phase and may overcome the problems associated with the use of rapidly metabolizable substrates. This method is also useful in cases where the initial viscosity of the medium is too high to permit higher substrate concentration or where the substrate is toxic to the fermenting organism at high concentration. In some cases, selective cell recycle is also possible with fed batch technique. Fed batch technique is successfully used in production of products like baker’s yeast. So, in a nutshell, the benefits offered by fed batch
fermentation are as follows:
Fed-batch offers many advantages over batch and continuous cultures. From the concept of its implementation it can be easily concluded that under controllable conditions and with the required knowledge of the microorganism involved in the fermentation, the feed of the required components for growth and/or other substrates required for the production of the product can never be depleted and the nutritional environment can be maintained approximately constant during the course of the batch. The production of by- products that are generally related to the presence of high concentrations of substrate can also be avoided by limiting its quantity to the amounts that are required solely for the production of the biochemical. When high concentrations of substrate are present, the cells get “overloaded”, this is, the oxidative capacity of the cells is exceeded, and due to the Crabtree effect, products other than the one of interest are produced, reducing the efficacy of the carbon flux. Moreover, these by-products prove to even “contaminate” the product of interest, such as ethanol production in baker’s yeast production, and to impair the cell growth reducing the fermentation time and its related productivity.
Sometimes, controlling the substrate is also important due to catabolic repression. Since this method usually permits the extension of the operating time, high cell concentrations can be achieved and thereby, improved productivity [mass of product/ volume/time]. This aspect is greatly favored in the production of growth-associated products.
Additionally, this method allows the replacement of water loss by evaporation and decrease of the viscosity of the broth such as in the production of dextran and xanthan gum, by addition of a water-based feed.
As previously mentioned, fed-batch might be the only option for fermentations dealing with toxic or low solubility substrates.
When dealing with recombinant strains, fed-batch mode can guarantee the presence of an antibiotic throughout the course of the fermentation, with the intent of keeping the presence of an antibiotic-marked plasmid. Since the growth can be regulated by the feed, and knowing that in many cases a high growth rate can decrease the expression of encoded products in recombinant products, the possibility of having different feeds and feed modes makes fed-batch an extremely flexible tool for control in these cases.
Because the feed can also be multisubstrate, the fermentation environment can still be provided with required protease inhibitors that might degrade the product of interest, metabolites and precursors that increase the productivity of the fermentation.
Finally, in a fed-batch fermentation, no special piece of equipment is required in addition to that one required by a
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batch fermentation, even considering the operating procedures for sterilization and the preventing of contamination.
` A cyclic fed-batch culture has an additional advantage: the productive phase of a process may be extended under controlled conditions. The controlled periodic shifts in growth rate provide an opportunity to optimize product synthesis, particularly if the product of interest is a secondary metabolite whose maximum production takes place during the deceleration in growth.
• So fed batch fermentations is an ideal mode for many products. All advantages and no drawbacks, is it? Unfortunately, no. The science of fed batch fermentations also comes with several disadvantages. Some of them are as follows:
• it requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity
• it requires a substantial amount of operator skill for the set- up, definition and development of the process
• in a cyclic fed-batch culture, care should be taken in the design of the process to ensure that toxins do not accumulate to inhibitory levels and that nutrients other than those
incorporated into the feed medium become limiting, Also, if many cycles are run, the accumulation of non-producing or low-producing variants may result.
• the quantities of the components to control must be above the detection limits of the available measuring equipment
• Right. Are there different types of fed batch fermentations, too?
Sure there are. Two basic approaches to the fed-batch fermentation can be used: the constant volume fed-batch culture - Fixed Volume Fed-Batch - and the Variable Volume Fed-
Batch. We will see the kinetics of the two types of fed-batch
culture subsequently. First let’s see what constant volume or fixed volume fed batch fermentations are.
In this type of fed-batch, the limiting substrate is fed without diluting the culture.
