Microbial Kinetics and Substrate
utilization in Fermentation
Turbidity (optical density)
9.0
7.0
ia l
De
a
onen
t
6.0
5.0
4.0
Lag
Time
th
Optical density
Stationary
Ex p
Log 10 CFU/ml
8.0
Batch culture and Kinetics of Microbial growth
in batch culture
• After inoculation the growth rate of the cells
gradually increases.
• The cells grow at a constant, maximum, rate
and this period is known as the log or
exponential, phase.
Growth of a typical microbial culture in
batch conditions
The rate of growth is directly proportional to cell
concentration or biomassi.e.
dx/dt
α
dx/dt = μX
X
----------1
Where,
X is the concentration of microbial biomass,
t is time, in hours
μ is the specific growth rate, in hours -1
• On integration of equation (1) from t=0 to t=t ,we
have:
xt = xo e μt
--------- 2
Where,
• Xo is the original biomass concentration,
• Xt is the biomass concentration after the time
interval, t hours,
• e is the base of the natural logarithm.
• On taking natural logarithms of equation (2)
we have :
In Xt = In Xo + μt
(3)
• Therefore, a plot of the natural logarithm of
biomass concentration against time should yield
a straight line, the slope of which would equal to
μ.
• During the exponential phase nutrients are in
excess and the organism is growing at its
maximum specific growth rate, ‘μmax ‘ for the
prevailing conditions.
• Typical values of μmax for a range of microorganisms are
given below in the Table.
Effect of substrate concentration on microbial growth
Whether the organism is unicellular or mycelia the growth is influenced by
consumption of nutrients and the excretion of products. The cessation of growth may
be due to the depletion of essential nutrient in the medium (substrate limitatioln), the
accumulation of some autotoxic product of the organism in the medium (toxin
limitation) or a combination of the substrate limitation and toxin limitation.
The nature of the limitation of growth may be discussed by growing the organism in
the presence of a range of substrate concentrations and plotting the biomass
concentration at stationary phase against the initial substrate concentration is shown
given below in fig 2:
FIG. 2. The effect of initial substrate concentration on the biomass concentration at the
onset of stationary phase, in batch culture.
From figure 2 it may be seen that over the zone A to B due to an
increase in initial substrate concentration gives a proportional
increase in the biomass occur at stationary phase. This relation
between increase in initial substrate concentration and proportional
increase in the biomass may be described by equation:
X = Y(SR - s) ---------(3)
Where,
X -is the concentration of biomass produced,
Y -is the yield factor (g biomass produced g-1 substrate consumed),
SR -is the initial substrate concentration, and
s -is the residual substrate concentration.
• Thus, equation (3) may be used to predict the production of biomass
from a certain amount of substrate
• In Fig. 2:• Over the zone A to B: s = 0; at the point of
cessation of growth.
• Over the zone C to D an increase in the initial
substrate concentration does give a
proportional increase in biomass due to the
exhaustion of another substrate or the
accumulation of toxic products
Monod Equation
The decrease in growth rate and the cessation of growth due to the depletion
of substrate, may be described by the relationship between μ and the
residual growth limiting substrate.
This relationship is represented by a equation given by Monod in1942 is
know as Monod equation.
Based upon Michaelish-Menten kinetics.
According to Monad equationμ = μmax . S /Ks + S
(4)
• Where,
• S is residual substrate concentration,
• Ks is substrate utilization constant, numerically equal
to substrate concentration when μ is half of μmax.
• Ks s a measure of the affinity of the organism with substrate
It tell about the relationship between specific growth rate ‘μ’ and growth
limiting substrate concentration ‘S’.
Fig: 3 The effect of residual limiting substrate concentration on specific growth rate of a
hypothetical bacterium.
In the above figure
The zone A to B is equivalent to the exponential phase in batch culture where
substrate concentration is in excess and growth is at μmax .
The zone C to A is equivalent to the deceleration phase of batch culture where the
growth of the organism is due to the depletion of substrate to a growth-limiting
concentration which will not support μmax .
Some representative values of Ks for a range of micro-organisms and substrates
Typical values of K, for a range of organisms and substrates are usually very
small and therefore the affinity for substrate is high.
• If the organism has a very high affinity for the limiting substrate (a low Ks value)
the growth rate will not be affected until the substrate concentration has
declined to a very low level. Thus, the deceleration phase for such a culture
would be short.
• However, if the organism has a low affinity for the substrate (a high Ks value)
the growth rate will be deleteriously affected at a relatively high substrate
concentration. Thus, the deceleration phase for such a culture would be
relatively long.
• The biomass concentration at the end of the exponential phase is at its highest
level. Therefore the decline in substrate concentration will be very rapid so that
the time period during which the substrate concentration is close to Ks is very
short.
