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Looking  for  a  green  sulfur  bacterium  that  oxidizes  ferrous  iron  Nancy Y. Kiang Microbial Diversity summer course, 2014 Marine Biological Laboratory, Woods Hole, MA Abstract. At School Street Marsh, Woods Hole, MA, an iron-enriched pool of streamwater was chemically characterized (salinity, contents of iron, oxygen, and sulfide) and spectral irradiance measured at the sediment surface to assess its favorability to harbor an as yet unknown iron-oxidizing green sulfur bacterium (GSB). Microsensors showed fast depletion of oxygen in the water column by the first 2 cm, absence of sulfide, and strong gradients in very acidic pH (4.6-6). The site’s high iron content in the water was confirmed with Scanning Electron Microscope (SEM)/Energy Dispersive X-ray Spectroscopy (EDS), and pigment extracts via HPLC revealed a bacteriochlorophyll-like spectrum with peaks at 526 and 746 nm, to be identified. Enrichments were made in anoxic, sulfide-free, Fe2+-rich media, PSII inhibitor DCMU, and incubation in 630 nm light to select for green sulfur bacteria. After 11 days of incubation, no evidence of iron oxidation was seen, but evidence of sulfate reduction was manifested in blackening of the medium precipitates, changing from grey siderite to black FeS. Due to logistics with various equipment delaying earlier use, site characterization later revealed low pH due to organic acids at the time of sampling likely to be unfavorable to GSB iron oxidation. However, this does not rule out the existence of such an organism during conditions of higher water flow and more neutral pH.

Introduction  This study was conducted as a project for the Microbial Diversity course at the Marine Biological Laboratory during the summer of 2014 (hereafter referred to as “MD2014”). My goal was to find an example of an iron-oxidizing green sulfur bacterium in Woods Hole, Massachusetts. Green sulfur bacteria (GSB) are classified in the family Chlorobiaceae, and are anoxygenic photoautotrophic obligate anaerobes known for using sulfide as their electron donor. Their reaction center (RC), P840, uses bacteriochlorophyll a (BChl a). Their antenna complexes include both chlorosomes with BChl c, d, and e, with peak absorbances that tune between 710-760 nm, as well as the Fenna-Matthew-Olson (FMO) antenna protein, with peak absorbance at 810 nm, sandwiched between the chlorosome and the RC (Blankenship, 2002). These light harvesting complexes enable GSBs to live at extremely low light, such that one species has been found living off hydrothermal vent radiation (Beatty et al., 2005). Unlike other phototrophs, the generation of NADH goes to a reverse TCA cycle to fix carbon, instead of the Calvin-Benson cycle. Only one example GSB has been found able to oxidize iron for an electron donor and at the same time unable to oxidize sulfide (Heising et al., 1999). Since GSBs as well as purple bacteria likely were the earliest photosynthetic organisms, there is interest in their

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potential role in creation of the Banded Iron Formations (BIFs) in the fossil rock record (Hegler et al., 2008) prior to the emergence of cyanobacteria and the oxygenation of the atmosphere. The aim of this study was to find another example of an iron-oxidizing GSB, by identifying environments with conditions favorable to the growth of such an organism, looking for evidence of BChl c, d, or e as potential biomarkers for their presence (note that green non-sulfur bacteria also have BChl c) in environmental samples, and doing lab enrichments and isolation.

