Optimization of levan production from bacterium Bacillus licheniformis and its application in copolymer synthesis.

U okviru ove doktorske disertacije proučavan je uticaj različitih uslova fermentacijena produkciju levana bakterije Bacillus lichenifomis NS032, ispitivana struktura dobijenogpolisaharida i sintetisani kalemljeni kopolimeri levana i polistirena (Lev-g-PS).U prvoj seriji eksperimenata optimizovane su podloge za produkciju levanatehnikom jedne promenljive u vremenu i metodom površine odgovora. Najviši prinosipolisaharida se dobijaju na temperaturi od 37 °C i bez mešanja. Upotrebom podloge sa196,75 g/L saharoze, 2,44 g/L amonijum-hlorida i inicijalnog pH od 6,97 dobija se 47,84g/L levana LS200, a na podlozi sa 397,65 g/L saharoze, 4,62 g/L amonijum-hlorida iinicijalnog pH od 7,31 dobija se 99,19 g/L levana LS400. U sledećim uslovima: 62,6 %melase, 4,66 g/L fosfata i inicijalnim pH od 7,2 dobija se 53,6 g/L levana LM.Strukturna ispitivanja (kisela hidroliza, metilaciona analiza, FTIR, 13C- i 1H-NMR,COSY, HSQC i HMBC) su pokazala da su dobijeni levani polifruktani sa β-(2,6)-glikozidnom vezom u osnovnom nizu i grananjem u položaju β-(2,1). Stepeni grananja su11,04 ± 0,57 %, 11,99 ± 0,63 % 12,68 ± 0,71 % za levane LS200, LS400 i LMrespektivno. Levani LS200 i LM relativno su visoke molekulske mase (opseg > 106 Da) saviskozitetima 0,25 dL/g i 0,24 dL/g, dok je levan LS400 niže molekulske mase (105 Da) iviskoziteta 0,14 dL/g.U poslednjoj seriji eksperimenata ispitivan je uticaj različitih reakcionih uslova nasintezu Lev-g-PS slobodnoradikalskim mehanizmom i upotrebom kalijum-persulfata kaoinicijatora. Maksimalni G (%) = 58,1 dobijen je na 70 °C, u atmosferi azota i upotrebomaminoaktivatora DMEA. FTIR, SEM, TG/DTA, XRD i 13C NMR spektroskopija dobijenogkopolimera su pokazali da je došlo do reakcije kalemljenja između levana i polistirena.Within this doctoral dissertation, the effects of various fermentation factors on levanproduction by Bacillus lichenifomis NS032 were studied, structure of obtainedpolysaccharide was investigated and grafted copolymers of levan and polystyrene (Lev-g-PS) were synthetized.In the first series of experiments, media for levan production were optimized usingone factor at a time and response surface methods. The highest levan yield was obtained at37 °C and without agitation. Using the medium with 196.75 g/L sucrose, 2.44 g/Lammonium chloride and pH 6.97, obtained amount of LS200 levan was 47.84 g/L and inmedium with 397.65 g/L sucrose, 4.62 g/L ammonium chloride and pH 7.31 amount ofLS400 levan was 99.19 g/L. In the following conditions: 62.6 % molasses, 4.66 g/Lphosphate and an initial pH 7.2, LM levan (53.6 g/L) was obtained.Structural characterization (acid hydrolisis, methylation analysis, FTIR, 13C- and1H-NMR, COSY, HSQC i HMBC) showed that investigated levans are polyfructans with β-(2,6)- glycosidic linkages in backbone and branching through β-(2,1) positions. The extentsof branching are 11.04 ± 0.57 %, 11.99 ± 0.63 % 12.68 ± 0.71 % for LS200, LS400 andLM levans, respectively. LS200 and LM have relatively high molecular weight (range >106 Da) and viscosity (0.25 dL/g and 0.24 dL/g), contrary to LS400 with lower molecularweight (105 Da) and viscosity (0.14 dL/g).Finally, influence of different reaction conditions on synthesis of Lev-g-PS by freeradical mechanism and potassium persulfate as initiator was investigated. Maximum G (%)= 58.1 was obtained at 70 °C, in nitrogen atmosphere and using the amine activator DMEA.FTIR, SEM, TG/DTA, XRD and 13C NMR spectroscopy of copolymer showed thatgrafting reaction of levan and polystyrene was successful