Two-stage alkali-oxygen pretreatment capable of improving biomass saccharification for bioethanol production and enabling lignin valorization via adsorbents for heavy metal ions under the biorefinery concept

Accepted Manuscript Two-stage alkali-oxygen pretreatment capable of improving biomass saccharification for bioethanol production and enabling lignin valorization via adsorbents for heavy metal ions under the biorefinery concept Kai Song, Qiulu Chu, Jinguang Hu, Quan Bu, Fuqiang Li, Xueyan Chen, Aiping Shi PII: DOI: Reference:

S0960-8524(18)31778-4 https://doi.org/10.1016/j.biortech.2018.12.107 BITE 20864

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

11 November 2018 27 December 2018 28 December 2018

Please cite this article as: Song, K., Chu, Q., Hu, J., Bu, Q., Li, F., Chen, X., Shi, A., Two-stage alkali-oxygen pretreatment capable of improving biomass saccharification for bioethanol production and enabling lignin valorization via adsorbents for heavy metal ions under the biorefinery concept, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.107

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Two-stage alkali-oxygen pretreatment capable of improving biomass saccharification for bioethanol production and enabling lignin valorization via adsorbents for heavy metal ions under the biorefinery concept

Authors: Kai Song a,b, Qiulu Chu a,*, Jinguang Hu c, Quan Bu a, Fuqiang Li a, Xueyan Chen a, Aiping Shi a

Affiliation: a School of agricultural equipment engineering, Jiangsu University, Zhenjiang, 212013, China b Department of Chemical and Biological Engineering, The University of British Columbia, 2360 East Mall, Vancouver, BC, V6T 1Z3, Canada c Department of Chemical and Petroleum Engineering, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1Z4, Canada * Corresponding author. E-mail address: [email protected] (Q. Chu).

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Abstract Converting lignin into value-added products in current lignocellulosic biorefineries has been challenging, which in turn restricts the commercialization of many lignocellulosic biorefineries. In this work, a two-stage alkali-oxygen assisted liquid hot water pretreatment (AlkOx) was proposed as the first step of biorefinery. This alkalioxygen pretreatment facilitated biomass fractionation by solubilizing majority of lignin in water-soluble fraction, while remaining most of cellulose and hemicellulose in waterinsoluble fraction. As a result, biomass saccharification was significantly improved by selective removal and oxidative modification of lignin through alkali-oxygen pretreatment. Moreover, lignin residues from both pretreatment hydrolysate and enzymatic hydrolysate were shown to be favorable adsorbents for Pb(II) ions, with adsorption capacity of 263.16 and 90.91 mg/g, respectively. Results demonstrated that this integrated process could not only improve biomass saccharification but also enable lignin valorization, which encouraged the holistic utilization of lignin residues as part of an integrated biorefinery.

Keywords: Biomass pretreatment; Enzymatic hydrolysis; Delignification; Lignin modification; Heavy metal adsorption

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1. Introduction Biorefinery refers to the techniques of utilizing renewable lignocellulosic biomass to produce a wide range of bio-based fuels, chemicals, and materials, which serves as an alternative to traditional petroleum refinery (Chu et al., 2018). However, the complex lignocellulosic structure, in particular the recalcitrant nature of lignin, not only impedes efficient biomass saccharification into a sugar platform for biofuels/chemicals production, but also hampers lignin valorization for value-added products under the biorefinery concept. It has been reported that one of the greatest challenges in biorefinery is to engineer lignin structures to not only reduce biomass recalcitrance but also enable lignin valorization (Ragauskas et al., 2014). Therefore, lignocellulosic pretreatment capable of improving biomass saccharification while enabling lignin valorization is essential for biorefinery. In this context, alkali-oxygen assisted liquid hot water (AlkOx) pretreatment, like the oxygen-delignification in pulp and paper industry, is proposed as a promising first step of biorefinery to process lignocellulosic biomass (Chu et al., 2017; Qing et al., 2016). Different from auto-catalyzed liquid hot water (LHW) pretreatment, the alkalioxygen pretreatment facilitates biomass fractionation by solubilizing majority of lignin in water-soluble fraction, while the lignin structure is modified with oxygen-functional groups upon oxidation (Jiang et al., 2017). Lignin removal has been proved to increase cellulose accessibility, and the selective lignin modification mostly relates to decreased unproductive adsorption of enzymes to lignin (Li et al., 2018). As a result, biomass

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saccharification can be significantly improved due to the lignin removal and modification. More fermentable sugars can be released through pretreatment and subsequent enzymatic hydrolysis for further utilization such as bioethanol production. Besides, there are two potential streams from which lignin can be valorized within a biorefinery: pretreatment hydrolysate lignin and enzymatic hydrolysis residue mainly consisting of lignin (EHR). Considering the abundance of lignin, its valorization is believed to be one of the determinant factors for the economically feasible biorefinery (Chen et al., 2018). It has been shown that producing lignin adsorbents can be a favorable approach for lignin valorization due to low cost, abundance and vast availability of lignin (Supanchaiyamat et al., 2018). Adsorption technologies are easy to operate and have been utilized on an industrial scale for the treatment of waste effluents (Neris et al., 2019). Previous work has suggested that developing bio-based adsorbents from lignin not only diversifies the final products, but also encourages the holistic utilization of wastes to expand the profit margin as part of the integrated biorefinery (Supanchaiyamat et al., 2018). The lignin residues that modified with oxygen-functional groups after the alkali-oxygen pretreatment, has a great potential for direct utilization as adsorbents for heavy metal ions, which favors lignin valorization. In this work, poplar wood pellets were chosen as the starting biomass, since wood pellets have advantages such as great uniformity and small particle size, high biomass density and low moisture content, thus promoting easy handling and reducing transportation and storage costs (Rijal et al., 2012). The pellets were subjected to a two-

