INVESTIGATION OF SUGAR PRODUCTION FROM UNWASHED PRETREATED LIGNOCELLULOSIC BIOMASS
LI WAN YOON*, ISHRAK SHARIAR RAFI
School of Engineering, Taylor’s University, Taylor's Lakeside Campus, No. 1 Jalan Taylor's, 47500, Subang Jaya, Selangor DE, Malaysia
*Corresponding Author: [email protected]
Abstract
Washing is an essential step to neutralise the pH of the deep eutectic solvent (DES) pretreated substrate prior to enzymatic hydrolysis. Elimination of washing stage could result in significant cost saving to produce bioethanol. This study aims to investigate the feasibility of applying unwashed DES-pretreated substrate for glucose analysis by finding a suitable DES producing high glucose content. Four different DES consisting of choline-chloride citric acid, choline-chloride glycerol, choline-chloride urea and choline-chloride malonic acid were used in pre-treating sugarcane bagasse. Chlorine-chloride malonic acid was chosen as the DES due its ability to produce the highest glucose concentration. To evaluate the feasibility of unwashed pretreated substrate, glucose production from enzymatic hydrolysis from both washed and unwashed pre-treated sugarcane bagasse was compared and evaluated. The washing step is proven to be non-essential as the glucose content measured by DNS method for the unwashed pretreated substrate (3.03mg/ml) has shown to be higher than the washed pretreated substrate (2.91 mg/ml). This study suggests that it is feasible to apply unwashed pre-treated substrate for bioethanol production which will further reduce the cost involved in the wastewater generation.
Keywords: bioethanol production, deep eutectic solvent, enzymatic hydrolysis, pretreatment, lignocellulosic biomass, washed substrate, unwashed substrate, glucose concentration.
1. Introduction
Dwindling of fossil fuels and high dependence on crude oil for energy consumptions has driven researcher to explore and develop alternative sources of energy and chemicals. To partly replace fossil-derived fuels, bioethanol plays a significant role due to its carbon-neutral renewability, which means a reduction of CO2 emission as it is a greenhouse gas contributing to the global warming [ CITATION EBD07 \l 17417 ]. Production of bioethanol have increased from 13.12 billion of gallons in 2007 to over 25.68 billion of gallons in 2017 [ CITATION ASR08 \l 17417 ]. A global estimate of up to 1.3 billion tons/year of food processing waste are produced which are lignocellulosic in nature [ CITATION ASR08 \l 17417 ]. Prominent feedstock for bioethanol is lignocellulosic biomass, such as agricultural residues, forest-based woody materials, and municipal waste which are plentifully available at a low cost. Its abundance is one of the aspects on why it’s seen as a potential sustainable solution to replace oil-based resources by transformation of lignocellulosic biomass into reducing sugar for bio-fuels or other value-added products.
Abbreviations
DES Deep Eutectic Solvent SCB Sugarcane Bagasse IL Ionic Liquid
200 billion tons of lignocellulosic biomass are produced annually, making it the most abundant renewable organic resource on earth that is readily available for conversion to biofuels and other value-added products [ CITATION Dan16 \l 17417 ]. Secondary plant biomass consists mainly of lignocellulose. However, lignocellulosic biomass requires adequate comprehension before it can be utilized for production of various important chemicals. The structure of lignocellulose is complex, and the content must be optimized distinctively to produce each chemical. Therefore, it is of great significance to understand what lignocellulose consists of and how to maximize and/or minimize the contents.
I.S Rafi and Y.L.Wan
Lignocellulose is made of three major components; cellulose, hemicellulose and lignin. It also contains some moisture content and small quantities of other minerals[1].
The common stages employed to convert lignocellulosic biomass into ethanol are pretreatment, hydrolysis and fermentation. Initially, lignocellulosic substrate undergoes pretreatment to modify its recalcitrant nature which results in a more porous structure accessible by enzymes. Pretreatment aids in reducing the lignin content and cellulose crystallinity depending on the type of pretreatment applied. After that, cellulose in pretreated biomass is converted into glucose monomers through enzymatic hydrolysis[4]. The sugars can then be further fermented into bioethanol by microorganism such as yeast. For the past few decades, many pre-treatment methods have been developed, including alkali treatment, ammonia explosion, and others. Many methods have been shown to result in high sugar yields that are above 90% of the theoretical yield for lignocellulosic biomasses such as woods, grasses, corn, and so on[ CITATION ASR08 \l 17417 ] .
