In this article we will discuss about:- 1. History and Design of Fermenters 2. Basic Functions of Fermenters 3. Types 4. Construction 5. Design and Operation 6. Aseptic Operation and Containment 7. Batch Fermentation 8. Fed-Batch Fermentation 9. Continuous Fermentation 10. Scale-Up of Fermentations.
History and Design of Fermenters:
De Becze and Liebmann (1944) used the first large scale (above 20 litre capacity) fermenter for the production of yeast. But it was during the First World War, a British scientist named Chain Weizmann (1914-1918) developed a fermenter for the production of acetone.
Since importance of aseptic conditions was recognized, hence steps were taken to design and construct piping, joints and valves in which sterile conditions could be achieved and manufactured when required.
For the first time, large scale aerobic fermenters were used in central Europe in the year 1930’s for the production of compressed yeast. The fermenter consisted of a large cylindrical tank with air introduced at the base via network of perforated pipes.
In later modifications, mechanical impellers were used to increase the rate of mixing and to break up and disperse the air bubbles (Fig. 20.1). This process led to the compressed air requirements. Baffles on the walls of the vessels prevented a vortex forming in the liquid (Fig. 20.2). In the year 1934, Strauch and Schmidt patented a system in which the aeration tubes were introduced with water and steam for cleaning and sterilization.
The decision to use submerged culture technique for penicillin production, where aseptic conditions, good aeration and agitation were essential, was probably a very important factor in forcing the development of carefully designed and purpose-built fermentation vessels.
In 1943, when the British Govt. decided that surface culture was inadequate, none of the fermentation plants were immediately suitable for deep fermentation. The first pilot fermenter was erected in India at Hindustan Antibiotic Ltd., Pimpri, Pune in the year 1950.
Basic Functions of Fermenters:
The main function of a fermenter is to provide a controlled environment for growth of a microorganism, or a defined mixture of microorganism, to obtain a desired product while bioreactors refer to production units of mammalian and plant cell culture. The following criteria are used in designing and constructing a fermenter.
The vessel should be capable of being operated aseptically for a number of days and should be reliable for long term operation. The adequate aeration and agitation should be provided to meet the metabolic requirements of the microbes. However, the mixing should not damage the microorganism. The power consumption should be low and temperature and pH control system should be provided.
The evaporation losses from the fermenter should not be excessive. The vessel should be designed to require the minimal use of labour in operation, harvesting, cleansing and maintenance. It should have proper sampling facility. The vessel should be constructed to infuse instead of flange joints. The cheapest and best material should be used and there should be adequate service provisions for individual plants.
Types of Fermenters:
i. Fluidized Bed Bioreactor:
It is more popular in chemical industry rather new to biochemical industries. These are mostly used in conjunction with immobilized cells or enzyme system and are operated continuously.
ii. Loop or Air Lift Bioreactor:
In the conventional bioreactor, oxygen is supplied by vigorous agitation of the bioreactor content. The heat is generated which is a problem in conventional type. In air lift fermenter, cooling becomes simpler due to the position of inner or outer loop.
iii. Membrane Bioreactor:
These consist of a semipermeable membrane made up of cellulose acetate or other polymeric materials. The primary purpose of the membrane is to retain the cells within the bioreactor, thus increasing their density, while at the same time allowing metabolic products to pass through the membrane.
iv. Pulsed Column Bioreactor:
The essential component of a pulsed column bioreactor is a column bioreactor generator connected to the bottom of the column. A pulsed column bioreactor can be utilized as an aerobic bioreactor, enzyme bioreactor or as a separation unit since its original successful application was in the extraction of uranium.
v. Bubble Column Bioreactor:
Multistage bubble column bioreactor are suitable in the equivalent batch process. It is necessary to vary the environmental conditions over the course of the reaction. In this bioreactor, it is possible to provide different environmental conditions in various stages. The system may not be suitable for fungal fermentation due to high oxygen demanding system.
vi. Photo Bioreactor:
For the growth and production of photosynthetic organisms, a light source is retired. In photo-bioreactor, there is an important ‘reactant’, the photons which must be absorbed in order to react and produce products. So the design of the light source is critical in the performance of this type of bioreactor.
