Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

Production and characterization of biosurfactant from marine bacterium Inquilinus limosus KB3 grown on low-cost raw materials

Inquilinus limosus strain KB3, isolated from marine sediment in the south of Thailand, was used to produce a biosurfactant from a mineral salts medium (MSM) with palm oil decanter cake (PODC) as a carbon source. It was found that cellular growth and biosurfactant production in MSM were greatly affected by the medium components. I. limosus KB3 was able to grow and to produce surfactant reducing the surface tension of medium to 28.2 mN/m and giving a crude surfactant concentration of 5.13 g/l after 54 h. The biosurfactant obtained was found to reduce the surface tension of pure water to 25.5 mN/m with the critical micelle concentration of 9 mg/l, and retained its properties during exposure to elevated temperatures (121 °C), high salinity (12 % NaCl), and a wide range of pH values. Chemical characterization by FT-IR, NMR, and ESI-MS revealed that the biosurfactant has a lipopeptide composition with molecular mass (m/z) of 1,032. The biosurfactant was capable of forming stable emulsions with various hydrocarbons and had the ability to enhance oil recovery, PAHs solubility, and antimicrobial activity.     

Antimicrobial biosurfactants from marine Bacillus circulans: extracellular synthesis and purification

Aims:  To purify the biosurfactant produced by a marine Bacillus circulans strain and evaluate the improvement in surface and antimicrobial activities.
Methods and Results:  The study of biosurfactant production by B. circulans was carried out in glucose mineral salts (GMS) medium using high performance thin layer chromatography (HPTLC) for quantitative estimation. The biosurfactant production by this strain was found to be growth-associated showing maximum biosurfactant accumulation at 26 h of fermentation. The crude biosurfactants were purified using gel filtration chromatography with Sephadex^® G-50 matrix. The purification attained by employing this technique was evident from UV–visible spectroscopy and TLC analysis of crude and purified biosurfactants. The purified biosurfactants showed an increase in surface activity and a decrease in critical micelle concentration values. The antimicrobial action of the biosurfactants was also enhanced after purification.
Conclusions:  The marine B. circulans used in this study produced biosurfactants in a growth-associated manner. High degree of purification could be obtained by using gel filtration chromatography. The purified biosurfactants showed enhanced surface and antimicrobial activities.
Significance and Impact of the Study:  The antimicrobial biosurfactant produced by B. circulans could be effectively purified using gel filtration and can serve as new potential drugs in antimicrobial chemotherapy.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater

The production and properties of a biosurfactant, synthesized by Bacillus subtilis LB5a strain, using cassava wastewater as substrate were investigated. The microorganism was able to grow and to produce surfactant on cassava waste, reducing the surface tension of medium to 26.6 mN/m and giving a crude surfactant concentration of 3.0 g/L after 48 h. The surface-active compound retained its properties during exposure to elevate temperatures (100 degrees C), high salinity (20% NaCl) and a wide range of pH values. The surfactant was capable of forming stable emulsions with various hydrocarbons. Preliminary chemical characterization revealed that the surfactant has a lipopeptide composition with a CMC value of about 33 mg/L. Cassava wastewater proved to be a suitable substrate for biosurfactant biosynthesis, providing not only bacterial growth and product accumulation but also a surfactant that has interesting and useful properties with potential for many industrial applications.     

Quantitative analysis of biosurfactants using Fourier Transform Infrared (FT-IR) spectroscopy

A quick and simple technique has been developed that is suitable to quantify the concentration of most types of biosurfactants in a typical growth medium using infrared spectroscopy. The area of the peak due to the carbonyl bond of the biosurfactant was measured and compared to the area of the nitrile peak in an internal standard (anthracenecarbonitrile). This ratio showed a linear trend with the concentration of biosurfactant in the medium for concentrations from 0.2 to 3.2 g biosurfactant l^–1. The presence of hydrocarbons in the media and variations in the pH of the media did not interfere with the measurements.     

Effect of culture conditions on fatty acids composition of a biosurfactant produced by Candida ingens and changes of surface tension of culture media

This work investigated the effect of culture medium composition on a biosurfactant production and their total fatty acids content, as well as the surface tension of media, and biomass production by Candida ingens. A factorial experimental design was used to evaluate the combined effect of C/P, C/N(inorganic), C/Fe, C/Mg ratios and yeast extract concentration. The highest biosurfactant production was reached when high C/Fe and high C/P ratio variables were combined; biosurfactant concentration increased by a 3.42 fold. The variable with the highest effect on net decrease surface tension (DeltaST) and fatty acids percentage of C. ingens biosurfactant was yeast extract. The average of DeltaST (25 mN/m) and fatty acids percentage (34.7%) values were enhanced at high yeast extract concentration of 1g/l. The main conclusion of this study was that the culture composition affected the biosurfactant production by C. ingens. It was also observed that the surface tension and total fatty acids of the biosurfactant were modified as the media composition changed.     

