Fermentation Processes

Fermentation Processes

A) Batch fermentations

  • A tank of fermentor is filled with the prepared mash of raw material to be fermented. The temperature and pH for microbial fermentations is properly adjusted, and occasionally nutritive supplements are added to the prepared mash.
  • The mash is steam sterilized in a pure culture process. The inoculum of a pure culture is added to the fermentor from a separate pure culture vessel.
  • Fermentation proceeds and after the proper time the contents of the fermentor are taken out for further processing.
  • The fermentor is cleaned and the process is repeated. Thus, each fermentation is a discontinuous process divided into batches.

B) Continuous fermentation

  • Continuous fermentations are those in which fresh nutrient medium is added either continuously or intermittently to the fermentation vessel, accompanied by a corresponding continuous or intermittent withdrawal of a portion of medium for recovery of cells or fermentation product.
  • In continuous fermentation, the substrate is added to the fermentor continuously at a fixed rate. This maintains the organisms in the logarithmic growth phase. The fermentation products are taken out continuously.
  • But the design and arrangements for continuous fermentation is somewhat complex than batch fermentation.
  • Whereas in batch fermentation large volume of nutrient medium is inoculated and growth and biochemical synthesis are allowed to proceed only until maximum yields have been obtained. This is because of the limitation of one or more of the essential nutrients.
  • At this point, the batch fermentation is stopped for product recovery.

Methods of continuous fermentation – Continuous fermentation can be conducted in various ways. It can be carried out as-

i) Single-stage fermentation – In this type of fermentation, the fermentor is inoculated and then kept in continuous operation by balancing the input and output of nutrient solution and harvested culture, respectively.

ii) Recycle fermentation – In recycle continuous fermentation, a portion of the withdrawn culture or of unused residual substrate plus the withdrawn culture is recycled to the fermentation vessel.

iii) Multistage fermentation –Multiple stages continuous fermentation involves two or more stages with the fermentors which are operated in sequence. The fermentation is divided into two phases, i) growth phase occurs in the first fermentor followed by ii) synthesis phase in the second fermentor. The multiple-stage continuous fermentation is particularly applicable to those fermentations in which the growth and synthetic activities of the cells are not simultaneous i.e. product formation occurs only after the cell multiplication rate has slowed.

  • There are different ways in which microbial activity in continuous culture can be controlled.
  • This includes a chemostat and turbidostat. In both approaches it is necessary to maintain a constant cell population in fermentor or fermentors.
  • In this connection, the feed of fresh nutrients is criticalas it is related to the generation time of organism.
  • Very low flow rate of feed can allow the culture to go into maximum stationary phase of growth and thus the continuous aspect of fermentation will not be maintained.
  • Whereas very high flow rate can dilute the cell population that and thus the continuous aspect of fermentation will not be maintained.

Advantages

1) The productivity of continuous fermentation is greater than that for batch fermentation.

2) Continuous fermentation appears to be better because of two factors, because the fermentation equipment is in constant usage with little shutdown time and after initial inoculation, further production of inoculum is not required.

Disadvantages

1) It requires through knowledge of the dynamic aspects of microbial behavior and growth which is deficient in most of the industrial fermentation process because of the complexities of the growth and synthetic pattern of the organism.

2) Contamination and mutation in producing strain are the distinct problems for the development of successful continuous fermentation process.

3) Continuous fermentation often wastes nutrient substrate. A fermentation broth is continuously withdrawn for product recovery contains a certain amount of the residual unused nutrients of the medium as well as portion of the fresh medium which is being continuously added.

4) In a process which employs the use of viscous media, adequate mixing of nutrients and inoculum is not possible.

5) Continuous fermentations become more complex and difficult to accomplish when a chemical product and not a microbial cell is the desired product. This is because the conditions which are optimal for growth are not optimal for the formation of a chemical fermentation product.

Example – Antibiotic fermentations.

i) Chemostat

  • Chemostat maintains the nutrient feed and harvest culture withdrawal rates at constant values, but always less than that which allows a maximum growth rate.
  • The growth rate is controlled by supplying only a limiting amount of critical growth factor in the feed solution.
  • Thus, cell multiplication cannot proceed at a rate greater than that allowed by the availability of this critical nutrient.
  • The controlling factor for growth can be a high concentration of toxic product of the fermentation and the pH value of temperature of incubation.

Advantages

The chemostat operation of continuous fermentation is more often used that turbidostat operation because.

1) Fewer mechanical problems are encountered.

2) Occurrence of less residual unused nutrient in the harvested culture.

ii) Turbidostat

  • In the turbidostat, the total cell population is held constant by employing a device that measures the culture turbidity so as to regulate both nutrient feed rate to the fermentor and the culture withdrawal rate from the fermentor.
  • If a population rate rises above a predetermined level, a greater amount of fresh medium is added to the fermentor so as to dilute the cell concentration.
  • Thus, there is no limiting nutrient consciously imposed with this process so that the cell growth rate should always be maximal and the growth is maintained in logarithmic phase.
Fermentation Processes
Turbidostat

Disadvantages

1) Since the culture is to be maintained in logarithmic phase, the fermentation is operated at lower cell concentration and thus may require longer period.

