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

Production and characterization of biosurfactant from marine bacterium Inquilinus limosus KB3... Ann Microbiol (2013) 63:1327–1339 DOI 10.1007/s13213-012-0592-7 ORIGINAL ARTICLE Production and characterization of biosurfactant from marine bacterium Inquilinus limosus KB3 grown on low-cost raw materials Atipan Saimmai & Satianpong Udomsilp & Suppasil Maneerat Received: 3 July 2012 /Accepted: 13 December 2012 /Published online: 8 January 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Inquilinus limosus strain KB3, isolated from ma- Introduction rine sediment in the south of Thailand, was used to produce a biosurfactant from a mineral salts medium (MSM) with The interest in biosurfactants has increased considerably in palm oil decanter cake (PODC) as a carbon source. It was recent years, as they are potential candidates for many found that cellular growth and biosurfactant production in commercial applications in the biomedical, pharmaceuticals, MSM were greatly affected by the medium components. I. petroleum, and food processing industries (Banat et al. limosus KB3 was able to grow and to produce surfactant 2010). The biosurfactants have several advantages over reducing the surface tension of medium to 28.2 mN/m and chemical surfactants including high ionic strength tolerance, giving a crude surfactant concentration of 5.13 g/l after 54 h. high temperature tolerance, higher biodegradability, lower The biosurfactant obtained was found to reduce the surface toxicity, lower critical micelle concentration (CMC), and tension of pure water to 25.5 mN/m with the critical micelle higher surface activity (Abdel-Mawgoud et al. 2009; Banat concentration of 9 mg/l, and retained its properties during et al. 2010; Gudina et al. 2011). In spite of the advantages, exposure to elevated temperatures (121 °C), high salinity fermentation must be cost competitive with chemical syn- (12 % NaCl), and a wide range of pH values. Chemical thesis, and many of the potential applications that have been characterization by FT-IR, NMR, and ESI-MS revealed that considered for biosurfactants depend on whether they can be the biosurfactant has a lipopeptide composition with molec- produced economically. The choice of inexpensive raw ular mass (m/z) of 1,032. The biosurfactant was capable of materials is important to overall economy of the process forming stable emulsions with various hydrocarbons and because they account for 50 % of the final product cost had the ability to enhance oil recovery, PAHs solubility, and also reduce the expense of waste treatment (Makkar and antimicrobial activity. and Cameotra 2002). In recent years, much work has been carried out towards efficient utilization of agro-industrial . . Keywords Inquilinus limosus Biosurfactant Palm oil residues such as cassava wastewater (Nitschke and Pastore . . decanter cake Oil recovery Polyaromatic hydrocarbon 2006), ground-nut oil refinery residue and corn steep liquor (Sobrinho et al. 2008), molasses (Joshi et al. 2008; Saimmai et al. 2011), and potato peels (Das and Mukherjee 2007). Palm oil decanter cake, a by-product from the palm oil milling decantation process, is an abundant and low cost A. Saimmai (*) S. Udomsilp agricultural waste residue. It is easily available in large Faculty of Agricultural Technology, quantities in the south of Thailand. It accounts for about Phuket Rajabhat University, Phuket 83000, Thailand e-mail: s4680108@hotmail.com 3 % of the weight of the empty fruit bunch of palm oil and is rich in oil residues and various vitamins and mineral ele- S. Maneerat ments (Yahya et al. 2010). However, palm oil decanter cake Department of Industrial Biotechnology, has not found any significant commercial application until Faculty of Agro-Industry, Prince of Songkla University, now and is generally disposed of in open areas, leading to Hat Yai, Songkhla 90112, Thailand 1328 Ann Microbiol (2013) 63:1327–1339 potentially serious environmental problems. It is thus nec- NH NO , peptone, and yeast extract were employed at a 4 3 essary to explore its industrial reutilization. concentration of 1 g/l with the optimum carbon source. The This study was carried out to explore the feasibility of C:N ratio (with optimized carbon and nitrogen sources) was using palm oil decanter cake as the substrate for the produc- varied from 5 to 40 by keeping a constant nitrogen source tion of biosurfactant by the marine bacterial Inquilinus concentration of 1 g/l. limosus KB3 isolated from marine sediment and the poten- tial application of biosurfactant obtained for enhanced solu- Recovery of biosurfactant bilization of hydrophobic compounds. Four solvent systems; a mixture of chloroform:methanol (2:1), cold acetone, dichloromethane, and ethyl acetate were Materials and methods examined for biosurfactant extraction (Saimmai et al. 2012b). The method showing the highest biosurfactant ac- Biosurfactant-producing strain tivity was used to recover biosurfactant from I. limosus KB3. Inquilinus limosus KB3 (accession number AB685266) was isolated from marine sediment collected from the southern Chemical analysis of biosurfactant part of Thailand, during a screening study for biosurfactant- producing bacteria in mangrove sediment (unpublished da- The chemical nature of the biosurfactants obtained was ta). I. limosus KB3 was maintained on nutrient agar plates determined with thin layer chromatography (TLC). The and transferred monthly. biosurfactant was spotted in triplicate on ready-made silica gel TLC plates (Merck, Darmstadt, Germany) using CHCl : Media and cultivation conditions CH OH:H O (65:15:1) as the solvent system. One of the 3 2 plates was put into a jar saturated with iodine vapor to detect Nutrient broth was used for preparation of the inoculum. lipids (Das et al. 2009). Another plate was sprayed with The composition of the nutrient broth used was as follows: anisaldehyde and ninhydrin reagent (0.2 % ninhydrin solu- beef extract 1.0 g, yeast extract 2.0 g, peptone 5.0 g, NaCl tion in acetone) and dried. It was then heated at 120 °C for 5.0 g in 1 l of distilled water. To make nutrient agar, 15.0 g 5 min for detection of sugars and peptides (Das et al. 2009), of agar was added to the nutrient broth. The culture was respectively. grown in this broth for 20–24 h at 30 °C. This was used as Fourier transform infrared spectroscopy (FT-IR) of the inoculum at the 3 % (v/v) level. For biosurfactant synthesis, biosurfactant obtained was done on a Nexus-870 FT-IR a mineral salt medium (MSM) with the following composi- spectrometer (Thermo Electron, Yokohama, Japan) by the tion (g/l) was utilized: K HPO , 0.8; KH PO , 0.2; CaCl , KBr pellet method. Further characterization of the biosur- 2 4 2 4 2 0.05; MgCl , 0.5; FeCl , 0.01; NaCl, 10.0 (Saimmai et al. factant was carried out using nuclear magnetic resonance 2 2 2012a). pH of the medium was adjusted to 7.0. Carbon and (NMR) using CDCl with an AMX 300 NMR spectrometer nitrogen sources were added separately. Cultivation was (500 MHz; Bruker). Final characterization of the compound performed in 250-ml flasks containing 50 ml medium at was performed by liquid chromatography-mass spectrosco- room temperature (30±3 °C), and shaking in a rotary shaker py (LC-MS)withanLCQ™ quadrupole ion-trap mass at 150 rpm for 48 h. spectrometer (Finnigan MAT, San Jose, CA, USA) which utilizes electrospray ionization (ESI) (Thavasi et al. 2008). Medium optimization Application of the biosurfactant in ULO removal The medium optimization was conducted in a series of from contaminated sand experiments changing one variable at a time, keeping the other factors fixed at a specific set of conditions. Three Biosurfactant suitability for enhancing oil recovery was factors were chosen aiming to obtain higher productivity investigated using 800.0 g of acid-washed sand impregnated of the biosurfactant: carbon source (C), nitrogen source (N), with 50.0 ml of ULO. Fractions of 20.0 g of the contami- and C:N ratio. The carbon sources used were 1 g/l of glu- nated sand were transferred to 250-ml flasks which were cose, glycerol, molasses, oleic acid, palm oil decanter cake, submitted to the following treatments: addition of 60.0 ml soybean oil, stearic acid, used lubricating oil (ULO), and distilled water (control), addition of 60.0 ml aqueous solu- used vegetable oil (UVO), with (NH ) SO as a nitrogen tions of the SDS, Triton X-100 and biosurfactant at the 4 2 4 source. For evaluation of the most appropriate nitrogen CMC level of each compound. The samples were incubated sources for the production of biosurfactants, beef extract, on a rotary shaker (200 rpm) for 24 h at 30 °C and centri- monosodium glutamate, NaNO ,(NH ) SO ,NH Cl, fuged at 3,354 g for 20 min for separation of the laundering 3 4 2 4 4 Ann Microbiol (2013) 63:1327–1339 1329 solution and the sand. The amount of oil residing in the sand Antimicrobial activity of surface active compound after the impact of biosurfactant was gravimetrically deter- mined as the amount of material extracted from the sand by The extracted compound was tested for antimicrobial hexane (Sobrinho et al. 2008). activity using the agar well diffusion method and the inhibition zone was measured (Candan et al. 2003). Extracted active compound was tested against pathogenic Laboratory experiment on biodegradation of ULO with microorganisms including Bacillus cereus, Candida albi- biosurfactant cans, Enterococcus faecium, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella An experiment was conducted to study the impact of the sp., Salmonella typhimurium, Staphylococcus aureus, biosurfactant isolated from I. limosus KB3 on biodegrada- Vibrio cholerae and Vibrio vulnificus. All strains were tion of ULO in natural seawater. Shake flask biodegradation obtained from Songklanagarind Hospital, Prince of experiments were carried out in 500-ml Erlenmeyer flasks Songkla University, Thailand. Briefly, the extract was with 100 ml of sterilized seawater. The experiment was dissolved in distilled water at a concentration of 10 mg/ conducted with four different sets: (1) bacterial cells