Characterization of hydrocarbon emulsification and solubilization occurring during the growth of Endomycopsis lipolytica on hydrocarbons

A comparative characterization of the phenomena of hydrocarbon emulsification and solubilization taking place during the growth of Endomycopsis lipolytica on n-alkanes and alkenes was made. Evidence was obtained for the cellular production of different factors involved in emulsification and solubilization of hydrocarbons. It was shown that the production of these factors closely followed cell growth. The inducible nature of the alkane solubilizing factor was demonstrated using actidione as inhibitor. Whereas emulsifying factor was demonstrated using actidione as inhibitor. Whereas emulsifying factors showed a broad affinity to some particular hydrocarbons, solubilizing factor was found to be highly specific for the particular hydrocarbon on which the cells were grown. The emulsifying factor was heat stable whereas the solubilizing factor was highly unstable even at ?4°C. Metal-ion chelating agents strongly inhibited the activity of both of the factors. A crude isolate of the alkane emulsifying factor was obtained and its peptide characteristics were demonstrated. Using EDTA as an inhibitor for the emulsification–solubilization activity, evidence was obtained for the predominent role played by the emulsification–solubilization mechanism in the uptake of alkane by yeast cells.     

Predominant role of hydrocarbon solubilization in the microbial uptake of hydrocarbons

Using EDTA and proteolytic enzymes to suppress hydrocarbon solubilization, direct evidence is presented in support of the mechanism of liquid hydrocarbon uptake by microbial cells predominantly from the solubilized or accommodated substrate. EDTA (2-5mM) strongly inhibited growth of three yeast species and one bacterial species on n-hexadecane and the inhibition was removed by surfactant-emulsified and surfactant-solubilized alkane and also by excess addition of Ca(2+). EDTA had no inhibitory effect on the growth of the organisms on soluble substrates such as sodium acetate and nutrient broth or on n-pentane, a volatile alkane which was primarily transported by diffusion from gas phase. EDTA was shown to have no significant effect on the adsorption of cells on alkane drops. EDTA inhibition of growth was considered to be due to suppression of alkane solubilization, brought about by the solubilizing factor(s) produced by cells. It was shown that this chelating agent did not inhibit the growth of yeast on solubilized alkane but strongly inhibited its growth on alkane drops. It was demonstrated that adherent capacity of microbial cell to oil phase was closely related to the state of hydrocarbon emulsification and had no relationship to the ability of organisms to grow on hydrocarbon. Certain proteolytic enzymes inhibited the growth of yeast on alkane, presumably by digesting the alkane solubilizing protein, but not on glucose, and the inhibition was removed by a supply of surfactant-emulsified and surfactant-solubilized alkane. Specific solubilization of various hydrocarbon types during growth of the prokaryotic bacterial strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited strain was demonstrated. The specific solubilization of hydrocarbon was strongly inhibited by EDTA, and the inhibition was removed by excess Ca(2+). It was concluded that specific solubilization of hydrocarbons is an important mechanism in the microbial uptake of hydrocarbons.     

The effect of temperature on eicosane substrate uptake modes by a marine bacterium Pseudomonas nautica strain 617: relationship with the biochemical content of cells and supernatants

Three hydrocarbon uptake modes (adherence, emulsification and solubilization) were identified and quantified in cells and supernatants of a mesophilic marine bacterium Pseudomonas nautica strain 617 grown on eicosane. The adherence capacity was related to the enrichment of cells with wax esters and glycolipids. The emulsifying activity was related to the presence of extracellular biosurfactants composed of proteins, carbohydrates and lipids (35:63:2). The intensity of substrate uptake modes was sensitive to temperatures currently found in the original environment of P. nautica (16°C, 20°C and 32°C). When temperature decreased, a significant increase in adherence and emulsifying activity was observed in relation to biochemical changes, whereas solubilizing activity decreased. The marine bacterium was able to degrade 53–59% eicosane at the end of exponential growth after 13, 5 and 3 days incubation at 16°C, 20°C and 32°C respectively.     

