Contents 1 pH-stat: Culture of Synechocystis sp. PCC 6803 1.1 Objectives of the culture 1.2 Establishing the culture under laboratory conditions. 1.2.1 Results and discussion 1.3 The temporal evolution of the pH in the culture 1.4 References

pH-stat: Culture of Synechocystis sp. PCC 6803

Objectives of the culture

General

By introducing a culture of Synechocystis sp cianobaceria. 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, so that in the artificial system created, as time passes, the colicins produced by Escherichia coli will increase their concentration in their habitat, being inactive until the pH reaches the optimum range of activation, which is when they ´ll get activated and will produce cell lysis and kill the pathogens.

Specific

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.

Establishing the culture under laboratory conditions.

The material and methods needed are :

• 18 Watts 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.

• Volumetric flasks, 100 ml and 200 ml

• Air Pumps

• Commercial Fertilizer Brand COMPO

• Distilled, tap and analytical water (Type II)

In addition, our cultures, to make low-cost bioreactor were performed in a fed-batch type, in which the addition of nutrientres occurs periodically, so that the objetive of this is to maintain its exponential growth phase without reaching the limit maximum load.

The first experiment consisted of a series of six cultures in which environmental conditions varied so as to know the habitat preferences of the cyanobacterium. We distinguished two different groups:

1. The first, consisting of 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.

1. The second group , which are C4, C5 and C6, without reflector or magnetic stirrer, but we kept the water in the same order as before.

Results and discussion

The other conditions remained constant in both cases, as are detailed in the following table:

The day after having inoculated the culture, the absorbency data were collected and the cells / ml counted with a Neubauer chamber. The absorbancy was measured at 440 nm and 750 nm as the first was the highest value according to the spectrophotometer after a sweep, and the second was taken according to references (Burrows, EH, et. Al., 2009, JF Allen, 2008). In the account we distinguished between simple cells, ie those which are not in the reproductive period, and those which are.

Cultures is seen as 1, 2, 4 and 5, tend to decrease in chlorophyll content, an indicator of the concentration of the substance diminishes over time in our culture, ie, they tend to die out. In contrast, cultures 3 and 6, not only survive, but also increases its concentration according to the evidence given by the spectrophotometer, showing a growth in them.

The following two graphs show the number of cells Synechocystis sp., counted with the Neubauer chamber and a microscope. The first picture shows the cells in the reproductive or Siamese, which, as noted, tend to increase in number while they are not reproducing tend to decrease, as shown in the second graph.

As can be seen, except for crops 3 and 6, all others aren´t in optimal conditions for growth, so they tend to stay rather than to grow.

After this experiment we concluded after observing the differences between them, we got rid of the remains and assemble new crops with the intention of taking them to higher concentrations, we got rid of the remains and started a new culture. This time we chose the least contaminated water whith better growth, analytical water, to which we added a higher concentration of fertilizer to stimulate growth. Besides ,we decided to stop using the reflector box as we had evidence that such a bright light caused photo-inhibition. The volumes and other conditions kept constant.

Synechocistys was observed to be viable/ feasible under laboratory conditions. The problem we found is that it is very easy for the culture to get contaminated , appearing unwanted cells pretty soon.

The temporal evolution of the pH in the culture

Experiments were carried out to verify the temporal variation of pH with the latest cultures we had established, those with the greatest concentration, and in which we could observe with the microscope that most cells were in the reproductive status. A new 4h/4h photoperiod culture imposed and we measured the pH 12 times, every hour. What we obtained was this:

In our culture, the total variations of pH were lower than expected ;a fact that might be due to the fact that the total concentration of Synechocystis in our culture was below the concentration obtained in another habitat, such as the BG11, recommended habitat for the growth of cyanobacteria (Portilla, A. et. al., 2009).

Thanks to a group of we knew that the variations in pH can increase more than 2 degrees between the dark and the light phase (Paula Tamagnini, fom IBMC, Porto. Personal comunication. Http://www.ibmc.up.pt/index. php? id = 447 # IBMC). These variations may be sufficient to activate or inactivate the colicins, and be used as a switch for our project. The results obtained by Paula Tamagnini, the IBMC, are shown below:

The culture show the results of which were in continuous culture, 6h/6h The photoperiod is used.

The pH values ​​in a measurement taken continuously for 295 hours, then observe the changes produced both between phases and the dark lighting and the general trend of increasing pH to more basic pH's. Between day and night variations is seen as reaching beyond two degrees, one strong enough variation to make it serve as a switch.

The overall trend is growing to the basicity, but tend to stabilize the pH variations between day and night with the passage of 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 rre15Photosynthesis. 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