High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover

Accepted Manuscript High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover Lei Qin, Zhi-Hua Liu, Mingjie Jin, Bing-Zhi Li, Ying-Jin Yuan PII: DOI: Reference:

S0960-8524(13)01167-X http://dx.doi.org/10.1016/j.biortech.2013.07.099 BITE 12144

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

30 May 2013 19 July 2013 22 July 2013

Please cite this article as: Qin, L., Liu, Z-H., Jin, M., Li, B-Z., Yuan, Y-J., High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.07.099

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High temperature aqueous ammonia pretreatment and post-washing enhance 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 61 62 63 64 65

the high solids enzymatic hydrolysis of corn stover

Lei Qin1, Zhi-Hua Liu1, Mingjie Jin2, Bing-Zhi Li1*, Ying-Jin Yuan1

1

Key Laboratory of Systems Bioengineering, Ministry of Education, School of

Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China 2

Biomass Conversion Research Lab, Department of Chemical Engineering and

Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910, USA

* Corresponding author. Fax: +86 22 27403888. E-mail addresses: [email protected]

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Abstract 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 61 62 63 64 65

Aqueous ammonia pretreatment was optimized and the limiting factors in high solids enzymatic hydrolysis were assessed. The recommended pretreatment condition to achieve high enzymatic yield was: 180°C, 20% (w/w) ammonia, 30 min, and 20% solids content. FT-IR and GC-MS results indicated that most of the lignin was degraded to soluble fragments after pretreatment. The pretreated solids after postwashing showed higher enzymatic digestibility at high solids loading than that without washing. The washed solids required lower cellulase and xylanase dosage than unwashed solids to achieve high sugar yield. Enzymatic conversions were declined with the increased solids loading of pretreated solids, pretreated-washed solids, and filter papers. The results indicated that solids loading in enzymatic hydrolysis was an important factor affecting sugar yield. The increasing concentration of glucose and ligno-phenolics mainly inhibited the enzymatic hydrolysis of aqueous ammonia pretreated corn stover.

Keywords Corn stover; Aqueous ammonia pretreatment; Post-washing; Enzymatic hydrolysis; High solids loading

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1. Introduction 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 61 62 63 64 65

Energy security and environmental stress enhance the development of biofuels as a substitute of fossil energy. Developing the technologies for large-scale production of ethanol from lignocellulosic feedstocks is probably a promising way to resolve those concerns (Zhong et al., 2010). Pretreatment of recalcitrant cellulosic materials is projected to be the single, most expensive processing step in lignocellulosic ethanol production, which is essential to achieve high yields. In addition, pretreatment affects virtually all other steps including biomass size reduction, hydrolysis, and fermentation, etc. (Yang and Wyman, 2008; Pryor et al., 2012). Pretreatments using ammonia, such as soaking in aqueous ammonia (SAA) (Ko at al., 2009), ammonia recycle percolation (ARP) (Kim et al., 2003), and ammonia fiber expansion (AFEX) (Li et al., 2010) have been widely applied to disrupt plant cell wall structure and increase enzymatic hydrolysis yields. One particular benefit of ammonia pretreatments is removal of lignin, which is a major cause of lignocellulosic biomass recalcitrance (Wyman et al., 2005). As ammonia recovery and lignin recovery process are developed (Sherman et al., 2012), pretreatment using ammonia is as competitive as other pretreatment methods. In addition, High solids content pretreatment has attracted more and more attention recently (Modenbach and Nokes, 2012; Zhang et al., 2011), which is able to reduce cost significantly (Zhu et al., 2010; Tao et al., 2011). Further improvements on overall yield, titer and productivity are required to make cellulosic ethanol more commercially-attractive. The efficient enzymatic saccharification of cellulose at low cellulase loadings continues to be a challenge for commercialization of bioethanol (Arantes and Saddler, 2010). Enzymatic hydrolysis at high solids loading is also identified as a bottleneck affecting overall ethanol yield (Lau and Dale, 2009). As known, high ethanol concentration is essential to reduce the