Mode of uptake of insoluble solid substrates by microorganisms. II: Uptake of solid n-alkanes by yeast and bacterial species

Using two species of yeast and one of bacterium, evidence has ben obtained which indicates that the microbial uptake of solid alkane powders occurs primarily through a substrate solubilization mechanism. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited the growth of these organisms on alkane powder; the inhibition could be removed vai a supply of artificially solubilized alkane. One of the yeast strians, which was a mutant incapable of growing on solid alkane powder and liquid alkane, could grow very well on artifically solubilized alkanes. It was demonstrated that the solid alkane solubilization rate during microbial growth could satisfactorily account for the maximal alkane uptake rate actully observed during growth. The specificity of solubilization for the solid alkane used as the growth substrate was demonstrated.     

Different modes of hydrocarbon uptake by two Pseudomonas species

The mode of n-hexadecane uptake by two organisms-Pseudomonas M 1 and Pseudomonas N 1-was studied. During the growth of Pseudomonas M 1 on n-hexadecane, no extracellular biosurfactant/bioemulsifier was produced, no significant n-hexadecane pseudosolubilization was observed, and the reduction of surface and interfacial tensions in the cell-free culture broth was negligible. EDTA, a known inhibitor of hydrocarbon pseudosolubilization, did not inhibit the growth of the organism on n-hexadecane. Normal hexadecane-grown cells showed strong surface-active properties and capacity to adhere firmly to hydrocarbon phase. It was concluded that in this organism, surface-active properties of the cells facilitate attachment of cells to the hydrocarbon-water interface generated by agitation, and promote substrate uptake and growth; no hydrocarbon pseudosolubilization or extracellular mediator is involved in the substrate uptake. Pseudomonas N 1 grew on n-hexadecane much faster than Pseudomonas M 1. Growth of this organism on n-hexadecane was associated with the extracellular production of biosurfactant-bioemulsifier and n-hexadecane pseudosolubilizing factor; the growth was strongly inhibited by 5 mM EDTA, indicating that hydrocarbon pseudosolubilization was the dominant factor in substrate uptake. The rate of n-hexadecane pseudosolubilization was high enough to account for the substrate up take rate. Hydrocarbon emulsifying and n-hexadecane pseudosolubilizing factors were isolated and tentatively characterized as lipoprotein and glycoprotein, respectively. Both factors act in a synergistic manner to provide enhanced hydrocarbon transport to cells through pseudosolubilization. It is proposed that this facility of mediated hydrocarbon transport is the basis for the relatively fast rate of growth of Pseudomonas N 1 on hydrocarbon.     

黑曲霉解磷能力的影响因素及培养条件优化

本文探讨了培养条件下黑曲霉解磷能力的主要影响因素。通过单孢株筛选得到解磷能力较强的菌株Xj-2,其在液体培养基中的解磷能力达到539.90 mg·L^-1。解磷发酵动力学模型模拟发现,其解磷能力在培养的第4 天达到平稳,可作为终止发酵时间。不同磷源培养液中菌株Xj-2的解磷量依次为磷酸钙(539.90 mg·L^-1)>磷酸锌(238.45 mg·L^-1)>磷酸铁(182.64 mg·L^-1)>磷矿粉(71.80 mg·L^-1)>磷酸铝(24.40 mg·L^-1)。通过单因素试验并结合响应面优化,研究了其解磷最佳条件。结果表明: 碳源对Xj-2的解磷能力影响最大,其次是菌群密度和培养液pH。当培养温度35 ℃、转速160 r·min^-1、培养液pH 6.0、氮源(尿素)浓度0.79 g·L^-1、碳源(葡萄糖)浓度10.00 g·L^-1、菌群密度3.8%、培养时间为4 d时,Xj-2的解磷能力最高,为616.81 mg·L^-1。     

