- Open Access
Lignosulfonate and elevated pH can enhance enzymatic saccharification of lignocelluloses
© Wang et al.; licensee BioMed Central Ltd. 2013
- Received: 31 July 2012
- Accepted: 13 September 2012
- Published: 28 January 2013
Nonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields and energy efficiencies. This study demonstrated the role of lignin sulfonation in enhancing enzymatic saccharification of lignocelluloses – lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses, contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding of cellulase.
The study found that lignosulfonate from SPORL pretreatment and from a commercial source inhibits enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding of cellulase. Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered as the concentrations of these two lignosulfonates increased. We hypothesize that lignosulfonate serves as a surfactant to enhance enzymatic hydrolysis at higher concentrations and that this enhancement offsets its inhibitive effect from nonspecific binding of cellulase, when lignosulfonate is applied to lignocellulosic solid substrates. Lignosulfonate can block nonspecific binding of cellulase by bound lignin on the solid substrates, in the same manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly proportional to the amount of lignosulfonate applied which is very important to practical applications. For a SPORL-pretreated lodgepole pine solid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g glucan with the application of its corresponding pretreatment hydrolysate coupled with increasing hydrolysis pH to above 5.5 compared with only 51% for the control run without lignosulfonate at pH 5.0. The pH-induced lignin surface modification at pH 5.5 further reduced nonspecific binding of cellulase by lignosulfonate.
The results reported in this study suggest significant advantages for SPORL-pretreatment in terms of reducing water usage and enzyme dosage, and simplifying process integration, i.e., it should eliminate washing of SPORL solid fraction for direct simultaneous enzymatic saccharification and combined fermentation of enzymatic and pretreatment hydrolysates (SSCombF). Elevated pH 5.5 or higher, rather than the commonly believed optimal and widely practiced pH 4.8-5.0, should be used in conducting enzymatic saccharification of lignocelluloses.
- Enzymatic hydrolysis/saccharification
- Nonspecific/nonproductive binding/adsorption
- Cellulase enzymes
The interactions between lignin and cellulase play a major role in enzymatic hydrolysis of lignocelluloses for sugar and biofuel production from biomass . These interactions can be described as (1) lignin physical blockage to limit cellulose accessibility to cellulase , and (2) nonspecific adsorption or binding of cellulase enzymes to lignin [3–7]. Reported studies indicate that these two mechanisms produced negative effects on enzymatic saccharification of lignocelluloses. Pretreatment of lignocelluloses such as Organosolv [8, 9] and SPORL  is able to partially remove lignin physical blockage by solubilizing a fraction of lignin into the hemicellulosic sugar stream (pretreatment spent liquor). However, further processing lignocelluloses to remove lignin blockage such as by delignification is not only expensive but also may not be necessary in terms of improving cellulose accessibility. It is probably more effective to address the issue of nonspecific binding of cellulase to lignin to further enhance enzymatic saccharification. One passive approach was to wash the solid fraction of pretreated lignocelluloses to eliminate nonspecific binding of cellulase to free lignin (referring to lignin separated from solid lignocellulosic substrate), as commonly described in the literature . Another passive approach is to use surfactant, protein and metal compound to block bound lignin (referring to lignin retained in solid lignocellulosic substrate) and free lignin, reducing their nonspecific binding to cellulase [3, 12–19]. However, washing is a significant environmental concern because the amount of water required is on the order of 10 m3 water/ton lignocellulose, based on pulp mill experience . The applications of surfactant and protein are both expensive at the required levels.
Hydrophobic interaction has been identified as the primary driving force for protein adsorption . Increasing the hydrophobicity of a substrate results in enhanced adsorption of protein or cellulase [1, 21]. This suggests that nonspecific binding (adsorption) of cellulase (made of protein) to lignin will be different for lignins of different hydrophobicities. Sulfonated lignin, has good hydrophilic properties. We can hypothesize that sulfonated lignin such as lignosulfonate in the sulfite pretreatment hydrolysates (spent liquor) [9, 10] may produce less nonspecific binding (adsorption) to cellulase enzymes. This hypothesis is indirectly corroborated by the excellent enzymatic digestibility of lignocellulosic substrates after sulfite pretreatment such as SPORL, sulfite pulping , and lignin sulfonation .
