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Sustainable production of bioethanol from renewable brown algae biomass
Biomass and Bioenergy 92 (2016) 70e75
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Biomass and Bioenergy journal homepage: http://www.elsevier.com/locate/biombioe
Review
Sustainable production of bioethanol from renewable brown algae biomass Ok Kyung Lee, Eun Yeol Lee* Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2015 Received in revised form 29 February 2016 Accepted 23 March 2016
Brown algae have been considered as renewable biomass for bioethanol production because of high growth rate and sugar level. Saccharification of brown algae biomass is relatively easy due to the absence of lignin. Among the major sugar components of brown algae, alginate cannot be directly used because industrial microorganisms are not able to metabolize alginate. This problem has been overcome by the development of metabolically engineered microbes to efficiently utilize alginate. This review analyzes and evaluates recent research activities related to bioethanol production from brown algae. This review mainly deals with the recent development and potential of a metabolically engineered microbial cell factory and bioethanol production from brown algae biomass including alginate as the main carbohydrate. Future researches for cost-effective bioethanol production from brown algae are discussed. © 2016 Published by Elsevier Ltd.
Keywords: Brown algae Alginate Bioethanol production Metabolically engineered microbes
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Brown algae as renewable and sustainable biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Bioethanol production from brown algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 The pros and cons of bioethanol production from brown algae biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Conclusion remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1. Introduction Due to the decline of petroleum reserves and a dramatic explosion in demand for energy, various types of alternative energy have been extensively investigated. Biofuel such as bioethanol, biodiesel and biohydrogen is one of the promising alternatives [1e3]. However, the production of biofuels from biomass has some drawbacks. In the case of first-generation biomass, there are moral issues with using food for biofuel production. In order to use second-generation lignocellulosic biomass, cost-intensive pretreatment is absolutely required before saccharification and fermentation [4].
* Corresponding author. E-mail address: [email protected] (E.Y. Lee). http://dx.doi.org/10.1016/j.biombioe.2016.03.038 0961-9534/© 2016 Published by Elsevier Ltd.
Macro- and microalgae are an example of third-generation biomass [5]. Algae present several advantages over other types of biomass. Algae are the fastest growing photosynthetic organisms [6]. Algae can efficiently remove carbon dioxide and synthesize polysaccharides or oil that can be used for biofuel production [7]. Generally, algal carbohydrates can be used for bioethanol fermentation after relatively easy saccharification due to the absence of lignin [5]. Oil can be trans-esterified into biodiesel [8]. In addition, algal biomass can be directly used for heat and power generation using anaerobic digestion and pyrolysis [9e11] (Fig. 1). There are three types of macroalgae, brown, green and red algae [12]. Brown algae such as sea mustard (Undaria pinnatifida) and kelp (Saccharina japonica) are one of the promising biomass for biofuel production because cultivation productivity based on area is the highest among three types of macroalgae [13,14],
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Fig. 1. Conceptual diagram of brown algae for production of biofuels and biochemicals.
approximately 40 kg wet biomass/m2 of gulf-weed (Sargassum muticum), compared to 2.3 and 6.6 kg/m2 of green laver (Ulva lactuca) and agar weed (Gelidium amansii), respectively. The largescale cultivation of brown algae is already practiced in several countries including Korea, China and Japan. Compared to microalgae, brown algae have a higher sugar and lower oil contents. Thus, brown algae are a more suitable feedstock for bioethanol production than biodiesel. With respect to bioethanol productivity, one report prepared for the US Department of Energy claimed that bioethanol productivity from macroalgae per hectare per year could theoretically be two times higher than sugarcane and five times higher than corn [15]. This review is intended to analyze and evaluate recent research activities related to biofuel production from brown algae, especially bioethanol. This review mainly deals with the recent development and potential of a metabolically engineered microbial cell factory and bioethanol production from brown algae biomass including alginate as the main carbohydrate. 2. Brown algae as renewable and sustainable biomass Brown algae have several advantages as a feedstock for production of biofuels. Firstly, brown algae avoid competition with food production. Brown algae requires no arable land and are cultivated in the ocean using aquaculture without needing the expensive nutrients, fertilizers or fresh water that are required for conventional agriculture. Brown algae have no lignin, and thereby no extensive pretreatment is used to release sugars, but just using simple operations such as milling or crushing. There is no moral issue associated with the use of brown algae for biofuel production. One particularly important advantage is that brown algae can offer high biomass yields per acre [13,14]. Brown algae contain high levels of carbohydrates, contributing up to 55% (w/w) of the dry biomass [16]. Therefore, brown algae are an ideal renewable and sustainable biomass due to its abundance and high sugar levels that can be used for production of bioethanol and chemicals. Roughly 70 million dry tons of macroalgae are cultivated and harvested worldwide in offshore and near-shore coastal farms [17]. This production scale is mostly for food applications. Cultured brown algae are harvested by both manual and mechanical methods. The harvested brown algae are then normally treated
with milling to reduce biomass sizes for efficient saccharification or alginate extraction [18] (Fig. 2). The saccharified broth can be used for bioethanol fermentation. 3. Bioethanol production from brown algae The most abundant sugars in brown algae are alginate, mannitol and laminarin. Mannitol and glucose from laminarin (a form of glucan in brown algae) are normal sugars that are efficiently used for bioethanol fermentation [19,20]. Laminarin and mannitol from Laminaria hyperborea extracts were fermented to bioethanol under oxygen-limiting conditions using Zymobacter palmae [21]. Bioethanol with a yield of 0.4 g ethanol/g of sugars was produced with ethanogenic Escherichia coli KO11 from Laminaria japonica hydrolysates mainly containing mannitol after chemo-enzymatic saccharification [22]. Ethanol of 7.7 g/L was produced from S. japonica biomass using the simultaneous saccharification and fermentation (SSF) method with a theoretical yield of 33.3% [23]. Alginate is a structural polysaccharide in the cell wall of brown algae [24,25]. In some brown algae, alginate constitutes up to 60% of the total sugars. Thereby, alginate, the most abundant carbohydrate, should be utilized for the production of bioethanol. Alginate is present in the insoluble calcium salt form [18]. In the alginate extraction process, calcium alginate is converted to alginic acid in an acid pre-extraction step, and then alginic acid is converted to soluble sodium alginate in alkaline extraction [26]. Alginate can be degraded into unsaturated uronate monosaccharides using alginate lyase [27]. Alginate lyase is the enzyme that catalyzes the b-elimination breakage of the glycosidic bond of alginate. Various alginate lyases have been cloned and characterized [28e30]. Recently, alginate lyase-based saccharification has been developed by employing exolytic alginate lyase as the biocatalyst with a 30% yield [31,32]. Theoretically, ethanol cannot be directly synthesized from unsaturated uronic monosaccharides under anaerobic conditions because no NADH is available for alcohol dehydrogenase to catalyze the conversion of acetaldehyde to ethanol. The NADH generated from glyceraldehyde-3-phosphate (G-3-P) to pyruvate is used for the conversion of 4-deoxy-L-erythro-hexoseulose uronate (DEH) to 2-keto-3-deoxygluconate (KDG) (Fig. 3). As a result, the net NADH
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Fig. 2. Processing of brown algae for bioethanol production.