The desire for better pretreatment has led to discovery of deep eutectic solvent (DES). It is one such alternative group ionic solvents pretreatment that has similar physicochemical properties as ionic liquid (IL). In principle, it is a liquid eutectic mixture formed by self-association (or hydrogen-bonding interaction) of two or three components. DESs are attractive solvents for the fractionation of lignocellulose and the valorisation of lignin, owing to the high solubility of lignin in DESs. It is made up of salts and hydrogen bond donors with melting points low enough that they can be used as solvents. Comparably, DES have proved to be a good alternative to traditional organic solvents and ionic liquids (ILs) in many biocatalytic processes. Some of the benign characteristics of DES with ILs includes low volatility, low flammability and low melting point, and in addition, they have their exclusive merits of easy preparation and low-cost owing to their renewable and available raw materials [ CITATION Luc18 \l 17417 ].
One major concern after DES pretreatment and prior to enzymatic hydrolysis is the requirement of washing to remove the DES remnants from the pretreated biomass and to neutralize the pH of the substrate. It is through washing that aids in the removal of inhibitory products for better enzymatic hydrolysis [10]. Therefore, it adds to another cost factor for bioethanol production which can be greatly reduced if the amount of wastewater generation is minimized. For example, a study by Dijeloue, et al., [ CITATION Die05 \l 17417 ] showed that at bench scale, 30 L of water are needed to successively remove inhibitory compounds while another study by Hodge et al.,[ CITATION Hod081 \l 17417 ] showed minimum 3 L of water are required for washing at the pilot scale. Although, limited studies have been conducted on bioethanol production without washing but the studies done such as by Menghui Yu et.al[ CITATION Men14 \l 17417 ] have advanced the idea that fermentable sugar conversion can have high yield without incorporating the washing step prior to enzymatic hydrolysis.
To the best knowledge of author, results of fermentable sugar from unwashed substrate pretreated by DES remains unknown. Fermentable sugar production from unwashed DES pretreated lignocellulosic substrate can further reduce the cost effectiveness of the production route and yield of bioethanol. Thus, this study aims to assess the feasibility of producing fermentable sugar from suitable DES pretreated biomass without incorporating the washing stage. In this study, the effect of glucose concentration at different time intervals during enzymatic hydrolysis will be investigated and compared for both washed and unwashed pretreated substrate.
2. Research Methodology 2.1. Determining Suitable DES
Four different DES are selected to investigate the suitability for the application of pretreatment of sugarcane biomass based on its glucose content. The highest glucose content after enzymatic hydrolysis of 24 hr is selected for the subsequent study of glucose production for washed and unwashed pretreated biomass.
2.1.1 Preparation of Sugarcane Bagasse(SCB)
Sugarcane bagasse is collected and dried in an oven for 48 hours at 60°C until a moisture content below 10% is achieved. It is later grinded and sieved and stored at a room temperature for subsequent usage.
2.1.2 Deep Eutectic Solvent (DES)
DES combination of choline chloride with urea, citric acid, glycerol, and malonic acid were prepared at a ratio of 1:2, 1:2, 1:1 and 1:1. Each of the components were weighed at 15g, 12.986 g, 16.798 g, 18.4 g and 24.3 g and transferred into a Schott bottle. The DES solution mixture then underwent heating using a hotplate with magnetic stirrer using 150
rpm at 80°C. A complete DES preparation is indicated when the mixture turned into homogenous colorless liquid phase. The synthesized DES is then cooled down before sealed tightly and kept in dehumidified chamber for protecting the DES from contamination of any atmospheric water vapor.
2.1.3 Preparation of Citrate Buffer
Citric acid monohydrate of 210 g was added in a large volume beaker containing 750ml of ultrapure water. After proper stirring of citric acid monohydrate into the ultrapure water, NaOH pellets were added until a pH of 4.3. was obtained.
