Bioethanol production from Cogongrass by sequential recycling of black liquor and wastewater in a mild-alkali pretreatment

Fuel 258 (2019) 116141

Contents lists available at ScienceDirect

Fuel journal homepage: www.elsevier.com/locate/fuel

Full Length Article

Bioethanol production from Cogongrass by sequential recycling of black liquor and wastewater in a mild-alkali pretreatment

T

Amir Goshadrou Department of Chemical Engineering, Faculty of Engineering, University of Isfahan, Isfahan 81746-73441, Iran

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

Keywords: Bioethanol Cogongrass Alkali pretreatment Black liquor recycling Wastewater reuse Surfactant-assisted hydrolysis

The present investigation evaluated Cogongrass as a prospective non-edible feedstock for second-generation bioethanol. An innovative recycling-based strategy was employed to minimize chemical consumption and wastewater generation during the mild-alkali pretreatment. The biomass was pretreated with 2% (W/V) sodium hydroxide (SH) solution at 85 °C for 90 min and subsequently subjected to separate saccharification and fermentation. Accordingly, the produced black liquor (BL) and waste wash-water (WWW) were sequentially withdrawn and partially fed back to the next pretreatment and washing processes, respectively. The results indicated that enzymatic hydrolysis of the biomass was substantially improved from 24.8% up to 90.8% following the alkali pretreatment; and afterward, it was progressively decreased to a minimum value of 66.4% upon recycling BL and WWW for ten times. Parallel hydrolysis experiments were conducted in the presence of three non-ionic environmentally-friendly surfactants at different concentrations (0.25–2% V/V). Notably, supplementation with 0.5% (V/V) Tween 80 significantly improved hydrolysis yield of recycled BL (RBL) pretreated substrate to a maximum value of ~88%. Fermentation of the RBL-pretreated Cogongrass did not reveal any cell inhibition and resulted in maximum ~76% ethanol production yield, while also saving 59% of the water consumption and over 45% of the alkali usage. Detailed analyses of the biomass using semi-quantitative techniques revealed that enhanced cellulose accessibility to the enzyme, decreased crystallinity, and extensive lignin and hemicellulose removal were the key factors contributing to the observed improvements.

Abbreviations: BL, Black liquor; WWW, Waste wash-water; RBL, Recycled black liquor; SH, Sodium hydroxide; WRV, Water retention value; SS, Simons’ stain; FTIR, Fourier transform infrared spectroscopy; SEM, Scanning electron microscopy; DB1, Direct blue 1; DO15, Direct orange 15; DAC, Dye adsorption capacity; DAR, Dye adsorption ratio E-mail address: [email protected]. https://doi.org/10.1016/j.fuel.2019.116141 Received 15 May 2019; Received in revised form 13 August 2019; Accepted 3 September 2019 0016-2361/ © 2019 Elsevier Ltd. All rights reserved.

Fuel 258 (2019) 116141

A. Goshadrou

1. Introduction

not only save more than 45% of the alkali reagent, but also maintain high conversion of sugarcane bagasse into fermentable sugars [26]. Although the exact mechanism of the surfactant action has not been established, it is believed that surfactants increase enzyme accessibility to cellulose by inhibiting the non-productive enzyme-lignin interactions [30]. It should be noted that a majority of wastewater gets produced during extensive washing of the pretreated materials to achieve neutral pH and water clarification. Although washing of the pretreated materials is necessary to remove inhibitory compounds prior to the enzymatic hydrolysis, it would be impractical to completely wash them with large quantities of wash water. Reducing the water consumption and recycling the spent waste water are the critical targets that must be taken into consideration for the establishment of a cost-effective bioethanol production process [31,32]. To our knowledge, no previous reports have investigated a sustainable approach to sequentially recycling BL and WWW in the alkali pretreatment of Cogongrass. Meanwhile, the effects of surfactant supplementation during enzymatic hydrolysis of the Cogongrass has not been investigated. The present investigation dealt with mild alkali pretreatment of the non-valuable Cogongrass as a prospective non-edible feedstock for second-generation bioethanol. The key focus of the study was to examine whether the major by-product of SH pretreatment (BL) could be sequentially recycled to pretreat Cogongrass and if so, how efficient it would be compared with the conventional SH pretreatment. In particular, the research also tried to minimize alkali consumption and wastewater generation via sequential BL and WWW recycling approach. Three non-ionic green surfactants polyethylene glycol 4000 (PEG), Tween 20, and Tween 80 at different concentrations were also employed as remarkable promoters for enzymatic hydrolysis of the RBLpretreated biomass. Moreover, semi-quantitative methods (e.g., Simons’ stain and water retention value) were employed to track the changes in the key features of the substrate upon pretreatment and provide a better understanding of its recalcitrance.

Bioethanol is the most widely commercialized liquid biofuel which can partially replace petroleum-derived gasoline to accommodate both the high energy demand and the greenhouse gas emission reduction. Ethanol production from abundant and renewable lignocellulosic feedstocks is an attractive option promoting its economic and environmental sustainability. Lignocellulosic materials are complex mixture of polysaccharides (cellulose and hemicellulose) firmly linked to the polyphenolic lignin through covalent and hydrogen bonds, which are highly robust and recalcitrant to enzymatic and biological attacks [1–6]. Imperata cylindrical or Cogongrass is an aggressive, perennial, and a globally distributed weed which acts as an ecological threat to many upland crops. It requires only a minimal amount of water, grows fast even in unfertile soils, dominates vast tracts of land, and competitively excludes other plants. Field burning is mostly preferred to remove weeds and to minimize pesticide overuse, but it produces large amounts of toxic air pollutants. On the other hand, it is an abundant non-valuable lignocellulosic waste which contains appreciable amounts of carbohydrate polymers such as cellulose. The average biomass yield of Cogongrass is 8–20 ton ha−1 year−1, which is comparable to switchgrass (~11 ton ha−1 year−1) and Miscanthus (10–15 ton ha−1 year−1) [7–11]. Utilizing Cogongrass as an alternative feedstock for bioethanol could also decrease pesticide consumption and, at the same time, minimize the use of edible food crops. Biomass pretreatment is crucial to break down the linkages among components, release carbohydrate polymers, and render the downstream enzymatic and microbial processing [12]. However, pretreatment is a capital- and energy-intensive unit operation posing a serious bottleneck when scaling-up the bioethanol production process. Several processes so far have been investigated which are generally physical, chemical, biological, or combined pretreatments [13–15]. Among various methods, alkali pretreatment using NaOH (as the most important alkali) has emerged as one of the front-runners. It has certain advantages over other pretreatments: (1) SH is non-toxic, non-corrosive, and almost low in price; (2) pretreatment process could be performed under relatively mild conditions; (3) and it is capable to modify or remove lignin and hemicellulose by saponification of intermolecular ester bonds [16–19]. However, its main drawback is high alkali and water consumption and a large amount of black liquor (BL) production which could limit the economic and environmental viability of the process. The BL is a toxic and hazardous waste that contains appreciable amounts of alkali and organic materials such as polysaccharides, lignin, and cross-linked macromolecules comprising many aromatic groups of related structures (e.g., phenols, catechols, etc.) [20,21]. Some of the constituents could also play a role in pretreatment. For example, phenol is an important monophenolic product from lignin depolymerization processes, and it has been proven effective in the delignification of wheat straw for improved enzymatic hydrolysis [22,23]. Likewise, Zhang et al. have reported delignification ability of the BL could be attributed to the appreciable amounts of organic compounds in the BL, which improved the solvation of lignin during corn stover pretreatment [24]. Several methods such as combustion, electrocoagulation, bioaugmentation, electrolysis, adsorption, and precipitation have been investigated to remove lignin from BL; however, most of them suffer from technical and economic problems [25]. A number of studies have also suggested BL recycling approach to minimize wastewater generation and chemical discharge during the alkali pretreatment. However, progressive drop in enzymatic hydrolysis yield following the sequential BL recycling is the next obstacle in practical applications [25–29]. Previous research has highlighted the promising effect of a non-ionic surfactant as an additive to enzymatic hydrolysis process following pretreatment with RBL. It has been proved that BL recycling approach together with the addition of surfactant at low concentration of 0.25% (V/V) could

