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When a fermentation is to be successfully carried out, it is essential that the culture used to inoculate it satisfies the following criteria:

1. Healthy, active state requiring minimum lag phase. 2. Availability in sufficiently large volumes

3. Stable morphological form 4. Free of contamination

5. Retaining maximum productivity

The process adopted to produce an inoculum meeting these criteria is called inoculum development. The importance of inoculum development cannot be really overemphasized. If we use a non-standard inoculum, the entire fermentation will go haywire regardless of the scale of the fermentation. Much of the variation observed in small scale laboratory fermentations is due to poor inocula being used. Such a programme not only aids consistency on a small scale but also is invaluable in scaling up the fermentation. It also forms an essential part in

programming a new process.

What could go wrong if the inoculum is not properly prepared? Use the space provided to express and justify your views.

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development. Is it the same to be used for the fermentation proper?

A critical factor in obtaining a suitable inoculum is the choice of the culture medium. It must be stressed that the suitability of an inoculum medium is determined by the subsequent performance of the inoculum in the production stage. The design of a production medium is determined by two factors; b. The nutritional requirements of the organism,

c. Maximum product formation.

Whereas the production of highest possible quantity of the desired metabolite is the aim of the fermentation proper, it is precisely that we do not want to happen during the inoculum

development stage. In other words, the inoculum medium should support the maximum growth of the organism and not the formation of product. It has been observed, though, growing the culture in the same or similar medium, in which the fermentation is carried out, minimizes the lag time in a fermentation. This is easy to understand. Organisms

acclimatized to the composition of the fermentation medium take minimum time for adaptation, thus reducing the lag phase considerably. Should we use a medium with major differences in pH, osmotic pressure and anion composition, it may result in very sudden changes in uptake rates, which, in turn, may affect viability. It has been also emphasized that for antibiotic fermentations the inoculum medium should contain sufficient carbon and nitrogen to support maximum growth until transfer, so that secondary metabolism remains repressed during growth of the inoculum. It has been shown that chemostat culture of Penicillium chrysogenum under carbohydrate-limited conditions led to a loss of penicillin synthesizing ability and an increase in the proportion of non- conidiated variants whereas this did not occur in ammonia-, phosphate- or sulphate limited conditions. Inoculum media are, generally, less nutritious than production media and contain a lower level of carbon.

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The quantity of inoculum normally used is between 3 and 10% of the medium volume but it may be as high as 20 %. A relatively large inoculum volume is used to minimize the length of the lag phase and to generate the maximum biomass in the production fermentor in as short a time as possible, thus increasing vessel productivity. Addition of too high inoculum size is usually associated with problems like excessive dilution of the production medium, unwanted pH changes and carryover of unwanted wastes or metabolic products. Thus, starting from a stock culture, the inoculum must be built up in a number of stages to produce sufficient biomass to inoculate the production-stage fermentor. This may involve two or three stages in shake flasks and one to three stages in

fermentors, depending on the size of the ultimate vessel. Throughout this procedure there is a risk of contamination and strain degeneration necessitating stringent quality-control procedures. The greater the number of stages between the master culture and the production fermentor the greater is the risk of contamination and strain degeneration. Therefore, a compromise may be reached regarding the size of the inoculum to be used and the risk of contamination and strain

degeneration. Another factor to be considered in the

determination of the inoculum volume is the economics of the process. A seed fermentor 10% of the size of the production fermentor represents a considerable financial investment and must be justified in terms of productivity. A large-scale

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continuous fermentation for the production would be expected to operate at steady state in excess of 100 days. Thus the number of times that the fermentor is inoculated should be very few compared with batch or fed-batch systems. In such circumstances it may be more economic to compromise on the size of the inoculum and to tolerate a relatively lengthy period of growth up to maximum biomass than to invest a large seed vessel, which would be used on very few occasions. This is particularly relevant for biomass continuous processes where one very large fermentor may be used and, thus, any seed vessel would only be servicing the one production vessel.

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Following steps are involved in the development of an inoculum:

1. The master culture is reconstituted and plated on to solid medium.

2. Approximately ten colonies of typical morphology of high producers are selected and inoculated on to slopes as the sub-master cultures, each sub-master culture being used for a new production run. At this stage, shake flasks may be inoculated to check the productivity of these cultures, the results of such tests being known before the developing inoculum eventually reaches the production plant.

3. A sub-master culture is used to inoculate a shake flask (250 or 500 ml containing 50 or 100 ml medium), which, in turn, is used as inoculum for a larger flask, or a laboratory fermentor, which may then be used to inoculate a pilot-scale fermentor.

4. Culture purity checks are carried out at each stage to detect contamination as early as possible. Although the results of these tests may not be available before the culture has reached the production plant, at least it is known at which stage in the procedure contamination occurred.

