Can We Reduce the Cellulase Enzyme Loading Required To Achieve

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Can we reduce the cellulase enzyme loading required to achieve efficient lignocellulose deconstruction by only using the initially absorbed enzymes? Jinguang Hu, Yiu Ki Mok, and John (Jack) N Saddler ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00004 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Sustainable Chemistry & Engineering

Can we reduce the cellulase enzyme loading required to achieve efficient lignocellulose deconstruction by only using the initially absorbed enzymes? Jinguang Hu, Yiu Ki Mok, John N. Saddler* Forest Products Biotechnology and Bioenergy Group University of British Columbia, Vancouver BC, Canada * corresponding author Jack N. Saddler [email protected]

Abstract: The cost-effective production of sugars from biomass continues to be challenging, partly due to the relatively high enzyme/protein loading required to achieve effective hydrolysis of the insoluble polysaccharides within the pretreated lignocellulosic substrates. Previous work has suggested that those enzymes that initially, strongly adsorb to the insoluble substrate are crucial for effective cellulose hydrolysis. However, most previous work in this topic area has used either purified enzymes or “older” generations of cellulase preparations acting on “model” cellulosic substrates. The results were, in several cases, contradictory or inconclusive. In the work reported here, the roles and functions of the initially adsorbed enzymes in determining the rate and extent of cellulose hydrolysis were assessed when using several different pretreated biomass substrates and the Novozyme enzyme preparation, Cellic CTec3. It was apparent that the initially adsorbed enzymes (irreversible bound to substrate after centrifugation) played a critical role as the removal of the “free/unadsorbed” enzymes in solution resulted in no significant decrease in the rate and extent of cellulose hydrolysis, regardless of the enzyme loading and the substrates used. By removing the initially, “free/unadsorbed” enzyme, the enzyme loadings required for an effective biomass deconstruction (>70% cellulose hydrolysis yields within 3 days) could be reduced by up to 50%, depending on the substrate used. Keywords: Enzyme adsorption, Enzymatic hydrolysis, Biomass pretreatment, Enzyme recycle, Biorefinery, High solid hydrolysis

Introduction: With increasing concerns about global sustainability, the transition from today’s fossil based economy towards a more sustainable bio-based economy has received significant interest across the world. 1,2 However, the cost-effective, enzyme mediated, deconstruction of lignocellulosic biomass is still challenging. This is partially due to the significant amount of enzymes needed to establish a biomass derived “sugar-platform” that could form the basis of a sustainable biofuels and biochemicals sector. 3 Although some reductions in enzyme dosage have been achieved through various strategies such as improving the various enzymes’ catalytic activity/thermostability, enhancing the synergism among cellulase enzymes and adding accessory enzymes/disrupting proteins to cellulase mixture, 4-6 it is clear that additional efforts will still be required if these processes are ever to become commercially successful. 3

Biomass recalcitrance and the challenge of enzyme accessibility to cellulose have been shown to be the major factors in limiting the effectiveness of enzyme mediated biomass deconstruction. 7,8 Key factors such as the irreversibility/reversibility of cellulase binding at various temperature conditions, the

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influence of cellulase synergism, the nature of the cellulosic substrate and the pretreatment used to “openup” the substrate are all known to effect the efficacy of enzymatic hydrolysis. 9-13 Due to the insoluble nature of lignocellulosic biomass, it is well-recognised that effective diffusion and adsorption of the cellulase enzymes to the substrate is a key prerequisite if efficient enzymatic hydrolysis, particularly at high substrate concentrations, is to be achieved. Although significant work has been carried out to try to better understand the dynamics of cellulases adsorption and its influence on cellulose hydrolysis, the exact mechanisms involved remain unclear 14-24. However, it has been observed that a relatively large proportion of the added enzymes (~30-60%) typically remains in solution after enzyme adsorption equilibrium is achieved (usually within 1st hour) (Table S1). Interestingly, it has also been reported that the removal of these “free” enzymes in solution seemed to have only a minimal effect on the hydrolysis of the cellulosic substrates 25-27. Thus, the use of only the initially adsorbed enzymes, by removing and recovering these “free/unadsorbed” enzymes in solution, might provide an opportunity to further reduce the high enzyme loadings currently required for effective cellulose hydrolysis. A review of the literature indicated that although there were many previous studies assessed the initial cellulase enzyme distribution/adsorption on various “model” and “realistic” cellulosic substrates (Table S1), the roles and functions of these initial adsorbed enzymes on cellulose hydrolysis has remained elusive. As result, the truly representative hydrolytic potential of initially adsorbed enzymes proved difficult to determine. Since enzyme adsorption and subsequent enzymatic hydrolysis is strongly influenced by the nature of enzyme mixtures and the substrate 28,29, in the work reported here, the roles and functions of the initially adsorbed enzymes (after the adsorption equilibrium reached within 1st hour) on the cellulose hydrolysis were assessed by selectively removing different portions of “free/unadsorbed” enzymes in the hydrolysis solution. The commercial cellulase enzyme mixture Cellic CTec3, at various enzyme loadings, was added to a range of industrial relevant pretreated lignocellulosic substrates. A major goal of this work was to evaluate whether this could be an effective strategy to reduce enzyme loadings, particularly when high substrate loadings were used to try and achieve high final sugar concentrations.