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stage alkali-oxygen assisted liquid hot water pretreatment, in order to promote selective lignin removal/modification and biomass saccharification. Then, the biomass fractionation, saccharification and adsorption capacity of the obtained lignin residues for heavy metal ions were investigated (Fig. 1). Heavy metals are hazardous contaminants of water pollution due to their toxic effects (Ogunsile and

Bamgboye,

2017). In this work, Pb(II) was selected as a model heavy metal to demonstrate the technical feasibility for aqueous heavy metal ion removal. Finally, mass balance was carried out to evaluate the process efficiency to coproduce fermentable sugars and lignin adsorbents for heavy metal ions.

2. Materials and methods 2.1. Preparation of the wood samples and chemicals used Poplar wood pellets were obtained from Gansu province, China. The moisture content of the wood pellets was determined by drying a known amount of chips in the oven at 105 oC overnight and measuring the weight loss. The moisture content of pellets was 6.38 ± 0.33% (measured in triplicate). The chemical composition was determined as follows: cellulose 46.65 %; hemicellulose 20.26 %; lignin 29.05 %. NaHCO3, NaOH, HNO3, NaNO3, Pb(NO3)2 and O2 (gas) were purchased from Sinopharm Chemical Reagent Co., Ltd. 2.2. Two-stage alkali-oxygen assisted liquid hot water (LHW) pretreatment of poplar wood pellets The pretreatment was carried out in a stainless Parr reactor containing 50 g poplar 5

wood pellets (dry mass, 20-100 mesh). Initially, pellets were impregnated in an aqueous sodium bicarbonate solution with charge of 18 % (w/w) based on dry mass, at 60 oC and an oxygen pressure of 100 psi (0.69 MPa) for 12 h. The solid-to-liquid ratio was 1:5 (w/v). After impregnation, the reactor was heated up to 130 oC and maintained for 30 min as the first stage pretreatment. Then, the temperature was rapidly raised to 210 oC and the mixture was cooked for 10 min as the second stage pretreatment. After that, the pretreatment was terminated immediately by cooling the reactor to room temperature in cold tap water. The two-stage liquid hot water (LHW) pretreatment was carried out as described above, but without the charge of sodium bicarbonate and oxygen. After pretreatment, the solid residue was separated from black liquor by filtration and washed with deionized water. The black liquor and washing water were collected for determination of sugars (water-soluble fraction, WSF), while the washed solid (waterinsoluble fraction, WIF) was kept at -4 oC for further use. 2.3. Enzymatic hydrolysis Enzymatic hydrolysis of the pretreated substrates was performed in acetate buffer (50 mM, pH 4.8) at 50 ºC and 180 rpm. The enzymes used for enzymatic hydrolysis were Cellic Ctec 2 and HTec (Novozymes, Franklinton, NC). Enzyme dosage was 20 mg protein of cellulase (Ctec 2) and 10 mg protein of xylanase (HTec) per gram cellulose (i.e. cellulase-to-xylanase ratio of 2:1). Enzymatic hydrolysis was conducted at substrate loading of 10 % (w/v) in a 100 mL Erlenmeyer flask. Samples were taken after 48 h enzymatic hydrolysis. Enzymes were inactivated by heating to 100 ºC for 5 min

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and subsequently stored at -18 ºC until sugar analysis was performed. All experiments were run in duplicate. 2.4. Preparation of lignin adsorbents The black liquor from two-stage alkali-oxygen pretreatment was collected. Nitric acid (0.25 M) was then added dropwise to the black liquor solution until pH dropped to between 2 to 3. The precipitated solids were separated from liquid fraction by filtration on Whatman No. 1 filter paper and washed three times with warm water (water-to-solid ration of around 1:50 each time for washing). The washed solid was then dried in a vacuum oven at 50 °C for 24 hours and milled in a mortar. The obtained solid was used as the adsorbent in the following adsorption experiments. 2.5. Batch adsorption studies The effect of pH on adsorption was investigated by first preparing 166 mg/L Pb(II) (0.8 mM) in 0.01 M NaNO3 solution. Then, 0.25 g of lignin were added to 100 mL of Pb(II) solution in 500 mL beaker. The pH was adjusted using 0.1 M HNO3 or 0.1 M NaOH as appropriate. Experiments were carried out at pH of 3.0, 4.0, 5.0, 6.0 and 7.0, respectively. At pH higher than 7, it was reported that the precipitation of metal ions occurred and resulted in a colloidal suspension. The mixtures were stirred at 20 °C, 80 rpm on a stirring plate to reach a homogenous environment for adsorption. After 6 h adsorption, the mixture was settled down for 5 min. Supernatant was taken and centrifuged at 5,000 rpm for 10 min. The residual metal ion analysis was performed by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 5300 DV,

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PerkinElmer). Effect of contact time on adsorption was investigated as follows: 0.25 g of lignin was mixed with 100 mL of aqueous solution with initial Pb(II) concentration of 166 mg/L. Samples were taken at intervals of 5, 10, 20, 30, 60, 90, 120 and 150 min. Effect of initial Pb(II) concentration on adsorption was also investigated: 0.25 g of lignin was mixed with 100 mL of aqueous solution with initial Pb(II) concentration ranging from 134.70 to 673.50 mg/L. Samples were taken at 60 min and centrifuged for residual metal ion analysis. Each experiment was carried out in duplicates. The adsorbed metal ions was determined as follows (Klapiszewski et al., 2017): 𝑞𝑒 =

