A comparison of different pre-extraction methods followed by steam pretreatment of bamboo to improve the enzymatic digestibility and ethanol production

Journal Pre-proof A comparison of different pre-extraction methods followed by steam pretreatment of bamboo to improve the enzymatic digestibility and ethanol production

Zhaoyang Yuan, Guodong Li, Weiqi Wei, Jiarun Wang, Zhen Fang PII:

S0360-5442(20)30263-2

DOI:

https://doi.org/10.1016/j.energy.2020.117156

Reference:

EGY 117156

To appear in:

Energy

Received Date:

13 November 2019

Accepted Date:

13 February 2020

Please cite this article as: Zhaoyang Yuan, Guodong Li, Weiqi Wei, Jiarun Wang, Zhen Fang, A comparison of different pre-extraction methods followed by steam pretreatment of bamboo to improve the enzymatic digestibility and ethanol production, Energy (2020), https://doi.org/10.1016/j. energy.2020.117156

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A comparison of different pre-extraction methods followed by steam pretreatment of bamboo to improve the enzymatic digestibility and ethanol production

Zhaoyang Yuana, Guodong Lib,* Weiqi Weic, Jiarun Wangd, Zhen Fanga,e,*

aDepartment

of Biochemistry & Molecular Biology, Michigan State University, 603 Wilson

Road, East Lansing, Michigan 48824, United States bState

Key Laboratory of Biobased Material and Green Papermaking, Qilu University of

Technology, Shandong Academy of Sciences, Jinan 250353, China cJiangsu

Co-Innovation Center of Efficient Processing and Utilization of Forest Resources,

Nanjing Forestry University, Nanjing 210037, China dDepartment

of Forestry, Michigan State University, 480 Wilson Road, East Lansing, Michigan

48824, United States eGreat

Lakes Bioenergy Research Center- Michigan State University, 164 Food Safety and

Toxicology Building, East Lansing, Michigan, 48824, United States

*Correspondence: [email protected] [email protected]

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Abstract: In this work, investigation of pre-extraction of bamboo under three different conditions (alkaline, neutral, and acidic) prior to acid catalyzed (AC) steam pretreatment on the conversion of polysaccharides into fermentable sugars and ethanol was reported. Pre-extraction process was demonstrated to significantly improve the enzymatic digestibility of bamboo and reduce the formation of inhibitory compounds for fermentation. Comparing to the pre-extraction of bamboo under neutral and acidic conditions, alkaline pre-extraction of bamboo yielded the highest monomeric sugar yields following enzymatic hydrolysis, which were approximately 86% of glucose and 82% of xylose (based on the initial sugar composition). The fermentation of sugars released during enzymatic hydrolysis with pentose-hexose fermenting Saccharomyces cerevisiae Lg8-1 strain achieved the final ethanol concentrations of 50.1, 46.3, and 49.0 g/L, corresponding to the ethanol yields of 70.5%, 68.9%, and 65.1% (based on theoretical ethanol yield of the initial bamboo) for the prep-extraction of bamboo under alkaline, neutral, and acidic conditions, respectively. The results indicate that the pre-extraction of extractives and ash followed by AC steam pretreatment is a promising approach to improve the bioconversion of bamboo biomass to biofuels.

Keywords: Bamboo; Enzymatic hydrolysis; Ethanol; Fermentation; Pre-extraction; Acid catalyzed steam pretreatment

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1. Introduction Bamboo is a quick-growing species that becomes harvest-ready in 3-5 years and has similar chemical composition (cellulose, hemicellulose, and lignin) to that of woody species [1,2]. Moreover, bamboo has lower ash content that of agricultural residues such as wheat straw and rice straw and is highly abundant in Asia and South America [3,4]. Additionally, it can grow in marginal land that reduces the competition with food crops [1]. Based on these advantages, bamboo has been recognized as a promising renewable alternative feedstock for the production of biofuels and chemicals besides its application in construction and furniture. Similar to the production of second-generation ethanol from other lignocellulosic biomasses, the production of biofuels from bamboo still generally requires a sequential process of pretreatment, enzymatic hydrolysis, and fermentation to convert polysaccharides into ethanol [5]. Also, a diverse of biomass pretreatment technologies (i.e. acidic/alkaline, steam, ionic liquid, biological, organosolv, or mechanical pretreatment) have been developed to reduce the recalcitrance of the plant cell wall and facilitate the liberation of structural sugars [6]. Among the pretreatment technologies, acid catalyzed (AC) steam, known for its simplicity as well as high efficiency to solubilize hemicellulose and substantially improve the enzymatic digestibility of retained cellulosic substrate, has been recently used in industry scale [7-10]. During AC steam pretreatment, the relocation/redistribution of lignin in the cell wall is considered to be the main factor that increases the enzymatic digestibility of cellulosic substrates [11]. The challenge of the one-stage AC steam pretreatment, however, might be the requirement of severe conditions; this will result in the formation of high amounts of inhibitory compounds such as acetic acid, furfural, 5-hydroxymethylfurfural, and phenolics that inhibit the fermentation process [11-13].

