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METHODS FOR DETERMINATION OF CHEMICAL COMPOSITION OF PLANT

BIOMASS

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 Journal SITA, 2015, 17(4], pp. 208-214

METHODS FOR DETERMINATION OF CHEMICAL COMPOSITION OF PLANT

BIOMASS
Michael Ioelovich
Designer Energy Ltd, 2 Bergman Str., Rehovot 7670504 (ISRAEL)

E-mail: bd895892@zahav.net.il

ABSTRACT
Improved methods for the quantitative analysis of polysaccharides and lignin in biomass samples
were developed by Designer Energy Ltd (DE). The DE method for determination of
polysaccharides in biomass samples is based on the isolation of holocellulose, i.e. total
polysaccharides containing both cellulose and hemicelluloses. After acid hydrolysis of holocellulose
under mild conditions hemicelluloses were removed, and as a result the content of cellulose can be
determined. The content of acid-insoluble lignin was measured by improved method after two-stage
acidic hydrolysis of the biomass. In order to prevent loss of the components, a centrifugation
technique was used for isolation of final products. The developed methods were used for analysis of
chemical composition of crude and pretreated samples of switchgrass and sugarcane bagasse. It has
been shown that DE methods provide more reliable results than conventional methods of chemical
analysis.

Keywords: Biomass, Chemical composition, Polysaccharides, Lignin, Quantitative analysis

INTRODUCTION
In recent years a considerable attention has been given to non-edible plant biomass as a
renewable source for production of green energy, bioproducts and biochemicals. The various
biomass types involve residues of agricultural plants (e.g. stalks, husks, cobs, etc.), forest
residues (e.g. sawdust, twigs, shrubs, etc.), waste of wood, textile, pulp, paper and cities, as well
as some plant species (e.g. Miscanthus, Switchgrass, Bermuda grass, etc.). Agriculture, forestry,
pulp and paper industry, as well as cities create vast amounts of lignocellulosic residues.
Moreover, huge amounts of algae are not utilized yet. These materials are related to abundant,
renewable and inexpensive biomass types. Total amount of the plant biomass that is accumulated
annually in the world is estimated in 10 billion tons at least. Only in USA annual accumulation
of the biomass is about 1 billion tons [1]. In general, the biomass can be used as feedstock for
production of liquid biofuel for vehicles and as solid fuel for burning and gasification in order to
generate heat, steam, and electricity, and also as feedstock for manufacturing of various
bioproducts and biochemicals [2, 3]. To choose the optimal path for utilization of a certain
biomass type it is necessary to know its chemical composition. Any type of the biomass consists
of three basic plant polymers: cellulose, hemicelluloses and lignin. The other components of the
biomass can be mineral substances, organic extractives (waxes, fats, oils), pectin, starch, proteins
and some other admixtures.
Cellulose is the most abundant organic matter on Earth [4]. The main sources of cellulose
are plants. The content of cellulose in herbaceous plants is 30 to 40%, in woods 45 to 50%, in
bast plants (flax, ramie, jute, etc.) 60 to 70% and in cotton fibers upwards of 90% [5]. Cellulose
is a linear, stereoregular, semicrystalline polysaccharide composed of D-glucopyranosic units
linked by chemical β-1,4-glycosidic bonds. The linear macromolecules joined by hydrogen
bonds form supermolecular structure of cellulose that consists of thread-like elementary
nanofibrils and their bundles called microfibrils. The cellulose microfibrils form layers and walls
of plant cells (fibers).
Hemicelluloses are attributed to non-cellulose polysaccharides. Their content in various
plants can be in the range 10 to 40% [6]. Hemicelluloses are hydrophilic amorphous
heteropolymers. In addition to physical bonding of cellulose, hemicelluloses also form ester
bonds with lignin. The chemical structure of hemicelluloses consists of chains of a variety of
acetylated links of pentoses or hexoses and backbones. Various plants and also its tissues contain
different types of hemicelluloses. Agricultural and herbaceous plants, as well as hardwoods
contain mainly the pentosan-type hemicellulose xylan, while softwoods are enriched with the
hexosan-type hemicellulose mannan.
Lignin is a rigid, aromatic, amorphous and hydrophobic polymer [7]. The content of lignin
in plant biomass ranges from 10% for corn cob or rice straw to 48% for olive husk [6, 8]. Lignin
is a complex polymer of phenylpropane units (guaiacyl, syringyl and hydroxyphenyl), which are
cross-linked to each other with a variety of different chemical bonds. In the lignified plant
biomass, one portion of lignin is localized inside the cell (fiber) wall in the form of thin
hydrophobic nano-layers, surrounding the cellulose microfibrils and hemicelluloses, while
another portion of lignin is localized between the cells (fibers) in the middle lamella.
The chemical composition of the various types of plant biomass can be determined by
conventional methods of chemical analysis. However researchers of different countries, different
methods and procedures are used in order to determine the composition of the biomass samples.
For example, the percentage of acid-insoluble lignin in the plant biomass can be determined by
means of TAPPI method T222 [9], NREL method [10], SCAN INNVENTIA method [11], etc.
The content of polysaccharides (cellulose, xylan and other hemicelluloses) can be found using
TAPPI method T249 [12], NREL method LAP 002 [13], SCAN method CM 71:09 [14], method
of holocellulose isolation [15, 16], etc. Since different methods may give different results, a
comparison of these methods is required.

