Team:Valencia/Engineering

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Bioreactor

CO2 Pump
br> To enhance a faster growth rate in our Synechococcus broth culture and taking advantage of the bacterium being photosynthetic, we decided to make a device with a CO2 pump in order to make the transformations quickly.

Materials:

• A bottle of water (1.5 litres) + a cap so the bottle remains hermetically sealed.
• PVC tubes.
• Rapid fixation glue.
• 30g of sugar.
• One spoon of yeast (Saccharomyces cerevisae).
• Water.
• Methylene blue.

(FOTO)

Construction:

First of all, we bored holes to the bottle and sealed the edges. Then we cut the tubes so they fit in the holes properly. Fill in one third of the botle approximately with water at 100ºC. Then add up the sugar. After that, shake the bottle so the sugar dissolves in the water. Add the spoon of yeast and shake again.

Fresh yeast is a living organism, a micro fungus able to perform fermentation: in absence of oxygen converts the sugar into alcohol and liberates CO₂.

At last, pour 1 litre of Methylene blue into another bottle, used as a contamination control device, in order to purify the air that got into our culture. And finally we connect the air pumps to keep shaking the coccus.

(FOTO)

The results of our experiment were not the expected, having proved that our cyanobacteria grew up faster if we provided an extra input of CO2,bu the culture didn’t develop well due to contamination problems. We do not know if the yeast caused the problem or the bottle just wasn’t properly sealed.

Advanced continuous coculture system

Our furthest goal is to build a continuous culture system with spatial and temporal decoupling from the photosynthetic and the bioluminescent module, totally autonomous.

Design:

Set 2 separate culture modules, a flat wide one for S. elongatus cscB as a solar module and a smaller compact one for A. fischeri as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO₂ sink), but with a Pasteurian design opening to avoid contamination from deposition.

Set tubing connecting both cultures, protecting each end with a syringe filter 0.45 microns of pore size. The fluid medium dynamics is borne by a pair of peristaltic reversible-flow pumps, which switch flow direction every 30-60 seconds to avoid collapsing any side of the membranes with jammed cells. The membranes allow the exchange of gases, ions, water, sucrose and AHL, but not cells, so that populations do not mix. This is fundamental to guarantee the light-efficiency of each compartment, without having cyanobacteria shading light emission from A. fischeri, and permitting a spatial decoupling of the photosynthetic module and the biolamp.

Cell density would be regulated by output tubes from each culture which would recycle broth after passing it through a filter/skimmer. This process would be regulated by a turbidimeter (in theory) or by sampling-and-testing, at least in the first experimental prototype.

Water loss due to evaporation would be solved by a water-level-wise lever opening a valve which would let in some distilled water from a small deposit, when the volume of the liquid falls below certain threshold.

A microcontroller would be used as hardware to coordinate pump flow switch, input signals from turbidimeters and control of the cell density control system.

All electric devices would be powered by a small solar panel, to preserve to the last detail the energetic autonomy of the system.

Routine analyses of the system would be carried out on sucrose, oxygen and AHL levels in the liquid medium. Luminescence would be measured at night to acquire an experimental Light/Time curve.

[Future design development: Achieve a knock-out/directed mutagenesis of the luxI autoinducer genes in A. fischeri to unable isolated autoinduction. This would render the system totally dependent on the AHL production of the transformed S. elongatus. In such situation, turning valves of different membrane pore size at the cell-stopping sections would result extremely useful, to manually switch on/off the diffusion of AHL from the Synechococcus culture to the biolamp. This enables an element of human control to turn on/off the lights over the normal day/night fashion].

Figure a: Advanced continuous cocultive device, showing in both culture compartments
with optical cell density monitors (green diodes), reversible-flow pumps at th sides,
bacteriological (Fb) and activated carbon (Fc) filtering and medium recycle system
at the middle. Evaporation loss automatic replenisher at the top (H₂O).

Results:

Construction of inter-flask pumping system with 0.45micron pore membranes.