Methods  

Sampling  Sites   The primary sampling location of interest was a puddle at School Street Marsh, Woods Hole, MA (latitude 41.525583, longitude -70.668507). This site has a freshwater stream that receives inputs of iron from a rusty set of metal stairs (across the driveway), accumulating in a fairly stagnant, low-flow pool with dense, decaying organic debris shaded by trees and shrubs (hereinafter referred to as “School Street Marsh Puddle,” SSMP). Brown biofilms with a rusty metallic sheen cover most of the water surface. Depth to the sediment layer is difficult to ascertain due to thick organic matter that is very soft, but is approximately 3.5 cm at arm’s length from the bank. Sediment is a fine, black soft, organic-rich material. The site has yielded isolates of bacteria with interesting iron utilization, including a magnetotactic bacterium (Sabrina Powell, Microbial Diversity 2000, Marine Biological Lab) and an iron-oxidizing purple non-sulfur bacterium, Rhodopseudomonas palustris Strain TIE-1 (Jiao et al., 2005). In addition, a separate stream has diurnal cycles of sulfide/oxygen gradients and exhibits white biofilms of sulfur-oxidizing Thiovulum along the mud edges of the stream (“School Street Marsh Stream,” SSMS). Samples were collected on August 6, 2014, at 2:00-3:30 pm EDT, during clear, sunny conditions, air temperature ~ 24 ˚C. These included: water from the very surface; water at the sediment interface (about 4 cm depth); organic matter and sediment at the sediment-water interface. On August 13, a 33 cm tall, ~10 cm diameter core through the water and sediment was taken, plugged from beneath before lifting it out of the water, then carried carefully back to the lab at Loeb Hall, with the top exposed to the air, and letting the sediments settle for 3 hours. In addition, samples were taken from Salt Pond, Falmouth, MA (41.543007, -70.626884) and at the outlet of the Trunk River, where freshwater drains to the ocean (latitude 41.534659, longitude -70.641149). At Salt Pond, the samples were at various distances/depths from the shore on August 11, 2014 at 5:20-6:00 pm EDT, at its southeast corner where a small drainage ditch flows through rip rap rock into the pond, bringing iron-enriched water (“Salt Pond,” SP). Rusty sand bordered the edges of the inlet, and black sediment could also be seen in a small shallow portion at the center of the inlet. On the same day, at the Trunk River, a sample was taken from the green layer of a microbial mat in the sandy banks about 15 m upstream from a small bridge (“Trunk River”, TR).

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The field samples for each location are listed in Table 1. School Street Marsh Puddle

School Street Marsh Stream

Salt Pond Trunk River

Figure 1. Field sites and close-up of sampled water and sediments.

Table 1. Field sites and water and sediment samples

Date/Location Site Name Lat./Long. Sample ID Sample Description 6 August 2014 School Street Marsh, Woods Hole, MA core:13 August 2014

School Street Marsh Puddle

41.525583, -70.668507

SSMP-1 Surface water SSMP-3 Bottom water ~4 cm SSMP-5 Bottom organic matter

and sediment SSMP-5X Water-sediment core

~23 cm School Street Marsh Stream

SSMS-7 Water ~10 cm SSMS-8 Mud with white biofilm

11 August 2014 Salt Pond, Falmouth, MA

Salt Pond 41.543007, -70.626884

SP-7 Sediment layer and water at 3 cm depth

SP-10 Black sediment at 2 cm depth

SP-12 Sediment at 45 cm depth

11 August 2014 Trunk River, Falmouth, MA

Trunk River 41.534659, -70.641149

TR-20 Microbial mat green layer

Site  characterization   For all sites, salinity was measured with a refractometer, temperature at the time with an alcohol-in-glass thermometer, and surface temperature with an infrared thermometer. For pH, for School Street Marsh, within an hour sampling, water samples in centrifuge tubes were measured with a pH meter back in the lab (in the “chem room”).

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A profile of a SSMP core was also measured at a later date (see below). In the field at Salt Pond and the Trunk River, pH was measured with Aldrich pH paper. For samples SSMP-1 and SP-10, elemental content of the water was measured with Scanning Electron Microscope (SEM) with Energy Dispersive X-ray Spectroscopy (EDS) for normalized mass fractions of C, N, S, Si, Fe, P, and Mn. Since dissolved salts were not washed first from the samples, typical elements for dissolved salts were excluded from the analysis. Elemental composition was summarized at 50X resolution to integrate over a large area of the sample. For School Street Marsh Puddle, the core SSMP-5X was measured with Unisense microsensors for profiles of O2, H2S, and pH. On a later date, on August 17, 2014 at 1:00-1:15 pm EDT, solar spectral irradiance was measured with and Ocean Optics JAZ-A field spectrometer at: an open parking lot of the site; the tree-shaded water surface at SSMP; and underwater at the sediment-water interface ~ 3 cm depth on that day, both in a clear area of the water, and under the brown biofilm. In addition, to look for evidence of anoxygenic photosynthetic bacteria in the water column prior to culture growth, the water was analyzed for photosynthetic pigments by HPLC (Agilent 1100, CR18 column). About 0.5 L of SSMP column water was concentrated by vacuum filtration onto a GF/F filter, and the filter cut up and submersed in methanol in a 15 ml centrifuge tube. About 1.0 ml of the extract was filtered through 0.22 µm filters into the HPLC injection vials. The column pressure stabilized for > 5 minutes with 5.0 ml/minute flow of 20% 50 mM ammonium acetate to 80% methanol buffer, then switched to 1.5 ml/minute flow. Samples were run with a solvent solution of 20% acetone and 80% methanol. As a pseudo control, to check for the existence of GSBs at all at these sites, samples SSMP-1 and SSMS-8 were inoculated into Pfennig bottles of MD2014 “Marine Sulfur-Phototrophs” medium and incubated in the 630 nm LED cabinet, suited for the enrichment of sulfide oxidizing GSBs.

