Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2014 www.elsevier.com/locate/jbiosc
Production of 16.5% v/v ethanol from seagrass seeds Motoharu Uchida,1, * Tatsuo Miyoshi,1 Masaki Kaneniwa,2 Kenji Ishihara,2 Yutaka Nakashimada,3 and Naoto Urano4 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi 2-17-5, Hatsukaichi-shi, Hiroshima 739-0452, Japan,1 National Research Institute of Fisheries Science, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 235-0452, Japan,2 Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan,3 and Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan4 Received 3 April 2014; accepted 22 May 2014 Available online xxx
Ethanol fermentation on seeds of seagrass Zostera marina was studied. The seeds were collected from the annual plant colony of Z. marina at Hinase Bay, Okayama. The seeds contained 83.5% carbohydrates including 48.1% crude starch on a dry weight basis, which is comparable to cereals such as wheat flour and corns. The seeds were saccharified with glucoamylase (50 C, 96 h) and 103.4 g/l concentration of glucose juice was obtained. The glucose juice was further fermented (23 Ce35 C, 15 days) with Saccharomyces cerevisiae strains NBRC10217T and Kyokai 7-go, and ethanol was obtained at a 65.0 g/l (82.3 ml/l) level by monographic double-fermentation and at a 130.4 g/l (165.1 ml/l) level by parallel double-fermentation. Fermented products of seagrass seeds containing such a high ethanol concentration as the present study have potential to be utilized not only for biofuel but also for foods and beverages in the future. Culturing of seagrass seeds as a crop may enable development of a new marine fermentation industry. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aquatic bioresources; Ethanol fermentation; Saccharomyces cerevisiae; Seagrass seed; Zostera marina]
Research in bioethanol production from aquatic plants and algae has increased after the 2000s (1e6). Choice of a suitable species as a substrate is one of key elements for effective ethanol production. Korean research groups are focusing on the utilization of red algae as a substrate (7,8). Red algae are known to contain galactan as a major component [20e51% on a dry weight basis (9)] besides glucan. Relatively higher quantity of fermentable sugars of galactose and glucose can be obtained by acid-hydrolisis of red algae [30.4% of dry matter on average (10)], and both of the sugars can be further metabolized to ethanol by yeast with a conversion efficiency at 51% (w/w) according to the following reaction: C6H12O6/2C2H5OH þ 2CO2. Park et al. (11) and Yanagisawa et al. (12) produced ethanol from agar weeds at 33 ml/l and 55 ml/l levels, respectively. Brown algae are another candidate as a suitable substrate for ethanol production (13). Brown algae contain alginate [3e38% on a dry weight basis (14)] as major components, but microorganisms rarely convert the alginate to ethanol. To overcome this problem, genetically engineered microorganisms were developed to convert alginate to ethanol, and the ethanol concentration in the fermentation culture was observed at 13 g/l (16.5 ml/l) utilizing alginate alone as a carbon source (15) and 47 ml/l utilizing whole components of Saccharina japonica (kombu) as carbon sources (16). Green algae are also a candidate as a substrate since some species such as Ulva spp. are known to make huge blooms in coastal waters. Isa et al. (17) demonstrated ethanol production from
* Corresponding author. Tel.: þ81 829 55 3580; fax: þ81 829 54 1216. E-mail address: [email protected] (M. Uchida).
green algae at a 10 g/l (12.7 ml/l) level. As for seagrass, Viola et al. (18) produced ethanol at a 47 ml/l level from eel grass (Zostera marina). As for freshwater plants, water-hyacinth (Eichhornia crassipes) is known to be suitable as a substrate for fermentation (19e21). Mishima et al. (22) produced ethanol at a 16.9 g/l (21.4 ml/ l) level from water-hyacinth. Microalgae have also been studied as substrates for ethanol fermentation (23e25). Harun et al. (26) demonstrated production of ethanol at a 3.83 g/l (4.8 ml/l) level from Chlorococcum sp. However, aquatic plants and algae are generally moisture rich and dry matter contents and fermentable sugars that are expected to be converted to ethanol are estimated to be only 12.5% (w/w) and 1.6 % (w/w) on average, respectively (10). Therefore, the ethanol concentration obtained from fermentation of aquatic plant and algae is limited to a low level [mostly less than 20 ml/l (1, 27, 28)]. For use of the ethanol product as a fuel, distillation and dehydration of ethanol to a 99.9% (v/v) concentration level is necessary. This distillation process needs a high energy input (29), and, therefore, it is advantageous to obtain a fermented product with a high ethanol concentration from the view-point of energy balance. It is mentioned in manuscripts (12) that the economically feasible concentration of ethanol contained in fermented products is at least larger than 4e5% (v/v). The present study focused on obtaining fermented products that contains ethanol at a high concentration and chose seeds of seagrass Z. marina as a substrate. Firstly, seagrass seeds were collected and the basic characteristics and sugar component of the seeds were clarified and compared with other substrates. Secondly, saccharification conditions were optimized to obtain glucose juice from the seeds. Thirdly, fermentation of the glucose juice was
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.05.017
Please cite this article in press as: Uchida, M., et al., Production of 16.5% v/v ethanol from seagrass seeds, J. Biosci. Bioeng., (2014), http:// dx.doi.org/10.1016/j.jbiosc.2014.05.017
2
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FIG. 1. Collection of seeds of seagrass Zostera marina. The flowering shoots were collected in early June, 2011 at Hinase Bay, Okayama. The shoots were submerged in water for five months for biodegradation and harvested. The harvested seeds were eliminated for debris and small animals, freeze dried, milled and used for saccharification and fermentation experiments.
