Module 5 ferm

• Culture used to inoculate a fermentation


  • Must be in a healthy, 'active state' thus minimizing the length of the lag phase in the subsequent fermentation
  • Must be available in sufficiently large volumes to provide an inoculum of optimum size
  • Must be in a suitable morphological form
  • Free of contamination
  • Retain its product-forming capabilities
• Inoculum medium


  The suitability of an inoculum medium is determined by the subsequent performance of the inoculum in the production stage
• Production medium


  The design of a production medium is not only determined by the nutritional requirements of the organism, but also the requirements for "maximum product" formation
• Seed medium


  The formation of product in the seed culture is not an objective during inoculum development so that the seed medium may be of different composition from the production medium
• Lag time in a fermentation


  Is reduced by growing the culture in the "final type" medium - also reduces fermentation time
• Quantity of inoculum used

  Between 3 and 10% of the medium volume
• Inoculum development


  1. From the stock culture, the inoculum is built up in a number of stages to produce sufficient biomass to inoculate the production stage fermenter

  2. This may involve 2 or 3 stages in shake flasks and 1 - 3 stages in fermenters, depending on the size of the ultimate (final) fermenter
• Throughout the inoculum development 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 fermenter the greater the risk of contamination and strain degeneration
• A typical inoculum development programme


  1. Master culture is reconstituted and plated onto solid medium

  2. Approximately 10 colonies of typical morphology of high producers are selected and inoculated onto slopes as the sub-master cultures, each sub-master being used for a new production run

  3. Shake flasks may be inoculated at this stage to check the productivity of these cultures, the results of such tests being known before the developing inoculum eventually reaches the production plant

  4. The submaster culture is used to inoculate a shake-flask (250 or 500 ml) which, in turn, is used as inoculum for a larger flask or laboratory fermenter, which may then be used to inoculate a pilot-scale fermenter

  5. Culture purity checks are carried out at each stage to detect contamination as early as possible
• Yeasts, bacteria, fungi have different requirements for inoculum development and these are dealt with separately
• Inoculum development programme for yeast processes: brewing


  1. It is common practice in the British brewing industry to use the yeast from the previous fermentation (pitch, in brewing terms) to inoculate a fresh batch of wort (liquid extracted from the mashing process during the brewing)

  2. Pitching is the process of adding yeast to wort to start fermentation

  3. Dangers inherent in this practice are the introduction of contaminants and the degeneration of the strain

  4. The most common degeneration being a change in the degree of flocculence (clumping together) and attenuating abilities of the yeast

  5. In breweries employing top fermentations these dangers are minimized by collecting yeast to be used for future pitching from middle skimmings

  6. During 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

  7. As the head of yeast develops, the surface layer (the most flocculent and highly contaminated yeasts) is removed and discarded and the underlying cells (the middle skimmings) are harvested and used for subsequent pitching

  8. Therefore the middle skimmings will contain cells which have the desired flocculence and which have been protected from contamination by the surface layer of the yeast head
• Preventing bacterial contamination in pitching yeast


  1. 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, Acid washing, Washing with ammonium persulphate, Treatment with antibiotics such as polymixin, penicillin and neomycin

  2. Despite these precautions, yeasts are rarely used for more than five to ten consecutive fermentations
• Commercial production of bakers yeast


  1. Involves the development of an inoculum through large number of stages

  2. Although the production stages of the process may not be operated under strictly aseptic conditions a pure culture is used for the initial inoculum thereby keeping contamination to a minimum in the early stages of growth

  3. Process involving five stages, the first two being aseptic while the remaining stages were carried out in open vessels

  4. First two stages were carried out in closed vessels without aeration or nutrient feeds; it is uneconomic to equip the smaller vessels with the necessary equipment
• Main objective of inoculum development for bacterial fermentations is to produce an active inoculum which will give as short a lag phase as possible in subsequent culture
• Inoculum size


  Normally ranges between 3 and 10% of the culture volume
• Bacterial inocula should be transferred in the logarithmic phase of growth, when the cells are still metabolically active
• Age of the inoculum


  Is 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
• Inoculum development programme for the production of proteases by Bacillus subtilis


  Inoculum for a seed fermenter was grown for 1 to 2 days on a solid or liquid medium and then transferred to a seed vessel where the organisms was allowed to grow for a further ten generations before transfer to the production stage
• Inoculum development programme for the production of bacitracin by B. subtilis


  1. Stage 1: 4-dm³ shake flask inoculated with a stock culture, incubation time 18 to 24 h

  2. Stage 2: Stage 1 culture inoculated into 750-dm³ fermenter, incubation time 6 h

  3. Stage 3: 750-dm³ culture inoculated into 6000-dm³ fermenter, grown to the point of greatest production of cells

  4. Stage 4: 6000-dm³ culture inoculated into 120,000-dm³ production fermenter
• Development of inocula for acetic acid bacteria


  1. Acetic acid bacteria used in the vinegar process are highly aerobic and are extremely sensitive to oxygen starvation

  2. Therefore, to avoid disturbing the system, the cells at the end of a fermentation are used as inoculum for the next batch by removing approximately 60% of the culture and restoring original level with fresh medium

  3. Advantage of a highly active inoculum apparently outweighs the disadvantages of possible strain degeneration and contamination
• Inoculum development programme for the clostridial acetone-butanol fermentation


  1. Stage 1: Reconstitution of the spore stock culture - 24 hour incubation

  2. Stage 2: Stage 1 culture inoculated into 600 cm³ of medium, incubated for 20-24 hours

  3. Stage 3: 90 cm³ of stage 2 culture inoculated into 3000cm³ medium in a 4000-cm³ Erlenmeyer flask

  4. Stage 4: Stage 3 culture inoculated into 25,000-dm³ fermenter

  5. Stage 5: Stage 4 culture inoculated into 300,000 to 2,500,00-dm fermenters at a 0.5 to 3% inoculum
• Preparation of inocula for fungal fermentations


  1. Most industrially important fungi are capable of asexual sporulation so it is common practice to use a spore suspension as seed during an inoculum development programme

  2. Three basic techniques have been developed to produce a high concentration of spores for use as an inoculum: Sporulation on solidified media, Sporulation on solid media, Sporulation in submerged culture
• Sporulation on solidified media


  • Roll-bottle technique for the production of spores of Penicillium chrysogenum
• Sporulation on solid media


  • Substrates such as barley, hard wheat bran and ground maize are all suitable for the sporulation of a wide range of fungi
  • Mass production of spores of several Aspergillus and Penicillium species on whole loaves of white bread
• Sporulation in submerged culture


  Conditions for the submerged sporulation of the griseofulvin-producing fungus, Pencillium patulum, and the medium utilized: Whey powder to give 3.5% Lactose, 0.05% Nitrogen, 0.4% KH2PO4, 0.05% KCI, Com-steep liquor solids to give approx. 0.04% N, 0.38%