Franck Porteous @FranckPrts
Julien Pichon @Ju_Pichon
Julie Le Bot @FdvJulie
Halobacterium salinarum and osmotic pressure.
Hello everyone,
we are “Bacteriano”, and we are fascinated by little organisms called Halobacterium salinarum. This specie of Archaea lives in very salty environment and it is this parameter that we want to study. Variation of environment salinity is a criteria of osmotic pressure. And to quantify the variation of salt in a media, and so variations of osmotic pressure, conductivity is a good tool.
We dream about a world where H. salinarum can give us the conductivity of a media. Maybe, we will show that this organism is more precise than a electronic sensor like a ohmmeter. So to sum up, is a biological sensor as an Archaea could give more precise information about a media than a electronic sensor ?
What is osmotic pressure?
Osmosis is a physical principle that occurs in a liquid at two different concentrations. To understand it, let say we have two media with two different concentrations of ions separate by a semi-permeable membrane.
The water can pass through the membrane but not the ions.
Due to Brownian movement, water diffuses towards the highest concentration of ions compartment, until both compartments have the same electrical charge.
If cells are in a water with not enough or too much ions, they lose or win water and die.
However, there is a mechanism called osmotic pressure that allows cells to survive. It prevents the water to move in or out cells.
Halobacterium sounds like bacterium
No! Halobacterium is an Archaea! It is an extremophile; this category of organism lives in, as it sounds, “extreme” environments, (high or low temperature, acidic or basic water... ). H.salinarum live in salted water like the Dead See.
Their characteristics are very
interessant to experiment conductivity. We can work with more salted media than other microorganism, so compare more higher conductivity.
However, the growth rate of H.salinarum (time until next generation of cell) depends of the osmotic pressure. So we can observe the size of a colony to know the concentration in ions, so the conductivity!
We decide to test different environment, in a range of salt to let our Archaea to grow, and in the same time to record its conductivity with an ohmmeter.
The DIY ohmeter (arduino)
We have our biosensor! Miss the electronic sensor now
To measure the conductivity, we use an Arduino device that we build ourselves.
So we build this wonderful ohmmeter with an Arduino and threads.
The value of conductivity is given on the computer.
We will use it to measure the conductivity of LB media (where H.salinarum lives) with 6 different concentration of salt.
To test if our device records a coherent value, we measure the conductivity on our media with an ohmmeter ( a no DIY one!).
Our DIY ohmmeter seems to saturate faster than the other one.
However, the more salty the more conductive.
The growth of H.salinarum
To measure the growth of H.salinarum, we use a spectrophotometer Tecan Infinity: a spectrophotometer measures the optical density. And, this optical density is in direct relation with the number of H.salinarum.
Indeed, with the results given by the spectrophotometer we have a precise monitoring of Archaea evolution during 24 hours. We incubate our Archaea at 42°C, their ideal temperature, in 6 different concentrations.According to literature, 4.3M (approximatively 250g per liter !) is their ideal concentration to grow.
To be able to read the growth rate, we plot every optical density per concentration (given by the spectrophotometer), and with R (a programming language) we graph the linear regression. The coefficient of linear regression gives us the actual growth rate per concentration. On the graph below, we observe the six concentrations and the linear regression for each one of them.
As you can see on the y scale, the optical density (ABS) is near 0 for every concentration. Indeed, Archaea are supposed to make a colony on 7 days, and we only observe them during 24 hour.
According these results, the concentration that is ideal for Archaea development is 3.9M. We can also notice that, in this experiment, 5.1M concentration slows Archaea growth.
H.salinarum versus DIY ohmeter : Conclusion
To conclude, in both biological and electronic sensor, salt, so osmotic pressure has an influence: for the Arduino, the conductivity is higher and for Archaea, the growth rate is not the same. However, there is a linear correlation between salt concentration and conductivity, but there is not with Archaea growth.
So if you want to test salt concentration of medium, and know if it is near 4M, use Archaea! If they grow, here you go. But if you want a more precise measure, we advise you to take a ohmmeter :)
If you want to know more, take a look on our PowerPoint presentation, for more graphs and schema of our experiment.
And if you want to see more precisely our data, Arduino and R codes, find all our resources on line, on GitHub.
And finally, during all our project, we twitted everyday (@Bacteruino) and we made little storify to sum up this intensive week.
Sources :
Leuko, Stefan, Mark J. Raftery, Brendan P. Burns, Malcolm R. Walter, et Brett A. Neilan. « Global Protein-Level Responses of Halobacterium Salinarum NRC-1 to Prolonged Changes in External Sodium Chloride Concentrations ». Journal of Proteome Research 8, no 5 (mai 2009): 2218‑25. doi:10.1021/pr800663c.
« Halohandbook_2008_v7 - Halohandbook_2008_v7.pdf ». Consulté le 25 janvier 2017. http://www.haloarchaea.com/resources/halohandbook/Halohandbook_2008_v7.pdf.
Kish, Adrienne, Patrick L. Griffin, Karyn L. Rogers, Marilyn L. Fogel, Russell J. Hemley, et Andrew Steele. « High-Pressure Tolerance in Halobacterium Salinarum NRC-1 and Other Non-Piezophilic Prokaryotes ». Extremophiles: Life Under Extreme Conditions 16, no 2 (mars 2012): 355‑61. doi:10.1007/s00792-011-0418-8.