Fig. 3. Metabolically engineered pathway of alginate monosaccharide degradation in a microbial cell factory for production of bioethanol from alginate and mannitol of brown algae.
is zero when an unsaturated monosaccharide is converted to 2 mol of pyruvate. Thus, alginate is considered to be a difficult-to-ferment sugar for ethanol fermentation.
A research group at Kyoto University reported that metabolic engineering of a native alginate-metabolizing Sphingomonas sp. A1 cells harboring pyruvate decarboxylase and alcohol dehydrogenase
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Fig. 4. Metabolic pathway of alginate monosaccharide degradation and biosynthesis of biofuels from alginate. DEH(4-deoxy-L-erythro-hexoseulose uronic acid), KDG(2-keto-3deoxy-D-gluconic acid), KDPG(2-keto-3-deoxy-6-phosphogluconic acid), G-3-P(glyceraldehydes-3-phosphate).
II from Zymomonas mobilis could produce bioethanol from alginate [33]. A pit in the range of 0.02e0.1 mm was formed on the surface of the cell in the presence of alginate. Alginate polymer was directly transported into the inner membrane of the cell through bacterial super channel and periplasmic alginate-binding proteins [34]. Alginate was transported into the cytoplasm via an ATP-binding cassette (ABC) transporter in the inner membrane [35]. The alginate was degraded into oligomer and monosaccharides by various alginate lyases inside the cytoplasm. The unsaturated monosaccharide was nonenzymatically converted to a-keto acid, DEH. In Sphingomonas sp. A1, DEH is converted to KDG, a metabolite of Entner-Doudoroff (ED) pathway, by NADPH-dependent uronate reductase [36] (Fig. 4). KDG was further degraded to pyruvate and glyceraldehyde-3-phosphate by kinase A1-K and aldolase A1-A. Pyruvate was converted to ethanol by pyruvate decarboxylase and alcohol dehydrogenase II [33]. Metabolically engineered Sphingomonas sp. A1 with a homoethanol pathway could produce 0.25 kg bioethanol/kg alginate with a volumetric productivity of 13 g/L. In contrast to alginate, an additional 1 mol of NADH is generated under fermentative conditions when mannitol is metabolized to 2 mol of ethanol (Fig. 3). One mole of NADH is generated from the conversion of mannitol to fructose-6-phosphate [21]. When fructose-6-phosphate is converted to two pyruvates, two NADHs are generated and these NADHs are consumed in the conversion of 2 mol of pyruvate to 2 mol of ethanol. Therefore, if 2 mol of mannitol and 1 mol of alginate monosaccharide are simultaneously utilized by cells under fermentative conditions, the amount of NADH is balanced (Fig. 3). Interestingly, a typical sugar ratio of mannitol to alginate in brown algae is approximately 8:5 [37]. Hence, these two sugar components in brown algae can be simultaneously used for ethanol fermentation. To utilize alginate and mannitol simultaneously in bioethanol
fermentation, the recruitment of alginate lyase enzymes for alginate uptake/degradation and reconstruction of metabolic pathway for converting the monosaccharide into ethanol are necessary. Recently, E. coli has been engineered to utilize all sugars from brown algae to make bioethanol [37]. Key genes for alginate uptake, degradation and metabolism of Vibrio splendidus 12B01 were successfully inserted into E. coli. In metabolically engineered E. coli, the alginate polymer was degraded into alginate oligomers by an extracellular alginate lyase from Pseudoalteromonas sp. SM0524. The oligomers were transported into the cytoplasm by an essential alginate transport system (putative sodium/solute symporter, termed ToaA) from V. splendidus. The oligomers were degraded to unsaturated monosaccharides by various alginate lyases, and then non-enzymatically isomerized DEH was converted to KDG by a putative NADH/NADPH-dependent alcohol dehydrogenase from V. splendidus 12B01. This alcohol dehydrogenase exhibited a preference for NADH rather than NADPH, and showed approximately 40% DNA sequence identity with NADPH-dependent uronate reductase from Sphingomonas sp. A1. The resulting KDG was further metabolized to pyruvate by kinase and aldolase of ED pathway in E. coli. Finally, the pyruvate was fermented to ethanol by relevant genes from Z. mobilis. Using the metabolically engineered microbial platform, ethanol at a final titer of approximately 4.7% (v/v) was produced from S. japonica biomass. Ethanol conversion was approximately 0.281 wt ethanol/wt dry biomass, equivalent to 80% of the maximum theoretical yield with an overall rate of 0.64 g/L/h. In this way, metabolic engineering enables the difficult-to-ferment alginate to be used as a renewable biomass for bioethanol fermentation, and thus makes brown algae an economically alternative feedstock. Recently, a synthetic yeast platform was developed for efficient bioethanol production from brown algae [38]. The DEH transporter
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Table 1 Comparison of annual production, sugar composition and bioethanol yields of algae. Macroalgae
Microalgae
Red algae
Brown algae
Green algae
Production (ton/year)
8,978,535a
6,784,93a
21,545a
Species
Kappaphycus alvarezii Eucheuma denticulatum Eucheuma sp. Gracilaria verrucosa Carrageenan Agar Cellulose
Laminaria japonica Undaria pinnatifida Sargassum fusiforme Phaeophyceae Laminarin, Mannitol Alginate Fucoidan Cellulose 0.152e0.196, 12.98 [45] 0.281, 37.8 [37] 0.362, 36.2 [38]
Codium fragile Caulerpa sp. Monostroma nitidum Enteromorpha clathrata Starch Cellulose
Sugars
Bioethanol yield and titer (g/g dry biomass, g/L) a b c
0.045, 0.5e0.83 [43] 0.236, 4.72 [44]
0.09, 9.31 [46]
10,000b 7,000c Chlorella Dunaliella Spirulina Glucose Galactose Xylose Arabinose Rhamnose 0.234, 11.7 [47]
Estimated in wet metric ton and adopted from FAO [40]. Total world production of dry algal biomass [41]. Total world production of dry algal biomass that are mostly produced in open systems [42].
