REPORT

2010-bt-chem-07
2010-bt-chem-08
2010-bt-chem-09

University of Engineering & Technology, Lahore

(KSK Campus)
 Contents

Description Page

1. Introduction of Bioreactor……………………………………………………….2

2. Types of bioreactor……………………………………………………………….2
2.1 Batch bioreactor………………………………………………………………2
2.2 Fed-batch bioreactor…………………………………………………………2
2.3 Continuous bioreactor……………………………………………………….2

3. Bioreactor Design……………………………………………………………………3
3.1 Bubble column bioreactor……………………………………………………3
3.2 Air lift bioreactor……………………………………………………………….3
3.3 Fluidized bed bioreactor……………………………………………………..3
3.4 Packed bed bioreactor………………………………………………………..4
3.5 Flocculated cell bioreactor…………………………………………………..5

3. Stirred batch bioreactor…………………………………………………………..5

4. Construction of bioreactor………………………………………………………..5
4.1 Vessel/reactor…………………………………………………………………..6
4.2 Motor agitator/impeller……………………………………………………….6
4.3 Air Sparger………………………………………………………………………6
4.4 Sensors…………………………………………………………………………..6
4.5 Cooling jackets…………………………………………………………………6
4.6 Valve, filter, ports……………………………………………………………..6

5. Pre-startup of bioreactor………………………………………………………….6
5.1 Clean in place………………………………………………………………….7
5.2 Check valves and probes……………………………………………………7
5.3 Check pressure holding……………………………………………………..7
5.4 Steam in place………………………………………………………………….8

5. Startup………………………………………………………………………………..9

6. Operation……………………………………………………………………………..9

7.1 Detail process of bioreactor………………………………………………. 10

7.2 Dilution Tank…………………………………………………………………. 10
7.3 Pre-bioreactor…………………………………………………………….. 10
7.4 Bioreactor…………………………………………………………………… 11
7. Troubleshooting………………………………………………………………….. 13
1

8. Shut down……………………………………………………………………………13
Page

9. Reference…………………………………………………………………………… 14

Page 1 of 14
 1- Introduction
A Bioreactor can be any device which is used to artificially produce a biological
environment in-vitro or which is capable of growth of organisms such as bacteria
or fungi in a biological environment under controlled conditions.

It can be used in varied applications such as in aerobic/anaerobic chemical

processes, in fermentation, in cell culture applications, in industrial applications
such as production of antibodies, vaccines, in bio-conversion for producing by-
products from raw materials. The environment within a bioreactor is maintained
and controlled in such a manner that constant homogeneous conditions are
maintained.

2- Types of Bioreactor
Based on the functioning of the Bioreactor it can be of 3 types:

2.1 Batch Bioreactor:

This bioreactor’s volume is constant
and cells and media are added for
performing the operation in batches.
The operation is run until a particular
set point in terms of time and
concentration is reached. Then the
used media is drained out to fill it
with a fresh new media.
In the beginning it starts as a batch
process. After reaching a particular
point, a input is given .Here the
volume of the bioreactor increases
after the input is provided.
2.3 Continuous Bioreactor:
Here there is a continuous flow of
media solution containing cells from
2

2.2 Fed-Batch Bioreactor: input end to the bioreactor. The used

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solution and product is also taken out automated as compared to batch and
continuously from the bioreactor. The fed-batch bioreactor where every time
rate of input and output is after batch the operation has to be
maintained in such a manner that the
started again.
volume of the bioreactor is constant.
Among all the bioreactors, continuous
bioreactor has the advantage that it is

3- Bioreactor design
-
The major types of bioreactors used in industry include:

3.1 Bubble column bioreactor

These are tall reactors which use air alone to mix the contents

3.2 Air lift bioreactor

These reactors are similar to bubble column reactors, but differ by the fact that
they contain a draft tube. The draft tube is typically an inner tube which improves
circulation and oxygen transfer and equalizes shear forces in the reactor.

