Pretreatments for Enhanced Enzymatic Hydrolysis of Pinewood: a Review

Pinewood is an abundant source of lignocellulosic biomass that has potential to be used as renewable feedstock in biorefineries for conversion into advanced biofuels and other value-added chemicals. However, its structural recalcitrance, due to the compact packing of its major components, viz. cellulose, hemicellulose and lignin, high lignin content, and high cellulose crystallinity, is a major bottleneck in its widespread use as a biorefinery feedstock. Typical chemical, thermal, and biological pretreatment technologies are aimed at removing lignin and hemicellulose fractions for improving enzyme accessibility and digestibility of cellulose. This review highlights common pine pretreatment procedures, associated key parameters and resulting enzymatic hydrolysis yields. The challenges and limitations are also discussed as well as potential strategies to overcome them, providing an essential source of information to realize pine as a compelling biorefinery biomass source.     

Bioconversion of Lignocellulose into Bioethanol: Process Intensification and Mechanism Research

Biofuels produced from lignocellulosic biomass can significantly reduce the energy dependency on fossil fuels and the resulting effects on environment. In this respect, cellulosic ethanol as an alternative fuel has the potential to become a viable energy source in the near future. Over the past few decades, tremendous effort has been undertaken to make cellulosic ethanol cost competitive with conventional fossil fuels. The pretreatment step is always necessary to deconstruct the recalcitrant structures and to make cellulose more accessible to enzymes. A large number of pretreatment technologies involving physical, chemical, biological, and combined approaches have been developed and tested at the pilot scale. Furthermore, various strategies and methods, including multi-enzyme complex, non-catalytic additives, enzyme recycling, high solids operation, design of novel bioreactors, and strain improvement have also been implemented to improve the efficiency of subsequent enzymatic hydrolysis and fermentation. These technologies provide significant opportunities for lower total cost, thus making large-scale production of cellulosic ethanol possible. Meanwhile, many researchers have focused on the key factors that limit cellulose hydrolysis, and analyzing the reaction mechanisms of cellulase. This review describes the most recent advances on process intensification and mechanism research of pretreatment, enzymatic hydrolysis, and fermentation during the production of cellulosic ethanol.     

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.     

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。     

Lignin Structural Alterations in Thermochemical Pretreatments with Limited Delignification

Lignocellulosic biomass has a complex and rigid cell wall structure that makes biomass recalcitrant to biological and chemical degradation. Among the three major structural biopolymers (i.e., cellulose, hemicellulose, and lignin) in plant cell walls, lignin is considered the most recalcitrant component and generally plays a negative role in the biochemical conversion of biomass to biofuels. The conversion of biomass to biofuels through a biochemical platform usually requires a pretreatment stage to reduce the recalcitrance. Pretreatment renders compositional and structural changes of biomass with these changes ultimately governing the efficiency of the subsequent enzymatic hydrolysis. Dilute acid, hot water, steam explosion, and ammonia fiber expansion pretreatments are among the leading thermochemical pretreatments with a limited delignification that can reduce biomass recalcitrance. Practical applications of these pretreatment are rapidly developing as illustrated by recent commercial scale cellulosic ethanol plants. While these thermochemical pretreatments generally lead to only a limited delignification and no significant change of lignin content in the pretreated biomass, the lignin transformations that occur during these pretreatments and the roles they play in recalcitrance reduction are important research aspects. This review highlights recent advances in our understanding of lignin alterations during these limited delignification thermochemical pretreatments, with emphasis on lignin chemical structures, molecular weights, and redistributions in the pretreated biomass.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Laccase applications in biofuels production: current status and future prospects

The desire to reduce dependence on the ever diminishing fossil fuel reserves coupled with the impetus towards green energy has seen increased research in biofuels as alternative sources of energy. Lignocellulose materials are one of the most promising feedstocks for advanced biofuels production. However, their utilisation is dependent on the efficient hydrolysis of polysaccharides, which in part is dependent on cost-effective and benign pretreatment of biomass to remove or modify lignin and release or expose sugars to hydrolytic enzymes. Laccase is one of the enzymes that are being investigated not only for potential use as pretreatment agents in biofuel production, mainly as a delignifying enzyme, but also as a biotechnological tool for removal of inhibitors (mainly phenolic) of subsequent enzymatic processes. The current review discusses the major advances in the application of laccase as a potential pretreatment strategy, the underlying principles as well as directions for future research in the search for better enzyme-based technologies for biofuel production. Future perspectives could include synergy between enzymes that may be required for optimal results and the adoption of the biorefinery concept in line with the move towards the global implementation of the bioeconomy strategy.     

