Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and... Ann Microbiol (2012) 62:753–763 DOI 10.1007/s13213-011-0315-5 ORIGINAL ARTICLE Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production Rashmi Rekha Saikia & Suresh Deka & Manab Deka & Ibrahim M. Banat Received: 22 December 2010 /Accepted: 6 July 2011 /Published online: 20 July 2011 Springer-Verlag and the University of Milan 2011 Abstract An efficient biosurfactant-producing native Pseu- tion of the biosurfactant was 90 mg/L. The absorption domonas aeruginosa RS29 has been isolated from crude oil bands of the FTIR spectra confirmed the rhamnolipid contaminated soil. Isolation was followed by optimization nature of the biosurfactant. The biosurfactant was thermo- of different factors to achieve maximum production of stable (up to 121°C for 15 min) and could withstand a wide biosurfactant in terms of surface tension reduction (STR) range of pH (2–10) and NaCl concentration (2%–10% w/v). and emulsification index (E24). The isolated strain pro- The extracted biosurfactant had good foaming and duced highest biosurfactant in the presence of glycerol after emulsifying activities and was of satisfactory quality in terms 48 h of incubation at 37.5°C, with pH range of 7–8 and at of stability (temperature, pH and salinity) and foaming salinity <0.8% (w/v). The extent of STR and the E24 of activity. medium with different nitrogen sources were investigated . . and found to be maximal for sodium nitrate (26.3 mN/m, Keywords Isolation Biosurfactant Pseudomonas E24=80%) and potassium nitrate (26.4 mN/m, E24=79%). aeruginosa RS29 Rhamnolipid 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 Introduction kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and Biosurfactants are diverse groups of surface-active mole- highest STR was achieved (27.0 mN/m) after 44 h of cules/chemical compounds synthesized by microorganisms growth. The biosurfactant was produced as a primary (Desai and Banat 1997). These are amphipathic molecules metabolite and 6 g/L crude biosurfactant was extracted by with both hydrophilic and hydrophobic domains. Biosur- chloroform:methanol (2:1). The critical micelle concentra- factants reduce surface tension and critical micelle dilution (CMD) in both aqueous solution and hydrocarbon mixtures. These properties create micro-emulsions in which micelle formations occur in which hydrocarbons can solubilize in R. R. Saikia (*) S. Deka water or water in hydrocarbons (Banat 1995). Micro- Life Sciences Division, Institute of Advanced Study organisms have beenreportedtoproduce severalclassesof in Science and Technology, Paschim Boragaon, biosurfactants, such as glycolipids, lipopeptides, phospholi- Guwahati 781035 Assam, India pids, neutral lipids or fatty acids and polymeric biosurfactants e-mail: rashmi.saikia@gmail.com (Cooper and Zajic 1980; Cooper 1986; Kosaric 1993). These compounds are metabolic products produced during M. Deka Department of Biotechnology, Gauhati University, the growth of microorganisms on water-soluble and water Guwahati 781014 Assam, India immiscible substrates (Sheppard and Mulligan 1987; Jenny et al. 1993; Ron and Rosenberg 2001). I. M. Banat The most commonly isolated and the best studied groups Faculty of Life and Health Sciences, University of Ulster, of biosurfactants are mainly glycolipids and phospholipids Coleraine BT52 1SA Northern Ireland, UK 754 Ann Microbiol (2012) 62:753–763 in nature. Nevertheless, rhamnolipids are glycolipid com- with fresh medium. Cultures from the last enriched flask pounds produced by Pseudomonas sp. that can reduce water were plated on nutrient agar using a serial dilution surface tension and emulsify oil (Babu et al. 1996;Dezielet technique. Morphologically different individual bacterial al. 1999;Lang and Wullbrandt 1999; Mata-Sandoval et al. colonies were isolated from the agar plates and streaked on 1999; Rahman et al. 2002; Perfumo et al. 2006). Biosurfac- nutrient agar to obtain pure cultures of the isolates. The tants are environmentally friendly and have potential isolates were grown in nutrient broth for 48 h at 35°C at industrial and environmental applications. 150 rpm and used as inoculum for further experiments. When compared to synthetic surfactants, biosurfactants Proper screening of the biosurfactant-producing bacterial have several advantages, including high biodegradability, isolates was carried out by adding 10 mL inoculum of low toxicity, low irritancy, and compatibility with human each isolate and growing them in 500 mL flasks skin (Banat et al. 2000; Cameotra and Makkar 2004). containing 100 mL sterilized mineral medium with Therefore they are superior to their synthetic counterparts. glucose as the sole carbon source. Cultivations were Many studies have been conducted with microorganisms performed in triplicate. The composition of the mineral to ensure the production and activity of biosurfactants, medium used was as follows (g/L): NH NO (4.0), KCl 4 3 emphasizing the importance of the need for these com- (0.1), KH PO (0.5), K HPO (1.0), CaCl (0.01), 2 4 2 4 2 pounds. However, we have failed to identify any literature MgSO ⋅7H O (0.5), FeSO ⋅7H O (0.01), Yeast extract 4 2 4 2 report dealing solely with native bacterial isolates. Explor- (0.1) and 10 mL of trace element solution containing (g/L): ing native strains for biosurfactant production could be of 0.26 g H BO ,0.5 gCuSO ⋅5H O, 0.5 g MnSO ⋅H O, 3 3 4 2 4 2 great importance since native strains can be assumed to 0.06 g (NH )6Mo O ⋅4H O and 0.7 g ZnSO ⋅7H O. The 4 7 24 2 4 2 perform better in their native environment than other exotic pH of the medium was adjusted to 7.0±0.2 and the flasks strains. Therefore, the present experiment was conducted to were kept in a shaking incubator at 35°C and 150 rpm for isolate potent biosurfactant-producing native bacteria and to 7 days. Screening was based on surface tension reduction identify the optimal environmental factors—temperature, (STR); surface tension was measured every day up to the pH, salinity, nitrogen source and carbon source—for 7th day using a K11 tensiometer (Kruss, Hamburg, Germany) maximum production of biosurfactant. Besides other and the plate method. All chemicals were purchased from objectives, the nature of the produced biosurfactant was Merck (Mumbai, India). also characterized, along with production kinetics and efficacy studies. Identification of efficient isolates The most efficient bacterial strain was identified by (1) Materials and methods studying the morphological and physiological character- istics (Cappuccino and Sherman 1999), and (2) sequencing Soil sample 16S rDNA and aligning the sequence in the NCBI GenBank and RDP database (performed by Bangalore Crude oil-contaminated soil samples were collected from GeNei, India). Following a standard protocol, genomic the Lakowa oil fields of Assam, India. Soil samples DNA was extracted from a pure culture, and a 16S rDNA were collected at a depth of 0–10 cm from the soil fragment of ∼1.5 kb was amplified using Taq DNA surface and stored at room temperature (25±2°C) for polymerase and consensus primers. The PCR product was subsequent use. bi-directionally sequenced using the forward, reverse and an internal primer. Sequence data was aligned and analyzed Isolation and screening of biosurfactant-producing bacteria to find the closest homolog of the isolated bacterial strain. Bacteria were isolated by adding 1 g collected soil sample Optimization of growth conditions to a flask containing nutrient broth and mineral salt solution at a 1:1 ratio and crude oil (2% w/v) was provided as a sole Different carbon sources were tested to select the one most carbon source; the enriched flask was incubated at 35°C for suitable for maximum STR. The carbon sources used in the 4 days in a rotary shaker Scigenics Biotech, ORBITEK experiment were glucose, glycerol, mannitol, n-hexadecane LJEIL at 150 rpm. From this original flask, 5 mL cultures and sludge. These were added to the mineral medium to were subsequently used to inoculate a second flask 2%. The inoculated mineral medium was incubated in a containing fresh medium, and this flask was maintained rotary shaker at 35°C and 150 rpm for 4 days and the under the same conditions. This process was repeated four surface tension was recorded every day. To compare STR, times, and each time same amount of culture was flasks with the same carbon sources but no bacterial culture withdrawn from the older flask and added to a new flask (control) were also studied. Ann Microbiol (2012) 62:753–763 755 A range of temperatures was investigated to determine nitrogen salts were used at 4% (w/v) concentration. A the optimum temperature for maximum production of control was also studied where no nitrogen salts were added biosurfactant. Production of biosurfactant was measured to the mineral medium. Variation in pH of the culture in terms of STR and emulsification index (E24). Efficient medium, the E24, OD as well as the surface tension of the strains growing in mineral medium with a suitable medium were measured. carbon source were incubated at the following temper- atures: 25, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5 and Biosurfactant extraction and characterization 45.0°C at 150 rpm. The initial pH of the mineral medium with a suitable carbon source was investigated by Crude biosurfactant was extracted from cell-free culture adjusting the pH of the medium within the range of 2– broth (CFCB) by chloroform:methanol (2:1) extraction 9. The pH of the medium was adjusted using 1 M HCl three times. CFCB was obtained by centrifuging 48-h-old and 1 M NaOH solutions. The cultures were kept in the bacterial culture grown on glycerol at 8,000 rpm for 20 min shaking incubator at the optimum temperature at at 4°C. The combined extracts were then transferred to a 150 rpm. Optimum salinity was determined by changing round-bottom flask connected to a rotary evaporator. The the concentration of NaCl of the mineral medium from concentration process was continued at 40°C until a 0.2% to 5% (w/v). The efficient strain was grown in yellowish-brown-colored viscous and consistent extract medium with a suitable carbon source. The initial pH of was obtained. The crude biosurfactant was then dried the medium was adjusted and the culture was incubated and weighed. in the rotary shaker at 150 rpm at the optimum The extracted biosurfactant was dissolved in distilled temperature. Surface tension and E24 were measured in water at concentrations ranging from 1.0 to 200 mg/L each flask. E24 was measured by mixing 3 mL whole for calculation of critical micelle concentration (CMC). bacterial culture with an equal volume of n-hexadecane and This is a direct measurement of surfactant concentration vortexing at high speed for 2 min. The sample was left corresponding to the concentration of an amphiphilic undisturbed for 24 h. The E24 is expressed as the percentage component at which the formation of micelles is initiated of the height of the emulsified layer (mm) divided by the in the solution (Abouseoud et al. 2008a). The CMC of total height of the liquid column (mm) (Cooper and the produced biosurfactant was determined following Goldenberg 1987;Abouseoudetal. 