Product Inhibition of Cellulases during Enzymatic Hydrolysis of the Pre-Treated Ligno-Cellulose
Integration of life sciences & engineering
Integration of Life Sciences & Engineering - Poster (T5-P)
Keywords: enzymatic hydrolysis, cellulases, product inhibition, ligno-cellulose, bio-reactor
The use of cellulose degrading enzymes (cellulases) for the hydrolysis of ligno-cellulosic biomass, as part of bio-ethanol process, is a promising, but also a very difficult and challenging task. Due to the problems with the inefficient stirring of the highly viscous feed mixture (pre-treated biomass and enzymes), crystalline structure of cellulose (resistant to enzymes action) and enzymes de-activation (by product inhibition, un-productive adsorption to lignin or shear stress), the application of cellulases in hydrolysis reactor is considered inefficient, and gives rise to the high contribution of enzymes costs to the overall bio-ethanol processing costs.
It is known that all three groups of enzymes found in cellulases (cellobiohydrolases, endoglucanases, β-glucosidases) can be inhibited by intermediate or final hydrolysis products, cellobiose and glucose, respectively. In particular, cellobiohydrolases are strongly and β-glucosidases very strongly inhibited by cellobiose, and glucose, respectively.
The consequence of the product inhibition is a significant loss in cellulose degrading activity and quantification of cellulase inhibition rate provides a useful tool in the hydrolysis reactor design. In general, there has been a lack of information on inhibition of commercially available enzymes acting on pre-treated ligno-cellulosic substrates, at high sugar concentrations, which are associated with a high dry matter contents.
The objective of this study is to quantify commercial cellulase (Celluclast®1.5L supplemented with Novozym®188) product inhibition rates during the enzymatic hydrolysis of pre-treated wheat straw, at high sugar concentration levels (up to 250 g/L), and investigate the possibility of reducing the inhibition. The product inhibition is studied in continuously stirred batch reactor by either addition of pure sugars or by sugar removal from the hydrolysis reactor with a diafiltration membrane. The results are analyzed in terms of glucose and cellobiose concentration and glucose yield, and compared with the same results from continuously stirred batch reactor without added or removed sugars.
The enzymatic hydrolysis experiments are done with wheat straw hydro-thermally pre-treated in 3 steps (60, 180, 195oC; 15, 10, 3 min, respectively) in a pilot plant operated by DONG A/S (Elsam). Cellulase activity measurement is done using standard FPU assay. Biomass compositional analysis is performed in accordance with NREL procedure. The reaction is performed at 50oC and pH=5, using enzymes Celluclast®1.5L and Novozym®188, produced by Novozymes A\S (enzyme loading 8 FPU/gsubstrate and 13 CBU/g substrate, respectively). The reaction mixture is analyzed for glucose and cellobiose concentration using Dionex HPLC system.
Presented Wednesday 19, 13:30 to 15:00, in session Integration of Life Sciences & Engineering - Poster (T5-P).