conducted with yeast using a monographic double-fermentation method, i.e., a method performing saccharification and fermentation separately. Furthermore, a parallel double-fermentation method was tested to obtain higher concentration of ethanol. The parallel double-fermentation method is a method performing saccharification and fermentation in parallel in a tank, and this method makes it possible to obtain approximately w20% (v/v) ethanol products during the manufacture of Japanese sake or rice wine. MATERIALS AND METHODS Collection and characterization of seagrass seeds The seeds of Z. marina were collected according to the method of Fukuda (30). Flowering shoots were harvested at the annual plant colony of Hinase Bay, Okayama, and housed in plastic mesh bags in early June 2011. The mesh bags were submerged in water by being hung down from oyster rafts and let to maturate and biodegrade. The biodegraded shoots were harvested after five month in October, the debris and degrading animals such as polychaetes and gastropods were removed, and wet seeds were obtained. The wet seeds were washed with tap water and freeze dried to obtain dry seeds, and stocked until use. The seeds were measured for weight and size. The seeds were milled by using a mixer (Panasonic, MK-K48P-W), and analyzed for proximate content according to the method by the Council for Science and Technology, Ministry of Education, Culture, Sports, Science, and Technology, Japan (31). Carbohydrate content % (w/w) was obtained by subtracting the values of protein%, lipid%, and ash% from 100%. Starch content was measured as follows: Aliquots (0.1e0.3 g) of milled seeds were weighed accurately and washed with 10 ml of 50% (v/v) ethanol to eliminate low molecular weight sugars. The pellet was collected and added with 1 ml of 99.5% (v/v) ethanol, 20 ml of water, and 2 ml of 10% (w/v) sodium hydroxide, and heated for 3 min in boiling water. The sample was cooled to room temperature, neutralized with 25% (v/v) hydrochloric acid, then further reacted with amyloglucosidase (Megazyme, Biocon Ltd., Nagoya, >480 U) for 2 h at 37 C. The produced glucose was measured with an enzymatic method (Roche Diagnostics K.K., Tokyo). Sugar composition was measured as follows: Aliquots (0.3e0.6 g) of milled seeds were weighed and incubated stirring with 2 ml of 72% (w/w) sulfuric acid for 1 h at room temperature, then diluted with 56 ml of distilled water (final conc. 4% (w/ w)) and autoclaved for 1 h at 121 C. The mixture was neutralized with 30% (w/w) sodium hydroxide, volume adjusted to 100 ml, and analyzed after passing through No.5B (Toyo Roshi Kaisha, Ltd., Tokyo) and 0.45 mm-mesh filters. The quantitative analysis of sugar compounds was performed on a Shimadzu high-performance liquid chromatography (HPLC) system model LC-20AD equipped with a Shimadzu RF-20AxS detector (Shimadzu Corporation, Kyoto). Twenty microliters of sample
was injected, and the chromatographic separation was performed on a TSKgel Sugar AXI (Tosoh Corporation, Tokyo) anion exchange column, 4.6 mm 150 mm at 60 C with a mobile phase of 0.5 M boric acid buffer solution (0.4 ml/min, pH 8.7). The sugars were coupled with 1% (w/v) L-alginin solution (0.7 ml/min) at 150 C to give a fluorophor, which is detected by a fluorometer with excitation at 320 nm and detection of emission measured at 430 nm. Saccharification of seagrass seeds Two grams of the seed powder, 0.06 g of enzymes, 4 ml of distilled water were placed in glass tubes (16 mm in diameter) and mixed. The glass tubes were set at a 30 inclination against horizontal line and reacted by shaking (120 rpm) at 50 C. The enzymes tested were Cellulase 12S (Yakult Honsha Co. Ltd., Tokyo, 12,000 U/g), Cellulase A Amano 3 (Amano Enzyme Inc., Nagoya, 30,000 U/g), Cellulase T Amano 4 (Amano Enzyme Inc., 40,000 U/g), Hemicellulase Amano 90 (Amano Enzyme Inc., xylanase 9000 U/g), Gluczyme AF6 (Amano Enzyme Inc., glucoamylase 1600 U/g), and Gluc 100G (Amano Enzyme Inc., glucoamylase 60,000 U/g and a-amylase 350,000 U/g). The produced glucose was measured periodically using a F-kit glucose. The test was conducted using duplicate tubes and the data is shown as mean standard deviation (SD) value. Fermentation of seagrass seeds Fermentation tests were conducted by both monographic double-fermentation and parallel double-fermentation methods. For monographic double-fermentation, a mixture of 12 g of the seed powder, 0.24 g of Gluczyme AF6, 0.042 g of Cellulase A Amano 3, 0.042 mg of Hemicellulase Amano 90, 24 ml of distilled water was reacted for 24 h at 50 C, and the saccharified juice was obtained by centrifuging for 20 min at 6000 g. Aliquots of the juice were taken and measured for glucose and galactose contents by enzymatic methods (F-kit glucose, F-kit galactose, Roche Diagnostics K.K.). The remaining juice was filtrated (GS, Millipore) and dispensed 1.2 ml each into six sterile two milliliters-volume polypropylene