(𝐶𝑜 ‒ 𝐶𝑒)𝑉

(1)

𝑀

while the metal ions removal percentage was calculated as (Klapiszewski et al., 2017): 𝑅𝑒𝑚𝑜𝑣𝑎𝑙 % =

100(𝐶𝑜 ‒ 𝐶𝑒)

(2)

𝐶𝑜

where qe (mg/g) was the equilibrium adsorption capacity, Co (mg/L) and Ce (mg/L) were the initial and residual metal ions concentration, respectively; M (g) was the dry mass of lignin adsorbent used and V (mL) was the volume of the Pb(II) solution. 2.6. Adsorption kinetics The pseudo-first-order kinetics was expressed as (Klapiszewski et al., 2017): (3)

𝑙𝑛(𝑞𝑒 ‒ 𝑞𝑡) = 𝑙𝑛𝑞𝑒 ‒ 𝑘1𝑡

The pseudo-second-order kinetics was expressed as (Klapiszewski et al., 2017): 𝑡 1 𝑡 = + 𝑞𝑡 𝑞𝑒 𝑘2·𝑞𝑒2

(4)

where qe and qt were the amounts (mg/g) of adsorbed Pb(II) ions on lignin at equilibrium and at time t, respectively; k1 was the rate constant of pseudo-first-order

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(1/min); k2 was the rate constant of pseudo-second-order (g/(mg·min)). 2.7. Adsorption isotherms The Freundlich isotherm was expressed as (Neris et al., 2019): 1

(5)

𝑙𝑛𝑞𝑒 = 𝑙𝑛𝐾𝑓 + 𝑙𝑛𝐶𝑒 𝑛

where qe was the amount of metal ions per unit mass of adsorbent in mg/g and Ce was the equilibrium concentration in mg/L. The Freundlich isotherm coefficients were determined by plotting ln qe against ln Ce. The slope

1 indicated the adsorption 𝑛

intensity or surface heterogeneity while the constant Kf was a measure of the adsorption capacity. The Langmuir isotherm was expressed as (Neris et al., 2019): 𝐶𝑒 𝑞𝑒

𝐶𝑒

1

(6)

= 𝑏· 𝑞 + 𝑞 𝑚

𝑚

where b was the adsorption equilibrium constant (L/mg) that was related to the apparent energy of adsorption, qm was the quantity of Pb(II) required to form a monolayer on unit mass of adsorbent (mg/g) and qe was the amount adsorbed on unit mass of the adsorbent (mg/g) when the equilibrium concentration was Ce (mg/L). A plot of would give a straight line of slope

𝐶𝑒 𝑞𝑒

versus Ce

1 1 and intercept · . 𝑞𝑚 𝑏 𝑞𝑚

2.8. Analytical methods All analyses were done in duplicate. The chemical components of raw material and pretreated samples were determined based on the procedure developed by the National Renewable Energy Laboratory for analyzing biomass material (Sluiter et al., 2008). The concentrations of monosaccharide were determined using a high performance liquid 9

chromatography system as described elsewhere (Chu et al., 2018). To assess the accessibility of cellulose, direct orange staining (Simon’s Stain technique) was performed as described by de Oliveira Santos et al. (2018). Fiber swelling was determined by the water retention value (WRV), which was measured according to the Technical Association of the Pulp and Paper Industry's (TAPPI) useful method UM 256 (Chu et al., 2018). Acid groups in substrates were determined by conductometric titration as described by Katz et al. (1984). The Fourier transform infrared (FTIR) spectra were performed using a Nicolet 560 spectrophotometer. The samples were prepared using KBr-disk method. Then, each sample was scanned ranging from 500 to 4000 cm−1 with a resolution of 1 cm−1.

3. Results and discussion 3.1. Effect of two-stage alkali-oxygen assisted liquid hot water (AlkOx) pretreatment on biomass fractionation In a biorefinery process, the main goals of the pretreatment step were to enhance the biomass fractionation and to recover cellulose, hemicellulose and lignin components in usable forms (Chu et al., 2017). The liquid hot water pretreatment was recognized as a promising technology for the pretreatment of lignocellulosic biomass, as it could fractionate and solubilize most of the hemicellulose components in the water-soluble fraction (WSF), and remain the cellulose and lignin components in the water-insoluble fraction (WIF) (Del Rio, 2012). As illustrated, the hemicellulose content dropped to 7.04 % after LHW

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pretreatment (Table. 1), primarily due to the hemicellulose solubilization. Only 27.39 % of the original hemicellulose was remained in the water-insoluble fraction (WIF), while 20.19 % was present in the water-soluble fraction (WSF). Results implied that more than 50 % of the original hemicellulose was degraded and resulted in the formation of soluble compounds that was inhibitory towards subsequent enzymatic hydrolysis and fermentation (Yuan et al., 2018). In general, a detoxification step is required to remove toxic components from the pretreatment liquor, which might remove a substantial fraction of sugars and increase the cost of biorefinery process. Besides, as a result of hemicellulose solubilization, lignin content in WIS increased to 33.85 %, which was reported to adversely affect the subsequent enzymatic hydrolysis by acting as a physical barrier or non-productive adsorbent of enzymes (Del Rio, 2012). Different from auto-catalyzed LHW pretreatment, the alkaline pretreatment could readily remove lignin, while exhibiting minor carbohydrate solubilization (Kallioinen et al., 2013). Moreover, the addition of oxygen to alkaline media was supposed to improve delignification degree as indicated in oxygen delignification of pulp and paper industry. It had been reported that the two-stage alkali-oxygen assisted steam pretreatment could enhance selective lignin modification and delignification of poplar wood chips while maximizing enzymatic hydrolysis of cellulose (Chu et al., 2017). In this section, poplar wood pellets were subjected to two-stage alkali-oxygen assisted LHW pretreatment with different severities, in order to promote biomass fractionation and to improve cellulose hydrolysis. As expected, 41.24 % of original lignin was removed from