The main purpose of this paper was to compare different methods of chemical analysis of
biomass in order to develop an improved scheme and analytical procedure for the quantitative
determination of lignin and polysaccharides in biomass samples.

EXPERIMENTAL
Materials
Two plant materials – switchgrass (Nott Farms, Canada) and bagasse of sugarcane (Cosan,
Brazil), were used as initial biomass samples. The initial biomass samples were dried, cut on
small pieces (5-10 mm) and pretreated with boiling 3 wt.% sulfuric acid and 2 wt.% alkali -
sodium hydroxide. About 100 g samples were put in 2 L lab glass beakers and then reagent
solution was added up to liquid/solid ratio of 7. The beakers containing the biomass and
reagent solution were placed on a heating plate, heated up to boiling and treated for 1h while
stirring. The pretreated samples were washed up to neutral pH, squeezed on a vacuum glass-
filter No1 and dried at 105oC to constant weight. Initial and pretreated biomass samples were
knife-milled and screened through a 40 mesh sieve to obtain the fraction 0.4 mm.

Besides, ashless Whatman filter paper No 42 containing 98% α-cellulose was used as a
standard sample.
Lab equipment
The following lab equipment was used: analytical balance readable to 0.1 mg; convection
oven with temperature control; desiccator containing anhydrous silica gel; plastic and glass ware
(PP-tubes, Erlenmeyer flasks, beakers, funnels, reflux condenser, etc.); water bath; heating
plates; centrifuge equipped with a rotor for 50 mL tubes that provides the rcf of 4000-5000 g
(e.g. Eppendorf centrifuge 5804 equipped with rotor A-4-44); electric muffle furnace; porcelain
crucibles; Soxhlet apparatus; rotary evaporator; etc.

Chemical analysis
Chemical analysis of biomass samples was performed by conventional TAPPI and NREL
methods. To determine content of acid-insoluble lignin, the TAPPI T222 method [9] provides
pre-hydrolysis of the extracted sample with 72 wt.% sulfuric acid at 20oC for 2 h; then the
concentrated acid was diluted with water, and the sample was hydrolyzed with dilute 3 wt.% acid
at boiling temperature for 4 h. After overnight, the acidic dispersion of lignin was filtered
through glass-filter having average pore diameter of 10 μm. Sediment of lignin was washed on
the filter with hot water up to pH=7 and then dried at 105 oC to constant weight. In contrast to
TAPPI T222 [9], the method of NREL LAP 003 [10] provides the use a low amount of the
sample and low volume of 72 wt.% sulfuric acid; besides the pre-hydrolysis was carried out at
30oC for 2 h. After diluting of concentrated acid with water to 4 wt.%, the sample was
hydrolyzed in crimp seal glass bottles at 121oC for 1 h. Then, the acidic dispersion of lignin was
filtered through glass-filter having average pore diameter of 10 μm. Sediment of lignin was
washed on the filter with hot water up to pH=7 and then dried at 105 oC to constant weight.
To determine the content polysaccharides, the TAPPI T 249 method [12] requires a multi-
step procedure: extraction of the extracted biomass sample; acid hydrolysis of polysaccharides;
filtration and neutralization of the hydrolyzate; reduction of monosaccharides with sodium
borohydride into alditols; acetylation of alditols; extraction of acetylated alditols with methylene
chloride and analysis by gas chromatography. The NREL method LAP 002 [13] is a simple: the
extracted sample was hydrolyzed into monosaccharides; the hydrolyzate was neutralized with
calcium carbonate and after filtration the concentration of glucose and other sugars was
measured using HPLC.
Furthermore, an improved DE method of chemical analysis was proposed by Designer
Energy Ltd (DE), which is described below. All samples were analyzed in duplicate by all used
methods to calculate the standard deviation (SD).

