International Journal of Environment and Bioenergy, 2012, 3(2): 88-97

International Journal of Environment and Bioenergy

ISSN: 2165-8951
Florida, USA
Journal homepage: www.ModernScientificPress.com/Journals/IJEE.aspx
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; E-Mail: dyah_styarini@yahoo.com; Tel.:

+62-21-7560929; Fax: +62-21-7560549.
Article history: Received 12 June 2012, Received in revised form 7 August 2012, Accepted 8 August
2012, Published 16 August 2012.

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.

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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 (CH3CH2OH) 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 20th century,
although the use of concentrated acid to release sugars from wood dates to the early 19th century.
Klason, in 1906 is often credited as the first to use sulfuric acid to isolate lignin from wood (Sluiter et
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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 40 - 60 mesh (250
- 400 m) in size.
2.2. 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:
2.2.1. Method A (NREL method): Determination of extractives, structural carbohydrate and lignin

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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% H2SO4 for two hours at 30 oC. 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 oC 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 oC 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% H2SO4 and then measured by UV/Vis spectrophotometry at 205
nm. In the analysis of sugars content, the hydrolyzate was neutralized by adding CaCO3 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 (300 7.8 mm, BIO RAD,
Hercules, CA) at 65 oC using 5 mM H2SO4 as mobile phase at a flow rate of 0.6 mL/min and detected
with an refractive index (RI) detector (Waters 2414) at 40 oC (Sluiter et al., 2011).
2.2.2. 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% H2SO4 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 oC for 15 min. The mixture
was filtered and the solid residue was dried at 105 oC 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% NaClO2 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 oC for one hour. The addition of 2.4 mL
of 25% NaClO2 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 oC for 16 to 24 h. The dried solid residue that is
holocelluloce was then analyzed to obtain the -cellulose content.
In the analysis of -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
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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 oC
for 16 to 24 h and after cooling it was weighed as -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 3.2.
3.1. 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 10.08 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
r2 value for glucose and xylose at range concentration that has mentioned above are 0.9999 and 0.9999
(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 oC
for 3 h and the ash content of the EFB sample was 6.69%. The results of compositional analysis are
given in Table 1.

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30.00

10.080

9.443

93

25.00

20.00

MV

15.00

10.00

5.00

0.00

-5.00

-10.00
7.00

8.00

9.00

10.00
Minutes

11.00

12.00

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
Extractives
Total lignin
Acid insoluble ash
Cellulose
Hemicellulose

Content (%, w/w)

1.34 (0.0)
31.68 (0.19)
0.98 (0.04)
37.26 (4.32)
14.62 (2.13)

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3.2. Result from Method B

In the method B that also known as wood analytical method, Klason lignin, holocellulose and
-cellulose were obtained gravimetrically while hemicelluloce content was calculated by difference
between holocelluloce and -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
Klason lignin
Cellulose
Hemicellulose

2.11 (0.03)
31.14 (0.26)
34.75 (0.12)
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.
3.3. 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 (tcrit) two tailed at 95% confidence level. The null hypothesis will be accepted when t < tcrit .

.............................................. (1)
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Due to t value is smaller than tcrit , 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 tcrit (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
Lignin
Cellulose
Hemicellulose

Mean (%)
Method A
Method B
31.68
37.26
14.62

31.14
34.75
21.7

tcrit

The difference

2.52
1.01
5.65

3.18
2.78
2.78

Not significant
Not significant
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 84914). 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.
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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 2010-2012 between Indonesian Institute of Sciences, Korea Institute of Science and
Technology and Changhae Energeering Co., LTD supporting by KOICA, Republic of Korea.

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