DDXCUGI

1. Fermentation Medium
Most fermentations require liquid media,
often referred to as broth,
although some solid-substrate
fermentations are operated.

2. What Does the Medium Need to
Do?
• Cause no problems with:
– Preparation and sterilisation
– Agitation and aeration
– Downstream processing
• Ingredients must have an acceptable:
– Availability
– Reliability
– Cost (including transport costs)

3. Types of media
• Two types of media was used
• Defined media
• Crude media

4. Defined medium
• Defined media are like those we use in the lab
e.g. minimal salts medium.
• Defined media is expensive but composition is
known and should not vary.
• Defined media is used for expensive low
volume products e.g. anticancer drugs.

5. Defined Media—Good properties
• Consistent
– Composition
– Quality
• Facilitate R & D
• Unlikely to cause foaming
• Easier upstream processing (formulation,
sterilisation etc.)
• Facilitate downstream processing (purification
etc.)

6. Defined Media— Bad properties
• Expensive
• Need to define and supply all growth
factors…only mineral salts present
• Yields and volumetric productivity can be
poor:
– Cells have to “work harder”…proteins etc. are not
present
– Missing growth factors…amino acids etc.

7. Crude media
• Crude media is made up of unrefined
agricultural products e.g. maize, barley.
• Crude media is cheap but composition is
variable.
• Crude media is used for large volume
inexpensive products e.g. biofuel from
agriwastes.

8. Crude Media – Good Properties
• Cheap
• Provide growth factors (even “unknown” ones)
• Good yields and volumetric productivity

9. Crude Media – Bad Properties
• Variability:
– Composition
– Quality
– Supply
– Cost (Agri-politics)
• Availability to organism
– (More detail follows)
• Unwanted components….iron or copper which can
often be lethal to cell growth.

10. Different technical objectives of media
formulation
• Inoculum (starter culture) propagation steps / pilot-
scale fermentations / main production fermentation
• Biomass or primary metabolites production /
secondary metabolite production

11. Considerations in seed culture media
formulation
• The seed stages are designed to give rapid and
reproducible growth without nutrient depletion,
autolysis, or an adverse change in pH.
• There is less concern with the cost of ingredients in
the seed stages since the volume is usually only 5%
of the fermentation volume and excellent uniformity
of medium ingredients is highly desirable.
• Some specially prepared dairy products have been
used quite extensively in primary and secondary
seeds.

12. Constituents of medium
• Water
• Carbon source / Nitrogen source / Sources of
phosphorous and sulfur / Minor and trace elements /
Vitamins such as biotin and riboflavin
• Oxygen: even some anaerobic fermentations require
initial aeration, e.g. beer fermentations
• Buffers or controlled by acid and alkali additions
• Antifoam agents
• Precursor, inducer or inhibitor compounds

13. Considerations in media design
Nutritional Requirements
Environmental Requirements
Techno-economic Factors

14. Nutritional requirements
• Nutritional requirements include elemental, specific
nutrient, and energy requirements
• Elemental requirements : the stoichiometry for
growth and product formation
C-source + N-source + O2 + minerals + specific
nutrients → cell mass + product + CO2 + H2O + heat
• Specific nutrient requirements:
Auxotroph: To use a complex medium or to identify
the specific nutrient

16. Elemental requirements
• Main elemental formula of microbial cells C4H7O2N
(dry weight basis 48% C, 7% H, 32% O, 14% N), e.g.
Baker’s yeast C3.72H6.11O1.95N0.61S0.017P0.035K0.056
• Average: C 45-55%, N 6-14%, K 0.5-2%, P 1-3%,
Mg 0.1-1%, S 0.02-1%, minor minerals (mg /100g
cell) Cu 0.1-1, Fe 1-10, Zn ~1, Mn 0-5 (e.g. 10g/L of
cell mass containing 0.4% magnesium will require at
least 0.04 g/L of Mg or 0.2 g/L of MgSO4 or 0.4 g/L
of MgSO4‧7H2O)
• Chemical composition of fermentation product
• Typical concentration of fermentation products in the
broth (dry wt / vol, %): lactic acid (13), citric acid
(12), glutamic acid (10), ethanol (8), baker’s yeast (5),
benzyl penicillin (3), riboflavin (1), vitamin B12
(0.002)

