Bioresource Technology 205 (2016) 90–96

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Reutilization of green liquor chemicals for pretreatment of whole rice waste biomass and its application to 2,3-butanediol production Ganesh D. Saratale, Moo-Young Jung, Min-Kyu Oh ⇑ Department of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul 136-713, South Korea

h i g h l i g h t s
NaC–NaS pretreatment for whole rice waste biomass (RWB) was investigated.
Intensified delignification of RWB facilitates enzymatic saccharification.
Reusability of NaC–NaS spent wash to treat RWB and its hydrolysis were evaluated.
Obtained RWB hydrolysates were used for BDO production by K. pneumonia.
Developed pretreatment strategy performed well for saccharification and biorefinery.

a r t i c l e

i n f o

Article history: Received 12 November 2015 Received in revised form 5 January 2016 Accepted 6 January 2016 Available online 20 January 2016 Keywords: Rice waste biomass Na2CO3–Na2SO3 pretreatment Reusability of chemicals 2,3-Butanediol

a b s t r a c t The performance of green liquor pretreatment using Na2CO3 and Na2SO3 and its optimization for whole rice waste biomass (RWB) was investigated. Incubation of Na2CO3–Na2SO3 at a 1:1 ratio (chemical charge 10%) for 12% RWB at 100 °C for 6 h resulted in maximum delignification (58.2%) with significant glucan yield (88%) and total sugar recovery (545 mg/g of RWB) after enzymatic hydrolysis. Recovery and reusability of the resulting chemical spent wash were evaluated to treat RWB along with its compatibility for enzymatic digestibility. Significant hydrolysis and lignin removal were observed for up to three cycles; however, further reuse of Na2CO3 and Na2SO3 lowered their performance. Significant 2,3butanediol (BDO) was produced by Klebsiella pneumoniae KMK-05 with the RWB enzymatic hydrolysate from each pretreatment cycle. BDO yield achieved using RWB-derived sugars was similar to those using laboratory-grade sugars. This pretreatment strategy constitutes an ecofriendly, cost-effective, and practical method for utilizing lignocellulosic biomass. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction In lignocellulosic biomass, cellulose and hemicellulose are present in thick bundles covered by layers of impermeable lignin; thus, initial treatment must be sufficiently harsh to remove this protective covering (Saratale et al., 2013). A large number of physical (comminution, milling, grinding), chemical (acid, alkali, oxidizing agents), biological, or a combination of these pretreatment approaches have been investigated for treating a variety of feedstocks (Saratale et al., 2008; Kumar et al., 2009). Several factors must be considered when selecting an efficient pretreatment method, such as the applicability to different raw materials, high digestibility, maximum sugar recovery, minimum inhibitor production, and non-energy-intensive process (Mosier et al., 2005).

⇑ Corresponding author. Tel./fax: +82 2 3290 3308. E-mail address: [email protected] (M.-K. Oh). http://dx.doi.org/10.1016/j.biortech.2016.01.028 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Efficient processes for pretreating lignocellulosic biomass are still limiting, despite numerous research efforts and great progress in the field. Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is considered the greenest saccharification process (Saratale et al., 2012). However, in the plant cell wall, lignin encases the cellulose and limits the rate and extent of enzymatic hydrolysis of lignocellulosic biomass (Saratale and Oh, 2015a). Complete delignification of biomass is not necessary to achieve maximum enzymatic digestibility, but removal of 20–65% of lignin is sufficient for improving saccharification (Ko et al., 2009; Gu et al., 2012). Thus, an effective pretreatment technology that can remove lignin effectively, disintegrate microfibrils, and improve enzymatic saccharification must be developed (Behera et al., 2014). Green liquor, mainly composed of Na2CO3 and Na2SO3, has been used for pretreatment of kraft pulping and biomass conversion, showing effective performance in maintaining pulp quality and enzymatic digestibility, respectively (Yang et al., 2013; Jin et al.,

