Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization

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Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization Ganesh Dattatray Saratale a, Rijuta Ganesh Saratale b, Sang Hyoun Kim c, Gopalakrishnan Kumar d,* a

Department of Food Science and Biotechnology, Dongguk University-Seoul, Ilsandong-gu, Goyang-si, Gyeonggi-do, 10326, Republic of Korea b Research Institute of Biotechnology and Medical Converged Science, Dongguk University-Seoul, Ilsandong-gu, Goyang-si, Gyeonggi-do, 10326, Republic of Korea c School of Civil and Environmental Engineering, Yonsei University, Seoul 03722, Republic of Korea d Green Processing, Bioremediation and Alternative Energies Research Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam

article info

abstract

Article history:

This work evaluated the effects of individual alkaline, sodium carbonate (Na2CO3 denoted as;

Received 3 May 2017

NaC), sodium sulfide (Na2SO3 denoted as; NaS) and combination of NaC þ NaS pretreatment

Received in revised form

for the saccharification of sugarcane bagasse (SCB). The effects of different pretreatments on

13 November 2017

chemical composition and structural complexity of SCB in relation with its saccharification

Accepted 29 January 2018

were investigated. For enzymatic hydrolysis of pretreated SCB we have utilized the produced

Available online xxx

crude enzymes by Streptomyces sp. MDS to make the process more cost effective. A enzyme dose of 30 filter paperase (FPU) produced a maximum reducing sugar (RS) 592 mg/g with 80.2%

Keywords:

hydrolysis yield from NaC þ NaS pretreated SCB under optimized conditions. The resulted

Sugarcane bagasse

enzymatic hydrolysates of each pretreated SCB were applied for hydrogen production using

NaC þ NaS pretreatment

Clostridium beijerinckii KCTC1785. NaC þ NaS pretreated SCB hydrolysates exhibited

Enzymatic hydrolysis

maximum H2 production relative to other pretreatment methods. Effects of temperature,

Clostridium beijerinckii

initial pH of culture media and increasing NaC þ NaS pretreated SCB enzymatic hydrolysates

Biohydrogen production

concentration (2.5e15 g/L) on bioH2 production were investigated. Under the optimized conditions, the cumulative H2 production, H2 production rate, and H2 yield were 1485 mL/L, 61.87 mL/L/h and 1.24 mmol H2/mol of RS (0.733 mmol H2/g of SCB), respectively. The efficient conversion of the SCB hydrolysate to H2 without detoxification proves the viability of process for cost-effective hydrogen production. © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction The primary energy consumption in the world is documenting rapid increase in last five decades due to the growing world

population. Fossil fuel resources are widely used for the development of industrial sector to meet the growing energy demand of the population [1]. However continuous depletion of fossil fuels, their increasing price, environmental and

* Corresponding author. Green Processing, Bioremediation and Alternative Energies Research Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam. E-mail address: [email protected] (G. Kumar). https://doi.org/10.1016/j.ijhydene.2018.01.187 0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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ecological point of views have stimulated the reawakened interest in developing sustainable and renewable energy sources to lessen the reliance on fossil fuels [2,3]. Hydrogen is viewed as a sustainable energy carrier with high-energy yield (122 kJ g1) [4,5]. To reduce the production cost, it is preferable to produce biohydrogen from lignocellulosic (LC) biomass, which are the most abundant, less expensive, and renewable resource available in nature that do not compete with food crops. Thus LC biomass signifies a huge potential in terms of renewable energy and has drawn tremendous amount of attention for the conversion of LC biomass to biofuels [1,6]. Sugarcane bagasse (SCB), the fibrous residue acquired after extracting the juice from sugar cane (Saccharum officinarum) which is about 280 kg per ton of sugarcane. Worldwide approximately 5.4  108 dry tons of sugarcane is processed annually thus generating SCB as a valuable byproduct [7]. Generally, the resulted SCB (~50%) is used in distillery plants for energy production by combustion which causes environmental pollution [8]. There is a great interest to develop biological process for fuel and chemicals production from SCB which can offer economic and environmental advantages [7,8]. However, SCB are usually not readily fermentable by microorganisms thus, pretreatment steps are often required for its conversion into biofuels. The pretreatment is required for removing the lignin, to increase cellulose availability by changing the chemical composition, surface area, and porosity by which it improves the enzymatic susceptibility of biomass [9,10]. An effective and economical pretreatment should use inexpensive chemicals, simple procedures and which must preserve the utility of the hemicelluloses and should evade the formation of fermentation inhibitors [11,12]. Various physical, chemical, physicochemical and biological technologies have been explored for biomass pretreatment taking into account of enhancing the yields of fermentable sugars and subsequently biohydrogen production using dark fermentation strategies [2,6,8,13,14]. The enzymatic hydrolysis represents a more inexpensive and ecofriendly perspective for the release of fermentable sugars from biomass using a multi-component enzyme system. There are many advantages of this process including, low energy requirement, no corrosion issues, less byproduct formation, better yield and process can run under mild environmental conditions [14,15]. Saccharification of LC biomass can be done by directly using pure commercial enzymes which normally result in faster hydrolysis rate and higher sugar yield [16]. However, due to the high cost arising from enzymes production and purification the application of commercial enzymes for hydrolysis limits the entire process. Thus extensive attention for cost-effective and high-efficiency hydrolytic (cellulose and hemicellulose degrading) enzymes production and their utilization for the saccharification of LC biomass is required. This approach will overcome the above limiting issues and could make the lignocellulosic biohydrogen production more cost effective and practically applicable. In lignocellulosic biohydrogen production mainly two approaches are used. First one is two stage processes and the other known as simultaneous saccharification and fermentation. In case of two stage processes enzymatic hydrolysis and H2 fermentation were conducted separately. The hydrogen yield was reported to be lower in simultaneous saccharification