centrifuge tubes. Fermentation was conducted under three experimental conditions: the duplicated tubes for each were incubated with Saccharomyces cereviciae NBRC10217T, S. cerevisiae Kyokai 7-go (K7, purchased from Brewing Society of Japan), and without yeast, respectively. Fresh yeast cells grown on GAM plates (Nissui Pharmaceutical Co. Ltd.) were collected and suspended in autoclaved 0.85% (w/v) NaCl solution at a concentration of OD660nm ¼ 0.5 (contain approximately 2 105 CFU/ml). Inoculation was conducted by adding 5% volume (¼ 60 ml) of the yeast cell suspension to culture water. Incubation was conducted for the initial five days at 37 C and further 10 days at 23 C. For parallel double-fermentation, a mixture of 500 g of the seed powder, 7.5 g of Gluczyme AF6, 1.5 g of Cellulase A Amano 3, 1.5 g of Hemicellulase Amano 90, and one liter of distilled water was reacted for 24 h at 50 C. The saccharified juice was autoclaved two times for 20 min at 90 C with a 24 h-interval for sterilization. The juice was dispensed 50 g (45 ml) each for four sterile screw-capped centrifuge tubes and 500 g (450 ml) each for two screw-caped plastic bottles. The 50-g scale (n ¼ 2) and 500-g scale (n ¼ 1) bottles were fermented after adding 1% volume of the two kinds of yeast cell suspensions. Fermentation was conducted for the initial five days at 37 C and a further 44 days at 23 C. Glucose and ethanol contents were measured periodically by using F-kit glucose and F-kit ethanol (Roche Diagnostics K.K.).
Please cite this article in press as: Uchida, M., et al., Production of 16.5% v/v ethanol from seagrass seeds, J. Biosci. Bioeng., (2014), http:// dx.doi.org/10.1016/j.jbiosc.2014.05.017
VOL. xx, 2014
ETHANOL FERMENTATION ON SEAGRASS SEEDS RESULTS
TABLE 2. Proximate contents of seagrass seeds, cereals, and seaweeds. Potential substrate
Collection of seagrass seeds Scheme of collection of seagrass seeds is shown in Fig. 1. The freeze dried seeds, 1.1 kg, were obtained from 3.1 kg of wet seeds, milled and stocked for use of saccharification and fermentation experiments. Characteristics of seagrass seeds Basic characteristics of seeds of Z. marina are shown in Table 1. The size is 3.78 0.37 mm (mean SD) in length and 1.98 0.13 mm in width (diameter). The weight was 12.4 2.1 mg on a wet weight basis or 4.7 0.8 mg on a dry weight basis. The fresh seeds contained 62.0% (w/w) of moisture. Proxy contents of the seeds are shown in Table 2 with those of reference materials (32). The seeds contained 83.5% carbohydrate, 10.1% protein, 1.3% lipid, and 5.1% ash on a dry weight basis. The richness of carbohydrate in seagrass seeds is comparable to those of cereals (82.5e91.2%). Sugar composition of the seeds was glucose (59.9%), xylose (3.5%), rhamnose (0.3%), and arabinose (0.2%), and starch content was 48.1% on a dry weight basis (Table 3). Saccharification The seeds were saccharified with enzymes in culture water containing 333 g of dried seeds/kg or 306 g of dry matter/kg (or per 0.9 l of culture water). The maximum glucose content was obtained at a 103.4 g/l level after reaction with Gluczyme AF6 (glucoamylase) for 96 h at 50 C (Table 4). The use of cellulase and hemicellulase were observed to be less effective. Gluc 100G is an enzyme mixture product designed for sakemanufacturing containing glucoamylase and a-amylase activities, but the effect was inferior to that of Gluczyme AF6. The reaction mixture was estimated to contain 203.7 g/l of total glucose (calculated by 306 g 0.599/0.9 l), and production of 103.4 g/l of glucose was regarded as 50.8% (calculated by 103.4 g/ 203.7 g 100) of saccharification efficiency. The saccharification experiment was conducted without a sterilizing treatment and production of gas and smell of alcohol was detected after 96 h of reaction as a result of activities of contaminant microbes. Therefore, experiments of the following ethanol fermentation were conducted using a newly prepared saccharified juice obtained after 24 h of enzymatic digestion before the growth of contaminant microbes. Ethanol fermentation The supernatant of the newly prepared saccharified juice containing 92 g/l of glucose was fermented with yeast strains of S. cerevisiae NBRC10217T and K7 for 15 days, i.e., by the monographic double-fermentation method, and production of ethanol was observed at 61.7 g/l (78.1 ml/l) and 65.0 g/l (82.3 ml/l) levels on average, respectively (Table 5). Furthermore, saccharified juice containing 87.3 g/l of glucose was fermented in a condition containing the solid part of seeds, i.e., by the parallel double-fermentation method. Production of ethanol was observed at 117.4 g/l (148.6 ml/l, for S. cerevisiae NBRC10217T) and 130.4 g/l (165.1 ml/l, for S. cerevisiae K7) levels, respectively (Table 5). This data is based on duplicate fermentation tanks, and the highest value of ethanol concentration was found for S. cerevisiae K7 at a 145.6 g/l (184.3 ml/l) level.
