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Industrial Microbiology

BTech. 6th Sem


PPT-1
Introduction to industrial biotechnology
Industrial microorganisms
Fermentor / Bioreactor Design

Dr. Arun Kumar Pradhan


Assistant Professor
CBT, SOA University
Industrial Microbiology
The Scope:

1. This course seeks to introduce students to those


aspects of applied microbiology which they are likely
to encounter in the Fermentation/Medicare sector.

2. Knowledge of the techniques for growing


microorganisms together with sterilization practices
contributes to Good Manufacturing Practice
Industry and Microbes

Industry wants……………

1. Large scale production

2. Within short time period

3. Low cost (Cost effective)


How does microbe full fill the industrial wants?
(Suitability of microbes in industrial processes)

1. Short generation time, High growth rate

2. Easy available and culturable

3. Small genome size, easy gene manipulation

4. Having secretory behavior, easy harvesting

5. Microbial cell itself acts as a good fermenter

6. Diversity
Microbes for various Industries
Industrial
Microbial source Role of enzyme
Application

Hydrolysis of starch into dextrins


Starch B.amyloliquefaciens,
forming a less viscous starch
conversion B.licheniformis
suspension

Bakery Converting starch in dough to smaller


B.stearothermophilus
Industry fermentable sugars
Detergent Digests starch containing foods to
B.licheniformis
Industry water soluble dextrin
Textile Used in removal of starch sizing agent
Bacillus sp
Industry from woven fabric

Converting starch in to smaller


Fuel alcohol
E.coli, B.subtilis fermentable sugars which are acted
Production
upon by yeast to produce alcohol
Sources of Microorganisms
Bacteria are ubiquitous in nature (omnipresent), but for industrial purpose,
the sources vary from bacterium to bacterium.

Type of Industry Bacteria/Fungi Probable sources


Bioleaching Acidithiobacillus thiooxidans, Mining areas
A. ferrooxidans
Enzyme (a-Amylase) Bacillus sp., Aspergillus various climates
worldwide,
contaminated food
Ethanol or solvent S. cerevisiae, E. coli various climates
worldwid
Organic acid Aspergillus niger contaminated food
Food (Cheese, Butter, S. cerevisiae, Lactobacillus Yogurt
bakeries)
Leather tanning Pseudomonas stutzeri Soil and marine waters
sewage work Putrifying bacteria (anerobic) various climates
Medicines (Antitoxins, Penicillium chrysogenum Dry bread, fruits and
antibiotics) nuts
Some commercially available microbial lipases
Sigmoid type curve
1. Lag phase
When a microorganism is introduced into the fresh medium, it takes some time to adjust
with the new environment (acclimatization). This phase is termed as Lag phase, in which
cellular metabolism is accelerated, cells are increasing in size, but the bacteria are not
able to replicate and therefore no increase in cell mass.

The length of the lag phase depends directly on the previous growth condition of the
organism. When the microorganism growing in a rich medium is inoculated into
nutritionally poor medium, the organism will take more time to adapt with the new
environment. The organism will start synthesising the necessary proteins, co-enzymes
and vitamins needed for their growth and hence there will be a subsequent increase in
the lag phase.

Similarly when an organism from a nutritionally poor medium is added to a nutritionally


rich medium, the organism can easily adapt to the environment, it can start the cell
division without any delay, and therefore will have less lag phase it may be absent.
2. Exponential or Logarithmic (log) phase
During this phase, the microorganisms are in a rapidly growing and dividing state. Their
metabolic activity increases and the organism begin the DNA replication by binary fission at a
constant rate.