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biomass after alkali-oxygen pretreatment without impregnation (UnImp AlkOx), increased by 33 % when compared to LHW pretreatment (Table. 1). Meanwhile, 57.67 % of original hemicellulose was remained in WIF, which was 30 % higher than that after LHW pretreatment. In order to evaluate the biomass fractionation through pretreatment in terms of lignin removal and carbohydrate (cellulose and hemicellulose) preservation, delignification selectivity was proposed as gram lignin removed per gram carbohydrate solubilized from the original biomass. For instance, the LHW pretreatment exhibited a low delignification selectivity of 0.14, as it mostly related to hemicellulose solubilization but not lignin removal. As expected, the alkali-oxygen pretreatment without pre-impregnation showed drastically higher delignification selectivity of 1.15, indicating the pretreatment efficacy of lignin removal more than carbohydrate solubilization. As reported, an impregnation step, which also was the first part of a Kraft cooking (Wedin et al., 2010), could enhance the pretreatment efficacy and sugar yield after subsequent enzymatic hydrolysis (Kim et al., 2014; Qing et al., 2017). In particular, the impregnation was considered essential prior to pretreatment when poplar wood pellets were used as feedstock. This was because the pelletization process to make durable pellets, especially the drying step, would cause the loss of pit pores and substrate permeability (Liu et al., 2014), resulting in decreased biomass susceptibility towards pretreatment. Thus, an impregnation step to reopen the tightly packed pore structure of substrate was required prior to pretreatment. It was observed that the alkali-oxygen

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treatment following impregnation at 60 ºC overnight enhanced lignin removal to 48.54 %, comparing to alkali-oxygen treatment without impregnation (Table 1). This improved delignification likely arose from the rewetting and swollen effect of impregnation, which promoted the uniform penetration of cooking liquors within woody biomass, bringing about homogeneous delignification. As a result of similar carbohydrate preservation and higher lignin removal, delignification selectivity of alkali-oxygen pretreatment increased to 1.34 (Table 1). In addition, anthraquinone (AQ), a widely used promoter in alkaline pulping process, was also used in the alkali-oxygen treatment. With the addition of 0.1 % (w/w) AQ in the alkali-oxygen pretreatment, the hemicellulose recovery in WIF increased to 63.47 % (Table 1). This was because AQ promoted the formation of aldonic acid in the polysaccharide end groups (de Menezes et al., 2017), contributing to (hemi)cellulose preservation. The relatively higher hemicellulose retention in the pretreated solids would be favorable to obtain higher total fermentable sugars concentration in the enzymatic hydrolysate, thus increasing the end-product titer after bioconversion. Meanwhile, the addition of AQ allowed for high delignification (60.36%, Table 1), primarily through extensive cleavage of β-O-4 linkages in lignin (de Menezes et al., 2017). The selective delignification was proved to increase substrate porosity and cellulose accessibility, resulting in improved ease of biomass saccharification (Del Rio et al., 2011). As a result of greater carbohydrate preservation and higher delignification, the delignification selectivity was significantly improve to 1.94 (Table 1).

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Moreover, to have an insight into the role of each stage during the two-stage pretreatment, the performance of first-stage-only pretreatment (First-stage AlkOxaq) was also evaluated for comparisons. It was found that the first-stage pretreatment resulted in hemicellulose recovery of 93.04 % with 86.40% in WIF (Table 1), which was significantly reduced to 81.17% (63.47 % in WIF) after two-stage pretreatment. This suggested that during the harsh second-stage pretreatment the hemicellulose components inevitably solubilized and degraded, probably due to the highly branched and easily hydrolyzed nature of hemicelluloses. As for lignin removal, first-stage pretreatment led to 32.08 % removal, while 60.36% lignin was removed after two-stage pretreatment (Table 1). In other words, 28.28 % of lignin was removed by second-stage pretreatment at 210 oC, indicating both stages performed comparatively in lignin removal. This is in agreement with previous work that reported the first-stage alkalineoxidation under mild conditions could remove easily-extracted lignin, followed by a harsh second-stage pretreatment to remove the more recalcitrant lignin from the cell walls (Liu et al., 2014). In summary, these results showed that auto-catalyzed LHW pretreatment fractionated biomass by solubilizing most hemicellulose in WSF, while retaining cellulose and lignin in WIF. As comparison, the alkali-oxygen pretreatment promoted biomass fractionation by solubilizing most lignin in WSF, while retaining most cellulose and hemicellulose in WIF. This fractionation strategy provided the potential to produce fermentable sugars mainly via enzymatic hydrolysis on WIF, and to recover

