STRUCTURE AND COMPOSITION OF SWEET SORGHUM STALK COMPONENTS

STRUCTURE AND COMPOSITION OF SWEET SORGHUM STALK COMPONENTS Evaggeli BILLA, Dimitris P. KOULLAS, Bernard MONTIES· and Emmanuel G. KOUKIOS Bioresource Technology Unit, Department of Chemical Engineering National Technical University of Athens, Zografou Campus GR - 15700 Athens, Greece tel: (+30-1) 7723287; fax: (+30-1) 7723163 e-mail: [email protected] • Laboratoire de Chimie Biologique (INRA), Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France

ABSTRACT Stem bark and pith of sweet sorghum were analysed with reference to their sucrose, simple reducing sugars, cellulose, hemicelluloses, lignin and associated phenolic acids contents. Moreover, lignin monomeric units (guaiacyl and syringyl) engaged in non-condensed structures were characterized by thioacidolysis, whereas cell wall associated phenolic acids (pcoumaric and ferulic acids) were estimated by alkaline hydrolysis at 170°C. The results obtained showed that bark and pith are heterogeneous as far as their chemical composition and the structure of their chemical components are concerned. In particular, the pith content in water soluble sugars is twice as high compared to the one in the bark, whereas bark is enriched in lignocellulosic fibres. Bark lignin is twice more important in content and less condensed in structure compared to pith lignin. P-coumaric acid is the predominant phydroxycinnamic acid associated to the cell walls, whereas ferulic acid is present in significant quantities.

KEYWORDS sweet sorghum, pith, bark, lignin structure, phenolic acids, sugars

INTRODUCTION Sweet sorghum (Sorghum bicolor {L.} Moench) is a C4 plant characterized by a high photosynthetic efficiency. It is a high biomass yield crop and compared to other species has one of the highest dry matter accumulation rates on a daily basis. Overall, out of many "new

1492

Structure and Composition ofSweet Sorghum

1493

crops" that are currently investigated as potential raw materials for energy and industry, sweet sorghum seems to be the most promising one (Gosse, 1996). Most of the existing studies concern the breeding and harvesting of the sorghum plant and recently, its conversion to industrial outlets. Nevertheless, very little is known about the chemical structure of the sorghum components which is critical for the optimisation of the different scenarios for industrial exploitation. In the context of the present work, the content in cellulose, hemicelluloses, lignin, sucrose, glucose and ash were determined in whole sweet sorghum stalks, as well as in the stalk pith and bark. In addition, the cell wall monosaccharide and phenolic acids composition were mesured. Finally, lignin monomeric composition was characterised in situ by thioacidolysis, a chemical degradation method which provokes the cleavage of the most prominent alkyl aryl ether (P-O-4) linkages in lignin (Lapierre, 1993). MATERIALS AND METHODS Material Sweet sorghum stalks provided fresh by the CRES were preserved in the freezer after removing the leaves, roots, soil, and impurities. The samples were ground in a laboratory fine grinder, and the fractions passing a 30 mesh screen were used for the chemical analysis. Quantitative saccharification. The lignocellulosic material was first quantitatively saccharified (Saeman et aI., 1945). After filtration, the hydrolysate was analysed for glucose (enzymically) and reducing sugars (Miller, 1959), in order to determine the cellulose and hemicellulose content, respectively. The latter was calculated from the difference between reducing sugars and glucose. Ash content was determined according to ASTM 0-1102. Glucose. reducing sugars and sucrose determination. Soluble sugars, i.e. glucose, reducing sugars and sucrose were determined after two successive extractions (10% wlv consistency) in water at 800C for 1 h each. Glucose concentration was determined with a glucose oxidasechromogen reagent and reducing sugars by the DNS method (Miller, 1959). Sucrose was determined after acid hydrolysis with cone. HCI to glucose and fructose at 600C for 15 min. After neutralization, the glucose produced was determined enzymically and sucrose was calculated by the difference between glucose content before and after hydrolysis. Preparation of cell wall residue. Separated ground sorghum stalk fractions were exhaustively extracted in a Soxhlet apparatus successively with toluene-ethanol (2: 1 v/v), ethanol 960/0 (vIv) and, finally, water. Determination of lignin content. Klason lignin (KL) was estimated according to a procedure similar to the one used by Eftland (1977). Thioacidolysis. Thioacidolysis was performed according to Lapierre et a1. (1989). Phenolic acids. Bound esterified and etherified p-coumaric and ferulic acids were analysed after alkaline hydrolysis at 170°C for 2 hours as described by Iiyama et a1. (1990) and Billa and Monties (1995a). Free phenolic acids were analysed by HPLC. Cell wall monosaccharides. The neutral monosaccharides were released by acid hydrolysis (H2S04 72%) of the cell wall polysaccharides. After reduction and acetylation, their alditol

