Fermentation vessels

Commercial-scale operations are conducted in batch, fed-batch, or continuous culture systems. Fermentation vessels include the conventional baffled aerated tank, with or without impeller agitation, and the ak-lift tower fermentors in which ak is sparged into an annular space between the... 

A typical bourbon fermentation continues for 72 hours at a fermentation temperature within the 31—35°C range. Many fermentation vessels are equipped with agitation and/or cooling coils that facHitate temperature control. Significant increases in yeast numbers occur during the first 30 hours of fermentation. Over 75% of the carbohydrate is consumed and converted to ethanol. Within 48 hours, 95% or more of the ethanol production is complete. 

Biological reactors play a valuable role in tlie conversion of substrates by microorganisms and mammalian cells into a wide range of products such as antibiotics, insulin, and polymers. Figures 11-12, 11-13, and 11-14 illustrate various types of biological reactor, and Figure 11-15 shows the physical characteristics of a typical coimuercial fermentation vessel. 

This material was made up with distilled water to provide 41 g per liter, and the mixture was adjusted to pH 7.0 with potassium hydroxide solution. To the mixture were added per liter 5.0 g of calcium carbonate and 7.5 ml of soybean oil. 2,000 ml portions of this medium were then added to fermentation vessels, equipped with stirrers and aeration spargers, and sterilized at 121°C for 60 minutes. After cooling the flasks were inoculated with a suspension of strain No. ATCC 11924 of Streptomyces lavendulae, obtained from the surface of agar slants. The flasks were stirred for 4 days at 28°C at approximately 1,700 rpm. At the end of this period the broth was found to contain cycloserine in the amount of about 250 C.D.U./ml of broth. The mycelium was separated from the broth by filtration. The broth had a pH of about 7.5. Tests showed it to be highly active against a variety of microorganisms. 

Brewing Austenitic steels are used in fermentation vessels, storage tanks and attemperators, in yeast processing, and for transport tanks and barrels. 

Typical units for productivity are kg m 3 h 1. Factors that influence productivity include the production time of the fermentation, the time required to dean and set up the reactor, the sterilisation time and the length of the lag phase of growth. Figure 2.2 shows how total productivity and maximal productivity can be calculated for a batch fermentation. The dedsion as to when the fermentation is terminated (maximum or total productivity) depends on the operating costs, which include the capacity of the fermentation vessel, energy costs and labour costs. 

A bioreactor is a vessel in which an organism is cultivated and grown in a controlled manner to form the by-product. In some cases specialised organisms are cultivated to produce very specific products such as antibiotics. The laboratory scale of a bioreactor is in the range 2-100 litres, but in commercial processes or in large-scale operation this may be up to 100 m3.4,5 Initially the term fermenter was used to describe these vessels, but in strict teims fermentation is an anaerobic process whereas the major proportion of fermenter uses aerobic conditions. The term bioreactor has been introduced to describe fermentation vessels for growing the microorganisms under aerobic or anaerobic conditions. 

There several DO probes available. Some well-known branded fermenters, like New Brunswick, Bioflo series and the B. Braun Biotstat B fermenters are equipped with a DO meter. This unit has a 2 litre fermentation vessel equipped with DO meter and pH probe, antifoam sensor and level controllers for harvesting culture. The concentration of DO in the media is a function of temperature. The higher operating temperature would decrease the level of DO. A micro-sparger is used to provide sufficient small air bubbles. The air bubbles are stabilized in the media and the liquid phase is saturated with air. The availability of oxygen is major parameter to be considered in effective microbial cell growth rate. 

In a chemostat and biostat or turbidostat, even with differences in the supply of nutrients and/or fresh media, constant cell density is obtained. The utilisation of substrate and the kinetic expressions for all the fermentation vessels are quite similar. It is possibile to have slight differences in the kinetic constants and the specific rate constants.3,4 Figure 5.9 shows a turbidostat with light sources. The system can be adapted for photosynthetic bacteria. 

At steady-state condition for chemostat operation, change of concentration is independent of time. Material balance for the fermentation vessel is ... 

Exponential growth in a batch culture may be prolonged by addition of fresh medium to the fermentation vessel. In a continuous culture the fresh medium has to be displaced by an equal volume of old culture, then continuous cell production can be achieved. 

With cell recycling, chemostat efficiency is improved. To maintain a high cell density the cells in the outlet stream are recycled back to the fermentation vessel. Figure 5.10 represents a chemostat unit with a cell harvesting system. The separation unit is used for harvesting the cells and recycling then to the culture vessel to increase the cell concentration. 