and fermentation as compared to two stage process under dark fermentation [17,18]. Some investigators utilized two stage process to enhance lignocellulosic biohydrogen production and thus the process becomes more economically feasible, less energy intensive and practically applicable [19e22]. In addition to this, microbial fermentation parameters including type of inoculum, type of substrate and operational and environmental factors (organic loading rate (OLR), initial pH, temperature, etc.) need to be optimized for better H2 production [23]. This study was aimed to explore the possibility of sugarcane bagasse as a feed stock for biohydrogen production. This paper focuses about the different physical, chemical and physicochemical pretreatment to prepare SCB feedstock. The pretreatment conditions for NaC þ NaS including chemical loading and their ratio, incubation time, and solid/liquid ratio were tested to govern the effectiveness of the pretreatment process. Moreover, the enzymatic saccharification of untreated and pretreated SCB was carried out through the produced crude enzymes by Streptomyces sp. MDS to make this process more cost effective and practically applicable. The optimization of saccharification parameters for maximum reducing sugar production were also evaluated. Then, the SCB hydrolysate was fermented by Clostridium beijerinckii KCTC 1785 to validate its potential as substrate for hydrogen production and also optimized the process parameters to enhance H2 yield. This work is anticipated to provide useful information for evaluating the feasibility of SCB as efficient feedstock for cellulosic biohydrogen production.

Material and methods Lignocellulosic substrates used Wheat straw was used for biomass hydrolyzing enzymes production under solid state fermentation and which was collected from the local farmers. Sugarcane bagasse was used for the pretreatment studies and was collected from local sugar industry, South Korea. The raw substrates were collected, then air dried, milled and sieved through a 0.2 mm and 0.5 mm screen before storing at room temperature.

Hydrolytic enzymes production under solid state fermentation Pure culture of isolated actinomycetes namely Streptomyces sp. MDS was used in this study and the detailed results of 16SrRNA sequencing and phylogentic analysis have been reported elsewhere [24]. Pure culture was maintained on Dubos salt medium (g/L): NaNO3, 0.5; K2HPO4, 1.0; MgSO4$7H2O, 0.5; KCl, 0.5; FeSO4$7H2O, 0.01; agar powder, 20; CMC (10 g/L); with pH 6.5, stored at 4  C and subcultured monthly. Solid state fermentation was carried out in 250 mL Erlenmeyer conical flasks containing 5 g of dry wheat straw moistened with Dubos salt medium by keeping the initial pH (5.0), at 30  C and moisture content ratio (1:3) for cellulase and hemicellulase production. For inoculation, 1 mL of spore suspension (absorbance ~1.0 at 600 nm) from 7 days old culture of Streptomyces sp. was used and prepared in sterile saline and further mixed well. After an appropriate time interval, the fermented substrate were aseptically

Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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removed and extracted the enzymes with McIlvaine's buffer (0.1 mol/L citric acid-0.2 mol/L phosphate buffer; pH 5) using the procedure reported earlier [15].

Enzyme assays Endoglucanase (CMCase), exoglucanase (avicelase), and xylanase activities were determined by following the procedures reported earlier [15,24]. Total cellulase activity (FPU) was assayed according to IUPAC recommendations using Whatman filter paper as the substrate [25]. Glucose and xylose standard curves were used to calculate cellulase and xylanase activities. One unit of enzyme activity was defined as the amount of enzyme required to release 1 mmol of reducing sugars per minute. Cellobiase activity by assaying the release of p-nitrophenol (pNP) was determined by using the procedure reported earlier [26]. One unit of cellobiase activity was defined as the amount of enzyme required to release 1 mmol of pNP per minute under the assay conditions. All enzyme assays were conducted in triplicate and the average rates were calculated to characterize the enzyme activity.

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used in further experiments. Two step enzymatic hydrolysis of untreated and chemically pretreated SCB biomass was performed at the biomass level of 2.0% (w/v) in 20 mL of 50 mM citrate buffer (pH 5.0) containing 0.005% (w/v) sodium azide with crude enzyme dosage of 30 IU filter paperase/g of untreated and pretreated SCB. The operational conditions for two step enzymatic hydrolysis were according to the procedure reported earlier [3]. We evaluated the effects of increasing substrate concentration (SCB: 5e25 g/L) while keeping the enzyme concentration constant (30 FPU/g of SCB) as well as the effects of increasing enzyme concentration (10e50 FPU/g of SCB) by keeping the SCB concentration (10 g/L) constant. The released reducing sugars, the overall hydrolysis yield, glucose yield and xylose yield after two steps of hydrolysis were calculated according to the following formulas [3,12]: Hydrolysis yield (%) ¼ reducing sugar (mg)  0.9  100 ÷ cellulose and hemicellulose content in the substrate (SCB) Glucose yield (%) ¼ glucose (mg)  0.9  100 ÷ cellulose content of substrate

Sugarcane bagasse and their pretreatment conditions Physical pretreatment i.e. auto-hydrolysis at neutral pH without any added chemicals which was considered as a control. In the preliminary investigation chemical pretreatment was carried out by subjecting SCB biomass to NaC and NaS with different concentration (1, 2, 3, 4 and 5% (w/v) individually as well as in combination) in an electrically heated water bath at 100  C for 6 h. The maximum hydrolysis yield and glucose yield of SCB was observed at 4% (w/v) NaC þ NaS concentration and thus selected this concentration to evaluate the maximum hydrolysis of SCB (data not shown). During the alkaline pretreatment, SCB biomass was soaked in 2% (w/ v) solutions of NaOH at 121  C for 30 min. The ratio of the solid phase to liquid phase in each pretreatment was maintained at 1:10. The treated biomass was thoroughly washed with tap water to remove impurities and further washed with distilled water, and dried at 60  C to reach a constant weight.