E. Billa et al.

1494

acetate derivatives where determined by gas chromatography (Englyst and Cummings, 1984; Blankeney et al., 1983). RESULTS AND DISCUSSION Sweet sorghum stalks are composed of pith and bark which are present in approximately equal quantities on a dry basis; pith accounts for 65% of the stem on fresh matter basis. As shown in table 1 fresh sweet sorghum is extremely wet with a moisture content in pith being 380/0 higher compared to that of bark. Cell wall residue yields were rather low (390/0 for whole stalks) compared for example to the wheat straw ones (80-850/0) (Billa 1994). Moreover, cell wall residue yield of bark is 1550/0 higher compared to pith. This is mainly due to the high content of sweet sorghum in water soluble sugars; this content is twice more important in the pith compared to the bark. Table I.Moisture content (0/0 on fresh matter basis) and yield of cell wall preparation (% on dry basis). Mean standard deviation between dublicate experiments was less than 10%. Moisture content (%)

cell wall residue (0/0)

sweet sorghum

70

39

pith

77

22

bark

56

56

Chemical composition Table 2: Chemical composition of sorghum, pith and bark. The results are expressed as percentage (%) of dry weight. Mean standard deviation between duplicates were less than 10%. whole sorghum

pith

bark

cellulose

12.4

8.7

19.2

hemicelluloses

10.2

6.3

17.5

lignin

4.8

0.6

8.8

sucrose

55

67.4

32.2

glucose

3.2

3.7

2.4

ash

0.3

0.2

0.5

Table 2 gives the chemical composition of whole stalks as well as pith and bark, expressed on a dry matter basis. The two sorghum stalk fractions exhibit substantial differences with respect to their composition. In particular, the pith is twice more rich in sucrose and glucose (71% of dry weight) compared to the bark (34,60/0). Moreover, total cellulose, hemicelluloses and lignin content of the bark approaches 45,5% , thus constituing a shelter of fibrous texture

Structure and Composition ofSweet Sorghum

1495

"protecting" the inner part of the plant and preventing the degradation of the "appetizing" pith components. Pith is poorly lignified compared to bark, which is also enriched in cellulosic fibres. Lignin composition Table 3 shows the thioacidolysis yields of guaiacyl (G) and syringyl (S) lignin monomeric units from extractive free sweet sorghum stalks, as well as pith and bark. The yields are expressed on the basis of Klason lignin content in the sample; Klason lignin content in pith cell walls is 8.7% compared to 15.7% in the bark, and 12.40/0 in whole stalk cell walls. The total yield is indicative of the amount of monomeric units engaged in P-O-4 linkages, i.e., the so called "non condensed" monomeric units. The p-hydroxyphenyl (H) units were found in quantities of less than 5% of the units engaged in the alkyl aryl ether structures, in good accordance with previous results (Chabbert et al., 1993). Table 3: Total yields and molar ratios of monomeric non condensed guaiacyl (G) and syringyl (S) units determined by thioacidolysis in bark, pith and sorghum cell wall residues. Yields are expressed in umoles per g of Klason lignin.

sorghum

G

s

S/G

G+S

404±13

461±15

1.1

865±28 705±60 955±19

pith

307±24

397±35

1.3

bark

459±8

494±11

1.1

The recovery yields of the monomeric products were higher in the bark compared to the pith thus indicating a less condensed lignin (fewer carbon-carbon bonds). Moreover, S/G ratio was found to be higher in the pith compared to the bark. Phenolic acids associated to sorghum cell walls Table 4 shows the contents in p-coumaric and ferulic acids associated through ester or ether bonds to the sorghum stalk cell walls as obtained by alkaline hydrolysis at 170°C (Iiyama et aI., 1990). In both, sorghum pith and bark, p-coumaric acid is the predominant phydroxycinnamic acid encounterd in amounts 2.5-2.8 times higher compared to ferulic acid. Moreover, p-coumaric content in bark is 43% higher compared to pith. This can be explained by the fact that p-coumaric acid accumulation in the case of grasses cell wall, is mainly correlated to the extend of lignification, whereas ferulic acid was reported to be mainly associated with polysaccharide deposition (He and Terashima, 1990; Chabbert et al., 1994). Table 4: Yields ofp-coumaric (PA) and ferulic acids obtained by alkaline hydrolysis at 170°C. Yields are expressed in umoles per gram of the cell wall residue (CWR). PA

FA

PA/FA

pith

84.8±1.9

33.3±O.2

2.5

bark

121.3±6.9

43.6±2.7

2.8

umoles/g CWR

E. Billa et al.

1496

P-coumaric and ferulic acids are considered responsible for cross-linkages through ester and ether bonds between lignin and hemicelluloses (Billa and Monties, 1995b; Lam et al., 1992; Scalbert et al., 1985). These cross-linkages can have dramatic effects on various plant cell wall technically important properties such as accessibility, extensibility, plasticity and digestibility (Ralph and Helm 1993).

