Advanced bioethanol production from nipa palm sap via acetic acid fermentation

GRADUATE SCHOOL OF ENERGY SCIENCE KYOTO UNIVERSITY ADVANCED BIOETHANOL PRODUCTION FROM NIPA PALM SAP VIA ACETIC ACID FERMENTATION NGUYEN VAN DUNG A thesis submitted for the degree of Doctor of Philosophy 2017 ADVANCED BIOETHANOL PRODUCTION FROM NIPA PALM SAP VIA ACETIC ACID FERMENTATION NGUYEN VAN DUNG Contents Chapter Introduction 1.1 Palm and palm sap 1.1.1 Origin and transportation of sap inside palm 1.1.2 Methods for tapping palm sap 1.1.3 Composition of palm sap 13 1.1.4 Traditional uses of palm sap 16 1.2 Nipa 18 1.2.1 Nipa palm 18 1.2.2 Nipa sap 20 1.3 Bioethanol production 22 1.3.1 Alcoholic fermentation 22 1.3.2 Bioethanol production via acetic acid fermentation 22 1.4 Research objectives 24 Chapter Effect of Gas Conditions on Acetic Acid Fermentation by Moorella thermoacetica 2.1 Introduction 26 2.2 Materials and methods 27 2.2.1 Materials 27 2.2.2 Reviving freeze-dried culture and preparation of inoculum 27 2.2.3 Batch fermentation 28 2.2.4 Analyses 29 2.3 Results and discussion 29 2.3.1 Fermentation of glucose by M thermoacetica under sparged N2 29 i 2.3.2 Fermentation of glucose by M thermoacetica under non-sparged N2 31 2.3.3 Fermentation of glucose by M thermoacetica under sparged CO2 32 2.3.4 Comparison of acetic acid yield and cell growth under gas conditions 32 2.4 Summary 34 Chapter Hydrolysis of Nipa Sap for Acetic Acid Fermentation 3.1 Introduction 35 3.2 Materials and methods 35 3.2.1 Materials 35 3.2.2 Acid hydrolysis 35 3.2.3 Enzymatic hydrolysis 36 3.2.4 Batch fermentation 36 3.2.5 Analyses 36 3.3 Results and discussion 37 3.3.1 Chemical composition of nipa sap 37 3.3.2 Acid hydrolysis 38 3.3.3 Enzymatic hydrolysis 43 3.3.4 Comparison of the catalysts for acetic acid fermentation 43 3.3.5 Acetic acid fermentation of hydrolyzed nipa sap by M thermoacetica 45 3.4 Summary 47 Chapter Fed-Batch Fermentation of Nipa Sap to Acetic Acid 4.1 Introduction 48 4.2 Materials and methods 48 4.2.1 Materials 48 4.2.2 Batch fermentation of standard sugars 48 ii 4.2.3 Fed-batch fermentation of hydrolyzed nipa sap 49 4.2.4 Analyses 50 4.3 Results and discussion 51 4.3.1 Chemical composition of nipa sap 51 4.3.2 Batch fermentation 51 4.3.3 Choice of substrate concentration and feeding time for fed-batch fermentation 53 4.3.4 Fed-batch fermentation 55 4.3.4 Comparison of fermentation performance during each feeding cycle 58 4.4 Summary 59 Chapter Minimal Nutrient Requirements for Acetic Acid Fermentation of Nipa Sap 5.1 Introduction 60 5.2 Materials and methods 60 5.2.1 Materials 60 5.2.2 Acetic acid fermentation 60 5.2.3 Analyses 61 5.3 Results and discussion 62 5.3.1 Fermentation of hydrolyzed nipa sap and standard sugars with/without nutrient supplement 63 5.3.2 Fermentation of hydrolyzed nipa sap without inorganics or yeast extract supplement 64 5.4 Summary 67 Chapter Evaluation of Advanced Bioethanol Production from Nipa Sap 6.1 Comparative study of bioethanol production by ethanologen and via acetogen 68 6.1.1 Introduction 68 6.1.2 Process for bioethanol production from nipa sap via M thermoacetica 68 iii 6.1.3 Comparison of ethanol production from nipa sap by ethanologen and via acetogen 69 6.1.4 Summary 69 6.2 Process simulation for bioethanol production from nipa sap by acetogen 70 6.2.1 Introduction 70 6.2.2 Methods 70 6.2.3 Results and discussion 71 6.2.4 Summary 73 Chapter Concluding Remarks 7.1 Conclusions 74 7.2 Prospects for future research 75 References 76 Acknowledgments 88 List of Publications 90 iv Chapter Introduction 1.1 Palm and palm sap Rapid depletions and increasing prices of fossil fuels to meet continuously increasing demands are of global concern [1] Petroleum-based fuels lead to environmental pollution, which results in global warming, health hazards, and ecological imbalances [2] The shift towards sustainable and environmentally friendly energy sources has generated significant interest in developing biofuel production from plant biomass [3] Arable land areas for crops such as corn and sugarcane are limited Agricultural expansion can result in deforestation, which is one of the main factors that is causing climate change [2] Planting, maintaining, replanting, and growing such crops for ethanol production require various fossil energy inputs such as fertilizers, herbicides, insecticides, machinery, irrigation, and electricity, which can cause social and environmental impacts [4, 5] The use of available plants that not require extensive maintenance and much fertilizer will be more appropriate for future biofuel production One such industrial plant is palm It can grow abundantly with little care and can yield sugary sap as a feedstock for bioethanol production [6] Palms are monocotyledonous angiosperms that belong to the Arecaceae family (also known as Palmae) They include six subfamilies, approximately 200 genera, and around 2,500–2,700 recognized species [7, 8] Geographically, most are native to tropical and subtropical regions from 44° north to 44° south [7] Sap from the palms is a sugar-rich exudate that can be obtained from wounded growing parts of a palm [9] As reviewed by Francisco-Ortega and Zona [10], ~40 global palm species are used commonly to produce sap by local people Coconut palm (Cocos nucifera), palmyra palm (Borassus flabellifer), sugar palm (Arenga pinnata), nipa palm (Nypa fruticans), kitul palm (Caryota urens), oil palm (Elaeis guineensis), date palm (Phoenix dactylifera), wild date palm (Phoenix sylvestris), and raffia palms (Raphia spp.) were reported as major sugar-yielding palms in Asia and Africa [11] Limited harvesting of these palms occurs for domestic utilization as a fresh beverage; in animal feed; and/or for the production of brown sugar, alcoholic beverages, and vinegar [4, 10] These saps contain a high amount of free sugars such as sucrose, glucose, and fructose that can be fermented to bioethanol much more easily than starchy or lignocellulosic materials [3] Thus, this chapter aims to review the properties of these palm saps for bioethanol production 1.1.1 Origin and transportation of sap inside palm 1.1.1.1 Origin of sugary sap in palm Many palm species (e.g., Arenga spp., Caryota spp., Corypha spp., and Metroxylon spp.) preserve their photosynthetic products from leaves as starch inside their stems [12] During flowering and fruiting, starch is converted into sugars and enters the nutrient flow to be transported toward the growing parts of the plants [9] The liquid that contains the nutrients and sugars constitutes the sap Photosynthesis, starch hydrolysis, and sap flow require water that may be taken up from the environment through the roots of standing palms or from the tissues of felled palms [13] In contrast, palm species such as C nucifera and N fruticans contain little starch in their stems [11, 14] To explain the sugar source in this case, Van Die and Tammes [9] proposed that soluble sugars from photosynthesis in the leaves are transported as the mobile phase of the sieve tube system throughout vegetative parts of the palms before they are used directly to form fruits or sap without starch accumulation Ranasinghe et al [15] found that soluble sugars are available in leaf and trunk tissues in sap- and nut-producing coconut palms (C nucifera) Sugary sap appears to be the major reserve in this palm rather than starch 1.1.1.2 Sap transportation in palm Figures 1-1a and b compare the anatomy of a typical tree trunk and an oil palm trunk Palms are monocotyledonous angiosperms and their anatomy differs from softwood and hardwood [16] As shown in Fig 1-1a, a typical tree has concentric vascular tissues: xylem includes sapwood and