Characterization of a Phosphate Solubilizing and Antagonistic Strain of Pseudomonas putida (B0) Isolated from a Sub-Alpine Location in the Indian Central Himalaya

The morphological, biochemical, and physiological characteristics of a phosphate solubilizing and antagonistic bacterial strain, designated as B0, isolated from a sub-alpine Himalayan forest site have been described. The isolate is gram negative, rod shaped, 0.8 x 1.6 microm in size, and psychrotrophic in nature that could grow from 0 to 35 degrees C (optimum temp. 25 degrees C). It exhibited tolerance to a wide pH range (3-12; optimum 8.0) and salt concentration up to 4% (w/v). Although it was sensitive to kanamycin, gentamicin, and streptomycin (<10 microg mL(-1)), it showed resistance to higher concentrations of ampicillin, penicillin, and carbenicillin (>1000 microg mL(-1)). The isolate showed maximum similarity with Pseudomonas putida based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (4-28 degrees C), and maximum activity (247 microg mL(-1)) was recorded at 21 degrees C after 15 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced chitinase, ss-l,3-glucanase, salicylic acid, siderophore, and hydrogen cyanide. The plant growth promotion and antifungal properties were demonstrated through a maize-based bioassay under greenhouse conditions. Although the bacterial inoculation was found to result in significant increment in plant biomass, it stimulated bacterial and suppressed fungal counts in the rhizosphere. The present study is important with respect to enumerating microbial diversity of the colder regions as well as understanding the potential biotechnological applications of native microbes.     

The uptake of pristane (2,6,10,14-tetramethylpentadecane) into phospholipid bilayers as assessed by NMR, DSC, and tritium labeling methods.

Unilamellar dioleoylphosphatidylcholine (DOPC) liposomes (250 microM) incorporated 2 mol% of [3H]pristane at 37 degrees C after addition of 50 microM pristane solubilized with beta-cyclodextrin. Conventional solubilization in dimethyl sulphoxide resulted in much lower uptake. Premixing of perdeuterated pristane with DOPC and dipalmitoylphosphatidylcholine (DPPC) prior to the formation of multilamellar liposomes resulted in homogeneous incorporation of up to 5 mol% pristane at 22 degrees C and 50 degrees C, respectively, as observed by 2H-NMR. Lipid order parameters measured by 31P and 2H-NMR remained unchanged after pristane uptake. Pristane induced the transformation of part of the dioleoylphosphatidylethanolamine (DOPE)/DOPC (3:1, mol/mol) liquid crystalline lamellar phase into an inverse hexagonal phase. 5 mol% pristane in DPPC bilayers decreased the midpoint of the main phase transition temperature of DPPC from 41.5 degrees C to 40.9 degrees C. Upon cooling in the temperature range from 41 degrees C to 36 degrees C, pristane was either displaced from the DPPC bilayer or the mode of incorporation changed. These results may aid in defining the mechanisms whereby pristane, an isoprenoid C19-isoalkane, induces plasmacytomagenesis in mice.     

Mode of uptake of insoluble solid substrates by microorganisms. I: Sterol uptake by an arthrobacter species

The mode of uptake of sterols, which are nearly insoluble in water by an Arthrobacter species, was studied on the basis of substrate transfer via the aqueous phase (solubilization/pseudosolubilization) and through direct contact with sterol particles. Growth of the organism, on stero powder was predominantly in nonlogarithmic in character, indicating a possible limitation of substrate transfer. Soluble sterol was shown to be the preferential form of the substrate for assimilation by the organism. Evidence was obtained for increased solubilizition of beta-sitosterol and cholesterol during microbial growth on these substrates. But the rate of solubilization of beta-sitosterol (3.06 mg L(-1) h(-1)) was too inadequate to account for the observed substrate uptake rare (107 mg L(-1) h(-1)) during growth. A cholesterol solubilization rate of 44 mg L(-1) h(-1) could, however, account to an appreciable extent for the observed cholesterol uptake rate of 140 mg L(-1) h(-1) during growth. Increasing attachement of cells to sterol particles during growth was observed by microscopic examination, indicating that growth may take place over the surface of sterol particles. By using the synthetic surfactant HYOXYD AAO (alkyl aryl polyglycol ether), which prevented attachment of cells to sterol particles without affecting the metabolic integrity of the cells, it was shown that growth indeed took place predominantly on the surface of the sterol particles. Increased generation of finer particles of sterol, which provides increased substrate surface area during growth, was demonstrated. It was concluded that with beta-sitosterol, growth takes place almost entirely by attachement, whereas with cholesterol, about 30% of the growth take place on solubilized substrate and the rest through attachament.     

