Investigation of bioethanol production from sugar beetmolasses by immobilised recombinant E.coli KO11

In this study, bioethanol production from sugar beet molasses as a carbon source has been investigated by using E. coli KO11 in continuous system. The studies have been conducted in three stages: hydrolysis, batch fermentation (with free and immobilized cells), and continuous (with immobilized cells). Sugar beet molasses is a by-product of the sugar industry with about 50 % sucrose in its content. Two acidic and one enzymatic hydrolysis methods were applied to convert glucose and fructose, which are the building blocks of sucrose existing in the structure. It has been determined that enzymatic hydrolysis is better than other hydrolysis methods used. Thus, the enzymatic hydrolysis conditions have been optimized by central composite design (CCD). The hydrolysis experiments have been carried out at 55 °C, in 50 ml working volume of 250 ml flasks, and at pH 4.5. Total 20 experiments have been designed by CCD. İnvertase enzyme has been used in the optimization experiments. The results of enzymatic hydrolysis have been determined as 87.8 g/L molasses concentration, 0.45 (%, v/v) enzyme concentration, and 15.93 h time. The stage of second, yield of bioethanol production have been investigated by using free and immobilization cells in a batch reactor. In free media, working volume of 50 ml has been used flasks with volume of 250 ml. As a results of experiments under conditions carried out incubation at 37 °C and 100 rpm, bioethanol was produced with a theoretical yield of 96 %. To obtain suitable bead structure for studies performed by immobilized microorganisms IX in the batch system, different alginate and CaCl2 quantities were tried. The best available bead structure has been obtained by using solution with alginate concentration of 3 % (w/v) and CaCl2 concentration of 5 % (w/v). Theoretical fermentation yield of 93% was achieved in the batch fermentation experiment using these beads. The final stage of this work, the continuous bioethanol production has been investigated by using immobilized E.coli KO11 strain obtained at the suitable immobilization conditions in the batch system. For this purpose, the continuous system column made of borosilicate glass has 3 mm of thick, 3 cm of inner diameter, and 20 cm of length. The continuous system column has a heating jacket from the outside. The working temperature of the column has been kept at 37 °C during the experiments. The pH of hydrolysates has been adjusted to 6, where E.coli KO11 can show activity. In continuous fermentation experiments, the effect of different dilution rates (column flow rate/column volume) on the bioethanol yield has been examined. 89 % of yield has been obtained from the experiments carried out at dilutin rate of 1.68 h-1. Compared with the theoretical yield of 93 % obtained by batch immobilization experiment results, it can be said that bioethanol is produced at high rate by continuous system. It is thought that the continuous system is clearly advantageous when the productivity of the continuous (36 hours) and continuous (60 hours) systems is compared while the maximum ethanol is produced in the continuous system. Keywords: Bioethanol, Sugar Beet Molasses, E.coli KO11, Optimization, Immobilized Cell Reactor.