Integrated enzymatic catalysis for biomass deconstruction: a partnership for a sustainable future
© De and Luque; licensee Springer. 2015
Received: 1 December 2014
Accepted: 17 March 2015
Published: 27 March 2015
Deconstruction of lignocellulosic biomass using enzymatic catalysis can offer several advantages as compared to chemical catalysis in terms of product selectivity, production cost and sustainability issues. This contribution aims to provide an account of current developments in the understanding of plant biomass microstructures and the impact of various enzymatic processes on cellulose decrystallization. Critical problems, including biomass recalcitrance, and operational factors, including potential solutions to improve their effectiveness as alternatives in future biorefineries, will be also discussed.
Plant cell walls represent a largely abundant source of renewable carbon in the biosphere. The inherent complexity of fractions including cellulose and lignin requires several deconstruction strategies to release structural polysaccharides for suitable applications. Cellulose and chitin are two highly abundant biopolymer resources with a significant physicochemical recalcitrance, which limits their rapid and cost-effective degradation. Plants indeed evolved considerable defense mechanisms against deconstruction of their cell wall polysaccharides into sugars, and, as such, lignocellulosics depolymerization to various simple fractions (e.g. sugars) for subsequent biological or catalytic conversion to fuels has been the focus of intensive research endeavors in past years.
Recent biocatalytic attempts to valorize lignocellulosic biomass for the production of chemicals and biofuels have focused on identifying enzymes with enhanced hydrolytic capabilities . In nature, many microorganisms produce “free enzyme” cocktails of individual enzymes that work synergistically to depolymerize biomass [2-4]. Cellulolytic and hemicellulolytic enzymes able to deconstruct celluloses and hemicelluloses into fermentable sugars can facilitate the use of a plentiful source of renewable carbon.
Scientists from National Renewable Energy Laboratory (NREL), USA have significantly contributed to advances in the field of biological conversion of lignocellulosic feedstocks into liquid fuels in the past decades. Between 1999 and 2012, NREL conducted extensive studies to quantify the economic implications associated with measured conversion-performance for the biochemical production of cellulosic ethanol. A goal for pilot-scale demonstration by 2012 of biochemical ethanol production was set at a price competitive with petroleum gasoline, which was successfully achieved through NREL’s 2012 pilot plant demonstration runs.
Why enzymatic deconstruction?
Three major approaches have been widely explored in cellulosic conversion: physical (e.g. high temperatures, pressures and various pre-treatment/conversion technologies such as milling), chemical (e.g. strong acid treatment) and biological procedures. Physical and chemical processes are generally more efficient in terms of total conversion but inherently energy consuming, with a large generation of by-products. Comparatively, enzyme-based biological processes can be performed under mild conditions with high specificity for the target product. Enzyme-assisted cellulosic conversion can therefore constitute an alternative green approach, which can potentially reduce experimental costs, reduce the formation of unwanted byproducts as well as enhance reaction efficiency (under optimum conditions) and specificity.
The use of one-pot protocols mediated by multiple enzymes without any need for isolation steps (so-called cascade reactions) and/or continuous flow processes can offer significant benefits with respect to physic-chemical conversion processes. These include decreased unit operations, decreased reactor volume, increased volumetric and space-time yields, and shortened cycle times, as well as inherent advantages of flow reactions such as simpler work-up and more controllable reaction conditions . Multi-enzyme systems are complex in nature, but unique synergistic effects and the coupling of steps may even push unfavorable equilibria towards the formation of desired products under cascade-type processes (i.e. continuous flow conditions). However, major problems of cascading enzymatic reactions need to be overcome, including a generally lower stability and recyclability of the enzymes (as different enzymes are used under different reaction conditions). Heterogeneous immobilization techniques can overcome these issues, which will be discussed later.
Pretreatment of lignocellulosics
Enzymes must work directly at the solid–liquid interface for the depolymerization of individual cellulose chains to hydrolyze carbohydrate polymers, due to the complex composition of rigid cell walls. The surface ablation process proceeds in a slower reaction rate than any freely diffusing enzymatic reactions due to limited substrate accessibility . Importantly, enzymatic hydrolysis yields are typically low (~20% of glucan in feedstock) in the absence of pretreatment . A pretreatment step is highly essential to open up the structure of hydrolysis-resistant lignocellulosic matrices to enzymatic or microbial biocatalysts able to convert the carbohydrates into soluble sugars. Another major concern relates to the removal of the lignin fraction (cross-linked with the polysaccharides to make a rigid hydrophobic network) as lignin was proved to play an important role as an inhibitor in enzymatic hydrolysis, fermentation and other downstream processes . Combined steps of pretreatment and enzymatic hydrolysis can easily overcome the biomass recalcitrance during a biochemical conversion process.
Dilute sulfuric acid pretreatment has been an extensively utilized pre-treatment to break down cellulosic structures. Recently, ionic liquids (ILs) were explored as novel pretreatment to effectively disrupt cellulose crystallinity . However, a major issue in an integrated process relates to the recovery of ILs and isolation of the dissolved lignin-hemicellulose after pretreatment as native cellulases are severely inhibited by trace amounts of residual ILs. More research is consequently needed to develop IL-tolerant enzymes as well as to achieve clear knowledge about cellulase activity, stability and structure-activity relationships in cellulose-dissolving ILs. Interestingly, some relevant advances have recently been made in discovering and developing cellulases and other glycosyl hydrolases with increased IL-tolerance . Besides, more research on designing enzyme-compatible cellulose-dissolving ILs and cellulose stabilization techniques have also been the focus of recent studies.
Immobilized catalytic systems
A critical problem of cellulases as biocatalysts relates to their quick deactivation by environmental factors (e.g. temperature), which greatly hinders their practical uses in industry. Immobilization of cellulases on suitable solid materials (i.e. amorphous or mesoporous silica, agarose gel etc.) has been reported to improve their stability and reusability without significantly reducing their catalytic activity [12-14]. Based on previous studies, mesoporous silica materials have attracted a great deal of attention due to their large specific surface area, high mechanical strength and tunable surface functionalities . The cellulase activity has been shown to be largely dependent on pore size and surface area of the support, different immobilization methods (i.e. physical adsorption and chemical binding), loading amount and stability of cellulase after immobilization. Trichoderma reesei cellulases chemically bound to various supports, including silicas and magnetic nanoparticles, exhibited excellent stability and catalytic activity, exceeding 80% glucose yield from biomass . Recent research efforts also disclosed an integrated sequential enzyme cascade technique to deconstruct cellulose into 5-hydroxymethylfurfural (HMF) in high yields (46.1%).
Despite all these advancements, the enzymatic decrystallization process is still critical and poorly understood in many cases, and thus considerable research is needed to enhance the performance of cellulase action. The mechanism of action of hydrolase enzymes in the context of the cellulose surface must be understood at the molecular level. Emerging technologies such as cascading techniques should be further investigated for more efficient biocatalytic conversions. New techniques in the microscale are also required to be developed for the quantitative large-scale screening of enzyme libraries for biomass hydrolysis. Numerous biopolymers and enzymatic processes exist in nature; we just need to find out the most effective processes and technologies with highest degree of compatibility between substrates and enzymes in terms of enzymatic action.
Sudipta De gratefully acknowledges the University Grants Commission (UGC), India and University of Delhi for the financial support and necessary journal access during this work. Rafael Luque gratefully acknowledges Consejeria de Ciencia e Innovacion, Junta de Andalucia for funding under project P10-FQM-6711.
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