heartwood parts with pith, whereas phloem is only a narrow layer separated from xylem by a vascular cambium In contrast, as shown in Figs 1-1b and c, xylem and phloem in palms are not concentric but are dispersed inside numerous vascular bundles These vascular bundles are embedded in ground parenchyma, which is a storage tissue in which starch, a sap source, can be detected [17] According to Berg [16], water and dissolved minerals flow in xylem, whereas phloem is used to transport aqueous solutions of sugars and other nutrients either from the leaves to the consumption and storage sites or from the storage to the growing sites Consequently, sap flow, which originates from leaves and/or storage sites, may be transported in the phloem to growing sites during flowering and fruiting Fig 1-1 Structure of (a) cross section of a typical tree trunk compared with (b) cross section of an oil palm trunk and (c) its vascular bundle [17] An early study by Molisch (cited in [13]) found many plugged xylem vessels in the inflorescence stalk This indicates that xylem vessels are unable to transport bleeding sap Later reports proved that sap is released from phloem only in a sieve tube system [9] The sap of deciduous trees such as the maple tree (Acer spp.) can be tapped in early spring and has a lower sugar content (3–5%) compared with palm sap (10–20%) [9, 11] In contrast with palm, the sap in maple trees flows in the xylem According to Essiamah and Eschrich (cited in [18]), starch accumulates in xylem parenchyma cells by late October During the winter and early spring, this reserve is converted into dissolved sucrose, which is believed to protect the trees from frost damage Consequently, xylem sap in maple trees can be exuded by drilling holes into the trunk Because of differences in structure and sap transportation, palm sap tapping is very different Humid areas of tropical South and Southeast Stalk Asia (e.g., India, Sri Lanka, Guam, Papua New Guinea, Indonesia, Thailand, Vietnam) India Sugar palm Wight's sago palm Arenga pinnata Arenga wightii African fan palm Stalk (peduncle) Inflorescence (spadix) Inflorescence Humid areas of South Asia (e.g., India, Sri Lanka, Malaysia, Indonesia, Philippines) Common to tropical lands Tropical rainforest of South and Southeast Asia (e.g., Sri Lanka, India, Myanmar, Thailand, Cambodia) Kitul palm Coconut palm Talipot palm Caryota urens Cocos nucifera Corypha umbraculifera Inflorescence India, Brunei, Malaysia, Myanmar, Indonesia, Thailand, Vietnam Clustering fishtail palm Caryota mitis Inflorescence (spadix) Tropical countries in Asia (e.g., Nepal, Sri Lanka, India, Malaysia, Indonesia, Phillipines, Vietnam) Palmyra palm Lontar palm Stem below terminal bud Sub-Saharan Africa (e.g., Senegal, Mali, Ivory Coast, Niger and Burkina Faso) Non-destructive Non-destructive Non-destructive Non-destructive Non-destructive Non-destructive Palm heart Destructive (apical meristem) Destructive Non-destructive Non-destructive Destructive Tapping method Tropical zone from West Africa through India and Southeast Asia to New Guinea and Australia Dry to slightly humid lowlands of American Crown meristem (e.g., Columbia) of felled palm Borassus flabellifer Borassus akeassii - Borassus aethiopum Attalea butyracea Yagua palm Terminal bud Tropical regions of the Americas (e.g., Mexico, Caribbean countries, Paraguay, Argentina) Macaw palm Coyol palm Acrocomia aculeata Inflorescence (spadix) Tapped part Distribution Common name Scientific name 90-120 40-45 60-90 - 90-180 Year–round 35-45 20-30 > 20 30-60 (Max ~365) 25 30-70 10-20 - 20-30 - 35 15-25 - 5-12 10-14 - 20 3-5 - 30 - - - - 2-5 - 20 1.7-4.3 45 - 6-10 0.5-10 1.8-4.1 10 1-3.7 12-15 (Max 33) [7, 35] [11, 32-34] [30, 31] [7, 8] [11, 28, 29] [26] [27] [25] [24] [7, 23] [21, 22] [19, 20] Tapping Age of first Years of Sap yield** Reference