2008a). standard methods (Kim et al. 1997; Bonilla et al. 2005). Optimization of growth conditions was followed by a The critical micelle dilution (CMD) is defined as the kinetic study of biosurfactant production as well as solubility of a surfactant in an aqueous phase and is production of biomass. These parameters were investigated commonly used to measure the efficiency of a surfactant −1 −2 (Desai and Banat 1997). CMD by growing the efficient strain in mineral medium with a and CMD were suitable carbon source and maintaining all the optimum determined by measuring the surface tensions of cell free conditions. The kinetics of biosurfactant production were supernatant diluted 10-times and 100-times in distilled studied in batch cultures over a period of 72 h. Surface water (Kosaric 1993). Cell free supernatant was obtained tension and optical density at 460 nm (OD )were by centrifuging a 48-h-old culture of the strain grown in measured at 4-h intervals. Surface tension was measured mineral medium with glycerol at 8,000 rpm for 20 min in tensiometer and OD was measured in a SHIMADZU at 4°C. UV-1800 UV-spectrophotometer. E24 was also recorded. Carbohydrate moieties in the biosurfactant molecule Dry cell biomass was determined by centrifugation of an were assayed using rhamnose (Dubois et al. 1956) and aliquot (100 mL) of 48-h-old bacterial culture at 8,000 rpm Molisch’s test. The rhamnose test was performed by adding for 20 min. The cell pellet was washed with n-hexane to 0.5 mL cell supernatant to 0.5 mL 5% phenol solution and remove any slimy materials attached to the cell surface that 2.5 mL sulfuric acid and incubating the sample for 15 min might cause error in the assessment. The washed cells were before measuring absorbance at 490 nm. Molisch’s test was resuspended in sterilized distilled water and centrifuged performed by adding 3 mL cell free supernatant to 1 mL again. The pellet was oven-dried at 105°C for 4 h and 10% α-naphthol. This was followed by the addition of weighed (Raza et al. 2007). 1 mL concentrated sulfuric acid to the sample without disturbing it. Evaluation of nitrogen sources The crude biosurfactant extracted with chloroform: methanol was analyzed by thin layer chromatography Different nitrogen sources, namely ammonium nitrate, (TLC). The TLC tank was filled with a solvent mixture of ammonium chloride, ammonium sulfate, sodium nitrate chloroform:methanol:acetic acid:water (25:15:4:2 v/v/v/v). and potassium nitrate, were investigated for maximum The chromatogram was sprayed with α-naphthol and production of biosurfactant. For this experiment, the sulfuric acid. The crude biosurfactant was also analyzed 756 Ann Microbiol (2012) 62:753–763 in a Bruker Vector 22 FTIR-Spectrometer. The spectral subtilis, the optimal production of biosurfactant was −1 −1 region used was 4,000–400 cm at a resolution 4 cm observed at pH 7 (Makkar and Cameotra 2002). Addition using a KBr plate of 0.26 mm thickness. of NaCl to the mineral medium had no influence on the enhancement of biosurfactant production. On the contrary, Activity characterization NaCl supplementation lowered production at concentra- tions <0.8% (w/v) (Fig. 3), suggesting that either the strain Foam was produced by hand shaking a 2-day-old culture cannot tolerate salinity or that it cannot produce biosurfac- supernatant for a few minutes. The stability of the foam was tant at higher salinity. This is in agreement with a result monitored by observing it for 48 h. Thermal stability of the reporting that limiting the concentrations of salts of biosurfactant was determined by autoclaving cell-free magnesium, calcium, potassium, sodium and trace elements culture supernatant at 121°C for 15 min. The effect of pH resulted in a better yield of rhamnolipid in P. aeruginosa and salinity on stability of the biosurfactant was evaluated DSM 2659 (Guerra-Santos et al. 1986). by altering the pH (2–10) and the concentration of NaCl The surface tension dropped rapidly at about 20 h of (2%–10%) of the cell free culture supernatant and measur- growth, reaching its lowest value (27.0 mN/m) during ing the surface tension (Bordoloi and Konwar 2008). exponential phase after about 44 h of growth. At the stationary phase of growth, no further decrease in surface tension was recorded. Surface tension gradually increased Results and discussion at this phase of bacterial growth (Fig. 4). As seen in Fig. 4, with the growth of the bacteria, the E24 gradually increased Isolation, screening and identification with the decrease in surface tension. The E24 reached a of biosurfactant-producing bacteria maximum (76%) at 68–72 h of growth and then began to decrease with the increase in surface tension. The E24 plot, a Table 1 lists 29 morphologically different bacterial isolates measure of the biosurfactant concentration, also showed that that were screened out from the collected soil samples. the surfactant was not present initially in sufficient amount to Among these, isolate no. 29, which could reduce the form micelles (Abouseoud et al. 2008b), but with the growth maximum surface tension of the mineral medium from of the bacterial culture, the concentration of surfactant in the 71.1 to 31.4 mN/m, was selected as the most potent medium increased, thus increasing the E24. These results biosurfactant-producing strain. The morphological and indicated that biosurfactant production in glycerol occurred physiological patterns of the strain showed a high similarity predominantly during the exponential growth phase, suggest- to Pseudomonas aeruginosa (99%). When the partial 16S ing that the biosurfactant was produced as a primary rDNA gene sequence was aligned with the NCBI GenBank metabolite accompanying cellular biomass formation and RDP databases, ten of the top ten matches were to (growth-associated kinetics). The dry cell biomass obtained Pseudomonas aeruginosa strains. Since the sequence was 3.7 g/L. similarities to Pseudomonas aeruginosa were uniformly 100%, the unknown strain was confirmed as Pseudomonas Evaluation of nitrogen sources aeruginosa RS29. Maximum STR of the medium was achieved in 24 h of Growth conditions optimization bacterial growth using sodium or potassium nitrate as nitrogen source (Table 3). With ammonium nitrate, maxi- Glycerol was found to be the best carbon source on which mum STR was obtained in 48 h; thereafter surface tension to reduce maximum surface tension (62%). Glucose and started increasing to a certain extent. The control with no mannitol could reduce the surface tension of mineral nitrogen source showed a gradual decrease in surface medium to 57% and 56%, respectively. Growth of the tension. STR for ammonium chloride- or ammonium strain on hexadecane resulted in a 37% decrease in surface sulfate-containing media was less prominent compared tension and a poor result was recorded for sludge (19%) with other nitrogen sources. (Table 2). These results indicated that the strain produces As seen in Table 3, the pattern of change in pH of the maximum biosurfactant on water-soluble substrates. Bio- mineral medium was different for different nitrogen surfactant production reached its maximum at 37.5°C sources. With ammonium chloride and ammonium sulfate, (Fig. 1), with an optimum initial pH range of 7.0–8.0 pH of the medium decreased abruptly from 7.5 to (Fig. 2). This is in agreement with a previous report that approximately 5.0 within 24 h of bacterial growth. In the maximum biosurfactant production by Pseudomonas aeru- control, the initial pH of the medium was retained almost ginosa 181 was achieved after 120 h of incubation at pH for the whole period of 96 h. With ammonium, sodium and 7.0 and 37°C (Al-Araji and Issa 2004). For Bacillus potassium nitrate, pH of the medium increased by 24 h then Ann Microbiol (2012) 62:753–763 757 Table 1 Surface tension of 29 bacterial isolates on glucose-containing mineral medium over a period of 7 days. Results are presented as mean ± SD of three replicates Isolate no. Day C 71.1±0.30 71.0±0.24 71.0±0.40 69.9±0.30 69.9±0.23 70.0±0.20 69.8±0.32 69.8±0.04 1 65.0±0.25 60.0±0.20 55.8±0.50 46.3±0.40 45.0±0.43 45.0±0.33 48.9±0.30 50.0±0.30 2 65.5±0.50 56.3±0.09 56.0±0.20 53.9±0.24 53.0±0.32 53.2±0.22 48.3±0.20 40.8±0.30 3 67.9±0.40 69.4±0.44 66.5±0.43 65.0±0.31 65.0±0.33 63.7±0.26 62.2±0.40 60.0±0.21 4 68.8±0.16 64.0±0.23 61.8±0.43 61.0±0.33 62.2±0.43 63.0±0.27 60.7±0.44 69.5±0.20 5 71.2±0.19 71.0±0.30 70.4±0.41 70.1±0.50 69.0±0.50 69.0±0.30 69.8±0.32 57.2±0.20 6 71.3±0.25 71.0±0.51 54.1±0.40 53.4±0.30 52.4±0.30 55.5±0.15 57.0±0.20 44.5±0.22 7 60.3±0.16 45.9±0.21 47.0±0.20 46.0±0.30 45.0±0.20 43.2±0.30 41.4±0.24 56.4±0.32 8 69.8±0.12 68.5±0.17 60.1±0.24 60.0±0.43 62.7±0.41 59.8±0.16 57.7±0.25 69.5±0.40 9 69.3±0.50 69.5±0.46 70.6±0.34 70.6±0.40 70.0±0.30 69.7±0.40 69.5±0.30 54.0±0.30 10 68.3±0.25 52.2±0.30 53.7±0.40 51.8±0.30 45.0±0.16 48.3±0.30 53.5±0.40 62.2±0.31 11 69.9±0.51 56.6±0.26 60.2±0.40 60.0±0.30 60.0±0.37 61.7±0.40 62.0±0.21 61.8±0.40 12 60.8±0.15 57.7±0.21 60.4±0.16 57.8±0.23 57.0±0.16 59.5±0.24 61.0±0.40 57.1±0.22 13 70.9±0.50 70.2±0.47 61.2±0.50 61.0±0.35 55.5±0.25 55.0±0.34 57.1±0.23 44.7±0.40 14 69.4±0.16 59.2±0.24 58.6±0.22 57.1±0.35 56.2±0.40 55.0±0.08 54.7±0.12 57.4±0.05 15 65.9±0.06 63.8±0.17 61.3±0.07 55.3±0.11 55.0±0.22 45.0±0.17 46.3±0.41 54.5±0.15 16 71.2±0.40 70.5±0.30 66.5±0.31 66.2±0.23 65.0±0.33 60.9±0.06 57.0±0.32 57.0±0.17 17 69.4±0.21 66.0±0.16 62.3±0.45 59.0±0.09 59.3±0.24 59.3±0.32 59.0±0.05 51.6±0.32 18 68.8±0.06 58.9±0.17 55.5±0.30 53.9±0.20 50.4±0.31 50.0±0.40 49.2±0.09 67.6±0.21 19 69.9±0.05 68.0±0.21 67.0±0.31 67.1±0.22 68.2±0.09 68.3±0.26 68.9±0.17 54.4±0.33 20 68.0±0.15 60.9±0.18 60.0±0.09 59.4±0.12 56.6±0.47 56.0±0.06 55.7±0.27 47.0±0.20 21 69.4±0.50 55.9±0.45 46.7±0.30 45.0±0.30 44.0±0.24 44.2±0.22 45.9±0.40 57.1±0.14 22 69.3±0.30 65.0±0.08 62.9±0.30 58.1±0.50 57.4±0.06 57.0±0.31 59.3±0.16 66.8±0.31 23 67.8±0.31 64.6±0.22 64.0±0.06 56.6±0.31 55.2±0.21 60.8±0.22 63.5±0.40 56.8±0.20 24 69.0±0.16 65.4±0.07 59.8±0.21 52.3±0.30 50.1±0.20 50.0±0.21 55.5±0.30 57.3±0.20 25 68.9±0.22 66.6±0.18 60.7±0.16 54.9±0.20 53.0±0.20 53.2±0.31 51.2±0.40 52.0±0.21 26 68.2±0.40 54.7±0.09 48.3±0.23 47.2±0.30 45.0±0.21 52.2±0.30 51.4±0.30 57.7±0.40 27 70.2±0.21 64.0±0.34 60.6±0.32 58.2±0.16 58.6±0.21 58.0±0.26 70.2±0.19 44.0±0.24 28 66.7±0.04 47.0±0.30 50.2±0.30 49.0±0.31 48.2±0.30 44.2±0.20 43.3±0.50 35.6±0.16 29 60.5±0.23 55.9±0.33 31.4±0.12 35.5±0.15 35.3±0.23 35.3±0.33 35.5±0.34 35.6±0.22 Abiotic control Isolate reducing surface tension to 31.4 mN/m, the lowest value amongst all isolates decreased at 48 h, with this change being more abrupt for again followed by an increase in pH of the medium in the ammonium nitrate than the other two N sources. This was subsequent hours for all three N sources. Table 2 Surface tension reduc- Carbon source Surface tension of control