Optimization of surfactin production by Bacillus subtilis ATCC 6633 in a miniaturized bioreactor

In recent years, biosurfactants due to wide applications in chemical, petroleum, food and pharmaceutical industries, have been widely considered by researchers. Biosurfactants are produced by a series of microorganisms, so it is important to screen culture medium and operating conditions in miniaturized bioreactors prior to scaling up to large bioreactors.In this study, using a kind of miniaturized bioreactor called ventilation flask, optimal production conditions, including filling volume and shaking frequency to produce a surfactin-type biosurfactant by Bacillus subtilis ATCC 6633, were examined. Moreover, the effect of oxygen transfer rate [OTR] on the surfactin production was investigated according to Amoabediny and Buchs model. The results indicated that the maximum biomass and biosurfactant yield which obtained under optimal conditions [filling volume of 15 mL and shaking frequency of 300 rpm] were evaluated 0.3 g/L/h and 0.0485 g/L/h, respectively. Also, at the same conditions, the amount of surface tension decreased from 60.5 mN/m to 31.7 mN/m and the maximum oxygen transfer rate [OTR[max]] obtained as 0.01 mol/L/h

Optimization of reactive blue 19 biodegradation by Phanerochaetechrysosporium

Problematic dyes extensively used in different industries such as textile, paper, food, plastics and cosmetics have undesirable environmental effects. White rot fungi demonstrating effective extracellular enzyme system, which is capable of degrading dyes and various xenobiotics. The aim of this study was to optimize decolorization of reactive blue 19 [RB19] dye using Phanerochaetechrysosporium. A Box- Behnkendesign and Response Surface Methodology [RSM] were used to study the effect of independent variables, namely glycerol concentration [15, 20 and 25 g/L], temperature [27, 30 and 40°C] and pH [5.5, 6.0 and 6.5] on color removal efficiency in aqueous solution. From RSM-generated model, the optimum conditions for RB19 decolorization were identified to be at temperature of 20°C, glycerol concentration of 120/L and pH: 6.5. At the optimum conditions, predicted decolorization was 98 percent. The confirmatory experiments were conducted, which confirmed the results by 99.8 percent color removal. Thus, the experimental investigation and statistical approach enabled us to improve reactive blue 19 biodegradation process by Phanerochaetechrysosporium up to 1.25 times higher than non-optimized conditions

Production of a whey-based beverage using several kefir microflora and assessment of its chemical and organoleptic characteristics

Kefir is one of the oldest traditional beverages produced by lactic-alcoholic fermentation of milk. On the other hand, milk whey, a by-product of the cheese-making industry, has high contents of valuables nutrients. However, it is usually discarded in Iran, which causes contamination of the environment around the milk processing plants, specially surface and underground waters. Whey fermentation by isolated kefir starter cultures could be a sensible solution towards solving this problem. In the present study, various ratios of starter culture of kefir grain were used for production of a fermented beverage using whey as the substrate. Acidic whey was used to produce a fermented beverage. Culture media were prepared using various ratios of lactic acid bacteria, yeasts, and acetic acid bacteria isolated from kefir strains and used in the production of the beverage. Keeping constant the incubation time [24 h] and temperature [250C], substrate [pasteurized whey], inoculation rate [3% - 5%], and mixing rate [90 rpm], samples of the beverage with various ratios of the cultures were produced and analyzed for sugar, fat, protein, riboflavin, alcohol, carbon dioxide, dry material and ash. Three different essences, namely, those of Mentha paprika, Anethum graveolens, and Thymus vulgaris were added to the beverage samples, which were then tested for organoleptic properties. Beverage samples were produced using an inoculation rate of 3% individual starter cultures and mixed cultures of Lactobacilli, Coccci, and yeasts. After finding the best ratio in each case, inoculation rates of 3% and 5% were used. Based on organoleptic and chemical [acidity and carbon dioxide] properties, out of the 61samples produced 8 were judged to be desirable. Based on the results it can be concluded that, with regard to color, flavor and aroma, the beverage with Mentha paprika essence is the most desirable. Also, results showed that beverage samples produced with 3% [v/v] lactic acid and acetic acid bacteria and 2% yeast culture [v/v] were the best with respect to quality and acceptability

Optimization of reactive blue 19 decolorization by ganoderma SP. using response surface methodology

