Enzyme Recycling by Adsorption during Hydrolysis ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Enzyme Recycling by Adsorption during Hydrolysis of OxygenDelignified Wheat Straw Oscar Rosales-Calderon, Heather L. Trajano,* Dusko Posarac, and Sheldon J.B. Duff Department of Chemical and Biological Engineering, University of British Columbia, 2360 East Mall, Vancouver, British Columbia V6T 1Z3, Canada S Supporting Information *

ABSTRACT: Enzyme recycling by adsorption from supernatant to fresh substrate is a promising strategy to reduce enzyme expenses and the production cost of lignocellulosic ethanol. The study was performed using oxygen-delignified wheat straw, and the effect of lignin content, enzyme loading, and hydrolysis time on recycling was determined. The percent of recycled cellulases, 0−35% of initial cellulase loading, increased with increasing enzyme loading and hydrolysis time but decreased with increasing lignin content. Cellulose conversions of 10−71% were achieved during the second hydrolysis round using only recycled cellulases indicating the existence of a highly active subset of enzymes. To achieve constant production of sugars during enzyme recycling, fresh cellulases were loaded before the second hydrolysis round to match the cellulase loading used in the first round. Subsequently, similar glucose, xylose, and protein concentrations were obtained at the end of the first and second rounds for all conditions. Recycling mass balances were developed to support future techno-economic analyses to determine the impact of enzyme recycling on the cost of ethanol. KEYWORDS: Enzymatic hydrolysis, Lignocellulose, Cellulases, Recycling, Biofuel, Mass balance

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for cellulose.2,4,9−11 Qi et al.4 applied this method and reported that hydrolysis yields decreased with each recycling round during three consecutive rounds. Applying a similar methodology, Tu et al.3 found that hydrolysis yields from ethanolpretreated mixed softwood decreased from 86% to 60% after two rounds. A downside of enzyme recycling by adsorption is that β-glucosidase cannot be simultaneously recycled as it does not adsorb to cellulose11,12 and must be added each round. The effectiveness of enzyme recycling depends on numerous factors, and predicting optimum recycling conditions is complex. Activity and amount of cellulase available for recycling depend on lignin content10 as lignin irreversibly adsorbs cellulases.1,13−15 The amount of cellulases available for recycling changes with time as cellulases adsorb and gradually desorb.10,16 Cellulase desorption is proportional to the amount of adsorbed cellulases, which is defined primarily by enzyme loading.16 Past recycling studies reported recycling performance as a function of cellulase activity,9,10 hydrolysis yields in subsequent rounds,3,11 or the difference in total protein concentration before and after adsorption.4,11 The mass recovery of cellulases,

INTRODUCTION Lignocellulosic ethanol can be produced via four steps: pretreatment, enzymatic hydrolysis, fermentation, and distillation.1 Enzymatic hydrolysis requires a cocktail of different enzyme functionalities. Endoglucanases (EGs) expose cellulose chain ends; cellobiohydrolases (CBHs) generate soluble polysaccharides from chain ends. EG and CBH, also known as cellulases, adsorb onto cellulose. β-Glucosidases cleave soluble polysaccharides into glucose.2 Lignocellulosic ethanol production remains expensive3,4 in part because of enzyme costs;2 estimates range from $0.03/L to $0.39/L ethanol.5 Enzyme recycling may reduce costs.6 Enzymes can be recovered and recycled from the supernatant or the residual solids after hydrolysis. Weiss et al.7 recycled enzymes associated with insoluble residues; under the most favorable conditions, constant glucose yields were achieved with 30% reduction in enzyme dosage. However, recycling insoluble residues leads to solids accumulation therefore increasing reactor size and slurry viscosity. Enzymes in the supernatant can be recovered by ultrafiltration; for example, Qi et al.8 recovered 74−89% of the protein content after hydrolysis. Disadvantages of ultrafiltration include membrane fouling and high operating pressures.9 An attractive alternative to recover enzymes from supernatant is addition of fresh substrate to the supernatant. Enzymes in the supernatant are adsorbed onto fresh substrate because of the affinity of cellulase © XXXX American Chemical Society