alone; ml and filter-sterilized using a 0.2-μm membrane filter. (2) with fertilizer and cells; (3) with cells and biosurfactants Each tested microorganism was grown in brain heart (0.1 %, w/v); and (4) with fertilizer and biosurfactant. infusion (BHI; Hi-Media Laboratories, Mumbai, India) Exactly 2.0 % (w/v) of ULO was added to the sterilized and diluted to a concentration of 10 CFU/ml. They were seawater, and inoculation was performed with 24-h-old cul- overlaid onto the surface of BHI agar. The agar plates 3 4 ture at the rate of 1 % (v/v, 10 –10 CFU/ml) concentration. were dried for 20 min at room temperature. Microbial Flasks were shaken at 150 rpm in dark at room temperature suspensions were plated on BHI agar plates, which were for a period of 168 h. The biodegradation of oil was esti- dried for 20 min at room temperature. The wells were mated fluorimeterically as described in Intergovernmental cut from the agar and 100 μl of extract solution were Oceanographic Commission (IOC) Manuals and Guide No. added to the wells. The plates were incubated at 37 °C 13 (1982). An uninoculated control was kept to assess the for 24 h; after incubation, the clear zone was measured. natural weathering of oil and degradation. Analytical methods Polyaromatic hydrocarbons (PAHs) solubilization assay Biomass determination was done in terms of the dry cell PAHs solubilization assay was done as described by Barkay weight. At different times of fermentation, samples were et al. (1999). Briefly, 0.6 μg each of the following PAHs mixed in pre-weighted tubes with chilled distilled water (anthracene, fluoranthene, fluorine, naphthalene, phenan- and centrifuged at 9,693 g for 30 min. The biomass obtained threne or pyrene; all from 0.6 mg/ml stock in acetone) were was dried overnight at 105 °C and weighed. distributed into glass test tubes (10 mm×170 mm) and kept Emulsification activity was performed according to Wu open inside an operating chemical fume hood to remove the et al. (2008). Briefly, 4 ml of hydrocarbon or oil was added solvent. Subsequently, 3.0 ml of assay buffer (20 mM Tris- to 4 ml of aqueous solution of culture supernatant in a screw HCl, pH 7.0) and the biosurfactant at increasing concentra- cap tube, and vortexed at high speed for 2 min. The emul- tions (0–50 mg/ml) obtained from the bacterial strain used in sion activity (E24) was determined after 24 h. E24 was this study. Assay buffer containing the biosurfactant, but no calculated by dividing the measured height of the emulsion PAH, was used as blank. Tubes were capped with plastic layer by the total height of the mixtures and multiplying by closures and incubated overnight at 30 °C with shaking 100. (200 rpm) in dark. Samples were filtered through 1.2-μm The surface tension of culture supernatant was measured filters (Whatman, Springfield Mill, UK) and 2.0 ml of this using a Model 20 Tensiometer (Fisher Science Instrument, filtrate was extracted with an equal volume of hexane. This PA, USA) at 25 °C. CMC was determined by plotting the emulsion was centrifuged at 9,693 g for 10 min to separate surface tension versus concentration of biosurfactant in the the aqueous and hexane phases. The concentration of the solution. PAHs was measured spectrophotometrically (Libra S22; All experiments were carried out at least in triplicate. Biochrom, Cambridge, UK) at the specific wave lengths of Two well-defined synthetic surfactants, Triton X-100 and each compound (Barkay et al. 1999). From a calibration SDS were used as positive controls, while distilled water curve of individual PAHs (in hexane), the concentration of and MSM medium were used as negative controls. each PAH was determined. Assay buffer with biosurfactant Statistical analysis was performed using the Statistical without PAH was identically extracted with hexane and Package for Social Science (SPSS 10.0 for Windows; served as blank. Chicago, IL, USA). 1330 Ann Microbiol (2013) 63:1327–1339 Results and discussion (Chayabutra et al. 2001) or toxicity toward bacterial cells (Li and Chen 2009). Effect of carbon source on growth and biosurfactant production Effect of nitrogen source on growth and biosurfactant production The literature has revealed that the type and concentra- tion of carbon and nitrogen substrates markedly affected After examining the most commonly used organic and inorgan- the production yield of biosurfactant (Wu et al. 2008). In ic nitrogen sources reported in the literature (Abdel-Mawgoud light of this, this study started with the investigation of et al. 2008), it was found that the type of nitrogen source carbon and nitrogen sources on biosurfactant production. affected the growth and biosurfactant production of I. limosus I. limosus KB3was grownoneachof9 typesofcarbon KB3 (Table 2). The highest biomass was obtained when yeast sources. As seen in Table 1, the type of carbon source extract was used. However, NaNO exhibited the highest sur- affected both the biosurfactant production and E24. Palm face tension reduction and biosurfactant yield (35.49 mN/m and oil decanter cake differed from the others in relation to 1.79 g/l, respectively). This yield was nearly 2-fold that the biosurfactant concentration and E24, being the most obtained from using (NH ) SO as the nitrogen source. 4 2 4 appropriate carbon source; surface tension reduction Moreover, using NaNO as the inorganic nitrogen source not reached 25.25 mN/m with 1.10 g/l and achieved an only increased the biosurfactant yield but also improved the E24 of 19.25 % toward xylene. Table 1 also shows that biomass and E24 at 4.12 g/l and 25.35 %, respectively. there seems to be clear trend between biomass and bio- surfactant yields which strongly depends on the carbon Effect of C:N ratio on growth and biosurfactant production source used. Although vegetable oils or glucose have been frequently used as the carbon substrates for biosur- The C:N ratio is also known as a vital factor influencing the factant production (Banat et al. 2010), I. limosus KB3 performance of biosurfactant production (Santos et al. attained a lower biosurfactant yield from soybean oil, 2002). As indicated in Table 3, the best biosurfactant activ- UVO, and glucose than that from palm oil decanter cake, ity in surface tension reduction and yield (44.35 mN/m and molasses, and glycerol (Table 1). Direct use of fatty 4.90 g/l, respectively) were obtained at a C:N ratio of 25; acids (i.e., oleic acid and stearic acid) as the carbon this yield was 2.6-fold that obtained from control (C:N ratio source did not improve biosurfactant production, sug- at 1:1). The productivity of biosurfactant tended to decrease gesting that hydrolysis of the oils was not the bottle- as the C:N ratio increased from 30 to 40, especially for C:N neck step. Moreover, ULO and UVO were also ineffi- ratio >35. Some reports mentioned that biosurfactant pro- cient in cell growth and biosurfactant production, result- duction is more efficient under nitrogen-limiting conditions ing in a low biosurfactant yield of only 0.50 and 0.48 g/ (Benincasa et al. 2002). The results show that a possible l, respectively, probably due to its poor biodegradability inhibitory effect on the bacterial metabolism may occur due Table 1 Effect of carbon source on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h (nitrogen source: 1 g/l of (NH ) SO ) 4 2 4 a a a a Carbon source (1 g/l) Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction No carbon source 0.06±0.01 72.01±1.27 70.01±0.04 1.51±0.04 0.05±0.01 0 Glucose 4.05±0.20 71.51±2.27 59.21±1.24 12.11±2.27 0.53±0.01 20.51±5.07 Glycerol 3.95±0.92 70.03±0.53 53.56±2.18 18.03±5.53 0.91±0.01 20.03±4.73 Molasses 4.54±1.62 65.08±2.57 44.64±1.03 20.08±2.57 1.08±0.08 20.08±2.67 Oleic acid 2.48±0.63 68.35±2.67 59.19±2.06 8.35±2.67 0.32±0.05 8.05±2.54 Palm oil decanter cake 3.72±0.63 69.25±1.58 45.21±4.18 25.25±1.58 1.10±0.02 19.25±4.50 Soybean oil 3.54±0.41 62.23±0.87 46.13±3.13 16.23±0.87 0.88±0.04 15.01±2.93 Stearic acid 1.04±0.12 70.23±0.36 60.25±0.15 8.23±0.36 0.32±0.02 10.03±4.57 Used lubricating oil 0.40±0.09 57.62±2.52 46.20±1.86 10.62±2.52 0.50±0.06 17.61±5.01 Used vegetable oil 0.61±0.02 65.61±2.05 56.25±4.04 9.61±2.05 0.48±0.04 12.12±4.07 Values are given as means ± SD from triplicate determinations Ann Microbiol (2013) 63:1327–1339 1331 Table 2 Effect of nitrogen source on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h (carbon source: 1 g/l of palm oil decanter cake) a a a a Nitrogen source (1 g/l) Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction No nitrogen source 0.21±0.03 70.01±1.27 60.10±0.14 9.20±0.56 0.45±0.04 4.52±0.47 Beef extract 5.59±1.50 70.51±2.27 59.21±1.24 15.75±2.56 0.89±0.05 27.72±3.07 Monosodium glutamate 5.02±1.00 68.03±0.53 43.56±2.18 25.49±2.08 1.19±0.13 20.74±2.78 NaNO 4.12±0.58 71.08±2.57 44.64±1.03 30.23±3.50 1.91±0.04 25.35±5.4 (NH ) SO 3.72±0.63 69.25±1.58 45.21±4.18 25.25±1.58 1.10±0.02 19.25±4.50 4 2 4 NH Cl 3.61±0.82 68.20±1.58 46.21±4.18 22.70±2.82 1.04±0.06 20.51±5.00 NH NO 3.05±0.81 69.23±0.87 58.13±3.13 10.41±3.67 0.81±0.04 25.51±5.60 4 3 Peptone 6.84±1.01 70.23±0.36 39.25±0.15 20.05±2.35 0.98±0.02 21.68±3.73 Yeast extract 6.91±0.71 69.62±2.52 46.20±1.86 22.50±2.02 1.35±0.03 24.01±0.90 Values are given as means ± SD from triplicate determinations to a likely nutrient transport deficiency. That is, nitrate first either biomass or biosurfactant production levels was ob- undergoes dissimilatory nitrate reduction to ammonium and served. Growth-associated production of biosurfactant has then is assimilated by glutamine–glutamate metabolism. It is been reported for Aeromonas sp. (Ilori et al. 2005), Bacillus likely that assimilation of nitrate as the nitrogen source is so subtilis (Abdel-Mawgoud et al. 2008), Leucobacter komaga- low, leading to a simulated nitrogen-limiting condition tae 183(Saimmaietal. 