TABLE 1. Characteristics of Zostera marina seeds. Characteristics
Mean
SD
Length (mm, n ¼ 20) Width (mm in diameter, n ¼ 20) Dry weight (mg/seed)a Wet weight (mg/seed)b Moisture (%, w/w)c
3.78 1.98 4.7 12.4 62.0
0.37 0.13 0.8 2.1 1.4
a b c
3
Measured for three sets of 100-dry seeds. Estimated from mean dry weight and moisture of fresh seeds. Measured for two sets of 1.5-kg fresh seeds.
Zostera marina (seed, seagrass) Rice (polished)a Rice (unpolished)a Wheat floura Corn (maize kernels)a Zostera marina (whole, seagrass) Eichhornia crassipes (freshwater plant) Ulva sp. (green seaweed) Gracilaria incurvata (red seaweed) Undaria pinnatifida (brown seaweed)
Proximate contents (% on a dry weight basis) Protein
Carbohydrate
Lipid
Ash
10.1 8.0 7.2 12.1 10.1 15.1 9.6
83.5 87.3 91.2 82.5 82.6 57.4 76.7
1.3 3.2 1.1 3.5 5.8 1.4 0.7
5.1 1.4 0.5 1.8 15.2 26.1 13.0
17.0 18.9
58.3 56.6
0.3 0.3
24.5 24.1
18.9
50.3
1.4
29.3
a Data are cited from Subdivision on Resources, The Council for Science and Technology, Ministry of Education, Culture, Sports, Science, and Technology, Japan (32).
DISCUSSION Z. marina is a widespread plant in Northern Hemisphere and the most commonly distributed seagrass species in Japanese coastal waters. Many groups of local governments and non-governmental organizations have been making efforts for preserving and restoring the seagrass bed in Japan. A skillful method has been developed to collect a large quantity of seagrass seeds for annual planting purpose during the joint activities of local government and fisherman’s association over 20 years at the Hinase Bay colony. This seagrass is typically perennial, however, annual colonies are also observed at several waters like the Hinase Bay colony spreading over 200 ha. For the case of perennial colonies, less than 10% of shoots are reproductive (33). In contrast, most of the shoots are reproductive and make seeds for the case of annual colonies. Therefore, it is advantageous to collect seeds at annual colonies making seeds at a 109 seeds/ha level (34). This production is equivalent to 4.7 t/ha of dry seeds (calculated by 4.7 mg per one dry seed 109 seeds) and 2.8 t/ha of glucose and starch (calculated by 4.7 t 59.9%). Harvest of barley, wheat, and corn is estimated to be 5.8, 7.2, and 6.9 t/ha, respectively (35). Harvest of seagrass is a little smaller than those of cereal resources, however, increase of harvest can be expected if the Z. marina is cultured as a crop in the future. When the reproductive shoots make seeds, the shoots will be detached and drift in May and June. Seeds may be able to be collected from these drifting shoots as an eco-harmonized manner, although seeds were collected from harvested shoots in the present study. The nutritional analysis clarified that the seagrass seeds are rich in starch at a level comparable to cereal resources. It is noteworthy that seagrass has a potential as a crop. Saccharification of seagrass seeds was studied in the present study, and it was observed that commercial enzyme products containing glucoamylase activity are
TABLE 3. Sugar composition of seagrass seeds. Sugars Glucose Xylose Rhamnose Arabinose Fructose Starch
Contents (% on a dry weight basis) 59.9 3.5 0.3 0.2
Production of 16.5% v/v ethanol from seagrass seeds Motoharu Uchida,1, * Tatsuo Miyoshi,1 Masaki Kaneniwa,2 Kenji Ishihara,2 Yutaka Nakashimada,3 and Naoto Urano4 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi 2-17-5, Hatsukaichi-shi, Hiroshima 739-0452, Japan,1 National Research Institute of Fisheries Science, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 235-0452, Japan,2 Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8530, Japan,3 and Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan4 Received 3 April 2014; accepted 22 May 2014 Available online xxx
Ethanol fermentation on seeds of seagrass Zostera marina was studied. The seeds were collected from the annual plant colony of Z. marina at Hinase Bay, Okayama. The seeds contained 83.5% carbohydrates including 48.1% crude starch on a dry weight basis, which is comparable to cereals such as wheat flour and corns. The seeds were saccharified with glucoamylase (50 C, 96 h) and 103.4 g/l concentration of glucose juice was obtained. The glucose juice was further fermented (23 Ce35 C, 15 days) with Saccharomyces cerevisiae strains NBRC10217T and Kyokai 7-go, and ethanol was obtained at a 65.0 g/l (82.3 ml/l) level by monographic double-fermentation and at a 130.4 g/l (165.1 ml/l) level by parallel double-fermentation. Fermented products of seagrass seeds containing such a high ethanol concentration as the present study have potential to be utilized not only for biofuel but also for foods and beverages in the future. Culturing of seagrass seeds as a crop may enable development of a new marine fermentation industry. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aquatic bioresources; Ethanol fermentation; Saccharomyces cerevisiae; Seagrass seed; Zostera marina]
