Production of baker's yeast by fermentation

Production of baker’s yeast by fermentation
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1. THEORY
The primary task of bioengineering is to design and operate bioreactors with the best
possible economic effect. Such optimization is not possible without solving material balances
and energetic balances of given bioprocess. At design and process control of production,
material balances enable optimization of effectiveness of substrate transformation to products.
Thus, the costs of raw materials could be optimized. Similarly, energetic balances help at
optimization of stirring costs and of costs of bioreactor cooling.
Material and enthalpy balances of bioreactors might not be formulated without so-called
source terms, which reflect the influence of biochemical process on balance of components
and energy. At classic chemical reactions, it is relatively straightforward to identify all the
reactions and their stoichiometry. However, the principles of classic stoichiometry are rather
hard to apply at biotechnological productions. The reason for this is that thousands of
reactions take place simultaneously in bioreactors. It is impossible to identify or quantify all
of these reactions. Thus, the description of stoichiometry and of bioprocess kinetics needs to
be simplified. This means that we describe only the transformation of key substrates to key
products. The key substrates are especially the sources of carbon, nitrogen, oxygen and also
the sources of phosphorus and sulfur. Solution of such stoichiometric equation is based on
balance of atoms of carbon, nitrogen, oxygen and hydrogen, which might be replaced by
balance of free electrons. Remaining equations, which are necessary for calculation of
stoichiometric coefficients, could be obtained from experimental data, such as the respiratory
quotient RQ or yield factors Yx/s, Yp/s, Y O2 /s.
For better illustration, an example is introduced: a stoichiometry of aerobic cultivation of onecell
proteins from bacteria Methyllococus capsulatus. As a source of carbon, methane was
used; ammonia was the source of nitrogen and no other product was formed in the liquid
phase. The overall process could be described by a simple equation:
s CH 4 + a NH 3 + b O 2 = c CH 1.8 O 0.5 N 0.2 + d CO 2 + e H 2 O (1)
Where the values s, a, b, c, d, e are modified stoichiometric coefficients related to 1 mole of
consumed substrate Yi/s (mol i/mol s). These are actually the yield factors defined as a ratio
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of reaction rates. In order to solve a stoichiometric equation with 6 unknown stoichiometric
coefficients, six equations are necessary.
These equations are the balances of atoms of C, N, H, O, s = 1 (modified coefficients related
to 1 mole of consumed methane). Another equation is one for calculation of average yield
factor Yx/s, which could be calculated from experimental measurement of rate of substrate
consumption and of biomass production (Yx/s = 0.4785).
Balance of elements’ atoms comprises a system of six algebraic equations, which could be
solved. Thus, it is possible to calculate e.g. the amount of oxygen and nitrogen necessary for
preparation of given amount of biomass.
C :
N :
O :
H :
Y
S:
x/s
:
1s
+ 0 + 0 − 1c
− 1d
+ 0 = 0
0 + 1a + 0 − 0.2c + 0 + 0 = 0
0 +
4s + 3a + 0 −1.8c
+ 0 − 2e = 0
0 + 0 + 0 −1c
+ 0 + 0 = 0.4785
1s +
0 + 2b − 0.5c − 2d −1e
= 0
0 + 0 + 0 + 0 + 0 = 1
2. Aim of the work
1. To perform an aerobic cultivation of baker’s yeast in a laboratory stirred batch
fermentor.
2. To experimentally monitor development of concentration of substrate, biomass and
oxygen during the process. To calculate average yield factors based on these
measurements.
3. To construct a stoichiometric equation of the process. To calculate the values of
modified stoichiometric coefficients and calculate the total amount of consumed
oxygen and produced CO 2 .
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3. METHODS
3.1 Preparation of the inoculum
The composition of inoculation medium (in g dm -3 ): d-glucose 25, yeast extract (empiric
formula CH 1.91 O 0.56 N 0.23 ) 10, peptone (empiric formula CH 1.795 O 0.5 N 0.2 ) 10, adjust pH to value
of 5.8. Dissolve the glucose in 20 cm 3 of redistilled water and sterilize separately at 120°C for
20 minutes. Dissolve other components of the medium in 60 cm 3 of redistilled water, adjust
pH to 5.8 and sterilize at 120 ºC for 20 minutes in a 500 ml Erlenmeyer flask. After the
sterilization, blend both parts of the medium aseptically (final volume of 80 ml). After
inoculation by one eye from inclined agar, which is used for preservation of the strain, the
inoculum is cultivated for 15 hours on a rotary shaker at 26 ºC and at stirring of 200 rpm.
