Enhanced High-Solids Fed-Batch Enzymatic Hydrolysis of Sugar

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Enhanced high-solids fed-batch enzymatic hydrolysis of sugarcane bagasse with accessory enzymes and additives at low cellulase loading Marie Rose Mukasekuru, Jinguang Hu, Xiaoqin Zhao, Fubao Fuelbiol Sun, Kaneza Pascal, Hongyan Ren, and Junhua Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01972 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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ACS Sustainable Chemistry & Engineering

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Enhanced high-solids fed-batch enzymatic hydrolysis of sugarcane bagasse with accessory

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enzymes and additives at low cellulase loading

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Marie Rose Mukasekuru,†,¶ Jinguang Hu,‡ Xiaoqin Zhao,† Fubao Fuelbiol Sun,†* Kaneza Pascal,† Hongyan

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Ren,§* Junhua Zhang╫

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†

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Biotechnology, Jiangnan University, Wuxi 214122, China

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‡

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Columbia, Vancouver, BC V6T 1Z4, Canada

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§

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of

Forestry Products Biotechnology / Bioenergy Group, Wood Science Department, University of British

Jiangsu Key Laboratory of Anaerobic Biotechnology, School of Environmental and Civil Engineering,

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Jiangnan University, Wuxi 214122, China

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¶

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Rwanda

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╫

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*Corresponding Author

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Fubao Fuelbiol Sun, PhD, Assoc Prof;

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Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of

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Biotechnology, Jiangnan University;

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[email protected] ; [email protected]

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*Corresponding Author

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Hongyan Ren, PhD, Assoc Prof;

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Jiangsu Key Laboratory of Anaerobic Biotechnology, School of Environment and Civil Engineering, Jiangnan

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University, Wuxi 214122,China;

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[email protected] ;

Department of Applied Biology, College of Science and Technology, Avenue de l’armée, Kigali 3900,

College of Forestry, Northwest A&F University, Yangling 712100, China

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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HIGHLIGHT

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hydrolysis

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High-solids hydrolysis (20%) produced ~160 g/L fermentable sugars with 83% cellulose and 90% xylan hydrolysis at 3 FPU/g substrate after 72 h

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Extra β-glucosidase addition is evidently unnecessary for Cellic CTec2 hydrolysing lignocellulosic substrate

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Mixed use of additives and accessory enzymes is highly instrumental for high-solids cellulase

al-AGO pretreated substrates present an industrially relevant hydrolysability

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ABSTRACT

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High cellulase loading is still a major impediment in the production of fermentative sugars from high-solids

4

enzymatic hydrolysis of lignocellulosic substrates in the enzyme-based “biorefinery” industry. This study

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attempted a high-solids (20%) enzymatic hydrolysis of lignocellulosic substrate at a very low cellulase

6

loading with mixed use of additives and accessory enzymes by fed-batch mode. To avoid the high initial

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biomass viscosity, the high-solids enzymatic hydrolysis of lignocellulosic substrates was initiated with a

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solids content of 8%. Thereafter, 4% of the additional substrates were consecutively fed into the hydrolysis

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system after 6, 12, and 18 h to reach a final solid content of 20%. Some additive mixtures (40 mg Tween 80

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+ 10 mg tea saponin + 20 mg/ g substrate BSA) were observed to enable this fed-batch hydrolysis to

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increase 30% of the glucose yield after the 48 h. The combination of these additives and accessory

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enzymes (2.4 mg/g substrate xylanase and 1 mg/g substrate AA9) in the high-solids hydrolysis system

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further boosted the sugar release. This allowed us to achieve an industrially relevant sugar yield (83%

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cellulose and 90% xylan hydrolysis) and fermentable sugar titer (~160 g/L) after 72 h, with a low cellulase

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enzyme loading (3 FPU/g substrate). Our results indicate that the fed-batch substrate addition process is a

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favourable model for high-solids enzymatic hydrolysis of lignocellulosic substrates. Moreover, the synergism

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between the additives and accessory enzymes can greatly boost the high-solids enzymatic hydrolysis of

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lignocellulosic substrates.

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KEYWORDS: Atmospheric glycerol organosolv-pretreated, Sugarcane bagasse, High solids content,

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Enzymatic hydrolysis, Low enzyme loading, Additive and accessory enzyme

22 23

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■INTRODUCTION

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A potent sugar platform comprising pretreatment and enzymatic hydrolysis is widely recognized in the

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lignocellulosic biorefinery industry as the key to the efficient bioprocessing of holocellulosic polysaccharides

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into fermentative sugars. This has led to extensive research on pretreatment processes including dilute

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acid/alkaline,1 steam explosion,2 liquid ammonia,3 and ionic liquid.4 To date, these employed pretreatment

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methods are still unsatisfactory in terms of component selectivity, holocellulose hydrolysability, and

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fermentation inhibitor production. In our laboratory, atmospheric glycerol organosolv (AGO) pretreatment

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was initiated ten years ago using industrial glycerol as a cooking solvent.5,

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Thereafter, dozens of

10

researchers investigated the AGO pretreatment method of various lignocellulosic biomass.7 To date, the

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AGO pretreatment has presented obvious features as below 7, 8: 1) organosolv pretreatment; 2) atmospheric

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operation process; 3) value-added organosolv lignin; 4) non-toxic solvent; 5) usable solvent residual as

13

microbial carbon source; 6) less production of furan inhibitors; 7) a bridge between bioethanol and biodiesel

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production. Besides, the AGO pretreatment deconstructs the recalcitrant cell wall of lignocellulosic biomass

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with good selectivity, rendering the substrate a robust hydrolysability and fermentability. AGO-pretreated

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wheat straw with a 2 % solids content reached 90% of glucose yield after 24-h enzymatic hydrolysis with 16

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FPU/g of cellulase loading. Recently, the 48-h enzymatic hydrolysis of AGO-pretreated sugarcane bagasse

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at a solids content of ≤10% reached >80% at 10 FPU/g.8 Additionally, this pretreatment process produced

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relatively few furan derivatives that are adverse to the subsequent microbial fermentation.8 Briefly, an

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AGO-pretreated substrate is likely to be a valid feedstock candidate for the ongoing industrially relevant

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enzyme-based biorefineries.