Surface tension of sample Surface tension tion (STR) with different carbon (mN/m) (mN/m) reduction (%) sources. Surface tension values represented mean ± SD of three Glucose 69.3±0.2 29.7±0.30 57 independent experiments Glycerol 70.2±0.14 28.4±0.33 60 Mannitol 68.7±0.31 30.4±0.12 56 n-Hexadecane 55.0±0.22 37.0±0.22 33 Sludge 47.0±0.32 39.0±0.34 17 758 Ann Microbiol (2012) 62:753–763 70 80 60 80 30 40 0 0 25 27.5 30 32.5 35 37.5 40 42.5 45 0 0 0 0.2 0.4 0.6 0.8 1 5 Temperature (°C) NaCl (%) Surface tension Emulsification index Surface tension Emulsification index Fig. 1 Effect of temperature on biosurfactant production Fig. 3 Effect of NaCl concentration on biosurfactant production Maximum bacterial growth was observed in ammonium nitrogen limitation enhances biosurfactant production nitrate-containing mineral medium, with almost equal growth (Suzuki et al. 1974; Ramana and Karanth 1989). being recorded for the rest of the four nitrogen sources. The In the case of ammonium nitrate, biomass growth of P. lowest bacterial growth was observed in the control (Table 3). aeruginosa RS29 was supported more than biosurfactant E24 was maximal, and almost the same, for sodium nitrate synthesis. After maximum STR of the medium, surface (80%) and potassium nitrate (79%) followed by ammonium tension again started to increase with time, indicating that nitrate (72%). A minimum value was observed for ammonium the concentration of biosurfactant decreased in the medium. chloride (36%) and ammonium sulfate (36%). The E24 for the The observed disappearance of biosurfactant might be control was 50% (Table 3). related to the development of competence. There are three In summary, P. aeruginosa RS29 could utilize all five possible mechanisms responsible for the decline in the tested nitrogen salts for growth. However, maximum biosurfactant concentration in stationary phase: (1) the growth (OD 4.0) was recorded in medium containing biosurfactant was degraded by the enzymes in the culture, ammonium nitrate as the nitrogen source. Although growth (2) the biosurfactant might be adsorbed on the cell surface, or of the strain was lower in sodium and potassium nitrate (3) the biosurfactant was reinternalized and processed than ammonium nitrate, the former two were best for intracellularly (Lin et al. 1993). Although ammonium sulfate production of biosurfactant in terms of STR and E24 and ammonium chloride supported bacterial growth, pro- (26.3 mN/m and 26.4 mN/m, with E24 of 80 and 79%, duction of biosurfactant was very poor. It can be deduced respectively). This might be because nitrogen was less that, at low pH of the culture medium, bacteria could not available from these two salts because nitrate first under- efficiently synthesize biosurfactant (Fig. 2). The increase in goes dissimilatory nitrate reduction to ammonium followed pH in the presence of ammonium-, sodium- and potassium- by assimilation by glutamine-glutamate metabolism. This nitrate implied accumulation of compounds like siderophores means that the assimilation of nitrate as a nitrogen source is in the culture medium (Varma and Chincholkar 2007). The so slow that it would simulate conditions of limiting color of the medium also changed to brown for these nitrogen (Hisatsuka et al. 1971; Itoh and Suzuki 1972; three salts. The change in color of the transparent Guerra-Santos et al. 1983). It had been reported that mineralmediumtobrown colorafter48hofincubation was also an indicator of the accumulation of siderophores in the medium (Nair et al. 2007). The pH of the medium 60 80 increased during the growth period, in accordance with the siderophore concentration, suggesting that alkalinity is important to avoid siderophore destruction (Díaz de Villegas et al. 2002). The control also exhibited a gradual 30 40 decrease in surface tension. The observed bacterial growth and biosurfactant production in the control was because of bacterial utilization of yeast extract in the mineral medium as a nitrogen source. 0 0 45 6789 Biosurfactant recovery and characterization Initial pH Surface tension Emulsification index The yield of biosurfactant (6 g/L) from P. aeruginosa RS29 Fig. 2 Effect of initial pH on biosurfactant production strain in the presence of glycerol as sole source of carbon Surface tension (mN/m) Surface tension (mN/m) Emulsification index (%) Emulsification index (%) Surface tension (mN/m) Emulsification index (%) Ann Microbiol (2012) 62:753–763 759 Fig. 4 Kinetics of biosurfactant 3.5 80 production by Pseudomonas aeruginosa RS29 under optimized conditions 2.5 1.5 0.5 0 0 0 8 16 24 32 40 48 56 64 120 Time (h) O.D. Surface tension Emulsification index Table 3 Effect of nitrogen Mineral medium with different 0 h 24 h 48 h 72 h 96 h source on surface tension nitrogen sources reduction (STR), pH, optical density (OD) and emulsification Control index (E24) of the mineral medium during biosurfactant Surface tension 62.0±0.12 51±0.15 36.1±0.62 32.2±0.3 28.9±0.8 production by Pseudomonas pH 7.5±0.02 6.7±0.5 6.8±0.4 7.1±0.30 7.0±0.30 aeruginosa RS29. Results OD 0.138±0.15 1.18±0.03 1.39±0.02 1.47±0.02 1.5±0.03 represent the mean ± SD E24 (%) 50±1.20 of three independent experiments Ammonium nitrate Surface tension 63.2±0.10 35.5±0.5 27.2±1.0 30.9±0.8 31.1±0.7 pH 7.5±0.03 8.2±0.12 5.54±0.20 8.3±0.40 8.3±0.45 OD 0.165±0.05 2.33±0.04 3.87±0.05 4.0±0.03 4.0±0.04 E24 (%) 72±1.0 Ammonium chloride Surface tension 62.0±0.02 50.5±0.15 52.2±0.5 50.6±0.3 47.0±0.40 pH 7.5±0.02 4.8±0.20 4.6±0.45 5.3±0.4 4.8±0.23 OD 0.162±0.1 2.039±0.02 2.12±0.02 2.15±0.04 2.15±0.03 E24 (%) 36±0.6 Ammonium sulfate Surface tension 62.3±0.05 49.3±0.34 51.5±0.4 52.6±0.42 51.1±0.50 pH 7.5±0.01 5.1±0.20 5.53±0.2 5.0±0.3 4.8±0.20 OD 0.165±0.22 1.96±0.02 2.06±0.01 2.12±0.02 2.11±0.02 E24 (%) 36±0.5 Sodium nitrate Surface tension 62.2±0.01 26.3±0.1 26.3±0.12 26.5±0.1 27.0±0.20 pH 7.5±0.02 8.5±0.10 7.4±0.15 8.6±0.2 8.65±0.20 OD 0.160±0.07 2.18±0.01 2.36±0.02 2.36±0.01 2.32±0.01 E24 (%) 80±1.0 Potassium nitrate Surface tension 62.1±0.03 26.4±0.2 26.4±0.4 26.5±0.4 26.8±0.30 pH 7.5±0.03 8.3±0.10 7.4±0.12 8.8±0.20 8.53±0.30 OD 0.163±0.2 2.08±0.01 2.28±0.02 2.31±0.01 2.25±0.01 E24 (%) 79±0.6 OD (460 nm) Surface tension (mN/m), Emulsification index (%) 760 Ann Microbiol (2012) 62:753–763 70 50 50 40 40 30 30 20 20 40 10 10 0 0 30 2468 10 NaCl (%) 20 Surface tension Emulsification index Fig. 7 Effect of salinity on biosurfactant activity tant molecules had begun to aggregate (Karsa et al. 1999; Meylheuc et al. 2001). The CMC obtained was in agreement with previous reports (Bordoloi and Konwar 2009). In the Conc. of biosurfactant (log of mg/L) case of P. aeruginosa SP4, the excreted biosurfactant in the Fig. 5 Critical micelle concentration (CMC) of extracted biosurfac- culture supernatant could decrease the surface tension of tant produced by Pseudomonas aeruginosa RS29 pure water from 72.0 to 28.3 mN/m, and the CMC was estimated to be 120 mg/L (Pornsunthorntawee et al. 2008). was higher than the reported biosurfactant production by P. For Pseudomonas fluorescens, the CMC recorded for the aeruginosa NM strain (5 g/L) grown in glycerol (Das and isolated biosurfactant was 290 mg/L and the corresponding Mukherjee 2005), and almost the same as with a previous surface tension was 32 dynes/cm. The CMC value of the report for P. aeruginosa M strain (Das and Mukherjee 2005) chemical surfactant sodium dodecyl sulphate (SDS) is and other Pseudomonas sp. (Pruthi and Cameotra 1995). 140 mg/L (Bordoloi and Konwar 2009). So, the biosurfactant As seen in Fig. 5, the surface tension is dependent on the produced by P. aeruginosa RS29 exhibits better properties in concentration of crude biosurfactant, and the CMC corre- terms of higher STR and a lower CMC. The results of −1 −2 sponded to a sudden change in the surface tension CMD and CMD of the biosurfactant containing cell-free (Abouseoud et al. 2008b). The CMC for the isolated medium were 28.3 mN/m and 40.0 mN/m, respectively, biosurfactant calculated from the breakpoint of surface depicting little change in efficiency. The results suggest that tension versus the log of its concentration curve was 90 mg/ a sufficient amount of biosurfactant was present in the L, and the corresponding surface tension was 27.8 mN/m. culture medium, and thus its surface activity was retained Biosurfactant concentrations above the CMC could not even at such a high dilution. These values are appreciably decrease the surface tension further, indicating that biosurfac- better than the values reported for P. aeruginosa strains Fig. 6 Fourier transform infrared 1.1 (FTIR) spectra of the biosurfac- tant extracted from Pseudomonas aeruginosa RS29 0.9 C-O 0.8 COO 0.7 C-O-C CH CH 2 3 0.6 0.5 3370 O-H 0.4 3900 3400 2900 2400 1900 1400 900 400 0.17 0.7 1.3 1.9 2.15 Surface tension (mN/m) Surface tension (mN/m) Emulsification index (%) Ann Microbiol (2012) 62:753–763 761 80 80 (MTCC8165, MTCC7815, MTCC7812 and MTCC7814) (Bordoloi and Konwar 2008). 60 60 The rhamnose test was positive, indicating that the separated biosurfactant could be of the glycolipid type. 40 40 Molisch’s test showing a clear purple ring between the 20 20 layers of solvent and the sample, indicating that the sample contained sugar moieties. Red spots appeared on the TLC 0 0 plate after spraying with α-naphthol and sulfuric acid, 2468 10 indicating the presence of carbohydrates in the sample. The pH production of glycolipid-type biosurfactant was previously Surface tension Emulsification index reported for Pseudomonas sp. (Persson et al. 1988; Wilson Fig. 8 Effect of pH on biosurfactant activity and Bradley 1996; Patel and Desai 1997). Spectral analysis (Fig. 6) showed strong absorption −1 bands at 3,370 cm . This was observed due to stretching vibration of the –OH group. The absorption stability. The effect of addition of NaCl on surface tension −1 band observed at 2,923 cm confirmed the presence of alkyl and E24 of biosurfactant is shown in Fig. 7. Little change (CH and CH ) groups. A carbonyl stretching band 2 3 was observed in either parameter with addition of up to 10% −1 characteristic of ester compounds was found at 1,720 cm (w/v) NaCl. The surface tension of the biosurfactant was −1 . The absorption at 1,629 cm was because of stretching of stable at different pH values ranging from 2 to 10, and pH the COO– group. The spectra also showed an absorption increase had a positive effect on E24 (Fig. 8). This could be −1 band at 1,045 cm due to stretching vibration of –C–O–C. caused by the higher stability of fatty acids-surfactant The pattern of absorption bands observed for this micelles in presence of NaOH and the precipitation of particular strain was reported previously for rhamnolipids secondary metabolites at higher pH values (Abouseoud et al. (Bordoloi and Konwar 2009). These latter authors 2008a). Similarfindingswerereportedfor P. aeruginosa −1 explained that strong absorption bands at 3,443 cm occurred isolate Bs20, which exhibited excellent stability at high due to stretching vibration of the –O–H group