• The stationary phase in batch culture is that point where the growth rate has
declined to zero. This phase is also known as the maximum population phase.
Growth Curve
Turbidity (optical density)
9.0
7.0
ia l
De
a
onen
t
6.0
5.0
4.0
Lag
Lag
Time
th
Optical density
Density
Optical
Stationary
Ex p
10
Log
LogCFU/ml
CFU/ml
8.0
Lag phase
Three causes for lag:
physiological lag
low initial numbers
appropriate gene(s) absent
growth approx. = 0
(dX/dt = 0)
Exponential phase
Nutrients and conditions are not limiting
growth = 2n
or
X = 2nX0
Where X0 = initial number of cells
X = final number of cells
n = number of generations
20
20
21
21
22
22
23
23
24
24
n
2
2n
Example: An experiment was performed in a lab flask growing cells on
0.1% salicylate and starting with 2.2 x 104 cells. As the experiment
below shows, at the end there were 3.8 x 109 cells.
This is an increase is 5 orders of magnitude!!
How many doublings or generations occurred?
X = 2nX0
1.73 x
=
105 =
2n(2.2
x
104)
2n
log(1.73 x 105) = nlog2
17.4 = n
1.0e+9
Viable Count (CFU/ml)
3.8 x
109
1.0e+10
1.0e+8
Cells grown on salicylate, 0.1%
1.0e+7
1.0e+6
1.0e+5
1.0e+4
0
20
40
60
Time (Hours)
80
100
Calculating growth rate during exponential growth
1.0e+10dX/dt
where u = specific growth rate (h-1)
= uX
Viable Count (CFU/ml)
1.0e+9 dX/X = udt
Rearrange:
1.0e+8 lnX = ut + C,
Integrate:
where C = lnX0
dX/dt = uX
where u = specific growth rate (h-1)
1.0e+7
lnX = ut + ln X0 or
X = X0eut
1.0e+6y
= mx + b (equation for a straight line)
1.0e+5
Note that u, the growth rate, is the slope of this straight line
1.0e+4
0
20
40
60
Time (Hours)
80
100
Calculating growth rate during exponential growth
dX/dt = uX
where u = specific growth rate (h-1)
Rearrange: dX/X = udt
Integrate: lnX = ut + C,
where C = lnX0
lnX = ut + ln X0 or
X = X0eut
y = mx + b (equation for a straight line)
Note that u, the growth rate, is the slope of this straight line
Find the slope of this growth curve
1.0e+10
Viable Count (CFU/ml)
1.0e+9
1.0e+8
or u = lnX – lnX0
t – t0
lnX = ut + ln X0
1.0e+7
u = ln 5.5 x 108 – ln 1.7 x 105
8.2 - 4.2
1.0e+6
1.0e+5
1.0e+4
0
20
40
60
Time (Hours)
80
100
= 2 hr-1
Now calculate the doubling time
If you know the growth rate, u, you can calculate the doubling time
for the culture.
lnX = ut + ln X0
For X to be doubled: X/X0 = 2
or: 2 = eut
From the previous problem, u = 2 hr-1,
2 = e2(t)
t = 0.34 hr = 20.4 min
What is fastest known doubling time? Slowest?
How can you change the growth rate???
When under ideal, nonlimiting conditions, the growth rate can only be
changed by changing the temperature (growth increases with increasing
temp.). Otherwise to change the growth rate, you must obtain a different
microbe or use a different substrate.
In the environment (non-ideal conditions), the growth rate can be
changed by figuring out what the limiting condition in that environment is.
Question: Is exponential growth a frequent occurrence in the
environment?
Growth Curve
Turbidity (optical density)
9.0
7.0
ia l
De
a
onen
t
6.0
5.0
4.0
Lag
Time
th
Optical density
Stationary
Stationary
Ex p
Log 10 CFU/ml
8.0
Stationary phase
nutrients become limiting and/or toxic waste products accumulate
growth = death
(dX/dt = 0)
Death phase
death > growth
(dX/dt = -kdX)
Monod Equation
The exponential growth equation describes only a part of the growth
curve as shown in the graph below.
The Monod equation describes the dependence of the growth rate on the
substrate concentration:
.
um S
Ks + S
u = specific growth rate
1.0e+10
1.0e+9
Viable Count (CFU/ml)
u =
(h-1)
um = maximal growth rate (h-1)
S = substrate concentration (mg
L-1)
1.0e+8
1.0e+7
1.0e+6
1.0e+5
1.0e+4
Ks = half saturation constant (mg L-1)
0
20
40
60
Time (Hours)
80
100
Combining the Monod equation and the exponential growth equation allows
expression of an equation that describes the increase in cell mass through
the lag, exponential, and stationary phases of growth:
dX/dt = uX
u =
um. S
Ks + S
u = dX/Xdt
Monod equation
Exponential growth equation
1.0e+10
dX/dt = um. S . X
Ks + S
Viable Count (CFU/ml)
1.0e+9
1.0e+8
1.0e+7
1.0e+6
Does not describe death phase!