RESULTS AND DISCUSSION

General scheme of chemical analysis of biomass samples is shown in Fig. 1. The biomass
samples were dried, milled and screened to obtain the 40 mesh fraction having total weight of 15
g. About 2 g of the each sample were used for determination of ash content in duplicate using
method of NREL LAP 005 [17]. Other portion of the samples, 13 g, was extracted with organic
solvents in order to remove extractives and determine their content in accordance with NREL
LAP 010 procedure [18]. The extracted samples were used to determine the content of lignin,
cellulose and hemicelluloses (hemi-cel) by various methods.
The experiments have shown that the determination of acid-insoluble lignin in biomass
samples by method of TAPPI T222 [9] requires a long time (two days); besides this method
gives a reduced content of lignin due to penetration of submicron particles of lignin through the
glass-filter. The method of NREL LAP 003 [10] is faster, but also this method does not prevent
the loss of submicron particles of lignin (Fig. 2).

Biomass

Drying

Ash Milling & Screening

Lignin Extraction Extractives

Holocellulose

Cellulose Hemi-Cel

Figure 1: Scheme of chemical analysis of biomass

To overcome the shortcomings of the conventional methods, Designer Energy Ltd (DE)
proposed an improved procedure to determine the content of acid-insoluble lignin in biomass
samples. The extracted biomass sample, 0.3 g, was mixed with 5 mL of 72 wt.% sulfuric acid in
100-ml Erlenmeyer flask and pre-hydrolyzed at 25oC for 2 h using a water bath. The
concentrated acid was diluted with 45 mL distilled water, and the sample was hydrolyzed with
dilute acid on a heating plate at boiling temperature for 2 h using a reflux condenser. After
cooling at room temperature for 30 min, the acidic dispersion of lignin was poured out into 50
mL PP-tubes and centrifuged at rcf of 4500 g for 15 min. The experiments have shown that
under the used centrifugation conditions the particles of lignin settle completely, and the liquid
phase becomes transparent. As follows from sedimentation experiments under the used
centrifugal force, a minimum particle radius of acid-insoluble lignin is estimated at 100 nm.
The sediment of lignin was washed with hot water (cca 50oC), 5 wt.% sodium bicarbonate
and finally with distilled water to a pH 7, separating the liquid phase from lignin by
centrifugation. The washed lignin was dried in the PP-tube at 105oC to constant weight. The
percentage of acid-insoluble lignin (AIL) in the extracted biomass sample was calculated by the
equation:

AIL = 100% (P - Pt)/Ps (1)

where P is weight of dry acid-insoluble lignin together with PP-tube; Pt is weight of empty PP-
tube; and Ps is weight of extracted and dried biomass sample.
 22

20

18

AIL, %
TAPPI
16
NREL
14 DE

12

10
SG BAG

Figure 2: Percentage of acid-insoluble lignin (AIL) in extracted switchgrass (SG) and bagasse (BAG)
samples measured by methods of TAPPI, NREL and DE

Through the use of centrifugation technique, the proposed DE method prevents the loss of
lignin particles, and therefore this method provides a more reliable content of acid-insoluble
lignin, which is higher than can be obtained by conventional methods (Fig. 2).
To test various methods for determination the polysaccharides in biomass, the experiments
were performed with Whatman filter paper (FP) containing 98% α-cellulose. The results showed
that NREL method [13] gave 92% cellulose, whereas TAPPI method [12] 88% cellulose only
(Fig. 3). Thus, the measurement of polysaccharides by multistage TAPPI method [12] is not
sufficiently reliable, which is probably due to decrease in the output of intermediate products
after each subsequent stage. The NREL LAP 002 method [13] is a simpler and more accurate;
but also this method has several shortcomings, such as (1) partial degradation of carbohydrates at
the step of high-temperature hydrolysis, and (2) non-productive adsorption of obtained sugars on
the surface of precipitated calcium sulfate.

100

95
Cellulose, %

90

85

80
TAPPI NREL DE

Figure 3: Percentage of cellulose in FP measured by methods of TAPPI, NREL and DE

 Due to shortcomings of conventional methods, Designer Energy Ltd (DE) developed its
own alternative method for determination polysaccharides in biomass samples, which is based on
the isolation of holocellulose, i.e. total polysaccharides containing both cellulose and
hemicelluloses.
The extracted biomass sample, 0.5 g, was placed into 100-ml Erlenmeyer flask, and then 40
mL distilled water, 0.5 g sodium chlorite (NaClO2) and 1 mL glacial acetic acid were added into
the flask. The flask covered with Petri dish was put into a water bath having temperature 90oC
and treated for 45 min while stirring. Then to the flask an additional portion of 0.5 g sodium
chlorite and 1 mL acetate buffer was added, and the treatment was continued again 45 min. After
cooling at room temperature for 30 min, a dispersion of holocellulose was poured out into 50 mL
PP-tubes and centrifuged at rcf of 4500 g for 10 min. The sediment of holocellulose was washed
with hot water (cca 50oC), and finally with distilled water to a pH 7, separating the liquid phase
from holocellulose by centrifugation. The washed holocellulose was dried in the PP-tube at
105oC to constant weight. The percentage of holocellulose (HC) in the extracted biomass sample
was calculated by the equation:
HC = 100% (P - Pt)/Ps (2)
where P is weight of dry holocellulose together with PP-tube; Pt is weight of empty PP-tube; and
Ps is weight of extracted and dried biomass sample.

