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

1. Preparation of Culturing Medium

Note: Information on the required equipment, chemicals and supplies for the preparation of the culture medium is listed in Table 1. Italic alphanumerical codes in brackets refer to the equipment itemized in Table 2 and shown in Figure 1.

1. Prepare 5 L of Marine Phototroph (MP) medium (referred to hereafter as "medium") following the protocol of Wu et al.¹⁶. Adjust the pH to 6.8 using anoxic and sterile 1 M HCl or 0.5 M NaCO₃. As a source for Fe(II), add 3.5 ml of a 1 M anoxic and sterile FeCl₂-solution to achieve a final Fe(II) concentration of 500 µM after filtering the medium solution in the next step.
2. Store the medium solution at 5 °C for 48 hr in order to precipitate Fe(II) carbonate and phosphate minerals. Fe(II) addition and Fe mineral precipitation results in a pH change, therefore readjust the pH back to 6.8. Filter the medium in an anoxic (100% N₂) glovebox through a 0.22 µm filter unit. Dispense the filtered medium into a sterile 5 L glass bottle (E.'1) inside a glovebox, and cap with a sterile butyl rubber stopper.
3. Flush the headspace of the medium bottle with N₂/CO₂(v/v, 90/10) by inserting one disposable needle connected to the gas line into the stopper, and a second needle that acts as a vent. Make sure to change the headspace volume 10 times. For example, with a constant gas flow of 10 ml/sec, flush the headspace volume of 50 ml for at least 50 sec (compare Hungate & Macy¹⁷).
4. Cover the medium bottle (E) that is now ready to use with aluminum foil and store at RT under dark conditions to prevent photooxidation of Fe(II). Allow 3 days for medium preparation.

2. Preparation of the Culture

Note: The culture of Synechococcus sp. PCC 7002 that is used in the column experiment is described as unicellular marine photoheterotrophic cyanobacteria genus¹⁸. It was provided by Dr. M. Eisenhut (Institute for Plant Biochemistry, University of Duesseldorf, Germany). For the current study the stock culture was grown on anoxic MP medium without additional Fe(II).

1. Prepare 100 ml MP medium following the protocol of Wu et al.¹⁶ but substitute ferric chloride with 1 ml/L ferric ammonium citrate at 6 mg/ml.
2. Under anoxic conditions (glovebox, 100% N₂), dispense the medium into one 120 ml sterile serum bottle, cap with a sterile butyl rubber stopper and crimp with an aluminum cap. Change the headspace to N₂/CO₂(v/v, 90/10) (compare Hungate & Macy¹⁷) and inoculate with 5% of the stock culture. Subsequently store the culture in a light incubator at 25 °C and 600 Lux from a Tungsten light bulb.
3. Since Synechococcus sp. PCC 7002 is photosensitive, following transfer, cover the serum bottle with a thin paper towel for the first 24 hr within the light incubator. Allow the culture to grow for 6-8 days. Photosynthetic activity will result in the oxidation of Fe(II) and Fe(II) will not be static for the timescale of cellular growth, therefore transfer the culture after 7 days to maintain Fe(II) in the medium and the cells adapted to Fe(II).
4. Monitor the cell density by taking samples for optical density (OD) measurements: The cell density (cells/ml) of the culture can be determined via the absorbance of the cell suspension sample in a photo-spectrometer at a wavelength of 750 nm¹⁹. A linear relationship between OD₇₅₀ and direct microscopic cell counts of a culture in log phase will determine the absolute cell density²⁰.
5. As soon as a cell density of  ̴10⁸cells/ml is reached, wrap the serum bottle with aluminum foil in order to stop oxygen production by photosynthesis.
6. Remove the O₂ in the cell suspension by using a 0.22 µm sterile syringe filter attached to a long (100 mm) disposable needle inserted into the liquid medium. Flush the headspace and bubble the culture with N₂/CO₂ for 5 min (compare Hungate & Macy¹⁷). Keep the sample in dark until inoculation into the column.

3. Preparation of Items and Individual Parts for Experimental Set-up

Note: Information on the required equipment for the experimental set-up, quantities and specifications are listed in Table 2. Parts of the items that will be used for the experimental set-up are prepared in advance and are individually labeled with a single capital letter (A-G), listed in Table 2 and shown as close-ups in Figure 1 as well. Italic alphanumerical codes in brackets in the protocol refer to the equipment itemized in Table 2 and are shown in Figure 1.