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Therefore, an approach that could reduce the generation of compounds that inhibit downstream conversion process while maintaining high overall sugar and ethanol yields should be identified. High amounts of non-structural components in bamboo biomass, such as inorganic minerals (i.e. ash) and organic components including phenols, proteins, lipids, terpenes, fatty acids and their esters, waxes, polyhydric alcohols, and other aromatic components, are known to reduce the efficiency of the downstream conversion processes of enzymatic hydrolysis and fermentation [14-16]. Hence, removal of these non-structural compounds could potentially improve the conversion of bamboo biomass into bioethanol. For instance, several methods including air classification and ash leaching with water or low concentration of acid or alkali were explored to remove the inorganic ashes [17,18]. Thus, the presence of up to 17% organic and inorganic nonstructural components (known as extractives and ash) in bamboo stem could presumably reduce the conversion efficiency of bamboo biomass [14,19]. Limited attention, however, has been focused on the removal of these non-structural compounds from bamboo prior to AC steam pretreatment for the improvement of the recovery of sugars and production of ethanol. In this study, bamboo chips were first subjected to a pre-extraction stage under three different conditions (alkaline, neutral, and acidic) to remove non-structural components. Subsequently, the pre-extracted bamboo chips were subjected to AC steam pretreatment for the increase of digestibility. Then, the two-stage pretreated bamboo substrates were subjected to enzymatic hydrolysis at a modest enzyme loading to evaluate the impact of these processing conditions on the sugar yields from bamboo. Afterwards, the fermentation of the enzymatic hydrolysate was performed using a metabolically engineered Saccharomyces cerevisiae Lg8-1 strain (designed for fermentation of both pentose and hexose sugars) to further determine the impact of precondition methods on the ethanol yield.

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2. Material and methods 2.1. Materials Bamboo (Phyllostachys edulis) trees at three-year old were collected from Chengdu, Sichuan Province, China. The obtained bamboo trees were washed with deionized water and air dried for 72 h at room temperature and then chipped to desired dimensions (approximately 1 cm × 1 cm × 0.5 cm). The air-dried bamboo chips (moisture content was about 16%) were stored at 4 °C prior to compositional analysis and subsequent experiments. All chemicals were reagent grade and purchased from Sigma-Aldrich (Beijing, China). All experiments in this study were performed at least in triplicate. 2.2. Pre-extraction of bamboo chips The pre-extraction experiments were conducted in a 2 L vessel equipped with a rotating reactor system (Greenwood Instruments, USA). The bamboo biomass pre-extraction was performed at 70 ℃ for 6h under three different conditions: 1) neutral wash with deionized water; 2) dilute acidic pre-extraction with 1% (w/w) sulfuric acid (H2SO4); 3) alkaline pre-extraction with 5% (w/w) sodium hydroxide (NaOH), respectively. The pre-extraction was performed by mixing 100 g (oven-dried weight) bamboo chips with 900 mL deionized water in the stainless vessel with a total liquid volume of 1 L by the addition of 100 mL deionized water, H2SO4 (1% w/w), or 5% (w/w) NaOH solution. At the end of the reaction, the solid and liquid fractions were separated by filtration. The solid fraction was thoroughly washed thoroughly with deionized water until the filtrate reached to neutral pH and stored at 4 ℃. 2.3. Acid-catalyzed steam pretreatment During AC steam pretreatment, oven-dried weight bamboo chips (50 g) and 150 mL deionized water were impregnated with 3% (w/w) gaseous sulfur dioxide (SO2, > 99% pure) (or

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0.3 g SO2 per gram dry bamboo) in a sealable plastic bag at room temperature for 24 h; this allowed the penetration of SO2 into bamboo chips for utilizing as the acidic catalyst. Then, the impregnated was carefully transferred into a 2 L stainless pretreatment reactor (QBS-80, Hebi Zhengdao Heavy Machine, Hebi, China) and mixed with deionized water to a final solid loading of 25% (w/v). High pressure steam, around 20 bar, was used to treat bamboo chips. AC steam pretreatment of the original and pre-extracted bamboo chips was performed at 190 ℃ for three different times (5, 10, and 15 min). Following AC steam pretreatment, the solid was separated by filtration. The collected solids were washed with room temperature deionized water (about 2 L) and stored at 4 ℃ for further experimentation. The pretreatment liquor was also collected from the measurement of chemical composition. 2.4. Enzymatic hydrolysis Enzymatic hydrolysis was performed in 125-mL Erlenmeyer shake flasks using a cellulolytic enzyme cocktail with a 2:1 protein mass ratio of Cellic CTec3 (Novozymes investment Co. Ltd, Bagsværd, Denmark) to β-glucosidase preparation (Novozym 188, Novozymes A/S Bagsværd, Denmark). The mixture of biomass substrate and enzyme cocktail were incubated at 50 ℃, pH 5 (50 mM sodium citrate buffer) for 72 h with an enzyme loading of 20 mg protein/g glucan using a shaking incubator (HNY-111C, Zhengzhou, China). Two different solid loadings, low (5% w/v) and high (20% w/v), were investigated during enzymatic hydrolysis. Moreover, the shake flasks used for enzymatic hydrolysis were pre-autoclaved empty at 121 ℃ for 15 min. The sugar concentrations were measured using a high-performance liquid chromatography (HPLC) system following a National Renewable Energy Laboratory (NREL) protocol [20]. In one series of experiments, the two-stage pretreated cellulosic substrate was mixed with the steam pretreatment liquor for enzymatic hydrolysis to determine the overall sugar yields.