Results and discussion: The influence of initially adsorbed enzymes on the hydrolysis of “pure” cellulosic substrate. To minimize the possible influence of hemicellulose and lignin, the roles and functions of adsorbed enzymes were first evaluated on the never dried, relatively “pure” cellulosic substrate, dissolving pulp (DsP). The enzyme loading was selected based on the minimal enzyme dose required to achieve effective cellulose hydrolysis yields (70-90%) and was calculated by using the simplified HCH-1 model (Figure 1 insert). The model was based on the linear relationship between cellulose hydrolysis (after 72 hours) and the natural logarithm of the initial enzyme loading 30. The model had been previously, successfully, tested on various cellulosic/lignocellulosic substrates across a wide range of enzyme and substrate loadings 31,32. In order to assess the roles and functions of initially adsorbed enzymes, various amounts of the “free/unadsorbed” enzymes in the liquid phase were removed from the hydrolysis system when the enzyme adsorption equilibrium was reached after 1 hour. As shown in Figure 1, the removed, “free/unadsorbed” enzymes appeared to only contribute marginally to cellulose hydrolysis as it was apparent that similar hydrolysis rates and yields were achieved even with the removal of up to 75% of the “free/unadsorbed” enzyme in solution (equivalent to ~50% of the total enzyme added). Briefly, cellulose hydrolysis yields of 93% could still be achieved when 25% of the free enzymes, which represented 18% of the initial protein added, were removed after one hour by centrifugation. When the amount of free enzymes/protein removed was further increased to 50% and 75%, more than 85% of the DsP was hydrolysed after 72 hours, corresponding to only a 10% decrease in yield despite removing 37% and 55% of the initial protein added.


