International Journal of Environment and Bioenergy, 2012, 3(2): International Journal of Environment and Bioenergy

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1 International Journal of Environment and Bioenergy, 2012, 3(2): International Journal of Environment and Bioenergy Journal homepage: ISSN: Florida, USA Article Comparison of Two Analytical Methods for Compositional Analysis of Lignocellulosic Biomass for Bioethanol Production Dyah Styarini 1, *, Lucky Risanto 2, Yosi Aristiawan 1, Yanni Sudiyani 1 1 Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspiptek Serpong, 15314, Indonesia 2 Research and Development Unit for Biomaterials, Indonesian Institute of Sciences, Cibinong Science Center, Indonesia * Author to whom correspondence should be addressed; dyah_styarini@yahoo.com; Tel.: ; Fax: Article history: Received 12 June 2012, Received in revised form 7 August 2012, Accepted 8 August 2012, Published 16 August Abstract: Empty fruit bunch (EFB) of oil palm is one of potential lignocellulosic biomass for bioethanol production. An accurate biomass compositional analysis is needed to evaluate the conversion yields and process economics due to changes in feedstock or process design. The chemical components of EFB of oil palm that will be used as biomass in bioethanol production was determined by using two analytical procedures in two laboratories. Two-stage sulfuric acid hydrolysis is the most common procedure to fractionate biomass both for gravimetric and instrumental analysis. Both methods employed two stages sulfuric acid hydrolysis to determine the lignin content. The differences between the procedure are the solvents used in the determination of extractive compounds, length of time required to hydrolize the biomass and the procedure for measuring the cellulose content that in one method employs HPLC-RI while the other using gravimetric method. The purpose of this inter-laboratory comparison was to get the reliable data of chemical components composition of raw EFB. There are no significant differences of composition values for two major chemical components (lignin and cellulose) of EFB sample resulted from both methods while for hemicellulose content is significant different. Keywords: lignocelulosic biomass; interlaboratory comparison; compositional analysis; HPLC-RI; gravimetry.

2 89 1. Introduction Energy consumption has increased steadily over the last century as the world population has grown and more countries have become industrialized. Crude oil has been the major resource to meet the increased energy demand (Sun and Cheng, 2002). In view of continuously rising petroleum costs and dependence upon fossil fuel resources, considerable attention has been focused on alternative energy resources. Production of ethanol or ethyl alcohol (CH 3 CH 2 OH) from biomass is one way to reduce both the consumption of crude oil and environmental pollution (Demirbas, 2005). Among potential bioenergy resources, lignocellulosics have been identified as the prime source of biofuels and other value added products. Lignocelluloses comprise a large fraction of municipal solid waste, crop residues, animal manures, woodlot arisings, forest residues or dedicated energy crops. Focusing on residuals, it can be stated that lignocelluloses such as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world (Kusch et al., 2009). Empty fruit bunch (EFB) of oil palm is one of potential lignocellulosic biomass for bioethanol production. Indonesia as one of the largest palm oil exporter in the world has an abundant source of EFB to be processed into bioethanol. Lignocellulosic biomass is typically nonedible plant material composed primarily of the polysaccharides cellulose and hemicellulose. The third major component is lignin, a phenolic polymer that provides structural strength to the plant. The minor components in biomass can include protein, ash, organic acid and other nonstructural materials (Sluiter et al., 2010). Processing of lignocellulosics to ethanol consists of four major steps: pretreatment, hydrolysis, fermentation, and product purification. Hydrolysis converts carbohydrate polymers into monomeric sugars which are then fermented to ethanol. The goal of all pretreatment is to break the lignin seal and to disrupt the crystalline structure of cellulose in order to make cellulose more accessible to enzymes that convert the carbohydrate polymers into fermentable sugars (Kusch et al., 2009). Production costs of bioethanol from lignocellulosic biomass are still high. Accurate feedstock compositional analysis enables evaluation of conversion yields and process economics due to changes in feedstock or process design. Accurate measurement of biomass carbohydrate content is of prime importance because it is directly proportional to ethanol yield (L/mg) in biochemical conversion process (Sluiter et al., 2010). Analytical methods for compositional analysis of biomass have been widely available and continue to be developed so that the method of analysis become more efficient and enables summative mass closure to account all components in the raw material. Two-stage sulfuric acid hydrolysis is the most common procedure to fractionate biomass both for gravimetric and instrumental analysis. The use of two stage sulfuric acid hydrolysis for the analysis of lignin dates to the turn of the 20 th century, although the use of concentrated acid to release sugars from wood dates to the early 19 th century. Klason, in 1906 is often credited as the first to use sulfuric acid to isolate lignin from wood (Sluiter et

