THE DOWNSTREAM PROCESSING IN MICROBIAL FERMENTATION

1. UPSTREAM PROCESSING IN FERMENTATION
BY
MUMTHAS P K
2nd MSC MICROBIOLOGY

3. UPSTREAM PROCESSING
Upstream processing encompasses any technology that leads to the
synthesis of a product. Upstream includes the exploration, development
and production.
The upstream process mainly includes ;
Culture isolation and screening
Inoculum preparation
Media formulation and sterilization
Equipment preparation and
sterilization

5. CULTURE ISOLATION AND SCREENING
 Strains of microorganisms may be obtained
from companies, universities, national or
international culture collections and
environment.

6. ISOLATION OF SOIL MICROORGANISMS
 Environment is the ultimate
source of many different
types of microorganisms.
 Novel and better strains can
be obtained. Costs may be
lower by using this
procedure.

7. The usual pure culture isolation
techniques includes :
 Streak plate method of isolation
 Pour plate method
 Spread plate method
 Micromanipulator

8. MICROMANIPULATOR METHOD
 The instrument is used in
conjunction with a microscope to
pick a single cell from a hanging
drop preparation.
 This method are the culture comes
from a single cell and one can
obtain strains with in the species.

9. SCREENING OF MICROORGANISM
The procedure of isolation, detection and separation of microorganisms of
our interest from a mixed population by using highly selective procedure
is called screening .

10. PRIMARY SCREENING
 It determines which microorganisms are able to produce a
compounds.
 Does not provide much idea about the production or yield potential of
microorganisms.
 It separate out a few microorganisms only few have commercial value
while discards the valueless microorganisms.
 It is based on the qualitative ability to produce the desired product like
antibiotic or amino acid or an enzyme etc.

11. PRIMARY SCREENING OF ORGANIC ACID PRODUCING
MICROORGANISMS
 The pH indicating dyes may be used for detecting microorganisms that are
capable of producing organic acids.
 These dyes undergo colour changes according to its pH.
 Neutral red , Bromothymol blue are added to the poorly buffered nutrient
agar media.
 Incorporation of CaCO3 in medium is also used to screen organic acid
producing microbes on the basis of formation of clear zone of dissolved
CaCO3 around the colony.

12. NUTRIENT AGER WITH CALCIUM CARBONATENEUTRAL RED INDICATOR

13. SCREENING OF ANTIBIOTIC PRODUCING ORGANISMS
 Crowded plate technique is used for
screening of antibiotic producing
organisms.
 Dilutions are made and then pouring
and spreading of soil samples that give
300 to 400 or more colonies per plate.
 Colonies showing antibiotic activity are
indicated by zone of inhibition around
the colony.
 The purified cultures are then tested to
find the microbial inhibition spectrum.

14. SCREENING OF EXTRACELLULAR METABOLITE PRODUCING
MICROORGANISMS
 Auxanography technique is employed for detecting microorganisms able
to produce growth factors , vitamins , amino acids etc.
 Two major steps used for this :
1. Preparation of first plate :
 A filter paper strip is put across the bottom of petri dish.
 The nutrient agar is prepared and poured on the paper disc and allowed
to solidify .
 Soil sample is diluted and proper dilutions are inoculated.

15.  A minimal media lacking the growth factors is prepared and seeded
with the test organism. It is poured onto fresh petri plate and is allowed
to set .
 The agar in the first plate is then lifted and placed on the second plate
without inverting.
 The growth factors produced on agar can diffuse into the lower layer
containing test organism.
 The zone of stimulated growth of test organism around colonies in an
indication that organism produce growth factor extracellularly.
2. Preparation of second plate :

16. ENRICHMENT CULTURE TECHNIQUE
 This was designed by Beijerinck to isolate the desired microorganisms
from heterogeneous microbial population.
 It consists of following steps :
1. Nutrient broth is inoculated with microbial source and incubated.
2. A small portion of all inoculums is plated onto the solid medium and
well isolated colonies are obtained .
3. Suspected colonies from the plate are sub cultured on fresh media
and subjected for further testing.

18. SECONDARY SCREENING
It is a systemic screening programme intended to isolate industrially
important or useful microorganisms.
 It is useful in sorting of microorganisms that have real commercial value.
The microorganisms having poor applicability in fermentation process
are discarded .
 It provides the information whether the product formed by
microorganisms is new or not. This may be accomplished by paper or
thin layer chromatographic techniques .It should whether the product
posses physical properties such as UV light absorption or fluorescence or
chemical properties that can be employed to detect the compound during
use of paper chromatography.