period* (day) tapping (yr) tapping (yr) (L/palm/day) Table 1-1 Distribution and tapping characteristics of various palms 14 Tamunaidu P, Kakihira T, Miyasaka H, Saka S (2011) Prospect of nipa sap for bioethanol production, in Zero-Carbon Energy Kyoto 2010, Yao T, Editor Springer: Japan p 159164 15 Ranasinghe C, Silva L (2007) Photosynthetic assimilation, carbohydrates in vegetative organs and carbon removal in nut-producing and sap-producing coconut palms Cocos 18: p 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bioethanol production from lignocellulosics Fuel 178: p 118-123 143 Cardona Alzate CA, Sánchez Toro OJ (2006) Energy consumption analysis of integrated flowsheets for production of fuel ethanol from lignocellulosic biomass Energy 31(13): p 2447-2459 87 Acknowledgments First and foremost, I would like to express my sincere thanks to my supervisor, Professor Shiro Saka, for his guidance, encouragement and kindness during my study at Saka laboratory, Energy Ecosystems, Department of Socio-Environmental Energy Science, Graduate School of Energy Science, Kyoto University From the bottom of my heart, I feel extremely fortunate to become a doctoral student of Saka sensei Under his supervision, I have never stopped being guided to become more professional in research and working style Saka sensei always provides best conditions for my experiments, gives insightful guidance on my research and offers great opportunity for me to attend academic conferences I also appreciate his consideration on my research progress to satisfy the time limit of my scholarship; otherwise, I cannot write this dissertation now I sincerely thank Special Assistant Professor Harifara Rabemanolontsoa for her great support on my research She teaches me experimental techniques, gives me valuable suggestions, and helps me to prepare reports and scientific papers Without her push on my research, my work would not be finished as expected I would like to express my gratitude to Associate Professor Haruo Kawamoto in Saka laboratory for his support on hydrogenation step I also thank Assistant Professor Eiji Minami and Dr Pinthep Sethapokin, presently in King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand, for their contribution on process simulation of advanced bioethanol production from nipa sap using Pro/IITM software My appreciation goes to the secretary of Saka laboratory, Ms Rie Nakanishi, for her kindness and support throughout my study I wish to express my gratitude to my tutor, Mr Masatsugu Takada, for his great help for my life in Kyoto I extend my sincere thanks to all members of Saka laboratory for their warm supports, encouragements and funny stories I am thankful to Dr Pramila Tamunaidu, the former Ph.D student in Saka laboratory and presently senior lecture in Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, for providing the nipa sap samples used in this study Her previous study on bioethanol production from nipa sap at Saka laboratory also provides a lot of useful knowledge for my current research In addition, I want to thank to Ms Le Thi Minh Thi for sharing the photographs of nipa palm in Vietnam and other copyright holders for providing permissions to reproduce their photographs in this dissertation 88 My special thanks to Japan International Cooperation Agency (JICA) for the financial support under the AUN/SEED-Net Project during my doctoral study at Kyoto University I am also grateful to Ms Eriko Miyashita, a staff of JICA Kansai, for her kind help Most importantly, I wish to express my sincere love and gratitude to my beloved family for their endless love and encouragement At last but not least, I would like to thank the others who are not mentioned here for their contribution on my doctoral research and my life in Japan Thank you Nguyen Van Dung 08 February 2017 89 List of Publications