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Ethanol Production from Vegetative Fronds and Turions of Wolffia Arrhiza

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Ethanol Production from Vegetative Fronds and Turions of Wolffia Arrhiza 133 ［Japanese Journal of Water Treatment Biology Vol.50 No.4 133－140 2014］ Ethanol Production from Vegetative Fronds and Turions of Wolffia arrhiza YUICHIRO TAKAI1, 2, DAISUKE MISHIMA1, MASANORI KUNIKI1, KAZUNARI SEI3, SATOSHI SODA1, and MICHIHIKO IKE1 1Graduate School of Engineering, Osaka University/2－1 Yamadaoka, Suita, Osaka, 565－0871, Japan 2Institute of Environment, Agriculture and Fisheries, Osaka Prefecture /442 Syakudo, Habikino, Osaka, 583－0862, Japan 3School of Allied Health Sciences, Kitasato University /1－15－1 Kitasato, Sagamihara-Minami, Kanagawa, 252－0373, Japan Abstract A duckweed, Wolffia arrhiza, was examined for its potential use as a feedstock for ethanol production. The starch contents of its turions, the plant’s dormant form, were 43%, whereas that of vegetative frond as the growth form was 13%. The ethanol yield from turions was 0.28 g－ethanol·g－biomass－1 in the simultaneous saccharification and fermentation mode (SSF) using a commercially available cellulase for enzymatic hydrolysis after alkaline / oxidative pretreatment. Ethanol was produced efficiently from starch-rich turions of W. arrhiza, in SSF using the amylase mixture with a comparable yield of 0.25 g－ethanol·g－biomass－1. Ethanol was produced from the vegetative fronds at the yield of 0.16 g－ethanol·g－biomass－1 in the SSF mode using cellulase, although that with the amylase mixture was only 0.07 g－ethanol/g－biomass. These results suggest that W. arrhiza, especially its turions, presents considerably high potential for use as an ethanol production feedstock. Keywords: ethanol production, duckweed, simultaneous saccharification and fermentation, turion, amylase mixture ethanol production because biofuel production INTRODUCTION from inedible biomass is regarded as a crucial Aquatic plants have been cultivated to requirement to minimize the land use conflict assimilate and remove nutrients from between food/feed production and energy eutrophic water1)－3) because water treatment feedstock production14). However, ethanol using aquatic plants demands less energy production from such lignocellulosic biomass than conventional microbiological processes has remained economically difficult because used for nutrient removal4). Moreover, the of the high complexity of the cell wall harvested biomass from water treatment component containing cellulose, hemicellulose, ponds and constructed wetlands can be and lignin15). Instead of such lignocellulosic converted to valuable products such as aquatic plants, a starch-rich aquatic plant is compost5), animal feed6), and the renewable desirable for more efficient ethanol production. energy resources7). Therefore, we have also specially examined Some aquatic plants such as water a species of duckweed, Wolffia arrhiza hyacinth8),9), water lettuce9), duckweed (rootless duckweed, about 2 mm long) for (Spirodela polyrrhiza)10),11), common reed12), nutrient removal and starch production16),17). and giant reed13) have been examined recently W. arrhiza grows quickly and absorbs large for their potential use as a feedstock for amounts of nutrients in its growth form 134 Japanese J. Wat. Treat. Biol. Vol.50 No.4 called ‘vegetative fronds’. In unfavorable g・l －1 glucose and 20 g・l －1 agar at 28 ℃ for 24 circumstances, the vegetative fronds are h. Obtained colonies were transferred to 100 transformed into the dormant form called ml of liquid YM medium (pH 6.2) containing ‘turion’, containing great amounts of starch 5.0 g・l －1 peptone, 3.0 g・l －1 yeast extracts, 3.0 (40－44% of biomass16)). Although duckweed g・l －1 malt extract and 20 g・l －1 glucose and species have high starch contents, few reports incubated at 28 ℃ and 120 rpm for 24 h on a have described ethanol production from rotary shaker before use as the inoculum. starch-rich duckweed10),11). Ethanol production in SHF At the In this study, ethanol production from enzymatic hydrolysis step in the SHF mode, vegetative fronds and turions of W. arrhiza 250－ml filter-sterilized cellulase (Sumitime biomass was demonstrated using sacchar- C; Shin Nihon Chemical Co. Ltd., Japan) ification enzymes, cellulase and amylase in solution (cellulase activity: 20 filter paper separated hydrolysis and fermentation (SHF) units (FPU)・g－1 substrate) in 0.1 M sodium and simultaneous saccharification and phosphate (pH 5.0) was added to Erlenmeyer fermentation (SSF) modes to evaluate the flasks (500 ml) containing 25 g of the potential of W. arrhiza as a feedstock for pretreated biomass of W. arrhiza. The renewable energy. reaction was performed at 45 ℃ and 120 rpm for 96 h for hydrolysis steps. After the MATERIALS AND METHODS enzymatic reaction, the hydrolysate was Plant materials and pretreatment W. centrifuged at 21,000 × g for 10 minutes. arrhiza was cultivated in 0.1% Hyponex 6－10 The supernatant was