5. For a sporulating organism the process may be modified to facilitate the use of a spore suspension as inoculum.

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and behavior of an inoculum?

The physiological condition of the inoculum when it is transferred to the next culture stage can have a major effect on the performance of the fermentation. Enough work has been done to indicate that the physiological state of the organism has a pronounced effect on the growth profile of the organism. This is proven by the fact that same inoculum size from organisms of different growth phases result in totally different growth profiles.

Similarly, the transfer of culture also must be done at the right time. The optimum time of transfer must be determined experimentally and then procedures established so that inoculation with an ideal culture may be achieved routinely. The most widely used criterion for the transfer of vegetative inocula is biomass and such parameters as packed cell volume, dry weight, wet weight, turbidity, respiration, residual nutrient concentration and morphological form have been used.

The physiological uniformity of the organisms is also an important parameter. The entire batch of selected organisms should be in a state of complete physiological uniformity. This is important in the sense that a single batch of

inoculum may be used to inoculate several fermentors and in such case, the physiological uniformity of the culture makes the entire fermentation operation much more predictable.

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your comments with a hypothetical example. Genetic stability of the organism during inoculum preparation is an extremely important aspect. Generally spontaneous mutations are rare and as such do not pose a major threat during the build up of inoculum. However, if the fermentation itself employs a genetically modified strain, there is always a chance of ‘back mutation’ resulting in proliferation of the native strain. So, alongwith the physiological check and morphological check, a genetic check of the organism should be carried out during the build up of the inoculum.

We all know that microbial fermentations are often employed for the production of industrial enzymes. Many of these enzymes are ‘adaptive’ or ‘induced’ enzymes which are produced only under the stimulus of presence of the specific substrate. Suppose we are producing enzyme amylase using Bacillus subtilis. If the medium does not contain starch, this enzyme will not be produced. So care must be taken to incorporate starch in the inoculum medium at least, and especially, during the final stages of the inoculum so as to ensure activation of the amylase producing machinery.

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incorporation of specific constituents is required for ‘triggering’ of the production mechanism?

Inoculum quality is most conveniently checked by using parameters that can be monitored on-line. These would include dissolved oxygen, pH and oxygen or carbon dioxide in the effluent gas. The use of the carbon dioxide

production rate (CPR) has been suggested as a transfer criterion, which requires analysis of the fermentor effluent air.

In recent years, probes have been developed for on-line assessment of biomass and these could be invaluable in estimating the time of inoculum transfer. In an innovative experiment, the use of a biomass sensor (the Bug meter) to control the yeast pitching rate (inoculum level) in brewing has been suggested. The probe measures the dielectric permittivity of viable yeast cells and is unaffected by the presence of dead cells, air bubbles or detritus, making it ideal for the routine monitoring of yeast inoculum. Using the probe, an automatic inoculum dispenser allowing preset viable yeast mass to be transferred from a yeast storage vessel to the brewery fermentation has been developed.

Now, let us see a typical example of inoculum development, namely that of yeast in brewing.

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Case study: development of Brewer’s yeast

It is common practice in the British brewing industry to use the yeast from the previous fermentation to inoculate a fresh batch of wort. The brewing terms used to describe this process are ‘crop’, referring to the harvested yeast from the previous fermentation, and ‘pitch’, meaning to inoculate. The dangers inherent in this practice are the introduction of contaminants and the degeneration of the strain, the most common degenerations being a change in the degree of flocculence and attenuating abilities of the yeast. In breweries employing top fermentations in open fermentors these dangers are minimized by collecting yeast to be used for future pitching from the middle of the fermentor. During the fermentation the yeast cells flocculate and float to the surface, the first cells to do this being the most flocculent and the last cells the least flocculent. As the head of yeast develops, the surface layer (the most flocculent and highly contaminated yeasts) is removed and discarded and the underlying cells are harvested and used for subsequent pitching. Therefore, the middle layer of the yeasts contain cells which have the desired flocculence and which have been protected from contamination by the surface layer of the yeast head. The pitching yeast may be treated to reduce the level of contaminating bacteria and remove protein and dead yeast cells by such treatments as reducing the pH of the slurry to 2.5 to 3, washing with water, washing with ammonium

persulphate and treatment with antibiotics such as polymixin, penicillin and neomycin.

However, traditional open vessels are becoming increasingly rare and the bulk of beer is brewed using cylindro-conical

fermentors. In these systems the yeast flocculates and collects in the cone at the bottom of the fermentor where it is subject to the stresses of nutrient starvation, high ethanol concentration, low water activity, high carbon dioxide concentration and high pressure. Thus, the viability and physiological state of the yeast crop would not be ideal for an inoculum. The viability of the crop may be assessed using a biomass probe of the type described earlier, thus ensuring that at least the correct amount of viable biomass is used to start the next fermentation. However, the physiological state of the biomass will not have been influenced by such monitoring procedures. The situation is further complicated by the fact that the harvested yeast is stored before it is used as inoculum. Metabolic activity is minimized during this time by chilling rapidly to about 1°, suspending in beer and storing in the absence of oxygen. If oxygen is present during the storage period then the yeast cells consume their stored glycogen, which renders them very much less active at the start of the fermentation.