2. Materials and methods 2.1. Raw materials Cogongrass (Imperata cylindrical) stalks and leaves were harvested from a local area of Abadan, Iran. The harvested materials were washed with tap water to remove the dust and heated at 45 °C until a constant weight was obtained. The shredded dried biomass was hammer-milled and screened to a particle size between 297 and 841 μm (20–50 mesh) and then kept in a refrigerator at 4 °C until used. A duplicate sample of Cogongrass was oven-dried at 105 °C overnight for determination of its moisture content [33]. The average moisture content of the biomass was found to be 5.1 ± 0.2%. 2.2. RBL pretreatment with WWW reuse An amount of 10 g (based on dry weight) of Cogongrass was immersed in 100 mL of 20 g/ L SH solution. Heating of the mixture was carried out on a water bath at 85 °C for 90 min, while it was manually mixed every 10 min. Upon completing the pretreatment, the suspension was separated into residual solids and BL using a Buchner funnel (Whatman No. 44). The solid fraction was washed with 300 mL water in three stages, and effluent WWW from the 1st, 2nd, and 3rd stages were collected and marked as WWW1, WWW2, and WWW3, respectively. The materials were then suspended in 60 mL water, and the pH was adjusted to 4.8 with 0.5 M sulfuric acid. The above-mentioned conventional SH pretreatment was used as the control pretreatment. The collected BL was also preserved for use in the forthcoming pretreatment process. Subsequently, 65 mL of BL was diluted with WWW1 to a final volume of 100 mL, replenished with 1% (W/V) SH and marked as RBL1. The 1st stage RBL pretreatment was performed using RBL1 at the same 2

Fuel 258 (2019) 116141

A. Goshadrou

Fig. 1. Sequential BL and WWW recycling strategy for pretreatment of Cogongrass.

condition described above, except that the 1st and 2nd washing process was conducted using WWW2, and WWW3, respectively. Likewise, the 2nd stage RBL pretreatment was carried out using the BL and WWW generated by the former pretreatment. As shown in Fig. 1, the same RBL pretreatment was repeated for ten times and the results were thoroughly compared with that of the one stage pretreatment with fresh SH [9,26]. All the materials were oven-dried (48 °C) and kept at 4 °C until used for subsequent hydrolysis experiments. A previous study has emphasized that it is necessary to maintain BL alkalinity in a pH range 12.6–13.3 to provide a substantial alteration in biomass structure [27]. The initial pH of the fresh SH solution (2% W/V) and the first BL were found to be 13.70 and 13.27, respectively. With SH supplementation (1% W/V) prior to the 2nd pretreatment stage, the pH raised again to 13.64. Each RBL was similarly replenished to maintain the pH value within the permissible limits throughout the investigation.

isolated using filtration by a 45 µm nylon membrane. The substrate was wrapped in a nonwoven bag and suspended in 10 mL deionized water for 2 h, then subjected to 15 min centrifugation at 3000×g [38–40]. The percent of WRV was calculated as follows:

% WRV =

Mw

Md Md

× 100

(1)

where Mw and Md are the wet and dry masses, respectively. 2.5. Simons’ stain (SS) The SS technique utilizes two direct dyes of different sizes for determination of enzyme accessibility to cellulose. The dyes Direct blue 1 (Pontamine Fast Sky Blue 6BX, or DB1) and Direct orange 15 (Pontamine Fast Orange 6RN, or DO15) were provided by Pylam Products Co. Inc. (Garden City, NY). The DO15 solution (1% W/V) was filtered (100 kDa MWCO) to remove its low molecular weight fraction. The materials were well mixed (10% W/V) with phosphate buffered saline solution (pH 6, 0.3 M PO4, 1.4 mM NaCl) and then supplemented with equal volumes (0.25–2 mL) of DB1 and DO15 stock solutions (10 mg/mL). The mixtures were incubated at 70 °C and 150 rpm after dilution to a final volume of 10 mL. Following 10 min centrifugation at 3000×g, the supernatants were collected and analyzed for DB1 and DO15 spectrophotometrically at 455 and 624 nm, respectively [41,42].

2.3. Chemical composition The presence of Klason lignin in the materials was determined gravimetrically after two-stage acid hydrolysis according to the procedure presented by the National Renewable Energy Laboratory [34]. The holocellulose fraction was isolated after delignification using sodium chlorite solution with some modifications. Subsequently, the cellulose content was determined according to the procedure developed by the Korean Standards Association [35–37].

