Sophorolipids (SLs) are glycolipidic biosurfactants suitable for various biological and physicochemical applications. The nonedible Jatropha oil has been checked as the alternative raw material for SL synthesis using
In terms of production volume, surfactants belong to the most important classes of industrial chemicals with a current total world production exceeding 13 million tonnes per year [
Sophorolipids (SLs) are a kind of microbial extracellular biosurfactants produced by nonpathogenic yeasts, such as
Economy is often the bottleneck of biotechnological processes. Biosurfactants have to compete with surfactants of petrochemical origin in three aspects: cost, functionality, and production capacity (Makkar and Cameotra 2002). The success of biosurfactant production depends on the development of cheaper processes and the use of low cost raw materials, which account for 10–30% of the overall cost [
Here we have explored the utility of the nonedible Jatropha oil to be used as raw material for SL synthesis. The oil is nonedible owing to the presence of antinutritional factors such as phorbol esters [
Fatty acid composition of Jatropha oil (adapted from [
Fatty acid | Weight % in Jatropha oil |
---|---|
Palmitic acid (C16:0) | 16.69 |
Stearic acid (C18:0) | 7.67 |
Oleic acid (C18:1) | 40.39 |
Linoleic acid (C18:2) | 33.09 |
Linolenic acid (C18:3) | 0.28 |
Owing to the amphiphilic nature of SLs, their applicability as an ingredient of laundry detergent has been identified previously [
Nonpathogenic yeast,
All chemicals and solvents used in this study were of analytical grade and supplied by either Himedia Pvt. Ltd., India, or Merck India Ltd. Jatropha oil was purchased from local market in Pune, India, in a single batch.
In addition to primary and secondary carbon sources, media contain nitrogen source, growth factors, buffer components, and other minerals which show significant effect on SL yields. Different media previously reported for maximum production of SL were tried. The media compositions have been listed in Table
Compositions of different media used during SL yield maximization experiments.
Medium A [ |
Medium B [ |
Medium C [ |
Medium D [ |
Medium E [ |
Medium F [ | |
---|---|---|---|---|---|---|
Glucose | 100 g/L | 100 g/L | 100 g/L | 150 g/L | 100 g/L | 50 g/L |
Yeast extract | 5 g/L | 1 g/L | 1 g/L | 4 g/L | — | 3 g/L |
Peptone | — | — | — | — | 5 g/L | 5 g/L |
Magnesium sulphate | 5 g/L | 0.3 g/L | 0.7 g/L | 0.3 g/L | 5 g/L | — |
Dipotassium hydrogen phosphate | — | — | 0.16 g/L | — | — | — |
Potassium dihydrogen phosphate | 1 g/L | — | 1 g/L | 6 g/L | 1 g/L | — |
Disodium hydrogen phosphate | — | 2 g/L | — | 2 g/L | — | — |
Sodium dihydrogen phosphate | — | 7 g/L | — | — | — | — |
Sodium citrate | — | — | 5 g/L | — | — | — |
Sodium chloride | 0.1 g/L | — | 0.5 g/L | — | 0.1 g/L | — |
Ammonium sulphate | — | 1 g/L | — | — | — | — |
Ammonium nitrate | 0.05 mol | — | — | — | 0.05 mol | — |
Ammonium chloride | — | — | 1.5 g/L | — | — | — |
Urea | — | — | — | 2 g/L | — | — |
Calcium chloride | 0.1 g/L | — | 0.27 g/L | — | 0.1 g/L | |
Malt Extract | — | — | — | — | — | 3 g/L |
Seed culture was prepared by inoculating 5 mL of respective media with
The SL was harvested by the procedure previously reported by Shah and Prabhune 2007. Culture medium was centrifuged at 5,000 rpm, at 10°C for 20 minutes. The cell pellet was washed with ethyl acetate to recover the SLs precipitated during centrifugation. The supernatant was extracted twice with equal volumes of ethyl acetate, organic layer was dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The yellowish brown semicrystalline product was washed twice with n-hexane and yields were determined [
The medium and incubation period giving maximum yield were fixed and the glucose concentrations were varied, namely, 5, 7 and 10% w/v. In case of secondary carbon source, oil feeding was varied within the range 1 to 5% v/v.
