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The basic steps for bioreactor design came from fermentation. As we know that products like bread, beer, wine and cheese are made by fermentation. These products are obtained by the bioreactions of microorganisms during fermentation. The famous microbiologist and French chemist Louis Pasteur studied details of fermentation process which is operated by the microscopic plant called yeast. Louis Pasteur’s work laid the foundation for today’s bioreactors. Thus the scope of bioengineering has grown from simple wine bottle microbiology to industrialization of fermented products like beer, wine, bread and cheese also along with biotechnology products like antibodies, steroid hormones, enzymes, sugars, vitamins and organic acids2.
Products of Bioreaction:
Products of bioreaction are formed by three processes such as:
Process to produce product from cells either by extracellular or intracellular
Process that produces a cell mass
Process by biotransformation
Process to produce Product from cells either by extracellular or intracellular:
In this process cells produce extracellular products like alcohols or citric acid or intracellular products like metabolite or enzyme. There are two types of cellular products produced by cells during their biological cycle. They are primary metabolites and secondary metabolites. Primary metabolites like amino acids, nucleic acids, nucleotides, proteins, lipids and carbohydrates are produced during the growth of cells and are important for the continuous growth of cells. Secondary metabolites are produced after growth of cells stops. Some of the examples of primary products used for industry are citric acid, acetone, butanol, lysine, vitamins and polysaccharides.
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Secondary metabolites are obtained from products of primary metabolism or from intermediate stage of primary metabolism. All microorganisms don’t produce secondary metabolites but majorly produced by fungi and plants. Secondary metabolites have toxic nature or have antibiotic properties. Industrial enzymes are used in brewing, baking, grain processing, dairy making and in the production of detergents, wines and other products2.
Processes that produce a cell mass :
Baker’s yeast is an example of produced cell mass, used in baking industry. Other example of produced cell mass includes single cell proteins, which is used as food source2.
Processes by biotransformation:
Biotransformation is defined as the “Process that modifies a compound that is added to the fermentation process”. Processes of biotransformation occur by inherent enzymatic ability of most cells. All types of cells are used to biocatalyze transformation of certain compounds by oxidation, dehydration, hydroxylation, ammination or isomerization. Generally enzymatic conversion requires lower activation energy and higher selectivity than their chemical counter parts. Products like steroids, antibiotics and prostaglandins are produced through biotransformation process.2
What is a Bioreactor?
Bioreactor is a cylindrical vessel of various sizes from liters to cube meters. These reactors are used to carry out chemical process of organisms, often these reactors are made of stainless steel. Designing a bioreactor is complex engineering challenge. Environmental conditions in the reactor vessel play a major role in the reproduction and growth of microbial cells7.
Basic design of a Laboratory Bioreactor:
The basic design of a laboratory bioreactor consists of various parts like Upper lid, Lid (lower), Baffles, Mixer, Spargers, Bioreactor vessel, condenser outlet valve & filter, and sampling.
Upper lid of bioreactor has the following parts:
Ports for electrodes (pH, oxygen, temperature and pressure)
Ports for the supply of titration and feeding medium
Pipes for sampling and chemostat
Ports for the connection of the outlet air and condenser and filter
Drive sealing (if the mixer’s upper drive is used)
All connection on lid port should ensure the air tightness and leak poorfness, sterility, convenient removal and installation of sensor and other elements8.
Lower cover of a bioreactor has the following ports and elements:
Mixer’s lower drive
Connections of all these ports secure air tightness and leak poorfness and prevent favorable conditions for infective agent’s reproduction8.
Mixers are placed on the mixer axis in bioreactor; location of mixer axis depends on whether the bioreactor has the upper or lower drive. For mechanically sensitive microorganisms mixing is recommended because axial flows cause even distribution of microorganisms8.
These are vertical plates located radially in a bioreactor. Baffles prevent funnel formation by rotational speed of mixer8.
Spargers are used to supply compressed air. Loop pipe with small holes in the lower part are used to supply air. These types of loop spargers with conical air outlet channel are used for mycelia culture8.
Generally steel or metal or combinations of these two are used in making bioreactor. Bioreactor vessel material plays a major role in the inhibition of microorganism growth along with other parts of the reactor like sensors, pipes etc present inside the reactor. Inner surface of the bioreactor is polished like a mirror surface for easy washing and sterilization process8.
Condenser outlet valve and filter:
Condenser is used to cool the liquid at the outlet for the separation of foam and other liquid particles. Condenser outlet is connected with the outer valve; this outer valve is used to regulate the necessary overpressure in the bioreactor vessel. The valve outlet is connected to a porous microbiological filter for filtration8.
For sampling, valve at the inlet of the bioreactor is used. Fermentation broths are introduced into the reactor as much as required and close the tap. To avoid infection on site, sample is sterilized by alcohol or steam.8
The purpose of bioreactors is to control and positively influence the biological reactions. So to obtain better results of bioreactors, chemical engineers must have to consider two areas.
Selecting suitable reactor parameters for biological, chemical and physical (macrokinetics) system. Physical system includes microbial growth and metabolite production. Bacteria, yeast, fungi and animals, plant, fish and insect cells are included in microbes.