from Asteromyces cruciatus was cloned and integrated into Saccharomyces cerevisiae genome to confer ability to uptake the unusual sugar. In order to synchronize the DEH and mannitol metabolic pathway, all relevant genes for alginate and mannitol metabolism were also chromosomally integrated. The engineered yeast produced bioethanol with a bioethanol titer of 4.6% (v/v) (36.2 g/L), efficiency of 75% based on total sugars, and productivity of 1.9 g/L/h from 1:2 M ratio of DEH and mannitol at 9.8% (w/v). Thus, manipulation of metabolic network of other microbial strain based on the synchronization of alginate and mannitol metabolic pathway enables the use of total sugars including alginate monosaccharide in brown algae for bioethanol production. Although the recombinant Sphingomonas, Escherichia and Saccharomyces strains have been successfully constructed for bioethanol production, some relevant issues need to be addressed. For the recombinant Sphingomonas strain, various genetic tools need to be developed for metabolic engineering to construct a microbial cell factory with enhanced bioethanol productivity. Optimization of fermentation conditions for the recombinant Sphingomonas strain should be conducted to enhance the bioethanol productivity. In the case of recombinant E. coli strain, uronate reductase should be functionally expressed because a large amount of reductase was expressed as insoluble form and reductase activity was rather low in E. coli. The recombinant Sphingomonas and Escherichia strains need to be engineered to be tolerant to high bioethanol concentration. The recruitment and functional expression of alginate-specific transporter is important for the recombinant Escherichia and Saccharomyces strains to increase bioethanol fermentation efficiency. Cell surface display or efficient secretion expression of various alginate lyases and other saccharifying enzymes is required for an efficient consolidating bioprocessing of brown algae biomass for bioethanol production. EMP, PP and ED pathways of microbial cell factory are required to be reconstructed and optimized to utilize all kinds of sugars in brown algae and enhance the bioethanol productivity. 4. The pros and cons of bioethanol production from brown algae biomass Bioethanol production from brown algae is compared with that from microalgae and other macroalgae (red and green algae), as shown in Table 1. Brown algae can be a good feedstock for bioethanol production based on high productivity. When recombinant E. coli and S. cerevisiae expressing alginate metabolizing genes and transporters were used for bioethanol fermentation, the
productivity of 0.28e0.36 g/g dry biomass were obtained [37,38]. Generally, the bioethanol productivity from red, green algae and microalgae were in the range of 0.045e0.236 g/g dry biomass [39,43e47]. There are many constraints in commercialization of bioethanol production from brown algae. Large-scale and cost-effective production of brown algae biomass is one of those hurdles in commercial bioethanol production. The current floating cultivation and non-automatic harvesting technology are not appropriate in terms of economics. At present, brown algae are harvested only for one to three months per year in a restricted area, and thus it is difficult to have large amount of brown algae biomass at low price [48]. In order to make brown algae-derived bioethanol in a practical scale, the development of mass production technology of brown algae biomass at low cost is absolutely required. Development of yearround multiple cultivation technology of brown algae can be a solution for cost-effective production of brown algae biomass. Recently, Song et al. suggested that the annual production of brown algae can be enhanced by combining the species [48]. They reported that species combination (e.g., Undria, Sargassum, and Ekulonia) could enhance annual productivity of brown algae by 2e4 times. In South Korea, a research program has been conducting on the mass cultivation of brown algae and the production of biofuel from the brown algae since 2008 [49]. They plan to demonstrate the brown algae biomass production with 50e60 ton (dry wt)/ha/yr productivity in offshore farms. Cost-effective production of brown algae biomass will be a key success factor for commercialization, together with the development of robust microbial cell factories. 5. Conclusion remarks At present, bioethanol production from brown algae is not yet economically-feasible [17]. Bioethanol production from brown algae is in the early stage of commercialization even though some distinct advances in metabolic engineering have been realized [37]. Bioethanol derived from brown algae is a drop-in fuel that can be used for existing engines. Thus, the commercialization of ethanol production from brown algae could become possible once costeffective methods for brown algae cultivation and integrated bioprocessing are developed. With respect to the practice of brown algae cultivation, it was estimated that more than 60 billion gallons of biofuel from brown algae could be produced across three percent of the world's coastlines [14,15]. This would result in the development of cost-effective and large-scale cultivation methods that
