Characterization of Lake Biwa Macrophytes in Their Chemical Composition

Journal of the Japan Institute of Energy, 91,J. 621-628 Jpn. Inst. (2012) Energy, Vol. 91, No. 7, 2012 621 Original Paper Characterization of Lake Biwa Macrophytes in their Chemical Composition Harifara RABEMANOLONTSOA, and Shiro SAKA （Received December 2, 2011） Macrophytes growing in Lake Biwa such as ofusa-mo (Myriophyllum aquaticum), sennin-mo (Potamogeton maackianus), okanada-mo (Egeria densa), kuro-mo (Hydrilla verticillata) and kokanada-mo (Elodea nuttallii) were characterized in their chemical composition in order to evaluate their potential as biorefinery feedstocks. As a result, cellulose and hemicellulose contents were found to be in a range, respectively, between 227 - 436 g/ kg and 88 - 194 g/kg, while lignin content was from 71 to 175 g/kg. In more detail, hemicelluloses were mostly composed of xylose, galactose, mannose and arabinose in relatively equal amounts, whereas lignin was composed of guaiacylpropane, syringylpropane and p-hydroxylphenylpropane moieties, while ash and protein were remarkably high to be 105-223 g/kg and 137-229 g/kg, respectively. Although inorganics as shown by ash might be a limitation in the utilization of the macrophytes as biorefinery feedstocks, cellulose, hemicellulose, lignin and protein are available as raw materials for the production of a wide range of value- added biobased products. Key Words Myriophyllum aquaticum, Potamogeton maackianus, Egeria densa, Hydrilla verticillata, Elodea nuttallii, Lake Biwa, Chemical composition 1. Introduction verticillata) as well as okanada-mo (Egeria densa) were also Biorefinery from terrestrial biomass feedstocks en- abundant in the basin 5). The large quantity of macrophytes countered problems related to food price and land use. Land especially in summer and early autumn engenders navi- occupies only about 30% of the earth surface, while the gation and odor issues so that the Shiga prefectural gov- remaining 70% is covered with water and, particularly ernment, Japan spent ¥ 70 million in FY 2007 to remove freshwater ecosystems were demonstrated to be some of 2,800 t of the macrophytes in conjunction with other aquatic the most productive ones on earth 1). In place of terrestrial plants and algae 6). plants, aquatic plants are therefore investigated as new The question then arises as to how to use efficiently candidates for renewable resources. those available biomass. Under such circumstances, the Aquatic plants play important roles in physico-chem- chemical composition of the macrophytes should serve as istry of lakes and their ecology with significant impact on a basis and an effective tool to define the most profitable the food chain 2) 3). They also present several uses as use of the biomass. Therefore, the aim of this study is to biosorbent for water purification to extract nutrients, heavy analyze the chemical composition of the major macrophytes metals or other toxic chemicals 2) 3). In the case of Lake Biwa, growing in the Lake Biwa in order to evaluate their poten- most macrophyte species are invasive and their quantity tial as biorefinery feedstocks. seems to increase drastically over the years 4). The total biomass on dried weight basis of macro- 2. Materials and methods phytes in the southern basin of the Lake Biwa was esti- Fig. 1 shows the various species of the macrophytes mated to be 10,735 ± 3,030 t in 2002 against 6,500 t in collected in the Lake Biwa, whereas Table 1 shows their 2001 and 3,940 t in 1936. In 2002, sennin-mo (Potamogeton taxonomical classification, their part studied, sampling time maackianus) was the dominant species, and kuro-mo (Hydrilla and sampling site in the Lake. The samples were rinsed Department of Socio-Environmental Energy Science, with water, air-dried, milled with a Wiley mill (1029-C, Graduate School of Energy Science, Kyoto University Yoshida Seisakusho Co., Ltd.), and sieved to retain particles Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan of 150-500 μm in size (30-100 mesh). For comparison, buna 622 J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012 or Japanese beech (Fagus crenata) as hardwood sample was In brief, ash was determined after incineration of the oven- also prepared in a similar way. dried samples at 600 ℃ for 4 h. For additional analyses, The samples were then oven-dried and the summative the samples were extracted with acetone until it was clear chemical composition was determined according to the of any color. On the extractives-free samples, holocellulose method by Rabemanolontsoa et. al 7) as summarized in Fig. 2. and lignin were, respectively, determined by modified Wise method 8) and Klason method 9) . Both were then ash- and protein-corrected by subtracting ash and protein contents of the residues from the total residues yielded. Cellulose content was determined as α -cellulose by extraction with 17.5 % aqueous sodium hydroxide of the holocellulose powder 10) and hemicellulose content was evaluated by the