Enhanced ethanol production by Saccharomyces cerevisiae fermentation post acidic and alkali chemical pretreatments of cotton stalk lignocellulose

International Biodeterioration & Biodegradation 147 (2020) 104869

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Enhanced ethanol production by Saccharomyces cerevisiae fermentation post acidic and alkali chemical pretreatments of cotton stalk lignocellulose

T

Kamran Malika,b, El-Sayed Salamaa,∗, Tae Hyun Kimc, Xiangkai Lib,∗∗ a

Department of Occupational and Environmental Health, School of Public Health, Lanzhou University, Lanzhou, 730000, Gansu Province, PR China MOE, Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, 730000, Gansu Province, PR China c Department of Materials Science and Chemical Engineering, Hanyang University, Ansan, Gyeonggi-do, 15588, South Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lignocellulose Cotton stalk Pretreatment Enzymatic saccharification Fermentation Bioethanol

Direct conversion of lignocellulose to biofuel without pretreatment always results in a low-ethanol yield owing to its highly rigid structure. The current study was performed to improve the effectiveness of two different chemical pretreatments, alkaline and acidic, prior to the enzymatic hydrolysis of cotton stalk, and the yeast fermentation process for ethanol production. Alkaline pretreatments used alkaline hydrogen peroxide (AHP) and sodium hydroxide (NaOH), while acidic pretreatments used sulfuric acid (H2SO4) and phosphoric acid (H3PO4) at concentrations of 1.0%, 3.0%, 5.0%, and 7.0%. The highest bioethanol production (3.956 g/L) was observed in the 1.0% AHP pretreated sample, while the reducing sugar yield after enzymatic hydrolysis was 178 mg/g. The highest reducing sugar yield after enzymatic hydrolysis was obtained from the samples pretreated with 7.0% NaOH and 7.0% AHP, which yielded 241 mg/g and 238 mg/g, respectively, and provided 3.798 g/L and 3.739 g/L of ethanol, respectively. Scanning electron microscopy and Fourier-transform infrared analyses of the biomass showed that the structure of the alkali-treated cotton stalk was more disrupted and distorted than the acid-treated cotton stalk. Therefore, alkaline chemical pretreatment is more effective for breaking down lignocellulose and enhancing the yield of reducing sugars and bioethanol production from cotton stalk.

1. Introduction Lignocellulosic biomass attracts increasing amounts of attention due to its abundance, affordability, and renewability (Bhatia et al., 2017; Schmetz et al., 2016). In addition, as they are inedible by humans, they can be used as a significant source for biofuel production (Kawaguchi et al., 2016). Lignocellulosic biomass is comprised of three primary constituents, cellulose, hemicellulose, and lignin, which firmly bond to each other, forming a rigid structure matrix. Consequently, the deconstruction of these constituents is difficult (Chen et al., 2017; Zabed et al., 2017). Conversion processes of lignocellulosic biomass to ethanol involves four major sequential steps: pretreatment, saccharification, fermentation, and product separation. In this conversion process, pretreatment is an essential step because it disrupts the outer cell wall of plants and makes the plant structure more susceptible to enzymatic hydrolysis (Zabed et al., 2017). In the second step, the pretreated biomass is converted into fermentable sugars using either acids or commercial enzymes. In the fermentation step, yeasts, bacteria, or fungal strains are employed to ferment the sugars obtained from hydrolysis

∗

into ethanol. The final product stream produced from the fermentation process is a mixture of water and ethanol, which needs to be distilled in order to retrieve and purify the ethanol (Guragain et al., 2014; Zhang et al., 2016a). An efficient pretreatment approach should enhance the availability of fermentable sugars, prevent the formation of inhibitors, and should be inexpensive (Schmetz et al., 2016). Pretreatment approaches can be classified as chemical, physical, physicochemical, biological, or a combination of them (Paudel et al., 2017; Qiao et al., 2016; Solarte-Toro et al., 2019). Chemical and physicochemical pretreatment approaches are often used for treating lignocellulosic biomass because of their distinct destructive action on the cell wall structure. Different pretreatments have distinctive outcomes; for example, dilute acid pretreatment hydrolyzes hemicellulose components, while alkaline pretreatments categorically target lignin removal from cell wall (Qiao et al., 2016; Zhang et al., 2016b). Acidic and alkaline pretreatment methods are found to be efficient in opening lignocellulosic structures (Dai et al., 2018; Sun et al., 2019). Generally, acids are considered to hydrolyze hemicellulose effectively in lignocellulosic biomass because the hydrogen ions (H+) in the acid

Corresponding author. Corresponding author. E-mail addresses: [email protected] (E.-S. Salama), [email protected] (X. Li).