Airlift bioreactors (ALB) are generally

classified as pneumatic reactors
without any mechanical stirring
arrangements for mixing and use the
expansion of compressed gas to bring
about the mixing.

The turbulence caused by the fluid

flow ensures adequate mixing of the
liquid.

The advantages of Airlift reactors are

the elimination of attrition effects
generally encountered in mechanical
agitated reactors

.
It is ideally suited for aerobic cultures since oxygen mass transfer coefficient are
quite high in comparison to stirred tank reactors.

3.3 Fluidized bed bioreactor

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In fluidized bed reactors, cells are "immobilized" small particles which move with
the fluid. The small particles create a large surface area for cells to stick to and
enable a high rate of transfer of oxygen and nutrients to the cells

Fluidized bed bioreactors (FBB) have
received increased attention in the
recent years due to their advantages
over other types of reactors.
Most of the FBBs developed for
biological systems involving cells as
biocatalysts are three phase systems
(solid, liquid & gas).
The FBBs are generally operated in
co-current up flow with liquid as
continuous phase and other more
unusual configurations like the
inverse three phase fluidized bed or
gas solid fluidized bed are not of
much importance.
In comparison to packed bed reactors
FBBs can be operated with smaller
size particles without the drawbacks
of clogging, high liquid pressure drop,
channeling and bed compaction.
The smaller particle size facilitates
higher mass transfer rates and better
mixing.
The volumetric productivity attained
in FBBs is usually higher than in
stirred tank and packed bed
bioreactors.

3.4 Packed bed bioreactor

In packed bed reactors, cells 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.

The immobilized biocatalyst is packed

in the column and fed with nutrients
either from top or from bottom.

One of the disadvantages of packed

beds is the changed flow
characteristic due to alterations in the
bed porosity during operation.

The packed bed reactors are widely

used with immobilized cells.

Several modifications such as tapered

beds to reduce the pressure drop
across the length of the reactor,
inclined bed, horizontal bed,
rotary horizontal reactors have been
tried with limited success.
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3.5 Flocculated cell reactors
Flocculated cell reactors retain cells by allow them to flocculate. These reactors are
used mainly in wastewater treatment.

3.6 Stirred tank reactor

In these reactors, mechanical stirrers (using impellers) are used to mix the reactor
to distribute heat and materials (such as oxygen and substrates).

4- Construction of Bioreactor
Bioreactor can come in any size and configuration. A typical bioreactor has got
many components such as:

1) Vessel or Chamber 6) Pressure sensor and valves

2) Motor agitator/Impeller 7) Flow meter

3) Gas Sparger 8) Cooling jacket

4) Temperature sensor 1) Input and Output port

5) PH sensor 2) Filters

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4.1 Vessel/Reactor
The vessel/chamber is the main housing where all these components will be
loaded and the media solution containing the cells will be placed. The vessel
should be biocompatible so that it can be autoclaved to remain contamination free.
Hence the material used can be stainless steel or glass. Glass can be used as it is
transparent and can give a clear view of the working of the components inside.

4.2 Motor agitator/impeller:

An agitator is used so as to mix all the contents in the bioreactor so that the
solution contains all the nutrients, gases in equal proportions and homogeneous
conditions with constant PH and temperature.

4.3 Air Sparger

The Sparger helps to introduce and mix gases into the solution. It maintains the
level of oxygen and other gases required for the bioreactor system.

4.4 Sensors:
Different sensors and valves to monitor the conditions of the bioreactor are used
such as temperature sensors, PH sensors, pressure sensors, flow meter etc.

4.5 Cooling jacket

The cooling helps to reduce and maintain the temperature by flow of a coolant
around the bioreactor.

4.6 Valves, filters, ports

Different ports are used to connect various components in the bioreactor system.
Various filters can be used in a bioreactor to either filter the gases entering or
leaving the system or filter the media solution leaving the system to maintain the
PH and keep it constant. Various valves can be used to control the flow of gases
and liquid solutions.