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2.5. Inoculum and medium Strain Saccharomyces cerevisiae Lg8-1 (cell stock stored at -70 ℃) was used to determine the fermentability of sugars in the enzymatic hydrolysate. The microorganism was cultured in medium containing glucose (60 g/L), xylose (15 g/L), yeast extract (10 g/L), and peptone (20 g/L) at 35 ℃ for 24 h with no agitation. The cell growth was determined by measuring the optical density at 600 nm. The pre-cultured cells were harvested by centrifugation to prepare a stock of 50 g/L S. cerevisiae cell concentration. 2.6. Fermentation Fermentation was conducted using 50 mL enzymatic hydrolysate in 125-mL serum bottles (Sigma-Aldrich, Canada) and initiated with 1.0 g/L initial cell concentration yeast (S. cerevisiae Lg8-1) then continued under anaerobic conditions at 35 ℃, pH 5.5 (adjusted with 4 M potassium hydroxide), 180 rpm for up to 72 h. During fermentation, samples (1 mL) were withdrawn periodically to monitor cell growth by measuring optical density at 600 nm and nitrogen was injected to maintain anaerobic conditions in the reactor after inoculation and sampling. At the end of fermentation, the ethanol concentration and the residual sugar content were determined by HPLC wherein the overall ethanol yield was calculated following Eq. 1 [21]. [𝐸𝑡𝑂𝐻] × 𝑉𝐿

Ethanol yield (%) = 0.51 × (𝑀𝐺 × 1.111 + 𝑀𝑋 × 1.136) (1) where MG and MX are the mass (g) of glucan and xylan in original bamboo, respectively, [EtOH] is the ethanol concentration in the liquid (g/L), VL is the volume of the liquid phase (L), 1.111 and 1.136 account for the stoichiometry of glucan conversion to glucose and xylan conversion to xylose, respectively, and 0.51 is the conversion factor for both glucose and xylose to ethanol based on stoichiometric biochemistry of yeast. 2.7. Analytic methods 7

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The chemical compositions of both raw and pretreated materials were determined following NREL protocol [20]. Briefly, following sulfuric acid (H2SO4) hydrolysis of the ground biomass, Klason lignin was separated by filtration and weighed after drying at 105 ± 2 °C. Acid-soluble lignin in the hydrolysate was measured at 205 nm using a UV-vis spectrophotometer (Unico SpectroQuest SQ2802, Unico Co., USA) and lignin content was calculated as the sum of above. The concentrations of monosaccharides and acetyl groups in the hydrolysates were determined using a Dionex ICS 5000+ HPLC system fitted with an AS-AP autosampler (Thermo Fisher Scientific, MA, USA) at 45 °C, against sugar standards, on a Dionex CarboPac SA 10 analytical column using 1 mM NaOH as the mobile phase at a flow rate of 1 mL/min. The sugars were quantified (internal standard: fucose) using electrochemical detection and Chromeleon software (Thermo Fisher Scientific, MA, USA). All analyses were performed in triplicate. The sugar composition in the liquid phase was measured using the same HPLC system and protocol following autoclave with 4% (w/w) H2SO4 for 60 min [20]. The concentrations of monomeric sugars in the enzymatic hydrolysates and the concentration of ethanol in the fermentation samples were analyzed using the same HPLC system (Dionex ICS 5000+) using a Biorad Aminex HPX-87H column (Bio-Rad Laboratories, USA) operating at 65 ℃ with a 5 mM H2SO4 mobile phase at a flow rate of 0.6 mL/min. The yields of glucose, xylose, and total sugar were calculated following Zhu et al. [22]. The concentrations of inhibitors, including furfural, 5hydroxymethylfurfural (HMF), levulinic acid, formic acid, and acetic acid were determined with the same HPLC system equipped with a refractive index detector (RefractoMax 521) using a standard curve generated by authentic samples. The ash content of bamboo samples was determined following TAPPI Standard Method T211 om-02. Briefly, about 5 g of 20-mesh milled dry biomass were completely ashed at 550 ℃

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for 12 h to a constant weight and the corresponding weight was used to determine the ash content. Water and solvent extractives of bamboo sample were determined by weighing the biomass before and after Soxhlet extraction for 20 h. Mineral composition of the ash obtained after incineration at 700 ℃ was determined by atomic emission measurements using an iCP spectrometer (iCAP 6000 Series, Thermo Scientific, MA, USA) by using nitric acid to prepare testing solutions. 2.8. Statistical analysis Unscrambler v10.5 software (CAMO software, Japan) was used to analyze the differences among data collected from triplicate experiments using one-way analysis of variance. Multiple comparisons among treatments were determined according to the Tukey post hoc test. P-values of 0.05 or lower was considered to be statistically significant. Error bars in all graphs refer to 95% confidence intervals.

3. Results and discussion 3.1. Impact of pre-extraction on the properties of bamboo Table 1 summarizes the chemical composition of bamboo chips (solid fraction) before and after pre-extraction. As shown in Table 1, the composition of 43.6% glucan and 18.4% xylan in original bamboo suggests that bamboo is a promising raw material for ethanol production. However, the content of non-structural components in original bamboo was approximately 12%, including 2.2% ash, 3.3% acetyl groups, and 6.8% extractives (Table 1), which again could depress subsequent enzymatic hydrolysis and fermentation processes. To alleviate the adverse effects caused by the non-structural components, pre-extraction of bamboo chips under different conditions was conducted. Following pre-extraction, the remained solid mass fraction ranged