2012b), and Pseudomonas sp. (Barber and Stuckey 2000). (Obayori et al. 2009). Tabatabaee et al. (2005)also docu- mented that a biosurfactant synthesized by a strain of Bacillus sp. was a primary metabolite produced during cellular Time course of growth and biosurfactant production biomass formation. From the obtained result, it can be seen that a cultivation time of 54 h gave the highest biosurfactant yield. The results in Fig. 1a show that biosurfactant production started early in the exponential phase and the production kinetics paralleled the biomass kinetics up to 2 days of Recovery of biosurfactant incubation. On the basis of these facts, it can be concluded that biosurfactant production is growth-associated. It was The ability of various solvent systems to recover surface- found that the maximum level of cell biomass was obtained active components from the culture supernatant of I. limosus after 48 h of incubation. However, maximum biosurfactant KB3 after 54 h of cultivation was examined. The use of concentration was obtained 6 h later (5.13 g/l), i.e., after ethyl acetate resulted in greater activity of crude extract 54 h of incubation. After those periods, a sharp reduction in against systems based on mixtures of chloroform and Table 3 Effect of C:N ratio on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h a a a a C:N ratio Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction Control 4.12±0.58 71.08±2.57 40.64±1.03 30.23±3.50 1.91±0.04 35.35±5.41 5 4.26±0.92 71.51±1.27 36.21±1.24 35.30±4.00 2.73±0.36 40.41±6.20 10 4.48±0.23 71.03±0.53 32.56±2.18 39.09±2.28 3.92±0.39 46.64±4.38 15 4.61±0.92 71.08±0.57 31.64±1.03 40.03±2.22 4.22±0.53 50.70±3.60 20 4.85±0.52 71.25±1.58 28.21±4.18 43.11±2.70 4.81±0.41 55.64±2.07 25 5.00±0.64 72.20±2.08 28.21±4.18 44.35±2.56 4.90±0.51 59.62±5.20 30 5.18±0.43 72.23±0.87 32.13±3.13 40.59±3.28 4.85±0.05 55.50±4.70 35 5.36±0.14 72.23±0.87 40.13±3.13 32.59±3.28 4.01±0.05 50.50±2.10 40 5.49±0.55 72.23±0.36 41.25±0.15 31.15±5.25 3.52±0.13 48.58±3.83 Values are given as means ± SD from triplicate determinations 1332 Ann Microbiol (2013) 63:1327–1339 Fig. 1 Time course of growth and biosurfactant production by 6.0 Inquilinus limosus KB3 in optimal medium at 150 rpm and 5.0 30 °C (a) and surface tension 60 versus biosurfactant 50 4.0 concentration produced by Inquilinus limosus KB3 (b). Bars indicate the standard 3.0 deviation from triplicate determinations 2.0 Emulsification activity (%) Biosurfactant (g/l) 1.0 Surface tension (mN/m) Dry cell weight (g/l) 0 0.0 0 6 12 18 24 30 36 42 48 54 60 66 72 Times (h) 02468 10 20 30 Biosurfactant concentration (mg/l) methanol, cold acetone or dichloromethane (Table 4). It Surface tension and critical micelle concentration (CMC) hasalsobeenreportedpreviouslythatthe extraction of bioproducts with considerably high polarity by ethyl acetate The relationship between surface tension and concentration solvent is rather efficient (Chen and Juang 2008). Because the of the crude extracted biosurfactant solution was investigat- recovery and concentration of biosurfactants from fermenta- ed (Fig. 1b). The biosurfactant produced exhibited excellent tion broth largely determines the production cost, ethyl acetate surface tension reducing activity. The surface tension of is a better choice than the highly toxic chloro-organic water of 72 mN/m decreased to 25.5 mN/m by increasing compounds. the solution concentration up to 9 mg/l. Further increases in Table 4 Effect extraction meth- a a Recovery method Yield (g/l) CMC (mg/l) Surface tension (mN/m) ods on yield, critical micelle concentration (CMC) and activ- Acetone precipitation 6.65±0.85 16 61.5±2.2 ity of biosurfactant produced by Inquilinus limosus KB3 Acid precipitation 7.34±1.24 30 50.0±3.4 Ammonium sulfate precipitation 3.58±0.23 25 31.5±0.5 Chloroform:methanol extraction 4.21±0.55 12 32.5±0.4 Dichloromethane extraction 5.00±0.15 15 29.0±0.5 Ethanol precipitation 3.27±0.27 24 33.5±0.5 Values are given as means ± SD Ethyl acetate extraction 5.13±0.31 9 28.2±0.7 from triplicate determinations Surface tension (mN/m) Surface tension (mN/m), E24 (%) Biosurfactant (g/l), Dry cell weight (g/l) Ann Microbiol (2013) 63:1327–1339 1333 the concentration of the biosurfactant solution did not re- Surface te ns ion (mN/m) Emuls ification activity (%) duce the surface tension of the water, indicating that the 55 65 CMC was reached at this concentration. The biosurfactant 50 60 from I. limosus KB3 showed a minimum surface tension and 45 55 CMC value compared with the biosurfactant from B. subtilis (26.7 mN/m, 10 mg/l, respectively) (Ghojavand et al. 2008), 40 50 from Lactobacillus paracasei (41.8 mN/m, 250 mg/l) (Gudina 35 45 et al. 2011), and from Pseudomonas aeruginosa Bs 20 (29.5 mN/m, 13.4 mg/l) (Abdel-Mawgoud et al. 2009). 30 40 25 35 Study of biosurfactant stability 20 30 30 40 50 60 70 80 90 100 110 121 The results obtained from thermal stability analysis of biosur- Te mpe rature C factant over a wide range of temperature (4–121 °C) revealed that the biosurfactant from I. limosus KB3 was thermostable Surface te ns ion (mN/m) Emuls ification activity (%) (Fig. 2a). Heating of the biosurfactant solution up to 100 °C 55 65 (or its autoclaving at 121 °C) caused little effect on the bio- 50 60 surfactant performance and its emulsification ability. The sur- 45 55 face tension activity and E24 were relatively stable at the temperatures used (ST≈30 mN/m, E24≈57 %), respectively. 40 50 The activity of the biosurfactant and its emulsification ability 35 45 were also affected by the pH. When the pH was acidic and set 30 40 to 2, 3, and 4, the biosurfactant activities were 50, 47, and 40 mN/m, respectively (Fig. 2b). Correspondingly, the emul- 25 35 sification ability was limited to the acid to neutral pH and 20 30 emulsification activity up to 36, 40, and 45 %, respectively, 23 45 67 8 9 10 11 12 was obtained. The optimum pH for both parameters, namely pH biosurfactant activity (ST=25.5 mN/m) and emulsification Surface te ns ion (mN/m) Emuls ification activity (%) activity (E24=60 %) was determined to be 7–10. However, 55 65 the emulsification activity was relatively stable between pH 8 and 12. Figure 2c demonstrates the effect of the addition of 50 60 sodium chloride on the surface tension and E24 of the bio- 45 55 surfactant obtained. As is shown, negligible changes occurred in surface tension activity with an increase in the NaCl con- 40 50 centration up to 12 %. Likewise, an increase in NaCl concen- 35 45 tration up to 15 % did not have a significant effect on E24. However, at the highest level of NaCl (21 %), E24 was 30 40 severely decreased to 42 % and surface tension was also 25 35 increased (48 mN/m). 20 30 Emulsification properties of biosurfactants 0 3 6 9 12 15 18 21 NaCl (%) Biosurfactant isolated from I. limosus KB3 showed a good Fig. 2 Effect of temperature (a), pH (b), and NaCl (c) on activity of emulsification against several hydrophobic substrates crude biosurfactant produced by Inquilinus limosus KB3. Bars indicate the standard deviation from triplicate determinations (Fig. 3a). The E24 of biosurfactant from I. limosus KB3 was higher than that of the chemical surfactants, since it more effectively emulsified aromatic and aliphatic hydro- the biosurfactant to stabilize the microscopic droplets of carbons and several plant oils. Olive oil, soybean oil, and these compounds. The explanation of these results could toluene were good substrates for E24 by the KB3 biosur- come from the structure of these compounds, consisting of factant, showing no significant differences. Benzene, hexa- a mixture of paraffin, naphthalene, and aromatic hydrocar- decane, hexane, kerosene, and motor oil also formed stable bon, which was difficult to emulsify by crude biosurfactant emulsions. Xylene and ULO differed from the others, result- (Muthusamy et al. 2008). The ability of the biosurfactant ing in poor emulsification, probably due to the inability of produced by I. limosus KB3 to emulsify various Surface tension (mN/m) Surface tension (mN/m) Surface tension (m N/m ) Emulsification activity (%) Em ulsification activity (%) Emulsification activity (%) 1334 Ann Microbiol (2013) 63:1327–1339 −1 hydrophobic substrates indicates that it has a good potential by the C-H stretching modes at 2,920–2,854 cm and −1 for applications in microbial-enhanced oil recovery, as a 1,460–1,057 cm . These results strongly indicate that cleaning and emulsifying agent in food industry, and also the biosurfactant contains aliphatic and peptide-like moi- for bioremediation. eties. The overall FT-IR spectrum of the biosurfactant from I. limosus KB3 was very similar to cyclic lipopep- Chemical characterization of the biosurfactant tides produced by bacilli-like surfactin (produced by B. subtilis) and lichenysin (produced by B. licheniformis) The chemical nature of the biosurfactant from I. limosus which are the most effective biosurfactants so far discov- KB3 was seen as a single spot on TLC. This fraction ered (Roongsawang et al. 2002; Joshi et al. 2008). showed positive reaction with ninhydrin reagent and rhoda- To further confirm the results of this study, a NMR analysis was performed (Fig. 4a, b). Results obtained from mine B reagent indicating the presence of peptide and lipid moieties in the molecule (data not shown). These results H-NMR indicated that the molecule is a lipopeptide. Almost all the backbone amide NH groups are in the region indicate the existence of lipopeptide biosurfactant. The FT- IR spectrum of the biosurfactant from I. limosus KB3 from 8.2 to 7.3 ppm downfield from tetramethylsilane. showed strong absorption bands, indicating the presence of Alpha hydrogens of the amino acids come into resonance −1 peptides at 3,360 cm resulting from N-H stretching mode from 5.1 to 3.9 ppm. A doublet at δ=0.90 ppm for the −1 (Fig. 3b). At 1,660 cm , the stretching mode of a CO-N (CH ) -CH group indicated terminal branching in the fatty 3 2 −1 bond was observed, and at 1,540 cm , the deformation acid component. Owing to the presence of CH at 1.2 ppm, mode of the NH bond combined with N-H stretching mode the ratio of the methylene and terminal groups could not be occurred. The presence of an aliphatic chain was indicated resolved. Other multiplets in the upfield region arise as a Fig. 3 Emulsification activity KB3 TitronX-100 Tween 80 of the biosurfactant produced by Inquilinus limosus KB3 (a) and Fourier transform infrared