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Bioluminescence

#EXPERIMENTS

ALGAE

Pyrocystic fusiformis is a marine dinoflagellate deriving their energy from the sun through photosynthesis.

P. fusiformis has a full life cycle of approximately 5–7 days and reproduces asexually. The reproduction phase creates 1 or 2 zoospores which grow inside of the parent’s cell wall until they become new cells.

LIGHT

P. fusiformis will only photosynthesize during daylight hours and mostly produce bioluminescence during night because of their circadian rhythm which controls both processes.

My research showed that you could simulate natural light using Cool White (4000-5000 K) fluorescent lightning set on a 12h cycles light / dark. They can be adjusted to new time regimes, but they will take about a week to adjust.

WATER / SEAWATER

P. fusiformis is a marine dinoflagellate living in seawater. To produce homemade seawater I used “Instant Ocean Salt” being told that this was the best to use in saltwater aquariums. Using 33 g / pr liter of water and a hydrometer to ensure gravity.

I decided to use destilled water from the pharmacy instead of tap water because of the use of chlorine in Oslo waters.

MICRO ALGAE GROWTH

Based on Guillard F2 fertilizer formula. This formula has been used in the aquaculture and scientific community for culturing a wide spectrum of unicellular microalgae for over 30 years.

Its possible to produce this formula yourself, but for me it was simpler to buy it ready made. Add to mixed 1 liter salt water 0,5 ml Micro Algae Growth.

STARTER CULTURE

Started the project with 1 x 250″Pyrocystis fusiformis” algae culture and “Photobacterium (Vibrio) phosphoreum” from Sciento UK.

They were hesitant of sending this to a private person without lab facilities, so I explained them I worked for the University of Oslo.

EQUIPTMENT

STARTER CULTURE 1 – SUBCULTURE

Florida Aqua farms used several weeks to process my order, I cancelled it and bought from ebay instead, but they send my order after 1 month anyway. This ment that it took 3 weeks after receiving the algae culture before I could subculture it. During this time the algae was living under natural light cycle – ca 12 h/12h. As time went the culture was decreasing in light intensity.

I subcultured ca 200 ml of Algae to 50 ml divided on 4 Erlend Meyers flasks in which I added:

  • 2x 200 ml of Destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
  • 2x 200 ml of Tap water + Instant Ocean Salt + 0,1 ml Micro Algae Growth

I set up the growth chamber in my living room first under the 45W light bulb, and then after activity decreased 30W light bulb.

RESULTS

As time went the algae decreased in both number and light intensity, before being assumed as “dead”. I was sure the experiment had failed due to any of the following causes, or a combination of them:

  • Poor home sterilization of flasks (200 degree in oven / 1 h)
  • The lights emitted to much heat
  • The temperature variations were too high
  • My living room was too cold
  • Wrong mixture and measurements
  • Waited too long for subculturing

16.05.2015. STARTER CULTURE no.2 – SUBCULTURE no.1

I ordered 2 more starter cultures – together with “Saltwater medium” from Sciento. Sciento warned me that saltwater medium was only for short time culturing – but I wanted to compare the 2 methods to see if I had failed to find a sufficiant medium mixture.

I set up 2 growth chambers to see if it had anything to do with the different strength light bulbs – and I moved the whole setup to the bathroom to have a warmer and more consistent temperature.

LARGE CHAMBER

  • 1. ca 200 ml algae + ca 800 ml of destilled water + Instant Ocean Salt + 0,5 ml Micro Algae Growth (Based on starter culture no.1)
    LOW/MEDIUM ACTIVITY
  • 2. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 3. 20 ml algae + 300 ml of Imsdal bottled water + Instant Ocean Salt + 0,15 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 4. 10 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,075 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 5. 50 ml algae + 500 ml of destilled water + Sciento Salt + (31.05.15 – 0,075 ml Micro Algae Growth)
    NO ACTIVITY
  • 6. 10 ml algae + 150 ml of destilled water + Sciento Salt + (31.05.15 – 0,025 ml Micro Algae Growth)
    NO ACTIVITY

SMALL CHAMBER

  • 7. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 8. 25 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 9. 15 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 10. 10 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,075 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 11. 5 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth
    LOW/MEDIUM ACTIVITY
  • 12. 5 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth
    LOW/MEDIUM ACTIVITY
  • 13. 50 ml algae + 200 ml of Imsdal bottled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 14. 40 ml alge + 500 ml of destilled water + Sciento Salt
    NO ACTIVITY
  • 15. 40 ml alge + 500 ml of destilled water + Sciento Salt (31.05.15 – 0,25 ml Micro Algae Growth)
    NO ACTIVITY
  • 16. 10 ml alge + 150 ml of destilled water + Sciento Salt (31.05.15 – 0,075 ml Micro Algae Growth)
    NO ACTIVITY
  • 17. 10 ml alge + 150 ml of destilled water + Sciento Salt
    NO ACTIVITY
  • 18. 5 ml alge + 90 ml of destilled water + Sciento Salt
    NO ACTIVITY

RESULTS

The second experiment tested previous theories, and proved the following.

