Contents 1 pH-stat: Controlling pH through photons 1.1 Objectives of the culture 1.2 Growing cyanobacteria under laboratory conditions 1.2.1 Study of growth parameters 1.3 The temporal evolution of the pH in the culture 1.4 References

pH-stat: Controlling pH through photons

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.

Why is this important?

Well, we wanted to use pH as a switch for the action of 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, absorbency 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 absorbency value after a spectrophotometer sweep and the second was taken according to references (Burrows, EH, et al., 2009 & JF Allen, 2008). We distinguished between simple cells, i.e. those which are not in the reproductive period and those which are.

Cultures C1, C2, C4 and C5 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