TYPES OF FERMENTATION

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Fermentation processes are broadly divided into two parts viz: liquid fermentation and solid fermentation. In liquid fermentation, the microbial cells are suspended in an aqueous nutrient medium. But in solid fermentation, the microbial cells are adsorbed to a solid and nutrient rich material that supports the growth of the organism. The volume of free liquid or aqueous medium is minimal in solid fermentation processes unlike in liquid fermentation processes where the free liquid is large. Only liquid fermentation processes will be elucidated in this section – since they are mostly applied in many fermentation activities. Fermentation occurs when microbes including yeasts, actinomycetes, moulds and bacteria consume, breakdown or metabolize organic substrate molecules as part of their own metabolic process; and in the process produce desired end-products or metabolites that are of great economic importance.

The growth of microbes in food is considered a problem especially when such microbial activities result in disease outbreak (as is obtainable in food borne diseases) and food spoilage. Nevertheless, some fermentative processes spurred by microbial activities are important to man, animals and the environment because they result in the production of desirable end-products including fermented foods, beverages, drugs and alcohols. These fermentative activities are usually carried out in bioreactors that support the growth of microbes via nutrient supplementation or addition. Fermentation processes are usually classified according to the ways or manner in which the substrate or fermentation nutrients are added and the desired end-product recovered or harvested from the process. To this end, there are usually different types of fermentation processes used in industrial/biotechnological productions especially those industrial processes that has to do with the production of foods, alcoholic beverages, drugs and pharmaceutical products. Batch fermentation, fed-batch fermentation, continuous fermentation and semi-continuous fermentation are usually the major types of liquid fermentation processes used in industrial microbiology productions.

BATCH FERMENTATION

Batch fermentation is defined as the liquid fermentation process in which the culture is inoculated into a sterile medium contained in a closed vessel. In batch fermentation process, there is no addition of nutrients once the fermentation process starts. This type of fermentation process can also be known as a closed-culture system – since there are no nutrient additions once the process is started. Environmental and/or physical parameters vital for the growth of the organism, such as pressure, temperature, pH and aeration (oxygen supply) are usually controlled and regulated in batch fermentation process.

In batch fermentation, the growth nutrients and other additives for the fermentation process are added in the required amounts in the beginning of the fermentation. Once the sterile medium in the fermentation vessel is inoculated with the appropriate culture or microorganism of interest, it passes through a number of growth phases including lag phase (where there is no growth; but acclimatization of the organism to the new environment it was introduced to); log/exponential phase (where there is increased microbial growth and buildup of microbial biomass); stationary phase (where growth ceases because of nutrient depletion) and decline/death phase (where the cells begin to lose their viability). The microorganisms grow at a rapid (exponential) rate due to the availability of excess nutrients in the fermentation vessel. There is no refill of nutrients once the fermentation process has started and the end-product is recovered at the end of the process. The microbes increase in number with rapid use of the available nutrients and simultaneously produce toxic metabolites. The growth of the microbial cells slow down during the end of the fermentation process; and this is usually due to nutrient depletion and the buildup of toxic metabolites – which affect microbial growth.

Once the batch fermentation process is completed, the fermentation vessel is cleaned properly, and then sterilized before it is used for another batch fermentation process. It is noteworthy that oxygen is usually added in a batch fermentation system (to activate and spur the growth of aerobic microbes). Anti-foaming agents (which takes care of foam formation) and acids or bases (which controls the pH) of the fermentation vessels can also be added even though nothing is usually added in the entire course of a batch fermentation process (closed-culture system). Batch fermentation is usually applied in fermentation processes in which one fermenter is used to make various products; in fermentation processes in which only small amounts of the desired product is produced; and in fermentation processes in which the product must be produced with minimal risk of possible contamination or any alteration in the genetic makeup of the microorganism. Generally, batch fermentation is characterized by an initial charging of the fermentation media with an appropriate inoculant (microorganism); and the product is withdrawn or recovered at the end of the fermentation process – without any addition of nutrient during the process. Batch fermentation is characterized by some merits and demerits.

MERITS OF BATCH FERMENTATION

  1. Batch fermentation is more flexible with many biological systems and/or products.
  2. There is reduced risk of contamination of the process – since nutrients are only added once at the start of the process.
  3. Mutation of the microbial cell is minimal since the process does not last for a long time.
  4. Batch fermentation process is cost effective and thus requires low capital to set up.
  5. There is usually a higher conversion of the raw materials in batch fermentation systems than in other fermentation processes. And this is usually because the growth process in batch (closed-culture) fermentation process is controlled.

DEMERITS OF BATCH FERMENTATION

  1. The frequent sterilization of the fermentation vessel used for batch fermentation makes the process to have an increased focus on instrumentation.
  2. Batch fermentation is capital intensive since it requires more labour and resources for process control.
  3. There is also a lower productivity levels as a result of the time it takes to sterilize, clean, empty and fill the fermentation vessel.
  4. Several subcultures are usually prepared for inoculating a batch fermentation vessel; and this increases the cost of operating the process.