and temperature (heating at 100°C for 1 h and autoclaving at −1 absorption at 1605–1625 cm due to either stretching 121°C for 10 min), salinities up to 6% NaCl, and pH values of –C=C or >C=O, i.e., stretching of the carboxylate anion. up to pH 13 (Abdel-Mawgoud et al. 2009). The properties of −1 It was also reported that absorption at 1,625 cm occurred the biosurfactant produced by native P. aeruginosa RS29 are due to presence of carboxylate anion, and absorption at promising for its application in different industries. −1 1,120 cm due to stretching vibration of –C–O–Cof the ether linkage of Rha-C8-C10 and Rha-C10-C8 molecules. Conclusion Activity characterization The isolated P. aeruginosa RS29 is a very potent Biosurfactant containing culture supernatant showed good biosurfactant-producing native strain. Strain RS29 produces foaming stability. The foam produced was stable for 48 h. maximum biosurfactant in sodium- or potassium-nitrate- Stable foam indicates that the produced biosurfactant can be containing medium where surface tension was reduced from used as a good foaming agent. Similar findings were reported 70.0 to 26.3 mN/m and 26.4 mN/m, respectively. The amount for 48-h-old culture of P. aeruginosa PTCC 1561 grown in of crude biosurfactant recovered (6 g/L) from the culture nutrient broth, which showed foam stability for 48 h medium is very promising. The CMC value (90 mg/L) of the (Noudeh et al. 2010). The biosurfactant produced by P. produced biosurfactant is superior to many other biosurfac- aeruginosa RS29 was shown to be thermostable. Autoclav- tants. The tensioactive properties and stability of the ing it at 121°C did not destroy its foaming properties. The biosurfactant to high temperature, pH and salinity reveal surface tension measured before autoclaving the sample was good prospects for this product in industrial applications. The 27.0 mN/m. The value was retained and recorded as emulsifying and foaming activity of the biosurfactant indicate 27.6 mN/m after autoclaving the sample. The E24 before that it can be used as a good emulsion-forming and foaming autoclaving the sample was 72% and 70% after autoclaving. agent in different industries. This indicated that STR and the E24 were stable up to quite a high temperature, in contrast to synthetic surfactants such Acknowledgments We thank the Director, Prof. Joyanti Chutia, IASST, as SDS, which exhibits a significant loss of emulsification Guwahati, India for permission and support. Moreover, this work has been activity above 70°C (Kim et al. 1997). Thus, our product is made possible through a project grant (BT/PR-9795/BCE/08/590/2007) better than synthetic surfactants in terms of temperature funded by Department of Biotechnology, Govt of India. We thank to Dr. N. Surface tension (mN/m) Emulsification index (%) 762 Ann Microbiol (2012) 62:753–763 Sen Sarma, Senior Assistant Professor, IASST for facilitating the FTIR microbial enhancement of oil recovery. US Department of analysis at IASST, Guwahati, India. Energy, Washington DC, pp 12–14 Guerra-Santos LH, Kappeli O, Fiechter A (1986) Dependence of Pseudomonas aeruginosa continuous culture biosurfactant pro- duction on nutritional and environmental factors. Appl Microbiol References Biotechnol 24:443–448 Hisatsuka K, Nakahara T, Sano N, Yamada K (1971) Formation of rhamnolipid by Pseudomonas aeruginosa: its function in Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NA (2009) Char- hydrocarbon fermentation. Agric Biol Chem 35:686–692 acterization of Rhamnolipid Produced by Pseudomonas aerugi- Itoh S, Suzuki T (1972) Effect of rhamnolipids on growth of nosa Isolate Bs20. Appl Biochem Biotechnol 157:329–345 Pseudomonas aeruginosa mutant deficient in n-paraffinutilizing Abouseoud M, Maachi R, Amrane A, Boudergua S, Nabi A (2008a) ability. Agric Biol Chem 36:2233–2235 Evaluation of different carbon and nitrogen sources in production of Jenny K, Deltrieu V, Kappelli O (1993) Lipopeptide production by biosurfactant by Pseudomonas fluorescens. Desalination 223:143–151 Bacillus licheniformis. In: Kosaric N (ed) Biosurfactants. Abouseoud M, Yataghene A, Amrane A, Maachi R (2008b) Biosurfactant Production, property, application. Dekker, New York, pp 135–156 production by free and alginate entrapped cells of Pseudomonas Karsa DR, Bailey RM, Shelmerdine B, McCann SA (1999) Overview: fluorescens. J Ind Microbiol Biotechnol 35:1303–1308 a decade of change in the surfactant industry. In: Karsa DR (ed) Al-Araji Y, Issa L (2004) Biosurfactant production by Pseudomonas Industrial applications of surfactants, vol 4. Royal Society of aeruginosa 181. PhD thesis, University Putra Malaysia Chemistry, London, pp 1–22 Babu PS, Vaidya AN, Bal AS, Kapur R, Juwarkar A, Khanna P (1996) Kim H, Yoon B, Lee C, Suh H, Oh H, Katsuragi T, Tani Y (1997) Kinetics of biosurfactant production by Pseudomonas aeruginosa Production and properties of a lipopeptide biosurfactant from strain BS2 from industrial waste. Biotechnol Lett 18:263–268 Bacillus subtilis C9. J Ferment Bioeng 84(1):41–46 Banat IM (1995) Biosurfactant production and possible uses in Kosaric N (1993) Biosurfactants. Production, property, application, microbial enhanced oil recovery and oil pollution remediation: surfactant sciences series, 48. Dekker, New York review. Bioresour Technol 55:1–12 Lang S, Wullbrandt D (1999) Rhamnose lipids, biosynthesis, Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial microbial production and application potential. Appl Microbiol applications of microbial surfactants. Appl Environ Microbiol Biotechnol 50:22–32 53:495–508 Lin SC, Sharma MM, Georgiou G (1993) Production and deactivation Bonilla M, Olivaro C, Corona M, Vazquez A, Soubes M (2005) of biosurfactant by Bacillus licheniformis JF-2. Biotechnol Prog Production and characterization of a new bioemulsifier from 9(2):138–145 Pseudomonas putida ML2. J Appl Microbiol 98:456–463 Mata-Sandoval JC, Karns J, Torrents A (1999) High performance Bordoloi NK, Konwar BK (2008) Microbial surfactant-enhanced liquid chromatography method for the characterization of mineral oil recovery under laboratory conditions. Colloids Surf rhamnolipid mixtures produces by Pseudomonas aeruginosa B Biointerfaces 63:73–82 UG2 on corn oil. J Chromatogr A 864:211–220 Bordoloi NK, Konwar BK (2009) Bacterial biosurfactant in enhancing Makkar RS, Cameotra SS (2002) Effects of Various Nutritional solubility and metabolism of petroleum hydrocarbons. J Hazard Supplements on Biosurfactant Production by a Strain of Bacillus Mater 170:495–505 subtilis at 45°C. J Surfactants Deterg 5:11–17 Cameotra SS, Makkar RS (2004) Recent applications of biosurfactants Meylheuc T, Heary JM, Bellon-Fontaine MN (2001) Les biosurfac- as biological and immunological molecules. Curr Opin Microbiol tants, des biomolécules à forte potentialité d’application. Sci 7:262–266 Aliments 21:591–649 Cappuccino JG, Sherman N (1999) Microbiology—a laboratory Nair A, Juwarkar AA, Singh SK (2007) Production and characteriza- manual. Addison-Wesley Longman, Harlow, pp 417–421 tion of siderophores and its application in arsenic removal from Cooper DG (1986) Biosurfactants. Microbiol Sci 3:145–149 contaminated soil. Water Air Soil Pollut 180:199–212 Cooper DG, Goldenberg BG (1987) Surface-active agents from two Noudeh GD, Noodeh AD, Moshafi MH, Behravan E, Afzadi MA, Bacillus species. Appl Environ Microbiol 53(2):224–229 Sodagar M (2010) Investigation of cellular hydrophobicity and Cooper DG, Zajic JE (1980) Surface-active compounds from surface activity effects of biosynthesed biosurfactant from broth microorganism. Adv Appl Microbiol 26:229–256 media of PTCC 1561. Afr J Microbiol Res 4(17):1814–1822 Das K, Mukherjee AK (2005) Characterization of biochemical Patel RM, Desai AJ (1997) Surface-active properties of rhamnolipids from properties and biological activities of biosurfactants produced Pseudomonas aeruginosa GS3. J Basic Microbiol 37:281–286 by Pseudomonas aeruginosa mucoid and non-mucoid strains Perfumo A, Banat IM, Canganella F, Marchant R (2006) Rhamnolipid isolated from hydrocarbon-contaminated soil samples. Appl production by a novel thermophilic hydrocarbon-degrading Microbiol Biotechnol 69:192–199 Pseudomonas aeruginosa AP02-1. Appl Microbiol Biotechnol Desai JD, Banat IM (1997) Microbial production of surfactants and 72(1):132–138 their commercial potential. Microbiol Mol Biol R 61(1):47–64 Persson A, Österberg E, Dostalek M (1988) Biosurfactant production Deziel E, Lepine F, Dennie D, Boismenu D, Mamer OA, Villemur R (1999) by Pseudomonas Xuorescens 378: growth and product character- Liquid chromatography/mass spectrometry analysis of mixture of istics. Appl Microbiol Biotechnol 29(1):1–4 rhamnolipids produced by P.aeruginosa strain 57RP grown on mannitol or naphthalene. Biochim Biophys Acta 1440:244–252 Pornsunthorntawee O, Arttaweeporna N, Paisanjit S, Somboonthanatea P, Díaz de Villegas ME, Villa P, Frías A (2002) Evaluation of the Abeb M, Rujiravanita R, Chavadeja S (2008) Isolation and siderophores production by Pseudomonas aeruginosa PSS. Rev comparison of biosurfactants produced by Bacillus subtilis PT2 Latinoam Microbiol 44:112–117 and Pseudomonas aeruginosa SP4 for microbial surfactant- Dubois M, Gills KA, Hamilton JK, Rebers PA, Smith F (1956) enhanced oil recovery. Biochem Eng J 42:172–179 Colorimetric method for determination of sugar and related Pruthi V, Cameotra SS (1995) Rapid method for monitoring maximum substances. Anal Chem 28:350–356 biosurfactant produced by acetone precipitation. Biotechnol Tech Guerra-Santos L, Kappeli O, Fiechter A (1983) Growth and 9:271–276 biosurfactant production of a bacterium in continuous culture. Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM (2002) In: Donaldson EC, Clark JB (eds) International conference on Rhamnolipid biosurfactant production by strains of Pseudomonas Ann Microbiol (2012) 62:753–763 763 aeruginosa using low-cost raw material. Biotechnol Prog Sheppard JD, Mulligan CN (1987) The production of surfactin by 18:1277–1281 Bacillus subtilis grown on peat hydrolysate. Appl Microbiol Ramana KV, Karanth NG (1989) Factors affecting biosurfactants Biotechnol 27:110–116 production using Pseudomonas aeruginosa CFTR-6 under Suzuki T, Tanaka H, Itoh S (1974) Sucrose lipids of Arthrobacteria, submerged conditions. J Chem Technol Biotechnol 45:249–257 Corynebacteria and Nocardia grown on sucrose. Agric Biol Raza ZA, Khan MS, Khalid ZM (2007) Physicochemical and surface- Chem 38:557–563 active properties of biosurfactant produced using molasses by a Wilson NG, Bradley G (1996) The eVect of immobilization on Pseudomonas aeruginosa mutant. J Environ Sci Health A Tox rhamnolipid production by Pseudomonas Xuorescens. J Appl Hazard Subst Environ Eng 42:73–80 Bacteriol 81(5):525–530 Ron EZ, Rosenberg E (2001) Natural roles of biosurfactants. Environ Varma A, Chincholkar S (eds) (2007) Microbial siderophores. Microbiol 3:229–236 Springer, New York, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

Download PDF
11 pages

Loading next page...