3.2 Preparation of the production medium
3.3
The composition of production medium (in g dm -3 ): d-glucose 25, yeast extract 10, peptone
10. Dissolve the extract and peptone in 1220 dm 3 of redistilled water and pour it into the
reactor. Dissolve glucose in 500 dm 3 of redistilled water and pour it into 1000 ml Erlenmeyer
flask. Check the completeness of the reactor and test-connect it according to scheme (Fig.1.)
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7
6
out-gassing
pO
T
air
3
2
5 4 1
pH
FERMENTOR
Fig.1. Scheme of connections of the fermentor
9
10
B
I
O
S
T
A
T
1 – safety vessel with glass wool, 2 – U-manometer, 3,5,7 – safety vessel, 4,8 – sterile air
microfilters, 6 – condenser, 9 – storage vessel containing the acid for adjustment of pH, 10 –
storage vessel for inoculum.
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3.4 Fermentation (conditions 28°C, 400 RPM, 1.5 VVM)
• All sensors, except for temperature sensor, are sterilizable and would be sterilized right
inside the reactor. The pH sensor must be calibrated before the sterilization in a
subprogram of the main menu CALIBRATION.
• Calibration of the pH sensor: use an arrow on the keyboard and set the cursor to second
row and switch the regime from “auto” to “man”. Switching is performed using button
ALTER and the choice is confirmed by button ENTER. The cursor relocates to
temperature; manually enter desired temperature. Press ENTER. Immerse the pH sensor
into pH 7 buffer and press ENTER. When the pH value stabilizes, the biostat would
record the value and it will automatically relocate the cursor to following row. Immerse
the electrode into pH 4 buffer and press ENTER. When the pH value stabilizes, the
regime is automatically switched to “auto” and the calibration is successfully finished.
CALIBRATION
pH : 7.2 actual pH value inside reactor
TEMP: 28 C man Temperature at calibration.
- auto: calibration is performed at actual
temperature inside reactor
- man: temperature is set manually
BUFZ: 7.00 pH ok Displaying of pH of “zero” buffer
Ok – confirmation of the calculation of zero point.
The cursor automatically skips to row BUFS
BUFS : 4.00 pH ok Displaying of pH value for calculation of the
slope.
Ok – after confirmation, the cursor skips to second
row and the regime automatically switches to
“auto”.
Connections of sensors at the head of reactor is displayed on Fig.2.
1 – inlet of air
2 – outlet of air
3 – condenser
4 – access to temperature sensor
5 – access to oxygen sensor
6 – access to pH sensor
7 – sampling port
8 – inlet of acid
9 – inoculum
10 – pouring of mediums
Fig.2. Scheme of the head of bioreactor
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• The reactor with its accessories is sterilized in a large autoclave. All parts of the reactor
might be sterilized with exception of the temperature probe, which would be inserted into
the device after sterilization at aseptic conditions. The duplicator must be filled with
water without air bubbles (CONTROL LOOP auto). At sterilization, it is important that
the ends of all hoses and all of metallic parts would be covered by aluminum foil; the
outlet of out-gassing from reactor must not be closed. After the sterilization and cooling,
place the reactor into inoculation box and pour inside the glucose solution under aseptic
conditions; insert the temperature probe and connect sterilized air filters to inlet and
outlet of air.
• Transport the reactor back to biostat; connect all sensors, cooling water, air supply
according to Fig.1. Turn on the cooling water from central supply and turn on the flow of
air, also from central supply. Carefully and in parallel, turn on the air supply on the
biostat and simultaneously open the squeezer on the air inlet line. Regulate the flowrate
of air using a rotameter, which is an accessory of the biostat. Set the value to 3 dm 3 min -1 .
After stabilization of temperature, adjust pH to value of 5.6 by solution of H 2 SO 4 .
Turn on the temperature control. In the subprogram CONTROL LOOPS on the first page,
change the regime from “Off” to “Auto”. Set the cursor to respective row, press “ALTER”
and then “ENTER”. Navigate between rows using the arrows in the main keyboard of the
biostat.
CONTROL LOOP
TEMP: 30 C
SETP : 28 C
MODE: Off
PARA :
Actual value in the reactor
Set value
Auto: automatic temperature control
Off: the control is turned off
Setting of PID controller parameters
Turn on the stirring control: Change the regime from “Off” to “Auto” in the same way.