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distillation energy and save the cost for cellulosic ethanol. Hence, a high solids 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 61 62 63 64 65

loading for enzymatic hydrolysis is necessary (Dien et al., 2003). Unfortunately, higher solids loading resultes in substantial reduction in the conversions to monomeric sugars (Zhong et al., 2009). The inhibitor to enzymes in enzymatic hydrolysate is the main reason for decrease of sugar conversion at high solids loading and low cellulase loading. However, there is no consistent conclusion on this phenomenon. Some researchers showed that all the degradation products somewhat inhibit the hydrolysis (Jing et al., 2009; Kim et al., 2011), while others supported that specific kind of inhibitor is the primary cause, which consists of: inhibition by lignophenolic fragments and extractives depositing on biomass surface (Ximenes et al., 2010); inhibition by high concentrations of glucose and cellobiose (Kristensen et al., 2009; Hodge et al., 2008); inhibition by xylooligomers (Qing et al., 2010); inhibition by organic acids (Panagiotou and Olsson, 2007); potential increasing viscosity (Hodge et al., 2008). To our knowledge, the relationship between sugar conversion and solids loading has not been studied when using aqueous ammonia pretreated biomass as the substrates. In addition, the concentration of kinds of inhibitors as the solids loading increasing is not clear, and what is the major inhibition factor during high-solids enzymatic hydrolysis is not known. Post-washing after pretreatment is usually employed to reduce or diminish the inhibition. Previous study reported the saccharification deficiency of pretreated solids without post-washing (Chundawat et al., 2007). In this study, we optimize the aqueous ammonia pretreatment conditions (temperature, time, ammonia concentration, solids content, with or without washing). In subsequent hydrolysis, the effects of cellulase and xylanase loading and the solids

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loading on digestibility of corn stover (CS) are examined, and the inhibition factors in 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 61 62 63 64 65

high solids enzymatic hydrolysis are also discussed.

2. Material and Methods 2.1 Materials Corn stover used for this study was obtained from the suburb of Tianjin, China, and then milled and screened. The fractions between 20 and 80 meshes were collected and air dried at room temperature. Air-dried CS had an average moisture content of 8%. The composition of CS (dry basis) determined following the Laboratory Analytical Procedure (LAP) of the National Renewable Energy Laboratory (NREL, 2008) is: 31.7% glucan, 17.1% xylan, 2.6% arabinan, 12.6% acid insoluble lignin (AIL), 23.5% extractives, and 4.3% ash. Whatman No.1 filter paper (consisting of 98% glucan and 1% xylan of dry matter) was used as the pure cellulose, which was cut into ~1×5 mm pieces for hydrolysis. The protein concentrations of the respective enzymes are as follows: Accellerase 1500 (Genencor, 89 mg/ml, 77 FPU/ml, where FPU stands for filter paper units), Novozyme 188 (Sigma-Aldrich, 67mg/ml, 850 CBU/ml, where CBU stands for cellobiose units), and Multifect xylanase (Genencor, 42 mg/ml). 2.2 Aqueous ammonia pretreatment Milled CS (15 g dry matter) was mixed with the ammonia solution to a final weight of 150 g, and subjected to a tube reactor heated by an oil bath, as previous described (Qin et al., 2012). The pretreated slurry was pressed through a filtration cloth to separate free liquid from hydrolyzed solids. The solid fraction was washed 3 times using 300 ml water totally, or without washing in some experiments. The solid fraction was dried at room

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temperature until the moisture was less than 10%, and then subjected to LAP 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 61 62 63 64 65

composition analysis and enzymatic hydrolysis process. 2.3 Enzymatic hydrolysis The NREL standard protocol (LAP-009) was followed for enzymatic hydrolysis. Experiments at varied glucan loadings were conducted with 20-ml reaction volume in 100-ml Erlenmeyer flasks, with commercial cellulase and beta-glucosidase at 50°C and 200 rpm. 50 mM citrate buffer (pH 4.8) with 40 mg/l tetracycline was used in enzymatic hydrolysis. Certain samples were also hydrolyzed using additional commercial xylanases (0~25 mg protein/g glucan). Samples with glucan loading greater than 3% were added in batches in a period of time, rendering the solid-liquid mixture fluid. After enzymatic hydrolysis, the mixture was centrifuged at 12,000 rpm for 10 min to separate hydrolysate from solid residue. The hydrolysates were frozen at -20°C for subsequent sugar analysis. The glucose (xylose) yield is defined as: Glucose (Xylose) yield = The amount of glucose (xylose) in hydrolysate × 0.9 (0.88) / The amount of glucan (xylan) in untreated CS × 100% Oligomeric sugars in the hydrolysate were acid-hydrolyzed and analyzed according to LAP-014 from NREL. 2.4 Response surface methodology The experimental data was analyzed by the statistical software Design Expert 8.0 (Stat-Ease Inc., USA) based on the Box-Behnken design. The study of the aqueous ammonia pretreatment was addressed by performing the experimental design in which pretreatment temperature, time, and ammonia concentration (defined as the proportion of ammonia in the solid-liquid mixture, w/w), as detailed in Table 1, were retained as

