Experiments

• 1: CO2 Sparging
• 2: Control Testing
• 3: Spirulina Batch
• 4: HOB Enrichment

1 - CO2 Sparging

Describes how to get started with CO2 sparging on the Pioreactor.

Kit List

Pioreactor

1. Pioreactor 20 mL (or via LabCrafter if you’re in the UK/EU)
2. Raspberry Pi Zero 2 W with header
3. Raspberry Pi Micro USB Power Supply (or via LabCrafter if you’re in the UK/EU)
4. Micro SD card (or via LabCrafter if you’re in the UK/EU)

CO2 sparging kit

1. Sodastream cylinder (classic blue, not quick connect pink)
2. Sodastream-to-regulator adapter (TR21-4 thread size in most of the world, CGA 320 in North America)
3. CO2 regulator with solenoid, e.g. FZONE
4. Barrel power cord with 2.1mm DC plug
5. Connector to connect barrel power cord to PWM output, e.g. TE Connectivity AMP connector (housing and socket contacts), or solder Dupont female square head wires to barrel power cord
6. ~20cm 4mm PU tubing
7. Check valve
8. 3/16" barb female Luer lock connector
9. 2 x 1/16" barb male Luer lock connectors
10. ~10cm 1/16" silicone tubing

3D printable parts

1. Pioreactor vial cap for electrolysis and CO2 sparging
2. Dovetail platform for Pioreactor
3. Dovetail platform for Sodastream cylinder holder

Tools required

1. Crimping pliers, if you’re going to use the TE Connectivity AMP connectors with the barrel power cord, or a soldering iron if you’re going to solder Dupont cables to the barrel power cord
2. Wrench for attaching adapter to Sodastream cylinder and regulator

Setup

Hardware

1. Connect the CO2 regulator to the Sodastream cylinder using the adapter and a wrench.

   

2. Connect one end of ~20cm PU tubing to the regulator and the other end to a check valve.

   

3. Connect another short piece of PU tubing to the other end of the check valve and attach a 3/16" barb female Luer lock connector. Attach two 1/16" barb male Luer lock connectors to each end of ~10cm of 1/16" silicone tubing and connect to the 3/16" connector and the Pioreactor port.

   

4. Connect the barrel power cord to PWM channel 4 on the one end. You can either solder Dupont female square head wires to the power cord, or crimp a TE Connectivity AMP connector as in the image.

   

5. Plug the other end of the power cord into the solenoid of the regulator.

   

6. Make sure that your Pioreactor supports 12V on the PWM channels and that a 12V power supply is connected to the barrel jack of the Pioreactor HAT.

Software

1. Install the pioreactor-relay-plugin plugin.
2. In your Pioreactor configuration, make sure that PWM channel 4 is set to relay:

[PWM]
# map the PWM channels to externals.
# hardware PWM are available on channels 2 & 4.
1=stirring
2=media
3=waste
4=relay
5=heating

3. Test that it works by manually turning on the relay in the Activities tab of the Manage screen of the Pioreactor UI. You should hear the solenoid turn on and CO2 rushing into the Pioreactor vial. You can adjust the amount of CO2 sparged using the dial on the regulator.

   

4. Create a new experiment profile and copy and paste the following into the profile:

experiment_profile_name: CO2 sparging every hour

metadata:
  author: Gerrit Niezen
  description: Turns on the relay for 10 seconds every hour

common:
  jobs:
    relay:
      actions:
        - type: repeat
          hours_elapsed: 1.0
          repeat_every_hours: 1.0
          actions:
            - type: log
              hours_elapsed: 0.0  # relative to the repeat loop, 1h
              options:
                message: "Sparging CO2 for 10 seconds"
                level: info
            - type: start
              hours_elapsed: 0.0
              options:
                start_on: True
            - type: stop
              hours_elapsed: 0.00278

When the experiment profile is running it should sparge CO2 for 10 seconds every hour.

2 - Control Testing

To ensure that the Pioreactors we’re using are working as expected, we ran an experiment where we used the exact same experimental conditions with two Pioreactors, with the hope to produce the same results.

The original experiment is described on the Pioreactor website.

Kit List

1. 2 x Pioreactor 20 ml - see our thoughts on Pioreactors
2. 2 x Raspberry Pi Zero 2 W with header
3. 2 x Raspberry Pi 15W USB-C Power Supply
4. 2 x Micro SD cards
5. 3 x 20mL vials
6. Another computer on the same network as the Raspberry Pi with a web browser
7. Pressure cooker to ‘autoclave’
8. Aluminium foil to cover the vessel ports
9. Distilled water
10. Baker’s yeast
11. Digital pocket scale
12. YPD broth
13. Measuring cup
14. Dropper/pipette

Protocol

Preparing and sterilizing the media

1. Prepare YPD broth media for three 15mL vials.
2. Pour 300mL tap water into the pressure cooker, place the three vials on the metal trivet in the pressure cooker and turn to a high setting.
3. Once the water in the cooker starts to boil, steam will come out of the open valve. Put the heaviest weight (15lb) weight on top of the valve.
4. The steam will lift the weight and start to escape. As soon as the steam starts to escape, start timing the sterilization and turn down the heat so that the steam is only just escaping and not rushing out. Aim to maintain a gentle hissing.
5. After 20 minutes turn off the heat and leave to cool.
6. Wait until pressure is completely reduced then lift the weight off the valve allowing any remaining steam to escape. Never open the pressure cooker until the steam valve has been opened to release the pressure.

Running the experiment

1. Dilute a small amount of baker’s yeast in 15mL of YPD broth media
2. Innoculate the three sterile vials with the same amount (just a drop) of culture from the stock solution.
3. Wipe the vials and place them in the Pioreactors.
4. Start a new experiment on your Pioreactor dashboard.
5. Select Manage all Pioreactors
6. Start Stirring activity
7. Start Temperature automation activity and set to 30°C
8. Start OD reading activity
9. Go back to the graphs and check that an Optical Density (OD) signal is being received.
10. Select Manage all Pioreators again and start Growth rate.

Results

The raw results from the experiment are available as CSV files on GitHub.

Report

3 - Spirulina Batch

Kit List

1. Pioreactor 20 ml - see our thoughts on Pioreactors
2. Super Bright White 5mm LED for photosynthesis
3. 5mm LED cables to connect the LED
4. Raspberry Pi 4B 8GB RAM - needn’t be 8GB, that’s just what I had from a prior project
5. Raspberry Pi 15W USB-C Power Supply
6. Micro SD - because Tom said
7. Ethernet cable - just because we use CAT8 in the studio for anything in live production
8. Another computer on the same network as the Raspberry Pi with a web browser
9. PH0 Screwdriver
10. Jewellers scales (to calibrate pipettor and weigh out growth media powder)
11. 10ml Volumetric flask to make up growth media
12. 10-100 ul Pipettor and 200 ul tips for inoculation
13. Weighing boat
14. Spatula
15. Pressure cooker to ‘autoclave’
16. cotton wool and foil to cover the vessel ports.
17. Distilled water
18. Spirulina inoculum & growth media - as it was the only Spirulina we could source in time.

Setup

Results

4 - HOB Enrichment

Kit List

We’re still optimising the kit list for Hydrogen Oxidising Bacteria (HOB) enrichment, please see this discussion on the Bill of Materials for the latest.

Media

We’re reasonably happy with our mesonutrients (other than the vitamin pill & perhaps the Calcium Chloride) but more work is required for us to be able to recommend an optimal food grade micronutrient solution. In the meantime there are many options if you’re happy to go non-food grade - see Trace element solution for our latest thoughts.

Setup