Introduction to

Fermentation
Aspergillus niger and Lactobacillus
 Introduction to Fermentation
• Aspergillus niger and Lactobacillus Delbruckii are the
microbes used to commercially produce citric acid and
lactic acid, respectively. The production takes place in a
batch fermenter. This tutorial will introduce you to the
following areas regarding batch fermentation
– Microbial Growth Kinetics
– Media for Industrial Fermentations
– Sterilization
– The Development of Inocula for Industrial Fermentations
– Design of a Fermenter
– Instrumentation and Control
– Aeration and Agitation
 Microbial Growth Kinetics
• Microbial Growth Kinetics
describe how the microbe
grows in the fermenter. This
information is important to
determine optimal batch times.
The growth of microbes in a
fermenter can be broken down
into four stages:
– Lag Phase
– Exponential Phase
– Stationary Phase
– Death Phase

(Growth curve is from Shuler p.

161)
 Microbial Growth Kinetics
• Lag Phase
– This is the first phase in the fermentation
process
– The cells have just been injected into a new
environment and they need time to adjust
accordingly
– Cell growth is minimal in this phase.
 Microbial Growth Kinetics
• Exponential Phase
– The second phase in the fermentation process
– The cells have adjusted to their environment
and rapid growth takes place
– Cell growth rate is highest in this phase
 Microbial Growth Kinetics
• Exponential Phase (Continued)
– At some point the cell growth rate will level
off and become constant
– The most likely cause of this leveling off is
substrate limited inhibition
• Substrate limited inhibition means that the
microbes do not have enough nutrients in the
medium to continue multiplying.
 Microbial Growth Kinetics
• Stationary phase
– This is the third phase in the fermentation
process
– The cell growth rate has leveled off and
become constant
– The number of cells multiplying equals the
number of cells dying
 Microbial Growth Kinetics
• Death phase
– The fourth phase in the fermentation process
– The number of cells dying is greater than the
number of cells multiplying
• The cause of the death phase is usually that the
cells have consumed most of the nutrients in the
medium and there is not enough left for
sustainability
Media for Industrial Fermentations
• The media is the feed solution
– It must contain the essential nutrients needed for the
microbe to grow
• Factors of consideration when choosing media
-Quality consistence and availability
-Ensure there are no problems with Media Prep or
other aspects of production process

Ex. Cane molasses, beet molasses, cereal grains

 Sterilization
• Sterilizing the feed solution is essential
because the media cannot contain foreign
microbes because this could severely
hinder the growth of the production
microbe
– Most popular method is heat sterilization of
the feed solution
 The Development of Inocula for
Industrial Fermentations
• The inoculum is the starter culture that is
injected into the fermenter
– It must be of sufficient size for optimal growth
kinetics
• Since the production fermenter in industrial
fermentations is so large, the inoculum volume
has to be quite large
- A seed fermenter is usually required to produce the
inoculum volume
-The seed fermenter’s purpose is not to produce
product but to prepare inoculum
 Design of a Fermenter
• Factors to consider when
designing a fermenter
– Aseptic and regulatory
capability, long-term
reliability
– Adequate aeration and
agitation
– Low power consumption
– Temperature and pH
controls
– Sampling facilities

(14 L fermenter shown is a

copyright of New
Brunswick Scientific)
 Instrumentation and Control
• The success of a fermentation process is
highly dependent on environmental
factors
– The fermenter needs to be able to control
such factors as temperature, pH, and
dissolved oxygen levels
 Aeration and Agitation
• Most industrial fermentations are aerobic
processes meaning that the production
microbe requires oxygen to grow
– The oxygen demand is met by sparging air
through the fermentation vessel and using an
agitator increase the amount of dissolved
oxygen
 References
• Stanbury, P.F., A. Whitaker, and S. J. Hall,
Principles of Fermentation Technology, 2nd
ed., Butterworth Heinemann, Oxford,
2000.
• Shuler, M. L. and F. Kargi. Bioprocess
Engineering Basic Concepts, 2nd ed.,
Prentice Hall, Upper Saddle River, NJ,
2002.
Microbial Fermentation
(Enzymology, Metabolic pathways
and Fermentation aspects)

OSAMA O. IBRAHIM, PH.D.