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The role of initially adsorbed enzymes on the cellulose hydrolysis of various pretreated lignocellulosic substrates. Although the removal of free enzymes/protein appeared to have little effect on the hydrolysis of DsP, it remained unclear if the same phenomenon extends to more complex and recalcitrant lignocellulosic substrates. Unlike DsP, lignocellulosic biomasses are naturally heterogeneous, containing hemicellulose and lignin in addition to cellulose. In previous work, it was suggested that the accessibility to cellulose by cellulases is one of the major factors that affects the ease of hydrolysis of lignocellulosic biomass. 7,13 This is in part due to the restriction of fibre swelling by both lignin and hemicellulose, which limit the penetration of the cellulase to the individual cellulose fibres. 13,33 Additionally, unproductive binding may also occur between cellulases and lignin through hydrophobic, electrostatic and hydrogen bonding interactions that occur within the lignocellulosic substrates. For example, it has been shown that during acid based pretreatment, pseudo-lignin may be produced (due to sugar degradation), which can further limit cellulose accessibility and unproductive bind cellulases. 48 We next wanted to further assess the roles and functions of the initially adsorbed enzymes on the hydrolysis of industrial relevant pretreated lignocellulosic biomass. As hemicellulose and lignin have both been shown to influence the adsorption of cellulase enzymes and subsequent enzymatic hydrolysis 34,35, a range of SO2 catalyzed steam pretreated lignocellulosic substrates were produced and used (Table 1). As expected, the lignin content in steam pretreated softwood lodgepole pine (LPP) was higher than hardwood poplar (SPP), while the low severity pretreated poplar SPP190 (T=190°C) retained six times more xylan compared to the substrate pretreated at a higher severity SPP200 (T=200°C). The total enzyme loading used to hydrolyse each substrate was calculated based on the HCH-1 model described earlier (Figure S1). Similar to what was observed with DsP hydrolysis, the removal of 75% of the “free/unadsorbed” enzymes in the solution resulted in minimal influence on the rate and extent of cellulose hydrolysis of all of the pretreated biomass substrates (Figure 2). Although it appeared that the removal of free enzymes (up to 75%) had only a minor to negligible influence on the rate and extent of hydrolysis of pure cellulosic and lignocellulosic substrates at various levels of enzyme loading (Fig. 1-3), it was apparent that a considerable proportion of the substrate was hydrolyzed within the first hour of adsorption/hydrolysis when using the complete enzyme mixture. To further elucidate if only the adsorbed enzymes were responsible for the majority of the cellulose hydrolysis, after 75% of the “free/unadsorbed” enzyme was removed, the substrates were further extensively washed with 50mM sodium acetate buffer to try to remove the remaining ~25% “free/unadsorbed” enzymes. In this approach, the initial enzyme adsorption was performed for 1 hour at 4°C instead of 50°C, to minimize initial cellulose hydrolysis. Again, the hydrolysis yields of the cellulosic substrates were only marginally influenced by this extensive “free/unadsorbed” enzymes removal process (Table 2). When the free/unadsorbed enzymes were replaced by buffer, this resulted in there being a lower amount of protein/enzymes in the overall reaction. Consequently, the efficiency of the hydrolysis process appeared to be improved. It also provided an opportunity to recycle/reuse the unabsorbed enzymes. The apparent faster hydrolysis of the SPP190 substrate compared to that observed with SPP200 was due to the higher enzyme loading used to hydrolyzing SPP190. The enzyme loading for each substrate was selected based on the minimal enzyme dose required to achieve effective cellulose hydrolysis yields (~80%) after 72 hours hydrolysis (Fig. S1).

The influence of initially adsorbed enzymes on cellulose hydrolysis, using different enzyme loadings. Having established that the removal of “free/unadsorbed” enzymes had only a minor to negligible effect on the rate and yield of cellulose hydrolysis over a range of cellulosic and lignocellulosic substrates, we next wanted to determine if this phenomenon was dependant on the initial enzyme loading that was used. Consequently, all of the substrates were hydrolyzed at various enzyme loadings with/without the removal of 75% of the “free/unadsorbed” enzymes (Figure 3). As expected, lower



enzyme loadings resulted in a significant reduction in cellulose hydrolysis after 72 hours. However, it was also apparent that the adsorbed enzymes continued to achieve similar cellulose hydrolysis yields as compared to the complete enzyme mixture across all of the substrates and enzyme loading tested (Figure 3). Thus, it appeared that efficient cellulose hydrolysis could be achieved using the initially adsorbed enzymes regardless of the enzyme loading used. The composition and nature of the “free/unadsorbed” enzymes. Although a considerable amount of the “free/unadsorbed” enzymes could be removed without influencing the efficacy of cellulose hydrolysis, it was not apparent whether specific types or amounts of enzyme activities were unadsorbed. It was apparent that the major cellulase enzyme activities in the “free/unadsorbed” fraction, the endoglucanase (CMCase), exoglucanase (p-NPCase), and β-glucosidase activities (p-NPGase), as well as the major hemicellulase activity (Xylanase) were present, with slightly decreased p-NPCase and increased CMCase activities as compared with the complete Cellic CTec3 preparation (Figure 4). The reduced pNPCase activity was expected as the major cellulase monocomponent Cel7A (p-NPCase) has been reported to have a higher binding affinity towards cellulosic substrates 36-38. The increased portion of CMCase activity in the “free/unadsorbed” enzyme fraction suggested that less endoglucanse activity might be required to hydrolyze the residual solid components in the hydrolysis system. The similar proportions of β-glucosidase activities (p-NPGase) in both the “free/unadsorbed” enzyme and originally added cellulase mixtures was likely a result of the previously observed high binding affinity of βglucosidase in the newer enzyme preparations that were used in this study. 39 Direct high solid loading hydrolysis using just the initially absorbed enzymes. Since the removal of 75% of the “free” enzymes after centrifugation also resulted in a relative high solid content biomass slurry (~10% w/w), we next assessed the potential of using just the absorbed enzymes to carry out high solids loading hydrolysis. The bound enzymes again showed a similar hydrolytic performance as compared to the results obtained when using the complete enzyme mixture, on both DsP and SPLP substrates (Figure 5). It was also apparent that the bound enzymes (after the removal of about 75% of the “free/unadsorbed” enzyme) resulted in a much improved hydrolytic performance, as compared to same amount of enzymes directly added to the 10% biomass substrate (more than 20% cellulose hydrolysis improvement) (Figure 5). It seemed that by removing the unadsorbed enzymes at the initial 2% solid loading (w/w) and subsequently increasing the substrate concentration to 10%, this resulted in enhanced enzyme distribution throughout the insoluble biomass as compared with directly applying the same amount of enzymes to the 10% solids (Direct 10% sold loading hydrolysis with only “bound” enzyme loading, Figure 5). It is well documented that one of the major challenges for high solid loading hydrolysis is inefficient enzymesubstrate mixing due to less free water being available at the initial stages of hydrolysis 40-42. Thus, it appeared that the removal of the “free/unadsorbed” enzymes not only provided a good way to reduce overall enzyme loading, it also facilitated effective hydrolysis at high solids loading. Current, ongoing work is looking at developing a more industrially relevant separation strategy which should allow an even higher solid biomass concentration being reached (15-20% W/V).

