Factors affecting the enzymatic hydrolysis of cellulose in batch and continuous reactors: Computer simulation and experiment

Abstract

The process of enzymatic cellulose saccharification has been widely investigated recently since cellulose is the most abundant renewable resource and glucose formed as a product of hydrolysis can be converted to ethanol, microbial products, and other chemicals. The efficiency of enzymatic cellulose hydrolysis is known to be affected by many factors related to the source of the cellulase preparation,14 composition and structure of cellulosic material, the method of its pretreatment,'-1° process conditions,"I2 and the reactor design.'",." Since these factors are closely interrelated, a complex study of hydrolysis and a quantitative estimation of the role of the individual factors affecting its efficiency are needed to optimize the process.P r e v i o~s l y , '~, '~ we have developed mathematical models for the enzymatic hydrolysis of cellulose by cellulases from Trichodermu longibrachiatum in batchstirred and plug-flow column reactors. The model for the batch reactor process14 allows for the component composition of the cellulase preparation, inhibition by hydrolysis products (glucose and cellobiose), decrease in cellulose reactivity, and enzyme inactivation in the course of hydrolysis. The column reactor model" also takes into account the adsorption and distribution of cellulases in layers of the reactor as well as the redistribution of the enzymes during the process.In the present communication, the models are used for the quantitative evaluation of factors affecting the enzymatic hydrolysis of cellulose in batch-stirred and continuous plug-flow column reactors. Their role and * To whom correspondence should be addressed. relative contribution to the process kinetics are discussed. MATERIALS AND METHODSA crude cellulase preparation from the fungus Ti+ chodermu longihruchiutum, obtained from a culture filtrate by precipitation with ethanol, having a filter paper activityI6 (FPA) of 28 IU/g (5OoC, pH 4.8), an endoglucanase a~tivity'.'~ of 76 IUig (4OoC, pH 4.3, and a cellobiase activity' of 0.5 IU/g (4OoC, pH 4.5) was used. Sulfite delignified cellulose from cotton stalks with an 8043% degree of ~rystallinity','~ was used as a substrate.The hydrolysis products were analyzed by high-performance liquid chromatography (HPLC)IX on a Du Pont-850 analytical chromatograph, using a Zorbax NH2 column (4.6 mm x 25 cm) and acetonitrile-water mixture (70:30) as a mobile phase. Sugars were detected by using a refractive index detector.Thermostatted glass vessels (100 mL volume) equipped with a magnetic stirrer were used as batchstirred reactors. The glass columns (8 and 40 mL total volume) equipped with a thermostat jacket and a porous glass filter at the outlet were used as plug-flow column reactors. Cellulose hydrolysis in the column has been described in detail in ref. 15. The concentration of cellulose in the column was 250 g/L. All the experiments were performed at 50°C and pH 4.5 (0.1M acetate buffer).The models for the enzymatic hydrolysis of cellulose in batch and plug-flow column reactors have been described in detail...

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

“…Typically, the rate of cellulose hydrolysis by enzymes decreases rapidly with conversion, leading to decreased yields, long processing times, and high enzyme usage; and the rate of soluble sugar formation per amount of adsorbed enzyme dramatically declines as hydrolysis progresses (Nutor and Converse, 1991;Wang and Converse, 1992). Many hypotheses have been presented to explain this observation, including thermal instability of cellulases (Caminal et al, 1985;Converse et al, 1988;Eriksson et al, 2002a,b;Gonzalez et al, 1989), hydrolysis product inhibition (Eriksson et al, 2002b;Gan et al, 2003;Gusakov and Sinitsyn, 1992;Holtzapple et al, 1990;Kadam et al, 2002;Todorovic et al, 1987), cellulase inactivation (Converse et al, 1988;Gusakov and Sinitsyn, 1992;Gusakov et al, 1987;Mukataka et al, 1983;Ooshima et al, 1990;Reese, 1982;Sinitsyn et al, 1986;Sutcliffe and Saddler, 1986), enzyme slowing down/stopping (Desai and Converse, 1997), substrate transformation into a less digestible form (Zhang et al, 1999), and/or the heterogeneous structure of the substrate (Nidetzky and Steiner, 1993;Zhang et al, 1999). ''Restart'' experiments have been used to identify factors that control the rate of cellulose hydrolysis (Desai and Converse, 1997;Gusakov et al, 1985;Nidetzky and Steiner, 1993;Ooshima et al, 1991;Valjamae et al, 1998;Zhang et al, 1999), and some results indicated that the drop in rate for continual hydrolysis of cellulose could be explained by declining substrate reactivity (Zhang et al, 1999).…”

Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes

“…Cellobiose is further hydrolyzed to glucose in solution by ␤-D-glucosidases. Inhibition of cellulases by cellobiose constitutes a major obstacle for achieving high product yields in the enzymatic hydrolysis of cellulose (3,4) and thus significantly impacts the efficiency of biomass conversion (5,6). Many studies have been designed to quantify cellulase product inhibition (7)(8)(9)(10)(11)(12)(13), but measured inhibition constants vary by orders of magnitude depending on factors such as temperature, pH, and substrate (3).…”

Colloidal catalysts on the base of iron(3+) oxides for oxidative treatment of biomass

“…It facilitates catalytic oxidative destruction of biomass by H 2 O 2 . The ability to bind with cellulose fiber [23,24] was assumed as highly important among factors affecting the enzymatic hydrolysis of cellulose by different cellulases. Oxidative treatment of biomass occurs, obviously, via mixed free radical -molecular mechanism, which includes the catalytic decomposition of Н 2 О 2 into free radicals (OH* and HOO*), and their reactions with biomass components, and chain oxidation of organic materials with oxygen in air.…”