Finally, the solution was diluted to 1L to make a stock solution of 1M of citrate buffer.
2.1.4 DES Pretreatment
Pretreatment was then carried out with 4% solid loading by mixing 0.5 g of SCB and 12 mL of DES respectively in each Schott Bottle. The samples were then heated at 130°C for 3.2 hours in an oven (Un-75, Memmert). 5 ml deionized water was then added to remove the suspended non-ionic substrate and transferred into a centrifuge tubes. The contents were centrifuged at 3500 rpm respectively for 10 mins. Washing and rinsing of biomass is then carried out using suction filtration. The supernatant (black liquid) obtained after the rinsing and washing was collected into a sampling bottle and stored at 4°C. The pretreated biomass was dried at 50°C for 8 hours in an oven to remove the minimize the moisture content and then kept in desiccator prior to hydrolysis process.
2.1.5 Enzymatic Hydrolysis
Enzymatic hydrolysis was performed by 2 wt% solid loading by mixing 0.2g of pretreated biomass with 10 ml of sodium citrate buffer(pH 4.3) which was added with 100mg of cellulase. The enzymatic hydrolysis process is then carried out as mentioned in NREL Protocol [94] using an orbital shaker set(LM-400D, Yihder) at 50°C for 24 hours at 150 rpm. After hydrolysis, the sample was centrifuged at 3500 rpm for 15 min, and the supernatant is collected for glucose analysis.
2.2 Sugar production from washed and unwashed pretreated substrate 2.2.1 Deep Eutectic Solvent(DES)
DES is prepared using the same procedure as mentioned earlier. The chosen DES was choline-chloride malonic acid with 1:1 in molar ratio, and hence a new DES solution with more volume was prepared. To prepare ChCl: malonic acid, 140g of choline chloride and 104 g of malonic acid was put into a Schott bottle and heated at 80°C using a hot plate with 180 rpm. The solution was stirred until a homogenous solution is obtained that took approximately 4 hours.
2.2.2 DES Pretreatment
DES pretreatment was conducted using the same procedure as mentioned earlier. It was carried out with 4 wt% solid loading by mixing 1.6g of SCB and 40 ml DES respectively in two Schott bottles. One for investigating washed substrate, and the other for unwashed substrate. The sample then under underwent heating at 130°C for 3.2 hours in an oven. 8ml of denionized water then added to the mixture after the heating to remove the suspended non-ionic substance.
2.2.3 Washed and unwashed substrate
Washing of the substrate was performed using a suction filtration with ultrapure water. A successful washing is indicated when the pH of the remnant from the suction filter is close to 7 pH and hence after every washing cycle, the pH of the remnant was continuously measured. For unwashed substrate, the suction filter was only used to remove the DES remnant without incorporating the washing stage and hence had an acidic pH. All the DES remnant was collected into a sampling bottle and stored at 4C.
I.S Rafi and Y.L.Wan
2.2.4 Enzymatic Hydrolysis
Enzymatic hydrolysis was performed in duplication for both the washed and unwashed substrate. Wet solid of 0.55g for washed substrate, and 0.59g for unwashed substrate was added to 25ml of citrate buffer with 5mg of cellulase in a 50ml conical flask. Enzymatic hydrolysis was then carried out using the same procedure as mentioned earlier for 48 hours.
1.5 ml sample was collected at 0,8,17,20,45,48 hr into a centrifuge tube. The sample was then boiled at 100°C for five min to deactivate the cellulase Centrifugation was performed at 3500 rpm, 10 min to remove the solid, the supernatant was then pipetted into a new tube. The sample was then tested for glucose content using DNS method.
2.3 Analytical Method
2.3.1 Moisture Content Analysis
The moisture content of the washed and unwashed biomass was determined to adjust the % solid loading in enzymatic hydrolysis. Once the washed and unwashed pretreated substrate have been filtered, it was put into a crucible and the content measured which is the initial weight. After that it was heated in oven for 105°C for one hour and then kept in desiccator to cool down for 5 minutes. The differences in the weight of the content was then measured and the % solid loading was adjusted accordingly.