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could be applied in the coastal environment and would certainly meet production goals. Metabolic engineering in the development of a microbial cell factory that utilizes various brown algae carbohydrates and converts those sugars to bioethanol will play a key role in the commercialization of bioethanol production from brown algae. Various metabolic engineering-related tools such as genomics, proteomics, and metabolomics enable us to develop microbial cell factories that contribute to the cost-effective production of ethanol. In addition to bioethanol, metabolic engineering makes it possible to expand product lines such as bio-butanol, as well as other valuable chemicals and biopolymers. Acknowledgments This work was supported by Ministry of Oceans and Fisheries, Republic of Korea. This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012R1A1A2008647). References [1] L.L. Beer, E.S. Boyd, J.W. Peters, M.C. Posewitz, Engineering algae for biohydrogen and biofuel production, Curr. Opin. Biotechnol. 20 (2009) 264e271. [2] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294e306. [3] Y. Lin, S. Tanaka, Ethanol fermentation from biomass resources: current state and prospects, Appl. Microbiol. Biotechnol. 69 (2006) 627e642. [4] Y. Sun, J.Y. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol. 83 (2002) 1e11. [5] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal biomass: a renewable source for bioethanol, Bioresour. Technol. 102 (2011) 186e193. [6] A. Singh, P.S. Nigam, J.D. Murphy, Mechanism and challenges in commercialisation of algal biofuels, Bioresour. Technol. 102 (2011) 26e34. [7] M. Aresta, A. Dibenedetto, G. Barberio, Utilization of macro-algae for enhanced CO2 fixation and biofuels production: development of a computing software for an LCA study, Fuel Process Technol. 86 (2005) 1679e1693. [8] A.B.M. Hossain, A. Salleh, A.N. Boyce, P. Chowdhury, M. Naqiuddin, Biodiesel fuel production from algae as renewable energy, Am. J. Biochem. Biotechnol. 4 (2008) 250e254. [9] A.V. Bridgewater, Biomass fast pyrolysis, Thermal Sci. 8 (2004) 21e50. [10] S. Wang, Q. Wang, X. Jiang, X. Han, H. Ji, Compositional analysis of bio-oil derived from pyrolysis of seaweed, Energy Convers. Manag. 68 (2013) 273e280. [11] V.N. Gunaseelan, Anaerobic digestion of biomass for methane production: a review, Biomass Bioenergy 13 (1997) 83e114. [12] M. Yanagisawa, K. Nakamura, O. Ariga, K. Nakasaki, Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable polysaccharides, Process Biochem. 46 (2011) 2111e2116. [13] M. Aizawa, K. Asaoka, M. Atsumi, T. Sakou, Seaweed bioethanol production in Japan-the ocean sunrise project, in: IEEE Conference Proceedings, Vancouver, Sept. 29eOct. 4 2007, 2007, pp. 1e5. [14] K.A. Jung, S.R. Lim, Y. Kim, J.M. Park, Potentials of macroalgae as feedstocks for biorefinery, Bioresour. Technol. 135 (2013) 182e190. [15] National algal biofuels technology roadmap. http://www1.eere.energy.gov/ bioenergy/pdfs/algal_biofuels_roadmap.pdf. [16] J. Adams, J. Gallagher, I. Donnison, Fermentation study on Saccharina latissima for bioethanol production considering variable pre-treatments, J. Appl. Phycol. 21 (2009) 569e574. [17] Macroalgae as a biomass feedstock: a preliminary analysis. http://www.pnl. gov/main/publications/external/technical_reports/PNNL-19944.pdf. [18] S.Y. Chee, P.K. Wong, C.L. Wong, Extraction and characterization of alginate from brown seaweeds (Fucales, Phaeophyceae) collected from Port Dickson, Peninsular Malaysia, J. Appl. Phycol. 23 (2011) 191e196. [19] J.Y. Lee, P. Li, J. Lee, H.J. Ryu, K. Oh, Ethanol production from Saccharina japonica using an optimized extremely low acid pretreatment followed by simultaneous saccharification and fermentation, Bioresour. Technol. 127 (2013) 119e125. [20] S.J. Horn, I.M. Aasen, K. Østgaard, Ethanol production from seaweed extract, J. Ind. Microbiol. Biotechnol. 25 (2000) 249e254. [21] S.J. Horn, I.M. Aasen, K. Østgaard, Production of ethanol from mannitol by Zymobacter palmae, J. Ind. Microbiol. Biotechnol. 24 (2000) 51e57. [22] N.J. Kim, H. Li, K. Jung, H.N. Chang, P.C. Lee, Ethanol production from marine algal hydrolysates using Escherichia coli KO11, Bioresour. Technol. 102 (2011) 7466e7469. [23] J.S. Jang, Y. Cho, G.T. Jeong, S.K. Kim, Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF)
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[40] FAO, Fishery and Aquaculture Statistics, 2012. ftp://ftp.fao.org/FI/CDrom/CD_ yearbook_2010/index.htm. [41] J.R. Benemann, Opportunities and Challenges in Algae Biofuels Production, 2008. http://www.fao.org/uploads/media/algae_positionpaper.pdf. [42] L.R.S. Gris, A.C. Paim, M. Farenzena, J.O. Trierweiler, Laboratory apparatus to evaluate microalgae production, Braz. J. Chem. Eng. 30 (2013) 487e497. [43] M.D.N. Meinita, B. Marhaeni, T. Winanto, G.T. Jeong, M.N.A. Khan, Y.K. Hong, Comparison of agarophytes (Gelidium, Gracilaria, and Gracilariopsis) as potential resources for bioethanol production, J. Appl. Phycol. 25 (2013) 1957e1961. [44] F.C. Wu, J.Y. Wu, Y.J. Liao, M.Y. Wang, I.L. Shih, Sequential acid and enzymatic hydrolysis in situ and bioethanol production from Gracilaria biomass, Bioresour. Technol. 156 (2014) 123e131. [45] H. Kim, Y. Cho, S.K. Kim, Ethanol production from seaweed (Undaria pinnatifida ) using yeast acclimated to specific sugars, Biotechnol. 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Contents lists available at ScienceDirect
Biomass and Bioenergy journal homepage: http://www.elsevier.com/locate/biombioe
Review
Sustainable production of bioethanol from renewable brown algae biomass Ok Kyung Lee, Eun Yeol Lee* Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 February 2015 Received in revised form 29 February 2016 Accepted 23 March 2016
Brown algae have been considered as renewable biomass for bioethanol production because of high growth rate and sugar level. Saccharification of brown algae biomass is relatively easy due to the absence of lignin. Among the major sugar components of brown algae, alginate cannot be directly used because industrial microorganisms are not able to metabolize alginate. This problem has been overcome by the development of metabolically engineered microbes to efficiently utilize alginate. This review analyzes and evaluates recent research activities related to bioethanol production from brown algae. This review mainly deals with the recent development and potential of a metabolically engineered microbial cell factory and bioethanol production from brown algae biomass including alginate as the main carbohydrate. Future researches for cost-effective bioethanol production from brown algae are discussed. © 2016 Published by Elsevier Ltd.