1. In order to prepare the sampling ports (D) for the column, close the ports with tight-fitting butyl rubber stoppers (A.3). Insert one stainless steel needle (D.') into the butyl rubber stopper. Make sure that the tip of the needle is in the center of the column.
   1. Connect the needle to a rubber tube (D.2) and seal the connection with a heat-shrink tube (D.3). Attach the other end of the tube to a small Luer lock tube connector ('D.5), seal the connection with a heat-shrink tube (D.3) and cover the tube connector with an appropriate plastic cap (D.6).
      Note: Depending on the desired number of sampling ports it is necessary to provide one main port with various sampling ports — therefore insert the stainless steel needles (D.1) in an oblique angle into the butyl rubber stopper.
2. In order to connect the medium and discharge bottles to the column, modify butyl rubber stoppers following the next steps:
   1. Insert two stainless steel capillaries (E.3; E.4) into the butyl rubber stopper (E.2). Connect the appropriate rubber tubes (E.6) to these capillaries and seal the connection with heat-shrink tubes (E.5).
   2. Add a tube connector (E.7) to the other end of the tube of the longer capillary (E.3) and also fix this connector with a heat-shrink tube (E.5).
   3. Connect a stainless steel needle (E.11) to the free end of the other tube attached to the shorter capillary (E.4), seal the connections with heat-shrink tubes (E.5), and insert the other end of the stainless steel needle into a smaller butyl rubber stopper (E.10).
   4. Insert two stainless steel capillaries (G.3) into a large butyl rubber stopper (G.2) and attach the appropriate rubber tubes (G.4; G.5). Connect the free end of the shorter rubber tube (G.4) to a medium discharge bottle capillary (w2). Equip the free end of the longer tube (G.5) with a small tube connector (G.6). Repeat these steps in order to prepare another butyl rubber stopper for a second discharge bottle.
3. Prepare the stopper for the medium distribution panel (F) by inserting two stainless steel needles (F.3) into a butyl rubber stopper (F.2) in order to produce two media supply lines for the column. Attach a rubber tube (F.4) to each of the needles and connect one medium bottle capillary (c1) to one of the rubber tubes, respectively.
4. In order to prepare the glands for the medium supply line (B), connect one medium supply capillary (c2) to a small tube connector (B.3) with a rubber tube (B.4). Repeat this step for another medium supply gland.
   1. Use the longer medium discharge capillary (w1) instead to prepare glands for the medium discharge line and follow the previous steps (compare (B) in Figure 1).
      Note: It is helpful to label inlet and outlet with different colors of tape to assist in proper assembly.
5. Assemble the parts for the headspace gas exchange panel (C) by inserting two long Luer lock stainless steel needles (C.2) into a rubber tube (C.1) and make sure that the tips of the needles reach 4 cm outside the other end of the tubing. Fill the tube with Polymers glue (C.8) and let the assembly dry for at least 6 hr.
   1. Loosely fill two Luer lock glass syringes (C.3) with cotton (C.4).
   2. Separately prepare two butyl rubber stoppers (C.5), one with a stainless steel needle inserted (C.6) and the other with a stainless steel needle (C.7). Do not connect to the glass syringes, yet.
6. Prepare a glass syringe (E.8) filled with cotton (E.9) for later use in line with the medium bottle and gas pack (gp).
7. Assemble the equipment for a 10 L gas pack (gp) by connecting a rubber tube (gp.1) onto the valve of the gas pack. Insert a tube connector (gp.2) into the free end of the rubber tube. Repeat this procedure for a second 10 L gas pack.

4. Sterilization of the Column and the Equipment

Note: Depending on the material properties, the equipment is sterilized by one of the following three methods:

1. Sterilize equipment made of glass by dry oven (180 °C for 4.25 hr):
   1. Sterilize the equipment for making medium (see Table 1 and section 1.2) separately and in advance. Therefore, prepare 2 x 5 L glass bottles and cover the opening and the bottleneck with aluminum foil. Pack 4 x 5 ml and 5 x 2 ml glass pipettes into a heat resistant container and oven-sterilize all the equipment.
   2. Sterilize the equipment for the column set up in a second step. Wrap the glass syringes (C.3; E.8; F.1 — prepared in section 3) with aluminum foil and make sure that the corresponding butyl rubber stoppers are removed.
   3. Place the glass beads (A.2) in a glass beaker and cover the top with aluminum foil. Also cover the transparent glass plate (A.4), 4 x large tube connectors (B.2) and the 3-way connector (G.7) with aluminum foil and oven-sterilize everything.
2. Sterilize autoclavable plastics and liquids by autoclave (120 °C, 10 bar, 20 min):
   1. In the first step, sterilize the Widdel flask with the medium, the NaHCO₃-buffer solution and 2 butyl rubber stoppers for the 5 L glass bottle.
      Note: Butyl rubber stoppers are first prepared by boiling in ultrapure water 3 times and then autoclaved in a glass beaker with a bit of water, covered with aluminum foil. The wet stoppers are easier to insert into glass bottles.
   2. In a second step sterilize the equipment for the experimental column set up. In order to prepare the column for sterilization in the autoclave, wrap the top opening, the media supply vents, the media discharge vents, and the headspace vent with aluminum foil before autoclaving.
   3. Cover the sampling ports (D.'5) with the appropriate plastic caps (D.'6) and make sure to remove the clamps (D.4) before autoclaving.
   4. Wrap the butyl rubber stoppers and the corresponding capillaries for the medium and discharge bottles (E.2; 2 x G.2 — prepared in section 3), the smaller stoppers for the glass syringes (2 x C.5; E.'10'; F.'2 — prepared in section 3) and the capillaries connected to the medium supply and discharge glands (s2; w1 — prepared in section 3.4) into aluminum foil and sterilize all the equipment in the autoclave.
   5. After sterilization, dry the sterilized column and equipment in an oven at 60 °C for another 4 hr.
3. Since the pump tubing (pt) is not autoclavable, sterilize them in an Ethanol (EtOH) solution (80% EtOH, 20% water). Fill an appropriate beaker with EtOH-solution and place the pump tubing in it, ensuring that the tube is completely filled with EtOH-solution. Take out after 3 hr and wrap directly into pre-sterilized aluminum foil (oven-sterilized) and let dry at RT for 2 hr.

