Fermentation Process

The fermentation process involves actual growth of the microorganism and formation of the product under agitation and aeration, to provide uniform environment and adequate oxygen to the cell for growth, survival, and product formation. Modes of Operation

A fermentation system is usually operated in one of the following modes: batch, fed batch, or continuous fermentation. The choice of the fermentation mode is dependent on the relation of consumption of substrate to biomass and products. Batch Fermentation Fermentation processes performed on a batch basis involve a sequence of operations, from development of inoculum from a stock culture, to seed for a production fermentor. The seed and production fermentors are the main concern in fermentation process development. A number of seed stages may be involved, but the production stage is usually performed in a single fermentor (100). The multistage system has the advantages of increased productivity resulting in lower total fermentor volume and the possibility for variation of the environmental conditions from stage to stage. The time required for batch fermentation varies from hours to weeks depending on the conversion attempted and conditions used. Growth rate in batch fermentation is normally uncontrolled and is highest at the start (101).

Productivity of batch fermentation: The productivity of batch fermentation is calculated by the final concentration of biomass or product being produced divided by the complete time of batch, which includes fermentation time and turnaround time (time for emptying, cleaning, sterilizing, and refilling). The batch fermentation setup and productivity for biomass production is shown in Figure 3.3 and Figure 3.4, respectively. Continuous Fermentation Continuous fermentation is an open system to maintain cells in a state of balanced growth by continuously adding fresh medium and removing the culture medium at the same rate. Essentially, the two modes of operation for continuous fermentation are chemostats and auxostats. The commonly used auxostats include turbidostats (102), the pHauxostat (103), and the nutristat (104). The above modes of operation have specific control configurations and applications as discussed below.

Chemostat: Presently, the chemostat is the most widely used apparatus for studying microorganisms under constant environmental conditions. It is a continuous fermentation process performed in a Continuous Stirred Tank Reactor (CSTR). A CSTR operates by

Chemostat Continuous Fermentation

Figure 3.3 Batch fermentation system

Chemostat Continuous Fermentation

Figure 3.4 Productivity in batch fermentation maintaining a growth rate through continuously feeding a growth limiting nutrient and withdrawing part of medium at the same rate, thereby achieving steady state growth. The growth limiting nutrient may be carbon, nitrogen, phosphorus, or any other essential nutrient, which influences the specific growth rate. A significant advantage of chemostat mode over batch mode is that by changing the feed rate of growth limiting nutrient, the growth rate can be varied. A schematic of chemostat (single stage) cultivation with and without cell recycle is shown in Figure 3.5 and Figure 3.6 respectively.

Auxostat: An auxostat is a continuous culture technique wherein the dilution rate is regulated based on an indication of the metabolic activity of the culture. A chemostat is essentially used for operation at moderate to low dilution rates, but an auxostat is used at high dilution rates. Population selection pressures in an auxostat lead to cultures that grow rapidly. Practical applications include high rate propagation, destruction of wastes with control at a concentration for maximum rate, open culturing because potential contaminating organisms cannot adapt before washing out, and operation of processes that benefit from careful balance of the ratios of nutrient concentrations. In a pHauxostat, the feed rate is regulated by measurement and control of the pH of the fermentation medium. This can be applied only if there is a change in pH consequent to the growth of microorganism. The pHauxostat has been used for continuous mass cultivation of bacteria for isolation of intra-cellular products (105,106).

Turbidostat Operation

Feed _ inlet

Figure 3.5 Continuous fermentation system

Turbidostat Continuous Fermentation
Figure 3.6 Continuous fermentation system with cell recycle

Turbidostat: The turbidostat controls the feed rate depending on the optical density (turbidity) of the fermentation broth, which is proportional to the biomass concentration. In this mode of operation, the culture cannot washout as in a chemostat. This mode of operation is ideal only when operated near maximum growth. The isolation of acid tolerant baker's yeast variants was developed in a turbidostat (107).

Nutristat: The nutristat involves regulation of the feed rate to maintain the residual substrate concentration during fermentation. The use of specific sensors for monitoring the residual substrate level during fermentation is employed. Ion selective electrodes (NH4+) have been used for control in the nutristat. However, the lack of reliable and accurate sensors for common substrates is a bottleneck to nutristat operation (108).

