Bacterial production of antimicrobial biosurfactants by Bacillus subtilis.

Bence, Keenan (2011-12)

Thesis (MScEng)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: Biosurfactants are microbially produced molecules that show excellent surface-active properties. Bacillus subtilis ATCC 21332 produces the biosurfactant, surfactin, which exhibits antimicrobial activity against bacteria as well as fungi. Although antimicrobial activity has been exhibited by a number of bacterially produced biosurfactants, notably the rhamnolipid from the pathogen Pseudomonas aeruginosa, the GRAS status B. subtilis makes the use of this organism preferable for large scale bioprocesses. The objectives of this study were to: (1) evaluate the effect of different nutrient conditions on growth and surfactin production; (2) evaluate the growth of B. subtilis ATCC 21332 and associated surfactin production on a hydrocarbon substrate; (3) evaluate the antimicrobial activity of surfactin against Mycobacterium aurum, and (4) to establish whether active growth of B. subtilis ATCC 21332 and associated surfactin production can be extended during fed-batch culture. B. subtilis ATCC 21332 was grown on low-nitrate; phosphate-limited and nutrient rich media with glucose as substrate during shake flask culture. Nitrate, phosphate, glucose and surfactin were quantified by HPLC analyses and growth via CDW and optical density measurements. Growth and surfactinproduction were further evaluated during shake flask cultureon a hydrocarbon substratereplacing the glucose in the nutrient rich medium with an equivalent amount of n-hexadecane. The antimicrobial activity was quantified by growth inhibition of M. aurum. Bioreactor batch and fed-batch studies were conducted to evaluate growth and surfactin production under controlled conditions. The fed-batch experiments included four constant dilution 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 these experiments 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 was 2.5- and 1.6-fold higher than that achieved in the phosphate-limited medium and the lownitrate medium respectively. A surfactin concentration of 652 mg/L was achieved in the nutrient rich medium, while a maximum surfactin concentration of 730 mg/L was achieved in the phosphate-limited medium. A surfactin concentration of only 172 mg/L was achieved in the low-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.