The growth medium is exploited at the maximal rate, the culture reaches the maximum growth
rate and the number of bacteria increases logarithmically (exponentially) and finally the single
cell divide into two, which replicate into four, eight, sixteen, thirty two and so on (That is 20, 21,
22, 23.........2n, n is the number of generations)

This will result in a balanced growth. The time taken by the bacteria to double in number during
a specified time period is known as the generation time. The generation time tends to vary with
different organisms. E.coli divides in every 20 minutes, hence its generation time is 20 minutes,
and for Staphylococcus aureus it is 30 minutes.
3. Stationary phase
As the bacterial population continues to grow, all the nutrients in the growth medium are used
up by the microorganism for their rapid multiplication. This result in the accumulation of waste
materials, toxic metabolites and inhibitory compounds in the medium. This shifts the
conditions of the medium such as pH and temperature, thereby creating an unfavourable
environment for the bacterial growth. The reproduction rate will slow down, the cells
undergoing division is equal to the number of cell death, and finally bacterium stops its division
completely. The cell number is not increased and thus the growth rate is stabilised.

4. Decline or Death phase


The depletion of nutrients and the subsequent accumulation of metabolic waste products and
other toxic materials in the media will facilitates the bacterium to move on to the Death phase.
During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to
die due to the unfavourable conditions and the death is rapid and at uniform rate. The number
of dead cells exceeds the number of live cells.

Some organisms which can resist this condition can survive in the environment by producing
endospores.
The generation (doubling) time can be calculated from the growth curve

The exactly doubled points from the absorbance readings were taken and, the points were
extrapolated to meet the respective time axis.

Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in minutes to obtain
the absorbance 0.2) = 90-60 = 30 minutes
Let No = the initial population number; Nt = population at time t

n = the number of generations in time t

Therefore,

Therefore,

n = Total time period / generation time period


Amoebae are known to double in 3 min. Two identical
vessels A & B, respectively contain one and two
amoebae to start with. The vessel B gets filled in 3
hours. When will A get filled?

Ans: 3 hr and 3 min


Exponential Growth Curve
Continuous culture with sufficient nutrient

“J” shaped

The equation for exponential population growth is dN 


dt rN
r = intrinsic rate of increase
Diauxie growth Curve
Diauxie is a Greek word coined by Jacques Monod to mean two growth phases. The word is
used to describe the growth phases of a microorganism in batch culture as it metabolizes a
mixture of two sugars.
Rather than metabolizing the two available sugars simultaneously, microbial cells commonly
consume them in a sequential pattern, resulting in two separate growth phases.

This is a photocopy of a figure from an old (1940s) Monod's doctoral thesis reproduced
in a later (1960s) article.
During the first phase, cells preferentially metabolize the sugar on which it can
grow faster (often glucose but not always). Only after the first sugar has been
exhausted do the cells switch to the second.

At the time of the "diauxic shift", there is often a lag period during which cells
produce the enzymes needed to metabolize the second sugar.
Bioreactor
An apparatus in which a biological reaction or process is carried out,
especially on an industrial scale.
Bioreactor (based on volume)

Shake flash Lab scale Large/ pilot scale


Bioreactor Bioreactor Bioreactor
(100-500 ml) (500 ml- 5 lit) (5-200 lit)
Components of Bioreactor
Agitator – used for the mixing of the contents of the reactor which
keeps the “cells” in the perfect homogenous condition for better
transport of nutrients and oxygen to the desired product(s).

Baffle – used to break the vortex formation in the vessel, which is


usually highly undesirable as it changes the center of gravity of the
system and consumes additional power.

Sparger – In aerobic cultivation process, the purpose of the sparger is


to supply adequate oxygen to the growing cells.

Jacket – The jacket provides the annular area for circulation of


constant temperature of water which keeps the temperature of the
bioreactor at a constant value
Process Parameters for Bioreactors
1. Temperature: the mesophiles survive in mesophilic temperature around 30°C to 40°C,
while thermophiles are considered the first microorganism existing at thermophilic
temperature around 50°C to 65°C

2. pH: The concentration range suitable for most organisms is 6.0–9.0. Methanogens in
wastewater treatment systems are most active in the neutral pH range (7.0).