∗∗

https://doi.org/10.1016/j.ibiod.2019.104869 Received 26 October 2019; Received in revised form 21 November 2019; Accepted 28 November 2019 0964-8305/ © 2019 Elsevier Ltd. All rights reserved.

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was neutralized. The pretreated aqueous solution was transferred into falcon tubes, and the solid substrates were collected and dried in an oven for 2 h at 60 °C. The dried substrate was sealed and stored in airtight bags (total weight 112 g) for the enzymatic hydrolysis test and further analysis.

solution break chemical bonds inside hemicellulose as well as between cellulose and hemicellulose, resulting in the release of xylose (Si et al., 2015; Yan et al., 2017). The acid hydrolysis of hemicellulose produces cellulose-rich substrates that are then effectively hydrolyzed into glucose by hydrolysis enzymes (Wang et al., 2015; Zhang et al., 2016a). First-generation feedstock for ethanol production includes crops with a high-sucrose content (sugar cane, sugar beet, and sweet sorghum) and crops with a high-starch content (corn, wheat, rice, potato, barley, and sweet potato). First-generation feedstocks are mostly edible crops, and there is a serious conflict between food production and fuel production. Second-generation feedstock, which comprises lignocellulosic biomass (wood, straw, and grasses), has no conflict with food production. Third-generation feedstock typically includes algae (Wong et al., 2017). Algal biomass has been attractive for biofuel production due to its high lipid content; however, it still has many issues, such as low biomass production yield and a high cost of experiment. Cotton is a vital economic crop in China, which has an output accounting for 25% of the total global cotton production. Cultivation results in the waste of cotton stalk (CS) amounting to 40 million tons annually (Du et al., 2013). The CS should be removed after cultivating the cotton because not only is it an environmental pollutant but also the cause of many cotton diseases, that can affect subsequent cultivation (Egbuta et al., 2017). CS can be used for biofuel production, as it is costeffective and contains abundant sugars, which can be converted into bioethanol (Keshav et al., 2016). Previous studies have discussed the effectiveness of pretreatment of CS for increasing the ethanol concentration to a satisfactory level in fermenters (Liu et al., 2019). Therefore, pretreatment methods that achieve high ethanol concentrations in the fermentation step of CS are still necessary. In this study, two different chemical pretreatments (alkaline and acidic) were performed prior to the enzymatic hydrolysis of CS, followed by yeast (Saccharomyces cerevisiae) fermentation for ethanol production. The soluble organic concentration, consumption of total reducing sugar, and ethanol production were analyzed. Spectroscopic analyses using scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) were also conducted to evaluate the chemical and structural changes of CS after chemical pretreatments.

2.3. Spectroscopic analysis The structural morphology of the CS biomass was examined using SEM. All samples, including untreated and pretreated, were fixed on adhesive tape and coated with gold-palladium particles in order to obtain clear images. The SEM images were visualized at 2000× magnification, 50-μm diameter, and 5.00 kV voltage using an Apera S SEM (Thermo Fisher Scientific Inc., Waltham, MA, USA). FTIR analysis was conducted following the procedure described by Du et al. (2013) using a Nicolet spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The FTIR spectra of the samples were in the range of 4000–500 cm−1. Compositional analysis of the pretreated and raw biomass was performed using the method reported by Omar et al. (2011). The soluble organic concentration of the samples (liquid fraction) was measured by using a standard solutions kit according to the manufacturers’ instructions (LianHua Tech, Foshan, Guangdong, China). Briefly, 2.5 mL of soluble sample was added to a glass tube, followed by the addition of two digestion solutions (provided with kit). The tube was then thoroughly mixed using a vortex. Samples were placed in a digester at 165 °C for 10 min. After digestion, the samples were aircooled for 2 min, and 2.5 mL of deionized water was added. The samples were cooled in water for 2 min before being analyzed in the spectrophotometer (Model: 5B–3B (V8), LianHua Tech, Foshan, Guangdong, China) at 480 nm. 2.4. Enzymatic saccharification/hydrolysis A commercial cellulase enzyme from Trichoderma reesei (Shanghai Boao Biotech. Corp., Shanghai, China, ≥ 700 units) was used for the enzymatic saccharification in this study. Sodium acetate buffer (50 mM) was prepared with its pH adjusted to 5.0 by acetic acid titration. Two grams of cotton substrate was added to a 100 mL buffer solution at a concentration of 2.0% (w/v). The cellulase enzyme (total enzyme activity of 60 FPU/g-Filter Paper Unit) was added at a quantity of 30 FPU/g-solid substrate (Wang et al., 2016). Sodium azide (0.65 g/ L) was used as an antibiotic to prevent microbial contamination. The mixture was incubated at 50 °C for 72 h with a circular rotation of 120 rpm. A 3.0 mL sample was extracted and centrifuged at 5500×g for 10 min. The supernatant was collected for reducing sugar analysis, and the debris was stored for compositional analysis. All experiments were conducted in triplicate. The reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) method (Hu et al., 2008; Miller, 1959). The results were calculated using the following equation (1) (Wang et al., 2016).

