The realm of cellulases in biorefinery development

Geopolitical concerns (unstable supply of gasoline, environmental pollution, and regular price hikes), economic, and employment concerns have been prompting researchers, entrepreneurs, and policy makers to focus on harnessing the potential of lignocellulosic feedstock for fuel ethanol production and its commercialization. The carbohydrate skeleton of plant cell walls needs to be depolymerised into simpler sugars for their application in fermentation reactions as a chief carbon source of suitable ethnologic strains for ethanol production. The role of cellulolytic enzymes in the degradation of structural carbodydrates of the plant cell wall into ready-to-fermentable sugar stream is inevitable. Cellulase synergistically acts upon plant cell wall polysaccharides to release glucose into the liquid media. Cellulase predominantly dominates all the plant cell wall degrading enzymes due to their vast and diverse range of applications. Apart from the major applications of cellulases such as in detergent formulations, textile desizing, and development of monogastric feed for ruminants, their role in biorefinery is truly remarkable. This is a major area where new research tools based upon fermentation based formulations, biochemistry, and system biology to expedite the structure–function relationships of cellulases including cellulosomes and new designer enzymatic cocktails are required. In the last two decades, a considerable amount of research work has been performed on cellulases and their application in biomass saccharification. However, there are still technical and economic impediments to the development of an inexpensive commercial cellulase production process. Advancements in biotechnology such as screening of microorganisms, manipulation of novel cellulase encoding traits, site-specific mutagenesis, and modifications to the fermentation process could enhance the production of cellulases. Commercially, cheaper sources of carbohydrates and modified fermentation conditions could lead to more cost-effective production of cellulases with the goal to reduce the cost of ethanol production from lignocellulosics. Implementation of integrated steps like cellulase production and cellulase mediated saccharification of biomass in conjunction with the fermentation of released sugars in ethanol in a single step so called consolidated bio-processing (CBP) is very important to reduce the cost of bioethanol. This paper aims to explore and review the important findings in cellulase biotechnology and the forward path for new cutting edge opportunities in the success of biorefineries.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Cellulase production from palm oil mill effluent in Malaysia: Economical and technical perspectives

The demand for cellulases has increased tremendously over the last few decades. This is due to its numerous applications in industry and also because it can be used to hydrolyze cellulosic materials into sugars that can be fermented into bioethanol and bio‐based products. This does not only open up a big and significant market for cellulases, but also provides another source of biofuel and bioenergy in the future. Nevertheless, the cost of the existing substrates for cellulase fermentation is very high if required for large‐scale production. Sustainable supplies and an economically feasible biomass are needed to reduce the cost of cellulase production. Palm oil mill effluent (POME) is rich in carbohydrates, proteins, nitrogenous compounds, lipids, minerals, cellulose, hemicelluloses and lignin. It can be used naturally as a fermentation medium, either for cellulase or other value‐added product fermentation. In Malaysia, a large and continually increasing amount of POME is produced every year because of the high global demand for palm oil. Hence, the development of cellulase production from POME is reviewed, covering the POME production, cellulase production and the major challenges together with the future prospects of these processes.     

Conversion of rice straw to bio-based chemicals: an integrated process using Lactobacillus brevis

Commercialization of lignocellulosic biomass as a feedstock for bio-based chemical production is problematic due to the high processing costs of pretreatment and saccharifying enzymes combined with low product yields. Such low product yield can be attributed, in large part, to the incomplete utilization of the various carbohydrate sugars found in the lignocellulosic biomass. In this study, we demonstrate that Lactobacillus brevis is able to simultaneously metabolize all fermentable carbohydrates in acid pre-processed rice straw hydrolysate, thereby allowing complete utilization of all released sugars. Inhibitors present in rice straw hydrolysate did not affect lactic acid production. Moreover, the activity of exogenously added cellulases was not reduced in the presence of growing cultures of L. brevis. These factors enabled the use of L. brevis in a process termed simultaneous saccharification and mixed sugar fermentation (SSMSF). In SSMSF with L. brevis, sugars present in rice straw hydrolysate were completely utilized while the cellulase maintained its maximum activity due to the lack of feedback inhibition from glucose and/or cellobiose. By comparison to a sequential hydrolysis and fermentation process, SSMSF reduced operation time and the amount of cellulase enzyme necessary to produce the same amount of lactic acid.     

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.     

里氏木霉产纤维素酶研究进展

木质纤维素类生物质被认为是重要且可持续的可再生能源,其主要组成部分是纤维素.纤维素酶是一种能将纤维素分解为葡萄糖的复合酶,能有效地降解木质纤维素生物质.真菌、细菌、放线菌、酵母等多种微生物均可以产生纤维素酶,其中里氏木霉具有完整的纤维素酶系结构,常作为生物技术领域中一个重要菌株,广泛应用于纤维素酶的商业生产.介绍了纤维...     

One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae

ABSTRACT: BACKGROUND: While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor. RESULTS: Ethanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8--12 FPU/ml throughout the one-pot process. When 50--300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7--46.3 g/l and 0.15--0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50--300 g SF/l, the ethanol concentration and yield were 9.5--35.1 g/l with their yields of 0.12--0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol. CONCLUSION: A. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid

Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.     

植物基因工程改善生物质能利用的研究进展

利用植物木质纤维资源发酵生产乙醇越来越受到人们的重视,但是要实现工业化生产仍然存在很多难题。最近,利用植物基因工程技术,改善植物自身性状,包括减少植物自身细胞壁中木质素含量、细胞中积累表达纤维素酶和木聚糖酶等方法,使自生产生的生物质更利于降解利用。目前,对这种新的能源转基因植物的研究取得了一定进展。     

Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.     

A 2.08 Å resolution structure of HLB5, a novel cellulase from the anaerobic gut bacterium Parabacteroides johnsonii DSM 18315

Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut, anaerobic bacteria are essential to the well‐being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here, we report the 2.08 Å resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for glycosidases. We anticipate that the information presented here will facilitate the identification of additional cellulases for which no homologues have been identified to date and enhance our understanding how these novel cellulases bind and hydrolyze their substrates.     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。     

Applications of computational science for understanding enzymatic deconstruction of cellulose

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.     

The challenge of enzyme cost in the production of lignocellulosic biofuels

With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.     

Synergistic Activity of Plant Extracts with Microbial Cellulases for the Release of Free Sugars

Agricultural lignocellulosic waste such as corn stover is a potential source of inexpensive, abundant, and renewable biomass for the production of bioethanol. The enzymatic process for the economically viable breakdown of cellulose to ethanol relies on the availability of inexpensive microbial cellulases. Although the cost of cellulase has decreased in recent years, current costs still preclude the production of economically viable bioethanol from lignocellulose. Substantive efforts in this lab are being directed to transgenic production of cellulases in maize in order to boost efficiency both of production of enzymes and degradation of corn stover. We serendipitously observed that the addition of non-transgenic maize seed extracts to cellulose and microbial enzymes potentiated free sugar release by as much as 20-fold. Further, this synergistic effect between cellulase enzymes and extract was seen with a variety of plant species and tissue extracts, but varied in efficiency, and was optimal at low concentrations of cellulases. Although the nature of the synergistic molecule is not known, the use of extracts to potentiate cellulose breakdown provides opportunities for a clearer mechanistic understanding of the degradation process as well as an economical way to improve the efficiency of cellulases to produce more cost-effective bioethanol from agricultural waste.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.