Bio-processing

Biotransformation

Conversion of a substrate to a product by living systems, lysates, or purified enzymes

How does biotransformation work with genetic engineering?

Scientists combine parts of metabolic pathways from various organisms to catalyze particular reactions

Fermentation (biochemically)

Cells use a partially-reduced source of carbon under anaerobic conditions

2 types of anaerobic fermentation

Lactic-acid fermentation

Alcohol fermentation

Lactic acid fermentation

Bacteria and fungi ferment glucose to lactic acid

Alcohol fermentation

Yeasts and molds ferment glucose into ethanol and CO2

Fermentation Biotechnology

Tissue culture cells, fungi or microorganisms growing under optimal conditions for maximum cell division and product production

Any process that produces biomass or performs a biotransformation

Anaerobic or aerobic growth

Biomass

Amount of living organisms

It can be the end product (yeast) or starting point (beta-gal)

Primary metabolites of fermentation

Produced by actively-growing organisms and are essential for growth and reproduction

intermediates or end products (vitamins, amino acids)

Secondary metabolites

not essential for rapid growth; often made for defense or survival (e.g. antibiotics, toxins)

Uses of enzymes

Use in vitro (restriction enzymes)

Use industrially to produce products more efficiently (cellulose for wines and beers)

Enzyme with most economic importance

Beta-galactosidase

Durable enzymes use and examples

Tolerate extreme environments

Used for laundry detergent (ex. protease digest protein)

Genetic engineering for industry

Can introduce genes for commercially important enzymes into inexpensive to grow organisms (e. coli)

Ex. Chymosin

Therapeutic proteins

Proteins produced for medical treatment purposes.

Some are toxins or derivates that inhibit certain enzymes (digitalis and snake venom)

Some are hormones, antibodies, or proteins we would normally make (ex. Insulin)

Antibiotics

Many fungi and microbes are sources

Kill bacteria or prevent them from growing or dividing

How do antibiotics work

Cell wall synthesis inhibitors (penicillin)

Transcription inhibitors (actinomycin D)

Translation inhibitors (streptomycin)

Fuels

Some microbes can be used to produce renewable fuel (methanogens make methane gas from organic waste)

Bioplastics

Organisms are able to produce energy storage polymers with quality similar to plastics (ex. Polylactic acid)

Bioadhesives

Bivalves (mussels) produce natural adhesives that they use to attach themselves to one another (genetically modified saccharomyces cerevisiae to produce it)

Pigments and dyes

E. Coli modified to produce nontoxic indigo

Upstream processing

All he work leading up to and including fermentation (ex. Selecting organism, mutation)

Downstream processing

All the work after fermentation (ex. Product purification, testing, packaging)

Unit operation

a single step in the sequence of steps required to transform the starting material into a final product (ex. Centrifugation, chromatography)

Process

Series of steps used to create a valuable product from raw materials

Unit operations can't change but what can?

The order which they are used

The conditions used

The materials used

2 categories of unit operation

Transformational unit operations

Physical unit operations

Transformation unit operations

Chemical changes to the sample (fermentation)

Upstream processing

Physical unit operations

Physical manipulations on the sample but do not cause chemical changes (drying)

Downstream processing

Goal when selecting organism for bioprocessing

Produce product as quick and cheap as possible

Organism sources

Wild-type (in nature)

Crossed (produced by breeding)

Mutated (randomly mutated then selected)

Engineered (purposefully modified in lab)

Advantages and disadvantages of bacteria as fermentor

Pros: well understood, easy to manipulate genetically, grows fast

Cons: prokaryotes limit protein modification, intracellular production requires extra downstream processing

Advantages and disadvantages of fungi as fermentor

Pros: Eukaryotic modifications possible, sources of commercially valuable substances, sexual reproduction

Cons: intracellular production and cell walls requires extra downstream processing

