ERT 211 BIOCHEMICAL ENGINEERING

1. ERT 211 BIOCHEMICAL ENGINEERING

2. Course Outcome • Ability to describe the usage and methods for cultivating plant and animal cell culture. • Ability to discuss the technologies available in bioconversion.

3. Chapter 5 Considerations in Using Plant and Animal Cell Cultures

4. Chapter 5(i) Outline • Usage of plant cell cultures • Potential products from plants cultures • Approaches to increase productivity • Difference of plant cells from microbes - implications of bioreactor design

5. Usage of plant cell cultures Control of supply of product independent of availability of the plant itself. Cultivation under controlled and optimized conditions. Strain improvement with programs analogous to those used for microbial systems. With the feeding of compounds analogous to natural substances, novel compounds not present in nature can be synthesized.

6. Control of supply of product independent of availability of the plant itself Many drugs contained active ingredients source from plant parts. For example, paclitaxel (taxol) an anticancer agent extracted from bark of Pacific yew tree (Taxus brevifolia). Three 100-year-old trees is needed to supply enough paclitaxel for one patient. The semi-synthetic productions concerns with environment contaminants from chemical processing.

7. Cultivation under controlled and optimized conditions The accumulation substances extracted from plants affected by many factors etc. condition of the plants and environment. Production of saponin via tissue culture technique from Panax ginseng was effected by the concentration of hormones used. 3.62% of total saponins was detected in callus cultivated in MS medium containing 5 mg/L 2,4-D and 1 mg/L kinetin, while 8.78% was produced in 10 mg/L and 1 mg/L kinetin medium.

8. Callus cultures of Oxalis reclinata are yellow in the light, but when they are transferred to the dark, they start producing red anthocyanin pigments.

9. Strain improvement with programs analogous to those used for microbial systems. The physiological characteristics of individual plant cells are not always uniform. In tissue culture, selection of cell lines for specific compounds production will increased in yield. A strain of Euphorbia milli was recognized to accumulate about 7 times higher amounts of anthocyanins than that of the parent strain after 24 selections.

10. With the feeding of compounds analogous to natural substances, novel compounds not present in nature can be synthesized. Addition to the culture media of appropriate precusors or related compounds sometimes stimulates secondary metabolite productions. Precursor can be synthetic or natural substances. This approach is advantageous if the precursors are inexpensive. The production of limonene is improved with the addition of mevalonic acid (primer precursor).

11. A diagram of limonene biosynthetic pathway.

12. Potential products from plants cultures

13. Shikonin and its derivatives such as acetyl shikonin and isobutyl shikonin accumulated in roots of Lithospermum erythrorhizon. They are reddish purple pigments and have been used in traditional dyeing and as a herbal medicine. Because of shortage of this plant, Mitsui Petrochemical company in Japan investigated mass cultivation of L. erythrorhizon cells to produce shikonin compounds. 1. Shikonin production

14. 1. Lithospermum erythorrhizon plant 2. L. erythorrhizon dry root 3. Suspension cultures of L. erythorrhizon in bioreactor 4. Cell agregates of L. erythorrhizon 3 1 4 2

15. First step was to established a cell line for shikonin production. Second step is to promote the cell growth in Linsmair-Skoog’s medium. Third step is to accumulate shikonin compound in White’s medium. This type of culture is called two-stage culture. Later they found that selecting the cell lines from the protoplasts is more efficient than from aggregated cells. They also feeding nutrients into the medium in order to increase the cell mass and therefore will increase the compounds productions.

16. Another researchers established a hairy root culture of L erythrorhizon with Agrobacterium rhizogenes.

17. Assignment 1 With labeled diagram, explain in details two-stage culture process for shikonin production

18. 2. Morphinan alkaloids • Codeine is an analgesic and cough-suppressing drug. • Morphine from Papaver somniferum L. (opium poppy) is a traditional commercial source. • Morphine can be converted to codiene. • Thebaine from capsules of P. bracteatum also can be converted to codeine. • Little success has been achieved from culture of undifferentiated cells of these plants for codiene production.

19. After many attempts, researchers still failed to get direct codiene and morphine from the cell cultures. Later, Japanese researchers (Furuya et al), studied the biotransformation of codeinone to codiene using immobilized cells of P. somniferum. The conversion yield was 70.4% of codiene produced and out of that, 88% was converted was excreted into the medium.

20. Assignment 2 Explain techniques that can be used to increase production of codein

21. 3. Berberine production 4. Rosmarinic acid production 5. Antocyanins production Reference Suri, S.S., Sharma, R., Ramawat, K.G. 1999. Production of secondary metabolites in bioreactor. In: Role of biotechnology in medicinal and aromatic plants. Volume II. Ukaaz Publications.