difference between holocellulose and cellulose contents. Crystallinity of the α-cellulose samples were analyzed with an X-ray diffractometry (RINT 2200V, Rigaku Denki). The operating voltage and current were 40 V and 30 mA, respectively. Additionally, monosaccharides composition was determined by a combined method 11). Glucose was quantified as hydrolysate from the Klason lignin procedure by 72 % sulfuric acid and analyzed with high-performance anion- exchange chromatography (HPEAC, Dionex ICS-3000 system) equipped with CarboPac PA-1 column (4mm × Fig. 1 Various species of the selected macrophytes alive in the Lake Biwa 250mm), whereas the other neutral sugars and the uronic Table 1 Taxonomical classification of the selected macrophytes, their part studied, sampling time and site in the Lake Biwa Classification Vernacular name Scientific name Part studied Sampling time Sampling site Angiosperm Dicotyledon Myriophyllum Entire plant Moriyama, Shiga, Japan Ofusa-mo 04/2010 aquaticum 35°2’ 41.65” N 135°55’ 3.24” E Monocotyledon Potamogeton Stem together Otsu, Shiga, Japan Sennin-mo 07/2010 maackianus with leaves 35°3’ 16.29” N 135°52’ 40.72” E Stem together Otsu, Shiga, Japan Okanada-mo Egeria densa 07/2010 with leaves 35°3’ 16.29” N 135°52’ 40.72"E Hydrilla Stem together Moriyama, Shiga, Japan Kuro-mo 07/2010 verticillata with leaves 35°1’ 37.91” N 135°54’ 54.93” E Stem together Moriyama, Shiga, Japan Kokanada-mo Elodea nuttallii 07/2010 with leaves 35°1’ 37.91” N 135°54’ 54.93” E Fig. 2 Analytical method applied to the selected samples to quantify their chemical composition 7) J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012 623 acids by acid methanolysis 12) using Hitachi G-7000M and rophytes. M-9000 gas chromatograph-mass spectrophotometer (GC- Acid-soluble lignin from ofusa-mo, a dicotyledonous MS) equipped with 30 m × 0.25 mm i.d., 0.25 μm CP-Sil 8 macrophyte was, thus, higher than that of the monocoty- CB-Low Bleed/MS capillary column. As for xylose, the high- ledonous ones studied. est value among the 2 procedures was taken. Acetyl group In addition to holocellulose and lignin, the macrophytes was quantified from the acetic acid obtained after the 72 % contained protein in a range from 137 to 229 g/kg. These sulfuric acid hydrolysis 9). Aminex HPX-87H column (Bio- values agree well with data from literature reported in Rad) was used with a refractive index detector and the tem- various macrophytes to be from 98 to 228 g/kg 16). These perature of the column oven was set at 85 ℃. Distilled wa- lines of evidence confirm that aquatic macrophytes are rich ter was utilized as the mobile phase at a flow-rate of in protein. 0.6 ml/min. On the content of inorganic constituents as shown by Furthermore, alkaline nitrobenzene oxidation was per- ash, it varied greatly with species. Kuro-mo, okanada-mo formed on the extractives-free samples according to Iiyama 13) and kokanada-mo presented the highest ash content to be with slight modifications. The oxidized products were, respectively 223, 201 and 158 g/kg, followed by ofusa-mo silylated using trimethylchlorosilane (TMCS), bis to be 112 g/kg and sennin-mo to be 105 g/kg. Ash content (trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine in of the aquatic macrophytes was generally high as compared a volumetric ratio of 2:1:7 and analyzed by gas chroma- to wood. This is probably due to the high capacity of the tography with veratraldehyde as an internal standard. macrophytes to uptake inorganics and heavy metals from In addition, starch and protein determinations from the water 17). extractives-free samples were, respectively, completed by Acetone extractives in the macrophytes were also stud- perchloric acid method 14) and Kjeldahl nitrogen method by ied. They mostly represent the extracellular components using a nitrogen factor of 6.25 15), whereas lipid was deter- that are not a part of the cell wall structure, being varied mined by Soxhlet extraction with hexane. from 26 to 67 g/kg in the macrophytes. They are therefore considered as minor constituents. However, they might 3. Results and discussion influence the quality of the biobased products to be derived 3.1 Quantitative assay for chemical composition of the from the macrophytes. macrophytes Other minor components present in the macrophytes The quantitative assay for chemical composition of the were starch and lipid. Those compounds represent the en- various macrophytes growing in the Lake Biwa was made ergy storage in the plants. Ofusa-mo recorded the highest in this study, and the obtained results are shown in Table lipid content as 28 g/kg with the lowest starch content to 2. Instead of expressing the chemical composition as wt % be 2 g/kg. The other macrophytes had their lipid fraction of the original oven-dried samples, g/kg was used as it is lower than 20 g/kg and starch varying from 16 to 23 g/ recently considered more adequate internationally for fur- kg as reported in Table 2.

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