spectrum (b) of the biosurfactant produced by Inquilinus limosus KB3. Bars indicate the standard deviation from triplicate determinations Hexadecane Hexane Kerosene Motor oil Olive oil Soybean oil Toluene ULO Xylene Benzene Emulsification index (%) Ann Microbiol (2013) 63:1327–1339 1335 result of the sidechain protons of the amino acids. methyl, methlene, ester, and the carboxyl group, respective- Remaining spectra clearly confirmed the presence of β- ly (Fig. 4b). Therefore, the biosurfactant produced by I. hydroxy fatty acid. The C-NMR spectrum showed strong limosus KB3 could be a lipopeptide, possibly an isoform signals at 14.0, 22.98–37.68, 171.4, and 174.2 ppm from of surfactin (Tang et al. 2007). C-(CH ) 3 2 CH-(-CH ) 2 9 CαH C-CH -CO Hαs of amino acids Backs bone amine NHs of amino acids CH Solvent CDCl CH C-OH -COOH 13 1 Fig. 4 C(a) and H nuclear magnetic resonance spectrum (b) of the biosurfactant produced by Inquilinus limosus KB3 1336 Ann Microbiol (2013) 63:1327–1339 Table 5 Dose-dependent solubilization of polyaromatic hydrocarbons by crude biosurfactant isolated from Inquilinus limosus KB3 Concentration of crude biosurfactant (mg/l) Solubility of PAHs (mg/l) Anthracene Fluoranthene Fluorene Naphtalene Phenantrene Pyrene 0 0.07±0.01 g 0.25±0.01 f 1.98±0.21 g 30.31±4.02 e 1.39±0.17 g 0.15±0.02 e 5 0.19±0.03 f 1.12±0.09 e 2.41±0.14 f 49.21±4.65 d 2.42±0.32 f 0.82±0.06 d 10 0.45±.009 e 1.81±0.23 d 3.27±0.35 e 58.21±5.10 d 2.41±0.31 e 1.68±0.31 c 15 0.79±0.04 d 2.91±0.34 c 3.82±0.11 d 64.16±4.03 c 2.58±0.42 d 2.01±0.36 c 20 1.07±0.11 c 3.31±0.09 b 4.51±0.41 c 76.45±4.63 bc 2.92±0.30 c 2.57±0.57 bc 25 1.32±0.05 b 4.81±0.31 a 5.61±0.25 b 84.34±5.38 ab 3.81±3.12 b 2.82±0.48 ab 30 1.51±0.09 a 4.98±0.20 a 6.41±0.62 a 91.47±9.23 a 3.98±0.58 a 3.05±0.32 a Different letters in the same column indicate significant differences (p<0.05) Values are given as means ± SD from triplicate determinations The above structure of the biosurfactant obtained was that the removal efficiency could deviate depending on the fully supported by its mass spectrometric analysis. characteristic of the contaminants and site characteristics. Analysis of the intact molecules with LCQ-MS revealed four molecular ion peaks with molecular masses [M+H] Laboratory experiment on biodegradation of ULO of 1,004, 1,018, 1,032, and 1,046, respectively (Fig. 5a). with biosurfactant The spectra clearly indicate the presence of higher and lower homologs of surfactants for the difference between promi- Biodegradation of crude oil in the laboratory-scale experi- nent M , peaks being around 14, corresponding to a differ- ment inferred that maximum biodegradation was found with ence in the number of methylene groups (CH ). This finding the biosurfactant (72.3 %), followed by the biosurfactant- was in accordance with Tang et al. (2007) who reported that and fertilizer-added set (65.2 %), fertilizer (52.4 %), and in a surfactin was a lipopeptide-type biosurfactant with a molec- normal setup (40.7 %). The improved biodegradation levels ular mass in the range of 1,007–1,072 Da. To our knowl- obtained with the biosurfactant indicated that it represents edge, this is the first report of the production of lipopeptide the most efficient accelerators for hydrocarbon biodegrada- from the genus Inquilinus. tion through increasing oil bioavailability (Maneerat 2009). The use of the biosurfactant in combination with fertilizer could reduce the actual amount of fertilizer to be added to Application of the biosurfactant in ULO removal polluted sites. In some studies, water-soluble fertilizers en- from contaminated sand countered problems such as being washed away and rapid dilution in aquatic environments. Thavasi et al. (2001) Petroleum hydrocarbon compounds bind to soil components reported that fertilizers only stimulate the early stage degra- and are difficult to remove and degrade (Sobrinho et al. dation rate of the oil and that the final degradation efficien- 2008). Biosurfactants can emulsify hydrocarbons, enhanc- cies with fertilizers were not significantly different from ing their water solubility, decreasing surface tension, and those where no fertilizers were used. It is important, how- increasing the displacement of oil substances from soil ever, to keep in mind that nutrient or fertilizer use may be particles (Banat et al. 2010). The ability of the biosurfactant essential in some environments with insufficient nutrient from I. limosus KB3 to enhance ULO removal from con- levels. taminated sand was examined in comparison with those of synthetic surfactants, i.e. a nonionic surfactant Triton X-100 Effect of biosurfactant on PAHs solubilization and anionic surfactants SDS. The biosurfactant of I. limosus KB3 and Triton X-100 could recover 25–30 % of ULO from Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminated sand at 25 °C; 50 % at room temperature (30± environmental polluants. Excessive inputs from anthropo- 2 °C); 65 % at 45 °C; and 85 % at 60 °C. The synthetic genic activities have caused serious contamination and ad- surfactant SDS was found to be less efficient. In the case of versely affect the health of aquatic and human through control (distillated water), very little recovery (5–18 %) could be obtained in the temperature range. These results Fig. 5 Mass spectrum (a) and microbial enhanced oil recovery (b)of have implications on the potential use of a biosurfactant the biosurfactant produced by Inquilinus limosus KB3 under different produced by I. limosus KB3 to enhance sorbed oil from temperatures. Bars indicate the standard deviation from triplicate the environment. However, it is important not to rule out determinations Ann Microbiol (2013) 63:1327–1339 1337 Triton X-100 Biosurfactant SDS Control 25 Room temperature 45 60 Temperature ( C) Oil recovery (%) 1338 Ann Microbiol (2013) 63:1327–1339 bioaccumulation. PAHs are hydrophobic and readily growth not only of microorganisms but also of viruses and adsorbed onto particulate matter; therefore, coastal and ma- cancer cells (Singh and Cameotra 2004). rine sediments become the ultimate sinks, and elevated concentrations have been recorded (Aniszewski et al. 2010). Solubilization of PAHs depends on the type and dose Conclusion of the surfactant, the hydrophobicity, the surfactant–soil interactions and the time that the contaminant has been in In the present study, the production of the biosurfactant from contact with the soil (Zhou and Rhue 2000). The effect of I. limosus KB3 which was isolated from marine sediment is the biosurfactant on the apparent aqueous solubility of PAHs reported. The growth characteristics were obtained and stud- was determined by test tube solubilization assays in the ies on the properties of the biosurfactant low-cost fermenta- presence of increasing concentrations of biosurfactant (0 to tive medium indicate the possibility of its industrial 50 mg/l) and is depicted in Table 5. In general, the biosur- application. The spectra obtained from FT-IR spectroscopy, factant obtained enhanced the apparent solubility of PAHs in NMR, and ESI-MS confirmed the presence of lipopeptide in a dose-dependent manner. However, solubilization of fluo- the sample. The properties of the biosurfactant obtained rene, naphthalene, or phenantrene by the biosurfactant from have potential application especially for microbial- this strain (about 3–5 times higher apparent solubility com- enhanced oil recovery and/or reducing the intensity of en- pared to control) was significantly lower (p<0.05) when vironmental contamination. Finally, biosurfactants are a compared with anthracene, fluoranthene, or pyrene affected suitable alternative to synthetic medicines and antimicrobial by biosurfactant (15–20 times higher compared to control). agents and may be used as safe and effective therapeutic In the present study, the crude biosurfactant showed ability agents. to solubilize PAHs in aqueous phase indicating its possible role in increasing the bioavailability of non-soluble organic Acknowledgments We are grateful to Phuket Rajabhat University for compounds for bacterial metabolism. These characteristics providing a scholarship to A.S. This work was supported by the Higher indicate the potential to use the biosurfactant obtained in Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission and the Post- environmental remediation. doctoral Fellowship from Prince of Songkla University. The work was also funded by the Graduate School, Prince of Songkla University. Antimicrobial activity of biosurfactant The crude biosurfactant of I. limosus KB3 was found to be an References antimicrobial agent, depending on the microorganism (data not shown). It was found that the biosurfactant obtained Abdel-Mawgoud AM, Aboulwafa AM, Hassouna NAH (2008) Opti- exhibited a high antimicrobial activity against B. cereus, C. mization of surfactin production by Bacillus subtilis isolate BS5. Appl Biochem Biotechnol 150:305–325 albicans, P. aeruginosa,and S. aureus at tested concentra- Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NAH (2009) Char- tions. In addition, it was observed that the biosurfactant acterization of rhamnolipid produced by Pseudomonas aerugi- obtained showed no antimicrobial activity against V. vulnificus nosa Isolate Bs20. Appl Biochem Biotechnol 157:329–345 and V. cholerae and low activity against E. faecium, E. coli, L. Anderson RC, Yu PK (2005) Factors affecting the antimicrobial activ- ity of ovine-dervied cathelicidins aginst Escherichia coli O157: monocytogenes, Salmonella sp. and S. typhimurium. H7. Int J Antimicrob Ag 25:205–210 Generally, surfactants having high surface-active proper- Aniszewski E, Peixoto SR, Mota FF, Leite SGF, Rosado SA (2010) ties show certain antimicrobial activities to some extent. Bioemulsifier production by Microbacterium sp. strains isolated Indeed, many lipopeptide biosurfactants show various bio- from mangrove and their application to remove cadmium and zinc from hazardous industrial residue. Braz J Microbiol 41:235–245 logical activities reflecting their structures (Banat et al. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, Fracchia 2010). The effect of the biosurfactant on bacteria appears L, Smyth TJ, Marchant R (2010) Microbial biosurfactants pro- more marked with Gram-positive bacteria than with Gram- duction, applications and future potential. 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Production and characterization of biosurfactant from marine bacterium Inquilinus limosus KB3 grown on low-cost raw materials