2.6. Fourier transform infrared spectroscopy (FTIR)

2.4. Water retention value (WRV)

The structural constituents of the selected materials were analyzed using FTIR (JASCO 6300, Japan) and a universal ATR (Attenuated Total Reflection) accessory. Sixty scans were acquired per sample at the resolution of 4 cm−1 in the range of 600 to 4000 cm−1. The TCI and LOI

Approximately, 1 g of the biomass was disintegrated in an excessive amount of deionized water. After 1 h presoaking, the substrate was 3

Fuel 258 (2019) 116141

A. Goshadrou

After 30 h cultivation at 32 °C and 120 rpm, the yeast biomass was aseptically separated. The hydrolysates were sterilized, supplemented with the same nutrients (without glucose) as used for inoculum culture (as stated just above), and inoculated with 5 g/L yeast biomass at 32 °C for ethanol production [48]. The concentration of ethanol was determined using a gas chromatograph (GC, Agilent 6890) apparatus with an Agilent HP-Innowax column. The ethanol production yield was calculated as follows:

Table 1 Chemical composition* and solid recovery (%) of untreated and pretreated Cogongrass. Substrate

Cellulose

Hemicellulose

Klason lignin

Solid recovery

Untreated SH-pretreated RBL1-pretreated RBL2-pretreated RBL3-pretreated RBL4-pretreated RBL5-pretreated RBL6-pretreated RBL7-pretreated RBL8-pretreated RBL9-pretreated RBL10-pretreated

41.5 60.1 57.2 57.7 55.5 54.4 53.6 53.0 53.3 52.4 50.9 51.3

22.5 10.6 11.2 11.5 14.7 16.1 15.2 15.4 14.3 15.0 15.1 14.6

20.4 ± 0.4 8.5 ± 0.2 13.2 ± 0.4 13.7 ± 0.5 14.4 ± 0.3 14.1 ± 0.3 14.5 ± 0.4 15.0 ± 0.4 14.9 ± 0.4 15.2 ± 0.3 15.5 ± 0.3 15.3 ± 0.2

100.0 66.1 69.8 70.3 71.1 70.7 71.5 71.8 72.3 72.9 72.4 73.0

± ± ± ± ± ± ± ± ± ± ± ±

1.2 1.4 1.5 1.0 0.9 1.1 0.9 1.0 1.6 1.4 1.1 1.0

± ± ± ± ± ± ± ± ± ± ± ±

0.7 0.3 0.3 0.3 0.4 0.5 0.4 0.5 0.3 0.4 0.5 0.3

% Ethanol yield =

2.10. Statistical analysis The experiments were conducted in duplicate and the significant differences between individual averages were evaluated by the SNK procedure (SAS, Version 9.2, NC, USA). The criterion level of significance was set at a probability P < 0.05.

crystallinity indices were estimated according to a previously described method [43,44]

3. Results and discussion

2.7. Scanning electron microscopy (SEM)

3.1. Effect of pretreatment on the composition of Cogongrass

SEM was investigated to observe morphological features of the selected materials before and after pretreatments. The samples were sputter-coated with gold before imaging under 15 kV and high vacuum mode SEM (Zeiss, Germany).

The cellulose, hemicellulose, and Klason lignin content of the materials are presented in Table 1. The raw Cogongrass contained 41.5% cellulose (the dominant constituent of biomass), 22.5% hemicellulose, and 20.4% acid-insoluble lignin. Cellulose fraction of the biomass was almost similar to that of sweet sorghum bagasse (~41%), but higher than rice straw (39%) and wheat straw (31%) [33,49,50]. The results indicated that SH pretreatment led to a significant increase (~44.8%) in cellulose content by removal of the hemicellulose and lignin parts. Moreover, substantial delignification was observed after alkali pretreatment, whereas the lignin fraction in the SH-pretreated biomass was substantially decreased by 58.3%. Similarly, hemicellulose, a heteropolymer of polysaccharides and polyuronides, could partially be removed (52.9%) by SH pretreatment. Hemicellulose and lignin are strong barriers to enzymatic hydrolysis as covering layers of them prevent direct contact of the enzyme with cellulose [13]. It is well accepted that, during pretreatment reactions, SH provides hydroxide ion (OH−) which: (1) effectively attacks the lignin-hemicellulose linkage in lignocellulosic structure by disrupting the ether and ester bonds, and (2) cleaves the ester and carbon-to-carbon bonds in lignin molecules, leading to enhanced hemicellulose dissolution and lignin degradation [16]. Lignin is a complex polymer of three primary units (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) that plays a cementing role to connect cellulose and hemicellulose in the lignocellulose structure. Various types of lignin with different amounts of methoxy groups are present in softwood, hardwood and grass-type plant species. Contrary to softwoods which contain mostly guaiacyl lignin (a polymer of coniferyl alcohol), hardwoods and grass species are mainly made up syringyl lignin with varying ratios of coniferyl and sinapyl alcohol type of units. Because alkali reagents such as SH cleave the dimer containing syringyl units more rapidly than guaiacyl-guaiacyl dimers, they are more effective for delignification of hardwoods and herbaceous biomass than softwoods [16]. Compared to the untreated substrate, a similar trend of compositional changes was observed after RBL pretreatments; however, the lignin content of RBL-pretreated materials (13.2–15.5%) was noticeably higher than that of SH-pretreated Cogongrass (8.5%). It might be due to either the gradual decrease of delignification efficiency of SH following recycling and/or cumulative aggregation of ligninoriginated degradation products on the residual biomass. No significant change in hemicellulose solubilization was observed after RBL pretreatments for five consecutive times. A possible explanation is that hemicellulose is more vulnerable by pretreatment than lignin and is more likely to experience most of its

2.8. Surfactant-assisted enzymatic hydrolysis Hydrolysis experiments were carried out in citrate buffer solution (50 mM, pH 4.8) at 3% (W/V) solid loading and 150 rpm for 72 h. The enzyme loading was 20 FPU cellulase (Celluclast 1.5L, Sigma) and 30 CBU β‐glucosidase (Novozyme 188, Sigma) per gram of dry substrate. The cellulase and β‐glucosidase activities were 77 FPU/mL and 560 CBU/mL, respectively, according to standard procedures [45–47]. Sodium azide at 0.05% (W/V) was added to the media to avoid possible bacterial contamination. Moreover, several parallel experiments were also conducted by adding different surfactants polyethylene glycol 4000 (PEG), Tween 20, and Tween 80 to the hydrolysis mixture. Accordingly, prior to hydrolysis of the 10th stage pretreated Cogongrass, the media were supplemented with 0.25%, 0.5%, 1%, 1.5% and 2% (V/ V) of the surfactants. The glucose concentration in the hydrolysates was determined by the enzymatic colorimetric method using glucose oxidase kit (Pars Azmoon Inc., Tehran, Iran). The hydrolysis yield was calculated as follows:

[G] [G]0 × 100 [S ] × F × 1.111

(3)

where [E] is the ethanol concentration (g/L).

*The relative standard deviation between replicates was less than 3.5% for composition analyses.

