Team:Valencia/Project2

As you can see on the following figure, we have effectively accomplished the control of pH through the autotrophic growth of Synechocystis sp. PCC 6803

. Thus opening the door to the construction of the CopH, the controller of pH. Why is this important?

Well, we wanted to use pH as a switch for the action of li bacteriocins.

How have we done this?

Short answer: cyanobacteria growing only on light, CO₂, commercial fertilizer and H₂O

Long answer: keep reading!

Objectives of the culture

By introducing a culture of cyanobaceria Synechocystis sp. PCC 6803 we intent to ensure that the pH changes resulting from growth and proliferation function as a switch to enable the denaturation of the colicins. The objective is that, as time goes by, the colicins produced by Escherichia coli will be produced but inactive until the pH reaches the optimum range of activation, thus getting activated and producing cell lysis and killing pathogens.

In order to develop what we have stated above, we need to know:

• How to establish the culture at the laboratory
• The temporal evolution of the pH in the culture

Growing cyanobacteria under laboratory conditions

The materials used were:

• 18W fluorescent tubes of white light, special tubes for aquariums that divide the spectrum mostly between the peaks of the visible red and blue light, which stimulate the photosynthesis.
• 100 ml and 200 ml flasks
• Air Pumps
• Commercial COMPO Fertilizer
• Distilled, tap and Type II analytical water
• Cardboard boxes
• Household aluminium foil

We decided to perform fed-batch growth, in which the addition of nutrientres occurs periodically, so that we could maintain cyanobacterial exponential growth phase as long as possible without reaching the maximum load limit.
We designed a low-cost photobioreactor using our lamps, cardboard boxes and aluminum foil in ordert to build a reflector box in which we made all our measurements

Study of growth parameters

Our first experiment consisted of a growth variables study so as to know the habitat preferences of the cyanobacterium. We made six cultures with different media conditions and we looked at its effects on growth. We distinguished two different groups:

1. First group, consisting of cultures C1, C2 and C3, which have tap, distilled and analytical water, respectively. It also uses a reflector box, which increases the irradiation of light on the culture, and a magnetic stirrer, which prevents the deposition of cells on the bottom.

2. The second one, made up of cultures C4, C5 and C6, without reflector box or magnetic stirrer.

The day after having inoculated the culture, absorbancy data were collected and cell concentration measured on a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest absorbancy value after a spectrophotometer sweep and the second was taken according to references (Burrows, EH, et al., 2009 & JF Allen, 2008).

Cultures C1, C2, C4 and C5 tend to decrease in chlorophyll content, an indicator of the concentration of cells diminishes over time, i.e., they are dying. In contrast, cultures C3 and C6, not only survive, but also increase their concentration, thus they grow.

We distinguished between simple cells, i.e. those which are in the reproductive period and those which are not. The following two graphs show the number of growing and non-growing Synechocystis cells, counted with the Neubauer chamber and a microscope. You can see

The first graph shows the cells in the reproductive or Siamese state, which, as noted, tend to increase in number while the ones non-growing tend to decrease, as shown in the second graph.

As can be seen, cultures 3 and 6 grow pretty well, all other cultures are not under optimal conditions.

From that we concluded that major factors for cyanobacterial growth were type of water and the reflector box. With that on mind, we chose to grow cells under Type II analytical water, to which we added a higher concentration of fertilizer to stimulate growth. Besides, we decided to drop the use of the reflector box as we had evidences that s much bright light might cause photo-inhibition. The volumes and other conditions were the same as Table above.

We accomplished to grow Synechocystis sp. PCC6803 on a low-cost photobioreactor under laboratory conditions. The major problem we found is that this culture gets easily contaminated as it does not use antibiotics.

Temporal evolution of the pH in the culture

Experiments were carried out to verify the temporal variation of pH in growing cultures. A 4h/4h photoperiod culture imposed and we measured the pH 12 times, every hour. Resulting in this:

This figure depicts a variation of up to a unit of pH. This might not be sufficient for colicin denaturation, but it's still significant, taking into account the low-cost growth device, the suboptimal growth media, the fed-batch growth mode and our inexperienced hands. We are confident that growing Synechocystis in chemostat mode and with BG11 growth medium (far better for cyanobacterial growth, Portilla, A. et. al., 2009) we could have had much better results.

In fact, partner groups in Porto and Sheffield have accomplished, in a very similar experimental design, but in continuous cultures and 6h/6h photoperiod, to shift pH to up to three units, from pH 8 to pH 11 and back (Prof. Paula Tamagnini, fom IBMC, Porto and Prof. Phillip Wright, Sheffield University, personal communication). These variations may be sufficient to activate and inactivate bacteriocins, allowing to use pH as a switch. The results they obtained are shown here:

The overall trend is towards basicity, stabilizing pH variations among day and night with time.

References

Burrows, E. H., et. al., 2009. Optimization of pH and Nitrogen for Enhanced Hydrogen Production by Synechocystis sp. PCC 6803 via Statistical and Machine Learning Methods. Wiley InterScience. 25: 1009-1018

Allen J.F., et. al., 2008. Evaluation of Acid Stress Tolerance in Synechocystis sp. PCC 6803 Mutants Lacking Signal Transduction-Related Genes sigB, sigD, and rre15. Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1519–1522.

Portilla, A. et. al., 2009. Evaluación del rendimiento de producción de aceite en cuatro microalgas nativas de las provincias ecuatorianas de Orellana, Esmeraldas, Imbabura y Pichincha. http://www3.espe.edu.ec:8700/bitstream/21000/427/1/T-ESPE-029605.pdf