FED-BATCH FERMENTATION

Fed-batch fermentation is defined as the liquid fermentation process in which growth nutrients are periodically added in the fermentation medium during fermentation. The process is akin to batch fermentation process except that small concentrations of nutrients are added at the beginning of the process, and there is a continuous addition of nutrients during the process in small amounts. In fed-batch fermentation, there is usually a predetermined or controlled addition of nutrients into the bioreactor at certain times of fermenter operations; and the process allows a temporal variation in the supply of growth nutrients to the culture. Fed-batch fermentation resembles batch fermentation and continuous fermentation systems but they all show variations in their mode of operations. Fed-batch fermentations are most suited for the production of compounds produced by organisms that exhibit slow growth. This system of fermentation is also useful when the microbial biomass or product yield is highest at low substrate concentrations. Fed-batch fermentations can also be used when the product formation is dependent on a specific nutrient composition such as a carbon to nitrogen ratio.

Fed-batch fermentations allow the operators to adjust the nutrient inflow into the vessel in such a way that it matches the current physiological state of the microbial cell; and it is widely used in most fermentation industries. They are suitable for the manufacture or production of recombinant proteins, baker’s yeasts, enzymes, antibiotics, amino acids, organic acids, growth hormones, vinegar, antibiotics and amylase. With fed-batch fermentation, the high concentration of microorganisms and possible feedback inhibition (catabolite repression) of the process could be controlled and reduced respectively. There is an increased production of bio-products in fed-batch fermentations since the exponential and stationary phase of growth is usually lengthened. A fed-batch fermentation is useful in achieving high concentration of products as a result of high concentration of cells for a relative large span of time; and it is 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 of the fermentation process. Fed-batch fermentation like batch fermentation process has advantages and disadvantages.

MERITS OF FED-BATCH FERMENTATION

  1. There is usually an increased opportunity for optimizing the environmental conditions of the organisms in line with their phases of growth.
  2. There is a higher yield of the product. And this is attributed to the well-defined cultivation period during which time no cells is added or removed.
  3. The fed-batch fermentation system is suitable for mutating microorganisms and those at risk of contamination since the process can be operated in a stationary state.

DEMERITS OF FED-BATCH FERMENTATION

  1. In fed-batch fermentation, a specific growth rate cannot be maintained.
  2. The dynamic process of control carried out in fed-batch fermentation systems makes the process to be capital intensive.
  3. There is usually a lower productivity levels experienced in this system of fermentation due to the time it takes to fill, sterilize, clean and empty the vessel.

CONTINUOUS FERMENTATION

Continuous fermentation is defined as the fermentation process in which sterile growth nutrients are added continuously to the fermentation vessel and an equal amount of converted nutrient solution (end-product) with microorganisms is simultaneously harvested in the process. It is also known as an open culture system – since it allows nutrients to be continuously added during the fermentation process. In batch cultures, nutrients are not renewed and so growth remains exponential for only a few generations. But this is not the case in continuous fermentations – in which a steady state or balance microbial growth can be obtained. Microbial population can be maintained in a state of exponential growth for a long time by using a continuous fermentation system or culture. The exponential growth of the culture is continuous until a time when the fermentation vessel is completely filled with the fermentation media.

The balanced microbial growth obtainable in continuous culture systems is maintained by supplying growth medium continuously; and the growth medium is designed or compounded in such a way that microbial growth is restricted by substrate and not by toxin buildup. And this allows an exponential growth of the organism by addition of new fresh medium. The rate of addition of fresh growth medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. The chemostat and turbidostat are typical examples of continuous fermentation systems used for the continuous culture of microbes in the laboratory. Chemostat is a continuous fermentation apparatus that feeds nutrient medium into the culture vessel at the same rate as the medium containing the microorganisms is removed (Figure 1). The medium in a chemostat contains one essential growth nutrient (e.g. amino acid) in a limiting amount; and because a sub-maximal amount of the essential growth nutrient is used at any given time, a constant population of the microbial cell is maintained in a constant volume.

Figure 1. Illustration of a chemostat.

Turbidostat is a continuous fermentation system that is fitted with a photocell that adjusts and regulates the flow of nutrient medium through the culture vessel in order to maintain a constant cell density or turbidity (Figure 2). Unlike the chemostat where the nutrient is limited, the nutrients are present in excess amounts in the turbidostat; and the cell density (turbidity of the medium) is monitored by the photocell device – which translates any change in turbidity to a mechanism that automatically reduces or increases the rate of the nutrient inflow and broth outflow as deemed necessary.

Figure 2. Illustration of a turbidostat set-up. 1. Reservoir of sterile nutrient. 2. Valve controlling flow of medium. 3. Photo cell. 4. Light source. 5. Outlet for spent medium.