  • Home sterilisation method proved successful
  • Lights did not emit too much heat
  • Temperature were too low / or variations to great in my living room
  • Measurements and mixture proved successful
  • Likely I waited too long to subculture
  • Sciento Salt proved to be unsuitable for long time culturing, as predicted

13.06.2015. STARTER CULTURE no.2 – SUBCULTURE no.2

Subcultured the surviving algae from previous experiment based on the Instant Ocean Salt + Micro Algae Grow mixture. Continued to use 2 chambers for growing – but changed the larger chamber with a larger box. The result of this was that the 40W bulb were closer to the bottles, than in the previous arrangement.

I choose not to subculture some of the algae to see how they would develop over time. I found recommendations for subculturing every 3 weeks – because of the amount of dead algae – and wanted to trial if this was the case.

I kept the Sciento Salt Mix for a while after, but this never came back to life.

LARGE CHAMBER

1. Subcultured Algae Mix 1 –
(ca 200 ml algae + ca 800 ml of destilled water + Instant Ocean Salt + 0,5 ml Micro Algae Growth (Based on starter culture no.1))

  • 1a. 100 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    NO ACTIVITY
  • 1b. 900 ml algae + 1100 ml of destilled water + Instant Ocean Salt + 0,85 ml Micro Algae Growth
    NO ACTIVITY

2. Subcultured Algae Mix 2 –
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 2a. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 2b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 2c. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 2d. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 2e. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    NO ACTIVITY

3. Subcultured Algae Mix 3
(20 ml algae + 300 ml of Imsdal bottled water + Instant Ocean Salt + 0,15 ml Micro Algae)

  • 3a. 320 ml algae + 1200 ml of Imsdal bottled water + Instant Ocean Salt + 0,75 ml Micro Algae Growth
    LOW/MEDIUM ACTIVITY

4. Subcultured Algae Mix 4
(10 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,075 ml Micro Algae Growth)

  • 150 ml algae + 600 ml of destilled water + Instant Ocean Salt + 0,37 ml Micro AlgaeGrowth
    NO ACTIVITY

SMALL CHAMBER

7. Subcultured Algae Mix 7 –
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 7a. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 7b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 7c. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 7d. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY
  • 7e. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM/HIGH ACTIVITY

8. Left Algae Mix 7 – to see further development
(25 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth)
MEDIUM/HIGH ACTIVITY

9. Left Algae Mix 9 – to see further development
(15 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth)
MEDIUM/HIGH ACTIVITY

10. Subcultured Algae Mix 10 –
(10 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,075 ml Micro Algae Growth)

  • 10a. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    MEDIUM/HIGH ACTIVITY
  • 10b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    NO/LOW ACTIVITY
  • 10c. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    NO ACTIVITY

11. Left Algae Mix 11 – to see further development
(5 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth)
MEDIUM/HIGH ACTIVITY

12. Left Algae Mix 12 – to see further development
(5 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth)
MEDIUM/HIGH ACTIVITY

13. Subcultured Algae Mix 13 –
(50 ml algae + 200 ml of Imsdal bottled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 13a. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    MEDIUM/HIGH ACTIVITY
  • 13b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    NO/LOW ACTIVITY
  • 13c. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (small bottle)
    NO ACTIVITY

RESULTS

It seems as if the success rate of the big chamber is reduced – this could be because the 40W light bulb is closer to the bottles in the new chamber and cause temperature change. Bottles further away from the light source shows better survival rate.

Furthermore it seems like the survival rate is reduced based on how full the bottles are. Bottles with less liquid shows a better survival rate than those filled up. This could be because of less surface area for oxygen in the bottles.

Another reason could be that the culture have been contaminated from the air. I have kept cotton pads as “stoppers”, but they sometimes fall off.

19.07.2015. STARTER CULTURE no.2 – SUBCULTURE no.3

Subcultured the surviving algae from previous experiment, continued to use 2 chambers for growing. Made sure to put bottles further away from the 40W light bulb based on the previous experiment.