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factors. The ratio liquid to solid or solid content in the pretreatment reactor was kept 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 61 62 63 64 65

constant at 10% (w/w). Glucose yield was determined as model response (Y). 2.5 Fourier transform infrared spectroscopy (FT-IR) The FT-IR spectra were obtained using a Nicolet IZ10 Fourier transform infrared spectroscopy and potassium bromide disc containing about 1% finely ground samples. The sample was pressed uniformly and tightly against the diamond surface using a spring-loaded anvil. Spectra were obtained by averaging 32 scans from 4,000 to 400 cm-1 at 1 cm-1 resolution. 2.6 Supernatant protein determination The total concentration of protein in supernatant was measured by the Bradford method with BSA as the protein standard to determine how much dissolved protein was not adsorbed on substrates (Tu et al., 2007). 2.7 Analysis of phenolic compounds in hydrolysate by GC-MS The hydrolysates were extracted by methyl tert-butyl ether (MTBE) before sampling, following the extraction procedure by Chen et al. (2006). Salicylic acid was used as the internal standard. The samples were then analyzed by GC-TOF-MS. One microliter sample was injected by Agilent 7683 autosampler into Agilent 6890 GC which was equipped with a fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm DB-5MS, J&W Scientific, Folsom, CA) with the following temperature program: after a 2 min delay at 70°C, the oven temperature was increased to 290°C at 8°C min-1, holding for 3min. Masses were acquired in the range of m/z 50-800. Peak detection was performed by Masslynx software (Version 4.1, Waters Corp., USA). Phenolics were identified by comparing their mass fragmentation with NIST library and Golm Metabolome Database.

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3. Results and Discussion 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 61 62 63 64 65

3.1 Optimization of temperature, ammonia concentration and time in aqueous ammonia pretreatment The aqueous ammonia pretreatment conditions were optimized according to the experimental design in Table 1. The pretreated samples were post-washed and enzymatic hydrolyzed at 6% glucan loading using Accellerase 1500 (60 FPU/g glucan) and Novozyme 188 (30 CBU/g glucan) for 168 h. In this case, the high level of enzyme loading and incubating time ensures the adequate access of enzymes to the pretreated solid. The statistic interpretation of the results in Table 1 was formulated by using the quadratic equation, which allows the influence of each factor on the responses as well as interactions among factors to be determined. The variance analysis results of glucose yield are summarized in Supplementary data, Table S1. The model F-value of 55.7 for glucose yield and the value of ―Prob > F‖ less than 0.05 for the model showed that the model is significant. The model also passed the ―lack of fit‖ test. All the potential parameters except the ―time‖ term showed a high significance. The high level of significance of regression together with the high R2 (0.986) and R2Adj (0.969) indicate model strength in representing the data. Good agreement between predicted and experimental values for glucose yield was shown in Supplementary data, Fig. S1. The glucose yield increased as the severity of the pretreatment condition increased, particularly as temperature and ammonia concentration increased (Fig. 1). However, the difference of glucose yield between the reaction times was rather small especially when temperature and ammonia concentration was fixed at 180°C and 20%, respectively, where final glucose yield was almost independent of reaction time. To be more industrially-relevant, relatively short time (30 min or even less) was used.

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Optimal condition for aqueous ammonia pretreatment was 20% ammonia, 30 min and 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 61 62 63 64 65