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2010). Weak alkaline sodium carbonate breaks the ester bridges of hydroxycinnamic acids of lignin and cross-links between lignin and hemicellulose (Jin et al., 2013). Sodium sulfide is an effective chemical agent for delignification. Sulfonation of lignin increases its hydrophilicity and improves the subsequent enzymatic hydrolysis of biomass (Zhu et al., 2009). Green liquor pretreatment showed better delignification efficiency and lower hydrolysis of polysaccharides (cellulose and hemicellulose) during pretreatment process, by which maximum sugar recovery after enzymatic hydrolysis could be achieved. Another important feature of green liquor pretreatment is the formation of lower amounts of toxic byproducts, such as furfural and acetic acid, that affect the fermentation process (Jin et al., 2010; Gu et al., 2012). However, elemental sulfur in the spent wash leads to the complex formation and environmental contamination (Yang et al., 2013). Therefore, from an economic perspective, re-usability of the chemicals of green liquor can significantly reduce the chemical cost and also make the process environmentally benign. This novel strategy for reutilization of the chemical spent wash for pretreatment of RWB was investigated in our study. 2,3-Butanediol (BDO) has potential industrial applications, such as in the production of cosmetic products, pharmaceuticals, antifreeze agents, synthetic rubber, fuel additives, and flavoring agents in food products (Ji et al., 2011; Wu et al., 2008). Fermentative BDO production using renewable resources is cost-effective, ecofriendly, and a promising alternative production process (Mazumdar et al., 2013; Jung et al., 2015). A number of microbial species including Klebsiella, Enterobacter, Bacillus, and Serratia have been studied for BDO production. Among them, Klebsiella pneumoniae showed a broad substrate spectrum and culture adaptability, indicating its potential advantages for BDO production (Kim et al., 2014). The metabolic engineering of the strain was a promising strategy for efficient conversion of renewable biomass containing mixtures of sugars into a higher valued product (BDO). We utilized an engineered K. pneumoniae strain (Jung et al., 2014) for the conversion of RWB hydrolysates into BDO. In the present study, we explored the potential of combined sodium carbonate and sodium sulfite chemical pretreatment on the digestibility of whole RWB and determined its hydrolysis yield. We demonstrated the feasibility of reusing the chemical spent wash for pretreating RWB and further conversion into BDO using the engineered K. pneumoniae strain. Furthermore, the chemical composition and structural features of pretreated biomass and its crystallinity were evaluated using various analytical techniques. Here, we report the reuse of the chemical spent wash to pretreat RWB biomass and for the efficient conversion of RWB hydrolysates to BDO.

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2.2. Chemical pretreatment Initially, pretreatment was carried out by combining RWB biomass individually with NaC, NaBC, and NaS at 2% and 4% (w/v) in an electrically heated water bath at 100C for 3 h. The effects of combined NaC + NaS and NaBC + NaS treatment by keeping each chemical concentration at 2% and 4% (w/v) on RWB hydrolysis was evaluated. The ratio of the solid phase to the liquid phase in each pretreatment was maintained at 1:10. The effects of acidified sodium chlorite (ASC) treatment following NaC (2% and 4%) pretreatment to RWB biomass was also evaluated. The ASC procedure was carried out using the procedure reported earlier (Saratale and Oh, 2015b). The effects of the molar ratio of NaC–NaS at each chemical charge (1:0, 2:1, 1:1, 1:2, and 0:1) on the hydrolysis of RWB was investigated. 2.3. Optimization of NaC–NaS pretreatment Various process parameters including the chemical charge of NaC–NaS (1:1 ratio) 4–14%; incubation temperature (30, 50, 60, 80, 100 °C, and autoclaving at 121 °C for 30 min), substrate concentration (2–20%), and incubation time (1–12 h) on the hydrolysis of RWB using a ‘‘one variable at a time” approach while keeping other conditions constant were systematically investigated. All treated biomass samples were extensively washed with tap water to remove impurities. The samples were then neutralized with distilled water and dried at 60 °C until a constant weight achieved and further subjected to enzyme hydrolysis analyses. 2.4. Reusability of spent wash of NaC–NaS treatment for pretreatment of RWB To establish a cost-effective pretreatment method, we repeatedly used the spent wash to pretreat RWB. Under optimized conditions after each NaC–NaS pretreatment of RWB, the spent chemical wash was collected by centrifugation at 8000 rpm for 15 min. The volume of the separated supernatant was measured. The reusability of the supernatant (spent liquor) was evaluated with 12% (w/w) of biomass loading according to obtained solution volume. The solution was maintained at 100 °C for 6 h during each cycle. After each pretreatment cycle, the pH of the spent liquor was recorded. The reusability of the spent wash for hydrolyzing RWB was evaluated for up to five cycles. The pretreated solid was washed with deionized water to remove the residual chemicals and then used to determine the weight loss of the total solid and biochemical composition prior to enzymatic hydrolysis. 2.5. Enzymatic hydrolysis of pretreated RWB biomass