One of the key physiological features of yeast inoculum is the level of sterol in the cells. Sterols are required for membrane synthesis but they are only produced in the presence of oxygen. Thus, we have the conflict of oxygen being required for sterol synthesis; yet anaerobic conditions are required for ethanol production. This anomaly is resolved traditionally by aerating the wort before inoculation. This oxygen allows sufficient sterol synthesis early in the fermentation to support growth of the cells throughout the process that is after the oxygen is

exhausted and the process is anaerobic. An alternative approach

to this has been suggested where the pitching yeast is vigorously aerated prior to inoculation. The yeast was then sterol rich and had no requirement for oxygen during the alcohol fermentation.

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It can’t be. The difficulties outlined above and the likelihood of strain degeneration and contamination necessitate frequent change of yeast culture being employed. An arrangement where yeast cells are continuously (and aerobically) produced has been suggested. This is called the yeast propagators.

Find out more about yeast propagators and make your notes in the space provided for:

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The main objective of inoculum development for traditional bacterial fermentations, or any fermentation for that matter, is to produce an active inoculum which will give as short a lag phase as possible in subsequent culture. A long lag phase is disadvantageous in that not only is time wasted but also medium is consumed in maintaining a viable culture prior to growth. The age of the inoculum is particularly important in the growth of sporulating bacteria, for sporulation is induced at the end of the logarithmic phase and the use of an inoculum containing a high percentage of spores would result in a long lag phase in a subsequent fermentation. Many workers have recommended the importance of addition of inocula from the logarithmic phase of growth, albeit in varying proportions. It has been also shown that, with the use of actively growing bacterial cells in the

inoculum it is possible to completely eliminate the lag phase in the fermentation vessel, at least in some cases.

There are some exceptions, of course especially if we are using recombinant strains. Plasmid stability and productivity in an E.coli biotin fermentation was greatly improved if stationary, rather then exponential, phase cells were used as inoculum.

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Well, the explanation provided is that the plasmid copy number may be higher in stationary cells than in exponential ones, resulting in a lower plasmid loss in the subsequent fermentation when a stationary culture is used as inoculum. It was also suggested that a stationary phase inoculum would result in a lag phase, but this disadvantage was more than compensated for by the considerable improvement in plasmid retention and biotin production compared with that obtained using an exponential inoculum.

In some cases, especially with the Clostridium species, it is necessary to briefly expose the spores to a mild to moderate hest treatment. This seems to ‘wake up’ these spores and allow them quick germination. This type of ‘pregermination’ does not seem to be necessary in fungi and actinomycetes. Still, the advantage of using inoculum from the exponential phase seems to be multidimensional. Take the case of lactic-

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acid bacteria. Here, the organism may be inhibited by lactic acid. Thus, production of lactic acid in the seed fermentation may result in the generation of poor quality inoculum. Continuous removal of lactate from the inoculum is found to reduce the length of the lag phase in the production fermentation.

Can you think of any other examples where the product produced at the end of the log phase acts to inhibit the growth of the producing organism? Feel free to use the space provided.

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It is common practice to use a spore suspension of fungi as seed during an inoculum development programme because the majority of industrially important fungi and

streptomycetes are capable of asexual reproduction by sporulation. A major advantage of a spore inoculum is its high viability especially as compared to the vegetative cells. Three basic techniques have been developed to produce a high concentration of spores for use as an inoculum. First one is the sporulation of these fungi on solidified media. Most fungi and streptomycetes will sporulate on suitable agar media but a large surface area must be employed to produce sufficient spores. The ‘roll-bottle’ technique for the production of spores of Penicillium chrysogenum has been described which uses thin coat of the agar medium on the inside of a bottle as a growth surface. Several such examples have been quoted.

Then there is the production of mycelial spores on solid media. Many filamentous organisms will sporulate profusely on the surface of cereal grains from which the spores may be harvested. Substrates such as barley, hard wheat bran, ground maize and rice are all suitable for the sporulation of a wide range of fungi. The sporulation of a given fungus is particularly affected by the amount of water added to the cereal before sterilization and the relative humidity of the atmosphere, which should be as high as possible during sporulation. Substrates as simple as cooked rice may be used for this purpose.

It has been found that very high spore count can be achieved using this technique.

The third technique employed uses sporulation in

submerged culture. Many fungi will sporulate in submerged culture provided a suitable medium is employed. This technique is more convenient than the use of solid or solidified media because it is easier to operate aseptically and it may be applied on a large scale. The technique was first