3 90 al., 2010). In recent years, sulfuric acid method has been developed that can be used to analyze the components of biomass as a feedstock for biofuel. Summative compositional analysis methods that have originated as wood lignin isolation procedures are empirical and differences in technique can affect the final results. It is very important to obtain reliable and comparable analytical result of lignocellulosic biomass composition as a feedstock for bioethanol production because it is related to the process that will be applied. In order to obtain reliable and comparable analytical result, analytical methods must be validated as fit for their purpose before used in the laboratory. Whenever possible validation should be achieved by means of collaborative trials that conform to a recognize protocol. Where possible all reported data should be traceable to reliable and well-documented reference materials, preferably certified reference material. Where certified reference material is not available, traceability to a definitive method should be established. Participating in proficiency testing schemes provide laboratories with an objectives means of assesing and demonstrating the reliability of the data they are producing (Thompson et al., 1993). The purpose of this inter-laboratory comparison was to get the reliable data of chemical components composition of raw EFB. At this interlaboratory comparison, each laboratory use a method of analysis commonly used in routine analysis. Both methods employed two stages sulfuric acid hydrolysis to determine the lignin content. The differences between the procedure are the solvents used in the determination of extractive compounds, length of time required to hydrolize the biomass and the procedure for measuring the cellulose content that in one method employs HPLC-RI while the other using gravimetric method. 2. Research Method 2.1. Raw Material EFB of oil palm was used as raw material. The raw material was air dried until its moisture content less than 10%, milled with milling machine, and sieved to retain particles of mesh ( µm) in size Analytical Method The chemical composition of the sample was determined by comparing two analytical methods that are NREL method and wood analytical method in a interlaboratory comparison. The description of each method is as follow: Method A (NREL method): Determination of extractives, structural carbohydrate and lignin

4 91 Raw EFB was extracted with ethanol for 8 h before hydrolysis in order to remove organic extractives (Tina,1994). The extractives free raw material of approximately 0.3 g was hydrolized with 3 ml of 72% H 2 SO 4 for two hours at 30 o C. After completing the first hydrolysis, the acid was then diluted to 4% by adding 84 ml of distilled water and second hydrolysis was completed in an autoclave at approximately 121 o C for one hour. After completion of the autoclave cycle, the sample was cooled at room temperature and then the mixture was filtered. The solid residue was dried and weighed to obtain the mass of solid residue. The dried solid residue was then placed to muffle furnace and heated at 575 o C for 3 h to get acid insoluble ash. The acid insoluble lignin content can be obtained by substracting the dry mass of solid with acid insoluble ash. Acid soluble liginin content was measured by diluting the hydrolizate with 4% H 2 SO 4 and then measured by UV/Vis spectrophotometry at 205 nm. In the analysis of sugars content, the hydrolyzate was neutralized by adding CaCO 3 and filtered with Minisart syringe filter before injected to HPLC system. Sugars content was measured by HPLC RI. Glucose and xylose were separated with an Aminex HPX 87H ( mm, BIO RAD, Hercules, CA) at 65 o C using 5 mm H 2 SO 4 as mobile phase at a flow rate of 0.6 ml/min and detected with an refractive index (RI) detector (Waters 2414) at 40 o C (Sluiter et al., 2011) Method B (wood analytical method): Determination of Klason lignin, holocellulose and alphacellulose Raw EFB was extracted with mixture of alcohol : benzene (1 : 2) for minimum 3 h before hydrolysis in order to remove organic extractives. The extractives free material will be analyzed to determine the Klason lignin and holocellulose content. In the analysis of Klason lignin, 0.5 g of free extractive sample was hydrolized with 7.5 ml of 72% H 2 SO 4 for 4 h at room temperature. After completing the first hydrolysis, the acid was then diluted by adding 280 ml of distilled water and second hydrolysis was completed in an autoclave at approximately 121 o C for 15 min. The mixture was filtered and the solid residue was dried at 105 o C for 16 to 24 h. The dried solid residue was then weighed. In the analysis of holocellulose, 90 ml of distilled water, 2.4 ml of 25% NaClO 2 and 0.12 ml of acetic acid glacial 100% were poured into an erlenmeyer containing 1.5 g of extractives free raw material. The mixture was then heated in the waterbath at 80 o C for one hour. The addition of 2.4 ml of 25% NaClO 2 and 0.12 ml of acetic acid glacial 100% was done twice untill the EFB powder colour change become white. The mixture was then filtered. The solid residue was washed with 250 ml of cold water and acetone and then dried at 105 o C for 16 to 24 h. The dried solid residue that is holocelluloce was then analyzed to obtain the α-cellulose content. In the analysis of α-cellulose, 12.5 ml of NaOH 17.5% was poured to an erlenmeyer containing 0.5 g of dried holocellulose. The mixture was stirred for 30 min and added with 12.5 ml of