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lignin from WSF for downstream utilization. 3.2. Effect of two-stage alkali-oxygen pretreatment on biomass saccharification Aside from lignin removal, the use of alkali and oxygen in the LHW pretreatment was reported to bring about the formation of acid groups in the lignin macromolecule, which had been proved to be another alternative to improve biomass saccharification (Rovio et al., 2012). Thus, the acid groups incorporation was evaluated in this work. The acid groups in raw pellets were determined as 22.88 mmol/kg, which decreased to 10.73 mmol/kg after LHW pretreatment (Fig. 2). This phenomenon was probably because during LHW pretreatment, acetyl groups were released from hemicellulose components to form acetic acid, thereby leading to the acid-induced lignin repolymerisation. This reaction would cause the loss of functional groups such as acid and hydroxyl groups in lignin structure, which hampers efficient enzymatic hydrolysis and lignin utilization (Nakagame et al., 2011; Pielhop et al., 2015). However, it was found that the two-stage alkali-oxygen pretreatment led to substantial amount of acid groups incorporation into substrate (AlkOxaq in Fig. 2, 136.60 mmol/kg), in which the first-stage AlkOxaq treatment contributed to 107.66 mmol/kg acid groups incorporation. This significant acrid groups incorporation was primarily due to oxidation reaction during the pretreatment. Results implied that the first stage made the major contribution to lignin modification. It seemed that the lignin in the mild first stage was more reactive and susceptible to alkali-oxygen pretreatment, and reactivity of the lignin towards oxidation in harsh second stage was compromised due to carbocation induced lignin

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repolymerisation at higher temperature (Chu et al., 2017). The acid groups incorporation had the potential to decrease the hydrophobicity of lignin, thus decreased the non-productive binding of cellulase enzymes to lignin, resulting in higher ease of enzymatic hydrolysis (Nakagame et al., 2011). Besides, the acid groups may have an important impact in promoting the fiber swelling, by forming a hydrophilic layer on fiber surface (Del Rio et al., 2011). The extent of fiber swelling could be estimated by calculating the centrifugal water retention value (WRV) of substrates. It was evident that highest WRV was obtained on AlkOxaq substrate (Fig. 2), indicating the maximum fiber swelling. Moreover, it was suggested that fiber swelling, together with selective delignification, could contribute to increase the accessibility of cellulose to enzymes (Del Rio, 2012). Cellulose accessibility, estimated as orange dye adsorption on fiber, was also illustrated (Fig. 2). It was apparent that the AlkOxaq substrate exhibited the most dye adsorption of 133.42 mg on gram fiber, indicating the highest cellulose accessibility. The cellulose accessibility is proposed as the key property of substrate governing the susceptibility of substrate to enzymatic hydrolysis (Del Rio, 2012). Lignin affected enzymatic hydrolysis in mainly two ways: restricting the cellulose accessibility to enzymes and acting as non-productive adsorbent of the enzymes. In this work, the lignin removal and fiber swelling upon the alkali-oxygen pretreatment increased cellulose accessibility, while lignin modification such as acid groups incorporation had the potential to minimize the non-productive binding of enzymes to

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lignin (Del Rio, 2012). By overcoming the limitations inflicted by lignin, improved ease of enzymatic hydrolysis could be expected, as proposed hypothesis model of two-stage alkali-oxygen pretreatment to improve enzymatic hydrolysis (Fig. 3). Enzymatic hydrolysis was also performed on either LWH or alkali-oxygen pretreated substrate. The result of cellulose hydrolysis showed a good agreement with that of cellulose accessibility (evaluated as orange dye adsorption in Fig. 2). After 48 h hydrolysis, 55.73 % of the cellulose in LHW substrate could be converted to glucose, while the two-stage alkali-oxygen pretreatment with anthraquinone (AlkOxaq) led to the highest cellulose hydrolysis yield of 82.18 %. Besides, the total sugar release from original biomass (or the sugar yield based on the original carbohydrate content in the starting material) reached 75.08 % after the two-stage alkali-oxygen pretreatment and subsequent enzymatic hydrolysis, which was 30 % higher than that of LHW pretreatment (Fig. 2). 3.3. Effect of two-stage alkali-oxygen assisted LHW pretreatment on adsorption capacity of obtained lignin for heavy metal ions Conventionally, using lignin from black liquor of alkali pretreatment to prepare lignin-based adsorbents usually requires extra carbonization, activation or lignin modification with functional groups, which inevitably offsets the profit margin produced by lignin valorization (Supanchaiyamat et al., 2018). In this work, the obtained lignin from black liquor could be directly utilized as adsorbents, since the lignin structure was believed to be modified with oxygen-containing functional groups.

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Thus, FTIR analysis was carried out to explore the potential functional groups in lignin from black liquor after two-stage alkali-oxygen pretreatment. Two sets of control were used: soda-AQ lignin extracted from LHW pretreated poplar pellets (LHW-Soda Lignin) (Tian et al., 2017a), and lignin from black liquor of two-stage alkali assisted LHW treatment without the addition of oxygen (Alkaline Lignin). A number of absorption peaks were shown in FTIR spectra that reflected lignin structure (Supplementary materials). It was found that all lignin samples showed a broad band at 3400 cm−1, attributed to the phenolic and aliphatic hydroxyl groups which were also considered as the major functional group for metal ion adsorption (Chen et al., 2018). In the carbonyl/carboxyl region, bands were found at 1720 and 1620 cm−1, originating from unconjugated carbonyl/carboxyl stretching (Tian et al., 2017b). Particularly, bands around 1675 cm−1 appeared after alkali-oxygen pretreatment, which could be associated with the conjugated C=O stretch (Garcia et al., 2010). This observation was likely ascribed to the oxidation at α-position of side-chain of lignin during pretreatment, which confirmed certain oxidative modification of lignin during alkali-oxygen pretreatment (Wen et al., 2013). Results agreed with previous report that, after the oxidative modification, the hydroxyl and carboxyl groups content of the lignin increased, whereas the lignin skeleton was maintained (Ge and Li, 2018). This increment in functional groups could significantly improve the adsorption capacity of lignin for heavy metal ions, because the adsorption of heavy metals on lignin was an ion exchange process in which the hydroxyl and carboxyl groups on lignin deprotonated