Keywords: Brown algae Alginate Bioethanol production Metabolically engineered microbes
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Brown algae as renewable and sustainable biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Bioethanol production from brown algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 The pros and cons of bioethanol production from brown algae biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Conclusion remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1. Introduction Due to the decline of petroleum reserves and a dramatic explosion in demand for energy, various types of alternative energy have been extensively investigated. Biofuel such as bioethanol, biodiesel and biohydrogen is one of the promising alternatives [1e3]. However, the production of biofuels from biomass has some drawbacks. In the case of first-generation biomass, there are moral issues with using food for biofuel production. In order to use second-generation lignocellulosic biomass, cost-intensive pretreatment is absolutely required before saccharification and fermentation [4].
* Corresponding author. E-mail address: [email protected] (E.Y. Lee). http://dx.doi.org/10.1016/j.biombioe.2016.03.038 0961-9534/© 2016 Published by Elsevier Ltd.
Macro- and microalgae are an example of third-generation biomass [5]. Algae present several advantages over other types of biomass. Algae are the fastest growing photosynthetic organisms [6]. Algae can efficiently remove carbon dioxide and synthesize polysaccharides or oil that can be used for biofuel production [7]. Generally, algal carbohydrates can be used for bioethanol fermentation after relatively easy saccharification due to the absence of lignin [5]. Oil can be trans-esterified into biodiesel [8]. In addition, algal biomass can be directly used for heat and power generation using anaerobic digestion and pyrolysis [9e11] (Fig. 1). There are three types of macroalgae, brown, green and red algae [12]. Brown algae such as sea mustard (Undaria pinnatifida) and kelp (Saccharina japonica) are one of the promising biomass for biofuel production because cultivation productivity based on area is the highest among three types of macroalgae [13,14],
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Fig. 1. Conceptual diagram of brown algae for production of biofuels and biochemicals.
approximately 40 kg wet biomass/m2 of gulf-weed (Sargassum muticum), compared to 2.3 and 6.6 kg/m2 of green laver (Ulva lactuca) and agar weed (Gelidium amansii), respectively. The largescale cultivation of brown algae is already practiced in several countries including Korea, China and Japan. Compared to microalgae, brown algae have a higher sugar and lower oil contents. Thus, brown algae are a more suitable feedstock for bioethanol production than biodiesel. With respect to bioethanol productivity, one report prepared for the US Department of Energy claimed that bioethanol productivity from macroalgae per hectare per year could theoretically be two times higher than sugarcane and five times higher than corn [15]. This review is intended to analyze and evaluate recent research activities related to biofuel production from brown algae, especially bioethanol. This review mainly deals with the recent development and potential of a metabolically engineered microbial cell factory and bioethanol production from brown algae biomass including alginate as the main carbohydrate. 2. Brown algae as renewable and sustainable biomass Brown algae have several advantages as a feedstock for production of biofuels. Firstly, brown algae avoid competition with food production. Brown algae requires no arable land and are cultivated in the ocean using aquaculture without needing the expensive nutrients, fertilizers or fresh water that are required for conventional agriculture. Brown algae have no lignin, and thereby no extensive pretreatment is used to release sugars, but just using simple operations such as milling or crushing. There is no moral issue associated with the use of brown algae for biofuel production. One particularly important advantage is that brown algae can offer high biomass yields per acre [13,14]. Brown algae contain high levels of carbohydrates, contributing up to 55% (w/w) of the dry biomass [16]. Therefore, brown algae are an ideal renewable and sustainable biomass due to its abundance and high sugar levels that can be used for production of bioethanol and chemicals. Roughly 70 million dry tons of macroalgae are cultivated and harvested worldwide in offshore and near-shore coastal farms [17]. This production scale is mostly for food applications. Cultured brown algae are harvested by both manual and mechanical methods. The harvested brown algae are then normally treated
with milling to reduce biomass sizes for efficient saccharification or alginate extraction [18] (Fig. 2). The saccharified broth can be used for bioethanol fermentation. 