15 Types of Reactors

Babita Khosla

16.1 Introduction:

The bioreactors are in use form the last thousand years in one way or another, although till nineteenth century their use was limited only to the production of alcohol. The first truly large scale aseptic reactor vessel was developed by Chaim Weizmann in Great Britain during World War I (191401918) for the anaerobic process of production of acetone using Clostridium acetobutylicum. The first large scale aerobic reactor vessel was used in Central Europe in 1930’s for the production of compressed yeast. It was only after 1940s, that fermentation as it is known today began to appear with the need to produce organic acids, enzymes, acetone, butyl alcohol and antibiotics.

The exact configuration of a bioreactor is usually process driven and can be classified into three modes:

1. Batch
2. Fed-batch
3. Continuous

Figure1: Various modes of operation of Reactors

16.2 Batch mode: In the batch mode of fermentation, which is a closed system all of the nutrients required for the organism’s growth and product formation are added initially before the start of the fermentation process in the vessel. The vessel can be a shake flask, single use disposable system at the laboratory scale and big stainless steel vessels at the industrial scale. The parameters such as rate of oxygen transfer, pH, temperature, rate of agitation etc. are controlled throughout the whole process. After the sterilisation of medium, the organism is inoculated into the vessel and allowed to grow in controlled set of conditions. The only material  allowed to be added and removed during the course of batch fermentation is the gases and pH control solutions and they too are made sterile before addition. In this mode of operation, conditions are continuously changing with time, and the fermenter is always in an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time. The batch is harvested when one or more of the following conditions have been achieved:

• (i) The microbial growth has stopped due to the depletion of the nutrients or the build of toxic/inhibitory metabolites.
• (ii) After a fixed period of time which is predetermined as per the pilot studies.
• (iii) The concentration of desired product has been achieved or the concentration of the reactants has decreased to minimum.

16.2.1 The Growth Curve in Batch Culture

When the microbial cells are grown in a batch culture, they will typically follow the growth curve as shown below (Figure 2) and proceed through four distinct phases namely lag phase, log phase, stationary phase and death/decline phase.

Figure2: A typical growth curve of microbes showing four distinct phases in batch mode.

Lag Phase: When microorganisms are introduced into fresh culture medium, usually there is ‘little or no growth” takes place at the beginning of the fermentation. This period is called the lag phase. The duration of the lag phase is variable depending upon the age of the culture and the environment following inoculation. The lag phase can be time consuming and costly in the industry and so it is highly desirable to minimise this phase for economic feasibility. This can be achieved by growing the inoculum in same medium and under similar growth conditions (pH, temperature, aeration and agitation rate etc.) as is present in the production tank. Also, a minimum of 5% volume by volume inoculum of cells at exponential phase should be used to minimise the lag phase.

Exponential Phase: During the exponential (log) phase, microorganisms are growing and doubling in number at regular intervals and the growth rate is constant during this phase. The population is more or less uniform so, the cultures in the exponential phase are usually used in biochemical and physiological studies.

Stationary Phase: In batch culture, which is a closed system the population growth reduces eventually. This phase of the curve is a horizontal line showing the total number of living cells constant. This condition may result when the rate at which cells are dividing is equal to rate of cell death (apoptosis), or the population may simply stop dividing but remain metabolically active.

Microbial populations enter the stationary phase for several reasons like nutrient limitation in the growth medium and the formation of metabolites, which act as inhibitors (ethanol, lactic acid, acetic acid, methanol, and aromatic compounds) and decelerate cell growth.

Death Phase: The decline in living cell counts following the stationary phase is described as the “death phase.” The changes in the environment such as limiting concentration of nutrients and the build-up of toxic wastes are supposed to be responsible for loss of cell viability. When the cells from this stage are transferred to fresh medium, no cellular growth was observed.

16.2.2 Advantages of Batch Culture

A batch culture is easy to operate and is less likely to have failure because of short runs. The production of secondary metabolites during the stationary phase of growth curve takes place in the batch culture.