Lignosulfonate functions as a surfactant due to its strong hydrophilicity. This property has been used to develop commercial surface modification products such as dispersants and plasticizers. Therefore, we can further hypothesize that the application of lignosulfonate can reduce nonspecific binding of cellulase to the bound lignin fraction in lignocelluloses in the same manner as other surfactants to result in a gain in enzymatic hydrolysis efficiency. Furthermore, it is possible that the application of certain lignosulfonate, e.g., spent liquors from SPORL pretreatments, with low nonspecific bindings of cellulase – hypothesized in the previous paragraph – may result in a net enhancement of enzymatic saccharification of lignocelluloses. The validation or confirmation of this argument has significant scientific and practical implications for sulfite pretreatment technologies, such as SPORL  that has been demonstrated robust performance for sugar and biofuel production with very high yields from woody biomass including very recalcitrant softwood species [24–26]. Specifically, the separation of the SPORL pretreatment hydrolysate (spent liquor) from the SPORL pretreated solid ligno cellulosic fraction and washing of the SPORL solid fraction would not be required in order to enhance enzymatic saccharification. This can significantly simplify biorefinery process integration and save a significantly amount of water.
Previously, we demonstrated that the application of divalent metal compound, such as Ca(II) applied for neutralizing pretreatment hydrolysate, can eliminate nonspecific cellulase binding to lignosulfonate in the unwashed SPORL pretreated aspen (hardwood) solids [18, 19]. The aspen lignosuflonate in the SPORL spent liquor had a low degree of lignin sulfonation because of a low sulfite loading of 3% on oven dry (od) wood used in pretreatment. This study is a step further in demonstrating the role of lignin sulfonation in enhancing enzymatic saccharification. SPORL spent liquor that contain lignosulfonate with high degree of sulfonation, produced from lodgepole pine (softwood) by SPORL at 8% sulfite loading on od wood, was directly mixed with SPORL pretreated lodgepole pine and dilute acid pretreated aspen. The objectives of the present study are: (1) to verify the two hypotheses proposed above by directly comparing enzymatic cellulose saccharification efficiencies of several SPORL-pretreated lignocellulosic substrates with and without the applications of a SPORL-pretreatment hydrolysate from lodgepole pine at different loadings; (2) to verify our previous finding [27, 28]: significant improvement in cellulose saccharification when enzymatic hydrolysis of lignocelluloses is conducted at an elevated pH 5.5 – 6.2 as oppose to pH 4.8 – 5.0 as exclusively used in the literature. The elevated pH study was conducted with the application of SPORL pretreatment hydrolysate for further enhancement of saccharification efficiency. This work lays the foundation for direct simultaneous enzymatic saccharification and combined fermentation (SSFCombF) of the whole lignocellulosic slurry at high solids loadings without a separation and washing step after pretreatment .
Enzymatic hydrolysis of Whatman paper with the addition of lignosulfonates
Our previous study indicated that a divalent metal can form complex with lignosulfoante to reduce its affinity to cellulase . We conducted a separate study by neutralizing the same SPORL-pretreatment hydrolysate used in the previous set of experiments (Figure 2a) using Ca(OH)2. The same trend of SED reduction and recovery with the addition of lignosulfonate as discussed in the previous paragraph was observed. However, slightly higher SEDs than that for the control run without lignosulfonate were obtained after 48 hours hydrolysis when lignosulfonate concentration was greater than 0.3 g/L as Klason lignin (Figure 2b). The difference in SED between runs using NaOH (Figure 2a) and Ca(OH)2 neutralization (Figure 2b) were within the error margin, suggesting the lodgepole pine lignosulfonate produced using a high sulfite dosage of 8% on wood may already has very low affinity to cellulase due to its high degree of sulfonation or hydrolphlicity. The formation of lignosulfonate-Ca(II) complex on lignin nonspecific binding to cellulase is not significant.