Research in bioethanol production from aquatic plants and algae has increased after the 2000s (1e6). Choice of a suitable species as a substrate is one of key elements for effective ethanol production. Korean research groups are focusing on the utilization of red algae as a substrate (7,8). Red algae are known to contain galactan as a major component [20e51% on a dry weight basis (9)] besides glucan. Relatively higher quantity of fermentable sugars of galactose and glucose can be obtained by acid-hydrolisis of red algae [30.4% of dry matter on average (10)], and both of the sugars can be further metabolized to ethanol by yeast with a conversion efficiency at 51% (w/w) according to the following reaction: C6H12O6/2C2H5OH þ 2CO2. Park et al. (11) and Yanagisawa et al. (12) produced ethanol from agar weeds at 33 ml/l and 55 ml/l levels, respectively. Brown algae are another candidate as a suitable substrate for ethanol production (13). Brown algae contain alginate [3e38% on a dry weight basis (14)] as major components, but microorganisms rarely convert the alginate to ethanol. To overcome this problem, genetically engineered microorganisms were developed to convert alginate to ethanol, and the ethanol concentration in the fermentation culture was observed at 13 g/l (16.5 ml/l) utilizing alginate alone as a carbon source (15) and 47 ml/l utilizing whole components of Saccharina japonica (kombu) as carbon sources (16). Green algae are also a candidate as a substrate since some species such as Ulva spp. are known to make huge blooms in coastal waters. Isa et al. (17) demonstrated ethanol production from
* Corresponding author. Tel.: þ81 829 55 3580; fax: þ81 829 54 1216. E-mail address: [email protected] (M. Uchida).
green algae at a 10 g/l (12.7 ml/l) level. As for seagrass, Viola et al. (18) produced ethanol at a 47 ml/l level from eel grass (Zostera marina). As for freshwater plants, water-hyacinth (Eichhornia crassipes) is known to be suitable as a substrate for fermentation (19e21). Mishima et al. (22) produced ethanol at a 16.9 g/l (21.4 ml/ l) level from water-hyacinth. Microalgae have also been studied as substrates for ethanol fermentation (23e25). Harun et al. (26) demonstrated production of ethanol at a 3.83 g/l (4.8 ml/l) level from Chlorococcum sp. However, aquatic plants and algae are generally moisture rich and dry matter contents and fermentable sugars that are expected to be converted to ethanol are estimated to be only 12.5% (w/w) and 1.6 % (w/w) on average, respectively (10). Therefore, the ethanol concentration obtained from fermentation of aquatic plant and algae is limited to a low level [mostly less than 20 ml/l (1, 27, 28)]. For use of the ethanol product as a fuel, distillation and dehydration of ethanol to a 99.9% (v/v) concentration level is necessary. This distillation process needs a high energy input (29), and, therefore, it is advantageous to obtain a fermented product with a high ethanol concentration from the view-point of energy balance. It is mentioned in manuscripts (12) that the economically feasible concentration of ethanol contained in fermented products is at least larger than 4e5% (v/v). The present study focused on obtaining fermented products that contains ethanol at a high concentration and chose seeds of seagrass Z. marina as a substrate. Firstly, seagrass seeds were collected and the basic characteristics and sugar component of the seeds were clarified and compared with other substrates. Secondly, saccharification conditions were optimized to obtain glucose juice from the seeds. Thirdly, fermentation of the glucose juice was
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.05.017
Please cite this article in press as: Uchida, M., et al., Production of 16.5% v/v ethanol from seagrass seeds, J. Biosci. Bioeng., (2014), http:// dx.doi.org/10.1016/j.jbiosc.2014.05.017
2
UCHIDA ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Collection of seeds of seagrass Zostera marina. The flowering shoots were collected in early June, 2011 at Hinase Bay, Okayama. The shoots were submerged in water for five months for biodegradation and harvested. The harvested seeds were eliminated for debris and small animals, freeze dried, milled and used for saccharification and fermentation experiments.