There are fewer possibilities of contamination as all of the materials required for the process are present in the vessel and sterilised before the start of the run.

It is easy to assign a unique batch number to each run, which is critically important in a food and pharmaceutical industries.

16.2.3 Disadvantages of Batch Culture

The culture ageing can be a specific problem in batch mode cultivation.

The build-up of toxic metabolites during the growth phase can inhibit cell growth and product formation as is the case of alcohol production.

The initial concentration of substrate has to be limited if the high concentration is having inhibition and repression effects. For example high concentration of sugars can inhibit the growth of yeast cells in alcoholic fermentations. So, it is not desirable to add the whole excess of the substrate at the start of the run but is repeatedly fed into the vessel called Fed-batch mode of culture.

The release of autolytic breakdown products during the decline phase affects the amount of product, its composition and potentially adding to downstream processing challenges.

The biggest disadvantage of batch mode is batch-to-batch variability in the product. The use of batch cultures in industrial systems can lead to an increased non-productive time period due to cleaning, re-sterilisation, filling and cooling of equipment between the first run and start of the next run.

The usage of the organism from one batch to seed another batch may cause degeneration or differentiation, which could affect the product formation. The use of batch cultures actually contributes to the complexity of the experiments since the cell population is heterogeneous and constantly changing.

16.3 Continuous Mode: Continuous culture mode of operation is a technique in which the exponential phase of an organism is prolonged by minimising the fluctuations in the concentration of nutrients, cell number or biomass during the process. This is also known as steady state. The fresh nutrients are added to the process and at the same time, spent medium plus cells from the system are removed. As a result, microbial cells continuously receive fresh medium and products and waste products and cells are continuously removed. This ensures that several factors like culture volume, biomass or cell number, product and substrate concentrations, as well as the physical parameters of the system such as pH, temperature and dissolved oxygen remain constant throughout the process for extended periods. The reactor can thus be operated for long periods of time without having to be shut down.

Continuous culture systems having low nutrient levels are mimicking the conditions present in natural environments and make possible the study of microbial growth in a more realistic way. The continuous culture techniques have been widely used in waste water treatment plants where continuously the sewage/ industrial effluents are fed and treated water is discharged. The major types of continuous culture systems commonly used are chemostats, turbidostats and auxostats.

16.3.1 Chemostat culture: The arrangement in which “the system depends upon the fact that the concentration of an essential nutrient (substrate) within the culture vessel will control the growth rate of the cells”. The concentration of substrate within the culture vessel is in turn controlled by the dilution rate. Any essential nutrient for growth of the cells can be used to control the size of cell population in the vessel, making chemostat a flexible tool to study cellular behaviour under various nutrient limiting conditions. The metabolism operates in a steady state.

The growth rate-limiting substrate added at a constant flow rate (f) in the inflowing medium and the total volume (V) of the culture is kept constant by overflow of effluent through a side port. Because one nutrient is limiting, growth rate is determined by the rate at which new medium is fed into the growth chamber; the final cell density depends on the concentration of the limiting nutrient. The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml).

D= f/V

The inverse of dilution rate is Residence time (t) and is measured in hours.

t= V/f

The continuous culture must go through four or five residence times before it can be considered to be in a steady state. A stirred tank bioreactor with moderate concentrations of cells present is assumed to be perfectly mixed and this assumption is used in the derivation of the continuous culture equations. Therefore the following equations are assumed throughout a chemostat when at steady state:

dX/dt= 0 and

dS/dt= 0

i.e. change in biomass (X) over time (t) is zero, and change in substrate concentration (S) over time (t) is zero, that is, no net accumulation of cell mass or substrate. The specific growth rate of a culture at steady state is set by the dilution rate (i.e., µ = D), which is in turn determined by the rate of flow of nutrient solution to the culture. Both population size and generation time are related to the dilution rate, and population density remains unchanged over a wide range of dilution rates. The generation time decreases (i.e., the rate of growth increases) as the dilution rate increases. The limiting nutrient will be almost completely depleted under these balanced conditions. If the dilution rate rises too high, microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate. This occurs because fewer microorganisms are present to consume the limiting nutrient.