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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10.1007/s13213-012-0592-7
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Abstract

Ann Microbiol (2013) 63:1327–1339 DOI 10.1007/s13213-012-0592-7 ORIGINAL ARTICLE Production and characterization of biosurfactant from marine bacterium Inquilinus limosus KB3 grown on low-cost raw materials Atipan Saimmai & Satianpong Udomsilp & Suppasil Maneerat Received: 3 July 2012 /Accepted: 13 December 2012 /Published online: 8 January 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Inquilinus limosus strain KB3, isolated from ma- Introduction rine sediment in the south of Thailand, was used to produce a biosurfactant from a mineral salts medium (MSM) with The interest in biosurfactants has increased considerably in palm oil decanter cake (PODC) as a carbon source. It was recent years, as they are potential candidates for many found that cellular growth and biosurfactant production in commercial applications in the biomedical, pharmaceuticals, MSM were greatly affected by the medium components. I. petroleum, and food processing industries (Banat et al. limosus KB3 was able to grow and to produce surfactant 2010). The biosurfactants have several advantages over reducing the surface tension of medium to 28.2 mN/m and chemical surfactants including high ionic strength tolerance, giving a crude surfactant concentration of 5.13 g/l after 54 h. high temperature tolerance, higher biodegradability, lower The biosurfactant obtained was found to reduce the surface toxicity, lower critical micelle concentration (CMC), and tension of pure water to 25.5 mN/m with the critical micelle higher surface activity (Abdel-Mawgoud et al. 2009; Banat concentration of 9 mg/l, and retained its properties during et al. 2010; Gudina et al. 2011). In spite of the advantages, exposure to elevated temperatures (121 °C), high salinity fermentation must be cost competitive with chemical syn- (12 % NaCl), and a wide range of pH values. Chemical thesis, and many of the potential applications that have been characterization by FT-IR, NMR, and ESI-MS revealed that considered for biosurfactants depend on whether they can be the biosurfactant has a lipopeptide composition with molec- produced economically. The choice of inexpensive raw ular mass (m/z) of 1,032. The biosurfactant was capable of materials is important to overall economy of the process forming stable emulsions with various hydrocarbons and because they account for 50 % of the final product cost had the ability to enhance oil recovery, PAHs solubility, and also reduce the expense of waste treatment (Makkar and antimicrobial activity. and Cameotra 2002). In recent years, much work has been carried out towards efficient utilization of agro-industrial . . Keywords Inquilinus limosus Biosurfactant Palm oil residues such as cassava wastewater (Nitschke and Pastore . . decanter cake Oil recovery Polyaromatic hydrocarbon 2006), ground-nut oil refinery residue and corn steep liquor (Sobrinho et al. 2008), molasses (Joshi et al. 2008; Saimmai et al. 2011), and potato peels (Das and Mukherjee 2007). Palm oil decanter cake, a by-product from the palm oil milling decantation process, is an abundant and low cost A. Saimmai (*) S. Udomsilp agricultural waste residue. It is easily available in large Faculty of Agricultural Technology, quantities in the south of Thailand. It accounts for about Phuket Rajabhat University, Phuket 83000, Thailand e-mail: s4680108@hotmail.com 3 % of the weight of the empty fruit bunch of palm oil and is rich in oil residues and various vitamins and mineral ele- S. Maneerat ments (Yahya et al. 2010). However, palm oil decanter cake Department of Industrial Biotechnology, has not found any significant commercial application until Faculty of Agro-Industry, Prince of Songkla University, now and is generally disposed of in open areas, leading to Hat Yai, Songkhla 90112, Thailand 1328 Ann Microbiol (2013) 63:1327–1339 potentially serious environmental problems. It is thus nec- NH NO , peptone, and yeast extract were employed at a 4 3 essary to explore its industrial reutilization. concentration of 1 g/l with the optimum carbon source. The This study was carried out to explore the feasibility of C:N ratio (with optimized carbon and nitrogen sources) was using palm oil decanter cake as the substrate for the produc- varied from 5 to 40 by keeping a constant nitrogen source tion of biosurfactant by the marine bacterial Inquilinus concentration of 1 g/l. limosus KB3 isolated from marine sediment and the poten- tial application of biosurfactant obtained for enhanced solu- Recovery of biosurfactant bilization of hydrophobic compounds. Four solvent systems; a mixture of chloroform:methanol (2:1), cold acetone, dichloromethane, and ethyl acetate were Materials and methods examined for biosurfactant extraction (Saimmai et al. 2012b). The method showing the highest biosurfactant ac- Biosurfactant-producing strain tivity was used to recover biosurfactant from I. limosus KB3. Inquilinus limosus KB3 (accession number AB685266) was isolated from marine sediment collected from the southern Chemical analysis of biosurfactant part of Thailand, during a screening study for biosurfactant- producing bacteria in mangrove sediment (unpublished da- The chemical nature of the biosurfactants obtained was ta). I. limosus KB3 was maintained on nutrient agar plates determined with thin layer chromatography (TLC). The and transferred monthly. biosurfactant was spotted in triplicate on ready-made silica gel TLC plates (Merck, Darmstadt, Germany) using CHCl : Media and cultivation conditions CH OH:H O (65:15:1) as the solvent system. One of the 3 2 plates was put into a jar saturated with iodine vapor to detect Nutrient broth was used for preparation of the inoculum. lipids (Das et al. 2009). Another plate was sprayed with The composition of the nutrient broth used was as follows: anisaldehyde and ninhydrin reagent (0.2 % ninhydrin solu- beef extract 1.0 g, yeast extract 2.0 g, peptone 5.0 g, NaCl tion in acetone) and dried. It was then heated at 120 °C for 5.0 g in 1 l of distilled water. To make nutrient agar, 15.0 g 5 min for detection of sugars and peptides (Das et al. 2009), of agar was added to the nutrient broth. The culture was respectively. grown in this broth for 20–24 h at 30 °C. This was used as Fourier transform infrared spectroscopy (FT-IR) of the inoculum at the 3 % (v/v) level. For biosurfactant synthesis, biosurfactant obtained was done on a Nexus-870 FT-IR a mineral salt medium (MSM) with the following composi- spectrometer (Thermo Electron, Yokohama, Japan) by the tion (g/l) was utilized: K HPO , 0.8; KH PO , 0.2; CaCl , KBr pellet method. Further characterization of the biosur- 2 4 2 4 2 0.05; MgCl , 0.5; FeCl , 0.01; NaCl, 10.0 (Saimmai et al. factant was carried out using nuclear magnetic resonance 2 2 2012a). pH of the medium was adjusted to 7.0. Carbon and (NMR) using CDCl with an AMX 300 NMR spectrometer nitrogen sources were added separately. Cultivation was (500 MHz; Bruker). Final characterization of the compound performed in 250-ml flasks containing 50 ml medium at was performed by liquid chromatography-mass spectrosco- room temperature (30±3 °C), and shaking in a rotary shaker py (LC-MS)withanLCQ™ quadrupole ion-trap mass at 150 rpm for 48 h. spectrometer (Finnigan MAT, San Jose, CA, USA) which utilizes electrospray ionization (ESI) (Thavasi et al. 2008). Medium optimization Application of the biosurfactant in ULO removal The medium optimization was conducted in a series of from contaminated sand experiments changing one variable at a time, keeping the other factors fixed at a specific set of conditions. Three Biosurfactant suitability for enhancing oil recovery was factors were chosen aiming to obtain higher productivity investigated using 800.0 g of acid-washed sand impregnated of the biosurfactant: carbon source (C), nitrogen source (N), with 50.0 ml of ULO. Fractions of 20.0 g of the contami- and C:N ratio. The carbon sources used were 1 g/l of glu- nated sand were transferred to 250-ml flasks which were cose, glycerol, molasses, oleic acid, palm oil decanter cake, submitted to the following treatments: addition of 60.0 ml soybean oil, stearic acid, used lubricating oil (ULO), and distilled water (control), addition of 60.0 ml aqueous solu- used vegetable oil (UVO), with (NH ) SO as a nitrogen tions of the SDS, Triton X-100 and biosurfactant at the 4 2 4 source. For evaluation of the most appropriate nitrogen CMC level of each compound. The samples were incubated sources for the production of biosurfactants, beef extract, on a rotary shaker (200 rpm) for 24 h at 30 °C and centri- monosodium glutamate, NaNO ,(NH ) SO ,NH Cl, fuged at 3,354 g for 20 min for separation of the laundering 3 4 2 4 4 Ann Microbiol (2013) 63:1327–1339 1329 solution and the sand. The amount of oil residing in the sand Antimicrobial activity of surface active compound after the impact of biosurfactant was gravimetrically deter- mined as the amount of material extracted from the sand by The extracted compound was tested for antimicrobial hexane (Sobrinho et al. 2008). activity using the agar well diffusion method and the inhibition zone was measured (Candan et al. 2003). Extracted active compound was tested against pathogenic Laboratory experiment on biodegradation of ULO with microorganisms including Bacillus cereus, Candida albi- biosurfactant cans, Enterococcus faecium, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella An experiment was conducted to study the impact of the sp., Salmonella typhimurium, Staphylococcus aureus, biosurfactant isolated from I. limosus KB3 on biodegrada- Vibrio