Received: April 26, 2017 Revised: July 17, 2017

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DOI: 10.1021/acssuschemeng.7b01294 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Composition of Raw and Pretreated Wheat Straw composition (wt %)

a

pretreatment conditions

substrate

Glua

Xylb

Arab.c

Gald

Manne

lignin

moisture content (wt %)

raw 30 min, 6% caustic, 120 °C 60 min, 10% caustic, 150 °C

R M S

35.8 49.9 55.3

20.1 23.6 24.2

3.2 3.2 2.2

0.9 0.7 0.3

0.8 1.2 0.0

15.8 9.0 4.7

6.85 81.70 81.95

Glu: Glucan. bXyl: Xylan. cArab.: Arabinan. dGal: Galactan. eMann: Mannan.

required for mass balances and process simulations, cannot be determined from these studies. A possible industrial lignocellulosic ethanol facility configuration is to have pretreatment and distillation operating continuously while enzymatic hydrolysis and fermentation operate in batch mode.17 For this configuration to be feasible, sugar production from each hydrolysis round must be constant; therefore, fresh cellulases must be supplemented prior to each round to ensure consistent enzyme and sugar concentrations before and after each round. This work’s objectives are to determine the recyclable mass fraction of enzymes and evaluate recycled cellulase performance in subsequent rounds. Finally, conditions will be defined whereby a uniform flow rate of sugars can be produced using enzyme recycling.

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Table 2. Enzymatic Hydrolysis Experimental Conditions at 5% Solid Concentration

EXPERIMENTAL SECTION

Materials. Wheat straw was ground to pass through a 1 mm mesh sieve and stored at 4 °C. Straw was warmed to room temperature prior to use. Moisture content was determined by drying biomass at 105 °C. Commercial enzyme preparations were used: Celluclast 1.5L [129.3 mg protein/mL, 30.7 cellobiose units per milliliter (CBU/mL), 63.8 filter-paper units per milliliter (FPU/mL)] and Novozyme 188 (102.2 mg protein/mL, 626.4 CBU/mL). The major cellulase monocomponents identified within Celluclast 1.5L are Trichoderma reesei Cel7A (CBH I), Cel6A (CBH II), Cel7B (EG I), and Cel5A (EG II).18,19 Additionally, T. reesei has been reported to produce at least three specific endo-β-1,4-xylanases: XYN I, XYN II, and XYN III.20−22 Enzymes were stored at 2 °C. Pretreatment. Wheat straw was pretreated by oxygen delignification as described previously.23 The reactor (Parr 4520) was charged with 500 g of aqueous caustic (NaOH) slurry at 4% (w/w) dry wheat straw. The reactor was sealed, purged with nitrogen, and heated to target temperature; oxygen was then continuously added. The reaction was stopped by stopping oxygen flow and cooling the reactor. Pretreated biomass was filtered and washed with water to remove pretreatment liquor. To investigate the effect of lignin content on enzyme recycling, pretreatment was carried out at mild (M) and severe (S) conditions (Table 1). Enzymatic Hydrolysis. Hydrolysis was performed in 250 mL Erlenmeyer flasks (50 mL total volume, 5 wt % dry biomass) at 50 °C in an incubator at 150 rpm. The reaction was carried out in 50 mM acetate buffer (pH 4.8) with 0.02% w/v tetracycline and 0.015% w/v cyclohexamide. The biomass slurry was preheated to 50 °C prior to enzyme addition. A sample of 0.5 mL was taken after hydrolysis and centrifuged (relative centrifugal force, RCF 16 904g; 10 min). A 0.1 mL sample of supernatant was used to determine protein content, and remaining supernatant was kept at −4 °C until analyzed for sugars. Enzyme recovery was conducted at times corresponding to three stages: high conversion rate, deceleration of conversion rate, and approaching maximum conversion.23 Conditions are summarized in Table 2. Novozyme 188, which contains primarily β-glucosidase, was loaded at a ratio of 1:5 FPU/CBU to avoid cellobiose inhibition of cellulase.23 Replicates were performed at select conditions and pooled standard deviation (Sp) was calculated.24 Enzyme Recycling. The enzyme recycling methodology is shown in Figure S1 of the Supporting Information. Solids and hydrolysis broth were separated using a glass microfiber membrane (Whatman