I also made sure not to fill the bottles, but leave more than half filled with air. The last option left me with a spatial problem, since now both chambers have reached the maximum amount of bottles – and I need to find a new solution for this.

Made sure the cotton pad stoppers would not fall off.

LARGE CHAMBER

1. Subcultured Algae Mix 2a
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth

  • 2aa. 250 ml algae + 1000 ml of destilled water + Instant Ocean Salt + 0,5 ml Micro Algae Growth (Rund stor flaske)
    BACTERIAL INFECTION

2. Subcultured Algae Mix 2b
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth

  • 2ba. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske A)
    GREEN ALGAE INFECTION
  • 2bb. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske A)
    MEDIUM ACTIVITY
  • 2bc. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske A)
    MEDIUM ACTIVITY
  • 2bd. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske A)
    MEDIUM ACTIVITY

3. Subcultured Algae Mix 2c
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth

  • 2ca. 125 ml algae + 500 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske B)
    GREEN ALGAE INFECTION
  • 2ca. 125 ml algae + 500 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth (Vanlig flaske B)
    GREEN ALGAE INFECTION

4. Left Algae Mix 2d – to see further development
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)
MEDIUM / HIGH ACTIVITY

5. Left Algae Mix 3a – to see further development
(320 ml algae + 1200 ml of Imsdal bottled water + Instant Ocean Salt + 0,75 ml Micro Algae Growth)
BACTERIAL INFECTION

6. Mixed 3 unsuccessful algae mix from previous experiment (SMALL CHAMBER) together
(10b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)
(10c. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)
+ some extra mix from previous tests. Ca 700 ml altogether.
BACTERIAL INFECTION

SMALL CHAMBER

7. Subcultured Algae Mix 7a –

(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 7aa. 250 ml algae + 1000 ml of destilled water + Instant Ocean Salt + 0,5 ml Micro AlgaeGrowth
    MEDIUM ACTIVITY

8. Subcultured Algae Mix 7b – (E)
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 7ba. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 7bb. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 7bc. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 7bd. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 2be. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY

9. Subcultured Algae Mix 7c –
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 2ca. 75 ml algae + 400 ml of destilled water + Instant Ocean Salt + 0,2 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 2cb. 75 ml algae + 400 ml of destilled water + Instant Ocean Salt + 0,2 ml Micro Algae Growth
    GREEN ALGAE INFECTION

10. Subcultured Algae Mix 7d –
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 2da. 125 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,2 ml Micro Algae Growth
    GREEN ALGAE INFECTION
  • 2db. 125 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,2 ml Micro Algae Growth
    MEDIUM ACTIVITY

11. Mixed the successful algae from “small bottles C” – a mix of destilled water and Imsdal bottled water.
(13a. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)
(13b. 50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 13a+b. 500 ml algae
    MEDIUM ACTIVITY

12. Left Algae Mix 7e – to see further development
(5 ml algae + 150 ml of destilled water + Instant Ocean Salt + 0,05 ml Micro Algae Growth)
MEDIUM ACTIVITY

13. Subcultured Algae Mix 10a.
(50 ml algae + 200 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth)

  • 10aa. 200 ml algae + 400 ml of destilled water + Instant Ocean Salt + 0,2 ml Micro Algae Growth
    MEDIUM ACTIVITY
  • 10ab. 100 ml algae + 50 ml of destilled water + Instant Ocean Salt + 0,1 ml Micro Algae Growth
    MEDIUM ACTIVITY

RESULTS

The smaller chamber had a far better result than the bigger one – but still – the algae did not reach density of the “test” flasks that has not been subcultured the previous times, and I couldn’t figure out what was wrong. The info I have found says that they will reach back in density over 3 weeks – and should be subcultured accordingly. But the small flasks have not been subcultured for about 2 months (?), so there is something thats not right about the growth rate.

29.08.2015. MOVING to THE UNIVERSITY

I found help at the Biology dept at University of Oslo – that offered me a far better growing environment for them in their algae lab. Yey! I also learned that some of my flasks have been infected with green algae – as I understood is a quite common problem when culturing algae. Another flask most likely with bacteria / fungus – so there is at least some of my answers in regards to the algae not growing / and dying.

At the university the algae has been placed in a growth chamber that holds a constant temperature of 21 degrees – and is lit with proper lighting. They think that this will help the growth rate and hope to see results next week.

RESULTS

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