CONSULTANT BIOTECHNOLOGY
GURNEE IL. 60031
BIOINNOVATION04@YAHOO.COM
 Agenda 17
 Introduction.
 Fermentation media.
 Industrial microorganisms.
 Types of fermentation.
 Batch fermentation.
 Fed-Batch fermentation.
 Growth rate.
 Continuous fermentation.
 Effect of flow rate on substrate concentration.
 Important factors for continuous fermentation.
 Classification of fermentation.
 Agenda (Cont.) 18
 Enzymes.
 Enzymes equilibrium state.
 Factors effects enzymes catalytic activity.
 Natural mechanisms for regulating enzyme activity.
 Enzymes activators.
 Classification of enzymes.
 Fermentor systems.
 Fermentation process.
 Microbial cell breakage systems.
 Introduction 19

 The fermentation industry is composed of

five major bio-ingredient categories.
 They are:
- Proteins & amino acids.
- Organic acids.
- Antibiotics.
- Enzymes.
- Vitamins & hormones.
 Introduction (cont.) 20

 Fermentation industry is driven by:

- The cost and availability of feed-stocks.
- The efficiency of industrial microorganism.
- Fermentation condition and optimization.
- Down stream process and end-product
recovery efficiency.
- Fermentation by-product utilization.
- Utility consumption and labor cost.
 Fermentation media 21
 Optimum balance of the media is mandatory for
cells propagation and for the maximum
production of target metabolite (end-product).
 Media compositions:
- Carbon source.
- Nitrogen source.
- Minerals.
- Growth factors.
- Precursors (mutants).
 Industrial microorganisms 22

Microbial screening.
93 C


- Wild strains.
 Microbial yield improvement
43 C

- Mutation.
- Recombinant DNA. 21 C

- Genetically engineered. 4C

 Microbial selection.
 Industrial microorganism
 Types of fermentation 23

 Solid State fermentation (SSF).

 LiquidState fermentation (LSF)

Surface culture & submerged
culture
 Solid State Fermentation (SSF) 24
 SSF process can be defined as microbial
growth on particles without presence of
free water.
 Particles are a solid culture substrate such
as rice or wheat bran saturated with water
and inoculated with (mold, yeast,
bacteria) in controlled room temperature.
 It is ideal for growing filamentous fungi.
 It has been used in Asia and developing
nations.
 It is more cost effective (smaller vessels
lower water consumption, reduced waste
water treatment costs, lower energy
consumption, and less contamination
problems).
 SSF process and applications 25
Applications:
 Potentially many high value
products such as extra-cellular
enzymes, primary metabolites,
and antibiotics could be
produced in SSF.
 It is estimated that nearly a third
of industrial enzyme produced in
Japan is made by SSF process.
 Production of organic and
ethanol from starchy substrates.
 Digestibility of fibers and
lignocelluloses materials for both
human and animal consumption.
Liquid State fermentation (LSF)26
[Submerged culture]
- Submerged culture is performed in tanks
which can reach in size for over 100,000
gallons.
- It is ideal for the growing unicellular
organisms such as bacteria and yeast.
LSF methods:
- Batch fermentation.
- Fed-batch fermentation.
- Continuous fermentation.
- Semi-continuous fermentation.
 Batch fermentation 27

 Considered to be a closed system.

 The sterilized media in the fermenter is
inoculated with the microorganism.
 Incubation is allowed under the optimum
conditions (aeration, agitation,
temperature).
 During entire fermentation nothing is
added except air, antifoam and acid/base.
 28
Fed-Batch fermentation
 It is enhancement of batch fermentation.
 Continue adding the nutrients (feeding) in a
small doses during the fermentation.
 The method in controlling nutrients feeding
process is by measuring methods.
 The main advantage of fed-batch
fermentation is the elimination of catabolite
repression (feed-back inhibition).
Microbial growth rate 29

Secondary
metabolites
Primary
metabolites
Batch fermenter system 30
 Continuous fermentation 31

 It is an open system.
 Continuously sterile nutrient is added and
the converted nutrient is taken out from
the fermentor.
 In continuous process cell loss as a result
of outflow must be balanced by growth
of the microorganism.
Effect of flow rate on substrate32
concentration

The relationship between biomass (X), the concentration of limiting nutrients (C) ,and
the dilution rate (D) are important factors in continuous
Continuous fermenter system 33
 Important factors for 34
continuous fermentation
 The system must be stable for at least 500
hours.
 Maintaining sterile conditions for all period of
fermentation time.
 The composition of nutrients must be
constant all the time.
 Maintaining the strain stability for constant
high production yield (concerning about
reverse mutation).
 Semi-continuous 35
fermentation
 Semi-continuous fermentations, in
which a fraction of a fermentation
is replaced with fresh media at
regular intervals.
Classification of fermentation 36
 Classification is according to product formation:
- TYPE I:
Ex
Substrate A P product

