Production of Bio-ethanol from sugar beet pulp using recombinant E. coli and S. cereviceae

Ethanol is one of the most important biofuels that can be produced from different renewable sources. Sugar beet pulp (SBP) is used as renewable and cheap raw material for ethanol production. SBP is the by-product of the sugar industry from sugar beet that is used as animal feed after processing (pressing, dehydration, and pelletizing). Ethanol from SBP will be more profitable value than the other uses as animal feed.The two highest cellulases producer isolates S11 and S88 from the previous work were subjected to DNA identification using the 16S rRNA gene. 16S rRNA is tool used to identify the origin, classification, evolutionary and relationship history. The isolates S11 ( Streptomyces sp. strain FDZH12) and S88 ( Streptococcus mitis strain FDZH16) had been submitted to EMBL and their accession numbers are OK033363 and OK033364, respectively. Cellulase gene from S11 Streptomyces FDZH12 then cloned into E.coli to produce superior strain for cellulases production.The recombinant E. coli was confirmed by colony PCR using gene-specific primers of cellulases. Ethanol production from SBP is achieved through three steps: first, acid-base treatment for SBP and then the resulting cellulose content hydrolyzed to fermentable sugar using genetically engineered E.coli cloned by cellulases enzyme. Finally, the fermentable sugar is fermented to ethanol using S.cereviciae FDZH2O The weight of dried SBP after acid-base treatment was 45.5 % of the original dried SBP. Cellulose contents of untreated SBP were 27.95 % and reached 84.22 % after acid-base treatment (842.2g/kg). The maximum yields of glucose by the recombinant E.coli after 24 hours of saccharification of treated SBP were 28.36 g/50 g of acid base treated SBP (67.52% of their cellulose content). _______________________________________ Each 100 ml saccharified solution has 5.672 g glucose. After fermentation, each 100 ml saccharified solution has 2.83 ethanol (0.5008 g/g sugar 98% of the theoretical value). The maximum yield of ethanol by S. cerevisiae FDZH2O (equal to 14.20 g ethanol / 50 g of hydrolyzed SBP which have 42.11 g cellulose) and achieved at pH 6, 30 ºC, and 10% inoculum size after 72 hours of fermentation. According to the mass balance in our study each 6.557 kg, wet beet pulp with the moisture of 86% produces 1 kg dried SBP (DSBP) with moisture of 7.92% then after acid-base treatment produces 455 g treated DSBP that saccharified by recombinant E. coli into 258 g glucose and fermented finally by S. Cerevisiae into 129.24 g ethanol. This level is relatively low and more experiments are still needed to increase the productivity of this bioprocess.