Transcript INTRODUCTION TO MYCOLOGY

MIC 303
INDUSTRIAL AND
ENVIRONMENTAL
MICROBIOLOGY
FERMENTATION
TECHNOLOGY
Mdm Aslizah Mohd Aris
STRUCTURE OF A BATCH
BIOREACTOR
Fermenter
Are structures designed to optimize the growth
conditions of the specific organisms that we
want
 Control oxygen, pH, medium, temperature
and nutrients antifoaming
 Stirred tank reactor
 Air lift reaction
STRUCTURE OF A BATCH
BIOREACTOR
Basic function of a fermenter:
 to provide a controlled environment for the
growth of microorganism or a defined mixture of
microorganism to obtain desired product.
Two basic types of fermenter are used:
1) A glass vessel with round or flat bottom and a
top flanged carrying plate.
2) A glass cylinder with stainless steel top and
bottom plates.
The thickness of construction materials increase with
scale.
It is important to select materials that can withstand
repeated steam sterilization.
Factors to be considered:
1) Vessel should be capable of being operated
aseptically- for a no of days/ long operation.
2) Adequate aeration and agitation- mixing should
not cause damage to the organism.
3) Power consumption- should be low as possible.
4) Temperature controlled system.
5) pH controlled system.
6) Sampling facilities should be provided.
7) Evaporation losses from the fermenter should not
be excessive.
8) Vessel should be designed to require the minimal
use of labour in operation, harvesting, cleaning
and maintenance.
9) The vessel should be suitable for a range of
process.
10) The vessel should be constructed to ensure
smooth internal surfaces, using welds instead of
flange joints whenever possible.
11) The vessel should be of similar geometry to both
smaller and larger vessels in the pilot plant or
plant to facilitate scale up.
12) The cheapest materials which enable
satisfactory results to be achieved should be
used
13) Should be adequate service provisions for
individual plants.
Side view
INDUSTRIAL FERMENTORS
Top view
Fermentor sizes for Industrial
Processes
Size of
Fermentor (L)
Product
1 - 20,000
Diagnostic enzymes, substances
for molecular biology
40 - 80,000
Some enzymes, antibiotics
100 - 150,000
200,000500,000
Penicillin, aminoglycoside
antibiotics, proteases, amylases,
steroid transformations, amino
acids, wine and beer
Amino acids (Glutamic acid),
Wine, Beer
Advantages of bioreactor over
large flask containers
Larger cultures volume can be grown.
Ability to control substrate environment.
Sterilization and cleaning are designed in place.
Improved aeration and mixing characteristics result in
improved cell growth and high final cell densities.
A high degree of automation is possible.
Process reproducibility is improved.
It offers aseptic sampling and harvest systems for inprocess sampling.
TYPICAL FERMENTATION PROCESS
STARTER CULTURES
STARTER CULTURE
Starter culture: Inoculum.
Isolation of Industrially Important Microorganisms
 1st Stage: screening of microorganism that are
potential in industrial application
 Purpose of isolation: to obtain either pure or mixed
cultures that are able to carry out the desired
reaction (organic acid transformation and
biodegradable plastic) or produce the desired
products (enzyme, antibiotic, amino acid etc)
Two main isolation methods:
1) Enrichment Liquid Culture
2) Solidified Media
Criteria of Inoculum
Culture must be in a healthy active state.
Must be available in sufficient large volume to
provide optimum inoculum size (normally 10%
from total volume).
Suitable morphological form.
Free from contamination.
Retain it product-forming capabilities
Important Criteria of Inoculum for
Fermentation Processes
Nutritional characteristics of the organism.
 Very cheap medium
 Pre-determined medium (ex: the use of methanol
as an energy sources)
Optimum temperature of the organism
 The use of an organism of optimum temperature
above 40ºC reduces considerably the cooling
cost of a large scale fermentation)
The reaction of organism with the equipment use
and the suitability of the organism to the type of
process to be used
 If an airlift fermentor to be used, culture of
microorganism with low viscosity should be used.
The stability of organism and its amendability to
genetic manipulation.
The productivity of the organism.
 Yield based (Yp/s) of the substrate consume (g
product/substrate) and overall productivity (g/L
product / h)
The ease of product recovery.
 Contamination during fermentation should be
taken into account
 Extracellular/Intracellular- determine the type of
product
Quality of the product
 Ex:
Glucoamylase
produce
by
different
microorganism produce different type of product
INOCULUM PREPARATION
Stage of Inoculum Preparation
Depend on the scale of production fermenter.
MEDIA FORMULATION
Media for Industrial Fermentations
The media is the feed solution.
 It must contain the essential nutrients needed
for the microbe to grow.
Factors to be considered:
 Quality consistence and availability.
 Ensure there are no problems with media prep
or other aspects of production process.
 Ex: Sugar cane, molasses
MEDIA FORMULATION
Basic requirement of microorganism:
 Water
 Source of energy – carbon, nitrogen, mineral
element, vitamin
 Oxygen (if aerobes)
The constituents of medium must satisfy:
1) Cell biomass requirement.
2) Metabolite production.
3) Energy supply – biosynthesis , cell maintenance.
Criteria of Fermentation Medium
Produce maximum yield of product or biomass per
gram of substrate used.
Produce maximum concentration of product / biomass.
Permit max rate of product formation.
Minimize yield of undesired products.
Consistent quality and be readily available throughout
the year.
Minimal problems during media making and sterilization.
Minimal problems in production process such as
aeration, agitation, purification and waste treatment.
Cheap substrate/ simpler procedure.
