Classification of microorganisms

Classification is the arrangement of organisms into groups or taxa

based on mutual similarity or evolutionary relatedness.

Benefits:

• It allows us to organize huge amounts of knowledge

• It allows us to make predictions and frame hypotheses for further

research based on knowledge of similar organisms

• Taxonomy places microorganisms in meaningful, useful groups

with precise names so that anyone can easily work with them and
communicate effectively

• Taxonomy is essential for accurate identification of

microorganisms
Basis of classification : mutual similarities between the organisms

Species, genera, families, orders, classes, phyla or divisions and kingdom or

domain
A procaryotic species is a collection of strains that share
many stable properties and differ significantly from other
groups of strains.

Strains: a group originating from a single culture

 Classification systems

Natural classification Anatomy

Phylogenetic classification Evolutionary
Phenetic classification Pynotypic relation
Numerical classification Presence or absence
or of a character
Numerical taxonomy
 Numerical taxonomy

Similarity matrix

Dendrogram
 Major Characteristics Used in Taxonomy

Morphological Characteristics
Physiological and Metabolic Characteristics
 Ecological Characteristics
Habitat : terrestrial, freshwater, marine, intestine, hot spring, etc.

Molecular Characteristics
Comparison of Proteins : protein sequence

Nucleic Acid Base Composition: G+C

Cultivation

Bacterial growth
Nutrition and other conditions for growth

Macro & micro elements

Growth factors
Specific pH
Specific temperature
Gaseous requirements
 Growth Media

Liquid media •Complex media

Solid media •Defined media

Semisolid media •Maintenance media

•Cultivation media

•Differential media
•Enrichment media
•Selective media
Cultivation techniques

•Batch

•Continuous
 Sterilization & disinfection

Sterilization is the process by which all living cells, viable spores,

viruses, and viroids are either destroyed or removed from an object or
habitat.

Disinfection is the killing, inhibition, or removal of microorganisms

that may cause disease.

Sanitization is closely related to disinfection. In sanitization, the

microbial population is reduced to levels that are considered safe by
public health standards.
The Pattern of Microbial Death
 Sterilization techniques

Physical : Heat :- Dry or Moist

 Effectiveness of sterilization

Decimal reduction time (D) or D value

Time required to kill 90% of the microorganisms or spores in a sample
at a specified temperature.

z value
Increase in temperature required to reduce D to 1/10 its value

F value
Time in minutes at a specific temperature (usually 250°F or 121.1°C)
needed to kill a population of cells or spores.
z = 12.5
The D value for Clostridium at 121°C is 0.204 minutes.
Therefore it would take 12D or 2.5 minutes to reduce 1012 spores to one
spore by heating at 121°C.

The z value for C. botulinum spores is 10°C—that is, it takes a 10°C

change in temperature to alter the D value tenfold.

If the cans were to be processed at 111°C rather than at 121°C, the D

value would increase by tenfold to 2.04 minutes and the 12D value to
24.5 minutes.
 Controlling Food Spoilage: industrial level

Removal of Microorganisms
Low Temperature
High Temperature
Water Availability
Chemical-Based Preservation
Radiation
Microbial Product–Based Inhibition
 Removal of Microorganisms

By filtration

Pre-filtrations and centrifuges improve the

effectiveness

Preserves aroma and nutritive value

Food products : fruit juices, wine, beer,

water, soft drinks etc.
 Low Temperature

Refrigeration
Refrigeration after pasteurization
 High Temperature

Canning (110–121°C)
Fruit juices, fruit pulps, fish,
meat etc.

Pasteurization
Low-temperature holding (LTH) :
62.8°C for 30 minutes

High-temperature, short-time
(HTST) : 71°C for 15 seconds

Ultra-high-temperature (UHT):
141°C for 2
 Water Availability

Dehydration: grains, fruits, meat, fishes, dry fruits etc.

