Physicochemical Characterization of Native and Steam Explosion Pretreated Wild Sugarcane ( Saccharum spontaneum )

. The technology of biomass conversion to bioethanol primarily based on pretreatment, enzymatic hydrolysis, and fermentation. This study was to investigate the effectiveness of the steam explosion pretreatment of Saccharum spontaneum L., which accomplishes the greater efficacy of physicochemical and structural properties. The collected plant material was processed and analyzed for ash, moisture, carbon content, and other elements. The cellulose content of pretreated biomass was increased to 54.31% when compared to native wild sugarcane 41.23% due to the removal of lignin. SEM and FTIR results identified the changes in structural and functional groups also the BET analysis confirmed the increased surface area of Pretreated biomass is 55.541m ² /g whereas the surface area of native biomass is 17.939 m ² /g, this is due to the increase in pore volume and pore diameter of pretreated wild sugarcane which is 0.260 cc/g and 9.712 nm when compared to pore volume and Pore Diameter Dv(d) of raw material is 0.040 cc/g and 3.650 nm. XRD crystallinity pattern of pretreated wild sugarcane showed an increase in the crystallinity index due to the breakage of lignin during pretreatment. This comparative study has been carried out to know the effect of steam explosion pretreatment over the physicochemical composition and structural changes of wild sugarcane for sustainable bioethanol production. ©2020. CBIORE-IJRED. All rights


Introduction
In recent years, lignocellulosic based bioethanol production has gained interest due to a variety of reasons like the high cost of petroleum-based fuel, sustainable energy, less pollution ). Bioethanol produced from lignocellulosic biomass serves as an alternative promising carbon-neutral biofuel (Sindhu et al, 2019). In India, agricultural crop residues were considered as the most abundant available feedstocks for bioethanol production ). The agricultural biomass as lignocelluloses characterizes an effective reservoir of carbon-rich material (Rebello et al., 2020). The lignocellulosic biomass conversion to biofuel provides economic and environmental sustainability compared to fossil fuels. The bioethanol production from lignocellulose biomass includes three significant steps, viz. biomass pretreatment, enzymatic hydrolysis, fermentation, and a distillation process. The pretreatment is considered a bottleneck in the process of obtaining a higher yield of bioethanol production. It involves hydrolyzing the carbohydrate to monomeric * Corresponding authors: suguraj200@gmail.com sugars through alteration of the structural and chemical composition of biomass.
Wild sugarcane (Saccharum spontaneum) is considered as a wasteland weed, which is a long perennial grass with deep roots. It is understood as a precursor of S. officinarum L. (cultivated sugarcane) and considered as a common regional weed (Sastri and Kavathekar, 1990;Hammond, 1998). S. spontaneum is suitable for biofuel production since its cell walls are rich in carbohydrates (67.85% on dry basis), (Chandel et al., 2009a, b;Scordia et al., 2010). The genus Saccharum comprised 13 species and found to be the same as the genus of Miscanthus; both this genus can be differed by the disposition of its spikelets (Scally et al., 1997). Also, they show a higher yield after the second generation of crop establishment whereas in the Miscanthus genus it happens only after thirdgeneration (Cosentino et al., 2006). The studies of effective pretreatment have been carried out on different sugarcane hybrids, but for wild species, no reports were found so far. However, the efficiency of hydrolysis for the conversion of carbohydrates into monomeric sugars depends on the utilization of biomass (substrate) which is a major step that includes the changes in its structure, size, and nature of the biomass (Moiser et al., 2005).
Generally, the steam explosion was considered as a successful pretreatment method due to the sudden explosive decompression (Chen and Liu, 2007a,b). NaOH is commonly used in the chemical pretreatment of lignocelluloses because of its ability to delignify biomass (Sindhu et al., 2015). In this process, the lignin was redistributed and removed from the biomass at high temperatures by the acetic acids whereas the hemicellulose was hydrolyzed (Mosier et al., 2002). The enzyme accessibility of cellulose microfibrils increases due to the removal of hemicelluloses by exposing the cellulose surface (Alvira et al., 2010). The objective of the present study was to analyze the efficiency of pretreatment using a steam explosion for efficient bioethanol production. To investigate the changes in biomass after steam explosion pretreatment, characterization studies of native and pretreated samples were carried out by SEM, FTIR, BET, and XRD spectrum (Ragavi et al., 2016).

