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. ther applications of the data for its quantification. The individual analysis is based on a quantitative ba- The obtained cellulose and hemicellulose contents of sis for the total biomass, thus the obtained total must be these macrophytes were found to be ranged, respectively, theoretically 1000 g/kg. Since the summative results for from 227 to 436 g/kg and 88 to 194 g/kg. On the results the constituents independently analyzed of the macro- of cellulose, they were slightly similar to the ones reported phytes were in a range between 985 and 998 g/kg, the by Boyd 16) on different aquatic plants to be in a range from results of all analyses must be valid, and most components 188 to 356 g/kg, although in this study, the highest cellu- have been evaluated. lose content was found in kokanada-mo to be 436 g/kg. Lignin as a sum of Klason lignin and acid-soluble lig- 3.2 Holocellulose composition of the macrophytes nin was the highest in ofusa-mo (175 g/kg), followed by Holocellulose is composed of cellulose and hemicellu- sennin-mo (149 g/kg). The three other species presented lose which further comprises hexosan and pentosan such similar results ranging from 71 to 79 g/kg. Thus, the total as glucomannan and xylan, respectively. lignin content of ofusa-mo, one of the dicotyledonous mac- Therefore, composition of the neutral sugars, uronic rophytes was higher than that of the monocotyledonous acids and acetyl group of the macrophytes was studied and ones studied. Similarly, spectral evaluation by UV spectros- the obtained results are shown in Table 3. Holocellulose copy resulted in 29 g/kg of acid-soluble lignin for ofusa- (1) was determined by the sodium chlorite procedure, in mo, against 15 to 18 g/kg for the monocotyledonous mac- which the residual ash, lignin and protein were all corrected, 624 J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012

Table 2 Chemical composition of the macrophytes studied (g/kg of the original oven-dried biomass basis)

Biomass Lignin Vernacular Scientific Cellulose a Hemicellulose b Klason c Acid- Protein Extractives Starch Lipid Ash Total name name soluble Myriophyllum Ofusa-mo 263 194 146 29 162 53 2 28 112 989 aquaticum Potamogeton Sennin-mo 347 88 131 18 229 40 20 19 105 997 maackianus Egeria Okanada-mo 262 181 54 17 226 26 16 15 201 998 densa Hydrilla Kuro-mo 227 141 64 15 228 67 18 9 223 992 verticillata Elodea Kokanada-mo 436 93 61 15 137 52 23 10 158 985 nuttallii

Buna Fagus crenata 439 284 210 30 6 19 5 - 6 999 a (Cellulose ) = (α -Cellulose) b (Hemicellulose) = (Holocellulose (1) in Table 3) - (α -Cellulose) c (Klason lignin) = (Acid-insoluble material after Klason lignin procedure) - (ash in the residue) - (protein in the residue)

Table 3 Monosaccharides and acetyl group composition of the macrophytes as compared to the one of Buna

Yield (g/kg of the original oven-dried biomass basis) c Biomass Hexoses Pentoses Acid sugars Holocellulose Holocellulose Acetyl Vernacular Scientific (1) a (2) b Glc Man Gal Rha Fru Xyl Ara Glc-A Gal-A group c name name Myriophyllum Ofusa-mo 457 434 289 41 24 13 22 32 37 3 13 9 aquaticum Potamogeton Sennin-mo 435 426 343 6 5 8 3 58 26 2 17 6 maackianus Egeria densa Okanada-mo 443 413 267 19 59 16 1 51 16 8 16 6

Hydrilla Kuro-mo 368 333 241 1 44 12 1 29 24 1 10 7 verticillata Elodea Kokanada-mo 529 501 422 31 26 8 0 25 14 5 21 5 nuttallii

Buna Fagus crenata 723 687 417 14 36 23 0 213 9 3 17 32

a Holocellulose (1) = Residue after sodium chlorite procedure － lignin in the residue － ash in the residue － protein in the residue

b Holocellulose (2) = 162/180 ∑ Hexoses ＋ 132/150 ∑ Pentoses ＋ 176/194 ∑ Acid sugars + 42/60 Acetic acid c Acetyl group = 42/60 Acetic acid d Glc : Glucose, Man : Mannose, Rha : Rhamnose, Xyl : Xylose, Ara : Arabinose, Glc-A : Glucuronic acid, Gal-A: Galacturonic acid

