Bioprocess Technology : SCBT 32113

Lecture 7

Development of Inocula for

Industrial Fermentations

Inoculum / Seed culture / Starter culture

G. Prabhakaran
 Bioprocess Technology

Upstream ---------- Fermentation ----------- Downstream

Bioreactor
Learning Objectives
1. Criteria or standards required for industrial inocula.

2. Various aspects of Inoculum Development

3. Inoculum development method for industrial applications for:

A) brewing (yeast),
B) penicillin (fungi)
C) enzyme production (bacteria)

4. Importance of asepsis (clean techniques) in inoculation of

fermenters.

5. Physiological aspects of inoculum.

6. Quality control aspects of inoculum.

7. Inoculum Development Program.

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 Concept Map

Criteria or STANDARDS
(based on 5 aspects)
Critical Factors in Inoculum devpt
Inoculum Devpt
program (medium, age, measurement
methods and types of inoculum,)
INOCULUM
(OR)
Inoculum Quality SEED Inoculum devpt methods for:
control methods beer (yeast), penicillin (fungi)
7 aspects and enzyme (bacteria)

Physiological aspects Inoculum production and

(age & critical ability) transfer methods

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 DEFINITION OF INOCULUM

Living organisms or an amount of material

containing living organisms (such as
bacteria or other micro-organisms) that is
added to initiate or accelerate a biological
process, i.e., Biological seeding.

The term : Seed culture, Starter culture , Inoculum are one and the same

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 Standards

1) Criteria / required for industrial inocula

(log phase)

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 2) Aspects of Inoculum Development
a) Critical factors

 A critical factor in obtaining a suitable inoculum is the choice of

the culture medium.

 Inoculum development medium should be sufficiently similar to

the production medium to minimize any period of adaptation of the
culture to the production medium, thus reducing the lag phase and
the fermentation time.

 Inoculum media are, generally, more nutritious than production

media and contain a lower level of carbon.

 The quantity of inoculum normally used is between 3 and 10% of

the medium volume.
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b) Criteria for the Transfer of Inoculum

 Optimum transfer time to Keep Physiological condition –“Age”

 Standardization of culture conditions & monitoring culture

 Yeast, fungal and streptomycete fermentations have different requirements

for inoculum development
 Biomass as a key parameter Cell mass

Methods for measurement of Cell Growth by :

Cell number
1. Packed cell volume
2. Dry weight
3. Wet weight
4. Turbidity
5. Respiration
6. Residual Nutrient Concentration
7. Morphological form 8
 1
2

1 / weight

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1

10
2

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Limitations in various methods for measurement of
Biomass / Cell Growth

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c) Types of Inocula

Pure Monocultures Vs Mixed Cultures

i) Pure Monocultures:

• Advantages:
easy to obtain (isolate, genetically modify, or purchase;
better control of products; can be patented

• Disadvantages:
subject to contamination and genetic change
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 Bioprocesses with monocultures
Examples of pure monoculture fermentations

- industrial ethanol
- alcoholic beverages
- fermented foods
- pharmaceuticals
- acetone-butanol
- acetic acid
- single cell protein
- industrial enzymes
- insulin, growth hormone
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 ii) Mixed Culture:

- Several bioprocesses require mixed cultures

- Issues in preserving mixed cultures & inoculum
development

Advantages:
obtained by enrichment or purchased; can't be patented;
contamination not as much of problem

Disadvantages:
control of culture and products is less definite.
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Bioprocesses with mixed cultures
Examples:

- breads: sour dough, soda cracker

- wines
- vegetables: pickles, sauerkraut
- dairy products: yogurt, sour cream
- ensiling
- composting
- anaerobic digestion
- soil and groundwater remediation
- bioleaching
- microbial enhanced oil recovery
- microbial metals recovery
- waste water treatment 16
 3) Inoculum Development
A) DEVELOPMENT OF YEAST INOCULUM
For Brewing Industry

 Hansen (1896) pioneered the use of 10% of pure yeast culture

as inoculum volume

 common practice to use the yeast from the previous fermentation

to inoculate a fresh batch of wort.

 brewing terms: used to describe this process are 'crop', referring

to the harvested yeast from the previous fermentation, and
'pitch', meaning to inoculate.

 the reduced cost of using yeast from a previous fermentation is an

attractive proposition
The inherent dangers are:

1. introduction of contaminants and

2. degeneration of the strain, the most common degenerations being a
change in the degree of flocculence and attenuating* abilities of the
yeast.

