A review of enzymes and microbes for lignocellulosic biorefinery and the possibility of their application to consolidated bioprocessing technology
Highlights
► Biorefinery approaches produce fuels and chemicals through biomass conversion. ► Consolidated bioprocessing of lignocellulose is desired for effective biorefinery. ► This review focuses on the development of microbes for consolidated bioprocessing. ► The production of bio-based chemicals and advances fuels is emphasized.
Introduction
To ensure a reliable future source of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. A biorefinery is a concept that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from biomass, instead of conventional oil refinery processes. Lignocellulosic biomass obtained as agriculture byproducts and industrial residues is an abundant, inexpensive, and renewable source of sugars, and is a desirable feedstock for the sustainable production of liquid fuels and chemical products through the biorefinery processes (Menon and Rao, 2012). After pretreatment of the biomass, cellulosic and hemicellulosic materials are enzymatically decomposed into simple sugars that can be metabolized by microorganisms and converted to desired chemical products, including alcohols, fatty acids, organic acids and amino acids in microbial fermentation. Bio-ethanol is currently one of the most promising alternatives to conventional petroleum-based transport fuels. However, the recalcitrant structure of lignocellulosic biomass makes the process involved in its bioconversion more complicated than that of starchy and sugary materials, because current sugar-platform technologies require enzymatic conversion of the substrate to fermentable sugars prior to initiating microbial fermentation (Mussatto et al., 2010).
Lignocellulosic materials are mainly composed of cellulose, hemicellulose, and lignin. In plant biomass, cellulose forms highly crystalline microfibrils consisting of homopolymers of β-1,4-linked glucose units embedded in a hemicellulose, pectin and lignin matrix, a confirmation that makes the structure resistant to saccharification by hydrolytic enzymes. In general, the chemical and physicochemical pretreatment of lignocellulose causes cellulose to swell, thereby increasing its accessibility to saccharification enzymes (Chandel et al., 2012, Hong et al., 2012, Menon and Rao, 2012). However, the hydrolysis of cellulose remains a major limiting factor for the efficient utilization of lignocellulosic materials (Matano et al., 2012, Olson et al., 2011).
To release soluble sugars from cellulose, the activities of multiple enzymes, including endoglucanase, exoglucanase, and β-glucosidase, are required (Chandel et al., 2012). As cellulase reactions are inhibited by their intermediary and final products, such as cellooligosaccharides and glucose, microbial fermentation processes that combine enzymatic hydrolysis with sugar consumption are preferential for the alleviation of cellulase activity inhibition (van Zyl et al., 2007). However, the large difference in optimum temperatures between saccharification and fermentation during simultaneous saccharification and fermentation (SSF) is a drawback of bio-ethanol production (Hasunuma and Kondo, 2012). To overcome this limitation, large amounts of saccharification enzymes by fungi and bacteria are required, which severely impacts the cost effectiveness of biorefinery from lignocellulosic materials.
The recent development of microorganisms capable of efficient cellulose hydrolysis and fermentation represents a significant step in reducing the requirement for enzyme addition into the SSF processes (Matano et al., 2012). Such consolidation of enzyme production, saccharification, and fermentation into a single process is increasingly recognized as having potential for the low-cost production of biofuels and bio-based chemicals, as the high costs of capital investment, raw materials, and equipment associated with microbial enzyme production can be avoided (Menon and Rao, 2012, Mussatto et al., 2010, Olson et al., 2011). To develop such organisms, researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic (Hasunuma and Kondo, 2011, Olson et al., 2011), as there is still no ideal organism to use in one-step biomass conversion.
Although early efforts toward achieving efficient biorefinery processes from plant biomass have typically focused on ethanol, recent advances in microbial metabolic engineering have enabled to perform a challenge for producing renewable chemicals and advanced bio-fuels that are compatible with existing engines and fuel distribution infrastructure (Zhang et al., 2011a). This review will focus on a discussion of microbial strains for use in consolidated bioprocessing (CBP) technologies. In particular, approaches for the conversion of lignocellulosic materials into bio-based chemicals and fuels, as well as bio-ethanol, through microbial fermentation are emphasized.
Section snippets
Cellulase enzymes
The hydrolysis of insoluble cellulose by microorganisms requires the production of either free or cell-associated extracellular cellulases. The biochemical analyses of cellulase systems from aerobic and anaerobic bacteria and fungi performed during the past two decades have revealed that multiple enzymatic activities are needed to hydrolyze cellulose into soluble sugar monomers that can be metabolized by microorganisms (van Zyl et al., 2007, Zhang and Lynd, 2004). At least three major types of
Recombinant cellulase expression
A number of microorganisms used in industry, including Escherichia coli, Bacillus sp., lactic acid bacteria, Corynebacterium glutamicum, and Saccharomyces cerevisiae, have been engineered to tolerate toxic compounds and metabolize a range of carbon sources such as arabinose and xylose present in hemicellulose as well as glucose. The availability of extensive genetic manipulation tools has enabled the engineering of metabolic pathways to not only improve production titers and yields, but also to
Cellulase-expressing strains
Microorganisms capable of utilizing diverse substrates would provide a clear competitive advantage in biorefinery processes. Natural plant-degrading microorganisms producing extracellular multiple enzyme systems with different substrate specificities, such as cellulase and xylanase, would be able to utilize cellulosic and hemicellulosic materials as carbon sources. Thus, CBP of lignocellulosic materials has been developed with native cellulolytic microorganisms, including Clostridia sp., fungi,
Synthesis
Recent progress in the development of microorganisms through evolutionary, metabolic, and synthetic engineering approaches have paved the way for utilizing lignocellulosic biomass as a bioresource for fuel and chemical production. However, the one-step fermentative conversion of cellulosic material to chemical products represents a major challenge to achieving this goal. When using recombinant cellulose-utilizing microbes, low cellulolytic activities of heterologous enzymes often result in
Perspectives for future CBP microorganisms
Establishing economically feasible fermentation processes requires markedly increasing final product titers due to the high energy demands of subsequent product recovery steps, as well as the capital and production costs associated with biorefinery equipment (Hasunuma and Kondo, 2011, Mussatto et al., 2010). Although high-yield production of target compounds by metabolically optimized microbes is necessary, achieving higher titers inevitably requires increased loading of solid lignocellulose in
Conclusions
In this review, recent advances in the development of microorganisms for the fermentative production of bio-based fuels and chemicals from cellulosic materials were highlighted. Direct conversion of cellulose to key commodities such as alcohols, fatty acids, isoprenoid and organic acids has been achieved through the engineering of metabolic pathways and cellulose hydrolysis abilities of microorganisms. Despite of the obvious requirements for increasing yields and lowering production costs, this
Acknowledgements
This work was supported by project P07015 of the New Energy and Industrial Technology Development Organization (NEDO) under the sponsorship of the Ministry of Economy, Trade, and Industry (METI) of Japan. This work was also supported by Grants-in-Aid for Young Scientists (B) to TH and KYH from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan and Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary
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