whereas holocellulose (2) was estimated, based on the data as compared to woods. However, not only the availability of the contents of neutral sugars, uronic acids and acetyl and abundance of the macrophytes but also the possible group. The obtained results of holocelluloses (1) and (2) are use of the other components might justify their utilization in good agreement with each other to be comparable, in as feedstocks for integrated biobased production. spite of the different methods used. The detailed monosaccharides composition of the According to these results, kokanada-mo had the high- holocellulose in Table 3 shows that glucose is evidently est holocellulose content, slightly over 500 g/kg for both the dominant sugar component, most probably coming from holocelluloses (1) and (2), whereas ofusa-mo, sennin-mo and the cellulosic portion of the samples. okanada-mo had holocellulose (1) between 435 and 457 g/ X-ray diffractometry of the α-celluloses from the mac- kg. Only kuro-mo had its holocelluloses (1) and (2) below rophytes as compared to the one from buna were under- 400 g/kg. On the whole, the holocellulose content of the taken and the diffraction patterns of the different species macrophytes, varying from 368 to 529 g/kg is quite low are shown in Fig. 3. The degree of crystallinity was calcu- J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012 625

galactose and xylose in rather equal amounts to be 26 and 25 g/kg, respectively, in Table 3. As acid sugars, uronic acids such as glucuronic and galacturonic acids of the macrophytes were similar among species in their contents. However, galacturonic acid was higher than glucuronic acid in all the analyzed macrophytes. Table 4 shows hexosan and pentosan composition of the macrophytes as compared to the one of hardwoods buna (Fagus crenata). It is apparent that although slightly higher, hexosan content of hardwood is comparable to those of the macrophytes, except for kuro-mo which presented hexosan Fig. 3 X-ray diffraction patterns of α -celluloses from the macrophytes as compared to the one from buna (F. crenata) below 300 g/kg. However, pentosan content in the macrophytes ranged between 34 and 74 g/kg. These values are lated according to Segal et. al 18). While buna and kokanada- remarkably low compared to 195 g/kg for buna, one of the mo had comparable cellulose contents, respectively, 439 and hardwoods. 436 g/kg, the degree of crystallinity for buna to be 71 % was much higher than the one of kokanada-mo to be 41 %. 3.3 Aromatic compounds from lignin The other samples showed even lower degree of crystal- The aromatic compounds produced from the extrac- linity with 34% for sennin-mo, 32 % for ofusa-mo and 29 % tives-free macrophytes by alkaline nitrobenzene oxidation for both okanada-mo and kuro-mo. That finding suggests are presented in Table 5. that as compared to buna hardwood, the macrophytes pre- The presence of p-hydroxybenzaldehyde, syring- sented more amorphous cellulose which would be much aldehyde and vanillin as the aldehydes of p- prone to the milder hydrolysis than crystalline ones. hydroxyphenylpropane (P), syringylpropane (S) and The non-cellulosic monosaccharides including the guaiacylpropane (G) units appears to indicate that the uronic acids and a minor part of glucose are considered as aquatic macrophytes have independently or associated to- the hemicellulosic part of the samples. As opposed to hard- gether with P, G and S types of lignin, characteristics of wood which comprises xylose-rich hemicellulose, ofusa-mo, monocotyledonous species such as rice and wheat etc. It is one of dicotyledonous species, had its hemicellulose com- interesting to note that, although ofusa-mo was a dicotyle- posed of mannose, xylose, arabinose and galactose in a donous species, its lignin seemed also to consist of P, G relatively equivalent amount, implying that hemicellulose and S moieties. The alkaline nitrobenzene oxidation results, of ofusa-mo had the typical characteristics of its family therefore, suggest that lignin structure of the macrophytes (Myrtales) containing specifically arabinogalactan 19), in ad- could be similar to those of herbaceous monocotyledonous dition to xylan. Okanada-mo and kuro-mo, monocotyledon- species. ous species, had a noticeable amount of galactose, respec- However, it was reported for herbaceous samples that tively, 59 and 44 g/kg as compared to their other monosac- a large proportion of p-hydroxybenzaldehyde and vanillin charides. Kokanada-mo had mannose as its predominant produced after the alkaline nitrobenzene oxidation could hemicellulosic monosaccharide to be 31 g/kg, followed by be formed respectively from p-coumaric acid and ferulic

Table 4 Hexosan and pentosan composition of the macrophytes as compared to that of buna (g/kg of the original oven-dried biomass basis)