180°C (among the tested condition ranges) according to the mathematic model. In this case, overall glucose yield is predicted to be reached to 90.5%, while xylose yield is 33.1%. These pretreatment conditions and sugar yields are quite close to the previous study (Kim et al., 2003). Interestingly, the optimal condition to achieve maximum delignification (180°C, 20% ammonia, and 90 min) was not accordant with the condition to achieve maximum glucose yield. Using ammonia to remove lignin has been known to improve cellulose digestibility for years. However, the glucan conversion in enzymatic hydrolysis was not well-dependent on the delignification of pretreated solids among the pretreatment conditions in this study (Supplementary data, Fig. S2), reflecting delignification is not the single influencing factor. Such high temperature (180°C) may also be helpful to improve digestibility of pretreated solids by swelling biomass (Tao et al., 2011). All in all, this optimal pretreatment condition exhibited high conversion of pretreated cellulose in enzymatic hydrolysis. 3.2 Effects of solids content on pretreatment Higher solids content pretreatment (solids content varied from 15% to 25%) was also carried out with comparison to the solids content of 10%. Pretreatment at high solids content can reduce both capital cost and operating cost, which increase the economic feasibility of the biorefinery process (Modenbach and Nokes, 2012). Previous study also emphasized using a high solid-to-liquid ratio to reduce thermal energy consumption for pretreatment (Zhu et al., 2010). It is suggested that some pretreatments (such as dilute acid and steam explosion pretreatment) at high solids content also achieved high enzymatic digestibility (Zhang et al., 2011; Li and Kim, 2011). The composition, recovery and sugars yield from different solids content pretreatments were shown in Table 2. For post-washed solids, only 5.6% decrease of

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glucose yield was detected when solids content during pretreatment increased from 10% 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 61 62 63 64 65

to 25%, while acid insoluble lignin (AIL) removal was decreased. By contrast, a greater decrease of glucose yield (16.0%) was obtained for unwashed solids. It seems that the digestibility and delignification was attributed to the ammonia loading (defined as the ratio of ammonia to untreated solid, w/w), because more ammonia solublized more lignin. Previous study also argued that, in fact, the ammonia loading obviously impacts the delignification and enzyme digestibility, while solid-to-liquid ratio did not exhibit significant effects (Li and Kim, 2011). As a result, pretreatment at the optimal condition with high solids content (20%) can be applied to achieve dryto-dry process and reduce energy. 3.3 Effects of post-washing on solids digestibility Table 2 also showed that digestibility and delignification were both significantly improved by washing. Results imply that the soluble lignin after pretreatment will reconsolidate and re-adhere to the surface of cellulose. The post-washed solids resulted in much more AIL removal than the unwashed solids due to the alkali-solubility of AIL. The composition and mass recovery of solids with and without washing from the optimized pretreatment at 20% solids content was especially discussed here to understand this variance. For the unwashed solid, there was no evident loss of glucan and xylan, while 39.0% of AIL was removed during pretreatment. Most of the glucan and 61.7% of the xylan was recovered in solid phase after post-washing. The removed xylan by washing was attributed to its improved solubility, which was caused by cleavage of hemicellulose-lignin complex linkages during pretreatment. Another 24.1% of lignin was washed out during the post-washing step. This extra removed AIL was likely to be alkali soluble in the washing stream. The removal of hemicellulose and lignin increased the cellulose content in pretreated solid and increased glucose yield.

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The removal of AIL resulted in enhanced enzymatic hydrolysis by reducing the 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 61 62 63 64 65

cellulase-lignin binding and increasing the accessibility of cellulose to the enzyme, which confirmed the result from previous study (Ko et al., 2009). We also observed that the glucose yield of a washed solid with 48.6% of AIL removal was higher than a solid without washing with 64.3% of AIL removal (Table 2). It suggests that besides AIL, some soluble materials, including ligno-phenolics, aliphatic acids or soluble polysaccharides, also clearly affects the digestibility. Considering results above, postwashing was regarded as a crucial step on enhancing enzymatic digestibility of pretreated CS by removing not only lignin but also other soluble inhibitors. The FT-IR spectra for aqueous ammonia pretreated CS with and without washing and for untreated CS were shown in Supplementary data, Fig. S3. The pretreated CS changed the 1720 cm-1 peak attributed to hemicellulose acetyl and uronic ester groups or linkages in lignin or ester hemicellulose feurilic and p-coumaric acid carboxylic groups (Liu at al., 2013), which means the ester linkages of hemicellulose–lignin complexes were dissociated by aqueous ammonia pretreatment. The intensity of the 1244 cm-1 band attributed to the C-O stretching band of guaiacyl units, which indicated that the relative content of guaiacyl lignin units have significant decreased after pretreatment compared with untreated ones. There is also a change in the relative intensity of the peak at 1670 cm-1, which are characteristic of amide linkages, possibly due to the ammonolysis of the acetyl groups in hemicellulose (Chundawat et al., 2007). The peaks at 1599, 1508, and 1451 cm-1 are from aromatic skeletal vibrations in the lignin, which were present in both untreated and pretreated unwashed solids. This information implied that lignin degraded to small fragments and adhered to the surface of pretreated solids without post-washing, which may