1.0e+5
1.0e+4
0
20
40
60
Time (Hours)
80
100
There are two special cases for the Monod growth equation
1.
At high substrate concentration when S>>Ks, the Monod equation
simplifies to:
dX/dt = umX
growth will occur at the
maximal growth rate.
2.
Ks
At low substrate concentration
when S<< Ks, the Monod equation
simplifies to:
dX/dt = um. S . X
Ks
growth will have a first order dependence on substrate concentration
(growth rate is very sensitive to S).
Which of the above two cases is the norm for environmental samples?
Growth in terms of substrate loss
In this case the growth equation must be expressed in terms of substrate
concentration. The equations for cell increase and substrate loss can be
related by the cell yield:
dS/dt = -1/Y (dX/dt)
where Y = cell yield
Y = g cell mass produced
g substrate consumed
Glucose (C6H12O6)
0.4
Pentachlorophenol (C6Cl5OH)
0.05
Octadecane (C18H38)
1.49
Growth in terms of substrate loss
dS/dt = -1/Y (dX/dt)
Combine with: dX/dt = um. S . X
Ks + S
dS/dt = - um (S . X)
Y (Ks + S)
Remaining
phenanthrene (%)
Which parts of this curve does the equation describe?
0
1
2
3
4
5
Time (days)
6
7
8
Fermentation technology for production of various
industrial compunds(vitamins,antibiotics,organic acid, etc)
Microbial products by fermentation
Technology
• Primary metabolites
Small molecules of living cells Intermediates or end products of
the pathway.
Related to synthesis of microbial cells in the growth
phase.Include alcohols, amino acids, nucleotides, organic acids,
polyols, vitamins, and enzymes
e.g. Lactic acid,citric acid
• Secondory metabolitesAccumulate following active growth.
Have no direct relationship to synthesis of cell material and
natural growth
Include antibiotics and toxins
Primary metabolites-
Secondory metabolites-
Primary metabolites
They are compounds made during the ordinary
metabolism of the organism during the growth phase. A
common example is ethanol or lactic acid, produced
during glycolysis. Citric acid is produced by some strains
of Aspergillus niger as part of the citric acid cycle to
acidify their environment and prevent competitors from
taking over. Glutamate is produced by
some Micrococcus species,and some Corynebacterium
species produce lysine, threonine, tryptophan and other
amino acids. All of these compounds are produced during
the normal "business" of the cell and released into the
environment. There is therefore no need to rupture the
cells for product recovery.
Secondory metabolitesThey are compounds made in the stationary phase; penicillin, for
instance, prevents the growth of bacteria which could compete
with Penicillium molds for resources. Some bacteria, such
as Lactobacillus species, are able to produce bacteriocins which
prevent the growth of bacterial competitors as well. These
compounds are of obvious value to humans wishing to prevent
the growth of bacteria, either as antibiotics or
as antiseptics (such as gramicidin S . Fungicides, such
as griseofulvin are also produced as secondary
metabolites.[Typically secondary metabolites are not produced in
the presence of glucose or other carbon sources which would
encourage growth,[8]and like primary metabolites are released
into the surrounding medium without rupture of the cell
membrane.
Vitamin production by Fermentation
Technology
• Vitamins are defined as essential micronutrients
that are required in trace quantity and are very
important compounds in diet
• Synthesized by prototrophic microorganisms
• Microbes excrete vitamins in excess of their
metabolic needs under highly specific and
artificial condition
• Vitamin B12
• Vitamin B2
• Vitamin C
• proVitamin A
Micro –organisms in industrial
production of vit. B12
Streptomyces griseus , S. olivaceus ,
Bacillus megaterium ,
B. coagulans , Pseudomonas
denitrificans ,
Propionibacterium freudenreichii , P.
shermanii and
a mixed fermentation of a Proteus spp
and a Pseudomonas sp
Vit.B12 production using
Streptomyces olivaceus NRRL B-1125
• Manufactured by submerged fermentation
• Aeration and agitation of medium essential
• Fermentation process completed in 3 to 5
days
Inoculum prepation
• Pure slant culture of Streptomyces olivaceus
NRRL B-1125 is inoculated and grown in 100 to
250 ml of inoculum medium.
• Seeded flask are kept on shaker for incubation .
• Flask cultures are used to inoculate large amount
of inoculum media arranged in series of tank .
• 2 or 3 successive transfers are made to obtain
required amount of inoculum cultures.
• Inoculum of production tank must be 5% of the
volume of production medium
Industrial production of vitamins
42
Production medium
• Consist of carbohydrate ,proteinaceous material , and
source of cobalt and other salts .
