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Split addition of enzymes in enzymatic hydrolysis at high solids concentration to increase sugar concentration for bioethanol production
Journal of Industrial and Engineering Chemistry 18 (2012) 707–714
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Split addition of enzymes in enzymatic hydrolysis at high solids concentration to increase sugar concentration for bioethanol production Ying Xue, Hasan Jameel *, Richard Phillips, Hou-min Chang Department of Forest Biomaterials, North Carolina State University, Raleigh, NC 27695, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 April 2011 Accepted 11 July 2011 Available online 11 November 2011
One challenge in making bioethanol production economical is to increase total solids in hydrolysis system while maintaining sugar conversion efficiency. Because the removal of excess water from hydrolysate requires enormous amounts of heat, large volume of reaction towers and high capital expenditure (CAPEX) for equipment, a lengthy operating time, and high operating costs. When solids loading in hydrolysis system increased from 5% to 20% with no mixing strategies, final sugar conversion decreased markedly. If cellulase is mixed with pulp at 5% solids and pressed to 20% solids, then 80% of the cellulase retained in the pulp thinned down the pulp mixture in 2 h. This thinning effect enabled additional cellulase, xylanase, and b-glucosidase to be mixed into the slurry. Sugar concentration was significantly improved; from 26 g/L to 121 g/L, while sugar conversion was remained as enzymatic hydrolysis with 5% total solids enzymatic hydrolysis. A US patent has been granted to NCSU for this concept and licenses have been granted to various companies. ß 2011 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: High total solids Enzymatic hydrolysis Split addition Sugar concentration Sugar conversion
1. Introduction Biomass has been considered as the only sustainable source of organic carbon. Biofuels, or fuels derived from plant biomass, are the only current sustainable source of liquid fuels [1–4]. Global biofuel production has been increased sharply, but still accounts less than 3% of the global transformational fuel supply [5]. The expanding demand of starch as energy source has driven food price to near record level [6]. Fuel from lignocellulosic waste, could be one of the solution to alleviate the impact of food price of biofuel production, and also relief fossil fuel crises and climate change [7]. Fuel ethanol from lignocellulosic biomass can be produced in three integrated stages: pretreatment, hydrolysis, and fermentation. The purpose of the pretreatment stage is to reduce the recalcitrance of lignocellulose for more effective hydrolysis. Pretreatment can include alkalis, acids, water, gases, enzymes, organic solvents, ionic solvents at a specific temperature, pressure, or physical forces [8]. The advantage of alkaline pretreatment is its ability to recover most of the chemicals used in process. Alkaline pretreatment in the presence of sodium sulfide (Na2S) can help alkali interact and dissolve lignin in the pretreatment liquor. The chemicals and dissolved lignin can be recovered through burning and causticization, which can tremendously decrease the cost of pretreatment
* Corresponding author. Tel.: +1 919 515 7739; fax: +1 919 515 6302. E-mail address: [email protected] (H. Jameel).
[9]. Recycling the chemicals not only decreases the cost of pretreatment, but also decreases the concentration of inhibitors present in enzymatic hydrolysis and fermentation, created by the pretreatment chemicals and byproducts. Therefore, it presented an opportunity for the study of enzymatic hydrolysis with high total solids. Therefore, alkaline pretreatment was selected for this study. The second stage of this study involves hydrolysis to convert the polysaccharides in biomass to fermentable mono sugars. To efficiently convert pretreated lignocellulosic biomass to fermentable sugars employing enzymatic hydrolysis, three main types of hydrolytic enzymes are involved: cellulase, hemicellulase (xylanase), and b-glucosidase. Cellulose, one of the major components of biomass, is a b-1,4 linked cellobiose chain. Cellulase is comprised of a family of different enzymes, including exo-1, 4-b-D-glucanase (cellobiohydrolase, also abbreviated as CBH) (EC 3.2.1.91), which cuts cellobiose units off from the ends. Two types of CBHs work for this function: one works from the reducing end (CBH I), and the other works from the non-reducing end (CBH II). Endo-1, 4-b-Dglucanase (EG) (EC 3.2.1.4) randomly cleaves b-1,4-glucosidic bonds inside the cellulose chain. 1,4-b-D-Glucosidase (cellobiase) (EC 3.2.1.21) hydrolyzes cellobiose into glucose and also cleaves off glucose units from cello-oligosaccharide chains. These enzymes work synergistically to hydrolyze cellulose by creating new accessible sites for each other, removing obstacles, and relieving product inhibition [10,11]. For example, as CBHs increase the concentration of cellobiose in the hydrolytic system, the activity of
1226-086X/$ – see front matter ß 2011 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry. doi:10.1016/j.jiec.2011.11.132
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CBH could be inhibited by cellobiose, its end product. Cellobiase can further digest cellobiose into glucose, and therefore reduce the end product inhibition of CBHs by cellobiose. Hemicellulose in biomass can hinder the reaction of cellulase with the cellulose chain. The function of hemicellulase is to depolymerize the heterogeneous hemicellulose into mono sugars. Hence, hemicellulase also works synergistically with cellulase as it removes hemicellulose from the biomass structure. Hemicellulose removal releases cellulose bundles and provides easier access to its reaction sites [12–14]. Typical enzymatic hydrolysis of woody biomass was conducted at 2–5% total solids substrate (w/w) to ensure proper contact between enzyme and substrate [15]. For enzymatic hydrolysis with 5% total solids, the sugar concentration can never exceed 50 g/ L. Inefficient mixing would occur above 10% total solids, during which no obvious whirling of liquid can be observed [16]. With high total solids, cellulose can swell to create a thick mixture, creating difficulty, for instance, in transfer between reactors. Moreover, the removal of excess water from hydrolysate requires an enormous amount of heat, large volume reaction tanks, and a high capital cost for equipment [17]. Further, distillation energy consumption decreases sharply when ethanol concentration increases to 4–5 wt.%, which is equivalent to about 100 g/L of sugar concentration [18,19]. Such a high sugar concentration requires an enzymatic hydrolysis process with high total solids, and high total solids in enzymatic hydrolysis, and it is therefore one of the key issues in decreasing bioethanol production costs. There are limited studies that discuss a enzymatic hydrolysis process with high total solids industry application, such as Cara et al. [20]. However, the hydrolysis efficiency at 20% total solids is 13–25% lower than observing 5% total solids at 48 h. Therefore, a novel enzymatic hydrolysis process for real biomass is needed for better economical feasibility of bioethanol production. This work is based on the concept of an enzyme binding onto its substrate. Most studies of adsorption were performed with cellulase mono components or domains under 2–8 8C, with pure cellulose as a substrate, while some of the studies were performed at room temperature [21–24]. Data related to adsorption of enzymes onto lignocellulose feedstock at higher temperatures is limited [15,25]. A higher temperature is preferable for industrial processes, given the fact that the substrate is usually at a high temperature from pretreatment before enzymatic hydrolysis [26]. Moreover, 40–50 8C is the range that most lignocellulytic enzymes digest substrates at the highest efficiency [27]. The adsorption of enzymes was investigated on pretreated biomass at 50 8C in this work. In this study, the adsorption behavior of enzymes onto pretreated biomass was measured, the feasibility of decreasing the amount of water used in the process as much as possible without hurting the hydrolysis performance of enzymes was assessed, the process development was presented, and the bestcase scenario was reported. The results suggested that it is beneficial to dose enzymes according to their adsorption characteristics, in order to develop a better process for an industrial application. 2. Materials and methods
Table 1 Pretreatment condition of wood chips for green liquor pretreated pulp. TTAa Sulfidityb H Factorc Liquor to wood ratio Cooking temp
16% 25% 800 4:1 160 8C
a Total of all viable sodium alkali compounds. Calculated as Na2O based on bone dry wood chips. b Sulfidity was defined as the ratio of Na2S to TTA. c When relative reaction rate was plotted against cooking time in hours, the area under the curve was defined as H factor.
