A simple method to study microbial coal solubilization using Pluronic F-127-amended media

Summary Microbial coal solubilization and the extraction of solubilized coal products were carried out in media amended with polyol (Pluronic F-127), an agent which gels above 18°C but reverts to a liquid state at low temperature (4°C). The solubilized coal products, the unsolubilized coal particles and the mycelial mat were separated effectively by centrifugation at 4°C. The amount of coal solubilization was 30–50% higher in polyol-amended media than in agar media regardless of the microorganism. On the other hand, the amount of coal solubilization in polyol-amended control media was less compared to agar-amended control media.     

Biodesulphurization of coal: rate enhancement by sulphur-grown cells

Pyritic sulphur was removed from coal by growing Thiobacillus ferrooxidans in a 250 ml batch bioreactor. Thiobacillus ferrooxidansgrown on sulphur and which was added 5 days after initial inoculation, enhanced the iron solubilization rate by 35% as compared to control (without addition of sulphur-grown cells). About 93% pyritic sulphur was removed in presence of sulphur-grown cells as compared to 77% in the control.     

Solubilization of coal by an extracellular product fromStreptomyces setonii 75Vi2

Summary Several low-ranked coals were solubilized when placed on the surface of agar cultures ofStreptomyces viridosporous T7A andS. setonii 75Vi2. When grown in submerged cultureS. setonii 75Vi2 produced an extracellular component that was capable of solubilizing coals. The extracellular coal solubilizing component had a molecular weight of <10000 and was heat stable since, after 1h at 121°C, only 30–40% of the activity was lost. Treatment with any of three proteases also appeared to be ineffective in decreasing activity. These results suggest that coal solubilization byS. setonii 75Vi2 is nonenzymatic.Research supported by the Fossil Energy Advances Research and Technology Program, managed by the Pittsburg Energy Technology Center, U.S. Department of Energy, under Contract No. DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.     

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.     

Colonization and degradation of oxidized bituminous and lignite coals by fungi

Summary APenicillium sp. previously shown to grow on lignite coals degraded an air-oxidized bituminous coal (Illinois #6) to a material that was more than 80% soluble in 0.5 N NaOH. Scanning electron microscopy of the oxidized Illinois #6 revealed colonization of the surface by thePenicillium sp., production of conidia, and erosion of the coal surface. The average molecular weight (MW) of Illinois #6 degraded by the fungus and base-solubilized was approximately 1000 Da. The average MW for base-solubilized Illinois #6 that was not exposed to the fungus was 6000 Da, suggesting solubilizing mechanisms other than base catalysis. A spectrophotometric assay to quantify the microbial conversion of biosolubilized coal was developed. Standard curves were constructed based on the absorbance at 450 nm of different quantities of microbe-solubilized coal. An acid precipitation step was necessary to remove medium and/or microbial metabolites from solubilized coal to prevent overestimation of the extent of coal biosolubilization. Furthermore, the absorption spectra for different coal products varied, necessitating construction of standard curves for individual coals.     

Efficient solubilization,purification of recombinant extracellular α-amylase from <Emphasis Type="Italic">pyrococcus furiosus</Emphasis> expressed as inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding the Pyrococcus furiosus extracellular α-amylase (PFA) was amplified by PCR from P. furiosus genomic DNA and was highly expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL. The recombinant α-amylase was mainly expressed in the form of insoluble inclusion bodies. An improved purification method was established in this paper. The solubilization of the inclusion bodies was achieved by 90°C treatment for 3 min in Britton–Robinson buffer at pH 10.5. The solubilized PFA was then diluted and subsequently purified by Phenyl Sepharose chromatography. The overall yield of the new purification method was about 58,000 U/g wet cells, which is higher than previously reported.     

Organic sulfur removal from coal by microorganisms from extreme environments

Abstract: Microbial samples were collected from sulfurous, near neutral pH, thermal waters of Yellowstone Park. Thermophilic mixed cultures were identified that removed 90% of pyritic and sulfate sulfur and 33% of the organic sulfur from North Dakota lignite. The 30–40% organic desulfurization barrier was studied for possible inhibitors to organic sulfur removal from coal.     