 
/lp/springer-journals/isolation-of-biosurfactant-producing-pseudomonas-aeruginosa-rs29-from-m40oHdKkZW

References (46)

Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-011-0315-5
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2012) 62:753–763 DOI 10.1007/s13213-011-0315-5 ORIGINAL ARTICLE Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production Rashmi Rekha Saikia & Suresh Deka & Manab Deka & Ibrahim M. Banat Received: 22 December 2010 /Accepted: 6 July 2011 /Published online: 20 July 2011 Springer-Verlag and the University of Milan 2011 Abstract An efficient biosurfactant-producing native Pseu- tion of the biosurfactant was 90 mg/L. The absorption domonas aeruginosa RS29 has been isolated from crude oil bands of the FTIR spectra confirmed the rhamnolipid contaminated soil. Isolation was followed by optimization nature of the biosurfactant. The biosurfactant was thermo- of different factors to achieve maximum production of stable (up to 121°C for 15 min) and could withstand a wide biosurfactant in terms of surface tension reduction (STR) range of pH (2–10) and NaCl concentration (2%–10% w/v). and emulsification index (E24). The isolated strain pro- The extracted biosurfactant had good foaming and duced highest biosurfactant in the presence of glycerol after emulsifying activities and was of satisfactory quality in terms 48 h of incubation at 37.5°C, with pH range of 7–8 and at of stability (temperature, pH and salinity) and foaming salinity <0.8% (w/v). The extent of STR and the E24 of activity. medium with different nitrogen sources were investigated . . and found to be maximal for sodium nitrate (26.3 mN/m, Keywords Isolation Biosurfactant Pseudomonas E24=80%) and potassium nitrate (26.4 mN/m, E24=79%). aeruginosa RS29 Rhamnolipid 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 Introduction kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and Biosurfactants are diverse groups of surface-active mole- highest STR was achieved (27.0 mN/m) after 44 h of cules/chemical compounds synthesized by microorganisms growth. The biosurfactant was produced as a primary (Desai and Banat 1997). These are amphipathic molecules metabolite and 6 g/L crude biosurfactant was extracted by with both hydrophilic and hydrophobic domains. Biosur- chloroform:methanol (2:1). The critical micelle concentra- factants reduce surface tension and critical micelle dilution (CMD) in both aqueous solution and hydrocarbon mixtures. These properties create micro-emulsions in which micelle formations occur in which hydrocarbons can solubilize in R. R. Saikia (*) S. Deka water or water in hydrocarbons (Banat 1995). Micro- Life Sciences Division, Institute of Advanced Study organisms have beenreportedtoproduce severalclassesof in Science and Technology, Paschim Boragaon, biosurfactants, such as glycolipids, lipopeptides, phospholi- Guwahati 781035 Assam, India pids, neutral lipids or fatty acids and polymeric biosurfactants e-mail: rashmi.saikia@gmail.com (Cooper and Zajic 1980; Cooper 1986; Kosaric 1993). These compounds are metabolic products produced during M. Deka Department of Biotechnology, Gauhati University, the growth of microorganisms on water-soluble and water Guwahati 781014 Assam, India immiscible substrates (Sheppard and Mulligan 1987; Jenny et al. 1993; Ron and Rosenberg 2001). I. M. Banat The most commonly isolated and the best studied groups Faculty of Life and Health Sciences, University of Ulster, of biosurfactants are mainly glycolipids and phospholipids Coleraine BT52 1SA Northern Ireland, UK 754 Ann Microbiol (2012) 62:753–763 in nature. Nevertheless, rhamnolipids are glycolipid com- with fresh medium. Cultures from the last enriched flask pounds produced by Pseudomonas sp. that can reduce water were plated on nutrient agar using a serial dilution surface tension and emulsify oil (Babu et al. 1996;Dezielet technique. Morphologically different individual bacterial al. 1999;Lang and Wullbrandt 1999; Mata-Sandoval et al. colonies were isolated from the agar plates and streaked on 1999; Rahman et al. 2002; Perfumo et al. 2006). Biosurfac- nutrient agar to obtain pure cultures of the isolates. The tants are environmentally friendly and have potential isolates were grown in nutrient broth for 48 h at 35°C at industrial and environmental applications. 150 rpm and used as inoculum for further experiments. When compared to synthetic surfactants, biosurfactants Proper screening of the biosurfactant-producing bacterial have several advantages, including high biodegradability, isolates was carried out by adding 10 mL inoculum of low toxicity, low irritancy, and compatibility with human each isolate and growing them in 500 mL flasks skin (Banat et al. 2000; Cameotra and Makkar 2004). containing 100 mL sterilized mineral medium with Therefore they are superior to their synthetic counterparts. glucose as the sole carbon source. Cultivations were Many studies have been conducted with microorganisms performed in triplicate. The composition of the mineral to ensure the production and activity of biosurfactants, medium used was as follows (g/L): NH NO (4.0), KCl 4 3 emphasizing the importance of the need for these com- (0.1), KH PO (0.5), K HPO (1.0), CaCl (0.01), 2 4 2 4 2 pounds. However, we have failed to identify any literature MgSO ⋅7H O (0.5), FeSO ⋅7H O (0.01), Yeast extract 4 2 4 2 report dealing solely with native bacterial isolates. Explor- (0.1) and 10 mL of trace element solution containing (g/L): ing native strains for biosurfactant production could be of 0.26 g H BO ,0.5 gCuSO ⋅5H O, 0.5 g MnSO ⋅H O, 3 3 4 2 4 2 great importance since native strains can be assumed to 0.06 g (NH )6Mo O ⋅4H O and 0.7 g ZnSO ⋅7H O. The 4 7 24 2 4 2 perform better in their native environment than other exotic pH of the medium was adjusted to 7.0±0.2 and the flasks strains. Therefore, the present experiment was conducted to were kept in a shaking incubator at 35°C and 150 rpm for isolate potent biosurfactant-producing native bacteria and to 7 days. Screening was based on surface tension reduction identify the optimal environmental factors—temperature, (STR); surface tension was measured every day up to the pH, salinity, nitrogen source and carbon source—for 7th day using a K11 tensiometer (Kruss, Hamburg, Germany) maximum production of biosurfactant. Besides other and the plate method. All chemicals were purchased from objectives, the nature of the produced biosurfactant was Merck (Mumbai, India). also characterized, along with production kinetics and efficacy studies. Identification of efficient isolates The most efficient bacterial strain was identified by (1) Materials and methods studying the morphological and physiological character- istics (Cappuccino and Sherman 1999), and (2) sequencing Soil sample 16S rDNA and aligning the sequence in the NCBI GenBank and RDP database (performed by Bangalore Crude oil-contaminated soil samples were collected from GeNei, India). Following a standard protocol, genomic the Lakowa oil fields of Assam, India. Soil samples DNA was extracted from a pure culture, and a 16S rDNA were collected at a depth of 0–10 cm from the soil fragment of ∼1.5 kb was amplified using Taq DNA surface and stored at room temperature (25±2°C) for polymerase and consensus primers. The PCR product was subsequent use. bi-directionally sequenced using the forward, reverse and an internal primer. Sequence data was aligned and analyzed Isolation and screening of biosurfactant-producing bacteria to find the closest homolog of the isolated bacterial strain. Bacteria were isolated by adding 1 g collected soil sample Optimization of growth conditions to a flask containing nutrient broth and mineral salt solution at a 1:1 ratio and crude oil (2% w/v) was provided as a sole Different carbon sources were tested to select the one most carbon source; the enriched flask was incubated at 35°C for suitable for maximum STR. The carbon sources used in the 4 days in a rotary shaker Scigenics Biotech, ORBITEK experiment were glucose, glycerol, mannitol, n-hexadecane LJEIL at 150 rpm. From this original flask, 5 mL cultures and sludge. These were added to the mineral medium to were subsequently used to inoculate a second flask 2%. The inoculated mineral medium was incubated in a containing fresh medium, and this flask was maintained rotary shaker at 35°C and 150 rpm for 4 days and the under the same conditions. This process was repeated four surface tension was recorded every day. To compare STR, times, and each time same amount of culture was flasks with the same carbon sources but no bacterial culture withdrawn from the older flask and added to a new flask (control) were also studied. Ann Microbiol (2012) 62:753–763 755 A range of temperatures was investigated to determine nitrogen salts were used at 4% (w/v) concentration. A the optimum temperature for maximum production of control was also studied where no nitrogen salts were added biosurfactant. Production of biosurfactant was measured to the mineral medium. Variation in pH of the culture in terms of STR and emulsification index (E24). Efficient medium, the E24, OD as well as the surface tension of the strains growing in mineral medium with a suitable medium were measured. carbon source were incubated at the following temper- atures: 25, 27.5, 30.0, 32.5, 35.0, 37.5, 40.0, 42.5 and Biosurfactant extraction and characterization 45.0°C at 150 rpm. The initial pH of the mineral medium with a suitable carbon source was investigated by Crude biosurfactant was extracted from cell-free culture adjusting the pH of the medium within the range of 2– broth (CFCB) by chloroform:methanol (2:1) extraction 9. The pH of the medium was adjusted using 1 M HCl three times. CFCB was obtained by centrifuging 48-h-old and 1 M NaOH solutions. The cultures were kept in the bacterial culture grown on glycerol at 8,000 rpm for 20 min shaking incubator at the optimum temperature at at 4°C. The combined extracts were then transferred to a 150 rpm. Optimum salinity was determined by changing round-bottom flask connected to a rotary evaporator. The the concentration of NaCl of the mineral medium from concentration process was continued at 40°C until a 0.2% to 5% (w/v). The efficient strain was grown in yellowish-brown-colored viscous and consistent extract medium with a suitable carbon source. The initial pH of was obtained. The crude biosurfactant was then dried the medium was adjusted and the culture was incubated and weighed. in the rotary shaker at 150 rpm at the optimum The extracted biosurfactant was dissolved in distilled temperature. Surface tension and E24 were measured in water at concentrations ranging from 1.0 to 200 mg/L each flask. E24 was measured by mixing 3 mL whole for calculation of critical micelle concentration (CMC). bacterial culture with an equal volume of n-hexadecane and This is a direct measurement of surfactant concentration vortexing at high speed for 2 min. The sample was left corresponding to the concentration of an amphiphilic undisturbed for 24 h. The E24 is expressed as the percentage component at which the formation of micelles is initiated of the height of the emulsified layer (mm) divided by the in the solution (Abouseoud et al. 2008a). The CMC of total height of the liquid column (mm) (Cooper and the produced biosurfactant was determined following Goldenberg 1987;Abouseoudetal. 