Titratable Alkaline as Na2O and the contents pulped to the target H Factor. Table 1 shows the pulping conditions that were used. The pulp yield was 78.4% based on wood. After pulping, the samples were washed overnight. The yield was measured by centrifuging the chip mass and measuring the total solids and total weight. The chips were then disintegrated using a refiner with a 0.005 in. gap and screened using a 0.008 in. screen plate. The rejects were refined with a disk gap of 0.001 in. and added back to the accepts. The pulp was then centrifuged and fluffed for further processing. 2.2. Raw material analysis Small scale TAPPI method was conducted for raw materials analysis [28]. Air-dried 0.1 OD gram pulp with 92–95% total solids was added with 1.5 mL of 72% sulfuric acid (Fisherbrand, Fair Lawn, NJ) in a room temperature water bath. The pulp was stirred every 15 min for the duration of the 2-h reaction and transferred into a serum bottle with 56 mL of deionized water. Sealed with an aluminum cap, the serum bottle with the sample solution was heated up to 120 8C at 1.25 atm pressure for 90 min in an autoclave. The autoclaved suspension was then filtered with a finesized crucible after being cooled down with running tap water. The filtrate was collected for acid-soluble lignin content analysis at 205 nm wavelength with a UV-Vis spectroscope (Perkin Elmer, Model Lambda XLS, Waltham, MA), and the residue in the crucible was oven dried for Klason lignin content analysis. The filtrate was also used for sugar analysis, which was performed on an Ion Chromatography with Carbo Pac PA 10 (Dionex, Sunnydale, CA). The component of the pulps are shown in Table 2. 2.3. Enzyme activity assays Enzyme activity for cellulase and xylanase test was tested according to the IUPAC standard method by Ghose [29]. A straight standard line was built with a Whatman No. 1 filter paper (Whatman, England). The 1 cm 6 cm strip reacted with diluted cellulase solution for 60 min in the working buffer (NaAc, NaN3, HAc were from Fisherbrand, Fair Lawn, NJ). The reducing sugar was then tested with 3,5-dinitrosalysilic acid solution (3,5-dinitrosalysilic acid, potassium sodium tartarate, and sodium metasulfite, were from Sigma Aldrich, St. Louis, MO and phenol were from Fluka, Switzerland) for color development by reading UV absorbance at 540 nm (PerkinElmer UV spectroscope, model Lambda XLS) and back-calculated for enzyme stock solution activity. When the cellulase activity was too low for the Ghose method (lower than 0.37 FPU/mL), a standard curve was created by reacting
2.1. Green liquor-pretreated pulps Mixed hardwood chips from a mill in the southeastern United States were used in this study. The chips were screened and the accepts from 3/8 in. and 5/8 in. holes were used for pretreatment. The pretreatments were done in a 7-L M&K Digester with 800 OD grams of chips. The green liquor (comprised of Na2CO3 and Na2S, Fisherbrand Fair Lawn, NJ) was charged, based on percent Total
Table 2 Raw materials analysis. Pulp
Glucan
Xylan
AISLa
ASLb
Balance
Error
GL16
61.1%
15.0%
20.0%
2.9%
99.0%
1.1%
a b
Acid insoluble lignin content. Acid soluble lignin content.
Y. Xue et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 707–714
different concentrations of cellulase with a 1 cm 6 cm strip of Whatman No. 1 filter paper for 60 min of incubation time. 2.4. Enzyme adsorption The buffer and enzymes were pre-incubated in a 50 8C water bath for 10 min. Enzymes and buffer were added into the pulp to make a mixture with 5% total solids, mixed well with a vortex mixer for 30 s. Then, the mixture was put back into the 50 8C incubator for 10 min. The liquid phase was separated by vacuum filtration with a C-sized crucible (Kimax, Vineland, MA). The consistencies of the residue for the pulps tested were 19–21%. The filtrate was tested for the enzyme concentration, and the difference between the original amount of enzyme added to the mixture and the enzyme in the filtrate was assumed to be the enzyme that remained in the residue. 2.5. Enzymatic hydrolysis Cellulase, xylanase, and b-glucosidase systems in separate solutions were used in this study in a mixing ratio of 10:3:3. The dosage of xylanase and b-glucosidase was recommended by the vendor. Enzymatic hydrolysis and enzyme adsorption were performed in 0.1 M acetate buffer at pH 4.8, with NaN3 as an antibiotic. All enzymatic hydrolysis was conducted at 50 8C in a water bath, and the agitation level was set to 90 rpm. After enzymatic hydrolysis, the hydrolysate was filtered through C-sized crucibles. Filtrate was collected for IC sugar analysis, and the residue in the crucible was dried in an oven overnight in an oven at 120 8C for weight loss monitoring. 2.6. Sugar test After obtaining the filtrate of enzymatic hydrolysate, it was filtered through a 0.45 mm Millipore syringe filter (Billerica, MA), and diluted to the desired range of concentration with Milli-Q water. A sugar test was performed with a Dionex IC 3000 system with a Carbo Pac PA 10 sugar separation column. The fluid was H2O, at the speed of 1.1 mL/min, which gives a pressure of 1800 psi. 200 mM NaOH was used as the eluent for cleaning purposes. Standard sugars D-glucose, D-mannose, D-xylose, Dgalactose, D-rhaminose, L-arabinose, and D-fructose were from Sigma Aldrich (St. Louis, MO), and chromatographic grade NaOH was from Fisherbrand (Fair Lawn, NJ). Sugar yield (sugar conversion) was defined as the ratio created by dividing the amount of sugar in the filtrate by the amount of sugar in the substrate. 3. Results and discussion There is always need to develop high-efficiency process for biofuel production [30,31]. One area in need of improvement is to increase the solids loading in enzymatic hydrolysis to decrease operating and capital cost. Three different strategies were developed and evaluated for enzymatic hydrolysis with high total solids: Scheme CXB (Fig. 1a): cellulase, xylanase, and b-glucosidase were mixed with the substrate to obtain the desired total solids. After 48 h the reaction was stopped by filtering the suspension with a course-sized crucible. Scheme C + XB (Fig. 1b): cellulase was added into the pulp containing 5% total solids. After 10 min of retention time, this mixture was filtered. The filtrate was collected and analyzed for sugar concentration. Thickened pulp was reacted in a water bath for another 2–8 h and supplementary xylanase and b-glucosidase