2. Materials and methods 2.1. Biomass and reagents An agricultural farm from Gansu province, China, provided the CS (Gossypium hirsutum), which was grown and harvested in 2017. The raw biomass was air-dried, cut into 6 cm long pieces, and powdered using a high-speed grinder. The ground biomass was then stored in sealed, airtight polythene bags (total weight 192 g) for further experiments. The reagents, alkaline hydrogen peroxide (H2O2), sodium hydroxide (NaOH), sulfuric acid (H2SO4), phosphoric acid (H3PO4), and sodium azide were purchased from Sigma Aldrich (China) and Macklin (Shanghai Macklin Biochemical Co., Ltd). Cellulase was obtained from Shanghai Boao Biotech. Corp. China (≥700 units).

Reducing sugar yield (mg/g dry mass) = (ρ × V) ⁄ m

(1)

where ρ represents the reducing sugar concentration (mg/mL) in the hydrolyzed sample, V represents the total sample volume (mL); m represents the initial dry weight (g) of untreated or pretreated CS.

2.2. Pretreatment conditions Pretreatment was conducted in this study with both alkalis (H2O2 and NaOH) and acids (H2SO4 and H3PO4) at concentrations of 1.0%, 3.0%, 5.0%, and 7.0% (%, v/v). To make H2O2 (commercial 30%) alkaline, it was titrated to pH 11.5 ( ± 0.27) with 5 M NaOH as described in a previous study (Banerjee et al., 2011). Four grams of CS was added into 100 mL of each chemical solution at a solid loading of 4% (w/v). The bottles were kept at room temperature for 72 h and shaken manually after equal intervals. The samples pretreated with reagents were filtered and repeatedly washed with distilled water until the pH

2.5. Fermentation process 2.5.1. Microbial growth conditions Saccharomyces cerevisiae (baker's yeast, ATCC, 204508) was used in the fermentation experiment. For the preparation of the inoculum, yeast cells were cultivated in 50 mL growth media containing 0.2 g yeast extract, 1.5 g glucose, and 0.1 g ammonium sulfate. The culture medium was incubated at 35 °C for 12 h in an incubator shaker (MQL2

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61R, YK instruments, Shanghai, China) at 120 rpm to activate the microbial culture. The fermentation test began with a low inoculum concentration of 0.25 g/L of yeast cells, as described in a previous study (Guerrero et al., 2018). 2.5.2. Fermentation design and conditions In this study, a separate hydrolysis and fermentation (SHF) design was adopted because it is a simpler method compared to other processes, such as simultaneous saccharification and fermentation. In SHF, enzymes hydrolyze solid residues into fermentable sugars, and then the yeast ferments the sugars to bioethanol in the following step (Gupta and Verma, 2015). The experiment was performed in a 250 mL flask reactor, containing a working volume of 100 mL. The pH of the fermentation media was adjusted to 5.0. No chemicals or mineral salts were added as supplements to the fermentation media. When the yeast inoculum was added to the medium, the screw caps of the bottles were replaced with rubber stoppers. The flasks were incubated in an incubator shaker at 35 °C. The thick rubber stoppers had two hollow glass rods; one was used for CO2 release, and the other used to withdraw a sample from the medium. Each reactor was flushed with nitrogen for 6 min to create an anaerobic environment. Samples were withdrawn at time intervals of 12, 24, 36, 48, 60, and 72 h to measure pH variation, reducing sugar consumption, and ethanol yield. For ethanol quantification, aliquots were centrifuged for 10 min at 12000 g followed by filtration using 0.45-μm filters (CAT: BS-PES-22, Biosharp, China). GCMS (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a capillary column (T-05, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for ethanol analysis. Nitrogen was used as a carrier gas at a flow rate of 1.42 mL/min. The temperature was maintained at 40 °C for 1 min, then increased to 250 °C at a rate of 15 °C/min. The injection volume of the samples was 2.0 μL. Ethanol standards were prepared in ethyl acetate at 0, 5x, 10x, 20x, 50x, and 100x.

