Optimizacija produkcije levana bakterije Bacillus licheniformis i primena u sintezi kopolimera

Abstract

U okviru ove doktorske disertacije proučavan je uticaj različitih uslova fermentacije na produkciju levana bakterije Bacillus lichenifomis NS032, ispitivana struktura dobijenog polisaharida i sintetisani kalemljeni kopolimeri levana i polistirena (Lev-g-PS). U prvoj seriji eksperimenata optimizovane su podloge za produkciju levana tehnikom jedne promenljive u vremenu i metodom površine odgovora. Najviši prinosi polisaharida se dobijaju na temperaturi od 37 °C i bez mešanja. Upotrebom podloge sa 196,75 g/L saharoze, 2,44 g/L amonijum-hlorida i inicijalnog pH od 6,97 dobija se 47,84 g/L levana LS200, a na podlozi sa 397,65 g/L saharoze, 4,62 g/L amonijum-hlorida i inicijalnog 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 su 11,04 ± 0,57 %, 11,99 ± 0,63 % 12,68 ± 0,71 % za levane LS200, LS400 i LM respektivno. Levani LS200 i LM relativno su visoke molekulske mase (opseg > 106 Da) sa viskozitetima 0,25 dL/g i 0,24 dL/g, dok je levan LS400 niže molekulske mase (105 Da) i viskoziteta 0,14 dL/g. U poslednjoj seriji eksperimenata ispitivan je uticaj različitih reakcionih uslova na sintezu Lev-g-PS slobodnoradikalskim mehanizmom i upotrebom kalijum-persulfata kao inicijatora. Maksimalni G (%) = 58,1 dobijen je na 70 °C, u atmosferi azota i upotrebom aminoaktivatora DMEA. FTIR, SEM, TG/DTA, XRD i 13C NMR spektroskopija dobijenog kopolimera su pokazali da je došlo do reakcije kalemljenja između levana i polistirena.

Within this doctoral dissertation, the effects of various fermentation factors on levan production by Bacillus lichenifomis NS032 were studied, structure of obtained polysaccharide 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 using one factor at a time and response surface methods. The highest levan yield was obtained at 37 °C and without agitation. Using the medium with 196.75 g/L sucrose, 2.44 g/L ammonium chloride and pH 6.97, obtained amount of LS200 levan was 47.84 g/L and in medium with 397.65 g/L sucrose, 4.62 g/L ammonium chloride and pH 7.31 amount of LS400 levan was 99.19 g/L. In the following conditions: 62.6 % molasses, 4.66 g/L phosphate and an initial pH 7.2, LM levan (53.6 g/L) was obtained. Structural characterization (acid hydrolisis, methylation analysis, FTIR, 13C- and 1H-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 extents of branching are 11.04 ± 0.57 %, 11.99 ± 0.63 % 12.68 ± 0.71 % for LS200, LS400 and LM 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 molecular weight (105 Da) and viscosity (0.14 dL/g). Finally, influence of different reaction conditions on synthesis of Lev-g-PS by free radical 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 that grafting reaction of levan and polystyrene was successful.