Monika Mathur

2014FST29D
  Introduction
 Fermentation

 Products

 Industrial scale

Contents  Types

 Advantages

 Disadvantages

 Summary
• FERMENTATION TECHNOLOGY
 microorganisms, grown on a large scale, to produce
valuable commercial products or to carry out important
chemical transformations.

• FERMENTATION

 Pasteur’s “life without air”,

 Latin word fervere, to boil

What is CONTINUOUS cell culture
 Substrate is added continuously to the fermenter, and
biomass or products are continuously removed at the
same rate.
 Under these conditions the cells remain in the
logarithmic phase of growth
 Homogenously mixed Bioreactors
In this type, the culture solution is homogenously
mixed
The bioreactors are of two types
 Chemostat bioreactors
The concentration of any one of the substrate is
adjusted to control the cell growth and maintain a steady
state.
 Turbidostat bioreactors
In this case, turbidity measurement is used to
monitor the biomass concentration.
The rate of addition of nutrient solution can be
appropriately adjusted to maintain a constant cell
growth.
• FED-BATCH FERMENTATION

 Substrate increments as the fermentation

progresses.
started as batchwise with a small substrate
concentration.
 Initial substrate is consumed, addition of
fermentation medium
• BATCH FERMENTATION

Sterile nutrient substrate , inoculated, grow until no more

of the product is being made, "harvested" and cleaned out
for another run.

 lag phase (adapt to their surroundings)

 exponential growth (grow in numbers)
 stationary phase (stop growing)
 death phase
 surface (solid state) submersion techniques.

• microorganisms microorganisms grow in a

cultivated on the Liquid medium liquid medium.
surface of a liquid or
solid substrate.
(biomass, protein,
• complicated and antibiotics, enzymes and
rarely used in sewage treatment) are
industry.
carried out by submersion
processes.
• Mushroom, bread,
cocoa, tempeh
 Solid-state fermentation (SSF) has been considered as a
useful tool for biomass energy conservation, solid waste
treatment and production of added-value molecules such
as enzymes, organic acids and biologically active secondary
metabolises
 One of the major advantages that SSF offers is in the
utilization of agro-industrial residues, which have no other
practical applications
 Solid state fermentation
 Solid state fermentation (SSF) is a process in which microorganisms
grow on or within solid substrates or supports, in the absence of free
water.
 The Solid State Fermentation (SSF) processes can be defined as
methods of culturing microorganisms on and/or within particles of
solid matrix. The liquid content bound with these particles must be at
a level corresponding to the water activity assuring correct growth and
metabolism of microorganisms, but not exceed the maximum water
holding capacity of solid matrix.
The wide range of solid matrix used in SSF can be
classified into two great categories :
 The particles are in the same time the support and the substrate. This is the case of all organic
materials 0ignocellulosic or starchy products),
 The solid matrix only play the part of support( mineral or synthetic materials) and must be
in this case wetted with different nutritive solutions for the microorganisms.
Similarly to submerged fermentation, when the objectives of the research on SSF are industrial
applications, studies on engineering and scale-up are absolutely necessary. Mainly the choice of a
reactor design and the elaboration of an efficient parameter control system are very important.
Unfortunately, in spite of resurgence of interest in SSF from last 15 years, the design of solid state
reactors has been almost entirely empirical. This is due to the lack of "knowledge as well in the
physiology of the microorganism as in engineering data.
So ntan.,~ different reactors and control systems are used (I). They can be summarized in three
main types :
 - reactors without agitation,
 - reactors v, ith occasional agitation.
 - reactors with continuous agitation or rotation.
 For each type, two alternatives are possible: the presence or not of a forced aeration. By forced
aeration we mean the air circulation through the culture layer and not only at the surface of the
solid medium.
 As a general rule, to well define reactor equipment,
some criteria might be taken into account :
 - the type of microorganisms involved,
 - the structure of the solid matrix used,
 - the environmental conditions needed for the process,
 - the type of use aimed (research, industrial
applications).
Fermentor is the basic
equipment used for
fermentation.

contains the media to

carry out fermentation,
and creates
environment for
fermentation at large
scale.