2008a). standard methods (Kim et al. 1997; Bonilla et al. 2005). Optimization of growth conditions was followed by a The critical micelle dilution (CMD) is defined as the kinetic study of biosurfactant production as well as solubility of a surfactant in an aqueous phase and is production of biomass. These parameters were investigated commonly used to measure the efficiency of a surfactant −1 −2 (Desai and Banat 1997). CMD by growing the efficient strain in mineral medium with a and CMD were suitable carbon source and maintaining all the optimum determined by measuring the surface tensions of cell free conditions. The kinetics of biosurfactant production were supernatant diluted 10-times and 100-times in distilled studied in batch cultures over a period of 72 h. Surface water (Kosaric 1993). Cell free supernatant was obtained tension and optical density at 460 nm (OD )were by centrifuging a 48-h-old culture of the strain grown in measured at 4-h intervals. Surface tension was measured mineral medium with glycerol at 8,000 rpm for 20 min in tensiometer and OD was measured in a SHIMADZU at 4°C. UV-1800 UV-spectrophotometer. E24 was also recorded. Carbohydrate moieties in the biosurfactant molecule Dry cell biomass was determined by centrifugation of an were assayed using rhamnose (Dubois et al. 1956) and aliquot (100 mL) of 48-h-old bacterial culture at 8,000 rpm Molisch’s test. The rhamnose test was performed by adding for 20 min. The cell pellet was washed with n-hexane to 0.5 mL cell supernatant to 0.5 mL 5% phenol solution and remove any slimy materials attached to the cell surface that 2.5 mL sulfuric acid and incubating the sample for 15 min might cause error in the assessment. The washed cells were before measuring absorbance at 490 nm. Molisch’s test was resuspended in sterilized distilled water and centrifuged performed by adding 3 mL cell free supernatant to 1 mL again. The pellet was oven-dried at 105°C for 4 h and 10% α-naphthol. This was followed by the addition of weighed (Raza et al. 2007). 1 mL concentrated sulfuric acid to the sample without disturbing it. Evaluation of nitrogen sources The crude biosurfactant extracted with chloroform: methanol was analyzed by thin layer chromatography Different nitrogen sources, namely ammonium nitrate, (TLC). The TLC tank was filled with a solvent mixture of ammonium chloride, ammonium sulfate, sodium nitrate chloroform:methanol:acetic acid:water (25:15:4:2 v/v/v/v). and potassium nitrate, were investigated for maximum The chromatogram was sprayed with α-naphthol and production of biosurfactant. For this experiment, the sulfuric acid. The crude biosurfactant was also analyzed 756 Ann Microbiol (2012) 62:753–763 in a Bruker Vector 22 FTIR-Spectrometer. The spectral subtilis, the optimal production of biosurfactant was −1 −1 region used was 4,000–400 cm at a resolution 4 cm observed at pH 7 (Makkar and Cameotra 2002). Addition using a KBr plate of 0.26 mm thickness. of NaCl to the mineral medium had no influence on the enhancement of biosurfactant production. On the contrary, Activity characterization NaCl supplementation lowered production at concentra- tions <0.8% (w/v) (Fig. 3), suggesting that either the strain Foam was produced by hand shaking a 2-day-old culture cannot tolerate salinity or that it cannot produce biosurfac- supernatant for a few minutes. The stability of the foam was tant at higher salinity. This is in agreement with a result monitored by observing it for 48 h. Thermal stability of the reporting that limiting the concentrations of salts of biosurfactant was determined by autoclaving cell-free magnesium, calcium, potassium, sodium and trace elements culture supernatant at 121°C for 15 min. The effect of pH resulted in a better yield of rhamnolipid in P. aeruginosa and salinity on stability of the biosurfactant was evaluated DSM 2659 (Guerra-Santos et al. 1986). by altering the pH (2–10) and the concentration of NaCl The surface tension dropped rapidly at about 20 h of (2%–10%) of the cell free culture supernatant and measur- growth, reaching its lowest value (27.0 mN/m) during ing the surface tension (Bordoloi and Konwar 2008). exponential phase after about 44 h of growth. At the stationary phase of growth, no further decrease in surface tension was recorded. Surface tension gradually increased Results and discussion at this phase of bacterial growth (Fig. 4). As seen in Fig. 4, with the growth of the bacteria, the E24 gradually increased Isolation, screening and identification with the decrease in surface tension. The E24 reached a of biosurfactant-producing bacteria maximum (76%) at 68–72 h of growth and then began to decrease with the increase in surface tension. The E24 plot, a Table 1 lists 29 morphologically different bacterial isolates measure of the biosurfactant concentration, also showed that that were screened out from the collected soil samples. the surfactant was not present initially in sufficient amount to Among these, isolate no. 29, which could reduce the form micelles (Abouseoud et al. 2008b), but with the growth maximum surface tension of the mineral medium from of the bacterial culture, the concentration of surfactant in the 71.1 to 31.4 mN/m, was selected as the most potent medium increased, thus increasing the E24. These results biosurfactant-producing strain. The morphological and indicated that biosurfactant production in glycerol occurred physiological patterns of the strain showed a high similarity predominantly during the exponential growth phase, suggest- to Pseudomonas aeruginosa (99%). When the partial 16S ing that the biosurfactant was produced as a primary rDNA gene sequence was aligned with the NCBI GenBank metabolite accompanying cellular biomass formation and RDP databases, ten of the top ten matches were to (growth-associated kinetics). The dry cell biomass obtained Pseudomonas aeruginosa strains. Since the sequence was 3.7 g/L. similarities to Pseudomonas aeruginosa were uniformly 100%, the unknown strain was confirmed as Pseudomonas Evaluation of nitrogen sources aeruginosa RS29. Maximum STR of the medium was achieved in 24 h of Growth conditions optimization bacterial growth using sodium or potassium nitrate as nitrogen source (Table 3). With ammonium nitrate, maxi- Glycerol was found to be the best carbon source on which mum STR was obtained in 48 h; thereafter surface tension to reduce maximum surface tension (62%). Glucose and started increasing to a certain extent. The control with no mannitol could reduce the surface tension of mineral nitrogen source showed a gradual decrease in surface medium to 57% and 56%, respectively. Growth of the tension. STR for ammonium chloride- or ammonium strain on hexadecane resulted in a 37% decrease in surface sulfate-containing media was less prominent compared tension and a poor result was recorded for sludge (19%) with other nitrogen sources. (Table 2). These results indicated that the strain produces As seen in Table 3, the pattern of change in pH of the maximum biosurfactant on water-soluble substrates. Bio- mineral medium was different for different nitrogen surfactant production reached its maximum at 37.5°C sources. With ammonium chloride and ammonium sulfate, (Fig. 1), with an optimum initial pH range of 7.0–8.0 pH of the medium decreased abruptly from 7.5 to (Fig. 2). This is in agreement with a previous report that approximately 5.0 within 24 h of bacterial growth. In the maximum biosurfactant production by Pseudomonas aeru- control, the initial pH of the medium was retained almost ginosa 181 was achieved after 120 h of incubation at pH for the whole period of 96 h. With ammonium, sodium and 7.0 and 37°C (Al-Araji and Issa 2004). For Bacillus potassium nitrate, pH of the medium increased by 24 h then Ann Microbiol (2012) 62:753–763 757 Table 1 Surface tension of 29 bacterial isolates on glucose-containing mineral medium over a period of 7 days. Results are presented as mean ± SD of three replicates Isolate no. Day C 71.1±0.30 71.0±0.24 71.0±0.40 69.9±0.30 69.9±0.23 70.0±0.20 69.8±0.32 69.8±0.04 1 65.0±0.25 60.0±0.20 55.8±0.50 46.3±0.40 45.0±0.43 45.0±0.33 48.9±0.30 50.0±0.30 2 65.5±0.50 56.3±0.09 56.0±0.20 53.9±0.24 53.0±0.32 53.2±0.22 48.3±0.20 40.8±0.30 3 67.9±0.40 69.4±0.44 66.5±0.43 65.0±0.31 65.0±0.33 63.7±0.26 62.2±0.40 60.0±0.21 4 68.8±0.16 64.0±0.23 61.8±0.43 61.0±0.33 62.2±0.43 63.0±0.27 60.7±0.44 69.5±0.20 5 71.2±0.19 71.0±0.30 70.4±0.41 70.1±0.50 69.0±0.50 69.0±0.30 69.8±0.32 57.2±0.20 6 71.3±0.25 71.0±0.51 54.1±0.40 53.4±0.30 52.4±0.30 55.5±0.15 57.0±0.20 44.5±0.22 7 60.3±0.16 45.9±0.21 47.0±0.20 46.0±0.30 45.0±0.20 43.2±0.30 41.4±0.24 56.4±0.32 8 69.8±0.12 68.5±0.17 60.1±0.24 60.0±0.43 62.7±0.41 59.8±0.16 57.7±0.25 69.5±0.40 9 69.3±0.50 69.5±0.46 70.6±0.34 70.6±0.40 70.0±0.30 69.7±0.40 69.5±0.30 54.0±0.30 10 68.3±0.25 52.2±0.30 53.7±0.40 51.8±0.30 45.0±0.16 48.3±0.30 53.5±0.40 62.2±0.31 11 69.9±0.51 56.6±0.26 60.2±0.40 60.0±0.30 60.0±0.37 61.7±0.40 62.0±0.21 61.8±0.40 12 60.8±0.15 57.7±0.21 60.4±0.16 57.8±0.23 57.0±0.16 59.5±0.24 61.0±0.40 57.1±0.22 13 70.9±0.50 70.2±0.47 61.2±0.50 61.0±0.35 55.5±0.25 55.0±0.34 57.1±0.23 44.7±0.40 14 69.4±0.16 59.2±0.24 58.6±0.22 57.1±0.35 56.2±0.40 55.0±0.08 54.7±0.12 57.4±0.05 15 65.9±0.06 63.8±0.17 61.3±0.07 55.3±0.11 55.0±0.22 45.0±0.17 46.3±0.41 54.5±0.15 16 71.2±0.40 70.5±0.30 66.5±0.31 66.2±0.23 65.0±0.33 60.9±0.06 57.0±0.32 57.0±0.17 17 69.4±0.21 66.0±0.16 62.3±0.45 59.0±0.09 59.3±0.24 59.3±0.32 59.0±0.05 51.6±0.32 18 68.8±0.06 58.9±0.17 55.5±0.30 53.9±0.20 50.4±0.31 50.0±0.40 49.2±0.09 67.6±0.21 19 69.9±0.05 68.0±0.21 67.0±0.31 67.1±0.22 68.2±0.09 68.3±0.26 68.9±0.17 54.4±0.33 20 68.0±0.15 60.9±0.18 60.0±0.09 59.4±0.12 56.6±0.47 56.0±0.06 55.7±0.27 47.0±0.20 21 69.4±0.50 55.9±0.45 46.7±0.30 45.0±0.30 44.0±0.24 44.2±0.22 45.9±0.40 57.1±0.14 22 69.3±0.30 65.0±0.08 62.9±0.30 58.1±0.50 57.4±0.06 57.0±0.31 59.3±0.16 66.8±0.31 23 67.8±0.31 64.6±0.22 64.0±0.06 56.6±0.31 55.2±0.21 60.8±0.22 63.5±0.40 56.8±0.20 24 69.0±0.16 65.4±0.07 59.8±0.21 52.3±0.30 50.1±0.20 50.0±0.21 55.5±0.30 57.3±0.20 25 68.9±0.22 66.6±0.18 60.7±0.16 54.9±0.20 53.0±0.20 53.2±0.31 51.2±0.40 52.0±0.21 26 68.2±0.40 54.7±0.09 48.3±0.23 47.2±0.30 45.0±0.21 52.2±0.30 51.4±0.30 57.7±0.40 27 70.2±0.21 64.0±0.34 60.6±0.32 58.2±0.16 58.6±0.21 58.0±0.26 70.2±0.19 44.0±0.24 28 66.7±0.04 47.0±0.30 50.2±0.30 49.0±0.31 48.2±0.30 44.2±0.20 43.3±0.50 35.6±0.16 29 60.5±0.23 55.9±0.33 31.4±0.12 35.5±0.15 35.3±0.23 35.3±0.33 35.5±0.34 35.6±0.22 Abiotic control Isolate reducing surface tension to 31.4 mN/m, the lowest value amongst all isolates decreased at 48 h, with this change being more abrupt for again followed by an increase in pH of the medium in the ammonium nitrate than the other two N sources. This was subsequent hours for all three N sources. Table 