2. Methods 2.1. Biomass collection and preparation Rice (Oryza sativa) plant biomass was collected from a local farm in South Korea. The whole plant body, except the roots, was used as the substrate for pretreatment studies. The raw materials were air-dried, chopped, washed with water, and then dried at 60 °C. The sample was then milled, sieved through 0.2-mm screens, and stored at 4 °C until use. Sodium carbonate (NaC), sodium bicarbonate (NaBC), sodium sulfite (NaS), Whatman filter paper No. 1, sodium chlorite and commercial Trichoderma reesei cellulase (Celluclast 1.5 L) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals used for pretreatment studies, enzymatic hydrolysis, and BDO fermentation experiments were of the highest purity available and of analytical grade.

RWB pretreated using different methods was subjected to enzymatic hydrolysis. Filter paperase activity (FPase) was measured according to IUPAC recommendations using Whatman filter paper as the substrate (Ghose, 1987). One unit of enzyme activity was defined as the amount of enzyme required to release 2 lmol of reducing sugars. The adsorption capacity of cellulase for cellulose affects the rate of enzymatic hydrolysis of the biomass (Saratale and Oh, 2015a). Thus, a two-step enzymatic hydrolysis process was developed which uncouples the enzymatic hydrolysis and the separation. Two-step enzymatic hydrolysis of untreated and pretreated RWB was performed at 2% (w/v) in 20 mL of 50 mM citrate buffer (pH 5.0) containing 0.005% (w/v) sodium azide. We evaluated the effects of increasing RWB concentration (5–25 g/L) while keeping the enzyme dosage constant (30 FPase/g of RWB) and the effects of increasing enzyme concentration (10–50 FPase/ g of RWB) by keeping the substrate concentration (20 g/L of RWB) constant. An enzyme solution equivalent to FPase activity

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of 30 U/g of untreated and pretreated RWB was added to Erlenmeyer flasks and performed the procedure as reported earlier (Saratale and Oh, 2015a). The reducing sugars released during the two steps of enzymatic hydrolysis were combined to calculate the overall hydrolysis yield (Saratale et al., 2012)

Hydrolysis yield ð%Þ ¼ Reducing sugar ðmgÞ  0:9  100  polysaccharides content of substrate Glucose yield ð%Þ ¼ glucose ðmgÞ  0:9  100  cellulose content of substrate Hydrolysis of polysaccharides involves water. For each mole of reducing sugar released, 1 mol of H2O is required. A correction factor of 0.9 was therefore included in the calculation of the amount of polysaccharides hydrolyzed.

metabolites in the fermented broth were analyzed using highperformance liquid chromatography with an ACME-9000 instrument (Young-Lin Instrument, Seoul, Korea) and a SH1011 column (Shodex, Tokyo, Japan) capable of detecting the refraction index as previously described (Saratale and Oh, 2015a: Jung et al., 2012). The substrates and metabolites were quantified by comparison with their respective standards of known concentration. 2.8. Statistical analysis Data were analyzed by one-way analysis of variance with Tukey–Kramer multiple comparisons test. Readings were considered significant when p was 60.05. 3. Results and discussion 3.1. Effects of chemical pretreatment on enzyme digestibility of RWB