Conclusion: Regardless of the enzyme loading and the nature of the biomass substrate, similar hydrolysis yields and overall hydrolytic performances were achieved when using either the initially adsorbed enzymes or the complete (the adsorbed and unadsorbed enzymes) cellulase enzyme mixture. The removal of “free/in solution” enzymes considerably reduced the required enzyme loading, which in turn, enhanced the effectiveness of cellulose hydrolysis. By using just the initially adsorbed enzymes associated with the substrate, rather than the whole enzyme system, it would only take 50-85% of the initial enzyme loading


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(depending on the nature of the cellulosic substrate) to accomplish the desired cellulose conversion. Although there was some preferential absorption of a few of the individual enzymes at the initial stages of the reaction, the fact that the majority of enzyme activities were detected in the supernatant suggested the potential to recycle/reuse these “free/unadsorbed” enzymes. Removing the “free/unadsorbed” enzymes also appeared to enhance the more even distribution of the adsorbed enzymes due to a large initial volume at the initial enzyme adsorption process, consequently increasing the effectiveness of cellulose hydrolysis as compared to same amount of enzymes (same as the adsorbed enzyme) which were directly added at high solids loadings.

Materials and Methods: Cellulosic and lignocellulosic substrates. The mountain pine beetle killed lodgepole pine (Pinus contorta) (LPP) and hybrid poplar (POP) were provided by the British Columbia Forest service and FPInnovations, respectively. Dissolving pulp (DP) produced from hardwood was obtained from Tembec (Montréal, Canada). Two lignocellulosic substrates, poplar and lodgepole pine, were pretreated via SO2catalyzed steam pretreatment using a 2L StakeTech III steam gun (Stake Technologies, Norvall, ON, Canada). Prior to steam pretreatment, poplar and lodgepole pine wood chips (DW; dry weight of 300 g) were first impregnated with SO2 in a re-sealable plastic bag and allowed to react overnight. The plastic bags containing unadsorbed SO2 were then opened and allowed to vent for 60 minutes. Subsequently, 50g of the biomass were pretreated under near optimal conditions in the steam gun based on previous studies. 43,44 After pretreatment, the water insoluble fractions of the pretreated biomass were separated from the liquid fractions by vacuum filtration and stored at -20°C until further analysis. Compositional analysis of pretreated biomasses. The chemical composition of the water insoluble fractions of the steam pretreated materials were determined using the Technical Association of the Pulp and Paper Industry (TAPPI) standard method T222 om-88 as previously described by Bura et al. 43. The monomeric sugars in the acid hydrolyzate were subsequently quantified by high performance liquid chromatography (HPLC) (Dionex DX-3000, Sunnyvale, CA) using a CarboPac PA1 column and fucose as the internal standard. Acid soluble lignin was also quantified at 205nm using a UV-Vis spectrometer (Varian Cary 50, Belrose, Australia) and acid insoluble lignin was quantified using gravimetric filtration using a medium coarseness Gooch type filtering crucible (Pyrex, Corning, NY). All compositional analyses were carried out in triplicates. Enzymatic Hydrolysis. Commercial cellulase enzyme mixtures Cellic CTec 3 were provided by Novozymes North America Inc. The enzymatic hydrolysis experiments were conducted at a total volume of 25mL in 50mL round-bottom centrifuge tubes centrifuge tubes (Pyrex, Corning, NY) at a solids loading of 2% or 10% w/v in 50mM (pH=4.8) sodium acetate buffer at 50⁰C with rotational mixing at 150 rpm using a hybridization incubator (FinePCR COMBI D-24, Korea). Hydrolysis was performed over a period of 72 hours with periodic sampling of 1mL volume at 1, 2, 5, 24, 48 and 72 hours. Each collected sample was centrifuged at 13000rpm at 4oC for 10 minutes to separate the solid and liquid fractions. The liquid fractions were heated at 100oC for 10 minutes and stored at 4oC for further analysis. Total sugar produced was quantified by high-performance liquid chromatography (HPLC) (Dionex DX3000, Sunnyvale, CA) as described elsewhere 45. All experiments were performed in duplicates and repeated at least twice. To assess the role of adsorbed and free enzymes in determining the hydrolysis kinetics and yield of lignocellulosic biomass, hydrolysis with the complete enzyme mixture was allowed to proceed for 1 hour to reach adsorption equilibrium between the liquid and solid phases. The resulting hydrolyzates were subsequently centrifuged at room temperature at 5000rpm for 15 minutes to separate the liquid and solid