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were added afterward. Collection of hydrolysate at 48 h was the same as the CXB Scheme. Scheme C + CXB (Fig. 1c): part of the cellulase was added to the pulp, to make a 5% total solids mixture. The pulp was thickened as described in the C + XB process after 10 min. The remaining cellulase was added with supplementary xylanase and b-glucosidase after 2 h of incubation time. Collection of filtrate at 48 h was the same as in CXB and C + XB procedures. Green liquor-pretreated hardwood pulp, representing a real lignocellulosic substrate, was used in the following studies for evaluating the process developed above. 3.1. Scheme CXB (conventional strategy) The disadvantage of low total solids in enzymatic hydrolysis is the low sugar concentration, which makes the amount of energy required for distilling alcohol from fermentation economically not feasible. In contrast, a high concentration of sugar can reduce the amount of energy required for distillation. It also decreases capital and operational costs for evaporation, fermentation, and distillation, since the equipment could be smaller. One method to increase sugar concentration is to reduce the amount of water used in enzymatic hydrolysis so that the sugar concentration could increase tremendously. However, the total solids in enzymatic hydrolysis influences sugar yield [20]. Total sugar yield decreased as the total solids increased, as shown in Fig. 2a. One reason for this may be due to inefficiently mixing the enzyme with the substrate. Although hydrolysis with 5% total solids produced a 64% sugar yield, the lower yield (54%) in hydrolysis with 10% total solids has a higher sugar concentration (46 g/L) than in hydrolysis with 5% total solids (26 g/L) at 20 FPU/g substrate enzyme loading. When the total solids was increased to 20%, sugar yield was decreased from 64% to 44% (Fig. 2b). However, in this same scenario, the sugar concentration increased from 26 to 84 g/L. A marked difference in sugar yield was measured at low enzyme dosages when comparing the efficiency of hydrolysis with 5% and 20% total solids with Scheme CXB. This difference decreased as the enzyme loading increased, so that at very high enzyme loading the difference in enzymatic hydrolysis was lower. However, high enzyme loadings are not practical because of the cost of enzymes. For example, sugar yield only decreased from 70% to 68% (40 FPU/g substrate). In comparison, sugar yield decreased from 40% to 19% when total solids were increased from 5% to 20% at 5 FPU/g substrate enzyme loading. The small difference of sugar yield with 5% and 20% total solids at high enzyme loading is more likely due to fast early stage hydrolysis. The cellulose was cut into smaller molecular weights [27], which gave enzymes a better chance to penetrate into bulk substrate earlier. In spite of the increased sugar concentration in hydrolysis with elevated total solids, the sugar yield from the substrate was not high enough to make this process commercially feasible. One problem caused by high total solids was inefficient mixing [32] of enzymes with substrate in a hydrolysis system, especially in the early stages of hydrolysis when the pulp was thick. Evenly distributed enzyme in the pulp suspension might help it thin more quickly, making thorough mixing in the later stages possible. 3.2. Scheme C + XB If conventional Scheme CXB did not yield high sugar conversion due to inefficient mixing of enzymes with substrates, it would be beneficial to mix the enzymes capable of adsorbing [33,34] onto fiber surfaces at low total solids before the pulp was thickened. After the thickened pulp was thinned again by the hydrolysis reaction with the cellulase enzymes adsorbed on the substrate [35,36], supplementary enzymes could then be blended into the
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a
Sugar
C,B, X
48 Hour Retention
Pulp
Filter
C = Cellulase B= β- Glucosidase X= Xylanase
b
Residue
C Liquor =9 Dilution/Mix Tank 5% TS
Sugars to Fermentation 19 10 min
4
Filter 20% TS
0-8 hour Retention
15
4 Mix X, B 40-48 Hour Retention C = Cellulase B= β- Glucosidase X= Xylanase
c
Lignin to Boiler
Filter
C Liquor =9 Dilution/Mix Tank 5% TS
Sugars to Fermentation 19 10 min
Filter 20% TS 15
4 2 hour Retention 4 Mix
C, X, B 46 Hour Retention C = Cellulase B= β- Glucosidase X= Xylanase
Lignin to Boiler
Filter
Fig. 1. (a) Scheme CXB for enzymatic hydrolysis. Cellulase, xylanase and b-glucosidase were added into pulp mixture at desired total solids level. After 48 h of incubation at 50 8C, the mixture was subjected to vacuum filtration to separate liquid phase and residue. (b) Scheme C + XB for enzymatic hydrolysis with 20% total solids. Cellulase was added to 5% total solids pulp and mixed well. After 10 min of mixing and retention, the pulp was thickened to 20% total solids by vacuum filtration. After various time intervals, supplementary xylanase and b-glucosidase were added into the thinned mixture, and incubated for a total 48 h. (c) Split addition of cellulase, part of the cellulase was added to pulp with 5% total solids for even distribution and thinning effect on pulp. The rest of the cellulase was added with xylanase and b-glucosidase after the pulp was thinned. (d) A whole view of the saccharification process. Filtrate from thickening process containing some of the sugar could be used as dilution water and washing liquid for fresh substrate, therefore the enzyme loss and the sugar loss could be minimized.
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C
d
Sugars to Fermentation Liq=7.3
Washer From Digester
Dilution/Mix Tank 5% K
19
11 4
4
Press 20% K
2 hour Retention
15
4
Sugars + residual enzymes
Mixer C, B, X
C = Cellulase B= bGlucosidase X= Xylanase
46 Hour Retention
Lignin Filter Lignin to Boiler
Fig. 1. (Continued ).
pulp mixture without any mixing problems. This process could create a better chance to achieve high sugar concentration without decreasing the sugar yield. Therefore, an enzyme adsorption study was performed to help investigate which enzyme adheres onto the pulp fiber surface best and should be added before thickening. It also identified which enzyme would not be absorbed on the pulp and should be added after the thickening process. Cellulase and xylanase were thoroughly mixed with hardwood pulps of different lignin contents at 5% total solids and a
Fig. 2. (a) Kinetics of total sugar yield over various enzymatic total solids for 48 h time range at 50 8C. The pulp total solids were 5%, 7.5%, and 10%, and the enzyme charge was 20 FPU/g substrate. (b) Sugar yield from hydrolysis Scheme C + XB at 20% total solids, compared with conventional enzymatic hydrolysis Scheme CXB with 5% and 20% total solids, which was conducted without a particular mixing strategy.
temperature of 50 8C. In this experiment, the enzymes were mixed into the pulps at 5% total solids and then thickened to 20%. During the thickening process, 20% of the liquid was retained in the pulp and 80% of the liquid was removed. If no adsorption occurred, the enzymes would be split in the same ratio. Fig. 3 shows the cellulase and xylanase adsorption onto pulps with a wide range of lignin content and demonstrates their different adsorption behaviors. Around 70–80% of FPU cellulase activity remained on residual hardwood alkaline-pretreated pulp with a 0–26% lignin content. Therefore, cellulase was preferentially adsorbed on the substrate. However, the birch xylanase adsorption activity test only demonstrated around 18–20% birch xylanase activity remaining on the substrate with the same conditions. Taking into consideration that around 20% of the liquor remained in the residue after filtration, the loss of birch xylan activity was almost proportional to the loss of liquor in the mixture. This data implies that no xylanase adsorption occurred. The birch xylan activity in the xylanase solution appeared to be retained only in the liquid rather than that adsorbed onto substrate, compared to obvious adsorption of cellulase FPU activity. This might due to the low xylan content in the pulp. Therefore the adsorption site available for the xylanase was low in the pulp. This was in accordance with Kurakake et al.’s observation that 7.7–34.2% xylanase adsorption
Fig. 3. Cellulase and xylanase adsorption onto alkaline pretreated hardwood pulp at different lignin contents. Adsorption was conducted at 50 8C, 5% total solids, 20 FPU/g substrate enzyme loading, xylanase was dosed as recommended by vendor and retention time was 10 min. *The adsorption percentage was based on activity.