Synthetic dyes are extensively used in different industries. Dyes have adverse impacts such as visual effects, chemical oxygen demand, toxicity, mutagenicity and carcinogenicity characteristics. White rot fungi, due to extracellular enzyme system, are capable to degrade dyes and various xenobiotics. The aim of this study was to optimize decolorization of reactive blue 19 [RB19] dye using Ganoderma sp. fungus. Response Surface Methodology [RSM] was used to study the effect of independent variables, namely glycerol concentration [15, 20 and 25 g/L], temperature [27, 30 and 33 °C] and pH [5.5, 6.0 and 6.5] on color removal efficiency in aqueous solution. From RSM-generated model, the optimum conditions for RB19 decolorization were identified to be at temperature of 27°C, glycerol concentration of 19.14 mg/L and pH=6.3. At the optimum conditions, predicted decolorization was 95.3 percent. The confirmatory experiments were conducted and confirmed the results by 94.89% color removal. Thus, this statistical approach enabled to improve reactive blue 19 decolorization process by Ganoderma sp. up to 1.27 times higher than non-optimized conditions

Cleaning oil-contaminated vessel by emulsan producers [autochthonous bacteria]

In a process for cleaning hydrocarbonaceous residues, including residual petroleum from laboratory made oil-contaminated vessels, several previously isolated bacteria from Ilam and Paydar oil reservoirs, were used. The isolated strains were compared with the standard sample of Acinetobacter calcoaceticus PTCC 1318 from Persian Type Culture Collection [PTCC]. This gram-negative bacterium grows on a variety of different substrates as sole carbon and energy sources, including crude oil, soy oil and ethanol. It is oxidase-negative, non-motile and strictly aerobic. Among the isolated strains, two autochthonous strains were found to produce an extracellular emulsifying agent when grown in Mineral Salt Medium containing soy oil, ethanol or local crude oil. The crude emulsifier of PTCC1318, Paydar-4 and Ilam-1 were concentrated from the cell-free culture fluid by ammonium sulfate precipitation to yield 1.89 g, 1.78 g and 1.69 g of bioemulsan, respectively. Although measuring the surface tension [ST] is not very applicable procedure in case of bioemulsan, but in order to prove this theory, ST was conducted.Further analysis of purified emulsion was performed to prove the molecular structure by Carbon13 Nuclear Magnetic Resonance, Proton1Nuclear Magnetic Resonance and Fourier Transform Infrared Radiation methods. These investigations showed that the molecular weight of emulsion produced by species isolated from Ilam and Paydar crude oil reservoirs are comparable with Acinetobacter calcoaceticus PTCC 1318

Emulsan analysis produced by locally isolated bacteria and Acinetobacter calcoaceticus RAG-1

Growth of previously isolated bacteria from Iranian oil reservoirs on different carbon and energy sources and under varying conditions have been used to produce a class of extracellular microbial protein-associated lipopolysaccharides named emulsan.Several Bacteria were previously isolated from Iranian oil reservoirs and designated as; Ilam-1 and Paydar-4. In present study, the isolated strains were compared with standard sample of Acinetobacter calcoaceticus RAG-1 from Persian Type Culture Collection [PTCC 1641], IROST. Among the isolated strains, two strains were found to produce an extracellular, emulsifying agent when grown in Mineral Salt Medium containing soya oil, ethanol or local crude oil. The isolated bacteria were cultured and further analysed using protein estimation, reducing sugar analysis, hemolytic activity, surface tension and emulsification activity tests. The crude emulsifier of RAG-1, PAYDAR-4 and ILAM-1 were concentrated from the cell-free culture fluid by ammonium sulfate precipitation to yield 1.89g, 1.78g and 1.69g of bioemulsan respectively. Emulsifying activity was observed over the entire production process. These investigations showed that emulsan produced by isolated Iranian crude oil reservoir were comparable with Acinetobacter calcoaceticus RAG-1 which is made of carbohydrate backbone as its hydrophilic part [N-acetyl-D-galactoseamine, N-acetylgalactoseamine uronic acid, diamino-6-deoxy-D-glucose] and fatty acid chain as its hydrophobic portion

Ability of indigenous bacillus licheniformis and bacillus subtilis in microbial enhanced oil recovery

