ERT: 314 BIOREACTOR SYSTEM

Experiment 1: Media Development and vessel

sterilization

Lee Lay Pei 101140407

Group members :
Afifah Fadhilah Binti Rosli 101140027
Tan Wan Ting 101141270
Moganeswari A/P Arumugam 101140484

Date of experiment : 25th February 2013

1.0 Objectives
1.1 To study the media development for yeast fermentation in a bioreactor.
1.2 To know the criteria indentify the areas for sterilization before run the experiment.

2.0

Introduction
Bioreactor is a vessel or container which carries out biochemical process that
involves microorganism. The selection and specific design of a bioreactor system include
a series of decision on ranging from basic microbiology and biochemistry to process
engineering. Bioreactor provides an optimal and controlled environment for the biological
system, operating at minimum cost in order to maximize the productivity and benefit of
the process. Fermentation is the conversion of a carbohydrate such as sugar into an acid or
an alcohol. More specifically, fermentation can refer to the use of yeast to change sugar
into alcohol. All atoms of carbon, hydrogen, oxygen, nitrogen and other elements are
consumed by new cells or excreted as product when fermentation. The growth and product
synthesis are well formulated by metabolic stoichiometry. This stoichiometry provides
information including mass ad energy balance of fermentation, comparison on the
theoretical and actual product yields, and consistency of experimental data and lastly the
formulation of the nutrient media. The sufficiency of nutrient media, oxygen, temperature
and pH is vital to support the cell growth and the produce the desired product.
Furthermore, the developed media in the bioreactor has to be sterilized to prevent
contamination by the unwanted microorganisms.

Figure 1 : Bioreactor
stucture of a bioreactor

Figure 2: Internal

3.0 Apparatus : Bioreactor, Autoclave, Weight balance, Erlenmeyer flask, magnetic stirrer,
measuring cylinder.
Chemicals : Distilled water, (NH4)2SO4, glucose monohydate, malt extract, yeast extract,
KH2PO4, MgSO4

4.0 Procedures
Experiment 1: Media Development
4.1.1 The concentration and total amount of glucose and (NH 4)2SO4 in the nutrient medium are
determined.
4.1.2 The required carbon and nitrogen sources are weighted.
4.1.3 The prepared media is pour into the 250 ml Erlenmeyer flask.
4.1.4 The media is sterilized for 121 C for 15 minutes and let it be cooled upon inoculation of
yeast.
4.1.5 The media is inoculated and the fermentation process is run for 48 hours.
4.2 Experiment 2: Vessel Preparation before Autoclave
Preparation of Bioreactor before Autoclave
4.2.1 The main power of the bioreactor unit is switched on and filled the water inside the jacket
heater to the maximum level.
4.2.2 The pH probe is taken out and calibrated it appropriately with pH 7 followed by pH 4 .The
probe immerse in KCI solution in a conical flask.
4.2.3 Each of the dosing pumps is calibrated to get the correct flow rate during the experiment.
4.2.4 The lid is removed and detached all the hosing connected are remove from the main touch
panel. All black hosing cannot be autoclaved, remove them before autoclave. Other colour of
hosing can be autoclave.
4.2.5 Motor all hosing and attached probes are removed from bioreactor.
4.2.6 The vessel is rinse with tap water as much time as necessary to make sure it clean and clear.
4.2.7 The electrolyte in the pO2 probe is checked whether its still having or not. This is to ensure
membrane inside the pO2 probe is protected during autoclave.
4.2.8 1L liquid media is prepared and pour into the vessel.
4.2.9 The lid reattach back to the vessel. The pH probe and pO2 probe are mounted back.
4.2.10 The pH, pO2, antifoam, level, temperature sensor and the hoses are covered with cotton and
aluminium foil.
2

4.2.11 The relief valve/filter at the condenser and sampling port are not covered with cotton, just
wrap with aluminium foil.