Sample preparation
The plant Saccharum spontaneum L (wild sugarcane) was collected from Ash Lake, Neyveli, Tamilnadu, India, and authenticated by the Botanical Survey of India (BSI), TNAU -BSI/SRC/2804. The stem part of the feedstock was collected by removing leaves and flowers, chopped into small pieces, and sun-dried. The sun-dried raw material was subjected to mechanical size reduction and stored at room temperature as shown in (Fig.1).

Apparatus and experimental procedure
Briefly, 5 g of the fresh sample was collected, the final biomass weight was noted after drying at 70°C in a hot air oven. The percentage of moisture content was determined by the method described by AOAC (1990).

Ash Content
In a crucible, 3 g of dried wild sugarcane sample was taken and heated to oxidize flames until smoke dissipated. The crucible was placed for 6 hrs in the muffle furnace at 550°C, then cooled and weighed in a desiccator. The composition of ash content was determined by the standard method (AOAC 1990).

Estimation of Cellulose
To the known quantity (0.5 g or 1 g ) of wild sugarcane biomass, 3 mL of acetic/nitric reagent (150 mL 80% acetic acid and 15 mL concentrated nitric acid ) were added in a test tube, mixed and placed at 100°C for 30 minutes in the boiling water bath. The content was cooled and then centrifuged for 15 -20 minutes at 5000 rpm. After centrifugation, the biomass residue was washed using distilled water and the supernatant was collected separately. One mL supernatant was diluted to 100 mL of the above solution.
Ten mL of anthrone reagent was applied to 1 mL of this diluted solution and blended well. The contents are kept for heating in the boiling water bath for 10 minutes and measured the color at 630 nm and the amount of cellulose (Norman and Jenkins 1933).

Acid Detergent Fibre (ADF)
One gram of wild sugarcane was added to a round bottom container then 100 mL of acid detergent solution was prepared and added to mixture (20 g CTAB in 1 L of 1 N sulfuric acid). The content was warmed to simmer for 5 to 10 min and refluxed until boiling for 1 hour. The contents were drained by suction through a pre-weighed sintered glass crucible and washed twice with acetone accompanied by hot water until the filtrate was colorless and kept for overnight drying at 100 °C. The fiber was weighted after cooling (Norman and Jenkins 1933).

Acid Detergent Lignin (ADL)
To the obtained acid detergent fiber (ADF), 25-50 mL of 72% H2SO4 and 1 g of asbestos was added and kept it to stand for 3 hours. The content was diluted with distilled water, filtered with pre-weighed filter paper Whatman No.1 and dried at 100°C. The filter paper was weighed after cooling. The filter paper was added to a pre-weighed silica crucible and kept in a muffle furnace for ashing for 3 hours at 550°C and the ash content was measured. For a blank, 1 g asbestos with 72% H2S04, was taken (Norman and Jenkins, 1933)

Estimation of hemicellulose
To 1 g of the powdered wild sugarcane sample, 10 mL of cold neutral detergent solution was added and kept in a refluxing flask. About 0.5 g of sodium sulfite and 2 mL of decahydronaphthalene were added to the mixture. The content was heated to the warm condition and refluxed for 60 minutes. The sample was washed with acetone and hot water by suction filtration using sintered glass crucible. The residue was kept in a silica crucible and dried at 100°C for eight hours. The crucible was cooled and weighed (O'dwyer, 1923).

EDAX analysis
The qualitative analysis of the chemical elements was performed using an energy dispersive spectrometer (EDAX System), which is coupled with SEM (JOEL -JSM 6390, Japan). For the EDX analysis, elements present in the sample absorb the X-ray beam, resulting in the movement of electrons from a ground state, causing dislocation of the electrons and thereby creating a hole that was filled by electrons from other higher energy state. This creates a difference in energy, which results in peak formation.

Estimation of CHN
CHNS analyzer (Thermo Finnigan FLASH EA 1112 series, Italy) was used to estimate the total carbon, hydrogen, and nitrogen content of the wild sugarcane at the SAIF, IIT Bombay. The CHNS(O) analyzer determined the percentages of carbon, hydrogen, nitrogen, sulphur and oxygen of organic compounds by instantaneous oxidation of the sample at 1800°C by "flash combustion". The combustion products were separated by a chromatographic column and detected by the thermal conductivity detector.

Pretreatment by steam explosion
The pretreatment was carried out for the powdered raw material as described by (Kaushik and Singh, 2011).