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resolve in the hydrolysate and affect the enzymes activity, confirming the previous 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 61 62 63 64 65

discussion of the role of post-washing. 3.4 Xylanase supplementary As shown above, CS after pretreatment with 20% solids content was subjected to enzymatic hydrolysis. The experiment of xylanase addition in the presence of 60 FPU of Accellerase 1500 and 30 CBU of Novozyme 188 per gram of glucan was conducted to enhance the enzymatic digestibility. As expected, the addition of xylanase increased both glucose and xylose conversion (Fig. 2A). Furthermore, the sugar conversion increased as the dosage of xylanase increased. Commercial hemicellulase preparations contain trace amount of cellulase and beta-glucosidase activities, which may help cellulose degradation. In addition, synergistic interaction between xylanases and cellulases might also play a role in the improvement of cellulose accessibility by increasing fiber swelling and porosity (Hu at al., 2011). Xylanase loading beyond the 12.5 mg of protein/g glucan did not significantly improve cellulose digestibility. As the Multifect Xylanase loading was increased from 0 to 25 mg of protein/g glucan, the xylose conversion for washed solid increased by 24.7%, while 5.8% of increase was observed for glucose conversion. Under the same condition, the glucose and xylose conversion from unwashed solid increased by 29.2% and 25.4%, respectively. This higher improvement compared with washed solid may be due to the higher xylan content and higher lignin content in unwashed solid. Previous studies suggested that the xylan content influenced the enzyme requirements for hydrolysis (Pryor et al., 2012). 3.5 Effects of cellulase loading on the hydrolysis of pretreated CS Experiments with cellulase loadings of 15, 30 and 60 FPU/g glucan were performed to give more insight into the mechanism of the hydrolysis of aqueous

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ammonia pretreated CS with and without washing. Novozyme 188 and Multifect 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 61 62 63 64 65

Xylanase were fixed at 30 CBU/g glucan and 12.5 mg protein/g glucan, respectively. Fig. 2B showed that the initial rates of xylose and glucose release increased with the increasing cellulase loading. The results supported the hypothesis that the initial rate of hydrolysis is dependent on the number of available reaction sites on cellulose and amount of enzyme available to occupy these sites (Öhgren et al., 2007). It took 5 days for washed solids to achieve no less than 60 g/l glucose in hydrolysis at three varied cellulase loadings. However, lower concentrations and hydrolysis rates of glucose were observed for unwashed solids (43.9, 49.4 and 61.5 g/l glucose for enzyme loadings of 15, 30 and 60 FPU/g glucan, respectively, after 7-days hydrolysis), indicating that competitive inhibition occurred in the hydrolysis for unwashed solids (Jing et al., 2009). The result of supernatant protein also confirmed this inference (Supplementary data, Fig. S4). Protein content was lower in the hydrolysate of the unwashed solids than the washed solids in the early stage of hydrolysis (about initial 3 h) for both 15 and 60 FPU cellulase loadings. After the initial fast adsorption, the enzymes began to desorb for the washed substrates. However, the enzymes in the hydrolysate of unwashed solids didn’t desorb and the amount of free enzymes continued to reduce until no protein was detected in the supernatant, reflecting the soluble material in the unwashed solids bonded to enzymes and caused them precipitation. In addition, different xylose concentrations were also observed for washed and unwashed solids (Fig. 2B). The lower xylose concentrations and hydrolysis rates of washed solids were due to the less xylan content in substrates.

3.6 Effects of solids loading on sugar yield in enzymatic hydrolysis

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To understand the yield determining factors at high solids loadings, enzymatic 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 61 62 63 64 65