Optimal temperature for
The fermentation, extraction procedures were essentially done the same as mentioned before. The experiments were carried out in triplicates in 250 mL Erlenmeyer flasks containing 50 mL of the production media.
SL is known to be the stationery phase metabolite. Hence in order to reduce the carbon consumption for biomass increase and cell maintenance purpose and direct it majorly towards SL production, the cells were pregrown in optimum medium, that is, medium F which also gave maximum biomass production and then subjected to the SL production medium containing the precursors for SL production, that is, glucose and Jatropha oil as per the optimized conditions.
Seed culture of
After the yield maximization experiments, the MALDI-MS (matrix assisted laser desorption/ionization-mass spectrometry), NMR analysis was performed to know about structural composition of the SL sample. Further the surface active properties of SLJO were evaluated.
Minimum surface tension and critical micelle concentration of SLJO were estimated using a KRUSS surface tensiometer K11 by Wilhelmy plate method.
Stock of SLJO was prepared in MilliQ water (pH = 7.0) and diluted appropriately to get different concentrations. The concentration range used was 0.95–850 mg/L. A clean, dry 100 mL glass beaker was filled with the different concentrations of SLJO solution and subjected to surface tension measurement one by one. The beaker was placed on the sample platform of the Kruss K11 tensiometer. The platinum surface tension probe was removed from the tensiometer hook and rinsed with deionized water and dried with the blue part of the flame from the propane torch. The probe was then air-cooled and reinserted onto the tensiometer hook. The surface tensions of the solutions of different concentrations were measured as described in the tensiometer operating manual. All surface tension measurements were the average of 4 readings recorded at an interval of 30 seconds.
To determine critical micelle concentration (CMC), the surface tension was measured as a function of surfactant concentration. Surface tension was then plotted versus log surfactant concentration. The resulting curve had a nearly horizontal portion at concentrations higher than the CMC and had a negative steep slope at concentrations less than the CMC. The CMC was calculated as that concentration of the curve where the flat portion and the extrapolated steep slope intersected. The surface tension beyond CMC was the value in the flat portion of the curve.
Emulsification activity and stability of the SLJO were tested with oleic acid as an organic solvent using a modified method of Cirigliano and Carman [
The effect of environmental parameters such as water hardness, pH, and temperature on emulsification activity was determined by varying the levels of the individual parameters one at a time by keeping the other parameters at a fixed level. The modified method of Daverey and Pakshirajan 2010 has been used [
To test the effect of hardness on emulsification activity and stability of SLJO, 1 mL SL solution (0.5 mg/mL) was prepared in moderately hard water and hard water and incubated for 1 hour at 30°C and then emulsification activity and stability were assessed according to the procedure mentioned earlier.
Protocol was followed to check the bacterial inhibition by SLJO. Appropriate dilution of bacterial cell suspension was exposed to different SL concentrations (50–500
In view of their intended use as detergent additive, wetting property and contact angle reduction were examined.
Contact angle measurements were performed using Goniometer: G-10 contact angle meter. SL stock solution of 1
In detergency test, the comparative performance of SLJO, a commercial detergent preparation, and SLJO in combination with commercial detergent has been evaluated against 4 different stains, namely, coffee, turmeric, oil, and poster color, on 2 different types of fabrics, namely, cotton and polyester.
Following method was practiced. Pieces of 2 × 2 inches were cut of cotton and polyester cloth. Cloth pieces were placed on saran wrap and stained, another piece of saran wrap was placed on it, and heavy weight was put on it for 5–10 minutes and then stains were allowed to dry overnight. Next day, stained pieces of cloth were soaked individually in 0.1 g% solution of SLJO and 0.1 g% solution of commercial detergent for 10 minutes. Soaked pieces of cloth were hand-washed for approximately 1-2 minutes. Excess water from cloth was squeezed out and cloth pieces were allowed to dry normally and results were visually noted [
Same procedure was carried out to test the stain removal capacity of SL in combination with commercial detergent, in 1 : 1 proportion with appropriate controls.