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on lignocellulose biomass [37]. b-Glucosidase works on soluble cellobiose [27] and it is therefore not necessary to be added at the thickening stage with insoluble substrate. A new process was developed according to these adsorption properties of different enzymes, to achieve better enzyme distribution and minimize enzyme loss. Because cellulase appeared to remain on the pulp while xylanase tended to leave the pulp mixture during water removal, cellulase was mixed into pulp at low total solids. After thorough mixing at a low solids concentration, water was removed with a vacuum filter. The time at which supplementary xylanase and bglucosidase are added can influence sugar conversion efficiency. Addition of xylanase and b-glucosidase at an earlier time, when the pulp is not thinned, will result in an uneven distribution in the pulp mixture. In contrast, late addition of the xylanase and bglucosidase will make the interaction time of the added enzymes with substrates too short for efficient hydrolysis. The lack of time for synergistic effect between xylanase (or b-glucosidase) with cellulase would also decrease final sugar yield, since xylanase can release cellulose chains from hemicellulose hindrance, and bglucosidase can eliminate end-product inhibition for cellulase. Thus, the proper time for the addition of these two enzymes after the pulp-thickening process to yield highest sugar conversion was investigated (Fig. 4). The difference of sugar conversion between 1 h and 2 h interval process is marginal. And 2 h interval would be a safer time point for supplementary enzyme addition, since the pulp should be thinner than 1 h interval and newly added enzymes could be well mixed into the system. Hence, 2 h interval might be the optimum time to add b-glucosidase and xylanase. This time may vary with the enzyme charge and the substrate recalcitrance. In the following experiments, a 2-h time interval was chosen for adding supplementary enzymes, to improve enzymatic hydrolysis efficiency. And of course, this process can be further optimized. Sugar yield that resulted from separately adding enzymes showed better conversion efficiency than simply mixing the enzyme cocktail with pulp with 20% total solids. In that case, the sugar conversion was 44% for Scheme CXB but 59% for Scheme C + XB at 20 FPU/g enzyme dosage, as is shown in Figs. 2b and 5b. And the sugar concentration was increased from 84 g/L to 114 g/L at 20 FPU/g enzyme dosage after 48 h as well. When comparing with 5% total solids Scheme CXB at the same hydrolysis conditions, the sugar concentration was increased from 26 g/L to 114 g/L, while the sugar conversion is a little lower than 5% total solids Scheme CXB case (59% compared with 64%). This process was especially beneficial for lower enzyme loading. For 5 FPU, when the total solids of conventional process was increased from 5% to 20%, the sugar conversion was decreased from 40% to 19%, as was mentioned in Section 3.1. However, when Scheme C + XB was applied, the 20% total solids yielded 38% sugar conversion.
Fig. 4. Addition of supplementary xylanase and b-glucosidase at different time intervals after cellulase adsorption stage.
Although around 4% sugar (Fig. 5a) and 20% cellulase FPU activity could be expected to be lost during mixing and thickening at 20 FPU enzyme loading in the filtrate. However, filtrate from the thickening filter (or press on an industrial scale) could be used for diluting or washing fresh feedstock, as is shown in Fig. 1d, to minimize sugar and enzyme loss. 3.3. Scheme C + CXB The cellulase addition strategy in high solids enzymatic hydrolysis schemes may benefit from further optimization. If all the cellulase was added into low total solids pulp at the beginning of the process, as was shown in Scheme C + XB (Fig. 1b), cellulase could be thoroughly mixed with pulp, and after water removal, xylanase and b-gludosidase may be more easily mixed into the bulk solid since the cellulase adsorbed on pulp helped hydrolyze part of the substrate and thinned down the system. However, in the Scheme C + XB, some fraction of cellulase was lost in the filtrate during the thickening process, especially enzyme proteins lacking in binding domains. Previous work observed that the molar ratio of cellulase components is an important factor that determines the efficiency of enzymatic hydrolysis [27,38,39]. As a result, the overall efficiency of sugar conversion would be low. But, if all cellulase is added to xylanase and b-glucosidase into a substrate with 20% total solids to avoid the loss of enzyme, enzymes could not be mixed well with substrate as was shown in the previous discussion. Because inefficient distribution of cellulase led to an inefficient first stage thinning effect, the sugar conversion was therefore expected to be low, although there was no enzyme lost. The scheme involving split addition of the cellulase enzyme was studied to optimize the effects of enzyme addition. (Fig. 6a–c). When adding half of the cellulase in the adsorption stage and the other half with supplementary xylanase and b-glucosidase, the sugar yield after 48 h would be almost the same as enzymatic
Fig. 5. (a) 10 min sugar yield in thickening filtrate in Scheme C + XB over a range of 5–40 FPU/g substrate enzyme loading. (b) Final sugar yield after 48 h for Scheme C + XB over a range of 5–40 FPU/g substrate enzyme loading.
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Table 3 Sugar yield and sugar concentration of schemes evaluated at 20 FPU/g substrate enzyme loading, 48 h incubation time, and 50 8C with pH 4.8 acetic acid buffer.
Scheme Scheme Scheme Scheme
CXB 5% CXB 20% C + XB 20% C + CXB 20%
Sugar yield (%)
Sugar concentration (g/L)
64 44 59 63
26 84 114 121
concentration was raised from 26 g/L to 121 g/L. Sugar concentration and sugar conversion of all schemes are summarized in Table 3. 4. Conclusion A more economically feasible process to run enzymatic hydrolysis with high total solids that can be implemented on a commercial scale was developed in this study. Using the scheme proposed in this paper the solids content can be increased to 20% while maintaining the enzymatic hydrolysis conversion efficiency comparable to that with 5% total solids. This increases the sugar concentration from 26 g/L to 121 g/L. The key to this process for increasing solids content in enzymatic hydrolysis is to mix a fraction of the cellulase with the pulp at 5% low total solids, thicken or press to 20% solids and allow the adsorbed cellulase to perform the necessary thinning effect which enabled thorough mixing of supplementary enzymes specially xylanase and beta-glucosidase. NCSU was granted a US patent for this concept in 2010 and licenses have been granted to companies. Acknowledgements This work was funded by the Wood-to-Ethanol Research Consortium (WERC). WERC members are: American Process, Andritz, Arborgen, KBR Engineers, Catchlight, Evolution Resources, and Japan Pulp and Paper Research Institute. Enzymes were kindly provided by Novozymes. The authors are grateful to WERC member companies and Novozymes. References Fig. 6. (a–c) Split addition of cellulase (Scheme C + CXB) at 10 FPU, 20 FPU, 40 FPU/g substrate enzyme loading at 20% total solids, compared with Scheme CXB containing 5% total solids.
