Ameliorated enzymatic saccharification of corn stover with a novel

Journal of Bioresources and Bioproducts. 2016, 1(1): 42-47

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ORIGINAL PAPER

Ameliorated enzymatic saccharification of corn stover with a novel modified alkali pretreatment Guang Yua,§ , Huanfei Xub,§, Chao Liua, Paul DeRousselc,Chunyan Zhangd, Yuedong Zhanga, Bin Lia,*, Haisong Wanga,*, Xindong Mua a) CAS Key Laboratory of Bio-based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China; b) College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China; c) Procter and Gamble Manufacturing, Procter and Gamble Co., Mason, Ohio, 45040, USA; d) Materials, Process and Delivery, Procter and Gamble Singapore Innovation Centre, Procter and Gamble Co., Singapore, 138648, Singapore. §: These authors contributed equally to this work. *Corresponding authors: [email protected]; [email protected]

ABSTRACT Enzymatic saccharification/hydrolysis is one of the key steps for the bioconversion of lignocelluloses into sustainable biofuels. In this work, corn stover was pretreated with a novel modified alkali process (NaOH + anthraquinone (AQ) + sodium lignosulfonate (SLS)), and then enzymatically hydrolyzed with an enzyme cocktail (cellulase (Celluclast 1.5L), β-glucosidase (Novozyme 188) and xylanase (from thermomyceslanuginosus)) in the pH range of 4.0-6.5. It was found that the suitable pH for the enzymatic saccharification process to achieve a high glucan yield was between 4.2 and 5.7, while the appropriate pH to obtain a high xylan yield was in the range of 4.0-4.7. The best pH for the enzymatic saccharification process was found to be 4.4 in terms of the final total sugar yield, as xylanase worked most efficiently in the pH range of 4.0-4.7, under the conditions in the study. The addition of xylanase in the enzymatic saccharification process could hydrolyze xylan in the substrates and reduce the nonspecific binding of cellulase, thus improving the total sugar yields. Keywords: Enzymatic saccharification; Hydrolysis pH; Xylanase; Corn stover; Biorefinery; Fermentable sugar; cellulase

1. INTRODUCTION Enzymatic saccharification/hydrolysis is one of the key steps for the bioconversion of lignocelluloses into sustainable biofuels.1 In this step, polysaccharides are hydrolyzed to monomeric sugars by cellulase enzymes for downstream fermentation or catylatic conversion.2, 3 However, enzymatic saccharification is highly dependent upon the properties of substrates (e.g. lignin content and distribution, hemicellulose and acetyl group content, cellulose crystallinity and degree of polymerization) and hydrolysis conditions (e.g. enzyme cocktail, hydrolysis pH, solid loading.4, 5 On the one hand, to achieve an efficient enzymatic hydrolysis, cost effective pretreatment (e.g. hydrothermal pretreatment, ammonia fiber explosion (AFEX), sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), etc.) is acquired to break the natural recalcitrance of lignocelluloses.6-8 Alkali-based pretreatment (e.g. NaOH pretreatment) is also a promising pretreatment technology, because it can efficiently remove lignin by breaking the cross-linking bonds between lignin and carbohydrates, obtain high final total sugar yields, and partially use the existing and mature pulping equipment as well as the chemical recovery and waste water treatment system.9 In our previous study, a modified alkali pretreatment (MAP) www.Bioresources-Bioproducts.com

process was established in both lab and pilot scale, based on the on-line production and utilization of sodium lignosulfonate (SLS) (Figure 1). SLS was produced by direct sulfonation of spent liquor of alkali pretreatment. The produced SLS was then reused as a surfactant to promote chemical (like anthroquinone (AQ)) penetration and lignin removal in the next cycle of MAP process, thus enhancing the enzymatic saccharification of pretreated corn stover.10 In this case, part of spent liquor of MAP process was more efficiently utilized. Compared to the conventional NaOH pretreatment, MAP could reduce NaOH dosage from 13-14% to 11% and lower the temperature from 140 ºC to 120 ºC to obtain about 80% of final total sugar yield. On the other hand, hydrolysis conditions are of great importance for the high efficiency of enzymatic saccharification. For instance, enzymes are pH-sensitive. The optimal pH of 4.8-5.0 for enzymatic hydrolysis of pure cellulose has been widely accepted.11 However, different substrate has different properties which may also influence the optimal pH for enzymatic saccharification. It was reported that the optimal pH of enzymatic saccharification for dilute acid pretreated or SPORL pretreated substrate was 5.5-6.0.12 In the present work, the NaOH pretreated and MAP pretreated corn stover were enzymatically hydrolyzed respectively with the same enzyme cocktail (cellulase (Celluclast 1.5L), β-glucosidase (Novozyme 188) and 42