Enzymatic hydrolysis of a SPORL-pretreated lodgepole pine with the application of SPORL-pretreatment hydrolysates
List of substrates produced along with the production conditions
Chemical charges on wood(%)
Duration @ T (min)
Lodgepole Pine – Solid cellulosic substrates
Lodgepole Pine – Liquid substrates: Pretreatment hydrolysates (spent liquor)
Aspen – Solid cellulosic substrates
Dilute acid (DA)
The gains in SED per unit mass of lignosulfonate loading, ΔSED/mlig, for the results presented in Figure 3a were calculated under different lignosulfonate concentrations and various enzymatic hydrolysis durations. The results indicate negative SED gains at low lignosulfonate loadings (< 0.2 g/L as Klason lignin) and short hydrolysis duration of 6 h (Figure 3b). This again confirmed that lignosulfonate does have inhibitive effect on enzymatic hydrolysis. However, there are two competing processes, lignosulfonate adsorbed by cellulase to reduce cellulase activity and lignosulfonate serves as a surfactant to block lignin on the solid substrate to prevent cellulase adsorption on the solid substrate lignin. The second process prevails at high lignosulfonate concentrations and longer hydrolysis duration. The results also indicate that the SED gain at 48 h per unit mass of lignosulfonate, ΔSED/mlig, initially decreased rapidly and then decreased very slowly at lignosulfonate concentration exceeding 0.2 g/L as Klason lignin. At a lignosulfonate dosage of 1.0 g/L as Klason ligin, ΔSED/mlig increased from approximately 2% L/g at hydrolysis time of 1 h to 25% L/g at 48 h. The slow-diminishing effect, i.e., ΔSED/mlig decreased very slowing with the increase in lignin concentration, has significant importance for simultaneous enzymatic saccharification and combined fermentation of the enzymatic and pretreatment hydrolysates (SScombF) using the whole slurry at a high solids loadings.
Hydrolysate neutralization using Ca(OH)2 showed consistently but only slightly increased enhancement of enzymatic saccharification at 72 h than those observed using NaOH by comparing the results in Figure 4a to those in Figure 4b. The fact that the differences in SED enhancements are pretty much within the measurement errors suggests the lignosulfonate produced at a high sulfite dosage of 8% on wood may already has a very low affinity to cellulase. The role of divalent metal to form complex with lignosulfonate to reduce lignosulfonate binding of cellulase [18, 19] is not very important. This is in agreement with that observed from Figure 2a and b.
Improved enzymatic hydrolysis of a lodgepople pine by combining with its pretreatment hydrolysate and using an elevated pH of 5.5
A similar study was also conducted using a dilute acid- (DA-AS) and SPORL- (SP-AS) pretreated aspen substrates with the addition of SPORL-pretreatment hydrolysate at lignosulfonate concentration of 1.0 g/L as Klason lignin, L-BD4-T85-3, at elevated pH of 5.5. The results indicate that using a high pH alone can enhance enzymatic saccharification as represented by SED gains (Figure 5b). With the application of L-BD4-T85-3, SED decreased slightly during the first 10 hours for both substrates (by comparing the solid symbols on solid line with the corresponding open symbols on dash line), suggesting lignoslfonate can be inhibitive to enzymes. But the lignosulfonate can enhance enzymatic hydrolysis after 24 hours. The SED of both substrates after 24 hours were further enhanced at elevated pH 5.5. When compared with the control run using buffer solution pH of 4.8 without the application of pretreatment hydrolysate (L-BD4-T85-3), the SED at 72 h were increased by 25 and 62% for the DA-AS and SP-AS, respectively. The pH effect on improving enzymatic saccharification is more pronounced for the SPORL-pretreated sample compared with the dilute acid pretreated sample, and agrees with our previous study .
Commercial lignosulfonate to enhance enzymatic saccharification of lignocellulose
This study demonstrates lignosulfonate has a low affinity to cellulase. As a surfactant, certain lignosulfonate, such as lignosulfonate from SPORL pretreatment of lodgepole pine, can be applied to significantly enhance enzymatic hydrolysis of lignocelluloses. Elevated pH can also enhance enzymatic saccharification of lignocelluloses. By directly mixing SPORL pretreatment hydrolysate of a lodgepole pine with the corresponding SPORL pretreated lodgepole pine solid substrate, 90% of enzymatic saccharification of pretreated lodgepole pine can be achieved at pH 5.5 and cellulase loading of 13 FPU/g glucan, or increased by approximately 75% compared with saccharification of the washed solid substrate alone at pH 4.8. These results demonstrate the feasibility of eliminating solid substrate washing for direct simultaneous enzymatic saccharification and combined fermentation of enzymatic and pretreatment hydrolysates (SSCombF) using SPORL technology with reduced water and enzyme use.