Most chemostat cultures become progressively more unstable as the dilution rate approaches the critical dilution rate (above which washout occurs).

Figure 3: Chemostat (Left) and Turbidostat (Right) type of Continuous culture system Image: http://www.biologydiscussion.com/bacteria/growth-of-bacteria/

16.3.2 Turbidostat culture: The second type of continuous culture system, the turbidostat measures the turbidity of the culture medium (absorbance/ optical density) in the growth vessel. The cell concentration in the vessel is maintained constant by monitoring the optical density of the culture using the photocell. By modulating the medium feed rate a set point optical density is maintained throughout the process. When the optical density rises above the set value, the feed rate is increased and the volume is maintained constant by an overflow device. The well mixed culture is diluted and the optical density approaches the set value. On the opposite side if the optical density decreases from the set value, the feed rate of medium is decreased to give sufficient time to the cells to build upo their number to set value. The metabolic state of the cells operates in a pseudo-steady-state.

The main utility of turbidostat is in controlling the growth rate near the maximum growth rate, an operating region in which chemostat is less stable. Although. the turbidostat is less used because of difficulties in continuously monitoring of cell concentration.

16.3.3 Auxostat: Auxostats use medium feed rate to control the variable, such as pH or dissolved oxygen, in continuous culture. The microorganisms themselves establish their own dilution rate as per the variable parameter. The auxostats tend to be much more stable than chemostats at higher dilution rates, especially at dilution rates approaching the maximum specific growth rate of the microorganism. The high dilution rates exert a selection pressure upon the microbial population, which leads to more rapidly growing cultures. This method of continuous culture control is therefore ideal for such applications as high-rate propagation and detoxification of waste materials at maximum rate concentrations.

16.3.4 Advantages of Continuous Culture:

Uses small reactors than batch mode to produce the same amount of product as well as the equipment required for all process can be at smaller scale.

The productivity and growth rate can be optimised by changing the nutrient feed rate and the longer periods of productivity with less down time (time duration between batch runs).

The immobilised cells/catalyst can be used in continuous mode, where the high cell concentrations can be maintenaned in the bioreactor even at low substrate concentrations.

The physiological state of the cells is uniform and effects of environmental factors are more easily analysed in a continuous system, where any change observed in the constant steady state can be attributed solely to the change in the factor.

The response of culture in response to the selective pressure in terms of physiological, metabolic or genetic change can be readily studied in continuous mode. Basically, continuous culture of a given strain allows us to ‘direct the evolution’ of the strain.

16.3.5 Disadvantages of Continuous Culture:

Culture mutation can easily occur in continuous process duration, which may vary between 500-1000 hours. Hence, some of the microorganism may lose their recombinant plasmid contruct often termed as ‘shedding’ genes required for product formation, or in the case of genetically engineered organisms, a ‘back mutation’ can occur. For example, Clostridium acetobutylicum loses its ability to make acetone butanol in continous culture and instead makes acetate and butyrate.

All products are not produced optimally in continuous processes, e.g. some fermented foods and beverages require cellular products released from different phases of batch culture growth for full flavour development.

Non-growth-associated products such as antibiotics, monoclonal antibodies and toxins are not produced well in continuous culture.

The US Food and Drug Administration (FDA) does not accept continuous culture in the production of therapeutic products as a Current Good Manufacturing Practice (cGMP).

The maintenance of sterile conditions on an industrial scale for longer periods is difficult. The contamination at any point can result in the wash out of the desired organism and therefore a loss of product.