cholerae and Vibrio vulnificus. All strains were tion of ULO in natural seawater. Shake flask biodegradation obtained from Songklanagarind Hospital, Prince of experiments were carried out in 500-ml Erlenmeyer flasks Songkla University, Thailand. Briefly, the extract was with 100 ml of sterilized seawater. The experiment was dissolved in distilled water at a concentration of 10 mg/ conducted with four different sets: (1) bacterial cells alone; ml and filter-sterilized using a 0.2-μm membrane filter. (2) with fertilizer and cells; (3) with cells and biosurfactants Each tested microorganism was grown in brain heart (0.1 %, w/v); and (4) with fertilizer and biosurfactant. infusion (BHI; Hi-Media Laboratories, Mumbai, India) Exactly 2.0 % (w/v) of ULO was added to the sterilized and diluted to a concentration of 10 CFU/ml. They were seawater, and inoculation was performed with 24-h-old cul- overlaid onto the surface of BHI agar. The agar plates 3 4 ture at the rate of 1 % (v/v, 10 –10 CFU/ml) concentration. were dried for 20 min at room temperature. Microbial Flasks were shaken at 150 rpm in dark at room temperature suspensions were plated on BHI agar plates, which were for a period of 168 h. The biodegradation of oil was esti- dried for 20 min at room temperature. The wells were mated fluorimeterically as described in Intergovernmental cut from the agar and 100 μl of extract solution were Oceanographic Commission (IOC) Manuals and Guide No. added to the wells. The plates were incubated at 37 °C 13 (1982). An uninoculated control was kept to assess the for 24 h; after incubation, the clear zone was measured. natural weathering of oil and degradation. Analytical methods Polyaromatic hydrocarbons (PAHs) solubilization assay Biomass determination was done in terms of the dry cell PAHs solubilization assay was done as described by Barkay weight. At different times of fermentation, samples were et al. (1999). Briefly, 0.6 μg each of the following PAHs mixed in pre-weighted tubes with chilled distilled water (anthracene, fluoranthene, fluorine, naphthalene, phenan- and centrifuged at 9,693 g for 30 min. The biomass obtained threne or pyrene; all from 0.6 mg/ml stock in acetone) were was dried overnight at 105 °C and weighed. distributed into glass test tubes (10 mm×170 mm) and kept Emulsification activity was performed according to Wu open inside an operating chemical fume hood to remove the et al. (2008). Briefly, 4 ml of hydrocarbon or oil was added solvent. Subsequently, 3.0 ml of assay buffer (20 mM Tris- to 4 ml of aqueous solution of culture supernatant in a screw HCl, pH 7.0) and the biosurfactant at increasing concentra- cap tube, and vortexed at high speed for 2 min. The emul- tions (0–50 mg/ml) obtained from the bacterial strain used in sion activity (E24) was determined after 24 h. E24 was this study. Assay buffer containing the biosurfactant, but no calculated by dividing the measured height of the emulsion PAH, was used as blank. Tubes were capped with plastic layer by the total height of the mixtures and multiplying by closures and incubated overnight at 30 °C with shaking 100. (200 rpm) in dark. Samples were filtered through 1.2-μm The surface tension of culture supernatant was measured filters (Whatman, Springfield Mill, UK) and 2.0 ml of this using a Model 20 Tensiometer (Fisher Science Instrument, filtrate was extracted with an equal volume of hexane. This PA, USA) at 25 °C. CMC was determined by plotting the emulsion was centrifuged at 9,693 g for 10 min to separate surface tension versus concentration of biosurfactant in the the aqueous and hexane phases. The concentration of the solution. PAHs was measured spectrophotometrically (Libra S22; All experiments were carried out at least in triplicate. Biochrom, Cambridge, UK) at the specific wave lengths of Two well-defined synthetic surfactants, Triton X-100 and each compound (Barkay et al. 1999). From a calibration SDS were used as positive controls, while distilled water curve of individual PAHs (in hexane), the concentration of and MSM medium were used as negative controls. each PAH was determined. Assay buffer with biosurfactant Statistical analysis was performed using the Statistical without PAH was identically extracted with hexane and Package for Social Science (SPSS 10.0 for Windows; served as blank. Chicago, IL, USA). 1330 Ann Microbiol (2013) 63:1327–1339 Results and discussion (Chayabutra et al. 2001) or toxicity toward bacterial cells (Li and Chen 2009). Effect of carbon source on growth and biosurfactant production Effect of nitrogen source on growth and biosurfactant production The literature has revealed that the type and concentra- tion of carbon and nitrogen substrates markedly affected After examining the most commonly used organic and inorgan- the production yield of biosurfactant (Wu et al. 2008). In ic nitrogen sources reported in the literature (Abdel-Mawgoud light of this, this study started with the investigation of et al. 2008), it was found that the type of nitrogen source carbon and nitrogen sources on biosurfactant production. affected the growth and biosurfactant production of I. limosus I. limosus KB3was grownoneachof9 typesofcarbon KB3 (Table 2). The highest biomass was obtained when yeast sources. As seen in Table 1, the type of carbon source extract was used. However, NaNO exhibited the highest sur- affected both the biosurfactant production and E24. Palm face tension reduction and biosurfactant yield (35.49 mN/m and oil decanter cake differed from the others in relation to 1.79 g/l, respectively). This yield was nearly 2-fold that the biosurfactant concentration and E24, being the most obtained from using (NH ) SO as the nitrogen source. 4 2 4 appropriate carbon source; surface tension reduction Moreover, using NaNO as the inorganic nitrogen source not reached 25.25 mN/m with 1.10 g/l and achieved an only increased the biosurfactant yield but also improved the E24 of 19.25 % toward xylene. Table 1 also shows that biomass and E24 at 4.12 g/l and 25.35 %, respectively. there seems to be clear trend between biomass and bio- surfactant yields which strongly depends on the carbon Effect of C:N ratio on growth and biosurfactant production source used. Although vegetable oils or glucose have been frequently used as the carbon substrates for biosur- The C:N ratio is also known as a vital factor influencing the factant production (Banat et al. 2010), I. limosus KB3 performance of biosurfactant production (Santos et al. attained a lower biosurfactant yield from soybean oil, 2002). As indicated in Table 3, the best biosurfactant activ- UVO, and glucose than that from palm oil decanter cake, ity in surface tension reduction and yield (44.35 mN/m and molasses, and glycerol (Table 1). Direct use of fatty 4.90 g/l, respectively) were obtained at a C:N ratio of 25; acids (i.e., oleic acid and stearic acid) as the carbon this yield was 2.6-fold that obtained from control (C:N ratio source did not improve biosurfactant production, sug- at 1:1). The productivity of biosurfactant tended to decrease gesting that hydrolysis of the oils was not the bottle- as the C:N ratio increased from 30 to 40, especially for C:N neck step. Moreover, ULO and UVO were also ineffi- ratio >35. Some reports mentioned that biosurfactant pro- cient in cell growth and biosurfactant production, result- duction is more efficient under nitrogen-limiting conditions ing in a low biosurfactant yield of only 0.50 and 0.48 g/ (Benincasa et al. 2002). The results show that a possible l, respectively, probably due to its poor biodegradability inhibitory effect on the bacterial metabolism may occur due Table 1 Effect of carbon source on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h (nitrogen source: 1 g/l of (NH ) SO ) 4 2 4 a a a a Carbon source (1 g/l) Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction No carbon source 0.06±0.01 72.01±1.27 70.01±0.04 1.51±0.04 0.05±0.01 0 Glucose 4.05±0.20 71.51±2.27 59.21±1.24 12.11±2.27 0.53±0.01 20.51±5.07 Glycerol 3.95±0.92 70.03±0.53 53.56±2.18 18.03±5.53 0.91±0.01 20.03±4.73 Molasses 4.54±1.62 65.08±2.57 44.64±1.03 20.08±2.57 1.08±0.08 20.08±2.67 Oleic acid 2.48±0.63 68.35±2.67 59.19±2.06 8.35±2.67 0.32±0.05 8.05±2.54 Palm oil decanter cake 3.72±0.63 69.25±1.58 45.21±4.18 25.25±1.58 1.10±0.02 19.25±4.50 Soybean oil 3.54±0.41 62.23±0.87 46.13±3.13 16.23±0.87 0.88±0.04 15.01±2.93 Stearic acid 1.04±0.12 70.23±0.36 60.25±0.15 8.23±0.36 0.32±0.02 10.03±4.57 Used lubricating oil 0.40±0.09 57.62±2.52 46.20±1.86 10.62±2.52 0.50±0.06 17.61±5.01 Used vegetable oil 0.61±0.02 65.61±2.05 56.25±4.04 9.61±2.05 0.48±0.04 12.12±4.07 Values are given as means ± SD from triplicate determinations Ann Microbiol (2013) 63:1327–1339 1331 Table 2 Effect of nitrogen source on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h (carbon source: 1 g/l of palm oil decanter cake) a a a a Nitrogen source (1 g/l) Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction No nitrogen source 0.21±0.03 70.01±1.27 60.10±0.14 9.20±0.56 0.45±0.04 4.52±0.47 Beef extract 5.59±1.50 70.51±2.27 59.21±1.24 15.75±2.56 0.89±0.05 27.72±3.07 Monosodium glutamate 5.02±1.00 68.03±0.53 43.56±2.18 25.49±2.08 1.19±0.13 20.74±2.78 NaNO 4.12±0.58 71.08±2.57 44.64±1.03 30.23±3.50 1.91±0.04 25.35±5.4 (NH ) SO 3.72±0.63 69.25±1.58 45.21±4.18 25.25±1.58 1.10±0.02 19.25±4.50 4 2 4 NH Cl 3.61±0.82 68.20±1.58 46.21±4.18 22.70±2.82 1.04±0.06 20.51±5.00 NH NO 3.05±0.81 69.23±0.87 58.13±3.13 10.41±3.67 0.81±0.04 25.51±5.60 4 3 Peptone 6.84±1.01 70.23±0.36 39.25±0.15 20.05±2.35 0.98±0.02 21.68±3.73 Yeast extract 6.91±0.71 69.62±2.52 46.20±1.86 22.50±2.02 1.35±0.03 24.01±0.90 Values are given as means ± SD from triplicate determinations to a likely nutrient transport deficiency. That is, nitrate first either biomass or biosurfactant production levels was ob- undergoes dissimilatory nitrate reduction to ammonium and served. Growth-associated production of biosurfactant has then is assimilated by glutamine–glutamate metabolism. It is been reported for Aeromonas sp. (Ilori et al. 2005), Bacillus likely that assimilation of nitrate as the nitrogen source is so subtilis (Abdel-Mawgoud et al. 2008), Leucobacter komaga- low, leading to a simulated nitrogen-limiting condition tae 183(Saimmaietal. 