2.6. BDO production media and cultivation conditions The metabolically engineered K. pneumoniae KMK-05 strain was used for BDO production (Jung et al., 2014). The fermentation medium containing (g/L): KH2PO4, 3; Na2HPO4, 6.8; KCl, 0.75; (NH4)2 SO4, 5.35; Na2SO4, 0.28; MgSO47H2O, 0.26; citric acid, 0.42; yeast extract, 5; casamino acid, 10; and 0.3 mL microelement solution containing (g/L) ZnCl2, 34.2; FeCl36H2O, 2.7; MnCl24H2O, 10; CuCl22H2O, 0.85 and H3BO3, 0.31 with initial pH 7.0 was used for BDO production (Jung et al., 2014). The NaC–NaS-pretreated RWB enzymatic hydrolysates were concentrated to a sugar concentration of 30 g/L in a rotatory evaporator. In the flask culture, the synthetic mixture of sugars (g/L: glucose; 16.72, xylose 5.25 and arabinose; 8.28) or filtration-sterilized RWB enzymatic hydrolysates were added to 50 mL of production medium in 250-mL Erlenmeyer flasks to achieve a final sugar concentration of 30 g/L. The effects of increasing concentrations of RWB hydrolysates (20 and 30 g/L) on BDO production were also studied. For pH neutralization of the flask cultivation solution, 5% CaCO3 was added to the medium before cultivation. The culture, in 50 mL medium in 250-mL flasks sealed with silicon stoppers, was incubated at 37 °C under shaking condition (250 rpm). Samples (1 mL) were withdrawn at specific time intervals and centrifuged (7500g, 15 min) for subsequent measurement of pH, BDO concentration, and residual sugar content. Experiments involving the mixture of sugars and RWB hydrolysates were conducted in triplicate. 2.7. Analytical methods The chemical composition (cellulose, hemicellulose, and lignin) of raw and pretreated RWB was estimated as described by Goering and van Soest (1970). The reducing-sugar content of the enzymatic hydrolysates was determined using the dinitrosalicylic acid method (Miller, 1959). Fourier-transform infrared (FTIR) spectroscopy (Cary 630; Agilent, Santa Clara, CA, USA) of raw and pretreated RWB was conducted in the mid-infrared region by averaging 32 scans in the range of 400–4000 cm1 at a resolution of 4 cm1 to detect changes in functional groups. Scanning electron microscopy (SEM) images of the particles coated with platinum were obtained using a JEOL JSM-6360A microscope (Tokyo, Japan) at an operating voltage of 20 kV as previously described (Saratale et al., 2012). Samples of the raw and pretreated RWB were analyzed by X-ray diffraction (XRD) using a D2 Phaser tabletop model (Bruker, Billerica, MA, USA) operating at 30 kV and 10 mA as described previously (Waghmare et al., 2014). The crystallinity index of cellulose was calculated according to the peak height method (Kim and Holtzapple, 2006). The residual sugar content in RWB enzymatic hydrolysates and the concentration of soluble

Feedstock pretreatment is one of the most critical steps in the biochemical conversion of lignocellulose biomass into valueadded products. The best pretreatment method must improve the enzymatic digestibility, which is greatly enhanced after significant lignin removal from biomass. In this case, a pretreatment method that focuses on lignin removal appears to be promising. In this study, lignin removal, hydrolysis yield, and glucan yield were used to describe the performance of pretreatment and enzymatic digestibility of a substrate. Initially, RWB was pretreated with individual chemical agents including NaC, NaBC, and NaS (each 2% and 4% concentration). NaC pretreatment showed better hydrolysis of RWB compared to NaS and NaBC pretreatment. Upon increasing the chemical concentration, sequential increase in sugar production and hydrolysis yield were observed. The maximum total sugar production and hydrolysis yield for NaC pretreatment (352 mg/g of RWB and 54.78%) were higher than those for NaS (305 mg/g of RWB and 47.25%) and NaBC (298 mg/g of RWB and 46.15%) pretreatment. NaBC treatment changed the properties of the cellulose surface and allowed the enzyme to degrade the cellulose micro-fibrils (Kahar et al., 2013). However, in this study, NaBC treatment was less effective in terms of saccharification and hydrolysis of RWB. These results suggest that NaC is a promising chemical agent for hydrolyzing RWB. Similarly, NaC pretreatment was also effective for the pretreatment of biomass from various waste sources to produce fermentable sugars (Khaleghian et al., 2015; Jin et al., 2013). To enhance the hydrolysis and improve the saccharification of RWB, experiments involving combined chemical pretreatment were conducted. Combinations of NaS with NaC or NaBC increased the hydrolysis yield and improved lignin removal of RWB compared to individual chemical treatments (Fig. 1A). Significant lignin removal (48.12%), maximum sugar recovery (465 mg/g of RWB), and glucan yield (80.45%) after enzymatic hydrolysis were observed following NaC–NaS (4% each) pretreatment (Fig. 1A). ASC treatment following NaC pretreatment was also effective for lignin removal (about 55%), but the hydrolysis yield (68.8%) and glucan yield (78.1%) of RWB were not as high as those with NaC– NaS combination (Fig. 1A). The effects of combination of different molar ratios of NaC and NaS on the hydrolysis of RWB were evaluated. Better lignin removal (48.12%) and maximum sugar production (465 mg/g of RWB), hydrolysis yield (73.10%), and glucan recovery (82.45%) were observed after enzymatic hydrolysis following NaC–NaS (1:1 ratio) pretreatment (Fig. 1B). Application of only NaC or NaS did not result in significant hydrolysis than combination. Even when the ratios of NaC and NaS were increased, lignin removal, hydrolysis yield, and glucan recovery of RWB were not significantly