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Fig. 1 The process for the enzymatic saccharification of corn stover with an alkali pretreatment

xylanase (from thermomyceslanuginosus)) in the pH range of 4.0 - 6.5, and the impact of key factorson enzymatic hydrolysis of MAP treated corn stover was investigated. 2. EXPERIMENTAL 2.1 Materials Corn stover was harvested from a village of Qingdao, Shandong province, China. The corn stover was cut into 3-5 cm in length, air dried, milled by a hammer mill, and then screened to obtain the fraction with the particle size larger than 0.25 mm and less than 1cm. The screened corn stover was stored in ziplocked plastic bags for moisture balance. The corn stover contained 31.22±0.53% of glucan, 17.66±0.09% of xylan, 1.91±0.02% arabinan, 15.05±0.23% of lignin, 22.61±0.07% extractives, and 6.89±0.18% of ash. Cellulase (Celluclast 1.5L), β-glucosidase (Novozyme 188), and xylanase (from thermomyceslanuginosus) were purchased from Sigma-Aldrich China Inc., and their corresponding enzyme activities were 52 FPU/mL, 741 IU/mL, and 365 IU/g, respectively. Enzyme activities were measured according to standard methods.13 All other chemicals were bought from Sinopharm Chemical Reagent Co. Ltd. and used as received. 2.2 Methods 2.2.1 Pretreatment Alkali pretreatment or modified alkali pretreatment (MAP) with the chemical combination of 11wt% NaOH + 2wt% SLS + 0.1wt% AQ were carried out in a cooking digester (PL1-00, Xianyang TEST Equipment Co., Ltd., Xianyang, China) with the size of 15 L, following the procedure reported previously.10, 14 The corn stover was pretreated at 120 ºC for 40 min, and the heating up time was 30 min. Upon completion of pretreatment, the digester was cooled down below 100 ºC and then the samples were taken www.Bioresources-Bioproducts.com

out and washed with tap water to neutral pH. The washed substrates were stored at 4 ºC for further use. 2.2.2 Disc refining The pretreated and washed corn stover was refined using a pilot scale pressurized refiner (ф300, Shandong Chenzhong Machinery Co., Ltd. China). The refining consistency was 10 wt% and the throughput of the refiner was 100 kg/h. The rotational speed, refining gap, and the steam pressure of the refiner were 2980 rpm, 0.2 mm, and 0.4 MPa, respectively. 2.2.3 Enzymatic hydrolysis The enzymatic hydrolysis of the refined substrates were performed in an air bath incubator shaker at 50 ºC for 48 h. The enzymes dosages were 20 FPU/g-substrate and 5 IU/g-substrate for cellulase and β-glucosidase respectively. 0.02% sodium azide and the desired amount of xylanase were also added for saccharification. After enzymatic hydrolysis, the hydrolyzate was cooled at 4ºC refrigerator and then filtered through a 0.22 μm membrane to be ready for sugar analysis. Each experiment was conducted at least in duplicate and the average was reported. 2.2.4 Chemical composition and sugar analyeis The chemical compositions of corn stover were analyzed using the National Renewable Energy Laboratory (NREL) procedure.15 A high performance liquid chromatography (HPLC, Model 1200, Agilent, USA) was used to measure the sugar concentration in enzymatic hydrolyzate. The column was a Bio-Rad Aminex HPX-87H column, and the mobile phase was the 0.005 M sulfuric acid. The calibration was performed with the standard sugar solutions. The effectiveness of enzymatic hydrolysis was evaluated by glucan yield, xylan yield, and final total sugar (glucan + xylan) yield as described previously.12

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Table 1 The pH range for the enzymatic hydrolysis processa Buffer pH