Components
Distillers solubles
Dextrose
CaCO3
COCl2.6H2O
Amount ( %)
4.0
0.5 to 1
0.5
1.5 to 10 p.p.m.
• Sterilization of medium batchwise or continuously .
• Batch – medium heated at 250°F for 1 hr
• Continuous – 330°F for 13 min by mixing with live
steam.
43
Temperature , pH , aeration and agitation
• Temperature : 80°F
• pH : At starting of process pH falls due to rapid
consumption of sugar, then rises after 2 to 4 due to
lysis of mycelium
pH 5 is maintained with H2SO4 and reducing agent
Na2SO4 .
• Aeration and agitation : Optimum rate of aeration is
0.5 vol air/vol medium/min. Excess aeration cause
foaming.
44
Antifoam agent , prevention of contamination
• Antifoam agent : soya bean oil , corn oil,
lard oil and silicones (sterilized before adding)
.
• Prevention of contamination : essential to
maintain sterility ,
contamination results in reduced yields ,
equipments must be sterile and all transfers
are carried out under aseptic conditions .
Industrial production of vitamins
45
Yields
• Yield of cobalamin are usually in the range of
1 to 2 mg. per litre in the fermented broth
Recovery
• Cobalamin associated with mycelium- boiling
mixture at pH 5 liberates the cobalamin
quantitatively from mycelium.
• Broth containing cobalamin is subjected to
further work up depending on type of
product to be produced
Recovery contd….
• Filtration - to remove mycelium.
• Filtered broth treated with cyanide –
(cobalamin to cyanocobalamin).
• Adsorption chromatography , ion exchange
chromatography – adsorbents : activated
charcoal , bentonite , fuller’s earth .
Bentonite
Fuller’s earth
• Elution : water, water-acetone and solution of sodium cyanide or
sodium thiocyanate .
• further extraction – countercurrent distribution b/w cresol,
amyl phenol or benzyl alcohol and water or single extraction into
organic solvent (phenol)
RecoveRy contd….
• To aqueous concentrates , dissolve a Zn salt in a slight acidic
solution & then rise the pH to bring about precipitation of
ZnOH(impurities are removed) .
• Chromatography on alumina & crystallization from
methanol-acetone , ethanol-acetone, or acetone-water.
Industrial production of vitamins
50
Recovery contd….
• To use as feed supplement , final fermented
broth is evaporated to dryness.
• Final broth contain 3% solids – in vacuo
evaporation (15 to 20 % solid content).
• Syrup – drum dried or spray dried.(contain 10
to 30 mg.lb. of cobalamin)
Beta- carotene or provitamin A
Provitamin A -----> Vitamin A (intestine)
•
•
•
Fat soluble
Deficiency leads to night blindness
Best source is liver and whole milk also coloured fruits and vegetables
•
•
•
Isoprene derivatives
Tetraterpenoids with eight isoprene residues
400 naturally occurring carotenoids: b-carotene, a-carotene, d-carotene, lycopene, zeaxanthin
Carotenoids Used as food colorants and animal feed supplements for poultry and
aquaculture, carotenoids play an increasing role in cosmetic and pharmaceutical
applications due to their antioxidant properties.
The pigments are often regarded as the driving force of the nutraceutical boom, since they
not only exhibit significant anticarcinogenic activities but also promote ocular health, can
improve immune response, and prevent chronic degenerative diseases.
Commercial production
Submerged Fermentation process
Microbial fermentation
Blakeslea trispora (high yeild; 7g/L)
Phycomyces blakesleeanus
Choanephora cucurbitarum
Corn starch, soyabean meal, b-ionone, antioxidants
stimulators
Trisporic acid: act as microbial sex hormone, improves yield
b-Ionone: incr b-carotene syn by incr enzyme activity
Purified deodorized kerosene increases solubility of hydrophobic substrates
Recovery: b- carotene rich mycelium used as feed additive
Mycelium is dehydrated by methanol, extracted in methylene chloride and
crystallized which is 70-85% pure
DSM Nutritional Products (Switzerland) and BASF (Germany) dominate
the market with their chemical synthesis processes, but Chinese
competitors are catching up.
Halophilic green microalgae Dunaliella salina. It accumulates the pigments in oil glo- bules in
the chloroplast interthylakoid spaces, protecting them against photoinhibition and
photodestruction.
Excessive pigment formation in D. salina is achieved by numerous stress factors like high
temperature, lack of nitrogen and phosphate but excess of carbon, high light intensity, and
high salt concentration, the latter two having the highest impact.
Dried D. salina biomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition
crystalline material obtained after extraction with edible oil is also sold.