Fig. 1. Structural characterization of CS pretreated with 3.0% AHP, 3.0% NaOH, 1.0% H2SO4, and 5.0% H3PO4.

concentration indicates that NaOH pretreatment can degrade a high quantity of lignin (Zhang et al., 2018). The 3.0% AHP treatment yielded a concentration of 6.47 g/L, which implies that AHP can also be a favorable choice for lignin degradation and enzymatic sugar release (Saha and Cotta, 2010). Soluble organic concentration was measured because lignin solubilization and degradability in the pretreatment liquor can be easily deduced using this measurement (Liu et al., 2015). The highest soluble organic concentration among the acid pretreatment liquors was 5.42 g/L in the 5.0% H3PO4 sample. The major function of lignin is to support plant structure against the permeation of microbes and deterioration, and usually, amorphous heteropolymers are insoluble in water (Hendriks and Zeeman, 2009). However, Li et al. (2016) reported that alkaline pretreatment is the most effective technique for non-cellulosic polymer removal. 3.2. Scanning electron microscopy

3. Results and discussion Scanning electron microscopy (SEM) of CS showed that the raw biomass (Fig. 2, left, a) contained a packed, rough, and non-uniform external structure. The external layer of the CS was mainly composed of lignin, ash, and hemicellulose that enfolded the inner structural cellulose fibers (Reddy and Yang, 2009). In comparison with untreated CS biomass, some apparent alterations were observed in the outer structure of pretreated CS. With regards to AHP (Fig. 2, left, b), the external surface of the cellulose fibers had hollow portions and exhibited more layering and scaling in the structure. This pretreatment not only increased the removal of lignin but also enhanced hemicellulose degradation (Zhang et al., 2008). The NaOH pretreated samples (Fig. 2, left, c) indicated clear abrasions, cracks, holes, and erosion on the cellulose surface compared to other pretreatments. The increased breakdown within the cell structure might be associated with the highalkaline effect on the CS biomass. Sulfuric acid-treated CS (Fig. 2, left, d) showed clear abrasion and split fibers; also, few layering and scaling were exhibited. The possible cause for this disruption may be the partial hemicellulose degradation after sulfuric acid pretreatment. Phosphoric acid pretreatment also caused a disruption in the cell structure of the substrate. The cell structure was significantly disturbed and showed clear scratches and cuts. The disintegration of the structure is shown in Fig. 2, left, e. After enzymatic hydrolysis, the SEM images of the biomass showed increased structural degradation and biodeterioration because the chemicals, as well as the cellulase enzyme, both contributed to the structural breakdown (Fig. 2, right).

3.1. Characterization of substrates before and after chemical pretreatment The atomic composition of raw cotton stalk (CS) biomass is presented in Table S1. The composition of the raw biomass shows that CS has sufficient carbohydrate content (including cellulose and hemicellulose) for ethanol production. In addition to the chemical constituents, there are more factors affecting the enzymatic digestibility of biomass, such as particle size, as well as lignin and cellulose content. Pretreatment was used to increase the digestibility of the biomass, disrupt hemicellulose, and alter the lignin structure, which generally results in an increased availability of cellulose for enzymatic hydrolysis. With regard to the pretreatment of agricultural wastes, alkaline pretreatment has been proven more effective than acidic pretreatment (Kaur et al., 2012). Lignin content in alkali pretreated CS (AHP and NaOH) decreased compared to untreated and acid pretreated biomass (Fig. 1). The lignin removal of alkali-treated CS samples was observed to be 18.14% and 19.46% in 3.0% AHP and 3.0% NaOH pretreatment, respectively (Fig. 1). The acidic pretreatments dissolved more hemicellulose than the alkaline pretreatments (15.81% in 1.0% H2SO4 and 16.02% in 5.0% H3PO4). The residual cellulose content after alkaline pretreatment was higher (54.5% in 3.0% AHP and 53.9% in 3.0% NaOH), while the cellulose content remained at 50.3% and 51.9% in the acid pretreated CS using sulfuric and phosphoric acid, respectively. The probable reason for the increase in lignin was due to carbohydrate loss, mainly from hemicellulose reduction during acid pretreatment. The development of pseudo-lignin during the dehydration of carbohydrates could also be a cause of this increase (Poulomi et al., 2011). The soluble organic concentration of the pretreated biomass is shown in Table 1. The biomass pretreated with 3.0% NaOH had the highest soluble organic concentration (17.20 g/L). This high