Microbially produced lipopeptide have been isolated and studied for microbial enhanced oil recovery. About 60 gram positive bacteria isolated from soil contaminated with crude oil, near the crude oil storage tank in Tehran Refinery, Tehran, Iran. However, most of these studies have produced lipopeptide by one of the pure-culture microbes isolated in a laboratory. Among the isolates, heamolytic tests revealed two biosurfactant producers. The isolated strains were designated as C2, E1. By using morphological, biochemical and molecular biology tests [16 SrRNA], the strains identified as Bacillus licheniformis and Bacillus subtitlis, respectively. Emulsification activity and measurement of surface tension indicated that, the isolates were high producers of biosurfactant. The product of C2 and E1 is mainly lipopeptide. This product reduce surface tension from 65 to 30 mN/m. Emulsified activity of crude oil was 92% for C2 and 90% in case of E1. This is the first report of indigenous Bacillus lichenifbrmis and Bacillus subtilis from a soil contaminated with oil in an Iranian refinery with ability to produce biosurfactant

Production of rhamnolipids by pseudomonas aeruginosa growing on carbon sources

Arhamnolipid producing bacterium, Pseudomonas aeruginosa was previously isolated from Iranian oil over years. Isolated strain was identified by morphological, biochemical, physiological and 16 sr RNA [1]. Glycolipid production by isolated bacterium using sugar beet molasses as a carbon and energy source was investigated. Biosurfactant production was quantified by surface tension reduction, Critical Micelle Dilution [CMD], Emulsification Capacity [EC], and Thin Layer Chromatogeraphy. biosurfactants during growth on waste Dates as the primary carbon and nitrogen sources, respectively. After 48 h of growth the culture supernatant fluid had a rhamnose concentration of 0.18 g/L and surface tension was reduced to 20 mN/m [%].[reduced the interfacial tension against crude oil from 21 mN/m to 0,47 mN/m] Result from the study showed that the growth of the bacteria using molasses as carbon sources is growth-associated. The specific production rate of rhamnolipid with 2%, 4%, 6%, 8% and 10% of molasses are 0.00065; 4.556; 8.94; 8.85; and 9.09. respectively The yield of rhamnolipid per biomass with 2%,4%,6%,8% and 10% molasses are 0.003;0.009;0.053;0.041 and 0.213 respectively. The production of rhamnolipid [0.0531 g rhamnolipid/g biomass] is higher compare to the culture grown in aerobic condition [0.04 g rhamnolipid/g biomass].The rhamnolipids were able to form stable emulsions with n-alkanes, aromatics, crude oil and olive oil. These studies indicate that renewable, relatively inexpensive and easily available resources can be used for important biotechnological processes

Cloning of protective antigen gene from Bacillus anthracis into Bacillus subtilis

Protective antigen [PA] of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 microg/ml. The objectives of this study were: to clone the gene of PA in an expression vector [pWB980] and then transformation into the B. subtilis WB600 strain. The pXOl plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5 alpha using CaC12 method. After that the gene was separated from the T-vector by enzymatic digestion [Sa1I and KpnI]. Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies [AMN1 and AMN3] was confirmed by restriction analysis and PCR. We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host

Isolation of Biosurfactant producing bacteria from oil reservoirs

Biosurfactants or surface-active compounds are produced by microoaganisms. These molecules reduce surface tension both aqueous solutions and hydrocarbon mixtures. In this study, isolation and identifi-cation of biosurfactant producing bacteria were assessed. The potential application of these bacteria in petroleum industry was investigated. Samples [crude oil] were collected from oil wells and 45 strains were isolated. To confirm the ability of isolates in biosurfactant production, haemolysis test, emulsification test and measurement of surface tension were conducted. We also evaluated the effect of different pH, salinity concentrations, and temperatures on biosurfactant production. Among importance features of the isolated strains, one of the strains [NO.4: Bacillus.sp] showed high salt tolerance and their successful production of biosurfactant in a vast pH and temperature domain and reduced surface tension to value below 40 mN/m. This strain is potential candidate for microbial enhanced oil recovery. The strain4 biosurfactant component was mainly glycolipid in nature

Production of biosurfactant from Iraninan oil fields by isolated Bacilli

Crude oil and water samples were collected from selected Iranian oil reservoirs. Experimental works were carried out in laboratory conditions. The samples have been grown on PYG medium and incubated at 30-80 0C. Thirty-six mesophile and thermophile bacterial strains have been isolated. All the isolates were able to grow at aerobic condition. Batch growth kinetic studies were carried out in a 500 ml. shake flask. Out of 36 isolated strains from 24 crude oil and water samples, 35 strains were gram positive rod. Shaped spore forming bacteria and one strain was coccid form. Eight out to 35 bacillus species were capable of producing surfactant. Production of biosurfactant was found to be cell growth associated. The ability of surfactant producing bacteria indicated by reduction of surface tension [ST] and interfacial tension [IFT] of the supernatant. Eight strains obtained the IFT reduction in crude oil, hexadecane, sucrose, glucose, fructose and mannose medium as a sole source of carbon and energy at 40 0C by 15-30 mN/m. Results showed that all the bacteria are producing more surfactant when glucose is the carbon source. Further screening of biosurfactant producer showed that three of the isolated strains resulted the maximum ST and IFT. Effect of temperature on these three isolates investigated at 30-80 0C, above 50 0C surfactant production was dramatically reduced. The isolated strains had the capacity to produce the surfactant at 3-5% NaCl a wide rang of pH [6.5-8.5]