Primary Metabolites: Organic Acids
Organic acids are produced by through metabolisms of carbohydrates. They accumulate in
the broth of the fermenter from where they are separated and purified.
Glycolysis
Krebs cycle
I. Terminal end products
lactic acid
(pyruvate, alcohol)
Propionic acid
II. Incomplete oxidation of sugars
(glucose)
Itaconic acid
citric acid
III. Dehydrogenation of alcohol with O2
Gluconic acid
acetic acid
Manufactured on large scale as pure products or as salts
CITRIC ACID: industrial uses
Flavoring agent
In food and beverages
Jams, candies, deserts,
frozen fruits, soft
drinks, wine
Antioxidants
preservative
and
Chemical industry
Antifoam
Treatment of textiles
Metal
industry,
pure
metals +citrate (chelating
agent)
Acidifyer
Flavoring
Chelating agent
Primary metabolite
Present in all organisms
Agent for stabilization of
Fats, oil or ascorbic acid
Stabilizer for cheese
preparation
Pharmaceutical industry
Trisodium citrate (blood
preservative)
Preservation of ointments
and cosmetics
Source of iron
Detergent cleaning industry
Replace polyphosphates
Commercial Production
Strains that can tolerate high sugar and low pH with reduced
synthesis of undesirable by products (oxalic acid, isocitric acid,
gluconic acid)
Glucose
Glucose
Pyruvate
Aspergillus niger
A. clavatus
Pencillium luteum
MEDIUM
CYTOPLASM
Pyruvate
CO2
Pyr carboxylase
OXA
Malate
Pyruvate
Pyr Dehydrogenase
Acetyl CoA
Malate
CO2
OXA
MITOCHONDRIA
Succinyl CoA
Fumarate
Citrate
synthase
citric acid
a-KG
100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate
Factors for regulation
 CARBOHYDRATE SOURCE:
sugar should be 12-25%
 Molasses (sugar cane or sugar beet)
 Starch (potato)
 Date syrup
 Cotton waste
 Banana extract
 Sweet potato pulp
 Brewery waste
 Pineapple waste
High sugar conc incr uptake and production of citric acid
 TRACE METALS:
 Mn2+, Fe3+, Zn2+ incr yield
 Mn2+ incr glycolysis
 Fe3+ is a cofator for enzymes like aconitase
 pH: incr yield when pH below 2.5, production of oxalic acid and gluconic acid is
suppressed and risk of contamination is minimal
 DISSOLVED O2: high O2, sparging or incr aeration can affect if interrupted
 NITROGEN SOURCE: addition of ammonium stimulates overproduction, molasses is
good source of nitrogen
Citric acid production
Surface fermentation
submerged fermentation
Solid
liquid
Stirred
Airlift
Bioreactor
bioreactor
N alkanes (C9-C23) can also be used to produce citric acid; can
result in excess production of isocitric acid
ACETIC ACID: industrial uses
ACETIC ACID
Incomplete oxidation of ethanol
Vinegar is prepared from alcoholic liquids since ceturies
NAD+
NADH +H+
NADP+
NADP +H+
CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 -------
CH3COOH
Ethanol
acetaldehyde
acetaldehyde hydrate
Alcohol
dehydrogenase
Acetaldehyde dehydrogenase
Gluconobacter, Acetobacter with acid tolerant A. aceti
One molecule of ethanol one molecule of acetic acid is produced
12% acetic acid from 12% alcohol
Clostridium thermoaceticum
It is an obligate anaerobe, Grampositive, spore-forming, rod-shaped,
thermophilic
organism
with
an
optimum growth temperature of 55–
60 o C
and
optimum pH
of
6.6–6.8.
VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters
flauoring agent in sauces and ketchups,
preservative also
Wine, malt, whey (surface or submerged fermentation process)
Surface: trickling generator; fermentale material sprayed over surface, trickle thro
shavings contaning acetic acid producing bacteria; 30oC (upper) and 35oC (lower).
Produced in 3 days.
Submerged: stainless steel, aerated using suction pump, production is 10X higher
Clostridium thermoaceticum (from horse manure) is also able to utilize fivecarbon sugars:
2C5H10O5 --- 5CH3COOH
A variety of substrates, including fructose, xylose, lactate, formate, and
pyruvate, have been used as carbon sources in an effort to lower substrate
costs. This factor is also important if cellulosic renewable resources are to be
used as raw materials.
Typical acidogenic bacteria are Clostridium aceticum, C. thermoaceticum,
Clostridium formicoaceticum, and Acetobacterium woodii. Many can also reduce
carbon dioxide and other one-carbon compounds to acetate.