3.3. Fourier-transform infrared analysis FTIR spectra of the native and pretreated CS are shown in Fig. 3a and b. The position of the absorption peaks was allocated to chemical components according to the related reported literature (Pandiyan et al., 2014). The FTIR spectra showed few bands between 1740 and 3

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Table 1 Soluble organic concentrations after alkaline and acidic pretreatments of CS. Pretreatment

1.0% 3.0% 5.0% 7.0% 1.0% 3.0% 5.0% 7.0% 1.0% 3.0% 5.0% 7.0% 1.0% 3.0% 5.0% 7.0%

AHP AHP AHP AHP NaOH NaOH NaOH NaOH H2SO4 H2SO4 H2SO4 H2SO4 H3PO4 H3PO4 H3PO4 H3PO4

0h

1.09 ± 0.1 0.92 ± 0.2 1.05 ± 0.1 1.00 ± 0.2 1.15 ± 0.1 1.25 ± 0.1 1.10 ± 0.1 1.20 ± 0.1 0.31 ± 0.2 0.53 ± 0.2 0.37 ± 0.2 0.59 ± 0.2 0.22 ± 0.1 0.45 ± 0.1 0.57 ± 0.2 0.53 ± 0.1

Concentration (g/L) 12 h

24 h

1.47 ± 0.5 1.59 ± 0.4 1.72 ± 0.6 1.67 ± 0.8 2.38 ± 0.9 2.22 ± 0.4 2.21 ± 0.3 2.33 ± 0.8 0.9 ± 0.3 0.81 ± 0.4 0.70 ± 0.8 0.66 ± 0.6 0.53 ± 0.4 0.97 ± 0.4 1.50 ± 0.8 1.29 ± 0.6

2.78 3.14 3.34 3.39 7.53 6.72 6.48 7.21 1.99 1.91 1.55 1.48 1.35 2.05 3.23 2.40

36 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.7 0.8 0.9 1.8 1.4 1.6 1.7 0.8 0.9 0.5 0.7 0.1 0.8 0.9 0.6

3.07 3.61 3.41 3.49 8.61 9.72 7.63 9.20 2.49 2.51 2.35 2.13 1.55 2.31 3.55 2.71

580 cm−1, which contained information about lignin. In contrast to the untreated CS biomass, the band absorptions (1740, 1640, 1040, and 623 cm−1) (Chung et al., 2004; Sun et al., 2015) in these regions were decreased in the pretreated CS, representing lignin removal after chemical pretreatment. The broad band at 3420-3340 cm−1 represents intramolecular and intermolecular hydrogen bonding. While, the peak at 2940 cm−1 with adjacent bands indicated aliphatic CH2 (Li et al., 2015). A major decrease in intensity of the 897 cm−1 band corresponding to β-D-cellulose linkages, which is related to amorphous cellulose removal, especially in hemicelluloses, and the band at 1740 cm−1, was detected after chemical pretreatment and enzymatic saccharification (Fig. 3a and b). These results confirm the loss of β-D-cellulose linkages and the clear removal of hemicellulose, both of which have a strong aqueous affinity. The breaking down of β-D-cellulose linkages initiated by cellulose degradation resulted in the susceptibility of cellulose to the cellulase enzyme, thus increasing enzymatic hydrolysis. The samples after hydrolysis did not contain the band at 897 cm−1, which confirmed the disintegration of cellulose during enzymatic hydrolysis. The band at 1040 cm−1 was ascribed to C–O extensions from guaiacyl-type lignin, hemicellulose, or cellulose (Rodríguez-Abalde et al., 2013). The decreased band at 1040 cm−1 in the pretreated CS biomass samples also indicated the breakdown of xylan (hemicellulose) in the chemical pretreatment samples. The sharp bands at 2350–2170 cm−1 indicated C]O bonds in ketone groups. The low absorption of bands at around 2350 cm−1 might be because of a slight removal of hemicellulose. In the reported literature, the bands at 2800–3000 cm−1 were associated with CH stretching, and the bands at 3550–3100 cm−1 were assigned to the hydrogen-bonding OH stretching of cellulose. The peaks at 2900 and 3370 cm−1 after all the pretreatments were considerably reduced, which indicated the breaking down of intermolecular hydrogen bonding between cellulose and hemicellulose.