The culture volume can also be maintained practically constant by feeding the growth limiting substrate in undiluted form, for example, as a very concentrated liquid or gas (ex. oxygen).
Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiation can be the growth limiting factor without affecting the culture volume.
A certain type of extended fed-batch - the cyclic fed-batch culture for fixed volume systems - refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the starting point for a further fed-batch process. Basically, once the fermentation reaches a certain stage, (for example, when aerobic conditions cannot be maintained anymore) the culture is removed and the biomass is diluted to the original volume with sterile water or medium containing the feed substrate. The dilution decreases the biomass concentration and result in an increase
in the specific growth rate (see mathematical description in section 6). Subsequently, as feeding continues, the growth rate will decline gradually as biomass increases and approaches the maximum sustainable in the vessel once more, at which point the culture may be diluted again.
• Ok, now tell me about variable volume fed batch fermentations.
As the name implies, a variable volume fed-batch is one in which the volume changes with the fermentation time due to the substrate feed. The way this volume changes it is
dependent on the requirements, limitations and objectives of the operator.
The feed can be provided according to one of the following options:
(i) the same medium used in the batch mode is added; (ii) a solution of the limiting substrate at the same
concentration as that in the initial medium is added; and (iii) a very concentrated solution of the limiting substrate is
added at a rate less than (i), (ii) and (iii) .
This type of fed-batch can still be further classified as repeated
fed-batch process or cyclic fed-batch culture, and single fed-batch process.
The former means that once the fermentation reached a certain stage after which is not effective anymore, a quantity of culture is removed from the vessel and replaced by fresh nutrient medium. The decrease in volume results in a increase in the specific growth rate, followed by a gradual decrease as the quasi- steady state is established.
The latter type refers to a type of fed-batch in which supplementary growth medium is added during the fermentation, but no culture is removed until the end of the batch. This system presents a disadvantage over the fixed volume fed-batch and the repeated fed-batch process: much of the fermentor volume is not utilized until the end of the batch and consequently, the duration of the batch is limited by the fermentor volume.
• All right. Now are there any special considerations to be made about fed batch fermentations?
Yes. First and foremost is the fermentation vessel or equipments used for fermentation. Actually no special piece of equipment is required over the equipment required for batch. However, some considerations should be made over the equipment used for fed-batch fermentation.
The vessels, particularly those used for the acid and base control, must be constructed from a non-toxic, corrosion- resistant material which is capable of withstanding repeated sterilization cycles. Figure 4.1. Illustrates two methods of assembling vessels for easy transfer of either inoculum or medium to the fermentor.
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Figure.4.1. Holding vessels. A. Screw-neck borosilicate glass vessel with medium/inoculum addition assembly. (a) Stainless steel rod; (b) Silicon tubing; (c) Silicon disc; (d) Hypodermic needle; (e) Air vent; (f) Screw cap; (g) Magnetic bar. B. Aspirator- type vessel for introducing an inoculum of filamentous fungi into the fermentor. (a) Cotton-wool plug; (b) Magnetic stirrer bar.
• What would be the next consideration to be made? Next would be the pumps required for the fermentation. There are two types of pumps which are suitable for the aseptic pumping of small volumes of culture media: the peristaltic pump and the diaphragm-dosing pump. Other pumps are unsuitable because they are difficult to sterilize and cannot be used for pumping small volumes.
The peristaltic pump is typically constituted by a main body that comprises both the drive motor and electrics, and the rotating unit of rollers. This unit of rollers occludes the tube which, as it recovers to its original size passes to the nest roller until is expelled, as the unit moves round. The flow rate can be varied by either the speed setting or by changing the diameter of the tube being used.
The diaphragm-dosing pump consists of a main body and a detachable heat-sterilizable head. The fluid is sucked in to the pump head. The suction inlet tube then closes and the pressure discharge tube opens and forces the fluid out. The suction and pressure forces in the pump head are generated by the reciprocating action of both the diaphragm plunger and the return spring.