http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
SOLID STATE FERMENTATION
 Most fermentation technologies using microbes
employ a fairly dilute medium where the substrate to
be fermented is diluted in water and then inoculated
with desired microbes.
 Some fungai can grow in conditions without free
water.
 In this case the moisture required by the fungus exists
than absorbed or complex form in the solid matrix ,
often ranging between 10% and 80% processes which
exploit the growth of fungai in this type of system are
designated solid state fermentation.
EXAMPLES FOR SOLID SUBSTRATE
FERMENTATION
EXAMPLE SUBSTRATE MICRO ORGANISMS

MUSHROOM STRAW, MANURE AGARICUS BISPORUS

PRODUCTS
SOY SAUCE SOYA BEANS AND ASPERGILLUS ORYZAE
WHEAT

CHEESE MILK CURD PENICILLUM

ROQUEFORTI

TEMPEH SOYA BEANS RHIBOPUS

OLIGOSPORES
LEACHING OF LOW GRADE ORES THIOBACILLUS SP….
METALS
ORGANIC ACIDS CANE SUGAR,MOLASSES ASPERGILLUS NIGER
FERMENTOR DESIGN
WINE MAKING FERMENTOR
FERMENTOR DESIGN
 A fermentor is a device in which the organisms are
cultivated and motivated to form a desired product

 Closed vessel or containment designed to give a right

environment for optimal growth and metabolic activity of
the organism

 Fermenter: for microbes/ Bioreactor : for eukaryotic cells

 Size variable ranging from 20-250 million litres or more.

 Large scale production (10-100L to1000-million L capacity)

 Regular monitoring for physical, chemical biological
parameters is done through controlled systems of the
fermentor, because these parameters influnces the
growth of the microbes.
 As per requirements and use of types of
microorganisms there are different types of fermentors
available.
 The most common among these is stirred tank
fermentor where impellers used to stirr the medium.
Helps to meet requirements of:
• pH
• temperature
• aeration
• agitation
• drain or overflow
• control systems
• sensors
• cooling to achieve maximum microbial yield
 The increasing concentration of microbial cells will
deplete the dissolved O2 concentration resulting in
creation of anaerobic conditions.
 The microbial growth will simultaneous decline in
product production.
 The forced aeration favours rapid growth of micro
organisms.
 A pH fitted with fermentor regularly monitors the pH
and maintains at optimum by adding acid or alkali.
Some important fermentation products
Product Organism Use

Ethanol Saccharomyces Industrial solvents,

cerevisiae beverages
Glycerol Saccharomyces Production of
cerevisiae explosives
Lactic acid Lactobacillus Food and
bulgaricus pharmaceutical
Acetone and Clostridium Solvents
butanol acetobutylicum
-amylase Bacillus subtilis Starch hydrolysis
25
 Pure culture: organism, quantity, physiological state

 Sterilised medium: for microorganism growth

 Seed fermenter: inoculum to initiate process

 Production fermenter: large model

 Equipment

 i) drawing the culture medium

ii) cell separation iii) collection of cell

iv) product purification v) effluent treatment.

Microbial cell (Biomass) Yeast

Microbial enzymes Glucose isomerase

Microbial metabolites Penicillin

Food products Cheese, yoghurt, vinegar

Vitamins B12, riboflavin

 P.F. STANBURY, A. WHITAKER AND S. J. HALL, PRINCIPLES OF FERMENTATION TECHNOLOHY
 Submerged Fermentation
(SmF)/Liquid Fermentation (LF)
 SmF utilizes free flowing liquid substrates, such as molasses and broths. The
bioactive compounds are secreted into the fermentation broth. The substrates
are utilized quite rapidly; hence need to be constantly replaced/supplemented
with nutrients. This fermentation technique is best suited for microorganisms
such as bacteria that require high moisture content. An additional advantage
of this technique is that purification of products is easier. SmF is primarily
used in the extraction of secondary metabolites that need to be used in liquid
form.
 Submerged liquid fermentations are traditionally used for the
production of microbially derived enzymes. Submerged fermentation
involves submersion of the microorganism in an aqueous solution
containing all the nutrients needed for growth.
Surface Fermentation
 The microorganisms are cultivated on the surface of a
liquid or solid substrate. These techniques are very
complicated and rarely used in industry.
 Surface fermentation is easy to control and to implement.
It needs no aeration or agitation of the fermentation broth,
so it needs no instrumentation for aeration and agitation.
 With surface fermentation, the fermentation broth is
concentrated due to a high evaporation rate during
fermentation. Thus, expenses and losses during recovery
and purification are low.
 disadvantages: Building investment costs are high.
 Personnel expenses are high in developed industrial countries
with extremely high wages.
 Fermentation time is long and therefore productivity is low.
Advantages of Solid State Fermentation
1. Higher volumetric productivity
2. Usually simpler with lower energy requirements
3. Might be easier to meet aeration requirements
4. Resembles the natural habitat of some fungi and bacteria
5. Easier downstream processing
6. The fungal hyphae are bathed in a liquid medium and do not run the risk of desiccation;
7. Temperature control is typically not overly difficult, such that the organism is exposed
to a constant temperature throughout its growth cycle;
8. The availability of O2 to the biomass can be controlled reasonably well at a particular
level of saturation of the medium
9. The availability of the nutrients to the organism can be controlled within relatively
narrow limits if desired, through the feeding of nutrient solutions.
10. Although shear forces do occur within mechanically stirred bioreactors, the nature and
magnitude of these forces are well understood and it is possible to use bioreactors that
provide a low-shear environment, if the organism is highly susceptible to shear
damage, such as bubble columns or air lift bioreactors;
11. pH control is relatively easy to provide.
Submerged fermentation is favored over surface
fermentation for the following reasons