In order to maximize the product yield, 6 media differing in the proportion of sugar, nitrogen source, presence of buffer components, and so forth were chosen. For media compositions, refer to Section
SL yield has been improved through the use of optimized parameters combined with resting cell method. SL production is associated with stationary phase of growth. Therefore the cells which were already used for synthesizing SLs could give satisfactory yields when supplemented with just glucose and Jatropha oil. 15.25 g/L of SL derived from Jatropha oil could be obtained with 1% v/v oil feeding. Resting cell method allowed the use of same biomass for up to 3 times, thus making the process still more efficient. With the second and third time use of the cells, 15.1 g/L and 13.25 g/L of SL yield were obtained, respectively. After that SL production dropped considerably.
Typical structure of SLs consists of a sophorose (dimeric sugar) linked
Comparative data on structural composition of SLJO.
SL structural forms | Mol. Wt. |
|
SLJO | |
---|---|---|---|---|
Relative abundance | Approximate % composition | |||
Nonacetylated SL of C18:0, acidic form | 623 | 647 | 1.86 | 0.67 |
Monoacetylated SL of C18:1, lactonic form | 645 | 669 | 6.75 | 2.45 |
Diacetylated SL of C16:0, lactonic form | 661 | 685 | 29.1 | 10.54 |
Monoacetylated SL of C18:1, acidic form | 663 | 687 | 6.41 | 2.32 |
Diacetylated SL of C16:0, acidic form | 679 | 703 | 8.47 | 3.07 |
Diacetylated SL of C18:2, lactonic form | 685 | 709 | 9.04 | 3.27 |
Diacetylated SL of C18:1, lactonic form | 687 | 711 | 100 | 36.22 |
Diacetylated SL of C18:0, lactonic form | 689 | 713 | 46.95 | 17.00 |
Monoacetylated SL of C20:0, acidic form | 692 | 716 | 1.19 | 0.43 |
Diacetylated SL of C18:2, acidic form | 703 | 727 | 15.21 | 5.51 |
Diacetylated SL of C18:1, acidic form | 705 | 729 | 46.41 | 16.81 |
Diacetylated SL of 20:0, acidic form | 735 | 759 | 4.73 | 1.71 |
MALDI/MS spectrum of the SLJO preparation.
The 1H NMR spectrum of the SLJO preparation was assigned to a typical glycolipid-type structure. And characteristic proton chemical shift peaks could be observed. Protons of (–CH3) of fatty acid resonated at 1.20. Protons of (–CH2) bonded to carboxylic group of fatty acid resonated at 1.99. Resonance of protons belonging to sophorose moiety resulted in peaks within the region 4–4.5. Appearance of peaks around 5.3 was attributed to the signals from (–CH=CH–), that is, unsaturation in the fatty acid chain. The data was found to be in agreement with previously reported SL-NMR data from relevant references [
Before proceeding to further analysis, surfactant property of the synthesized SL was qualitatively confirmed by oil displacement test. The surface active properties were checked using different experimental and analytical methods.
It was seen that SLJO reduced the surface tension of distilled water from 70.714 mN/m to 33.512 mN/m at the CMC value of 9.5 mg/L as depicted in Figure
Minimum surface tension and critical micelle concentration of SLJO.
The emulsification activity and stability of SLJO and synthetic surfactants, namely, SDS and Triton X-100, have been noted in Table
Emulsification activity and stability of SLJO and synthetic surfactants.
Emulsification activity ( |
Decay constant ( |
|
---|---|---|
SLJO | 1.9725 |
|
Triton X-100 | 0.789 |
|
SDS | 2.250 |
|
As an ingredient of detergent, SLJO has to perform satisfactorily in extreme physical conditions and different water qualities. Therefore the effect of different parameters on emulsifying property was explored.
Effect of water hardness on emulsifying property and stability of SLJO and Triton X-100.
Hardness | SLJO | Triton X-100 | ||
---|---|---|---|---|
Emulsification activity ( |
Decay constant ( |
Emulsification activity ( |
Decay constant ( |
|
Distilled water | 1.9725 |
|
0.789 |
|
Moderately hard water | 1.846 |
|
1.0665 |
|
Hard water | 0.779 |
|
1.531 |
|
The experiment was repeated twice and the emulsification indices were calculated from the average
It was observed that emulsions formed by SLJO were stable within the pH range of 5.0–8.0. The decay constant of SLJO is −11.3184 at pH 4.0 (refer to supplementary information for details). The results are in agreement with those of the report by Daverey and Pakshirajan, 2009, wherein they have used sugarcane molasses and soybean oil for SL synthesis and found that emulsifying activity was maximum at pH 7.0 and highest stability at pH 8.0 [
Effect of temperature on emulsification activity and stability of SLJO.