Review paper Nguyen DV, Rabemanolontsoa H and Saka S (2016) Sap from various palms as a renewable energy source for bioethanol production Chemical Industry and Chemical Engineering Quarterly, 22 (4): 355−373 Original papers Nguyen DV, Sethapokin P, Rabemanolontsoa H, Minami E, Kawamoto H and Saka S (2016) Efficient production of acetic acid from nipa (Nypa fruticans) sap by Moorella thermoacetica (f Clostridium thermoaceticum) International Journal of Green Technology, 2:1-12 Nguyen DV, Rabemanolontsoa H and Saka S Fed-batch fermentation of nipa sap to acetic acid by Moorella thermoacetica (f Clostridium thermoaceticum), Chemical Industry and Chemical Engineering Quarterly (In press, DOI:10.2298/CICEQ170103003N) Rabemanolontsoa H, Nguyen DV, Jusakulvijit P and Saka S Effects of gas condition on acetic acid fermentation by Clostridium thermocellum and Moorella thermoacetica (C thermoaceticum), Applied Biochemistry and Biotechnology (Under review) Nguyen DV, Rabemanolontsoa H and Saka S Minimal nutrient requirement for acetic acid fermentation of nipa sap by Moorella thermoacetica (f Clostridium thermoaceticum), Bioresources and Bioprocessing (To be submitted) Related papers in biofuel field Nguyen HN, Nguyen DV and Tran MTT (2011) Synthesis of biodiesel from rubber seed oil on solid catalysts Vietnam Journal of Chemistry, 49(2ABC): 111-115 Nguyen HN, Trieu AT, Nguyen DV, Nguyen LQ (2011) Conversion of high FFA rubber seed oil to biodiesel on solid catalysts Vietnam Journal of Science and Development, 49(6C): 319-326 90 Abstracts of International Conferences Nguyen DV, Rabemanolontsoa H and Saka S (2015) Biological conversion of nipa sap to acetic acid as an important biorefinery precursor Abstracts of the 2nd International Biotechnology, Chemical Engineering and Life Science Conference, July 20-22, 2015, Hokkaido, Japan, 399 Nguyen DV, Rabemanolontsoa H and Saka S (2015) Advanced bioethanol production from nipa sap via acetic acid fermentation Abstracts of the International Chemical Congress of Pacific Basin Societies 2015, December 15-20, 2015, Hawaii, United States 10 Nguyen DV, Rabemanolontsoa H and Saka S (2016) Efficient acetic acid production from nipa sap by fed-batch fermentation using Moorella thermoacetica (f Clostridium thermoaceticum) Abstracts of the 3rd International Biotechnology, Chemical Engineering and Life Science Conference, August 2-4, 2016, Okinawa, Japan, 25 11 Nguyen DV, Sethapokin P, Rabemanolontsoa H, Minami E, Kawamoto H and Saka S (2016) Process simulation for advanced bioethanol production from nipa sap through acetic acid fermentation Abstracts of the 3rd International Biotechnology, Chemical Engineering and Life Science Conference, August 2-4, 2016, Okinawa, Japan, 225 Abstract of Domestic Conferences 12 Nguyen DV, Sethapokin P, Rabemanolontsoa H, Minami E, Kawamoto H and Saka S (2016) Process simulation for efficient acetic acid production from nipa sap Abstracts of the 68th Annual Meeting of the Society for Biotechnology, Japan, September 28-30, 2016, Toyama, Japan, 199 13 Nguyen DV, Rabemanolontsoa H and Saka S (2017) Potential and sustainability of various palm saps as raw material for bioethanol production Abstracts of the 67th Annual Meeting of the Japan Wood Research Society, March 17-19, 2017, Fukuoka, Japan 91 ... yield from nipa sap reached 0.48 g ethanol/g sugars only 1.3.2 Bioethanol production via acetic acid fermentation 1.3.2.1 Proposed process for bioethanol production from nipa sap via acetic acid fermentation. . .ADVANCED BIOETHANOL PRODUCTION FROM NIPA PALM SAP VIA ACETIC ACID FERMENTATION NGUYEN VAN DUNG Contents Chapter Introduction 1.1 Palm and palm sap 1.1.1 Origin... for acetic acid fermentation 43 3.3.5 Acetic acid fermentation of hydrolyzed nipa sap by M thermoacetica 45 3.4 Summary 47 Chapter Fed-Batch Fermentation of Nipa Sap to Acetic