Bacterial production of antimicrobial biosurfactants by Bacillus subtilis.

[摘要] ENGLISH ABSTRACT: Biosurfactants are microbially produced molecules that show excellent surface-activeproperties. Bacillus subtilis ATCC 21332 produces the biosurfactant, surfactin, which exhibitsantimicrobial activity against bacteria as well as fungi. Although antimicrobial activity has beenexhibited by a number of bacterially produced biosurfactants, notably the rhamnolipid from thepathogen Pseudomonas aeruginosa, the GRAS status B. subtilis makes the use of this organismpreferable for large scale bioprocesses.The objectives of this study were to: (1) evaluate the effect of different nutrient conditions ongrowth and surfactin production; (2) evaluate the growth of B. subtilis ATCC 21332 andassociated surfactin production on a hydrocarbon substrate; (3) evaluate the antimicrobialactivity of surfactin against Mycobacterium aurum, and (4) to establish whether active growthof B. subtilis ATCC 21332 and associated surfactin production can be extended during fed-batchculture.B. subtilis ATCC 21332 was grown on low-nitrate; phosphate-limited and nutrient rich mediawith glucose as substrate during shake flask culture. Nitrate, phosphate, glucose and surfactinwere quantified by HPLC analyses and growth via CDW and optical density measurements.Growth and surfactinproduction were further evaluated during shake flask cultureon ahydrocarbon substratereplacing the glucose in the nutrient rich medium with an equivalentamount of n-hexadecane. The antimicrobial activity was quantified by growth inhibition ofM. aurum.Bioreactor batch and fed-batch studies were conducted to evaluate growth and surfactinproduction under controlled conditions. The fed-batch experiments included four constantdilution rate (D=0.40h-1; D=0.15h-1; D=0.10h-1 and D=0.05h-1) and two constant feed rate(F=0.40L/h and F=0.125L/h) fed-batch strategies. The nutrient rich medium was used for theseexperiments and also as the feed medium for fed-batch experiments.A CDW of 12.6 g/L was achieved in the nutrient rich medium during shake flask culture and was2.5- and 1.6-fold higher than that achieved in the phosphate-limited medium and the lownitratemedium respectively. A surfactin concentration of 652 mg/L was achieved in thenutrient rich medium, while a maximum surfactin concentration of 730 mg/L was achieved inthe phosphate-limited medium. A surfactin concentration of only 172 mg/L was achieved in thelow-nitrate medium. Subsequently, growth and surfactin production were evaluated on n-hexadecane as sole carbon source. After inoculation, the CDW did not increase over a period of 119 h, which indicated that B. subtilis ATCC 21332 was unable to utilize n-hexadecane for growth and surfactin production.The maximum CDW (27 g/L) and maximum surfactin concentration (1737 mg/L) achieved in the bioreactor batch experiments were 2.1- and 2.6-fold higher respectively than that achieved in the nutrient rich medium during shake flask experiments. These results served as a benchmark for further fed-batch experiments. During the fed-batch phase of the D=0.40h-1 experiment, the biomass further increasedby 9 g/h, which was 3.5-, 3.1- and 5.3-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Similarly, the biomass increased by 10.7 g/h during the fed-batch phase of the F=0.40L/h experiment, which was 4.6-fold higher than that of the F=0.125L/h experiment. The average rate of surfactin production was 633 mg/h during the fed-batch phase of the D=0.40h-1 experiment, 29.4-, 5.4- and 34.2-fold higher compared to the fed-batch phases of the D=0.15h-1, D=0.10h-1 and D=0.05h-1 experiments respectively. Analogously, the average rate of surfactin production (544 mg/h) of the F=0.40L/h experiment was 9.4 fold higher than that of the F=0.125L/h experiment.The antimicrobial assay showed that surfactin inhibits M. aurum growth. An inhibition zone diamater of 4mm was measured at a surfactin concentration of 208 mg/L, which linearly increased to 24mm at a surfactin concentration of 1662 mg/L.High feed flow rate strategies achieved higher rates of biomass increase and surfactin production and will thus decrease the production time required for large scale surfactin production.The antimicrobial activity of surfactin against M. aurum indicates that this biosurfactant has the potential to be used against M. tuberculosis, and as such has the potential to be used in the medical industry to reduce the spread of this, and other deadly diseases.

[发布机构] Stellenbosch University