CONTROL LOOP
STIRR: 250 rpm Actual value in the reactor
SETP : 250 rpm Set value
MODE: Off Auto: automatic control of stirring
Off: the control is turned off
PARA :
Setting of PID controller parameters
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Calibration of the oxygen sensor: switch air supply to supply of nitrogen. Navigate to second
page in subprogram CALIBRATION (navigate between pages using button ENTER). Set
cursor to second row using the arrows on the keyboard and switch regime from “man”
(manual) to “auto”. Switching between regimes is performed using button ALTER; confirm
the choice by button ENTER. The cursor relocates to temperature; manually enter desired
temperature. Press ENTER. Navigate the cursor to NITR and press ENTER. When the value
of concentration of dissolved nitrogen stabilizes at value of approximately 0%, navigate the
cursor to NITR and press ENTER. Biostat records a stable value and the cursor automatically
relocates to AIR. Switch the supply of nitrogen to supply of air and wait until the value of
concentration of dissolved oxygen grows to value of approximately 100%. Press ENTER to
record a stable value corresponding to saturation state. The cursor relocates to row TEMP and
the regime switches automatically to “Auto”. The calibration is successfully finished.
CALIBRATION
2 nd page pO 2 : 87.2 % Actual value of pO 2 in the reactor
TEMP: 28 C man Temperature at calibration.
- auto: calibration is performed at actual
temperature inside reactor
- man: the temperature is set manually
NITR : 0.0 % ok Displaying of “zero” pO 2
Ok – confirmation of zero point calculation. The
cursor relocates to row AIR.
AIR : 100 % ok Displaying of pO 2 value for calculation of the
slope.
Ok – after confirmation, the cursor relocates to the
second row and the regime switches automatically
to “auto”.
After calibration of oxygen sensor, disconnect the reactor and place it into inoculation box.
Transfer 80 ml of 15-hour (old) inoculum under sterile conditions into separator connected to
bioreactor. Reconnect the reactor to biostat. Turn on the inlet of cooling water and air. Turn
on the temperature control and stirring control and, after checking the completeness of the
device and connections, inoculate the reactor.
Inoculation: Pump the inoculum aseptically to reactor using a peristaltic pump, or inoculate
the fermentor under flame through nozzle 10 (Fig.2.).
Pump the inoculum to reactor at time zero, turn on the data collection.
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Turn on the data sampling to computer: turn on the computer and start the program Biostat B
Data Collection. The computer starts to sample the data only after the Biostat relays the signal
to it. The sampling rate could be altered only on Biostat in subprogram MAINTENANCE.
Enter the data sampling rate and then alter the mode by “ALTER” and confirm by “ENTER”.
MAINTANANCE
2 nd page Printer
CYCLE : 1 min
MODE: Off
PARAM :
Data sampling interval
Start: Data collecting is turned on
Off: Data collection is turned off
Setting of parameters
Software: PROCESS VALUES – it displays the actual values of monitored process quantities
TRENDY – it displays the development of monitored quantities during
fermentation process
PRINTED VALUES – it displays the printing layout
The program continuously stores the data into file printer.txt. For use in program EXCEL, it is
better to export given data to a file in form of a database.
Data exporting: Within the window TRENDY, press the icon of a diskette located in lower
right corner “Copy to file” and store the data in a file “name.dbf”.
3.5 Sampling
Sample extraction:
1. During cultivation, take an approximately 6 ml sample every hour; during exponential
phase of growth, take sample every 45 minutes.
Sample processing:
Use 5 ml of sample for measurement of optical density.
Centrifuge 0,8 ml of sample in an Eppendorf vial (300 rpm, 10 minutes). After centrifugation,
use micropipette to carefully draw clear supernatant into a clean Eppendorf vial and freeze it.
At the end of cultivation, all sampled supernatants would be used to determine the
concentration of the substrate – the glucose.
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3.6 Measurement of optical density
1. 5 ml of sample centrifuge (3000 RPM min -1 , 10 minutes). Wet biomass rinse gradually
3 times with isotonic solution and centrifuge at 3000 RPM min -1 , 10 minutes. Then
add accurately 5 ml of isotonic solution, mix well a measure optical density of cells
against isotonic solution at wavelength of 600 nm. If the sample absorbance is greater
than 0.7, the sample would be properly diluted by isotonic solution. The concentration
of wet biomass would be determined during growth using a calibration line.
Construction of calibration line
1. Prepare 100 ml of standard biomass solution of concentration of 3 g/l in a isotonic
solution. Dilute the standard biomass solution with physiological solution to reach
concentrations of: 0.3, 0.15, 0.12, 0.075, 0.06, 0.05 g/l (each solution with volume of
10 ml). Measure absorbance of all solutions in comparison with absorbance of isotonic
solution at 600 nm. Using the measurements of absorbance of standard biomass
solutions, construct a calibration dependence cx = f (A600), calculate the parameters
of calibration equation and calculate the regression coefficient.