Optimization,production and characterization of glycolipid biosurfactant from the marine actinobacterium,Streptomyces sp. MAB36

A potential glycolipid biosurfactant producer Streptomyces sp. MAB36 was isolated from marine sediment samples. Medium composition and culture conditions for the glycolipid biosurfactant production by Streptomyces sp. MAB36 were optimized, using two statistical methods: Plackett–Burman design was applied to find out the key ingredients and conditions for the best yield of glycolipid biosurfactant production and central composite design was used to optimize the concentration of the four significant variables, starch, casein, crude oil and incubation time. Fructose and yeast extract were the best carbon and nitrogen sources for the production of the glycolipid biosurfactant. Biochemical characterizations including FTIR and MS studies suggested the glycolipid nature of the biosurfactant. The isolated glycolipid biosurfactant reduced the surface tension of water from 73.2 to 32.4 mN/m. The purified glycolipid biosurfactant showed critical micelle concentrations of 36 mg/l. The glycolipid biosurfactant was effective at very low concentrations over a wide range of temperature, pH, and NaCl concentration. The purified glycolipid biosurfactant showed strong antimicrobial activity. Thus, the strain Streptomyces sp. MAB36 has proved to be a potential source of glycolipid biosurfactant that could be used for the bioremediation processes in the marine environment.     

Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites

Biosurfactant-producing bacteria were isolated from terrestrial and marine samples collected in areas contaminated with crude oil or its byproducts. Isolates were screened for biosurfactant/bioemulsifier production in different carbon sources (glucose, fructose, sucrose and kerosene) using the qualitative drop-collapse test. Glucose produced the highest number of positive results (17 of 185 isolates). All 17 isolates produced emulsions with kerosene and 12 exhibited high emulsion-stabilizing capacity, maintaining 50% of the original emulsion volume for 48 h. Eight of the 17 isolates reduced the growth medium surface tension below 40 mN m(-1) with 5 exhibiting this capacity in cell-free filtrates. Onset of biosurfactant production differed among the isolates, with some initiating synthesis during the exponential growth phase and others after the stationary phase was reached. Increasing temperature from 25 to 35 degrees C accelerated onset of biosurfactant production in only two isolates while pH (6.5-7.6) had no effect in any isolate tested. Isolation from petroleum contaminated sites using the screening protocol presented proved to be a rapid and effective manner to identify bacterial isolates with potential industrial applications.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by <Emphasis Type="Italic">Pseudomonas nitroreducens</Emphasis> isolated from soil

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from petroleum-contaminated soil was investigated. The effects of carbon, nitrogen and carbon to nitrogen ratio on biosurfactant production were examined using mineral salts medium as the growth medium. The tenso-active properties (surface activity and critical micelle concentrations of the produced biosurfactant were also evaluated. The best carbon source, nitrogen source were glucose and sodium nitrate giving rhamnolipid yields of 5.28 and 4.38 g l⁻¹, respectively. The maximum rhamnolipid production of 5.46 g l⁻¹ was at C/N (glucose/sodium nitrate) of 22. The rhamnolipid biosurfactant reduced the surface tension of water from 72 to ~37 mN/m. It also has critical micelle concentration of ~28 mg l⁻¹. Thus, the results presented in our reports show that the produced rhamnolipid can find wide applications in various bioremediation activities such as enhanced oil recovery and petroleum degradation.     

Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis 20B

A biosurfactant producing strain, Bacillus subtilis 20B, was isolated from fermented food in India. The strain also showed inhibition of various fungi in in-vitro experiments on Potato Dextrose Agar medium. It was capable of growth at temperature 55 degrees C and salts up to 7%. It utilized different sugars, alcohols, hydrocarbons and oil as a carbon source, with preference for sugars. In glucose based minimal medium it produced biosurfactant which reduced surface tension to 29.5 mN/m, interfacial tension to 4.5 mN/m and gave stable emulsion with crude oil and n-hexadecane. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations for five days. Oil displacement experiments using biosurfactant containing broth in sand pack columns with crude oil showed 30.22% recovery. The possible application of organism as biocontrol agent and use of biosurfactant in microbial enhanced oil recovery (MEOR) is discussed.     

