3 Main Stages Involved in Fermentation | Microbiology

Industrial fermentation processes require two most important stages to complete product fermentation. These are up-stream process and down-stream process, which are abbreviated as USP and DSP respectively.

Stage # 1. Up-Stream Process:

This process comprises of obtaining a desired microorganism, and its improvement so as to enhance the productivity and yield. It also includes the maintenance of strain purity, preparation of inocula and further efforts to improve the economic efficiency of the process.

There are two distinct processes involved in USP but both the processes run simultaneously. The medium is prepared for the culture (inoculum) preparation as well as for the production of desired product. The vessel of the fermenter is filled with production media and inoculated with suitable microbial culture for the industrial product formations as shown in Fig. 20.4.

Stage # 2. Down-Stream Process:

This process includes the selection of suitable fermentation media, optimization of important fermentation condition to give rise maximum yield. The main objective of this process is to get reproducibility of result as far as possible besides safe recovery of the target product.

Further processing is carried out depending upon whether the product is intracellular or present inside the cell or membrane bound or release outside the cell or extracellular in nature. This leads to recovery of the product in pure form.

The presence of undesirable products (by products), impurities in media, antifoam agents, etc. affect the DSP steps. Therefore, each step is required thorough check up in both USP as well as in DSP. For example, a cheap carbon and energy source may increase DSP cost.

At commercial level use of existing equipment’s, ancillaries, etc. are more advantageous than that of creating new facilities because the later step requires more investment which may lead to escalation of cost of the product and processes.

The DSP consists of series of distinct unity or processes which are connected to each other for getting the final product as shown in Fig 20.5.

For the precautionary measures it is necessary to keep this unit process minimum. This will not only save the cost of the plant but will also ensure not to loose the product during individual steps (Fig. 20.5). Now-a-days, most preferred DSP is to link with the fermentation. This increases productivity and reduce time and cost.

There are two types of major processes for product formation. The product formation is coupled with growth i.e. primary metabolites and another where product is formed after growth as in case of antibiotics. In case of primary metabolites, higher productivity is achieved by using integrated system of fermentation with DSP. This kind of system maintains a high cell density through cell retention of recycling.

If product is inhibitory, various methods have been employed to partition fermenter as in case of formation of glucose by breakdown of cellulose. This allows the rapid in-situ removal of product by extraction, adsorption or stripping.

The process can be ex-situ where the product is removed outside the fermenter and the processed medium is returned to the fermentation. Such processes have been successfully used for removing alcohols, solvents, proteins, etc. as shown in Fig. 20.5. Following are the major steps for the product recovery.

The following steps are employed for the recovery of the product(s) obtained after downstream processing:

(a) Cell Harvesting:

Since, microbial cells are in liquid medium or broth, solid-liquid separation method is influenced by the size and morphology of the microorganisms (single cells, aggregates or mycelia).

Further, the process also depends upon the location of product whether it is intracellular or extracellular it has been secreted into the medium. Besides, other factors such as specific gravity, viscosity, and rheology of the medium also influence cell harvesting method.

(b) Broth Conditioning Technique:

This technique allows separation of cells from large volume of liquid medium. In this case, cells are allowed to form floccules or precipitate by using chemical, physical and biological treatments.

Coagulating materials such as simple electrolytes, acids, bases, salts, multivalent ions etc. are added to form small floes from dispersed colloids or suspended materials. Certain floes precipitation methods are also used at the end of many traditional beer and wine fermentation processes for the precipitation of yeast cells.

(c) Sedimentation:

This process is widely used for yeast separation during alcohol production and in waste-water treatment. The rate of particle sedimentation is a function of both size and density. If, larger the size, greater its density, and the faster rate of sedimentation. Therefore, for quick separation, the difference in density between the particle and the medium must be large with low viscosity.

(d) Centrifugation:

This process allows small particles or cells to separate from liquid. It is also suitable for some liquid-liquid separations. Similar to sedimentation, its effectiveness also depends upon cell size, density difference between the cells and the medium, and its viscosity.

Higher speed of centrifugation requires for the separation of smaller microorganisms, such as bacteria. In case of yeast cells present in beer, relatively, low centrifugation effectively recovers residual yeast cells.

(e) Filtration:

There are certain filters such as clothes, glass wool or cellulose that retain the solids and pass on the liquid. The solids accumulate above the filter. Such technique is useful in case of fungal mycelia separation.

There are two types of filters used in industrial process: plate and frame filters or filter press, and rotatory vacuum filter, which help in harvesting the fungal mycelia during antibiotic manufacture, bakers yeast production and in dewatering of sewage sludge.

(f) Disruption of Microbial Cells:

When the product is intracellular, it is necessary to disrupt the cells/cell membrane so as to release the product. Cell disruption can be achieved by mechanical and non-mechanical methods. Some of the methods used are ultra-sonication and application of lysozyme. The ‘French-press’ is often used in laboratories, while Manton and Gaulin homogenizer (high pressure homogenizer) is employed for pilot scale cell disruption.

These are used for disruption of bacterial, yeast cells and fungal mycelia. The non-mechanical cell disruption is carried out by cell permeabilization. This can be accompanied by autolysis, osmotic shock, ruptures by ice crystal or heat shock treatment. Some organic solvents such as acetone, butanol, chloroform and methanol have been used to release enzymes from microorganisms.

Simple treatment with sodium dodecyl sulfate (SDS) or Triton X-100 is also found effective. Lysozyme is useful for Gram-positive organisms but addition of EDTA improves the effectiveness of lysozyme of Gram-negative bacteria. Some antibiotics namely penicillin and cycloserine may also be used to lyse the actively growing bacterial cells. Chitosan is effective for yeast cells.