Combining hot-compressed water and ball milling pretreatments to improve the efficiency of the enzymatic hydrolysis of eucalyptus

Background  

Lignocellulosic biomass such as wood is an attractive material for fuel ethanol production. Pretreatment technologies that increase the digestibility of cellulose and hemicellulose in the lignocellulosic biomass have a major influence on the cost of the subsequent enzymatic hydrolysis and ethanol fermentation processes. Pretreatments without chemicals such as acids, bases or organic solvents are less effective for an enzymatic hydrolysis process than those with chemicals, but they have a less negative effect on the environment.     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

Novel Single-step Pretreatment of Steam Explosion and Choline Chloride to De-lignify Corn Stover for Enhancing Enzymatic Edibility

It is important to develop efficient and economically feasible pretreatment methods for lignocellulosic biomass, to increase annual biomass production. A number of pretreatment methods were introduced to promote subsequent enzymatic hydrolysis of biomass for green energy processes. Pretreatment with steam explosion removes the only xylan at high severity but increases lignin content. In this study, corn stover soaked in choline chloride solution before the steam explosion is economically feasible as it reduced cost. Enzymatic hydrolysis of de-lignified corn stover is enhanced by combinatorial pretreatments of steam explosion and choline chloride. Corn stover pretreated with choline chloride at the ratio of 1:2.2 (w/w), 1.0 MPa, 184 °C, for 15 min efficiently expelled 84.7% lignin and 78.9% xylan. The residual solid comprised of 74.59% glucan and 7.51% xylan was changed to 84.2% glucose and 78.3% xylose with enzyme stacking of 10FPU/g. This single-step pretreatment had ∼ 4.5 and 6.4 times higher glucose yield than SE-pretreated and untreated corn stover, respectively. Furthermore, SEM, XRD and FTIR indicated the porosity, crystalline changes, methoxy bond-cleavage respectively due to the lignin and hemicellulose expulsion. Thus, the released acetic acid during this process introduced this novel strategy, which significantly builds the viability of biomass in short pretreatment time.     

Structural features affecting biomass enzymatic digestibility

The rate and extent of enzymatic hydrolysis of lignocellulosic biomass highly depend on enzyme loadings, hydrolysis periods, and structural features resulting from pretreatments. Furthermore, the influence of one structural feature on biomass digestibility varies with the changes in enzyme loading, hydrolysis period and other structural features as well. In this paper, the effects of lignin content, acetyl content, and biomass crystallinity on the 1-, 6-, and 72-h digestibilities with various enzyme loadings were investigated. To eliminate the cross effects among structural features, selective pretreatment techniques were employed to vary one particular structural feature during a pretreatment, while the other two structural features remained unchanged. The digestibility results showed that lignin content and biomass crystallinity dominated digestibility whereas acetyl content had a lesser effect. Lignin removal greatly enhanced the ultimate hydrolysis extent. Crystallinity reduction, however, tremendously increased the initial hydrolysis rate and reduced the hydrolysis time or the amount of enzyme required to attain high digestibility. To some extent, the effects of structural features on digestibility were interrelated. At short hydrolysis periods, lignin content was not important to digestibility when crystallinity was low. Similarly, at long hydrolysis periods, crystallinity was not important to digestibility when lignin content was low.     

Modeling cellulase kinetics on lignocellulosic substrates

The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass to yield biofuel. This hydrolysis by cellulases, a heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion. To render the process economically viable, increases in hydrolysis rates and yields are necessary and require improvement both in enzymes (via protein engineering) and processing, i.e. optimization of reaction conditions, reactor design, enzyme and substrate cocktail compositions, enzyme recycling and recovery strategies. Advances in both areas in turn strongly depend on the progress in the accurate quantification of substrate–enzyme interactions and causes for the rate slowdown. The past five years have seen a significant increase in the number of studies on the kinetics of the enzymatic hydrolysis of cellulose. This review provides an overview of the models published thus far, classifies and tabulates these models, and presents an analysis of their basic assumptions. While the exact mechanism of cellulases on lignocellulosic biomass is not completely understood yet, models in the literature have elucidated various factors affecting the enzymatic rates and activities. Different assumptions regarding rate-limiting factors and basic substrate–enzyme interactions were employed to develop and validate these models. However, the models need to be further tested against additional experimental data to validate or disprove any underlying hypothesis. It should also provide better insight on additional parameters required in the case that more substrate and enzyme properties are to be included in a model.     

Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted. Biotechnol. Bioeng. 2013; 110: 729–736. © 2012 Wiley Periodicals, Inc.     