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SC SC 4% 2% 4% 2% aS aS aS aS +A +A 2% 4% +N +N +N +N C C % % % % 2 4 Na Na C2 C4 BC BC Na Na Na Na

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Hydrolysis yield (%) Glucan yield (%) Lignin removal (%)

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RWB concentration (%) 0 :0) :1) :2) :1) :1) S (1 S (2 S (1 S (1 S (0 :Na :Na :Na :Na :Na C C C C C Na Na Na Na Na

Molar ratio Fig. 1. Effects of combination of (A) NaC + NaS, NaBC + NaS, and NaC + ASC chemical pretreatment (B) at different chemical molar ratios of NaC and NaS on lignin removal, hydrolysis yield, and glucan yield from RWB after enzymatic hydrolysis (30 FPase/g of RWB).

improved (Fig. 1B). Similarly, some studies reported the effectiveness of NaC–NaS pretreatment for the effective degradation of biomass (Yang et al., 2013; Gu et al., 2012). These results indicate a synergistic effect of both chemicals, leading to significant delignification and increase in enzyme accessibility for the hydrolysis of polysaccharides and better saccharification of RWB. In addition, we studied the effects of increasing RWB concentration and of increasing enzyme concentration on the hydrolysis yield of RWB to achieve better saccharification. The optimal hydrolysis yield was obtained by using 20 g/L biomass with 30 FPase/g of RWB (Fig. S1A and B). This condition was used to check the enzymatic digestibility of pretreated RWB. 3.2. Optimization of NaC–NaS pretreatment conditions We further optimized the pretreatment conditions, including chemical charge, substrate loading, reaction temperature, and reaction time on lignin removal, enzymatic digestibility, and glucan recovery of RWB. During pretreatment, an increasing chemical charge (4–14%) led to enhanced delignification from 25.1% to 61.2% (Fig. 2A). The optimal chemical charge at 10% of NaC–NaS resulted in higher solid yield (55–60%), lignin removal (58.2%), and glucan yield (88%) (Fig. 2A). At relatively low chemical charge, partial delignification of RWB clearly inhibited the enzymatic digestibility.

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Hydrolysis yield (%) Glucan yield (%) Lignin removal (%)

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Lignin removal (%)

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Hydrolysis yield, Glucan yield (%)

Hydrolysis yield, Glucan yield (%)

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Chemical concentration (%)

Type of pretreatment

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80 Hydrolysis yield (%) Glucan yield (%) Lignin removal (%)

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(A) Hydrolysis yield, Glucan yield (%)

80 Hydrolysis yield (%) Glucan yield (%) Lignin removal (%)

Lignin removal (%)

Hydrolysis yield, Glucan yield (%)

120

20 20

0

0 30

50

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121

Reaction temperature (oC) Fig. 2. Effects of (A) NaC–NaS chemical charge (4–14%), (B) RWB concentration (2–20%), and (C) reaction temperature (30–100 °C and autoclaving at 121 °C for 30 min) on lignin removal, and hydrolysis yield and glucan yield from RWB after enzymatic hydrolysis (30 FPase/g of RWB).

