Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen. As oxygen is ubiquitous in air, retaining oxygen-free conditions during cultivation is a pre-requisite for anaerobic culturing. The applied technique represents a simple and inexpensive method to provide anoxic conditions to cultivate a mixed microbial population in liquid culture.

This technique can also be applied for the cultivation of pure cultures. However, for highly oxygen sensitive microorganisms, this procedure might not be applicable. Start by weighing medium ingredients in an appropriate flask, and dissolving them with half volume of distilled water.

Then, add milliliter of REDOX indicator, followed by vitamins in trace element solution. Adjust the pH according to organism requirements, and bring the final volume to 1 liter with distilled water. The color of REDOX indicator is pH dependent, and might require some time to adjust.

Fill 120 milliliter serum flasks with 50 milliliter of medium, then incubate them in a 100 degree Celsius water bath for 20 to 30 minutes, to reduce the solubility of the oxygen in the liquid phase. Flush the head space with nitrogen. Take care of appropriate room ventilation when working with nitrogen.

Close the flasks with butyl rubber septa, and fix the aluminum caps. Next, add 0.1 milliliters of reducing agent to each flask, to further reduce the REDOX potential. And autoclave the flasks for 20 minutes at 121 degrees Celsius.

An autoclave certified for the sterilization of closed vessels has to be used when applying this protocol. Otherwise, overpressure derived from temperature increase might cause serum flasks to explode. To prepare inoculum from anaerobic digester, add 400 milliliters of distilled water to a flask and bring it to boil.

Cool it down to approximately 30 degrees Celsius while permanently flushing the head space with nitrogen. Add approximately 100 grams of sludge, derived from an anaerobic digester, making sure to records the exact mass of the sludge for determination of dilution. Exchange the flask's head space with nitrogen, and close it with a butyl rubber septum.

Shake the flask for 30 minutes at 120 RPM. Let larger sludge particles sediment, then use a syringe and canula to remove 5 milliliters of inoculum and inject it into a previously prepared serum flask. Incubate the inoculated serum flask at an appropriate temperature for the experiment, making sure to drain the over pressure caused by the temperature increase, approximately 15 to 30 minutes after beginning incubation.

To evaluate bio gas production and composition during incubation time, record the current atmospheric pressure and prepare a manometer to evaluate the pressure within the flask. Shake the flasks and remove 1 milliliter of head space gas using the syringe and canula. Measure the hydrogen, oxygen, methane, and carbon dioxide concentration using gas chromatography.

Use 1 milliliter of liquid from the flask to monitor the concentrations of all tall fatty acids, according to manuscript directions, and drain the flasks over pressure after returning it to the appropriate temperature. This protocol was used to create miniature bioreactors conducting anaerobic digestions. Tryptophan, tyrosine, phenylalanine, meat extract, and casein were added at 3 different concentrations, and methane production was monitored over a 28 day incubation period.

Methanogens were visualized by radiating the co-enzyme F420, which is an electron carrier in methanogenesis, and exhibits a blue-green fluorescence with an absorption maximum at 420 nanometers. Concurrent with gas analysis, samples were analyzed for volatile fatty acid, and phenyl acid concentrations during the incubation period. Molecular biological methods targeting the abundance of specific microorganisms and/or composition of the microbial community, at a certain point of the experiment, can be applied using the described procedure.