Industrial cultures

Fermentation

and scale up processing

Handling and preservation of industrial
microorganism
Sterility in industrial microbiology
Microbiological industrial application

 Industrial

microbiology and microbial

biotechnology, are they in similar field?

 Major

microbial used:

fungi (yeast and mold)

Certain prokaryote: streptomyces

Industrial

microorganism are metabolite

specialists, capable of synthesizing one or
more product in high yield
Genetically alter strains of industrial microbe
by mutations or recombinant - achieved
higher yield

 Sources

microbe:

ATCC- American type culture collection

DSMZ, germany Deutsche Sammlung von
Microorganism

Process:

New biocatalyzed process (patented)

deposited into national collection

 Microbe

suitable for industrial process must

have other feature beside just being able to
produce the substance of interest:

Organism must capable to growth and product

formation in large scale culture
Produce spore or some reproductive cell form so
that it can be easily inoculated into large scale
fermentor
Grow rapidly and produce desire product in a
relatively short period of time
Able to grow in a relatively inexpensive liquid
culture medium obtainable in bulk quantities

Not be pathogenic esp to humans or economically

important animals or plants
Can be amenable to genetic manipulation.

Example

of industrial product:

Microbial cell yeast

Substances produced by cell

Enzyme(glucose isomerase)
Commodity chemical ethanol, citric acid, etc

Homolactic
Pyruvic acid
metabolized in
the absence of
oxygen

Definasi

Pyruvic lactic
acid
No gas are produce
Lactobacilli,
streptococci
Eg. In cheeses
making

CO2 released
from Pyruvic
acid to form
acetaldehyde
ethyl alcohol
Yeast
fermentation
Eg in bread,
wine

Alcoholic

 Vessel

in which industrial process is carried

oout: fermentor
Fermentation:

Any large scale microbial process whether or not

it is biochemically fermentation
Mostly involve aerobic

size

varies depend on the process and how it

is operated.

5-10 L : laboratory scale

500000 L : industrial scale

Fermentor size (L)

Product

1-20 000

Diagnosis enzymes, substances

for molecular biology

40-80 000

Some enzyme, antibiotics

100-150 000

Penicilin, aminoglycoside
antibiotics, proteases, amylases,
steroid transformations, amino
acid, wine, beer

200 000 500 000

Amino acid (glutamic acid),

wine, beer

Industrial

fermentor: anaerobic and aerobic

process

Anerobic: require little special equipment except

for removal of heat generated
Aerobic: more elaborate equipment for better
mixing and adequate earation

 A-exhaust
B-impeller
C-sparger

Cooling water in

(high
pressure air)
D-harvest
E- culture broth
F-cooling jacket
Sterile air

 Installation.

Note:

External cooling coils

 Installation.

Note:

Location of mezzanine
floor

 Top

(mezzanine
floor). Note:

Agitator motor

 Top

(mezzanine
floor). Note:

Control panel (now

superseded by
microprocessor/comp
uter control)

 Top

(mezzanine
floor). Note:

Inspection hatch

 Interior

view from
bottom. Note:

Agitators

 Interior

view from
bottom. Note:

Baffles

 Interior

view from
bottom. Note:

Inspection hatch and

ladder

 Fermenter

Building

Air mixed fermenters are taller/thinner than systems

with agitators

 Top

Note lack of agitator motor

 Base

 CIP

(clean in place) and in situ sterilisation

Constructed in stainless steel:

Inert and strong

Cooling:

Jacket or coils (internal or external)

 General

View

 Control

Consol.

Note:

Microprocessor logging
and control

 Control

consol.

Note:

Microprocessor logging
and control
Gas supply rotameters

 Control

consol.

Note:

Microprocessor logging
and control
Gas supply rotameters
Pumps for pH control,
antifoam, nutrient
feed etc

 Fermenter

vessel.

Note:

Detachable stirrer
motor

 Fermenter

vessel.

Note:

Detachable stirrer
motor
pH/oxygen electrodes

 Fermenter

vessel.

Note:

Detachable stirrer
motor
pH/oxygen electrodes
Exhaust gas condenser

 Fermenter

Note:

vessel.

Detachable stirrer
motor
pH/oxygen electrodes
Exhaust gas condenser
Dialysis unit (not
usual!)

 Measure

of growth and product formation

Control the process by altering environment
parameters : temperature, pH, cell mass,
level of key nutrients, product concentration
Obtain data in real time:

Eg:alter one or more environmental parameter as

fermentation progress or to feed a nutrient at a
rate that exactly balance growth

Computer

process of data on line and then

response accordingly

 Scale

up:

The transfer of a process from small scale

laboratory equipment to large scale commercial
eqipment

Biocatalyst

processes rarely behave the same

way in large scale fermentor.
Needed of biochemical engineer: familiar
with gas transfer, fluid dynamic, mixing,
thermodynamics

Laboratory
fermentor(110L)
-small scale

Laboratory
flask
-indication of
possible process
for commercial
interest product

fermentor
- First effcort of
scale up
- Test variation in
medium, temp, pH
etc (costing)

Piloty plant
stage (3003000L)
- Condition
more closely
approach to
commercial
scale

Commercial
fermentor(10
000-500000L
-

Isolatio
n of the
microbe
from
nature
resourc
es

Identifica
tion of
desire
microbe

Charact
erizatio
n of the
microbe

Screenin
g of the
desire
microbe

Inoculum
preparati
on

Strain
improve
ment

fermentation

Microbial preservation
microorganism or virus has been selected or
created to serve a specific purpose, it must be
preserved in its original form for further use and
study

Objective of preservation:
to ensure optimal long-term viability and genetic
stability

 The

preservation methods used in the

collections may differ.
Each collection must maintain detailed
protocols of the applied preservation
methods and their application to specific
groups of microorganisms.
After every preservation of a microbial
strain, controls are necessary. e.g viability
and purity, and where appropriate, the
identity of the preserved culture have to be
checked immediately after preservation.

