There is truly no such thing as 'totally oxygen free' i.e. there is always some (trace) amount of dissolved oxygen. As Ramesh noted, you can flush the solution with N2 or argon. I would suggest bubbling with argon because argon is more dense than atmosphere and will therefore lower the rate of O2 diffusion from atmosphere. I suggest bubbling through your solution, decreasing surface area to volume ratio as much as possible, and stirring vigorously. You'll need a rubber septum that can vent excess gas throughout the degassing (a needle works for us), and prevent O2 diffusion. Make sure to remove the needle before stopping the degassing, and you probably want a slight excess of argon in your head space so there is positive pressure in your reaction vessel.
Following this question. Should be possible to keep a low concentration of Dissolved Oxygen in an experimental tank (with bivalves) by adding CO2 and O2 at the same time? It seems quite hard to keep low levels of DO while individuals are breathing in a fluctuating way (sometimes individuals stop breathing even several hours).
If someone has already tested something similar i would appreciate advices about the methodology.
Dear Sergio, may be you can purge (O2) gas into the solution at certain flow rate with maintaining certain temperature, but along with CO2 I don't have any idea !
I would flush the water column with N2 (in a water tank with a small surface-to-volume ratio, ideally covered) and counter-balance the pH changes in the water column with a controlled flow of CO2.
Sodium sulfite is primarily used in water treatment as an oxygen scavenger agent, to treat water being fed to steam boilers to avoid corrosion problems
I have a similar problem in my anaerobic fermentations (glucose fermentation by C.beijerinckii). I flush the fermenter with N2 (0.5 l/min) entire time and stir.
I prepare my anaerobic media accordingly so that there is no dissolved oxygen in the begining of the fermentation. However, during exponential growth phase, dissolved oxygen level increases drastically, up to 60%. The problem is not the probe nor the equipment. I observed the same pattern in different setups (Aplikon fermenters and microreactor fermentations).
Does anyone experience a similar thing?
You suggest a reducing agent such as the sulfides. Is there any paper about the effect of the reducing agent on the fermentation?
Hi Cansu - I don't know if this is useful for you, but in the context of in vitro fermentation assays with rumen microbiota, there has been some research suggesting these reducing agents consisting of sulphides may impair microbial growth of certain fermenters (Fukushima et al., 2003) so Mould et al. (2005) recommend using cysteine HCl as a reducing agent instead.
Papers referenced:
Fukushima, R.S., Weimer, P.J., Kunz, D.A., 2003. Use of photocatalytic reduction to hasten preparation of culture media for saccharolytic Clostridium species. Bras. J. Microbiol. 34, 22–26.
Mould, F.L., Morgan, R., Kliem, K.E. and Krystallidou, E., 2005. A review and simplification of the in vitro incubation medium. Animal Feed Science and Technology, 123, pp.155-172
Robert Johannes Meier , I think is a wonderful idea, but how much I need to add, do you know, or could you tell me where I can find that information???
Oleksandr Goncharov Do you have a formula or method for calculating the dosing of sodium metabisulfite to water for the removal of dissolved oxygen. I'm trying to figure a ratio for a hydrogeochemical pilot test, and want to make sure I doing it correctly (based on temperature, in particular). Most of the methods I've found only provide information for boiler environments (involving hot water), which is the opposite of what I need. Thanks!
Dear Samantha Smith, you have to add an excess of sodium metabisulfite (pyrosulfite). This reaction is comparatively slow (for room temperature water) and depends on quantity of reagent. You can increase the reaction velocity by stirring of the solution. Anyway, to completely remove the oxygen you have to use at least 0.4-0.5 ml of 33% solution of metabisulfite per 1 liter of water. But it will take about 17 minutes for non-catalized solution. For more fast reaction you can use more pyrosulfite. I did such experiments. You can read more using the following link: https://www.researchgate.net/publication/334671693
You can use the freeze /thaw method to remove dissolved oxygen from a solution. This is the best way to get rid of oxygen. First you freeze your reaction mixture using liquid nitrogen, then pull vacuum for few min while it is frozen. At this temperature your mixture will be a solid and your gasses are still in the gaseous form. By pulling vacuum all the gases can be removed. Then you have to thaw the reaction to room temperature. do the same thing for two more times for accurate results.