Optimization of NaC þ NaS pretreatment The effects of molar ratio of NaC þ NaS at each chemical charge (w/v) (1:0, 2:1, 1:1, 1:2, and 0:1) on hydrolysis of SCB was examined. Various process parameters including incubation temperature (60  C, 80  C, 100  C and at 121  C for 30 min), substrate concentration (4%, 8%, 10%, 12%, 14% and 16%), and chemical dosage (4%, 8%, 10%, 12%, 14% and 16%) by keeping incubation time constant (6 h) on hydrolysis of SCB were systematically investigated. During optimization studies “one variable at a time” approach was used. The pretreated residues were further washed to neutral pH and dried at 60  C till constant weight and further subjected to enzyme hydrolysis analyses.

Application of produced crude enzyme complex for the hydrolysis of untreated and pretreated SCB biomass The optimal temperature (at 50  C) and initial pH of the buffer (5.0) for better enzymatic hydrolysis were determined and was

The hydrolysis of polysaccharides involves water. For each mole of reducing sugar released, 1 mol of H2O is needed. The correction factor 0.9 was therefore included in the formula for the amount of polysaccharides hydrolyzed.

Biohydrogen production using pretreated SCB enzymatic hydrolysates by C. beijerinckii Clostridium beijerinckii KCTC 1785 was evaluated for dark hydrogen fermentation using SCB enzymatic hydrolysates. The medium for dark H2 fermentation containing untreated and chemically pretreated SCB enzymatic hydrolysates was (g/L): yeast extract, 4.0; NH4HCO3, 6.72; NaHCO3, 5.24; K2HPO4, 0.125; MgCl2$6H2O, 0.1; MnSO4$6H2O, 0.015; FeSO4$7H2O, 0.025; CuSO4$5H2O, 0.005; CoCl2$5H2O, 0.00012. Initially, we have evaluated the biohydrogen production performance by keeping pH of 6.5, temperature of 35  C and by taking enzymatic hydrolysates of different pretreated SCB containing 5.0 g/L reducing sugar (RS).

Optimization of environmental conditions for biohydrogen production To optimize the biohydrogen production initial pH of 5.5e7.5, incubation temperature of 28e40  C, by taking enzymatic hydrolysates of NaC þ NaS pretreated SCB containing 5.0 g/L reducing sugar (RS) was used. The 24 h old preculture and 10% (v/v) inoculum size was used to start the fermentation experiments. As a control experiment, biohydrogen fermentation was also conducted using autoclaved SCB without any chemical treatment. During the course of fermentation, cell concentration, pH, residual reducing sugar, production of bioH2 and soluble metabolites were monitored with respect to culture time. Moreover, the effects of increasing concentration of NaC þ NaS pretreated SCB hydrolysates RS (2.5e15 g/L) was also evaluated on biohydrogen production under optimized conditions.

Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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240

Time (h) Fig. 1 e Cellulase and hemicellulase production by Streptomyces sp. MDS using wheat straw (30  C, initial pH 5.0, moisture content 1:3) at different incubation period under solid state fermentation.

Analytical methods The presence of sugars (glucose, xylose, and arabinose) and soluble products (volatile fatty acids and ethanol) in fermented broth and the concentration of fermentation inhibitors such as furfural, hydroxymethyl furfural, acetic acid and formic acid in the hydrolysates after each chemical treatment were detected by high-performance liquid chromatography (HPLC) with refraction index detection (RID-10A, Shimadzu, Japan) in the filtered (0.2 mm) supernatant of culture broth during bioH2 production. The gas products (mainly H2 and CO2) was analyzed by gas chromatography (GC-17A, Shimadzu, Kyoto, Japan) using a thermal conductivity detector. The detailed procedures for HPLC and GC analyses were described in our previous work [21]. Cellulose, hemicellulose and lignin contents of SCB were estimated by the method of Goering and Van Soest, [27]. Reducing sugars (RS) content was determined by using the dinitrosalicylic acid (DNS) method [28]. FTIR spectroscopic (Agilent, Cary 630; USA) analysis of native and pretreated SCB were executed in the mid-IR region of 400e4000 cm1 with a resolution of 4 cm1 and 32 scan speed. SEM images of the particles coated with platinum were recorded using a JEOL JSM-6360A microscope with operating voltage 20 kV. Samples of the raw and pretreated SCB were analyzed by X-ray diffraction (XRD) using a D2 Phaser tabletop

43.5 48.9 54.1 49.2 ± 1.36 45.2 ± 1.45 55.6 ± 1.42 75.6 ± 2.78 78.2 ± 2.88 88.5 ± 3.10 60.5 ± 2.25 66.2 ± 2.44 70.5 ± 2.45

39.5 49.2 8.20 ± 0.36 43.7 ± 0.98

Xylose yield (%) Glucose yield (%)

15.4 ± 0.55 74.5 ± 2.44 12.2 ± 0.65 66.4 ± 2.36

CrI Hydrolysis yield (%)

Values are the mean of three experiments ± SEM. Statistics were determined by one-way ANOVA with TukeyeKramer Multiple Comparisons Test.