To minimize the dangers:

1. 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.

2. Pre treatment of the Pitch.

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* Ability to transform sugar to alcohol
Pre treatment of the Pitch

 The pitching yeast may be treated to reduce the level of contaminating

1) bacteria 2) remove protein and 3) dead yeast cells by:

 reducing the pH of the slurry to 2.5 to 3.0,

 washing with water,
 washing with ammonium persulphate and
 treatment with antibiotics such as polymixin, penicillin and
neomycin

 One of the key physiological features of yeast inoculum is the level of

sterol in the cells.

 The likelihood of strain degeneration and contamination mean that

yeast are rarely used for more than five to ten consecutive
fermentations

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B) DEVELOPMENT OF FUNGAL INOCULUM

 Common practice to use a fungal spore suspension as

seed during an inoculum development program.

 Three basic techniques to produce a high concentration

of spores for use as an inoculum.

i. Sporulation on solidified media (agar medium)

ii. Sporulation on solid media (of cereal grains)
iii. Sporulation in submerged culture (Liquid medium)

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 i) Sporulation 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.

 Parker (1950) described the 'roll-bottle' technique for the

production of spores of Penicillium chrysogenum.

 300 ml medium with 3% agar sterilized in 1 liter cylindrical bottles

and rotated on a roller mill.

 The bottles inoculated with a spore suspension from

a sub-master slope and incubated at 24° for 6 to 7 days.

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 ii) Sporulation 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

1) amount of water added to the cereal before sterilization and

2) relative humidity of the medium incubation

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Sporulation on solid media on cereal grains

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iii) 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.

 Submerged sporulation was induced by inoculating 600 ml of the

medium, in a 2-liter shake flask, with spores from a well-sporulated
agar culture and incubating at 25°C for 7 days.

 The resulting suspension of spores used as a 10% inoculum for a

vegetative seed stage in a stirred fermenter.

 The seed culture subsequently providing a 10% inoculum for the

production fermentation.
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 Innoculum Preparation
Aim:
To provide a pure inoculum in fast growing log phase
For penicillin production
Primary source of spores stored on soil

One or more growth stages on solid medium

Shaken flask culture

Seed stage
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C) DEVELOPMENT OF BACTERIAL INOCULUM

 Bacterial inocula should be transferred in the logarithmic

phase of growth, when the cells are still metabolically
active.

 Production of bacterial enzymes, the lag phase in plant

fermenters could be almost completely eliminated by
using inoculum medium of the same composition as
used in the production fermenter and employing large
inocula of actively growing seed cultures.
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4) THE ASEPTIC INOCULATION OF PLANT FERMENTERS

The inoculation of plant-scale fermenters may involve the transfer of

culture from a laboratory fermenter, or spore suspension vessel, to a plant
fermenter, or the transfer from one plant fermenter to another.

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ASEPTIC INOCULATION OF PLANT FERMENTORS

Transfer from seed tank to plant-scale reactor is carried out

aseptically.

CRITICAL POINT IN THE PROCESS and INVOLVES;

 Opening and closing a series of valves in a defined

Sequence

 Sterilizing pipes\valves (usually with steam) in a defined

sequence

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 5) PHYSIOLOGICAL ASPECTS

a) AGE OF THE CULTURE USED AS INOCULUM

 Lag phase – represents dead time with respect to process;

true lag = all of the population is retarded

apparent lag = part of population dead/ normal

Lag period - may be due to;

1. Change in nutrients on transfer

2. Change in physical environment e.g. pH, O2
Log Phase
3. Presence of inhibitor e.g. trace elements culture is
4. Spore germination the best
5. Viability of culture on transfer
6. Size of inoculum

Number of generations during the growth cycle;

for example 6 - 7% biomass as inoculum gives 100% final biomass 32

after 4 generations (doubling times)
 B) INSTABILITY (e.g Recombinant cultures/ plasmids)

• Organism has tendency to lose ability to produce product or some

desirable characteristic (e.g. yeast --> ability to flocculate)

• Can occur at any stage during inoculum protocol (e.g. preservation,

storage, recovery from storage, in inoculum development unit or in
production.