Productivity of continuous fermentation: For a continuous fermentation, there is no emptying, cleaning, sterilizing, and refilling component. The productivity of a continuous fermenter is calculated by multiplying the dilution rate (D) by the concentration of product in the outlet stream:

The productivity in continuous fermentation for biomass formation is represented in Figure 3.7. The commonly employed commercial applications of continuous culture include baker's yeast, vinegar, gluconic acid, acetone, butanol, and ethanol fermentation systems. Fed Batch Fermentation Fed batch fermentation, which is a technique in between batch and continuous fermentation, is a more recent development in industrial fermentation systems. Neither batch nor continuous fermentation is suitable for non growth associated products. In order to produce such products, it is first necessary to build up a high concentration of cells in the growth or batch phase, and then switch the metabolism of the cell to arrest cell growth by feeding product precursors, carbon, and oxygen at a rate sufficient to meet the maintenance and product synthesis requirements. Essentially, fed batch fermentation involves two phases: growth phase and production phase. After the initial growth phase, one or more of the nutrients are supplied to the fermentor while cells and product remain in the fermentor.

The rationale for fed batch fermentation is to match the organism's demand for nutrients by feeding an appropriate amount of that nutrient. To determine the fed batch approach, nutrient demand has to be ascertained. This demand is a function of cell mass and cell yield on the nutrient. Though batch fermentation may be considered simple, fed batch fermentation

Cell productivity = D.X (kg cells/m3/h) Product productivity = D.P (kg product/m3/h)

Dilution rate

Figure 3.7 Cell productivity in continuous fermanation system

Dilution rate

Figure 3.7 Cell productivity in continuous fermanation system offers the convenience of better control over substrate concentration variations and differentiation of growth, leading to improved overall productivity with essentially the same equipment used for batch fermentation. Fed batch systems can be superior to continuous systems due to problems involving contamination during fermentation.

Fed batch fermentation is well suited for production of compounds during very slow growth where there is no possibility of cell washout. Fed batch fermentation is well suited for producing product or cells when: 1) Substrate is inhibitory and there is a need to maintain low substrate concentration to avoid the cells being inhibited (e.g., citric acid, amy-lase), and 2) Product or biomass yields at low substrate concentrations are high (e.g., baker's yeast, antibiotic production).

There are two methodologies in the fed batch approach, namely fixed volume fed batch and variable volume fed batch (109). In fixed volume fed batch fermentation, a very concentrated feed nutrient is fed to the fermentor so that there is no appreciable increase in volume. The specific growth rate decreases with time and the biomass increases directly with time. In variable volume fed batch fermentation, there is an increase in volume due to nutrient inflow and no outflow. The specific growth rate is solely dependent on the concentration of the limiting nutrient.

Productivity of fed batch fermentation: The productivity of a fermentation process is better in the fed batch mode compared to a batch mode of operation (110). In batch fermentation of S. cerevisiae, a dry cell weight of 10 g/L was obtained, whereas in fed batch mode, with respiratory quotient (RQ) as the control parameter for glucose feed, a final dry cell weight of 31-56 g/L was obtained (111). Agitation

In stirred tank bioreactors, mixing and dispersion of air in the fermentation broth is achieved by mechanical agitation. The presence of impellers on the agitator shaft brings about uniform mixing of microorganisms and nutrients, and dispersion of air in the nutrient solution, resulting in efficient mass and heat transfer. Mixing by aeration or gas movement alone cannot be used in highly viscous systems due to coalescence of the gas bubbles. The heavy mycelial growth produced in antibiotic fermentations is typical of such a condition and hence mechanical agitation is essential for optimum production (112).

A turbine or a propeller, when rotated in a vessel without baffles, imparts circular flow due to swirling of the liquid around the vortex, which renders the motion unconducive for top to bottom mixing. In a baffled tank, on the other hand, the flow pattern may be radial or axial, promoting lateral flow and vertical flow currents, with the application of large amounts of power. Baffles reduce the unproductive tangential velocity component of all impellers to produce a more efficient radial or axial flow. Positioning and design of baffles, along with the placement, type, and design of heating coils has a significant impact on the flow patterns (113).

The types of impellers used in bioreactors are broadly classified based on flow pattern as radial flow (e.g., turbine) and axial flow (e.g., high efficiency impeller).

The Rushton turbine is the most common type of radial flow impeller. It consists of a number of short blades attached to a central shaft. The diameter of a turbine is normally between 30 and 50% of the tank diameter and there are usually between four to six blades. Turbines with flat blades are good for gas dispersion, where the gas is introduced just below the impeller on the axis and is drawn up to the blades and broken up into fine bubbles (114).

An axial flow impeller generates an axial flow pattern with the fluid flowing down the central axis and up on the sides of the tank. This flow pattern is also called down pumping because the impellers circulate the fermentation broth downward against the flow of the rising air, thereby holding the gas in the system longer. In multiphase fermentation systems involving solids, the axial component of the flow due to agitation is useful to keep the solids in suspension (115).