19.  It is conducted on agar plates, in flasks or in small fermenter containing
liquid media.
 It gives an idea about the economic position of the fermentation process
involving the use of a newly discovered culture.
 It helps in providing information regarding the product yield potentials of
different isolates.
 It determines the optimum conditions for growth or accumulation of a
product associated with a particular culture.
 Chemical, physical and biological properties of a product are also
determined during secondary screening. Moreover it reveals whether a
product produced in the culture broth occurs in more than one chemical
form.

20.  It detects gross genetic instability in
microbial cultures. This type of
information is very important since
microorganism tending to undergo
mutation or alteration is some way lose
their capability for maximum
accumulation of the fermentation
products.
 It tells about the chemical stability of the
fermentation products.
 It can be qualitative or quantitative in its
approach.

21. PRIMARY SCREENING
 Desired microorganisms are
isolated and detected.
 Isolated microorganisms are used
for secondary screening.
 Basic information about the
isolated colony is obtained.
SECONDARY SCREENING
 Industrially important organisms are
characterize by using highly selective procedure.
 The organisms that are capable to give high
yield of product by using cheap raw material are
screened.
 Detail information of isolated colony is obtained
and determined whether it can be used on
industrial scale.
 Here the capabilities and yield potential of
microbes is determined.
 Here valueless organisms are discarded and
valuable organism that are capable to produce
production on industrial scale are used further

22. PREPARATION OF INOCULUM
Inoculum is the mixture of cultured microbes along with media in which it
is growing. The fermentation process involve an inoculum development
step, which is the preparation of a population of microorganisms from a
stock dormant culture to a state useful for inoculating a final production
fermenter.
 Inoculum production is a critical stage in an industrial fermentation
process.
 One loop of cell line requires a prolonged period , if it is directly
introduced in to fermentation.
 Thus, inoculum is prepared as a stepwise sequence employing increasing
volumes of media , that helps to make the organism in an active stage so
that the initial time required for establishing the organism in to the new
place must be reduced.

23. CONSTITUENT OF INOCULUM MEDIA
 Chemical composition ;
 The inoculum media must have a suitable chemical composition
.Generally
the medium should contain a source of carbon, nitrogen, growth factors
and mineral salts.
 Maintenance of optimum pH and temperature must be needed. Mainly
CaCO3 act as the buffering agent for pH control.
 Defoamers should be added for controlling the foam. ( eg: oil mixed
octadecanol for penicillin fermentation )
 Proper aeration and agitation must needed.
Inoculation is an important process as it is necessary to adopt and made
culture in growing condition before feeding in to fermentation.

24. INOCULUM PREPARATION

25. MEDIA CHARECTERISTICS
The composition of fermentation medium can be simple or complex
depending on the particular organism and fermentation process.
 Is can be synthetic media or crude media .
 The synthetic media consists of all the constituents that are specifically
defined and known. Every constituent is relatively pure and exact
amount incorporated in to the medium are known.
 Crude media contains a rough composition of media required for
fermentation. It gives high yield of product and contain undefined
sources of ingredients.
 It contains high level of nutrients, vitamins, proteins, growth factors,
anti-forming agents and precursors etc.

26. CARBON SOURCE
 A carbon source is required for all biosynthetic leading to reproduction,
product formation and cell maintenance. In most fermentation it also
serves as the energy source.
 Molasses
 Malted barley
 Starch and dextrins
 Sulphite waste liquor
 Alkanes and alcohol
 Oils and fats
Factors influencing the carbon source;
 Cost of the product
 Rate at which it is metabolized
MOLASSES
SULPHATE WASTE LIUOR

27. Different carbon sources

28. NITROGEN SOURCE
Most industrial microbes can utilize both inorganic and
organic nitrogen sources.
 Inorganic nitrogen may be supplied as ammonium
salts, often ammonium sulphate and diammonium
hydrogen phosphate or ammonia. Ammonia can also
be used to adjust PH of the fermentation.
 Organic nitrogen sources mainly include amino acids,
protein and urea.
 Corn steep liquor
 Yeast extract
 Peptones
 Soya bean meal
YEAST EXTRACT
SOYA BEAN MEAL

29. MINERALS
 All microorganisms require certain mineral
elements for growth and metabolism.
 In many media , magnesium, phosphorus,
potassium, sulphur, calcium and chlorine
are essential components and must be
added.
 Other such as cobalt, copper, iron,
manganese, molybdenum, and zinc are
present in sufficient quantities in the water
supplies and as impurities in other media
ingredients.