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from 88% to 94% (based on the oven-dried mass of untreated biomass) due to the removal of different amounts of bamboo components (Table 1). As shown in Table 1, the glucan, xylan and lignin content in the pre-extracted solid materials slightly increased compared to that of the original bamboo due to the solubilization of other bamboo components (i.e. ash, acetyl groups, and extractives). The contents of galactan and arabinan, which were low initially, did not change substantially following the three pre-extraction methods (Table 1). The content of extractives decreased from 6.8% (w/w) in original bamboo to 1.2%, and 3.2% (w/w) after pre-extraction under alkaline and neutral conditions, respectively, while complete removal of extractives was achieved under acidic pre-extraction conditions (Table 1); this is in accordance with previous studies on the pre-extraction of phenolics and extractives from cotton stalk and bamboo with water or dilute acid [15,23]. Compared to neutral pre-extraction, alkaline and acidic pre-extraction removed more extractives as a result of alkaline saponification of esters and solubilization of acid-soluble materials, respectively [23-25]. In addition, the content of acetyl groups was under the detection limit in the alkaline pre-extracted bamboo, while about 30% of acetyl groups was extracted during acidic pre-extraction. In contrast, the content of acetyl groups did not change significantly during neutral pre-extraction (p > 0.05). The removal of the acetyl groups from bamboo following alkaline and acidic pre-extraction might reduce the inhibitory effects caused by acetic acid during the downstream fermentation process [21]. In addition, compared to neutral and acidic pre-extraction, higher amount of ash was removed during alkaline pre-extraction. As shown in Table 1, the ash content of alkaline, neutral, and acidic pre-extracted biomasses was 0.8%, 1.8%, and 1.4% (w/w), respectively (Table 1). Compositional analysis of the mineral ingredients in the biomass was determined to better understand the removal of each species in pre-extraction process (Table 2). For example, during

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pre-extraction with neutral water, the majority loss of minerals was potassium, phosphorus, and magnesium, of which the contents decreased by 88%, 80%, and 27%, respectively. Following acidic pre-extraction, the contents of calcium, magnesium, manganese, zinc, and potassium in bamboo significantly decreased (p ≤ 0.05). During alkaline pre-extraction, potassium and silicon were primarily removed, while contents of other minerals such as calcium, magnesium, manganese, zinc, and iron only slightly changed (Table 2). This could be associated with different mineral solubility under different pHs [26]. Notably, the decrease of silicon content after alkaline pre-extraction could be attributed to the conversion of insoluble silica into soluble silicates (formed from reaction with NaOH) and these silicates could further be recovered as amorphous silica particles for value-added products [27,28]. To further assess the effect of pre-extraction on the properties of the pre-extracted bamboo chips, enzymatic hydrolysis of the pre-extracted bamboo was performed at 5% solid loading with an enzyme loading of 20 mg protein/g glucan for 72 h (Fig. 1). As shown in Fig. 1, compared to untreated bamboo chips, significant increase in the yield of glucose and xylose was observed following enzymatic hydrolysis. For example, enzymatic hydrolysis of original bamboo, produced 16.2% glucose and 15.6% xylose (based on initial sugar composition) while from neutral water pre-extracted bamboo the glucose and xylose yields were increased to 28.7% and 29.9%, respectively. The pre-extraction under alkaline and acidic pre-extraction resulted in even higher yields of glucose and xylose (Fig. 1). As shown, for alkaline pre-extracted bamboo, the glucose and xylose yields were 38.2% and 36.1%, respectively. For acidic pre-extracted bamboo, the glucose and xylose yields were 36.4% and 36.2%, respectively (Fig. 1). This is presumably attributed to the removal of more extractives and minerals which increased the accessibility of polysaccharides and reduced the inhibitive effects of the non-structural components on enzyme 11

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[29,30]. Moreover, pre-extraction was expected to increase the porosity of the biomass, thereby offering an option to apply less severe AC steam pretreatment. 3.2. Acid catalyzed steam pretreatment of bamboo Table 3 shows that prolonging the AC steam pretreatment time, the remaining solid from original and pre-extracted bamboo both significantly decreased (p ≤ 0.05), which is not surprising as increasing the pretreatment severity could increase the solubilization and degradation of bamboo components such as xylan and glucan. For example, during the AC steam pretreatment of original bamboo, the fraction of removed xylan increased from 45.6% to 74.6% (based on the initial xylan) when the treatment time prolonged from 5 to 15 min (Table 3). Moreover, compared to original bamboo, the pre-extraction greatly increased the dissolution of xylan during the AC steam pretreatment stage. As shown in Table 3, AC steam treatment of the pre-extracted bamboo samples (under the three investigated pre-extraction conditions) from 5 to 15 min solubilized 59% to 89% of xylan (based on the initial xylan) (Table 3). This agrees with the fact that removal of organic extractives and inorganic ash during the pre-extraction could weaken the xylan structure and increase the exposure of xylan to degradation during the AC steam pretreatment [31,32]. The relative percentage of glucan of AC steam pretreated substrate was considerably increased due to the solubilization of xylan. Moreover, the pre-extraction of bamboo prior to AC steam pretreatment also contributed to achieve higher glucan percentage than that of the sample pretreated by AC steam only. In addition, the solubilized glucan in the liquid fraction was calculated as 1.5-6.0% (based on the initial glucan), which is in agreement with previous studies on steam pretreatment of other lignocellulosic feedstocks such as hardwood, wheat straw, and corn stover [33-35]. Following AC steam pretreatment, the content of lignin in pre-extracted bamboo also increased (Table 3). This could be attributed to the