Production and properties of a biosurfactant synthesized byArthrobacter protophormiae — an antarctic strain

This study reports the production of biosurfactant by a psychrophilic strain ofArthrobacter protophormiae during growth on an immiscible carbon source, w-hexadecane. The biosurfactant reduces the surface tension of the medium from 68.0 mN/m to 30.60 mN/m and exhibits good emulsification activity. The strain could grow and produce biosurfactant in the presence of high NaCl concentrations (10.0 to 100.0 g/1). Although the biosurfactant was isolated by growing the organism under psychrophilic conditions (10‡C) it exhibited stable activity over a wide range of temperature (30‡C to 100‡C). It retained its surface-active properties at p^H2 to 12. The biosurfactant was effective in recovering up to 90% of residual oil from an oil saturated sandpack column, indicating its potential value in enhanced oil recovery.     

Enhancement of tPA production under hyperoxic conditions by BHK cells in serum-free and serum-containing cultures

Effects of a wide range of dissolved oxygen concentration (DO, 0.5–28 mg/l) on anchorage-dependent BHK cell growth, metabolism and tPA production were examined with both serum-containing and serum-free media. In the range of DO from 0.5 to 5 mg/l, tPA production increased with an increase in DO in both media. Cell growth was higher at 5 mg/l DO than that at 0.5 or 2 mg/l DO in serum-containing medium, but it did not vary in serum-free medium in this DO range. Further investigation under hyperoxic conditions (DO > 6.8 mg/l) revealed that specific rates of tPA production were enhanced by 2-fold in serum-containing and 1.7-fold in serum-free media, although cell growth depressed above 5 mg/l of DO. Slight increases in specific rates of lactate accumulation and glucose consumption were observed in both media under hyperoxic conditions. In serum-free medium, cells were found to be less tolerant to hyperoxic conditions than those in serum-containing medium. A DO shift-up with shifting time of 4 h in serum-containing medium was found to influence significantly both cell growth and tPA production.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source.

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source

Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.     

Production of a biosurfactant exhibiting excellent emulsification and surface active properties bySerratia marcescens

A biosurfactant exhibiting excellent emulsification activity and surface properties was isolated during growth ofSerratia marcescens on 2% (w/v) sucrose. Reduction in surface tension values and increase in the yield of biosurfactant during the late log phase of growth indicates that the biosurfactant is a secondary microbial metabolite. The biosurfactant formed stable emulsions with a wide variety of hydrocarbons. The isolated surface-active compound has a potential application in enhanced oil recovery and is stable over a wide range of temperatures (10-120‡C) and pH (2-12). This is the first report of effective and stable emulsion formation by a strain ofSerratia marcescens.     

Optimization of <Emphasis Type="Italic">R</Emphasis>-(+)-α-terpineol production by the biotransformation of <Emphasis Type="Italic">R</Emphasis>-(+)-limonene

R-(+)-limonene is an abundant and non-expensive by-product of the citrus industry and is, therefore, a suitable starting material for the production of natural flavor and fragrance compounds. The biotransformation of R-(+)-limonene to R-(+)-alpha-terpineol by Fusarium oxysporum 152b has already been reported, although the influence of the main process parameters on the production has not yet been evaluated. In this paper, a Plackett-Burman screening design was used to define the effects of the medium composition (glucose, peptone, yeast extract, malt extract and pH), the presence of a co-substrate (biosurfactant), the cultivation conditions (temperature, agitation), the substrate concentration and the inoculum/culture medium ratio on the absolute amount of R-(+)-alpha-terpineol resulting from this biotransformation. The process conditions were further optimized applying response surface methodology (RSM). The volatiles were extracted using a SPME device and were subsequently quantified by GC-FID and identified by GC-MS. The best results were obtained using 0.5% (v/m) R-(+)-limonene in pure distilled water as the culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72 h cultivation at 26 degrees C/240 rpm. Under these conditions the concentration of R-(+)-alpha-terpineol in the culture medium reached 2.4 g L(-1), a production almost six times greater than in earlier trials. The presence of a biosurfactant (0-500 mg L(-1)) did not significantly increase the yield.     

Natural cashew apple juice as fermentation medium for biosurfactant production by Acinetobacter calcoaceticus

The success of biosurfactant production depends on the development of cheaper processes based on the use of low cost raw materials, which account for 10–30% of the overall process cost. In Brazil, the cashew apple agroindustry plays an outstanding role in the local economy. However, only a small part of the pseudofruit produced is used industrially and the amount wasted (about 94%) presents high potential as fermentation media, since it is rich in carbohydrate, fibers, vitamins and minerals salts. In this work, the performance of cashew apple juice (CAJ) as a complex medium for Acinetobacter calcoaceticus growth and production of biosurfactant was investigated. The microorganism was able to grow and to produce biosurfactant on a defined culture medium and on CAJ, reducing the surface tension of both media. The biosurfactant also achieved a maximum emulsion index of 80% for kerosene, when defined medium was used.