216

17.6 ± 0.28 16.3 ± 0.35 11.2 ± 0.25

192

25.3 ± 0.48 21.2 ± 0.65 18.4 ± 0.38

168

46.2 ± 0.68 48.4 ± 0.55 55.7 ± 0.68

144

60.5 ± 1.54 55.5 ± 2.12 54.5 ± 1.65

96

NaC NaS NaC þ NaS

72

Lignin

48

24.1 ± 0.58 16.2 ± 0.32

0

30.2 ± 0.45 22.4 ± 0.45

0

Hemicellulose

50

Cellulose

100

38.8 ± 0.65 50.2 ± 0.66

150

95.8 ± 4.36 45.5 ± 1.36

200

No pretreatment 2% (w/v) NaOH, 121  C, 30 min 4% (w/v) NaC 100  C, 6 h 4% (w/v) NaS 100  C, 6 h 4% (w/v) NaC þ 4% (w/v) NaS 100  C, 6 h

Endoglucanase Cellobiase Xylanase Amylase

Control Alkali

Enzyme actvity U/gds of wheat straw

0 250

After two step enzymatic hydrolysis

5

Biochemical composition (%)

10

Biomass recovery (%)

15

Pretreatment conditions

Exoglucanase FPU

20

Pretreatment

Enzyme actvity U/gds of wheat straw

25

Table 1 e Effect of individual and combined pretreatment of SCB on biomass recovery, biochemical composition and hydrolysis yield, glucose yield and xylose yield after two step enzymatic hydrolysis (enzyme dosage 30 FPU/g of SCB).

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Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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model (Bruker, Germany) operating at 30 kV and 10 mA using the procedure reported earlier [12]. The crystallinity index of cellulose was calculated according to the peak height method [26].

Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey-kramer multiple comparisons test. Readings were considered significant when P was 0.05.

Results and discussion Hydrolytic enzymes production by Streptomyces sp. MDS using wheat straw under solid state fermentation Lignocellulose-degrading enzymes production under solid state fermentation using wheat straw by isolated Streptomyces sp. MDS was evaluated. The optimal growth and significant production of cellulase and xylanase by Streptomyces sp. was

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observed at moisture ratio 1:3, at temperature 30  C, with initial pH set at 5. Under optimized conditions, the maximum expression of endoglucanase (105.4 U/gds), exoglucanase (20.85 U/gds), cellobiase (92.8 U/gds), Filter paperase (18.75 U/ gds), amylase (200.5 U/gds) and xylanase (218.2 U/gds) by Streptomyces sp. was achieved after 8 days of incubation when the cells entered in the log phase (Fig. 1). The obtained cellulase and xylanase activity was found to be higher than other studies [25,29e31] which increased the potential applicability of Streptomyces sp. for hydrolytic enzymes production. After extraction we have utilized the crude enzyme source (without purification) for the hydrolysis of untreated and pretreated SCB biomass in an attempt to make the process more cost effective and practically applicable.

Effects of chemical pretreatment on enzymatic hydrolysis of SCB LC biomass symbolizes one of the most abundant, renewable resources that can ease the sustainable production of biofuels. However, because of its recalcitrance nature pretreatment

Fig. 2 e Effects of (A) different chemical molar ratios of NaC and NaS, (B) reaction temperature (60e100  C and autoclaving at 121  C for 30 min), (C) NaC þ NaS chemical charge (4e16%), and (D) SCB concentration (4e16%) on hydrolysis yield and glucose yield from SCB after two step enzymatic hydrolysis (30 FPU/g of SCB). Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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step is required which is crucial step for the conversion of LC biomass into fermentable sugars [32]. In the present study, we have evaluated individual alkali, NaC, NaS and combined NaC þ NaS pretreatment options to increase the saccharification of SCB. The pretreatment efficiency was demonstrated by calculating the total sugars released after enzymatic hydrolysis of the pretreated SCB biomass. NaOH pretreatment effectively outbreaks the linkage between lignin and hemicellulose in particular by saponification of ester bonds interconnecting the components of lignocellulose. The sodium hydroxide pretreatment also showed ability to increase the surface area, porosity and swelling capacity of biomass by which it improves the enzymatic accessibility of substrates [3,33]. It was reported that NaC treatment can change the native structure of biomass by removing the linkage between lignin and hemicellulose whereas, NaS increase the hydrophilicity of lignin and thus higher delignification can be achieved [34,35]. We have also checked the combined performance of NaC þ NaS pretreatment to enhance the saccharification of SCB biomass. The main chemical components viz., cellulose, hemicellulose and lignin content of the untreated and pretreated SCB biomass with alkali, NaC, NaS and NaC þ NaS pretreatment are represented in Table 1. Among the studied pretreatments, individual alkaline and NaC þ NaS combined pretreatment showed higher increase in the cellulose content and significant delignification as compared to individual NaC and NaS pretreatment. In alkaline and NaC þ NaS pretreatment the cellulose content was increased from 38.8% to 50.2% and 55.7%, respectively whereas, about 33% and 54% of delignification rate was observed (Table 1). In case of individual NaC and NaS pretreatment moderate delignification of SCB about 27% and 33%, respectively was observed. In each pretreatment after enzymatic hydrolysis of pretreated biomass increase in hydrolysis yield, glucose yield and xylose yield was observed. However, maximum hydrolysis yield (70.5%) with significant glucose yield (88.5%) and xylose yield (55.6%) was recorded in NaC þ NaS pretreated SCB biomass (Table 1). Similarly, some studies reported the effectiveness of NaC þ NaS pretreatment for the effective degradation of biomass [35,36]. Moreover moderate hydrolysis yield of SCB in alkaline and in NaS pretreatment was observed. The results of each treatment are depicted in Table 1. In NaC þ NaS pretreatment, the maximum hydrolysis yield and glucose yield of SCB was observed so further studies on optimization of various operational parameters to enhance the hydrolysis of SCB was conducted.