hydrolysis at different solids loadings was conducted. Whatman No.1 filter paper was used as control due to its similar crystal form of cellulose (cellulose II) with ammonia pretreated solids. Glucan loadings from 1% to 9% of unwashed solids and that from 1% to 12% of washed solids were plotted against the glucan conversions and glucose concentrations with cellulase loading of 15 FPU/g glucan in Fig. 3. Solids loading higher than these ranges in hydrolysis led to hard liquefaction of the mixture. For each substrate, sugar concentration increased (Fig. 3A) but conversion continuously decreased (Fig. 3B) as the glucan loading increased, which was agreed with previous studies (Zhong et al., 2009). The conversion of filter paper to glucose also decreased when glucan loading increased, implying that high glucose concentration caused endproduct inhibition to cellulase, which is supported by previous study (Kristensen et al., 2009). It was reported that the proportion of adsorbed enzymes decreased with increasing solids loading when using filter paper as the substrate, because the cellulose binding domain of cellulase are affected by glucose and cellobiose (Kristensen et al., 2009). The glucan conversion at 1% glucan loading for all substrates was virtually over 95%. This suggested that the pretreated solids from the optimal condition had high digestibility, while post-washing apparently not affected digestibility at such low solid loading. However, as the solids loading increased (≥2%), the glucan conversion of unwashed solids decreased much more than that of washed solids and filter papers (Fig. 3), supporting that some soluble compounds in the unwashed solids inhibited enzyme activities. The decrease of xylan conversion to xylose was similar compared to glucan, while conversions to xylooligomer increased with greater glucan loading as previous reported (Zhong et al., 2009), rendering the total yields of monomeric and oligomeric

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xylose between 80%~100% for all the solids loadings (Fig. 4A). It implied that more 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 61 62 63 64 65

beta-xylosidase activity is needed in the commercial enzyme preparations for high solid loading hydrolysis. Accumulation of xylooligomers during high solids loading enzymatic hydrolysis was also observed and considered as an issue for AFEX pretreated biomass (Jin at al., 2013). It was reported that xylooligomers were stronger inhibitors to cellulase than either xylan or xylose at an equivalent amount, and the inhibition increased with concentration of these compounds (Qing et al., 2010). However, neither the higher xylose concentration nor the higher xylooligomer concentration (Fig. 4B) obviously reduced the glucan conversion, elucidated by the comparison between filter papers and washed solids (Fig. 3). The glucan conversion of the washed solids was slightly lower than that of filter paper with each glucan loading (less than 5%), implying the xylooligomer and xylose had little impact on enzymatic hydrolysis for each solids loading. The ligno-phenolic components in enzymatic hydrolysate were determined by GC-TOF-MS. Guaiacol, syringol, p-hydroxyl-benzaldehyde, vanillin, syringaldehyde, p-acetylphenol, acetoguaiacon and acetosyringone were detected as the main lignin degradation components (Fig. 5).These small molecule components were formed due to hydrolytic/oxidative cleavage at the high severe pretreatment conditions. Phenolic aldehydes and ketone are more likely to be produced in alkaline conditions. The amount of most phenolics increased as the solids loading increased for unwashed solids, while trace amount of phenolics was found for washed solids. It is reported that the soluble phenolic compounds were strongly inhibitory to cellulase, reduced both rate and yield of cellulose hydrolysis (Ximenes et al., 2010). Ligno-compounds inhibit the enzymes by non-productively adsorption and cause the precipitation of enzymes (Yang and Wyman, 2006; Kim et al., 2011). Thus, this effect reduced the

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amount of active enzymes on the solids and free enzymes in hydrolysate, which 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 61 62 63 64 65

agreed with the observation in Fig. S4. . Linear correlations were observed between glucose yield and phenolics concentration in enzymatic hydrolysates (e.g. vanillin in Fig. 6A), suggesting some phenolics directly affected the glucose yield. Fig. 6B showed that the functions of glucose yield and xylooligomer concentration between washed and unwashed solids was misaligned, reflecting xylooligomer was not the only inhibitor in hydrolysis. Partial correlation analysis was employed to estimate the impact of individual factor (including glucose, xylose, xylooligomer and vanillin) on the glucose yield of both pretreated solids with and without washing (Table 3). Glucose concentration and vanillin concentration had significant correlation factors (0.723 and 0.801, respectively) with glucose yield, while the correlations of xylooligomer and xylose on glucose yield were not significant. These results showed that glucose and phenolics were the main causes of glucose yield decrease as the solids loading increased. In addition, mass transfer limitations for enzymes and the solid substrate may be another factor to the deficiency in hydrolysis with high solid loading. However, Hodge et al. (2008) reported mass transfer limitation played a role only when soluble solids loading was more than 25%. It was reported that the external mass transfer was insignificant in comparison to internal diffusion of enzyme molecule into the matrix structure of the substrate solids (Gan et al., 2003). Since the substrates were added in batches to ensure adequate free water in hydrolysate in this study, the external mass transfer was not considered as the key limitation factor. Besides, other undiscovered inhibitors may also affect the enzymatic hydrolysis yield. It is certain that inhibition was various from pretreatment methods and enzymes diversity. In the actual hydrolysate, the inhibition was normally came from multiple

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and interactive factors, including physical and chemical ones. In order to reduce 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 61 62 63 64 65

inhibition and increase the enzymes activity, detoxification after pretreatment is an efficient approach. Even though post-washing, addition of non-productive protein or surfactant, and simultaneous saccharification and co-fermentation is effective to reduce inhibitions from phenolics or glucose, the mechanism of the inhibition of phenolics and glucose on enzymes needs to be further studied.