Temperature (°C) | Emulsification activity ( |
Decay constant ( |
---|---|---|
20 | 1.902 |
|
40 | 2.010 |
|
60 | 2.249 |
|
80 | 1.807 |
|
SLs are known to possess antimicrobial properties [
The minimum inhibitory concentration required to inhibit 90% of the organisms;S that is, MIC90 values were determined. Against
Antibacterial action of SLJO (a) against
The contact angle value is dictated by the interaction between surfactant molecule and the solid surface. SLJO was able to improve spreading and reduce the contact angle. SLJO brought down the value on teflon (95° to 56°) and stainless steel (85° to 42°). No much change in contact angle was seen in case of glass surface. Thus water droplets will spread evenly on teflon and stainless steel to give low
Dose dependent wetting performance of SL was assessed using canvas disc method. At 0.01% concentration of SL, 8.8 minutes were required for sinking, while at 1 g% concentration sinking time decreased to 1.15 minutes. In the first phase of washing, textile fibres and soil must be wetted as thoroughly as possible by the wash liquor. Wetting is a complex process which is determined by the interaction of the different interfacial tensions between the solid surface, the liquid, and the gas phase. A contact angle
Sinking time required in case of combinations of SLJO with SDS and Triton X-100 was less than the time required for individual SLJO, SDS, and Triton X-100 (refer to Figure
Effect of SLJO addition to improve wetting property of synthetic surfactants. Wetting improved with increasing proportion of SLJO.
Figure
Detergency test results-cleaning performances of SLJO and commercial detergent and their combination against coffee stain. (a) Coffee stained fabrics (b) washed with commercial detergent, (c) washed with SLJO, and (d) washed with SLJO and commercial detergent 1 : 1 (e) unstained fabric.
Similar results were observed in case of remaining 3 types of stains, turmeric, oil, and poster color (refer to electronic supplementary material). In cases where total cleaning was not achieved with the prescribed washing protocol, visibly SLJO performance almost matched that of the commercial detergent. And the combination SLJO-detergent worked best.
In the detergency test, stains differing in their chemical nature have been used which are considered to be notorious such as caffeic acid, a yellow solid containing phenolic, acrylic group in coffee stain, and curcuminoids in turmeric. Conventionally bleach or acids are used for these kinds of tough stains which damage the fabric; on the other hand, SLs are skin friendly.
Hence the results can be summed up as there is an indication that, for majority of stains, SLJO can work as good as detergent. And through standardization of the washing procedures, there can be improvement. When combined with detergent, SLJO enhances their action. This way, reduction in the detergent load to half is really attractive and will have big positive impact.
In the present paper SL, a type of biosurfactants, has been produced using nonedible Jatropha oil derived from seeds of
The SLJO was found to work at low CMC value, that is, 9.5 mg/L. Other desirable properties of surfactants to work as good detergent such as wetting property, contact angle reduction, antibacterial action, and so forth have also been confirmed with the SLJO. Emulsification property of SLJO has been evaluated with special reference to changes in environmental parameters pertaining to different water qualities. Also we are reporting the use of SL for cleaning the fabric stains in comparison with commercial detergent formulation. SL acts as efficient stain cleaner. When used with detergent, it showed improved performance, thus reducing the load and harm caused to environment. This is specifically advantageous as the half-life of detergent can be up to 16 days which is detrimental to aquatic life and badly affects the ecological balance of water bodies. On the contrary, the biosurfactant SLs are biodegradable, ecofriendly, and nontoxic.
Sophorolipid
Jatropha oil derived sophorolipid
Sodium dodecyl sulphate
Critical micelle concentration.
The authors have declared no conflict of interests.
Kasturi Joshi-Navare would like to thank University Grants Commission for the fellowship. The assistance with surface tension determination and contact angle reduction experiments at Institute of Chemical Technology, Matunga, Mumbai, is gratefully acknowledged. They thank CMC Division, NCL, for providing MALDI-MS facility.