AFRIKAANSE OPSOMMING: Biosurfaktante is oppervlak-aktiewe molekules wat deur sekere mikro-organismes geproduseer word. Bacillus subtilis ATCC 21332produseer ‘n biosurfaktant genaamd surfactin, wat antimikrobiese eienskappe toon teen bakterieë sowel as fungi.Menige bakterieël geproduseerde biosurfaktante toon antimikrobiese eienskappe, vernaam die rhamnolipied van die patogeen Pseudomonas aeruginosa, maar die algemene veiligheids-status van B. subtilis gee voorkeur aan hierdie organisme vir grootskaalse bioprosesse. Die doelwitte van hierdie studie was: (1) om die effek van verskillende medium samestellings (in terme van voedingstowwe) ten opsigte van bakteriële seldigtheid en surfactin-produksie te evalueer; (2) om die bakteriële seldigtheid van B. subtilis ATCC 21332 en geassosieerde surfactin produksie vanaf ‘n alkaan-substraat te evalueer; (3) om die antimikrobiese aktiwiteit van surfactin teen Mycobacterium aurum te evalueer; (4) om vas te stel of die aktiewe groei van B. subtilis ATCC 21332 en geassosieerde surfactin-produksie gedurende voer-lot kultuur verleng kan word. B. subtilis ATCC 21332 was op lae-nitraat; fosfaat-beperkte en voedingstofryk-media met glukose as substraat in skudflesse gekultiveer. Nitraat, fosfaat, glukose en surfactin was deur hoëdruk vloeistofchromatografie gekwantifiseer en die seldigtheid deur middel van seldroëmassa en optiese digtheid metings. Verder was die groei van B. subtilis, en geassosieerde surfactin produksie, vanaf ‘n alkaan-substraat in skudflesse ge-evalueer deur die glukose in die voedingstofryke medium met ‘n ekwivalente hoeveelheid van n-heksadekaan te vervang. Die antimikrobiese aktiwiteit van surfactin was deur die geїnhibeerde groei van M. aurum gekwantifiseer. Bioreaktor lot en voer-lot studies was uitgevoer om die groei en surfactin produksie onder beheerde toestande te evalueer. Die voer-lot eksperimente het vier konstante verdunningstempos (D=0.40h-1; D=0.15h-1; D=0.10h-1 en D=0.05h-1) en twee konstante voertempos (F=0.40L/h and F=0.125L/h) ingesluit. Die voedingstofryke medium was vir hierdie eksperimente en ook as die voermedium vir dievoer-lot eksperimente gebruik. ‘n Seldigtheid van 12.6 g/L is bereik gedurende skudfleskultuur in die voedingstofryk-media en was 2.5- en 1.6-voud hoër as die seldigthede wat in die fosfaat-beperkte en lae-nitraat media bereik is. ‘n Surfactin konsentrasie van 652 mg/L is bereik in die voedingstofryke medium, terwyl ‘n maksimum surfactin konsentrasie van 730 mg/L in die fosfaat-beperkte medium bereik is. ‘n Surfactin konsentrasie van slegs 172 mg/L is in die lae-nitraat medium bereik.Hierna was bakteriële seldigtheid en surfactin produksie geuvalueer met slegs n-heksadekaan as die enigste koolstof bron. Die bakteriële seldigtheid het geen verandering getoon na inokulasie nie, wat aangedui het dat B. subtilis ATCC 21332 nie die vermoë beskik om n-heksadekaan vir groei en surfactin produksie te gebruik nie. Die maksimum seldigtheid (27 g/L) en maksimum surfactin konsentrasie (1737 mg/L) bereik in die bioreaktor lot eksperimente was 2.1- en 2.6-voud hoër onderskeidelik as dit bereik in die voedingstofryke medium gedurende skudfles eksperimente. Hierdie resultate dien as ‘n basis vir verdere voer-lot eksperimente. Gedurende die voer-lot fase van die D=0.40h-1 het die biomassa verder verhoog teen 9 g/h, wat 3.5-, 3.1- en 5.3-voud hoër was as dit van die D=0.15h-1, D=0.10h-1 en D=0.05h-1 eksperimente onderskeidelik. Die biomassa het soortgelyk tydens die voer-lot fase van die F=0.40L/h eksperiment teen 10.7 g/h verhoog, wat 4.6-voud hoër was as dit van die F=0.125L/h eksperiment. Die gemiddelde tempo van surfactin produksie was 633 mg/h gedurende die voer-lot fase van die D=0.40h-1 eksperiment, 29.4-, 5.4- en 34.2-voud hoër vergeleke met die voer-lot fases van die D=0.15h-1, D=0.10h-1en D=0.05h-1 eksperimente onderskeidelik. Die gemiddelde tempo van surfactin produksie (544 mg/L) was soortgelyk 9.4-voud hoër gedurende die voer-lot fase van die F=0.40L/h eksperimente, vergeleke met die die F=0.125L/h eksperiment. Die antimikrobiese toetse van surfactin teen M. aurum het positief getoets, wat aandui dat surfactin die groei van hierdie organisme inhibeer. ‘n Inhibisie sone deursnee van 4mm was gemeet teen ‘n surfactin konsentrasie van 208 mg/L, wat lineêr verhoog het tot 24 mm teen ‘n surfactin konsentrasie van 1662 mg/L. Hoë voertempo strategieë het hoër biomassa verhogingstempos en surfactin produksie tempos getoon en sal dus die produksietyd aansienlik verkort tydens grootskaalse surfactin produksie. Die antimikrobiese aktiwiteit van surfactin teen M. aurum toon dat hierdie biosurfaktant die vermoë het om gebruik te word teen M. tuberculosis. Daarom het surfactin die potensiaal om gebruik te word in die mediese industrie om die verspreiding van Tuberkulose, en ander dodelike patogene, te voorkom.

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