Biotechnological Approaches for Enhanced Production of Biosurfactantants by Bacillus Subtilis SNW3

Abstract

Biological surface-active agents or “biosurfactants” are the compounds that can reduce the surface or interfacial tension between two same or different phases (liquid, gas and solid). The present study relates to the screening of biosurfactant-producing bacteria isolated from the Fimkassar oil field, Chakwal, Pakistan. The molecular screening for two important genes srfA and rhlB responsible for production of surfactin and rhamnolipid biosurfactants, respectively and biosurfactant production by using different growth substrates. In total, 38 out of 70 different bacterial isolates showing growth on crude-oil-containing media were screened for biosurfactant production. Evidently, 34.2% (n = 13) of the isolates were found to have the srfA gene, while 15.8% (n = 6) of the isolates contained the rhlB gene. Subsequently, 16S ribosomal RNA sequence homology studies confirmed the gene-positive isolates to be the species of genera Bacillus, Brevundimonas, Alcaligenes, Pseudomonas, Serratia, Proteus and Stenotrophomonas. The Presence of the srfA gene in Brevundimonas spp. and the rhlB gene in Alcaligenes faecalis involved in biosurfactant (surfactin and rhamnolipid) production, and the similarly unusual presence of both genes in Stenotrophomonas rhizophilia and Alcaligenes faecalis indicates the possibility of horizontal gene transfer and retention or presence of gene orthologs. All the genepositive isolates showed biosurfactant production under submerged fermentative conditions. Maximum production in terms of biosurfactant activities (E24 59.5± 4.0%; SFT 27.2 ± 1.1 mN/m; ODA 3.5 ± 0.2 cm) was revealed by Bacillus subtilis strain SNW3 (SWW1). Surfactin nature of biosurfactant produced was confirmed by thin-layer chromatography, Fourier transform infrared spectroscopy and LC-MS. In this study, a 2-level factorial model, Plackett-Burman design, was used to screen eleven different carbon sources affecting biosurfactant production by Bacillus subtilis SNW3. From these carbon sources, four were selected from the Plackett-Burman design on the basis of maximum reduction of surface tension of culture broth and emulsification index. These included molasses, pulses, red beans and potato peels. Further they were used in various combinations to check their combined effect with different inducers such as urea, yeast extract and amino acids. Analyzing all combinations on the basis of ODA, E24 and SFT, it was found that yeast extract could be replaced with red bean, potato starch and urea in combination as cheap carbon and nitrogen sources for the biosurfactant (surfactin and fengycin) production by Bacillus xii

subtilis SNW3. Lowering the C:N ratio by providing nitrogen by addition of red bean and urea has a profound effect on biosurfactant production especially using RB+PS+U (6+0.5+0.4%) in the medium resulting in 1.2 g/L surfactin and 300 mg/L fengycin. Optimization studies of temperature, agitation speed, inoculum size and age of culture revealed maximum production of surfactin (1.37 g/L) and fengycin (700 mg/L) at 23 °C (room temperature), 120 rpm, 2 % inoculum of 36 hours old culture by using the combination RB+PS+U (6+0.5+0.4%). Heat treatment (autoclavation) was found to have a positive effect on extraction of amino acids and sugars that led to a higher amount of surfactin and fengycin production as compared to the extract of red bean that was prepared directly. Red bean extract (prepared by autoclavation) produced 792 mg/L surfactin and 546 mg/L fengycin, while 329 mg/L surfactin and 197 mg/L fengycin was produced by red bean extract. Batch experiments were performed in a 13-L bioreactor. Maximum production of surfactin 1512 mg/L and 1236 mg/L fengycin (surfactin +fengycin) (named VITO Surf) was observed at the 7th day of incubation by Bacillus subtilis SNW3 at 23 °C pH 6.8 and 120 rpm. Biosurfactant production was found to be improved by using mutant M-20 and M-40 (Mutagenesis was performed by UV treatment) with reduced incubation time. LC/MS showed very interesting results that M-20 produced 1000 mg/L surfactin and M-40 produced 824 mg/L surfactin after 26 hours of incubation and immediately the concentration of surfactin decreased while the parent strain could produce about 300-400 mg/L at the same time of incubation. Similarly both mutants produced only surfactin. Providing an increased amount of red bean powder (100 mg/L) in the culture medium of both mutants, M-20 and M-40, resulted in an increased amount (1507 mg/L) of surfactin. Fed batch fermentation was performed to check addition of red bean powder and urea during fermentation using the mutants. At the 20th day of experiment addition of red bean powder and urea in the culture broth of M-20 and M-40 resulted in further production of surfactin. Downstream processing was performed by two methods in the current study. Using a two-step recovery process (evaporation and precipitation) resulted in 70.6% recovery of surfactin and 79.5% fengycin at a large scale volume using ethanol for extraction. While using another two-step recovery process (centrifugation and precipitation), % recovery of surfactin and fengycin was 70.2 % and 72.1 % respectively.