Enrichment   To enrich for iron-oxidizing GSBs, 30 mM NaCOO-OH was used for a carbon source, 8 mM FeCl2 for an Fe(II)2+ electron donor. This iron salt precipitates into siderite (FeCO3), grey-green powder. To prevent the growth of sulfide oxidizing purple sulfur bacteria or sulfide use by GSBs, 1.0 mM Na2SO4 was used for a sulfur source, and LED light panels at 630 nm at 15-25 µmol photons/m2/s to select for GSBs. To inhibit cyanobacteria, DCMU was added to the medium. For the Salt Pond and Trunk River inoculants, the stock medium was enhanced to achieve 24 %o salinity. The detailed procedure for medium preparation is given in the Appendix. Note that, in fact, the FeCl2⋅4H20 powder for stock solution S8 was partially oxidized with red coatings around green clumps. Filtering removed these oxidized particles, but the final solution was a yellow color, rather than green, indicating likely content of Fe(III). Therefore, the actual concentration of Fe(II)2+ was less than 8 mM. However, we considered 5 mM sufficient for enrichment, and the actual concentration was likely above this, sufficient for enrichment. In an anoxic chamber (3.3% H2), serum bottles of 40 ml volume each, containing 20.6 ml of the enrichment medium, 1 ml of sample water inoculants were aseptically injected with hypodermic needles (using ethanol as a disinfectant) into each serum bottle. The augmentation of salinity for the Salt Pond and Trunk River inoculants brought the

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volume per bottle to 25 ml plus 1 ml inoculant. Incubations in the LED cabinet were at room temperature. To check for growth of iron-oxidizing bacteria, the inoculated bottles were checked regularly for signs of red color due to iron oxide formation.

Results  

Site  characterization   Measurements of salinity and pH (and also temperature) are show in Table 2. It was interesting to find that Salt Pond’s salinity, 21-22 %o, is much lower than ocean waters, at 35%o. Also, despite the Trunk River’s outlet close to the ocean and supposed regular influx of seawater at high tide, the salinity at low tide was not much elevated above normal freshwater. The pH meter in the “chem room” in Room 207 of the Microbial Diversity course appeared unreliable (difficulty stabilizing) on a core that had been taken much earlier, so the low pH numbers were considered suspect, due to age of the core as well as unreliability of the sensor, but these numbers are entered here for the record. Values from pH paper were coarse resolution but gave consistently higher values with a difference higher by as much as 0.5 to 1.0 of pH. The pH values for the core SSMP-5X as measured by the Unisense sensor (#3383) gave similar pH to the chem room pH sensor, and the steep curve in the trend through the water column does show remarkably low pH. Table 2. Salinity and pH, day of sampling

Site Name Sample ID

Sample Description Temperature ºC

Salinity (%o) pH

School Street Marsh Puddle

SSMP-1 Surface water 24 3 5.88 SSMP-3 Bottom water ~4 cm 5.75 SSMP-5 Bottom organic matter

and sediment

SSMP-5X Water-sediment core ~20 cm

NA See Figure 2

School Street Marsh Stream

SSMS-7 Water ~10 cm 5 6.29 SSMS-8 Mud with white biofilm

Salt Pond SP-7 Sediment layer and water at 3 cm depth

27.3-27.8 21-22 6.5

SP-10 Black sediment at 2 cm depth

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SP-12 Sediment at 45 cm depth

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Trunk River TR-20 Microbial mat green layer

28 6 NA

As expected, O2 declines quickly with depth (in the upper 1.4 cm). Sensors were unable to detect any sulfide down to 9.0 cm (Figure 2).