4.3 Maintenance And Safety Precautions

4.3.1 All operating instructions is supplied with the unit must be carefully read and understood
before attempting to operate the unit.
4.3.2 The stirrer, pH electrode, pO2 electrode, antifoam, pump at the bioreactor are turned off.
4.3.3 When running the bioreactor must be assisted by trained PLV at all times.
4.3.4 All safety appliances are worn when operating a high pressure gas line.

5.4 General Shut Down Procedures

5.4.1 The agitator, temperature, dosing pump are turned off.
5.4.2 The air sparger valve is closed and the main switch of the touch panel is turned off.
5.4.3 The mixer motor is removed from top of the bioreactor and place on top of the apparatus.
5.4.4 The pH and pO2 probe are carefully removed and place in the KCI solution for pH probe,
while in the distilled water for pO2 probe.
5.4.5 Any hosing attached with the bioreactor are removed and the bioreactor carefully bring to
the sink.
5.4.6 The cover is removed and the cover place on top of the bench so that it will not roll off.
5.4.7 The reactor media is pour into the sink and wash thoroughly. The vessel rinse with distilled
water.
5.4.8 The cover place back to the vessel, and put back the vessel to its position.

5.0 Results and Calculation

No
1

RIGHT
All hosing connected from main touch

WRONG
Autoclave without detach all the hosing,

panel is removed before autoclaved.

Motor is removed and all probes are

especially the black hosing.

Motor and probes is left in the

removed.
The vessel is rinsed with tap water for

bioreactor.
The vessel is autoclaved without prior

several times.
pO2, antiform, level, pH electrode,

rinse with water.

pO2, antiform, level, pH electrode,

temperature sensors and the hoses are

temperature sensors and the hose are

covered by cotton and aluminium foil.

Relief valve or filter, at the condenser is

exposed to air.
Relief valve or filter at the condenser is

wrapped with aluminium foil only.

covered with cotton and then wrapped

The tubing which connected with air

with aluminium foil.

The tubing connected with air spurger,

spurger, sampling pot and from

sampling pot are not clipped.

corrective bottles are clipped to prevent

liquid from flowing out.

Results from Group A1 and A2

Time
(hour)
OD
values

15

16

17

18

19

20

21

41

0.266

0.690

0.694

0.749

0.835

0.875

1.124

0.187

0.146

Graph of Optical Density Versus Time (hour)

1.2
1
0.8

Optical Density

0.6
0.4
0.2
0
0

10

15

20

25

30

Time (hour)

35

40

45

Result from group A3 and A4

Time (hr)

Optical Density at 495nm

0.182

16

1.144

17

1.567

18

1.582

19

1.609

20

1.620

21

1.629

22

1.650

24

1.700

41

1.726

43

1.725

44

1.719

Graph of Optical Density versus Time

Optical Density at 495nm

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

10

15

20

25

30

Time (hr)

Calculation
6

35

40

45

50

Determine the concentration and total amount of glucose and (NH4)2SO4 in the nutrient medium.
Concentration of glucose

MW glucose 1 mol glucose

50dw/L
MW Yeast
0.48 mol yeast
198.17 g /mol 1 mol glucose

50dw/L
144 g /mol
0.48 mol yeast

= 143.35 g/L glucose.

Concentration of (NH4)2SO4

MW ( NH 4 ) 2 SO 4 0.48 mol NH 3 1mol

50dw/L
MW Yeast
0.48 mol yeast 2mol

132 g/mol 0.48 mol NH 3 1mol

50dw/L
144 g / mol 0.48 mol yeast 2mol

=22.9g/L (NH4)2SO4

To prepare 150ml of nutrient media,

Molecular weight of NH4)2SO4 = 132g/mol
Molecular weight of glucose (C6H12O6) = 198g/mol
Molecular weight of yeast (C6H10NO3) = 144g/mol
Final concentration = 50g/L
Seed culture 10% from working volume, bioreactor volume capacity is 2L,
The working volume = 70% of working volume
= 2L X 70%
= 1.4L
The growth of beakers yeast on glucose can be written by the following equation:
C6H12O6 + 3O2 + 0.48NH3 0.48C6H10NO3 + 4.32H2O + 3.12CO2
Yeast
7

