Kinetics of the extracellular cellulases of Clostridium thermocellum acting on pretreated mixed hardwood and Avicel

Initial hydrolysis rates were examined for mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 °C (PTW220) and Avicel. Linear rates were observed for fractional conversion relative to the theoretical up to 0.2 for PTW220 and 0.4 for Avicel. Initial rates were essentially unaffected by the presence of growth medium components over a range of pH values. Avicel-hydrolyzing activity was inhibited linearly by ethanol, with a 50% rate reduction at 8 wt.% ethanol. Rate saturation with either substrate or enzyme was observed in a manner qualitatively consistent with previously reported adsorption data. Although somewhat less reactive than Avicel at very low enzyme loadings, much higher reaction rates were observed for PTW220 at moderate and high enzyme loading because of its higher capacity to bind cellulase. At equal subtrate concentrations (as potential glucose) and fractional substrate coverage of 0.09, the initial rate of pretreated wood hydrolysis exceeded that of Avicel by 15-fold. For fractional substrate coverage values up to 0.09 (the maximum value achieved for PTW220), the initial rate was proportional to adsorbed enzyme for PTW220. However, the rate per adsorbed enzyme declined sharply with increasing fractional coverage for Avicel hydrolysis.     

Effect of Urea on the Enzymatic Hydrolysis of Lignocellulosic Substrate and Its Mechanism

Effect of hydrogen bond breaker (urea) addition on the enzymatic hydrolysis of Avicel and eucalyptus pretreated by dilute acid (Eu-DA) was investigated. Urea enhanced the enzymatic hydrolysis of Eu-DA at 50 or 30 °C when the concentration of urea was below 60 g/L, while it inhibited the hydrolysis of Avicel. Low concentration urea (<?240 g/L) had little effect on the cellulase spatial structure and its activity. But it decreased cellulase binding to cellulose surface to inhibit the cellulose hydrolysis. Meanwhile, urea obviously prevented the adsorption of cellobiohydrolase I (CBHI) on the lignin in spite of little effect on the adsorption of β-glucosidase (BGL) and two endoglucanases (EGIII and EGV) on lignin. It was proposed that urea enhanced the enzymatic efficiency of Eu-DA by decreasing the cellulase adsorption on lignin surface.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Inhibition of enzymatic hydrolysis by residual lignins from softwood--study of enzyme binding and inactivation on lignin-rich surface

Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS-PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.     

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

Application of cellulase and hemicellulase to pure xylan, pure cellulose, and switchgrass solids from leading pretreatments

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.     

Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-beta-1,4-glucanase from blue mussel, Mytilus edulis.

A cellulase (endo-beta-1,4-D-glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 degrees C. Another unusual feature is that the enzyme retains 55-60% of its maximum activity at 0 degrees C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 degrees C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication).     

Mild pretreatment of yellow poplar biomass using sequential dilute acid and enzymatically-generated peracetic acid to enhance cellulase accessibility

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.     

Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates

The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.     

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.     

Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca²⁺ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.     

Fractionating recalcitrant lignocellulose at modest reaction conditions

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.     

Kinetic study on enzymatic hydrolysis of cellulose by cellulose from Trichoderma viride

Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellubiose and glucose) inhibition to cellulose on the hydrolysis rate were quantitatively investigated. While these factor had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: a overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kind of cellulosic materials: Avicel and tissue paper.     

Fractal kinetic analysis of polymers/nonionic surfactants to eliminate lignin inhibition in enzymatic saccharification of cellulose

The profile of enzymatic saccharification of Avicel in the presence and absence of lignin has been described with a fractal kinetic model (Wang and Feng, 2010), in which the retarded hydrolysis rate of enzymatic saccharification of cellulose has been represented with a fractal exponent. The lignin inhibition in the enzymatic saccharification of cellulose is indexed by the increase of fractal exponent, which can not be fully counterbalanced by high cellulase loading due to the high fractal exponent at high cellulase loading. On the contrary, fractal kinetic analysis indicates that an addition of some nonionic surfactant/polymers decrease the fractal exponent to the original values of enzymatic saccharification of Avicel without lignin and the corresponding toxicity of nonionic surfactants/polymers on the consecutive ethanol fermentation strain Saccharomyces cerevisiae is also examined.     

Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis

A rapid and accurate method for determining the number-average degree of polymerization (DP(n)) was established for insoluble cellulose and soluble cellodextrins as the ratio of glucosyl monomer concentration determined by the phenol-sulfuric acid method divided by the reducing-end concentration determined by a modified 2,2'-bicinchoninate (BCA) method. The modified BCA method, featuring incubation at 75 degrees C for 30 min, did not result in beta-glucosidic bond cleavage, whereas substantial cleavage was observed at higher temperature. Solubilization of insoluble cellulose in cold phosphoric acid prior to measurement of the reducing-end concentration by the BCA method was found not to be necessary for several model celluloses such as microcrystalline cellulose, but such solubilization was required for large fibers of cellulose such as Whatman No. 1 filter paper. The phenol-sulfuric acid method can be used for measuring the glucosyl monomer concentration of soluble cellodextrins, and also for insoluble cellulose if preceded by a liquefaction step. Standard deviations of < or =2% were obtained for both reducing and glucosyl monomer determination and of < or =3% for overall determination of DP. By use of the reported method, hydrolysis of phosphoric acid-swollen cellulose (PASC) by the Trichoderma reesei cellulase system was shown to result in a rapid decrease in DP as hydrolysis proceeded. By contrast, the DP of Avicel remained nearly constant during hydrolysis. The specific enzymatic cellulose hydrolysis rate is 100-fold higher for PASC as compared to Avicel.