hydrolysis with 5% total solids. For 10 FPU/g substrate enzyme loading, a 43% sugar yield could be achieved with 5% total solids. The split addition of enzymes (Scheme C + CXB) to pulp with 20% total solids however, could yield 46% sugar conversion. At 20 FPU enzyme loading, 64% sugar yield was achieved in 5% total solids hydrolysis, while 63% sugar yield was achieved by Scheme C + CXB hydrolysis with 20% total solids. At 40 FPU, sugar yield was 70% at 5% total solids, and also 70% from Scheme C + CXB hydrolysis with 20% total solids. The lower water content of a hydrolysis system with high total solids made the hydrolysate sugar concentration 4– 5 times higher than the concentration obtained by a system with low total solids. The sugar concentration for 20 FPU/g substrate enzyme loading could achieve 121 g/L in enzymatic hydrolysis with 20% total solids, compared to 26 g/L in enzymatic hydrolysis with 5% total solids. Through the optimization of the process studied in this work, sugar concentration increased by 95 g/L (fourfold). Through Scheme C + CXB, sugar yield was 63%, and the sugar concentration increased to 121 g/L at 20 FPU/g substrate enzyme loading, compared to the conventional 5% total solids Scheme CXB. Overall enzymatic hydrolysis efficiency was the same, while sugar
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Contents lists available at SciVerse ScienceDirect
Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Split addition of enzymes in enzymatic hydrolysis at high solids concentration to increase sugar concentration for bioethanol production Ying Xue, Hasan Jameel *, Richard Phillips, Hou-min Chang Department of Forest Biomaterials, North Carolina State University, Raleigh, NC 27695, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 April 2011 Accepted 11 July 2011 Available online 11 November 2011
One challenge in making bioethanol production economical is to increase total solids in hydrolysis system while maintaining sugar conversion efficiency. Because the removal of excess water from hydrolysate requires enormous amounts of heat, large volume of reaction towers and high capital expenditure (CAPEX) for equipment, a lengthy operating time, and high operating costs. When solids loading in hydrolysis system increased from 5% to 20% with no mixing strategies, final sugar conversion decreased markedly. If cellulase is mixed with pulp at 5% solids and pressed to 20% solids, then 80% of the cellulase retained in the pulp thinned down the pulp mixture in 2 h. This thinning effect enabled additional cellulase, xylanase, and b-glucosidase to be mixed into the slurry. Sugar concentration was significantly improved; from 26 g/L to 121 g/L, while sugar conversion was remained as enzymatic hydrolysis with 5% total solids enzymatic hydrolysis. A US patent has been granted to NCSU for this concept and licenses have been granted to various companies. ß 2011 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.
Keywords: High total solids Enzymatic hydrolysis Split addition Sugar concentration Sugar conversion
1. Introduction Biomass has been considered as the only sustainable source of organic carbon. Biofuels, or fuels derived from plant biomass, are the only current sustainable source of liquid fuels [1–4]. Global biofuel production has been increased sharply, but still accounts less than 3% of the global transformational fuel supply [5]. The expanding demand of starch as energy source has driven food price to near record level [6]. Fuel from lignocellulosic waste, could be one of the solution to alleviate the impact of food price of biofuel production, and also relief fossil fuel crises and climate change [7]. Fuel ethanol from lignocellulosic biomass can be produced in three integrated stages: pretreatment, hydrolysis, and fermentation. The purpose of the pretreatment stage is to reduce the recalcitrance of lignocellulose for more effective hydrolysis. Pretreatment can include alkalis, acids, water, gases, enzymes, organic solvents, ionic solvents at a specific temperature, pressure, or physical forces [8]. The advantage of alkaline pretreatment is its ability to recover most of the chemicals used in process. Alkaline pretreatment in the presence of sodium sulfide (Na2S) can help alkali interact and dissolve lignin in the pretreatment liquor. The chemicals and dissolved lignin can be recovered through burning and causticization, which can tremendously decrease the cost of pretreatment
* Corresponding author. Tel.: +1 919 515 7739; fax: +1 919 515 6302. E-mail address: [email protected] (H. Jameel).
[9]. Recycling the chemicals not only decreases the cost of pretreatment, but also decreases the concentration of inhibitors present in enzymatic hydrolysis and fermentation, created by the pretreatment chemicals and byproducts. Therefore, it presented an opportunity for the study of enzymatic hydrolysis with high total solids. Therefore, alkaline pretreatment was selected for this study. The second stage of this study involves hydrolysis to convert the polysaccharides in biomass to fermentable mono sugars. To efficiently convert pretreated lignocellulosic biomass to fermentable sugars employing enzymatic hydrolysis, three main types of hydrolytic enzymes are involved: cellulase, hemicellulase (xylanase), and b-glucosidase. Cellulose, one of the major components of biomass, is a b-1,4 linked cellobiose chain. Cellulase is comprised of a family of different enzymes, including exo-1, 4-b-D-glucanase (cellobiohydrolase, also abbreviated as CBH) (EC 3.2.1.91), which cuts cellobiose units off from the ends. Two types of CBHs work for this function: one works from the reducing end (CBH I), and the other works from the non-reducing end (CBH II). Endo-1, 4-b-Dglucanase (EG) (EC 3.2.1.4) randomly cleaves b-1,4-glucosidic bonds inside the cellulose chain. 1,4-b-D-Glucosidase (cellobiase) (EC 3.2.1.21) hydrolyzes cellobiose into glucose and also cleaves off glucose units from cello-oligosaccharide chains. These enzymes work synergistically to hydrolyze cellulose by creating new accessible sites for each other, removing obstacles, and relieving product inhibition [10,11]. For example, as CBHs increase the concentration of cellobiose in the hydrolytic system, the activity of
1226-086X/$ – see front matter ß 2011 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry. doi:10.1016/j.jiec.2011.11.132
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CBH could be inhibited by cellobiose, its end product. Cellobiase can further digest cellobiose into glucose, and therefore reduce the end product inhibition of CBHs by cellobiose. Hemicellulose in biomass can hinder the reaction of cellulase with the cellulose chain. The function of hemicellulase is to depolymerize the heterogeneous hemicellulose into mono sugars. Hence, hemicellulase also works synergistically with cellulase as it removes hemicellulose from the biomass structure. Hemicellulose removal releases cellulose bundles and provides easier access to its reaction sites [12–14]. Typical enzymatic hydrolysis of woody biomass was conducted at 2–5% total solids substrate (w/w) to ensure proper contact between enzyme and substrate [15]. For enzymatic hydrolysis with 5% total solids, the sugar concentration can never exceed 50 g/ L. Inefficient mixing would occur above 10% total solids, during which no obvious whirling of liquid can be observed [16]. With high total solids, cellulose can swell to create a thick mixture, creating difficulty, for instance, in transfer between reactors. Moreover, the removal of excess water from hydrolysate requires an enormous amount of heat, large volume reaction tanks, and a high capital cost for equipment [17]. Further, distillation energy consumption decreases sharply when ethanol concentration increases to 4–5 wt.%, which is equivalent to about 100 g/L of sugar concentration [18,19]. Such a high sugar concentration requires an enzymatic hydrolysis process with high total solids, and high total solids in enzymatic hydrolysis, and it is therefore one of the key issues in decreasing bioethanol production costs. There are limited studies that discuss a enzymatic hydrolysis process with high total solids industry application, such as Cara et al. [20]. However, the hydrolysis efficiency at 20% total solids is 13–25% lower than observing 5% total solids at 48 h. Therefore, a novel enzymatic hydrolysis process for real biomass is needed for better economical feasibility of bioethanol production. This work is based on the concept of an enzyme binding onto its substrate. Most studies of adsorption were performed with cellulase mono components or domains under 2–8 8C, with pure cellulose as a substrate, while some of the studies were performed at room temperature [21–24]. Data related to adsorption of enzymes onto lignocellulose feedstock at higher temperatures is limited [15,25]. A higher temperature is preferable for industrial processes, given the fact that the substrate is usually at a high temperature from pretreatment before enzymatic hydrolysis [26]. Moreover, 40–50 8C is the range that most lignocellulytic enzymes digest substrates at the highest efficiency [27]. The adsorption of enzymes was investigated on pretreated biomass at 50 8C in this work. In this study, the adsorption behavior of enzymes onto pretreated biomass was measured, the feasibility of decreasing the amount of water used in the process as much as possible without hurting the hydrolysis performance of enzymes was assessed, the process development was presented, and the bestcase scenario was reported. The results suggested that it is beneficial to dose enzymes according to their adsorption characteristics, in order to develop a better process for an industrial application. 2. Materials and methods
Table 1 Pretreatment condition of wood chips for green liquor pretreated pulp. TTAa Sulfidityb H Factorc Liquor to wood ratio Cooking temp
16% 25% 800 4:1 160 8C
a Total of all viable sodium alkali compounds. Calculated as Na2O based on bone dry wood chips. b Sulfidity was defined as the ratio of Na2S to TTA. c When relative reaction rate was plotted against cooking time in hours, the area under the curve was defined as H factor.
