Holocellulose Determination in Biomass

Harifara Rabemanolontsoa and Shiro Saka

Abstract For determination of holocellulose, the residual yield and its chemical
composition during delignification by acidified sodium chlorite have been studied
for bamboo (Phyllostachys heterocycla) and Sargassum (Sargassum horneri).
It was then found that along with the extended number of chlorination, the residue
became yellowish, and then whitish. Accordingly, the holocellulose yield was
reduced. Thus, in order not to lose any part of the holocellulose, the number of
chlorination was found to be minimized so as for the residue to remain yellowish.
Subsequently, lignin and ash corrections were made on the yellowish residue to
determine the accurate holocellulose content. Such a modified procedure for the
holocellulose determination was proposed in this study.

Keywords Ash correction Carbohydrate Lignin correction Lignocellulosics

Sodium chlorite

1 Introduction

Sustainable and economic growth requires replacement of the massive use of fossil
resources to renewable ones such as biomass for production of fuels, chemicals and
materials. For those applications, one of the most interesting components of
biomass is carbohydrate which is composed of cellulose and hemicellulose, and
their sum is called holocellulose. By definition, holocellulose is composed of the
total polysaccharide fraction of extractives-free biomass and it is the starting-point
of most work in carbohydrate study of biomass. Its accurate determination is
therefore very important.

H. Rabemanolontsoa S. Saka (*)

Graduate School of Energy Science, Kyoto University,
Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
e-mail: saka@energy.kyoto-u.ac.jp

T. Yao (ed.), Zero-Carbon Energy Kyoto 2011, Green Energy and Technology, 135
DOI 10.1007/978-4-431-54067-0_14, # Springer 2012
136 H. Rabemanolontsoa and S. Saka

Holocellulose assays were proposed by Van Beckum and Ritter [1] as the
chlorineethanolamine procedure, by Wise et al. [2] as the chlorite method, and
by Poljak [3] as the peracetic acid method but the Wise chlorite technique has been
the most widely used. It consists of performing successive treatments of acid
chlorination to the sample in order to remove lignin. Improvements of the Wise
method were suggested [4] and the number of chlorination for hardwoods and
softwoods were settled to be respectively three and four times [5]. As chlorite
method was initially dedicated for wood species, the question arises on the extent of
chlorination to be applied on other non-woody biomass species.
Therefore, in this work, the chemical composition of the residual yield has been
studied during delignification by acidified chlorite for bamboo (Phyllostachys
heterocycla) as well as Sargassum (Sargassum horneri) and a revised new method
on holocellulose determination in various biomass species was, thus, proposed.

2 Materials and Methods

Bamboo (Phyllostachys heterocycla) and Sargassum (Sargassum horneri) as

respective representatives of monocotyledonous angiosperms and algae were used
in this study. Sargassum, collected from the western Wakasa bay in the Japan Sea
was washed with fresh water and then freeze-dried, while bamboo collected from
Kyoto, Japan was air-dried.
The samples were ground with Wiley mill (1029-C, Yoshida Seisakusho Co.,
Ltd.), and sieved to retain particles of 150500 mm (35100 mesh) and then
Soxhlet-extracted with acetone until the solvent was clear of any color. On the
extractives-free samples, holocellulose was quantified with sodium chlorite treat-
ment according to the procedure of Wise et al. [2] as adapted by Timell [6] with
slight modifications. In brief, 150 ml of 0.2 M acetic acid buffer was poured on 2.5 g
of extractives-free sample. Then, 1 g of sodium chlorite was added, followed by
0.2 ml of glacial acetic acid and the sample was put in a water bath for 1 h at
7080 C. Sodium chlorite with acetic acid was further repeated for successive
cycles of chlorination. After the necessary number of chlorination, the solution
was filtered, washed with 500 ml of cold water followed by 50 ml of acetone and the
holocellulose content as the remaining residue was determined gravimetrically.
Lignin in holocellulose as the sum of Klason and acid-soluble lignin was deter-
mined according to a modified Klason method [7], while ash content was quanti-
fied as the sample residue after ignition at 600 C for 4 h. Protein was also measured
according to the Kjeldahl nitrogen method by using a nitrogen factor of 6.25 [8].
Holocellulose Determination in Biomass 137

3 Results and Discussion

3.1 Aspects of Chlorite-Delignified Residues

Figure 1 shows examples of chlorite-delignified residues after different degrees of

chlorination. For bamboo, the sample had a pronounced yellow color after the first
treatment of chlorination. However, as chlorination has been repeated, it became
less yellowish and the residue finally became whitish after nine times of chlorina-
tion with yield of 55.2 wt%.
As for Sargassum, the first chlorination generated a yellow-greenish residue,
while second chlorination produced yellowish residue, and additional treatments
of chlorination resulted in whitish, and finally clear white residue after seven times
of chlorination, as observed in Fig. 1. It is, therefore, understandable that pure white
residues could only be recovered after nine times of chlorination for bamboo and
seven times for Sargassum.
For holocellulose determination, some authors recommended to repeat chlorina-
tion until the sample turns whitish [9, 10]. However, it was widely demonstrated
that lignin removal during chlorite delignification might also engender some loss of
the polysaccharide portion [11]. Therefore, in order to define an accurate
holocellulose determination, the pattern of the biomass components removal during
chlorite treatment must be studied.