3. Bioethanol production from brown algae The most abundant sugars in brown algae are alginate, mannitol and laminarin. Mannitol and glucose from laminarin (a form of glucan in brown algae) are normal sugars that are efficiently used for bioethanol fermentation [19,20]. Laminarin and mannitol from Laminaria hyperborea extracts were fermented to bioethanol under oxygen-limiting conditions using Zymobacter palmae [21]. Bioethanol with a yield of 0.4 g ethanol/g of sugars was produced with ethanogenic Escherichia coli KO11 from Laminaria japonica hydrolysates mainly containing mannitol after chemo-enzymatic saccharification [22]. Ethanol of 7.7 g/L was produced from S. japonica biomass using the simultaneous saccharification and fermentation (SSF) method with a theoretical yield of 33.3% [23]. Alginate is a structural polysaccharide in the cell wall of brown algae [24,25]. In some brown algae, alginate constitutes up to 60% of the total sugars. Thereby, alginate, the most abundant carbohydrate, should be utilized for the production of bioethanol. Alginate is present in the insoluble calcium salt form [18]. In the alginate extraction process, calcium alginate is converted to alginic acid in an acid pre-extraction step, and then alginic acid is converted to soluble sodium alginate in alkaline extraction [26]. Alginate can be degraded into unsaturated uronate monosaccharides using alginate lyase [27]. Alginate lyase is the enzyme that catalyzes the b-elimination breakage of the glycosidic bond of alginate. Various alginate lyases have been cloned and characterized [28e30]. Recently, alginate lyase-based saccharification has been developed by employing exolytic alginate lyase as the biocatalyst with a 30% yield [31,32]. Theoretically, ethanol cannot be directly synthesized from unsaturated uronic monosaccharides under anaerobic conditions because no NADH is available for alcohol dehydrogenase to catalyze the conversion of acetaldehyde to ethanol. The NADH generated from glyceraldehyde-3-phosphate (G-3-P) to pyruvate is used for the conversion of 4-deoxy-L-erythro-hexoseulose uronate (DEH) to 2-keto-3-deoxygluconate (KDG) (Fig. 3). As a result, the net NADH
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Fig. 2. Processing of brown algae for bioethanol production.
Fig. 3. Metabolically engineered pathway of alginate monosaccharide degradation in a microbial cell factory for production of bioethanol from alginate and mannitol of brown algae.
is zero when an unsaturated monosaccharide is converted to 2 mol of pyruvate. Thus, alginate is considered to be a difficult-to-ferment sugar for ethanol fermentation.
A research group at Kyoto University reported that metabolic engineering of a native alginate-metabolizing Sphingomonas sp. A1 cells harboring pyruvate decarboxylase and alcohol dehydrogenase
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Fig. 4. Metabolic pathway of alginate monosaccharide degradation and biosynthesis of biofuels from alginate. DEH(4-deoxy-L-erythro-hexoseulose uronic acid), KDG(2-keto-3deoxy-D-gluconic acid), KDPG(2-keto-3-deoxy-6-phosphogluconic acid), G-3-P(glyceraldehydes-3-phosphate).
II from Zymomonas mobilis could produce bioethanol from alginate [33]. A pit in the range of 0.02e0.1 mm was formed on the surface of the cell in the presence of alginate. Alginate polymer was directly transported into the inner membrane of the cell through bacterial super channel and periplasmic alginate-binding proteins [34]. Alginate was transported into the cytoplasm via an ATP-binding cassette (ABC) transporter in the inner membrane [35]. The alginate was degraded into oligomer and monosaccharides by various alginate lyases inside the cytoplasm. The unsaturated monosaccharide was nonenzymatically converted to a-keto acid, DEH. In Sphingomonas sp. A1, DEH is converted to KDG, a metabolite of Entner-Doudoroff (ED) pathway, by NADPH-dependent uronate reductase [36] (Fig. 4). KDG was further degraded to pyruvate and glyceraldehyde-3-phosphate by kinase A1-K and aldolase A1-A. Pyruvate was converted to ethanol by pyruvate decarboxylase and alcohol dehydrogenase II [33]. Metabolically engineered Sphingomonas sp. A1 with a homoethanol pathway could produce 0.25 kg bioethanol/kg alginate with a volumetric productivity of 13 g/L. In contrast to alginate, an additional 1 mol of NADH is generated under fermentative conditions when mannitol is metabolized to 2 mol of ethanol (Fig. 3). One mole of NADH is generated from the conversion of mannitol to fructose-6-phosphate [21]. When fructose-6-phosphate is converted to two pyruvates, two NADHs are generated and these NADHs are consumed in the conversion of 2 mol of pyruvate to 2 mol of ethanol. Therefore, if 2 mol of mannitol and 1 mol of alginate monosaccharide are simultaneously utilized by cells under fermentative conditions, the amount of NADH is balanced (Fig. 3). Interestingly, a typical sugar ratio of mannitol to alginate in brown algae is approximately 8:5 [37]. Hence, these two sugar components in brown algae can be simultaneously used for ethanol fermentation. To utilize alginate and mannitol simultaneously in bioethanol
fermentation, the recruitment of alginate lyase enzymes for alginate uptake/degradation and reconstruction of metabolic pathway for converting the monosaccharide into ethanol are necessary. Recently, E. coli has been engineered to utilize all sugars from brown algae to make bioethanol [37]. Key genes for alginate uptake, degradation and metabolism of Vibrio splendidus 12B01 were successfully inserted into E. coli. In metabolically engineered E. coli, the alginate polymer was degraded into alginate oligomers by an extracellular alginate lyase from Pseudoalteromonas sp. SM0524. The oligomers were transported into the cytoplasm by an essential alginate transport system (putative sodium/solute symporter, termed ToaA) from V. splendidus. The oligomers were degraded to unsaturated monosaccharides by various alginate lyases, and then non-enzymatically isomerized DEH was converted to KDG by a putative NADH/NADPH-dependent alcohol dehydrogenase from V. splendidus 12B01. This alcohol dehydrogenase exhibited a preference for NADH rather than NADPH, and showed approximately 40% DNA sequence identity with NADPH-dependent uronate reductase from Sphingomonas sp. A1. The resulting KDG was further metabolized to pyruvate by kinase and aldolase of ED pathway in E. coli. Finally, the pyruvate was fermented to ethanol by relevant genes from Z. mobilis. Using the metabolically engineered microbial platform, ethanol at a final titer of approximately 4.7% (v/v) was produced from S. japonica biomass. Ethanol conversion was approximately 0.281 wt ethanol/wt dry biomass, equivalent to 80% of the maximum theoretical yield with an overall rate of 0.64 g/L/h. In this way, metabolic engineering enables the difficult-to-ferment alginate to be used as a renewable biomass for bioethanol fermentation, and thus makes brown algae an economically alternative feedstock. Recently, a synthetic yeast platform was developed for efficient bioethanol production from brown algae [38]. The DEH transporter
