THE DIVERSITY AND BIOTECHNOLOGICAL APPLICATION OF MARINE MICROBES PRODUCING OMEGA-3 FATTY ACIDS BY JINWEI ZHANG A thesis presented for the degree of DOCTOR OF PHILOSOPHY March 2011 SCHOOL OF MARINE SCIENCE AND TECHNOLOGY NEWCASTLE UNIVERSITY NEWCASTLE Copyright statement This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognize that its copyright rests with its author and no quotation from the thesis and no information derived from it may be published without the author’s prior written consent. Acknowledgements Acknowledgements I would like to acknowledge my sincere thanks to my supervisors, without whom this thesis would never have been completed. Professor Grant Burgess, Professor Keith Scott and Dr. Gary Caldwell gave me their valuable scientific guidance, advice, suggestions and discussion throughout this work. My sincere thanks also go to Professor Alan Ward, Professor Michael Goodfellow, Dr Ben Wigham, Dr Jarka Glassey and Dr Michael Hall (Newcastle University), to Dr Keith Layden, Dr Surinder Chahal and Dr Robin Mitra (Croda Enterprises Ltd, England), and Dr Craig Hurst (Croda Europe Ltd - Leek) for their helpful discussions. In particular, I would like to thank Mr John Knowles, Mr Tristano Bacchetti De Gregoris, Mr William Reid and Miss Nithyalakshmy Rajarajan, who are my good colleagues in the laboratory and have given me great help during my working period at Newcastle University. I would also like to express my great thanks to Dr Enren Zhang for his inspiration and help on the microbial fuel cell project. Particularly, I am grateful for the encouragement from my friends and family. Thank you, dear parents, elder brother, sister-in-law, niece and Jane Wu for your support and endless love. 1 Abstract Abstract Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) play a role in the modulation and prevention of human diseases, in particular cardiovascular diseases. The omega-3 family is found mainly in fish, of which wild stocks are becoming limited. Therefore production of omega-3 PUFAs by marine microbes may provide an alternative source of such componds. The diversity of marine microbes was studied using 16S/18S rRNA gene sequencing of different marine biota with 1500 bacterial strains and 50 microalgae were isolated. The diversity of culturalbe microorganisms inhabiting Mid-Atlantic Ridge (MAR) non-vent sediments was examined for the first time in this area with findings of high diversily of Gram-positive strains, good production of squalene by an unusual strain Bacillus sp. MAR089 and the highest yield of EPA ever recovered from strain Shewanella sp. MAR441. North Sea sponge associated Vibrio sp. strain NSP560 produced considerable levels of EPA, whereas no PUFAs producers were found from tropic Caribbean marine sponge associated bacteria. Photobacterium sp. strain MA665, isolated from the coast of North Sea, was described for the first time of this genus and could be cultured easily under atmospheric conditions with appreciable levels of EPA with up to 25 % of total fatty acids (TFA) (or 10.6 mg g-1 in dried cell). Strain MAR441 was identified as a new species, designated as Shewanella dovemarina sp. nov. (Type strain MAR441T). The level of EPA production of strain MAR441 has been optimized by varying fermentation conditions, and 15-25 % EPA of TFA (or 17-30 mg g-1 in dried cell) could be achieved with 40 % improvement. In order to understand the PUFAs biosynthesis pathways and better predict the maximum EPA production, EPA gene clusters (pfaA, pfaB, pfaC, pfaD and pfaE) were cloned and sequenced from the following three species Shewanella, Vibrio and Photobacterium. Great potential was found in marine algae Phaeodactylum tricornutum strain M7 with lipid content of 10 % in dry wt biomass and 22-30 % EPA of TFA when it was cultured outdoors under local weather conditions in UK. Under anaerobic conditions, strain MAR441 contained less amount of EPA and produced electricity of ~100 mW m-2. Enhanced electricity production using artificial consortia of estuarine bacteria grown as biofilms was observed with power generation of ~200 mW m-2. In conclusion, bacteria taxonomic resolution based on complete cell fatty acid composition is possible and marine microbes with considerable production of EPA could be potential candidates for industrial production of PUFAs. 2 Memorandum Memorandum Except where acknowledgement is given this thesis is the unaided work of the author. The material presented has never been submitted to Newcastle University or to any other educational establishment for purpose of obtaining a higher degree. March 2011 Jinwei Zhang 3 Table of Contents Table of Contents Acknowledgements.......................................................................................................1 Abstract.........................................................................................................................2 Memorandum ...............................................................................................................3 Table of Contents .........................................................................................................4 List of Figures.............................................................................................................12 List of Tables ..............................................................................................................18 Abbreviations .............................................................................................................21 Chapter 1. Introduction ................................................................................................24 1.1 Overview ............................................................................................................... 24 1.2 Structural diversity and physiological functions of fatty acids and lipids ............. 26 1.2.1 Fatty acid nomenclature............................................................................... 26 1.2.2 Introduction to lipids..................................................................................... 29 1.2.3 Biological functions of lipids ......................................................................... 29 1.2.4 Physiological and medical effects of omega‐3, ‐6 and ‐9 fatty acids ........... 30 1.2.5 Omega‐3 fatty acids and their metabolism in plants and animals .............. 32 1.2.6 Dietary sources of omega‐3 fatty acids........................................................ 34 1.3 Marine sources of PUFAs ...................................................................................... 37 1.3.1 EPA and DHA from fish ................................................................................. 37 1.3.2 Marine algae ................................................................................................ 39 1.3.3 Marine fungi ................................................................................................. 41 1.3.4 Marine yeast................................................................................................. 42 1.3.5 Marine bacteria ............................................................................................ 43 1.4 Fatty acid biosynthesis .......................................................................................... 50 1.4.1 Fatty acid synthetase system (FAS) .............................................................. 52 1.4.2 Polyketide (anaerobic) pathway................................................................... 54 4 Table of Contents 1.4.3 PUFAs Polyketide synthase gene .................................................................. 58 1.5 Bacteria adaptive to low temperature.................................................................. 60 1.5.1 The role of PUFA in the bacterial cold adaptive response ............................ 61 1.6 The importance of PUFAs in marine food web ..................................................... 62 1.7 Advantages of microbial omega‐3 PUFAs ............................................................. 65 1.8 Thesis aims and objectives.................................................................................... 66 Chapter 2. Fatty acid production by microbial communities from non-vent Mid-Atlantic Ridge sediments ...........................................................................................................68 2.1 Abstract ................................................................................................................. 68 2.2 Introduction .......................................................................................................... 68 2.3 Materials and Methods......................................................................................... 70 2.3.1 Sample collection.......................................................................................... 70 2.3.2 Isolation of psychrophilic and psychrotrophic bacteria................................ 71 2.3.3 MFC construction and operation .................................................................. 72 2.3.4 Strain growth................................................................................................ 72 2.3.5 Lipid extraction, transesterification, gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) ........................................................ 72 2.3.6 Preparation of genomic DNA and