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2 0.8 0.9 0.8 1.9 1.9 1.2 1.9 1.4 1.2 0.9 1.3 1.2 0.8 0.7 0.9

48 h

60 h

72 h

3.62 ± 1.4 4.48 ± 1.2 4.15 ± 1.4 4.38 ± 0.9 10.58 ± 1.7 13.38 ± 2.1 10.72 ± 1.8 12.20 ± 1.9 3.26 ± 1.5 3.07 ± 1.7 2.80 ± 1.3 2.33 ± 1.3 1.85 ± 0.9 2.80 ± 1.3 4.45 ± 1.3 3.35 ± 1.2

4.19 ± 0.9 5.32 ± 1.5 5.08 ± 1.3 5.31 ± 1.5 11.43 ± 2.1 15.92 ± 2.4 12.38 ± 1.8 14.20 ± 1.9 4.06 ± 1.5 3.55 ± 0.9 3.15 ± 1.3 2.78 ± 1.4 2.01 ± 1.1 3.16 ± 1.4 5.15 ± 1.7 3.85 ± 1.1

4.66 ± 1.6 6.47 ± 1.3 5.52 ± 1.1 6.15 ± 1.4 12.24 ± 1.9 17.20 ± 2.5 13.38 ± 2.1 15.22 ± 2.2 4.43 ± 1.1 3.76 ± 0.9 3.50 ± 1.2 3.03 ± 1.1 2.46 ± 0.8 3.63 ± 0.9 5.42 ± 1.6 4.66 ± 1.5

NaOH showed the highest reducing sugar yield as compared to the untreated biomass, followed by the 7.0% AHP sample (Fig. 4a and b). The reducing sugars released by acidic pretreatment (H2SO4 and H3PO4) notably reduced hemicellulose from the biomass but displayed lower reducing sugar yields in comparison with alkaline pretreatments (Fig. 4c and d). These results corresponded well with those reported in previous studies (Silverstein et al., 2007). The reducing sugar content after microbial pretreatment as reported by Shi et al. (2009) was only 10–55.6 mg/g, indicating that the reducing sugar content in the current study is high. The alkali pretreatment dissolved lignin and hemicellulose and caused de-esterification of their cross linkages. During alkali pretreatment, the dissociation of hydroxide ions (OH−) occurred, and the high number of (OH−) ions increased the hydrolysis rate; thus, more fermentable sugars were produced. In the current experiment, alkaline pretreatment produced more sugars as compared to acidic pretreatment, which is in accordance with the previous studies (Wang et al., 2016). 3.5. Reducing sugar consumption and ethanol fermentation after chemical pretreatments The total reducing sugars (TRS) of CS pretreated with 1–7% AHP and NaOH during fermentation resulted in enhanced TRS from ~200 to > 500 mg/g after 12 h of fermentation (Fig. 5). The consumption of reducing sugars decreased gradually as fermentation continued. The increased reducing sugar content from low initial concentrations to high concentrations may be attributed to the degradation of polysaccharides into mono- and disaccharides during fermentation. The same trend in total reducing sugars was reported by Cruz et al. (2018) during alcoholic fermentation. The highest TRS at 12 h was observed in the 1.0% AHP and 1.0% NaOH pretreated samples, which was higher than the control by 2.74 and 2.54 folds, respectively (Fig. 5a and b). The TRS concentration decreased with the time, and at the end of the fermentation step (72 h), it was reduced to 12.85 mg/g in the case of CS pretreated with 1.0% AHP (Fig. 5c and d). The bioethanol concentration in the 1.0% AHP sample, after 48 h, reached its maximum (3.956 g/L). TRS in the 3.0% AHP and 3.0% NaOH samples were increased by 1.39 and 2.34 folds, respectively, compared to the CS control and then decreased to 77.94 mg/g after 72 h of fermentation in the 3.0% AHP sample. The final ethanol concentration of the 3.0% AHP pretreated and 3.0% NaOH pretreated CS was 2.995 and 3.252 g/L, respectively. The TRS of the 5.0% AHP and NaOH samples were 1.62 and 1.84 folds at 12 h, compared to the CS control, and then decreased after 72 h of fermentation. The ethanol concentration was 3.23 g/L in the 5.0% AHP sample and 3.14 g/L in the 5.0% NaOH sample. The