fractions. Various volumes of the liquid fraction, representing 25, 50 and 75% of the free enzymes in solution (based on protein concentration) were removed and replaced with an equivalent volume of 50mM sodium acetate buffer (pH=4.8) unless otherwise specified. The re-suspended solids were then hydrolyzed for a further 71 hours for a total of 72 hours with the adsorbed and residual free enzymes. For the 10% solid loading hydrolysis, the initial enzyme adsorption was performed at a 2% solid loading. Once enzyme adsorption reached equilibrium (after 1 hour at 4 °C), about 75% of the free, enzyme in solution, was removed with some of the liquid fraction, via centrifugation. The solid fraction, which was now at a 10% solids concentration due to the removal of some of the reaction media with the enzymes in solution, was hydrolyzed for another 71 hours without adding any additional buffer and enzymes in the hydrolysis system. Periodic sampling and sugar quantification were conducted. To further elucidate the role of only adsorbed enzymes, the initial 1 hour adsorption was maintained at 4oC to limit the initial hydrolysis, the liquid and solids fractions were then separated by centrifugation at 5000rpm for 15 minutes at 4oC. After centrifugation, the liquid fractions were removed and the solid fractions were re-suspended with 50mM sodium acetate buffer (pH=4.8) equivalent to the volume of liquid removed. This liquid-solid separation process was repeated until the total quantity of protein contained in the removed liquid fractions contained less than 5% of the initial protein added. The washed solids containing only adsorbed enzymes were then re-suspended with 50mM sodium acetate buffer (pH=4.8) to the initial starting hydrolysis volume and incubated at 50oC for 71 hours for a total of 72 hours. For comparison, hydrolysis experiments under the same conditions were also conducted with a complete enzyme mixture at the same enzyme loadings without liquid-solid separation. Sampling was conducted at 1 and 72 hours followed by sugar quantification as described above. All of the experiments were performed in triplicate and the mean values and error bars are reported. Protein concentration and enzyme activates. The xylanase and carboxymethyl cellulose activities (CMCase) were assessed as described by Hu et al., 46. The cellobiohydrolase and β-glucosidase activities were determined using p-nitrophenyl-β-Dcellobioside (p-NPC), p-nitrophenyl-β-D-glucopyranoside (pNPG), respectively, according to Hu et al., (2016). The protein content was measured by the modified Ninhydrin assay using bovine serum albumin (BSA) as the protein standard. 47

Supporting Information: Initial distribution of cellulase enzymes across different model and lignocellulosic substrates and total conversion obtained after hydrolysis (Table S1); relationship between enzyme loading and total glucan conversion on various steam pretreated lignocellulosic substrates (Figure S1)