2012b), and Pseudomonas sp. (Barber and Stuckey 2000). (Obayori et al. 2009). Tabatabaee et al. (2005)also docu- mented that a biosurfactant synthesized by a strain of Bacillus sp. was a primary metabolite produced during cellular Time course of growth and biosurfactant production biomass formation. From the obtained result, it can be seen that a cultivation time of 54 h gave the highest biosurfactant yield. The results in Fig. 1a show that biosurfactant production started early in the exponential phase and the production kinetics paralleled the biomass kinetics up to 2 days of Recovery of biosurfactant incubation. On the basis of these facts, it can be concluded that biosurfactant production is growth-associated. It was The ability of various solvent systems to recover surface- found that the maximum level of cell biomass was obtained active components from the culture supernatant of I. limosus after 48 h of incubation. However, maximum biosurfactant KB3 after 54 h of cultivation was examined. The use of concentration was obtained 6 h later (5.13 g/l), i.e., after ethyl acetate resulted in greater activity of crude extract 54 h of incubation. After those periods, a sharp reduction in against systems based on mixtures of chloroform and Table 3 Effect of C:N ratio on biosurfactant production by Inquilinus limosus KB3, which were cultivated in 250-ml flasks containing 50 ml MSM medium at 30 °C in a shaking incubator at 150 rpm for 48 h a a a a C:N ratio Dry cell weight (g/l) Surface tension (mN/m) Biosurfactant (g/l) E24 (%) Initial Final Reduction Control 4.12±0.58 71.08±2.57 40.64±1.03 30.23±3.50 1.91±0.04 35.35±5.41 5 4.26±0.92 71.51±1.27 36.21±1.24 35.30±4.00 2.73±0.36 40.41±6.20 10 4.48±0.23 71.03±0.53 32.56±2.18 39.09±2.28 3.92±0.39 46.64±4.38 15 4.61±0.92 71.08±0.57 31.64±1.03 40.03±2.22 4.22±0.53 50.70±3.60 20 4.85±0.52 71.25±1.58 28.21±4.18 43.11±2.70 4.81±0.41 55.64±2.07 25 5.00±0.64 72.20±2.08 28.21±4.18 44.35±2.56 4.90±0.51 59.62±5.20 30 5.18±0.43 72.23±0.87 32.13±3.13 40.59±3.28 4.85±0.05 55.50±4.70 35 5.36±0.14 72.23±0.87 40.13±3.13 32.59±3.28 4.01±0.05 50.50±2.10 40 5.49±0.55 72.23±0.36 41.25±0.15 31.15±5.25 3.52±0.13 48.58±3.83 Values are given as means ± SD from triplicate determinations 1332 Ann Microbiol (2013) 63:1327–1339 Fig. 1 Time course of growth and biosurfactant production by 6.0 Inquilinus limosus KB3 in optimal medium at 150 rpm and 5.0 30 °C (a) and surface tension 60 versus biosurfactant 50 4.0 concentration produced by Inquilinus limosus KB3 (b). Bars indicate the standard 3.0 deviation from triplicate determinations 2.0 Emulsification activity (%) Biosurfactant (g/l) 1.0 Surface tension (mN/m) Dry cell weight (g/l) 0 0.0 0 6 12 18 24 30 36 42 48 54 60 66 72 Times (h) 02468 10 20 30 Biosurfactant concentration (mg/l) methanol, cold acetone or dichloromethane (Table 4). It Surface tension and critical micelle concentration (CMC) hasalsobeenreportedpreviouslythatthe extraction of bioproducts with considerably high polarity by ethyl acetate The relationship between surface tension and concentration solvent is rather efficient (Chen and Juang 2008). Because the of the crude extracted biosurfactant solution was investigat- recovery and concentration of biosurfactants from fermenta- ed (Fig. 1b). The biosurfactant produced exhibited excellent tion broth largely determines the production cost, ethyl acetate surface tension reducing activity. The surface tension of is a better choice than the highly toxic chloro-organic water of 72 mN/m decreased to 25.5 mN/m by increasing compounds. the solution concentration up to 9 mg/l. Further increases in Table 4 Effect extraction meth- a a Recovery method Yield (g/l) CMC (mg/l) Surface tension (mN/m) ods on yield, critical micelle concentration (CMC) and activ- Acetone precipitation 6.65±0.85 16 61.5±2.2 ity of biosurfactant produced by Inquilinus limosus KB3 Acid precipitation 7.34±1.24 30 50.0±3.4 Ammonium sulfate precipitation 3.58±0.23 25 31.5±0.5 Chloroform:methanol extraction 4.21±0.55 12 32.5±0.4 Dichloromethane extraction 5.00±0.15 15 29.0±0.5 Ethanol precipitation 3.27±0.27 24 33.5±0.5 Values are given as means ± SD Ethyl acetate extraction 5.13±0.31 9 28.2±0.7 from triplicate determinations Surface tension (mN/m) Surface tension (mN/m), E24 (%) Biosurfactant (g/l), Dry cell weight (g/l) Ann Microbiol (2013) 63:1327–1339 1333 the concentration of the biosurfactant solution did not re- Surface te ns ion (mN/m) Emuls ification activity (%) duce the surface tension of the water, indicating that the 55 65 CMC was reached at this concentration. The biosurfactant 50 60 from I. limosus KB3 showed a minimum surface tension and 45 55 CMC value compared with the biosurfactant from B. subtilis (26.7 mN/m, 10 mg/l, respectively) (Ghojavand et al. 2008), 40 50 from Lactobacillus paracasei (41.8 mN/m, 250 mg/l) (Gudina 35 45 et al. 2011), and from Pseudomonas aeruginosa Bs 20 (29.5 mN/m, 13.4 mg/l) (Abdel-Mawgoud et al. 2009). 30 40 25 35 Study of biosurfactant stability 20 30 30 40 50 60 70 80 90 100 110 121 The results obtained from thermal stability analysis of biosur- Te mpe rature C factant over a wide range of temperature (4–121 °C) revealed that the biosurfactant from I. limosus KB3 was thermostable Surface te ns ion (mN/m) Emuls ification activity (%) (Fig. 2a). Heating of the biosurfactant solution up to 100 °C 55 65 (or its autoclaving at 121 °C) caused little effect on the bio- 50 60 surfactant performance and its emulsification ability. The sur- 45 55 face tension activity and E24 were relatively stable at the temperatures used (ST≈30 mN/m, E24≈57 %), respectively. 40 50 The activity of the biosurfactant and its emulsification ability 35 45 were also affected by the pH. When the pH was acidic and set 30 40 to 2, 3, and 4, the biosurfactant activities were 50, 47, and 40 mN/m, respectively (Fig. 2b). Correspondingly, the emul- 25 35 sification ability was limited to the acid to neutral pH and 20 30 emulsification activity up to 36, 40, and 45 %, respectively, 23 45 67 8 9 10 11 12 was obtained. The optimum pH for both parameters, namely pH biosurfactant activity (ST=25.5 mN/m) and emulsification Surface te ns ion (mN/m) Emuls ification activity (%) activity (E24=60 %) was determined to be 7–10. However, 55 65 the emulsification activity was relatively stable between pH 8 and 12. Figure 2c demonstrates the effect of the addition of 50 60 sodium chloride on the surface tension and E24 of the bio- 45 55 surfactant obtained. As is shown, negligible changes occurred in surface tension activity with an increase in the NaCl con- 40 50 centration up to 12 %. Likewise, an increase in NaCl concen- 35 45 tration up to 15 % did not have a significant effect on E24. However, at the highest level of NaCl (21 %), E24 was 30 40 severely decreased to 42 % and surface tension was also 25 35 increased (48 mN/m). 20 30 Emulsification properties of biosurfactants 0 3 6 9 12 15 18 21 NaCl (%) Biosurfactant isolated from I. limosus KB3 showed a good Fig. 2 Effect of temperature (a), pH (b), and NaCl (c) on activity of emulsification against several hydrophobic substrates crude biosurfactant produced by Inquilinus limosus KB3. Bars indicate the standard deviation from triplicate determinations (Fig. 3a). The E24 of biosurfactant from I. limosus KB3 was higher than that of the chemical surfactants, since it more effectively emulsified aromatic and aliphatic hydro- the biosurfactant to stabilize the microscopic droplets of carbons and several plant oils. Olive oil, soybean oil, and these compounds. The explanation of these results could toluene were good substrates for E24 by the KB3 biosur- come from the structure of these compounds, consisting of factant, showing no significant differences. Benzene, hexa- a mixture of paraffin, naphthalene, and aromatic hydrocar- decane, hexane, kerosene, and motor oil also formed stable bon, which was difficult to emulsify by crude biosurfactant emulsions. Xylene and ULO differed from the others, result- (Muthusamy et al. 2008). The ability of the biosurfactant ing in poor emulsification, probably due to the inability of produced by I. limosus KB3 to emulsify various Surface tension (mN/m) Surface tension (mN/m) Surface tension (m N/m ) Emulsification activity (%) Em ulsification activity (%) Emulsification activity (%) 1334 Ann Microbiol (2013) 63:1327–1339 −1 hydrophobic substrates indicates that it has a good potential by the C-H stretching modes at 2,920–2,854 cm and −1 for applications in microbial-enhanced oil recovery, as a 1,460–1,057 cm . These results strongly indicate that cleaning and emulsifying agent in food industry, and also the biosurfactant contains aliphatic and peptide-like moi- for bioremediation. eties. The overall FT-IR spectrum of the biosurfactant from I. limosus KB3 was very similar to cyclic lipopep- Chemical characterization of the biosurfactant tides produced by bacilli-like surfactin (produced by B. subtilis) and lichenysin (produced by B. licheniformis) The chemical nature of the biosurfactant from I. limosus which are the most effective biosurfactants so far discov- KB3 was seen as a single spot on TLC. This fraction ered (Roongsawang et al. 2002; Joshi et al. 2008). showed positive reaction with ninhydrin reagent and rhoda- To further confirm the results of this study, a NMR analysis was performed (Fig. 4a, b). Results obtained from mine B reagent indicating the presence of peptide and lipid moieties in the molecule (data not shown). These results H-NMR indicated that the molecule is a lipopeptide. Almost all the backbone amide NH groups are in the region indicate the existence of lipopeptide biosurfactant. The FT- IR spectrum of the biosurfactant from I. limosus KB3 from 8.2 to 7.3 ppm downfield from tetramethylsilane. showed strong absorption bands, indicating the presence of Alpha hydrogens of the amino acids come into resonance −1 peptides at 3,360 cm resulting from N-H stretching mode from 5.1 to 3.9 ppm. A doublet at δ=0.90 ppm for the −1 (Fig. 3b). At 