The turbidostat system consists of an optical-sensing device (the photo cell) which measures the absorbance of the culture density (turbidity) in the growth vessel. Changes in turbidity retard (or increase) passage of light (from the photo cell) through the culture; and these changes activate mechanisms that control the flow of nutrients in the turbidostat system as well as the flow of waste materials out of the main culture vessel. Chemostat and turbidostats are the two types of continuous fermentation vessels used to ensure steady states of microbial cells during a fermentation process. In the chemostat, the steady state is ensured by adjusting the concentration of one substrate; and this controls the cell growth. Cell growth is kept constant in the turbidostat by using turbidity to monitor the biomass concentration and the rate of inflow of nutrients is also adjusted in the process. A constant chemical environment is maintained in a chemostat while a constant cell concentration is maintained in a turbidostat.

The parameters that initiate a stationary phase of growth in a fermentation vessel include nutrient depletion, accumulation of toxic substances and accumulation of excess cells in the vessels. These parameters are usually out ruled in the open system of culture since nutrients are continuously added to the system to maintain a steady microbial growth over a long period of time. Generally, continuous fermentation involves addition of substrates in an unbroken stream and the withdrawal of fermentation medium in the same manner. Continuous fermentation has advantages and disadvantages.

MERITS OF CONTINUOUS FERMENTATION

  1. Continuous systems allow microbial growth to be regulated and maintained over a long period of time.
  2. It provides opportunity for studying the metabolic processes of organisms.
  3. It can be used to obtain a steady-state of microbial growth.
  4. Continuous culture results in higher productivity per unit volume. And this is because time consuming tasks such as sterilization, filling, cleaning and emptying of the vessel are out ruled.
  5. It results in the production of high quality products because of the steady-state of continuous culture.

DEMERITS OF CONTINUOUS FERMENTATION

  1. The major drawback in the use of continuous fermentation is in the nature of its operation. If the fermentation step in a process is operated continuously, it is most desirable to have all other steps of preparation and product recovery also in continuous operation. And in such a continuous process, a failure in one step will force a complete shutdown of the entire process.
  2. Continuous culture systems are prone to contamination because it is operated over a long period of time.
  3. It is not reliable in maintaining the growth of filamentous organisms such as fungi because of the viscosity and heterogeneous nature of the mixture in the vessel.
  4. The original product strain could be lost over time during the fermentation process; and this usually occurs when a faster growing strain overtakes it.
  5. Steady-state of microbial growth could be prevented in continuous cultures due to wall growth and cell aggregation in the fermentation vessel.

SEMI-CONTINUOUS FERMENTATION

Semi-continuous fermentation is defined as the fermentation process in which the substrate is added and the product removed at intervals. It combines some of the features of batch and continuous fermentation. In semi-continuous cultures, a fixed volume of the fermented medium is usually taken out from the fermentation vessel, and the same volume of nutrients is simultaneously added to the fermenter – in order to keep the volume of the fermentation medium at the same level as well as replenish the depleted nutrients for microbial growth. Semi-continuous fermentation process can be used to maintain the microorganisms in the same phase of growth over some period of time.

SIGNIFICANCE OF FERMENTATION

  • Fermentation increases the shelf-life of a finished product.
  • It improves the varieties of tastes and flavours of finished products.
  • It improves the nutritional value of foods and beverages.
  • It decreases the toxicity of finished products.
  • It improves the therapeutic value of finished products. For example, some fermented products such as yoghurts contain some beneficial microbes that improve the health status of the gut flora; and this goes a long way in improving the general health of the body.
  • Fermentation increases digestibility in the host when fermented finished foods are consumed.
  • Fermentation processes produce substances such as acids and alcohols that inhibit the overgrowth of spoilage and pathogenic microbes in the food.
  • Fermentation processes produce industrially useful end-products such as organic acids, alcohols, citric acid, alkaloids, aldehydes and ketones that are used for the production of other important products. For example, citric acid is used in the food industry, in medicine, in pharmacy and in various other industries. It is used in the food industry for example, as a major food acidulant used in the manufacture of soft drinks and sweets. It is used in medicine as sodium citrate in blood transfusion to prevent blood clotting. Citric acid has a low pH, and thus it is used in hair rinses, after shave lotions and in wig setting fluids.  

Further reading

Bushell M.E (1998). Application   of   the   principles   of   industrial   microbiology   to   biotechnology (ed. Wiseman, A.) Chapman and Hall, New York.

Byong H. Lee (2015). Fundamentals of Food Biotechnology. Second edition. Wiley-Blackwell, New Jersey, United States.

Frazier W.C, Westhoff D.C and Vanitha N.M (2014). Food Microbiology. Fifth edition. McGraw-Hill Education (India) Private Limited, New Delhi, India.

Jay J.M (2005). Modern Food Microbiology. Fourth edition. Chapman and Hall Inc, New York, USA.

Bushell M.E (1998). Application   of   the   principles   of   industrial   microbiology   to   biotechnology (ed. Wiseman, A.) Chapman and Hall, New York.

Farida A.A (2012). Dairy Microbiology. First edition. Random Publications. New Delhi, India.

Nduka Okafor (2007). Modern industrial microbiology and biotechnology. First edition. Science Publishers, New Hampshire, USA.

Roberts D and Greenwood M (2003). Practical Food Microbiology. Third edition. Blackwell publishing Inc, USA.

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