2 Surface tension reduc- Carbon source Surface tension of control Surface tension of sample Surface tension tion (STR) with different carbon (mN/m) (mN/m) reduction (%) sources. Surface tension values represented mean ± SD of three Glucose 69.3±0.2 29.7±0.30 57 independent experiments Glycerol 70.2±0.14 28.4±0.33 60 Mannitol 68.7±0.31 30.4±0.12 56 n-Hexadecane 55.0±0.22 37.0±0.22 33 Sludge 47.0±0.32 39.0±0.34 17 758 Ann Microbiol (2012) 62:753–763 70 80 60 80 30 40 0 0 25 27.5 30 32.5 35 37.5 40 42.5 45 0 0 0 0.2 0.4 0.6 0.8 1 5 Temperature (°C) NaCl (%) Surface tension Emulsification index Surface tension Emulsification index Fig. 1 Effect of temperature on biosurfactant production Fig. 3 Effect of NaCl concentration on biosurfactant production Maximum bacterial growth was observed in ammonium nitrogen limitation enhances biosurfactant production nitrate-containing mineral medium, with almost equal growth (Suzuki et al. 1974; Ramana and Karanth 1989). being recorded for the rest of the four nitrogen sources. The In the case of ammonium nitrate, biomass growth of P. lowest bacterial growth was observed in the control (Table 3). aeruginosa RS29 was supported more than biosurfactant E24 was maximal, and almost the same, for sodium nitrate synthesis. After maximum STR of the medium, surface (80%) and potassium nitrate (79%) followed by ammonium tension again started to increase with time, indicating that nitrate (72%). A minimum value was observed for ammonium the concentration of biosurfactant decreased in the medium. chloride (36%) and ammonium sulfate (36%). The E24 for the The observed disappearance of biosurfactant might be control was 50% (Table 3). related to the development of competence. There are three In summary, P. aeruginosa RS29 could utilize all five possible mechanisms responsible for the decline in the tested nitrogen salts for growth. However, maximum biosurfactant concentration in stationary phase: (1) the growth (OD 4.0) was recorded in medium containing biosurfactant was degraded by the enzymes in the culture, ammonium nitrate as the nitrogen source. Although growth (2) the biosurfactant might be adsorbed on the cell surface, or of the strain was lower in sodium and potassium nitrate (3) the biosurfactant was reinternalized and processed than ammonium nitrate, the former two were best for intracellularly (Lin et al. 1993). Although ammonium sulfate production of biosurfactant in terms of STR and E24 and ammonium chloride supported bacterial growth, pro- (26.3 mN/m and 26.4 mN/m, with E24 of 80 and 79%, duction of biosurfactant was very poor. It can be deduced respectively). This might be because nitrogen was less that, at low pH of the culture medium, bacteria could not available from these two salts because nitrate first under- efficiently synthesize biosurfactant (Fig. 2). The increase in goes dissimilatory nitrate reduction to ammonium followed pH in the presence of ammonium-, sodium- and potassium- by assimilation by glutamine-glutamate metabolism. This nitrate implied accumulation of compounds like siderophores means that the assimilation of nitrate as a nitrogen source is in the culture medium (Varma and Chincholkar 2007). The so slow that it would simulate conditions of limiting color of the medium also changed to brown for these nitrogen (Hisatsuka et al. 1971; Itoh and Suzuki 1972; three salts. The change in color of the transparent Guerra-Santos et al. 1983). It had been reported that mineralmediumtobrown colorafter48hofincubation was also an indicator of the accumulation of siderophores in the medium (Nair et al. 2007). The pH of the medium 60 80 increased during the growth period, in accordance with the siderophore concentration, suggesting that alkalinity is important to avoid siderophore destruction (Díaz de Villegas et al. 2002). The control also exhibited a gradual 30 40 decrease in surface tension. The observed bacterial growth and biosurfactant production in the control was because of bacterial utilization of yeast extract in the mineral medium as a nitrogen source. 0 0 45 6789 Biosurfactant recovery and characterization Initial pH Surface tension Emulsification index The yield of biosurfactant (6 g/L) from P. aeruginosa RS29 Fig. 2 Effect of initial pH on biosurfactant production strain in the presence of glycerol as sole source of carbon Surface tension (mN/m) Surface tension (mN/m) Emulsification index (%) Emulsification index (%) Surface tension (mN/m) Emulsification index (%) Ann Microbiol (2012) 62:753–763 759 Fig. 4 Kinetics of biosurfactant 3.5 80 production by Pseudomonas aeruginosa RS29 under optimized conditions 2.5 1.5 0.5 0 0 0 8 16 24 32 40 48 56 64 120 Time (h) O.D. Surface tension Emulsification index Table 3 Effect of nitrogen Mineral medium with different 0 h 24 h 48 h 72 h 96 h source on surface tension nitrogen sources reduction (STR), pH, optical density (OD) and emulsification Control index (E24) of the mineral medium during biosurfactant Surface tension 62.0±0.12 51±0.15 36.1±0.62 32.2±0.3 28.9±0.8 production by Pseudomonas pH 7.5±0.02 6.7±0.5 6.8±0.4 7.1±0.30 7.0±0.30 aeruginosa RS29. Results OD 0.138±0.15 1.18±0.03 1.39±0.02 1.47±0.02 1.5±0.03 represent the mean ± SD E24 (%) 50±1.20 of three independent experiments Ammonium nitrate Surface tension 63.2±0.10 35.5±0.5 27.2±1.0 30.9±0.8 31.1±0.7 pH 7.5±0.03 8.2±0.12 5.54±0.20 8.3±0.40 8.3±0.45 OD 0.165±0.05 2.33±0.04 3.87±0.05 4.0±0.03 4.0±0.04 E24 (%) 72±1.0 Ammonium chloride Surface tension 62.0±0.02 50.5±0.15 52.2±0.5 50.6±0.3 47.0±0.40 pH 7.5±0.02 4.8±0.20 4.6±0.45 5.3±0.4 4.8±0.23 OD 0.162±0.1 2.039±0.02 2.12±0.02 2.15±0.04 2.15±0.03 E24 (%) 36±0.6 Ammonium sulfate Surface tension 62.3±0.05 49.3±0.34 51.5±0.4 52.6±0.42 51.1±0.50 pH 7.5±0.01 5.1±0.20 5.53±0.2 5.0±0.3 4.8±0.20 OD 0.165±0.22 1.96±0.02 2.06±0.01 2.12±0.02 2.11±0.02 E24 (%) 36±0.5 Sodium nitrate Surface tension 62.2±0.01 26.3±0.1 26.3±0.12 26.5±0.1 27.0±0.20 pH 7.5±0.02 8.5±0.10 7.4±0.15 8.6±0.2 8.65±0.20 OD 0.160±0.07 2.18±0.01 2.36±0.02 2.36±0.01 2.32±0.01 E24 (%) 80±1.0 Potassium nitrate Surface tension 62.1±0.03 26.4±0.2 26.4±0.4 26.5±0.4 26.8±0.30 pH 7.5±0.03 8.3±0.10 7.4±0.12 8.8±0.20 8.53±0.30 OD 0.163±0.2 2.08±0.01 2.28±0.02 2.31±0.01 2.25±0.01 E24 (%) 79±0.6 OD (460 nm) Surface tension (mN/m), Emulsification index (%) 760 Ann Microbiol (2012) 62:753–763 70 50 50 40 40 30 30 20 20 40 10 10 0 0 30 2468 10 NaCl (%) 20 Surface tension Emulsification index Fig. 7 Effect of salinity on biosurfactant activity tant molecules had begun to aggregate (Karsa et al. 1999; Meylheuc et al. 2001). The CMC obtained was in agreement with previous reports (Bordoloi and Konwar 2009). In the Conc. of biosurfactant (log of mg/L) case of P. aeruginosa SP4, the excreted biosurfactant in the Fig. 5 Critical micelle concentration (CMC) of extracted biosurfac- culture supernatant could decrease the surface tension of tant produced by Pseudomonas aeruginosa RS29 pure water from 72.0 to 28.3 mN/m, and the CMC was estimated to be 120 mg/L (Pornsunthorntawee et al. 2008). was higher than the reported biosurfactant production by P. For Pseudomonas fluorescens, the CMC recorded for the aeruginosa NM strain (5 g/L) grown in glycerol (Das and isolated biosurfactant was 290 mg/L and the corresponding Mukherjee 2005), and almost the same as with a previous surface tension was 32 dynes/cm. The CMC value of the report for P. aeruginosa M strain (Das and Mukherjee 2005) chemical surfactant sodium dodecyl sulphate (SDS) is and other Pseudomonas sp. (Pruthi and Cameotra 1995). 140 mg/L (Bordoloi and Konwar 2009). So, the biosurfactant As seen in Fig. 5, the surface tension is dependent on the produced by P. aeruginosa RS29 exhibits better properties in concentration of crude biosurfactant, and the CMC corre- terms of higher STR and a lower CMC. The results of −1 −2 sponded to a sudden change in the surface tension CMD and CMD of the biosurfactant containing cell-free (Abouseoud et al. 2008b). The CMC for the isolated medium were 28.3 mN/m and 40.0 mN/m, respectively, biosurfactant calculated from the breakpoint of surface depicting little change in efficiency. The results suggest that tension versus the log of its concentration curve was 90 mg/ a sufficient amount of biosurfactant was present in the L, and the corresponding surface tension was 27.8 mN/m. culture medium, and thus its surface activity was retained Biosurfactant concentrations above the CMC could not even at such a high dilution. These values are appreciably decrease the surface tension further, indicating that biosurfac- better than the values reported for P. aeruginosa strains Fig. 6 Fourier transform infrared 1.1 (FTIR) spectra of the biosurfac- tant extracted from Pseudomonas aeruginosa RS29 0.9 C-O 0.8 COO 0.7 C-O-C CH CH 2 3 0.6 0.5 3370 O-H 0.4 3900 3400 2900 2400 1900 1400 900 400 0.17 0.7 1.3 1.9 2.15 Surface tension (mN/m) Surface tension (mN/m) Emulsification index (%) Ann Microbiol (2012) 62:753–763 761 80 80 (MTCC8165, MTCC7815, MTCC7812 and MTCC7814) (Bordoloi and Konwar 2008). 60 60 The rhamnose test was positive, indicating that the separated biosurfactant could be of the glycolipid type. 40 40 Molisch’s test showing a clear purple ring between the 20 20 layers of solvent and the sample, indicating that the sample contained sugar moieties. Red spots appeared on the TLC 0 0 plate after spraying with α-naphthol and sulfuric acid, 2468 10 indicating the presence of carbohydrates in the sample. The pH production of glycolipid-type biosurfactant was previously Surface tension Emulsification index reported for Pseudomonas sp. (Persson et al. 1988; Wilson Fig. 8 Effect of pH on biosurfactant activity and Bradley 1996; Patel and Desai 1997). Spectral analysis (Fig. 6) showed strong absorption −1 bands at 3,370 cm . This was observed due to stretching vibration of the –OH group. The absorption stability. The effect of addition of NaCl on surface tension −1 band observed at 2,923 cm confirmed the presence of alkyl and E24 of biosurfactant is shown in Fig. 7. Little change (CH and CH ) groups. A carbonyl stretching band 2 3 was observed in either parameter with addition of up to 10% −1 characteristic of ester compounds was found at 1,720 cm (w/v) NaCl. The surface tension of the biosurfactant was −1 . The absorption at 1,629 cm was because of stretching of stable at different pH values ranging from 2 to 10, and pH the COO– group. The spectra also showed an absorption increase had a positive effect on E24 (Fig. 8). This could be −1 band at 1,045 cm due to stretching vibration of –C–O–C. caused by the higher stability of fatty acids-surfactant The pattern of absorption bands observed for this micelles in presence of NaOH and the precipitation of particular strain was reported previously for rhamnolipids secondary metabolites at higher pH values (Abouseoud et al. (Bordoloi and Konwar 2009). These latter authors 2008a). Similarfindingswerereportedfor P. aeruginosa −1 explained that strong absorption bands at 3,443 cm occurred isolate Bs20, which exhibited excellent stability at high due to stretching vibration