Abdominal gland secretion ofBledius rove beetles as an effective defence against predators

Recent investigations indicated thatBacillus thuringiensis delta-endotoxins (DET) possess aphidicidal activity in an artificial diet bioassay. Crystalline preparations of CryIIA, CryIIIA and CryIVD solubilized in a slightly alkaline sucrose/amino acid diet clearly imparted toxicity toward adults of potato aphid,Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) after 4–5 days of continuous feeding. No obvious feeding deterrence was noted in these assays, as copious honeydew was produced and aphids often died in a feeding position. CryIIIA which was solubilized in aphid diet, but filtered to remove spores or crystalline toxin lacked aphidicidal activity. Spores from an acrystalliferous strain (EG2205) were not toxic by themselves at 7.75×10⁵ spores/ml aphid diet, but did restore toxicity to the filtered CryIIIA solution. Therefore, low levels of spores may be very effective in concert with DET for aphicidal activity. Results also clearly demonstrated that a suspension of crystalline CryIIIA alone, without spores, exhibited toxicity. Therefore, DET may be more toxic to the aphids when imbibed as a fine suspension, perhaps indicating the need for slow solubilization into the aphid midgut.     

Phosphate solubilization potential and stress tolerance of rhizobacteria from rice soil in Northern Thailand

Plant growth-promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. A total of 216 phosphate-solubilizing bacterial isolates were isolated from different rice rhizospheric soil in Northern Thailand. These isolate were screened in vitro for their plant growth-promoting activities such as solubilization of inorganic phosphate, ammonia (NH₃), catalase and cell wall-degrading enzyme activity. It was found that 100% solubilized inorganic phosphate, 77.77% produced NH₃ and most of the isolates were positive for catalase. In addition, some strains also produced cell wall-degrading enzymes such as protease (7%), chitinase (1%), cellulase (3%) and β-glucanase (3%), as evidenced by phenotypic biochemical test and quantitative assay using spectrophotometry. The isolates could exhibit more than two or three plant growth-promoting (PGP) traits, which may promote plant growth directly or indirectly or synergistically. Part of this study focused on the effect of NaCl, temperature, and pH on a specific the bacterial isolate Acinetobacter CR 1.8. Strain CR 1.8 was able to grow on up to 25% NaCl, between 25 and 55°C, and at pH 5–9. Maximum solubilization of tricalcium phosphate and aluminium phosphate was obtained at neutral pH, and 37°C. Strain CR 1.8 had protease activity but no cellulase, β-glucanase and cellulase activities.     

Bioleaching of cobalt from smelter wastes byThiobacillus ferrooxidans

Summary The bioleaching of cobalt from domestic, industrial smelter wastes was studied.Thiobacillus ferrooxidans solubilized Co from sulfidic dross furnace mattes. At pulp densities of 4% (w/v) up to 600 mg of Co per liter of leaching solution was released from nickel matte, corresponding to removal of about two-thirds of the original amount of Co in the matte. Bioleaching methods may be useful as a component of a process for solubilization and recovery of Co from sulfidic smelter mattes.     

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.     

Leaching of copper converter slag withAspergillus niger culture filtrate

Leaching of copper converter slag of M/s Hindustan Copper Ltd, Ghatshila (Bihar, India) was carried out usingAspergillus niger culture filtrate. The effects of the duration of leaching, temperature, pulp density and the addition of hydrochloric acid were studied.A. niger culture filtrate solubilized metals from the converter slag at levels of 18.70% copper, 7.40% nickel and 4.00% cobalt. Addition of hydrochloric acid was found to improve copper, nickel and cobalt solubilization to 46.52, 27.90 and 37.96%, respectively. HPLC analysis of the fungal culture filtrate revealed the presence of succinic and citric acids. Therefore, leaching of the slag was also carried out with matching concentrations of these organic acids individually as well as with both mixed together. Results are discussed.     

