Selection of microalgae species suitable for lipid production and nutritious biomass masters thesis major sciences and management of the environment

THE JOINT ACADEMIC PROGRAM OF EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF THE ENVIRONMENT BETWEEN INDUSTRIAL UNIVERSITY OF HOCHIMINH CITY AND LIÈGE UNIVERSITY DO NGUYEN MINH CHAU SELECTION OF MICROALGAE SPECIES SUITABLE FOR LIPID PRODUCTION AND NUTRITIOUS BIOMASS Major: EXECUTIVE MASTER IN SCIENCES AND MANAGEMENT OF THE ENVIRONMENT MASTER’S THESIS HOCHIMINH CITY, YEAR 2018 The project was completed at The Industrial University of Hochiminh City Supervisors: Gauthier Eppe, Prof Trinh Ngoc Nam, PhD The thesis was taken at The Industrial University of Hochiminh City date month year Committee members (name): - Committee Chair - Commissioner - Secretary (Write full name and signature) COMMITTEE CHAIR DEAN OF INSTITUTE OF ENVIRONMENTAL SCIENCE, ENGINEERING AND MANAGEMENT ACKNOWLEDGEMENTS First of all, I would like to deeply express my appreciation to people who supported and helped me immensely in this study: Prof Le Hung Anh, PhD Trinh Ngoc Nam and Prof Gauthier Eppe - my thesis’s supervisors They support me by their invaluable assistance during the time of doing this study, thanks for their reviews, constructive advices, patience and encouragement This study could be finished successfully with their instructions and motivation Also, I am so grateful for the support and an active and friendly environment that Renewable project gave me Additionally, all the member of Renewable project and Institute of Biotechnology and Food Technology help me with encouragement as well Furthermore, with all my deep gratitude for Prof Jean-Luc Vasel, I highly appreciate his valuable documents as well as all the help he gave me in Belgium Also, thanks to my dear colleague, Ms Trang Kim Ly who has been with me since the beginning of our work, Mr Nguyen Tri Phuong who encourage and support me mentally and academically and PhD Thien who support me a lot in this field of study, I could have conducted this thesis effectively Finally, there is no doubt that I would like to give my deepest thanks to my family for their unconditional love, support and faith in me Ho Chi Minh City, August 2018 Author ABSTRACT Microalgae are considered as a third generation of biofuel supply and nutrition biomass In Vietnam, microalgae have high biodiversity and high application potential but the current use is limited This study performs exploit indigenous microalgae species with potential for biofuel production Nine strains were identified as Chlorella vulgaris (Li9, Li14, Li21, TH3, TH6, TH14), Chlorella sorokiniana TH21, Halamphora coffeaeformis TH13, Chlorella sp TH38, TH35 strain is belong to Bacillariophyceae Two strains gave the highest total FAME results are Chlorella sorokiniana TH21 and Chlorella vulgaris Li9 respectively 11.51% and 3.26% In addition, CN and CFPP values indicate that lipid quality is suitable for biofuel production The corresponding CN values are 69.84 and 190.48 compared to standard EN 14214 with minimum value is 51 and CFPP respectively 16.2C and -15.8C compared to RD 61/2006 with a maximum value is -10C The mineral analysis values of microalgae showed low concentrations, but provided that heavy metals such as Cu, Cr, Cd and Pd were below acceptable level TABLE OF CONTENTS ACKNOWLEDGEMENTS ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION 10 The reason for choosing the topic 10 Objectives of the study 11 2.1 General Objective 11 2.2 Specific Objectives 11 Subject and scope of the study 11 3.1 Research subject 11 3.2 Research scope 12 The meaning of the topic 12 4.1 Significance of research 12 4.2 Practical implication of research 12 The methodology 12 5.1 Collecting information 12 5.2 Method of sampling analysis 12 5.3 Comparative method 13 5.4 The main techniques used 13 CHAPTER LITERATURE REVIEW 14 1.1 Microalgae 14 1.1.1 Types of cell organization 14 1.1.2 Cellular organization 15 1.1.3 Ultrastructure and cell division 16 1.1.4 Microalgal systematics 18 1.2 Lipid metabolism in microalgae 24 1.3 Application of microalgae on biofuel 27 1.4 Application of Microalgae on Cattle feeding 28 1.5 Condition of aquaculture industrial – shrimp farming: 2010-2014 29 1.6 Characteristics of industrial shrimp waste water 31 CHAPTER MATERIAL AND METHODS 33 2.1 Material 33 2.1.1 Samples 33 2.1.2 Instruments -Chemical -Laboratory materials 35 2.2 Experiment 37 2.3 Methods 37 2.3.1 Medium culture and Isolation method 37 2.3.2 Identification 38 2.3.3 Analysis of fatty acid composition 42 2.3.4 Analysis of mineral composition 43 CHAPTER RESULTS AND DISCUSSIONS 44 3.1 Isolation and Preliminary identification by Morphology 44 3.2 Identification by DNA sequence 53 3.3 Lipid content 81 3.4 Mineral content 83 3.5 Discussions 84 CHAPTER CONCLUSION 89 REFERENCES 91 APPENDIX 94 APPENDIX 102 APPENDIX 109 LIST OF TABLES Table 1.1 Physical and chemical characteristics of shrimp pond in Can Gio District, Ho Chi Minh.