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Important Dates
Quiz 1 – 6 Oct 2011 (Chapter 1-2) - Done
Midterm Test 1 – 21 Oct, Friday (Chapter 1-2)
Assignment 1 (Posted in Portal) – Due 13 Oct 2011 -
Done
2.2 Design, Formulation and Optimization
of Media
Growth and production of metabolites (product) by
organisms (microbes, plant, animal cells) are result of the
interactions between intra- and extracellular effectors
Intracellular effectors – specific to organism including
genome and control mechanisms of replication,
transcription and translation of genetic info
Extracellular effectors – physical (temperature, viscosity,
aeration/agitation) and chemical (nutrients) conditions
Fundamental steps of typical bioprocess
Cell
• Intracellular effectors
Medium
• Extracellular effectors
Bioreactor
• Extracellular effectors
Downstream Processing
Products
Introduction
Role of medium ingredients have provided evidence that nutrient deficiencies can be the major limiting steps in bioprocesses
For example, growth rate of E. coli is reported to be depended on the flux of precursor, metabolites and monomers derived from them, not the flux of ATP or rate of protein synthesis (HOW?)
Stimulatory effect has been shown by using high concentrations of organic nitrogen sources (yeast extract), which its metabolic role is yet to be understand well
Use of elicitors has becoming popular to enhance metabolite production in plant cell bioprocesses
2.2.1 Microbial Processes
Nutritional Requirements for growth and Product
Formation
Other additives
Biochemical Mechanisms
Upstream and Downstream Processes
Bioavailability of Ions
Cost, Availability and Sustainability of Medium Ingredients
Mass Composition of Cells, Yield, and Stoichiometry of
Growth and Product Formation
Optimization
Nutritional Requirements for Growth and
Product Formation
Macroelements
Constitute 90-95% of dry weight of microbial biomass: C, O, H, S, P, K and Mg
Carbon substrate acts as carbon and energy source, part of it contribute in cell carbon and rest provide energy such as carbohydrates, alcohol, organic acids, hydrocarbons
Nitrogen sources – inorganic ((NH4)2SO4) and organic (yeast extract and pepton) (HOW?)
L-amino acid also can be used as nitrogen source (HOW & EXAMPLE)
Microelements
Essentials for growth such as Ca, Mn, Fe, Cu, Co, and Zn whereas B, Cr, Mo and others are rarely used
Usually added as mineral salts
Deficiencies and toxic effects of microelements were found to limit the growth rate of microbes in fermentation (HOW and SOLUTIONS?)
Nutritional Requirements for Growth and
Product Formation
Growth Factor (GF)
Specific nutritional factors which microbes cannot synthesize
3 types of GF – vitamins, amino acids and miscellaneous
compounds (oleic acid and ergosterol)
Role of vitamins as GF (IMPORTANT!!!)
Influence of Physical Factors
Nutritional requirement are affected by physical conditions
such as temperature and water activity
For example: High temp for growth of yeast, 37°C instead of
30°C will make pathothenic acid content increased
Toxin production of B. thuringiensis increases as osmolarity of
the medium increased
Other Additives
These compounds serve different functions – increasing yields and avoiding precipitation, foam formation or inhibitory effects
Classified according to their functions – precursors, stimulants, protectants, antifoams, chelators, stabilizers & neutralizing agent
Precursors such as phenylacetic acid (penicillin), chloride (chlorotetracycline production)
Stimulants such as methanol (citric acid production), methionine (antibiotic)
Antifoams such as fatty acids, polyglycols, higher alcohols and silicones
Chelators such as EDTA (cannot be metabolized), citrate (assimilated by bacteria and fungi)
Stabilizers such as antibiotics and uracil (avoid loss of plasmid)
Biochemical Mechanisms
Microbial metabolism and its regulation is essential for medium design
Effect of carbon catabolite repression and phosphate and nitrogen metabolite regulation - nutritional repression
Carbon catabolite repression is significant due to many metabolites (enzymes and antibiotics) can be regulated
Glucose ,glycerol and citrate produces carbon catabolite repression
Ammonia is rapidly assimilated as nitrogen sources in synthesis of antibiotics
Phosphate concentration is crucial factor in any antibiotic fermentation (WHY?)
Upstream and Downstream Processes
Upstream processes – pretreatment of raw materials,
conversion steps of starch or cellulosic material
(liquefaction and saccharification) and effects of
sterilization on medium composition
Sterilization often produce undesirable chemical changes in
medium such as precipitation of di- and trivalent metals and
phosphates (SOLUTION?) and Maillard reaction
(SOLUTION?) between carbohydrate and nitrogen source
Some amino acids should be sterilized by filtration
Downstream processing should consider the influence of
medium components (products or byproducts) on the
separation and purification steps
Cost, Availability and Sustainability of
Medium Ingredients
Cost and availability of regular supply are essential affecting the suitability of any raw material used in medium preparation
Many raw materials of animal and vegetable origin are subject to price fluctuations depending on many factors which are difficult to control (SOLUTION?)