30. VITAMINS AND GROWTH FACTORS
 Many bacteria can synthesize all necessary vitamins from basic elements.
For some bacteria, filamentous fungi, and yeasts they must be added as
supplements to the fermentation medium.
CHELATORS
 Many media cannot be prepared without precipitation during autoclaving.
Hence some chelating agents are added to form complexes with metal ions
which are gradually utilized by microorganisms. Eg ; EDTA, citric acid,
polyphosphate etc.
 In many media these are added separately after autoclaving or yeast
extract, peptone complex with these metal ions.

31. PRECURSORS
 Precursors are defined as substances added
prior to or simultaneously with the
fermentation which are incorporated without
any major change into the molecule of the
fermentation product and which generally
serve to increase the yield or improve the
quality of the product.
 They are required in certain industrial
fermentation and are provided through crude
nutritive constituents .

32. INDUCER AND ELICITORS
 If product formation is dependent upon the presence of a specific
inducer compound or a structural analogue, it must be incorporated
into the culture medium or added at a specific point during the
fermentation.
 The majority of enzymes of industrial interest are inducible. Inducers
are often substrates such as starches or dextrins for amylase.
 In plant cell culture the production of secondary metabolites, such as
flavonoids can be triggered by adding elicitors.

33. INHIBITORS
 Inhibitors are used to redirect
metabolism towards the target
product and reduce formation
of other metabolic
intermediates.
 Others halt a pathway at a
certain point to prevent further
metabolism of the target
products.


34. WATER
 All fermentation process, except SSF require vast quantities of water.
 Not only is water a major components of all media, but also it is
important for heating, cooling, cleaning and rinsing.
 A reliable source of large quantities of clean water, of constituent
composition is essential.
 It is also influence;
 pH
 Dissolved salts
 Effluent contamination
 Reuse of water is also important that reduce water cost by 50% and
effluent treatment cost by 10 fold .

35. ANTIFOAMS
 Antifoams are necessary to reduce foam formation during fermentation.
 Foaming is largely due to media proteins that become attached to the
air-broth interface where they denature to form a stable foam skin that
is not easily disrupted.
 An idea of antifoam should have the following properties;
 Disperse readily and have fast action.
 Active at low concentration.
 Long acting in preventing new foam.
 Should not be metabolized.
 Should not be toxic to microorganisms, human etc.
 Cheap, should not cause problem in fermentation.

37. STERILIZATION OF MEDIA
 For successful fermentation, it is necessary to ensure the sterility of
the media containing nutrients and sterility of incoming and out going
air and the sterility of bioreactor.
 Media must be free from contamination before entering in fermenter.
 Sterilization of media , is commonly achieved by heat, other physical
methods like filteration, radiation and biochemical treatments.

38. HEAT STERILIZATION
 It is most widely used sterilization technique. It influences ;
 Composition of the media
 PH of the media.
 Size of the suspended particles
 Media is sterilized by boiling or passing live steam through medium or
by subjecting medium to steam under pressure.
 Sterilization of large batches of medium is done by passing through
heated retention tubes containing steam jet heaters.
 Synthetic media require shorter sterilization time than crude media.

39. BATCH STERILIZATION
The culture media are subjected to sterilization at 121˚C in batch volume,
it can be done by direct or indirect method.
Direct method involve injecting the steam in to the media, steam should be
pure and free from all chemical additives. Direct injection results in
accumulation of condensate and results in increase of volume of media.
Indirect method involves injecting steam into interior coils.
CONTINUES STERILIZATION
 It is carried out at 140˚C for 30-120 sec. it is carried out by directly
injecting steam or by means of heat exchanger. Due to condensation the
nutrient solution get diluted.
 This solution is pumped through an expansion valve in to a vaporise
and condensate is remained through vacuum pump.

40. Moist heat destroys microorganisms by the irreversible denaturation
of enzymes and structural proteins. The temperature at which denaturation
occurs varies inversely with the amount of water present.
The recommendation for sterilization in an autoclave is 15 minutes at
121°C (200 kPa).
The temperature should be used to control and monitor the process; the
pressure is mainly used to obtain the required steam temperature.
Dry heat sterilization uses high temperatures over extended periods of time
to kill bacterial spores and microorganisms from objects. Several types
include hot air oven, incineration, and flaming.