 Lower total investment costs;

 Improved process control;
 Reduced fermentation time;
 Reduced floor space requirements;
 Lower labour costs;
 Simpler operations; and
 Easier maintenance of aseptic conditions on an
industrial scale.
Reactors : THE INRA-DIJON
REACTORS
 Lab Scale and Preoilot Reactors.
 Taking into account the necessity during the culture :
 - to maintain temperature and moisture content of
the medium,
 - to ensure a good aeration in the bulk,
 we elaborate a system included in a control board to
regulate these important parameters by means of a
circulation of a thermostated air (Figure I ).
 The inlet dry air coming from a compressor is first passed,
after cartridge sterilization through a sparger in a
humidifier.
 The humidification is done by bubbling air in
thermostated sterile water.
 In the next step, the humidified air is heated by passing in
a heating box containing several small resistive heaters.
 Two probes, placed in the air circuit, control, via regulators,
the temperature and the relative
 humidity of the air before going into the different reactors.
 By the modifications of the setpoints
 on temperature and relative humidity regulators or by
modification of the airflow, it is possible
 during the culture to maintain temperature and moisture
content of the medium.
The lab scale reactors are wide mouthed jars in glass
(total ~.olume 2L) and hermetically closed by a cap.
The thermostated air is introduced at the bottom,
under a sieve which supports the solid medium
(maximum height layer about 15cm) and goes out by
the top. In the middle of the height, a small
thermometer indicates the medium temperature. To
isolate from external temperature variations, these jars
are put in a ventilated incubator. Up to 10 jars can be
connected on the same air circuit, the airflow can be
adjusted at different values for each of them.
The prepilot reactors, in stainless steel and insulated,
have a total volume of 50L permitting
culture on 30cm height. Each cylindrical reactor
contains a removable basket which can have
partitions allowing the use of different media in each
part. A unit is composed of two reactors
aerated strictly in the same conditions. At the bottom of
each fermentor a valve allows the airflow
adjustment (Figure 2).
Pilot Reactor.
 This reactor, in stainless steel and insulated, is rectangular with a
worldng volume of 1.6m 3.
 A one meter thick layer of substrate is supported on a wire mesh.
 It is possible to agitate by means of 3 vertical screws mounted on a
conveyor which moves them back and forth across the reactor.
 The design of the screws depends upon the substrate. The whole
reactor is supported by weight gauges.
 The thermostated air is forced by a variable speed fan through the
layer. Taking into account the volume of this reactor, the aeration
system is slightly different
 - a cooler containing water and glycol for drying and cooling the inlet
air;
 - a heater supplied with steam boiler;
 - humidification by steam injection in the air stream or by
thermostated water spraying under the wire mesh.
 Probes are located in the substrate layer and in the air
stream, allowing a continuous measurement of key
process variables, (0 2 consumption, CO 2 production,
medium temperature) enabling process control. In
some cases (2), pH can be measured directely in the
medium and adjusted by spraying solutions during the
agitation. A computer controls the temperature,
relative humidity and flow rate of the incoming air, the
weight, which can be used to regulate the temperature
and water content of the substrate mass
Application
 Animal Feeding
 Enzyme Biosynthesis
 Biological Control