% Hydrolysis yield =

[E ] × 100 [S ] × F × 1.111 × 0.51

(2)

where [G] and [G]0 are the final and initial concentration of glucose (g/ L), respectively, [S] the solid loading (g/L), and F the glucan fraction. 2.9. Ethanol production Based on the most promising results from the hydrolysis experiments, some of the samples were selected for subsequent fermentation. Accordingly, similar hydrolysis experiments were conducted for the selected samples, except using an autoclave for sterilization (121 °C, 20 min) instead of supplementation with sodium azide. Consequently, the hydrolysates were anaerobically fermented to ethanol by the Saccharomyces cerevisiae (PTCC 5052). The microorganism was cultivated at 30 °C on agar slants contained (g/L): glucose, 20; peptone, 10; yeast extract, 10; agar, 20. Subsequently, the yeast was transferred to a medium (pH 5.5) contained (g/L): glucose, 50; yeast extract, 5; (NH4)2SO4, 7.5; MgSO4·7H2O, 0.75; K2HPO4, 3.5; and CaCl2·2H2O, 1. 4

Fuel 258 (2019) 116141

A. Goshadrou

Fig. 2. FTIR spectra of untreated and selected pretreated Cogongrass.

losses during the reaction with SH. As expected, simultaneous hemicellulose and lignin removal led to decreased solid recovery yields (66.1–73.0%) which was more pronounced for the SH-pretreated materials.

could conversely decrease the index by forming less-ordered cellulose fibers. The results indicated that all the band intensities which are especially sensitive to the state of lignin and hemicellulose (1218, 1240, 1510, 1600, and 1735 cm−1) were substantially reduced, suggesting extensive lignin and hemicellulose removal during the pretreatments. Thus, decreasing of the LOI and TCI upon SH and RBL pretreatments must be most likely associated with partial disintegration of crystalline cellulose. Decrystallization efficiency of the alkali solutions was almost unchanged even after BL recycling continued for ten runs. It should be noted that accurate crystallinity measurement through conventional methods such as FTIR would be challenging because: (1) the crystallinity calculation is based on the entire material rather than the actual amorphous cellulose [51], and (2) drying of the samples prior to the analysis could lead to partial recrystallization of the lignocellulose [52].

3.2. Effect of pretreatment on the structure of Cogongrass FTIR analysis was investigated to study the structural change of Cogongrass caused by alkali pretreatments. The spectra of untreated and selected pretreated materials are shown in Fig. 2. Compared to the intact Cogongrass, both SH- and RBL10-pretreated materials underwent significant decreases in the band intensities at 896, 1158, 1319, 1337, 1375, 1425, 2920 cm−1, implying that highly ordered hydrogen bonds in crystalline regions of Cogongrass were disrupted after the pretreatments. The intensities at 1425 and 896 cm−1 are associated with the cellulose I and cellulose II, respectively. Compared to the crystalline cellulose I which is an array of parallel chains, cellulose II is amorphous and is thermodynamically more stable. The amorphous region tends to be hydrolyzed much more rapidly than crystalline portion and cellulose crystallinity indices are generally defined based on the ratio of two crystalline and amorphous cellulose regions. Lateral order index (LOI), which is calculated as the band intensity ratio A1425/A896, substantially dropped from 0.635 to 0.582 and 0.574 upon SH and RBL10 pretreatments (Table 2), respectively [43]. The total crystallinity index (TCI, A1375/A2920) of the untreated Cogongrass was 1.113, which was significantly decreased to 0.868 and 0.920 as a result of SH and RBL10 pretreatments, respectively. Moreover, alkali pretreatments resulted in shortening of O–H stretching band at 3350 cm−1 which is associated with weaker intra- and intermolecular hydrogen bonding and a lower degree of crystallinity. The main reactions during alkali pretreatment can bring about changes in the substrate properties such as crystallinity index, which are likely attributed to: (1) dissolution of lignin and hemicellulose, and (2) disruption of structural inter/intra hydrogen bonding of cellulose chains. The former should primarily cause of rising crystallinity index by removal of amorphous regions, whereas the latter,

3.3. Effect of pretreatment on the morphology of Cogongrass SEM analysis was used to provide insight into the changes in surface morphological features caused by pretreatments. The SEM images for untreated, SH-, and RTL10-pretreated materials are presented in Fig. 3. The images of the intact Cogongrass (Fig. 2a, d) showed a highly compact, smooth, and uniformly-arranged fibrillar structure, which was comparable to the typical nature of grass-type plants. However, Figs. (2b, e) showed that SH pretreatment resulted in severe structural damage to the surface. The observation revealed that SH pretreatment seriously disintegrated the rigid fiber bundles into small irregular fragments, and created a new pattern with a rougher surface, more cracks and voids, and improved surface area. Presence of small broken pieces with irregular texture on the surface should be attributed to the extensive lignin and hemicellulose destruction, and cellulose decrystallization during the alkali pretreatment. The morphology of and SHand RBL10-pretreated materials was almost similar and both pretreatments efficiently opened up the rigid structure of Cogongrass. However, it was appeared that the former could generate a more sponge-like texture with a higher accessible surface area.

Table 2 Crystallinity index for untreated and selected pretreated Cogongrass. Crystallinity index

LOI (A1425/A896) TCI (A1375/A2920)

3.4. Effect of pretreatment on Cogongrass accessibility to enzyme

Pretreatment Untreated

SH

RBL10

0.635 1.113

0.582 0.868

0.574 0.920

SS technique was applied to evaluate the total cellulosic surface area and its accessibility to enzyme. Both dyes (Direct blue 1 and Direct orange 15) exhibit high affinity for cellulose and bind specifically to it. During the staining experiments, small DB1, with a molecular diameter 5

Fuel 258 (2019) 116141

A. Goshadrou

Fig. 3. Scanning electron micrographs of (a, d) untreated, (b, e) SH-, and (c, f) RBL10-pretreated Cogongrass.

of ~1 nm, occupies pores with a diameter larger than 1 nm, while the large DO15 molecules (5–36 nm) only penetrate pores large enough to accommodate them. As DO15 has a much higher affinity for the hydroxyl groups on the cellulosic surface, it could competitively push off DB1 molecules inside the large pores. Assuming that the size of catalytic core domain of cellulase is comparable to that of the DO15 molecule, total amount of dyes adsorbed by the biomass (DO15 + DB1) and the adsorption ratio (DO15/DB1) could be considered as relevant measures of total and accessible cellulosic surface area (a measure of the large-tosmall pore ratio), respectively [41,53]. The results (Fig. 4) revealed that dye adsorption capacity (DAC) significantly increased by 1.2- to 2.8fold upon pretreatments. Interestingly, DAC of intact Cogongrass which was only 34.0 (mg/g substrate) reached a maximum value of 94.3 (mg/

g substrate) following SH pretreatment. Besides, the pretreatment sharply enhanced dye adoption ratio (DAR) from 0.46 to 1.92, which was corresponded to almost 4.2-fold improvement of accessible cellulosic surface area. It could be attributed to the extensive delignification and cellulose decrystallization which opened up internal pores, enriched the relative proportion of cellulose, and consequently provided more active sites for cellulose-dye interaction. In addition, DO15 could achieve further access to the cellulosic surface after pore enlargement caused by the pretreatments. Almost similar trend of DAC and DAR enhancement was found when the biomass was pretreated with the RBL. However, the results indicated that both DAC and DAR noticeably decreased once BL recycling was initiated. The smallest decrease was observed for the 1st RBL-, then for the 2nd RBL-, and finally the largest 6