Advantages and disadvantages of plants as fermentor

Pros: eukaryotic modifications possible, sources of commercially valuable substances, sexual reproduction, fruit as transportation

Cons: cell walls means extra downstream processing, wont synthesize some proteins, eukaryotic modifications possible may be incompatible for humans

Advantages and disadvantages of animals for fermenting

Pros: products compatible for human use, eukaryotic modifications possible may possible, sexual reproduction, transportation (milk, egg, meat)

Cons: high contamination risk, complex nutritional requirements, slow growth

Bioreactor

A vessel or container in which living cells or their products are used to make a product

Solid substrate bioreactor

Organisms grown under artificially controlled conditions on solid substrates (saw dust, rice) not submerged in liquid. Kept moist not wet

Ex. Mushrooms

Liquid media used for

Single celled organisms or cells in tissue culture

Batch culture

a closed-system microbial culture of fixed volume

Incubate uninterrupted to completion

Undergoes growth curve

Which bioreactor undergoes growth curve

Batch

Growth curve cycle

Lag

Exponential

Stationary

Death

Lag phase

Cells may be damaged, nutrient depleted, and need time to activate

Exponential growth phase

Produces primary metabolites

Cells reproduce rapidly

Stationary phase

Secondary metabolites produced

Nutrients used up, waste builds up, death=division rate

Death phase

Cells die due to lack of nutrients and waste accumulation

May produce products and secondary metabolites

Batch culture cons

Not ideal for products that depend on continuous production of biomass (secondary metabolites)

Production cycle interrupted to clean everything (time and money)

Fed batch culture

a modified form of batch culture in which nutrients are added at intervals during the fermentation process, and equal amount of old media is removed

Goal of fed batch culture

Keep cells in stationary phase (good for producing secondary metabolites)

Continuous culture

Frequent or ongoing addition of fresh media and removal of old media

Goal of continuous culture

Maintain exponential growth (primary metabolites)

Benefit of continuous culture over batch and fed-batch

Do not need to interrupt the process to collect biomass/product (clean and sterilize system)

Primary metabolites produced

Continuous culture bioreactors

Chemostat

Turbidostat

Chemostat

Nutrients are supplied at a constant flow rate (and removed at the same rate)

Turbidostat

The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity or cell density

Anaerobic reactors must

Allow access to media without introducing air

Aerobic reactors must

Incorporate a way of mixing air into the media without contaminating the culture

Types of aerobic reactors

Stirred tank

Airlift

Immobilized

Stirred tank bioreactor

Most common

Use an agitator to circulate air into the media

Airlift bioreactor

Bubble air into the media using a sparger or aerator

Advantages of Airlift Bioreactors

Can be used for plant/animal cells

Sterility easier to maintain

Pressure increases oxygen solubility

Large tanks are easier to cool

Bioreactor important components**

Impeller

Baffle

Air sparger

Foam breaker

impeller

Fan like mixer to ensure homogenous conditions

Baffle

Vertical metal strip in bioreactor that disruptions rotational flow, ensuring proper mixing

Filtration

Separate solid particles from a fluid-solid mixture by drawing it through a filter with a vacuum or positive pressure

Filter cake

Concentration of solids in the filter

Filtrate

liquid that has passed through a filter

Non-Newtonian fluids

A fluid which changes its viscosity under a force to become either more liquid or more solid.

Filtration pressure drop

Differential pressure across the filter

Can remain constant - filtration rate will continue to decrease as the filter cake increases resistance

Increase to maintain filtration rate - not common

Filtration rate

Rate at which filtrate is collected on the other side of the filter

Filtration rate depends on

Surface area of filter cloth

Viscosity of fluid

Pressure difference across filter

Resistance due to cloth

Resistance due to filter cake

Ways to improve filtration rate

Increasing the filter area

Increasing the filtration pressure drop (only for reduced compressibility filter cakes)

Reducing the filter cake mass

Reducing viscosity of mixture

Reducing resistance due to filter cake (increase porosity with filter air, reduce specific surface area)