9

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Nevertheless, most of the current enzymatic hydrolysis processes produced from the AGO-pretreated

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substrate at a dilute solids content (≤10%) yield low glucose titers (10 FPU/g).11 Briefly, the current high-solids

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enzymatic hydrolysis is not yet desirable in terms of “three-high” (titer, yield, and productivity) demand in the

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conventional starchy ethanol fermentation industry.17 Therefore, it is crucial to discover other effective

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strategies that enhance the “three-high” level of this process.

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As reported extensively in the literature, some additives [surfactants, bovine serum albumin (BSA), and

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polymers] and accessory enzymes [β-glucosidase, xylanase, and auxiliary activity 9 (AA9)] have improved

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the enzymatic hydrolysis of lignocellulosic substrates.17-21 Zhang et al. revealed that the addition of BSA and

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Tween 80 in an enzymatic hydrolysis with a solids content of 2% increased the glucose yield by 72.4% and

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68.8%, respectively.18 In our earlier study, the addition of accessory enzymes (BG, xylanase, and AA9)

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significantly enhanced enzymatic hydrolysis.19, 20 These additives22, 23 and accessory enzymes

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been investigated in high-solids enzymatic hydrolysis. Ma et al. demonstrated that the addition of 2 g/L

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Tween 80 allowed a 30% increase of enzymatic hydrolysis with a solids content of 25%.22 Hu et al.

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discovered that the combination of xylanase and AA9 increased the hydrolysis rate by 30% for SPP and 5


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have also


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20% for SPCS substrates at 20% solids loading.24 These studies have evidenced that the addition of

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additives and accessory enzymes promotes high-solids enzymatic hydrolysis. Nevertheless, to date,

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information on the recombination of these additives and accessory enzymes in the high-solids enzymatic

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hydrolysis of lignocellulosic substrates is scarce.

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Based on the above factors, this study attempted to improve the high-solids (20% solids content)

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enzymatic hydrolysis of an alkaline-catalysed atmospheric glycerol organosolv (al-AGO)-pretreated

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substrate at low enzyme loading. To our knowledge, this is the first time that the high-solid enzymatic

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hydrolysis of AGO-pretreated lignocellulosic substrate was reported, especially that with a mixed use of

9

additives and accessory enzymes. First, some key variables, i.e., enzyme loading and initial solid content,

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were selected for the high-solids enzymatic hydrolysis of al-AGO-pretreated sugarcane bagasse. Next, the

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high-solids hydrolysis of the substrates was explored using the fed-batch mode, and additives and

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accessory enzymes were subsequently selected for this hydrolysis process. Finally, the high-solids

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fed-batch enzymatic hydrolysis of the al-AGO-pretreated substrate was performed and characterized

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successfully to efficiently achieve high-titer fermentable sugars.

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■MATERIAL AND METHODS

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Material. Sugarcane bagasse (41.2% cellulose, 20.6% hemicellulose, and 23.0% lignin) was purchased

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from Guangxi Province, China. It was dried to constant weight at 105 °C and subsequently stored in a

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polyethylene plastic container. Industrial glycerol, purchased from a chemical plant in Jiangsu Province,

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China, was of commercial grade (95% purity). Tea saponin (95% purity) was technical grade, commercially

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purchased from Shanghai Shifeng Biological Technology Co., Ltd. Cellulase preparations Cellic® CTec2

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(CTec2; 141 FPU/g) and beta-glucosidase (54.21 U/mL) were a generous gift from Novozymes (China)

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Investment Co., Ltd. Endo-xylanase solid powder (150 mg protein/g) was acquired from Vland Biotech,

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China. Recombinant lytic polysaccharide monooxygenases (NCBI: XM_001907667.1; AA9) was produced

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in Pichia pastoris GS115 from Podospora anserina in our laboratory according to a previously reported

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method.25

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al-AGO Pretreatment of Sugarcane Bagasse. In a typical run, 100 g of dry sugarcane bagasse was 6


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directly mixed with 1000 g of 95.0% industrial glycerol and 0.2% (w/w) NaOH (3 g) as catalyst. And the

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mixture was then cooked at 240 °C for 30 min in a three-necked round-bottom flask (5 L). The pretreatment

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condition with alkaline addition was selected in our laboratory as reported in our earlier work, 5,8 which would

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be reported elsewhere. The main components of the al-AGO-pretreated sugarcane bagasse comprised

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(w/w): 57% cellulose, 25% hemicellulose, and 12% lignin.

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Selection of Key Variables and Additives for Fed-batch Enzymatic Hydrolysis. Each sample,

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equivalent to 4.50 g dried al-AGO-pretreated substrate (10%, w/v) was loaded in a 250-mL Erlenmeyer flask

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supplemented with citric buffer (0.05 M, pH = 4.8) to acquire 45 g of slurry. Next, different CTec2 loadings

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were added at 1, 2, 3, 4, 5, and 6 FPU/g dried substrate to select the desirable enzyme loading.

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Subsequently, the enzymatic hydrolysis processes of the al-AGO-pretreated substrates [6, 8 10, 12, and

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14% (w/v) solids contents] were carried out to select an initial solid content, in 250-mL Erlenmeyer flasks

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supplemented with citric buffer (0.05 M, pH 4.8), to acquire 45 g of slurry. To test an additive, 60 mg/g of the

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surfactant (Tween 80, Tween 20, PEG (Polyethylene glycol) 4000, PEG 6000, PEG 10000, and PEG 20000),

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non-catalytic protein [bovine serum albumin (BSA), casein, corn steep liquor (CSL), and peptone), or

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polymer [tea saponin and cationic polyacrylamide (c-PAM)] was added to the enzymatic hydrolysis mixture

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(10%, w/v) at the selected enzyme loading. Once the additive was identified, different amounts of it were

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further examined under the same hydrolysis condition to establish the additive amount. The enzymatic

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hydrolysis process was conducted at 50 °C for 48 h with a shaking speed of 180 rpm. For the sugar assay,

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samples were collected after 6 h, 12 h, 24 h, and 48 h and centrifuged at 10,000 rpm for 5 min.