3.4. Comparison of pretreatments and their effect on enzymatic saccharification During the enzymatic hydrolysis phase of separate hydrolysis and fermentation (SHF), the concentration of glucose increased steadily and reached a maximum concentration at 72 h (241 mg/g in 7.0% NaOH). At this stage of the reaction, the hydrolysis medium was inoculated by activated yeast. The solids in the medium were not removed from the medium because microorganisms were added at the end of the enzymatic hydrolysis (Guerrero et al., 2018). The total reducing sugars released after enzymatic saccharification of native CS was significantly lower than the pretreated biomass samples. CS pretreated with 7.0% 4

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Fig. 2. SEM graphs of untreated and pretreated CS biomass before hydrolysis (left) and after hydrolysis (right): (a) Native cotton stalk; CS pretreated with: (b) 3.0% AHP; (c) 3.0% NaOH; (d) 1.0% H2SO4; (e) 5.0% H3PO4. All images were obtained at the same scale bar (50 μm).

5

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Fig. 3. FTIR spectra of raw CS and CS pretreated with 3.0% AHP, 3.0% NaOH, 1.0% H2SO4 and 5.0% H3PO4: (a) before hydrolysis; (b) after hydrolysis.

Fig. 4. Reducing sugars after enzymatic hydrolysis by different pretreatment conditions: (a) AHP; (b) NaOH; (c) H2SO4; (d) H3PO4.

reducing sugars released from the 7.0% AHP and NaOH treated CS during fermentation at 12 h were 1.03 and 2.01 folds than that of CS control, respectively, and steadily decreased after 72 h. However, the ethanol concentration in the 7.0% AHP and 7.0% NaOH samples was at 3.739 and 3.798 g/L, respectively, at the end of the fermentation test. TRS and bioethanol production in the acidic pretreated 1.0% H2SO4 sample was increased by 1.19 compared to the control, while the 1.0%

H3PO4 sample was decreased compared to the control by 1.12 folds. The total reducing sugars (glucose) were effectively consumed during fermentation and remained at 12.85 mg/g (Fig. 5c and d). The total reducing sugar content in the acidic pretreatment of 1.0% H2SO4 and H3PO4 were ranged from 1.2 to 0.9 folds compared to the control at 12 h, and the ethanol concentration was 2.48 and 3.21 g/L, respectively. TRS in the 3.0% H2SO4 and H3PO4 samples were 0.95 and 1.25 6

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Fig. 5. Total Reducing sugars during fermentation by different pretreatment conditions: (a) AHP; (b) NaOH; (c) H2SO4; (d) H3PO4.

saccharification, an improvement to fermentation strategies in order to utilize hemicellulose is also required from further studies.

folds compared to the control at 12 h of fermentation and reduced to 34.36 mg/g in the case of H2SO4. From the acidic pretreatments, the highest ethanol concentration obtained from the SHF test was 3.429 g/L from the 7.0% H3PO4 pretreated sample. TRS in the 5.0% H2SO4 and H3PO4 samples at 12 h were 1.3 fold compared to the control with an ethanol concentration of 2.64 g/L in H2SO4-treated CS and 2.95 g/L in H3PO4-treated CS. TRS content in the 7.0% H2SO4 and H3PO4 pretreated samples were 1.49 and 1.74 folds compared to control, and the bioethanol concentrations were 2.66 and 3.39 g/L, respectively. The maximum ethanol production in the 5.0% H3PO4 pretreatment sample remained at 3.429 g/L. The initial pH of all the reactors was set at 5.0, as it is reported to be the best pH for successful fermentation (Gao et al., 2018). During the fermentation process, the pH dropped to 4.0 after 24 h and then gradually increased after 48 h until the end of the fermentation experiment. CO2 was produced in the initial 24 h; thereafter, little or no CO2 was observed in the reactor due to the low suspension pH. The TRS concentration in the alkaline pretreatment (549.04 mg/g) at 12 h was the highest among the pretreatments. The TRS progressively decreased in all the pretreatments, and finally, at 72 h of fermentation, the minimum sugars concentration was recorded as 12.85 mg/g in both the acidic and alkaline pretreatments. Various lignocellulosic biomass (including sugarcane bagasse, corn stover, CS, and cellulosic material) were used in the reported studies for ethanol production after chemical pretreatment (Table 2). The purpose of this study was to formulate potential pretreatment conditions for CS. Studies related to the consumption of fermentable sugars (pentose and hexose) in bioethanol production using CS are not enough; hence, in addition to enhancing pretreatment techniques and enzymatic