All experiments were carried out according to the experimental process flow similar to previous described except the pretreatment spent liquor was pH adjusted before added to the solid fraction to conduct enzymatic saccharification .
A lodgepole (Pinus contorta) tree killed by mountain pine beetle (Dendroctonus ponderosae) (estimated infestation age of 4 years, abbreviated BD4) was harvested from the Canyon Lakes Ranger District of the Arapaho–Roosevelt National Forest, Colorado. The details of the tree were described in our previous studies [31, 33]. Fresh aspen (Populus tremuloides) wood logs were obtained from northern Wisconsin, USA. All wood logs were shipped to the U.S. Forest Products Laboratory, Madison, Wisconsin, and chipped using a laboratory chipper. The wood chips were then screened to remove all particles greater than 38 mm and less than 6 mm in length. The thickness of the accepted chips ranged from 1 to 5 mm. The chips were kept frozen at a temperature of about −16°C until used.
Celluclast 1.5 L and Novozyme 188 (β-glucosidase) were generously provided by Novozymes North America (Franklinton, NC). Sodium acetate buffer, sulfuric acid, and sodium bisulfite were ACS reagent grade and used as received from Sigma-Aldrich (St. Louis, MO).
Several lignocellulosic solid substrates were produced from both the lodgepole pine and aspen wood chips using pretreatment methods with different process chemistries. Table 1 lists the various substrates produced along with production process conditions. Because dilute acid is a widely studied pretreatment and effective on many feedstocks except softwoods such as lodgepole pine used in this study. We also produced solid substrates from aspen (hardwood) using dilute acid and SPORL (Table 1) to evaluate the effectiveness of the application of lignosulfonate to enhance saccharfication of these aspen substrates. A laboratory wood pulping digester of capacity of 23 L was used to conduct pretreatment as described in our previous study . The digester was heated by a steam jacket and rotated at 2 rpm for mixing. The oven dry (od) weight of wood chips in each pretreatment was 2 kg. The pretreatment liquid to wood ratio (L/W) was kept at 3 (v/w). The chemical charges, reaction temperature, and duration of different pretreatments are listed in Table 1.
Chemical compositions and yields of the untreated and pretreated lignocellulosic substrates listed in Table 1
K lignin (%)
Solids yield (wt%)
Glucose and lignosulfonate concentrations in the pretreatment hydrolysates (hemicellulosic sugar stream) listed in Table 1
Lignosulfonate as Klason lignin(g/L)
The pH of the pretreatment hydrolysates were adjusted to 4.8 using NaOH before applied to the suspension of solid substrate to conduct enzymatic hydrolysis. For comparison purposes, the pH of the pretreatment hydrolysates were also adjusted to 4.8 using Ca(OH)2 in some hydrolysis experiments as indicated. The glucose was measured using the glucose analyzer described in the “Analytical methods” section.
A pure cellulosic substrate, a commercial Whatman filter paper (Grade 3, Cat No 1003 150, Whatman International, England) was also used. The manufacturer specified ash content is 0.06%. High purity lignosulfonate D748 from softwood sulfite pulping was donated by LignoTech USA (Rothschild, WI).
Enzymatic hydrolysis was conducted using commercial enzymes at 2% substrate solids (w/v) in 50-mL of buffer solutions on a shaker/incubator (Thermo Fisher Scientific, Model 4450, Waltham, MA) at 50°C and 200 rpm. Unless indicated, the pH of the buffer solution of sodium acetate was 4.8. Celluclast 1.5 L loadings varied between 7.5 to 13 FPU/g glucan. The ratio of Novozyme 188 (β-glucosidase) loading (in CBU) to Celluclast 1.5 L loading (FPU) was maintained at 1.5 for all experiments. Selected hydrolysis experiments were carried out in duplicates to ensure experimental repeatability. Hydrolysate was sampled periodically for glucose concentration analysis. Each data point is the average of two replicates. The average relative standard deviation was approximately 2%.