Composition of Fermentation
Medium
Carbon source
Nitrogen source
Mineral Element
Chelators
Growth Factors
Buffers
Precursors
Inhibitors/ Inducers
Carbon source
Factors influencing the choice of carbon source:
1) The rate which carbon source is metabolized
influence:
 Formation of biomass.
 Formation of primary metabolite.
 Formation of secondary metabolite.
2) The main product of a fermentation process also
determine the choice of carbon source.
3) The purity of carbon source – avoid undesirable
product.
Ex: metallic ion must be removed from
carbohydrate source used in some citric acid
process.
4) The method of media preparation particularly
sterilization may affect the suitability of
carbohydrates for individual fermentation process.
Ex: Sterilize sugar separately from nitrogen source +
mineral- to avoid reaction of ammonium ions and
amino acid to form black nitrogen (blackening
agent) containing compounds which will partially
inhibit the growth of microorganisms.
5) Government legislation.
Ex: European Economy Community (EEC): the use of
beet sugar and molasses is encouraged and the
minimum price controlled.
Examples of carbon source:
1) Carbohydrates:
 Starch (maize, potato, cassava)
 Barley grains
 Sucrose (Sugar cane and sugar beet)
 Molasses (Beet or cane molasses)
 Lactose and crude lactose (Milk whey powder)
 Corn steep liquor (by product after starch
extraction from maize)
2) Oils and Fats
 Oils: carriers for antifoams in antibiotic processes,
contain fatty acids, oleic, linoleic and linolenic
acid.
 Ex: vegetables oils (olive, maize, cotton seed,
tinseed, soya bean etc)
3) Hydrocarbon and derivatives.
 Using n-alkanes for production of organic acid,
amino acid, vitamin, protein etc.
Nitrogen source
Divided into:
1) Organic: amino acid, protein, urea
2) Inorganic: ammonia gas, ammonium salts or nitrates
Factors influencing the choice of nitrogen source:
1) Metabolic regulation.
Ex: control mechanism of nitrate reductase enzyme
involved in conversion of nitrate to ammonium ion,
repressed by ammonia)
2) Type of product.
Ex: Antibiotic production is influenced by the type of
nitrogen source, may be inhibited by ammonium,
nitrate or certain amino acid.
3) Pre-determined aspects of process.
Ex: Limiting substrate.
Mineral Element
Microorganism require certain mineral element for:
 Growth
 Metabolism
Macro element: Magnesium, phosphorus,
potassium, calcium and chlorine.
 Essential component of media
 Added as distinct component.
Trace element: Copper, iron, molybdenum and
zinc.
 Essential but usually present as impurities in other
major ingredients.
Chelators
Insoluble metal phosphate may be eliminated
by incorporating low concentration of chelating
agents such as EDTA (ethylene diamine tetra
acetic acid), citric acid, polyphosphates.
Form complexes with metal ions – may be
gradually utilized by microorganisms
Growth Factors
Some microorganism cannot synthesize a full
complement of cell component
- thus required pre-formed compounds
Ex: Vitamin, specific amino acid, fatty acid,
sterols.
Many natural carbon and nitrogen sources
contain some required growth factors.
Buffers
Functions:
1) Agent for control pH
2) As nutrient source
Ex: Calcium carbonate (pH 7), Phosphate,
balance of carbon and nitrogen source.
Precursors
Some chemical when added to certain
fermentation are directly incorporated into the
desired product.
Ex: Phenylacetic acid (precursor in Penicillin
production).
Inhibitors
Functions:
1) Production of specific products and inhibit
undesirable product.
2) Accumulation of metabolic intermediate.
Example:
Product
Glycerol
Inhibitor
Main Effect
Sodium
bisulphite
Acetaldehyde
production,
repressed
chlortetracycline
and cell wall
permeability
Microorganism:
A. niger
Inducers
Majority enzymes which are of industrial interest
are inducible.
Induced enzymes are synthesized only in
response to the presence of an inducer, often
substrate.
Ex: pulllulan for pullulanase, pectin for pectinase
MEDIUM STERILIZATION
Steam is used almost universally for the sterilization of
fermentation media.
Problem occur to the medium in a result of heat
treatment:
1) Interaction of the nutrient component in the medium.
Ex: Mallard type browning reaction results in the lost of
medium colour and nutrient.
2) Destruction of component that are heat sensitive ex
vitamin and amino acid
Therefore, need to design sterilization based on:
1) Batch sterilization process
2) Continuous sterilization process
* Involve in specific calculation, will not covered in this
course
SCALLING UP VS SCALLING
DOWN PROCESS
SCALING UP
Scaling up fermentation process is carried out at the
laboratory or even pilot plant scale fermentors to
yield data that could be used to extrapolate and
build the large scale industrial fermentors with
sufficient confidence it will function properly with all
its behaviours anticipated.
Increasing the scale of fermenter.
 Increase in volume
 Problem of process scale up are due to different
ways in which process parameters are affected
by the size of the unit.
Ex: Laboratory scale
pilot scale
production scale
Upstream Processing Equipment
Lab-Scale Bioreactor
3 liters
Large-Scale Bioreactor
25,000 liters
SCALING DOWN
Scaling down fermentation process is carried out
studies on smaller bioreactors in order to gain data
and confidence and predict the behaviour how
things actually will behave in large production
fermentor.
Scalling down fermentation process also will be
performed to troubleshoot or trying to optimize the
industrial scale fermentation.
This method is called the fermentation monitoring
experiment (mimic the industrial conditions).
This approach important in the development of:
 New product.
 Improvement of an existing full-scale fermentation.
Downstream Processing Equipment
Large Scale
Chromatography System
Lab Scale
Chromatography System