Preparation of Steam Exploded fibers
Alkaline treatment was done out in two steps. In the first step, 10g samples were soaked overnight in 2% NaOH solution and then autoclaved for four hours at 15 lb pressure after treating with 10% NaOH. The pressure was then immediately released and the residues were cleaned. In the first treatment, fibers are swollen due to the removal of excess impurities from the surface which makes further treatment easy. Lignin was removed in the second treatment from the fiber. The fibers were then washed in distilled water for the removal of alkali.

Preparation of Steam exploded bleached fiber
The pulp obtained after alkali treatment was kept for overnight soaking in 8% H2O2 (v/v) for bleaching. This bleached pulp was washed with distilled water to get rid of acid.

Treatment of Steam exploded bleached fiber in Acidic medium
After bleaching, the pulp was kept in ultrasonicator for mixing at a temperature of around 60 ± 1°C for 5 hr after treating with 10% HCl (1N) solution. Fibers were dried (Fig. 1) after neutralizing the final pH by washing with distilled water.

BET Analysis
The BET surface area, pore-volume, and porosity of wild sugarcane were measured using the (Brunauer−Emmett−Teller) BET surface area analyzer. BET-N2 adsorption experiments were carried out manometrically using an Autosorb (Quanta Chrome Crop). Before the measurements of gas adsorption, the samples were kept in a vacuum condition at 200°C for a period of 24 hrs at least for degassing. Nitrogen adsorption isotherms were measured at a series of different pressures at -196°C. And the BET surface area was determined using the standard BET equation as described by (Kibami et al., 2017).

Scanning electron microscopy
The SEM images were taken using a scanning electron microscope (JOEL -JSM 6390, Japan) to study the differences in the morphology of both pretreated and native wild sugarcane. The images were acquired at 20kV of an accelerating voltage at 500X magnification.

X-ray diffraction
X-ray diffraction analysis was performed using (XRD-6000 Shimadzu diffractometer) for the native and pretreated wild sugarcane. Diffraction patterns were reported utilizing 40 kV and 30 mA Cu-Kα radiation and 10-30 grade scale with a 0.03 phase scale. The crystallinity index was determined based on the empirical method (Segal et al., 1959).

Fourier Transmission Infrared Analysis
FT-IR spectroscopy (Shimadzu Spectrometer, Japan) was done to study the difference in structural and functional groups through the FT-IR spectrum which acts as a molecular fingerprint of the sample. This will detect the changes in functional groups that may have been caused during pretreatment. The FTIR analysis was performed with the detector at 4 cm -1 resolution, 50 scans per sample, and the spectrum between 4000 and 400 cm -1 . Discs were prepared by using 300 mg of spectroscopic grade KBr to 5 mg of dried specimens and mixed in an agate mortar. The obtained mixture was pressed for 3 min at 10 Mpa (Sindhu et al., 2014).

Effect of steam explosion pretreatment on wild sugarcane
Steam explosion pretreatment favors the reduction of lignin and hemicellulose by increasing the amount of cellulose in the biomass. Table 1 summarizes the dry basis composition of native and pretreated S. spontaneum L. The result shows the changes in biochemical composition of native and pretreatedS. spontaneum. The cellulose, hemicelluloses, and lignin composition in native was found to be 41.23%, 23.26%, 11.21%, whereas after pretreatment the composition was found as 54.31% of cellulose, 17.22% of hemicellulose and 7.14% of lignin, respectively. After pretreatment, the results showed increased cellulose content, reduced hemicellulose and lignin content compared to native raw material. Results from repeated measurements of wild sugarcane composition prior and after pretreatment showed excellent reproducibility. The pretreated carbohydrate content in the S.spontaneum biomass is highly comparable to other lignocellulosic substrates such as agricultural residue and other woody biomass which make this feedstock as a suitable substrate for the production of bioethanol.  (c) (d)

BET Surface characterization and nitrogen sorption of wild sugarcane
BET analysis revealed the measurement of the surface area of both native and pretreated wild sugarcane. The surface area of pretreated biomass increased to be 55.54 m²/g whereas the surface area of native biomass is 17.94 m²/g. Fig. 4a and 4b represent the adsorption−desorption isotherms of both native and pretreated biomass. Fig.4c and 4d represent the pore size distribution curve of native and pretreated wild sugarcane respectively. From the results, it was found that the pore volume and pore diameter-Dv(d) of raw material is 0.040 cc/g and 3.650 nm,respectively, whereas the pore volume and pore diameter-Dv(d) of pretreated biomass is 0.260 cc/g and 9.712 nm,respectively and found that pore volume was increased when compared with the untreated. The hydrolysis process will be more optimal due to the increase in specific surface area, this is because of the effective interaction between native and pretreated biomass. The pretreatment acts as a swelling agent and simultaneously removes the lignin, this results is an increase in a specific area that is suitable for enzyme action (Saha et al., 2005). The internal specific surface area is observed based on the capillary structure of cellulosic fiber; while the external specific surface area depends on the particle size and shape.