22. Approaches to increase productivity Optimization of Cultural Conditions explain on the use of two-stage culture under medium Selection of High-producing Strains Addition of precursors Biotransformation Elicitor Treatment

23. Optimization of Cultural Conditions The cultural conditions includes medium, surrounding environment (temperature, pH light & O2) and culture density. The most important is the medium that influence both the growth of cells and yield of desirable products. Various basal medium have been used and Murashige & Skoog (MS) is among the most widely applicable. Sucrose and glucose are carbon source for plant tissue cultures and affects cell growth and yield of products.

24. Phytohormones such as auxins and kinetins have shown the most remarkable effects on growth and productivity of plant metabolites. Generally, a temperature of 17-25°C is used for induction of callus tissues and growth of cultured cells. The pH is usually adjusted between 5-6 before autoclave. The use of light depends on the type of culture and the desire products. Oxygen is not critical for plant cultures but still has an effect on the growth and production.

25. The use of high cell density cultures in a suitable bioreactors found to increase yield in some cultures. Some plant cells needs different media for the cell growth and secondary metabolite production. This cultural condition is called two-stage culture. It means that 2 types of culture medium are used. both culture medium may differ in the concentration or types of nutrient, hormone or vitamins used.

26. two types of medium were used because the medium used for promoting growth is not suitable for promoting metabolites production. In such system, the first medium is used to promote the growth of cells in the culture system. the second culture is to promote the production of metabolites in the culture. Cells of plant will be first cultivate in the first medium and after certain period, they will be transferred into the second growth until harvesting period.

27. 2. Selection of High-producing Strains The physiological characteristics of individual plant cells are not always uniform. Therefore a rapid assay method is crucial in the selection of a high yielding cell line. The specific cell line is obtained from the selection a number of strains producing high level of desirable product . The strains then were subjected to further cell cloning to increase the level of secondary metabolites.

28. 3. Addition of precursors Precursors are related compounds sometimes stimulates secondary metabolites. This approach is advantageous if the precursors are inexpensive. For exp. Phenylalanine is one of the biosynthetic precursors of rosmarinic acid. Addition of this amino acid to Salvia officialis suspension cultures stimulated the production of rosmarinic acid.

29. 4. Biotransformation • Almost similar to precursor addition where, a suitable substrate compound may be biotransformed to a desired product using plant cell. • Biotransformation has been extensively applied in the fermentation using microorganisms and their enzymes. • For example, L-aspartic acid and L-malic acid can be biotransformed from fumaric acid using microorganisms.

30. Using plant cells, for exp. Digitalis lanata, the biotransformation of -mrthyldigitoxin to -methyldigoxin has been investigated. • Digoxin has a large market as a cardiac glycoside. • This approach beside precursors feeding are the most commercially realistic approaches because of economic reasons.

31. 5. Elicitor Treatment • Elicitor is an agent of microbial infections on intact plants that cause the synthesis of specific secondary metabolite. • Some studies reviewed possible correlations between stress and secondary metabolism in cultured cells. • Some suggested that upon infection, plants shows their defense mechanism by secreting secondary metabolite. • Elicitors that have been used in plant cell cultures are yeast extract, chitosan, inorganic and organic molecules and many more.

32. Plants grow under stress condition also show elicitation effects. Phosphate limitation in hairy root cultures of Hyoscyamus muticus had increased production of the sesquiterpene solavetivone. • Examples of inorganic compounds used are sodium chloride, potassium chloride, sorbitol and abscisic acid. • For economical use of the elicitors, they should be cheap and easy to obtain.

33. Implications of bioreactor design

35. Factors for growth in bioreactor • Gas-liquid mass transfer • Shear • Mixing

36. Assignment 3 Compare the plant cells with microbes and discuss the implications for bioreactor design.

37. Shear Stress Microorganisms cells are less sensitive to plant cells. Plant cells may require operation under low-shear conditions.

38. Aeration and Agitation • In aerobic fermentation processes, microorganisms require high aeration compare to plant cells. • Require to change the bioreactor system that provide high aeration rate. • This system can supply necessary oxygen to the microorganisms because they have high metabolic activities. • system can be equipped with mechanical devices used to agitate the liquid broth or by air compressor.

39. Size Plant cells are large compared to microorganisms. Cell often grow as aggregates or clumps. The large size, rigid cellulose-based cell wall, and large vacuole making plant cells sensitive to shear stress. (fan and balloon in a room). Cell often grow as aggregates or clumps and may have mass transfer limitations that limit the availability of nutrients to cells within the aggregate.

40. Duplication times Duplication time in microorganisms is within days compare to plants within weeks. To contain the rapid multiplication of cells, larger bioreactor volumes is needed and usually in the range of 10,000 – 100,000 L. Products are produce within shorter period. Shorter period in the culture systems means less contamination and less cost for the maintenance.