One of the most interesting photochemical reactors is the annular reactor. In this source of radiation is a cylinder with an annular section, which encloses the lamp completely. The nutrient passing from the product is removed from the top. This is used for Spirulina (SCP) and other algal protein production.
vii. Packed Tower Bioreactor:
It consists of cylindrical column packed with inert material like wood shavings, twigs cake, polyethylene or sand. Initially, both medium and cells are fed into the top of the packed bed. Once the cells adhered to the support and were growing well as a thin film, fresh medium is added at the top of the packed bed and the fermented medium removed from the bottom of the column.
This is used for vinegar production, sewage effluent treatment and enzymatic conversion of penicillin to 6-amino penicillanic acid. The design of fermenter involves the co-operation between experts in microbiology, biochemistry, mechanical engineering and economics.
Construction of Fermenters:
The criteria considered before selecting materials for constructions of a fermenter are:
(a) The materials that have no effect of sterilization, and
(b) Its smooth internal finish – discouraging lodging of contamination. The internal surface should be corrosion-resistant.
There are two types of such materials:
(i) Stainless steel, and
(ii) Glass which are used in fermenter.
According to American Iron and Steel Institute (AISI), if a steel contains 4% chromium, it is called stainless. The long and continuous use of stainless steel sometimes shows pitting. It is also important to consider the materials used for aseptic seal.
Sometimes it is made between glass and glass, glass and metal and metal joints between a vessel and detachable top or base plate. On pilot scale, any material to be used will have to be assessed on their ability to with stand pressure, sterilisation, corrosion and their potential toxicity and cost.
(i) Control of Temperature:
Since heat is produced by microbial activity and mechanical agitation, then it is sometimes necessary to remove it. On the other hand, in certain processes extra heat is produced by using thermostatically controlled water bath or by using internal heating coil or jacket meant for water circulation.
(ii) Aeration and Agitation:
The main purpose of aeration and agitation is to provide oxygen required to the metabolism of microorganisms. The agitation should ensure a uniform suspension of microbial cells suspended in nutrient medium.
There are following necessary requirements for this purpose:
(a) The agitator (impeller) for mixing;
(b) Stirrer glands and bearings meant for aseptic sealing;
(c) Baffles for checking the vortex resulting into foaming;
(d) The sparger (aeration) meant for introducing air into the liquid.
(a) The Agitator (Impeller):
The size and position of the impeller in the vessel depends upon the size of the fermenter. In tall vessels, more than one impeller is needed if adequate aeration agitation is to be obtained. Ideally, the impeller should be 1/3 or 1/2 of the vessel diameter (D) above the base of the vessel. The number of impeller may vary from size to size to the vessel (Fig. 20.1 and 20.2).
(b) Stirrer Gland and Bearing:
Four basic types of seals assembly have been used: the packed gland seal, the simple bush seal, the mechanical seal and the magnetic drive.
The baffles are normally incorporated into agitated vessel of all sizes to prevent a vortex and to improve aeration efficiency. They are metal strips roughly one-tenth of the vessel diameter and attached radially to the walls.
A sparger may be defined as a device for introducing air into the liquid in a fermenter. It is important to know whether sparger is to be used on its own or with mechanical agitation as it can influence equipment design to determine initial bubble size. Three basic types of sparger have been used and may be described as the porous sparger, the orifice sparger and the nozzle sparger.
(e) Microbial Biosensors:
A microbial sensor consists of a microorganism immobilized on a membrane and an electrode. The principle of working of biosensor is the change in respiration or the amount of metabolites produced as a result of the assimilation of substrate by the microorganism. A wide range of thermophilic microbes have been used for the manufacturing of microbial sensors as given in Table 20.1.
Immobilized yeast, Trichosporon cutaneum has been used to develop an oxygen probe for BOD estimation in sewage and other water samples. The BOD sensor includes an oxygen electrode that consists of a platinum cathode and an aluminum anode bathing in salt KCL solution and a Teflon membrane.
Immobilized yeast cells are crapped between the pores of porous membrane and the Teflon sensor can measure BOD at 3-60/mg/litre.
