Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. From the point of view for integrated utilization of lignocellulosic biomass, organosolv pretreatment provides a pathway for biorefining of biomass. This review presents the progress of organosolv pretreatment of lignocellulosic biomass in recent decades, especially on alcohol, organic acid, organic peracid and acetone pretreatments, and corresponding action mechanisms. Evaluation and prospect of organosolv pretreatment were performed. Finally, some recommendations for future investigation of this pretreatment method were given.     

Structural change in wood by brown rot fungi and effect on enzymatic hydrolysis

The effects of biological pretreatment on Pinus radiata and Eucalyptus globulus, were evaluated after exposure to two brown rot fungi Gloephylum trabeum and Laetoporeus sulphureus. Changes in chemical composition, structural modification, and susceptibility to enzymatic hydrolysis in the degraded wood were analyzed. After eight weeks of biodegradation, the greatest loss of weight and hemicellulose were 13% and 31%, respectively, for P. radiata with G. trabeum. The content of glucan decreased slightly, being the highest loss of 20% for E. globulus with G. trabeum. Consistent with degradation mechanism of these fungi, lignin was essentially undegraded by both brown rot fungi. Both brown rot fungi cause a sharp reduction in the cellulose degree of polymerization (DP) in the range between 58% and 79%. G. trabeum depolymerized cellulose in both wood faster than L. sulphureus. Also, structural characteristic of crystalline cellulose were measured by using two different techniques - X-ray diffraction (XRD) and infrared spectroscopy (FT-IR). The biological pretreatments showed an effect on cellulose crystallinity structure, a decrease between 6% and 21% was obtained in the crystallinity index (CrI) calculated by IR, no changes were observed in the XRD. Material digestibility was evaluated by enzymatic hydrolysis, the conversion of cellulose to glucose increased with the biotreatment time. The highest enzymatic hydrolysis yields were obtained when saccharification was performed on wood biopretreated with G. trabeum (14% P. radiata and 13% E. globulus). Decreasing in DP and CrI, and hemicellulose removal result in an increase of enzymatic hydrolysis performance. Digestibility was better related to DP than with other properties. G. trabeum can be considered as a potential fungus for biological pretreatment, since it provides an effective process in breaking the wood structure, making it potentially useful in the development of combined pretreatments (biological-chemical). A viable alternative to pretreatment process that can be used is a bio-mimetic system, similar to low-molecular complexes generated by fungi such as G. trabeum combined pretreatments (biological-chemical).     

The effect of high intensity mixing on the enzymatic hydrolysis of concentrated cellulose fiber suspensions

Enzymatic hydrolysis of lignocellulosic biomass in a high shear environment was examined. The conversion of cellulose to glucose in samples mixed in a torque rheometer producing shear flows similar to those found in twin screw extruders was greater than that of unmixed samples. In addition, there is a synergistic effect of mixing and enzymatic hydrolysis; mixing increases the rate of cellulose conversion while the increased conversion facilitates mixing. The synergy appears to result in part from particle size reduction, which is more significant when hydrolysis occurs during intense mixing.     

Eucalyptus globulus wood fractionation by autohydrolysis and organosolv delignification

This work provides an assessment on the fractionation of Eucalyptus globulus wood by sequential stages of autohydrolysis (to cause the solubilization of hemicelluloses) and organosolv pulping (to dissolve lignin, leaving solids enriched in cellulose). With this approach, valuable products (hemicellulose-derived saccharides, sulphur-free lignin fragments and cellulosic substrates with low contents of residual hemicelluloses) are obtained in separate streams, according to the biomass refinery approach. Autohydrolysis was carried out under optimized operational conditions, and organosolv pulping was performed using uncatalyzed ethanol-water solutions. The effects of the most influential operational variables (autohydrolysis severity, delignification temperature and ethanol concentration in the organosolv stage) on solid yield, solid composition, cellulose susceptibility and recovery of the various fractions was assessed using statistical methods, which enabled the identification of the most favourable operational conditions.     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Effect of particle size on the rate of enzymatic hydrolysis of cellulose

The effect of particle size on enzymatic hydrolysis of cellulose has been investigated. The average size of microcrystalline cotton cellulose has been reduced to submicron scale by using a media mill. The milled products were further subjected to hydrolysis using cellulase. High cellulose concentration (7%) appeared to retard the size reduction and resulted in greater particles and smaller specific surface areas than those at low concentration (3%) with the same milling time. Initial rate method was employed to explore the rate of enzymatic hydrolysis of cellulose. The production rate of cellobiose was increased at least 5-folds due to the size reduction. The yield of glucose was also significantly increased depending upon the ratio of enzyme to substrate. A high glucose yield (60%) was obtained in 10-h hydrolysis when the average particle size was in submicron scale.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood

Softwood constitutes the main source of lignocellulosic material in Sweden which can be used for ethanol production from renewable resources. To make the biomass-to-ethanol process more economically feasible, it is preferable to include the sugar-rich prehydrolysate, i.e. the liquid obtained after the pretreatment step, in the enzymatic hydrolysis of the solid fraction. This study shows that the prehydrolysate inhibits cellulose conversion in the enzymatic hydrolysis step. When the prehydrolysate was included in the enzymatic hydrolysis, the cellulose conversion was reduced by up to 36%. However, this inhibition can be overcome by fermentation of the prehydrolysate prior to enzymatic hydrolysis.     

Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose

To investigate the prebiotic properties of neoagaro-oligosaccharides (NAOS), obtained from enzymatic hydrolysis of agarose, the in vitro and in vivo effects of NAOS on bacterial growth were studied. In vitro NAOS were found to be highly resistant to enzymes of the upper gastrointestinal tract, which remained intact after 24h incubation with different amylolytic enzymes. NAOS significantly stimulated the growth of bifidobacteria and lactobacilli in Man-Rogosa-Sharp (MRS) medium, anaerobically. Compared with fructo-oligosaccharides (FOS), 1% (w/v) NAOS promoted the specific growth rate of beneficial bacteria by about 100%. The decreases of media pH with NAOS were almost the same as that with FOS. In vivo, NAOS significantly increased the numbers of lactobacilli and bifidobacteria (P<0.05) in fresh feces or cecal content while reducing putrefactive microorganisms. Mice fed with 2.5% (w/v) NAOS for 7 days had larger increases in colonic beneficial bacteria population than those fed with even 5% (w/v) FOS for 14 days. No side effects, such as eructation and bloating, were found. Interestingly, NAOS with higher degrees of polymerization (DP) showed better prebiotic activity. These results indicated that NAOS had great prebiotic effect, which could be beneficial to the host.     

Optimized delignification of wood‐derived lignocellulosics for improved enzymatic hydrolysis

One of the major bottlenecks in the bioconversion of lignocelluosic feedstocks to liquid ethanol is the recalcitrance of residue following pretreatment, specifically softwood derived residues. Peroxide delignification has previously been shown to effectively aid in the removal of condensed lignaceous moieties from substrates following pretreatment, and thereby improve the hydrolyzability of the polymeric carbohydrates to their monomeric constituents. Despite the effectiveness of peroxide, drawbacks in this system still remain, as the concentration of peroxide required for adequate hydrolysis performance is currently uneconomical. In an attempt to improve the efficacy of the delignification process, we evaluated other post‐treatment operations and concurrently attempted to limit the decomposition of peroxide loading; with the over arching aim to improve the efficiency of the bioconversion process. By employing several peroxide stabilizers and pre‐chelating the steam exploded recalcitrant substrates, we were able to substantially improve the delignification treatment of Douglas‐fir wood chips, and to reduce peroxide loading by more than 50% without negative effects on the hydrolysis rates and yield. Biotechnol. Bioeng. 2010;106: 884–893. © 2010 Wiley Periodicals, Inc.     

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     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of alkali/peracetic acid-pretreated sugarcane bagasse for ethanol and 2,3-butanediol production

The enzymatic digestibility of alkali/peracetic acid (PAA)-pretreated bagasse was systematically investigated. The effects of initial solid consistency, cellulase loading and addition of supplemental β-glucosidase on the enzymatic conversion of glycan were studied. It was found the alkali-PAA pulp showed excellent enzymatic digestibility. The enzymatic glycan conversion could reach about 80% after 24 h incubation when enzyme loading was 10 FPU/g solid. Simultaneous saccharification and fermentation (SSF) results indicated that the pulp could be well converted to ethanol. Compared with dilute acid pretreated bagasse (DAPB), alkali-PAA pulp could obtain much higher ethanol and xylose concentrations. The fermentation broth still showed some cellulase activity so that the fed pulp could be further converted to sugars and ethanol. After the second batch SSF, the fermentation broth of alkali-PAA pulp still kept about 50% of initial cellulase activity. However, only 21% of initial cellulase activity was kept in the fermentation broth of DAPB. The xylose syrup obtained in SSF of alkali-PAA pulp could be well converted to 2,3-butanediol by Klebsiella pneumoniae CGMCC 1.9131.     

Two-stage fungal biopulping for improved enzymatic hydrolysis of wood

A novel two-stage, whole organism fungal biopulping method was examined for increasing the yield of enzymatic hydrolysis of wood into soluble glucose. Liriodendron tulipifera wood chips (1 g) were exposed to liquid culture suspensions of white rot (Ceriporiopsis subvermispora) or brown rot (Postia placenta) fungi and incubated at 28 °C, either alone in single-stage 30 day (one fungal species applied) or two-stage 60 day (both fungal species applied in alternative succession) treatments. Fungi grew in all treatments, but did not significantly decrease the percent carbohydrate content of the wood. Two-stage treatments differed significantly in mass loss depending on order of exposure, suggesting additive or inhibitory fungal interactions occurred. Treatments consisting of C. subvermispora followed by P. placenta exhibited 6 ± 0.5% mass loss and increased the yield of enzymatic hydrolysis by 67-119%. This significant hydrolysis improvement suggests that fungal biopulping technologies could support commercial lignocellulosic ethanol production efforts if further developed.     