scenario

substrate

cellulase loading (FPU/g cellulose)

hydrolysis time (h)

M20 M20 M20 M40 M40 M40 S20 S20 S20 S40 S40 S40

M M M M M M S S S S S S

20 20 20 40 40 40 20 20 20 40 40 40

24 48 72 5 24 48 12 24 48 5 24 48

grade GF/A). The filtrate was sampled and centrifuged (RCF 16 904g, 10 min). A 0.1 mL sample of supernatant was used to determine protein content, and remaining supernatant was kept at −20 °C until analyzed for sugars. For recovery of cellulases from filtrate, fresh substrate equivalent to the amount used in the first hydrolysis (round 1) was combined with filtrate in a 250 mL flask. The flask was incubated at 50 °C and 150 rpm for 20 min. It was previously found that the majority of cellulases are adsorbed in 1 h.25 Furthermore, our previous results showed that, during enzymatic hydrolysis with 5 wt % dry biomass, 76−85% of cellulases are adsorbed in 11−16 min;23 therefore, we judged that 20 min was sufficient to achieve high enzyme adsorption. After adsorption, filtration with a glass microfiber membrane was repeated. Samples of 0.5 mL were taken before and after filtration to determine protein and sugar concentration and to calculate enzyme mass adsorbed on fresh substrate. The fresh substrate with adsorbed cellulases was then combined with fresh buffer for a second hydrolysis round (round 2). In the first set of experiments, fresh β-glucosidase was added to round 2 at the same protein loading as round 1 (Figure S1A), and each round lasted 48 h. Sugar and enzyme concentrations were determined after round 2. Replicates were performed at select conditions, and Sp was calculated.24 To implement enzyme recycling at an industrial scale, it is necessary to maintain uniform sugar and protein concentrations before and after each round. In the second set of experiments, round 2 was carried out by adding fresh β-glucosidase and cellulases to match initial concentrations in round 1 (Figure S1B). Sugar and protein concentrations were determined after round 2. Replicates were performed at select conditions, and Sp was calculated.24 Accessible Liquid in Substrate. Cellulases and β-glucosidase have similar diameter (approximately 5.9 nm)26,27 and molecular weight (Celluclast 1.5L, 52−62 kDa;25,28 Novozyme 188, 50−120 kDa29). Both are larger than sugars released during hydrolysis. As Novozyme 188’s components do not adsorb to substrate,30 Novozyme 188 was used to estimate the enzyme-inaccessible fraction of liquid contained in substrate. Solutions of Novozyme 188 at protein concentrations similar to those used during hydrolysis (0.34, 1.71, and 2.65 g/L) were prepared. A 5 g portion of wet substrate S (82.0% moisture content) was added to 5 mL of Novozyme 188 solution and B


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Figure 1. Percent of recycled cellulases (left axis) as calculated by eq 3 achieved in each scenario [mild (M) and severe (S) pretreated wheat straw with cellulase loading of 20 and 40 FPU/g cellulose] at different times. Mass of recycled cellulases can be determined from the right axis. It is noted that pooled standard deviation SP = 1.17%. incubated at 20 °C and 150 rpm along with control flasks containing 5 mL of Novozyme 188 solution. Total protein concentration in each flask was monitored for 1 h. The protein mass in the Novozyme 188− substrate and control flasks was equal, but enzyme-accessible liquid in the substrate caused measured protein concentration in Novozyme 188−substrate flasks to decrease. Cellulase Distribution. It was assumed that Celluclast 1.5L is composed only of cellulases as approximately 78% of total protein is cellulases.18,28 Cellulases readily adsorb to substrate11,12 while proteins in Novozyme 188 do not; thus, cellulase concentration in solution [EL] (g/L) can be determined by subtracting Novozyme 188 protein concentration [N] (g/L) from total measured protein concentration [T] (g/L):