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29. Hu, J.; Arantes, V.; Pribowo, A.; Gourlay, K.; Saddler, J. N. Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass. Energy Environ. Sci. 2014, 7, 2308-2315. DOI: 10.1039/C4EE00891J 30. Holtzapple, M. T.; Caram, H. S.; Humphrey, A. E. The Hch-1 Model of Enzymatic Cellulose Hydrolysis. Biotechnol. Bioeng. 1984, 26, 775-780. DOI: 10.1002/bit.260260723 31. O'Dwyer, J. P.; Zhu, L.; Granda, C. B.; Holtzapple, M. T. Enzymatic hydrolysis of lime-pretreated corn stover and investigation of the HCH-1 Model: Inhibition pattern, degree of inhibition, validity of simplified HCH-1 Model. Bioresour. Technol. 2007, 98, 2969-2977. DOI: 10.1016/j.biortech.2006.10.014 32. Zhu, L.; O'Dwyer, J. P.; Chang, V. S.; Granda, C. B.; Holtzapple, M. T. Multiple linear regression model for predicting biomass digestibility from structural features. Bioresour. Technol. 2010, 101, 4971-4979. DOI: 10.1016/j.biortech.2009.11.034. 33. Davis, M. F.; Ishizawa, C.; Jeoh, T.; Adney, W. S.; Himmel, M. E.; Johnson, D. K. Chemical and Physical Properties of Pretreated Biomass That Affect Enzyme Accessibility and Digestibility. In ACS National Meeting Book of Abstracts; 2007. 34. Ramos, L. P.; Saddler, J. N. Enzyme Recycling during Fed-Batch Hydrolysis of Cellulose Derived from Steam-Exploded Eucalyptus-Viminalis. Appl. Biochem. Biotechnol. 1994, 45-6, 193-207. 35. Mansfield, S. D.; Mooney, C.; Saddler, J. N. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol. Prog. 1999, 15, 804-816. DOI: org/10.1021/bp9900864 36. Kipper, K.; Valjamae, P.; Johansson, G. Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as 'burst' kinetics on fluorescent polymeric model substrates. Biochem. J. 2005, 385, 527-535. DOI: 10.1042/BJ20041144 37. Cruys-Bagger, N.; Elmerdahl, J.; Praestgaard, E.; Tatsumi, H.; Spodsberg, N.; Borch, K.; Westh, P. Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. J. Biol. Chem. 2012, 287, 18451-18458. DOI: 10.1074/jbc.M111.334946. 38. Maxim Kostylev, D. W. Synergistic interactions in cellulose hydrolysis. Future Science, Biofuels 2012, 3, 61-70. 39. Haven, M. Ø.; Jørgensen, H. Adsorption of β-Glucosidases in Two Commercial Preparations onto Pretreated Biomass and Lignin. Biotechnol. Biofuels 2013. DOI: org/10.1186/1754-6834-6-165 40. Di Risio, S.; Hu, C. S.; Saville, B. A.; Liao, D.; Lortie, J. Large-scale, high-solids enzymatic hydrolysis of steam-exploded poplar. Biofuels Bioproducts & Biorefining-Biofpr 2011, 5, 609-620. DOI: org/10.1002/bbb.323 41. Viamajala, S.; McMillan, J. D.; Schell, D. J.; Elander, R. T. Rheology of corn stover slurries at high solids concentrations - Effects of saccharification and particle size. Bioresour. Technol. 2009, 100, 925-934. DOI: org/10.1016/j.biortech.2008.06.070



42. Jorgensen, H.; Vibe-Pedersen, J.; Larsen, J.; Felby, C. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng. 2007, 96, 862-870. DOI: 10.1002/bit.21115 43. Bura, R.; Bothast, R. J.; Mansfield, S. D.; Saddler, J. N. Optimization of SO2-catalyzed steam pretreatment of corn fiber for ethanol production. Appl. Biochem. Biotechnol. 2003, 105, 319-335. 44. Kumar, L.; Chandra, R.; Chung, P. A.; Saddler, J. Can the same steam pretreatment conditions be used for most softwoods to achieve good, enzymatic hydrolysis and sugar yields? Bioresour. Technol. 2010, 101, 7827-7833. DOI: org/10.1016/j.biortech.2010.05.023 45. Hu, J.; Gourlay, K.; Arantes, V.; Van Dyk, J. S.; Pribowo, A.; Saddler, J. The Accessible Cellulose Surface Influences Cellulase Synergism during the Hydrolysis of Lignocellulosic Substrates. ChemSusChem. 2015. DOI: 10.1002/cssc.201403335. 46. Hu, J.; Arantes, V.; Pribowo, A.; Saddler, J. N. The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific. Biotechnol. Biofuels 2013, 6, 112. DOI: 10.1186/1754-6834-6-112. 47. Starcher, B. A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal. Biochem. 2001, 292, 125-129. DOI: 10.1006/abio.2001.5050 48. Sannigrahi, P.; Kim, D.H.; Jung, S.; Ragauskas, Pseudo-lignin and pretreatment chemistry. Energy Environ. Sci. 2011, 4, 1306-1310. DOI: 10.1039/C0EE00378F