studies, increase in the temperature enhances the hydrolysis performance and maximum hydrolysis yield and glucose yield was observed at 100  C however autoclaving found not effective this may be because of adherence of inhibitors on the biomass surface even after washing which directly affects the enzymatic hydrolysis performance (Fig. 2B). In this study, maximum hydrolysis yield (80.5%) and glucose yield (97.5%) was achieved when SCB substrate concentration was10% and 12% chemical dosage where NaC (4%) and NaS (8%) was present (Fig. 2C and D). Thus the optimized conditions; 10% of SCB concentration, chemical dosage 12%, at 100  C for 6 h of incubation were found effective and which were applied in further studies. In addition, we have determined the effects of increasing SCB concentration and enzyme concentration on the hydrolysis yield and to attain better saccharification of SCB. The optimal hydrolysis yield was achieved by using 10 g/L SCB biomass with 30 Filter paperase/g of SCB enzyme concentration (Fig. 3A and B). This optimized condition was used to check the enzymatic digestibility of pretreated SCB. After 30 Filter paperase enzyme dosage there is no significant change in the hydrolysis yield as well as reducing sugar production was observed. Under the optimized conditions after enzymatic hydrolysis of NaC þ NaS pretreated SCB the maximum

Optimization NaC þ NaS pretreatment During chemical pretreatment of biomass many operational factors influence on the hydrolysis of biomass. Considering this, we have studied various pretreatment conditions, including, NaC and NaS chemical charge and their ratio, substrate loading, and reaction temperature by keeping incubation period constant (6 h) on hydrolysis yield and glucose yield of SCB after enzymatic hydrolysis. The maximum hydrolysis yield and glucose yield was observed at NaC:NaS (1:2 ratio) however other ratio found less effective to hydrolyze SCB biomass (Fig. 2A). It was supposed that NaS plays a crucial role in the delignification of SCB. In case of temperature

Fig. 3 e Effects of (A) increasing enzyme concentration (10e50 FPU/g of SCB), and (B) increasing substrate concentration (5e25 g/L) on the hydrolysis yield and reducing sugar production.

Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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reducing sugar (RS) released was about 592 mg/g of SCB. The obtained RS was found to be significantly higher than the hydrolysis of wheat straw and sunflower hull by crude enzyme extract from Fomitopsis sp. RCK2010 and Trichoderma reesei RUT-C 30 which released RS 214.044 and 288 mg/g of substrate, respectively [25,37]. Moreover, the hydrolysis yield (80.5%) or saccharification efficiency of our crude enzymatic complex was found to be higher than cellulase by consortium (Agaricus arvensis and Sistotrema brinkmannii) and commercial enzymes (Celluclast 1.5 L and Novozyme 188) [38,39]. The obtained hydrolysis yield (80.5%) and glucose yield (97.5%) of SCB was found to be higher than NaC þ NaS pretreated rice straw which is about 74.5% and 82.7% after enzymatic hydrolysis with enzyme loading of 20 FPU/gcellulose [36]. Similarly, the obtained hydrolysis yield and glucose yield of SCB was significantly higher than sodium carbonate pretreated rice straw and sulfite pretreated robust of spruce and red pine reported earlier [35,40]. Moreover the hydrolysis yield and glucose yield was also found higher than our previous studies [12]. The foregoing results suggest that

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combined NaC þ NaS pretreatment strategy was effective in terms of delignification, better saccharification and glucose yield of SCB. However, further research need to be focused on the recovery and reusability of chemicals to minimize the cost of pretreatment process.

Structural characteristics of pretreated SCB biomass The morphological changes in SCB before and after each chemical pretreatment was observed in SEM images. SEM observations of native SCB displayed a smooth surface, whereas surface roughness was increased in the NaC and NaS pretreated SCB (Fig. 4A). However, in alkaline and NaC þ NaS pretreated SCB, the SEM results suggested that the integrated lignin and hemicellulose portion were significantly removed, by which cellulose fibrils appeared to be separated and accessible to the enzymes (Fig. 4A). Similar structural changes were recorded in sugarcane bagasse after microwave assisted pretreatment and in rice waste biomass after alkaline pretreatment [11,41].

Fig. 4 e (A) SEM photographs, (B) chemical changes as determined by FTIR, and (C) X-ray diffraction pattern of untreated and after chemical pretreatment of SCB. Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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FTIR spectroscopic analysis was performed to examine the structural changes in lignocellulosic biomass occurred due to various chemical treatments. The relative change in the absorbance at specific band positions was evaluated for control (untreated) and after chemical pretreatment of SCB. From the spectrum it was observed that the relative intensities of peaks of alkali, NaC, NaS, and NaC þ NaS treated SCB was found different (Fig. 4B). The change in their peak intensity indicates their structural changes due to change in their functional group in the LC biomass (SCB) as compared to the control (untreated). The region between 3400 and 3200 cm1 is related to the crystalline structure of cellulose. After various chemical treatment (NaC, NaS, Alkali and NaC þ NaS), the position of bands did not show any significant change however, broadening of peaks in this region especially in NaC þ NaS treated SCB was seen from spectrum and which is assigned for the OeH stretching (Fig. 4B). The weak peak at 2910 cm1 is due to the stretching of CeH denotes the characteristic group of cellulose. The bands at 1400e1600 cm1 in the spectrum denotes the C]C stretching of the aromatic rings in lignin which indicates the significant decrease or removal of lignin and hemicellulose content (Fig. 4B). The absorption peak at 1060 cm1 is attributed to the stretching vibration of CeO. The peak at 868 cm is characteristics of glycosidic bond b-(1e4) cellulose.