5. Assembly of the Column and the Equipment

1. Place the column (A) on a flat surface and stabilize with a laboratory stand and clamps. Make sure to work under sterile conditions (e.g., within 40 cm of a Bunsen burner or under a laminar-flow hood). Carefully remove the aluminum foil from top opening and fill in the sterilized glass-beads (A.2).
   1. Prepare the watch glass (A.4) in order to tightly close the column by applying the Polymers glue (A.5) to the inner surface in the area where it will be in contact with the column. Lightly press the watch glass in place at the top of the column in order to glue both parts tightly together. Allow at least 6 hr for the installation to dry.
      Note: It is possible to place a sterile, fine pliable wire between the column edge and the watch glass, in order to easily remove the watch glass after the experiment.
2. While working under sterile conditions attach the following parts to the column:
   1. Connect the rubber tubes (B.1) and the corresponding tube connectors (B.2) to the medium supply and discharge vents (compare (B) in Figure 1).
   2. Connect the tube connectors of the glands (B.3) for media supply and discharge (prepared in section 3.4) to the corresponding connectors at the column (compare (B) in Figure 1).
   3. Attach the headspace gas exchange panel (compare (C) in Figure 1 — prepared in section 3.5) to the headspace vent at the column and connect the glass syringes (C.3) to the corresponding stainless steel needles (C.2) and insert the appropriate butyl rubber stoppers (C.6; C.7 — prepared in section 3.5.2) into the cotton-filled glass syringes (C.3).
3. Insert the butyl rubber stoppers for the discharge bottles (2 x G.2 — prepared in section 3.2) into two sterile 3 L glass bottles (G.1), and connect the tube connectors of the bottles (G.6) to the 3-way connector (G.7).
4. Connect the free end of the medium discharge gland capillary (w1) to the free end of a discharge bottle capillary (w2) with pump tubing (pt). Repeat this procedure for the second medium discharge gland capillary and the second discharge bottle.
5. Connect the free end of one medium bottle capillary (s1) to the free end of the medium supply gland capillary (s2) with the pump tubing (pt). Repeat this procedure for the second medium bottle capillary and the second medium supply gland capillary.
6. Assemble the medium distribution panel by inserting the stopper with the corresponding capillaries (F.2 — prepared in section 3.3) into the glass syringe of the medium distribution panel (F.1).
7. Connect the medium distribution panel to the connector of the butyl rubber stopper for the medium bottle (E.7 — compare F で Figure 1).
8. Connect the N₂/CO₂ gas line to the headspace gas exchange panel to the Luer lock stainless steel needle (see position (LLF) in Figure 1), and flush the column installation and capillary system with N₂/CO₂ at low pressure (< 10 mbar). Maintain an outflow of gas at the open ends of the medium bottle capillary (E.3), the 3-way connector (G.7), and the sampling ports (D.5). Therefore, make sure to have the caps of the sampling ports (D.6) slightly open, to maintain sterile conditions through an overpressure of gas flowing out.
   1. Flush the complete installation for at least 20 min.
      Note: In addition, it is possible to close the outgassing needle (C.6) of the headspace gas exchange panel with an appropriate rubber tube and a clamp in order to increase the efficiency of flushing the complete installation.
9. Meanwhile, fill a 10 L gas bag (gp) with N₂/CO₂(v/v, 90/10), following 10 rounds of filling and deaerating (using a vacuum pump) the entire volume to ensure the bag is completely anoxic. Make sure to close the valve of the gas pack after the final fill. Repeat this procedure with a second gas pack but close the valve after deaeration (i.e., leave it empty).
   Note: The N₂/CO₂ filled gas pack will be connected to the medium bottle in order to compensate the increasing headspace volume due to medium loss by pumping. The N₂/CO₂ flushed, but empty gas pack, will later be connected to the discharge bottles in order to allow gas to escape the headspace due to increasing liquid discharge volume.
10. Insert the butyl rubber stopper (E.10 — prepared in section 3.2.3) into the sterile glass syringe filled with cotton (E.8 — prepared in section 3.6).
11. Place the filled and closed medium bottle (E — prepared in section 1) on the lab bench next to the stopper for the medium bottle (E.2), and prepare to connect the stopper to the medium bottle using the following procedure:
    1. Close the N₂/CO₂ gas flow into the capillary (E.3) by closing the corresponding rubber tube (E.5) with a hose clamp.
    2. Lightly lift the butyl rubber stopper off the medium bottle and flush the headspace with N₂/CO₂(50 mbar) by hanging a sterilized, cotton -filled syringe with a bent, long metal needle (1 mm x 140 mm) into the bottleneck of the medium bottle (compare Hungate & Macy¹⁷).
    3. Quickly remove the butyl rubber stopper from the medium bottle and insert the prepared butyl rubber stopper (E.2) into the medium bottle (E).
    4. Change the long metal needle of the syringe (previously used for flushing the headspace) to a disposable needle (0.9 mm x 45 mm; widely available) and inject into the stopper in order to flush the headspace of the medium bottle with N₂/CO₂.
    5. Make sure to have a slight outflow of gas at the open end of the gas syringe (E.8 — assembled in section 5.10) connected to the stopper of the medium bottle. Flush the headspace of the medium bottle (E) and the glass syringe (E.8) for at least 4 min.
12. Meanwhile, tighten the plastic caps (D.6) of the sampling ports (D.'5) and close the corresponding rubber tubing (D.2) with a clamp (D.4).
    Note: If necessary, open the outgassing needle (C.6) of the headspace gas exchange panel again in order to maintain an outflow of gas at the open end of the needle.
13. Place the 10 L gas pack, filled with N₂/CO₂(prepared in section 5.9), on the lab bench. Ensure that the tube connector is in a position next to the open end of the glass syringe (E.8) connected to the medium bottle.
14. After flushing the headspace of the medium bottle (E) for at least 4 min, close the N₂/CO₂ gas line of the flushing syringe and quickly pull the injection needle out of the stopper (E.2). The remaining overpressure of gas within the headspace of the medium bottle will be released through the glass syringe (E.8).
    1. Quickly open the valve of the gas pack and lightly press on the bag to maintain an outflow of N₂/CO₂ in order to flush the tube and the connector of the gas pack.
    2. As soon as the overpressure is released from the medium bottle, quickly connect the connector of the gas pack (gp.3) to the corresponding connector of the glass syringe (E.8).
15. Connect an empty 10 L gas pack (gp) that was previously flushed 10 times with N₂/CO₂ (prepared in section 5.9) to the free port of the 3-way connector (G.7) connected to the discharge bottles. Make sure to keep the valve of the gas pack closed.
16. Reduce the pressure of the N₂/CO₂ gas line (<0.1 mbar) in the headspace gas exchange panel (C で Figure 1; position LLF) to maintain an outflow of gas from the outgassing needle (C.6).
17. Insert the pump tubing (pt) into the pump (P) and remove the hose clamp from the corresponding rubber tube (E.6) of the medium bottle (E).
    1. Open the valve of the gas pack (gp) connected to the discharge bottles (G) to allow air to be released into the gas pack while the discharge bottles fill up with outflow medium.
18. Start the pump and monitor the medium distribution panel (see (F)) filling up with medium. Make sure to remove the remaining gas in the panel as it fills up by holding it in an inverted position. Gas is released through the capillaries that are connected to the pump.
19. Monitor the column as it fills with medium, and adjust the pump to a proper pumping rate of 0.45 L/day, which can be converted to a vertical flux of 10 mM Fe(II)/m²/d, in order to simulate the chemical flux of interest.
20. Install the light source (L) 2 cm above the upper end of the column and cover the upper 10 cm around the column with a dark tape and/or aluminum foil in order to prevent the light from radiating out of the top of the column and illuminating the lower part of the column. Cover the whole installation with a dark textile cover in order to prevent illumination of the column from external light sources.