2.3.2 3,5 Dinitrosalicylic Acid(DNS) Analysis
3,5-dinitrosalicylic acid(DNS) reagent with 1%(w/v) 3,5-dinitrosalicyclic acid and 0.2% (w/v) phenol and 1%(w/v) NaOH was prepared and stored in dark bottle. Prior to reducing sugar analysis, 1%(w/v) of sodium metabisulfite was added to the DNS reagent. DNS solution in 3ml was then added into the test tube that contained 1.5 ml of sample solution. The mixture was then boiled and placed in cold water and added with 1 mL of 40% Rochelle salt (potassium sodium tartrate tetrahydrate) immediately. After cooling the mixture, 0.1 mL of reaction mixture was diluted by adding 1.25 mL of ultrapure water and the absorbance of the diluted reaction mixture was measured at the wavelength of 540 nm by UV-Vis spectrophotometer (PRIM, Secoman). The reducing sugar concentration is determined by first constructing a standard curve by using D(+) glucose (Merck) solution.
3 Results and Discussion
3.1 Selection of suitable DES for pretreatment
All the sample for four different DES were carried out at fixed pretreatment, temperature duration and solid loading at 130°C, 3.2 hours and 4% solid loading for the selection of suitable DES. The performance of these DES pretreatment
was analyzed by comparing the glucose content after 24 hours.
Figure 1 Glucose Yield for different types of DES
From Figure 1, ChCl-malonic acid is chosen as DES for subsequent studied as it shows the highest amount of glucose concentration at which is 3.7g/l(0.075g/g SCB) compared to other DES. The average value is taken as all the samples were duplicated for producing high precision result. There are different factors such as pH, temperature, chemical structure, water content, viscosity that could contribute to the differences in glucose concentration for each respective DES. Based on the nature of DES used in the experiment, urea is shown to be slightly alkaline while glycerol is almost neutral and citric acid and malonic acid are the most acidic. From the result obtained, it can be deduced that glucose concentration is dependent on the acidity and alkalinity of the reagent. On the other hand, DES with neutral pH have lowest amount of glucose as shown in the result by ChCl-glycerol. The finding is further substantiated by various studies done on the effect of pH in different DES. Based on a study [ CITATION Ver14 \l 17417 ], it is shown that acidity in DES affect the extraction of various structures of lignin compounds. Other studies[ CITATION Maj13 \l 17417 ][ CITATION Nam \l 17417 ], demonstrated the significance of pH of DES in the role of biomass pretreatment, a pH of less than 7 result in hydrolysis of the hemicelluloses to monomeric sugars and minimize the need for hemicellulase. While a pH of 7 lead to solubilization of most of hemicelluloses and does not result in total conversion into monomeric sugars which explains why ChCl-glycerol had the lowest glucose concentration. The neutrality of ChCl-glycerol could probably be caused by the presence of alcohol in the DESs structure. For alkaline pH part of hemicellulose are in solid fraction, and hence solid hemicullalses are needed for both solid and dissolved fraction of hemicelluloses[9].
Regarding the study done on pH on DES, it is shown that low pH can achieve high product yields depending on the structure of biomass. However, a low acidity can increase the capital cost of reactors and equipment due to corrosive nature which is not applicable when a low pH DES used as the metal oxide formed in DES will not react if a non-metal equipment is used. The low acidity favorable can also be explained by the study done by Trajano et.al., [ CITATION Tra09 \l 17417 ]that states that a low pH facilitate the solubilization of cellulose by degrading its high crystallinity and strong molecular chain-to-chain affinity. The finding from the experiment is also aligned with a study done by DP that showed a high sugar was obtained by acidic pretreatment compared to alkaline pretreated biomass[ CITATION Zhe16 \l 17417 ]
Figure 2. Effect of pH versus temperature[ CITATION And19 \l 17417 ]
Viscosity of DES is another factor that can contribute to the difference s in efficacy, however with increased temperature, the viscosity can decrease and improve the glucose yield. Figure 2 shows how pH is affected with its increasing temperature and as can be seen the pH values decreases steadily with increasing temperature for all DESs used in the experiment. Such behavior of deep eutectic solvent proves that hydrogen-bond influences the effect of resultant pH, which explains why acid as hydrogen bond acid results to low pH having while alcohol as a hydrogen bond donor gives a close to neutral pH. Therefore, the results obtained from Figure 1 were found to be reliable as supported by various study. The subsequent study was carried out using Ch-Cl-Malonic Acid. Besides, the ability of
I.S Rafi and Y.L.Wan
producing high glucose content, choline-chloride malonic acid is also shown to be recyclable and reusable three times without the loss of its efficiency that further renders its economic viability.