For the design of a CSTR with inhibition, consider the following rate is valid for a CSTR as a fermentation vessel. 

High agitation and aeration cause major problems such as foaming, which may lead the fermentation vessel to unknown contamination. Antifoam cannot be always added for the reduction of foam it may have inhibitory effects on the growth of microorganisms, so the simplest devices have rakes mounted on the stirrer shaft and located on the surface of the fluid. 

In designing a bioreactor, material balance is used for all the streams associated with the fermentation vessel. The biomass at inlet, outlet and the generated biomass must be balanced while the fermentation proceeds. The cell balance without any cell accumulation is shown in the following equation ... 

The concentration of enzyme is veiy low, about several hundred milligrams per litre in the fermentation broth. Solvent extraction is a suitable process to recover a small amount of enzyme. The chance of some enzyme being intracellular is high, therefore cells are ruptured to liberate enzyme, which can then interact with organic solvents. Figure 7.1 shows a simple diagram for a jacketed fermentation vessel for operation at constant temperature. 

It is very simple to perform batch fermentation in a small flask with a volume of say 200 ml. Now our target is to use a 2 litre B. Braun fermenter. All accessories are shown in Figure 10.5. The fermentation vessel only, as shown in Figure 10.6, with about 250 ml of media without any accessories but with some silicon tubing attached with a filter for ventilation is autoclaved at a 131 °C for 10 minutes at 15psig.9 After that, the system is handled with special care and all accessories attached. Media is separately sterilised and pumped into the vessel. Inoculum is transferred and the batch experiment is started right after the inoculation of seed culture. An initial sample is withdrawn for analysis. 

Fig. 10.6. Geomeh ical dimensions of B. Braun fermentation vessel.

The batch experiment had neither incoming fresh media nor any product stream leaving the fermentation vessel. A complete experimental set up with a B. Braun Biostat, is shown in the above laboratory experimental set up. The continuous flow of media requires a feed tank and product reservoir. The batch process has many disadvantages such as substrate and product inhibition, whereas in the continuous process the fresh nutrients may remove any toxic by-product formed. 

All feed streams are sterilised before being metered into the fermentation vessel. Contaminants resistant to the antibiotic rarely find their way into the fermenter. When they find a way to contaminate media, their effects are so catastrophic that prevention is of paramount importance. A resistant, (3-lactamase producing, fast-growing bacterial contaminant can destroy the penicillin.5 The contaminants not only consume nutrients intended for the fungus, but also cause loss of pH control and interference with the subsequent extraction process. 

The control of these and any other parameters is most usually done in fermenter vessels specifically designed for the purpose and accommodating various working volumes, depending on the yield and production requirements. Laboratory-scale vessels could have a capacity of just 10 litres or less whereas clinical trials and production vessels may be as large as several thousand litres. 

The conventional batch fermenter and the important parts of the fermentation vessel are shown in Figure 11.5. 

At steady-state condition, change of oxygen concentration with time approaches zero. The value of KLa should be estimated by a correlation developed for various sizes of fermentation vessel. 

Height of liquid media was assumed to be 1.2 times the diameter of the fermentation vessel. 

Tlie power for the fermentation vessel was projected by the following general equation... 

Producing the kilograms of tPA necessary to satisfy the world s therapeutic needs requires the special skills possessed by modern biochemical engineers. Sophisticated engineering of the fermentation vessels, culturing conditions, and media compositions is required to culture thousands of liters of mammalian cells. In addition, new extremes of purity must be achieved in order to assure the safety of proteins derived from mammalian cells. The cost of the starting materials and the capacity constraints of the present-day equipment require that yields from each fermentation batch be as high as possible. 

Figure 5.9 Design of a generalized microbial cell fermentation vessel (a) and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences compared with microbial fermentation vessels. Note in particular (i) the use of a marine-type impeller (some animal cell bioreactors-air lift fermenters-are devoid of impellers and use sparging of air-gas as the only means of media agitation) (ii) the absence of baffles (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture. Note that various additional bioreactor configurations are also commercially available. Reprinted with permission from Proteins Biochemistry and Biotechnology (2002), J. Wiley Sons...

The initial approach to recombinant insulin production taken entailed inserting the nucleotide sequence coding for the insulin A- and B-chains into two different E. coli cells (both strain K12). These cells were then cultured separately in large-scale fermentation vessels, with subsequent chromatographic purification of the insulin chains produced. The A- and B-chains are then incubated together under appropriate oxidizing conditions in order to promote interchain disulfide bond formation, forming human insulin crb ... 

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