 Reduction

of temperature of growth
Dehydration
Reduction of nutrients

Reduction of microbial metabolite

 Preservation

on agar with ordinary refrigeration

(4 10C)
Organisms growing on suitable agar at normal growth
temperatures attain the stationary phase and begin to
die because of the release of toxic materials and the
exhaustion of the nutrients. Agar-grown organisms are
therefore refrigerated as soon as adequate growth
Aerobic microbial Agar slant and petri disk
Anaerobic microbial - oil overlay and agar stabs
which are then sealed with sterile molten
petroleum jelly,

Preservation in Deep Freezers at about -20C, or

between -60C and -80C
store microorganisms in either type of deep freezers in
the form of agar plugs or on sterile glass beads coated
with the organism to be stored
Preservation on glass bead

placed in broth containing cryoprotective compounds such

as glycerol, raffinose, lactose, or trehalose. Sterile glass
beads are placed in the glass vials containing the bacterial
cultures. The vials are gently shaken before being put in
the deep freezer

Storage of agar cores with microbial growth

placing agar plugs of confluent growth of bacteria and

yeasts and hyphe of moulds or actinomycete in glass vials
containing a suitable cryoprotectant and freezing the vials
in deep freezers as above

 Storage

in low temperature liquid or vapor

phase nitrogen (-156C to -196C)

method for organisms which will not survive

freeze-drying
Some of the most commonly used
cryoprotectants are (vol/vol) 10-20% glycerol and
5-10% dimethyl sulfoxide (DMSO) in broth culture
of the organism in vials which are then frozen in
liquid nitrogen

Drying on silica gel

Screw-cap tubes half-filled silica gel are sterilized in

an oven. On cooling a skim-milk suspension of spores
and the cells of the fungus or actinomycetes is placed
over the silica gel and cooled. They are dried at
25C, cooled and stored in closed containers
containing desiccants.

Preservation on sterile filter paper

placing drops of broth containing the spores on sterile

filter paper in a Petri dish and drying in a low
temperature oven or in a dessicator.
After drying the filter paper may be placed in sterile
screw caps bottles and stored either at room
temperature or in the refrigerator.

Freeze-drying (drying with freezing), lyophilization

organism is first frozen. Subsequently, water is removed

by direct vaporization of the ice with the introduction of
a vacuum.
As the suspension is not in the liquid state, distortion of
shape and consequent cell damage is minimized.
At the end of the drying the ampoule containing the
organism may be stored under refrigeration although
survival for many years has also been obtained by
storage at room temperature

L-drying (liquid drying, drying without refrigeration)

organisms are not frozen, but dried from the liquid state
to preserve non-spore formers sensitive to freeze-drying,
such as Cytophaga, Spirillum and Vibrio.
Liquid drying has been effectively used to preserve
organisms such as anaerobes that are

 Many

organisms die in distilled water

because of water absorption by osmosis.
However some have been known to survive
for long periods in sterile distilled water.
Usually such storage is accompanied by
refrigeration;

 Basic

of loss by contamination
Method of achieving sterility (physical and
chemical)
Aspects of sterilization in industry

Sterilization of the fermentor and it accessories

Medium sterilization

 The

contaminant may utilize the components

of the fermentation broth to produce
unwanted end-products and therefore reduce
yield.

e.g in beer industry when lactic acid bacteria

contaminate the fermentating wort, they utilize
sugars present therein to produce unwanted
lactic acid which renders the beer sour

contaminant

may alter the environmental

conditions such as the pH or oxidationreduction potential of the fermentation and
render it unsuitable for maximum production
of the required product.

 Contamination

by lytic organisms such as

bacteriophages or Bdellovibrio could lead to
the entire destruction of the producing
organism.
The result could be losses in manpower time
needed to devise means of dealing with the
product

 Physical

method

Asepsis
Filteration
Heat
Radiation

Chemical

method

Chemosterilants
Gaseous sterilants
Phenol and chlorine

Steam is used to sterilize the medium in situ in the fermentor but

sometimes the medium may be sterilized separately in a retort or
autoclave and subsequently transferred aseptically to a
fermentor.
In order to avoid microbial growth within the fermentor when not
in use, crevices and rough edges are avoided in the construction
of fermentors, because these provide pockets of media in which
undesirable microorganisms can grow. These crevices and rough
edges may also protect any such organisms from the lethal
effects of sterilization.
saturated steam should be used and should remain in contact
with all parts of the fermentor for at least half an hour.
Pipes which lead into the fermentor should be steam-sealed using
saturated steam.
The various probes used for monitoring fermentor activities,
namely probes for dissolved oxygen, CO2, pH, foam, etc., should
also be sterilized