 Chemical-Based Preservation

GRAS preservatives
 Radiation

UV: surfaces of utensils

Gamma irradiation from a cobalt-60: food particles

More efficient in moist conditions – production of peroxides

Factors affecting the efficiency of radiation

•The numbers of organisms (or spores) originally present.
•Some constituents [e.g., proteins, catalase, and reducing
substances (nitrites, sulfites, and sulfhydryl compounds)] may be
protective.
•The presence or absence of oxygen.
•The physical state of the food during irradiation.
•The condition of the organisms.
 Microbial Product–Based Inhibition

Antimicrobial peptide (AMP)

Eukaryotic AMP: antimicrobial with wide range of activity

toxic against prokaryotic and eukaryotic cells
Prokaryotic AMP: bacteriocin
narrow range
effective even at high temperature
Eg. Colicins, Nissin,
can be coupled with probiotic organisms
Strain improvement through mutation

UV
X ray
Mustard gas
Acridine dyes
Nitrous acid
Hydroxylamine
Alkylating agents
Protoplast fusion

Protoplasts are prepared by growing the cells in an isotonic solution while treating
them with enzymes such as cellulase and beta-galacturonidase

The protoplasts are then regenerated using osmotic stabilizers such as sucrose

A major advantage of the protoplast fusion technique is that protoplasts of different

microbial species can be fused, even if they are not closely linked taxonomically
 Insertion of Short DNA Sequences

Site-directed mutagenesis to protein engineering

This approach may lead to improved transformation of

recalcitrant materials, or even the degradation of materials that
have previously not been amenable to biological processing.
Transfer of Genetic Information between Different Organisms

“superbug,” patented by A. M. Chakarabarty in 1974

 Modification of Gene Expression

By changing gene transcription, fusing proteins, creating hybrid

promoters, and removing feedback regulation controls

metabolic pathway engineering (MPE)

 Natural Genetic Engineering

Forced evolution & Adaptive mutations

Through specific environmental stress microorganisms are forced to

mutate and then adapt
 Bacterial Growth

Growth

Bacterial population growth is analyzed by a growth curve

 The Growth Curve

Graphical representation of bacterial population in a batch culture

plotted as logarithm of number of cells versus time
Lag Phase
No increase in cell number immediately after inoculation - lag phase.

Reasons for lag phase

• The cells may be old and depleted of ATP

• Essential cofactors and ribosomes must be synthesized before

growth can begin

• The medium may be different from the one the microorganism was
growing in previously. Here new enzymes would be needed to use
different nutrients.

• Possibly the microorganisms have been injured and require time to

recover.
The lag phase varies considerably in length with the condition
of the microorganisms and the nature of the medium

Old culture as inoculum Long lag phase

Refrigerated culture Long lag phase

Different medium Long lag phase

Young culture as inoculum Short lag phase

Dense inoculum Short lag phase

Rich medium/same medium Short lag phase

Exponential Phase or Log phase
Growth and division of microorganisms will be possible maximum rate

Features of Log phase

• Their rate of growth is constant
• Growth curve rises smoothly
• The population is most uniform in terms of chemical and physiological
properties during this phase
• Exponential growth is balanced growth
• Limiting factor of growth in log phase is nutrients
 Stationary Phase

Features of stationary phase

• In the stationary phase the total number of viable microorganisms remains

constant
• population may simply cease to divide though remaining metabolically active
• produce a variety of starvation proteins, which make the cell much more
resistant to damage in a variety of ways
(i) Increase peptidoglycan cross-linking and cell wall strength
(ii) DNA-binding protein from starved cells protects the DNA
(iii) Chaperones prevent protein denaturation and drenature damaged
proteins
• Starved cells become harder to kill
(i) Salmonella typhimurium and some other bacterial pathogens become
more virulent when starved
Reasons for stationary phase
Limited nutrient availability
Limited O2 availability (aerobic culture)
Accumulation of toxic waste products (aerobic and anaerobic)
eg. Increased acidity and depletion of sugar in Streptococcal cultures
 Death Phase

Detrimental environmental changes like nutrient deprivation and the

buildup of toxic wastes lead to the decline in the number of viable cells
characteristic of the death phase.