Fig. 1 Delignified residues of (a) bamboo and (b) Sargassum after different degrees of
chlorination
138 H. Rabemanolontsoa and S. Saka

a b
100 100
90
Bamboo Lignin 90
Sargassum
80 Ash 80 Lignin
70 70
Ash
60 60
50 50
Protein
40 40
Holocellulose
30 30
20 20
10 10 Holocellulose
0 0
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
Number of chlorination

Fig. 2 Yield change of the cell wall components in (a) bamboo and (b) Sargassum during chlorite
delignification

3.2 Yield Change of Cell Wall Components During Chlorite

Delignification

The chemical composition of the residues from bamboo and Sargassum during the
course of chlorite delignification is shown in Fig. 2. The values corresponding to
zero chlorination represent the chemical composition of the untreated samples.
The residue from chlorite delignification, also called crude holocellulose, is
expressed in wt% of the original oven-dried sample. The holocellulose yield was
calculated by subtracting the lignin, ash and protein in the residue from the total
crude holocellulose. However, as bamboo had negligible protein, protein was not
studied in its crude holocellulose.
In Fig. 2, the crude holocellulose decreased in its yield, while the number of
chlorination was augmented. However, it is apparent that chlorite treatment on
bamboo dissolved lignin selectively only for the first five times of chlorination, and
on further treatments, the holocellulose decreased noticeably in its yield,
confirming that extended chlorination removes carbohydrate as well in bamboo,
as already reported in wood [11]. On the contrary, inorganics as ash remain in the
crude holocellulose.
As it is apparent from Fig. 2, the trends on the components recovery in Sargas-
sum are quite different from the ones in bamboo. In Sargassum, crude holocellulose
retained a considerable amount of protein, ash as well as lignin, and ash content is
getting even higher as chlorination was extended, supporting the fact previously
demonstrated [12] as the chlorite treatment contaminates the samples with
inorganics. In addition, carbohydrate loss in Sargassum is noticed to some extent
after two times of chlorination, and become drastic after seven times of
chlorinations when most of the lignin was dissolved.
To prevent such carbohydrate loss from the samples, chlorite treatment of bam-
boo and Sargassum was, therefore, respectively minimized to five and two times of
Holocellulose Determination in Biomass 139

Oven-dried sample

Acetone extraction

Extractives-free sample
NaClO2

Crude holocellulose
Lignin, Ash
Protein

Holocellulose

Fig. 3 Revised new procedure for holocellulose determination

chlorination. Furthermore, lignin and ash were all independently determined on the
residue. Additionally, protein correction was done on crude holocellulose from
Sargassum. As a result, as reported previously [12], the holocellulose content was
determined to be 70.5 wt% in bamboo and 23.9 wt% in Sargassum.

3.3 Holocellulose Determination in Biomass

For an adequate determination of holocellulose, chlorination should be enough to

remove lignin but not excessive to avoid a loss of carbohydrates. Therefore, the
degree of chlorination should be minimized so as for the sample to be still
yellowish. However, such yellowish residue possibly contains lignin and ash etc.,
such corrections should be done on the residue. In addition, for samples with high
protein content, protein correction has to be done also. Such method is illustrated in
Fig. 3, representing the revised new procedure for holocellulose determination.

4 Concluding Remarks

The results of this study showed that along with the chlorite delignification, parts of
the carbohydrates were lost, while inorganics and protein still remained in the
residues. Therefore, for biomass characterization, the treatment should be minimized
so as for the sample to be still yellowish. Ash and lignin corrections as well as protein
correction if necessary should subsequently be performed on the yellowish residue to
obtain reliable results.

Acknowledgement This work was accomplished under financial support from Kyoto University
Global COE Energy Science Program. The authors wish to thank the Kyoto prefectural marine
research center for kindly providing the Sargassum sample.
140 H. Rabemanolontsoa and S. Saka

References

1. Van Beckum WG, Ritter GJ (1937) Rapid methods for the determination of holocellulose and
cross and bevan cellulose in wood. Pap Trade J 104(19):4950
2. Wise LE, Murphy M, DAddieco AA (1946) Chlorite holocellulose, its fractionation and
bearing on summative wood analysis and on studies on the hemicelluloses. Pap Trade J 122
(2):3543
3. Poljak A (1948) Holzaufschluss mit peressigsaure. Angew Chem 60(2):4546
4. Timell TE (1964) Wood hemicelluloses: part 1. Adv Carbohydr Chem 19:247302
5. Kurada K (2000) Mokuzai Bunseki. In: Yoshida H (ed) Mokushitsu kagaku jikken manual.
Buneido shuppan, Tokyo, pp 8798
6. Timell TE (1961) Isolation of galactoglucomannans from the wood of gymnosperms. Tappi
44:8896
7. Yoshihara K, Kobayashi T, Fujii T, Akamatsu I (1984) A novel modification of klason lignin
quantitative method. Jpn Tappi 38:466475
8. Thiex NJ, Manson H, Anderson S, Persson JA (2002) Determination of crude protein in animal
feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam
distillation into boric acid: collaborative study. J AOAC Int 85(2):309317
9. Carson JF, Maclay WD (1948) Esters of lima bean pod and corn cob hemicelluloses. J Am Chem
Soc 70(1):293295
10. Samuel R, Pu Y, Foston M, Ragauskas AJ (2010) Solid-state NMR characterization of
switchgrass cellulose after dilute acid pretreatment. Biofuels 1(1):8590
11. Ahlgren PA, Goring DAI (1971) Removal of wood components during chlorite delignification
of black spruce. Can J Chem 49(8):12721275
12. Rabemanolontsoa H, Ayada S, Saka S (2010) Method applicable to analyze various biomass
species in their chemical composition. In: The 60th annual meeting of Japan Wood Research
Society, Miyazaki, Japan