Titratable Alkaline as Na2O and the contents pulped to the target H Factor. Table 1 shows the pulping conditions that were used. The pulp yield was 78.4% based on wood. After pulping, the samples were washed overnight. The yield was measured by centrifuging the chip mass and measuring the total solids and total weight. The chips were then disintegrated using a refiner with a 0.005 in. gap and screened using a 0.008 in. screen plate. The rejects were refined with a disk gap of 0.001 in. and added back to the accepts. The pulp was then centrifuged and fluffed for further processing. 2.2. Raw material analysis Small scale TAPPI method was conducted for raw materials analysis [28]. Air-dried 0.1 OD gram pulp with 92–95% total solids was added with 1.5 mL of 72% sulfuric acid (Fisherbrand, Fair Lawn, NJ) in a room temperature water bath. The pulp was stirred every 15 min for the duration of the 2-h reaction and transferred into a serum bottle with 56 mL of deionized water. Sealed with an aluminum cap, the serum bottle with the sample solution was heated up to 120 8C at 1.25 atm pressure for 90 min in an autoclave. The autoclaved suspension was then filtered with a finesized crucible after being cooled down with running tap water. The filtrate was collected for acid-soluble lignin content analysis at 205 nm wavelength with a UV-Vis spectroscope (Perkin Elmer, Model Lambda XLS, Waltham, MA), and the residue in the crucible was oven dried for Klason lignin content analysis. The filtrate was also used for sugar analysis, which was performed on an Ion Chromatography with Carbo Pac PA 10 (Dionex, Sunnydale, CA). The component of the pulps are shown in Table 2. 2.3. Enzyme activity assays Enzyme activity for cellulase and xylanase test was tested according to the IUPAC standard method by Ghose [29]. A straight standard line was built with a Whatman No. 1 filter paper (Whatman, England). The 1 cm 6 cm strip reacted with diluted cellulase solution for 60 min in the working buffer (NaAc, NaN3, HAc were from Fisherbrand, Fair Lawn, NJ). The reducing sugar was then tested with 3,5-dinitrosalysilic acid solution (3,5-dinitrosalysilic acid, potassium sodium tartarate, and sodium metasulfite, were from Sigma Aldrich, St. Louis, MO and phenol were from Fluka, Switzerland) for color development by reading UV absorbance at 540 nm (PerkinElmer UV spectroscope, model Lambda XLS) and back-calculated for enzyme stock solution activity. When the cellulase activity was too low for the Ghose method (lower than 0.37 FPU/mL), a standard curve was created by reacting
2.1. Green liquor-pretreated pulps Mixed hardwood chips from a mill in the southeastern United States were used in this study. The chips were screened and the accepts from 3/8 in. and 5/8 in. holes were used for pretreatment. The pretreatments were done in a 7-L M&K Digester with 800 OD grams of chips. The green liquor (comprised of Na2CO3 and Na2S, Fisherbrand Fair Lawn, NJ) was charged, based on percent Total
Table 2 Raw materials analysis. Pulp
Glucan
Xylan
AISLa
ASLb
Balance
Error
GL16
61.1%
15.0%
20.0%
2.9%
99.0%
1.1%
a b
Acid insoluble lignin content. Acid soluble lignin content.
Y. Xue et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 707–714
different concentrations of cellulase with a 1 cm 6 cm strip of Whatman No. 1 filter paper for 60 min of incubation time. 2.4. Enzyme adsorption The buffer and enzymes were pre-incubated in a 50 8C water bath for 10 min. Enzymes and buffer were added into the pulp to make a mixture with 5% total solids, mixed well with a vortex mixer for 30 s. Then, the mixture was put back into the 50 8C incubator for 10 min. The liquid phase was separated by vacuum filtration with a C-sized crucible (Kimax, Vineland, MA). The consistencies of the residue for the pulps tested were 19–21%. The filtrate was tested for the enzyme concentration, and the difference between the original amount of enzyme added to the mixture and the enzyme in the filtrate was assumed to be the enzyme that remained in the residue. 2.5. Enzymatic hydrolysis Cellulase, xylanase, and b-glucosidase systems in separate solutions were used in this study in a mixing ratio of 10:3:3. The dosage of xylanase and b-glucosidase was recommended by the vendor. Enzymatic hydrolysis and enzyme adsorption were performed in 0.1 M acetate buffer at pH 4.8, with NaN3 as an antibiotic. All enzymatic hydrolysis was conducted at 50 8C in a water bath, and the agitation level was set to 90 rpm. After enzymatic hydrolysis, the hydrolysate was filtered through C-sized crucibles. Filtrate was collected for IC sugar analysis, and the residue in the crucible was dried in an oven overnight in an oven at 120 8C for weight loss monitoring. 2.6. Sugar test After obtaining the filtrate of enzymatic hydrolysate, it was filtered through a 0.45 mm Millipore syringe filter (Billerica, MA), and diluted to the desired range of concentration with Milli-Q water. A sugar test was performed with a Dionex IC 3000 system with a Carbo Pac PA 10 sugar separation column. The fluid was H2O, at the speed of 1.1 mL/min, which gives a pressure of 1800 psi. 200 mM NaOH was used as the eluent for cleaning purposes. Standard sugars D-glucose, D-mannose, D-xylose, Dgalactose, D-rhaminose, L-arabinose, and D-fructose were from Sigma Aldrich (St. Louis, MO), and chromatographic grade NaOH was from Fisherbrand (Fair Lawn, NJ). Sugar yield (sugar conversion) was defined as the ratio created by dividing the amount of sugar in the filtrate by the amount of sugar in the substrate. 3. Results and discussion There is always need to develop high-efficiency process for biofuel production [30,31]. One area in need of improvement is to increase the solids loading in enzymatic hydrolysis to decrease operating and capital cost. Three different strategies were developed and evaluated for enzymatic hydrolysis with high total solids: Scheme CXB (Fig. 1a): cellulase, xylanase, and b-glucosidase were mixed with the substrate to obtain the desired total solids. After 48 h the reaction was stopped by filtering the suspension with a course-sized crucible. Scheme C + XB (Fig. 1b): cellulase was added into the pulp containing 5% total solids. After 10 min of retention time, this mixture was filtered. The filtrate was collected and analyzed for sugar concentration. Thickened pulp was reacted in a water bath for another 2–8 h and supplementary xylanase and b-glucosidase
709