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Table 1. Steam pretreatment conditions and chemical composition of pretreated lignocellulosic substrates

Substrates

Steam pretreatment conditions

Composition of the substrates (%)a Ara

Gal

Glu

Xyl

Man

AIL

Poplar

190oC, 5min, 3% SO2

0.1

0.2

58

4.9

1.1

30 ±

± 0.02

± 0.03

±1

± 0.3

± 0.3

2

Poplar

200oC, 5min, 3% SO2

0.1

0.1

58

1.5

0.9

34 ±

± 0.01

± 0.02

±2

± 0.2

± 0.1

1

Abbreviation SPP190 SPP200

Lodgepole 200oC, 5min, 4% 2.1 0.3 41 ± 0.5 52 1.2 LPP200 2 ± 0.1 ± 0.1 ± 1 ± 0.2 pine SO2 ± 0.3 a Ara, arabinan; Gal, galactan; Glu, glucan; Xyl, xylan; Man, mannan; AIL, acid insoluble lignin. The compositional analyses were carried out in triplicates and the mean values and error bars are reported.

Table 2. The cellulose hydrolysis yield of the complete enzyme mixture and the initially adsorbed enzymes (after extensively washing) after 72 h hydrolysis. Cellulose hydrolysis yield (%) Substrates

Complete enzyme mixture

Adsorbed enzymes

DsP

76 ± 2

66 ± 2

POP200

67 ± 0.4

66 ± 0.3

POP190

94 ± 2

91 ± 0.3

LPP200

80 ± 2

71 ± 1

DsP: dissolving pulp; SPLP: steam pretreated lodgepole pine; SPP200: steam pretreated poplar at 200C; SPP190: steam pretreated poplar at 190C. The initial enzyme adsorption on various substrates was performed for 1 hour at 4°C. The hydrolysis experiments were performed in triplicate and the mean values and error bars are reported.



Figure 1. Time course of hydrolysis of dissolving pulp (DsP) with/without the selective removal of different proportions of “free/unadsorbed” enzymes after the 1st h incubation at 50°C. Relationship between enzyme loading and total glucan conversion (inset).


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Figure 2. Time course of hydrolysis of various steam pretreated lignocellulosic substrates with/without the selective removal 75% “free/unadsorbed” enzymes after the 1st h incubation at 50°C. SPLP: steam pretreated lodgepole pine; SPP200: steam pretreated poplar at 200C; SPP190: steam pretreated poplar at 190C.



Figure 3. Cellulose hydrolysis of a range of lignocellulosic biomass at different enzyme loadings after 72 hours by using a complete enzyme mixture as compared to using the adsorbed enzymes (removal of 75% of the “free/unadsorbed” enzymes after the 1st h incubation at 50°C). DsP: dissolving pulp; SPLP: steam pretreated lodgepole pine; SPP200: steam pretreated poplar at 200C; SPP190: steam pretreated poplar at 190C.


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Figure 4. The proportion of major cellulase and xylanase enzymes activities of the complete CTec3 enzyme preparation and the 75% fraction of “free/unadsorbed” enzymes from the supernatants of the dissolving pulp (DsP) and steam pretreated lodgepole pine (SPLP) substrates after 1st h incubation at 50°C.



Figure 5. Enzymatic hydrolysis of the dissolving pulp (DsP) and steam pretreated lodgepole pine (SPLP) substrates after 72 h at 10% solid loading when using either the complete or initially adsorbed enzyme mixtures (removal 75% “free” enzymes after the 1st h incubation at 4°C). The control (direct 10% solid loading hydrolysis with only “bound” enzyme loading) was performed using initial enzyme loading equivalent to the residual amount of enzymes after 75% “free/unadsorbed” enzyme removal.


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Can We Reduce the Cellulase Enzyme Loading Required To Achieve

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