1. Fungal products and processes Course 27033 BioCentrum-DTU Technical University of Denmark January 2006 By, Marta Karolina Mustafa Shanshan Vijaya Krishna

2. List of contents 1.Introduction  Aim of the experiment  Design  Measurements 2.Results  Morphology  Intracomparisions  Intercomparisions 2.Conclussions 3.Scale up 4.Questions

3. Intoduction  Aim of the experiment  Design of the experiment  Measurements

4. Aim of the Experiment  Optimisation of growth media for α-amylase production  Investigation of effect of carbon source on α-amylase production  Investigation of the effect of carbon source concentration on α- amylase production

5. Design of the Experiment  Three different complex media were used -Pure glucose -Pure starch (10g/L) -Pure starch (15g/L)  Five flasks were prepared for each medium type  pH is adjusted to 5.5  A. Oryzae spores were harvested from rice  Each flask was inoculated with 2.0x10 8 spores  Incubated at 30 ºC, 250rpm

6. Measurements  Samples were taken after 16, 19, 22, 25 and 40 hours  Enzymatic assays were performed for starch and glucose concentration, and α-amylase activity  HPLC analysis was performed for the samples without starch  Volume of each sample and DW content measured  Starch analysis was made by using a kit  α-amylase activity was determined by using microtitre plate and starch plate assay  Morphology was examined for all the samples

7. Results 1.Morphology 2.Results based on the same carbon source used 3.pH comparison 4.Comprisons between different batch cultivations

8. Morphology Samples & Time  There are three samples A- starch 10g/L ; B- starch 15g/L ; C-glucose 10g/L  Time of sampling - sample1-16:00h (8am), sample2-19:00h (11am), sample3-22:00h (2pm), sample4- 25:00 (5pm), sample5-40:00h (next day 8am)

9. Staining of filamentous fungi  Looking at the morphology under the microscope  Distinguish viable and non-viable hyphal elements  Non-viable hyphal elements are stained by methylene blue  Viable cells excluded the methylene

10. Sample A Starch 10g/L A1(10X) A2(20X) A4(20X)

11. Sample B Starch 15g/L B1 (4x) B4 (20x) B5 (20X)

12. Sample C Glucose 10g/L C1(10X) C4(8X) C5(20X)

13. Intracomparisions - Starch 10 g/L

14. Starch 15 g/L

15. Glucose 10 g/L

16. pH Time h A Starch 10 g/L B Starch 15 g/L C Glucose 10 g/L 225.175.23.9 254.944.93.4 404.255.995.5

17. Time h pH Biomass concentration [g/L] Amylase produced [g/L] A Starch 10 g/L 254.941.914.5 A Starch 10 g/L 405.94.18.1 A Starch 10 g/L 405.55.711.3

18. Intercomparisions y = 0.3532*e^0.0925x y = 0.0552*e^0.1479x y = 0.1051*e^0.1325x

19. Maximum Specific Growth Rates μ Starch 10 g/l0.1479 Starch 15 g/l0.1325 Glucose 10g/l0.0925

21. Specific Production Rates

22. Conclusions  The product and the rate of production was higher and also obatined in shorter time for the medium starch with concentration of 10g/l  Higher maximum specific growth rate was also observed for the same starch medium with concentration of 10g/l  Highest amount of biomass formed was observed for glucose medium as contradictory to what was expected because of the glucose repression  pH was well maitained for the starch medium compared a decrease in pH for the glucoe medium  Efficient and early pellet formation was observed for the starch medium

23. Scale UP  The density or the biomass concentartion should be kept at low level inorder to reduce problem with mixing for good mass transfer  Monitering techniques need to more advanced compared to the lab scale  Decrease the chances of contamination or operate at high sterility level  Continuous or fed-batch preferred over batch because of the ease of maintaining a comparitively low or optimal glucose concentartion  pH need to monitered and maintained

24. QUESTIONS ????