Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

The overall goal of this experimental approach is to simulate a ferruginous upwelling system and to detect geochemical profiles of oxygen and aqueous iron in the water column when cyanobacteria were present and produces oxygen by photosynthesis. This newly developed method can help answer key questions in the field of earth's history such as the oxygenation of the atmosphere and the deposition of banded iron formations. The main advantage of this technique is that geochemical conditions still might have existed in a Precambrian ocean can be simulated on lab scale being investigated and seen to with modern methods.

Though this method can provide insight into early earth studies, it can also be applied to other modern environments such as for geo-microbial investigations and chemical fluctuating in sediment bodies. Generally, individuals new to this method will struggle because setting up the experiment is not trivial but the video will provide information on the most critical steps performed in the protocol. Bacteria culturing, early assembly steps of the set up, and notes on sterilizing materials are all described in the text protocol.

Note that the cell solution for Synechococcus 7002 must be anoxic for later experiments. Therefore, remove the soluble oxygen by bubbling the solution with anoxic nitrogen carbon dioxide via a 0.22 micron-filtered syringe, having a second needle in the stopper to release over pressure. In order to stop photosynthesis and the production of oxygen, cover the culture bottle.

After five minutes of bubbling, flush the head space. To assemble the column, place it on a flat surface and stabilize it with a laboratory stand and clamps. Make sure to work under sterile conditions and carefully remove the aluminium foil from the top opening.

Then fill the column with sterilized glass beads. Next, prepare a watch glass to tightly close the column. Apply glue to the inner surface where it will contact the column.

Then lightly press it to the top of the column. Let it set for at least six hours. Proceed with making attachments.

First, attach the head space gas exchange panel to the head space vent. Then insert the appropriate butyl rubber stoppers into the cotton filled glass syringes. Following this, connect the Luer Lock glass syringe to the corresponding stainless steel needle.

Then connect the anoxic gas line to the head space gas exchange panel using another Luer Lock stainless steel needle. After making a few more connections, be sure to connect the medium distribution panel to the butyl stopper for the medium bottle. Flush the column instillation and capillary system at low pressure with anoxic gas.

Let gas escape via the medium bottle capillary, the three way connector, and the sampling ports. Flush gas through the instillation for at least 20 minutes. To connect the stopper to the medium bottle, first close the anoxic gas flow with a hose clamp on the corresponding tube.

Then lightly lift the butyl rubber stopper off the medium bottle. Next, insert a sterile cotton filled syringe with a bent needle into the head space of the medium bottle blowing gas as 50 millibar and flush the head space with anoxic gas. Then quickly remove and replace the rubber stopper with the prepared stopper with the capillary.

This must be performed quickly and with strict attention to sterility to avoid adding oxygen and bacteria. Next, remove the long metal needle to close the lid. Then change the long metal needle of the glass syringe to a disposable needle and inject anoxic gas into the stopper to flush the head space of the medium bottle.

Do this for at least four minutes. Then close the gas line and pull the injection needle out of the stopper. The remaining over pressure of gas will escape via the glass syringe.

To proceed, connect the syringe to a gas pack loaded with anoxic gas while putting light pressure on the gas pack. The gas pack will compensate for the volume loss in the bottle when medium is pumped out of it. Next, connect an empty pre-flushed ten liter gas pack to the free port of the three way connector that is attatched to the discharge bottles.

Make sure to keep the valve of the gas pack closed. Now reduce the pressure of the anoxic gas to below 0.1 millibar to maintain a steady outflow of gas from the out-gassing needle. Then set up the pump and release the clamp on the hose to access the media.

Now open the valve of the empty gas pack and start the pump. The pack will collect air displaced from the discharge bottles as they are filled with medium. Monitor the medium distribution panel filling up with medium and remove the remaining gas from the panel as it fills by holding it in an inverted position.

The gas is released from the capillaries connected to the pump. Adjust the pump to 0.45 liters per day and monitor the column it fills with medium. Lastly, install the light source.

Position it two centimeters above the upper end of the column. Then cover the upper ten centimeters of the column with dark tape or aluminium foil. Then cover the whole instillation with a dark textile.

For the inoculation, have six syringes with needles long enough to reach the center of the column body. Begin with sterilizing the outside of the butyl rubber stoppers on the six main sampling ports. Use 80%ethanol and flaming.

Then flush a syringe with anoxic gas. Next, draw up one milliliter of the anoxic cell solution and inject it into the center of the column through the butyl rubber stoppers on the six sampling ports. If necessary, use sterile tweezers to stabilize the needle for injection.

Every 24 hours, take samples from the column. Start at the top port and work down. First, flush the syringe with anoxic gas.

Then fill it with anoxic gas. Next, quickly remove the plastic cap and put the syringe almost onto the tube connector. Then eject the gas in the syringe and firmly connect the syringe to the connector.

Now remove the clamp and take a one milliliter sample from the column. Reattach the clamp before withdrawing the syringe. Then firmly close the connector with it's cap.

Now repeat this process at the next port going down the column. A ten day control experiment with no inoculated bacteria demonstrated consistently low oxygen concentrations with no significant fluctuations in soluble iron throughout the upwelling water column. At day five, the lowest oxygen levels were below 15 milligrams per liter which is essentially anoxic.

Prior to inoculation on the day zero, no precipitates were visible thus no oxygen was present that could oxidize soluble iron into iron precipitates. The soluble iron concentrations were constant throughout the upwelling water column. 84 hours after inoculation with cyanobacteria, a green color developed within the top two centimeters of the column indicative of growth.

An orange band at negative three centimeters was indicative of precipitating iron due to oxygen production by the cyanobacteria. While prior to inoculation the initial oxygen concentration was essentially zero, after 84 hours of growth, the oxygen profile of the upper column rose significantly. Conversely, a drop in soluble iron from the upper depths was also measured.

After watching this video, you should have a good understanding of how to set up and perform the experiment. The collection of liquid samples will allow you both to quantify geochemical parameters and perform cell analysis. Once mastered, the experiment can be set up within one day.

After starting the run, changes in the water column can be monitored over a period of several weeks if it is performed properly. Following in this procedure, alternative set ups can be used to answer additional questions regarding insidious reductions and microbial distributions within sediment bodies.