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solubilization of a substantial amount xylan. Moreover, the formation of “pseudo-lignin” through the polymerization/condensation reactions from liberated sugars and their degraded products might also result in the increase of lignin content [36]. The formation of “pseudo-lignin” might negatively affect the enzymatic hydrolysis of pretreated bamboo by creating unproductive binding to enzymes and blocking the active cellulose surface binding sites to enzymes [36]. The results in Table 3 also suggest that more than 95% of organic extractives (based on initial extractives) were solubilized during AC steam pretreatment in all cases and this is in agreement with previous studies on steam pretreatment of both woody and non-woody materials [37,38]. Moreover, the sequential alkaline and AC steam pretreated bamboo had the lowest ash content (0.1-0.2% w/w) compared to other pretreated bamboo substrates (1.5-1.6% w/w). This could be attributed to the high silica content in original, neutral and acidic pre-extracted bamboo and it was difficult to remove during AC steam pretreatment [39]. In contrast, other minerals retained in alkaline pre-extracted bamboo was solubilized under acidic conditions during AC steam pretreatment. To further evaluate the impact of pre-extraction on the degradation of bamboo components, the concentrations of dissolved materials such as sugars and fermentation inhibitory compounds (such as HMF, furfural, acetic acid, and phenolics) in the AC steam pretreatment liquors were also analyzed (Table 4). The low concentration of glucose (0.6-2.6 g/L) in all water-soluble fractions confirmed the poor solubilization of glucan during the steam pretreatment of both original and pre-extracted bamboo. Contents of galactan and arabinan, which were low in original bamboo, led low concentrations of galactose and arabinose in all steam pretreatment liquors (Table 4). Xylose was the primary sugar detected in the AC steam pretreatment liquor and its concentration increased when increasing the steam pretreatment time. In addition, after

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steam pretreatment, pre-extracted bamboo (under alkaline, neutral, and acidic conditions) generated statistically higher concentration of xylose than original bamboo (p ≤ 0.05). For example, steam-treated original bamboo (190 °C for 10 min) produced 13.8 g/L xylose while in contrast, under the same steam pretreatment conditions the concentration of xylose was 18.9 g/L, 18.2 g/L, and 19.2 g/L for pre-extracted bamboo under alkaline, neutral, acidic conditions, respectively. Pre-extraction of non-structural components presumably improved the accessibility of xylan, thereby increasing the xylan solubilization during AC steam pretreatment. Notably, the concentration of xylose from alkaline and acidic pre-extracted bamboo was higher than that of neutral pre-extracted bamboo, which is likely due to the removal of more organic extractives and inorganic ash during pre-extraction that facilitated the hydrolysis of the xylosidic bonds [40]. The inhibitory components, such as HMF, furfural, penolics, and acetic acid were also detected in the AC steam pretreatment liquors (Table 4). The concentrations of phenolics, HMF and furfural increased when steam treatment extended and agreed with previous studies on steam pretreatment of hardwood and softwood [41,42]. Acetic acid, another type of inhibitory compound, was detected in the liquors generated from AC steam pretreatment of original, and neutral and acidic bamboo, but not detected in the liquor from alkaline pre-extracted bamboo due to the effective deacetylation during alkaline pre-extraction. These results indicate that the preextraction step could substantially facilitate the efficiency of steam treatment, again offering an opportunity to reduce the severity of steam pretreatment while still achieving a high enzymatic digestibility. 3.3. Enzymatic hydrolysis of pretreated bamboo at low solid loading After AC steam pretreatment, the solid substrates from all original and pre-extracted bamboo were subjected to enzymatic hydrolysis. The enzymatic hydrolysis was preformed using an

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enzyme loading of 20 mg protein/g glucan at 5% (w/v) solid loading (based on the initial bamboo) and 50 ℃ for 72 h (Fig. 2). Following enzymatic hydrolysis, the solid fraction from the two-stage (pre-extraction followed by AC steam pretreatment) pretreatment process achieved higher glucose yields [82%, 72%, and 78% (based on the initial glucan) for alkaline, neutral, and acidic pre-extracted bamboo, respectively] compared to that of the AC steam pretreated sample (67%, based on the initial glucan). The increase in enzymatic digestibility for pre-extracted bamboo could be associated with the removal of extractives and ash improved the deconstruction of biomass during AC steam pretreatment [43]. In addition, compared to the pre-extraction under neutral and acidic conditions, alkaline pre-extracted substrate had the highest glucose yields. This is likely due to the extraction of silica could reduce silica-enzyme interactions and increase the accessibility of polysaccharides by deconstructing the silica layer in bamboo, thereby facilitating the enzymatic hydrolysis of the bamboo biomass [44-46]. Fig. 2 also reveals that the glucose yields increased with prolonged AC steam pretreatment time for both original and pre-extracted bamboo. For example, the glucose yield from alkaline pre-extracted bamboo improved from 75% to 82% (based on initial glucan content) when steam treatment increased from 5 min to 10 min; no further significant increase in glucose yields when increasing the AC steam pretreatment from 10 to 15 min (p > 0.05). Similar results were also observed for acidic pre-extracted bamboo. However, continuous growth of glucose yields was observed for original and neutral water pre-extracted bamboo when AC steam pretreatment extended from 5 to 15 min (p ≤ 0.05). The results also indicate that the enzymatic digestibility of alkaline and acidic pre-extracted bamboo was improved faster by AC steam pretreatment than the original and neutral water pre-extracted bamboo. In summary, the data shown in Tables 3 and