Whereas, reactions with chemical charge at 12% and 14% showed higher solubilization of lignin and hemicellulose of RWB, resulted in higher glucan yield (about 95%) and lignin removal (61%). However, the hydrolysis yield and total sugar production were not significantly increased compared to that with 10% chemical charge, due to higher loss of hemicellulose. Therefore, we have selected 10% as an optimum chemical concentration for further experiments. Keeping the chemical concentration at 10%, the significant performance in terms of lignin removal, hydrolysis yield, and glucan yield was observed up to 12% RWB concentration. At 12% substrate concentration, significant lignin removal (58.2%) with higher sugar

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recovery (545 mg/g of RWB) and glucan yield (88%) after enzymatic hydrolysis were observed. However, a further increase in substrate concentration decreased lignin removal and hydrolysis yield significantly (Fig. 2B). On the basis of the obtained results, 10% chemical load and 12% RWB concentration were selected for further optimization studies. The effects of different reaction temperatures on pretreatment at constant RWB (12%) and NaC–NaS; (1:1, 10%) concentration were evaluated. Lignin removal and glucan yield were increased at higher reaction temperatures (Fig. 2C). Maximum hydrolysis of RWB with higher sugar production (545 mg/g of RWB), lignin removal (58.2%), and glucan yield (88%) were observed at 100 °C. Autoclaving the sample did not effectively degrade RWB, and even higher removal of lignin was observed (62.5%). After physicochemical pretreatment, larger amount of inhibitors may be produced and influence enzymatic hydrolysis, but this hypothesis requires further investigation (Fig. 2C). Reaction time is another critical factor influencing pretreatment effectiveness (Kim et al., 2014). A noticeable increase in lignin removal was observed from biomass pretreated for 3 h (45.7%) and 12 h (60.2%), but a small increase at between 6 h (58.2%) and 12 h (60.2%). Between 6 h and 12 h, the enzymatic digestibility and sugar recovery remained the same, indicating that 6 h was a critical reaction time for the NaC–NaS pretreatment of RWB (data not shown). The optimal conditions for maximum sugar recovery and lignin removal of RWB (12%) were application of an NaC–NaS chemical charge (10%, 1:1 ratio), at 100 °C, and 6 h of incubation using enzyme loading of 30 FPase/g of RWB. Approximately 58.2% of delignified RWB was conserved. Before and after treatment of RWB, we determined the biochemical composition of cellulose (35.15% and 36.25%), hemicellulose (30.65% and 27.15%), and lignin (22.40% and 9.40%). These results suggest that the cellulose concentration in the biomass was not affected, but a small loss of hemicellulose and significant lignin removal after pretreatment was observed. Under optimized conditions, significant delignification of RWB can liberate and expose the internal structure of biomass, allowing the enzymes to penetrate into the fibers and hydrolyze the polysaccharides to monomeric sugars (Saratale et al., 2008; Sun et al., 2000). 3.3. Reusability of chemical spent wash for pretreatment of RWB The production of biofuels or value-added products using renewable resources is a cost-effective and environmentally benign process that is in high-demand. However, the cost of the chemicals used during pretreatment in biomass conversion accounts for a large portion of the total cost. Additionally, the generated chemical spent wash creates environmental pollution. The recovery and reutilization of chemicals may reduce the cost of pretreatment and make the process more environmentally sound. These two points must also be commercially and practically applicable. In this study, repeated-batch operations were performed to examine the reusability of the resulting spent wash generated after NaC–NaS pretreatment under optimized conditions. During reutilization of the chemical spent wash, consistent hydrolysis performance and significant delignification of RWB was observed (Table 1; Fig. S2). After enzymatic hydrolysis of the pretreated biomass, the total sugar production performance was also significant. After NaC–NaS pretreatment, the resulting spent wash may have contained lower concentrations of inhibitors or toxic byproducts such as furfural and acetic acid, compared to other pretreatment methods (Jin et al., 2010; Gu et al., 2012). Until the III cycle, significant lignin removal (about 55%) and enzymatic digestibility of RWB was observed (Table 1). However, the IV and V cycles showed decreased delignification, hydrolysis, and enzymatic digestibility. The lower performance after cycle III may have resulted from a