Biomass Total Hexosan a Pentosan b Vernacular name Scientific name Ofusa-mo Myriophyllum aquaticum 411 350 61 Sennin-mo Potamogeton maackianus 403 329 74 Okanada-mo Egeria densa 385 326 59 Kuro-mo Hydrilla verticillata 316 269 47 Kokanada-mo Elodea nuttallii 472 438 34 Buna Fagus crenata 636 441 195

a Hexosan = 162/180 ∑ Hexoses b Pentosan = 132/150 ∑ Pentoses 626 J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012

Table 5 Aromatic compounds produced from extractives-free macrophytes by alkaline nitrobenzene oxidation (g/kg of original oven- dried sample)

Ofusa-mo Sennin-mo Okanada-mo Kuro-mo Kokanada-mo Buna Aromatic compounds Myriophyllum Potamogeton Egeria Hydrilla Elodea Fagus aquaticum maackianus densa verticillata nuttallii crenata Guaiacyl moiety Vanillin 0.21 2.69 0.15 0.29 0.07 7.84 Vanillic acid 0.18 0.35 0.00 1.45 0.25 - Coniferyl aldehyde 0.90 1.54 3.11 10.43 1.91 - Acetovanillin 0.25 2.76 0.83 1.10 0.22 - Ferulic acid 0.29 0.65 2.71 1.75 0.00 - Syringyl moiety Syringaldehyde 0.25 1.87 0.13 0.23 0.10 15.69 Syringic acid 0.00 0.28 0.68 1.06 0.17 - p-Hydroxyphenyl moiety p-hydroxybenzaldehyde 0.22 0.73 0.05 0.11 0.05 - p-hydroxybenzoic acid 0.19 0.42 0.56 0.66 0.00 - Coumaric acid 0.25 0.38 0.18 1.66 0.00 -

260. Such absorbance is due to the simultaneous presence of guaiacyl and syringyl moieties of lignin 21) 22). Typical UV spectra of guaiacyl, syringyl and p-hydroxyphenyl model compounds were respectively presented by Aulin-Erdtman 23), Pew 24) and Lang 25). Both syringyl and guaiacyl model compounds showed a minimum absorbance at 250 to 260 nm with maximum at 270 to 280 nm, while p-hydroxyphenyl model compound gave a strong peak at 260 nm with minimum absorbance in wavelengths lower than 240 nm. Musha and Goring 22) stated that if some p-hydroxybenzoic residues are associated with syringyl residues, the peak at 280 nm should flatten because of the strong ab- Fig. 4 UV spectra of the acid-soluble lignins from different mac- sorbance at low wavelength of the p-hydroxybenzoic acid rophytes and Japanese beech (Fagus crenata) for comparison group and that the minimum absorbance at 250-260 nm is likely to shift to the lower wavelength. Those facts can be acid esterified or etherified with lignin, and not from P observed in the difference between the UV absorbances and G moieties in the lignin polymer itself 13) 20). Since a large of buna and the macrophytes, confirming the statement amount of p-coumaric acid and ferulic acid were found in above as the studied macrophytes contained p- the samples as accounted in Table 5, it is possible that a hydroxybenzoic residues. This conclusion is in good agree- major part or all the p-hydroxybenzaldehyde and vanillin ment with the results obtained from the alkaline nitroben- recovered was originated from p-coumaric acid and feru- zene oxidation results. lic acid. Apart from the lignin phenylpropane units, phenolic Therefore, in order to confirm the lignin structures, acids, for instance, ferulic acid and coumaric acid were also UV spectra of the acid-soluble lignins from the macrophytes detected as shown in Table 5. They might represent the were studied in Fig. 4 and compared with that from buna. phenolic compounds linking lignin with polysaccharides, Spectra for ofusa-mo and kuro-mo are not shown but they which may play an important role in reinforcing the me- were similar to those of the other macrophytes shown in chanical strength of the plant cell walls as demonstrated Fig. 4. in grass species 26). The spectrum found for buna is typical of hardwood lignin showing a maximum absorbance at a wavelength between 270 and 280 nm, with minimum between 250 and J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012 627