5-Pre-Startup of Bioreactor

Step-3: Check
Step-1: pressure
Clean the holding
place
Step-4:
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Steam in
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Step-2: Check place

the probes
and valves Page 6 of 14
5.1- Step-1 (Clean in place)
CIP (clean in place) is always the first step in preparing a bioreactor for use. CIP
consists of a series of detergent washes, followed by water rinses that leave all
product-contact surfaces free of any dirt and organic debris left in the vessel from
the previous batch.

The CIP cycle consists of two washes: In order to clean the vessel properly,
the CIP solution must reach all areas
Phosphate-free alkaline liquid of the vessel, including the head
detergent containing potassium plate, addition valves and piping,
hydroxide (base). sample valves, impeller blades, and
exhaust piping.
Acidic liquid detergent containing
phosphoric acid. Spray balls are made of stainless
steel. Holes are drilled in the spray
CIP solution is injected into the
ball to ensure the inside of the vessel
bioreactor through both:
gets complete spray coverage.
The ring sparger, at the bottom of the
Some bioreactors or tanks have
vessel. multiple spray balls.
One or more spray balls, which are
typically mounted near the top of the
bioreactor

Following the CIP solution step, the liquid is drained and the bioreactor is rinsed
with water

5.2- Check Probes and valves

DO pH and CO2 probes are important tools that monitor the cell growth
environment Be careful not to damage the probes when putting them into the
bioreactor ports (pH probes are glass and will break) Ensure they are screwed (or
clamped) into the ports well to ensure sterility of the bioreactor is maintained

Addition and sample valves need to be installed on the vessel prior to the pressure
hold and the SIP cycle. The pressure hold will check to ensure there are no leaks
in the piping and the SIP cycle will sterilize the valves to prevent contamination.

5.3- Check Pressure Holding

7

Its purpose is to check the integrity of the sterile boundary. The areas that are
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checked include:

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• Bioreactor vessel / head plate connections

• Gas inlet and exhaust filter housings

• All piping between the inlet and exhaust filters

• Addition and sample valves added to the vessel

• Probe connections

Procedure
– Bioreactor is pressurized to a certain set-point

– Pressure is allowed to stabilize for a number of minutes

– Pressure is then held for a number of minutes, and pressure drop is measured

– The total pressure drop cannot exceed a predetermined criteria

– At a constant temperature, any pressure drop above the maximum allowed can
be attributed to a leak in the system.

– If a leak is detected, the bioreactor should not be used, it should be checked for
the leak source.

– The bioreactor must pass pressure hold prior to use.

5.4- Steam in place.

The last step in the preparation of a bioreactor, prior to media filling and
inoculation, is SIP (steam in place).

SIP sterilization is a time-proven and economical process for killing

microorganisms through the application of moist heat in the form of saturated
clean steam under pressure.

The rate by which microbial organisms are thermally inactivated depends on the
temperature and duration of heat exposure.

The amount of time the bioreactor is exposed to the desired SIP temperature set-
point is called the hold time or exposure time.

This time and temperature is determined during the validation of the vessel

Typical hold times range from 30 – 45 minutes

Typical SIP temperature set-point is ≥ 121 °C

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 6- Startup of bioreactor

Step-1 Step-3

Check and ensure the probes and ON power supply of agitator, probes
valves and blower.

Step-2

Fill the reactor with media.

Step-4

Start agitator and blower

Step-5

Start circulation of cooling water and

steam in jackets.

Step-6

Add cells/enzyme, acid/base and

anti-foaming agent.

7- Operation:-

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7.1- DETAIL PROCESS OF BIOREACTOR
In this section primarily Molasses (sucrose) undergoes hydrolysis into glucose and
fructose by an enzyme called invertase present in the yeast.