decrease in the reactivity of the chemical solution at this point as a decrease in the pH of the solution was also noted (13.5 to 10.0). In addition, inhibitory compounds (furfural, hydroxymethyl furfural, etc.) generated after lignin degradation may persist in the biomass even after washing because of their higher concentrations. This may have had a toxic effect on the hydrolytic enzymes and decreased the hydrolysis performance and saccharification of RWB. The relative hydrolysis yield (70.6% and 64.7%) in IV and V pretreated RWB biomass was observed compared to the first cycle (control) (Table 1). 3.4. Structural characteristics After each repeated cycle, morphological changes in RWB were observed in SEM images. SEM observations of native RWB showed a smooth surface, whereas surface roughness was increased in the pretreated RWB (Fig. S3). The SEM results suggested that up to cycle III, significant destruction of biomass and structural morphological deformation occurred compared to in the control (Fig. S3B– D). The SEM images clearly showed that following NaC–NaS pretreatment, the integrated lignin and hemicellulose portion were significantly removed, by which cellulose fibrils appeared to be separated and accessible to the enzymes, resulting in better saccharification. However, in cycles IV and V, SEM images showed less destruction of RWB, supporting the results of lower hydrolysis yield in these cycles (Fig. S3E and F). Among several factors, crystallinity was found to significantly affect the enzymatic saccharification of glucan (Waghmare et al., 2014; Kim and Holtzapple, 2006). XRD analysis is a useful technique for determining the crystallinity index (CrI) of biomass. XRD diffractograms of RWB treated with NaC–NaS showed moderate increase in the CrI in treated (48.7%) RWB compared to untreated RWB (39.5%) (Fig. S4A). Similar CrIs and diffraction patterns in the five cycles of RWB biomass pretreatment were observed, suggesting lower destruction of cellulose and hemicellulose components during NaC–NaS pretreatment (Table 1). FTIR measurements were conducted using raw and treated samples of RWB to determine the influence of NaC–NaS treatment. Characteristic peaks associated with cellulose and hemicellulose were observed at 3300 cm1 (OH stretching), 2900 cm1 (C–H and CH2 stretching), and 1029 cm1 (C–O stretching vibration in hemicellulose) (Waghmare et al., 2014; Saratale and Oh, 2015a). Bands at these peaks did not appear to change significantly after NaC–NaS pretreatment (Fig. S4B). However, a strong peak at 1743 cm1, a characteristic C–O stretching vibration, decreased in intensity, suggesting significant removal of lignin side chains from the RWB biomass after treatment (Fig. S4B). Characteristic peaks related to lignin were also distinguished at 1357 cm1 and 1238 cm1 (asymmetric bending in CH3 in lignin), indicating that the major effect resulted from lignin removal. Based on the FTIR spectral analysis, NaC–NaS pretreated biomass showed the significant removal of lignin from RWB biomass, by which polysaccharides were more accessible to the hydrolysis reaction, leading to improved sugar recovery. 3.5. BDO production using RWB enzymatic hydrolysates The availability of a biocatalyst capable of utilizing a mixture of sugars in the lignocellulosic biomass hydrolysate is important, as it provides a more economical route for BDO production. We demonstrated the potential of NaC–NaS pretreated RWB enzymatic hydrolysates for fermentative BDO production using the K. pneumoniae KMK-05 strain under batch cultivation. The strain performed well and showed complete consumption of sugars from RWB enzymatic hydrolysates. With increasing sugar concentration in RWB hydrolysates, BDO production were increased (Fig. 3). The

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Table 1 Effects of repeated use of chemical spent wash on the biomass recovery yield, lignin removal, crystallinity index (CrI) of biomass, hydrolysis yield, and total sugar recovery from pretreated RWB after enzymatic hydrolysis (30 FPase/g of RWB). Repeated batch cycle

pH of spent wash

Recovery yield of biomass (%)

Hydrolysis yield (%)

Lignin removal (%)

Total sugar concentration (mg/g of biomass)

CrI (%)

Relative hydrolysis yield (%)

I II III IV V

13.5 12.5 11.5 10.5 10.0

55 ± 2.25 55 ± 2.10 60 ± 2.45 65 ± 2.54 68 ± 2.20

85 ± 1.25 84 ± 1.28 70 ± 1.05 60 ± 0.88 55 ± 0.85

58.2 ± 0.85 56.5 ± 0.78 55.2 ± 0.80 51.2 ± 0.65 50.0 ± 0.68

545 ± 4.45 545 ± 4.25 448 ± 4.15 388 ± 4.05 352 ± 3.45

48.7 ± 0.25 48.7 ± 0.25 46.7 ± 0.26 44.6 ± 0.22 41.2 ± 0.21

100 ± 4.45 98.2 ± 4.24 82.3 ± 3.85 70.6 ± 3.54 64.7 ± 3.45

Values are the mean of three experiments ± SEM. Statistics were determined by one-way ANOVA with Tukey–Kramer multiple comparisons test.