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Table 1 Comparison of annual production, sugar composition and bioethanol yields of algae. Macroalgae
Microalgae
Red algae
Brown algae
Green algae
Production (ton/year)
8,978,535a
6,784,93a
21,545a
Species
Kappaphycus alvarezii Eucheuma denticulatum Eucheuma sp. Gracilaria verrucosa Carrageenan Agar Cellulose
Laminaria japonica Undaria pinnatifida Sargassum fusiforme Phaeophyceae Laminarin, Mannitol Alginate Fucoidan Cellulose 0.152e0.196, 12.98 [45] 0.281, 37.8 [37] 0.362, 36.2 [38]
Codium fragile Caulerpa sp. Monostroma nitidum Enteromorpha clathrata Starch Cellulose
Sugars
Bioethanol yield and titer (g/g dry biomass, g/L) a b c
0.045, 0.5e0.83 [43] 0.236, 4.72 [44]
0.09, 9.31 [46]
10,000b 7,000c Chlorella Dunaliella Spirulina Glucose Galactose Xylose Arabinose Rhamnose 0.234, 11.7 [47]
Estimated in wet metric ton and adopted from FAO [40]. Total world production of dry algal biomass [41]. Total world production of dry algal biomass that are mostly produced in open systems [42].
from Asteromyces cruciatus was cloned and integrated into Saccharomyces cerevisiae genome to confer ability to uptake the unusual sugar. In order to synchronize the DEH and mannitol metabolic pathway, all relevant genes for alginate and mannitol metabolism were also chromosomally integrated. The engineered yeast produced bioethanol with a bioethanol titer of 4.6% (v/v) (36.2 g/L), efficiency of 75% based on total sugars, and productivity of 1.9 g/L/h from 1:2 M ratio of DEH and mannitol at 9.8% (w/v). Thus, manipulation of metabolic network of other microbial strain based on the synchronization of alginate and mannitol metabolic pathway enables the use of total sugars including alginate monosaccharide in brown algae for bioethanol production. Although the recombinant Sphingomonas, Escherichia and Saccharomyces strains have been successfully constructed for bioethanol production, some relevant issues need to be addressed. For the recombinant Sphingomonas strain, various genetic tools need to be developed for metabolic engineering to construct a microbial cell factory with enhanced bioethanol productivity. Optimization of fermentation conditions for the recombinant Sphingomonas strain should be conducted to enhance the bioethanol productivity. In the case of recombinant E. coli strain, uronate reductase should be functionally expressed because a large amount of reductase was expressed as insoluble form and reductase activity was rather low in E. coli. The recombinant Sphingomonas and Escherichia strains need to be engineered to be tolerant to high bioethanol concentration. The recruitment and functional expression of alginate-specific transporter is important for the recombinant Escherichia and Saccharomyces strains to increase bioethanol fermentation efficiency. Cell surface display or efficient secretion expression of various alginate lyases and other saccharifying enzymes is required for an efficient consolidating bioprocessing of brown algae biomass for bioethanol production. EMP, PP and ED pathways of microbial cell factory are required to be reconstructed and optimized to utilize all kinds of sugars in brown algae and enhance the bioethanol productivity. 4. The pros and cons of bioethanol production from brown algae biomass Bioethanol production from brown algae is compared with that from microalgae and other macroalgae (red and green algae), as shown in Table 1. Brown algae can be a good feedstock for bioethanol production based on high productivity. When recombinant E. coli and S. cerevisiae expressing alginate metabolizing genes and transporters were used for bioethanol fermentation, the
productivity of 0.28e0.36 g/g dry biomass were obtained [37,38]. Generally, the bioethanol productivity from red, green algae and microalgae were in the range of 0.045e0.236 g/g dry biomass [39,43e47]. There are many constraints in commercialization of bioethanol production from brown algae. Large-scale and cost-effective production of brown algae biomass is one of those hurdles in commercial bioethanol production. The current floating cultivation and non-automatic harvesting technology are not appropriate in terms of economics. At present, brown algae are harvested only for one to three months per year in a restricted area, and thus it is difficult to have large amount of brown algae biomass at low price [48]. In order to make brown algae-derived bioethanol in a practical scale, the development of mass production technology of brown algae biomass at low cost is absolutely required. Development of yearround multiple cultivation technology of brown algae can be a solution for cost-effective production of brown algae biomass. Recently, Song et al. suggested that the annual production of brown algae can be enhanced by combining the species [48]. They reported that species combination (e.g., Undria, Sargassum, and Ekulonia) could enhance annual productivity of brown algae by 2e4 times. In South Korea, a research program has been conducting on the mass cultivation of brown algae and the production of biofuel from the brown algae since 2008 [49]. They plan to demonstrate the brown algae biomass production with 50e60 ton (dry wt)/ha/yr productivity in offshore farms. Cost-effective production of brown algae biomass will be a key success factor for commercialization, together with the development of robust microbial cell factories. 