Presence of herbicides or pesticides reduce alteration in composition of molasses affecting quality and suitability for microbial processes
Nitrogen and carbon sources must be stable and uniform (WHY?)
Complex substrates like peptone and corn steep liqour are not stable enough for storage for extended period (SOLUTION?)
Mass Composition of Cells, Yield, and
Stoichiometry of Growth and Product Formation
Medium is formulated based on mass composition of cells,
yield coefficients, and stoichiometry of growth and
product formation
The stoichiometry of biomass and product formation is
useful guide for formulation of minimal media for growth
and product formation
General equation can be written as:
ODHCOyNOCHy
NOCHyNHBOAOCH
sCOsp
sxba
22/'''/
/32
2
Carbon and Nitrogen Sources Used in Typical
Microbial Processes
Process / product Carbon sources Nitrogen sources
Single-cell protein Methanol
Ethanol
Molasses
Whey
n-Paraffins
Ammonia
Urea
Citric acid Molasses
Sucrose
Glucose
n-Alkanes
Ammonium nitrate
Penicillin Glucose
Lactose
Molasses
Vegetable oils
Ammonium sulfate
Corn-steep liqour (proteins, 24%)
Cottonseed meal (32%)
Soybean meal (protein, 42%)
Note:
Cane molasses (sucrose, 30-40%, reducing sugars, 15-20%), beet molasses (sucrose, 48-55%)
Solid whey (protein, 13%; lactose, 75%), acid whey (protein, 12.5%; lactose, 67%)
2.2.2 Plant Cell Processes
Medium Requirement for Growth and Product Formation
Other additives
Medium Preparation
Guidelines for the Design and Formulation of Media for
Plant Cell Suspension Culture
Plant Cell Processes
Higher plants are potential sources for natural products such as flavors, fragrances and pharmaceuticals (tongkat ali, misai kucing, kacip fatimah)
Plant cell can be cultured in several ways with different purposes such as:
Tissue culture – for cultivation of seedlings, embryos and organs
Cell tissue suspension culture – submerged cultivation of individual cells and lumps in liquid media
Immobilized plant cell culture – physical restriction of cells on a fixed support
Constraints in plant cell suspension cultures (Important!)
Solution/Improvements to the constraints (Important!!)
Plant Cells vs Microbial Cells
Characteristic Microbial Cells Plant Cells
Size 1-2 x 2-7 µm (Bacterial)
3.5 µm (Yeast)
20-40 x 20-200 µm
Aggregation Often single cells Normally in clumps
Doubling time <1h >24h
Water content 75-80% 90-95%
Shear stress Insensitive Often sensitive
Stability Normally stable Unstable
Oxygen consumption 5-90 mmol/l·h 1-4 mmol/l·h
Product Normally extracellular
(Media)
Generally intracellular
(Vacuole)
Mutations Possible Require haploids
Plant Cells vs Microbial Cells
From the aspects of fermentation technology, the key problem areas are: Plant cells are too fragile compared to microbial cells
Plant cells takes too long to grow in fermentors compared to microbial cells
Oxygen consumption is lower compared to microbial cells
As for product formation it will be a downstream problems of fermentation technology. The consequences of the three main points above are: Since plant cells are shear sensitive supplying air or mixing will be a
problems as those processes are intense shear generating forces
Too long a growth time in the fermentor will lead to the potential problem of microbial contamination to occur and fermentation disaster
Oxygen supply will have to be provided by a more suitable and controlled system
Plant Cell Processes
Species Specific growth
rate, µ (d-1)
Growth yield
coefficient (g/g)
Maintenance
coefficient, m (h-
1)
Catharantus roseus 0.26
0.45
0.58
(with sucrose)
0.0084
(with glucose)
Nicotina tabacum 0.68 0.43
(with sucrose)
0.0076
(with glucose)
Digitalis lanata 0.15 – 0.20 0.29 – 0.40
(with glucose)
-
0.11 – 0.32 0.41
(with sucrose)
-
Strawberry 0.12 – 0.14 0.39 – 0.44
(with sucrose)
-
Medium Requirement for Growth and
Product Formation
Media used in plant cell cultures can be 2 types:
Growth media – for producing plant cell biomass
Production media – for enhancing secondary metabolite
accumulation
Medium requirement for growth and product formation
in plant:
Macroelement
Microelement
Vitamins
Phytohormones
Medium Requirement for Growth and
Product Formation
Macroelements
Carbon sources – Sucrose and Glucose
Nitrogen sources – must be in a mixture of NO3 and NH4 (WHY?)
P, Ca, S, Mg and K sources – P has to be limiting substrate (WHY?), Ca concentration used higher than in microbial cell (WHY?)