1,660 cm , the stretching mode of a CO-N (CH ) -CH group indicated terminal branching in the fatty 3 2 −1 bond was observed, and at 1,540 cm , the deformation acid component. Owing to the presence of CH at 1.2 ppm, mode of the NH bond combined with N-H stretching mode the ratio of the methylene and terminal groups could not be occurred. The presence of an aliphatic chain was indicated resolved. Other multiplets in the upfield region arise as a Fig. 3 Emulsification activity KB3 TitronX-100 Tween 80 of the biosurfactant produced by Inquilinus limosus KB3 (a) and Fourier transform infrared spectrum (b) of the biosurfactant produced by Inquilinus limosus KB3. Bars indicate the standard deviation from triplicate determinations Hexadecane Hexane Kerosene Motor oil Olive oil Soybean oil Toluene ULO Xylene Benzene Emulsification index (%) Ann Microbiol (2013) 63:1327–1339 1335 result of the sidechain protons of the amino acids. methyl, methlene, ester, and the carboxyl group, respective- Remaining spectra clearly confirmed the presence of β- ly (Fig. 4b). Therefore, the biosurfactant produced by I. hydroxy fatty acid. The C-NMR spectrum showed strong limosus KB3 could be a lipopeptide, possibly an isoform signals at 14.0, 22.98–37.68, 171.4, and 174.2 ppm from of surfactin (Tang et al. 2007). C-(CH ) 3 2 CH-(-CH ) 2 9 CαH C-CH -CO Hαs of amino acids Backs bone amine NHs of amino acids CH Solvent CDCl CH C-OH -COOH 13 1 Fig. 4 C(a) and H nuclear magnetic resonance spectrum (b) of the biosurfactant produced by Inquilinus limosus KB3 1336 Ann Microbiol (2013) 63:1327–1339 Table 5 Dose-dependent solubilization of polyaromatic hydrocarbons by crude biosurfactant isolated from Inquilinus limosus KB3 Concentration of crude biosurfactant (mg/l) Solubility of PAHs (mg/l) Anthracene Fluoranthene Fluorene Naphtalene Phenantrene Pyrene 0 0.07±0.01 g 0.25±0.01 f 1.98±0.21 g 30.31±4.02 e 1.39±0.17 g 0.15±0.02 e 5 0.19±0.03 f 1.12±0.09 e 2.41±0.14 f 49.21±4.65 d 2.42±0.32 f 0.82±0.06 d 10 0.45±.009 e 1.81±0.23 d 3.27±0.35 e 58.21±5.10 d 2.41±0.31 e 1.68±0.31 c 15 0.79±0.04 d 2.91±0.34 c 3.82±0.11 d 64.16±4.03 c 2.58±0.42 d 2.01±0.36 c 20 1.07±0.11 c 3.31±0.09 b 4.51±0.41 c 76.45±4.63 bc 2.92±0.30 c 2.57±0.57 bc 25 1.32±0.05 b 4.81±0.31 a 5.61±0.25 b 84.34±5.38 ab 3.81±3.12 b 2.82±0.48 ab 30 1.51±0.09 a 4.98±0.20 a 6.41±0.62 a 91.47±9.23 a 3.98±0.58 a 3.05±0.32 a Different letters in the same column indicate significant differences (p<0.05) Values are given as means ± SD from triplicate determinations The above structure of the biosurfactant obtained was that the removal efficiency could deviate depending on the fully supported by its mass spectrometric analysis. characteristic of the contaminants and site characteristics. Analysis of the intact molecules with LCQ-MS revealed four molecular ion peaks with molecular masses [M+H] Laboratory experiment on biodegradation of ULO of 1,004, 1,018, 1,032, and 1,046, respectively (Fig. 5a). with biosurfactant The spectra clearly indicate the presence of higher and lower homologs of surfactants for the difference between promi- Biodegradation of crude oil in the laboratory-scale experi- nent M , peaks being around 14, corresponding to a differ- ment inferred that maximum biodegradation was found with ence in the number of methylene groups (CH ). This finding the biosurfactant (72.3 %), followed by the biosurfactant- was in accordance with Tang et al. (2007) who reported that and fertilizer-added set (65.2 %), fertilizer (52.4 %), and in a surfactin was a lipopeptide-type biosurfactant with a molec- normal setup (40.7 %). The improved biodegradation levels ular mass in the range of 1,007–1,072 Da. To our knowl- obtained with the biosurfactant indicated that it represents edge, this is the first report of the production of lipopeptide the most efficient accelerators for hydrocarbon biodegrada- from the genus Inquilinus. tion through increasing oil bioavailability (Maneerat 2009). The use of the biosurfactant in combination with fertilizer could reduce the actual amount of fertilizer to be added to Application of the biosurfactant in ULO removal polluted sites. In some studies, water-soluble fertilizers en- from contaminated sand countered problems such as being washed away and rapid dilution in aquatic environments. Thavasi et al. (2001) Petroleum hydrocarbon compounds bind to soil components reported that fertilizers only stimulate the early stage degra- and are difficult to remove and degrade (Sobrinho et al. dation rate of the oil and that the final degradation efficien- 2008). Biosurfactants can emulsify hydrocarbons, enhanc- cies with fertilizers were not significantly different from ing their water solubility, decreasing surface tension, and those where no fertilizers were used. It is important, how- increasing the displacement of oil substances from soil ever, to keep in mind that nutrient or fertilizer use may be particles (Banat et al. 2010). The ability of the biosurfactant essential in some environments with insufficient nutrient from I. limosus KB3 to enhance ULO removal from con- levels. taminated sand was examined in comparison with those of synthetic surfactants, i.e. a nonionic surfactant Triton X-100 Effect of biosurfactant on PAHs solubilization and anionic surfactants SDS. The biosurfactant of I. limosus KB3 and Triton X-100 could recover 25–30 % of ULO from Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminated sand at 25 °C; 50 % at room temperature (30± environmental polluants. Excessive inputs from anthropo- 2 °C); 65 % at 45 °C; and 85 % at 60 °C. The synthetic genic activities have caused serious contamination and ad- surfactant SDS was found to be less efficient. In the case of versely affect the health of aquatic and human through control (distillated water), very little recovery (5–18 %) could be obtained in the temperature range. These results Fig. 5 Mass spectrum (a) and microbial enhanced oil recovery (b)of have implications on the potential use of a biosurfactant the biosurfactant produced by Inquilinus limosus KB3 under different produced by I. limosus KB3 to enhance sorbed oil from temperatures. Bars indicate the standard deviation from triplicate the environment. However, it is important not to rule out determinations Ann Microbiol (2013) 63:1327–1339 1337 Triton X-100 Biosurfactant SDS Control 25 Room temperature 45 60 Temperature ( C) Oil recovery (%) 1338 Ann Microbiol (2013) 63:1327–1339 bioaccumulation. PAHs are hydrophobic and readily growth not only of microorganisms but also of viruses and adsorbed onto particulate matter; therefore, coastal and ma- cancer cells (Singh and Cameotra 2004). rine sediments become the ultimate sinks, and elevated concentrations have been recorded (Aniszewski et al. 2010). Solubilization of PAHs depends on the type and dose Conclusion of the surfactant, the hydrophobicity, the surfactant–soil interactions and the time that the contaminant has been in In the present study, the production of the biosurfactant from contact with the soil (Zhou and Rhue 2000). The effect of I. limosus KB3 which was isolated from marine sediment is the biosurfactant on the apparent aqueous solubility of PAHs reported. The growth characteristics were obtained and stud- was determined by test tube solubilization assays in the ies on the properties of the biosurfactant low-cost fermenta- presence of increasing concentrations of biosurfactant (0 to tive medium indicate the possibility of its industrial 50 mg/l) and is depicted in Table 5. In general, the biosur- application. The spectra obtained from FT-IR spectroscopy, factant obtained enhanced the apparent solubility of PAHs in NMR, and ESI-MS confirmed the presence of lipopeptide in a dose-dependent manner. However, solubilization of fluo- the sample. The properties of the biosurfactant obtained rene, naphthalene, or phenantrene by the biosurfactant from have potential application especially for microbial- this strain (about 3–5 times higher apparent solubility com- enhanced oil recovery and/or reducing the intensity of en- pared to control) was significantly lower (p<0.05) when vironmental contamination. Finally, biosurfactants are a compared with anthracene, fluoranthene, or pyrene affected suitable alternative to synthetic medicines and antimicrobial by biosurfactant (15–20 times higher compared to control). agents and may be used as safe and effective therapeutic In the present study, the crude biosurfactant showed ability agents. to solubilize PAHs in aqueous phase indicating its possible role in increasing the bioavailability of non-soluble organic Acknowledgments We are grateful to Phuket Rajabhat University for compounds for bacterial metabolism. These characteristics providing a scholarship to A.S. This work was supported by the Higher indicate the potential to use the biosurfactant obtained in Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission and the Post- environmental remediation. doctoral Fellowship from Prince of Songkla University. The work was also funded by the Graduate School, Prince of Songkla University. Antimicrobial activity of biosurfactant The crude biosurfactant of I. limosus KB3 was found to be an References antimicrobial agent, depending on the microorganism (data not shown). It was found that the biosurfactant obtained Abdel-Mawgoud AM, Aboulwafa AM, Hassouna NAH (2008) Opti- exhibited a high antimicrobial activity against B. cereus, C. mization of surfactin production by Bacillus subtilis isolate BS5. Appl Biochem Biotechnol 150:305–325 albicans, P. aeruginosa,and S. aureus at tested concentra- Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NAH (2009) Char- tions. In addition, it was observed that the biosurfactant acterization of rhamnolipid produced by Pseudomonas aerugi- obtained showed no antimicrobial activity against V. vulnificus nosa Isolate Bs20. Appl Biochem Biotechnol 157:329–345 and V. cholerae and low activity against E. faecium, E. coli, L. 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Journal

Annals of Microbiology – Springer Journals

Published: Jan 8, 2013

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