conducted with yeast using a monographic double-fermentation method, i.e., a method performing saccharification and fermentation separately. Furthermore, a parallel double-fermentation method was tested to obtain higher concentration of ethanol. The parallel double-fermentation method is a method performing saccharification and fermentation in parallel in a tank, and this method makes it possible to obtain approximately w20% (v/v) ethanol products during the manufacture of Japanese sake or rice wine. MATERIALS AND METHODS Collection and characterization of seagrass seeds The seeds of Z. marina were collected according to the method of Fukuda (30). Flowering shoots were harvested at the annual plant colony of Hinase Bay, Okayama, and housed in plastic mesh bags in early June 2011. The mesh bags were submerged in water by being hung down from oyster rafts and let to maturate and biodegrade. The biodegraded shoots were harvested after five month in October, the debris and degrading animals such as polychaetes and gastropods were removed, and wet seeds were obtained. The wet seeds were washed with tap water and freeze dried to obtain dry seeds, and stocked until use. The seeds were measured for weight and size. The seeds were milled by using a mixer (Panasonic, MK-K48P-W), and analyzed for proximate content according to the method by the Council for Science and Technology, Ministry of Education, Culture, Sports, Science, and Technology, Japan (31). Carbohydrate content % (w/w) was obtained by subtracting the values of protein%, lipid%, and ash% from 100%. Starch content was measured as follows: Aliquots (0.1e0.3 g) of milled seeds were weighed accurately and washed with 10 ml of 50% (v/v) ethanol to eliminate low molecular weight sugars. The pellet was collected and added with 1 ml of 99.5% (v/v) ethanol, 20 ml of water, and 2 ml of 10% (w/v) sodium hydroxide, and heated for 3 min in boiling water. The sample was cooled to room temperature, neutralized with 25% (v/v) hydrochloric acid, then further reacted with amyloglucosidase (Megazyme, Biocon Ltd., Nagoya, >480 U) for 2 h at 37 C. The produced glucose was measured with an enzymatic method (Roche Diagnostics K.K., Tokyo). Sugar composition was measured as follows: Aliquots (0.3e0.6 g) of milled seeds were weighed and incubated stirring with 2 ml of 72% (w/w) sulfuric acid for 1 h at room temperature, then diluted with 56 ml of distilled water (final conc. 4% (w/ w)) and autoclaved for 1 h at 121 C. The mixture was neutralized with 30% (w/w) sodium hydroxide, volume adjusted to 100 ml, and analyzed after passing through No.5B (Toyo Roshi Kaisha, Ltd., Tokyo) and 0.45 mm-mesh filters. The quantitative analysis of sugar compounds was performed on a Shimadzu high-performance liquid chromatography (HPLC) system model LC-20AD equipped with a Shimadzu RF-20AxS detector (Shimadzu Corporation, Kyoto). Twenty microliters of sample
was injected, and the chromatographic separation was performed on a TSKgel Sugar AXI (Tosoh Corporation, Tokyo) anion exchange column, 4.6 mm 150 mm at 60 C with a mobile phase of 0.5 M boric acid buffer solution (0.4 ml/min, pH 8.7). The sugars were coupled with 1% (w/v) L-alginin solution (0.7 ml/min) at 150 C to give a fluorophor, which is detected by a fluorometer with excitation at 320 nm and detection of emission measured at 430 nm. Saccharification of seagrass seeds Two grams of the seed powder, 0.06 g of enzymes, 4 ml of distilled water were placed in glass tubes (16 mm in diameter) and mixed. The glass tubes were set at a 30 inclination against horizontal line and reacted by shaking (120 rpm) at 50 C. The enzymes tested were Cellulase 12S (Yakult Honsha Co. Ltd., Tokyo, 12,000 U/g), Cellulase A Amano 3 (Amano Enzyme Inc., Nagoya, 30,000 U/g), Cellulase T Amano 4 (Amano Enzyme Inc., 40,000 U/g), Hemicellulase Amano 90 (Amano Enzyme Inc., xylanase 9000 U/g), Gluczyme AF6 (Amano Enzyme Inc., glucoamylase 1600 U/g), and Gluc 100G (Amano Enzyme Inc., glucoamylase 60,000 U/g and a-amylase 350,000 U/g). The produced glucose was measured periodically using a F-kit glucose. The test was conducted using duplicate tubes and the data is shown as mean standard deviation (SD) value. Fermentation of seagrass seeds Fermentation tests were conducted by both monographic double-fermentation and parallel double-fermentation methods. For monographic double-fermentation, a mixture of 12 g of the seed powder, 0.24 g of Gluczyme AF6, 0.042 g of Cellulase A Amano 3, 0.042 mg of Hemicellulase Amano 90, 24 ml of distilled water was reacted for 24 h at 50 C, and the saccharified juice was obtained by centrifuging for 20 min at 6000 g. Aliquots of the juice were taken and measured for glucose and galactose contents by enzymatic methods (F-kit glucose, F-kit galactose, Roche Diagnostics K.K.). The remaining juice was filtrated (GS, Millipore) and dispensed 1.2 ml each into six sterile two milliliters-volume polypropylene