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4, and Fig. 2 suggest that AC steam pretreatment at 190 °C for 10 min could be suitable for enhancing the enzymatic digestibility of bamboo. 3.4. Generation of high-sugar content hydrolysate for ethanol production During the production of ethanol from lignocellulosic biomass, increase of the sugar concentration and reduction of water/energy consumption were recognized as the promising strategies to increase the feasibility of commercialization [47]. In this series of experiments, the whole AC steam pretreated materials (no separation of solid and liquid fractions) were subjected to enzymatic hydrolysis at a solid consistency of 20% (w/v) using an enzyme loading of 20 mg protein/g glucan for 72 h. As shown in Fig. 3, compared to original bamboo, all the pre-extracted bamboo biomasses achieved much higher yields of both glucose and xylose. Moreover, the alkaline pre-extracted bamboo achieved the highest glucose yield, up to 85.8% (based on the initial glucan), and glucose yields for neutral and acidic pre-extracted bamboo were 76.1% and 82.4% (based on the initial glucan), respectively. These results provided further evidence that the removal of silica, acetyl groups, and extractives during the pre-extraction stage could significantly improve the bioconversion efficiency. The overall xylose yield for original bamboo was 71.2% (based on the initial xylan), which was much lower than that of pre-extracted bamboo (78-82%, based on the initial xylan) while alkaline pre-extracted bamboo achieved the highest overall monomeric sugar yields for ethanol fermentation. The slight difference on xylose yields among the three pre-extraction approaches could be presumably due to the solubilization of about 70% xylan during steam pretreatment (Table 3). Following enzymatic hydrolysis, the fermentability of the resulting enzymatic hydrolysates was evaluated using a metabolically engineered S. cerevisiae Lg8-1 strain. Fig. 4 shows the results of the fermentation of glucose and xylose into ethanol through the batch fermentation

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process. As shown in Fig. 4a, compared to pre-extracted bamboo, slightly longer time was required to consume 99% of glucose for hydrolysate from original bamboo as presumably due to the inhibitory effects caused by HMF, furfural, acetic acid, and phenolics (refer to Table 3) but glucose concentration still decreased to near zero after 48 h fermentation (Fig. 4a), indicating that the utilized S. cerevisiae Lg8-1 strain could efficiently convert hexose sugars into ethanol. Compared to the consumption of glucose, fermentation of xylose was much slower (Fig. 4b). As shown in Fig. 4b, after 72 h fermentation, only about 63% of the total xylose content was consumed in the original bamboo hydrolysate. Neutral water pre-extraction increased the xylose consumption to about 78%, while acidic and alkaline pre-extraction further promoted the xylose consumption to approximately 84% and 85%, respectively. These results are in accordance with previous studies on the improvement of ethanol production from poplar, rice straw, and corn stover by conducting alkaline or acidic deacetylation prior to acidic or alkaline pretreatment [45,48,49]. Fig. 4c shows that the pre-extraction of bamboo significantly increased the ethanol concentration (p ≤ 0.05) due to the presence of lower levels of inhibitory compounds in the hydrolysate which could increase monomeric sugar release. It should be noted that concentrations of inhibitory compounds (furfural, HMF, acetic acid, and phenolics) in the fermentation broth were 6.0, 1.9, 3.6, and 3.0 g/L for the untreated, alkaline, neutral, and acidic pre-extracted bamboo, respectively. Pre-extraction of bamboo under alkaline condition achieved the highest ethanol concentration (50.1 g/L), and the ethanol concentration for original and, neutral and acidic pre-extracted bamboo were 38.2, 46.3, and 49.0 g/L, respectively. Ethanol concentration higher than 40 g/L is critical in the distillation step as it can be desire-concentrated without costing excessive energy [50]. Based on ethanol concentration, the ethanol yields for original and, alkaline, neutral, and acidic pre-extracted bamboo were calculated to be 53.7%,

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70.5%, 65.1%, and 68.9% (based on the theoretical yield from initial sugar content in original bamboo) (calculations not shown), respectively. This further confirmed the results that the preextraction (especially alkaline pre-extraction) prior to steam pretreatment has a great potential to improve the bioconversion of bamboo into ethanol. To provide an overview of this biomass-to-ethanol process, a schematic diagram illustrating the process mass balance was provided based on 10 kg of dry bamboo biomass (Fig. 5). Preextraction of bamboo under alkaline condition was used as an example (Fig. 5). As shown, following the sequential process of alkaline pre-extraction (5% w/w NaOH, 70 °C, 6 h), AC steam pretreatment (3% w/w SO2, 190 °C, 10 min), enzymatic hydrolysis (enzyme loading of 20 mg protein/g glucan), and fermentation, 2.5 kg ethanol could be produced.

4. Conclusions In this work, the pre-extraction of bamboo under alkaline, neutral, and acidic conditions prior to acid catalyzed steam pretreatment was investigated to improve the production of fermentable sugar and ethanol. The utilization of pre-extraction facilitated the increase of enzymatic digestibility of bamboo during the AC steam pretreatment stage and substantially reduced the formation of inhibitory compounds. Following enzymatic hydrolysis of the whole AC steam pretreated substrates, the pre-extracted bamboo under alkaline condition achieved the highest yields of glucose (~86%, based on the initial glucan) and xylose (~82%, based on the initial xylan) compared to the original, and neutral and acidic pre-extracted bamboo. After fermentation of the enzymatic hydrolysates, a final ethanol concentration of 50.1 g/L at an ethanol yield of 68.9% was successfully achieved by using two-stage pretreatment approach combining alkaline pre-extraction and AC steam pretreatment.