18. Environmental requirements
• Effect of growth temperature on cell yield / below
optimal temperature for growth
• Effect of water activity (Aw = Ps/Pw) on growth rate,
vapor pressure of water in solution (Ps) or in pure
water(Pw)
• Combined effect of temperature and pH on growth /
opt pH for growth and production is not always the
same
• Environmental effect of substrate

20. Environmental effect of substrate
• Substrate concentration: Monod equation, μ
= μm S / (Ks + S)
Ks for C-source 1 ~ 10 mg/L, when S = 10 ~ 100
mg/L, μ ≈ μm; Ks for amino acid 0.003 ~ 0.2 mg/L; Ks
for ammonia 0.1 ~ 1.0 mg/L
• Substrate inhibition: carbohydrate 50 to 100 ~ 150
g/L (osmotic pressure); phenol, toluene, butanol a
few g/L (damage cell membrane); ammonia 3 ~ 5 g/L
• Catabolite repression
• NO3
- → NO2
- toxic effect
• Phosphate repression and sulfate repression

21. Techno-economic factors that affect the choice
of individual raw materials:
• Cost: transport and storage, e.g. temperature control
• Availability: consistent quality and year round
availability
• Ease of handling: solid or liquid forms
• Sterilization: thermal damage and inhibitory byproduct
• Operational characteristics: formulation, mixing,
complexing and viscosity characteristics that may
influence agitation, aeration, foaming and recovery
• Supply: the concentration of target product attained, its
rate of formation and yield per gram of substrate utilized
• Purification: levels and range of impurities, potential
for generating undesired products
• Pollution control
• Health and safety implications

22. Cost analysis
• Raw materials (consumed in
production or recovery) constitute
a major part of the manufacturing
cost
• 30 to 80% of the production cost
for biologically based production
system
• 10 to 50% of the production cost
for conventional chemical
production plants
• Nutrients: up to 60% of the
production cost
• Examples

23. Example

26. Major C sources

27. Molasses
• Byproduct of cane or beet sugar production / residues
remaining after most of the sucrose has been
crystallized from the plant extract
• Dark colored viscous syrup containing 50-60% (w/v)
carbohydrate, primarily sucrose, with 2% (w/v)
nitrogenous substances, along with some vitamins
and minerals.
• Overall composition varies depending upon the plant
source, the location of the crop, the climatic
conditions under which it was grown, and the factory
where it was processed
• The carbohydrate concentration may be reduced
during storage by contaminating microorganisms
• Hydrol molasses, containing primarily glucose, is a
byproduct of maize starch processing

29. Malt extract
• Concentrated aqueous extracts of malted barley to
form syrups / particularly useful for the cultivation of
filamentous fungi, yeasts and actinomycetes
• App. 90% carbohydrate (w/w) and some vitamins and
app. 5% nitrogenous substances, proteins, peptides and
amino acids / carbohydrate comprising 20% hexoses
(glucose and small amounts of fructose), 55%
disaccharides (maltose and traces of sucrose), 10%
maltotriose, and additionally contain 15-20% branched
and unbranched dextrins, which may or may not be
metabolized, depending upon the microorganisms
• Careful sterilization to prevent over-heating /Maillard
reaction products (brown condensation products
resulting from the reaction of amino groups and
carbonyl groups) when heated at low pH / color change,
loss of fermentable materials, some toxic products

31. Starch and dextrins
• Can be directly metabolized by amylase-producing
microorganisms, particularly filamentous fungi
• Maize starch is most widely used
• To allow use in a wide range of fermentations, the
starch is usually converted into sugar syrup,
containing mostly glucose. It is first gelatinized and
then hydrolyzed by dilute acids or amylolytic
enzymes, often microbial glucoamylases that operate
at elevated temperatures

