Bioreactor Set-Up

11
Exit gas
Motor

Air Pump 10
12
DO Controller
9
DO Meter
8
pH Controller

13
pH Meter

Temp Control
7
3
2
6
sterile 1 4
glucose
5

Elements of the Apparatus

1. Glass vessel
2. Dissolved Oxygen (DO) Probe
3. Sampling tube
4. Impeller
5. Sparger
6. pH Probe
7. Temperature probe
8. Sample bottle
9. Cooling water inlet
10. Cooling water outlet
11 & 12 Sterile filters
13. Base feed

Basic Principle
The fermentor apparatus is designed to contain all culture within the confines of the bioreactor
vessel, and to allow sampling without exposing the contents of the tank to the open atmosphere
which would allow any extraneous organisms to enter. In addition, pH, temperature and DO are all
controlled at user-specified set-points, so that the production of by-products will not have adverse
effects on the culture environment. The vessel is to be adequately stirred and aerated so as to
prevent any stagnant or oxygen-poor zones.

Description of the System

I. Dissolved Oxygen (DO) Control: Oxygen has a very low solubility in water, only about 7 PPM
at standard temperature and pressure. Hence, oxygen is usually the limiting nutrient for organism
growth, unless a feed strategy is designed to limit the growth rate via a different nutrient of choice.
Above a critical O2 concentration, growth rate is independent of DO concentration. Oxygen
limitations usually result in the production of by-products because of incomplete substrate
oxidation to CO2. Molecular O2 consumed by most organisms via respiration can only be used as a
terminal electron acceptor and therefore, must be converted to CO2.

glucose + 36 Pi + 36 ADP + 6 O2 6 CO2 + 6 H2O + 36 ATP

Air is fed to the vessel from an air compressor, through the DO controller and into the vessel
through a tube with fine holes in it (the sparger). The mixing energy of the impeller causes the air
bubbles to dissolve somewhat into the culture broth.

The DO probe must be calibrated prior to the fermentation run. Because DO probes are often
unreliable, it is a good idea to calibrate the probe prior to autoclaving to ensure its proper function.
Autoclaving the probe usually requires readjustment prior to inoculation. Probe calibration is as
follows:
1. Turn on the DO meter and controller
2. Blow filtered N2 into a hole in the head plate of the vessel while stirring. Observe the DO
meter, and when the value reaches a minimum (no further change) turn the zero knob until
the meter reads 0.0. Remove the N2 hose from the vessel and shut off the N2 tank valve
completely. Do not leave pressure in the regulator.
3. Turn on the air compressor and blow filtered air through the sparger into the tank. Observe
the DO meter and when a maximum is reached, adjust the cal knob until the value reads
100.
4. Set the controller to the desired set-point between zero and 100.

The exit gas from the fermentor goes through a condenser, in order to remove humidity from the
warm air, and out through a sterile filter. Sterile filters should have a pore size of 0.2 µm. The
purpose of the sterile filter is to prevent organisms from the room to find their way into the
bioreactor. The filters tend to get clogged during the run from humidity that is not condensed, as
well as from particulates that are in the exit stream (Foam quickly finds its way into exit line filters.
Clogged exit line filters result in back pressure build-up in the vessel. Large fermentors have a
pressure guage on the exit line to measure the pressure.

To think about...
During the fermentation run, what is the significance of a reading of "20" on the DO meter?
During the fermentation run, under what 2 conditions may the DO meter read 0.0?
How will back pressure have an effect on the DO control?

II. pH Control: All microorganisms that grow in culture are sensitive to the pH of the medium.
The pH in the cytoplasm of the organism is normally a neutral value, since proteins depend on
hydrogen bonding for their proper folding and function. Most, but not all culturable organisms
grow best around neutral pH (between pH 5.5 to 8). High cell densities in the culture result in the
rapid depletion of one or more nutrients, which often results in the production of organic acids:
acetic acid, succinate, formate and lactate. Likewise, production of CO2 results in the dissolution
of CO2 in the broth and the formation of bicarbonate. To maintain the pH in the vessel, base is
added in response to a low offset from a pH set-point. The bases most often used for pH control
are NH4OH (which also contributes Nitrogen in a usable form to the bacteria), KOH, NaOH, or
bicarbonate.