3. Hydraulic Retention Time (HRT): HRT also known as hydraulic residence time is a
measure of the average length of time that a soluble compound remains in a constructed
bioreactor. Hydraulic retention time is the volume of the aeration tank divided by the influent
flow rate:

where HRT is hydraulic retention time (d) and usually expressed in hours (or sometimes
days), the 𝑉 is the volume of aeration tank or reactor volume (m3), and 𝑄 is the influent
flow rate (m3/d).
4. Sludge Retention Time (SRT): SRT is known to be the key
parameter affecting biochemical and physical properties of sludge

5. Particle Size Distribution: The particle-size distribution (PSD) of


a powder, granular material, or particles dispersed in fluid is a list
of values or a mathematical function that defines the relative
amount, typically by mass, of particles present
according to size.

Most studies indicate that smaller media size gives more efficient
removal
Type of Bioreactors (BR)

Mechanically Air Driven /


Agitated BR Pneumatically Agitated BR Non-Agitated BR
(using compressed air)

Stirred Tank reactor Bubble column reactor Packed Bed


Rotary Drum Tank reactor Airlift reactor Fluidized Bed
Propeller loop reactor Membrane reactor
Stirred Tank Reactor
Batch stirred tank:

A batch stirred tank reactor is the simplest type of reactor. It is composed of


a reactor and a mixer such as a stirrer, a turbine wing or a propeller.

The batch system is generally suitable for the production of rather small
amounts of chemicals.

This reactor is useful for substrate solutions of high viscosity and for
immobilized enzymes with relatively low activity.

However, a problem that arises is that an immobilized enzyme tends to


decompose upon physical stirring.
Continuously Stirred Tank Reactor (CSTR)

Run at steady state with continuous flow of reactants and products; the feed
assumes a uniform composition throughout the reactor, exit stream has the same
composition as in the tank

The continuous stirred tank reactor is more efficient than a batch stirred
tank reactor but the equipment is slightly more complicated.
(Copyright New Brunswick Scientific, Edison, NJ)
Continuously Stirred Tank Reactor (CSTR)

(Copyright New Brunswick Scientific, Edison, NJ)


Fermentors are CSTRs used in biological processes in many industries, such as brewing,
antibiotics, and waste treatment. In fermentors, large molecules are broken down into
smaller molecules, with alcohol produced as a by-product.

The industrial fermentor on the left has a capacity of 500 L, while the one on the right
holds 3.0 L.
Used for Drum screen / for wastewater treatment
Bubble Column Bioreactors
The air or gas is introduced at the base of the column through
perforated pipes or plates.

The bubble column bioreactors may be fitted with perforated plates


to improve performance called as spargers .

The flow rate of the air/gas influences the performance factors —


O2 transfer, mixing.

The vessel used for bubble column bioreactors is usually cylindrical


with an aspect ratio of 4:6 (i.e., height to diameter ratio).

The bubble column is particularly useful in reactions where the gas-


liquid reaction is slow in relation to the absorption rate.

Used in various types of chemical reactions like wet oxidation, or as


algae bioreactor.
Types of Bubble Column
Bubble Column Bioreactors
Advantages:
•Highly distributed gas bubbles
•Suitable for low viscosity broths
•Provide a higher energy efficiency than STR
•Provide a low-shear environment.
•Absence of mechanical agitation reduces cost and eliminates one
potential entry point for contaminants.

Disadvantages:
•less vigorous mixing capabilities than STR
•Mixing may not be possible in highly viscous broths.
•Less flexible than STR.
•Work over a rather narrow range of gas flow rates (foaming ,bubble
coalescence ,nature of the broth)
Airlift Reactor

Typical motionless bioreactor where the internal circulation and


mixing are achieved by bubbling air.

Employ forced/ pressurized air to circulate cells and nutrient medium.

Can be used to culture cells that highly shear-sensitive.

Gas stream facilitate exchange of material between the gas phase and
the medium.

Oxygen is usually transferred to the liquid, products are removed


through exchange with the gas phase.
STRUCTURE/ DESIGN…

Entire reactor is divided into 2 halves by a Draft tube:


inner gassed region ( Riser)
outer ungassed region ( Down comer)

Riser has gas injection connected- air moves upwards.

Down comer region has degassed media and cells.