32. Sulfite waste liquor
• Sugar containing wastes derived from the paper
pulping industry are primarily used for the cultivation
of yeasts
• Waste liquors from coniferous trees contain 2-3%
(w/v) sugar, 80% hexoses (glucose, mannose and
galactose) and 20% pentoses (mostly xylose and
arabinose) / Liquors derived from deciduous trees
contain mainly pentoses
• Usually the liquor requires processing before use as it
contains sulfur dioxide / The low pH is adjusted with
calcium hydroxide or calcium carbonate, and these
liquors are supplemented with sources of nitrogen and
phosphorus

33. Cellulose
• Predominantly as lignocellulose (composed of
cellulose, hemicellulose and lignin)
• Available from agricultural, forestry, industrial and
domestic wastes
• Relatively few microorganisms can utilize it directly /
The cellulose component is in part crystalline,
encrusted with lignin, and provides little surface area
for enzyme attack
• At present, mainly used in solid-substrate
fermentations (e.g. mushrooms)
• Potentially a very valuable renewable source of
fermentable sugars once hydrolyzed, particularly in
the bioconversion to ethanol for fuel use

34. Whey
• An aqueous byproduct of the dairy industry / Annual
worldwide production is over 80 million tons,
containing over 1 million tons of lactose and
0.2 million tons of milk proteins
• Expensive to store and transport / Lactose
concentrates are often prepared for later fermentation
by evaporation of the whey, following removal of
milk proteins for use as food supplements
• Lactose is less useful than sucrose / e.g. S. cerevisiae
does not ferment lactose
• Formerly used extensively in penicillin fermentation /
Still employed for producing ethanol, single cell
protein, lactic acid, xanthan gum, vitamin B12 and
gibberellic acid

35. Alkanes and alcohols
• n-Alkanes (C10-C20): readily metabolized by certain
microorganisms / industrial use is dependent upon the
prevailing price of petroleum
• Methane: utilized by a few microorganism, but its
conversion product methanol is often preferred for
industrial fermentations
• High purity methanol is readily obtained / completely
miscible with water / has a high per cent carbon
content and is relatively cheap / only limited organisms
will metabolize methanol / only low conc., 0.1-1% (v/v)
are tolerated by microorganisms / oxygen demand and
heat of fermentation are high, but this is even more
problematic when growing on alkanes
• Ethanol is less toxic than methanol / used as a sole or
cosubstrate / too expensive for general use as a carbon
source / its biotransformation to acetic acid remains a
major fermentation process

36. Fats and oils
• Hard animal fats (composed mainly of glycerides of
palmitic and stearic acids) are rarely used in
fermentation
• Plant oils (primarily from cotton seed, linseed, maize,
olive, palm, rape seed and soy) and occasionally fish
oil, may be used as the primary or supplementary
carbon source, especially in antibiotic production /
Plant oils are mostly composed of oleic and linoleic
acids, but linseed and soy oil also have a substantial
amount of linolenic acid
• Oils contain more energy per unit weight than
carbohydrates / Oils can be particularly useful in fed-
batch operations than carbohydrates (aqueous
solutions less than 50%, w/v; occupy a greater volume)

37. Major N sources

38. Corn steep liquor
• Byproduct of starch extraction from maize / first
use in fermentations for penicillin production in the
1940s
• Exact composition varies depending on the quality
of maize and the processing conditions /
Concentrated extracts generally contain about 4%
(w/v) nitrogen, including a wide range of amino
acids, along with vitamins and minerals / Any
residual sugars are usually converted to lactic acid
(9-20%, w/v) by contaminating bacteria
• Can sometimes be replaced by liquor derived from
potato starch production

39. Yeast extract - 1
• Produced from waste baker’s and brewer’s yeast, or
other strains of S. cerevisiae / Or Kluyveromyces
marxianus (formerly K. fragilis) grown on whey and
Candida utilis cultivated using ethanol, or wastes
from wood and paper processing
• Extracts used in the formulation of fermentation
media are normally salt-free concentrates of soluble
components of hydrolyzed yeast cells / Extracts with
sodium chloride concentrations greater than 0.05%
(w/v) cannot be used in fermentation processes due to
potential corrosion problems
• Yeast cell hydrolysis is often achieved by autolysis,
which can be initiated by temperature or osmotic
shock, causing cells to die but without inactivating
their endogenous enzymes

