CELL CULTIVATION

- FERMENTATION -

Fermentation

Since ancient time Latin fermentum fervere (boil) to describe the metabolism of sugars by microorganisms Fruit fermentation beer brewing soy sauce Before WW II mainly applied commercially in food; after ? Substrates any material that supports microbial growth Culture microorganism grew in controlled environment

Categories & Applications of fermentation

Processing

Upstream processing all involved factors in fermentation : microorganism/cell, medium and fermentation process/condition
Downstream processing process folowing the fermentation : harvesting, separation , purification to obtain certain product level

Phases in development of fermentation process

Living Cell

BACTERIA
Unicellular, about 1500 species Diameter 0.5 to 1m, vary greatly in length Shape: cocci (spherical/ovoid), bacilly (cylindrical/rod), spirilla (helically coiled)

 Reproduction: asexual reproduction steps: 1) cell elongation, 2)invagination of the cell wall, 3)distribution of nuclear material, 4)formation of the transverse cell wall,5 )distribution of cellular material into two cells, and 6)separation into two new cells

FUNGI

Plant devoid of chlorophyll unable to synthesize their own foods range in size and shape from singlecelled yeasts to multicellullar mushrooms YEAST & MOLD

Yeast

widely distributed in nature (food, soil, in the air, on the skin and in the intestines of animals) depend on higher plants and animals for their energy Unicellular spherical to ovoid Size: 1 to 5 m in width; 5 to 30 rn in length The cell wall quite thin in young cells but thickens with age.

Asexual reproduction BUDDING

A small bud (or daughter cell) is formed on the surface of a mature cell. The bud grows and is filled with nuclear and cytoplasmic material from the parent cell. When the bud is as large as the parent, nuclear apparatus in both cells is reoriented and the cells are separated. The daughter cell may cling to the parent cell, often even after the cells are divided Saccharomeces cerevisiae wine, beer, leavening of bread

Mold

filamentous fungi A single cell or spore (conidia) is germinated to form a long thread, hyphae, which branches repeatedly as it elongates to form a vegetative structure called a mycelium. Since a mycelium is capable of growing indefinitely, it can attain macroscopic dimensions.

Find everywhere Aspergillus, penicillium, rhizopus

used in the production of antibiotics, enzymes, food and food additives

CELL CULTIVATION

Cells are removed from animal/plant tissue & cultivated in nutritional medium outside the donor's body mammalian cell culture human growth hormones, viral vaccines, cancer cells, transplantation, antibodies, tumor

Plant cell cultivation

Plants source of various chemical compounds) known as secondary

metabolites

Interface between the plants and environment, (as adaptations to stresses or chemical defenses against microorganisms, and higher predators) Synthesize? too difficult & costly Cultivate exogenous plant tissue instead of a whole plant as a culture in an aseptic condition, nutritious medium, filtered air As cultures grow sliced off and transferred to new media (subcultured) to allow for growth

Advantages of plant cultivation

1. regardless of weather and geographical conditions; no need to ship or store bulky raw materials. 2. The product quality and yields can be controlled by eliminating the problems encountered in the processing of botanicals, such as the quality of the raw material, uniformity within and among lots, disease, pest 3. some metabolic products can be produced from culture in higher quantities than that observed in whole plants 4. produce of multiples of plants in the absence of seeds or necessary pollinators to produce seeds 5. production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits

differences between plant and microbial cells

1. Plant cells are 10 to 100 times larger than bacterial and fungal cells (20-40 m in diameter and 100-200 m long) 2. The metabolism of plant cells is slower than microbial cells in the one order of magnitude the maintenance of sterility for a longer period of time. 3. Plant cells tend to grow in clumps which cause sedimentation, poor mixing, plugging the inlet and outlet lines, wall growth, and so on. 4. Plant cells are more sensitive to shear than microbial cells. 5. Metabolic production in plant cells is subject to more complex regulatory mechanisms than metabolic production in microbial cells. 6. Plant cells are more genetically unstable than microbial cells.

Plant secondary metabolite production

3 essential categories: alkaloid, essential oil and glycosides. Alkaloids are physiologically active and used in the pharmaceutical Industry, e.g codeine, nicotine, caffeine, and morphine. Essential oils consist of mixtures of terpenoids and used as flavorents, fragrances, and solvents. Glycosides include phenolics, tannins and flavonoids, saponins, and cyanogenic glycosides, some of which can be utilized as dye, food flavors, and pharmaceuticals.

Plant products of commercial interest

Cell immobilization

SmF cells attached to large particles or on surfaces ADVANTAGES: process design can be simplified cells are easily separated from product, fermenter is easier to be controlled, ensures continuous fermenter operation without the danger of cell washoutprovide conditions conducive cell-to-cell communication, high yields of secondary metabolites protect cells; decrease problems related to shear forces.

Cell immobilise methods

The methods

Attachment To Surface provide a large surface area for cell attachment Entrapment Within a Porous Matrix cell diffuse into preformed porous in which they will grow and be trapped advantages preformed support materials are more resistant to disintegration in packed beds or stirred vessels than other support materials, and the entrapment is not usually harmful to the cells. disadvantage difficult to reach a high cell concentration due to the limited pore volume

Containment Behind a Barrier immobilized within micro-capsules with either a permanent or nonpermanent semipermeable membrane advantage of encapsulation is the large surface area for contact of substrate and cells. The semipermeable membrane also selectively passes only low molecular weight components The disadvantage high cost, difficult in aeration Self-aggregation Self-aggregated or flocculated cells immobilized cells; large size similar advantages as other methods. While molds will form pellets naturally, some bacteria or yeast cells require flocculation