BIOREACTORSTHE STATE OF THE ART

PROF.DR/SAID SAAD SOLIMAN

By / Ahmed Shehata Ali Gail

DEFINITIONS 1 -bioreactor is a vessel in which a chemical process is carried out which

involves organisms (mainly microbes-viruses or bacteria, fungi and yeasts –traditionally designated as „fermenters“) or biochemically active substances (enzymes, e.g.) derived from such organisms –in opposite to fermenters frequently considered as „true“ bioreactors. this process can either be aerobic or anaerobic.

2 -an apparatus, such as a large fermentation chamber, for growing organisms such as bacteria or yeast that are used in the biotechnological production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste .

DEFINITIONS a bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. cell culture is the process by which cells are grown under cultivated conditions (animal cells, plant cells, algae).

INTRODUCTION

• the bioreactor´s environmental condition like gas (oxygen, nitrogen, carbon dioxide) and liquid flow rates, temperature, ph, concentration of substrate and products, cells number and their composition (proteins and nucleic acids), dissolved oxygen levels,, and agitation speed (or circulation rate) need to be closely and continuously monitored and controlled. in many cases, strictly aseptic conditions have to be maintained .

INTRODUCTION

• in an aerobic process, optimal oxygen transfer is perhaps the most difficult task to accomplish .

•there are, limits to the speed of agitation, due both to high power consumption and to the damage to organisms caused by excessive tip speed .

CLASSIFICATION

ON the basis of mode of operation 1 -batch

2 -fed batch 3 -continuous

CLASSIFICATION

on the basis of mode of flow of fluids 1 -cstr bioreactor (continuous flow stirred reactor-the content of the bioreactor is

ideally mixed)2 -bioreactor with piston flow and bioreactors with non- ideal flow of fluids

(cascade of ideal mixtures3 -dispersed flow of fluids .)

- the quality of flow of fluids significantly influences the rate of grow of cells and the degree of conversion of substrate

CLASSIFICATION

On the basis of a number of phases treated1 -Homogeneous bioreactors (e.G. One phase tubular bioreactor with enzyme

diluted in the liquid substrate) ( e.G. Two phase solid-liquid bioreactor like the column type bioreactor2-

heterogeneous bioreactors with immobilized enzyme and liquid substrate and/or three phase bioreactors with submersed culture: gas (air bubbles)-liquid (substrate)-solids (cells) .

SUMMARY OF BIOREACTOR SYSTEMS

Products Cell Systems used Bioreactor product design

SCP, Enzymes, Secondary metabolites, Surfactants

Bacteria, Yeast and other fungi

Air-Lift Bioreactor

Ethanol, Secondary , metabolites, Wastewater treatment

Immobilized bacteria, yeast and other fungi, Activated

sludge

Fluidized-Bed Bioreactor

Interferons, Growth factors,Blood factors, Monoclonalantibodies, Vaccines, Proteases, Hormones

Immobilized (anchored) mammalian cells on solid particles

Microcarrier Bioreactor

SUMMARY OF BIOREACTOR SYSTEMSProducts Cell Systems used Bioreactor product

design

Interferons, Growth factors,Blood factors , Monoclonal antibodies, Vaccines, Proteases, Hormones

mammalian, tissue growth on solid surface, tissue engineering

Surface Tissue Propagator

Ethanol, Monoclonal anti-bodies, Interferons, Growth factors

Bacteria, Yeasts, Mammalian cells, Plant cells

Membrane Bioreactors, Hollow fibers and membranes used, products Rotorfermentor

Ethanol, Monoclonal anti-bodies, Interferons, Growth factors

Immobilized Bacteria, Yeast, Plant cells

Modified Stirred Tank Bioreactor

SUMMARY OF BIOREACTOR SYSTEMS

Products Cell Systems used Bioreactor product design

Ethanol, Enzymes, Medicinal products

Immobilized Bacteria, Yeasts and other fungi

Modified Packed Bed Bioreactor

Single Cell Protein (SCP) Bacteria, Yeasts Tower and Loop Bioreactors

Ethanol, Volatile products

Bacteria, Yeasts, Fungi Vacuum Bioreactors

SUMMARY OF BIOREACTOR SYSTEMS

Products Bioreactor product design

Commodity products,SCP Bacteria, Yeasts, Fungi Cyclone Bioreactors

SCP, Algae, Medicinal plant products Monoclonal antibodies,Vaccines, Interferons

Photosynthetic bacteria Algae, Cyanobacteria, Plant Cell culture, r-DNA plant cells