Fuel 258 (2019) 116141

A. Goshadrou

Fig. 4. DAC (mg dye/g substrate), and DAR (DO15/DB1) of untreated and pretreated Cogongrass.

decrease for the 10th RBL-pretreated sample. The unfavorable drop of DAC and DAR gradually continued when recycling continued for three runs and then reached plateau values around 46 (mg/g substrate) and 0.92, respectively. It should be recalled that the present study used the minimum water usage for washing of the pretreated materials. Therefore, incomplete washing process might not efficiently remove lignindegradation products from the solid surface, leading to limited cellulose accessibility to the dyes. In addition, post-acidification for rapid pH adjustment could enhance lignin deposition on the substrate [54]. The observed plateau after three BL recycling steps should be most likely due to the surface saturation coverage of lignin on the substrate.

materials exhibited lower saccharification yield than the SH-pretreated one, whereas the initial and final hydrolysis yields were in the range of 19.6–44.4% and 66.4–88.7%, respectively. Recycling of BL for the first time resulted in a maximum hydrolysis yield of 88.7%, which underwent a downward trend by further recycling. The observed decay pattern was in good agreement with the conclusion drawn from SS which suggested progressive lignin deposition on RBL pretreated materials. It should be noted that the hydrolysis process occurs via layer by layer surface reaction and unidirectional enzyme movement along each layer. Hence, surface lignin coverage could impede hydrolysis by blocking enzyme traveling along the cellulose chain, and/or by limiting cellulase accessibility to sublayers. Moreover, lignin may chemically block the surface layer through the lignin-carbohydrate complex. The results also indicated that the negative impact of BL recycling on hydrolysis yield was more pronounced in the early- than in the late-phase of saccharification. The drop of lignin inhibition during prolonged hydrolysis time might be attributed to its migration from cellulose surface to bulk liquid phase. Although it is very challenging to explain the observed results, some hypotheses have been proposed to address the driving force of the lignin migration [55,56]. According to one, during the short-time hydrolysis, lignin disturbance on the cellulosic surface retards enzymatic digestion by stopping and/or slowing down the enzyme mobility; however, at extended hydrolysis times, heavy traffic jam of cellulases around the lignin may lead a “push” and subsequently restart and/or speed up hydrolysis reaction. The other one is that at prolonged hydrolysis times, the cellulase may facilitate lignin detachment by hydrolyzing the adjacent cellulose chains and loosening the blocked area. Therefore, the lignin inhibition would be gradually released when all the initial surfaces have been hydrolyzed. However, lignin migration from the solid phase and its accumulation in the supernatant may also inhibit hydrolysis reaction by non-productive binding to the enzyme [52,55,57]. According to the previously reported findings and the present results, it was speculated that the hydrolysis yield should be improved either by applying high cellulase loading and activity and/or by using lignin-blocking additives (e.g., surfactants) [58,59]. Consequently, further experiments were undertaken to assess the impact of three different surfactants on enzymatic susceptibility of the RBL10-pretreated Cogongrass.

3.5. Effect of pretreatment on enzymatic hydrolysis of Cogongrass The substrates were enzymatically hydrolyzed for 72 h, and the results are shown in Table 3. For the intact biomass, only 10.1% and 24.8% glucose yield was observed following 24 h and 72 h hydrolysis, respectively. After alkali pretreatments, enzymatic hydrolysis of the materials experienced substantial improvement, while the maximum yield (~91%) was achieved after 72 h hydrolysis of the SH-pretreated biomass. At 24 h, hydrolysis of the SH-pretreated biomass resulted in almost 65% cellulose conversion. However, the RBL-pretreated Table 3 Yield of enzymatic hydrolysis (%) of untreated and pretreated Cogongrass. Substrate

Untreated SH-pretreated RBL1-pretreated RBL2-pretreated RBL3-pretreated RBL4-pretreated RBL5-pretreated RBL6-pretreated RBL7-pretreated RBL8-pretreated RBL9-pretreated RBL10-pretreated

Hydrolysis time 24 h

72 h

10.1 64.9 44.4 37.2 35.5 29.8 25.4 22.6 19.6 19.7 19.7 21.9

24.8 90.8 88.7 85.3 86.4 80.8 76.5 76.8 74.2 71.6 68.3 66.4

7

Fuel 258 (2019) 116141

A. Goshadrou

Table 4 Yield of enzymatic hydrolysis (%) of RBL10-pretreated Cogongrass. Surfactant

Conc. (% V/V)

Table 5 WRV of untreated and pretreated Cogongrass.

Hydrolysis time 24 h

72 h

Tween 20

0.25 0.5 1 2

29.1 30.9 28.6 31.3

73.5 74.1 77.9 78.2

Tween 80

0.25 0.5 1 2

35.7 40.2 44.7 38.0

75.5 87.8 90.1 85.3

PEG

0.25 0.5 1 2

30.1 33.3 41.6 33.6

74.7 83.1 85.9 78.4

Substrate

WRV (g/g dry material)

Untreated SH-pretreated RBL1-pretreated RBL2-pretreated RBL3-pretreated RBL4-pretreated RBL5-pretreated RBL6-pretreated RBL7-pretreated RBL8-pretreated RBL9-pretreated RBL10-pretreated

0.74 1.79 1.42 1.11 1.16 1.14 1.05 1.04 1.08 1.12 1.10 1.13

substantially enhanced to the maximum of 44.7% with surfactant assistance, which was almost 2-fold higher than that without surfactant assistance. Comparison of the 24 and 72 h intervals (except for Tween 20) demonstrated a similar trend of the yield variation versus surfactant concentration. The high sensitivity of the short-time yield to surfactant assistance may contribute to facilitated “pushing off” and/or “peeling off” the deposited lignin barrier from the surface layer during the early stages of hydrolysis [55,57].