In order to find the content of cellulose, the obtained holocellulose was hydrolyzed with
dilute hydrochloric acid to remove hemicelluloses. The dried holocellulose sample was mixed
with 45 mL of 2 wt.% HCl in 100-ml Erlenmeyer flask, and the sample was hydrolyzed with the
dilute acid at boiling temperature for 2 h using a reflux condenser. After cooling at room
temperature for 30 min, an acidic dispersion of cellulose was poured out into 50 mL PP-tubes
and centrifuged at rcf of 4500 g for 10 min. The sediment of cellulose was washed with hot
water (cca 50oC), 1 wt.% sodium bicarbonate and finally with distilled water to a pH 7,
separating the liquid phase from cellulose by centrifugation. The washed cellulose was dried in
the PP-tube at 105oC to constant weight. The percentage of cellulose (C) and hemicelluloses (H)
in the extracted biomass sample was calculated by the equations:
C = HC (P - Pt)/Ps (3)
H= HC – C (4)

where HC is percentage of holocellulose; P is weight of dry cellulose together with PP-tube; Pt is

weight of empty PP-tube; and Ps is weight of extracted and dried biomass sample.
The experiment with Whatman filter paper (FP) showed that DE method gave 97.6%
cellulose, which was close to content of α-cellulose 98% in FP (Fig. 3).
Using the proposed DE methods for determination of acid-insoluble lignin (AIL), cellulose
(C) and hemicelluloses (H), as well as conventional NREL methods for determination of
extractives (E), ash (A) and acid-soluble lignin (ASL), a full analysis of chemical composition of
the investigated biomass samples was performed (Table 1, 2).

Table 1: Chemical composition of initial (SG), acid (SG-AC) and alkali (SG-AL) pretreated
switchgrass samples
Sample C, % H, % AIL, % ASL, % E, % A, %
SG 38.2 28.3 18.5 3.2 4.8 5.6
SG-AC 56.3 7.0 27.1 1.3 3.1 3.2
SG-AL 64.1 21.2 10.8 1.0 1.4 1.0
Table 2: Chemical composition of initial (BAG), acid (BAG-AC) and alkali (BAG-AL)
pretreated bagasse samples

Sample C, % H, % AIL, % ASL, % E, % A, %

BAG 39.1 27.3 20.2 3.1 4.5 5.2
BAG-AC 53.2 9.1 28.0 2.0 1.8 4.3
BAG-AL 62.0 20.2 14.3 1.1 1.2 1.0

As can be seen from the results, initial bagasse contains some more acid-insoluble lignin
than initial switchgrass. The acidic pretreatment of the biomass samples caused the removal of
main part of hemicelluloses and forming cellolignin having an increased content of lignin and
also cellulose. In contrast to acidic pretreatment, alkaline pretreatment of the samples caused the
decrease both of hemicelluloses and lignin; simultaneously, appreciable increasing of cellulose
content was observed.

CONCLUSION
In this paper a comparative study of various methods for analysis of chemical composition
of biomass has been carried out. Improved methods for the quantitative analysis of
polysaccharides and lignin in biomass samples were developed by Designer Energy Ltd (DE).
The DE method for determination of polysaccharides in biomass samples is based on the
isolation of holocellulose, i.e. total polysaccharides containing both cellulose and hemicelluloses.
After acid hydrolysis of holocellulose under mild conditions hemicelluloses were removed, and
as a result the content of cellulose can be determined. The content of acid-insoluble lignin was
measured by improved method after two-stage acidic hydrolysis of the biomass. In order to
prevent loss of the components, a centrifugation technique was used for isolation of final
products. The developed methods were used for analysis of chemical composition of crude and
pretreated samples of switchgrass and sugarcane bagasse. As can be seen from the results, initial
bagasse contains some more acid-insoluble lignin than initial switchgrass. The acidic
pretreatment of the biomass samples caused the removal the main part of hemicelluloses and
forming cellolignin having an increased content of lignin and also cellulose. In contrast to acidic
pretreatment, alkaline pretreatment of the biomass caused the decrease both of hemicelluloses
and lignin; simultaneously, appreciable increasing of cellulose content was observed. It has been
shown that DE methods provide more reliable results than conventional methods of chemical
analysis.

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