• That was about instrumentation. Now tell me about the control part of the fermentation.
Sure.
In case of fed batch fermentations, the control system adapted is called
adaptive control. It is the name given to a control system in which the controller learns about the process by acquiring data from a certain process and keeps on updating a control model. A parameter estimator monitors the process and estimates the process dynamics in terms of the parameters of a previously defined mathematical model of the process. A control design algorithm is then used to generate controller coefficients from those estimates, and a controller sets up the required control signals to the devices controlling the process. An extremely important feature of an adaptive
controller is the structure of the model used by the parameter estimator to analyze estimates of process dynamics. The process can be described by a set of mass balance equations, whose quantities can be measured directly or indirectly. The following figure describes schematically the concept.
Figure.5.1. Adaptive control: the controller compares the estimates from a mathematical model applied to the system to the readings obtained from the fermentation process. The controller then sends the signal to the device controlling the fermentation, for example, by increasing or decreasing a flow rate.
The optimal strategy for the fed-batch fermentation of most organisms is to feed the growth-limiting substrate at the same rate that the organism utilizes the substrate; this is, to match the feed rate with demand for the substrate.
This can be compared to making a boy work and feeding him such amount of food that will generate exactly the same amount of energy required for that work!
Four basic approaches have been used in attempts to balance substrate feed with demand (listed in order of increasing accuracy and/or complexity):
(i) open-loop control schemes in which feed is added according to historical data or predicted data;
(ii) indirect control of substrate feed based on non-feed source parameters such as pH, offgas analysis, dissolved O2 or concentrations of organic products;
(iii) indirect control schemes based on mass balance equations, the values of which are calculated from data obtained by sensors; and
(iv) direct control schemes based on direct, on-line measurements substrates.
Better and more flexible control may be obtained when there is direct measurement of substrate or an excreted metabolite in the medium, which can be used to influence feeding rates to the fermentation. This can be done off-line or semi-on-line, but on-line measurements are more useful because of
• the shorter analysis required,
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• a reduced chance of fermentor contamination.
Regardless the type of control, the design is strongly influenced by both mathematical model availabilities and measurement possibilities.
• Tell me, if the direct analysis of the control parameters is available, why do we resort to the mathematical models?
Control and optimization of bioreactors is strongly influenced by the quality of the sensors available for crucial response variables. Of primary importance is the ratio of the dynamic parameters of the sensor to those of the process. When these variables cannot be measured easily or quickly enough, a mathematical model must be used in some way in place of feedback information.
When an exact mathematical model is at disposal, an open-loop process control can be proposed which generally proves to be insufficient. The advantage of a feedback control is that a response to unforeseen and unexpected conditions during the fermentation is achieved and the process is controlled within the desired limits.
An indirect feedback control utilizes an observable parameter, such as dissolved oxygen, pH, respiratory quotient, and partial pressure of CO2, culture fluorescence or by-product formation, which is closely related to the course of microbial fermentation. As examples of fed-batch systems using this concept, one can mention the pH-stat - a system in which the feed is provided depending on the pH, - and the DO-stat - a system in which the feed is provided depending on the reading of the dissolved oxygen.
A direct feedback controller uses the concentration of limiting substrate in the culture medium as a feedback feed -related parameter for control. A direct feedback control can have the disadvantage of not being very feasible due to the difficulty associated with obtaining accurate on-line measurements of substrate concentrations or even by the absence of on-line sensors for the important compound to control. The advantage of a feedback control is that a response to unforeseen and unexpected conditions during the fermentation is achieved and the process is controlled within the desired limits.
A feedback control can be implemented accordingly to not only a single measurement, but also to obtain a finer control action in a dual-level system. Turner at al., describes a control method applied to a fed-batch culture of recombinant Escherichia coli in which a two-level control was preferred because it provided much greater flexibility and better control over the substrate concentration in the medium and the production of by- products.
As compared with the batch fermentation, two more parameters need to be specified to determine the operating conditions of a fed-batch fermentation: feed and initial feeding time.