Mean density gradient between riser and downcomer


regions causes continuous circulation.
The airlift reactors were used without draft tube to conduct
experiments on fungal cultivation on vinasse (by product of
the sugar industry) in the bubble column reactor
PARTS OF ALR…

RISER: Connected gas injection-upward air flow.

DOWNCOMER: degassed media+cells.

BASE: Connected to Perforated nozzle bank/ plate/ Sparger to


pump pressurized air.

HEAD SPACE: Gas release region, flocculation, foam accumulation


etc.

GAS SEPARATOR: Facilitates gas/liquid recirculation maximizes gas


residence time reduces gas friction in downcomer.
Airlift Reactor Types:

INTERNAL LOOP ALR: baffles placed strategically in a single vessel


create the channels required for the circulation

Shortest path that a bubble cover from the riser to the downcomer is
a straight line

EXTERNAL LOOP ALR: circulation takes place through separate and


distinct conduits (a tube )

There is usually a minimum horizontal distance to be covered,


increases the chances of disengagement of the bubbles
ADVANTAGES OF ALRs…

Hence, homogenous mixing/ distribution of gas/ oxygen.

Simple design with no moving parts or agitator for less maintenance,


less risk of defects.

Easier sterilization (no agitator shaft parts).

Low Energy requirement Homogenous distribution of nutrients and


shear force.

DISADVANTAGES

Greater air throughput and higher pressures needed.

Inefficient break the foam when foaming occurs.

NO bubbles breaker.


USES of ALR….

Mammalian cell cultures.

Waste water treatment

Biological processes involving biocatalysts as solids

To produce biopharma proteins etc from fragile cells/


broken cells.
Propeller Loop Reactor Jet Loop Reactor
Packed Bed Bioreactor

In packed bed reactors, cells / enzymes are immobilized on


large particles.

These particles do not move with the liquid.

Packed bed reactors are simple to construct and operate


but can suffer from blockages and from poor oxygen
transfer.
Packed Bed Bioreactor

Batch PBB Continuous PBB


A continuous packed bed reactor has the following
advantages over a batch packed bed reactor:

1. Easy, automatic control and operation

2. Reduction of labour costs

3. Stabilization of operating conditions

4. Easy quality control of products


There are three substrate flow possibilities in a packed bed and they
are illustrated below:
1. Downward flow method
2. Upward flow method
3. Recycling method

For industrial applications, upward flow is generally preferred over


downward flow because it does not compress the beds in enzyme
columns as downward flow does. When gas is produced during an
enzyme reaction, upward flow is preferred.

The recycling method is advantageous when the linear velocity of the


substrate solution affects the reaction flow rate. This is because the
recycling method allows the substrate solution to be passed through
the column at a desired velocity. Leaching purpose
Structured packing for distillation column
AFB Fine inert carriers provide large surface area for adherence and
growth of anaerobic microorganism are added in the bioreactor.
Basic principles
The solid substrate (the catalytic material upon which chemical species react)
material in the fluidized bed reactor is typically supported by a porous plate, known
as a distributor.
The fluid is then forced through the distributor up through the solid material.

At lower fluid velocities, the solids remain in place as the fluid passes through the
voids in the material. This is known as a packed bed reactor.

As the fluid velocity is increased, the reactor will reach a stage where the force of the
fluid on the solids is enough to balance the weight of the solid material. This stage is
known as incipient fluidization and occurs at this minimum fluidization velocity.

Once this minimum velocity is surpassed, the contents of the reactor bed begin to
expand and swirl around much like an agitated tank or boiling pot of water. The
reactor is now a fluidized bed.

Depending on the operating conditions and properties of solid phase various flow
regimes can be observed in this reactor.
Advantages of fluidized bed reactor
Uniform Particle Mixing: Due to the intrinsic fluid-like behavior of the solid material,
fluidized beds do not experience poor mixing as in packed beds. This complete mixing
allows for a uniform product that can often be hard to achieve in other reactor
designs. The elimination of radial and axial concentration gradients also allows for
better fluid-solid contact, which is essential for reaction efficiency and quality.