E1 E2 E3 E4
Substrate A B C D P product

- Type II:
E1 E2 E3 E4
Substrate A B C D P-Primary Metabolites

Es1
Es2 Es3 Es4
E F G P-Secondary Metabolites
 Enzymes 37

 Enzymes are active biological molecules responsible for

thousands of metabolic process that sustain life.
 Most enzymes are proteins, although some catalytic
enzymes are RNA molecules.
 In enzyme reactions, the molecules at the beginning of
the process, called substrates and converted into
different molecules that called end-products.
 Enzymes are very specific as which reaction they
catalyze and the substrate that involved in the reaction.
 Depends on enzyme activity, the bioconversion to end-
products can be faster and reached the equilibrium
state rapidly.
 Enzymes 38
equilibrium state

E+S ES E+P
Substrate binding Catalytic step
E= enzyme E=enzyme
S= Substrate P= product

E+S E+P
Enzyme/substrate 39
interaction
 Factors Effects Enzymes
Catalytic Activity 40
 Temperature: The optimum is generally 40-600C.
Some enzymes exhibit an optimum at almost 1000C.
 Value of PH: The optimum generally in the range
from 5-7. Extreme values of 1.5-10.5 have been
found.
 Activation: Many chemical activates the catalytic
enzymes activity, Such as inorganic ions.
 Inhibitors: Many chemical inhibits the catalytic
enzymes activity
 There are two types of enzymes inhibition:
Irreversible inhibitors (competitive inhibition) and
reversible inhibitors (uncompetitive inhibition).
 Substrate inhibition: High concentration of substrate
may inhibit the catalytic activity of an enzyme.
 End-product inhibition: In the case of multi enzyme
system (catalytic inhibition).
Factors effecting enzymes 41
activity
 Activators
42
(Cofactors and Coenzymes)
 Some enzymes do not need any additional
components to show full activity.
 Cofactors can be either inorganic (metals) or
organic compounds (flavin and heme).
 Coenzymes include NAD+, NADP+ and ATP.
 These coenzymes transfer chemical group between
enzymes.
 The chemical groups carried by the hydride ion (H+)
carried by NADH or NADPH.
NAD+ + 2H + + 2e- NADH / NADP+ + 2O-2 + 2H+ NADPH + 2O2

 Or phosphate groups carried by ATP.

ATP +H2o ADP+ P1 (-7.3kcal/ mole) / ATP +H2o AMP + PP1 (-14kcal/mole)
Enzymes inhibitors and 43
activators mechanism
Feedback inhibition and 44
precursor activation
 Natural mechanisms for 45
regulating enzyme activity
 Classification of enzymes 46
 The International of Biochemistry and Molecular Biology
developed a nomenclature for enzymes (EC number).
 Each enzyme is classified by sequence of four numbers
preceded by EC. (E.C. 5.3.1.18 Glucose isomerase)
 The top-level classification is:
- EC1 Oxidoreductases (catalyze oxidation/reduction
reactions).
- EC2 Tranferases (transferee a functional group).
- EC3 Hydrolases (catalyze the hydrolysis of various bonds).
- EC4 Lyases (cleave bonds by mean of hydrolysis /oxidation).
- EC5 Isomerases (isomerization within same molecule).
- EC6 Ligase ( join two molecules with covalent bonds).
 Enzymes production 47
Inoculums Flask Seed tank Fermentor Enzyme
recovery
• Constitutive enzymes: The microorganism produce the
enzyme in minimal fermentation media.
• Inducible enzymes: The microorganism require
adding inducible agents in the media to produce the target
enzyme.
• Extracellular enzymes: The microorganism secrete the
enzyme in the fermentation media.
• Intracellular enzymes: The microorganism produce the
enzyme inside the cell.
Microbial cell breakage systems48
 Enzymes (Conclusion) 49
 Enzymes are usually sold based on the activity
(u/ml or u/gm).
 If the efficiency of enzymes are considered, their
cost, is based on active enzyme protein u/mg
protein (specific activity).
 The commercial exploitation of enzymes range from
high-volume but low cost (industrial enzymes) to low
volume, but high cost (enzymes for medical,
scientific and analytical use).
 Workers handling industrial enzymes should use
protective clothing and eye protection.
 Food enzymes if foods processing have bees shows
to be safe through many years of manufacturing
practice.
 50

Thank You for your

attention