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and adsorbed a heavy metal cation through ionic binding (Chen et al., 2018). Adsorption behavior of different lignin samples for heavy metal ions was compared to evaluate the influence of pretreatments on adsorption performance of lignin. The effect of pH on adsorption was first investigated, as pH of heavy metal ions solution would influence the surface charge of lignin and the degree of ionization, thereby determining the adsorption performance of lignin (Liang et al., 2013). All lignin samples showed higher adsorption when pH increased, and the optimum adsorption occurred in a pH range of 6 to 7 (Fig. 4a). The adsorption processes reached equilibrium within 60 min, at pH 6 and 20 oC (Fig. 4b). Moreover, the initial Pb(II) concentration was showed to play an important role on the adsorption efficiency and amount of metal ions adsorbed (Fig. 4c). Adsorption capacity was shown in the following order: AlkOxaq Lignin > AlkOx Lignin > Alkaline Lignin > AlkOxaq EHR > LHW-Soda Lignin (Fig. 4). The LHW-Soda Lignin led to the lowest adsorption of Pb(II), likely because of the repolymerised lignin structure (Tian et al., 2017b). And the adsorption capacity of lignin was significantly improved by the change of lignin structure during alkali-oxygen pretreatment, as expected. It was also illustrated that the adsorption capacity of all lignin samples had lower growth at high initial Pb(II) concentrations (Fig. 4c). That was because at higher Pb(II) concentrations, the active sites on adsorbents surface were saturated and blocked (Sadeek et al., 2015). The adsorption kinetic parameters of the lignin for Pb(II) were calculated according to the pseudo-first-order model and pseudo-second-order model (Table 2). It

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was clear that the pseudo-second-order model displayed high correlation coefficients (R2) close to 1.0 for all lignin samples. The qe values calculated using the pseudosecond-order model were close to the experimental data (qe,exp). Results indicated that the kinetics of Pb(II) adsorption on lignin followed a pseudo-second-order model, suggesting that the adsorption process involved chemisorption as the rate limiting step (Klapiszewski et al., 2017). Table 2 also showed the parameters obtained from the Freundlich and Langmuir isotherm models. In this work, n values were all higher than 1, implying that adsorption intensity was favorable at high concentrations (Wang et al., 2017). However, higher correlation coefficient R2 was observed on Langmuir adsorption isotherm (Table 2), indicating that Pb(II) ions adsorption on lignin followed a monolayer coverage. Also, it suggested a homogeneous nature and distribution of the active sites of the adsorbents (Wang et al., 2017). The calculated saturation capacity (qm) according to the Langmuir model was 263.16 and 90.91 mg/g for AlkOxaq lignin and AlkOxaq EHR, respectively, indicating that both lignin streams from alkali-oxygen pretreatment-based biorefinery were favorable adsorbents of heavy metal ions. 3.4. Integrated process to coproduce fermentable sugars and lignin adsorbents for heavy metal ions To evaluate the overall production of the integrated biorefinery process, mass balance analysis was performed. LHW pretreatment facilitated biomass fractionation by solubilizing and recovering hemicellulose in water-soluble fraction (WSF), while leaving the more accessible cellulose in water-insoluble fraction (WIF) with the

20

presence of large amount of lignin (upper part of Fig. 5). However, results implied that the cellulose hydrolysis was probably restricted by lignin. Besides, it is still challenging to convert the hemicellulose-derived sugars in WSF and the repolymerised lignin in WIF into value-added products in current lignocellulosic biorefineries (Tian et al., 2017b; Yuan et al., 2018). On the other hand, we looked into fractionating biomass through alkali-oxygen pretreatment, thus to solubilize most of lignin in WSF, while retaining cellulose and hemicellulose in WIF (lower part of Fig. 5). Results showed that biomass saccharification was significantly improved by selective lignin removal and modification resulted from alkali-oxygen pretreatment. As illustrated in lower part of Fig. 5, 36.98 g glucan and 10.11 g xylan were converted to fermentable sugars after alkali-oxygen pretreatement on 100 g pellets and subsequent enzymatic hydrolysis. These values obviously outclassed the total sugar yield from pellets after LHW pretreatment and enzymatic hydrolysis (i.e. 24.86 g glucan and 2.15 g xylan in enzymatic hydrolysate and 4.09 g hemicellulose-derived sugars in WSF). Moreover, lignin could be partially solubilized through pretreatment and recovered in the watersoluble fraction (15.37 g, Fig. 5) with high adsorption capacity for heavy metal ions (263.16 mg/g, Table 2), while the lignin residue after enzymatic hydrolysis (23.24 g, Fig. 5) could also be utilized as adsorbents for heavy metal ions with decent adsorption capacity (90.91 mg/g, Table 2). Both lignin streams from this integrated process showed favorable adsorption capacity for Pb(II), considering that the capacity of unmodified lignin was reported to be less than 30 mg/g (Tian et al., 2017b; Yuan et al., 2018).

21

Results suggested that two-stage alkali-oxygen assisted liquid hot water (AlkOx) pretreatment was a promising first step of biorefinery to process lignocellulosic biomass, which was capable of not only improving biomass saccharification, but also enabling lignin valorization.