Enhanced enzymatic hydrolysis of rapeseed straw by popping pretreatment for bioethanol production

The objective of this study was to find a pretreatment process that enhances enzymatic conversion of biomass to sugars. Rapeseed straw was pretreated by two processes: a wet process involving wet milling plus a popping treatment, and a dry process involving popping plus dry milling. The effects of the pretreatments were studied both in terms of structural and compositional changes and change in susceptibility to enzymatic hydrolysis. After application of the wet and dry processes, the amounts of cellulose and xylose in the straw were 37-38% and 14-15%, respectively, compared to 31% and 12% in untreated counterparts. In enzymatic hydrolysis performance, the wet process presented the best glucose yield, with a 93.1% conversion, while the dry process yielded 69.6%, and the un-pretreated process yielded <20%. Electron microscopic studies of the straw also showed a relative increase in susceptibility to enzymatic hydrolysis with pretreatment.     

Correlating the effect of pretreatment on the enzymatic hydrolysis of straw

Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120 degrees C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (X(max)), and the enzymic hydrolysis time corresponding to 50% of X(max) (t(1/2)). An empirical correlation between X(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.     

The effect of biological pretreatment with the selective white-rot fungus Echinodontium taxodii on enzymatic hydrolysis of softwoods and hardwoods

Selective white-rot fungi have shown potential for lignocellulose pretreatment. In the study, a new fungal isolate, Echinodontium taxodii 2538, was used in biological pretreatment to enhance the enzymatic hydrolysis of two native woods: Chinese willow (hardwood) and China-fir (softwood). E. taxodii preferentially degraded the lignin during the pretreatment, and the pretreated woods showed significant increases in enzymatic hydrolysis ratios (4.7-fold for hardwood and 6.3-fold for softwood). To better understand effects of biological pretreatment on enzymatic hydrolysis, enzyme–substrate interactions were investigated. It was observed that E. taxodii enhanced initial adsorption of cellulase but which did not always translate to high initial hydrolysis rate. However, the rate of change in hydrolysis rate declined dramatically with decreasing irreversible adsorption of cellulase. Thus, the enhancement of enzymatic hydrolysis was attributed to the decline of irreversible adsorption which may result from partial lignin degradation and alteration in lignin structure after biological pretreatment.     

沙柳原料高浓度底物酶解发酵乙醇工艺

为了提高沙柳生物转化过程的经济可行性,考察了沙柳原料经过蒸爆、超微粉碎+稀酸、超微粉碎+稀碱预处理后高浓度底物补料酶解的效果,并对其高浓度水解糖液进行了乙醇发酵。结果表明:蒸爆处理法水解效果最好,通过补料酶解,底物质量分数可以达到30%,酶解液中总糖质量浓度达到132 g/L,葡萄糖质量浓度105 g/L;超微粉碎+稀酸预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度达到123 g/L,葡萄糖质量浓度73 g/L;超微粉碎+稀碱预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度133 g/L,葡萄糖质量浓度77 g/L。3种预处理使沙柳原料的酶解糖液都可以较好地被酿酒酵母利用发酵产乙醇,蒸爆处理原料的酶解糖液乙醇发酵效果最好,乙醇质量浓度达到47 g/L。     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

Hydrothermal processing and enzymatic hydrolysis of sorghum bagasse for fermentable carbohydrates production

Untreated and hydrothermally treated sorghum bagasse (SB) was hydrolyzed to simple sugars by the synergistic action of cellulases and hemicellulases produced by the fungi Fusarium oxysporum and Neurospora crassa. Synergism between the two lignocellulolytic systems was maximized with the application of higher fraction of N. crassa enzymes.Hydrothermolysis of SB was studied at a wide range of treatment times and temperatures. At intense pretreatment conditions (210 °C for 20 min; logR₀ = 4.54), the residual hemicellulose percentage was 17.45%, while formation of inhibitory products, 5-hydromethyl-furfural (HMF), furfural, acetic and formic acid, (0.21, 0.51, 3.36 and 1.80 g/l, respectively) remained in acceptable levels.Maximum conversion of cellulose and total polysaccharides of the untreated SB were 23.18% and 18.79%, respectively. Combining hydrothermal treatment and enzymatic hydrolysis of released oligosaccharides and insoluble solids resulted in improvement of cellulose (approximately 15% increase) and total polysaccharides (two fold) hydrolysis compared to that of untreated SB.