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Master of Science Thesis in Industrial Biotehcnology

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Master of Science Thesis in Industrial Biotehcnology MASTER OF SCIENCE THESIS IN INDUSTRIAL BIOTEHCNOLOGY PRODUCTION OF FATTY ACIDS IN MICROBIAL SYSTEMS Ganesh Kumar Radhakrishnan Supervisor and Examiner: Gen Larsson School of Biotechnology Department of Bioprocess Technology KTH Royal Institute of Technology Stockholm, Sweden, June 2014. ABSTRACT Poly unsaturated fatty acid such as ω-3 and ω-6 fatty acids possess numerous health benefits and are associated with many health disorders. Currently fish is the main source to obtain ω-3 fatty acids and fish resources are very much limited by massive exploitation. Microorganisms naturally produce these ω-3 fatty acids and in fact they are the primary producers in the food chain. Marine micro algae such as Thraustochytrium, Schizochytrium and Crypthecodinium cohnii accumulates ω-3 fatty acids such as docosahexaenoic acid in large proportions. While fungi Morteriella are good sources of ω-6 fatty acids such as arachidonic acid. This study describes the biosynthesis pathway of the fatty acids, depicts the major factors affecting the production and presents a comprehensive review of the production process in microbial systems. CONTENTS 1. INTRODUCTION ..............................................................................................................................5 1.1. Biosynthesis of Microbial PUFAs ........................................................................................................................ 6 1.2. Fermentation techniques ................................................................................................................................... 7 1.2.1. Submerged fermentation .............................................................................................................................................. 7 1.2.2. Solid State fermentation ............................................................................................................................................... 8 1.3. Downstream processing ..................................................................................................................................... 8 1.4. Microbial production of ω-6 fatty acids ........................................................................................................... 10 1.4.1. γ- Linolenic acid ........................................................................................................................................................... 10 1.4.2. Arachidonic acid .......................................................................................................................................................... 11 2. Microbial production of DHA ........................................................................................................ 15 2.1. Potential microorganisms ................................................................................................................................ 15 2.2. Biosynthesis of DHA ......................................................................................................................................... 15 2.2.1. Elongation and Desaturation pathway ........................................................................................................................ 16 2.2.2. Polyketide Synthase Pathway (PKS) ............................................................................................................................ 17 2.3. Factors affecting DHA production .................................................................................................................... 19 2.3.1. Nutritional factors ....................................................................................................................................................... 19 2.3.2. Environmental factors ................................................................................................................................................. 21 2.3.3. Cultivation parameters ................................................................................................................................................ 24 2.4. Reactor types ................................................................................................................................................... 26 2.4.1. Stirred Tank Bioreactor ............................................................................................................................................... 26 2.4.2. Airlift Bioreactor .......................................................................................................................................................... 26 2.5. Cultivation strategies........................................................................................................................................ 27 2.5.1. Batch cultivation ......................................................................................................................................................... 27 2.5.2. Fed Batch Cultivation .................................................................................................................................................. 27 2.5.3. Continuous Cultivation ................................................................................................................................................ 28 2.6. Industrial Production of DHA ........................................................................................................................... 28 2.7. Downstream Processing of DHA....................................................................................................................... 29 3. Microbial production of EPA ......................................................................................................... 32 3.1. Potential Microorganisms ................................................................................................................................ 32 3.2. Biosynthesis of EPA .......................................................................................................................................... 32 3.2.1. Desaturation and elongation pathway ........................................................................................................................ 32 3.2.2. PKS pathway ................................................................................................................................................................ 34 3.3. Factors affecting EPA production ..................................................................................................................... 35 3.3.1. Nutritional factors ....................................................................................................................................................... 35 3.3.2. Environmental factors ................................................................................................................................................. 36 3.4. Cultivation systems .......................................................................................................................................... 37 3.5. Cultivation strategies........................................................................................................................................ 38 3.6. Industrial Production of EPA ............................................................................................................................ 39 3.7. Downstream processing ................................................................................................................................... 39 4. DISCUSSION ................................................................................................................................. 41 5. CONCLUSION................................................................................................................................ 44 6. BIBLIOGRAPHY ............................................................................................................................. 45 1. INTRODUCTION Fatty acids are long chain aliphatic carbons with a carboxylate group in the end. The fatty acids that are produced by microorganism vary in their length from 12 carbons to 22 carbons and they are either saturated or unsaturated. In general, the ratio of unsaturated fatty acids to saturated fatty acids is 2:1 in microorganisms. Polyunsaturated fatty acids (PUFAs) in general are indispensable cell membrane components and they play a major role in offering important membrane features such as flexibility, fluidity and selective permeability.1 They co-ordinates and moderates the functioning of membrane bound receptors, ATPases, transport proteins and ion channels. Along with these functions, they also play an inevitable role in the fatty acid biosynthesis by acting as precursor for a broad spectrum of metabolites.2 The various sources of PUFAs include plants, animals and wide variety of microorganisms. Cultivating microorganisms to the production of PUFAs have several advantages such as process control, seasonal independence, simple extraction and purification process and genetic modifications.3 The common PUFAs have 18 to 22 carbon atoms in their chain and they are categorized as ω-3 and ω-6 depending on the position of double bond from the methyl end of the acyl chain (ω end).4 The structures of the PUFAs are illustrated shown in the Figure 1. An overall view of occurrence and production of various PUFAs in microorganisms are described
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