C12H22O11 + H2O C6H12O6 + C6H12O6

Secondly, Glucose is converted into ethanol and carbon dioxide by the enzyme
zymase present in the yeast.

C6H12O6 2C2H5OH + 2CO2

In this process Carbon dioxide from pre-bioreactor and Bioreactor and Sludge from
Bioreactor and sedimentation tank is the by-product. This sludge is further utilize
in biogas unit in the digester.

We divide this section into 3 major parts.

o Dilution Tank
o Pre-bioreactor / Buffer tank / Pre-fermenter
o Bioreactor / Fermenter

7.2- Dilution Tank:

Before entering in the dilution tank molasses of R.310 tank and process water mix
in the static screw mixer and then enters in the dilution tank where sulphuric acid
mix with this dilute molasses to maintain their pH to 4.During this process heavy
mash is formed. In dilution tank concentration is 28 brixs.The proess is basically
hydrolysis of molasses.

7.3- Pre-bioreactor / Pre-fermenter:

After processing in the dilution tank this mixture further mix with
process water to decrease its brix or concentration. Mixing is again done in the
static screw mixer in the line after mixing with process water the brix of the
dilution product decreases from 28 to 15 brix with in the line. This 15 brix
solution enter in the pre-bioreactor through the line of the pump which circulates
the mixture in the pre-bioreactor to make it homogenous. In pre-bioreactor we
only grow the bacteria (Saccharomyces cerevisiae bacteria) by providing optimum
10

pH (4.0) and temperature (30-320C) which is required for the bacterial growth in
the pre-bioreactor. We increase number of these bacteria’s up to 110 in Pre-
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bioreactor during every batch. DAP(Di-ammonium phosphate) is added in it after
that Yeast is added in it.

DAP is acting as food and provide nutrients(i.e. Nitrogen and Phosphorous) to the
bacteria which is essential for growth. On the other yeast provides the enzymes
which act as a catalyst for the aerobic fermentation. After 2 to 3 hours of addition
the volume of the reaction mixture increases and brix decreases to 7 after this
feeding process starts and the mixture of Sodium fluoride and Electoral added in
it. This mixture is used to kill the anti-cell which kill the bacteria’s or formed
resistance in the growth of bacteria’s.

During this process air continuously supplied in it and maintain their pH 4.0 by
the addition of H2SO4 it also provide sulfur content to the yeast for bacterial
growth. We also maintain temperature by heating with steam or cooling in plate
and frame heat exchanger. Steam and air is supplied through the coil which is
hanging with in the pre-bioreactor tank.

Mixture circulates continuously throughout the whole process through pump to

attain uniformity. At the end of the process the reaction mixture brix decreases to
7.0.After every batch pre-bioreactor can be washed with water to remove anti-cells
which is attached with the inner wall surface of the tanks.

Material of construction of the tank also effect on the bacterial growth.Pre-

bioreactor is made up of mild steel or stainless steel. Mild steel surface roughness
is greater than that of Stainless Steel. Due to greater roughness of MS some anti-
cells will remain on the wall which cannot be removed with washing and decreases
the efficiency. Another factor which decreases the efficiency is uniformity. Because
of large size of the pre-bioreactor the reaction mixture cannot completely uniform
which decreases its efficiency.

Some reaction also take place in the pre-bioreactor and produce 5% alcohol and
CO2.CO2 continuously evolve from bioreactor and this CO2 send towards the
absorption column. When working of the pre-bioreactor has been completed then
reaction mixture move towards the Bioreactor. Pre-bioreactor is also called buffer
tank, because in pre-bioreactor we resist the change in the pH of reaction mixture
by adding some additives.
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7.4- Bioreactor:
All the Bioreactor do the same work. We use five Bioreactor because
continuous operation has done.20% of the heavy mesh from dilution tank is filled
in the Bioreactor after this 60% of the pre-bioreactor product mix with it in 16-18
hours(mixing time). This 7.0 brix solution enter in the Bioreactor through the line
of the pump which circulates the mixture in the Bioreactor to mix it with heavy
mesh of 28 brix and to make it homogenous.

