Two basic approaches to the fed-batch fermentation can be ...



Basic Modes of Operation

Batch culture

Batch fermentation refers to a partially closed system in which most of the materials required are loaded onto the fermentor, decontaminated before the process starts and then, removed at the end. The only material added and removed during the course of a batch fermentation is the gas exchange and pH control solutions. In this mode of operation, conditions are continuously changing with time, and the fermentor is an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time.

The principal disadvantage of batch processing is the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermentor vessel, the cleaning, sterilization and re-start process.

Continuous culture

Continuous culture is a technique involving feeding the microorganism used for the fermentation with fresh nutrients and, at the same time, removing spent medium plus cells from the system. An unique feature of the continuous culture is that a time-independent steady-state can be attained which enables one to determine the relations between microbial behavior (genetic and phenotypic expression) and the environmental conditions.  

Fed-batch processes

The fed-batch technique was originally devised by yeast producers in the early 1900s to regulate the growth in batch culture of Saccharomyces cerevisiae. Yeast producers observed that in the presence of high concentrations of malt, a by-product - ethanol - was produced, while in low concentrations of malt, the yeast growth was restricted. The problem was then solved by a controlled feeding regime, so that yeast growth remained substrate limited.

The concept was then extended to the production of other products, such as some enzymes, antibiotics, growth hormones, microbial cells, vitamins, amino acids and other organic acids. Basically, cells are grown under a batch regime for some time, usually until close to the end of the exponential growth phase. At this point, the reactor is fed with a solution of substrates, without the removal of culture fluid. This feed should be balanced enough to keep the growth of the microorganisms at a desired specific growth rate and reducing simultaneously the production of by-products (that can be growth or product production inhibitory and make the system not as effective).

These by-products may also affect the culture environment in such a way that might lead to early cell death even though sufficient nutrients are available or are still being provided.

A fed-batch is useful in achieving high concentration of products as a result of high concentration of cells for a relative large span of time. Two cases can be considered: the production of a growth associated product and the production of a non-growth associated product. In the first case, it is desirable to extend the growth phase as much as possible, minimizing the changes in the fermentor as far as specific growth rate, production of the product of interest and avoiding the production of by-products.

For non-growth associated products, the fed-batch would be having two phases: a growth phase in which the cells are grown to the required concentration and then a production phase in which carbon source and other requirements for production are fed to the fermentor.

This case is also of particular interest for recombinant inducible systems: the cells are grown to high concentrations and then induced to express the recombinant product.

Also, considering that plasmid stability is very often guaranteed by the presence of an antibiotic marker gene and that the lifetime of this antibiotic in a fermentor can be limited, it might be of interest to use the fed-batch concept to feed this same antibiotic continuously so that the presence of the plasmid in the cells is more of a reliable fact.

Fed-batch fermentations can be the best option for some systems in which the nutrients or any other substrates are only sparingly soluble or are too toxic to add the whole requirement for a batch process at the start.

Finally, in fermentations such as mycelial culture the increase of viscosity with time can be compensated by the addition of relatively small quantity of water during the fermentation time, although the efficacy of this protocol is controversial among reserachers.

Many factors are involved in the regulation of a fed-batch reactor. This topic will be discussed in section 3.7. As an example, however, the feed rate can be varied to control the concentrations of nutrients in the bioreactor.

Fed-batch processes

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. The kinetics of the two types of fed-batch culture will be described in section 3.6.

Fixed volume fed-batch

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 3.6). 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.

Variable volume fed-batch

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) 21.

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.

Advantages and disadvantages of the fed-batch reactors  

Fed-batch fermentation is a production technique in between batch and continuous fermentation. A proper feed rate, with the right component constitution is required during the process.

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 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.

 

As a summary of what was described above, fed-batch mode of fermentation has the following features:

Advantages:

• production of high cell densities due to extension of working time (particularly important in the production of growth-associated products)

• controlled conditions in the provision of substrates during the fermentation, particularly regarding the concentration of specific substrates as for ex. the carbon source

• control over the production of by-products or catabolite repression effects due to limited provision of substrates solely required for product formation

• the mode of operation can overcome and control deviations in the organism's growth pattern1 as found in batch fermentation

• allows the replacement of water loss by evaporation

• alternative mode of operation for fermentations leading with toxic substrates (cells can only metabolize a certain quantity at a time) or low solubility compounds

• increase of antibiotic-marked plasmid stability by providing the correspondent antibiotic during the time span of the fermentation

• no additional special piece of equipment is required as compared with the batch fermentation mode of operation

       Disadvantages:

• 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

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