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Acknowledgements The authors greatly acknowledge the support from National Natural Science Foundation of China (Grant No. 21506105). The authors are grateful to Novozymes for the donation of the enzyme preparations.

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Figure captions: Fig. 1. Yields of glucose and xylose following enzymatic hydrolysis of the original and pre extracted bamboo. Error bars represent ± standard deviations of the means. Fig. 2. Glucose yields from steam pretreated bamboo following enzymatic hydrolysis. Enzymatic hydrolysis was performed at 5% (w/v) solid consistency using an enzyme loading of 20 mg protein/g glucan for 72 h. Error bars represent ± standard deviations of the means. Fig. 3. The overall yields of glucose and xylose following enzymatic hydrolysis of the whole steam pretreated materials. AC steam pretreatment was conducted at 190 °C for 10 min. Enzymatic hydrolysis of pretreated bamboo substrate was performed at 20% (w/v) solid consistency using an enzyme loading of 20 mg protein/g glucan for 72 h. Error bars represent ± standard deviations of the means. Fig. 4. Batch fermentation of the enzymatic hydrolysates using Saccharomyces cerevisiae Lg8-1 strain, a) glucose concentration, b) xylose concentration, c) ethanol concentration. Fermentation was performed at 35 ℃, pH 5.5, and 180 rpm for 72 h. Error bars represent ± standard deviations of the means. Fig. 5. Schematic flow diagram on the mass balance of the production of ethanol from bamboo through the process of alkaline pre-extraction, AC steam pretreatment, enzymatic hydrolysis, and fermentation.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights    

Different pre-extraction strategies prior to AC steam pretreatment were compared. Pre-extraction reduced the formation of inhibitors during AC steam pretreatment. Pre-extraction improved the recovery of monomeric sugar yields from bamboo. Alkaline pre-extraction achieved the highest ethanol fermentation yield.

Table 1. Chemical composition of original and pre-extracted bamboo chips. Solid Chemical composition (%)b Pre-extraction remaining Glucan Xylan Galactan Arabinan Lignin Ash Acetyl Extractives (%)a Originalc N/A 43.6 ± 1.1 18.4 ± 0.3 0.6 ± 0.1 0.8 ± 0.1 23.9 ± 0.9 2.2 ± 0.1 3.3 ± 0.2 6.8 ± 0.4 Alkalined 88.7 ± 0.7 48.1 ± 0.9 19.9 ± 0.6 0.6 ± 0.1 0.6 ± 0.1 26.5 ± 0.7 0.8 ± 0.1 N/D 1.2 ± 0.3 e Neutral 93.9 ± 0.4 46.0 ± 1.4 19.0 ± 0.8 0.6 ± 0.1 0.5 ± 0.1 25.3 ± 0.5 1.8 ± 0.1 3.2 ± 0.1 3.2 ± 0.3 Acidicf 89.2 ± 0.6 47.9 ± 1.4 19.6 ± 0.8 0.5 ± 0.2 0.7 ± 0.1 26.4 ± 1.1 1.4 ± 0.1 2.6 ± 0.4 N/D N/A: Not Applicable. N/D: Not Detected. Values are expressed as average ± standard deviation. aWeight

percentage of recovered mass (oven-dried weight) after pre-extraction to original biomass.

bWeight

percentage based on oven-dried weight of pre-extracted biomass.

cOriginal:

original bamboo biomass.

dAlkaline:

bamboo biomass pre-extracted with alkali (NaOH).

eNeutral: fAcidic:

bamboo biomass pre-extracted with deionized water.

bamboo biomass pre-extracted with dilute acid (H2SO4).

Table 2. Mineral element contents of original and pre-extracted bamboo biomasses. Pre-extraction Elements (µg/g biomass) Ca P Fe Original 4487 ± 84 484 ± 25 42.7 ± 5.8 Alkaline 4836 ± 125 241 ± 18 43.2 ± 3.7 Neutral 4124 ± 112 98.7 ± 13 51.9 ± 6.1 Acidic 2148 ± 133 197 ± 18 45.4 ± 2.8

Cu 297 ± 13 292 ± 15 294 ± 12 145 ± 8

Mg 744 ± 62 726 ± 60 542 ± 43 N/D

Mn 8.4 ± 0.7 6.9 ± 0.6 7.2 ± 0.4 N/D

N/D: Not Detected. Values are expressed as average ± standard deviation.

K 3102 ± 128 1020.6 ± 78 356 ± 32 N/D

Na 65.6 ± 0.3 71.2 ± 0.3 65.8 ± 0.3 68.7 ± 0.3

Zn 25.4 ± 0.5 24.8 ± 0.8 25.3 ± 1.2 N/D

Si 5784 ± 134 30.1 ± 0.7 5837 ± 140 5716 ± 125

Table 3. Chemical composition of bamboo obtained from AC steam pretreatment under different conditions. Preextraction Original Alkaline Neutral Acidic

Time (min) 5 10 15 5 10 15 5 10 15 5 10 15

Solid remaining (%)a 84.8 ± 0.6 78.6 ± 0.7 74.4 ± 1.1 74.6 ± 0.8 69.9 ± 0.7 65.8 ± 0.6 80.2 ± 0.7 75.9 ± 0.9 70.6 ± 0.9 74.1 ± 1.2 70.6 ± 0.6 66.3 ± 0.7