centrifuge tubes. Fermentation was conducted under three experimental conditions: the duplicated tubes for each were incubated with Saccharomyces cereviciae NBRC10217T, S. cerevisiae Kyokai 7-go (K7, purchased from Brewing Society of Japan), and without yeast, respectively. Fresh yeast cells grown on GAM plates (Nissui Pharmaceutical Co. Ltd.) were collected and suspended in autoclaved 0.85% (w/v) NaCl solution at a concentration of OD660nm ¼ 0.5 (contain approximately 2 105 CFU/ml). Inoculation was conducted by adding 5% volume (¼ 60 ml) of the yeast cell suspension to culture water. Incubation was conducted for the initial five days at 37 C and further 10 days at 23 C. For parallel double-fermentation, a mixture of 500 g of the seed powder, 7.5 g of Gluczyme AF6, 1.5 g of Cellulase A Amano 3, 1.5 g of Hemicellulase Amano 90, and one liter of distilled water was reacted for 24 h at 50 C. The saccharified juice was autoclaved two times for 20 min at 90 C with a 24 h-interval for sterilization. The juice was dispensed 50 g (45 ml) each for four sterile screw-capped centrifuge tubes and 500 g (450 ml) each for two screw-caped plastic bottles. The 50-g scale (n ¼ 2) and 500-g scale (n ¼ 1) bottles were fermented after adding 1% volume of the two kinds of yeast cell suspensions. Fermentation was conducted for the initial five days at 37 C and a further 44 days at 23 C. Glucose and ethanol contents were measured periodically by using F-kit glucose and F-kit ethanol (Roche Diagnostics K.K.).
Please cite this article in press as: Uchida, M., et al., Production of 16.5% v/v ethanol from seagrass seeds, J. Biosci. Bioeng., (2014), http:// dx.doi.org/10.1016/j.jbiosc.2014.05.017
VOL. xx, 2014
ETHANOL FERMENTATION ON SEAGRASS SEEDS RESULTS
TABLE 2. Proximate contents of seagrass seeds, cereals, and seaweeds. Potential substrate
Collection of seagrass seeds Scheme of collection of seagrass seeds is shown in Fig. 1. The freeze dried seeds, 1.1 kg, were obtained from 3.1 kg of wet seeds, milled and stocked for use of saccharification and fermentation experiments. Characteristics of seagrass seeds Basic characteristics of seeds of Z. marina are shown in Table 1. The size is 3.78 0.37 mm (mean SD) in length and 1.98 0.13 mm in width (diameter). The weight was 12.4 2.1 mg on a wet weight basis or 4.7 0.8 mg on a dry weight basis. The fresh seeds contained 62.0% (w/w) of moisture. Proxy contents of the seeds are shown in Table 2 with those of reference materials (32). The seeds contained 83.5% carbohydrate, 10.1% protein, 1.3% lipid, and 5.1% ash on a dry weight basis. The richness of carbohydrate in seagrass seeds is comparable to those of cereals (82.5e91.2%). Sugar composition of the seeds was glucose (59.9%), xylose (3.5%), rhamnose (0.3%), and arabinose (0.2%), and starch content was 48.1% on a dry weight basis (Table 3). Saccharification The seeds were saccharified with enzymes in culture water containing 333 g of dried seeds/kg or 306 g of dry matter/kg (or per 0.9 l of culture water). The maximum glucose content was obtained at a 103.4 g/l level after reaction with Gluczyme AF6 (glucoamylase) for 96 h at 50 C (Table 4). The use of cellulase and hemicellulase were observed to be less effective. Gluc 100G is an enzyme mixture product designed for sakemanufacturing containing glucoamylase and a-amylase activities, but the effect was inferior to that of Gluczyme AF6. The reaction mixture was estimated to contain 203.7 g/l of total glucose (calculated by 306 g 0.599/0.9 l), and production of 103.4 g/l of glucose was regarded as 50.8% (calculated by 103.4 g/ 203.7 g 100) of saccharification efficiency. The saccharification experiment was conducted without a sterilizing treatment and production of gas and smell of alcohol was detected after 96 h of reaction as a result of activities of contaminant microbes. Therefore, experiments of the following ethanol fermentation were conducted using a newly prepared saccharified juice obtained after 24 h of enzymatic digestion before the growth of contaminant microbes. Ethanol fermentation The supernatant of the newly prepared saccharified juice containing 92 g/l of glucose was fermented with yeast strains of S. cerevisiae NBRC10217T and K7 for 15 days, i.e., by the monographic double-fermentation method, and production of ethanol was observed at 61.7 g/l (78.1 ml/l) and 65.0 g/l (82.3 ml/l) levels on average, respectively (Table 5). Furthermore, saccharified juice containing 87.3 g/l of glucose was fermented in a condition containing the solid part of seeds, i.e., by the parallel double-fermentation method. Production of ethanol was observed at 117.4 g/l (148.6 ml/l, for S. cerevisiae NBRC10217T) and 130.4 g/l (165.1 ml/l, for S. cerevisiae K7) levels, respectively (Table 5). This data is based on duplicate fermentation tanks, and the highest value of ethanol concentration was found for S. cerevisiae K7 at a 145.6 g/l (184.3 ml/l) level.