Methanotrophic bacteria namely Methylomonas Flagellata used in measuring methane as well as oxygen according to the reaction given below:
CH4 + NADH2 + O2 → CH3OH + NAD+ + H2O
Similarly, ammonia and nitrate biosensors consist of immobilized nitrifying bacteria, Nitrosomonas europaea and a modified oxygen electrode. This is used to determine ammonia in waste water based on the conversion of nitrate to N2O by an immobilized denitrifying Agrobacterium sp. The nitrate biosensor has been used to measure nitrate profiles in biofilm.
Microbial biosensors have several uses in clinical analysis, general health care monitoring, veterinary and agricultural applications, industrial product processing and monitoring besides control of environmental pollution. In addition to low cost and small size, these are easy to use sensitive and selective in nature.
A large number of bacteria and eukaryotic cell culture are used in manufacturing new products. The monitoring of these items is essential by using microbial biosensors. This allows in minimizing the cost of production. Biosensors have immense importance to military and defense in detection of chemical and biological species used in weapons.
Design and Operation of Fermenter:
These are designed to provide support to the best possible growth and biosynthesis for industrially important cultures, and to allow ease of manipulation for all operations associated with the use of the fermenters.
These vessels must be strong enough to resist the pressure of large volume of agitating medium. The product should not corrode the material nor contribute toxicity to the growth medium. This involves a meticulous design of every aspect of the vessel parts and other openings, accessories in contact, etc.
In fennentations, provisions should be made for the control of contaminating organisms, for rapid incorporation of sterile air into the medium in such a way that the oxygen of air is dissolved in the medium and therefore, readily available to the microorganisms and CO2 produced from microbial metabolism is flushed from the medium.
Some stirring devices should be available for mixing the organism through the medium so as to avail the nutrients and oxygen. The fermenter has a possibility for the intermittent addition of antifoam agent. Some form of temperature control efficient heat transfer system is also there for maintaining a constant predetermined temperature in the fermenter during the growth of organism.
The pH should be detected. Other accessories in the fermenter consist of additional inoculum tank or seed tank in which inoculum is produced and then added directly to the fermenter without employing extensive piping which can magnify contamination problems (Fig. 20.1 and 20 .2).
Use of Computer in Fermenter:
Computer technology has produced a remarkable impact in fermentation work in recent years. Integration of computers into fermentation systems is based on the computers capacity for process monitoring, data acquisition, data storage, and error-detection.
Some typical on-line data analysis functions include the acquisition measurements, verification of data, filtering, unit conversion, calculations of indirect measurements, and differential integration calculation of estimated variables, data reduction, and tabulation of results, graphical presentation of results, process stimulation and storage of data.
Aseptic Operation and Containment of Fermenter:
Containment involves prevention or escape of viable cells from a fermentor or downstream equipment and is much more recent in origin. Containment guidelines were issued in 1970s.
Criteria to assess risk was explained by Collins (1992) in the following manner:
(a) The known pathogenicity of microorganism,
(b) The virulence or level of pathogenicity of the microbes are the diseases it causes mild or serious,
(c) The number of organisms required to initiate an infection,
(d) The route of infection,
(e) The known incidence of infection in the community and the existence locally of vectors and potential reserve,
(f) The amounts or volume of organism in the fermentation process,
(g) The technique or process used,
(h) Ease of prophylaxis and treatment.
Batch fermentation, is a process in which a large volume of nutrient medium is inoculated to proceed for the harvest and recovery of the product. This ends the batch fermentation as the vessel is cleansed and re-sterilized for the subsequent batches. This is a close system in which all the nutrients are initially added to the vessel and inoculated.
Subsequent treatments include maintaining adequate aeration by providing stirrers and pH control by the addition of an acid or alkali. In aerated systems, antifoam agents such as palm oil or soybean oil are added. Large scale growth of microorganisms tends to generate heat in the system. Temperature control is maintained by providing water circulation system around the vessel for heat exchange.
In large fermenter, the fed-batch method is employed to control the concentration of critical nutrients. For example, penicillin production is controlled by maintaining low glucose level in the medium because its accumulation in the medium results into catabolite repression of secondary metabolite.
In batch fermentation, the growth of microorganism follows the characteristics growth curve with a lag phase followed by a log phase, finally reaching the stationary phase due to limitation of nutrients and other factors.