The death of a microbial Population is usually logarithmic

.
 Measurement of microbial growth

Measurement of Cell Numbers:

Petroff-Hausser counting chamber

Bacteria/ml = no of bacteria X 25X 50 X 103

Measurement of Cell Numbers:
Plate count or TVC

A viable cell is defined as a cell which is able to divide and form a population (or
colony).
Measurement of Cell Numbers:
Membrane filter
Measurement of Cell Mass
 Continuous sterilizer design

• Direct : steam injector

• Indirect : spiral heat exchanger

Spiral heat exchanger:

Advantages

• No mixing of the sterile and non-sterile medium

• No use of cooling agents

• Heat conservation

• Reduce risk of sedimentation or burning

Direct : steam injector

Injects steam directly into the unsterile broth

Advantages:
• Very short time heating is required
• Efficient for media with suspended particles
• Easy cleaning
• High steam utilization efficiency

Disadvantages :
Foaming may occur
Chances of contamination are more
 Sterilization of the fermenter

By heating the jacket or coils of fermenter with steam and sparging

steam into the vessel through all openings (15psi 20 min.).

It was followed by passing sterile air into the system.

 Sterilization of the feed

1. Method of sterilization depends of the nature and quantity of the

feed
Continuous method of sterilization is preferred for large quantity,
however holding time varies with the material used.
 Filters

Methods
• Inertial impaction
• Diffusion
• Electrostatic attraction
• Interception
Filters:
Absolute filters : pore size is smaller than the particle (100%
efficient), also called as fixed pore filters
Depends on interception
Depth filters : pore size is larger than the particle, also
called as non-fixed pore filters
Depends on inertial impact, diffusion and electrostatic
attraction
 Filter sterilization of the media

Criteria for efficient filtration system

• Filtered medium must be free from contamination

• Minimal adsorption of proteins on the filters
• Filtered medium should be free from virus
• Filtered medium should be free from endo-toxins

In-order to fulfill all four criteria series of filters are used

(fig. 5.14)
 Filter sterilization of Air

Fixed pore filters

Mostly used are Polytetrafluoroethylene (PTFE) pleated membrane cartridges

Steam sterilizable, ammonia resistant

Air filtration system consists of

1. Prefilter – removes dust, carbon partilces and moisture

2. barrier filters (membrane cartridges) removes contaminants (fig 5.18 )

.
 Validation of sterilization

Validation: Documented evidence that the manufacturing process consistently

produces product that meets predetermined specifications

Sterilization is an absolute value; either sterile or non-sterile

Basic principles in validation process

1.Theoretical approach is (based on installation and operational qualification)

2.Performance of validation or performance qualification
3.Analysis and documentation of the validation data
Installation qualification (IQ) and operational qualification (OQ)

During the IQ and OQ phases, various features of the autoclave are

examined and tested for proper functioning.

Tests of the chamber design, pressure vessel, and door safety interlock
system and inspections of the chamber jacket, steam traps, electrical
circuits, pressure and temperature indicators, vacuum pumps, vent filters,
and steam supply to the chamber.

Performance qualification (PQ)

Objectives:
• to demonstrate the uniformity and effectiveness of the process
• to demonstrate the reproducibility of the process
• to demonstrate the compatibility of the process with the
items to be sterilized
Major steps involved in performance validation

1.Define an attribute of the final product Example: The

product will be sterile.
2.Specification of the attribute,
The product will be sterilized by a sterilization process sufficient to produce a
probability of nonsterility of one out of 1 million containers
3.Select appropriate method, method to estimate the bacterial count, select
appropriate autoclave, thermalcouples
4. Develop and conduct the test
Determine microbial load counts prior to container filling.
Determine D and Z values of biological indicator organism.
Perform heat distribution studies of empty and loaded autoclave.
Perform heat penetration studies of product at various locations in the batch.

5. Examine the test procedure to ensure the accuracy and reliability

Accuracy of thermocouples
Repeatability of the autoclave cycle in terms of temperature and F value
consistency.
A challenge of the sterilization cycle with varying levels of bioindicator
organisms.
Reliability of cleaning processes to produce consistent low-level
product bioburdens
 Bioreactors
Basic functions:
• Should be capable of reliable aseptic continuous operation for
several days
• Power consumption should be very low
• Should have provision for sampling, aeration, pH adjustment
and temperature checking
• Should be easy to operate, harvest, clean and maintain
• Should be similar in geometry as that of lab fermenter and pilot
plant
The performance of any bioreactor depends on

• Sterile conditions
• Biomass concentration
• Nutrient supply
• Metabolisms/microbial activities
• Effective agitations
• Aeration
• Heat removal
• Product inhibition
• Correct shear conditions
• Product removal
 TYPE OF BIOREACTOR