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High-solids Hydrolysis of Substrates via the Fed-batch Mode. Once the initial solids content and

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cellulase loading were selected, high-solids hydrolysis was carried out for 72 h with different feeding

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amounts (3 and 4%) and times (6, 12, 18, 24, and 30 h) to build the fed-batch enzymatic hydrolysis process.

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Then, an AL4 (43) orthogonal experiment with three variables (Tween 80, BSA, and tea saponin) and four

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levels was designed to determine the amount of additive mixtures.20 Next, to evaluate the role of the

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accessory enzymes (BG, endo-xylanase, and AA9), different enzyme loadings were assessed along with the

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additives in the fed-batch cellulase hydrolysis. The enzymatic hydrolysis process was also performed at

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50 °C for 72 h with a shaking speed of 180 rpm. Samples were drawn at the same intervals as those used 7



1

Page 8 of 32

for the sugar assay.

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Analytical Procedures. The glucose titer in the enzymatic hydrolysate was measured with a commercial

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SBA-40E biosensor (H2O2 electrode sensor) using glucose as standard. The xylose titer was assayed with

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high performance liquid chromatography equipped with an Aminex HPX-87H column (300 × 7.8 mm,

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BioRad, US) at a column temperature of 65 °C; 5 mM H2SO4was used as the mobile phase at a flow rate of

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0.6 mL min−1. The glucose and xylose yields were determined and calculated, respectively, from the

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equations (1) and (2):

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Glucose yield (%) = 0.9× [g in glucose] × 100 / [g in cellulose]

(1)

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Xylose yield (%) = 0.88 × [g in xylose] × 100 / [g in xylan]

(2)

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In this paper, except for specific descriptions, the hydrolysis refers to glucose yield. All the samples were

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analysed in duplicate and mean values were calculated with standard deviations of ˂ 4%.

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■RESULTS AND DISCUSSION

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Key Variables of the High-solids Enzymatic Hydrolysis Process. Reasonable cellulase loading is

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perquisite for the economic and efficient enzymatic hydrolysis of lignocellulosic biomass.26 In this study, to

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run the hydrolysis process at a low cellulase loading, different enzyme loadings (1, 2, 3, 4, 5, and 6 FPU/g

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dry substrate) were employed for the batch hydrolysis of al-AGO-pretreated sugarcane bagasse at 10%

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solids content with a shaking speed of 180 rpm (Figure 1). At an enzyme loading of 1 FPU/g and 2 FPU/g,

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the glucose yield from the enzymatic hydrolysis was 34% and 40% after 48 h, respectively. At 3

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FPU/g, >60% of the enzymatic hydrolysis was attained after 48 h, and at 4 FPU/g, glucose yield reached

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68% after 48 h. Results showed that the enzymatic hydrolysis increased with a high enzyme loading.

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Especially, the enzyme loading of 3 FPU/g contributed to a fast increase of the glucose yield. Meantime, the

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substrate liquefaction was observed with naked eyes. For the enzyme loading of 1 FPU/g and 2 FPU/g, the

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substrate took a long liquefaction and completely liquefied at 24 h. Contrarily, the substrate tended to liquefy

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when the enzyme loading was higher than 4 FPU/g. This observation indicated that an enzyme loading that

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is too low is adverse to substrate hydrolysis, resulting in a long liquefaction time. On the other hand, an

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enzyme loading that is too high can contribute towards a fast process but is less economically attractive 8


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owing to the high enzyme cost.15, 27 Considering that 3 FPU/g is of an extremely low cellulase loading level

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and rendered the enzymatic hydrolysis a relatively short liquefaction time (< 12 h), 3 FPU/g substrate of

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cellulase loading was selected to initiate the study. -----Figure 1------

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Initial Solids Content. The aim of this experiment was to select a suitable initial solids content for the

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high-solids hydrolysis process. Different solids contents [6, 8, 10, 12, and 14% (w/v)] were examined in a

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batch hydrolysis process for 48 h with a shaking speed of 180 rpm at 3 FPU/g substrate of cellulase loading.

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As shown in Figure 2, the glucose yield from enzymatic hydrolysis at different solid contents increased with

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the hydrolysis time. Obviously, the increase of solid content led to a gradually low profile of glucose yield.

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When the solid content was not more than 8%, the substrate took a short liquefaction time of 6 h by

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observation with naked eyes, whereas 12 h for the enzymatic hydrolysis at ≥ 10% of the solid content. Thus,

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it is evident that selecting 8% as the initial solid content was reasonable. In regards to the glucose titer

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(Figure S1), the glucose released from enzymatic hydrolysis process first increased and subsequently

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decreased. It can be guessed that the initial increase of solid content should supply more accessible

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opportunity of substrate to the cellulase enzymes and enhance the interaction of enzyme and substrate,

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thereby releasing more sugars. At a solids content of 8%, the glucose content reached a peak level of 42 g/L,

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suggesting that the interaction of enzyme and substrate reached a maximized level. Thereafter, the

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increased solid content was mainly responsible for the sticky broth, resulting in a long liquefaction time and

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thus low hydrolysis. And even, the hydrolysis process failed in liquefaction at 48 h when the solid content

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exceeded 18% (Data not shown). A similar phenomenon was reported in other studies.12, 16 They suggested

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that the unreasonable increase in solids content in the enzymatic hydrolysis process contributes towards a

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low hydrolysis yield. Accordingly, based on the liquefaction time and final glucose titer, an initial solids

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loading of 8% (w/v) was selected.

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------Figure 2--------

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---- Figure S1-------

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Exploration of the High-solids Hydrolysis of Substrates in the Fed-batch Mode. For most varieties of

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lignocellulosic substrates, it is generally acknowledge that high-solids enzymatic hydrolysis denotes solids 9



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concentrations >15% dry matter (w/w).10,

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extremely sticky slurry and mass-transferring difficulty, which was likely responsible for the above long

3

liquefaction time of substrate hydrolysis with a high initial solid content.