4. Conclusions Alkaline and acidic chemicals were used for the pretreatment of cotton stalk (CS) prior to enzymatic hydrolysis, and fermentation for ethanol production. SEM and FTIR analysis showed vast structural alterations in the pretreated CS compared to the untreated biomass. The soluble organic concentration after alkaline (3% NaOH) pretreatment of CS was increased by 13.7-folds, compared to control. The highest reducing sugar yield after enzymatic hydrolysis was obtained from the 5 and 7% NaOH pretreated samples, which showed an increase by 2-folds compared to the control. The reducing sugar content and ethanol production results confirm that alkaline pretreatment is an effective step in CS pretreatment. Declaration of competing interest No conflicts of Interest. Acknowledgments The present study was financed by Fundamental Research Funds for the Central Universities grant (No: lzujbky-2017-br01) and Gansu province major science and technology projects (No: 17ZD2WA017). National Natural Science Foundation of China Grant (No: 31870082). This research was also supported by the startup fund for the construction of the double first-class project (No. 561119201), Lanzhou 7

International Biodeterioration & Biodegradation 147 (2020) 104869

Wang et al. (2016)

This study

This study

Wirawan et al. (2012)

Carrillo-Nieves et al. (2017) Jin et al. (2012) Dimos et al. (2019)

Barrera et al. (2016)

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ibiod.2019.104869.

3.32 g/L

3.739 g/L

3.798 g/L

5.52 g/L

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Improving the bioenergy production from wheat straw with alkaline pretreatment. Biosyst. Eng. 140, 59–66. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Omar, R., Idris, A., Yunus, R., Khalid, K., Isma, M.A., 2011. Characterization of empty

Alkaline hydrogen peroxide

High pressure-assisted alkali pretreatment (HPAP) (3.0% NaOH, 121 °C, 130 kPa, 40 min).

Cotton stalk

Cotton stalk

CBP= Consolidated Bioprocessing, AFEX = Ammonia Fiber Expansion.

Sodium Hydroxide

Bagasse

Cotton stalk

Incubation 50 °C for 72 h, 120 rpm. Cellulase enzyme, 30 FPU/g Incubation 50 °C, 72 h, 120 rpm. Cellulase enzyme, 30 FPU/g Incubation 50 °C, 48 h, 120 rpm. Cellulase enzyme, 30 FPU/g

Z. mobilis ATCC 29191 immobilized in Ca-alginate and polyvinyl alcohol beads Saccharomyces cerevisiae, fermentation, 30 °C, 72 h, 150 rpm Saccharomyces cerevisiae, fermentation at 30 °C, 72 h, 150 rpm rotation Saccharomyces cerevisiae, fermentation at 30 °C, 48 h Accellerase enzyme

7 g/L 32.3 g/L CBP, Clostridium phytofermentans Dry yeast

AFEX commercial enzyme mixture Organo-solvent and hydrothermal treatment (175 °C, 2 h, N2) Phosphoric acid Corn stover Cotton stalk

24 h, 50 °C, 150 rpm, 15 FPU enzyme loading AFEX commercial enzyme mixture 20% solid loading 3.0% NaOH, 15 min, 120 °C, 21% solid loading

24 h, 30 °C, 150 rpm, S. cerevisiae Turbo strain

58 gallon/metric ton 48 gallon/metric ton 58.8% Ozonolysis

Blue Agava bagasse Sugarcane bagasse Mango bark residue

72 h, 28 °C, 1% yeast 5 h, 50 °C, 0.011 kg enzyme/kg bagasse

Pretreatment used

Enzymatic Hydrolysis

University, China.

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Substrate used

Table 2 Comparison of the results obtained in this study to those reported in previous studies.

Fermentation conditions

Ethanol concentration

Reference

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