The chemical compositions of the original and pretreated biomass were analyzed by the Analytical and Microscopy Laboratory of the Forest Products Laboratory as described previously . All lignocelulosic samples were Wiley milled (model #2, Arthur Thomas Co, Philedelphia, PA). The milled sample of 20 mesh (~1 mm) in size was hydrolyzed in two stages using sulfuric acid of 72% (v/v) at 30°C for 1 h and 3.6% (v/v) at 120°C for 1 h. The hydrolysate supernatant and remaining solids are then filtered through a Gooch Crucible lined with a 21 mm Whatman filter into a volumetric flask. The supernatant was used for carbohydrate analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) . Klason lignin (acid insoluble) retained on the filter paper was quantified gravimetrically after drying.
The saccharides in the pretreatment hydrolysates (spent liquors) were analyzed using a Dionex HPLC system (ICS-3000) equipped with integrated amperometric detector and Carbopac™ PA1 guard and analytical columns at 20°C. Eluent was provided at a rate of 0.7 mL/min, according to the following gradient: 0 → 25 min, 100% water; 25.1 → 35 min, 30% water and 70% 0.1 M NaOH; 35.1 → 40 min, 100% water. To provide a stable baseline and detector sensitivity, 0.5 M NaOH at a rate of 0.3 mL/min was used as post-column eluent. For fast analysis, glucose in the enzymatic hydrolysate was measured in duplicate using a commercial glucose analyzer (YSI 2700S, YSI Inc., Yellow Springs, OH).
The lignosulfonate in the SPORL hydrolysate was calculated as Klason lignin based on Klason lignin yield loss through SPORL-pretreatment with the assumption that all Klason lignin losses were solublized as lignosulfonate into the SPORL hydrolysate. The amount of lignosulfonate produced from the acid soluble lignin was not accounted for.
Wang and Lan were visiting Ph. D students at the USDA Forest Service(USDA-FS), Forest Products Lab (FPL), from South China University of Technology, GuangZhou, China. Wang is currently with Key Lab of Paper Science & Technology, Shandong Polytechnic University, Jinan, China. Zhu is a Scientific Team Leader at the USDA-FS-FPL. He is a co-inventor the SPORL pretreatment process and publishes extensively in woody biomass bioconversion for biofuel, fiber, and nanocellulosic materials. Zhu is an elected Fellow of the International Academy of Wood Science (IAWS) and an officer of the Forest Products Division of the American Institute of Chemical Engineering (AIChE) and the Technical Association of the Pulp and Paper Industry (TAPPI). He serves on the editorial boards of several technical journals. This work was conducted on official government time of Zhu.
This work was partially supported by a USDA Small Business Innovative Research (SBIR) Phase II project (Contract Number: 2010-33610-21589) to Biopulping International, Inc. This project provided partial financial support to Wang and full support to Lan for their visiting appointments at the US Forest Service (USFS), Forest Products Laboratory (FPL). The Chinese Scholarship Council provided the other partial support for Wang. We acknowledge Fred Matt (FPL) for carrying out carbohydrate analyses.