Based on aeration and agitation

1. Non-stirred, non-aerated system

2. Non-stirred, aerated system

3. Stirred and aerated systems

Based on the process

4. Batch

5. Fed batch

6. Continuous
Batch fermentation process

A tank of fermenter is filled with the prepared medium and steam sterilized

The inoculum of a pure culture is added to the fermenter, from a separate pure

culture vessel

The temperature and pH for microbial fermentation is monitored and adjusted,

and occasionally nutritive supplements are added to the medium

After the proper time the contents are harvested

The fermenter is cleaned and the process is repeated

Thus each fermentation is a discontinuous process divided into batches

Fed- Batch fermentation process

Principle: under nutrient stress microbes gives better yield of certain

products
Continuous Fermentation Process –

In continuous fermentation, the substrate is added to the fermenter

at a fixed rate.
This maintains the organisms in the logarithmic growth phase.
The fermentation products are taken out continuously.
 Solid State Fermentation
SSF involves the growth of micro-organisms on moist-solid particle
with the absence or nearly absence of free water

The solid particle acts as substrate and an anchorage for the cells
The inter-particle space is filled by the gas phase and majority of the water
in the system is absorbed within the moist-solid particles
•Koji method: oldest SSF

Rice fermentation for the production of the rice wine called “sake” in
Japan.

Aspergillus oryzae or Aspergillus awamori of a crude amylolytic

preparation (koji) which is a fermented mash of steamed rice. This
process breaks down starch into glucose that is then fermented in a
secondary stage by yeast in bamboo boxes

•Moldy Bran

It is based in a rotatory drum supplied with wheat bran fortified with a

mineral solution. Moldy bran was used for the production of cellulases,
amylases, and pectinases and was considered an extension of koji
technology.
•Other substrates

Nonbiodegradable materials

• amberlite
• polystyrene
• polyurethane
 General steps in SSF process.

Steps Process
Inoculum Preparation •Selection of pure strain
•Develop an inoculum of sufficient size and high
Viability
Substrate Preparation • Cutting or granulation to obtain apropriate size.
• Addition of water and nutritional supplements or pre-
treat with heat to increase the availability of nutrients.
• Sterilization

Inoculation , loading and Inoculation occurs either prior or after loading

fermentation
Downstream processing Depending on process

Waste Disposal
 Present industrial SSF systems

1. Fixed Bed Koji Methods

• Koji production is undertaken in shallow fixed beds of fermentation mash

distributed in trays.

• Air supply and cooling is provided by natural convection currents between

the mash and the environment

• Trays may be covered to prevent contamination

• Many trays can be put in stacks in large air-conditioned rooms.

 2. Fermentation with Fixed Beds and Forced Aeration

Zymotis reactor, which is a series of vertical, parallel, heat exchanger plates

with forced aeration through the fermentation mash

This allows for heat exchange to be at least partially dissociated, from the
mass transfer of oxygen, water, and carbon dioxide

3. Solid Fermentation with Mechanical Stirring and Aeration

Plafractor TM
Advantages of SSF over SmF
Disadvantages of SSF compared to SmF
 Filter press

Usually performed for extra-cellular products

Solid particles are separated from a fluid–solid mixture by forcing the

fluid through a filter medium or filter cloth, which retains the solid
particles.

The clear solution obtained are called as filtrate

The solid particle deposited on the filter after filtration is called as

filter cake
rate of filtration = driving force/resistance
The filtration rate depends on the area of the filter cloth, the viscosity
of the liquid, the pressure drop across the filter and filter cake
resistance

Filtration can be performed using either

(i) Vacuum
(ii) Positive-pressure equipment

Filter aids: It is a pre-coated filter medium to prevent blockage or blinding

of the filter by solids

Addition of coagulants
 Rotary vacuum filter
It Consists of a drum rotating in a tub of liquid to be filtered
 CENTRIFUGATION

Centrifugation is used to separate materials of different densities when a force

greater than gravity has been implemented, such as centrifugal forces.
 Measurement and control of bioprocess parameters
Success of fermentation technology depends on the proper control of bioprocess
parameters
Parameters
Temperature In the fermenter or pipes
Pressure
rpm
Foam
biomass
pH
Redox
DO
CO2
Viscosity
Measurement and control of bioprocess parameters : Temperature