3.8

4.2

4.5

4.9

5.2

5.5

6.0

Mixed pH 4.01 4.42 4.72 5.15 5.38 5.76 6.28 :buffer pH is the pH of buffer solution; mixed pH is the pH of mixture after adding substrate, enzymes, additives and buffer

6.3 6.50

a

3. RESULTS AND DISCUSSION 3.1 Impact of pH on the enzymatic hydrolysis The pH range for enzymatic hydrolysis is listed in Table 1. As can be seen, the pH of the mixture after mixing substrate, enzymes, additives and buffer was slightly higher than the pH of buffer solution. The impact of the pH on glucan yield, xylan yield, and final total sugar yield is presented in Figure 2a, 2b, and 2c, respectively. It was seen from Figure 2c that, the highest final total sugar yields of 79.54% were obtained at pH 4.42, and the suitable pH for enzymatic saccharification of MAP treated corn stover to get a high final total sugar yield (75~80%) was 4.2-5.76. The suitable pH for achieving high glucan yield was also in the range of 4.2-5.76 as shown in Figure 2a, while the appropriate pH for obtaining high xylan yield was between 4.0 and 4.7, and the highest xylan yield of 61.82% was got at pH 4.42 (Figure 2b). This may due to the optimum pH for xylanase in cellulase was in the range of 4.0-4.7. The cellulase contained some hemicellulases (e.g. xylanase), which could simultaneously hydrolyze hemicelluloses (e.g. xylan) during glucan hydrolysis.16 Due to the MAP treated corn stover contained about 25% xylan and more xylan recovered could reduce cellulase accessibility,10 adding more xylanase in enzymatic hydrolysis could further promote the enzymatic hydrolysis of cellulose.17

In Figure 3a, 66 U xylanase/g-substrate was added in the beginning of enzymatic hydrolysis. After 24 h, cellulase and β-glucosidase were added for another 24 h hydrolysis. As can be seen, the addition of xylanase could obviously improve the final total sugar yields (about 20% increase) in the pH range of 4.0-5.75. Figure 3b displays the effect of addition approach of xylanase on enzymatic hydrolysis at different pH. For the blue curve, xylanase was added in the beginning of hydrolysis. After the first 24 h, cellulase and β-glucosidase were added and the total hydrolysis duration was 48 h. Regarding to the pink curve (Figure 3b), xylanase, cellulase and β-glucosidase were added together in the beginning of hydrolysis. As shown in Figure 3b, for the one with xylanase alone in the first 24 h, a high final total sugar yields (85~90%) could be obtained in a wider pH range (4.0~5.75) compared to the one (pH 4.3~4.7) by adding the three enzymes together in the beginning of saccharification

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Fig. 2 Impact of pH on glucan yield (a), xylan yield (b), and final total sugar yields (c) (Enzymatic hydrolysis conditions: 20 FPU cellulase, 5 IU β-glucosidase, 50 ºC for 48 h, 2% solid loading)

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. Thus, adding xylanase 24 h before adding cellulase and β-glucosidase was more beneficial than later addition, except for the one at pH of 4.3~4.7. This was possibly because the xylanase in cellulase had the highest activity in the pH around 4.5. Akpinar et al. 18 conducted xylan hydrolysis using xylanase derived from T. longibrachiatum also at pH 4.6. The addition of xylanase first could hydrolyze xylan in the MAP treated substrates, thus increasing the accessibility of cellulase. On the other hand, the addition of xylanse could reduce the nonspecific binding of cellulase, because both cellulase and xylanse had a higher adsorption affinity to xylan than glucan.17 Fig. 4 Impact of temperature on the enzymatic hydrolysis (20 FPU cellulase, 5 IU β-glucosidase, 66 U xylanase, 48 h hydrolysis at pH 4.4, 2% solid loading, three enzymes were added together in the beginning of enzymatic hydrolysis)

Fig. 3 Impact of adding xylanase (a) and the addition sequence of xylanase (b) on the enzymatic hydrolysis (20 FPU cellulase, 5 IU β-glucosidase, 66 U xylanase, 50 ºC for 48 h, 2% solid loading)

3.2 Impact of temperature on the enzymatic hydrolysis Shown in Figure 4 is the impact of temperature on enzymatic hydrolysis of MAP treated corn stover. It was seen that, the suitable temperature was between 45 ºC and 55 ºC, which was in line with the typical temperature (~50 ºC) for enzymatic saccharification.2, 19 3.3 Effect of xylanase dosage and solid loading on the enzymatic hydrolysis Figure 5 shows the impact of xylanase dosage on final total sugar yields with different solid loading for enzymatic hydrolysis. As can be seen, the final total sugar yields were