4

effects derived from the high-solids enzymatic hydrolysis, we next adopted a substrate fed-batch method to

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realize the high-solids (20%) enzymatic hydrolysis process. The substrate feeding mode was assessed with

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an initial solids content of 8% by feeding unequal amounts (3% and 4%) at different times (6, 12, 18, 24, and

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30 h). The enzymatic hydrolysis of Mode 3 with feeding for three times was 51% at 72 h, a value that is

8

slightly higher than that observed for Mode 1 (49%) with feeding for four times (Table 1). Likewise, the

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comparison of Mode 4 and Mode 1 also showed that feeding for three times was better than feeding for four

10

times in improving the enzymatic hydrolysis of lignocellulosic substrate. When considering the feeding time,

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the hydrolysis at Mode 1 was better than that at Mode 2, while Mode 3 was superior to Mode 4. This

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comparison suggested that feeding the substrate at an earlier stage was favourable. Conversely, a delay in

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substrate feeding produced low hydrolysis yields, probably due to enzyme inactivity.12 Thus, a solids content

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of 4% (w/v) was selected, at feeding times of 6, 12, and 18 h, to achieve a total solids content of

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20%.Nevertheless, several studies have indicated that compared to the batch mode, the fed-batch mode

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hindered the high-solids enzymatic hydrolysis of lignocellulosic substrates.26, 29 These contradictory results

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are very probably attributed to the different stages in which the substrates are fed. A typical enzymatic

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hydrolysis process initially displays a relatively rapid rate that subsequently decreases. Thus, if the substrate

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is fed too early, the cellulase enzymes have not completed the initial substrate hydrolysis and not yet

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desorbed from the initial cellulosic substrate.26 And the viscosity in the hydrolysis system tends to increase

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with the substrate feeding, which is adverse to the mass transfer, hence a low hydrolysis yield.28 More

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seriously, the substrate feeding can result in occurrence of the hydrolysis break-off. Late addition is helpful

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to avoid the high viscosity in the broth as the initial substrate has made relatively sufficient liquefaction and

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hydrolysis. Nevertheless, the late addition is also less effective since the cellulase deactivity often occurs in

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the hydrolysis process and end product inhibition by the liberated glucose.13, 29 Thus, both the method and

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stage of substrate feeding are critical to maintain a fast liquefaction and thus a low viscosity. 28 Unfortunately,

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this is complicated by substrate characteristics, enzyme type/loading, and hydrolytic environment so that

Nevertheless, the high solid content tends to result in

16

10


Thus, to decrease the negative

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feeding scenarios are currently still elusive. Consequently, the way forward for the development of the

2

substrate fed-batch mode will require deeper and more precise knowledge of the lignocellulosic hydrolysis

3

course. Thus, the substrate fed-batch mode should be considered as an exclusively robust pathway to

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facilitate mass transfer (mixing) inside the reactor.11, 29 -----Table1------

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Enhanced High-solids Enzymatic Hydrolysis with Additives. Several studies have revealed that the

7

addition of surfactants to the cellulase mixture can significantly improve the enzymatic hydrolysis of

8

lignocellulosic substrates.19-21 In this experiment, the hydrolysis of al-AGO-pretreated substrates (3 FPU/g

9

substrate of cellulase loading) was supplemented with 60 mg/g substrate of surfactant to examine their

10

effect on batch hydrolysis. Figure 3A reveals that both Tween 80 and 20 significantly enhanced the

11

enzymatic hydrolysis process; addition of high molecular weight PEG also aided the process. Among the

12

three surfactants, Tween 80 afforded the highest cellulose hydrolysis, reaching 80% after 48 h. additionally,

13

compared to the process with no surfactant added, Tween 80 enhanced both the 24-h and 48-h enzymatic

14

hydrolyses by 30%. Accordingly, Tween 80 was selected as surfactant for subsequent analyses. Further, the

15

addition of 40 mg/g of Tween 80 produced a maximum enzymatic hydrolysis of 84%, 45% higher than that

16

observed with the control. Such a remarkable performance was attributed to the capability of Tween 80 to

17

act as a specific activator to alleviate the inhibition of lignin, hemicelluloses, and their derivatives in some

18

key cellulase components (cellobiohydrolase).21 Consequently, we concluded that the addition of 40 mg/g

19

substrate of Tween 80 as substrate was adequate to improve the hydrolysis of al-AGO-pretreated

20

sugarcane bagasse.

21

----Figure 3A----

22

Selection of Protein and Polymer. Some additives such as BSA and tea saponin significantly facilitate the

23

enzymatic hydrolysis of lignocellulosic biomass.31, 30 Notably, the tea saponin is a newly found additive

24

useful for the lignocellulosic hydrolysis, which is a type of natural non-ionic biosurfactant or biopolymer with

25

a weight-average molecular weight of ~ 800 g/mol.30 The tea saponin is commonly extracted from tea-oil

26

processing byproducts of the Camellia oleifera Abel. crop that is planted widely for extrusion of nutritional

27

edible oils in some Asian countries, mainly in China.32 In this experiment, accordingly, proteins (BSA, casein, 11



Page 12 of 32

1

corn steep liquor, and peptone) and polymers (tea saponin and c-PAM) were investigated in the batch

2

enzymatic hydrolysis of al-AGO-pretreated substrates at 3 FPU/g (Figure 3B). Among these additives, the

3

individual addition of BSA and tea saponin enhanced enzymatic hydrolysis and increased 48-h cellulose

4

hydrolysis by 13% and 11%, respectively. Thus, BSA and tea saponin were selected as additives with

5

respective concentrations of 40 mg/g and 10 mg/g. At these polymer concentrations, 48-h enzymatic

6

hydrolysis approached 90%. Both additives displayed an active role in the enzymatic hydrolysis of the