Other important area of designing a bioreactor involves bioreactor parameters such as:
Sufficient substrates of carbon sources like sugar, proteins and fats
Salts for nutrition
Oxygen (for aerobic processes) and
Product and byproduct removal
Along with all these parameters it’s also important to design bioreactors to promote the growth of required organisms and to eliminate the unwanted organisms from contamination or mutation of the organism. Designing a bioreactor to maintain controlled environmental conditions for biological activity and eliminating unwanted activities presents a greater challenge for engineers2.
Types of Bioreactors:
Bioreactors are used to carry out chemical process of organisms. Various types of bioreactors are categorized as below:
Basic types of bioreactors
Stirred tank bioreactor
Plug flow bioreactor
Bioreactors based on mode of operation
Fed batch bioreactor
Continuous stirred tank bioreactor
Cell based bioreactors
Membrane stirred tank bioreactor
Spin filter stirred tank bioreactor
Fluidized bed bioreactor
Hollow fiber bioreactor
Multiple membrane plate bioreactor
Ceramic system bioreactor
Basic types of bioreactors:
Like chemical reactors bioreactors are also classified based on their mixing properties.
Stirred tank bioreactor:
This reactor is also known as well mixed stirred tank reactor. In an ideal stirred tank reactor mixing takes place without forming clumps and there is no flow bypass from the reactor. When the substrate is added from the inlet, substrate is distributed throughout the reactor and makes homogenous mixture. For aerobic reactions gas is introduced by spargers and gas is distributed throughout the reactor by agitators. In these reactors the medium is homogenous throughout the reaction1.
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Plug Flow bioreactor:
In plug flow reactor the liquid moves with the same velocity throughout the reactor. Nutrient concentration decreases from inlet to outlet but concentration of metabolites increases. Due to decreasing of nutrients and increasing of metabolites the length of plug flow reactor is limited. In these types of reactors the medium is heterogeneous throughout the reaction1.
Operating mode of Bioreactors:
The first step in batch bioreactor is the sterilization of the reactor, followed by the inoculation of sterile culture media with microorganisms for better results. For aerobic cultures oxygen is introduced and byproduct CO2 is removed simultaneously. For controlling pH acid or alkali are added depending on the requirement and antifoaming agents are added to maintain the foam level based on the foam sensor indications2.
Due to relatively brief growth in batch reactor there is less risk of contamination or cell mutation.
Batch bioreactor is more flexible for variable products or biological system.
Batch bioreactor requires less capital investment compared to continuous process of same volume2.
Lower productivity levels of product because lot of time takes for filling, heating, sterilization, cooling, emptying and cleaning the reactor.
Products obtained are having minimal risk of contamination or organism mutation.
Only small amount of product is produced from the process.
Due to frequent sterilization, focus on instrument needed.
Large industrial hygiene risks due to potential contact with pathogenic microorganisms or toxins2.
Fed batch bioreactor:
Fed batch bioreactors are majorly used in multipurpose and multi product facilities because of flexibility, scalability and simplicity. In fed batch cultures cells with lower viable cell density are inoculated into the medium and cells are allowed to grow without altering the external manipulations. When cells reach exponential growth phase by using the medium, at this stage some portion of cells and medium are drawn for intermittent fed batch reactors then it is replaced by new medium and cells. This process is repeated by several times1.
Continuous Stirred tank reactor (CSTR):
In continuous stirred tank bioreactors the medium is inoculated with or without microorganisms (sterile medium) and product also drawn from the reactor to maintain steady state. In continuous stirred tank reactors control parameters and reaction variables are consistent by establishing a time constant state within the reactor. CSTR requires investigation and analysis of the system so that minor changes of physical or chemical changes can be evaluated2.
Consistent product quality due to invariable operating parameters.
This bioreactor can be operated automatically which can decrease the labor expenses and toxicity to them.
For emptying, filling and sterilizing the reactor requires less non-productive time2.
For controlling and automation of equipment takes high investment and for sterilization of the media the cost increase further.
Risk of contamination and cell mutation is more because of brief cultivation period.
Continuous replacement of non soluble, solid substrate like straw requires more processing cost2.
Cell Culture Bioreactor :
Various types of bioreactors are in use for culturing animal cell for research.
In air-lift reactors the liquid is circulated by introducing air from the bottom of the column by spargers. Density of sparged section is less compared to the bubble-free section, this difference in hydrostatic pressure causes liquid to move upwards and down words for better mixing. Design of air-lift bioreactors is simple and there is no mechanical agitation, so creates low shear force on cells. Airlift reactors with various size spargers are used depending on the bubble size1.
Air-lift reactors have simple design with no moving parts which requires less maintenance with less risk of defects and easy for sterilization.
Enhanced oxygen solubility achieved in large tanks with more pressure by increasing mass transfer.
Due to less shear force these reactors are used to grow both plant and animal cells.
Air-lift bioreactors have well controlled flow and efficacy mixing.2
An air-lift reactor requires high capital investment due to large scale process.