Chemical composition (%)b Glucan Xylan Galactan 50.6 ± 1.0 11.8 ± 0.5 0.2 ± 0.1 54.2 ± 0.8 8.7 ± 0.3 0.2 ± 0.1 56.6 ± 1.2 6.3 ± 0.6 N/D 55.2 ± 1.1 8.7 ± 0.8 0.1 ± 0.1 58.4 ± 1.2 6.2 ± 0.7 N/D 61.7 ± 0.9 3.8 ± 0.7 N/D 52.8 ± 0.9 9.3 ± 0.7 0.1 ± 0.1 55.1 ± 0.8 6.4 ± 0.8 ND 58.3 ± 0.7 5.0 ± 0.6 ND 55.9 ± 1.2 7.9 ± 0.5 0.2 ± 0.1 57.9 ± 0.4 5.2 ± 0.4 N/D 61.2 ± 0.9 2.9 ± 0.6 N/D

Arabinan 0.3 ± 0.1 0.2 ± 0.1 N/D 0.1 ± 0.1 N/D N/D 0.1 ± 0.1 N/D N/D N/D N/D N/D

Lignin 27.7 ± 0.6 29.7 ± 0.5 30.6 ± 0.4 30.1 ± 0.6 31.7 ± 0.7 32.8 ± 0.7 28.7 ± 0.7 29.9 ± 0.6 31.5 ± 0.7 30.6 ± 0.5 31.5 ± 0.8 33.0 ± 0.4

N/D: Not Detected. Values are expressed as average ± standard deviation. aWeight

percentage of recovered biomass (oven-dried weight) after AC steam pretreatment to original biomass.

bWeight

percentage based on oven-dried weight of AC steam pretreated bamboo biomass.

Ash 1.5 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 ~ 0.1 1.6 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.5 ± 0.1 1.6 ± 0.1

Acetyl ~ 0.2 ~ 0.1 N/D N/D N/D N/D ~ 0.1 N/D N/D N/D N/D N/D

Extractives 0.4 ± 0.2 ~ 0.1 N/D 0.1 ± 0.1 N/D N/D N/D N/D N/D N/D N/D N/D

Table 4. Chemical composition of water-soluble fraction from AC steam pretreatment of bamboo under different conditions. Preextraction Original Alkaline Neutral Acidic

Time (min) 5 10 15 5 10 15 5 10 15 5 10 15

pH 1.87 1.72 1.68 1.82 1.64 1.61 1.74 1.66 1.64 1.68 1.62 1.53

Chemical composition (g/L) Glucose Xylose Galactose Arabinose 0.6 ± 0.2 10.7 ± 0.5 0.4 ± 0.1 0.3 ± 0.1 0.9 ± 0.1 13.8 ± 0.3 0.3 ± 0.2 0.4 ± 0.1 1.5 ± 0.3 16.9 ± 0.6 0.4 ± 0.1 0.5 ± 0.2 1.8 ± 0.1 15.6 ± 0.8 0.4 ± 0.2 0.5 ± 0.1 2.4 ± 0.4 18.9 ± 0.7 0.3 ± 0.1 0.3 ± 0.1 2.6 ± 0.3 21.2 ± 0.7 0.4 ± 0.1 0.5 ± 0.2 0.9 ± 0.2 14.5 ± 0.7 0.3 ± 0.1 0.4 ± 0.1 1.7 ± 0.4 18.2 ± 0.8 0.4 ± 0.1 0.3 ± 0.1 2.4 ± 0.3 20.1 ± 0.6 0.2 ± 0.2 0.3 ± 0.1 1.5 ± 0.4 15.9 ± 0.5 0.5 ± 0.2 0.6 ± 0.2 2.3 ± 0.2 19.2 ± 0.4 0.4 ± 0.1 0.3 ± 0.1 2.5 ± 0.3 21.2 ± 0.6 0.3 ± 0.1 0.4 ± 0.2

N/D: Not Detected. HMF: 5-hydromethylfurfural. Values are expressed as average ± standard deviation.

Furfural 1.1 ± 0.1 1.8 ± 0.2 2.1 ± 0.3 0.7 ± 0.2 0.9 ± 0.1 1.5 ± 0.1 1.1 ± 0.2 1.2 ± 0.2 1.5 ± 0.3 1.0 ± 0.2 1.0 ± 0.1 1.5 ± 0.1

HMF 0.9 ± 0.1 1.1 ± 0.1 1.2 ± 0.2 0.7 ± 0.1 0.6 ± 0.2 1.0 ± 0.1 0.6 ± 0.0 0.7 ± 0.1 1.1 ± 0.0 0.9 ± 0.0 0.8 ± 0.1 1.2 ± 0.2

Phenolics 1.7 ± 0.1 2.2 ± 0.1 2.4 ± 0.1 0.9 ± 0.2 0.9 ± 0.2 1.2 ± 0.2 0.9 ± 0.1 1.1 ± 0.1 1.3 ± 0.1 0.8 ± 0.2 0.9 ± 0.2 1.0 ± 0.2

Acetic acid 1.8 ± 0.2 2.0 ± 0.1 2.1 ± 0.3 N/D N/D N/D 1.7 ± 0.2 1.8 ± 0.1 2.0 ± 0.1 0.6 ± 0.2 1.1 ± 0.3 1.1 ± 0.2