Recycle of cellulases and the use of lignocellulosic residue for enzyme production after hydrolysis of steam-pretreated aspenwood

Summary Various modes of substrate and enzyme addition were used to hydrolyze a 10% concentration (w/v) of steam-exploded, water-and-alkali extracted aspenwood withTrichoderma harzianum E58 cellulases. Although cellulose conversion was high (94–100%), enzyme recovery was low in all cases. Low enzyme recovery was due to a combination of thermal inactivation and adsorption of the cellulases onto the lignocellulosic residue. Enzyme recycle was not feasible as the activity of the recovered cellulases towards crystalline cellulose was low. However, the residual material from enzyme hydrolysis was a suitable carbon source for cellulase enzyme production byT. harzianum based on enzyme yield and hydrolytic potential. These residues could only be used up to a 1% substrate concentration, since at higher substrate loadings cellulase production was reduced, likely because of lignin inhibitors.     

纤维素酶与木质纤维素生物降解转化的研究进展

利用纤维素酶将预处理后的秸秆降解成可发酵性单糖,然后发酵生产所需的液体燃料及化工产品的技术,对于我国解决能源、环境、人口就业等难题有着巨大的积极影响。在木质纤维素生物降解转化工艺中,减少纤维素酶用量及提高酶解效率是降低木质纤维素降解成本的关键。纤维素酶系和木质纤维素酶水解技术的改进需要深入了解纤维素酶系统的组成及其协同作用、纤维素酶的结构与功能以及纤维素酶的生产技术。将就以上几个方面的研究进展进行讨论,并深入探讨了纤维素酶糖化能力的评价方法。     

Kinetics of the enzymatic hydrolysis of cellulose: 2. A mathematical model for the process in a plug-flow column reactor

The kinetics of enzymatic cellulose hydrolysis in a plug-flow column reactor catalysed by cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma longibrachiatum adsorbed on cellulose surface have been studied. The maximum substrate conversion achieved was 90–94%. The possibility of enzyme recovery for a reactor of this type is discussed. A mathematical model for enzymatic cellulose hydrolysis in a plug-flow column reactor has been developed. The model allows for the component composition of the cellulase complex, adsorption of cellulases on the substrate surface, inhibition by reaction products, changes in cellulose reactivity and the inactivation of enzymes in the course of hydrolysis. The model affords a reliable prediction of the kinetics of d-glucose and cellobiose formation from cellulose in a column reactor as well as the degree of substrate conversion and reactor productivity with various amounts of adsorbed enzymes and at various flow rates.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process

Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end-product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood-derived cellulose stream limited the number of times the cellulose containing residue could be recycled. (c) 1996 John Wiley & Sons, Inc.     

Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations

The conversion of lignocellulosic biomass to fuel ethanol typically involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a beta-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative 13C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (< or = 1.7-fold). Of all the enzymes tested, beta-glucosidase was least affected by lignin.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion — especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase inhibition mechanisms and kinetics. The data show that new strategies that place the bioreactor design at the center stage are required to alleviate the product inhibition and in turn to enhance the efficiency of enzymatic cellulose hydrolysis. Accomplishment of the enzymatic hydrolysis at medium substrate concentration in separate hydrolysis reactors that allow continuous glucose removal is proposed to be the way forward for obtaining feasible enzymatic degradation in lignocellulose processing.     

Novel enzymes for the degradation of cellulose

ABSTRACT: The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix.     

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady-state expressions that can be used to describe the pseudo-steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme-substrate affinity at which the pseudo-steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.     

Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.     

Parameter determination and validation for a mechanistic model of the enzymatic saccharification of cellulose‐Iβ