5. Conclusion remarks At present, bioethanol production from brown algae is not yet economically-feasible [17]. Bioethanol production from brown algae is in the early stage of commercialization even though some distinct advances in metabolic engineering have been realized [37]. Bioethanol derived from brown algae is a drop-in fuel that can be used for existing engines. Thus, the commercialization of ethanol production from brown algae could become possible once costeffective methods for brown algae cultivation and integrated bioprocessing are developed. With respect to the practice of brown algae cultivation, it was estimated that more than 60 billion gallons of biofuel from brown algae could be produced across three percent of the world's coastlines [14,15]. This would result in the development of cost-effective and large-scale cultivation methods that
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could be applied in the coastal environment and would certainly meet production goals. Metabolic engineering in the development of a microbial cell factory that utilizes various brown algae carbohydrates and converts those sugars to bioethanol will play a key role in the commercialization of bioethanol production from brown algae. Various metabolic engineering-related tools such as genomics, proteomics, and metabolomics enable us to develop microbial cell factories that contribute to the cost-effective production of ethanol. In addition to bioethanol, metabolic engineering makes it possible to expand product lines such as bio-butanol, as well as other valuable chemicals and biopolymers. Acknowledgments This work was supported by Ministry of Oceans and Fisheries, Republic of Korea. This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012R1A1A2008647). References [1] L.L. Beer, E.S. Boyd, J.W. Peters, M.C. Posewitz, Engineering algae for biohydrogen and biofuel production, Curr. Opin. Biotechnol. 20 (2009) 264e271. [2] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294e306. [3] Y. Lin, S. Tanaka, Ethanol fermentation from biomass resources: current state and prospects, Appl. Microbiol. Biotechnol. 69 (2006) 627e642. [4] Y. Sun, J.Y. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour. Technol. 83 (2002) 1e11. [5] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal biomass: a renewable source for bioethanol, Bioresour. Technol. 102 (2011) 186e193. [6] A. Singh, P.S. Nigam, J.D. Murphy, Mechanism and challenges in commercialisation of algal biofuels, Bioresour. Technol. 102 (2011) 26e34. [7] M. Aresta, A. Dibenedetto, G. Barberio, Utilization of macro-algae for enhanced CO2 fixation and biofuels production: development of a computing software for an LCA study, Fuel Process Technol. 86 (2005) 1679e1693. [8] A.B.M. Hossain, A. Salleh, A.N. Boyce, P. Chowdhury, M. Naqiuddin, Biodiesel fuel production from algae as renewable energy, Am. J. Biochem. Biotechnol. 4 (2008) 250e254. [9] A.V. Bridgewater, Biomass fast pyrolysis, Thermal Sci. 8 (2004) 21e50. [10] S. Wang, Q. Wang, X. Jiang, X. Han, H. Ji, Compositional analysis of bio-oil derived from pyrolysis of seaweed, Energy Convers. Manag. 68 (2013) 273e280. [11] V.N. Gunaseelan, Anaerobic digestion of biomass for methane production: a review, Biomass Bioenergy 13 (1997) 83e114. [12] M. Yanagisawa, K. Nakamura, O. Ariga, K. Nakasaki, Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable polysaccharides, Process Biochem. 46 (2011) 2111e2116. [13] M. Aizawa, K. Asaoka, M. Atsumi, T. Sakou, Seaweed bioethanol production in Japan-the ocean sunrise project, in: IEEE Conference Proceedings, Vancouver, Sept. 29eOct. 4 2007, 2007, pp. 1e5. [14] K.A. Jung, S.R. Lim, Y. Kim, J.M. Park, Potentials of macroalgae as feedstocks for biorefinery, Bioresour. Technol. 135 (2013) 182e190. [15] National algal biofuels technology roadmap. http://www1.eere.energy.gov/ bioenergy/pdfs/algal_biofuels_roadmap.pdf. [16] J. Adams, J. Gallagher, I. Donnison, Fermentation study on Saccharina latissima for bioethanol production considering variable pre-treatments, J. Appl. Phycol. 21 (2009) 569e574. [17] Macroalgae as a biomass feedstock: a preliminary analysis. http://www.pnl. gov/main/publications/external/technical_reports/PNNL-19944.pdf. [18] S.Y. Chee, P.K. Wong, C.L. Wong, Extraction and characterization of alginate from brown seaweeds (Fucales, Phaeophyceae) collected from Port Dickson, Peninsular Malaysia, J. Appl. Phycol. 23 (2011) 191e196. [19] J.Y. Lee, P. Li, J. Lee, H.J. Ryu, K. Oh, Ethanol production from Saccharina japonica using an optimized extremely low acid pretreatment followed by simultaneous saccharification and fermentation, Bioresour. Technol. 127 (2013) 119e125. [20] S.J. Horn, I.M. Aasen, K. Østgaard, Ethanol production from seaweed extract, J. Ind. Microbiol. Biotechnol. 25 (2000) 249e254. [21] S.J. Horn, I.M. Aasen, K. Østgaard, Production of ethanol from mannitol by Zymobacter palmae, J. Ind. Microbiol. Biotechnol. 24 (2000) 51e57. [22] N.J. Kim, H. Li, K. Jung, H.N. Chang, P.C. Lee, Ethanol production from marine algal hydrolysates using Escherichia coli KO11, Bioresour. Technol. 102 (2011) 7466e7469. [23] J.S. Jang, Y. Cho, G.T. Jeong, S.K. Kim, Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF)
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