Weight of Glucose needed =143.35 g/Lglucose1.4L = 200.7 g glucose

Weight of (NH4)2SO4 needed =22.9g/L (NH4)2SO41.4L = 32g

6.0 Discussion
Bioreactor is a vessel in which a chemical process is carried out which involves
organisms or biochemically active substances which derived from such organisms. This process
can either be aerobic or anaerobic. In this experiment, the bioreactor used is stirred tank reactor
(STR) which is the most common type used in industry. Typically, it is filled up with 70-80%
medium of total volume as during fermentation process, bubbles may form and fill up the
headspace of bioreactor. To make thing worst this situation may cause explosion. STR containing
stirrer, sparger, 3 flat-blade turbine, baffle and foam breaker.
For media preparation, glucose monohydrate and inorganic (NH 4)2SO4 act as carbon and
nitrogen source respectively. Carbon substrate acts as carbon and energy source, part of it
contributes in cell carbon and the rest provide energy. The nutrient media is prepared by using 1
liter of distilled water, macroelement which are 32g of (NH 4)2SO4, 200.7g glucose monohydrate,
and trace elements which are 1.4g of malt extract, 1.4g of yeast extract, 4.2g of KH 2PO4 and
0.28g of MgSO4. They are mixed in a schott bottle.
The bioreactor is autoclaved at 121 C before medium is pour into reactor in order to
prevent unwanted microorganism remain in it. To autoclave the bioreactor, several preparation
steps are needed. Firstly, all the hosing should be removed, especially the black hosing. Then the
motor and the electronic probes is also removed, because most of them are not temperature
resistant. Besides, the relief valve or filter, male connector and the pH, antifoam, level,
temperature sensor are cover with cotton or aluminium foil to avoid spoilage of the probes. Lastly,
the tubing is clipped to prevent back flow of liquid. In term of sterility, there are 3 main ways in
which a fermentation run may be approached which are firstly no selective condition,
fermentation has to perform under strict aseptic conditions. Then partly selective conditions exist,
whereby other organisms will proliferate and lastly very selective conditions exist, so there is
hardly any chance of contaminating microbes. The longer the duration of the fermentation, more
stringent demand on the bioreactor design for aseptic conditions.
After sterilizing, STR is cooled down to room temperature and then medium is added into
it. The fermentation process is started when the pH is calibrated. This experiment is done for 41
8

hours and samples are collected and the absorbance values are recorded by using spectrometer at
495nm wavelength. A graph of optical density (OD) versus time is plotted as shown in the result
from group A1 and A2. OD values are increasing at the beginning until a certain point then it
drops sharply. It can be explained that cells grow rapidly at the beginning and then cells denatured
at the end. Most probably is because depletion of nutrients. Compare to the results from group A3
and A4, cells grow rapidly at the beginning then it reached stationary point after 17 hours.

7.0 Question
7.1

List down the factors that can cause contamination to your culture.
The factors that can cause contamination are sterilization is not properly conducted,
environmental factors which atmosphere contains many unwanted microorganisms. While
weighting the material is contaminated by foreign substances such as dust.

7.2

Why pH probe needs to be calibrating before autoclave start and pO2 probe after
autoclave?
Since microbes are very sensitive to pH, therefore pH probe needs to be calibrating before
autoclave because need to make sure the pH at neutral which is pH 7 followed by pH4 and
the pO2 probe is calibrated after autoclave to get saturated oxygen concentration.