3.6 Determination of glucose concentration by a glucose test
Principle
The product of the enzymatic reaction – the glucose – is quantified using a glucose test GLU
GOD 6 x250. The basis of glucose quantification is formation of colored complex, whose
absorbance is measured spectrophotometrically. This complex is formed by a selective
oxidation of glucose to hydrogen peroxide and gluconate using enzyme glucose-oxidase.
Formed hydrogen peroxide is quantified by an oxidation reaction with substituted phenol and
4-aminoantipyrine catalyzed by enzyme peroxidase; a red colored product is formed.
Principle of quantification of released glucose:
β − D − glucose + H O + O ⎯⎯⎯⎯⎯→ D − gluconate + H O
(2)
glucose−oxidase
2 2 2 2 2
H O + dye ⎯⎯⎯⎯→ dye + H O
(3)
peroxidase
2 2 reduced
oxidized 2
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Method
1. Heat the glucose-test solution to 30°C. Transfer 1 ml of this solution into each
Eppendorf vial using a micropipette. The number of Eppendorf vials is equal to
number of glucose standards + 3x the number of samples + 1 (blind experiment).
2. Add 10 µl of water into the first Eppendorf vial (blind experiment) and add 10 µl of
glucose standards or samples into the remaining vials. Immediately after addition, stir
the mixture thoroughly.
3. Store the rack with Eppendorf vials in a dark place and let them incubate for 30
minutes.
4. After the end of incubation, take the samples and measure absorbance of each sample
at absorbance maximum of quantified product (500 nm). Use water as a reference
solution.
5. To construct a calibration line, use already prepared standard solutions of glucose of
concentration 0 – 0.02 mol/l.
3.7 Determination of cell dry mass
After fifth hour of cultivation, take approximately 13 ml sample and use 12 ml to
determine the dry mass. Centrifuge the 12 ml of sample and rinse it 3-times by isotonic
solution. Dissolve centrifuged biomass in approximately 2 ml of isotonic solution and dry
it at 80°C until a constant weight is reached.
4. EVALUTATION OF EXPERIMENTAL DATA
1. Construct the calibration dependence using measured absorbance of standard glucose
solution; calculate the parameters of calibration equation and the regression coefficient.
2. Calculate the concentration of remaining glucose in samples from fermentation using a
calibration line.
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3. Construct a calibration dependence using the measurement of standard solutions of
biomass; calculate the parameters of calibration equation and the regression coefficient.
4. Calculate the concentration of wet biomass and of dry cells in samples from fermentation
using the calibration line of the biomass.
5. The concentration of oxygen in equilibrium with partial pressure of oxygen in the gaseous
phase (c O2
*) would be calculated using Henry equation:
O2
*
O2cO2
p = H
(4)
where
p O2 is the partial pressure of O 2 in the gaseous phase, H O2 is Henry’s constant
for oxygen,
*
c O2 is the equilibrium concentration of oxygen in water ( H O2 = 8,0107*10 4 Pa
m 3 mol -1 at 25ºC; Nielsen: Bioreaction Engineering principles, 2003).
6. Using experimental data (% of equilibrium oxygen concentration in water), determine the
molar volumetric concentration of oxygen (mol/m 3 ) in water. Use following equation:
1.
c
c (%)
100
O2 *
O2 = ⋅cO2
(5)
7. Visualize graphically the development of all monitored parameters during fermentation.
8. Describe the cultivation by a single total stoichiometric equation; determine the values of
stoichiometric coefficients and calculate the amount of consumed oxygen and produced
CO 2 at fermentation.
Note:
- If the top of pO 2 probe is replaced, the probe must be activated for 24 hours while
connected to switched-on biostat. If only the electrolyte is being replaced, one-hour
duration is sufficient. It is better to store the probe without electrolyte in a dry state.
- Overpressure in the reactor: 79 mm.
- The pH probe is being screwed tight in place only by hand, not by a wrench.
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5. BIOSTAT OPERATIONS MANUAL
5.1 Structure of the main menu
Main functions
o PROCESS VALUES
Functions
: it displays the actual values of process variables
o CALIBRATION : calibration of sensors and pumps (pH, pO 2 , acid, base, anti-foam agent /
substrate 1, level of liquid / substrate 2)
o CONTROL LOOPS : control of process variables
o MAINTENANCE
: configuration of output signals, setting of time and date, setting of data
sampling interval
5.2 Process values
The menu comprises of four pages; the navigation between them is performed by arrows. It
displays on-line measured values and their actual values in the reactor.
2 nd page
TEMP : 40.5 ºC
STIRR: 1200 rpm
pH : 10.2 pH
pO 2 : 80.4 %
3 rd page
ACID :
BASE :
AFOAM:
LEVEL :
4 th page
FOAM : off
LEVEL: on
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