Photochemical Bioreactors

Cell Systems used

BASIC BIOREACTOR DESIGN CRITERIA

1 -Microbiological and biochemical characteristics of the cell system (microbial, mammalian, plant)

2 -Hydrodynamic characteristics of the bioreactor3 -Aeration and oxygen, mass and heat transfer characteristics of the

bioreactor4 -Kinetics of the cell growth and product formation5 -Genetic stability characteristics of the cell system

BASIC BIOREACTOR DESIGN CRITERIA

6 -Aseptic equipment design7 -Control of bioreactor environment (both macro-and micro-

environment)8 -Implications of bioreactor design on downstream products

separation9 -Capital and operating costs of the bioreactor

10 -Potential for bioreactor scale-up

BIOREACTOR MAIN DESIGNS

1 -Stirred tank 2 -air lift reactors3 -bubble column

4 -packed bed reactors5 -trickle bed reactors

6 -fluidized bed reactor

STIRRED TANK REACTORClick icon to add picture

mixing method: mechanical agitationhigh input requiredbaffles are constructed within the built-in.applications include production of antibiotics and free/immobilized enzymesdraw back is that high shear forces may break the cells

AIR LIFT REACTORS Click icon to add picture

Mixing method: airlift

Central draft tubeUp-flowing stream and down flowing streamHomogenization of all components presentApplications include bacterial, animal, plant, fungi and yeast cells.

BUBBLE COLUMN REACTOR

Click icon to add picture

Mixing method: gas spargingSimple designGood heat and mass transfer ratesLow energy inputGas-liquid mass transfer coefficients depend largely on bubble diameter and gas hold-up

PACKED BED REACTORClick icon to add picture

Column with attached biofilmBiocatalystsPump is required to make fluid move through the packed bedApplications include waste water treatment

FLUIDIZED BED REACTOR

Click icon to add picture

When the packed beds are operated in up-flow mode, the bed expands at high liquid flow rates due to upward motion of the particles .

Energy is requiredWaste water treatment

TRICKLE BED REACTORSClick icon to add picture

Liquid is sprayed onto the top of the packing and trickles down through the bed in small rivulets.In the process, the gaseous pollutants on the surface of the carriers is adsorbed and immediately biologically mineralized (degraded) by the microorganisms.

Introduction -A bioreactor is a device or vessels which are designed to obtain an effective

environment for conversion of one material into some product by appropriate biochemical reactions

-conversion is carried out by …… enzymes, microorganisms, cells of animals and plants, or sub cellular structures such as chloroplasts and mitochondria .

-Plants can be used as cheap chemical factories that require only water, minerals, sun light and carbon dioxide to produce thousands of chemical molecules with different structures .

Design gene for high level

expression

Plant transformation

Regeneration of Cell

Selection of transgenic

Growth of plants in field

Harvesting of plant materials

Purification of product

Biosafety & Functionality test

Where they are produced?

Shown are various intracellular organelles or extracellular spaces (ES) that can be used to store the recombinant proteins expressed in a plant bioreactor.

G - Golgi; PSV - Protein storage vacuole; OB - Oil body;C - Chloroplast; ES - Extracellular space; PVC - Prevacuolar compartment.

TYPES OF PLANT REACTORS

-seed-based plant bioreactors -plant suspension cultures

-hairy root system bioreactor -chloroplast bioreactor

SEED-BASED PLANT BIOREACTORS

An example is the successful expression of the human lysosomal enzyme alpha-l-iduronidase in arabidopsis thaliana seeds.