Purification and characterization of alkane solubilizing factor produced by Pseudomonas PG-1

The normal hexadecane emulsifying and solubilizing factor (PG-1 ESF C₁₆) produced by Pseudomonas PG-1 during growth on n-hexadecane was isolated and purified. The factor was composed of protein, carbohydrate and lipid, which were largely undialyzable. Ca²⁺ was necessary for activation and heat stability of the factor. Particle size of the factor was less than 10 nm. All the protein along with 68–74% of the carbohydrate in the factor was obtained in a single protein peak by gel filtration chromatography using Biogel P-30. The isolated protein fraction showed a 1–5 fold increase in n-hexadecane solubilizing activity. The isolated protein was shown to be a homogeneous, monomeric protein with a molecular weight of approximately 11,000 daltons by SDS-PAGE. The protein and carbohydrate moieties in the isolate were separated by DEAE-cellulose chromatography. Neither purified protein nor carbohydrate showed n-hexadecane solubilizing activity separately, but when these were mixed full activity was restored. Hydrocarbon emulsifying activity was confined to the lipid fraction, which was isolated to the extent of 85% from the Biogel P-30 column by ethyl ether extraction.     

Role of soil microorganisms in improving P nutrition of plants

Phosphorus (P) is one of the major plant growth-limiting nutrients although it is abundant in soils in both inorganic and organic forms. Phosphate solubilizing micro-organisms (PSMs) are ubiquitous in soils and could play an important role in supplying P to plants in a more environmentally friendly and sustainable manner. Although solubilization of P compounds by microbes is very common under laboratory conditions, results in the field have been highly variable. This variability has hampered the large-scale use of PSMs in agriculture. Many reasons have been suggested for this variability, but none of them have been extensively investigated. In spite of the importance of PSMs in agriculture, the detailed biochemical and molecular mechanisms of P solubilization are not known. Recent work in our laboratory has shown that the conditions employed to isolate PSMs do not reflect soil conditions and that PSMs capable of effectively releasing P from soil are not so highly abundant as was suggested in earlier studies. These studies have also indicated that the mineral phosphate solubilizing (mps) ability of microbes could be linked to specific genes, and that these genes are present even in non P solubilizing bacteria. Understanding the genetic basis of P solubilization could help in transforming more rhizosphere-competent bacteria into PSMs. Further research should also focus on the microbial solubilization of iron (Fe) and aluminum (Al) phosphates, as well as mobilization of the organic phosphate reserves present in the soils.     

Solubilization and partial purification of ATPase from a rose cell plasma membrane fraction

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tested was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2′,5′-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase.     

Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture

Two bacterial strains, Pseudomonas aeruginosa K1 and Rhodococcus equi P1, were used to degrade cyclo-alkanes (such as decalin) by a co-oxidation mechanism. Both strains possessed the capacity to degrade a broad range of n-alkane mixtures (C7 to C28) within 24 h of incubation. Strain P1 rapidly degraded 10 gl-1 pristane within 24 h of incubation (mu = 0.36 h-1 and Yx/s = 0.6). The addition of hexadecane as a growth substrate (above 0.5%, v/v) resulted in complete degradation of 1% (v/v) decalin by strain P1 via a co-oxidation mechanism. Co-oxidation to degrade decalin or pristane by strain K1 proved unsuccessful. Strain P1 was able to degrade decalin totally in a saturated hydrocarbon mixture. Strain K1 was only able to degrade hexadecane from the hydrocarbon mixture, but its degradation rate was higher than that of strain P1. Therefore, there was competition for the hexadecane needed to co-oxidize decalin. As a result, degradation of the hydrocarbon mixture, especially decalin, was incomplete in a mixed culture of strain P1 and K1. Serial addition of hexadecane (twice) allowed complete degradation of the remaining decalin by strain P1. Also, the biodegradation rate of the hydrocarbon mixture by a microbial population from gasoline-contaminated soil was delayed by addition of strain K1 to the population, while the addition of strain P1 resulted in an increase in the biodegradation rate.     