5 92 distilled water. The mixture was then filltered. The solid residue was washed with distilled water, 20 ml of acetic acid glacial 10% and 500 ml of hot water. The solid residue obtained was dried at 105 o C for 16 to 24 h and after cooling it was weighed as α-cellulose (Mokushitsu Kagaku Jiken Manual, 2000). 3. Results and Discussion Two analytical methods are compared utilize a strong sulfuric acid solution in a primary hydrolysis followed by second hydrolysis by dilution with water and heating at high temperature. The procedures hydrolize the polymeric carbohydrates into soluble monosacharides and leaving behind a lignin-rich residue that is vacuum filtered and measured gravimetrically (Sluiter et al., 2010). The results of compositional analysis obtained from both analytical methods compared are decribed in the sections 3.1 and Result from Method A Method A has been verified in order to determine total lignin, acid insoluble ash and sugars (glucose and xylose). Seven replicates of raw EFB sample derived from different batch of the sample used in the interlaboratory comparison were analyzed. The repeatability (%RSD) of the method for the total lignin, acid insoluble ash, glucose and xylose from seven replicate of analysis are 1.45, 2.73, 1.00 and 3.17%, respectively. Verification of the analytical instrument, HPLC-RI, was done. The chromatogram of glucose (retention time 9.44 min) and xylose (retention time min) can be seen in Fig. 1. The precision of the area of glucose and xylose were 0.23 and 0.26%, and the precision of the retention time of glucose and xylose were 0.01 and 0.01%, respectively. Response of the RI detector has been evaluated for the glucose and xylose solutions at range concentration of 0.10 to 1.10 %. The r 2 value for glucose and xylose at range concentration that has mentioned above are and (Fig. 2). The concentrations of glucose and xylose were used for calculating the cellulose and hemicellulose contents respectively. The concentrations of polymeric sugars were calculated from the concentration of the corresponding monomeric sugars, using an anhydro correction of 0.88 for C-5 sugars (xylose) and a correction of 0.90 for C-6 sugar (glucose) (Sluiter, et al., 2011). The EFB sample used in the interlaboratory comparison was analyzed in three replicates. The moisture content of the sample that is 8.69% was determined to calculate the chemical component of the sample in dry base. The ash of the sample was determined by ashing 0.5 g of EFB sample at 575 o C for 3 h and the ash content of the EFB sample was 6.69%. The results of compositional analysis are given in Table 1.

6 MV Minutes Figure 1. Chromatogram of glucose and xylose. Figure 2. Respon of RI detector for glucose and xylose at range concentration of 0.1 to 1.2%. Table 1. Mean of chemical composition of the raw EFB with the standard deviations in parentheses from method A Component Content (%, w/w) Extractives 1.34 (0.0) Total lignin (0.19) Acid insoluble ash 0.98 (0.04) Cellulose (4.32) Hemicellulose (2.13)

7 Result from Method B In the method B that also known as wood analytical method, Klason lignin, holocellulose and α-cellulose were obtained gravimetrically while hemicelluloce content was calculated by difference between holocelluloce and α-cellulose content. The moisture content of the sample derived from triplicate analysis was 5.07%. Three portions of sample were analyzed by using method B. The results of compositional analysis from method B are given in Table 2. Table 2. Mean of chemical composition of the raw EFB with the standard deviations in parentheses from method B Component Content (%, w/w) Extractives 2.11 (0.03) Klason lignin (0.26) Cellulose (0.12) Hemicellulose 21.7 (0.4) The comparison of compositional analysis results from both methods is shown in Fig. 3. Based on the chart it can be seen that the contents of lignin, cellulose and extractives resulted from both methods are almost the same, while hemicellulose results from both method are slightly different. In general, the results are comparable. Statistical tool is needed to ensure whether there is an significant difference among the results from both methods or not Statistical Tools for Significant Test T-test is a statistical significant test that can be used to compare two mean values. In order to decide whether the difference between sample means is significant, that is to test null hypothesis. The term null is used to imply that there is no difference between the mean values other than that which can be attributed to random variation (Miller et al., 1993 ). It can be written as is follow: t-statistic value (t) can be calculated by using equation 1. The t-statistic is then compared to critical value (t crit ) two tailed at 95% confidence level. The null hypothesis will be accepted when t < t crit.... (1)