4. Conclusions An integrated process based on two-stage alkali-oxygen assisted liquid hot water pretreatment was proposed to coproduce fermentable sugars and lignin adsorbents from poplar wood pellets. Results showed that liquid hot water pretreatment mainly catalyzed hemicellulose solubilization, while alkali-oxygen pretreatment mostly targeted at lignin removal and modification. Comparing with auto-catalyzed LHW pretreatment, the addition of alkaline and oxygen in pretreatment significantly improved biomass saccharification and adsorption capacity of the lignin residues for lead ions. Besides, during two-stage alkali-oxygen pretreatment, both stages performed comparatively in lignin removal, while the first stage made the major contribution to lignin modification.

Acknowledgements This project was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Foundation of Jiangsu University (17JDG034).

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Figures caption: Fig. 1. Flowchart of integrated process to coproduce fermentable sugars and lignin

26

absorbents under the biorefinery concept. Fig. 2. Substrate characteristics of LHW or alkali-oxygen pretreated biomass including total acid groups incorporation, fiber swelling (water retention value) and cellulose accessibility (orange dye adsorption), hydrolysis yield of cellulose and total sugar yield from biomass after pretreatment and enzymatic hydrolysis. LHW: liquid hot water pretreatment; UnImp AlkOx:

alkali-oxygen pretreatment without impregnation;

AlkOx: alkali-oxygen pretreatment; AlkOxaq: alkali-oxygen treatment with addition of anthraquinone (AQ); First-stage AlkOxaq: first-stage alkali-oxygen treatment with addition of anthraquinone. Fig. 3. Hypothesis model of two-stage alkali-oxygen pretreatment to improve the enzymatic hydrolysis of biomass: “+” and “-” indicated for improving and suppressing substrate characteristics and enzymatic hydrolysis. Fig. 4. Adsorption of heavy metal ions to obtained lignin residues: (a) effect of pH on Pb(II) adsorption to lignin; (b) effect of contact time on Pb(II) adsorption to lignin; (c) effect of initial Pb(II) concentration on adsorption capacity of lignin. AlkOxaq lignin: lignin residues after alkali-oxygen pretreatment with addition of anthraquinone (AQ); AlkOx lignin: lignin residues after alkali-oxygen pretreatment; Alkaline lignin: lignin residues after alkali pretreatment; AlkOxaq EHR: enzymatic hydrolysis residues (EHR) after alkali-oxygen pretreatment with addition of anthraquinone (AQ) and subsequent enzymatic hydrolysis; LHW-Soda lignin: Soda-AQ lignin extracted from liquid hot water pretreated substrate.

27

Fig. 5. Mass balance of LHW or alkali-oxygen pretreatment to coproduce fermentable sugars and lignin adsorbents for heavy metal ions.

28

OH COOH

Precipitation Lignin absorbent for heavy metal ions

Black liquor Alkali-oxygen pretreatment Biomass HO

COOH

Enzymatic hydrolysis Solid residue Cellulose Hemicellulose Lignin

Fermentable sugars

Enzymes Fermentable sugars Heavy metal ions

Lignin residue from enzymatic hydrolysis for adsorption of heavy metal ions

Modified lignin

Fig. 1

29

3.0

2.5

120

Water retention value (WRV)

Total acid groups (mmol/kg) Orange dye adsorption (mg/g) Cellulose hydrolysis yield (%) Total sugar yield (%)

150

2.0 90

1.5 60

1.0 30

0.5

0

0.0 LHW

UnImp AlkOx Total acid groups

AlkOx

Cellulose hydrolysis yield Water retention value (WRV)

Fig. 2

30

AlkOxaq

First-stage AlkOxaq Orange dye adsorption Total sugar yield

Biomass

Two-stage alkali-oxygen assisted

Enzymatic

liquid hot water pretreatment

hydrolysis

+ Lignin modification (Acid groups incorporation)

+ Lignin removal

+ +

Fiber swelling

-

+ Cellulose accessibility

+

Non-productive binding of cellulases

+

Enzymatic hydrolysis of biomass

Fig. 3

31

Fermentable sugars

Pb(II) adsorption to lignin (mg/g)

75

(a)

60

45

30 AlkOxaq Lignin AlkOx Lignin

15

Alkaline Lignin AlkOxaq EHR LHW-Soda Lignin

0 2.0

Pb(II) adsorption to lignin (mg/g)

75

4.0 6.0 pH value of adsorption

8.0

(b)

60

45

30 AlkOxaq Lignin AlkOx Lignin

15

Alkaline Lignin AlkOxaq EHR LHW-Soda Lignin

0 0

270

(c)

30

60 90 Time (min)

120

150

AlkOxaq Lignin

Pb(II) adsorption to lignin (mg/g)

AlkOx Lignin Alkaline Lignin

220

AlkOxaq EHR LHW-Soda Lignin 170

120

70

20 100

300 500 Initial Pb(II) concentration (mg/L)

Fig. 4

32

700

LHW pretreatment

Water-soluble fraction (WSF)

Cellulose-derived sugars: 0.10 g Hemicellulose-derived sugars: 4.09 g

Poplar Solid: 100g Cellulose: 46.65 g Hemicellulose: 20.26 g Lignin: 29.05 g

Water-insoluble fraction (WIF)