The reaction is too much vigorous, highly exothermic, foaming generates and
reaction mixture expands. In this situation we add antifoam and cool the reaction
mixture in the plate and frame heat exchanger to control the reaction and foaming.
In bioreactor we also maintain the temperature at 32-330C by using heat
exchanger and steam. Steam and air is supplied through the coil which is hang
within the Bioreactor.

Supply of air stops which can be adjusted with the weight of the reaction
mixture.20% of space remains empty within the Bioreactor the reaction mixture
needed this space when reaction mixture expand. After mixing time (16-18hours)
reaction stops and reaction mixture becomes dead and after this 14 hours rest
time is provided to the reaction mixture.

One more thing we notice that when fermentation complete foaming disappear and
solution becomes clear and brix of the solution stops between 12 to 13.During this
fermentation process glucose converted into alcohol and CO2 and fructose
converted into alcohol,CO2 and glycerol.

CO2 continuously evolve from Bioreactor and this CO2 send towards the
absorption column. In last 2 hours of the rest time we stop mixing pump and add
some calgon. Calgon is coagulants which neutralize the charge particles and
particles settle down. In last 1 hour purge stream valve open and slurry drain out.
When 14 hours rest time completed product contains 8-9% alcohol and this
product send towards the wine decanter through pump.

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 8- Troubleshooting

Problem Cause Troubleshooting

Enzyme Growth Issue 1. Cell density too Maintain require
low. condition for Enzyme.
2. Missing a Clean up reactor before
component. startup.
3. Agitation OFF. Insure before startup any
4. PH of media too low component not missed.
or high. Add acid/base to
maintain PH.
Mechanical Issue 1. Leakage in steam Isolate steam valve
valve To prevent pump strain
2. Broken pump. must be attach before
3. Leakage in pump.
mechanical seal of Isolate reactor and also
reactor. check before startup.
Foaming Issue 1. Low density By adding antifoaming
particles agent (Silicon FD 50).
Attach foam breaker
inside reactor.

9- Shutdown:

Step-1 Step-2

Stop adding media, Cells/enzyme, Close circulation of cooling water and

acid/base and anti-foaming agent. steam in jackets.

Step-3

OFF agitator and blower

Step-4

OFF power supply of agitator, probes

and blower.

Step-5
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Open discharge valve

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 Reference:
Links visited at 24 October,2013.

https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=r
ja&ved=0CDQQFjAB&url=http%3A%2F%2Fwww.ncsu.edu%2Fbiosucceed%2Fcour
ses%2Fdocuments%2FBioreactorEngineering.pptx&ei=TY6VUvWFCMf8ywOiqYDw
Dg&usg=AFQjCNFEGMSfWi05PjBaklQS0cT418nauQ&bvm=bv.57155469,d.ZG4

http://www.authorstream.com/Presentation/manoj1.-1361238-bioreactors/

https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=r
ja&ved=0CEEQFjAD&url=http%3A%2F%2Fsite.iugaza.edu.ps%2Fkelkahlout%2Ffil
es%2F2011%2F10%2Fchapter-
5.ppt&ei=TY6VUvWFCMf8ywOiqYDwDg&usg=AFQjCNEJQnLASey-
PAirNHES7hcWi7TMWg&bvm=bv.57155469,d.ZG4

https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&cad=r
ja&ved=0CGAQFjAI&url=http%3A%2F%2Fwww.cheric.org%2Fippage%2Fe%2Fipd
ata%2F2004%2F02%2Ffile%2Fe200402-
901.pdf&ei=TY6VUvWFCMf8ywOiqYDwDg&usg=AFQjCNGAb3D4c1g-
T_VYyup1a2U-PC-CIw&bvm=bv.57155469,d.ZG4

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