Max. H2 production rate

100

H2 yield

1200

80

900

60

600

40

300

20

0

1.6

0 Untreated

NaOH

NaC

NaS

1.2

0.8

0.4

200

Soluble metabolites (mg/L)

Cumulative H2 production (mL/L)

1500

(A)

Max. H2 production rate (mL/L/h)

120 Cumulative H2 production

H2 yield (mmol H2/ mol of RS)

1800

Overall comparing with the control (untreated) it can be seen that various chemical treated and mainly NaC þ NaS treated the intensity of OeH decreased and showed effective degradation and also broadened region is also indicative of swelling of the chemically delignified biomass which increases the enzyme accessibility that led to higher saccharification. It was well known fact that crystallinity significantly influences the enzymatic saccharification of biomass mainly cellulose component [26]. XRD analysis is a suitable technique for determining the crystallinity index (CrI) of biomass. XRD diffractograms of SCB treated with alkali, NaC, NaS and NaC þ NaS there is an increase in the CrI was observed relative to untreated SCB (Fig. 4C, Table 1). The significant increase in CrI was observed in NaC þ NaS (54.1%) and alkaline pretreated biomass (49.2%) whereas, moderate increase was observed in case of individual NaC (43.5%) and NaS (48.9%) pretreated biomass than that of the untreated RWB biomass (39.5%). The actual diffractograms after different treatments are shown in Fig. 4C. The maximum increase in CrI indicates that NaC þ NaS combined pretreatment strategy showed ability to remove amorphous part mainly hemicellulose and lignin fraction as compared to crystalline part mainly cellulose fraction of the SCB biomass. Similar observations have previously been reported [12,26].

Acetate (mg/L) Lactate (mg/L) Butyrate (mg/L) Formate (mg/L) Ethanol (mg/L)

150

(B)

100

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250

Concentration (mg/L)

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Acetate (mg/L) Formate (mg/L) Furfural (mg/L) HMF (mg/L)

(C)

150

100

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0 Untreated

NaOH

NaC

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Sugarcane bagasse hydrolysate

Fig. 5 e (A) Performance of dark fermentative H2 production, (B) soluble metabolites concentration (C) fermentation inhibitors concentration from untreated and pretreated SCB enzymatic hydrolysates (5.0 g RS/L) by Clostridium beijerinckii KCTC 1785.

Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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Dark fermentative H2 production from SCB hydrolysates

(180 mL/L) was observed with untreated SCB hydrolysate, suggesting the need of biomass pretreatment for maximum H2 production (Fig. 5A). The soluble metabolites formed along with H2 production from SCB hydrolysates mainly comprised of acetate, lactate, followed by butyrate and formate (Fig. 5B). At the end of fermentation dominant ethanol production was observed. The higher acid concentration and ethanol production was probably due to the complex composition of SCB. The resulted soluble metabolites decreased the pH of fermentation media which inhibited the bacterial growth and activity because of lower ATP levels [1,20,42]. Similarly, dominant ethanol production is also one of the reason for lower hydrogen production since it consumes additional electrons from NADH [1,11]. The results propose that biohydrogen production efficiency was influenced by sugar concentration as well as the soluble metabolites produced during fermentation. The literature survey reported that after chemical pretreatment, the hydrolysates of LC biomass contains potential

Hydrogen is an auspicious substitute to fossil fuels with many social, economic and environmental benefits to its credit. It was observed that after each pretreatment of SCB gave different hydrolysis yield and glucose yield. In the preliminary investigation, we have utilized all pretreated SCB enzymatic hydrolysates for hydrogen production by Clostridium beijerinckii. Among which NaC þ NaS pretreated SCB hydrolysates C. beijerinckii exhibited maximum cumulative H2 production 1204 mL/L with maximum H2 production rate 50.24 mL/L/h and H2 yield of 1.15 mmol H2/mol of RS, respectively (Fig. 5A). Alkali and NaC pretreated SCB hydrolysates showed moderate hydrogen production whereas, NaS pretreated SCB hydrolysate found less effective to convert into hydrogen (Fig. 5A). It was supposed that in NaS pretreatment after lignin degradation the solubilized furfural derivatives, which creates a negative environment for fermentative biohydrogen production [34,35]. Moreover, very less amount of H2 production

120

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0 28

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Max. H2 production rate

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Cumulative H2 production

Max. H2 production rate (mL/L/h)

Cumulative H2 production (mL/L)

1800

0.0

40

Culture temperature (oC)

Soluble metabolites (mg/L)

200 Acetate (mg/L) Lactate (mg/L) Butyrate (mg/L) Formate (mg/L) Ethanol (mg/L)

150

(B)

100

50

0 28

30

32

35

37

40

Culture temperature (oC) Fig. 6 e Effects of culture temperature on (A) cumulative H2 production, H2 yield, and H2 content, (B) soluble metabolites formation from NaC þ NaS pretreated enzymatic hydrolysates (5.0 g RS/L) by Clostridium beijerinckii KCTC 1785. Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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even after washing because of their high concentrations and thus it is essential to detoxify SCB hydrolysates.