• All right. Now tell me how we develop mathematical formulae for fixed volume fed batch fermentation. See, in developing the mathematical models for fixed volume fed batch fermentations, we have to assume the following:
• The feed is provided at a constant rate
• The production of mass of biomass per mass of substrate is constant during the fermentation time and
• A very concentrated feed is being provided to the fermentor in such a way that the change in volume is negligible. The equations that describe the system in terms of specific growth rate, biomass and product concentration (for both growth and non-growth associated products) with time are the following:
Mathematical modelling of fixed volume fed-batch.
Parameter Equation Equation # Specific Growth Rate u = (F . Yx/s) / x (3.6.1.1)
Biomass (as a function
of time) xt = xo + F . Y x/s . t (3.6.1.2) Product Concentration (non-growth associated) P= Pi + qp . xo . t + qp . F . Y x/s . t2 /2 (3.6.1.3) Product Concentration (non-growth associated) P= Pi + rp . t (3.6.1.4)
• x is the biomass [mass biomass/volume]
• xo is the biomass in the beginning of the fermentation [mass biomass/volume]
• t is time
• F is the substrate feed rate [mass substrate/(volume.time)] and
• Y x/s is the yield factor [mass biomass/mass substrate]
• u is the specific growth rate [time-1]
• P is the product concentration {mass product/volume] and
• qp is the specific production rate of product [mass product/ (mass biomass . time)
• rpis the product formation rate [mass product/(volume . time)]
From equations (6.1.1) and (6.1.2), it can be observed that (i) the specific growth rate decreases with time because the
biomass (in the denominator) is increasing with time and (ii) the biomass increases linearly with time.
The product variation with time will depend on its being growth or non-growth associated, this is, will depend on qp (specific product formation defined as the product formation rate divided by the biomass) being dependent on the specific growth rate or not, respectively.
The following figure depicts the typical behavior of a fixed- volume fed-batch culture.
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Time profiles for a fixed-volume fed-batch culture. u= specific growth rate, x = biomass concentration, S(GLS) = growth limiting substrate, SN = any other substrate other than the S(GLS), P(nga) is the non-growth associated product and P(ga) is the growth associated profile for product concentration.
• And how about the variable volume fed batch fermentations?
In variable volume fed-batch fermentation, an additional element should be considered: the feed. Consequently, the volume of the medium in the fermenter varies because there is an inflow and no outflow. Again, it is going to be considered that the growth of the microorganism is limited by the concentration of one substrate.
For the mathematical developments that will be presented, the assumptions are
• Specific growth rate is uniquely dependent on the concentration of the limiting substrate
• The concentration of the limiting substrate in the feed is constant
• The feed is sterile
• The yields are constant during the fermentation time The following table summarizes the equations that apply to this situation. These relations are the base for all further calculations and specific cases of variable volume fed-batch fermentation.
Mass balances for the main components for a fed-batch reaction.
Component Mass Balance Equation Equation #
Overall F = dV/dt (3.6.2.1)
Biomass dx/dt = x . (u? . V -– Kd . V -– F) /
V (3.6.2.2)
Substrate ds/dt = F . (so -– s)/V -– u. x/ Yx/s (3.6.2.3) Product dP/dt = qp . x -–P . F / V (3.6.2.4)
• V is the volume of the fermentor
• t is the time
• F is the feed rate [volume/time].
• x is the biomass concentration [mass biomass/volume]
• u is the specific growth rate [time-1]
• Kd is the specific death rate [time-1]
• s is the substrate concentration in the fermentor [mass substrate/volume]
• so is the substrate concentration in the feed [mass substrate/ volume]
• Y x/s is the yield factor [mass biomass/mass substrate]
• P is the product concentration {mass product/volume] and
• qp is the specific production rate of product [mass product/ (mass biomass . time)
• What are the various control and analytical techniques used with fed batch fermentations?
There are many. Let’s see them one by one.