8 95 Due to t value is smaller than t crit, two major components of EFB that are lignin and cellulose contents resulted from both methods are not significant different, while the other component, hemicellulose, is significant different because the t value is bigger than t crit (Table 3). Figure 3. Comparison of compositional analysis results from both methods. Table 3. t-test result for components result from methods A and B Component Mean (%) t t crit The difference Method A Method B Lignin Not significant Cellulose Not significant Hemicellulose significant These biomass compositional analysis methods are empirical so that the final results depend on how the method is run. Accuracy of the method can be evaluated by using reference material (RM). Due to the lack of RM, these methods were not verified for the accuracy parameter yet. In the future, verification of these methods using RM have to be done, to evaluate the performance of the method and the system in the laboratory including analysts and analytical instrument that used. Empirical methods can be compared using reference materials (RM) such as the National Institute of Standards and Technology (NIST) biomass reference materials (RMs ). It is difficult to compare method errors (measured difference from a true value) without an accepted true standard. Taylor and Kuyatt state, A measurement result is complete only when accompanied by a quantitative statement of its uncertainty. The uncertainty is required in order to decide if the result is adequate for its intended purpose and to ascertain if it is consistent with other similar results (Templeton, et. al., 2010). The results of compositional analysis from both methods are comparable. Both Klason lignin method and NREL method can be applied in every laboratory adjusted to the facility of each laboratory.

9 96 4. Conclusions In this work, interlaboratory comparison was conducted in order to get the reliable data for chemical component of EFB. Two different methods were used in the compositional analysis of EFB. The three major components that are lignin, cellulose, hemicellulose, and extractive content derived from both methods were compared to each other. In order to evaluate the difference between mean value for each component, t-test was performed. Two major components of EFB that are lignin and cellulose contents resulted from both methods are not significant different, while the other component, hemicellulose, is significant different. Both two methods are reliable for getting the information of the composition of biomass feedstock that to be used in bioethanol production. The description of analytical procedure, analysis result and discussion in this paper is expected can be used for the laboratory as a relevant consideration in choosing the relevant analytical method that is suitable to be applied. This is closely related to the facilities in the laboratory, including the available instrumentation, the analysis time, sample loading capacity and human resources. Acknowledgment This work is part of the Research Laboratory for Energy, Environment and Natural Substances project between Indonesian Institute of Sciences, Korea Institute of Science and Technology and Changhae Energeering Co., LTD supporting by KOICA, Republic of Korea. References Demirbas, A. (2005). Bioethanol from cellulosic materials: A renewable motor fuel from biomass. Ener. Sourc., 27: Harifara, R., Sumiko, A., and Shiro, S. (2011). Quantitative method applicable for various biomass species to determine their chemical composition. Biomass Bioener., 35: Kusch, S., and Morar, M. V. (2009). Integration of lignocellulosic biomass into renewable energy generation concepts. ProEnvironment, 2: Miller, J. C., and Miller, J. N. (1993). Statistics for Analytical Chemistry, 3rd Ed. Ellis Horwood, Chichester, p Mokushitsu Kagaku Jiken Manual. (2000). Japan Wood Research Society Publisher. Sluiter, J. B., Ruiz, R. O., Scarlata, C. J., Sluiter, A. D., and Templeton, D. W. (2010). Compositional analysis of lignocellulosic feedstocks. 1. Review and description of methods. J. Agric. Food Chem., 58: Sluiter, B., Hames, R., Ruiz, C., Scarlata, J., Sluiter, D., Templeton, M., and Crocker, D. (2011). Determination of structural carbohydrates and lignin in biomass. Technical report NREL/TP-510-

10 Sung, Y., and Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: A review. Biosour. Technol., 83: Templeton, D. W., Scarlata, C. J., Sluiter, J. B., and Wolfrum, E. J. (2010). Compositional analysis of lignocellulosic feedstock. 2. Method uncertainties. J. Agric. Food Chem., 58: Thompson, M., and Wood, R. (1993). The international harmonized protocol for the proficiency testing of (chemical) analytical laboratories. Pure Appl. Chem., 65: Tina, E. (1994). Standard method for the determination of extractives in biomass. NREL Laboratory Analytical Procedure, LAP-010.