• Can be major reason to reject a culture at industrial scale.

• Any increase in scale (followed by an increased number of generations)

will pose greater problems if culture tends to degenerate.

• Major problem with recombinant cultures.

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5) Quality Control of preserved cultures used
for inoculum development

• Each batch preserved stock cultures must be routinely tested.

• Whatever method is used in preservation of stock cultures it is

important to assess the quality of the stocks.

• Each batch of cultures should be routinely checked to ensure the

propagated strains retain the correct

a)growth characteristics,
b)morphology and
c)product forming properties.

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6) INOCULUM QUALITY CONTROL METHODS

A. PURE CULTURE - TESTS

Cultural methods - slow
 Loop dilution
 Streak plates
 Differential / selective plating

Direct methods - rapid (process requirement)

 Yeast; Morphology, granulation, cell shape and size
 Bacteria; Shape, Gram reaction

B. TEST FOR VIABILITY

Viable stain e.g methylene blue, DEFT* etc (viable cells exclude
the dye by metabolizing it. Do not stain. Dead cells stain)

C. TEST FOR CELL CONCENTRATION

Example from brewing - Sedimented volume
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* Direct epifluorescent filter technique (DEFT)
The Direct Epifluorescent Filter Technique (DEFT) is a rapid method for
enumerating bacteria. Used widely in the dairy industry for milk and milk
products, it has also been applied to beverages, foods, clinical specimens
and in environmental research.

The technique involves capturing bacterial cells on the surface of

polycarbonate membrane filters, staining with a fluorochrome such as
acridine orange, and visualisation using epifluorescence microscopy.

Enumeration of bacteria is greatly facilitated by image analysis and

Perceptive Instruments has great expertise on DEFT and similar direct
epifluorescence microscopy techniques.
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Example

Inoculum Quality Control in Brewing

Traditional plate counts at every stage of process

More rapid identification now commonplace eg ATP

Bioluminescence

D-Luciferin / Luciferase + ATP + O2 +MG2+

Light generation (562nm)

(More light = more viable cells)
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 Example

1) Detection of lactic acid bacteria in yeast cultures

Employs nested PCR were an initial PCR is carried out using a broad
spectrum primer which is followed by a second PCR on the first
amplified product.

The primers used in the second stage bind exclusively to lactic acid
bacteria and are specific for certain genera.

2) Non-brewing yeasts of Saccharomyces cerevisiae

3) General microbiological analysis of beer

Nested PCR which can detect 100-1000 bacterial cells in 20 x 106

yeast cells
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 Purity Control
• Pure yeast strains are prerequisites for good brewing performance
and product uniformity.

• Two different types of yeast are used by the brewers, one for ale (top
fermenting yeast) production and another for lager(bottom fermenting
yeast) beer.

• Ale yeasts have much in common with distiller's and baker's yeast
while lager yeasts seem to originate from an ancient species
hybridization.

• The purity of brewer's yeast is most precisely analyzed by DNA

fingerprints.
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 Strain Purity

Detection of the URA3 gene fragments on size-separated DNA from five

Saccharomyces brewer's yeasts. Lager yeasts L1, L2 and L3 and L4 contain a long
URA3 fragment IV together with one, two or none of the shorter fragments I-III. Ale
strains (A) never exhibit band IV. 42
7) Inoculum Development Program

 Master culture
 Sub-master culture – for production run
 Shake flask culture – (for check productivity)
 Large flask / Lab fermenter
 Pilot-scale fermenter
 At each stage – Culture Purity Check for
contamination
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 Formulation of inocula applied in dynamic environments
- delivery systems

Challenges:
• Laboratory / bioreactor prepared inocula (mixed culture) for waste
treatment, bioremediation, dairy and food, agricultural and
environmental systems

• Such inocula systems will require an efficient formulation and delivery

system to minimize the lag period and maximizing competitive
advantage.

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https://slideplayer.com/slide/1527178/
Mode of Assessment:

Continuous Assessment : 30% (Tests 10%, Lab reports 15% and Assignment presentation 5%)

Friday, 10 Dec ’21 Mid Term Examination

 Lecture 1 to 6 for CA Exam

 20 MCQA (0.5 x 20 = 10 marks)

 Time : 11 am to 12 noon. Duration 1 hour