Enzymatic hydrolysis

Enzymatic hydrolysate

Enzymatic hydrolysis residue

Solid: 78.83 g Cellulose: 44.60 g Hemicellulose: 5.55 g Lignin: 26.68 g

Liquid fraction

Solid: 51.82 g Cellulose: 19.74 g Hemicellulose: 3.40 g Lignin: 26.68 g

Cellulose-derived sugars: 0.04 g Hemicellulose-derived sugars: 3.13 g

Precipitation

Water-soluble fraction (WSF) AlkOxaq pretreatment

Glucose as glucan: 24.86 g Xylose as xylan: 2.15 g

Cellulose-derived sugars: 0.10 g Hemicellulose-derived sugars: 4.09 g Lignin: 17.53 g

Lignin adsorbent

Solid: 15.37 g Cellulose: 0.01 g Hemicellulose: 0.24 g Lignin: 15.12 g

Poplar Solid: 100g Cellulose: 46.65 g Hemicellulose: 20.26 g Lignin: 29.05 g

Enzymatic hydrolysis

Enzymatic hydrolysate

Water-insoluble fraction (WIF) Enzymatic hydrolysis residue (EHR)

Solid: 70.33 g Cellulose: 45.00 g Hemicellulose: 12.86 g Lignin: 11.51 g

Fig. 5

33

Glucose as glucan: 36.98 g Xylose as xylan: 10.11 g Solid: 23.24 g Cellulose: 8.01 g Hemicellulose: 2.75 g Lignin: 11.52 g

OH COOH

Precipitation Lignin absorbent for heavy metal ions

Black liquor Alkali-oxygen pretreatment Biomass HO

COOH

Enzymatic hydrolysis Fermentable sugars

Solid residue Cellulose Hemicellulose Lignin

Enzymes Fermentable sugars Heavy metal ions

Lignin residue from enzymatic hydrolysis for adsorption of heavy metal ions

Modified lignin

Highlights  An alkali-oxygen (AlkOx) assisted liquid hot water pretreatment was proposed.  This pretreatment enabled coproduction of fermentable sugars and lignin adsorbents.  Biomass saccharification was significantly improved by lignin removal/modification.  Lignin residues from biorefinery were proved favorable adsorbents for lead ions.  Lignocellulosic biomass was holistically utilized without any waste.

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Table 1. Chemical components, sugar recovery and lignin removal after pretreatments Pretreatments

Cellulose Hemicellulose Lignin (%) (%) (%)

LHW UnImp AlkOx AlkOx AlkOxaq First-stage AlkOxaq

Cellulose Hemicellulose recovery b Lignin Delignification a Recovery removal (%) selectivity c WSF (%) WIF (%) Total (%) (%)

56.58 60.77 61.51 63.98

7.04 15.85 16.41 18.28

33.85 23.15 20.66 16.37

95.82 96.28 95.57 96.57

20.19 18.31 13.95 17.70

27.39 57.67 58.61 63.47

53.13

20.51

23.12

97.36

6.64

86.40

LHW: liquid hot water pretreatment; UnImp AlkOx:

47.58 75.98 72.56 81.17 93.04

8.14 41.24 48.54 60.36

0.14 1.15 1.34 1.94

32.08

2.29

alkali-oxygen pretreatment

without impregnation; AlkOx: alkali-oxygen pretreatment; AlkOxaq: alkali-oxygen treatment with addition of anthraquinone (AQ); First-stage AlkOxaq: first-stage alkalioxygen treatment with addition of anthraquinone. a Cellulose

recovery is the sum percentage of initial glucose present in the liquid and the

solid fractions after pretreatment. b

Hemicellulose recovery is the sum of the percent recovery of mannose, galactose,

xylose, and arabinose (monomeric and oligomeric sugars) in the water-soluble fraction (WSF) and water-insoluble fraction (WIF). c

Delignification selectivity is defined as gram lignin removed per gram carbohydrate

removed.

35

Table 2. Experimental and calculated data for the pseudo-first-order and pseudosecond-order kinetics models and the Freundlich and Langmuir isotherms for adsorption of Pb(II) on lignin Model

Pseudo-first-order

Parameter

AlkOxaq Lignin

AlkOx Lignin

Alkaline Lignin

AlkOxaq EHR

LHW-Soda Lignin

qe,exp (mg/g)

68.91

65.00

57.12

45.27

31.15

R2

0.9471 0.1382

0.9193 0.1282

0.8304 0.1179

0.9118

(min-1)

0.0770

0.9519 0.0901

qe (mg/g)

39.54

35.08

26.05

31.13

22.59

1.0000 0.0162 69.44

1.0000 0.0110 66.67

1.0000 0.0139 58.48

0.9997

0.9999 0.0072 33.56

0.9693 15.81 2.28

0.9886 14.77 2.47

0.9850 10.52 2.65

0.957

0.9999 263.16 0.0095

0.9970 208.33 0.0091

0.9997 129.87 0.0084

0.9996

K1

R2 Pseudo-second-order K2 (g/(mg·min)) qe (mg/g) R2 Freundlich

Kf (mg/g) n R2

Langmuir

qm (mg/g) b (L/mg)

0.0057 45.62 6.92 2.41 90.91 0.0143

0.9698 10.78 4.11 0.9996 52.63 0.0176

AlkOxaq lignin: lignin residues after alkali-oxygen pretreatment with addition of anthraquinone (AQ); AlkOx lignin: lignin residues after alkali-oxygen pretreatment; Alkaline lignin: lignin residues after alkali pretreatment; AlkOxaq EHR: enzymatic hydrolysis residues (EHR) after alkali-oxygen pretreatment with addition of anthraquinone (AQ) and subsequent enzymatic hydrolysis; LHW-Soda lignin: Soda-AQ lignin extracted from liquid hot water pretreated substrate.

36