When complex nutrient solutions are used, two lag phases frequently occurs separated by a second log phase. This phenomenon is called diauxy and arises due to one of the substrates utilized preferentially. The presence of one substrate represses the breakdown of other substrate.
In such (fed-batch) cases, substrate is added in increments as the fermentation progresses. The formation of many secondary metabolites is subject to catabolite repression by high concentration of glucose, other carbohydrates, or nitrogen compounds.
In such situations, in the fed-batch method the critical elements of the nutrient solution are added in small concentration in the beginning of the fermentation and these substances (substrates) continue to be added in small doses during the production phase.
It is difficult to measure the substrate concentration directly and continuously during the fermentation. Indirect parameters, which are correlated with the metabolism of the critical substrates, have to be measured in order to control the feeding process. For example, in the process of organic acids the pH is to be used to determine the rate of glucose feeding. Sometimes dissolved O2 or the CO2 content in the exhaust air are monitored.
Fermentation may be carried out as batch, fed-batch or continuously in a vessel or fermenter. During continuous fermentation, some part of the components (include media and inoculum) of upstream process are withdrawn intermittently and replacement or withdrawn substances are made by adding the fresh medium or nutrients.
The withdrawn part from the fermenter is used for recovery of the products. During continuous fermentation, the equipment is always in use and secondly inoculum is not required in subsequent addition of the nutrients.
It is carried out in three different processes as given below:
(i) Single Stage:
In which a single fermenter is inoculated then kept in continuous fermentation. “Recycle” fermentation in which, a portion of the withdrawn culture or the residual unused substrate including withdrawn culture, is recycled in fermenter. During this process, the substrate is further utilized for product formation and inoculum can also be recycled.
(ii) Multiple-Stage Fermentation:
It involves two or more stages with the use of two or more fermenters in sequence. In such instances, the microbial growth occurs in first stage fermenter followed by a synthetic stage in the next fermenter. This happens in case those metabolites that are not related with growth. The microbial activity in continuous fermentation can be controlled either of the following.
In this the total cell population is held constant by employing a device that measures the culture turbidity so that it regulates both the nutrient feed rate and the culture withdrawn rate from the fermenter.
Sometimes, the inoculum added is multiplied so quickly that its level increased too much; hence medium is added further so as to dilute the inoculum. However, by adding the nutrient, the microbial growth should be maintained in the log phase. During this process unused nutrient is lost from the withdrawal/harvested culture.
On the other hand, in the chemostat the nutrient and harvest culture withdrawal rate at constant values. This growth rate can be controlled by toxic product formation during the fermentation, pH and even the change of temperature.
Actually, it is necessary to maintain a constant cell population in the fermenter. The flow rate also related to the growth of organism. If it is low than it can allow the culture to go into maximum stationary phase. Too high a flow rate can adversely affect a number of microorganisms namely, Streptomyces, Chlorella, Aerobacter, Azotobacter, Bacillus, Brucella, Clostridium, Salmonella, Penicillium, Saccharomyces, Torula etc. which are being used in continuous process of fermentation.
Some of the products such as beer, has been commercialized. The sewage treatment by activated sludge system has been considered as commercialized continuous fermentation, a process in which mixed microbial population acts on a heterogeneous substrate.
There are certain requirements for continuous fermentation. To know the microbial growth, its behaviour is requisite. To evaluate the contamination and mutation, prolonged incubation may cause contamination except Torula yeast on sulphate waste liquor. To check the contamination problems, antibiotics or other chemicals are added to continuous fermentation to hold the level of contaminant growth.
The multiple continuous fermentation is advantageous for checking mutation. It is therefore, necessary to reduce their rate of occurrence so that these cells do not multiply. Since every time, fresh medium is being added hence it is observed quite often that some part of it remain utilized. In certain cases, it is possible to separate the residual nutrient substrate from the harvested culture so that it is recycled through the fermenter.
Fermentation media sometimes require strong mixing if the media is viscous. Adequate mixing is a problem. To overcome the problem, mathematical assumptions are necessary while processing for continuous fermentation. It is also necessary to evaluate the microbial cells do not adhere/attach to the surface of the fermenter.