Microfiltration required for

Yeast and bacteria because of their shape and small size

Filter aids

Solid, highly porous particles that improve filtering efficiency by increasing the permeability of the filter cake

Ex. Diatomaceous earth

Ways to apply filters aids

Pre-coat filter before sample is applied to prevent cells from blocking the filter by becoming wedged in pores

Added to fermentation broth before filtration so its distributed through the filter cake as it forms (if extracellular fermentation product)

Cons of filter aids

Increase cost

Minimum quantity required for desired result

Absorbs liquid so some product might be lost

Reduced filtrate clarity

Handling problems when filter cake is contaminated with filter aid

Waste filtrate cannot be used before filter aid is removed

Filter cloth

Different types of fibers layered to create pores that allow liquid to flow through while collecting solid particles

Plate filters

Used for small batch sizes, need to be opened and cleared of filter cake periodically

Rotary-drum vacuum filters

Continuous filters that are used for larger, continuous processes. Most widely used

Vacuum applied on the interior of drum

Filter cake washed, then dried by vacuum

Sedimentation

Separation of cell biomass from fermentation broth

Centrifugation is used to

Remove cells from fermentation broth

To remove cell debris

To collect precipitates

To clarify media before fermentation

How to improve sedimentation rate

Increase speed

Increase particle diameter

Increase density difference between the particles and the liquid

Decrease viscosity of fluid component

How is centrifuge time increased in a continuous flow centrifuge

Slowing down the rate that the sample is fed into the centrifuge

Cons of centrifugation

More expensive

Pros of centrifugation

Can get smaller particles than filtration

Steam-sterilizable centrifuges are used when

Pellet or supernatant is returned to the bioreactor

Contamination must be prevented

Continuous flow centrifuges

Tubular-bowl centrifuge

Disc-stack bowl centrifuge

Tubular-bowl centrifuge

Sample fed continuously through nozzle at the bottom, particles are spun out and collide with walls of bowl, liquid flows out of top, solids removed separately

Disc-stack bowl centrifuge

Sheets of metal discs stacked on top of each other, disks rotate splitting liquid into layers, sample fed into bottom, heavier particles pushed outwards liquid inwards

Mechanical cell disruption

Grinding with abrasives

High speed agitation

High-pressure pumping

Sonication

Non-mechanical cell disruption

Osmotic shock

Freezing and thawing

Enzymatic digestion of cell walls

Treatment with solvents or detergents

Gaulin homogenizer

Cells forced through an adjustable opening at high pressure and then enter an area of low pressure, pressure change causes them to burst

Most labs use ____ for cell disruption

High-pressure homogenization

Chemical processing

Uses chemistry to produce a product

Bioprocessing

Uses biochemistry )living cells and components of cells) to produce a product

Process parameter

Measurable variable that affects the output of a process

Types of process parameters and examples

Physical (time, temperature)

Chemical (pH, enzyme activity)

Biological (cell concentration, optical density)

Process parameter analyzers

Hygrometers (humidity)

Level (liquid levels)

Conductivity (ability to carry electrical current)

PH meter (acidity/alkalinity)

Manometers (pressure)

Refractometer (refraction to determine concentration)

Sight flow indicators (flow and condition of gases/liquids/granular solids)

Spectrophotometers (light wavelengths for composition)

Thermometers

Turbidimeters (clarity)

Viscometers (viscosity)

Coulter counter (cell count and cell volume)

Direct cell growth measurement based on

Cell optical density

Total cell counters

Coulter counter

Cell dry weight

Packed cell volume

Indirect cell growth measurement based on

Cellular components

Measurements of ATP

Bioluminescence

Substrate consumption

Product formation

Oxygen uptake rate

Respiration rate

Heat production

Temperature of bioreactor controlled by

Fluid-filled jacket that surrounds the chamber filled with water or oil that can be heated or cooled to adjust the temperature