6. Inoculation of Bacteria into the Column

Note: For an abiotic control experiment this step is skipped.

1. Since the cell culture will be directly injected into the column through the butyl rubber stoppers of the main sampling ports along the side of the column, make sure to have prepared six syringes with needles that are long enough to reach the center of the column body.
2. Sterilize the outside of butyl rubber stoppers of the six main sampling ports at the column (A.3) with an EtOH solution (80%).
3. Have the serum bottle with the culture for inoculation ready (prepared in section 2) and sterilize the butyl rubber stopper by flaming several drops of EtOH solution. Flush the syringe with sterile N₂/CO₂ before taking an aliquot of the culture. Take 1 ml of the anoxic cell solution and inject it into the center of the column through the butyl rubber stoppers of the main sampling ports (A.3).
   Note: Depending on the bacterial growth, it may be necessary to adjust the pumping rate (or even stop pumping) during the first days of the lag-phase for cell growth and development to prevent the cultures from being washed out.

7 . Sampling

Note: In order to collect samples across the chemical gradients that develop inside the column, it is necessary to start sampling from the top sampling ports prior to the deeper ports, as volume loss occurs. Make sure to maintain sterile conditions (e.g., by working within 40 cm of a Bunsen burner or under a laminar-flow hood).

1. Collect the first samples 24 hr after inoculation. Flush the syringe that will be used for sampling with sterile N₂/CO₂, in order to avoid oxygen injection into the sampling ports (D). Make sure to fill the syringe with N₂/CO₂ before sampling.
2. Quickly remove the plastic cap (D.6) and insert the sampling syringe into the tube connector (D.5). Maintain a small gap between syringe and tube connector. Release the gas from the syringe and flush the tube connector with N₂/CO₂.
   1. Firmly connect the syringe to the tube connector. Remove the clamp (D.4) and start taking a sample of about 1 ml.
3. After drawing the sample and before removing the syringe, attach the clamp (D.4). Then remove the sampling syringe and firmly close the tube connector (D.5) with the corresponding plastic cap (D.6).
4. Immediately repeat steps 7.1-7.3 for sampling from every port.
5. Collect the next set of samples every 24 hr.
   Note: For additional samples at different time steps, it is necessary to remove a small amount of sample (e.g., 0.2-0.4 ml) initially before the sample can be taken, in order to remove residual media inside the sampling tube and to achieve representative samples from inside the column.

8. Methods of Analysis

1. Oxygen quantification:
   Note: The oxygen concentration within the flow-through medium and the headspace of the column is quantified non-invasively and non-destructively using the optical oxygen sensors and oxygen sensitive fluorescent foil patches of 0.5 x 0.5 cm (so called optodes), glued along the inner glass wall of the column using silicon glue (A.6). It was ensured that the oxygen sensitive foil patches are insensitive for Fe species in order to achieve reliable readings.
   1. Make sure to calibrate the computer software with the appropriate calibration parameters for the optodes that are used in the column. The optical oxygen sensors come with a specific calibration for the measurement using a corresponding PC-controlled fiber optic oxygen meter.
   2. Start the measurement and take care to hold the polymer optical fiber of the oxygen meter at a right angle to the oxygen sensitive foil that is glued inside the transparent glass wall of the column.
   3. Repeat the measurement for every single O₂ measuring point throughout the column.
2. Fe(II) and total Fe analysis of aqueous samples:
   1. Since Fe(II) is rapidly oxidized by oxygen in air at neutral pH, stabilize the liquid samples for aqueous Fe(II) quantification immediately in 1 M HCl solution. For a final sample volume of 1 ml mix 0.5 ml liquid sample with 0.5 ml 2 M HCl.
   2. Quantify total Fe after incubating an aliquot of the 1 M HCl-stabilized sample with hydroxylamine hydrochloride (10% wt/v, in 1 M HCl) for 30 min. This reagent reduces all Fe(III) to Fe(II), which can then be quantified via the Ferrozine assay²¹.
   3. Perform a Ferrozine Assay using a micro-titer plate reader. Measure the absorbance at a wavelength of 562 nm. Make sure to have standards within the range for detectable Fe(II) and total Fe concentrations.
      Note: If Fe concentrations in the liquid sample exceed standard calibrations, it is necessary to dilute the stabilized sample with 1 M HCl.
   4. Calculate the concentration of Fe(III) by the difference between total Fe and Fe(II).