of the –O–H group and temperature (heating at 100°C for 1 h and autoclaving at −1 absorption at 1605–1625 cm due to either stretching 121°C for 10 min), salinities up to 6% NaCl, and pH values of –C=C or >C=O, i.e., stretching of the carboxylate anion. up to pH 13 (Abdel-Mawgoud et al. 2009). The properties of −1 It was also reported that absorption at 1,625 cm occurred the biosurfactant produced by native P. aeruginosa RS29 are due to presence of carboxylate anion, and absorption at promising for its application in different industries. −1 1,120 cm due to stretching vibration of –C–O–Cof the ether linkage of Rha-C8-C10 and Rha-C10-C8 molecules. Conclusion Activity characterization The isolated P. aeruginosa RS29 is a very potent Biosurfactant containing culture supernatant showed good biosurfactant-producing native strain. Strain RS29 produces foaming stability. The foam produced was stable for 48 h. maximum biosurfactant in sodium- or potassium-nitrate- Stable foam indicates that the produced biosurfactant can be containing medium where surface tension was reduced from used as a good foaming agent. Similar findings were reported 70.0 to 26.3 mN/m and 26.4 mN/m, respectively. The amount for 48-h-old culture of P. aeruginosa PTCC 1561 grown in of crude biosurfactant recovered (6 g/L) from the culture nutrient broth, which showed foam stability for 48 h medium is very promising. The CMC value (90 mg/L) of the (Noudeh et al. 2010). The biosurfactant produced by P. produced biosurfactant is superior to many other biosurfac- aeruginosa RS29 was shown to be thermostable. Autoclav- tants. The tensioactive properties and stability of the ing it at 121°C did not destroy its foaming properties. The biosurfactant to high temperature, pH and salinity reveal surface tension measured before autoclaving the sample was good prospects for this product in industrial applications. The 27.0 mN/m. The value was retained and recorded as emulsifying and foaming activity of the biosurfactant indicate 27.6 mN/m after autoclaving the sample. The E24 before that it can be used as a good emulsion-forming and foaming autoclaving the sample was 72% and 70% after autoclaving. agent in different industries. This indicated that STR and the E24 were stable up to quite a high temperature, in contrast to synthetic surfactants such Acknowledgments We thank the Director, Prof. Joyanti Chutia, IASST, as SDS, which exhibits a significant loss of emulsification Guwahati, India for permission and support. Moreover, this work has been activity above 70°C (Kim et al. 1997). Thus, our product is made possible through a project grant (BT/PR-9795/BCE/08/590/2007) better than synthetic surfactants in terms of temperature funded by Department of Biotechnology, Govt of India. We thank to Dr. N. Surface tension (mN/m) Emulsification index (%) 762 Ann Microbiol (2012) 62:753–763 Sen Sarma, Senior Assistant Professor, IASST for facilitating the FTIR microbial enhancement of oil recovery. US Department of analysis at IASST, Guwahati, India. Energy, Washington DC, pp 12–14 Guerra-Santos LH, Kappeli O, Fiechter A (1986) Dependence of Pseudomonas aeruginosa continuous culture biosurfactant pro- duction on nutritional and environmental factors. Appl Microbiol References Biotechnol 24:443–448 Hisatsuka K, Nakahara T, Sano N, Yamada K (1971) Formation of rhamnolipid by Pseudomonas aeruginosa: its function in Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NA (2009) Char- hydrocarbon fermentation. Agric Biol Chem 35:686–692 acterization of Rhamnolipid Produced by Pseudomonas aerugi- Itoh S, Suzuki T (1972) Effect of rhamnolipids on growth of nosa Isolate Bs20. Appl Biochem Biotechnol 157:329–345 Pseudomonas aeruginosa mutant deficient in n-paraffinutilizing Abouseoud M, Maachi R, Amrane A, Boudergua S, Nabi A (2008a) ability. Agric Biol Chem 36:2233–2235 Evaluation of different carbon and nitrogen sources in production of Jenny K, Deltrieu V, Kappelli O (1993) Lipopeptide production by biosurfactant by Pseudomonas fluorescens. Desalination 223:143–151 Bacillus licheniformis. In: Kosaric N (ed) Biosurfactants. Abouseoud M, Yataghene A, Amrane A, Maachi R (2008b) Biosurfactant Production, property, application. Dekker, New York, pp 135–156 production by free and alginate entrapped cells of Pseudomonas Karsa DR, Bailey RM, Shelmerdine B, McCann SA (1999) Overview: fluorescens. J Ind Microbiol Biotechnol 35:1303–1308 a decade of change in the surfactant industry. In: Karsa DR (ed) Al-Araji Y, Issa L (2004) Biosurfactant production by Pseudomonas Industrial applications of surfactants, vol 4. Royal Society of aeruginosa 181. PhD thesis, University Putra Malaysia Chemistry, London, pp 1–22 Babu PS, Vaidya AN, Bal AS, Kapur R, Juwarkar A, Khanna P (1996) Kim H, Yoon B, Lee C, Suh H, Oh H, Katsuragi T, Tani Y (1997) Kinetics of biosurfactant production by Pseudomonas aeruginosa Production and properties of a lipopeptide biosurfactant from strain BS2 from industrial waste. Biotechnol Lett 18:263–268 Bacillus subtilis C9. J Ferment Bioeng 84(1):41–46 Banat IM (1995) Biosurfactant production and possible uses in Kosaric N (1993) Biosurfactants. Production, property, application, microbial enhanced oil recovery and oil pollution remediation: surfactant sciences series, 48. Dekker, New York review. Bioresour Technol 55:1–12 Lang S, Wullbrandt D (1999) Rhamnose lipids, biosynthesis, Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial microbial production and application potential. Appl Microbiol applications of microbial surfactants. Appl Environ Microbiol Biotechnol 50:22–32 53:495–508 Lin SC, Sharma MM, Georgiou G (1993) Production and deactivation Bonilla M, Olivaro C, Corona M, Vazquez A, Soubes M (2005) of biosurfactant by Bacillus licheniformis JF-2. Biotechnol Prog Production and characterization of a new bioemulsifier from 9(2):138–145 Pseudomonas putida ML2. J Appl Microbiol 98:456–463 Mata-Sandoval JC, Karns J, Torrents A (1999) High performance Bordoloi NK, Konwar BK (2008) Microbial surfactant-enhanced liquid chromatography method for the characterization of mineral oil recovery under laboratory conditions. Colloids Surf rhamnolipid mixtures produces by Pseudomonas aeruginosa B Biointerfaces 63:73–82 UG2 on corn oil. J Chromatogr A 864:211–220 Bordoloi NK, Konwar BK (2009) Bacterial biosurfactant in enhancing Makkar RS, Cameotra SS (2002) Effects of Various Nutritional solubility and metabolism of petroleum hydrocarbons. J Hazard Supplements on Biosurfactant Production by a Strain of Bacillus Mater 170:495–505 subtilis at 45°C. J Surfactants Deterg 5:11–17 Cameotra SS, Makkar RS (2004) Recent applications of biosurfactants Meylheuc T, Heary JM, Bellon-Fontaine MN (2001) Les biosurfac- as biological and immunological molecules. Curr Opin Microbiol tants, des biomolécules à forte potentialité d’application. Sci 7:262–266 Aliments 21:591–649 Cappuccino JG, Sherman N (1999) Microbiology—a laboratory Nair A, Juwarkar AA, Singh SK (2007) Production and characteriza- manual. Addison-Wesley Longman, Harlow, pp 417–421 tion of siderophores and its application in arsenic removal from Cooper DG (1986) Biosurfactants. Microbiol Sci 3:145–149 contaminated soil. Water Air Soil Pollut 180:199–212 Cooper DG, Goldenberg BG (1987) Surface-active agents from two Noudeh GD, Noodeh AD, Moshafi MH, Behravan E, Afzadi MA, Bacillus species. Appl Environ Microbiol 53(2):224–229 Sodagar M (2010) Investigation of cellular hydrophobicity and Cooper DG, Zajic JE (1980) Surface-active compounds from surface activity effects of biosynthesed biosurfactant from broth microorganism. Adv Appl Microbiol 26:229–256 media of PTCC 1561. Afr J Microbiol Res 4(17):1814–1822 Das K, Mukherjee AK (2005) Characterization of biochemical Patel RM, Desai AJ (1997) Surface-active properties of rhamnolipids from properties and biological activities of biosurfactants produced Pseudomonas aeruginosa GS3. J Basic Microbiol 37:281–286 by Pseudomonas aeruginosa mucoid and non-mucoid strains Perfumo A, Banat IM, Canganella F, Marchant R (2006) Rhamnolipid isolated from hydrocarbon-contaminated soil samples. Appl production by a novel thermophilic hydrocarbon-degrading Microbiol Biotechnol 69:192–199 Pseudomonas aeruginosa AP02-1. Appl Microbiol Biotechnol Desai JD, Banat IM (1997) Microbial production of surfactants and 72(1):132–138 their commercial potential. Microbiol Mol Biol R 61(1):47–64 Persson A, Österberg E, Dostalek M (1988) Biosurfactant production Deziel E, Lepine F, Dennie D, Boismenu D, Mamer OA, Villemur R (1999) by Pseudomonas Xuorescens 378: growth and product character- Liquid chromatography/mass spectrometry analysis of mixture of istics. Appl Microbiol Biotechnol 29(1):1–4 rhamnolipids produced by P.aeruginosa strain 57RP grown on mannitol or naphthalene. Biochim Biophys Acta 1440:244–252 Pornsunthorntawee O, Arttaweeporna N, Paisanjit S, Somboonthanatea P, Díaz de Villegas ME, Villa P, Frías A (2002) Evaluation of the Abeb M, Rujiravanita R, Chavadeja S (2008) Isolation and siderophores production by Pseudomonas aeruginosa PSS. Rev comparison of biosurfactants produced by Bacillus subtilis PT2 Latinoam Microbiol 44:112–117 and Pseudomonas aeruginosa SP4 for microbial surfactant- Dubois M, Gills KA, Hamilton JK, Rebers PA, Smith F (1956) enhanced oil recovery. Biochem Eng J 42:172–179 Colorimetric method for determination of sugar and related Pruthi V, Cameotra SS (1995) Rapid method for monitoring maximum substances. Anal Chem 28:350–356 biosurfactant produced by acetone precipitation. Biotechnol Tech Guerra-Santos L, Kappeli O, Fiechter A (1983) Growth and 9:271–276 biosurfactant production of a bacterium in continuous culture. Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM (2002) In: Donaldson EC, Clark JB (eds) International conference on Rhamnolipid biosurfactant production by strains of Pseudomonas Ann Microbiol (2012) 62:753–763 763 aeruginosa using low-cost raw material. Biotechnol Prog Sheppard JD, Mulligan CN (1987) The production of surfactin by 18:1277–1281 Bacillus subtilis grown on peat hydrolysate. Appl Microbiol Ramana KV, Karanth NG (1989) Factors affecting biosurfactants Biotechnol 27:110–116 production using Pseudomonas aeruginosa CFTR-6 under Suzuki T, Tanaka H, Itoh S (1974) Sucrose lipids of Arthrobacteria, submerged conditions. J Chem Technol Biotechnol 45:249–257 Corynebacteria and Nocardia grown on sucrose. Agric Biol Raza ZA, Khan MS, Khalid ZM (2007) Physicochemical and surface- Chem 38:557–563 active properties of biosurfactant produced using molasses by a Wilson NG, Bradley G (1996) The eVect of immobilization on Pseudomonas aeruginosa mutant. J Environ Sci Health A Tox rhamnolipid production by Pseudomonas Xuorescens. J Appl Hazard Subst Environ Eng 42:73–80 Bacteriol 81(5):525–530 Ron EZ, Rosenberg E (2001) Natural roles of biosurfactants. Environ Varma A, Chincholkar S (eds) (2007) Microbial siderophores. Microbiol 3:229–236 Springer, New York,

Journal

Annals of Microbiology – Springer Journals

Published: Jul 20, 2011

There are no references for this article.