Experiments

Plant Choice Materials and Method

A quick google search suggested radish and lettuce could be suitable as are observed to be fast growing plants.
Seeds were bought from a local garden centre along with growing boxes, and smaller growing containers (all with clear Perspex lids allowing for ventilation).
Soil was taken from the garden and distributed into the large growing box to a depth of 150mm.
Radish seeds were planted in 3 rows with approximately 80-90mm between them.
Lettuce seeds were planted in 3 rows with approximately 80-90mm between them.
30mL of water was added to each seeded area initially and this was supplemented every 2nd day with an additional 20mL of water.
The grow box was positioned in the same area continuously to both observe identical sunlight exposure and to minimise temperature fluctuations from drafts, shadows etc.
Perspex lid was maintained in place with ventilation slots left open (except for days when additional rainfall would affect water volume to seeds) and seeds were inspected daily.
Growth was allowed to continue for 21 days post seeding.

Biofrag 1.0 Seed Growth Ability Testing

Both internal and external Biofrag 1.0 were filled with dense sponge.
Seeds were added into the Biofrag via a small tunnel cut into the foam centre. This tunnel centred on holes within the inner Biofrag (which would hope to allow both stem and root growth of seed to be unimpeded as it exited the Biofrag structure).
Biofrag was planted within a glass jar (see pic) so upper surface was level with soil and 30mL of water was gently administered directly over it (to not potentially disturb its position).
An additional 20mL of water was applied every 2nd day.
The glass jar was positioned in the same area continuously to both observe identical sunlight exposure and to minimise temperature fluctuations from drafts, shadows etc.
Growth was allowed to continue for 21 days post seeding.

Alternate Biofrag 1.0 Seed Growth Ability Testing

See Biofrag 1.0 Seed Growth Ability Testing for initial steps of Biofrag setup.
Biofrag was then planted in the same manner but within the grow box and 30mL of water was gently administered directly over it (to not potentially disturb its position).
An additional 20mL of water was applied every 2nd day.
The grow box was positioned in the same area continuously to both observe identical sunlight exposure and to minimise temperature fluctuations from drafts, shadows etc.
Perspex lid was maintained in place with ventilation slots left open (except for days when additional rainfall would affect water volume to seeds) and seeds were inspected daily.
Growth was allowed to continue for 21 days post seeding.

Biofrag 2.0 Seed Growth Ability Testing

The inner frag was filled with soil surrounded by a thin layer of dense foam. Foam was arranged as to not interfere with the newly incorporated portals on both poles of the inner Biofrag This was accomplished by placing a hose pipe section through the portals creating space within the foam matrix that could be filled with soil afterwards.
The outer Biofrag halves were lined with dense foam and the inner frag was placed into one half with the protruding hose section lined up with the larger portal. The adjoining half was then secured to it with the other end of the hose section lined up with the adjoining portal.
The hose was filled with earth and the Biofrag was buried into soil (within a grow box) where the hose was removed allowing the earth within to fill the previous aperture made. Additional soil was added post hose removal to fill the Biofrag to the portal level.
One radish seed was placed into the portal opening to a depth of approximately 20mm and had 30mL of water added onto it.
The grow box was positioned in the same area continuously to both observe identical sunlight exposure and to minimise temperature fluctuations from drafts, shadows etc.
Perspex lid was maintained in place with ventilation slots left open (except for days when additional rainfall would affect water volume to seeds) and seeds were inspected daily.
20mL of additional water was added in the same way every 2nd day until completion of test.
Controls involving radish seeds being planted as normal around the Biofrag were used to compare potential growth differences.
This whole assay was repeated in a second grow box and growth was allowed to continue for 21 days post seeding.

Seed Growth in Varying Perchlorate (ClO4¯) Concentrations.

Soil Preparation

Oven tray filled with soil approximately 15mm in depth.
Oven pre heated to 130ºC and tray inserted on middle shelf for 60mins.
Tray was removed and allowed to cool before soil transferred to blender and blended at high speed for 60 seconds.

Falcon tube filling and Autoclaving

Five Falcon tubes were found to be 12.5g (mean) when weighed.
Each tube was filled with 8g of prepared soil (approx 25mL).
All weighing performed on laboratory scales (total weight:20.5g) and this was repeated for all 24 tubes needed for testing.
All tubes had the cap loosely screwed on and were placed inside an autoclaving bag and autoclaved for 15mins at 121°C and 15psi.
Once autoclaved, bag was placed into a biosafety cabinet before all tubes had their caps tightened to prevent potential contamination.

ClO4¯ Concentration	RODI	DMM
1%	3	3
0.5%	3	3
0.25%	3	3
0% (control)	3	3
Total tubes	12	12

Table 1: Falcon Tube Sample Numbers

Soil: Fluid Ratio (for ClO4¯ testing)

Two of the filled falcon tubes were set aside and 5mL of water was added to the first, the tube was vortexed for 20 seconds and inspected for soil/moisture homogenisation.
This was repeated until desired consistency was produced (20mL water).
The second Falcon tube had 20mL of water added in one go, was vortexed for 20 seconds, and inspected for soil/water homogenisation (same result).

Davis Minimal Media Broth (DMM) Preparation

2.9g of DMM powder was weighed out on a plastic weigh boat and transferred into a 500mL conical flask. 250mL of RODI was added via a volumetric flask and then placed into a microwave. The media was boiled for 1 minute and allowed to cool before use.

Soil ClO4¯ Concentrations

There was 60mL of each perchlorate concentration (1%, 0.5%, and 0.25%) needed to be made up in RODI and DMM.

The 1% ClO4¯ Soil Concentration needed 0.08g ClO4 ̄/8g soil.
20mL fluid (RODI or DMM) x 3 tubes = 60mL.
0.08g ClO4¯ x 3 tubes = 0.24g ClO4¯ .
0.24g ClO4¯ + 60mL DMM = 1% ClO4¯ solution.
0.5% solution = 0.12g ClO4¯ /60mL DMM.
0.25% solution = 0.06g ClO4¯ /60mL DMM.
All concentrations were duplicated in RODI.

RODI= Reverse Osmosis Deionised Water

ClO4¯ Concentration	ClO4¯ (g)	RODI (mL)	DMM (mL)
1% ClO4¯ (DMM)	0.24	N/A	60
0.5% ClO4¯ (DMM)	0.12	N/A	60
0.25% ClO4¯ (DMM)	0.06	N/A	60
1% ClO4¯ (RODI)	0.24	60	N/A
0.5% ClO4¯ (RODI)	0.12	60	N/A
0.25% ClO4¯ (RODI)	0.06	60	N/A
Ctrl: 0% ClO4¯ (DMM)	0	N/A	60
Ctrl: 0% ClO4¯ (RODI)	0	60	N/A

Table 2: Medium Perchlorate concentrations for soil testing

Falcon tube seed planting

Each tube was labelled with the date, ClO4¯ concentration, and either RODI or DMM. Using a pipette, 20mL of each liquid medium was transferred into its corresponding tube. The tube was vortexed for 20 seconds ensuring complete homogenisation of perchlorate and soil. Using a disposable loop, a small furrow was made into the soil and one seed was inserted at a depth of approximately 15mm. The cap was reattached, and this was repeated for each tube. Tubes were placed upright into a grow box and allowed to grow for 10 days.

Team:MTU-CORK/Experiments