Leaching of zinc from an industrial filter dust withPenicillium,Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

Summary Heterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Efficient phosphorus solubilization by mutant strain of Xanthomonas campestris using different carbon, nitrogen and phosphorus sources

The phosphate solubilization activity of Xanthomonas campestris was measured in both the wild type and mutant strains using various carbon and nitrogen sources. Glucose was found to be the best in both (wild 39.9%; mutant 67.1%) strains followed by sucrose (46.8%) in the mutant and molasses (36.0%) in the wild type. Ammonium sulphate was the best nitrogen source for both the strains, followed by ammonium nitrate and urea. Dicalcium phosphate (DCP) was solubilized maximally by both the strains followed by tricalcium phosphate (TCP) and rock phosphate (RP) when various concentrations of different phosphate sources were tested.     

Comparative studies of phenotypic and genetic characteristics between two desulfurizing isolates of Rhodococcus erythropolis and the well-characterized R. erythropolis strain IGTS8

Two Rhodococcus erythropolis isolates, named A66 and A69, together with the well-characterized R. erythropolis strain IGTS8 were compared biochemically and genetically. Both isolates, like strain IGTS8, desulfurized DBT to 2-hydroxybiphenyl (2-HBP), following the 4S pathway of desulfurization. Strain IGTS8 showed the highest (81.5%) desulfurization activity in a medium containing DBT at 30 °C. Strain A66 showed approximately the same desulfurization activity either when incubated at 30 °C or at 37 °C, while strain A69 showed an increase of desulfurization efficiency (up to 79%) when incubated at 37 °C. Strains A66 and A69 were also able to grow using various organosulfur or organonitrogen-compounds as the sole sulfur or nitrogen sources. The biological responses of A66, A69 and IGTS8 strains to a series of mutagens and environmental agents were evaluated, trying to mimic actual circumstances involved in exposure/handling of microorganisms during petroleum biorefining. The results showed that strains A69 and IGTS8 were much more resistant to UVC treatment than A66. The three desulfurization genes (dszA, dszB and dszC) present in strains A66 and A69 were partially characterized. They seem to be located on a plasmid, not only in the strain IGTS8, but also in A66 and A69. PCR amplification was observed using specific primers for dsz genes in all the strains tested; however, no amplification product was observed using primers for carbazole (car) or quinoline (qor) metabolisms. All this information contributes to broaden our knowledge concerning both the desulfurization of DBT and the degradation of organonitrogen compounds within the R. erythropolis species.     

Characterization of free and immobilized (<Emphasis Type="Italic">S</Emphasis>)-aminotransferase for acetophenone production

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355–362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.     

Phosphate-solubilizing microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an abandoned rock phosphate mine

The abandoned “Monte-Fresco” rock phosphate mine in Táchira, Venezuela, was sampled to study the biodiversity of phosphate-solubilizing microorganisms (PSM). Rhizosphere and bulk soils were sampled from colonizer plant species growing at a mined site where pH and soluble P were higher than the values found at a near by unmined and shrubby soil. Counting and isolating of PSM choosing strains showing high solubilization halos in a solid minimal medium with hydroxyapatite as phosphate source were evaluated using ammonia or nitrate as nitrogen sources and dextrose, sucrose, and mannitol as carbohydrate sources. A larger number of PSM were found in the rhizospheric than in the bulk soil. Six fungal strains belonging to the genus Penicillium and with high hydroxyapatite dissolution capacities were isolated from bulk soil of colonizer plants. Five of these strains had similar phenotypes to Penicillium rugulosum IR-94MF1 but they solubilized hydroxyapatite at different degrees with both nitrogen sources. From 15 strains of Gram-negative bacteria isolated from the rhizosphere of colonizer plants, 5 were identified as diazotrophic free-living encapsulated Azotobacter species able to use ammonium and/or nitrate to dissolve hydroxyapatite with glucose, sucrose and/or mannitol. Different nitrogen and carbohydrate sources are parameters to be considered to further characterize the diversity of PSM.