1mol
2moles
1mol
2moles
1mol
CODH
These enzymes are metalloproteins; for example,
CODH contains nickel, iron, and sulfur; FDH
contains iron, selenium, tungsten, and a small
quantity of molybdenum; and the corrinoid enzyme
(vitamin B12 compound) contains cobalt. C.
thermoaceticum does not have any specific amino
acid requirement; nicotinic acid is the sole essential
vitamin
LACTIC ACID: industrial uses
Technical grade
20-50%
>90%
Food additive
(sour flour and
dough)
Ester manufacture
Textile industry
Glucose
G3P
G3P dehydrogenase
Pharmaceutical grade
Food grade
>80%
1,3-biphosphoglycerate
Intestinal treatment
(metal ion lactates)
Lactic acid
NAD+
LDH
(Lactate
NADH
+H+dehydrogenase)
Pyruvate
LACTIC ACID
2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid
First isolated from milk
Toady produced microbial
Heterofermentation
Homofermentation
Other than lactate products
only lactate as product
Lactobacillus
Mostly one isomer is produced
L. delbrueckii
Glucose
L. leichmanni
L. bulgaricus
L.helvetii
(lactose)
L.lactis
------L.amylophilus --------
Whey
Maltose
Starch
LACTIC ACID: production process
1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced
Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients)
pH 5.5-6.5/temp 45-50oC/75h
Heat to dissolve Ca lactate
Addition of H2SO4
(removal of Ca SO4)
Filter and concentrate
Addtion of Hexacyanoferrant
(removes heavy metal)
Purification (Ion exchange)
Concentration
Lactic acid
Antibiotics by Fermentation
Technology
Antibiotics are produced industrially by a
process of fermentation, where the source
microorganism is grown in large containers
(100,000 – 150,000 liters or more) containing a
liquid growth medium.
Streptomycin
Secondary metabolite
produced by Streptomyces
griseus.
Change in environment
condition and substrate
availability influence final
product.
In fermentation a soyabean
based medium is used with
glucose as carbon source.
Nitrogen source is combined in
soyabean meal, limits growth.
After growth the antibiotic
levels in the culture begin to
increase.
Source: www.indiamart.com
Phases during fermentation of
streptomycin
PHASE 1: Rapid growth producing
mycelial biomasss.Little production
of Streptomycin is obtained.
Proteolytic activity of the microbe
releases NH3 to the medium from the
soybean meal, causing a rise in pH
The glucose and NH3 released are
consumed during this phase.The pH
remains fairly constant-between 7.6
and 9.0.
PHASE 2: Additional production of
mycelium.Streptomycin accumulates in
the medium.
PHASE 3: Process has completed.Finally
the mycelium is separated by filtration
and antibiotic recovered.
PRODUCTION OF PENICILLIN
• Penicillin was the first important commercial
product produced by an aerobic, submerged
fermentation
• First antibiotic to have been manufacture in
bulk.
• Used as input material for some semi
synthetic antibiotics.
• It is fermented in a batch culture
• When penicillin was first made at the end of
the second world war using the fungus
Penicillium notatum, the process made 1 mg
dm-3.
• Today, using a different species (P.
chrysogenum) and a better extraction
procedures the yield is 50 g dm-3.
• There is a constant search to improve the
yield.
The yield of penicillin can be increased by:
• Improvement in composition of the medium
• Isolation of better penicillin producing mold
sp. Penicillium chrysogenum which grow
better in huge deep fermentation tank
• Development of submerged culture technique
for cultivation of mold in large volume of
liquid medium through which sterile air is
forced.
Primary and Secondary Metabolites
• Primary metabolites are produced during
active cell growth, and secondary metabolites
are produced near the onset of stationary
phase.
Commercial Production Of Penicillin
• Like all antibiotics,
penicillin is a
secondary
metabolite, so is
only produced in
the stationary
phase.
INDUSTRIAL PRODUCTION OF ANTIBIOTICPENICILLIN
• The industrial production of penicillin was
broadly classified in to two processes namely,
• Upstream processing
• Downstream processing
UPSTREAM PROCESSING
• Upstream processing encompasses any
technology that leads to the synthesis of a
product. Upstream includes the exploration,
development and production.
DOWNSTREAM PROCESSING
• The extraction and purification of a
biotechnological product from fermentation is
referred to as downstream processing.
UPSTREAM PROCESSING
INOCULUM PREPARATION
• The medium is designed to provide the
organism with all the nutrients that it requires.
• Inoculation method- submerged technique
• Spores -major source of inoculum
RAW MATERIALS
•
•
•
CARBON SOURCES:
Lactose acts as a very satisfactory carbon compound, provided that is used in a
concentration of 6%. Others such as glucose & sucrose may be used.
NITROGEN SOURCES:
Corn steep liquor (CSL)
Ammonium sulphate and ammonium acetate can be used as nitrogenous sources.
MINERAL SOURCES:
Elements namely potassium, phosphorus, magnesium, sulphur, zinc and copper are
essential for penicillin production. Some of these are applied by corn steep liquor.