Recent developments in impeller design have led to the emergence of high efficiency impellers like MIG and INTERMIG, which require 25% and 40% less power input to get the same degree of mixing as a turbine impeller. Recently, high solidity hydrofoil impellers with an up pumping mode of operation were reported to increase mass and heat transfer, to and improve blending. Blending, which is less complicated than mass transfer, is not efficient on a large scale compared to small lab scale fermenters due to the formation of dead zones between impellers. Hence, optimal blending is essential for maximum yield and productivity in large scale fermentations (116).

Multiple impeller bioreactors (with impellers fixed at various heights on the central shaft) are becoming increasingly popular due to efficient gas distribution, higher gas phase residence time, increased gas hold up, superior liquid flow (plug flow) characteristics, and lower power consumption per impeller as compared to single impeller systems (117,118). Using both Rushton turbines and high efficiency axial flow impellers on the same shaft is reported to improve fermenter performance with a decrease in the total power consumption (119). Aeration

The cheapest mode of supplying oxygen to the fermentation media is air. The aeration system essentially comprises an air compressor, an air filter (prefilter and sterile filter) and a sparger. The air compressor used for fermentation may be a positive displacement or a nonpositive displacement unit, with the former being a reciprocating or rotary compressor and the latter a centrifugal compressor. The choice of compressor depends upon such factors as the discharge pressure, type of drive, capacity, and cost (120). Equipment sizing of the compressor is usually dependent on the maximum airflow requirement of a full production scale for all the fermentors. Careful scheduling of fermentations can frequently reduce the gap between peak and average demands and result in more economical use of the equipment.

The discharge pressure of the air from the compressor is subjected to pressure drop along the route at predictable locations, such as valves and pipefittings along the supply lines, air filters, the sparger, hydrostatic head, and static pressure, before it reaches the fermentor. The diameter of the supply line is so chosen that the maximum airflow may be enabled without undue pressure loss. A pressure drop of 1-2 p.s.i. per 100 ft of pipe may be used as a first approximation. The first three pressure drops discussed above increase with increasing flow rate, but the hydrostatic head and static pressure are independent of airflow. The hydrostatic head is the depth of nonaerated liquid above the sparger, corrected for density, and usually represents quite a large fraction of the total pressure loss. Air, filtered using sterile air filters, is passed into the fermentor from immediately after sterilization during the cooling cycle until the end of fermentation to maintain a positive pressure and circumvent contamination.

In most aerobic fermentations, peak oxygen consumption occurs over very short periods near the end of fermentation. It is possible to increase the oxygen uptake rate by providing high levels of oxygen transfer rate (OTR). OTR is the rate at which oxygen can be transferred from the air to the fermentation broth. It can be expressed as:

where kLa is the volumetric mass transfer coefficient, c* is the dissolved oxygen concentration in equilibrium with the gas phase, and cL is the dissolved oxygen concentration in the liquid.

The oxygen mass transfer coefficient, kLa, is directly related to power input by aeration and aeration rate by (121):

where Pg/ V is the gas power input by aeration per unit volume and vs is the superficial gas velocity.

A high kLa (volumetric mass transfer coefficient) indicates a high oxygen transfer rate (OTR) in the fermentation process. Since oxygen uptake rate is the key for a successful aerobic fermentation, a high kLa is required. However, providing higher levels of oxygen transfer usually requires very powerful drives, which add to mechanical problems and costs, as well as additional heat loads. OTR varies with the nature and the scale of the fermentation. For a 1m3 vessel, at an OTR between 250 and 300 mmol/L/h, heat transfer is not a major problem. However, for a 10m3 vessel, OTR at the above rates pose significant problems in terms of heat removal from the fermentor, indicationd the need for the use of internal cooling coils and low temperature coolants, which adds considerable operational costs. Process Monitoring and Control

The complexity of metabolic biosynthesis involved in fermentation necessitates the use of process control parameters for monitoring and control to ensure optimum process performance. The process control variables in a fermentation process may be broadly classified as physical, chemical, and biological. The physical variables include temperature, impeller speed, aeration rate, and pressure. The chemical variables include pH, dissolved oxygen, dissolved carbon dioxide, and redox potential. The biological variables include biomass, oxygen uptake rate, carbon dioxide production rate, and respiration quotient. While the physical variables are usually measured by inline sensors forming an integral part of the fermentation equipment, the chemical or process variables are measured by online sensors crucial for the successful operation of a fermentation process. The biological variables are process indicators depending on proper monitoring and control of process variables in a fermentation process. A modern technique for bioprocess monitoring and control uses biosensors. A biosensor is a device that detects, transmits, and records information regarding a physiological or biochemical change resulting in the generation of electronic signals. The main function of a biosensor is integration of a biological component with an electronic transducer to convert the biochemical changes into a quantifiable electrical response. Biosensors make use of a variety of transducers such as electrochemical, optical, acoustic and electronic (122).