[2] 32 Table 3.1 Strains of Microalgae [1] 44 Table 3.2 Sequence results [1] 57 Table 3.3 Define species [1] .79 Table AP.1 The composition of Guillard F/2 medium .95 Table AP.2 Number and volume of samples from Ninh Thuan .95 Table AP.3 Mineral compound 96 Table AP.4 Fatty acid compound 98 Table AP.5 Compare the sequences of strain TH30 and TH36 99 Table AP.6 Sequence used for phylogeny tree .100 LIST OF FIGURES Figure 1.1 Electron micrograph of a diving cell of Synechococcus sp.[5] .15 Figure 1.2 Electron micrograph of a cell of Chlorella vulgaris.[5] 16 Figure 1.3 Electron micrograph of a cell of Microcystis sp.[5] 20 Figure 1.4 Basic overview of the pathway of carbon capture and lipid biosynthesis [7] 25 Figure 1.5 The fatty acid and TAG biosynthesis [8] 27 Figure 2.1 Location of Ninh Thuận in Vietnam .33 Figure 2.2 Sampling diagram [40] 34 Figure 2.3 Experiment procedure [1] .37 Figure 2.4 Diagram of DNA sequence analysis of microalgae [1] 40 Figure 3.1 Electrophoresis results [1] .55 Figure 3.2 Phylogenetic tree were created using PAUP software after 1000 rounds of bootstrap [1] .71 Figure 3.3 Phylogenetic tree were created using PAUP software after 1000 rounds of bootstrap [1] .72 Figure 3.4 Compare of morphology [1] 76 Figure 3.5 Morphology comparison of TH35 78 Figure 3.6 Compare of morphology of strain TH30 and TH36 [1] 78 Figure 3.7 Comparative chart of mineral composition in microalgae [1] 83 Figure AP.1 Sequencing and comparison results on Blast of Li9 102 Figure AP.2 Sequencing and comparison results on Blast of Li14 102 Figure AP.3 Sequencing and comparison results on Blast of Li21 103 Figure AP.4 Sequencing and comparison results on Blast of TH3 103 Figure AP.5 Sequencing and comparison results on Blast of TH6 104 Figure AP.6 Sequencing and comparison results on Blast of TH13 104 Figure AP.7 Sequencing and comparison results on Blast of TH14 105 Figure AP.8 Sequencing and comparison results on Blast of TH17 105 Figure AP.9 Sequencing and comparison results on Blast of TH21 106 Figure AP.10 Sequencing and comparison results on Blast of TH24 106 Figure AP.11 Sequencing and comparison results on Blast of TH30 107 Figure AP.12 Sequencing and comparison results on Blast of TH35 107 Figure AP.13 Sequencing and comparison results on Blast of TH36 108 Figure AP.14 Sequencing and comparison results on Blast of TH38 108 Figure AP.15 Lobocystis planctonica [41] .109 LIST OF ABBREVIATIONS ACC Acetyl-coa carboxylase ACP Acyl-carrier protein BLAST Basic local alignment search tool CFPP Cold flow properties CN Cetane number DGAT Diacylglycerol acyltransferase EDTA Ethylenediaminetetraacetic acid ER Endoplasmic reticulum FAME Fatty acid methyl ester G3P Glycerol-3-phosphate GCMS Chromatography–mass spectrometry ICP-OES Inductively coupled plasma optical emission spectrometry KAS Ketoacyl-acp synthetizes LCSF Long chain saturated factor LPA Lysophosphatidate MGDG Monogalactosyl diglycerid NCBI National center for biotechnology information search database PAUP Phylogenetic analysis using parsimony and other methods APPENDIX Table AP.1 The composition of Guillard F/2 medium F2 medium Chemical NaNO3 Mg/L 75 NaH2PO4 Na2SiO3 Na2EDTA CoCl2 4.36 0.1 FeCL3.6H2O 3.15 CuSO4 0.1 0.18 30 MnCl2 Na2MoO4 ZnSO4 0.006 0.022 Thiamin Biotin 0.1 0.005 B12 0.005 Table AP.2 Number and volume of samples from Ninh Thuan Sample collection/ repeat time per year Pond Pond Pond Pond Pond Point 500 mL 500 mL 500 mL 500 mL 500 mL Point 500 mL 500 mL 500 mL 500 mL 500 mL Point 500 mL 500 mL 500 mL 500 mL 500 mL Point 500 mL 500 mL 500 mL 500 mL 500 mL Point 500 mL 500 mL 500 mL 500 mL 500 mL Total volume at pond 250 mL 250 mL 250 mL 250 mL 250 mL 1250 mL Total samples per time 95 TH17 TH13 Chlorellae Chlorellaf Spirulinag 36.5 47.5 314 - 30 3.5