Neutral component sugars The results of acid hydrolysis of sweet sorghum pith and bark cell walls and the determination of their neutral monosaccharides by gas chromatography of their alditol acetates are given in table 5. According to these data, pith is enriched in arabinose and galactose compared to bark whereas xylose is present in higher quantities in bark. The glucose and fucose contents do not present significant differences between the two fractions of sweet sorghum stalks. Table 5: Neutral component sugars content of bark and pith sorghum cell walls. The results are expressed as percentage (0/0) of dry weight. bark

pith

arabinose

2.2±O.1

4.4±O.1

xylose

23.3±0.4

15.1±0.4

glucose

49.7±O.4

59.2±1.4

galactose

O.5±O.Ol

1.2±O.O2

fucose

O.4±O.O3

O.54±O.O7

76±1.2

80.4±1.8

total

CONCLUSIONS Sweet sorghum stalk pith and bark are highly heterogeneous as far as their chemical composition, as well as the structure of their chemical components are concerned. Pith is rich in water-soluble sugars, whereas bark is enriched in lignin and phenolic acids. Lignin content in the bark cell walls is twice as high and less condensed compared to pith. Furthemore, pcoumaric and ferulic acids (the former being the predominant one) are found to be associated to the cell walls, creating cross linkages between lignin and hemicelluloses. These data indicate that bark, rich in lignocellulosic fibres could be a promising source for paper pulp and cellulose industry, whereas pith is a valuable substrate for bioconversion owing to its high sugars and low lignin contents. ACKNOWLEDGEMENTS This work was performed in the framework of the Greek-French research cooperation PLATON programme (contract no. 95057). We are also grateful to Mrs C. Vallet at the Laboratoire de Chimie Biologique of INRA at Grignon for her valuable help on the analysis of the neutral component sugars.

Structure and Composition ofSweet Sorghum

1497

REFERENCES Billa E., 1994. Presence, Structure et Reactivite des Lignines, des Acides Phenoliques et de la Silice de la Paille de BIe et des Pates a Papier a haut Rendement Correspondantes. These, Institut National de la Recherche Agronornique, Grignon, Paris, France. Billa E. and Monties B., 1995a. Structural variability of lignins and associated phenolic acids in wheat straw. Res. Cellulose Chern. Technol., 29, 305-314. Billa E. and Monties B., 1995b. Molecular Variability of Lignin Fractions Isolated from Wheat Straw. Res. Chern. Intermed., 21(3-5), 303-311. Blankeney A.B., Harris P.J., Henry R.J. and Stone B.A., 1983. A Simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr. Res., 113, 291-299. Englyst H.N. and Cummings J.H., 1984. Simplified method for the measurement of total nonstarch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst, 109, 937-942. Chabbert B., Tollier M.T. and Monties B., 1993. Lignin variability among different brown midbrid sorghum lines. In: Proceedings, Seventh International Synposium on wood and pulping chemistry, Beijing, R.P. China, May 25-28, Vol. 1,462-468. Chabbert B., Tollier M.T. and Monties B., 1994.Biological variability in lignification of maize: expression of the brown mibrid bm3 mutation in three maize cultivars. J Sci Food Agric, 64, 349-355. Effland M.J., 1977. Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi, 60, 143-144. Gosse G., 1996. Overview on the different routes for industrial utilisation of sorghum. In: Abstracts book, First European Seminar on Sorghum for Energy and Industry, Toulouse, France 1-3 April, pp. 2. Iiyama K., Lam T.B.T. and Stone B.A., 1990. Phenolic acid bridges between polysaccharides and lignin in wheat straw internodes. Phytochern. 29(3), 733-737. Lam T.B.T., Iiyama K. and Stone B.A., 1992. Cinnamic acid bridges between cell wall polymers in wheat and phalaris internodes. Phytochern., 31(4), 1179-1183. Lapierre C., 1993. Application of new methods for the investigation of lignin structure. In: Forage cell wall structure and digestibility (Jung H.G., Buxton D.R., Hartfield R.D. and Ralph J. Ed.), pp. 133-166. Lapierre C. louin D. and Monties B., 1989. Lignin Characterization of Wheat Straw Samples as Determined by Chemical Degradation Procedures. In: Physico-chemical Characterization of Plant Residues for Industrial and Feed Use (A. Chesson and E.R. Orskov, Ed.), Elsevier Applied Sci., 118-130. Miller G. L., 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Anal.Chem., 31(3),426-428. Ralph J. and Helm R.F., 1993. Lignin/hydroxycinnamic acid/polysaccharide complexes: synthetic models for regiochemical characterization. In: Forage cell wall structure and digestibility (Jung H.G., Buxton D.R., Hartfield R.D. and Ralph J.,Ed.), pp. 133-166. Saeman J. F., Bubl J. L. and Harris E. E., 1945. Quantitative saccharification of Wood and Cellulose. Industr. Engineer. Chem., 17 (1), 35-37 Scalbert A., Monties B., Lallemend J-Y., Guittet E. and Rolando C., 1985. Ether linkage between phenolic acids and lignin fractions from wheat straw. Phytochem., 24(6), 1359-62. He L. and Terashima N., 1990. Formation and structure of lignin in monocotyledons. III. Heterogeneity of sugarcane (saccharum officinarum L.) lignin with respect to the composition of structural units in different morphological regions. J. Wood. Chern. Techno/., 104, 435-459.