Microelements
Consist of trace elements such as I, B, Mn, Zn, Mo, Cu, Co, Fe
Affect growth of plant cells and production of 2nd metabolites
Eg. Increase in Cu concentration, has increased shikonin production; decrease boron concentration, has increased production of phenolic compounds
Medium Requirement for Growth and
Product Formation
Vitamins
Cell growth can be very slow if vitamins are not present in
medium
Thiamine HCl
Phytohormones
Also known as plant growth regulators
Natural – auxins, cytokinins, gibberellic acid, abscisic acid,
indoleacetic acid (IAA)
Synthetic – dichlorophenoxyacetic acid (2,4-D),
naphthaleneacetic acid (NAA)
Also shown remarkable effects on growth and product
formation
Other additives
Precursors If these compounds are supplied with the medium, the rate of
product synthesis can be increased
Examples – Phenylalanine, methylputrescine, isocapric acid, tropic acid
Elicitors – are microbe-derived molecules which stimulate 2nd metabolism Biotic – whole fungal mycelium, fungal cell wall material, glucan
polymers, glycoproteins and low molecular weight organic acids
Abiotics – UV irradiation, salts of heavy metals, and chemicals
Antibiotics Used as additives to media because their beneficial effects for
controlling microbial contamination
Many antibiotics have proved to be cytotoxic to certain plant cell species
Medium Preparation
Preparation of stock solutions for micronutrients,
vitamins, CaCl2, IK and phytohormones
The rest of components are added and dissolved in
distilled high purity water
The heat-stable GH such as kinetin and 2,4 –D are added
at the end, pH is adjusted, and medium is autoclaved
Heat-labile compounds like indoeacetic acid, indolebutric
acid or gibberellic acid are added to the medium before
autoclaving
Guidelines for the Design and Formulation
of Media for Plant Cell Suspension Culture
Considerations for design and formulation of media (IMPORTANT!!)
1. Use two-steps processes, first using growth medium and second, production medium
2. Sucrose is preferentially used for carbon source, increase in concentration might increase growth and production, but >10% might result in carbon catabolite repression
3. NO3 or NH4 as the sole nitrogen sources may produce inhibition of growth and product formation and their concentration are generally reduced in production step
4. PO4 as a regulatory compound of 2nd metabolite formation and its concentration has to be reduced in production step
5. Selection of the most efficient phytohormones is essential for improving yields
6. Addition of precursors or elicitors to the media for enhancing 2nd metabolite formation
Guidelines for the Design and Formulation
of Media for Plant Cell Suspension Culture
Guides for design, formulation and optimization of growth and production media for plant cell bioprocess (IMPORTANT!!)
1. Growth medium 1. Select basal medium such as MS, B5, W or other
2. Test the medium in shake flasks and in bioreactor and optimize it for maximal biomass production in continuous culture
2. Production medium, by maintaining the same carbohydrate concentration of growth medium (20-30 g/l), test the following modifications
1. Reduce P and N content. In some cases, N content can be reduced to nil
2. Modify the vitamins and iron contents
3. Find adequate phytohormones levels and ratios
4. Add a precursor and find the correct time for its addition
5. Test a biotic and abiotic elicitor
6. Optimize the medium by a fractional factorial methodology followed by simplex technique
Animal/Mammalian Cell Processes
Animal cells is more expensive than microbial cells due to higher medium costs, longer runs and lower yield
Why still use animal cells?
Certain mammalian cells can provide very specific posttranslational modifications of interest protein, desired functionality plus stability and pharmacokinetics
Yield of mammalian cell process – function of interaction of cells, bioreactor ad medium
The effects of media components on downstream operations based on potential to contaminate the product at all stages of purification steps has to be considered
Initial Medium Selection Serum free media
Media designed to grow a specific cell type or perform a specific application in the absence of serum
The use of serum-free media (SFM) represents an important tool, that allows cell culture to be done with a defined set of conditions as free as possible of confounding variables
Protein free media Protein-Free Media contain no proteins, but may contain plant or yeast
hydrolysates. Many are animal-origin-free
Advantages of SFM Increased definition.
More consistent performance.
Easier purification and downstream processing.
Precise evaluations of cellular function.
Increased growth and/or productivity.
Better control(s) over physiological responsiveness.
Enhanced detection of cellular mediators
Initial Medium Selection
Selection of basal medium start with basal medium that’s
supports the growth of parent cell line
Initial medium contains fetal bovine serum for practical
reasons and is easy plus convenient to implement for
initial production desired product
All mammalian cell used for manufacturing processes are
adapted to serum-free media and protein-free media
Multiple commercial available serum-free media –
convenient alternatives for fast and easy adaptations to
serum-free media (Table 9)
Medium Development
Eliminating phenol red from medium formulations (WHY?)
After selection of preliminary serum-free formulation, quantitative analyses of spent medium to balance the formulation for components such as amino acids, carbohydrates and lipids
Medium optimization has to be done depending on type of bioreactor used as well as mode of fermentation
Also include study on the effects of metabolic-by product and physicochemical parameters like temperature, pH and agitation because it may significantly affect the cell ability to take up nutrients and their overall performance
Medium Preparation and Use
Use powdered medium is common practice in industry
Concentrated medium formulations are available and
their use is increasing
It can reconstituted to single strength either in batches or
in continuous mode using a steady supply of sterile water
and eliminating the need for large mixing and storage tank
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