Bioreactor Startup and Shutdown Procedures

Startup Procedure
1. Autoclave all autoclaveable equipment, ethanol disinfect the rest.
2. Secure the vessel using two clamps one just below the liquid outlet and one
just above the sparger inlet.
3. Add the bottom cap to the bottom of the glass vessel by simply sliding in (the
O ring inside the cap should provide the seal). If the O ring does not slip on
easily, try using water, if not, source some grease from wyss workshop
(although do not use too much of this we dont want it to slip back off!).
4. First: check that there is no residual water in any of these connections.
Connect the 3/8 Mcmaster Tygon pipe to the air inlet and connect to 3/83/16 reducer. Connect 3/16 end to 3/16 pipe (black pipe) and feed this
pipe into the rotameter inlet (bottom). Using another 3/16 pipe (black pipe),
feed over the outlet of the rotameter (top). Feed this 3/16 pipe into the
3/16-1/8 reducer and attach a 1/8 Mcmaster Tygon pipe to the other end.
Feed this pipe into the valve and use another 1/8 Tygon pipe for the outlet.
Feed this pipe into the filter, and using another 1/8 tygon pipe, attach to
filter outlet. Feed this outlet through a 1/8-6mm reducer, and attach 6mm ID
pipe to the other end. Feed 6mm-3/8 reducer over this pipe and attach a
3/8 pipe to the other end. Feed 3/8 pipe over the sparger tube.
5. Insert and secure the sparger to the vessel using the chemthread tightener.
6. (Optional) Hold the air inlet piping system with a clamp (when you open the
air inlet, the pipes will move due to the pressure, so holding it in place will
help prevent any harsh movements).
7. The sparger is designed to introduce air through only a very small area on
one side of the sparger tip. We want this to be facing up, to aid the aid
distribution mechanism from the fritter plate. Thus, you may want to set up a
test tube filled with water, to identify the air inlet area, and then ensure this
area is facing up for the air inlet in the reactor. Reseal with the chemthread
before moving on.
8. Attach the water feed pipe to the first inlet opening of the reactor and
alkaline feed to the second opening.
9. Connect these to the respective pumps and connect the feeds to the pump.
10.Attach 6mm diameter pipe to the liquid outlet from the reactor, and feed into
the liquid outlet container. It seems the combination of low flow and plastic
pipe causes a lot of resistance at the water outlet. To achieve a constant
outflow, make sure 9mm outlet pipe is securely attached, and pull the pipe
down to be as vertical as possible. This will create a line of flow downwards
for the water to continuously follow (rather than building up and falling in
droplets). You may need to hold the pipe down with clamps.
11.Place inoculated disks into the reactor in the most ordered fashion which is
reasonably possible (using forceps). A geometry similar to the one in my
COMSOL model would be great, if not, as high a packing density as possible
would be preferred.
12.Add the top cap and ensure the level of the band is level with halfway of the
outlet pipe.
13.Unpackage each probe and uncap. Check each probe thoroughly. Each probe
should be crack free and membranes should not be damaged. Check O rings

are undamaged. For DO probe: make sure there is sufficient electrolyte at

the bottom of the probe, else readings are not reliable approximately 2mL
should be minimum (but check the EDGE instructions).
14.Calibrate pH and DO probes using instruction manual found in Hanna
Instruments EDGE box.
15.Insert the desired probe through the hole in the cap and ensure the probe
dips below the required level (there is a dash on each probe indicating lowest
level).
16.Check all fittings are tightly sealed (especially air lines as any loss of air will
significantly affect DO inside the reactor). Beware: rubber tube attached to
lab air is very prone to air leakage when you adjust the air flow rate using the
valve (due to pressure build up).
17.Switch on the water feed pump and slowly (step-wise) increase flow rate to
desired (1L/day). Must be slow at this point as steady state must be achieved
at each flow rate before increasing to the next step-flow.
18.Simultaneous to step 17, slowly feed air inlet till the flow rate reaches ~1-2x
flow of water (this will be hard to accurately determine since the rotameter
only reads 10L/day min). The best way to do this is to increase the flow of
the air inlet through the lab air valve until it reaches a high readable level on
the rotameter (between 1-2 L/h). This is important as the air from the sparger
must overcome the pressure of the water, which doesnt happen at a low flow
rate. Also be aware that the flow may take a while to pass through due to low
velocity, plus having to pass through the filter etc. Once you see bubbling in
the reactor, control using the attached needle valve to reduce to 0.1 L/h (this
needle valve is also not accurate). Depending on your water flow rate, you
may not need to reduce this flow rate if we are going for 1L/day water flow
rate then 0.1 L/h should be fine. There should be a very fine stream of
bubbles from the sparger, which will then dissipate through the fritted plate.
19.Check outlet pipe has a continuous flow rather droplet flow. If it does not, you
may need to induce a single, connected flow of liquid. To do this you will
need to increase the flow rate to a much higher value than we are aiming for
(e.g. a reading of 50 on the pump). This will create the single line of flow
down the outlet pipe. Slowly reduce the flow rate of the water to keep this
flow for the low flow rate which we are looking at.
20.Allow constant flow at 1L/day for 24 hours.
21.Once steady, adjust for pH and DO levels to ensure optimum conditions.
22.Once optimum conditions are achieved, allow biofilm to grow for 48 hours.
Check the pH/DO every couple of hours or so to ensure that the pH, DO or
temp have not deviated from the optimum. If they have: heat/cool, add NaOH
or change air flow accordingly.
23.Once grown, the system is ready for the first experiment.

Shutdown Procedure
1. Stop the air inlet first do this relatively slowly.
2. Slowly reduce the liquid inlet down to 0. Allow the reactor to drain.
3. There will be a small amount of liquid left below the fritted glass plate
remove the water and NaOH inlet, and the water should drip. In case there is

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still any more water locked in (there may still be a small pool at the bottom),
remove the bottom cap to allow the reactor to fully drain.
Once reactor is drained, remove the sparger from the system and rinse with
water (to prevent clogging of the sintered glass and glass tube).
Remove the probe from the system and follow EDGE instructions to clean and
store the probe.
Take apart all pipes and fittings (including liquid inlets and outlets).
Rinse all pipes and fittings with water to prevent residue build up after the
water dries out.
Remove the packing material from the system using forceps.
Unsecure the vessel from the clamps and rinse.