Emulsifier production byAcinetobacter calcoaceticus strains

Nondialyzable bioemulsifiers were found in the extracellular fluid of 16 different strains ofAcinetobacter calcoaceticus following growth on ethanol-salts medium. The amount of emulsifying activity, its specific activity, and hydrocarbon substrate specificity varied from one strain to another. In general, strains that grew well on the ethanol medium (2.4–2.6 mg cell dry wt/ml) produced high emulsifying activities (88–239 units/ml), whereas strains that grew more poorly (1.0–1.7 mg cell dry wt/ml) also produced less emulsifying activity (14–52 units/ml). With one exception, hexadecane/2-methylnaphthalane mixtures were emulsified more efficiently than pure hexadecane or 2-ethylnaphthalane.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

The novel and efficient method for isolating potassium solubilizing bacteria from rhizosphere soil

Potassium (K) is the third major essential macronutrient for plant growth and more than 90% of potassium in the soil exists in the form of insoluble rocks and silicate minerals. 150 potassium solubilizing bacterial (KSB) strains were isolated from rhizosphere soil using Aleksandrov medium containing insoluble mica powder. Ten efficient KSB strains were selected and quantification studies showed that higher K solubilization (50.6?mg L^?1) was observed in the strain HMP27 followed by strain WHP47 (46.4?mg L^?1) in liquid medium. Potassium solubilization by the bacterial strains is determined by measuring zone of clearance around the bacterial colony. This procedure requires 10–15?days incubation. Therefore, a simple, rapid, and user-friendly method has been developed for screening of potassium solubilizing bacteria using the bromothymol blue dye in modified Aleksandrov medium. Microorganisms possessing potassium solubilization property developed a clear zone around bacterial colony and changed the colour of dye from greenish blue to yellow after two days incubation. High-performance liquid chromatography analysis of the filtrates showed the presence of oxalic, tartaric, citric, and succinic acid, which could be responsible for solubilization of potassium. This method will allow researchers to readily isolate new potassium solubilizing strains adapted to specific environments.     

The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 microg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.     

Determination of the solubilizing activity of a cellulase complex with dyed substrates

When the solubilizing activity of a microbial cellulase complex (e.g.,Trichoderma viride) is determined with conventional methods based on formation of reducing sugars, the results depend on the concentration ratio of cellobiose and glucose in the reaction mixture and thus on the β-glucosidase present and on the type of measurement of reducing sugars. The use of dyed substrates is one way to avoid this problem. The release of coloured compounds from a dyed substrate is proportional to the solubilization.     

Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger

Three isolates of Aspergillus tubingensis and two isolates of Aspergillus niger isolated from rhizospheric soils were tested on solubilization of different rock phosphates. All the isolates of Aspergillus were capable of solubilizing all the natural rock phosphates. A. tubingensis (AT1) showed maximum percent solubilization in all the rock phosphates tested in this study when compared to other isolates. This isolate also showed highest phosphorus (P) solubilization when grown in the presence of 2% of rock phosphate. A. tubingensis (AT1) seems to be more efficient in solubilization of rock phosphates compared to other isolates reported elsewhere. This is the first report of rock phosphate solubilization by A. tubingensis and might provide an efficient large scale biosolubilization of rock phosphates intended for P fertilizer.