[E L] = [T ] − [N ]

where [N0] is initial protein concentration of Novozyme 188, k1″ = 0.016 h−1, and ks = 0.008 L g−1 h−1. Compositional Analysis. Substrate composition was characterized according to Sluiter et al.31 Sugar concentrations were quantified using a high-performance liquid chromatograph equipped with an ion exchange PA1 column (Dionex), a pulsed amperometric detector with a gold electrode, and a Spectra AS 3500 autoinjector (Dionex DX-500, Dionex). Fucose was used as an internal standard. Table 1 summarizes the composition of raw and pretreated wheat straw. Analysis of Enzyme Activity and Protein Concentration. Cellulase activity was measured and reported as FPU/mL.32 βGlucosidase activity was measured and reported as CBU/mL.33 Protein concentration was measured by the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Lyophilized powdered cellulases (C8546, Sigma-Aldrich) and β-glucosidase (9033-06-1, Sigma-Aldrich) were used as calibration standards.34 Interference of sugars in the assay was determined by measuring protein concentration of enzyme controls at 0, 4, and 20 mg glucose/mL. Negligible percent deviation (1.8%) indicated that there was no sugar interference. Hydrolysis broth samples were mixed with Bio-Rad dye, and absorbance was measured with a UV-spectrophotometer (UV-1800, Shimadzu) at 595 nm.

(1)

Novozyme 188 protein concentration decreases at 50 °C because of aggregation and precipitation, and can be predicted as a function of time, t, by30 [N ] =

[N0](ks[N0] + k1″e−t(k1″ + ks[N0])) k1″ + ks[N0]

(2) C


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Figure 2. Cellulose conversion, xylan conversion, and percent recycled cellulases without addition of fresh cellulases to round 2. Conversion after 48 h as a percentage of theoretical maximum at different hydrolysis conditions [mild (M) and severe (S) pretreated wheat straw with cellulase loading of 20 and 40 FPU/g cellulose] for the first (black ■) and second (gray ■) hydrolysis rounds. The percent of recycled cellulase is calculated from eq 3. It is noted that pooled standard deviation for cellulose conversion SP = 3.76%, xylan conversion SP = 4.48%, and recycled cellulase SP = 4.06%.

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adsorption. Qi et al.4 reported 74.8% enzyme recovery for dilute-alkali-pretreated wheat straw after 48 h of hydrolysis. Percent cellulase recovery was calculated for comparison. Cellulase recovery refers to the difference in cellulase concentration, excluding β-glucosidase and cocktail proteins, after hydrolysis and after adsorption. Conditions in scenario S20 are closest to those of Qi et al.;4 70% cellulase recovery was achieved in this study, and this is in close agreement with Qi et al.4 Cellulase recovery describes the efficiency of the readsorption process. However, the development of mass balances requires a measure of the recyclable fraction of cellulases initially loaded, therefore this paper reports the percentage of recycled cellulases. The percent of recycled cellulases is defined as

RESULTS AND DISCUSSION Dilution by Biomass Moisture Content. Previously, the fraction of enzymes recycled was determined from the difference in total protein concentration before and after cellulase adsorption on fresh substrate.4,11,35 However, dilution by liquid contained in fresh substrate must be considered. In this work, pretreated substrates had a moisture content of 82.0%, but not all substrate liquid is enzyme-accessible. Pores smaller than the diameter of cellulases and β-glucosidase, 5.9 nm,26,27 are inaccessible. By measuring dilution of Novozyme 188 protein concentration by substrate liquid, it was determined that 81.8% of biomass moisture is enzymeaccessible. Effect of Hydrolysis Conditions on Enzyme Recycling. Enzyme recovery has been reported previously and refers to the difference in total protein concentration (cellulases, βglucosidase, and cocktail proteins) after hydrolysis and after

recycled cellulases (%) = 100 D

ER E0

(3)