- Nakagame S, Chandra RP, Saddler JN: The influence of lignin on the enzymatic hydrolysis of pretreated biomass substrates. In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass. Edited by: Zhu JY, Zhang X, Pan XJ. Washington, DC: American Chemical Society; 2011:145-167.View ArticleGoogle Scholar
- Mooney CA, Mansfield SD, Touhy MG, Saddler JN: The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Bioresour Technol 1998,64(2):113-119. 10.1016/S0960-8524(97)00181-8View ArticleGoogle Scholar
- Eriksson T, Borjesson J, Tjerneld F: Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzym Microb Technol 2002,31(3):353-364. 10.1016/S0141-0229(02)00134-5View ArticleGoogle Scholar
- Mansfield SD, Mooney C, Saddler JN: Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Prog 1999, 15: 804-816. 10.1021/bp9900864View ArticleGoogle Scholar
- Sewalt VJH, Glasser WG, Beauchemin KA: Lignin impact on fiber degradation.3. Reversal of inhibition of enzymatic hydrolysis by chemical modification of lignin and by additives. J Agric Food Chem 1997,45(5):1823-1828. 10.1021/jf9608074View ArticleGoogle Scholar
- Berlin A, Balakshin M, Gilkes N, Kadla J, Maximenko V, Kubo S, Saddler J: Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations. J Biotechnol 2006,125(2):198-209. 10.1016/j.jbiotec.2006.02.021View ArticleGoogle Scholar
- Palonen H, Tjerneld F, Zacchi G, Tenkanen M: Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin. J Biotechnol 2004,107(1):65-72. 10.1016/j.jbiotec.2003.09.011View ArticleGoogle Scholar
- Pan XJ, Arato C, Gilkes N, Gregg D, Mabee W, Pye K, Xiao Z, Zhang X, Saddler J: Biorefining of softwoods using ethanol organosolv pulping: Preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products. Biotechnol Bioeng 2005,90(4):473-481. 10.1002/bit.20453View ArticleGoogle Scholar
- Iakovlev M, van Heiningen A: Efficient fractionation of spruce by SO2-ethanol-water treatment: Closed mass balances for carbohydrates and sulfur. ChemSusChem 2012,5(8):1625-1637. 10.1002/cssc.201100600View ArticleGoogle Scholar
- Zhu JY, Pan XJ, Wang GS, Gleisner R: Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine. Bioresour Technol 2009,100(8):2411-2418. 10.1016/j.biortech.2008.10.057View ArticleGoogle Scholar
- Nagle NJ, Elander RT, Newman MM, Rohrback BT, Ruiz RO, Torget RW: Efficacy of a hot washing process for pretreated yellow poplar to enhance bioethanol production. Biotechnolo Prog 2002, 18: 734-738. 10.1021/bp0155078View ArticleGoogle Scholar
- Borjesson J, Peterson R, Tjerneld F: Enhanced enzymatic conversion of softwood lignocellulose by poly(ethylene glycol) addition. Enzym Microb Technol 2007,40(4):754-762. 10.1016/j.enzmictec.2006.06.006View ArticleGoogle Scholar
- Tu MB, Zhang X, Paice M, McFarlane P, Saddler JN: Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine. Biotechnol Prog 2009, 25: 1122-1129. 10.1002/btpr.198View ArticleGoogle Scholar
- Ooshima H, Burns DS, Converse AO: Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression. Biotechnol Bioeng 1990, 36: 446-452. 10.1002/bit.260360503View ArticleGoogle Scholar
- Yang B, Wyman CE: BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol Bioeng 2006,94(4):611-617. 10.1002/bit.20750View ArticleGoogle Scholar
- Pan XJ, Xie D, Gilkes N, Gregg DJ, Saddler JN: Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content. Appl Biochem Biotechnol 2005, 121: 1069-1079.View ArticleGoogle Scholar
- Zheng Y, Pan Z, Zhang R, Wang D, Jenkins B: Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass. Appl Biochem Biotechnol 2008, 146: 231-248. 10.1007/s12010-007-8035-9View ArticleGoogle Scholar
- Liu H, Zhu JY, Fu S: Effects of lignin-metal complexation on enzymatic hydrolysis of cellulose. J Agric Food Chem 2010, 58: 7233-7238. 10.1021/jf1001588View ArticleGoogle Scholar
- Liu H, Zhu JY: Eliminating inhibition of enzymatic hydrolysis by lignosulfonate in unwashed sulfite-pretreated aspen using metal salts. Bioresour Technol 2010,101(23):9120-9127. 10.1016/j.biortech.2010.07.035View ArticleGoogle Scholar