Morphological analysis of wild sugarcane
SEM images show that most surface (lignin) substances have been removed, and linear cellulose crystal segments have been exposed after pretreatment. Pretreatment of the cellulosic biomass will make it much more available to enzymes for the degradation of cellulose and hemicellulose. Pretreatment with a steam explosion caused more damage to the microstructure of the cellular matter. The original hard structure was broken after steam explosion pretreatment with a high temperature and the individual fibers vanished (Fig. 5). Such morphological modifications led to increased external surface area and porosity of the cellulosic material similar to previous NaOH pretreatment experiments in rice straw and sugar cane bagasse (Lin et al., 2013;Zhang et al., 2008). The effects of the steam explosion pretreatment were recorded by observing the difference between the ultra-structure of the native plant cell wall and the possible disruption of the pretreated S. spontaneum cell wall. The SEM image reveals the short anatomy of chopped and untreated S. spontaneum, with the sheath leaves surrounded by straw itself as thick-walled fiber cells. Exposing cellulose by structural modification or alteration of hemicelluloses and lignin tends to be a critical factor in saccharification of the remaining fraction of carbohydrate contained in S. spontaneum cell walls. Similar observations were reported earlier regarding the exposure of fibrous cell walls by pretreatment of barley hull (Kim et al., 2008) and corn stover (Kim et al., 2003).

Crystallinity analysis of wild sugarcane
XRD spectrum of the native and pretreated samples was presented on (Fig. 6). The crystallinity index of biomass has a major impact during hydrolysis (Kim et al., 2003). Xray diffractograms results show the changes in the crystallinity index before and after the steam explosion pretreatment of wild sugarcane. The crystallinity index of native and pretreated samples was 36.13% and 41.1% respectively. The increase in the crystallinity index after pretreatment is based on the increase of amorphous polymer it is due to the hydrolysis of glycosidic linkages of cellulose fibers. The amorphous region of cellulose is easy for enzymes to digest. The increase in the crystallinity index after pretreatment of lignocellulose has been observed in other studies, such as sugarcane tops pretreated with acid (Sindhu et al., 2011), pretreatment of switchgrass with acid (Sindhu et al., 2012).

FT-IR analysis of wild sugarcane
The FT-IR spectroscopy is commonly used for the characterization of lignocellulose, as it provides a relatively easy tool for direct information on chemical change during different chemical pretreating processes (Ristolainen et al., 2002). The FTIR spectra of native and pretreated biomass revealed a significant difference was found in the fingerprint region (1659.13 -895 cm -1 ) for lignin's IR spectrum (Fig. 7). The peak at 1462.69 cm -1 , shows that the acetyl group linkage was reduced in this band which is present in between lignin and hemicellulose due to the breakage of the ester group. A significant reduction was also observed at the peak 1287.01 cm -1 which indicates the C-O stretching in lignin and hemicellulose. A sharp peak at 895 cm -1 is characteristic of β-glycosidic linkages between the sugar units (Gupta et al., 1987). The pretreated substrate also showed a reduction in the linkage of hemicellulose-lignin and C=O stretching due to carbohydrate linked with lignin via shift and decrease of the band at 3115.57 cm -1 and 2578.17cm -1 , respectively.

Conclusion
Saccharum spontaneum is considered as low-cost biomass since it is a wasteland weed which can be used as a renewable and potential source of feedstock for ethanol production. In this study, the steam explosion pretreatment of wild sugarcane increases the cellulose content by the removal of lignin and hemicellulose compared to the native wild sugarcane. The structural changes in the SEM image will enhance the biomass reaction during hydrolysis. Also, the release of lignin was detected in FTIR in pretreated biomass. The XRD results confirmed the increase in the crystallinity index after pretreatment than the native, this is due to the increase of amorphous nature of cellulose which improves the accessibility of cellulose surface which improves more cellulose hydrolysis to enable more theoretical yield by providing free b-glucosidic sites. The changes in the physicochemical, biochemical, and structural composition of wild sugarcane indicated that it is a suitable feedstock for bioethanol production.