were added afterward. Collection of hydrolysate at 48 h was the same as the CXB Scheme. Scheme C + CXB (Fig. 1c): part of the cellulase was added to the pulp, to make a 5% total solids mixture. The pulp was thickened as described in the C + XB process after 10 min. The remaining cellulase was added with supplementary xylanase and b-glucosidase after 2 h of incubation time. Collection of filtrate at 48 h was the same as in CXB and C + XB procedures. Green liquor-pretreated hardwood pulp, representing a real lignocellulosic substrate, was used in the following studies for evaluating the process developed above. 3.1. Scheme CXB (conventional strategy) The disadvantage of low total solids in enzymatic hydrolysis is the low sugar concentration, which makes the amount of energy required for distilling alcohol from fermentation economically not feasible. In contrast, a high concentration of sugar can reduce the amount of energy required for distillation. It also decreases capital and operational costs for evaporation, fermentation, and distillation, since the equipment could be smaller. One method to increase sugar concentration is to reduce the amount of water used in enzymatic hydrolysis so that the sugar concentration could increase tremendously. However, the total solids in enzymatic hydrolysis influences sugar yield [20]. Total sugar yield decreased as the total solids increased, as shown in Fig. 2a. One reason for this may be due to inefficiently mixing the enzyme with the substrate. Although hydrolysis with 5% total solids produced a 64% sugar yield, the lower yield (54%) in hydrolysis with 10% total solids has a higher sugar concentration (46 g/L) than in hydrolysis with 5% total solids (26 g/L) at 20 FPU/g substrate enzyme loading. When the total solids was increased to 20%, sugar yield was decreased from 64% to 44% (Fig. 2b). However, in this same scenario, the sugar concentration increased from 26 to 84 g/L. A marked difference in sugar yield was measured at low enzyme dosages when comparing the efficiency of hydrolysis with 5% and 20% total solids with Scheme CXB. This difference decreased as the enzyme loading increased, so that at very high enzyme loading the difference in enzymatic hydrolysis was lower. However, high enzyme loadings are not practical because of the cost of enzymes. For example, sugar yield only decreased from 70% to 68% (40 FPU/g substrate). In comparison, sugar yield decreased from 40% to 19% when total solids were increased from 5% to 20% at 5 FPU/g substrate enzyme loading. The small difference of sugar yield with 5% and 20% total solids at high enzyme loading is more likely due to fast early stage hydrolysis. The cellulose was cut into smaller molecular weights [27], which gave enzymes a better chance to penetrate into bulk substrate earlier. In spite of the increased sugar concentration in hydrolysis with elevated total solids, the sugar yield from the substrate was not high enough to make this process commercially feasible. One problem caused by high total solids was inefficient mixing [32] of enzymes with substrate in a hydrolysis system, especially in the early stages of hydrolysis when the pulp was thick. Evenly distributed enzyme in the pulp suspension might help it thin more quickly, making thorough mixing in the later stages possible. 3.2. Scheme C + XB If conventional Scheme CXB did not yield high sugar conversion due to inefficient mixing of enzymes with substrates, it would be beneficial to mix the enzymes capable of adsorbing [33,34] onto fiber surfaces at low total solids before the pulp was thickened. After the thickened pulp was thinned again by the hydrolysis reaction with the cellulase enzymes adsorbed on the substrate [35,36], supplementary enzymes could then be blended into the
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710
a
Sugar
C,B, X
48 Hour Retention
Pulp
Filter
C = Cellulase B= β- Glucosidase X= Xylanase
b
Residue
C Liquor =9 Dilution/Mix Tank 5% TS
Sugars to Fermentation 19 10 min
4
Filter 20% TS
0-8 hour Retention
15
4 Mix X, B 40-48 Hour Retention C = Cellulase B= β- Glucosidase X= Xylanase
c
Lignin to Boiler
Filter
C Liquor =9 Dilution/Mix Tank 5% TS
Sugars to Fermentation 19 10 min
Filter 20% TS 15
4 2 hour Retention 4 Mix
C, X, B 46 Hour Retention C = Cellulase B= β- Glucosidase X= Xylanase
Lignin to Boiler
Filter
Fig. 1. (a) Scheme CXB for enzymatic hydrolysis. Cellulase, xylanase and b-glucosidase were added into pulp mixture at desired total solids level. After 48 h of incubation at 50 8C, the mixture was subjected to vacuum filtration to separate liquid phase and residue. (b) Scheme C + XB for enzymatic hydrolysis with 20% total solids. Cellulase was added to 5% total solids pulp and mixed well. After 10 min of mixing and retention, the pulp was thickened to 20% total solids by vacuum filtration. After various time intervals, supplementary xylanase and b-glucosidase were added into the thinned mixture, and incubated for a total 48 h. (c) Split addition of cellulase, part of the cellulase was added to pulp with 5% total solids for even distribution and thinning effect on pulp. The rest of the cellulase was added with xylanase and b-glucosidase after the pulp was thinned. (d) A whole view of the saccharification process. Filtrate from thickening process containing some of the sugar could be used as dilution water and washing liquid for fresh substrate, therefore the enzyme loss and the sugar loss could be minimized.
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711
C
d
Sugars to Fermentation Liq=7.3
Washer From Digester
Dilution/Mix Tank 5% K
19
11 4
4
Press 20% K
2 hour Retention
15
4
Sugars + residual enzymes
Mixer C, B, X
C = Cellulase B= bGlucosidase X= Xylanase
46 Hour Retention
Lignin Filter Lignin to Boiler
Fig. 1. (Continued ).
pulp mixture without any mixing problems. This process could create a better chance to achieve high sugar concentration without decreasing the sugar yield. Therefore, an enzyme adsorption study was performed to help investigate which enzyme adheres onto the pulp fiber surface best and should be added before thickening. It also identified which enzyme would not be absorbed on the pulp and should be added after the thickening process. Cellulase and xylanase were thoroughly mixed with hardwood pulps of different lignin contents at 5% total solids and a
Fig. 2. (a) Kinetics of total sugar yield over various enzymatic total solids for 48 h time range at 50 8C. The pulp total solids were 5%, 7.5%, and 10%, and the enzyme charge was 20 FPU/g substrate. (b) Sugar yield from hydrolysis Scheme C + XB at 20% total solids, compared with conventional enzymatic hydrolysis Scheme CXB with 5% and 20% total solids, which was conducted without a particular mixing strategy.
temperature of 50 8C. In this experiment, the enzymes were mixed into the pulps at 5% total solids and then thickened to 20%. During the thickening process, 20% of the liquid was retained in the pulp and 80% of the liquid was removed. If no adsorption occurred, the enzymes would be split in the same ratio. Fig. 3 shows the cellulase and xylanase adsorption onto pulps with a wide range of lignin content and demonstrates their different adsorption behaviors. Around 70–80% of FPU cellulase activity remained on residual hardwood alkaline-pretreated pulp with a 0–26% lignin content. Therefore, cellulase was preferentially adsorbed on the substrate. However, the birch xylanase adsorption activity test only demonstrated around 18–20% birch xylanase activity remaining on the substrate with the same conditions. Taking into consideration that around 20% of the liquor remained in the residue after filtration, the loss of birch xylan activity was almost proportional to the loss of liquor in the mixture. This data implies that no xylanase adsorption occurred. The birch xylan activity in the xylanase solution appeared to be retained only in the liquid rather than that adsorbed onto substrate, compared to obvious adsorption of cellulase FPU activity. This might due to the low xylan content in the pulp. Therefore the adsorption site available for the xylanase was low in the pulp. This was in accordance with Kurakake et al.’s observation that 7.7–34.2% xylanase adsorption
Fig. 3. Cellulase and xylanase adsorption onto alkaline pretreated hardwood pulp at different lignin contents. Adsorption was conducted at 50 8C, 5% total solids, 20 FPU/g substrate enzyme loading, xylanase was dosed as recommended by vendor and retention time was 10 min. *The adsorption percentage was based on activity.