40. Yeast extract - 2
• Temperature and pH are controlled throughout an
optimal and standardized autolysis process /
Temperature control is particularly important to
prevent loss of vitamins
• Autolysis (50-55oC for several hours before the
temperature is raised to 75oC to inactivate enzymes),
plasmolysis or mechanical disruption of cells /
filtration or centrifugation to remove cell wall
materials and other debris / rapid concentration
• Extracts are available as liquids containing 50-65%
solids, viscous pastes or dry powders
• They contain amino acids (35-40%, w/v), peptides
(30-45%, w/v), water-soluble vitamins and some
glucose derived from the yeast storage carbohydrates
(trehalose and glycogen)

42. Peptones
• Peptones are usually too expensive for large-scale
industrial fermentations
• Prepared by acid or enzyme hydrolysis of high
protein materials: meat, casein, gelatin, keratin,
peanuts, soy meal, cotton seed, etc.
• Amino acids compositions vary depending upon the
original protein source / Gelatin-derived peptones are
rich in proline and hydroxyproline, but almost devoid
of sulfur-containing amino acids / Keratin peptone is
rich in both proline and cystine, but lacks lysine
• Plant peptones invariably contain relatively large
quantities of carbohydrates

44. Soya bean meal
• Residuals after extraction of soy oil
• Composed of 50% protein, 7% non-protein
nitrogenous compounds, 30% carbohydrates and
1% oil
• Often used in antibiotic fermentation because the
components are only slowly metabolized, thereby
eliminating the possibility of repression of product
formation

45. Water
• Use for media, cleaning, cooling ?
• A reliable source of large quantities of clean water, of
consistent composition, is essential
• Before use, removal of suspended solids, colloids and
microorganisms is usually required
• “Hard” water is treated to remove salts such as
calcium carbonate
• Iron and chlorine may also require removal
• Water is becoming increasingly expensive / recycle /
reuse wherever possible / minimizes water costs and
reduces the volume requiring waste-water treatment

46. Antifoams -1
• Foaming is largely due to media proteins that
become attached to the air-broth interface where
they denature to form a stable foam
• If foaming is minimized, then throughputs can be
increased
• Three approaches to controlling foam production:
modification of medium composition, use of
mechanical foam breakers and addition of chemical
antifoams
• Chemical antifoams are surface-active agents which
reduce the surface tension that binds the foam
together

47. Antifoams -2
• Ideal antifoam: 1. readily and rapidly dispersed with
rapid action; 2. high activity at low concentration; 3.
prolonged action; 4. non-toxic to fermentation
microorganisms, humans or animals; 5. low cost; 6.
thermostable; 7. compatibility with other media
components and the process , i.e. having no effect on
oxygen transfer rates or downstream processing
operations (e.g. some may adversely affect membrane
filtration)
• Natural antifoams include plant oils (e.g. from soy,
sunflower and rapeseed), deodorized fish oil, mineral
oils
• Synthetic antifoams are mostly silicon oils, poly
alcohols and alkylated glycols

48. Special compounds -1
• Precursors: phenylacetic acid or phenylacetamide as
side-chain precursors in penicillin production / D-
threonine in L-isoleucine production by Serratia
marsescens / anthranillic acid for L-tryptophan
production by yeast Hansenula anomala
• Inducers and elicitors: Inducers are often necessary
for genetically modified microorganisms (GMMs) /
Production of secondary metabolites, such as
flavonoids and terpenoids, in plant cell culture can be
triggered by adding elicitors, which may be isolated
from various microorganism, particularly plant
pathogens

49. Special compounds -2
• Inhibitors: 1. Used to redirect metabolism towards
the target product and reduce formation of other
metabolic intermediates (e. g. sodium bisulfite in
production of glycerol by S. cerevisiae) 2.
Antibiotics for some GMMS containing plasmids
bearing an antibiotic resistance gene
• Cell permeability modifiers: e.g. penicillins and
surfactants added to amino acid fermentations,
including processes for producing L-glutamic acid
by members of the genera Corynebacterium and
Brevibacterium