Cost‐effective production of fuels and chemicals from lignocellulosic biomass often involves enzymatic saccharification, which has been the subject of intense research and development. Recently, a mechanistic model for the enzymatic saccharification of cellulose has been developed that accounts for distribution of cellulose chain lengths, the accessibility of insoluble cellulose to enzymes, and the distinct modes of action of the component cellulases [Griggs et al. (2012) Biotechnol. Bioeng., 109(3):665–675; Griggs et al. (2012) Biotechnol. Bioeng., 109(3):676–685]. However, determining appropriate values for the adsorption, inhibition, and rate parameters required further experimental investigation. In this work, we performed several sets of experiments to aid in parameter estimation and to quantitatively validate the model. Cellulosic materials differing in degrees of polymerization and crystallinity (α‐cellulose‐I_β and highly crystalline cellulose‐I_β) were digested by component enzymes (EG_I/CBH_I/ ) and by mixtures of these enzymes. Based on information from the literature and the results from these experiments, a single set of model parameters was determined, and the model simulation results using this set of parameters were compared with the experimental data of total glucan conversion, chain‐length distribution, and crystallinity. Model simulations show significant agreement with the experimentally derived glucan conversion and chain‐length distribution curves and provide interesting insights into multiple complex and interacting physico‐chemical phenomena involved in enzymatic hydrolysis, including enzyme synergism, substrate accessibility, cellulose chain length distribution and crystallinity, and inhibition of cellulases by soluble sugars. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1237–1248, 2015     

Ionic-liquid induced changes in cellulose structure associated with enhanced biomass hydrolysis

The effects of varying ionic liquid pretreatment parameters on various sources of lignocellulosic biomass have been studied using X-ray powder diffraction, X-ray fiber diffraction, and compositional analysis. Comparative enzymatic hydrolysis and sugar analysis were used to relate the observed changes in cellulose structure to biomass digestibility. In this study, the factor most clearly associated with enhanced biomass hydrolysis is the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase.     

Biomass-Derived Inhibitors of Holocellulases

Enzymes constitute a major monetary cost in the bioconversion of holocellulose to ethanol. Identifying enzyme inhibitors and moderating their effects is one approach that may help to overcome this issue. Most inhibitors that reduce the hydrolysis activity of holocellulases are released as the holocellulosic biomass is broken down in the pretreatment and hydrolysis steps. Recent reports in the literature have shown that the major inhibitors or deactivators of cellulases are phenols and xylooligosaccharides. The bioconversion of hemicelluloses by hemicellulases also has important practical applications in various agro-industrial processes in addition to the conversion of hemicellulosic biomass to fuels and chemicals. Hemicellulases, such as β-xylosidases, may also help alleviate the inhibitory effect of xylooligosaccharides to cellulases. However, compared to cellulases, less is known about the inhibition or deactivation of hemicellulases and pectinases, especially for inhibitors that are generated during pre-treatment and the hydrolysis of lignocellulosic substrates. Considering the importance of such enzymes for the complete degradation of lignocellulosic substrates, this review provides a broad view of the effect of inhibitors of holocellulases (cellulases, hemicellulases, and pectinases).     

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.     

Relation between filter paper activity and saccharifying ability of a Trichoderma cellulase system

Summary Tests made to study the relation between filter paper activity and actual saccharifying ability of Trichoderma cellulases show that 30 IU/g of cellulose were sufficient to achieve over 80% hydrolysis of a 25 g/L cellulose suspension in 24 h. With the same enzyme/substrate ratio, but double the concentration of substrate, about 60% hydrolysis was achieved. End- product inhibition is one factor which seriously limits the degree of hydrolysis and therefore the concentration of sugars achievable by enzymatic hydrolysis at high levels of substrate concentration or enzyme/substrate ratio.     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.     

A mechanistic model for enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition

The projected cost for the enzymatic hydrolysis of cellulosic biomass continues to be a barrier for the commercial production of liquid transportation fuels from renewable feedstocks. Predictive models for the kinetics of the enzymatic reactions will enable an improved understanding of current limitations, such as the slow-down of the overall conversion rate, and may point the way for more efficient utilization of the enzymes in order to achieve higher conversion yields. A mechanistically based kinetic model for the enzymatic hydrolysis of cellulose was recently reported in Griggs et al. (2011) (Part I). In this article (Part II), the enzyme system is expanded to include solution-phase kinetics, particularly cellobiose-to-glucose conversion by β-glucosidase (βG), and novel adsorption and product inhibition schemes have been incorporated, based on current structural knowledge of the component enzymes. Model results show cases of cooperative and non-cooperative hydrolysis for an enzyme system consisting of EG(I) and CBH(I). The model is used to explore various potential rate-limiting phenomena, such as substrate accessibility, product inhibition, sterically hindered enzyme adsorption, and the molecular weight of the cellulose substrate.