2) This causes a greater loss of residual unused nutrient with the withdrawn harvested culture.

Applications of continuous culture

1) Turbidostat provides a constant source of cells in an exponential phase of growth, and they allow cultures to be grown continuously at extremely low concentration of substrate.

2) Growth at low substrate concentrations is valuable in studies on the regulation of synthesis or catabolism of the limiting substrate, in selection of various classes of mutants, and in ecological studies.

Fed-batch fermentation

Two basic approaches to the fed-batch fermentation can be used: the constant volume fed-batch culture – i) Fixed Volume Fed-Batch – and the ii) Variable Volume Fed-Batch.

i) 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.
  • 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.

ii) 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.
  • In this type of fermentation 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 an increase in the specific growth rate, followed by a gradual decrease as the quasi-steady-state is established.
  • 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.
  • In this type of fermentation, 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 a fresh nutrient medium.
  • The decrease in volume results in an increase in the specific growth rate, followed by a gradual decrease as the quasi-steady-state is established.

Single fed-batch process

This 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 of the fed-batch reactors

i) 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.

ii) 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.

iii) Sometimes, controlling the substrate is also important due to catabolic repression. Since fed-batch 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.

iv) 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.

v) Fed-batch process is ideal in fermentations dealing with toxic or low solubility substrates.

vi) In the case of recombinant strains, fed-batch mode can guarantee the presence of an antibiotic throughout the course of the fermentation. 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.

vii) Because the feed can also be multi-substrate, 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.

viii) In fed-batch fermentation, no special 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.

Disadvantages of the fed-batch reactors

i) 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.

ii) When high concentrations of a 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.

Advantages of cyclic fed-batch

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

ii) Controlled conditions in the provision of substrates during the fermentation, particularly regarding the concentration of specific substrates as for example 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 pattern as found in batch fermentation

ii) It allows the replacement of water loss by evaporation

iv) 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.

v) No additional special equipment is required as compared with the batch fermentation mode of operation.

Disadvantages of cyclic fed batch

i) It requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity

ii) It requires a substantial amount of operator skill for the set-up, definition and development of the process

iii) 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.

iv) The quantities of the components to control must be above the detection limits of the available measuring equipment.

C) Synchronous culture

Synchronous culture is a culture in which all cells divide simultaneously with constant division rate so that all the cells are in the same stage of growth phase and all the cell are dividing at the same time, this pattern of growth is known as synchronous growth.Many problems concerning cell multiplication which cannot be studied on conventional cultures may be solved by using the cells dividing more or less in similar ways. There are many laboratory methods by which one can manipulate the growth of cells so that they are all in same phase of their growth cycle.

Methods of obtaining synchrony

Several means of obtaining synchrony have been devised which are as follows —

I) Induction method ID Selection of cells

1) Low temperature

2) Effect of single temperature shift

3) Cyclic temperature shift

4) Thymine starvation

5) Inhibition of RNA and protein synthesis

6) Glucose starvation, periodic supply of growth factors

I) Induction methods

1) Low temperature

i) A log culture of Pseudomonas which is taken from37-25°C, held at low temperature for 155minutes has been found in phase with two respects-

a) during 5 minute, following 25°C about 2/3″ of the cells divide for two generations and thereafter cell division remain partially synchronized

b) after returning the cells to 37°C, their susceptibility to the transforming effect of Pneumococcal DNA change in cyclic manner.

ii) In case of Bacillus megaterium it was further observed that freezing young cells produce a characteristics compact nuclear composition and as well as synchrony uniformly in all cells. Thus a culture of Bacillus megaterium growing at 34°C which is cooled to 15°C and held for 30 minutes at that temperature and then returned to 34°C will divide synchronously after a lag of about 40 minutes. This division may be followed by a new lag and then a second burst of cell division. The cells will continue to grow but for many hours the cells will not divide and DNA: RNA ratio slowly increases and growth is underbalanced for some times.

2) Effect of single temperature shift

In case of Salmonella tphimurium when temperature is rapidly increased from 25°C to 37°C the following dissociation pattern occurs. The rate of cell vision remains unchanged for about 30 minutes, after 30 minutes a burst of cell division occur. The rate of RNA synthesis and mass increased almost immediately at that level. the DNA synthesis is greatly enhanced and the total DNA increase by about 50%.

3) Cyclic temperature shift (25°C to 37°C)

Cell division is particularly dependent upon temperature, so at lower temperature, growth of cells is arrested and gives a chance for more tardy members to catch with others i.e. to come toother’s level and when temperature is suddenly changed or raised, the barrier of cell division is lifted and virtually entire population proceed to divide so by repeating several stages entire population can be maintained. The primary effect of shift from 25°C to 37°C is specific stimulation of DNA synthesis.