7

substrates. BSA can be irreversibly and competitively adsorbed onto the lignin coating the cellulose, thereby

8

preventing lignin adsorption to cellulose.31 On the other hand, tea saponin can promote the adsorption of

9

cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes, thus improving the

10 11

hydrolytic performance of cellulose.30 ----Figure 3B----

12

Orthogonal Optimization of Additives. Recent research has evidenced that some additives enhanced

13

enzymatic hydrolysis performed at high solids contents.22, 23 Therefore, based on the above batch enzymatic

14

hydrolysis results, an orthogonal design (Tween 80, BSA, and tea saponin) was employed for the high-solids

15

fed-batch enzymatic hydrolysis process. The reaction was carried out with 8% of the initial solids content

16

and 4% substrate feeding at 6, 12, and 18 h to achieve a total solids content of 20% (w/v). Table 2 illustrates

17

that mixtures of these additives (Tween 80, tea saponin, and BSA) at different proportions significantly

18

enhanced the process. The order of additive influence was Tween 80 > Tea saponin > BSA (Table S1). The

19

reasonable combination of these additives was determined as 40 mg/g Tween 80, 10 mg/g tea saponin, and

20

20 mg/g BSA. With this determined combination, the high-solids enzymatic hydrolysis reached 58% and

21

released 74 g/L of glucose after 48 h. Compared to the control, this translates to ~30% enhancement.

22

---- Table 2-----

23

---- Table S1----

24

Figure 4 describes the profile of the high-solids fed-batch enzymatic hydrolysis process with the

25

optimized combination of additives. The substrate [4% (w/v) total solids content] was fed separately at 6, 12,

26

and 18 h of the processes with and without additives. This contributed towards slow liquefaction and thus,

27

low hydrolysis and even a decline at the initial stage of the 24-h process. Thereafter, both enzymatic 12


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1

hydrolysis processes increased. At 72 h, the hydrolysis processes with and without additives reached 62%

2

and 51%, respectively. Concomitantly, both glucose titers in the hydrolysate broth increased with an

3

increase in hydrolysis time. Notably, this increase was more pronounced when the established mixture of

4

additives was employed. The glucose titer in the hydrolysate broth with additives was 78 g/L, >20% higher

5

than that observed for the process with no additives. The data manifested that the mixture of these additives

6

was indeed robust to improve the high-solids enzymatic hydrolysis of the al-AGO-pretreated sugarcane

7

bagasse. The result is in accordance with some previously reported studies.33 Monschein et al. suggested

8

that compared to their individual addition, the combination of PEG 8000 and urea in a 24-h enzymatic

9

hydrolysis process enhanced the reaction by ~20%.33 This study clearly confirms that compared to the

10

addition of the individual additives, a mixture of several additives can effectively increase the enzymatic

11

hydrolysis process. Nonetheless, the effectiveness of additive combination has been mainly verified in

12

enzymatic hydrolysis at low solids contents. At high solids contents, there is an increasing tendency towards

13

using single additives.22, 23 Ma et al. indicated that the addition of Tween 80 (2 g/L) at a solids content of 25%

14

increased cellulose conversion by 30%.22 Unfortunately, there is extremely limited information on the use of

15

additive combination in hydrolysis processes at high solids contents. Thus, this study underlined the positive

16

role of a combination of several additives in the enzymatic hydrolysis process at a high solids content.

17

Consequently, the above combination of additives can be used for further experimentation to facilitate

18

fed-batch enzymatic hydrolysis.

19

-----Figure 4-----

20

Enhanced High-solids Hydrolysis of al-AGO-pretreated Substrates: Addition of Accessory

21

Enzymes. Previous studies have indicated that the supplementation of cellulases with BG can minimize the

22

inhibition of the end-products (mainly cellobiose) towards cellulases and thus, improve the hydrolysis of the

23

cellulose embedded in the lignocellulosic biomass.10, 34 Such beneficial effects would be more pronounced

24

in a high-solids enzymatic hydrolysis process where the cellulase enzymes experience multiple stresses in

25

the hydrolysis system. Therefore, in this study, different amounts of BG were supplied along with the

26

optimized additives to assess the role of BG in the cellulase hydrolysis of al-AGO-pretreated sugarcane

27

bagasse at a high-solids content (Figure 5A). However, the addition of extra BG did not boost the 13



Page 14 of 32

1

high-solids (20%) enzymatic hydrolysis, even at a high BG loading of 45 U/g dried substrate (Fig. 5). This

2

was attributed to the already high BG activity in the new Novozyme CTec preparation.19, 35 Our earlier work

3

revealed that the addition of BG (1–8.0 mg/g glucan) to Cellic CTec1 produced no beneficial effects on the

4

enzymatic hydrolysis of SO2-catalysed steam exploded sweet sorghum bagasse.19 Since the CTec2

5

preparation contains even higher BG activity (1200 U/mL) than that of CTec1 (210 U/mL),19 it follows that the

6

supplementation of BG to CTec2 is unnecessary for the hydrolysis of al-AGO-pretreated lignocellulosic

7

substrates, even at high solids loading. ------Figure 5A----

8 9

Addition of Endo-xylanase. Previous studies have shown that the supplementation of xylanase to a

10

cellulase preparation can effectively facilitate the liquefaction of various types of pretreated lignocellulosic

11

substrates at high solids contents.24 Therefore, we next assessed the addition of endo-xylanase in the

12

high-solids (20% of the total solid loading) fed-batch hydrolysis of al-AGO-pretreated substrates at a

13

relatively low cellulase loading (3 FPU/g substrate). As expected, the addition of xylanase (2.4 mg protein/g

14

substrate) significantly enhanced the cellulose hydrolysis by ≤ 30% after 48-h hydrolysis (from 56% to 71%,

15

Figure 5B). The result is consistent with our previous findings, whereby the addition of xylanase greatly

16

contributed towards the hydrolysis of SO2-catalysed steam-pretreated sweet sorghum bagasse to produce a

17

30% increase.19 In this study, the al-AGO pretreated substrate contained rich hemicellulose, high up to 25%.

