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

Pyruvic acid metabolized in the absence of oxygen

Definasi

Pyruvic lactic acid

No gas are produce Lactobacilli,

streptococci

Eg. In cheeses making

Homolactic CO2 released

from Pyruvic acid to form acetaldehydeethyl 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

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

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

A-exhaust

B-impeller

C-sparger (high

pressure air)

D-harvest

E- culture broth

F-cooling jacket

Sterile air

Cooling water in

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 vessel. Note: 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 flask

-indication of possible process for commercial interest product

Laboratory fermentor(1-10L)

-small scale fermentor

- First effcort of scale up

- Test variation in medium, temp, pH etc (costing)

Piloty plant stage (300-3000L)

- Condition more closely approach to commercial scale

Commercial fermentor(10 000-500000L

-

Isolation of the microbe

from nature resourc

es

Identification of desire

microbe

Characterization of the microbe

Screening 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 oxidation-reduction 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