The development of miniaturized in situ sensors for online monitoring and control of pH and dissolved oxygen has been driven by fermentation processes and is shown in Figure 3.8 (123).

pH: Fermentation at the optimum pH is essential for cell growth and product formation. A sterilizable pH probe which functions based on a potentiometric principle consists of a glass electrode and a reference electrode. The reference electrode system consists of an Ag/AgCl electrode and a KCl electrolyte. A constant voltage of the reference electrode system is provided by the electrolyte. A ceramic diaphragm maintains the electrolytic contact between the reference electrode and fermentation broth. Based on the response from the pH probe to the pH controller, the acid or base pump is triggered for correction of pH. Steam sterilizable glass electrodes have the limitation of low mechanical stability over a period of repeated sterilizations. They can be replaced with optical sensors based on absorbance or fluorescence from pH-sensitive dyes (124).

Food Biotechnology Images

Figure 3.8 Bioprocess control of fermentation process

Figure 3.8 Bioprocess control of fermentation process

Dissolved oxygen: Monitoring of the dissolved oxygen (DO) concentration is essential, because sparingly soluble oxygen is one of the limiting substrates for fermentation. Two types of DO electrodes used in fermentation: polarographic and galvanic, with the former more commonly used as it is reliable and resistant to sterilization, while the latter is not widely used due to poor stability. The polarographic or amperometric electrode (—0.7 V) needs an external voltage between a Pt cathode and an Ag/AgCl anode. The Pt cathode is in contact with KCl solution encapsulated by an oxygen permeable Teflon membrane. The DO from the broth diffuses across the membrane to be reduced at the Pt electrode surface. The DO concentration in a fermentor can be manipulated by two physical variables: impeller speed and aeration rate. The DO saturation (% pO2) for a particular fermentation can be controlled individually, sequentially, or simultaneously manipulating the variables based on a feedback from the DO controller. The controller compares the deviation of the signal from the DO electrode with the set point and adjusts the impeller speed controller or the airflow controller for maintenance of DO saturation. Another possibility of DO control in fermentation exists by variation of the oxygen concentration in the gas by application of three proportional valves for controlling the flows of air, oxygen, and nitrogen (125). The steam sterilizable DO electrode (126) is widely used for online bioprocess monitoring of the dissolved oxygen concentration during fermentation. An alternative based on quenching of fluorescence has recently been introduced (127).

Dissolved carbon dioxide: Dissolved carbon dioxide (DCD) is another indicator of the metabolic status of the respiring cell. It is commonly used for animal cell cultivation where DCD concentration helps to maintain the carbon dioxide saturation during the bio-process. DCD electrode operates on the principle of change in pH of an electrolyte buffer.

DCD sensors with bicarbonate buffers are not sterilizable, and have the limitations of drift and interference from changes in the medium composition. An in situ sterilizable DCD sensor with radiometric fluorescent dye HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) has been introduced (128).

Gas analysis: The exit gas composition from the fermentor, particularly oxygen and carbon dioxide, can be analyzed by a gas analyzer using paramagnetic and infrared detectors respectively. Mass spectrometry can also be used to analyze all gaseous components, including organic volatiles and lower alcohols, of the exhaust gas stream (129). From an oxygen to carbon dioxide gas balance across the fermentor, the oxygen uptake rate (OUR) or carbon dioxide production rate (CPR) can be determined. From a knowledge of the OUR and CPR during the fermentation process, the respiratory quotient (RQ = CPR/ OUR) can be determined.

Redox potential: The redox potential of fermentation broth is related to the overall availability of free electrons in the solution. Redox potential operates on the principle that when a noble metal electrode (Pt, Au or Ag) is immersed into a redox system, a potential is developed the magnitude of which depends on the oxidation-reduction concentration ratio. The potential formed at the noble metal electrode is tapped off by a reference electrode, which is also immersed in the solution. The value of the potential is a measure of the concentration of oxidizing or reducing agents. Redox values are found to vary significantly with changing pH and are expressed in mV. The redox potential decreases with increasing pH value of the measured solution. The metabolic activity of microorganisms is strongly influenced by the redox potential of the culture environment. Monitoring and control of the redox potential before inoculation facilitates controlled addition of reducing agents to ensure the proper range for initiation of growth. Control of the redox potential is a vital tool in determining its influence on the metabolic pathways of microorganisms, substrate utilization, or production of specific metabolites (130).