3.4 Lake Biwa macrophytes as potential biorefinery feed- lower as compared to the ones of buna, a hardwood. stocks The detailed monosaccharides study revealed that the A macrophyte is an aquatic plant that grows in or near macrophytes contained glucose in a range between 241 and water and is either emergent, submergent or floating 27), 422 g/kg, while the xylose contents were low as compared being classified into both dicotyledons and monocotyledons to hardwood buna. Accordingly, the contents of pentosan in angiosperms. To clarify its chemical composition, vari- in the macrophytes were rather small. ous macrophytes growing in Lake Biwa were selected and Although cellulose and hexosan contents of buna and studied. Consequently, holocellulose composed of cellulose kokanada-mo were comparable, the degree of crystallinity and hemicellulose was rather low in about 450 g/kg on of α -celluloses from kokanada-mo and the other studied average. In addition, lignin as the sum of Klason lignin and macrophytes were much lower than the one from buna. acid-soluble lignin was also low in content varying from Additionally, alkaline nitrobenzene oxidation on the 71 to 175 g/kg. On the other hand, protein and ash con- macrophytes suggested that their lignin consisted of P, G tents were found to be rather high in a range between 137- and S moieties. Such result was further confirmed by UV 229 g/kg and 105-223 g/kg, respectively. Furthermore, spectrophotometry on the acid-soluble lignin of the macro- extractives, starch and lipid were found in total to be 57- phytes. 94 g/kg. Therefore, it might be concluded that the macro- Characterization of the Lake Biwa macrophytes in their phytes are composed of various chemical constituents. This chemical composition revealed that those species had low finding suggests that the macrophytes can be of high po- potential for the traditional ethanol production. However, tential to various applications for biorefinery. However, cellulose, hemicellulose, protein, lignin, starch and ash are those macrophytes are not good candidates as raw materi- available materials for the production of extensive variet- als for the traditional bioethanol production, in spite of the ies of biobased products. claiming that the macrophytes in Lake Biwa can be good raw materials for ethanol production 28). Acknowledgement In more details on the saccharides content, hemicellu- This work was accomplished under financial support loses in the macrophytes were essentially composed of from Kyoto University Global Center of Excellence (GCOE) xylose, mannose, galactose and arabinose in relatively equal Energy Science Program by the Ministry of Education, amounts, thus not rich in pentosan (34-74 g/kg), while lig- Culture, Sports, Sciences, and Technology, Japan (MEXT). nin was characterized by the presence of p-hydroxyphenylpropane moiety in addition to syringylpropane and References guaiacylpropane moieties. 1）Likens, G., Hum. Ecol., 1（4）, 347-356（1973） Even though the macrophytes studied were individu- 2）Bhadra, R., Spanggord, R. J., Wayment, D. G., Hughes, J. ally different, their overall chemical compositions were B., Shanks, J .V., Environ. Sci. Technol., 33（19）, 3354-3361 basically similar, which allows their exploitation together （1999） without sorting. While ash content of the macrophytes 3）Bishop, P. L., Eighmy, T. T., J. Water Pollut. Control Fed., 61 might be a constraint in their use, lignin, cellulose, hemi- （5）, 641-648（1989） cellulose and protein represent promising raw materials for 4）Hamabata, E., Kobayashi, Y., Lakes Reservoirs: Res. Manage., a broad range of value-added biobased products that will 7（4）, 331-338（2002） reduce dependence on fossil resources and will enhance 5）Haga, H., Ohtsuka, T., Matsuda, M., Asiya, M., Jpn. J. Limnol., local development. 67（2）, 69-79（2006） The Lake Biwa aquatic macrophytes are low-cost, lo- 6）朝日新聞，琵琶湖の「悪者」をバイオ燃料に水草から cal and regionally available feedstocks. However, although 精製，成功, 2009. Retrieved April 4, 2011, http:// they are classified as perennial plants, a large quantity of www.asahi.com/eco/OSK200904020061.html their biomass is only available during summer and early 7）Rabemanolontsoa, H., Ayada, S., Saka, S., Biomass Bioenergy, autumn. Therefore, the installation of a biorefinery unit only 35（11）, 4630-4635（2011） for those species would not be much economically profit- 8）Timell, T. E., Tappi, 44, 88-96（1961） able. 9）Yoshihara, K., Kobayashi, T., Fujii, T., Akamatsu, I., Jpn. Tappi, 38, 466-475（1984） 4. Conclusion 10）Whistler, R. L., in Methods in carbohydrate chemistry, The studied macrophytes had high protein and ash Whistler, R. L. and Wolfrom, M. L., eds,（ Academic Press: contents. Consequently, their carbohydrate and lignin were New York）, 56-87（1962） 628 J. Jpn. Inst. Energy, Vol. 91, No. 7, 2012

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