16.4 Design of Stirred type reactor:

Continuous stirred type reactor (CSTR) is defined as well mixed model with continuous addition and removal of material and energy, no spatial variations in temperature, concentration, fluid properties and reaction rates. The properties of the exit stream may be considered same as throughout the vessel. Although, the ideal conditions are never attained, the vessel is designed to provide good mixing through selection of operating conditions and geometries of vessel, baffle and impeller. This stirred type reactors consist of a cylindrical vessel made up of stainless steel or glass with an aspect ratio (height to diameter ratio) of around 3-5. The mixing system in the vessel is a motor driven central shaft that supports one or more agitators. The agitator speed generally does not exceed 120 rpm for microbial cells. The shaft may enter from the top or bottom of the reactor vessel. The head plate will have ports that allow for the addition of probes, reagents and gas as well as the removal of samples. The microbial culture vessels are generally provided with baffles (4 baffles along the side walls of the vessel) to prevent vertex formation in the fluid during agitation. The width of the baffle strips is usually 1/10 to 1/12 of the vessel internal diameter. However, vessels designed for culturing animal cell lines are devoid of baffles to reduce turbulence which may damage the cell. At the bottom of the vessel below the agitator blade, a perforated ring or a single hole sparger is introduced to sparge the reactor liquid with gas, whereas the impellers mounted on the shaft disperse the gas/air bubbles. The temperature of the reactor is maintained either through vessel jacketing or internal cooling coils provided for the means of heat transfer.

16.5 Packed Bed Reactor: Packed bed reactors, also known as fixed bed reactors, are often used with particulate or immobilised catalysts/cells. The packed bed reactor is a vertical cylindrical column packed with pieces of some relatively inert material, e.g. wood shavings, twigs, coke, an aggregate or polythene.   have proved to be useful for long term culture of attached dependant cells. Initially both medium and cells are fed into the top of the packed bed. Once the cells have adhered to the support and are growing well as a thin film, fresh medium is added at the top of the column and the fermented medium is removed from the bottom of the column. Upon entering the reactor the reactants flow through the packed bed of catalyst. By contacting with the catalyst pellets, the reactants react to form products, which then exit the reactor on the bottom. When designing a packed bed reactor one must take into account the active life of the catalyst. This will affect the length of time a bed of catalyst may be used and thus how long the reactor may be run before the catalyst needs to be regenerated.

The best known example is the vinegar generator, in which ethanol was oxidized to acetic acid by strains of Acetobacter aceti supported on beech shavings.More recently, packed towers have been used for sewage and effluent treatment. In treatment of gas liquor, a column was packed to a height 7.9 m with ‘Dowpac’, a polystyrene derivative. The main advantages compared with other methods of effluent treatment being its simplicity of operation and a saving in land because of the increased surface areas within the column.

Figure 5: Packed bed Reactor Image: http://www.essentialchemicalindustry.org/processes/chemical-reactors.html

16.6 Fluidized bed reactors: The term “fluidised bed” is used to define those physical systems composed of a solid phase in the form of individual particles that move within a liquid phase and not in continuous contact with each other. Fluidization of solid particles is reached when the flow of fluid through the bed is high enough to compensate their weight and in order to keep them in reactor preventing washing out. The solid particles swirl around the bed rapidly, creating excellent mixing among them. The fluidized material is always a solid and the fluidizing medium is either a liquid or a gas. As compared to packed bed reactor, a fluidized bed has advantages such as better control of temperature, uniform microbial cells distribution and longer life of the microbial cells/ catalyst.