Biomass concentration: Biomass concentration is an important biological variable, which indicates the progress of the fermentation. The common offline cell mass determination methods are based on dry cell weight and packed cell volume. Direct online characterization of cell populations in bioreactors in terms of morphological changes can be determined by in situ microscopy equipped with an image analyser (131). Indirect online methods for determination of cell concentration may be based on electrical or optical approach. Electrically, capacitance of the medium measures the fraction of fluid held by polarizable membranes (132). Optically, cell mass is often measured by light absorbance (turbidity) or scattering (nephelometry) continuously in the visible and near infrared ranges (133). The advantages of optical sensors for bioprocess monitoring are that they are very sensitive, give specific and reversible measurements, have a rapid response and versatility, and involve easy maintenance. The disadvantage of the optical method is that scattering and absorption are not linearly dependent on the cell density and are vulnerable to interference from particulates and gas bubbles. A biological receptor, namely an enzyme, microorganism, or antibody that produces an optical signal such as NADH fluorescence (NADH upon UV radiation at 366 nm emits fluoroscence at about 460 nm) in an optical biosensor (134). The signal is converted by an electronic transducer into an electrical signal. Bioluminescent sensors have also been developed, which consist of a bioluminescent enzyme and an optical transducer. Cell mass in animal cell cultivations can also be predicted by software sensors (135). Direct online biomass determination in fermentation processes is limited by the presence of suspended solids. The productivity of a fermentation process depends specifically on the biomass concentration achieved. Maximization of biomass concentration to high cell densities (136) is dependent on operating the fermentor at the optimum controlled process conditions.

Substrate and product concentration: The determination of the exact concentration of substrate and product during fermentation is critical for the maintenance of the optimum feed concentrations required for maximum productivity and efficient substrate utilization.

Flow Injection Analysis (FIA) is a method for online detection of concentration with a suitable detector, which may use amperometry, potentiometry, NADH-fluorescence, che-miluminescence, or UV/Vis spectrophotometry, and involves an analysis time lasting only a few minutes (137). FIA coupled with an immobilized 6-galactosidase biosensor has been reported for monitoring of lactose in a fermentation process (138). Biosensors used for FIA do not need sterile conditions for analysis. FIA systems inject low amounts of samples into the carrier stream and the probability of cell growth and protein precipitation is low because of the high dilution (139). Enzyme based biosensors have been used for monitoring and control of substrates and products involved in glutamate and penicillin fermentation processes (140). Chromatographic techniques like gas chromatography, high performance liquid chromatography, fast protein liquid chromatography, and membrane chromatography have been employed for process monitoring of fermentation processes (141). Recombinant protein product can be determined spectroscopically by attaching the gene for green fluorescent protein (GFP) to the product gene. The product concentration can then be determined by analyzing the fluorescence intensity of the GFP in the culture (142).

Mass spectrometric analysis of gases such as oxygen and carbon dioxide in the exit gas paved the way for determination of dissolved gases such as oxygen, carbon dioxide, methanol, ethanol, butanol, and acetone by the use of appropriate sampling systems (143). The accuracy of analysis is higher than observed with online probes. Sampling for offline analysis is crucial from the point of view of sterility and proper representation of the medium. Many sampling devices based on microfiltration, ultrafiltration, and dialysis have been used in the fermentation industry (144). A powerful analytical tool for noninvasive analyisis of intracellular metabolites is nuclear magnetic resonance (NMR) spectroscopy. In vivo determination of metabolites and metabolic processes by NMR spectroscopy serves as a vital tool for metabolic flux analysis and metabolic engineering (145). Fourier-Transform-Near-Infrared (FTNIR) spectroscopy, a standard technique used in the food industry, is now used in biotechnology for online process monitoring (146). Artificial or electronic noses, with their broadband sensing capabilities, prove useful for the online monitoring of both airborne and liquid borne compounds. With the aid of artificial nose technology it may be possible to devise two array sensors to monitor the concentrations of various important components in both the gas and liquid phases in the fermentor, eliminating the need to use a large number of sensors to monitor complex mixtures (147).

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  • margaret
    Why are chemostat not widely used to produce commercial products?
    2 years ago
  • elias
    What is Chemostart an application of Industrial Fermentation process?
    2 years ago

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