Uniform Temperature Gradients: Many chemical reactions require the addition or


removal of heat. Local hot or cold spots within the reaction bed, often a problem in
packed beds, are avoided in a fluidized situation such as an FBR. In other reactor types,
these local temperature differences, especially hotspots, can result in product
degradation. Thus FBRs are well suited to exothermic reactions. Researchers have also
learned that the bed-to-surface heat transfer coefficients for FBRs are high.

Ability to Operate Reactor in Continuous State: The fluidized bed nature of these
reactors allows for the ability to continuously withdraw product and introduce new
reactants into the reaction vessel. Operating at a continuous process state allows
manufacturers to produce their various products more efficiently due to the removal
of start up conditions in batch processes.
Disadvantages of fluidized bed reactor
Increased Reactor Vessel Size: Because of the expansion of the bed materials in the reactor, a
larger vessel is often required than that for a packed bed reactor. This larger vessel means that
more must be spent on initial capital costs.
Pumping Requirements : The requirement for the fluid to suspend the solid material
necessitates that a higher fluid velocity is attained in the reactor. In order to achieve this, more
pumping power and thus higher energy costs are needed.
Particle Entrainment: The high gas velocities present in this style of reactor often result in fine
particles becoming entrained in the fluid. These captured particles are then carried out of the
reactor with the fluid, where they must be separated. This can be a very difficult and expensive
problem to address depending on the design and function of the reactor. This may often
continue to be a problem even with other entrainment reducing technologies.
Erosion of Internal Components: The fluid-like behavior of the fine solid particles within the bed
eventually results in the wear of the reactor vessel. This can require expensive maintenance and
upkeep for the reaction vessel and pipes.
Pressure Loss Scenarios: If fluidization pressure is suddenly lost, the surface area of the bed may
be suddenly reduced. This can either be an inconvenience (e.g. making bed restart difficult), or
may have more serious implications, such as runaway reactions (e.g. for exothermic reactions in
which heat transfer is suddenly restricted).
Membrane bioreactor (MBR)

Membrane bioreactor (MBR) is the combination of a membrane process like


microfiltration or ultrafiltration with a suspended growth bioreactor, and is
now widely used for municipal and industrial wastewater treatment with plant
sizes up to 80,000 population equivalent (i.e. 48 million liters per day).
Conventional activated sludge process (Top) Vs external (sidestream)
membrane bioreactor (Bottom)
PHA can be accumulated in the anaerobic/anoxic bioreactor when
the carbon source is in excess.

Process optimization can reduce PHA consumption in the aerobic


bioreactor.

The PHA-rich WAS (waste activated sludge) in the anaerobic


digester can enhance methane production and reduce sludge
production, thereby forming an economically attractive and
environmentally friendly method to enable maximized resource
recovery in the form of methane.

WAS: waste activated sludge.


Photobioreactors
Raceway Ponds as Photobioreactors
Raceway pond for microalgae, CSIR-IMMT, Bhubaneswar
Photobioreactors by silentcenter
Assignment 1
1. What is industrial biotechnology?
2. How does microbe full fill the needs of industry?
3. Give two examples of bacteria with their industrial importance.
4. Describe about batch culture bacterial growth curve with diagram.
5. Describe about continuous culture bacterial growth curve with diagram.
6. What is diauxie growth?
7. 5M, 500 ml stock solution is with you. Diagrammatically show the serial
dilution to get 5 mM 100 ml solution.
8. What is the need of serial dilution?
9. 100 μl sample taken from 1000 time diluted solution gives 6 CFU of Bacteria.
Calculate the bacteria (in CFU) present in 5 ml of original sample.
10. Describe about 4 important components of bioreactor.
11. Draw a labeled bioreactor diagram.
12. Classify bioreactors (only schematic).
13. Discuss about advantages and disadvantages of stirred tank bioreactor.
14. Discuss about advantages and disadvantages of bubble column bioreactor.
15. Describe about membrane bioreactor and its utility.
16. Describe about photo bioreactor and its uses.

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