Fig. 5 Impact of xylanase dosage on final total sugar yields (20 FPU cellulase, 5 IU β-glucosidase, 50 ºC for 48 h at pH 4.4, three enzymes were added together in the beginning of enzymatic hydrolysis. (a) 2% solid loading; (b) 8~9% solid loading linearly

related to the xylanase dosage, and about 86~89% of final total sugar yields could be obtained at the solid loading of 8~9% with the xylanase dosage of 66 U/g-substrate. But the final total sugar yields decreased when the solid loading increased. This was probably due to the mass transfer limitations between enzymes and substrates as well as the product inhibition in higher solid 45

Journal of Bioresources and Bioproducts. 2016, 1(1): 42-47 loading enzymatic hydrolysis.20 Therefore, sufficient mixing between enzymes and substrates is highly needed for high solid loading enzymatic hydrolysis. It has been reported that specially designed mixer (e.g. horizontal reactor using free-fall mixing)21 and fed-batch2 for high solid loading (≥15 wt%) enzymatic hydrolysis were effective and scalable. 3.4 Enzymatic hydrolysis duration The glucan and xylan yields (for MAP treated substrates) as the function of enzymatic hydrolysis time are presented in Figure 6a and 6b, respectively. Figure 6 shows that both glucan and xylan yields increased quickly in the first 24 h hydrolysis (particularly in the first 8 h). After 24 h, glucan yields increased slowly, but xylan yields were still in

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Figure 6 also displays that the addition of surfactants (i.e. Tween60 or SLS) could facilitate enzymatic hydrolysis. For instance, by adding 3% SLS, the glucan and xylan yields were improved 3.5% and 3.7% respectively after 72 h hydrolysis compared to the control, and the corresponding final total sugar yields were improved about 4%. This was due to the fact that surfactant could block the nonspecific binding of cellulase by bounding lignin on the solid substrates.23 Thus, the SLS produced by direct sulfonation of spent liquor from MAP process could also be added in enzymatic hydrolysis for further boosting the final total sugar yields. 4. CONCLUSIONS The impact of the key factors was investigated on the enzymatic hydrolysis of the corn stover pretreated with a novel modified alkali pretreatment (MAP). Results showed that the suitable pH for achieving high xylan yield was in the range of 4.0-4.7, and the highest final total sugar yield was obtained at pH 4.4. This was due to fact that the optimum pH for xylanase to work efficiently with cellulase was around 4.4 under the conditions. Also, the addition of xylanase or surfactant could promote the enzymatic hydrolysis by reducing the nonspecific binding of cellulose. The sodium lignin sulfonate (SLS) generated by direct sulfonation of the spent liquor from the MAP process could be added as surfactant in both the modified alkali pretreatment and the enzymatic hydrolysis to further ameliorate the final total sugar yield. Therefore, part of the spent liquor from the MAP could be more efficiently utilized in the overall process. ACKNOWLEDGMENTS

Fig. 6 Gluan yield (a) and xylan yield (b) of the MAP treated corn stover after the enzymatic hydrolysis (20 FPU cellulase, 5 IU β-glucosidase, 5 U xylanase, 50 ºC for 48 h at pH 4.4, 8% solid loading, three enzymes were added together in the beginning of enzymatic hydrolysis. Four replicates for each test were performed.)

clearly increasing tendency, even after 72 h hydrolysis. This was likely associated with the large amount of xylan recovered (about 78% of recovery rate of xylan) in MAP treated substrates.10 Similar phenomenon was also reported for the enzymatic saccharification of the hydrotropic pretreated corn stover.22

The authors are grateful for the great support of research funding from Procter and Gamble Co. This work was also partially supported by the National Natural Science Foundation of China (Grant No.31370582, Grant No. 21306216, and Grant No. 31470609), Shandong Provincial Natural Science Foundation for Distinguished Young Scholar (China) (Grant No. JQ201305), as well as the National High Technology Research and Development Program (“863” program) of China (Grant No. 2012AA022301). REFERENCES 1.

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