Sometimes, filamentous fungi grow in piping and valve. This is immobilized state and cultures do not expose to nutrient added. This condition makes difficult a proper regulation of inflow and outflow for the fermentation. It is also observed that conditions for the microbial growth and product formation differ. Hence, the optimum conditions for microbial growth do not align with the optimum conditions required for product formation.
In a single stage fermentation, a compromise must be laid in the nutrient and physical conditions during the fermentation, growth in the first stage and product formation in the second or succeeding stages are required. The multistage chemostat is useful in the utilization of multiple C source in the production of secondary metabolites. This system is complex hence in industries limited applications are those as in continuous brewing.
Scale-Up of Fermentations:
The determination of the proper incubation conditions to be employed with large scale production tanks as based on information obtained with various sized smaller tanks is called “scale- up”. This process allows to carry out laboratory procedure at industrial scale. It is the best way to obtain fermentation information for production tanks directly in large tanks.
However, this is not practical for:
(a) New fermentation,
(b) Variation studies on a fermentation already in production, and
(c) Valid experiment cannot be carried out with only a single tank; one or more tanks are required as experimental controls. Aside from these considerations, costs, media also affect the scale-up.
Use of Erlenmeyer Flasks:
The conventional methods provide a poor production because of the poor aeration characteristics associated with this vessel.
Use of Baffle Flasks:
In this, flask with glass baffles projecting into the medium from bottoms or sides provide better aeration than Erlenmeyer flask.
Use of Small Laboratory Fermenters:
One to ten to twelve litre size fermenters are most ideal for this type of studies, since their aeration and agitation conditions can be varied and the overall fermentation conditions of these tanks more closely resemble with those of the larger production tanks.
These tanks allow fermentation studies on a scale that has meaning in relation to production tank but without too great an expense for media, labour, power input, etc. Experience with particular fermentation equipment and previous fermentations are the only real guide in translating scale up.
(i) Sterilization of Gases and Nutrient Solutions:
In virtually all fermentation processes, it is mandatory for a cost effective operation to have contamination free seed culture at all stages from the preliminary culture to the production fermenter. A fermenter or bioreactor can be sterilized by destroying the organism with some lethal agents or by removing the viable organisms by a physical process such as filtration.
Nutrient media as initially prepared contain a variety of different cells and spares derived from the constituents of the culture medium, the water and the vessel. These must be eliminated by a suitable means before inoculation. A number of means are recommended but in practice for large scale sterilisation, heat is the main mechanism used.
(ii) Stock Cultures:
It is extremely important to maintain microorganisms for extended periods in viable conditions, and in situation which did not alter their desired product formation capacity. This condition is also true for strains used in biological assays. Thus, microbial species procured from various culture collection centres are maintained in viable conditions and known as stock-culture collection.
The stock culture generally retains all the characteristics initially described. Stock culture collection centres have been established throughout the world to help microbiologists in obtaining cultures for various studies. These centres also help in classifying a newly isolated organism.
There are two types of stock cultures: working stocks and primary stocks:
(a) Working Stocks:
These stocks are used frequently and they must be maintained in vigorous and uncontaminated conditions on agar slants, agar stabs, spore preparations, or broth culture, and one held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination.
(b) Primary Stocks:
The cultures that are held in reserve for presently practical or new fermentation for comparative purposes, for biological assays, or for possible later screening programmes. These are not maintained in a state of physiological activity. Transfers from these cultures are made only when a new working stock culture is required, or when the primary stock culture is sub-cultured to avoid death of the cells.
Thus, primary stock cultures are stored in such a manner as to require the least possible numbers of transfers over a period of time. Further, these are stored at room temperature, are maintained in sterile soil, or in agar or broth over-layed with sterile mineral oil. Agar and broth culture without mineral oil also are refrigerated. The culture in milk of agar are maintained frozen at low temperature.
Finally, primary stock cultures are lyophilized or frozen-dried, and stored at low temperature. The culture of Blakeslea trispora used in β – carotene production, cannot be stored at refrigeration temperature because they die relatively quickly. However, at room temperature transfers are being made to fresh medium when the cultures become nearly dried out.