Bioreactor System

ERT 314

Sidang 1 2011/2012

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

Chapter 2:Biological Systems

and Media Design

Week 2 - 3

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

Tissue Culture

Tissue Culture

Cell Tissue Suspension Cultures

Immobilized Plant Cell Culture

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

2.2.3 Animal Cell Processes

Initial Medium Selection

Medium Development

Medium Preparation and Use

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

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