18

The xylan dominated hemicellulose not only acts as a coating to restrict the accessibility of cellulose to

19

cellulase enzymes,19, 24 but also serves as a cellulose microfibril cross linker to maintain an integrated cell

20

wall structure. Accordingly, the xylanase addition exerted a beneficial hydrolysis-boosting effect on the

21

xylan-enriched substrate. It can assumed that this boosting effect should be attained by synergizing the

22

enzyme interaction between xylanase and cellulase, and increasing the cellulose accessibility to the

23

cellulase with the xylan coating removal.36 In addition, due to the hydrophilicity of the xylan polymer, the

24

removal of xylan by xylanase enzymes reduces the viscosity of the biomass slurry, thus improving the

25

liquefaction of high-solids loading hydrolysis.24 In brief, our data have indicated that the addition of

26

endo-xylanase robustly enhanced the enzymatic hydrolysis of al-AGO-pretreated substrates at high solids

27

loadings. 14


Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-----Figure 5B -----

1 2

Addition of AA9. Recent studies have emphasized that lytic polysaccharide monooxygenases (LPMOs)

3

such as AA9 can significantly improve cellulose hydrolysis by oxidative cleavage of the highly organized

4

crystalline cellulose region and thus, improve cellulose accessibility to cellulase enzymes.19 However,

5

previous studies have only assessed the boosting effects of AA9 at relatively low solids contents. The

6

negative charges created by AA9 oxidative cleavage induce fibre repulsion,37 thereby facilitating biomass

7

liquefaction by avoiding fibre aggregation at high solids contents. To verify this, different amounts of AA9

8

were added in dilute cellulase (3 FPU/g) with the optimized additives and endo-xylanase (2.4 mg/g) to

9

hydrolyse an al-AGO-pretreated substrate (20%, w/v) (Figure 5C). The initial enzymatic hydrolysis

10

increased significantly with the addition of AA9. Thus, when 1.0 mg/g AA9 was added, the 48-h cellulose

11

hydrolysis reached a plateau of 76%, a 10% increase over that observed with the control. Thus, 1.0 mg/g

12

substrate of AA9 was selected in our study.

13

----Figure 5C-----

14

High-solids Hydrolysis with Additives and Accessory Enzymes. Figure 6 depicts the course of 72-h

15

high-solids enzymatic hydrolysis processes with established additives (40 mg/g Tween 80, 10 mg/g tea

16

saponin, and 20 mg/g BSA) and accessory enzymes (2.4 mg/g endo-xylanase and 1 mg/g AA9). All the

17

processes produced more glucose and xylose with a longer hydrolysis time. With just 3 FPU/g of cellulase,

18

the 48-h fed-batch enzymatic hydrolysis of the al-AGO-pretreated substrate was 44% with a glucose titer of

19

56 g/L. This value is much higher than that observed for the batch enzymatic hydrolysis (25% and 31 g/L,

20

respectively; data not shown). Similarly, Wang et al. reported that the fed-batch enzymatic hydrolysis (20

21

FPU/g glucan) at 20% solids loading produced a glucose titer of 66.0 g/L, >20% higher than that observed

22

for the batch hydrolysis.38 Evidently, such a fed-batch mode is an effective way to run the high-solids

23

enzymatic hydrolysis process, especially at low enzyme loadings.39

24

As for the glucose yield (Fig. 6A), all the profiles fluctuated at early stage (24 h) of the hydrolysis with

25

feeding the substrate and subsequently presented a climbing tendency till up to the end. The solid content

26

increased by feeding the substrates in the hydrolysis system, whereby the access and interaction between

27

substrate was presumably intensified, finally releasing more fermentative sugars (Fig. 6B). Interestingly, the 15



Page 16 of 32

1

change of glucose yields in four types of hydrolysis was obviously different at the early stage. For the

2

hydrolysis with CTec2 alone, the first substrate feeding reduced the glucose yield of 12 h. With the

3

introduction of the additive and xylanase (Xyl), the first substrate feeding increased the glucose yield, and

4

the second and third substrate feedings lowered the glucose yield at 12-24 h of hydrolysis time. Further, the

5

addition of AA9 changed such the downward profile of glucose yield. Undoubtedly, the stepwise addition of

6

additives, xylanase, and AA9 in the cellulase increasingly improved the high-solids enzymatic hydrolysis

7

process. Finally, the high-solids hydrolysis process produced glucose titers of 78, 95, and 105 g/L,

8

respectively, with corresponding enzymatic hydrolysis reaching 62%, 76%, and 83%. That is, the single use

9

of additives, xylanase, and AA9 enabled the enzymatic hydrolysis to increase by 22%, 23%, and 9%,

10

respectively. On the other hand, with the combination of these additives and accessory enzymes, enzymatic

11

hydrolysis increased by > 60%. Moreover, the xylose titer augmented concurrently with the enzymatic

12

hydrolysis running so that a xylose titer of 51 g/L was attained after 72 h, > 90% xylan hydrolysis. Further,

13

when no additives and accessory enzymes were employed, the high-solids fed-batch enzymatic hydrolysis

14

required ~6 FPU/g enzymatic loading to achieve an equivalent hydrolysis level (Figure S2). Our earlier

15

study demonstrated that the 72-h batch enzymatic hydrolysis of substrates at 18% solids content required

16

10 FPU/g of cellulase loading to approach an equivalent level.8 These results indicate that the combination

17

of additives and accessory enzymes lowers cellulase loading during enzymatic hydrolysis; this can stand out

18

in high-solids fed-batch enzymatic hydrolysis processes. Accordingly, the combination of additives and

19

accessory enzymes has presented a superior advantage in high-solids fed-batch hydrolysis of

20

lignocellulosic substrates.