7.3

What are the difference between impeller design for microbial,mammalian and plant
bioreactor and why they are designed in different ways?
For microbial bioreactor, their blades are flat and set vertically along an agitation shaft
which produces a unidirectional radial flow. All impellers are designed to homogenously
mix cells, gases, and nutrients throughout the culture vessel. The mixing action evenly
distributes oxygen and nutrients to cells for healthy growth, keeps them from settling to
the bottom of the vessel, and helps to maintain a uniform culture temperature.
For Mammalian bioreactor, the pitched-blade impeller is used which low shear impeller
designed to gently mix the content of culture without causing cell damage. In order to
improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller
9

consisting of a double-screen concentric cylindrical cage impeller was designed and its
mass transfer rate evaluated. This new impeller design increases the specific screen area,
and the convective mass transfer rate through the annular cage was significantly increased.
For plant bioreactor, since plant cells are too fragile and it takes too long to grow in
fermentation compared to microbial cells, the blade impeller is used which can be flat or
convex to produce an axial flow and used application require gentle mixing without
causing cell rupture and damage.

7.4

Why microsparger is being used in mammalian bioreactor?

Mammalian bioreactor is used to improve gas transfer rates, enabling medium
oxygenation in the headspace. It provides significantly higher gas medium interface and
therefore higher O2 mass transfer coefficients with the same gas flow rate, which
effectively reduces the overall superficial gas flow rate.

7.5

Give some comment on design for sparger, impeller, baffle and heating element for these
three types of bioreactor.
For plant bioreactor, the use of impeller is marine blade impeller. Plant bioreactor the
location of the sparger in the flow direction of the impeller guarantees mass and
temperature homogeneity and optimal gas dispersion.
Mammalian bioreactor, pitched-blade impeller is used.
For microbial reactor, Ruston or flat bladed turbine impeller is used. The sparger is used to
deliver gas or air which will depend on quantity used of this three bioreactor. The rotation
of those ports creates a low-differential pressure at the base of the impeller tube, lifting
microbial up through the tube and expelling them out through its ports. This continuous
recirculation loop keeps cells uniformly dispersed throughout a vessel. Gases are
introduced through a ring sparger, which generates bubbles that pass along the impeller
between the exterior of the inner tube and an outer membrane, known as the aeration
cage.

10

7.6

How much yeast biomass will be produced theoretically from the stoichiometric equation
above?
C6H12O6 + 3O2 + 0.48NH3 0.48C6H10NO3 + 4.32H2O + 3.12CO2
0.48mol144g/mol = 69.2g

7.7

Determine the yield coefficients Yx/s (biomass/glucose) and Yx/o2(biomass/oxygen).

biomassg/ L 50 g /L
=
glucose g/ L 130 g /L

(i) Yx/s (biomass/glucose) =

(ii)Yx/o2(biomass/oxygen)
concentration of O2 =

MWO 2
no . of mole ofO 2

50dw/L
MW yeast no . of mole of yeast
32 g /mol
3 mol O2

50dw/L
144 g /mol 0.48 mol yeast

= 69.4 g/L O2
Yx/o2

biomassg /L
O 2 conc g /L

50 g /L
= 69.4 g / L
= 0.72 biomass/gO2

7.8

What will be happened if you have mistakenly tighten the lid screw and not clipped the
hose for aeration during sterization?
If mistakenly tighten the lid screw, the air cannot be pass through the hose. If not clipped

water will clog in the hose causes the medium cant flow out.

11

8.0 Conclusion
As a conclusion, media development is done by proper procedures and calculation from
the metabolic stoichiometry. Besides that, sterilization process is a very important step to avoid
any unwanted microorganisms to contaminate the culture medium.

9.0 Reference
Doran, P.M. (1995). Bioprocess Engineering Principles. Academic Press, London.
http://www.bioprocessintl.com/journal/2009/January/Which-Impeller-Is-Right-for-Your-CellLine-183538
http://www.ncbi.nlm.nih.gov/pubmed/18601112

12