supplemented with －5, a commercial liquid fertilizer (Hyponex additional nutrients to give a basal medium Japan Corp., Ltd., Japan) at daytime mean composition of 2.0 g・l －1 yeast extract, 0.2 －1 －1 temperature 30 ℃ for 3 weeks in a greenhouse g・l (NH4)2HPO4, 0.02 g・l MgSO4 (initial at Osaka University. Turion formation was pH 5.0). The 80 ml of hydrolysate was induced in a 0.001% Hyponex 6－10－5 solution. transferred to 125－ml Erlenmeyer flask that Vegetative fronds on the water surface and had a rubber cap and a sampling needle. turions, which sank at the bottom, were Then it was autoclaved again to stop collected separately. The collected plant enzymatic reactions and to sterilize the bodies were washed manually using tap supernatant. For fermentation, 4 ml of the water, dried at 60 ℃; then powdered to pass yeast preculture was inoculated aseptically a 0.8 mm－mesh sieve. Alkaline/oxidative into the flask. Fermentation was conducted pretreatment (A/O pretreatment)9) was for 96 h at 30 ℃ and 120 rpm on a rotary applied to the dried biomass as described shaker. All experiments were done at least briefly below. The samples were reacted in twice. 1% (w/v) NaOH at room temperature for 12 h Ethanol production in SSF The SSF with subsequent addition of 31% H2O2 (w/v) reaction mixtures consisted of 8.0 g of the to the final concentration of 1% (w/v). The pretreated biomass of W. arrhiza with filter- resultant suspension was left to react for sterilized cellulase (20 FPU・g－substrate－1) or another 12 h. The pretreated samples were the amylase mixture (containing 47.2 mg・g－ collected and washed with tap water using a substrate－1 of α－amylase (A9857; Sigma－ 38－µm－mesh sieve until the pH value of the Aldrich Corp., USA) and 0.625 mg・g－ drained water was neutral. Then the samples substrate－1 amyloglucosidase (A1602; Sigma－ were dried at 60 ℃ and powdered. The Aldrich Corp., USA)) in 0.1 M sodium pretreated samples were used for ethanol phosphate, and with the basal medium production. described in the prior paragraph to constitute Fermenting yeast Saccharomyces cerevisiae a working volume of 80 ml. The yeast NBRC2346 was used as a fermenting yeast preculture was inoculated to give the same strain. Before fermentation experiments, S. concentration as that of the SHF experiment. cerevisiae was cultured on solid YM medium Fermentation was conducted for 60 h at 30 ℃ (pH 6.2)9) containing 5.0 g・l －1 peptone, 3.0 and 120 rpm on a rotary shaker. All g・l －1 yeast extract, 3.0 g・l －1 malt extract, 20 experiments were conducted at least twice. Ethanol Production from Vegetative Fronds and Turions of Wolffia arrhiza 135 Analytical procedures During fermenta- Table 1 Sugar composition of W. arrhiza (g·g－ －1 tion experiments, samples were withdrawn biomass ) Vegetative periodically for chemical analysis. The Turion quantification of ethanol, sugars, and frond a byproducts was performed using HPLC Glucose 0.40 0.62 (Shimadzu LC－10AT; Shimadzu Corp., Starch 0.13 0.43 Sum of mannose, galactose Japan) with a refractive index detector 0.01 0.00 and xylose (RID－10A; Shimadzu Corp.). Quantification was performed with the carbohydrate analysis Arabinose 0.00 0.00 column (Aminex HPX－87H 300 × 7.8 mm; a: Glucose obtained by acid hydrolysis of the cell component including cellulose, hemicellulose, and starch Bio－Rad Laboratories Inc., Richmond, CA) using 5 mM sulfuric acid as the mobile phase at a flow rate of 0.6 lm ・minute－1 9). Mannose, respectively, 0.40 g・g－biomass－1 and 0.62 xylose and galactose (man/xyl/gal) were not g・g－biomass－1. The respective starch contents separable using this method, but the sum of of vegetative fronds and turions were about these sugars was quantified approximately. 0.13 g・g－biomass－1 and 0.43 g・g－biomass－1. The cellulase activity was estimated on filter Mannose, galactose, xylose, and arabinose, paper as FPU18). The sugar component of the which cannot be used for ethanol production plant biomass was measured using HPLC by the fermenting yeast, were also detected after acid hydrolysis as the following: 200 mg at low concentrations (< 0.02 g・g－biomass－1) of the biomass samples was hydrolyzed using in the acid-hydrolysate. Although mannose, 2 ml of 72% H2SO4 for 1 h at 30 ℃, with galactose and xylose were not separated subsequent autoclaving at 120 ℃ for 1 h after through the HPLC column used for this addition of 56 ml of water19). For solubilizing study, the sum of sugars was quite low. Since starch in W. arrhiza biomass, 20 ml of it can be inferred that the xylose concentration dimethyl sulfoxide and 5 ml of 8 M HCl were was very low, the hemicellulose contents of added to 100 mg of the dried vegetative both the vegetative frond and the turion were fronds and turions, and stirred moderately at apparently negligible. Considering these 60 ℃ for 30 minutes. After they were results, the cellulose contents were estimated solubilized, starch contents were estimated from the total glucose contents without starch using F－kit starch (R－Biopharm AG, contents.
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