As summarized in Table 4, noticeable improvement in hydrolysis yield was achieved following the assistance of surfactants. The final yield was significantly increased by 1.1- to 1.4-fold depending on the type and concentration of the surfactants. The results showed that addition of surfactants at only 0.25% (V/V) enhanced the conversion yield to 73.5–75.5%. For the PEG and Tween 80, increasing the surfactants loading from 0.25% to 0.5% (V/V) significantly increased the scarification yields and after that reached a peak at 1% (V/V). However, it was appeared that Tween 80 and PEG at 2% (V/V) negatively affected the hydrolysis yield. In the case of Tween 20, a slight upward trend of the yield versus surfactant loading was also detected. When compared to the Tween 20 and PEG, the observations revealed the more positive effect of Tween 80 on digestibility of the RBL10-pretreated materials, whereas the yield peaked at 90.1% after addition of 1% (V/V) Tween 80. A likely explanation is that surfactant could promote enzyme stability, modify the biomass structure, and positively affect enzymesubstrate interactions. However, a number of studies have confirmed that the latter approach was mainly responsible for impressive role of surfactants [59]. Almost all Trichoderma reesei cellulases have hydrophobic amino acids modules docked on the surface for optimal interaction with cellulose chains. However, the amino acid residues in cellulase could also attach to the functional groups in lignin through three major non-covalent forces, including hydrophobic, electrostatic, and hydrogen bonds interactions. Hydrophobic interactions have been proposed as a major feature in the non-productive binding of cellulase to lignin which negatively impact cellulase activity and hydrolysis performance [60]. Atomic-detail molecular dynamics simulation also confirmed that lignin binds preferentially to the tyrosine residues of Cel7A CBM, which plays a crucial role in cellulose binding [61]. Several strategies have been suggested to minimize or overcome the unproductive cellulase-lignin interactions (e.g., lignin chemical modification, enzyme engineering, and lignin blocking). Lignin blocking by non-ionic surfactants/polymers (e.g., Tween series, PEG, Triton X100) has been examined repeatedly by a great many research groups [62]. Basically, they are composed of both hydrophobic hydrocarbon chains and hydrophilic ethylene oxide head groups. The hydrophobic hydrocarbon tails of the surfactant can strongly bind to the lignin's hydrophobic sites and functional groups to prevent cellulase adsorption [63]. Eriksson et al. comprehensively investigated mechanism of various surfactants (e.g. Tween 20, Tween 80 and PEG) effect in enzymatic hydrolysis of lignocellulose and reported that hydrophobic interaction of Tween 80 with lignin reduced the unproductive enzyme-lignin interactions and consequently improved enzymatic hydrolysis of the lignocellulose [59]. The hydrophilic head groups most likely facilitate lignin removal from the surface by decreasing the surface tension between phases [64]. The results also showed a more pronounced effect of surfactants during the early stage of hydrolysis. At 24 h, the yield was

3.6. Effect of pretreatment on porosity of Cogongrass The swelling ability of the materials was estimated by analyzing their WRV that can be considered as a sensitive probe for measuring cell wall porosity. The results (Table 5) showed noticeable improvements in the swelling capacity of Cogongrass after pretreatments, while more than 1.4-fold increase in WRV was achieved for the SH-pretreated biomass. It is acknowledged that water molecules on hydrophilic surfaces are more constrained than on hydrophobic surfaces. A possible explanation for the improved capacity of SH-pretreated materials to uptake water molecules might be severe delignification, and cellulose enrichment occurred after pretreatment, leading to higher hydrophilicity of the surface layer. Furthermore, observations from SS and SEM imaging revealed an enhanced specific surface area which most likely assisted water diffusion inside the pores. Compared to the SHpretreated Cogongrass, RBL-pretreated materials appeared to lose their water absorption affinity, whereas the WRV was declined from 1.79 to 1.42 and 1.11 following the first and second round of BL recycling, respectively. It may be associated with the higher abundance of hydrophobic lignin relocated onto the first layer, and consequent loss of the accessible surface area [65]. A gradual decrease in the WRV with increasing BL recycling times was expected, however; this was not the case and no significant drop in WRV was experienced by further recycling of the BL. Also, the WRV variation trend was not quite as predicted by the SS and hydrolysis experiments. The technique suffers from the fact that the molecular size of water is much smaller than that of DO15 and cellulase enzyme used in the SS and hydrolysis experiments, respectively. Therefore, it is likely that the smaller pores of biomass could accommodate the water molecules but not the DO15 and enzyme molecules, leading to over-prediction of the results. Besides, water molecules are not specific for cellulose and the presence of hydrophilic hemicellulose might virtually prevent drop of WRV caused by lignin surface coverage. To sum up, the method was not sensitive enough to differentiate between water holding capacities of the RBL-pretreated materials and consequently predict the ease of hydrolysis. 3.7. Ethanol production The most promising pretreated materials in the hydrolysis stage were subjected to fermentation by the yeast S. cerevisiae, and the results 8

Fuel 258 (2019) 116141

A. Goshadrou

Fig. 5. Ethanol yield (% theoretical ethanol yield) and ethanol production (g/kg substrate).

process would use 200 g SH and 46 L fresh water (Fig. 6). By comparing, the BL-recycling approach could successfully save almost 59% of the wash water consumption and more than 45% of the alkali usage. 4. Conclusions For the first time, the non-valuable invasive Cogongrass was sequentially pretreated with environmentally hazardous BL, washed with recycled WWW, and subsequently subjected to SHF to ethanol. Rapid semi-quantitative methods revealed that pretreatment rendered the biomass more accessible to the cellulase owing to the lignin removal and substantial structural changes. Noticeably, BL recycling for ten times could not only save large amounts of water and alkali consumption, but achieved major improvement in ethanol production (17.9-fold) through surfactant-assisted SHF, when compared to the untreated biomass. On the other hand, quality of the BL tends to be gradually decreased upon sequential recycling. Therefore, further environmental and economic studies should be required to accurately determine the optimum number of recycling steps for developing a costeffective technology that minimize effluent discharge and maintain the ethanol productivity as high as possible.

Fig. 6. Water consumption (L) and alkali usage (g) per one kg of the initial Cogongrass subjected to SH and RBL10 pretreatment.

are shown in Fig. 5. Anaerobic fermentation of the intact Cogongrass resulted in just 8.3% of the theoretical ethanol yield, which reached a maximum value of 84.9% after 90 min SH pretreatment at 85 °C. However, without surfactant assistance, a noticeable decrease (~28%) in fermentability was observed for the RBL10-pretreated sample. It might be attributed to the toxin lignin-degradation products that could largely inhibit the fermentation process for bioethanol production [66]. Surprisingly, Tween 80 at a loading of 0.5% (V/V) was highly effective for improving ethanol yield from the RBL10-pretreated sample, whereas the hydrolysate supplemented with Tween 80 exhibited significantly higher ethanol yield (75.6%) than that without Tween 80 (60.7%). Also, it was appeared that supplementation with higher Tween 80 concentration contributed to slight inhibition in downstream fermentation. Based on the same amount of initial Cogongrass (1 kg) fed to the pretreatment stage, almost 174, and 134 g ethanol was produced from the SH-, and RBL10-pretreated materials, respectively, while it was only 7.5 g ethanol from the untreated biomass. Besides, it was estimated that the non-recycling alkali pretreatment followed by limited washing