Effects of operational conditions on biohydrogen production In dark fermentation the operational factors influencing on the growth rate and metabolic activities of hydrogen producing bacteria as well as hydrogen production and thus the optimization of these parameters are essential to enhance LC biohydrogen production. In the present investigation various operational parameters including, temperature, initial pH of media and substrate concentration on biohydrogen production by C. beijerinckii from NaC þ NaS pretreated SCB hydrolysates were determined.

Effects of temperature Temperature is a vital environmental factor that impacts on the growth of H2 producing bacteria and dark fermentative H2

1.6

(A)

Max. H2 production rate

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H2 yield

1200

80

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20

0

0 5.5

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Max. H2 production rate (mL/L/h)

Cumulative H2 production (mL/L)

120 Cumulative H2 production

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0.4

H2 yield (mmol H2/ mol of RS)

fermentation inhibitors such as furfural, hydroxymethylfurfural (HMF), acetic acid, formic acid etc. which greatly affects biohydrogen production. After pretreatment, furfural and HMF are produced from pentoses and hexoses whereas, acetic acid found in hemicellulose hydrolysates and formic acid is obtained from sugar and lignin degradation. Moreover, the degradation products formed after pretreatment mainly depends on type of biomass and pretreatment conditions applied [43]. We have determined the concentration of furfural, HMF, acetic acid and formic acid concentration after each pretreatment. It was observed that the concentration of all inhibitors were found higher in NaOH, and NaC þ NaS pretreated SCB hydrolysates (Fig. 5C). However, in individual NaS pretreatment higher concentration of all inhibitors relative to individual NaC pretreated SCB hydrolysates was observed (Fig. 5C). The foregoing results suggest that inhibitory compounds generated after lignin and hemicellulose degradation of SCB may persist in the biomass

0.0

7.5

Initial pH of culture media

Soluble metabolites (mg/L)

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(B)

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Initial pH of culture media Fig. 7 e Effects of initial pH of culture media on (A) cumulative H2 production, H2 yield, and H2 production rate, (B) soluble metabolites formation from NaC þ NaS pretreated enzymatic hydrolysates (5.0 g RS/L) by Clostridium beijerinckii KCTC 1785. Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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many LC-biohydrogen production studies were performed under mesophilic conditions as compared to thermophilic conditions [1,13].

Effects of initial pH of fermentation media The effect of initial pH of the fermentation medium (5.5, 6.0, 6.5, 7.0 and 7.5) on H2 production and soluble metabolites production from SCB hydrolysates RS (5.0 g/L) by C. beijerinckii at 35  C were examined. The H2 production rate was improved with increasing initial pH, with a maximum H2 production rate of 50.24 mL/L/h at pH 7.0 (Fig. 7A). The maximum H2 yield and H2 production of 1.18 mmol H2/mol of RS and 1210 mL/L were obtained at initial pH 7.0, respectively (Fig. 7A). Similar trend was observed in soluble metabolites production during fermentation. However, at higher pH 7.5 there is a sharp decrease in H2 and soluble metabolites production was noticed. The results exhibited that the initial pH significantly

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production [11,42]. The effect of incubation temperature from 28 to 40  C on growth, hydrogen and soluble metabolites production by C. beijerinckii using SCB hydrolysates were determined. Hydrogen production of C. beijerinckii were observed at temperatures ranging from 28  C to 40  C with an optimal temperature of 35  C (Fig. 6A). The lower hydrogen evolution and soluble metabolites production was observed at 28  C and there was an increase in their production along with increase in temperature up to 35  C and then further decreased. At higher temperature (40  C) sharp decrease in the H2 production was noted and this may be due to the inhibition of cell growth and denaturation of enzymes system required for fermentative H2 production [11,21]. However, the soluble metabolites concentration was not affected very well. The results are depicted in Fig. 6B. Similarly, maximum H2 production using sucrose at 35  C by Clostridium beijerinckii RZF1108 was observed [44]. The literature survey proves that

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SCB hydrolysate (g of reducing sugar /L)

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200

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SCB hydrolysate (g of reducing sugar /L) Fig. 8 e Effects of increasing NaC þ NaS pretreated enzymatic hydrolysates concentration (2.5e15 g/L) on (A) cumulative H2 production, H2 yield, and H2 production rate, (B) soluble metabolites formation from by Clostridium beijerinckii KCTC 1785. Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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affected the hydrogenase activity and the metabolism pathway which directly influences the H2 productivity [1,13]. These results were consistent with previous studies in which H2 production and cell growth were dependent on initial pH [42]. The maximum H2 production and H2 yield was observed at initial pH of media (7.0) and temperature (at 35  C) (Fig. 5A and B) and thus selected as the optimal conditions for H2 production in the following experiments.

Effects of increasing SCB hydrolysates concentration It was proved that type of substrate, its composition and concentration directly influences on biohydrogen production. The effects of increasing SCB hydrolysates concentration on H2 production and soluble metabolites concentration by keeping temperature at 35  C and initial pH 7.0 were systematically investigated. It was observed that both H2 yield and H2 production rate increased with increasing SCB hydrolysates concentration from 2.5 g/L to 10.0 g/L, reached a maximum of 1.24 mol H2/mol RS and 61.87 ml H2 mL/L/h, and then further decreased (Fig. 8A). The cumulative H2 production was also improved along with the increase in the concentration of SCB hydrolysates. The highest cumulative H2 production (1485 mL/ L) occurred at the concentration of 10.0 g RS/L (Fig. 8A). Similarly, continuous increase in soluble metabolites concentration in particular lactate, butyrate, formate and ethanol concentration was observed. However after 5.0 g/L decrease in acetate concentration was observed (Fig. 8B). When the concentration was increased to 15.0 g RS/L, significant reduction

in the H2 production and H2 yield was observed this might be due to the partial utilization of SCB hydrolysates and substrate inhibition (Fig. 8A). Based on the results obtained, 10.0 g RS/L of SCB hydrolysates was found to be optimal concentration for better conversion into H2. Similar results were acquired in various H2 production studies including Clostridium butyricum [8], C. beijerinckii RZF-1108 [44], C. saccharoperbutylacetonicum N1-4 [45] and C. butyricum CGS5 [46] where increased substrate concentration after certain level inhibit the fermentation process.