•
Calcium can be added in the form of chalk to counter the natural acidity of CSL
•
PAA- precursor
FERMENTATION PROCESS
• The medium is inoculated with a suspension
of conidia of Penicillium chrysogenum.
• The medium is constantly aerated and
agitated, and the mould grows throughout as
pellets.
• After about seven days, growth is complete,
the pH rises to 8.0 or above, and penicillin
production ceases
STAGES IN DOWNSTREAM PROCESSING
Removal of cells
• The first step in product recovery is the
separation of whole cells and other insoluble
ingredients from the culture broth by
technique such as filtration and centrifugation.
ISOLATION OF BENZYL PENICILLIN
• The PH is adjusted to 2-2.5 with the help of phosphoric or sulphuric
acids.
• In aqueous solution at low PH values there is a partition coefficient in
favor of certain organic solvents such as butyl acetate.
• This step has to be carried out quickly for penicillin is very unstable at
low PH values.
• Antibiotic is then extracted back into an aqueous buffer at a PH of 7.5,
the partition coefficient now being strongly in favor of the aqueous
phase. The resulting aqueous solution is again acidified & re-extracted
with an organic solvent.
• These shifts between the water and solvent help in the purification of
penicillin.
The main stages of Penicillin production are:
Fermented Meat
87
1.Introduction
Meat is the flesh (muscle tissue ) of warm-blooded
animals,but fermented specialties from poultry
( sausages as well as cured and smoked fermented
poultry) are available.
What is fermented sausage?
A sausage is fermented if
-its pH below 5.6 and D-lactic acid content above 0.2%
-its colour is heat-stable
-its texture is no longer crumble
-its aroma is typical
-lactic acid bacteria predominate
-Enterobacteriaceae counts are low
88
a) Nutritional Role of Meat in the Human Diet:
• essential component of the human diet to ensure
optimal growth and development.
• as a concentrated source of a wide range of nutrients.
• high digestibility required relatively smaller guts.
• meat and meat products has increased with the affluence
of the consumer.
• fat content of meat as consumed is around 2to5%.
• protein of high biological value.
• micronutrient such as iron, zinc, vitamin B1, niacin
equivalents, and vitamin B12 significantly contribute to
the nutritional value of meat.
• red meat contains 50-60% of iron in the hame from (from
hemoglobin and
myoglobin).
89
Table. 2 Classification of fermented sausages
90
2. The history and culture related
to fermented meat.
• Meat is extremely susceptible to microbial spoilage.
• meat as a substrate are optimal for the growth of bacteria.
• water activity and pH are 0.96 to 0.97 and 5.6 to 5.8,
respectively
• nutrients and growth factors are abundantly available.
• storage and preservation of meat is necessary for the
suppression of microbial
growth or the elimination of
microorganisms and prevention of recontamination.
91
2. The history and culture related
to fermented meat
• The traditional methods which comprise reduction 1) water activity ( drying, salting) and/ or pH
(fermentation, acidification)
2) smoking, storage at refrigeration or freezing
temperatures,
3) use of curing aids (nitrite and nitrate)
• meat may also contain bacterial food pathogens.
• meat has to be of high quality with regard to hygiene
and microbial counts.
92
3. The fermentation process
Fermentation process : two types
-foods from a comminuted matrix
-whole meat products.
93
A. Fermentation of a Comminuted meat matrix
a) Variables in sausage production
Variables include:
• The particle size of the comminuted meat and fatty
tissue (1 and 30 mm)
• The selection of additives (curing salt, nitrate,
ascorbic acid, sodium glutamate and
glucono-∂lactone -source glucose.
• The temperature /humidity (below 2to 3℃, the
temperature is raised usually to ＞20℃ and ＞28℃,
but maximum higher temperatures (32 to 38℃).
94
•
•
•
•
The diameter of the sausages
The nature of the casings smoking
Heating after fermentation
Supporting the development of mold growth on
the surface or establishing a
special tight
surface film (e. g. coating with a titanium dioxide
film)
• Dipping in antifungal preparations ( sorbic acid or
pimaricin)
• pH-4.8 to 5.4
95
Table. 3
96
Species Employed in Meat Starter Cultures
• Bacteria: Lactic Acid Bacteria such as Lactobacillus acidophilus, Lb.
alimentarius, Lb. curvatus, Lb. plantarum etc, Lactococcus lactis,
Pediococcus acidilactici, P. pentosaceus
• Actinobacteria : Kocuria
Bifidobacterium spp.
varians,
Streptomyces
griseus,
• Staphylococci: Staphylococcus xylosus, S. carnosus ssp.