Example – When a culture of Salmonella typhimurium is subjected at fixed intervals to alternate growth cycle at 25°C and 37°C. the entire population will divide synchronously. Cell mass increases at both temperature but rate at 25°C(20-30 minutes) is low as 37 °C (8 minutes) is closer to optimal one where the synchrony can persist for several generation.

4) Thymine starvation

  • This method is based on observation that certain proteins are required before a round of replication can be initiated. However, once started the round runs to completion even if the cell is temporarily unable to synthesize proteins.
  • The experiment can be done by starving the culture for a required amino acid (thymine) long enough for all cells to complete replication then the culture is switched over to the medium permitting protein but not DNA synthesis.
  • Immediate effect of withdrawal thymine is prevention of DNA synthesis without affecting RNA and protein synthesis.
  • During the second starvation of 30 min in glucose salt medium, protein synthesis is progressed thus preparing the cell for replication and if thymine is added back on right time.
  • Replication synchrony ensues and after short lag a burst of cell division observed, thus when protein synthesis is blocked, replication terminates very soon in some and much later in other cells.
  • The DNA: Mass ratio, therefore remain almost in the former, whereas if nearly doubled in cells that go through most of their replication cycle while starved of thymine.
  • Thymine starvation has certain effects in common with cyclic pretreatments to obtain division synchrony, in both systems pre-treatment allows cells to grow an abnormal and causes than to under large rapid division.

5) Inhibition of RNA and protein synthesis

  • In bacteria, DNA synthesis will continue for sometime after RNA and protein synthesis has been initiated.
  • This inhibition can be effected by transferring cells growing in rich media to minimal media by withdrawing required was or by addition of chloromphenicol to media.

a) DNA produced under protein and RNA inhibition represents the completion of alreadyinitiated replication.

b) It seems that protein and RNA synthesis is required to initiate new replication cycle.

This theory predicts that the cells with at the time of inhibition had almost finished replication and continue DNA synthesis for short time, whereas cells with had just began replication should continue synthesis for longer time and effectively double the DNA under conditions of inhibition of protein and RNA synthesis.

6) Glucose starvation

Division synchrony has been obtained in yeast culture which after glucose has been exhausted, diluted by factor of two with fresh medium at regular intervals. Division synchrony can be obtained by transferring cells to medium in which carbon source, glucose have been exhausted to fresh glucose salt medium. This lag regularly observed before first division. Transition from resting phase to exponential growth under certain condition can be made to above with fair degree of division synchrony. A combination of chilling and glucose starvation was used to obtain two rather than synchronized division cycle. Pretreatment may cause a fractionation of cell to keep division at certain time.

II) Selection of cells

1) Filtration (Hemstelfer Cumming’s)

This method involves the continuous collection of young cells coming from the lower surface of a filter onto which a parent population is adsorbed. In this Millipore cellulose acetate filter of same pore size are used. The pore size is so adjusted that it permits selectively passage of smallest cells of same size. The synchronous culture is observed on filter paper pad, then filter is removed and placed in such a way that bacteria facing to lower side, fresh medium is supplied, loosely attached cell will be washed out and attached cells remain and divide. Thus filtrate collects young cells of same size and age. But filtrate should be collected within 1 minute otherwise cell division will start and large age difference may occur.

2) Centrifugation (sucrose gradient)

When concentrated cell suspension of E.coli is layered on top of sucrose solution of specific gravity, 1.245 column then centrifuge, the smallest cell tends to accumulate at interface, immediately after centrifugation, large cell fraction is nonviable due to osmotic pressure but incubation in small without glucose for 20 minute result in partial recovery subsequent addition of glucose produce a fairly synchronous growth after a lag of 20 minutes. Because of the osmotic injury suffered during centrifugation, however this proved

Significance of synchronous culture

  • Measurement made on randomly dividing population. Do not permit any conclusion about the growth behavior of individual cells, because distribution of individual cell’s size and age is completely random but synchrony maintains entire population uniformly with respect to growth phase. Thus measurements made on such culture are similar to the measurement made on individual cells.
  • Synchrony facilitate to analyze growth behavior i.e. differentiation, organization, macromolecule synthesis of uniform culture which also implies same at individual cells level thus time course pattern ofvarious macromolecules (DNA/protein synthesis) synthesis can be studied by removing portions of synchronously dividing culture and analyzing the cells for the component or enzyme activity under investigation.
  • Usually synchrony is short lived but can be maintained indefinitely for longer time.

Applications of synchronous culture

1) Studies on kinetics of biosynthesis of macromolecules – Such induction to obtain synchronous growth is used in the study of kinetics of biosynthesis of macromolecules.

2) Studies on effect of disinfectant on macromolecules – Such culture can also be used in study of certain disinfectant effect on protein. DNA, RNA and other macromolecules.

3) Metabolism and research studies – Synchronous culture is useful for metabolism and research studies.


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  2. Design of a Fermentor
  3. Types of Fermentors
  4. Fermentation Media