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ACS Sustainable Chemistry & Engineering where E0 (g) and ER (g) are the mass of cellulases initially loaded, and recycled, respectively. Percent recycled cellulases as a function of time is presented in Figure 1. Cellulase concentration in solution varies with pretreatment conditions, enzyme loading, and hydrolysis time23 and determines the amount of cellulases available for recycling. For cases M40, S20, and S40, the concentration of cellulases increased with time because of the gradual desorption of cellulases. This is in accordance with previous results.4,11,23 We hypothesize that further increases in time will decrease the amount of cellulases available for recovery as thermal denaturation of Celluclast 1.5L’s enzymes and proteins will cause protein concentration to decrease after 4 days at 50 °C.30 The effect of pretreatment severity can be observed by comparing scenarios S20 and M20. Substrates M and S exhibit different cellulase adsorption−desorption profiles,23,30 likely due to differences in lignin content. Substrate M, containing more lignin, irreversibly adsorbed more cellulases than substrate S. Moreover, the desorption rate of cellulases from substrate S was faster than that from substrate M.23 This may be caused by the larger surface area created by increased delignification. High surface area increases reaction rate and depletion of cellulose; thus cellulases desorbed more quickly. Accordingly, a larger amount of cellulases were recycled during case S20 (0−19%) than case M20 (0−2%). A greater fraction of cellulases were recycled in scenarios M40 and S40 (5−35%) than in M20 and S20 (0−19%). Doubling enzyme loading may saturate the substrate surface with cellulases, increasing the amount of cellulases in solution available for recycling. The cellulase concentration profiles for scenarios S20 and S40 reported previously show that 100% and 93% of loaded cellulases are adsorbed onto substrate at the beginning of hydrolysis, respectively.23 Therefore, while the cellulase loading in scenario S40 is sufficient to saturate all accessible substrate surfaces, only 0.16 g cellulases/L remains in solution.23 Given that cellulases desorb as hydrolysis progresses and that it has been previously shown that doubling enzyme loading increases conversion rate,23 the increase in recycling may be due to increased polysaccharide conversion. Furthermore, it has been shown that cellulase monocomponents exhibit different adsorption affinities to cellulose. For example, Palonen et al.36 showed that 60−70% of CBH II (Cel6A) irreversibly adsorbs onto cellulose while more than 90% of CBH I (Cel7A) was reversibly adsorbed. Increasing enzyme loading and saturating the surface may shift the interactions between adsorption, hydrolysis, and desorption of the monocomponents leading to increased desorption. Enzyme Recycling without Supplementation. Enzyme recycling provided unexpected insight into Celluclast 1.5L activity. Enzyme recycling was initially conducted without adding fresh cellulases to round 2. Conversion after rounds 1 and 2, each lasting 48 h, and percent cellulases recycled are shown in Figure 2. The cellulose conversion achieved during round 2 of S40 is remarkable: 78% of the first hydrolysis yield was obtained during round 2 using only 32% of the initial cellulases mass. This is all the more remarkable given that the recycled cellulases retained this activity after 48 h at 50 °C. Others have made similar observations of high activity by recycled enzymes. For example, Lu et al.10 reported that 93% of the first hydrolysis yield was obtained in the second hydrolysis round and that 78% of initial enzyme activity was recovered. Characterization of the highly active recycled enzymes may lead to improved enzyme performance.