• Halomonadaceae : Halomonas elongata
• Fungi: Penicillium nalgiovense, P. chrysogenum, P. camemberti
• Yeasts: Debaryomyces hansenii, Candida famata
97
B. Fermentation of Whole Meat Products (HAM)
• curing by salting (with or without the use of nitrite and/or
nitrate)
• to achieve a water activity of ∠0.96 (equivalent to 4.5%
sodium chloride)
• temperatures (50C)―the salt will diffuse to the deepest
part of meat
• overcoming the food poisoning through Clostridium
botulinum contamination.
• after equilibrating the salt concentration and flavor
development, the temperature is raised to 15 to 250C to
ripen the ham.
• optimum flavor has no changed at least 6 to 9 months,
maximum 18th month.
• at the end of ripening step, the moisture has been
reduced by 25% and salt 4.5 to 6%)
98
4. Composition and changes during fermentation
• growth of LAB and concomitant acidification of the
product.
• reduction of nitrates to nitrites and formation of
nitrosomyoglobin
• solubilization and gelification
sarcoplasmic proteins
of
miofibrillar
and
• degradation of proteins and lipids
• dehydration
99
a) Fermentation Microflora
• sausage minces favor the growth of Micrococcacea and
Lactobacilli (5×108 to 109 CFU/g)
• Micrococcacea such as Kocuria varians, Staphylococcus
carnosus or S. xylosus
grow to cell counts 106 to 107
CFU/g, when nitrate cure is applied.
• inhibited the growth of organism
• the predominant microorganism is isolated
• growth of Staphylococcus occurs
• Penicillium constituted 96% of the microflora
• the nontoxigenic species Penicillium nalgiovense was most
frequently isolated
• the halotolerant yeast (Debaryomyces hansenii) is the
predominant
100
b) Acidification, Dehydration, and Microbial Antagonism
• isoelectric point of meat proteins (pH 5.3 to 5.4)
• increase the ionic strength
• sodium chloride and lactate in fermented sausages develop taste of
the product.
• acidification and drying are importance for inhibition of the growth of
pathogens.
• low pH and water activity exert an inhibitory effect towards
pathogens.
• lactic and acetic acids are the major fermentation products
• the dry matter content 50-75%
• the water activity values .86-.92 depend on ripening
101
c) Proteolytic and Lipolytic Degradation during fermentation
• Peptides and amino acids accumulate to levels of about 1% dry
matter
• Peptides and amino acids act as flavor enhancers and synergists.
• excess proteolysis may result in bitter and metabolic off-flavor
• amino acids and peptides are utilized by microorganisms for the
conversion to
flavor volatiles
• the bioactive peptides is influenced by lactic fermentation
• Kocuria varians is inhibited by environmental conditions
• Lb. casei utilizes peptides released from pork muscles
• fat content 40-60% of dry matter
• long chain fatty acids are released from triglycerides and
phospholipids
• free fatty acids are found 5% of the total fatty acids.
• polyunsaturated fatty acids is higher than saturated fatty acids. 102
d) Generation of Flavor volatiles
•
•
•
•
Routes:
by lipolysis and hydrolysis of phospholipids, followed by
oxidation of free fatty acids.
microorganisms produce organic acids: convert amino
acids and peptides to
flavor-active alcohols,
aldehydes, and acids
modify products of lipid oxidation
aroma is determined by the addition of spices, smoking,
or surface-ripening with yeasts or molds.
103
e) Biogenic amines
• histamine, tyramine, phenylethylamine, tryptamine,
putrescine and cadaverine not exceeding 100mg/kg.
• are mainly derived from bacterial decarboxylation of
amino acids
• putrescine and cadaverine are produced by the Gramnegative spoilage flora
• starter cultures inhibit rapidly metabolism of Gram
negative bacteria
• effectively reduce tyramine levels in fermented sausages
104
Product Diversity and Sensory Properties
• The main desirable effects of starter micro-organisms on
flavor and taste of fermented meats are
• formation of lactic acid
• transformation of compounds from abiotic breakdown of
lipids
• degradation of peptides and amino acids formed by meat
proteases
• Indirect effects are
• consumption of oxygen
• reduction of nitrate
• protein degradation by mould proteases
105
Sucuk
• One of the most important and widely
consumed traditional Turkish meat product,
• Dried, uncooked, cured and fermented
sausage,
• Produced from beef or buffalo meat Consist
of ground meat and sheep tail fat and curing
agents (nitrite and nitrate), with various spices
including cumin, garlic, salt, and black and red
pepper
106
Sucuk processing stages
Stuffing sausage mixture into natural sausage casings
Fermentation at 22-23ºC by either microorganisms
naturally present or added starter cultures
Drying for several weeks at ambient temperature and
humidity
due to fermentation, the final product has an
increased shelf life as a consequence of the
inhibition of the pathogenic and spoilage
bacteria,
107