3.2 Enzymatic Hydrolysis of washed and unwashed DES pretreated substrate
Figure 3. Glucose concentration for unwashed substrate
Figure 4. Glucose concentration for washed substrate
From the figures above, it can be seen the sugar concentration of unwashed substrate is slightly higher by 3.09%. The amount of glucose produced from washed substrate is 2.91 mg/ml while for unwashed substrate is 3.00 mg/ml. This proves that the feasibility of producing higher glucose concentration from unwashed substrate is achieved, however a low glucose yield can be explained due to the washing cycle used. Typically, washing involves cycles ranging from washing with one cycle to five, depending on the amount of desired inhibition product removal. For the experiment, only one washing cycle is used. The other reasons that could contribute to the factors are the residual left in the unwashed pretreated substrate could be at an insignificant level that did not attain maximum inhibitor impact to the action of cellulase. Besides, the effect of buffer in enzymatic hydrolysis could also be another factor due to the change in pH when the buffer is added to the unwashed substrate that is acidic in nature due to the remnant of DES solution. As such, adjusting the pH can be suggested prior to enzymatic hydrolysis for a better glucose concentration. Other than that, studies has shown that enzymatic hydrolysis can be enhanced by the application of certain metal compounds, namely Ca(II) and mg(II) , that can associate with lignin by reducing affinity of lignin to cellulase enzyme as such metal salt can be used to further increase enzymatic hydrolysis of unwashed pretreaated substrate. Different pretreatment such as sulphite pretreatment have shown to have high glucose yield for unwashed substrate that is comparable to washed substrate when metal compounds such as Ca(II) and Mg(II) are added [ CITATION Hao10 \l 17417 ].
The rate of hydrolysis is also compared with washing and unwashed substrate as shown in the table below.
Table 1 Rate of hydrolysis for washed and unwashed substrate Tim
e Washed substrate concentration(mg/ml )
Unwashed substrate concentration(mg/ml )
Rate of hydrolysi
s for
washed substrate
Rate of hydrolysi
s for
unwashed substrate
0 0.15 0.09 - -
8 1.67 0.98 0.19 0.11
17 2.21 1.9 0.06 0.08
20 2.42 2.1 0.07 0.09
45 3 2.8 0.024 0.028
48 2.98 3.0 0.082 0.091
From the table above, rate of hydrolysis for both the substrate are faster initially in the first 8 hours, and then with more time, the rate of hydrolysis is reduced. Comparing both the washed and unwashed substrate, for unwashed substrate the kinetic rate of hydrolysis is not affected by time when compared with washed substrate. Thus, it can be further proved that rate of hydrolysis for unwashed substrate is not affected by time to get max sugar.
4 Conclusions
This study has demonstrated the application of using unwashed pretreated DES substrate for sugar production. The best DES for that produced highest glucose from sugarcane bagasse has been determined which is choline-chloride malonic acid. DES that is acidic in nature has been proved to be more suitable for producing high glucose yield, which although depends on the types of biomass that is being used. The glucose yield from enzymatic hydrolysis of unwashed pretreated biomass is comparable and higher than washed pretreated biomass. Besides, enhancement of enzymatic hydrolysis for unwashed pretreated biomass has been suggested such as using certain metal compounds which will further improve the glucose yield. With the feasibility of applying unwashed pretreated biomass, investigation on bioethanol production can be carried out in the future as it will be more economically viable as wastewater generation is minimized by the elimination of washing stage.
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