiting Weathered Shale

Microbial pyrite oxidation is an important driver of biological weathering within shale, making a significant contribution toward biogeochemical cycling, bedrock expansion, and soil formation. These processes are of global importance, both within natural systems and in anthropogenic environments. Despite its significance, there is a lack of research that directly investigates microbe– pyrite interactions within shale. In this study, we use both field and laboratory approaches to inspect microbial pyrite oxidation in weathered shale environments within North Yorkshire, UK. Incubation of polished pyrite samples within iron-oxidizing enrichment cultures in vitro resulted in extensive colonization and surface pitting, demonstrating the weathering potential of shale microbial communities. Mineral samples were buried for 1 year within the floor of a shale rock mine, to explore pyrite bioweathering in situ. Image analysis revealed the formation of dissolution channels by microbial filaments, a novel mechanism of pyrite oxidation that broadens the taxonomic range of known microbe-pyrite interactions in weathered shale.     

Experimental Study of Microbial Pyrite Oxidation: A Suggestion for Geologically Useful Biosignatures

The oxidation of pyrite in cultures of Acidithiobacillus ferrooxidans (A.f) was studied. The experiments were performed at an initial pH of 2.5 at 28°C. The concentrations of total dissolved iron in solution and the pH were monitored during the first 36 days. Pyrite surfaces were examined by scanning electron microscopy and energy-dispersive spectrometry (SEM-EDS) after 100 days. The concentrations of total dissolved iron and hydrogen ions increased significantly in the presence of bacteria. SEM examination indicated that the crystal surfaces were subjected to two types of dissolution phenomena. Cracks were observable on the of crystal surfaces under both biotic and abiotic conditions, whereas rounded and polygonal pits appeared additionally on the surfaces under biotic conditions. The co-occurrence of the rounded and polygonal pits on the crystal surfaces and the presence of A.f at the pyrite surface suggests that A.f promotes pyrite oxidation by a contact mechanism. We propose that the rounded and polygonal pits be considered to represent a practical biosignature for tracing the evolution of microbial iron oxidation in the remote past.     

Degradation of massive pyrite: Physical,chemical, and bacterial effects

Massive pyrite was shown to produce soluble iron, hydrogen, and sulfate ions on exposure to air and water. The rate of this process was directly proportional to the surface area of the mineral; it was unaffected by a drop in the pH and the presence of the ferrous and sulfate ions formed. Cupic ion had no effect but ferric ion accelerated pyrite degradation until all the ferric ion was consumed, in accordance with FeS₂ + 2Fe³⁺ —>‐3Fe²⁺ + 2S°. Thiobacillus ferrooxidans increased pyrite degradation considerably; the presence of Thiobacillus thiooxidans had no influence on pyrite degradation.     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical. Received: 20 April 1998 / Received revision: 27 August 1998 / Accepted: 3 September 1998