Topic : Bio-Ethanol Advisor : Prof. Jo-Shu Chang NURHAYATI / 林海亞 N PAPER REVIEW

Due to date : 09 November 2012 Title: Producing bioethanol from cellulosic hydrolyzate via co-immobilized cultivation strategy Authors: Yu-Kuo Liu, Chih-An Yang, Wei-Chuan Chen, Yu-Hong Wei Journal: Bioscience and Bioengineering Year: 2012 Impact Factor: 1.793

Discussion The aim of this study is to develop and establish a new and highly effective co-immobilization system that uses co-cultivated Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis for direct conversion of agricultural wastes into bioethanol. To enable simultaneous saccharification and fermentation of this system, a modified bioreactor was applied. This figure exhibits the schematic diagram of modified bioreactor used in this study. Microorganisms and Media The T. reesei and A. niger stock culture was incubated at 30 o C for 7 days on potato dextrose agar (PDA) and then stored at 4 o C. While the Z. mobilis stock culture was grown on yeast extract (5.0 g/L), reducing sugars (20.0 g/L) and agar (20.0 g/L) at pH 6.8 and incubated at 30 o C for 2 days. Also, to cultivate these three strains, BHSE medium containing 5 g/L CMC was utilized. Cell Immobilization  Using Na-alginate 4.0%w/v, CaCl 2 3.0%, phosphate buffer Cultivation  Containing 200 mL medium, at 30 o C and 200 rpm. Polyurethane (PU) carrier in the upper aerobic reactor was applied.

Discussion Modified Bioreactor Configuration The top-side and bottom-side of this bioreactor were aerobic and anaerobic, respectively. The PU carrier applied both as a bacterial carrier and as an enzyme filter during bioethanol fermentation. Microbial proliferation in the upper aerobic bioreactor was increased by cultivating T. reesei and A. niger on a PU carrier with fresh BHSE medium. While Z. mobilis put in the lower anaerobic bioreactor. Efficiency of Co-cultivated A. niger and T. reesei in Suspension Cultivation This figure proves that the lignocelluloses (LC) conversion was more efficient in co-cultivated than in T. reesei alone. The best reducing sugar concentration and LC conversion rate were 1.29 g/L and 23.27%, respectively.

Discussion Effects of Inoculum Ratio and Concentration in Suspension Cultivation This figure shows that the 5/5 ratio of T. reesei and A. niger gave the best results. In this condition, reducing sugar production reached 3 g/L and 60% after 72 h and LC conversion reached 3.12 g/L and 62.4% after 120 h. There were five total inoculum concentrations (2%, 6%, 10%, 14% and 18%) of T. reesei and A. niger were compared. This figure exhibits the reducing sugar content and the LC conversion rate were 2.34 g/L and %, respectively by using the optimal total inoculum 18%.

Discussion Inoculum Ratio and Effects of Different T. reesei and A. niger Concentrations in Co-immobilized System Figure A depicts the total enzyme activity peaked after 42h cultivation with 1/1 ratio of T. reesei and A. niger. While figure B shows that total enzyme activity was optimal when the total inoculum concentration of T. reesei and A. niger reached 6.5 x 10 6 spores/mL. Effect of Z. mobilis Alginate Bead Size on Bioethanol Production When the 2.15 mm and 2.48 mm of gel particles diameter were used, the reducing sugar conversion rate reached 40% and 70% after 6h and 12h cultivation, respectively. However, over 12h of cultivation, the 2.15 mm gel particles obtained a higher reducing sugar conversion rate compared to the 2.48 mm gel particles.

Discussion Bioethanol Production by A Modified Bioreactor Figure A (full medium): after 24h cultivation, bioethanol production reached g/L and the reducing sugar conversion rate reached 4.26%. Figure B (the total medium volume was immediately reduced to half, so the exposed volume of PU carrier in the air was increased): after 24h cultivation, bioethanol production reached 0.56 g/L and the reducing sugar conversion rate reached 11.2%.  aeration strategy Figure C (one-third the height of the PU carrier was exposed to air after each 20h pre-cultivation, so after 60h the PU carrier was completely exposed to air): the bioethanol production peaked at 0.41 g/L after 24h cultivation. But, the amount of cellobiose was not converted to reducing sugars after the medium volume reduced.

Critical Thinking Even though this strategy can enhance the bioethanol production by simultaneous saccharification fermentation, but it seems less simply at fermentation time started. In this system, the ethanol producing bacteria were put in the lower anaerobic reactor in the following step preceding by hydrolysis of lignocelluloses in the upper aerobic reactor. It allows the potential of system will be contaminated when cell loading larger.