Bioprocessing Laboratory

Transcription

1 Efficient Exopolysaccharide Production by Ganoderma lucidum in Submerged Culture Nor Zalina Bte Othman Bioprocessing Laboratory Chemical Engineering Pilot Plant

2 Ganoderma lucidum Ganoderma lucidum (Ling Zhi) is a basidiomycete white rot macro fungus As a health food and medicine in the Far East also known as The mushroom of immortality

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4 Contain approximately 400 different bioactive compounds with therapeutics properties which mainly: Triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, proteins/peptides and trace elements

5 Polysaccharides Polysaccharides, especially beta-d-glucans, have been known to possess anti-tumor effects through immunomodulation and anti- angiogenesis. Polysaccharides have a protective effect against free radicals and reduce cell damage caused by mutagens.

6 The market size of immunomodulators :USD$ 43 billion in 2006 Expected to grow at a compound annual growth rate (CAGR) of 13% to reach US$ 80 billion by 2011 (Research and Market, 2007)

7 Therapeutics effects Immunomodulator Antiatherosclerotic Antiinflammatory Analgesic Antitumor Antibacterial Sleep promoting Antiviral (including anti- HIV) Antioxidative and radical- Anti-aging Hypoglycaemic scavenging Estrogenic activity

8 Traditionally cultivated in solid media: Disadvantages: 1. Time- (4-6 months) 2. Quality- different strain 3. Environmental factors-contamination

9 Current trend cultivation strategies of Ganoderma lucidum Solid substrates: wood logs and on sawdust in plastic bags or bottles Stationary liquid medium Submerged cultivation

10 Objectives of the research To ocultivate aethe egucdu G.lucidum in submerged culture u in bioreactor for optimal yield of mycelium biomass and exopolysaccharides (EPS). Problem during cultivation in submerged cultivation: The morphology of the mycelium that will affect the production of bioactive compounds and the cell growth

11 Links between morphology of Mushroom mycellium in submerged culture for the production of exopolysaccharide (EPS) Max size of pellet 400µm oxygen transfer/nutrients Long mycelium hypae good for EPS production May contribute to viscosity of the fermentation broth-oxygen transfer/nutrients

12 The Flow Diagram of the Research Cell Banking: Master Cell Bank Working Cell Bank Media Screening Medium optimization study in shake flask : Glucose and Nitrogen sources Process Parameter in Bioreactor-16L (500 rpm, 28 C,pH 5.5) - Controlled ph - Uncontrolled ph Biomass and EPS Production

13 Cultivation Procedure Working Cell Culture Agar Plate Shake Flask Centrifuge 16-L Bioreactor

14 Mushroom morphology of different medium Medium 1 Medium 2 Medium 3 Medium 4 Medium 4 Medium 5

15 Cell dry weight, EPS weight, EPS yield and final ph in 6 different media after 16 days of fermentation at 28 C in Erlenmeyer shake flasks (250mL) Medium 2: Max. EPS yield =0.156 g/g Medium 1 the highest biomass Composition of medium 2(g/L): Glucose : 30 Corn steep liquor: 5.0 NH 4 Cl : 2.0 KH 2 PO 4 : 2.0 K 2HPO 4 : 1.0 MGSO 4.7H 2 0 : 1.0 Yeast powdr : 1.0

16 0 g/l glucose 10 g/l glucose 20 g/l glucose 30 g/l glucose 40 g/l glucose 50 g/l glucose 60 g/l glucose 70 g/l glucose 80 g/l glucose

17 Effect of Glucose Concentration on Production of Biomass and EPS Mycellium biomass increased when concentration of glucose increased EPS yield decreased when glucose concentration increased Optimal glucose concentration- 30 g/l: EPS yield 0 18 g EPS/ g EPS yield 0.18 g EPS/ g biomass

18 Effect of Different Concentration Corn Steep Liquor Concentration on Production of Biomass and EPS Optimal lcsl conc.: 25 ml/l Max biomass : 11.2 g/l Max EPS Yield : 0.26 g EPS/g biomass Max EPS production: 2.97 Max EPS production: 2.97 g/l

19 Morphology of Cell Pellet in Different Concentration of Corn Steep Liquor 0ml/L 20ml/L 5ml/L 25ml/L 10ml/L 30ml/L 15ml/L 35ml/L

20 Growth Curve of Ganoderma lucidum in Unoptimized and Optimized Media ph Y P/X [g g -1 ] CDW [g L -1 ] tim e [days] EPS [g L -1 ] Un-optimized Optimized %Difference Max. cell concentration (g/l) Max. EPS production (g/l) Max. EPS yield (g EPS/ g biomass)

21 Growth Curve of Ganoderma lucidum when cultivated in the 16-L stirred tank bioreactor ; uncontrolled ph and controlled /ph5.5 Uncontrolled ph Controlled ph % Difference Max. cell concentration (g/l) Max. EPS production (g/l) Max. EPS yield (g EPS/ g biomass)

22 Morphology of Cell Pellet when cultivated in the 16-L stirred tank bioreactor ; uncontrolled ph Day 1 Day 3 Day 5 Day 7 Day 9 Day 12 Day 15 Day 17 Day 19 Day 21 Day 22 Day 24

23 Morphology of Cell Pellet when cultivated in the 16-L stirred tank bioreactor; ; controlled ph-5.5 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Day 15 Day 18 Day 20 Day 22 Day 25

24 Conclusion Optimized medium composition (in g/l): Glucose,60 ; Corn Steep Powder, 10ml; NH 4 Cl, 1.53; K 2 HPO 4, 2.0; KH 2 PO 4, 2.0; MgSO 4.7H 2 O, 1.0 ; Yeast Powder, 1.0 Fermentation at a controlled ph of 5.5 gave significantly btt better EPS yield ildcompared dto that thtof uncontrolled lldph. Decrease of mycelial density with some long hyphae protruding from their surface, indicating an improvement of the transport of nutrients to the inner parts of the pellet and more efficient with respect to the production of exopolysaccahride..

25 ACKNOWLEDGEMENTS Prof. Ramlan Aziz Prof. Dr Mohamad Roji Sarmidi Prof. Dr. rer. Nat. Hesham Ali El-Enshasy Mr Koh Zwin Zjeh Mrs. Nurul Shifa Ismail BIOPROCESSING LABORATORY CHEMICAL ENGINEERING PILOT PLANT UNIVERSITI TEKNOLOGI MALAYSIA