The advantage of these systems is that, proteins do not degrade at ambient temperature and are stable for long term storage .

Plant Suspension Cultures

express recombinant proteins, secondary metabolites and antibodies transported to subcellular organelles. for example, is the expression of 80-kda human lysosomal protein

Hairy Root System Bioreactor

it offers extreme biosynthetic stability and is suitable for making biopharmaceuticals as for example scopolamine in hyoscyamus muticus l. hairy root culture.

CHLOROPLAST BIOREACTOR

-insulin, interferon and other biopharmaceutical proteins can be made using chloroplast bioreactor .

-foreign genes are inserted into nuclear chromosomes and with peptides target expressed proteins into chloroplast .

-an example is the high yield in the expression of human serum albumin protein in chloroplast.

PLANTS GENETICALLY ENGINEERED TO MAKE PRODUCTS THAT ARE NOT OF PLANT ORIGIN

PRODUCTS: vaccines antigens

therapeutics products nutritional components

industrial products bio plastics

Vaccine antigens:antigens like insulin, rotavirus enterotoxin, anthrax lethal factor, hiv antigen, foot and mouth disease virus antigen, heat stable toxin have been produced in plants.

Therapeutic products:the first successful production of a functional antibody, namely a mouse immunoglobulin iggi in plants, was reported in 1989.

In 1992, C.J. Amtzen and co-workers expressed hepatitis B surface antigen in tobacco to produce immunologically active ingredients via genetic engineering of plants

Nutritional components:β-carotene (naqvi et

al., 2009), lycopene (fraser et

al., 2002),flavonoid (butelli et

al., 2008), nutraceuticals (kang et

al., 2009), fatty acid (hoffmann et

al., 2008), vitamins (nunes et

al., 2009), minerals (lee & an,

2009) & carbohydrates (Regina et

al., 2006).

Biodegradable plastics: Polyhydroxyalkanoates: biodegradable polymers which occur

naturally in plants.Plant was engineered to produce PHAs or PHBs in the various plant cell compartments.

When PHB expression targeted to cytoplasm, accumulation level was low.

Expression was increased by targeting plastids, (40% of dry weight was obtained).

• Industrial products:

Most expensive Drug – HgchST (Human somatotropin)

rHLF (Recombinant human lactoferrin)

Synthetic fiber: Produced from Potato and tobacco.

ADVANTAGES AND DISADVANTAGES

Low cost source. Simple & Cost effective. Plant pathogens do not infect humans or animals. Produce large biomass. Easy storage for long time.

Plant proteins have different sugar residues from human or animal proteins.

Problems need to be addressed

-Storage issues related to transgenic fruits or leaves. -Most inserted genes are expressed at very low level in plants.

-Enhancing the stability of products obtained. -Standardization of dosage in case of edible vaccine.

-Examining issues related to commercialization. -Issues relating to the ethical, social, biosafety and environmental

impact. -Some plants produce allergenic compounds.

Photobioreactors

-micro-algae are source of unique metabolites that can be used to produce novel high-added value bioactive compounds with industrial potential in medical technologies or as food, feed or cosmetic ingredients or as potential source of biofuels. - among these substances play important role polyunsaturated fatty acids (pufas). production of novel pufas by micro algae is highly challenging as current production processes from fish oil threatens natural marine organism’s populations.

algae can accumulate large amounts of polysaccharides, lipids and proteins with potential as nutrients/energy or biofuel source

Photobioreactors - experimental

Algae does not need expensive reactors to be cultivated

Photobioreactors – plastic bags

Photobioreactors - experimental

PHOTOBIOREACTORS

Advantages - controlled, optimized conditions - contamination can be minimized

- high rates of production Disadvantages

- expensive

Flat panel photobioreactors

Tubular photobioreactors

Open ponds

Czech“ way Open inclined Thin Layer Flat Plate Photobioreactor with high rates of algae suspension on the top of the plates. Harvest concentration 10-30 g/l.