Natural deep eutectic solvents for lignocellulosic biomass pretreatment: Recent developments,challenges and novel opportunities

Conversion of lignocellulosic biomass to fuels and chemicals has attracted immense research and development around the world. Lowering recalcitrance of biomass in a cost-effective manner is a challenge to commercialize biomass-based technologies. Deep eutectic solvents (DESs) are new ‘green' solvents that have a high potential for biomass processing because of their low cost, low toxicity, biodegradability, easy recycling and reuse. This article discusses the properties of DESs and recent advances in their application for lignocellulosic biomass processing. The effectiveness of DESs in hydrolyzing lignin-carbohydrate complexes, removing lignin/hemicellulose from biomass as well as their effect on biomass deconstruction, crystallinity and enzymatic digestibility have been discussed. Moreover, this review presents recent findings on the compatibility of natural DESs with enzymes and microorganisms.     

Epidemic based modeling of enzymatic hydrolysis of lignocellulosic biomass

An epidemic based model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, dilute sulfuric acid pretreated corn stover. The process of substrate getting adsorbed and digested by enzyme was simulated as susceptibles getting infected by viruses and becoming removed and recovered. This model simplified the dynamic enzyme “infection” process and the catalysis of cellulose into a two‐parameter controlled, enzyme behavior guided mechanism. Furthermore, the model incorporates the adsorption block by lignin and inhibition effects on cellulose catalysis. The model satisfactorily predicted the enzyme adsorption and hydrolysis, negative role of lignin, and inhibition effects over hydrolysis for a broad range of substrate and enzyme loadings. Sensitivity analysis was performed to evaluate the incorporation of lignin and other inhibition effects. Our model will be a useful tool for evaluating the effects of parameters during hydrolysis and guide a design strategy for continuous hydrolysis and the associated process control. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1021–1028, 2014     

The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover,Cellulase Structure,and Adsorption

Enzymatic hydrolysis of lignocellulosic biomass is limited by rapid cellulase deactivation, consequently requiring large amounts of enzyme to maintain acceptable biomass conversion. In this study, a new approach to improve lignocellulose hydrolysis was investigated. Performing enzymatic hydrolysis of corn stover (CS) in the presence of polymeric–surfactant micelles (PMs) was demonstrated to improve hydrolysis yield to a greater extent than using only surfactant micelles. Application of 2 % (w/w) of polyethylene glycol (PEG 6000) with casein, Tween-20, and Triton X-100 at levels above the critical micelle concentrations increased the hydrolysis yield of CS containing high-bound lignin (extrusion-pretreated) by up to 87.8, 11.7, and 7.5 %, respectively. These PMs were not effective during enzymatic hydrolysis of biomass lacking lignin (Avicel) or alkali-pretreated CS (7.2 % lignin). The main reasons for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween, and PEG-Triton were enhanced cellulase solubilization; reformation of α-helix substructure; and combination of induced cellulase solubilization, α-helix reformation, and chemical changes in the microstructure of biomass, respectively. Deformation of the cellulase substructure during hydrolysis of biomass and its subsequent reformation in the presence of surfactants were shown in this study for the first time. Chemical changes in the microstructure of biomass (e.g., lignin side changes, C–O bonds, and amorphous cellulose) were found to be another potential reason for the effectiveness of surfactants when they are incubated at above 6 g/L for 72 h with biomass.     

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

Cellulosic ethanol production: Progress,challenges and strategies for solutions

Lignocellulosic biomass is a sustainable feedstock for fuel ethanol production, but it is characterized by low mass and energy densities, and distributed production with relatively small scales is more suitable for cellulosic ethanol, which can better balance cost for the feedstock logistics. Lignocellulosic biomass is recalcitrant to degradation, and pretreatment is needed, but more efficient pretreatment technologies should be developed based on an in-depth understanding of its biosynthesis and regulation for engineering plant cell walls with less recalcitrance. Simultaneous saccharification and co-fermentation has been developed for cellulosic ethanol production, but the concept has been mistakenly defined, since the saccharification and co-fermentation are by no means simultaneous. Lignin is unreactive, which not only occupies reactor spaces during the enzymatic hydrolysis of the cellulose component and ethanol fermentation thereafter, but also requires extra mixing, making high solid loading difficult for lignocellulosic biomass and ethanol titers substantially compromised, which consequently increases energy consumption for ethanol distillation and stillage discharge, presenting another challenge for cellulosic ethanol production. Pentose sugars released from the hydrolysis of hemicelluloses are not fermentable with Saccharomyces cerevisiae used for ethanol production from sugar- and starch-based feedstocks, and engineering the brewing yeast and other ethanologenic species such as Zymomonas mobilis with pentose metabolism has been performed within the past decades. However strategies for the simultaneous co-fermentation of pentose and hexose sugars that have been pursued overwhelmingly for strain development might be modified for robust ethanol production. Finally, unit integration and system optimization are needed to maximize economic and environmental benefits for cellulosic ethanol production. In this article, we critically reviewed updated progress, and highlighted challenges and strategies for solutions.