21

----Figure 6-------

22

----Figure S2------

23

In summary, high-solids fed batch hydrolysis of sugarcane bagasse with a solids content of 20% was

24

successfully carried out with 3 FPU/g dried substrate of cellulase preparation CTec2 combined with several

25

additives (40 mg/g Tween 80, 10 mg/g tea saponin and 20 mg/g BSA) and accessory enzymes (2.4 mg/g

26

endo-xylanase and 1 mg/g AA9). The 72-h enzymatic hydrolysis process released 105 g/L of glucose and

27

51 g/L xylose, with 83% and 93% cellulose-to-glucose and xylan-to-xylose yields, respectively. Several 16


Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

studies have also reported high-solids enzymatic hydrolysis at ~20% solids contents (Table 3).On

2

comparing the other lignocellulosic substrates, al-AGO-pretreated sugarcane bagasse presented relatively

3

high holocellulose (57% cellulose and 25% hemicellulose) and low lignin (12%) contents that produced

4

high-titer fermentable sugars via high-solids enzymatic hydrolysis. After a 72-h fed-batch hydrolysis, the

5

substrate yielded 83% glucose and 90% xylose, rendering 156 g/L of fermentable sugars (105 g/L glucose +

6

51 g/L xylose). This was an unprecedentedly high level of sugar output as compared to that of previously

7

reported processes with similar solids contents. Thus, the enzymatic hydrolysis process studied herein

8

achieved an interestingly high fermentative sugars productivity of 2.2 g/L/h (= 156 (g/L) / 72 (h)). Notably, the

9

high-solids enzymatic hydrolysis was implemented at an extremely low cellulase loading (3 FPU/g substrate

10

compared to commonly reported values > 10 FPU/g).To the best of our knowledge, this is the lowest level

11

reported to date. ---Table 3---

12

13

■CONCLUSIONS

14

A fed-batch mode is indeed beneficial to run high-solids enzymatic hydrolysis of lignocellulosic substrates, in

15

which early feeding of all the substrates (< 24 h) facilitates the process. al-AGO pretreated lignocellulosic

16

substrates present an industrially relevant hydrolysability, rendering 83% of glucose yield and 90% of xylose

17

yield at 72 h from high-solids substrate (20%) with 3 FPU/g substrate of the cellulase loading. The

18

combination of additives (Tween 80, tea saponin, and BSA) and accessory enzymes (endo-xylanase and

19

AA9) can effectively enhance the high-solids enzymatic hydrolysis. Extra β-glucosidase addition is evidently

20

unnecessary for the cellulase preparation CTec2 hydrolysing lignocellulosic substrate. The customization of

21

a cellulase preparation on the specific substrate shows potential for application to high-solids hydrolysis of

22

lignocellulosic substrates to lower the enzyme cost in the ongoing lignocellulosic biorefining industry. 

23



24

■ASSOCIATED CONTENT

25

Supporting Information

26

The Supporting Information is available free of charge on the ACS Publications website 17



1

■AUTHOR INFORMATION

2

Corresponding Authors

3

*E-mail: [email protected] ; [email protected] (F.B.S)

4

*E-mail: [email protected] ; (HYR)

5

ORCID

6

Fubao Fuelbiol Sun: 0000-0002-1208-469X

7

Notes

8

The authors declare no competing financial interest.

9

■ACKNOWLEDGEMENTS

Page 18 of 32

10

The work was funded by the National Natural Science Foundation of China (21776114; 21176106), together

11

with the Jiangsu Provincial Natural Science Foundation of China (SBK2018021515). Part of the work was

12

also supported by the Jiangsu Province “Six Talent Peak” (XNY-010) and the China Postdoctoral Science

13

Foundation (2016T90419). The authors also give thanks to the national first-class discipline program of

14

Light Industry Technology and Engineering (LITE2018-01), the 111 Project (No. 111-2-06) and the Jiangsu

15

province "Collaborative Innovation Center for Advanced Industrial Fermentation" industry development

16

program.

17

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20


Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Table and Figure legend

3

Figure 1. Selection of a suitable cellulase loading for the high-solids enzymatic hydrolysis of an

4

al-AGO-pretreated substrate. Batch enzymatic hydrolysis was conducted at a solids content of

5

10% (50 °C and 180 rpm) in citric buffer solution (45 mL, 50 mM, pH = 4.8) for 48h.

6

Figure 2. Selection of the initial solids content for the fed-batch enzymatic hydrolysis. The process was

7

carried out using 3 FPU/g substrate of cellulase loading in citric buffer solution (45 mL, 50 mM,

8

pH = 4.8) for 48h.

9

Figure 3. Effect of surfactants (A) and proteins and polymers (B) on the enzymatic hydrolysis of an

10

al-AGO-pretreated substrate. The enzymatic hydrolysis [10% (w/v) solids content] was

11

conducted using 3 FPU/g substrate of cellulase loading and 60 mg/g substrate of the initial

12

additives under the above mentioned conditions.

13

Figure 4. Hydrolysis profile of the al-AGO-pretreated substrate [20% (w/v) solids content] with 3 FPU/g

14

cellulase loading supplemented with 40 mg/g Tween 80, 20 mg/g BSA, and 10 mg/g tea

15

saponin. The fed-batch hydrolysis was initiated with an initial solids content of 8%, followed by

16

feeding with 4% substrate, separately at 6, 12, and 18 h.

17

Figure 5. Evaluation of the effect of the accessory enzymes on the high-solids enzymatic hydrolysis

18

process [20% (w/v) solids content]. Hydrolysis was implemented as mentioned above. A) BG

19

addition; B) endo-xylanase addition; and C) AA9 addition (2.4 mg/g endo-xylanase).

20

Figure 6. High-solids enzymatic hydrolysis of al-AGO-pretreated substrates at 3 FPU/g for 72 h with the

21

combination of additives (40 mg/g Tween 80, 20 mg/g BSA, and 10 mg/g tea saponin) and

22

accessory enzymes (2.4 mg/g endo-xylanase and 1 mg/g AA9). A) glucose yield; B) sugar titer.

