Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2

ABSTRACT: BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

Bioemulsifier production by Streptomyces sp. S22 isolated from garden soil

Out of 45 actinomycetes isolated from garden soil, pond water and air; fifteen showed good emulsification activity. Streptomyces sp. S22 isolated from garden soil produced maximum bioemulsifier with 0.5% (v/v) sunflower oil during stationary phase at 37 degrees C, pH 6 and 250 rev/min. Emulsification activity was maximum (320 EU/ml) with sunflower oil as substrate. Partially purified bioemulsifier from Streptomyces sp. S22 was a peptidoglycolipid containing lipid (51.25%), protein (30%), non-reducing sugar (17.75%) and reducing sugar (1%). The yield of partially purified bioemulsifier was 1.6 g/l and reduced the surface tension of water by 23.09 mN/m. The bioemulsifier produced by Streptomyces sp. S22 was stable at room temperature for seven days.     

Statistical media design for efficient polyhydroxyalkanoate production in Pseudomonas sp. MNNG-S

Polyhydroxyalkanoate (PHA) is a promising polymer for various biomedical applications. There is a high need to improve the production rate to achieve end use. When a cost-effective production was carried out with cheaper agricultural residues like molasses, traces of toxins were incorporated into the polymer, which makes it unfit for biomedical applications. On the other hand, there is an increase in the popularity of using chemically defined media for the production of compounds with biomedical applications. However, these media do not exhibit favorable characteristics such as efficient utilization at large scale compared to complex media. This article aims to determine the specific nutritional requirement of Pseudomonas sp. MNNG-S for efficient production of polyhydroxyalkanoate. Response surface methodology (RSM) was used in this study to statistically design for PHA production based on the interactive effect of five significant variables (sucrose; potassium dihydrogen phosphate; ammonium sulfate; magnesium sulfate; trace elements). The interactive effects of sucrose with ammonium sulfate, ammonium sulfate with combined potassium phosphate, and trace element with magnesium sulfate were found to be significant (p??1 to 4.56?g?L^?1).     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.     

Staining human lymphocytes and onion root cell nuclei with madder root.

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5-10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Some observations on oscillatory changes in the growth rate of Saccharomyces cerevisiae in aerobic continuous undisturbed culture.

Oscillatory changes in the growth rate were observed in undisturbed continuous culture of Saccharomyces cerevisiae on sugar-cane molasses media when nitrogen sources (2.56 to 6.17 g/liter of ammonium sulfate or 1.22 g/liter of urea) were added to the feeding mash and when the air rate was 1.3 to 1.6 v/v/m. The oscillations were not affected by the addition of yeast extract. The suppression of the nitrogen source during the continous test leads to a nonoscillatory transient state. No oscillations occured at all when no nitrogen source was added to the medium and/or the air rate was equal to zero or equal to about 3.3 v/v/m. The oscillatory responses of the system were affected by a previous anaerobic continuous cultivation of the yeast.     

Production of a peptidoglycolipid bioemulsifier by Pseudomonas aeruginosa grown on hydrocarbon

A strain of Pseudomonas aeruginosa isolated from a polluted soil was found to produce an extracellular bioemulsifier when cultivated on hexadecane as sole carbon source. The emulsifier was precipitated with acetone and redissolved in sterile water. Dodecane, crude oil and kerosene were found to be good substrates for emulsification by the bioemulsifier. Growth and bioemulsifier production reached the optimal levels on the fourth and fifth day, respectively. Emulsifying activity was observed over a pH range of 3.5 to 10.0 with a maximum at pH 7.0. The activity of the bioemulsifier was heat stable up to 70 degrees C while about 50 percent of its activity was retained at 100 degrees C. The components of the bioemulsifier were determined, it was found to contain carbohydrate, protein and lipid. The protein complex was precipitated with ammonium sulphate and fractionated on a Sephadex G-100. Gel electrophoresis of the bioemulsifier showed a single band whose molecular weight was estimated as 14,322 Da. The bioemulsifier was classified as a peptidoglycolipid. Certain strains of P. aeruginosa produce peptidoglycolipid in place of rhamnolipid.     

黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。     

工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的条件

为了研究工业纤维素诱导里氏木霉RUT C-30产葡聚糖酶的最佳条件,根据单因素实验结果,以工业纤维素、(NH4)2SO4和生物素为实验因素,滤纸酶活为响应值,进行中心组合设计,建立一个二次多项式数学模型,进行响应面优化,寻找最优产酶结果.经过优化,选出工业纤维素、(NH4)2SO4和生物素的添加量分别为39.485 g/L、6.232 g/L和249.872 μg/L,最高的滤纸比酶活为6.298 U/mL,实验验证,滤纸比酶活为6.118 U/mL,与预测值相差了2.86%.     

Microbiological Production of Gibberellic Acid in Glucose Media

Gibberellic acid production from various substrates was studied in 43 strains of Fusarium, among which F. moniliforme strain IOC-3326 was selected as the best producer. Experiments were carried out in shaker flasks and pilot plant fermentors. The results indicate that the best substrate for gibberellic acid production with this strain is composed of the following: glucose, 20 g; corn steep liquor, 25 g; ammonium nitrate, 2.6 g; monopotassium phosphate, 0.5 g; potassium sulfate, 0.2 g; and water, 1000 ml. Glucose, ammonium nitrate, and corn steep liquor were found to be critical. With this medium, maximal yields of 1196 mg per liter in shaker flasks and 997 mg per liter in fermentors were produced.     

Evaluation of bioemulsifier mediated Microbial Enhanced Oil Recovery using sand pack column

Bacillus licheniformis K125, isolated from an oil reservoir, produces an effective bioemulsifier. The crude bioemulsifier showed 66% emulsification activity (E(24)) and reduced the surface tension of water from 72 to 34 mN/m. It contains substantial amount of polysaccharide, protein and lipid. This bioemulsifier is pseudoplastic non-Newtonian in nature. It forms oil in water emulsion which remains stable at wide range of pH, temperature and salinity. It gave 43+/-3.3% additional oil recovery upon application to a sand pack column designed to simulate an oil reservoir. This is 13.7% higher than that obtained from crude lipopeptide biosurfactants produced by the standard strain, Bacillus mojavensis JF2 and 8.5% higher than hot water spring isolate, Bacillus licheniformis TT42. The increased oil recovery obtained by using the crude bioemulsifier can be attributed to its combined surface and emulsification activity. Its mechanism of oil recovery must be similar to the mechanism exhibited by surfactant-polymer flooding process of chemical enhanced oil recovery.     

Extracellular Formation of Alanine by Anaerobes

The possibility of extracellular formation of amino acids by anaerobes was investigated. In general, anaerobes were able to produce 20 to 50 mg of alanine per dl of medium extracellularly. Clostridium saccharoperbutylacetonicum, the anaerobic bacterium for acetone-butanol fermentation, accumulated 100 mg of alanine per dl of medium containing 5 g of glucose, 1 g of ammonium acetate, 0.1 g of potassium dihydrogen phosphate, 0.04 g of magnesium sulfate, 0.001 g of ferrous sulfate, 1 μg of biotin, 0.1 g of yeast extract and 1 g of calcium carbonate in tap water.

Relationships between alanine formation and solvent yields or sporulation were investigated. Spore formation was not active in the medium for alanine formation and yield of acetone increased in this medium.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Effect of emulsifiers released by yeasts multiplying on n-alkane media on their growth and microscopic organization

Bioemulsifiers and ether extracts from the cultural broth of yeast cells were used to study their effect of Candida lipolytica growth and ultrastructural organisation. When the emulsifiers and ether extracts were added to the growth medium, the lag phase was reduced but the growth rate did not change. The ether extracts increased the growth rate of C. lipolytica 374/2 and the final biomass yield of C. lipolytica 704. The ultrastructural organisation of C. lipolytica 374/2 cells changed under the action of the bioemulsifier added to the growth medium.     

Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon,nitrogen and metal ions

To investigate the feasibility of using fresh water from Mangueira Lagoon (Rio Grande do Sul, Brazil) for biomass production in open raceway ponds (0.7 m long, 0.18 m wide, 0.075 m deep) we studied the influence of nutrient addition (carbon as sodium bicarbonate, nitrogen as urea, phosphate, sulfate, ferric iron, magnesium and potassium) on the growth rate of the cyanobacteria Spirulina platensis using a 22 factorial design. In unsupplemented lagoon water production of S platensis was 0.78 +/- 0.01 g/l (dry weight basis) while the addition of 2.88 g/l of sodium bicarbonate (without added urea, phosphate, sulfate or metal ions) resulted in 0.82 +/- 0.01 g/l after 400 hours of culture. The further addition of phosphate and metal ions resulted in growth for up to 750 h and a final S. platensis biomass of 1.23 +/- 0.04 to 1.34 +/- 0.03 g/l.     

Studies on bioemulsifier production by Acinetobacter strains isolated from healthy human skin

AIMS: In recent years, interest has been growing in the search for novel bioemulsifiers. Many bacterial genera including Acinetobacter have been reported to produce bioemulsifiers. The present study aims to screen Acinetobacter isolates from healthy human skin for bioemulsifier production. Methods and Results: Acinetobacter junii SC14 produced maximum bioemulsifier in the presence of almond oil during stationary growth phase at 37 degrees C and pH 7.2. Partially purified, nondialysable bioemulsifier from SC14 was a proteoglycan. The protein and polysaccharide fractions resulted in 95.2% reconstitution of the emulsification activity. The role of esterase in the release of cell-bound emulsifier and the contribution of capsular polysaccharide to the emulsification activity were observed. CONCLUSION: Acinetobacter strains from human skin exhibited better emulsification activity than that by burn wound or soil isolates, owing to the inherent differences in chemical microenvironment of their habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of skin commensals, especially acinetobacters, would lead to the discovery of novel bioemulsifiers with interesting properties. Attempts of screening and strain improvement directed towards skin commensals will open up new avenues for strains producing bioemulsifier on a commercial scale.     

Effect of nutritional factors on cellulase enzyme and microbial protein production by Aspergillus terreus and its evaluation

The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation, the crude protein content, biomass protein recovery, water-soluble carbohydrate, bagasse cellulose utilization, CMCase, and FPase activities were 32.8%, 20.1 g/100 g of bagasse, 6.2%, 82.7%, 1.0. and 0.08 U/mL, respectively. The final biomass recovered contained 32.8% crude protein content and had an in vitro rumen digestibility (IVRD) coefficient of 68.8%. The biomass contained almost all the essential and nonessential amino acids and was comparable with FAO reference protein. It is concluded that a fermentation time of 72 h gave a faster rate of protein production of 16.9 g/100 g of bagasse with 69.8% bagasse cellulose utilization with 76.0% IVRD. and contained almost all the essential and nonessential amino acids.     

Screening of bioemulsifier-producing micro-organisms isolated from oil-contaminated sites

Eighty-eight micro-organisms were isolated from oil-contaminated soils and checked for their extracellular bioemulsifier producing potential. The micro-organisms were screened on the basis of oil spread, drop collapse and emulsification index. Most efficient strains were characterized as Lysinibacillus sp. SP1025 and Bacillus cereus SP1035. In Lysinibacillus sp. SP1025, the E-24 index, surface tension and production of crude bioemulsifier were found to be 83.3 % with diesel, 34.20?±?0.03 mN/m and 3.07?±?0.62 g/L, respectively, whereas in the case of Bacillus cereus SP1035, the E-24 index, surface tension and production of crude bioemulsifier recorded were 76.5 % with diesel, 43.42?±?0.03 mN/m and 3.90?±?0.3 g/L. Crude biomemulsifier produced by selected micro-organisms was stable, withstanding a wide temperature and pH range with an E-24 Index value greater than 50 %. All emulsions formed were oil-in-water type. Emulsions formed with tested aliphatic and aromatic hydrocarbons, except those formed with ester based oils, were 100 % stable with the entire organic layer converted into emulsion. To the best of our knowledge this is the first report for bioemulsifier production from the genus Lysinibacillus.