TABLE 1. Characteristics of Zostera marina seeds. Characteristics
Mean
SD
Length (mm, n ¼ 20) Width (mm in diameter, n ¼ 20) Dry weight (mg/seed)a Wet weight (mg/seed)b Moisture (%, w/w)c
3.78 1.98 4.7 12.4 62.0
0.37 0.13 0.8 2.1 1.4
a b c
3
Measured for three sets of 100-dry seeds. Estimated from mean dry weight and moisture of fresh seeds. Measured for two sets of 1.5-kg fresh seeds.
Zostera marina (seed, seagrass) Rice (polished)a Rice (unpolished)a Wheat floura Corn (maize kernels)a Zostera marina (whole, seagrass) Eichhornia crassipes (freshwater plant) Ulva sp. (green seaweed) Gracilaria incurvata (red seaweed) Undaria pinnatifida (brown seaweed)
Proximate contents (% on a dry weight basis) Protein
Carbohydrate
Lipid
Ash
10.1 8.0 7.2 12.1 10.1 15.1 9.6
83.5 87.3 91.2 82.5 82.6 57.4 76.7
1.3 3.2 1.1 3.5 5.8 1.4 0.7
5.1 1.4 0.5 1.8 15.2 26.1 13.0
17.0 18.9
58.3 56.6
0.3 0.3
24.5 24.1
18.9
50.3
1.4
29.3
a Data are cited from Subdivision on Resources, The Council for Science and Technology, Ministry of Education, Culture, Sports, Science, and Technology, Japan (32).
DISCUSSION Z. marina is a widespread plant in Northern Hemisphere and the most commonly distributed seagrass species in Japanese coastal waters. Many groups of local governments and non-governmental organizations have been making efforts for preserving and restoring the seagrass bed in Japan. A skillful method has been developed to collect a large quantity of seagrass seeds for annual planting purpose during the joint activities of local government and fisherman’s association over 20 years at the Hinase Bay colony. This seagrass is typically perennial, however, annual colonies are also observed at several waters like the Hinase Bay colony spreading over 200 ha. For the case of perennial colonies, less than 10% of shoots are reproductive (33). In contrast, most of the shoots are reproductive and make seeds for the case of annual colonies. Therefore, it is advantageous to collect seeds at annual colonies making seeds at a 109 seeds/ha level (34). This production is equivalent to 4.7 t/ha of dry seeds (calculated by 4.7 mg per one dry seed 109 seeds) and 2.8 t/ha of glucose and starch (calculated by 4.7 t 59.9%). Harvest of barley, wheat, and corn is estimated to be 5.8, 7.2, and 6.9 t/ha, respectively (35). Harvest of seagrass is a little smaller than those of cereal resources, however, increase of harvest can be expected if the Z. marina is cultured as a crop in the future. When the reproductive shoots make seeds, the shoots will be detached and drift in May and June. Seeds may be able to be collected from these drifting shoots as an eco-harmonized manner, although seeds were collected from harvested shoots in the present study. The nutritional analysis clarified that the seagrass seeds are rich in starch at a level comparable to cereal resources. It is noteworthy that seagrass has a potential as a crop. Saccharification of seagrass seeds was studied in the present study, and it was observed that commercial enzyme products containing glucoamylase activity are
TABLE 3. Sugar composition of seagrass seeds. Sugars Glucose Xylose Rhamnose Arabinose Fructose Starch
Contents (% on a dry weight basis) 59.9 3.5 0.3 0.2