Environmental importance of rhamnolipid production from molasses as a carbon source

Rhamnolipid has been known as biosurfactant which is produced by Pseudomonas aeruginosa in fermentation process. Several carbon sources such as ethanol, glucose, vegetable oil and hydrocarbon have been used to produce rhamnolipid. In this study, we are trying to use molasses which is a waste product from sugar industry as carbon source to produce rhamnolipid. The bacterium which was previously isolated from Iranian oil over years Glycolipid production by isolated bacterium using sugar beet molasses as a carbon and energy source was investigated. Result from the study showed that the growth of the bacteria using molasses as carbon sources is growth-associated. The specific production rate of rhamnolipid with 2%, 4%, 6%, 8% and 10% of molasses are 0.00065, 4.556, 8.94, 8.85, and 9.09 respectively. The yield of rhamnolipid per biomass with 2%, 4%, 6%, 8% and 10% molasses are 0.003, 0.009, 0.053, 0.041 and 0.213 respectively. The production of rhamnolipid [0.0531 g. rhamnolipid/g biomass] is higher compare to the culture grown in aerobic condition [0.04 g. rhamnolipid/g biomass]. These studies indicate that renewable, relatively inexpensive and easily available resources can be used for important biotechnological processes

Chromiun bioremoval from tannery industries effluent by Aspergillus oryzae

Recently laboratory studies had recognized the capability of alge, fungi, and bacteria in the removal of heavy metals from industrial effluent. In this research, growth of Aspergillus oryzae in the tanning house effluent, and its capability in chromium bioremoval were assessed. Aspergillus oryzae can grow in different concentration of Cr+, 120-1080 mg/l. Maximum biomass growth and chromium removal rate at pH, 3.3, Cr+3 concentration equal to 240 mg/l and inoculum size equal to 0.12% [dry weight] were 0.25% [dry weight] and 94.2%, respectively. Effects of various factors such as pH, temperature, shaking velocity and nutrients were also investigated. At optimum conditions [ie: pH=5; temperature=30?C, shaking velocity = 150 rpm, and nitrogen source of dihydrogen ammonium phosphate concentration=0.3%], biomass growth and chromium removal rate were found as 0.45% of dry weight and 99.8%, respectively. Effect of detention time showed that after 30h, biomass growth and chromium removal rate were 0.28% and 97.6%, respectively. Statistical studies on factors such as pH, temperature, shaking velocity, type and concentration of nutrients on the "biomass growth" and "residual chromium", showed that all of the factors had significant effects [alpha = 0.05, P < 0.001]. Therefore A.niger capable grow in the tannery industries effluent with 240 mg/l chromium and 97.6% chromium removal rate

Application of white rot fungi living cells in bioremoval of chromium from tannery industries effluent by using of bench scale

Tannery industries effluent disposal into environment causes hazardous effects in environment. The goals were to determine the tolurance limit P. chrysosporium in the tanning house effluent and potential of chromium removal in the bench scale. At first, sampling of tanning house effluent, in order to effluent quality evaluation was carried out. Then adjusting carbon to nitrogen ratio from 10-12. Dry weight of fungi mass [0.35% - 2.1%] inoculated to sterile samples with Cr[+3] concentration 120 - 1080 mg/I. Samples were put in shaker incubator. Also effect of pH, temperature, shaking velocity and nutrients for finding optimum conditions, biomass growth and chromium removal rate were studied. Findings showed that maximum biomass growth and chromium removal rate were occurred in the sample that chromium concentration and inoculum size were 240 mg/I and 0.07% [dry weight], respectively. Also biomass growth and chromium removal rate were 0.04823% and 76.7%, respectively. Biomass growth and chromium removal in best condition and 26h detention time rate were 0.2934% and 95.8%, respectively. This study showed by optimizing environmental conditions, with 95% confidence [P<0.001], after 26h. biomass growth and chromium removal rate would be 0.29% and 95.8% respectively