4. Conclusions The optimized conditions for aqueous ammonia pretreatment exhibited an excellent performance on the improvement of enzymatic digestibility. The inhibition of substrates in high solids loading hydrolysate is more evident than that in low solids loading hydrolysate due to higher inhibitor concentrations. End-product inhibited the hydrolysis elucidated by the hydrolysis of filter paper. The digestibility of unwashed solids was much lower than that of washed solids, indicating that the increased concentrations of phenolics significantly reduced the enzymatic hydrolysis yield. Xylose and xylooligomer were not the main causes to inhibit enzymes as the solids loading increased in enzymatic hydrolysis.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Major International Joint Research Project: 21020102040), the National High Technology Research and Development Program ("863"Program: 2012AA02A701), the National Basic Research Program of China (―973‖ Program: 2013CB733601), International Joint Research Project of Tianjin (11ZCGHHZ00500). We thank Genecor International Corporation (Suzhou, China) for kindly providing the enzymes.

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Figure legends

Fig. 1 The surface response plot of the effects of temperature, time and ammonia concentration on the glucose yield. (A) The pretreatment time was fixed at 60 min. (B) The ammonia concentration was fixed at 15% (w/w). (C) The pretreatment temperature was fixed at 160°C. Enzymatic hydrolysis conditions: 6% glucan loading, 60 FPU cellulase /g glucan, 30 CBU beta-glucosidase /g glucan, digestibility at 168 h.

Fig. 2 Effects of xylanase (A) and cellulase (B) dosage on the enzymatic hydrolysis of pretreated CS with and without post-washing. Pretreatment conditions: 180°C, 30 min, 20% (w/w) ammonia, 20% (w/w) solids loading. Enzymatic hydrolysis conditions: 6% glucan loading and digestibility at 168 h. Values are means of duplicate experiments (Standard error < 5%).

Fig. 3 Effects of glucan loading in enzymatic hydrolysis on glucose concentration (A) and glucan conversion (B). Pretreatment conditions: 180°C, 30 min, 20% (w/w) ammonia, 20% (w/w) solids loading. Enzymatic hydrolysis conditions: 15 FPU cellulase/g glucan, 30 CBU beta-glucosidase/g glucan, 7.5 mg xylanase/g glucan, digestibility at 168 h. Values are means of duplicate experiments.

Fig. 4 Effects of glucan loading in enzymatic hydrolysis on xylan conversion (A) and xylose/xylooligomer concentration (B). The pretreatment and enzymatic hydrolysis

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conditions were illustrated in Figure 3. Xylose and xylooligomer conversion were calculated based on the pretreated solids. Values are means of duplicate experiments.

Fig. 5 Solids loading versus ligno-phenolic components in enzymatic hydrolysate. Yaxis: relative abundance; X-axis: glucan loading. Values are means of duplicate experiments.

Fig. 6 Glucose yields versus vanillin concentration (A) and xylooligomer concentration (B). The calculation of glucose yield was based on the glucan in pretreated solids. Values are means of duplicate experiments.

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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 61 62 63 64 65

Table 1 Experimental design and results of the Box-Behnken design Run

Variables

Response

Temperature (°C)

Time (min)

Ammonia conc. (%, w/w)

Glucose yield (%)a

1

140

60

10

59.2

2

180

60

10

71.5

3

140

60

20

53.0

4

180

60

20

86.8

5

140

30

15

59.5

6

180

30

15

66.8

7

140

90

15

54.8

8

180

90

15

80.6

9

160

30

10

53.8

10

160

30

20

75.4

11

160

90

10

65.1

12

160

90

20

64.8

13

160

60

15

60.3

14

160

60

15

58.3

15

160

60

15

59.8

16

160

60

15

61.7

17

160

60

15

61.9

Note: Solids content in the pretreatment reactor was kept constant at 10% (w/w). A post-washing process of pretreated CS was conducted. a

Enzymatic hydrolysis conditions: 6% glucan loading, 60 FPU cellulase/g glucan, 30

CBU beta-glucosidase /g glucan, pH 4.8, 50°C, 200 rpm, digestibility at 168 h.