High xylan yields in round 2 were also unexpected as it is unlikely that xylanases were responsible. Xylanases do not adsorb in significant quantities, and their activity is lost after 24−72 h.25 Xylan hydrolysis during round 2 was likely due to EG I (Cel7B) as it is the only cellulase previously reported to have activity on xylan.37 These results are in agreement with the higher adsorption affinity of cellulases to xylan relative to cellulose reported by Qing and Wyman.38 High enzyme loading increases the amount of enzymes that adsorb to substrate and catalyze hydrolysis. For both substrates, increasing enzyme loading increased cellulose and xylan conversion by 18−21% and 5−7%, respectively, during round 1. Cellulose and xylan conversion in round 1 increased by 5− 8% and 2−4%, respectively, by increasing pretreatment severity from mild to severe. Since substrate S has less lignin than substrate M, cellulose in substrate S is likely more accessible, thus explaining the greater yields from substrate S. In addition, removal of cellulose and hemicellulose promotes desorption of cellulases; therefore, increased recycling may also be due to increased polysaccharide conversion. Total protein concentration measured after each round and predicted Novozyme 188 protein concentration are shown in Figure 3. At the start of round 1, some cellulases irreversibly

Figure 3. Protein concentrations after enzymatic hydrolysis lasting 48 h. Total protein concentration (cellulases and Novozyme 188) at the end of the first (black ■) and second (gray ■) hydrolysis round, and Novozyme 188 protein concentration (⧄) as predicted by eq 2. It is noted that pooled standard deviation SP = 0.073 g/L.

adsorb on lignin, and thus do not participate in hydrolysis.39 Another portion of cellulases is reversibly adsorbed by accessible cellulose and xylan;16 once accessible surfaces are saturated, the balance of cellulases remains in solution. As hydrolysis proceeds, more substrate is exposed, and more cellulases are adsorbed. On the basis of the work by Chylenski et al.,40 it is expected that cellulases, most likely CBH I (Cel7A), CBH II (Cel6A), and EG I (Cel7B), are precipitating because of thermal instability. During hydrolysis, cellulases’ active sites are inhibited by glucose, but the binding domain is unaffected.16 Therefore, during adsorption, active and inactive cellulases may adsorb. Without supplementation, most cellulases are adsorbed at the start of round 2. The cycle of cellulase desorption from carbohydrates, reabsorption to newly accessible substrate, and precipitation then repeats. Ultimately, E


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ACS Sustainable Chemistry & Engineering recycled cellulases are either adsorbed or precipitated; thus primarily Novozyme 188 remains in solution after round 2. This hypothesis is supported by Figure 3 which shows that total protein concentration after round 2 was approximately equal to the predicted Novozyme 188 concentration; thus enzyme recycling for more than two rounds is not viable for the presented system. Others have reported that hydrolysis yield decreases with each round.4,10,35 Because of cellulase losses, cellulases must be supplemented to achieve consistent conversion after each round. Enzyme Recycling with Supplementation. Fresh cellulases and β-glucosidase were added prior to round 2 to achieve the same protein loading as at the start of round 1 with the objective of matching sugar concentrations after both rounds. Enzymes adsorbed to substrate and unadsorbed enzymes contained in substrate liquid were considered when calculating the mass of enzymes for supplementation. Figure 4 shows sugar and total protein concentrations after both rounds for scenarios S20 and S40. In scenario S20, the second round of hydrolysis contained 19% recycled cellulases and 81% fresh cellulases. In scenario S40, the second round of hydrolysis contained 32% recycled cellulases and 68% fresh cellulases. The time course of sugar and total protein concentrations from our previous enzymatic hydrolysis study (without recycling) are shown as solid symbols.23 In Figure 4, sugar and total protein concentrations after rounds 1 and 2 are within standard deviation of the time course profiles. Almost no enzyme is recycled after round 1 in scenario M20 (Figure 2), and thus round 2 is performed with essentially 100% fresh cellulase and β-glucosidase; sugar concentrations after both rounds were equivalent (data not shown). These results indicate that the production of consistent sugar concentrations is possible despite the interruptions (i.e., filtration, adsorption, filtration, and resuspension) associated with recycling. From these results and model assumptions, a mass balance of enzyme recycling was constructed. For the first time, cellulase distribution during hydrolysis and recovery was determined. Enzymes and sugars contained in substrate liquid after adsorption were included in the calculations. An illustrative mass balance with enzyme recycling after 48 h, scenario S40, is shown in Figure 5. The mass of fresh cellulases required to carry out one hydrolysis round is EI (g). With recycling, the same hydrolysis can be performed using a makeup amount of cellulases, EM (g), given by stream 6. Recycling reduces cellulase use by (EI − EM) × 100/EI. EI is the sum of streams 4 and 6; thus recycling reduces cellulase use by 35% in scenario S40. Additionally, the amount of Novozyme 188 used is reduced by 6% as some Novozyme 188 is recycled with substrate liquid. This mass balance can be used within process simulation software to simulate a lignocellulosic ethanol facility with pretreatment and distillation operating continuously while enzymatic hydrolysis, enzyme recycling, and fermentation are operated in batch mode. Such simulations will enable evaluation of the economic potential of enzyme recycling.