Image: https://bioprocessing.weebly.com/types-of-fermenters.html

Fluidized bed reactors in wastewater treatment are relatively recent innovation. The support matrix used (sand, anthracite, reticulated foam) is having large surface area to which adhered grow the microbial cells as biofilm. So, they are able to operate at high biomass concentration with high rates of treatment. This allows the treatment of heavily loaded wastewaters in small reactors. They are also useful for the treatment of industrial wastewaters when variable loadings are encountered. The support matrix is fluidized by the up-flow of effluent through the reactor, and the degree of bed expansion is controlled by the flow rate of wastewater. The treated effluent can thus be decanted off without loss of the support matrix and with careful operation a secondary sedimentation tank may not be needed. The support matrix is regularly withdrawn to remove excess biomass. Fluidized-bed systems can be operated aerobically, anaerobically or anoxically for denitrification. This reactor provides easy loading and removing of microbial biomass/catalysts. This is advantageous when the solids bed needs to be removed and replaced frequently. The support matrix (sand, anthracite, reticulated foam) in this type of reactor has a large surface area on which the biofilm adheres and thus they are able to operate at high biomass concentrations with high rates of treatment.

16.7 Suspended and attached growth reactors:

The reactors are classified into attached growth and suspended growth based upon the phase of the microbial cells.

16.7.1 Suspended-growth reactor: This type of system involves the process in which free flowing microorganisms gathering into biological flocs, the waste flows around and through the free-floating microorganisms that settle out of the wastewater. The processes involving suspended growth are activated sludge processes. The conventional activated sludge process is a suspended growth process comprising of microbial consortia capable of removing organic contaminants and transform wastewater into environmentally acceptable quality. In this process the wastewater is introduced to the vessel along with air. The rising air bubbles provide mixing and the resulting mixture of microorganisms and wastewater are sent to a clarifier where the microorganisms settled down as sludge and returned to the aeration vessel to increase the population of microorganisms therein. Once the desired concentration of microorganisms is reached through the activated sludge recycle, the surplus microorganisms are discarded from the system.

16.7.2 Attached-growth reactors: Attached-growth reactors also known as fixed-film reactors are used for the biological wastewater treatment processes. In these types of reactors the microbial biomass grows attached to some type of support matrix/media. The supporting matrix normally found in wastewater treatment plants are gravel, peat moss, ceramic, plastic materials, textile media, rock, and slag. The growth formed on the supporting matrix is a consortia of mainly aerobic microorganisms. These organisms are similar to those found in other secondary biological treatment processes. The microorganisms include free-swimming and stalked ciliates, rotifers, nematodes, and many others. As the biomass thickens, it loses its ability to adhere to the media and is sloughed. Attached-growth processes are easy to operate and resilient to shock loads, however they are less flexible for process control than activated-sludge process. Trickling filters and rotating biological contactors (RBCs) are two common types.

The rotating biological contactors are mechanically operated secondary treatment systems having the discs that support the growth of bacteria and micro-organisms which degrade/break down the organic pollutants present in the wastewater.

Trickling Filter: The treatment of waste water or other waste with a trickling filter type is one of the oldest and most processing technology is well marked.

In this type of attached growth system the removal of pollutants from the waste stream involves both absorption and adsorption of organic compounds by microbial biofilm. The porous media filters are usually selected to provide a very high surface area to volume. As a biofilm layer thickens, eventually sloughs off into the effluent to be treated and then part of the secondary sludge. Typically, trickling filter followed by a clarifier or sedimentation tank for the separation and removal of the decay.

Figure 9: Anaerobic Hybrid reactor Image: https://upload.wikimedia.org/wikipedia/commons/thumb/e/e0/Trickle_Filter.svg/

16.8 Hybrid Reactors

The hybrid reactor is an anaerobic system which is a combination of two processes namely up-flow anaerobic sludge bed (UASB) and upflow fixed-film (UFF) and retains the benefits of each technology. The lower part of the reactor has the granular sludge bed where a bed of anaerobic microbial biomass degrades the organic matter present in the wastewater. While the upper part of the reactor has a cross-flow type media to provide an extensive surface area for the growth of fixed microbial biofilm. The packing media in the upper part of the reactor also improves the separation of solids from the reactor effluent by intercepting the sludge bed and raw influent solids.