References [1] Morais RR, Pascoal AM, Pereira-Júnior MA, Batista KA, Rodriguez AG, Fernandes KF. Bioethanol production from Solanum lycocarpum starch: a sustainable non-food energy source for biofuels. Renewable Energy 2019;140:361–6. [2] Jiao J, Li J, Bai Y. Uncertainty analysis in the life cycle assessment of cassava ethanol in China. J Cleaner Prod 2019;206:438–51. [3] Loaiza SS, García CA, Alzate CAC. Bioethanol production: Advances in technologies and raw materials. Bioenergy and biofuels. CRC Press; 2018. p. 239–72. [4] Nguyen QA, Yang J, Bae H-J. Bioethanol production from individual and mixed agricultural biomass residues. Ind Crops Prod 2017;95:718–25. [5] Zhang J, Liu J, Kou L, Zhang X, Tan T. Bioethanol production from cellulose obtained from the catalytic hydro-deoxygenation (lignin-first refined to aviation fuel) of apple wood. Fuel 2019;250:245–53. [6] Mączyńska J, Krzywonos M, Kupczyk A, Tucki K, Sikora M, Pińkowska H, et al. Production and use of biofuels for transport in Poland and Brazil-the case of bioethanol. Fuel 2019;241:989–96. [7] Fojas JJR, Del Rosario EJ. Optimization of pretreatment and enzymatic saccharification of Cogon grass prior ethanol production. In: Proceedings of world academy of science, engineering and technology. World Academy of Science, Engineering and Technology (WASET); 2013:631.

9

Fuel 258 (2019) 116141

A. Goshadrou [8] Lin YS, Lee WC. Simultaneous saccharification and fermentation of alkali-pretreated cogongrass for bioethanol production. Bioresources 2011;6(3):2744–56. [9] Haque MA, Barman DN, Kim MK, Yun HD, Cho KM. Cogon grass (Imperata cylindrica), a potential biomass candidate for bioethanol: cell wall structural changes enhancing hydrolysis in a mild alkali pretreatment regime. J Sci Food Agric 2016;96(5):1790–7. [10] Makarova EI, Budaeva VV, Kukhlenko AA, Orlov SE. Enzyme kinetics of cellulose hydrolysis of Miscanthus and oat hulls. 3 Biotech 2017;7(5):317. [11] Songsomboon K, Crawford R, Crawford J, Hansen J, Cummings J, Mattson N, et al. Recurrent phenotypic selection for resistance to diseases caused by Bipolaris oryzae in switchgrass (Panicum virgatum L.). Biomass Bioenergy 2019;125:105–13. [12] Wyman CE, Cai CM, Kumar R. Bioethanol from lignocellulosic biomass. In: Kaltschmitt M, editor. Energy from organic materials (biomass). Encyclopedia of sustainability science and technology series. New York, NY: Springer; 2019. p. 997–1022. [13] Shafiei M, Kumar R, Karimi K. Pretreatment of lignocellulosic biomass. Lignocellulose-based bioproducts. Springer 2015:85–154. [14] Hassan SS, Williams GA, Jaiswal AK. Emerging technologies for the pretreatment of lignocellulosic biomass. Bioresour Technol 2018;262:310–8. [15] Sun SN, Chen X, Tao YH, Cao XF, Li MF, Wen JL, et al. Pretreatment of Eucalyptus urophylla in γ-valerolactone/dilute acid system for removal of non-cellulosic components and acceleration of enzymatic hydrolysis. Ind Crops Prod 2019;132:21–8. [16] Kim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour Technol 2016;199:42–8. [17] Yoo HY, Lee JH, Kim DS, Lee JH, Lee SK, Lee SJ, et al. Enhancement of glucose yield from canola agricultural residue by alkali pretreatment based on multi-regression models. J Ind Eng Chem 2017;51:303–11. [18] Kumar MN, Ravikumar R, Thenmozhi S, Kumar MR, Shankar MK. Choice of pretreatment technology for sustainable production of bioethanol from lignocellulosic biomass: bottle necks and recommendations. Waste and Biomass Valorization 2019;10(6):1693–709. [19] Liu H, Pang B, Zhao Y, Lu J, Han Y, Wang H. Comparative study of two different alkali-mechanical pretreatments of corn stover for bioethanol production. Fuel 2018;221:21–7. [20] Bajpai P. Properties, composition, and analysis of black liquor. In: Bajpai P, editor. Pulp and paper industry: chemical recovery. Elsevier Science; 2017. p. 25–38. [21] Cardoso M, de Oliveira ÉD, Passos ML. Chemical composition and physical properties of black liquors and their effects on liquor recovery operation in Brazilian pulp mills. Fuel 2009;88(4):756–63. [22] Kleinert M, Barth T. Phenols from lignin. Chem Eng Technol 2008;31(5):736–45. [23] Zacchi G, Skoog K, Hahn-Hägerdal B. Economic evaluation of enzymatic hydrolysis of phenol-pretreated wheat straw. Biotechnol Bioeng 1988;32(4):460–6. [24] Xu J, Zhang X, Cheng JJ. Pretreatment of corn stover for sugar production with switchgrass-derived black liquor. Bioresour Technol 2012;111:255–60. [25] Li Y, Qi B, Luo J, Wan Y. Alkali recycling from rice straw hydrolyzate by ultrafiltration: fouling mechanism and pretreatment efficiency. Ind Eng Chem Res 2015;54(32):7925–32. [26] Wang W, Wang Q, Tan X, Qi W, Yu Q, Zhou G, et al. High conversion of sugarcane bagasse into monosaccharides based on sodium hydroxide pretreatment at low water consumption and wastewater generation. Bioresour Technol 2016;218:1230–6. [27] Rocha GJM, Nascimento VM, da Silva VFN, Corso DLS, Gonçalves AR. Contributing to the environmental sustainability of the second generation ethanol production: delignification of sugarcane bagasse with sodium hydroxide recycling. Ind Crops Prod 2014;59:63–8. [28] Chen X, Kuhn E, Nagle N, Nelson R, Tao L, Crawford N, et al. Recycling of dilute deacetylation black liquor to enable efficient recovery and reuse of spent chemicals and biomass pretreatment waste. Front Energy Res 2018;6:51. [29] Cha Y-L, Yang J, Seo S-i, An GH, Moon Y-H, You G-D, et al. Alkaline twin-screw extrusion pretreatment of Miscanthus with recycled black liquor at the pilot scale. Fuel 2016;164:322–8. [30] Alhammad A, Adewale P, Kuttiraja M, Christopher L. Enhancing enzyme-aided production of fermentable sugars from poplar pulp in the presence of non-ionic surfactants. Bioprocess Biosyst Eng 2018;41:1133–42. [31] da Costa Nogueira C, de Araújo Padilha CE, de Jesus AA, de Santana Souza DF, de Assis CF, de Sousa Junior FC, et al. Pressurized pretreatment and simultaneous saccharification and fermentation with in situ detoxification to increase bioethanol production from green coconut fibers. Ind Crops Prod 2019;130:259–66. [32] Zhuang X, Wang W, Yu Q, Qi W, Wang Q, Tan X, et al. Liquid hot water pretreatment of lignocellulosic biomass for bioethanol production accompanying with high valuable products. Bioresour Technol 2016;199:68–75. [33] Goshadrou A, Karimi K, Taherzadeh MJ. Bioethanol production from sweet sorghum bagasse by Mucor hiemalis. Ind Crops Prod 2011;34(1):1219–25. [34] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, et al. Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory (NREL) Analytical Procedures, NREL/TP-510-42618 2008. [35] Jung SJ, Kim SH, Chung IM. Comparison of lignin, cellulose, and hemicellulose contents for biofuels utilization among 4 types of lignocellulosic crops. Biomass Bioenergy 2015;83:322–7. [36] Kim SJ, Kim MY, Jeong SJ, Jang MS, Chung IM. Analysis of the biomass content of various Miscanthus genotypes for biofuel production in Korea. Ind Crops Prod

