Studium produkce polyhydroxybutyrátu u bakterií

Abstract

Predložená diplomová práca je zameraná na štúdium produkcie polyhydroxybutyrátu (PHB) u baktérií. Po stručnej charakteristike PHB ako najvýznamnejšieho predstaviteža širokej skupiny polyhydroxyalkanoátov (PHA) v teoretickej časti, bola prakticky sledovaná produkcia polyméru PHB, resp. kopolyméru P(HB-co-HV), u vybraných bakteriálnych kmeňov. V prvej časti bola študovaná produkcia PHB baktériou Bacillus megaterium na syntetickom médiu. Dosahované koncentrácie PHB v bunkách boli navýšené až prevedením kultúry do limitovaného média, na úkor rastu. Prídavok etanolu do média podporil nielen syntézu produktu, ale aj rast (21 % PHB z 1,8 g/l biomasy). Následne bolo otestované produkčné BM médium s prídavkom glukózy (8 g/l). Produkcia PHB bola viac ako 1 g/l pri výraznom náraste kultúry oproti syntetickému médiu. Baktéria B.megaterium okrem glukózy preukázala schopnosť utilizovať xylózu a maltózu. Ďalšie kultivácie boli prevedené s baktériou Azotobacter vinelandii syntetizujúcou kopolymér P(HB-co-HV). Maximum rastu a produkcie kopolyméru bolo dosiahnuté na Burkovom médiu s 30 g/l glukózy. Prídavok peroxidu do média vyvolal nárast v obsahu P(HB-co-HV) – 46 % kopolyméru z 2,6 g/l biomasy. Baktéria A.vinelandii najlepšie rástla na maltóze, dokonca aj v porovnaní s glukózou (54 % kopolyméru z obsahu biomasy). V závere bola sledovaná produkcia PHB s využitím priemyselného odpadného produktu – srvátky. Modifikácia srvátkového média bola vykonaná pomocou štatistickej optimalizačnej metódy Plackett Burman. Baktéria B.megaterium vyprodukovala na upravenej srvátke 0,5 g/l PHB, 32 % z biomasy.Presented work is focused on study of polyhydroxybutyrate production in bacteria. In theoretical part short characterization of PHB was given and the most common representative of wide group of polyhydroxyalkanoates (PHA) were described. Then, production of PHB and copolymer P(HB-co-HV) in selected bacterial strains was experimentally proven. First, PHB production in Bacillus megaterium using synthetic medium was studied. The PHB content in cells was increased during cultivation under limiting conditions, despite low growth. Addition of ethanol into production media resulted in increased PHB synthesis as well as biomass production (21 % PHB of 1,8 g/l biomass). Further, BM medium containing 8 g/l glucose was tested. PHB production was more than 1 g/l at significant growth increase when compared with synthetic medium. The bacteria B.megaterium showed, except glucose, ability to utilize maltose and xylose. Another cultivations were tested with bacterial strain Azotobacter vinelandii, which is capable of copolymer P(HB-co-HV) synthesis. Maximal growth and copolymer content was reached on Burk's medium with 30 g/l of glucose. Addition of peroxide to growth medium influenced P(HB-co-HV) synthesis to 46 % of 2,6 g/l biomass. Bacteria A.vinelandii showed the best growth on maltose, even compared with glucose (54 % copolymer of biomass content). Finally, PHB production on industrial waste product – whey was monitored. Using Plackett-Burman design for statistical media optimization, the whey content was modified. B.megaterium grown on adjusted whey reached 0,5 g/l PHB, 32 % of cell's content.