Like the DO probe, the pH probe should be calibrated both before and after autoclaving. One
approach is to calibrate the probe with pH control buffers before inserting into the tank, add growth
media that is slightly below the optimum growth pH, then after autoclaving, do the following:
1. remove a small amount of medium in a sample bottle.
2. compare the pH reading on the bioreactor pH meter with that on a separate, calibrated pH
meter.
3. IF THE readings are the same (medium in the vessel should be at room temperature, then
while stirring and at the growth temperature-- allow the pH controller to automatically
adjust the pH to the desired growth pH set-point.
4. If the readings are different by less than 0.5 pH units, adjust the set-point of the desired
growth pH to account for the 0.5 pH unit difference, then allow the controller to
automatically adjust the pH.
5. If the readings are significantly different, adjust the set-point of the desired growth pH to
account for the pH difference, then allow the controller to automatically adjust the pH. Be
sure and take a sample of the broth once the pH set-point is reached and check the final pH.
If it is different from the desired growth pH, adjust the set-point again and either allow the
controller to add base from the system, or add sterile acid to drop the pH (this is an
undesirable outcome, as it adds additional ions to the broth). Be sure to measure the pH
during the run with an external pH meter and adjust the setpoint as needed.

Both a set-point and an offset (minimum and maximum allowable values) need to be specified. For
some organisms, a small change in the pH (≤ 0.1 pH unit) may have a significant impact on the
growth characteristics of an organism.

To think about...
If an organism is known to produce acid byproducts, what would be the cause of a sudden increase
in the culture pH during a run?

III. Temperature Control: Bugs tend to grow faster at higher temperatures until an optimum
temperature is reached, higher than which undesirable effects are produced. Temperature may also
be an important parameter for optimization of the production of a desired product. Heat production
is a direct result of metabolism, and in particular, of respiration. Hence, while it may be necessary
to add heat to the bioreactor just after inoculation, once a critical culture density is reached, it is
usually necessary to cool the reactor to maintain the optimum temperature for the organisms.

Temperature control is easily achieved in most bioreactors with a cooling water jacket or internal
cooling coils, as is done in very large industrial fermentors. A temperature probe (thermocouple) is
inserted into the culture vessel such that the temperature in the center of the vessel is measured.
The probe is connected to a control box which adjusts a heating (or cooling element) until there is
no offset from the set-point.

The Applikon Fermentor in the SJSU Bioprocess Engineering Laboratory has both heating and
cooling capabilities. A electric heating jacket is wrapped around the outside of the vessel to add
heat, and cooling water is run through the reactor when it needs to be cooled down. A chiller
generates cold water that is recirculated through the bioreactor control system and used for both the
condenser and the cooling water coil.

IV. Sampling the Vessel: The sampling assembly is designed to prevent long exposure of the
system to the open air. ***Most contaminated cultures are the result of poor sampling
technique.*** Once the sample line is filled with growth medium, stray bacteria from the air can
begin to grow there. For this reason, large fermentors that are designed for extended runs may have
valved-off steam lines attached to the sampling assembly so that decontamination can be performed
before and after each sample is taken.

A sample is taken as follows:

1. A clean sample bottle should be attached to the sampling assembly. That bottle was put
there immediately after the previous sample was taken.
2. First, remove the syringe from the connector and fill it with air. Replace the air-filled
syringe.
3. Open the sample valve and blow the air by pressurizing the syringe piston until bubbles
appear at the bottom of the sample tube.
4. Reverse flow in the syringe by pulling the piston until culture broth is seen flowing into the
sample vial. When 3 - 5 mls of culture fill the sample vial, reverse the flow again until
bubbles are seen again at the bottom of the sample tube.
5. Close the sampling valve. Remove the sample vial and immediately replace with a fresh
vial.

V. Nutrient feeding: Bacteria are sensitive to the concentration of glucose (or other nutrient) in the
culture broth. In addition, they consume the nutrient. Hence, a method to control the concentration
of glucose in the fermentor must be employed. Fermentations involving the addition of nutrients
are called fed-batch and can be optimized for extended running, for achieving high cell densities
(on the order of 50 to 200 g/liter of dry cell weight) and other desired outcomes.

The most common control strategy is the loose loop control, which requires the manual sampling of
culture, measurement of glucose concentration, and subsequent adjusting of feed rate, to maintain
the desired concentration. Fortunately for biochemical engineers, Yellow Springs Instruments
(YSI) built a convenient and overpriced Glucose Analyzer 2700, which quickly and easily
measures the glucose concentration. This device requires only 25 µl of sample, but the sample
should be free of bacteria. If the glucose concentration in the reactor is 2 to 5 g/l, one ml of liquid
sample can be put into an eppendorf tube and centrifuged at 16,000 RPM for 2 minutes. Follow the
instructions with the analyzer for further instructions. For higher glucose concentrations, the
sample should be diluted 1:10 with clean water prior to centrifugation.