2012;38:46–9. [37] Rabemanolontsoa H, Saka S. Holocellulose determination in biomass. In: Yao T, editor. Zero-carbon energy Kyoto 2011. Tokyo: Springer; 2012. p. 135–40. [38] Chandra RP, Chu Q, Hu J, Zhong N, Lin M, Lee J-S, et al. The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis. Bioresour Technol 2016;199:135–41. [39] Goshadrou A, Karimi K, Lefsrud M. Characterization of ionic liquid pretreated aspen wood using semi-quantitative methods for ethanol production. Carbohydr Polym 2013;96(2):440–9. [40] Luo X, Zhu JY. Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses. Enzyme Microb Technol 2011;48(1):92–9. [41] Chandra R, Ewanick S, Hsieh C, Saddler JN. The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, Part 1: a modified Simons’ staining technique. Biotechnol Prog 2008;24(5):1178–85. [42] Meng X, Wells T, Sun Q, Huang F, Ragauskas A. Insights into the effect of dilute acid, hot water or alkaline pretreatment on the cellulose accessible surface area and the overall porosity of Populus. Green Chem 2015;17(8):4239–46. [43] Carrillo A, Colom X, Sunol JJ, Saurina J. Structural FTIR analysis and thermal characterisation of lyocell and viscose-type fibres. Eur Polym J 2004;40(9):2229–34. [44] Haque MA, Barman DN, Kang TH, Kim MK, Kim J, Kim H, et al. Effect of dilute alkali pretreatment on structural features and enhanced enzymatic hydrolysis of Miscanthus sinensis at boiling temperature with low residence time. Biosyst Eng 2013;114(3):294–305. [45] Adney B, Baker J. Measurement of cellulase activities. National Renewable Energy Laboratory, NREL/TP-510-42628 2008. [46] Dien BS, Ximenes EA, O’Bryan PJ, Moniruzzaman M, Li XL, Balan V, et al. Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers’ grains and their conversion to ethanol. Bioresour Technol 2008;99(12):5216–25. [47] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59(2):257–68. [48] Goshadrou A, Lefsrud M. Synergistic surfactant-assisted [EMIM]OAc pretreatment of lignocellulosic waste for enhanced cellulose accessibility to cellulase. Carbohydr Polym 2017;166:104–13. [49] Karimi K, Emtiazi G, Taherzadeh MJ. Production of ethanol and mycelial biomass from rice straw hemicellulose hydrolyzate by Mucor indicus. Process Biochem 2006;41(3):653–8. [50] Xu X, Wu P, Wang T, Yan L, Lin M, Chen C. Synergistic effects of surfactant-assisted biodegradation of wheat straw and production of polysaccharides by Inonotus obliquus under submerged fermentation. Bioresour Technol 2019;278:43–50. [51] Kumar R, Mago G, Balan V, Wyman CE. Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresour Technol 2009;100(17):3948–62. [52] Sathitsuksanoh N, Zhu ZG, Wi S, Zhang YHP. Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass. Biotechnol Bioeng 2011;108(3):521–9. [53] Esteghlalian AR, Bilodeau M, Mansfield SD, Saddler JN. Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes? Biotechnol Prog 2001;17(6):1049–54. [54] Karuna N, Zhang L, Walton JH, Couturier M, Oztop MH, Master ER, et al. The impact of alkali pretreatment and post-pretreatment conditioning on the surface properties of rice straw affecting cellulose accessibility to cellulases. Bioresour Technol 2014;167:232–40. [55] Li H, Pu Y, Kumar R, Ragauskas AJ, Wyman CE. Investigation of lignin deposition on cellulose during hydrothermal pretreatment, its effect on cellulose hydrolysis, and underlying mechanisms. Biotechnol Bioeng 2014;111. [56] Yang Q, Pan X. Correlation between lignin physicochemical properties and inhibition to enzymatic hydrolysis of cellulose. Biotechnol Bioeng 2016;113(6):1213–24. [57] Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, et al. Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science 2011;333(6047):1279–82. [58] Qing Q, Yang B, Wyman CE. Impact of surfactants on pretreatment of corn stover. Bioresour Technol 2010;101(15):5941–51. [59] Eriksson T, Börjesson J, Tjerneld F. Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb Technol 2002;31(3):353–64. [60] Liu H, Sun J, Leu SY, Chen S. Toward a fundamental understanding of cellulaselignin interactions in the whole slurry enzymatic saccharification process. Biofuels Bioprod Biorefin 2016;10(5):648–63. [61] Vermaas JV, Petridis L, Qi X, Schulz R, Lindner B, Smith J. Mechanism of lignin inhibition of enzymatic biomass deconstruction. Biotechnol Biofuels 2015;8(1):217. [62] Saini JK, Patel AK, Adsul M, Singhania RR. Cellulase adsorption on lignin: a roadblock for economic hydrolysis of biomass. Renewable Energy 2016;98:29–42. [63] Ooshima H, Sakata M, Harano Y. Enhancement of enzymatic hydrolysis of cellulose by surfactant. Biotechnol Bioeng 1986;28(11):1727–34. [64] Yu Z, Gwak KS, Treasure T, Jameel H, Chang HM, Park S. Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass. ChemSusChem 2014;7(7):1942–50. [65] Yan Z, Li J, Chang S, Cui T, Jiang Y, Yu M, et al. Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse. Fuel 2015;158:152–8. [66] Jin W, Chen L, Hu M, Sun D, Li A, Li Y, et al. Tween-80 is effective for enhancing steam-exploded biomass enzymatic saccharification and ethanol production by specifically lessening cellulase absorption with lignin in common reed. Appl Energy 2016;175:82–90.

10