It’s impossible to maintain constant levels of nutrients, substrate and oxygen with the organisms circulating in the reactor and changing conditions.
It’s not possible to separate gas-liquid phases when foaming occurs.
This reactor requires high air throughput and higher pressure for large scale operation.2
Membrane stirred tank bioreactor:
In membrane stirred tank reactors long micro porous membrane made up of polypropylene covers the rotating rod. These micro pores expand by adjusting pressure in propylene tube which makes direct contact with medium. Gas pressure is altered to increase the pore size of membranes while not bursting to become spargers. Gentle agitation of micro carriers or suspended solids is obtained by rotation of tubing and foaming is also avoided at high serum concentration1.
Spin filter stirred tank bioreactor:
Spin filter stirred tank bioreactor contains a central piece of wire cage which is fixed on the agitation shaft. Sometime in other type of reactors this cage is fixed on shaft but rotated by motor on the top. Bottom of the wire cage is solid and cage sides are made up of wire with opening ranging from 25µm- 60 µm. Generally cell size ranges from 10µm-15µm. New medium is added from the inlet and culture medium is drawn from the inside of the cage simultaneously. Optimal range of agitation causes cell retention in spin filter stirred tank bioreactors1.
Vibromixer contains perforated disc which moves in vertical direction at high speed for mixing liquid throughout the perforated holes for better mixing. Using Vibromixer for suspension cell cultures is old. Sometimes it’s used for isolate cells attached to micro-carrier by suspending in suspension cultures for isolating cells in trypsinization step1.
Fluidized bed bioreactors:
This reactor is also known as expanded bed reactor or a circulating bed reactor. Particles used in this reactor are known as biofilm carriers. This reactor depends on the attachment of microorganisms to particles which are maintained in suspension by a high upward flow rate of the fluid to be treated. The carriers used in this reactor are sand grains, granular activated carbon, and diatomaceous earth or other solids that are resistant to abrasion. Carriers with higher amount of biomass are lighter and move higher in the reactor by upward velocity of fluid3.
Membranes are defined as engineered semi permeable barriers that allow only some materials to pass through it (e.g. water) not other materials (e.g. suspended solids). Materials pass through the membrane based on the permeability of membrane. Several factors influence in classifying membrane bioreactors such as 5
Pore features like size, orientation, density and surface charge
Primary separation mechanism (sieving, diffusion)
Driving force (pressure, osmosis)
Material of construction (organic, inorganic)
Various types of membrane bioreactors are:
Hollow fiber bioreactor:
These types of bioreactors contain a bundle of hollow fibers sealed inside a cylindrical tube know as cartridge. These bioreactors are used in biomedical research to produce more cells or cellular products. Fibers of hollow fiber bioreactor are made up of porous material which allows the medium to go through it but not cells and other high molecular weight compounds. Cells are seized in extracellular space between exterior of the fibers and cartridge body and fresh medium recalculates in the extracellular space to supply nutrients for cells growth4.
Multiple membrane plate bioreactor:
These reactors are used to avoid scaling problems of the reactor and medium flow distribution problems of cartridge bioreactors. In this reactor, various fibers are used, one for transferring oxygen and other for medium distribution etc. Manufacturing multiple membrane bioreactors suffers from practical problems in manufacturing a sealed reactor for long term aseptic conditions1.
Ceramic system bioreactor:
This reactor consists of a cylindrical ceramic core with many channels passing through the longitudinal section of the core. Cells are introduced through one of the channel and medium from another channel. Introduce cells may trap into the ceramic pores or adhere to the material. Fresh medium is circulated through the channel and harvested medium is passed into the medium reservoir. In these reactors product is directly introduced into the medium1.
Applications of Bioreactors:
Batch bioreactors are used in vaccine industry and in pre production scales of rDNA protein production.
Fed batch bioreactors are used in the production of recombinant proteins and antibodies.
Stirred tank bioreactors are widely used in culturing suspension cells. By using microcarriers or macroporous as a support for adherent cells which can also be grown in suspension cultures.
Airlift bioreactors are successfully used in culturing suspension cells of BHK21, human lyphoblastoid, CHO, hybridomas and insect cells.
Spin filter stirred tank bioreactors are used in aggregate or microcarrier cultures.
Vibromixers are used in 1960’s for the cultivation of suspension cells and virus production.
Fluidized bed bioreactors are used in chemical catalysis and also in adsorption column in bioseperation.
Hollow fiber bioreactors are used to study various metabolisms in research and to cultivate anchorage dependent or highly aggregated cells.
Anaerobic bioreactors are used in ethanol production, wine making beer brewing and waste water treatment1.
Combinations of biological reactors with membrane technology are used in the treatment of municipal and industrial waste6.
Bioreactors play a major role in producing various types of industrial products by aerobic or anaerobic processes. As though design and operation of a bioreactor requires more investment we are benefitted by the reactors. Bioreactors are integrated unit operation with both upstream and downstream unit operation. In future we may hope to see more modernized bioreactor with minimal contamination of products by technology advancement.
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