41. AIR STERILIZATION
Industrial fermentation are carried out under vigorous and continues
aeration.
Microorganism present in the air or other gases can be sterilized by
filtration, heat, radiation etc.
STERILIZATION BY FILTERATION
 The liquid are filtered through bacterial filter to remove any microbes
present. The method is very useful for sterilization of heat sensitive
liquids.
 The chances of clogging and long time duration for the process to
happen are drawbacks.
1. Membrane filters
2. Seitz filters

42. 1. MEMBRANE FILTERS
 Thin filter which are made up of cellulose,
polyvinylchloride, nylon and other
cellulose derivatives.
 can be employed for sterilization during
injection by placing the membrane
between the syringe and needle.
 The are highly efficient to sterilize liquid,
solvents, and gases.
 The chances of rupture of membrane
leading to improper sterilization.
MEMBRANE FILTER

43. 2. SEITZ FILTERS
Consists of two parts;
 Lower part fitted with a perforated plate over
which compressed asbestos pad is placed. Both
pats are joined together by nuts.
 Do not rupture during filtration. The solution
might get absorbed by the filter itself.
 No risk of contaminating the filtrate. More
suitable for viscous solution.
SEITZ FILER

44. 3. CANDLE FILTERS
They are made up of clay like diatom’s mud. Thus
special mud has minute pores, the microbes get
stuck during their travel through the pore in the
candle.
Candle filter
RADIATION STERILIZATION
Exposing the packed materials to radiation for sterilization. There are
two types of radiations available ;
1.Non ionic radiation
2. Ionic radiation

45. CHEMICAL STERILIZATION
 The article are subjected to sterilization by using toxic gases . The gas
penetrate quickly into the material like steam so that the sterilization is
effective.
 The chances of explosion and cost factors are to be considered.
 The gas used for sterilization are very poisonous. The commonly used gas is
;
 Ethylene oxide with a combination of CO2. The CO2 Added to minimize
the chances of explosion. Ethylene oxide kills all microorganisms such as
bacteria, virus and fungi.
 It is used to treat radiation sensitive substance.

46.  Fermenter has to be sterilized separately before the sterile medium is
added to it.
 This is achieved by heating the jacket or coils of the fermenter with
steam and sparging steam into the vessel through all entities apart
from air outlet.
 The steam under high pressure is normally followed.
 It is essential that sterile air is sparged into the fermenter after the
cycle is complete and a positive pressure is maintained, otherwise a
vacuum may develop and unsterile air be drawn into the vessel.
STERILIZATION OF FERMENTOR

47. FERMENTATION PROCESS

48.  Industrial fermentation uses microorganisms, typically grown on
large scale, to produce valuable commercial transformations.
 In industrial fermentation processes microorganism uses oxygen and
metabolites like CHO completely.
 Aerobic fermentation
 Anaerobic fermentation
Types of fermentations mainly include :
• Batch type fermentation
• Fed batch fermentation
• Continuous fermentation

49. BATCH FERMENTATION
The medium and culture are initially fed in to the vessel and it is closed, after
that no components are added . It is a discontinues process and the fermenter
has to be cleaned after each process and a fresh batch starts. In include the
following steps;
• Medium addition Sterilization of fermenter
• Inoculum addition Fermentation completion
• Product extraction
 After the onset of stationary phase, culture is disbanded for the recovery of
biomass and then a further new batch is set up.
 All nutrient are added before inoculation. Biomass production limited by
C/N load and production of toxic waste substances.

50. CONTINUOUS FERMENTATION
 The media is added continuously to
the fermenter at the fixed rate. The
organisms are maintained at the log
state by recovering products
continuously and hence
maintaining a particular density for
the media.
 Fermentation in this type is called
flow through fermentation.

52. FED- BATCH FERMENTATION
 This fermentation is an intermediate of both batch
and continuous fermentation.
 In fed batch system, a fresh aliquot of the medium is
continuously or periodically added without removal
of the culture fluid.
 The fermenter is designed to accommodate the
increasing volumes. A system is always at quasi-
steady state.
 Initially medium concentration is very low in media
but sterile nutrients are added in increments and
products are harvested only once at the end of
fermentation.

53. OVERALL VIEW OF FERMENTATION

54. REFERENCE
 Industrial biotechnology by casida
 Upstream processing solutions from research to production by Sartorius
stedim- biotech
 Food biotechnology by Anthony pometto, kalidas Shetty , gopinadhan
paliyath, Robert E Levin
 http://bioprocessint.com/manufacturing/continuesbioprocessing

55. THANK YOU