Figure 4. Glucose, xylose, and total protein concentrations during enzymatic hydrolysis with and without enzyme recycling. Glucose (circle symbol, pooled standard deviation SP= 0.540 g/L), xylose (square symbol, SP= 0.305 g/L), and total protein (diamond symbol, SP = 0.036 g/L) concentration at the end of the first hydrolysis round (open symbol) and second hydrolysis round (crossed symbol) at different recycling times. The second round of hydrolysis contained 19% and 32% recycled cellulases for scenario S20 and S40, respectively. The balance was fresh cellulases. Previously reported experimental concentrations of glucose (●), xylose (■), and total protein (◆) as a function of time are also shown; error bars represent the standard deviation of triplicate runs of enzymatic hydrolysis.23

Recycled cellulases were remarkably active during round 2. Unexpectedly high carbohydrate conversions were observed using one third of the initial enzyme loading. Insight into these highly active cellulases may generate substantial improvements in enzyme performance and reduce required cellulase application. Fresh cellulases must be added to support continuous enzyme recycle. The mass of fresh cellulases required to achieve equal initial conditions for each round was calculated resulting in equal carbohydrate conversion during each round. Mass balances were developed to support future process simulations and economic analyses.

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CONCLUSIONS This work’s objectives were to determine the fraction of enzymes that could be recycled and to develop refined mass balances. Dilution by substrate liquid and Novozyme 188 protein precipitation were considered to accurately determine the amount of cellulases recycled. Low lignin content, long hydrolysis times, and high enzyme loadings favored cellulase recovery. F


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Figure 5. Enzyme recycling mass balance. Mass balance of the primary components during a 48 h enzymatic hydrolysis with enzyme recycling for scenario S40 [severe (S) pretreated wheat straw with cellulase loading of 40 FPU/g cellulose].

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Notes

ASSOCIATED CONTENT

The authors declare no competing financial interest.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01294. Additional schematic diagrams of enzyme recycling methodology (PDF)

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by Consejo Nacional de Ciencia y Tecnologiá of Mexico, Agriculture and Agrifood Canada, and the Natural Science and Engineering Research Council of Canada Discovery Grant Program (RGPIN 138356-08). H.L.T. thanks the University of British Columbia Chemical and Biological Engineering department for their generous start-up funding.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+1) 604-827-1823. Fax: (+1) 604-822-6003. ORCID

(1) Jørgensen, H.; Kristensen, J. B.; Felby, C. Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels, Bioprod. Biorefin. 2007, 1, 119−134. (2) Kristensen, J. B.; Börjesson, J.; Bruun, M. H.; Tjerneld, F.; Jørgensen, H. Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzyme Microb. Technol. 2007, 40 (4), 888−895.

Heather L. Trajano: 0000-0002-8534-8211 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All authors contributed equally. G


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