Total sugar concentration (g/L)

Sugar consumption (20 g/L) Sugar consumption (30 g/L) BDO titer (RS: 20g/L) BDO titer (RS: 30g/L)

30

16

12 20 8 10 4

0

2,3 Butanediol titer (g/L)

20

40

0 0

4

8

12

18

24

Time (h) Fig. 3. Effects of increasing NaC–NaS enzymatic hydrolysate concentration (20– 30 g/L) on sugar consumption and BDO production using the Klebsiella pneumoniae KMK-05 strain for different incubation times.

maximum BDO titer was approximately 11.44 g/L and the yield was 0.381 g/g of sugar when we utilized RWB enzymatic hydrolysates (30 g/L). Finally, RWB enzymatic hydrolysates generated after reuse of the same spent wash for five cycles were utilized for BDO production. The enzymatic hydrolysates were concentrated to 30 g/L. Until the third repeated batch of RWB hydrolysates, complete utilization of sugar as well as similar BDO production and BDO yield were observed (Table 2). In each treated biomass hydrolysate (without detoxification), complete sugar consumption with higher BDO yield was observed. However, in the fourth and fifth batches of RWB hydrolysates, sugar utilization and consequently BDO production and BDO yield were reduced (Table 2). This may be because of the small amount of fermentative inhibitors present in the enzymatic hydrolysate. Importantly, the sugar consumption and BDO yield were similar to those of the mixture of synthetic sugars, increasing the practical applicability of this method (Table 2). The obtained BDO yield was comparable and higher than the reported literature of BDO production using lignocellulosic biomass (Table S1).

According to these results, we conclude that repeated use of NaC–NaS in the chemical spent wash was effective for enhancing the enzymatic saccharification of RWB and subsequent BDO production. This process can be used for the bioconversion of all carbohydrates present in various lignocellulosic biomasses for sustainable bio-based production and to produce bioenergy. Further studies should examine the process in continuous mode operation by designing small scale reactor and evaluate its commercial applicability in BDO production. 4. Conclusion Combined NaC–NaS pretreatment showed excellent adaptability for the pretreatment of RWB to improve its enzymatic digestibility. The corresponding delignification ratio of pretreated solid and glucan yield were 58% and 88%, respectively, after enzymatic hydrolysis. The repeated utilization of spent wash for the pretreatment of RWB and their utilization for BDO production were investigated. Up to the third cycle, significant hydrolysis and lignin removal were observed. The resulting enzymatic hydrolysates were effectively used for BDO production. The developed pretreatment strategy has many advantages for its commercialization for lignocellulosic biorefineries with low technological and environmental risks and barriers. Acknowledgements This study was supported by a grant from the New & Renewable Energy Program of the Korea Institute of Energy Technology Evaluation and Planning (No. 20143030091040), South Korea. It was also supported through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015M3D3A1A01064919). The authors acknowledge the technical help from Mr. Ji-Woong Jang from Korea University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.01. 028.

Table 2 Utilization of resulted enzymatic hydrolysates of RWB biomas generated during repeated use of chemical spent wash for 2,3 butanediol production and fermentation parameters. Repeated batch cycle

Total sugar concentration (g/L)

Sugar utilization (%)

pH after 24 h of fermentation

BDO titer (g/ L)

Volumetric BDO productivity (g/L/ h)

BDO yield (g/g of sugar)

I II III IV V Mixture of sugar

30 30 30 30 30 30

100 100 100 90 85 100

6.75 6.75 6.75 6.75 6.75 6.75

11.44 ± 0.55 11.37 ± 0.54 11.06 ± 0.48 8.92 ± 0.48 7.93 ± 0.44 11.54 ± 0.46

0.476 ± 0.07 0.473 ± 0.06 0.460 ± 0.06 0.371 ± 0.05 0.330 ± 0.05 0.480 ± 0.07

0.381 ± 0.015 0.379 ± 0.015 0.368 ± 0.012 0.330 ± 0.011 0.311 ± 0.010 0.384 ± 0.018

Values are the mean of three experiments ± SEM. Statistics were determined by one-way ANOVA with Tukey–Kramer multiple comparisons test.

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