It is usually necessary to adjust the rate of nutrient feed during the run since the cell density and
growth rate both change over the course of the fermentation. The following parameters can be
determined, and the feed rate adjusted so that the two be maintained approximately equal:
average nutrient consumption rate (g/l/h) = ∆concentration in samples (g/l)
time between samples (hr)

nutrient feed rate (g/l/hr) = (feed bottle concentration (g/l)) x (pump flow rate (liters/hr))
fermentor volume (liters)
The nutrient consumption rate will depend on the cell density, as well as the growth rate. Typical
yield factors (YX/S, yield of biomass on substrate) are 0.4 to 0.6 on glucose for aerobic organisms.
This can be used to estimate a feed rate for starters if a correlation between the dry cell weight and
optical density is known.

VI. Growth of a starter culture: An inoculum is made by growing the organism of interest in a
shake flask culture in growth medium. This medium may be different from what is in the
fermentor, considering the shake flask provides no means for pH or DO control. Temperature
control in the shaker is usually adequate for cultures up to 500 ml. To get adequate oxygen supply
in a shaker flask, the volume of liquid in the flask should be about 1/5 of the volume of the flask.
Baffles in the flask enable more oxygen entrainment in shaker cultures.

For hearty organisms such as wild-type E. coli, an overnight culture grown to stationary phase can
be used to inoculate a fermentor. More sensitive organisms may require a culture in exponential
phase to be used for the inoculum.

VII. Autoclaving the fermentor vessel:

1. Detach the vessel, filled to the desired level with growth medium, from all electrical
instrumentation.
2. All tubes, such as air and sample lines, connected to tubes that extend below the liquid level
should be tightly clamped off before autoclaving.
3. All open ends, including sterile filters, should be covered as tightly as possible with foil.
4. Glucose feed bottle can be connected to the vessel with a clamp on the feed line.
5. Condenser should be supported and not hanging freely next to the glass vessel.
6. Screw the caps on the top of probes if they exist. Otherwise cover tightly with foil.
7. Place the vessel, glucose bottle, and antifoam bottle (if you have one) in a secondary
container with enough volume to contain the contents of the vessel should it erupt.
8. Before placing in autoclave, loosen the screws of the top plate so that it is still resting on the
vessel. This is to allow air to escape as the pressure builds up.
9. Autoclave for 20 minutes at 121°C.
10. When cool enough to remove from the autoclave, do so, then screw tight the head plate
screws. Allow to come to room temperature. While waiting for it to cool, plug in the pH
and DO probes to the meter boxes, as it is necessary for many probes to be plugged in for
24 hours prior to operation to enable a fast response time.

VIII. Inoculation and sampling: When the pH and DO probes are recalibrated and the vessel is at
the desired growth temperature and other controller set-points, the vessel is ready to inoculate.
Typically, the inoculum is 0.01 to 0.1 times the volume of the growth medium in the vessel,
depending on the desired outcome of the experiment. For studying the growth or metabolism of the
culture, less inoculum may be required to avoid artifacts. At the time of the inoculation, a timer
should be set to zero for recording the run time. Immediately following the inoculation, a sample
can be drawn as a zero point.

Samples should be placed immediately on ice to stop the growth. In a high cell density sample, the
pH will drop quickly if not chilled right away. Measurements of optical density (OD), pH, and
glucose concentration should all be taken. If the absorbance is measured to be above 0.3 AU at 550
or 600 nm, the sample should be diluted so that the reading is between 0.1 and 0.3. When the
culture reaches exponential growth phase, it should be checked under the microscope to ensure that
the culture is not mixed, but the bugs have the physical characteristics of the starting organism.
***Collect all sample waste in a red biowaste bag. Empty all liquid culture waste into a labeled
container and soak the sample vials in a bucket with clorox in it. Leave them 2 hours before
washing out.

VIIIa. Dry Cell Weight: When the OD is above about 10, the dry cell weight can be measured. A
25 ml sample should be taken, immediately chilled, and the OD measurement taken in triplicate,
appropriately diluted. The cells should be spinned down in a superspeed, refrigerated centrifuge at
8000 RPM for 10 minutes. The supernatant should be carefully aspirated off and the cells
resuspended by vortexing in some neutral buffer(e.g. Phosphate Buffer). Repeat the washing twice
with distilled water. Finally, resuspend the cells in 5 mls of distilled water and place on a pre-
weighed pan and dry completely, preferably in a vacuum oven. Weigh the pan + cells and calculate
the weight of 25 mls of cells at the OD measured. This measurement is prone to error, so several
measurements should be made a different optical densities.

IX. Completing the run: When no further information is desired from the experiment, shut off the
temperature, pH and DO controllers and add clorox to the fermentor to a concentration of 10% by
volume. Continue to stir the fermentor for an hour before shutting down. Leave overnight. Put
order in the lab.