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Table 2 Effects of solids content and post-washing in aqueous ammonia pretreatment 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 61 62 63 64 65

of CS on solids composition, recovery and sugars yield Solids Content

Recovery (%)a

Composition (%)

Yield (%)b

Glucan

Xylan

AIL

Glucan

Xylan

AIL

Glucose

Xylose

10%

63.7

19.2

5.4

98.0

54.6

21.0

84.9

34.6

15%

60.3

19.1

8.7

97.1

56.7

35.0

83.7

34.2

20%

57.9

19.7

8.7

98.0

61.7

36.9

82.9

34.5

25%

56.1

20.1

11.2

102.0

67.5

51.4

81.3

39.1

10%

51.4

17.8

7.5

99.6

62.3

35.7

67.4

33.7

15%

42.5

19.4

7.9

101.1

84.9

47.1

63.6

36.7

20%

36.8

19.3

8.8

100.4

98.1

61.0

56.0

33.5

25%

36.2

19.4

10.6

103.7

102.0

75.7

51.4

31.6

With Washingc

Without washing

Note: The data in the table show the mean value (n = 2; Standard error < 5%). Other pretreatment conditions: 180°C, 30 min, 20% (w/w) ammonia. a

Calculations of recovery was based on the raw materials.

b

Calculations of yields was based on the untreated biomass. Enzymatic hydrolysis

conditions: 6% glucan loading, 60 FPU cellulase/g glucan, 30 CBU beta-glucosidase /g glucan, pH 4.8, 50°C, 200 rpm, digestibility at 168 h. c

Washing of pretreated CS was conducted at a ratio of 1 g dry CS to 20 ml of water.

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Table 3 Partial correlation analysis of effect of inhibitors on glucose yield 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 61 62 63 64 65

Control Variables

Variables

Correlation factor

X & OX & V

Y&G

0.723**

G & OX & V

Y&X

-0.143

G&X&V

Y & OX

-0.304

G & X & OX

Y&V

0.801**

Note: X stands for xylose concentration, OX stands for xylooligomer concentration, V stands for vanillin concentration, G stands for glucose concentration, Y stands for glucose yield. ** means significant on 0.01 level.

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Fig. 1

1

A

100%

Sugar conversion after enzymatic hydrolysis

90% 80%

70% 60% 50%

40% 30% 20%

10% 0% 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

Xylanase addition ( mg protein/g glucan )

B

Glucose Without washing

Glucose Washing

Xylose Without washing

Xylose Washing

70

Sugar Concentration ( g/l)

60

50

40

30

20

10

0 0

24

48

72

96

120

144

168

time (h) 15 FPU G Without washing 15 FPU G Washing 15 FPU X Without washing 15 FPU X Washing

30 FPU G Without washing 30 FPU G Washing 30 FPU X Without washing 30 FPU X Washing

Fig. 2

2

60 FPU G Without washing 60 FPU G Washing 60 FPU X Without washing 60 FPU X Washing

A

110 100

Filter paper

Glucose Concentreation (g/l)

Without washing 90

Washing

80 70 60 50 40 30 20 10 0 0% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13%

Glucan loading

B

Filter paper Without washing Washing

Conversion of Glucan to Glucose

100%

90%

80%

70%

60%

50%

40% 0% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% 11% 12% 13%

Glucan loading

Fig. 3

3

A

100%

Xylooligomer Xylose

90%

Coversion

80% 70% 60% 50% 40% 30% 20% 10% 0%

Without washing

Washing Glucan loading

B

60

Concentration (g/l)

50

Xylooligomer Xylose

40

30

20

10

0

Without washing

Washing Glucan loading

Fig. 4

4

Fig. 5

5

A

100% Without washing Washing

Glucose yield

90% 80%

Inhibited by glucose and phenolics

70% Inhibited by glucose

60% 50% 40% 0

10

20

30

Vanillin concentration (Relative abundance)

B

100% Without washing Washing

Glucose yield

90% 80% 70%

60% 50% 40% 0

10

20

30

Xylooligomer concentration (g/l)

Fig. 6

6

40

Highlights Post-washing was important to remove inhibition in enzymatic hydrolysis. Effects of solids loading on monomeric and oligomeric sugar yields were studied. Enzymatic conversion decrease was mainly due to inhibition of glucose and phenolics.