Hydrogen production under optimal conditions In the present investigation the better H2 production and H2 yield was recorded under optimal conditions of SCB hydrolysates concentration 10.0 g/L of RS; temperature at 35  C; and initial pH of media at 7.0. Under these optimal conditions, the highest cumulative H2 production 1485 mL/L, with H2 production rate 61.87 mL/L/h, and H2 yield 1.24 mmol H2/mol of RS (0.733 mmol H2/g of SCB) was observed. The H2 yield obtained from our studies was found to be better or comparable with that obtained by other reported results (Table 2). The foregoing results proves that SCB hydrolysate could be used as a fermentation media for H2 production. However, further research work need to be addressed including suitable bioreactor design, optimization of operation conditions to favor the H2 production metabolism to reduce the cost of bioH2 production.

Table 2 e Comparison of biohydrogen production performance with literature using cellulosic hydrolysates under dark fermentation. H2 producer

Clostridium butyricum Clostridium butyricum CGS5

Clostridium butyricum CGS5

Clostridium pasteurianum

Clostridium beijerinckii KCTC1785 Clostridium beijerinckii KCTC 1785 Clostridium butyricum CGS5 Clostridium saccharoperbutylacetonicum N1-4 Clostridium thermocellum 27405

Substrate (g/L)

Sugarcane bagasse (20 g COD/L) Sugarcane bagasse hydrolysates (RS 1.545 g/L) Hydrolysate of NaOH pretreated rice straw (100 g/L) Hydrolyzed carboxymethyl cellulose (10 g/L) Food waste (50 g COD/L) Sorghum husk Chlorella vulgaris ESP6 (RS 9 g/L) Rice bran (100 g/L)

Operation Temp pH Maximum H2 p Maximum H2 yield Reference mode (0C) roduction (mL H2/L) Batch

37

5.5

1611a

Batch

37

7.5

72.6

Batch

37

7.5

Batch

35

Batch

1.73 mmol H2/mol hexose 1.91 mmol H2/g sugarcane bagasse

[8]

1006

0.76 mmol H2/mol xylose

[47]

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23.8

1.21 mmol H2/mol hexose

[38]

40

5.5

1368.5

Batch Batch

35 37

6.5 5.5

1117 246

[42] 128 mL H2/g COD degraded 1.051 mmol H2/mol RS [11] 1.15 mol/mol RS [46]

Batch

30

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1132

Delignified wood fibers (0.1 g/L) Clostridium acetobutylicum X9 Microcrystalline cellulose (10 g/L) Thermotoga neapolitana (DSM 4359) Cellulose (5 g/L)

Batch

60

7.0

2528

Batch

37

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755

Batch

75

7.0

39e49

Clostridium beijerinckii KCTC 1785

Batch

35

7.0

1485

a

Sugarcane bagasse hydrolysates (RS 10 g/L)

3.37 mmol H2/mol hexose 1.6 mmol H2/mol hexose 0.188 mmol H2/mol hexose 1.07 mmol H2/mol hexose 1.24 mmol H2/mol RS

[21]

[45] [48] [49] [50] This study

mL H2/L/day.

Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187

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Conclusions [7]

This study revealed that the combination of NaC þ NaS pretreatment to be a promising option to achieve better saccharification of SCB biomass and enhanced bioH2 production. The crude enzymes produced under solid state fermentation could effectively hydrolyze SCB biomass releasing maximum reducing sugar with higher hydrolysis and glucose yield. This approach increases the practical applicability and makes the process cost effective. The resulted SCB hydrolysates were used for H2 production by Clostridium beijerinckii using dark fermentative approach. We have also optimized various operational conditions to enhance H2 evolution from NaC þ NaS pretreated SCB hydrolysates. Under optimal conditions, the cumulative H2 production, production rate, and yield were 1485 mL/L, 61.87 mL/L/h, and 1.24 mmol/mol RS, respectively. These results proved that combined NaC þ NaS pretreatment followed by enzymatic hydrolysis is a promising technology to increase bioH2 production from SCB biomass. Similarly, this technology could be beneficial to utilize other LC biomass and their conversion into hydrogen.

Acknowledgements This research was supported by Dongguk University-Seoul, South Korea under research fund 2016e2018. This work was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agricultural-Bio Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (710003-07-7-SB120, 116075-3) and funded by Korea Environmental Industry & Technology Institute (A11700197-0703-0). Authors would like to thank Ton Duc Thang University, Ho Chi Minh City, Viet Nam, for the financial assistance of this study.

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

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Please cite this article in press as: Saratale GD, et al., Screening and optimization of pretreatments in the preparation of sugarcane bagasse feedstock for biohydrogen production and process optimization, International Journal of Hydrogen Energy (2018), https:// doi.org/10.1016/j.ijhydene.2018.01.187