- Norde W: Adsorption of proteins from solution at the solid–liquid interface. Advances in Colloid and Interface Science 1986,25(C):267-340.View ArticleGoogle Scholar
- Hansen J, Ely K, Horsley D, Herron J, Hlady V, Andrade JD: The adsorption of lysozymes: A model system. Makromol Chem Macromol Symp 1988, 17: 135-154. 10.1002/masy.19880170110View ArticleGoogle Scholar
- Zhang X, Tu M, Paice M, Sacciadis G, Jiang Z, Jemaa N, Thibault A: Bioconversion of knot rejects from a sulfite pulp mill stream. BioResources 2010,5(1):23-42.Google Scholar
- Kumar L, Chandra R, Saddler JN: Influence of steam pretreatment severity on post-treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings. Biotechnol Bioeng 2011,108(10):2300-2311. 10.1002/bit.23185View ArticleGoogle Scholar
- Zhu JY, Gleisner R, Scott CT, Luo XL, Tian S: High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: A comparison between SPORL and dilute acid pretreatments. Bioresour Technol 2011,102(19):8921-8929. 10.1016/j.biortech.2011.07.047View ArticleGoogle Scholar
- Tian S, Luo XL, Yang XS, Zhu JY: Robust cellulosic ethanol production from SPORL-pretreated lodgepolep pine using an adapted strain S. cerevisiae without detoxification. Bioresour Technol 2010, 101: 8678-8685. 10.1016/j.biortech.2010.06.069View ArticleGoogle Scholar
- Lan TQ, Gleisner R, Zhu JY, Dien BS, Hector RE: High titer ethanol production from SPORL-pretreated lodgepole pine by simultaneous enzymatic saccharification and combined fermentation. Bioresour Technol 2013, 127: 291-297.View ArticleGoogle Scholar
- Zhang C, Zhu JY, Gleisner R, Sessions J: Fractionation of Forest Residues of Douglas-fir for Fermentable Sugar Production by SPORL Pretreatment. Bioenergy Research 2012,5(4):978-988. 10.1007/s12155-012-9213-3View ArticleGoogle Scholar
- Lan TQ, Lou H, Zhu JY: Enzymatic saccharification of lignocelluloses should be conducted at elevated pH 5.2 to 6.2. BioEnergy Research 2013., 6: 10.1007/s12155-012-9273-4Google Scholar
- Reinikainen T, Teleman O, Teeri TT: Effects of Ph and High Ionic-Strength on the Adsorption and Activity of Native and Mutated Cellobiohydrolase-I from Trichoderma-Reesei. Proteins-Structure Function and Genetics 1995,22(4):392-403. 10.1002/prot.340220409View ArticleGoogle Scholar
- Kim Y, Ximenes E, Mosier NS, Ladisch MR: Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass. Enzym Microb Technol 2011,48(4–5):408-415.View ArticleGoogle Scholar
- Luo X, Gleisner R, Tian S, Negron J, Horn E, Pan XJ, Zhu JY: Evaluation of mountain beetle infested lodgepole pine for cellulosic ethanol production by SPORL pretreatment. Ind Eng Chem Res 2010,49(17):8258-8266. 10.1021/ie1003202View ArticleGoogle Scholar
- Zhu W, Houtman CJ, Zhu JY, Gleisner R, Chen KF: Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF). Process Biochem 2012, 47: 785-791. 10.1016/j.procbio.2012.02.012View ArticleGoogle Scholar
- Zhu JY, Luo X, Tian S, Gleisner R, Negrone J, Horn E: Efficient ethanol production from beetle-killed lodgepole pine using SPORL technology and Saccharomyces cerevisiae without detoxification. TAPPI Journal 2011,10(5):9-18.Google Scholar
- Zhu W, Zhu JY, Gleisner R, Pan XJ: On energy consumption for size-reduction and yield from subsequent enzymatic sacchrification of pretreated lodgepole pine. Bioresour Technol 2010,101(8):2782-2792. 10.1016/j.biortech.2009.10.076View ArticleGoogle Scholar
- Zhu JY, Wang GS, Pan XJ, Gleisner R: Specific Surface to Evaluate the Efficiencies of Milling and Pretreatment of Wood for Enzymatic Saccharification. Chem Eng Sci 2009,64(3):474-485. 10.1016/j.ces.2008.09.026View ArticleGoogle Scholar
- Davis MW: A rapid modified method for compositional carbohydrate analysis of lignocellulosics by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD). Journal of Wood Chemistry and Technology 1998,18(2):235-352. 10.1080/02773819809349579View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.