23

G, glucose; Xyl, xylose

24

21


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Table 1. Selection of a substrate fed-batch mode for the high-solids enzymatic hydrolysis of the al-AGO-pretreated substrate. Enzymatic hydrolysis was initiated with a solids content of 8% and 3 FPU/g substrate of cellulase loading in citric buffer solution (45 mL, 50 mM, pH = 4.8) for 72 h. Enzymatic hydrolysis Substrate feeding

48 h

72 h

6h

12 h

18 h

24 h

30 h

Titer (g/L)

Yield (%)

Titer (g/L)

Yield (%)

Mode1

3%

3%

3%

3%

-

56 ± 0.7

44 ± 0.6

61 ± 1.4

49 ± 1.1

Mode 2

-

3%

3%

3%

3%

48 ± 1.4

39 ± 0.3

56 ± 0.7

45 ± 1.4

Mode 3

4%

4%

4%

-

-

58 ± 0.7

46 ± 0.6

64 ± 0.7

51 ± 0.7

Mode 4

-

4%

4%

4%

-

52 ± 1.4

42 ± 1.1

59 ± 1.4

47 ± 1.1

22


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Table 2. Optimization of additives using the orthogonal experiment for the high-solids enzymatic hydrolysis process. The hydrolysis was carried out with an initial solids content of 8%.Feeding with 4% of each substrate was performed at 6, 12, and 18 h to achieve a total solids content of 20% (w/v). Additives (mg/g)

Enzymatic hydrolysis

Tween 80

BSA

Tea saponin

Control

0

0

1

40

2

Glucose titer (g/L)

Glucose yield (%)

6h

12 h

24 h

48 h

6h

12 h

24 h

48 h

0

20 ± 0.7

28 ± 0.7

45± 1.1

58 ± 0.7

40 ± 0.5

35 ± 0.9

36 ± 0.8

46 ± 0.2

20

10

24 ± 0.0

38 ± 0.7

54 ± 0.7

74 ± 1.4

47 ± 0.0

48 ± 0.7

43 ± 0.6

58 ± 1.1

40

20

20

23 ± 0.4

35 ± 1.4

52 ± 1.4

72 ± 0.7

45 ± 0.7

44± 1.8

41 ± 1.1

57± 0.2

3

20

40

20

22 ± 1.4

32 ± 0.7

48 ± 0.4

66 ± 1.1

43 ± 2.8

40± 0.8

38 ± 0.3

52 ± 0.8

4

20

20

10

22 ± 0.7

33 ± 1.8

50 ± 1.4

68 ± 1.4

43 ± 1.0

42± 2.3

40 ± 1.1

54 ± 1.1

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Table 3. High-solids (~20%) enzymatic hydrolysis of various lignocellulosic substrates Substrate components (%)

Enzymatic hydrolysis

Hydrolysis

Substrate source

Source C

H

L

ABPERC

68

3

26

SEP wheat straw

52

14

SEP olive tree

57

AGO-pretreated SCB

Cellulase loading

Mode

Time (h)

Titer (g/L)

Yield (%)

46 FPU/g

Batch

114

126

84

[14]

26

13.5 FPU/g + 67.5 U/g BG

Batch

96

76

60

[9]

2

-

15 FPU/g + 15 CBU/g BG

Batch

72

65

52

[30]

60

13

14

10 FPU/g

Batch

72

96

80

[5]

SEP SCB

55

4

32

8.25 FPU/g

Batch

96

77

69

[31]

OP poplar

92

1

2


Batch

96

158

85

[25]

EP Poplar

49

13

34


Batch

48

71

83

[38]

NaCl-pretreated wood

48

19

29

22 FPU/g + 68 U/g BG+ 1% Tween 80

Batch

120

81

40

[37]

SEP SSB

49

5

41

20 FPU/g +0.175mL/g solids Tween80

Fed-batch

120

66

46

[34]

FP SCB

86

-

-

10 FPU/g

Fed-batch

144

150

80

[35]

AHP-pretreated SCB

65

14

13

15 FPU/g + 25 CBU/g substrate BG

Fed-batch

48

82

57

[36]

NaCl-pretreated wood

48

19

29

22 FPU/g + 68 U/g BG+ 1% Tween 80

Fed-batch

120

127

64

[37]

Al-AGO-pretreated SCB

57

25

12

3 FPU/g

Fed-batch

72

64

51

This work

3 FPU/g + additives + accessory enzymes

Fed-batch

72

105

83

This work

Abbreviations: C, cellulose; H, hemicellulose; L, lignin; FPU, filter paper unit; CBU, cellobiase unit, SCB, sugarcane bagasse; SSB, sweet sorghum bagasse; BG, beta-glucosidase; UBHW, unbleached hardwood pulp; AA9, lytic polysaccharide monooxygenase, AHP, alkaline hydrogen peroxide, al-AGO,

alkaline-catalysed

atmospheric

glycerol

organosolv;

GLP,

green

liquor-pretreated;

SEP,

steam

explosion-pretreated;

organosolv-pretreated; FP, formiline-pretreated; ABPERC, acid bisulfite-pretreated eastern red cedar; DAP, dilute sulfuric acid-pretreated

24


OP,

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Figure 1.

25



Figure 2.

26


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Figure 3A.

27



Figure 3B.

28


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Figure 4.

29



Figure 5. 30


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Figure 6.

31



GRAPHICAL ABSTRACT

Note: Picture adopted from the old Chinese fable of Yugong Yishan (The Old Fool Who Moved Mountains). C, cellulase; A, additives; AE, accessory enzymes; G / X, glucose and xylose, respectively. The high-solids substrate enzymatic hydrolysis process is just like moving the mountains, Tai-hang Mountain and Wang-wu Mountain. The additives and accessory enzymes (assisting laborer) are helping the cellulase (elite laborer) to hydrolyse collaboratively the cellulose and hemicellulose components (two mountains) consisting mainly of the al-AGO pretreated substrate into fermentative glucose and xylose sugars (little stones).

Figure Cellulase enzymatic hydrolysis of alkaline-catalysed atmospheric glycerol organosolv-pretreated substrate with mixture of additives and accessory enzymes


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Enhanced High-Solids Fed-Batch Enzymatic Hydrolysis of Sugar

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