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on lignocellulose biomass [37]. b-Glucosidase works on soluble cellobiose [27] and it is therefore not necessary to be added at the thickening stage with insoluble substrate. A new process was developed according to these adsorption properties of different enzymes, to achieve better enzyme distribution and minimize enzyme loss. Because cellulase appeared to remain on the pulp while xylanase tended to leave the pulp mixture during water removal, cellulase was mixed into pulp at low total solids. After thorough mixing at a low solids concentration, water was removed with a vacuum filter. The time at which supplementary xylanase and bglucosidase are added can influence sugar conversion efficiency. Addition of xylanase and b-glucosidase at an earlier time, when the pulp is not thinned, will result in an uneven distribution in the pulp mixture. In contrast, late addition of the xylanase and bglucosidase will make the interaction time of the added enzymes with substrates too short for efficient hydrolysis. The lack of time for synergistic effect between xylanase (or b-glucosidase) with cellulase would also decrease final sugar yield, since xylanase can release cellulose chains from hemicellulose hindrance, and bglucosidase can eliminate end-product inhibition for cellulase. Thus, the proper time for the addition of these two enzymes after the pulp-thickening process to yield highest sugar conversion was investigated (Fig. 4). The difference of sugar conversion between 1 h and 2 h interval process is marginal. And 2 h interval would be a safer time point for supplementary enzyme addition, since the pulp should be thinner than 1 h interval and newly added enzymes could be well mixed into the system. Hence, 2 h interval might be the optimum time to add b-glucosidase and xylanase. This time may vary with the enzyme charge and the substrate recalcitrance. In the following experiments, a 2-h time interval was chosen for adding supplementary enzymes, to improve enzymatic hydrolysis efficiency. And of course, this process can be further optimized. Sugar yield that resulted from separately adding enzymes showed better conversion efficiency than simply mixing the enzyme cocktail with pulp with 20% total solids. In that case, the sugar conversion was 44% for Scheme CXB but 59% for Scheme C + XB at 20 FPU/g enzyme dosage, as is shown in Figs. 2b and 5b. And the sugar concentration was increased from 84 g/L to 114 g/L at 20 FPU/g enzyme dosage after 48 h as well. When comparing with 5% total solids Scheme CXB at the same hydrolysis conditions, the sugar concentration was increased from 26 g/L to 114 g/L, while the sugar conversion is a little lower than 5% total solids Scheme CXB case (59% compared with 64%). This process was especially beneficial for lower enzyme loading. For 5 FPU, when the total solids of conventional process was increased from 5% to 20%, the sugar conversion was decreased from 40% to 19%, as was mentioned in Section 3.1. However, when Scheme C + XB was applied, the 20% total solids yielded 38% sugar conversion.
Fig. 4. Addition of supplementary xylanase and b-glucosidase at different time intervals after cellulase adsorption stage.
Although around 4% sugar (Fig. 5a) and 20% cellulase FPU activity could be expected to be lost during mixing and thickening at 20 FPU enzyme loading in the filtrate. However, filtrate from the thickening filter (or press on an industrial scale) could be used for diluting or washing fresh feedstock, as is shown in Fig. 1d, to minimize sugar and enzyme loss. 3.3. Scheme C + CXB The cellulase addition strategy in high solids enzymatic hydrolysis schemes may benefit from further optimization. If all the cellulase was added into low total solids pulp at the beginning of the process, as was shown in Scheme C + XB (Fig. 1b), cellulase could be thoroughly mixed with pulp, and after water removal, xylanase and b-gludosidase may be more easily mixed into the bulk solid since the cellulase adsorbed on pulp helped hydrolyze part of the substrate and thinned down the system. However, in the Scheme C + XB, some fraction of cellulase was lost in the filtrate during the thickening process, especially enzyme proteins lacking in binding domains. Previous work observed that the molar ratio of cellulase components is an important factor that determines the efficiency of enzymatic hydrolysis [27,38,39]. As a result, the overall efficiency of sugar conversion would be low. But, if all cellulase is added to xylanase and b-glucosidase into a substrate with 20% total solids to avoid the loss of enzyme, enzymes could not be mixed well with substrate as was shown in the previous discussion. Because inefficient distribution of cellulase led to an inefficient first stage thinning effect, the sugar conversion was therefore expected to be low, although there was no enzyme lost. The scheme involving split addition of the cellulase enzyme was studied to optimize the effects of enzyme addition. (Fig. 6a–c). When adding half of the cellulase in the adsorption stage and the other half with supplementary xylanase and b-glucosidase, the sugar yield after 48 h would be almost the same as enzymatic
Fig. 5. (a) 10 min sugar yield in thickening filtrate in Scheme C + XB over a range of 5–40 FPU/g substrate enzyme loading. (b) Final sugar yield after 48 h for Scheme C + XB over a range of 5–40 FPU/g substrate enzyme loading.
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713
Table 3 Sugar yield and sugar concentration of schemes evaluated at 20 FPU/g substrate enzyme loading, 48 h incubation time, and 50 8C with pH 4.8 acetic acid buffer.
Scheme Scheme Scheme Scheme
CXB 5% CXB 20% C + XB 20% C + CXB 20%
Sugar yield (%)
Sugar concentration (g/L)
64 44 59 63
26 84 114 121
concentration was raised from 26 g/L to 121 g/L. Sugar concentration and sugar conversion of all schemes are summarized in Table 3. 4. Conclusion A more economically feasible process to run enzymatic hydrolysis with high total solids that can be implemented on a commercial scale was developed in this study. Using the scheme proposed in this paper the solids content can be increased to 20% while maintaining the enzymatic hydrolysis conversion efficiency comparable to that with 5% total solids. This increases the sugar concentration from 26 g/L to 121 g/L. The key to this process for increasing solids content in enzymatic hydrolysis is to mix a fraction of the cellulase with the pulp at 5% low total solids, thicken or press to 20% solids and allow the adsorbed cellulase to perform the necessary thinning effect which enabled thorough mixing of supplementary enzymes specially xylanase and beta-glucosidase. NCSU was granted a US patent for this concept in 2010 and licenses have been granted to companies. Acknowledgements This work was funded by the Wood-to-Ethanol Research Consortium (WERC). WERC members are: American Process, Andritz, Arborgen, KBR Engineers, Catchlight, Evolution Resources, and Japan Pulp and Paper Research Institute. Enzymes were kindly provided by Novozymes. The authors are grateful to WERC member companies and Novozymes. References Fig. 6. (a–c) Split addition of cellulase (Scheme C + CXB) at 10 FPU, 20 FPU, 40 FPU/g substrate enzyme loading at 20% total solids, compared with Scheme CXB containing 5% total solids.
hydrolysis with 5% total solids. For 10 FPU/g substrate enzyme loading, a 43% sugar yield could be achieved with 5% total solids. The split addition of enzymes (Scheme C + CXB) to pulp with 20% total solids however, could yield 46% sugar conversion. At 20 FPU enzyme loading, 64% sugar yield was achieved in 5% total solids hydrolysis, while 63% sugar yield was achieved by Scheme C + CXB hydrolysis with 20% total solids. At 40 FPU, sugar yield was 70% at 5% total solids, and also 70% from Scheme C + CXB hydrolysis with 20% total solids. The lower water content of a hydrolysis system with high total solids made the hydrolysate sugar concentration 4– 5 times higher than the concentration obtained by a system with low total solids. The sugar concentration for 20 FPU/g substrate enzyme loading could achieve 121 g/L in enzymatic hydrolysis with 20% total solids, compared to 26 g/L in enzymatic hydrolysis with 5% total solids. Through the optimization of the process studied in this work, sugar concentration increased by 95 g/L (fourfold). Through Scheme C + CXB, sugar yield was 63%, and the sugar concentration increased to 121 g/L at 20 FPU/g substrate enzyme loading, compared to the conventional 5% total solids Scheme CXB. Overall enzymatic hydrolysis efficiency was the same, while sugar
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