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Figure 2. Microsensor profiles of O2, pH, and H2S in School Street Marsh Puddle (core sample SSMP-5X, on 8/13/15).

The SEM/EDS analysis of elemental content confirmed that the School Street Marsh Puddle is highly enriched in iron, at 24.5% of mass (normalized) for C, N S, Si, Fe,

P, and Mn (Figure 3. The School St. Marsh Puddle was also high in Ca. Oddly, the EDS scan did not identify the black sediment particles from School Street as distinctly any particular elements, and they did not show as distinct structures in the single element maps, as would happen for a sand grain or diatom that were clearly silicon. Possibly they were coated with dissolved salts and washing might reveal their composition. Figure 3. SEM/EDS normalized mass percents for C, N, S, Si, Fe, P, and Mn for Salt Pond black sediment and water, and for School Street Puddle.

Solar irradiance measurements (Figure 4) show the strong reduction of light through the overlying tree canopy and the vegetation red-edge spectral transmittance, such that the spectral irradiance to the water is relatively enriched in red to near-infrared light. For that date and time of day, the available photosynthetically active radiation (PAR) over 400-840 nm at the sediment interface was 0.03 – 0.14 µmol/m2/s without an overlying biofilm, and 0.03-0.04 µmol/m2/s underneath a brown biofilm.

Figure 4. Solar spectra irradiance at School Street Marsh Puddle, in the open, at the water surface under the tree canopy, and under water at the sediment interface.

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The HPLC chromatogram of the SSMP-1 water sample showed a spectrum that eluted about 8 minutes later than the Chl a peak. The peaks, in Figure 5, are not easily identified as any particular bacteriochlorophyll from in vitro spectral available to compare at the time of this writing. Figure 5. HPLC spectrum for an unknown bacteriochlorophyll, from sample SSMP-1.

Meanwhile, the bottles with thiosulfate medium yielded growth of green bacteria from the School Street Marsh inoculants within 8 days. The SSPM-3 bottle showed loose clumps of green, which the fiber optic spectrometer was unable to detect, while the SSMP-8 bottle showed well-distributed green growth that clearly identified with in vivo spectra as BChl c (Figure 6)

Figure 6. Growth of School Street Marsh inoculants after 10 days in MD2014 “Marine Sulfur-Phototroph” medium with 1 M thiosulfate and 630 nm light. left: left bottle, inoculant SSMP-3 and right bottle, inoculant SSMS-8. right: Absorbance spectra of SSMS-8 + thiosulfate bottle, showing BChl c.

Enrichments   Enrichments showed no signs of iron oxidation after 11 days of incubation. However, on the 6th day of incubation, the precipitates in the SSMS-7 School Street Marsh Stream inoculant (of just the water sample) turned black, and by day 10, two Puddle incubations also started to turn black. These would start turning darker gray one day and be fully black within 24 hours. For the Salt Pond incubations, it took only 4 days for SP-10, the inoculant from the black sediments, to turn black. Absorbance spectra of these bottles via fiber optic spectrometer did not show clear differences due to the overwhelming turbidity by the precipitates.

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After 6 days

After 11 days

Figure 7. School Street Marsh incubations after 6 days and 11 days, exhibiting formation of FeS. Grey sediments are siderite, unchanged from initial conditions. In order from left to right: SSMP-1, SSMP-3, SSMP-5, SSMS-7, SSMS-8.

Figure 8. Salt Pond and Trunk River incubations after 4 days. From left to right: SP-7, SP-10, SP-12, TR-20.

 

Discussion  

Role  of  pH   Anoxic conditions, lack of sulfide, and enrichment in iron would make this site favorable for GSBs, but low pH does not. If the acidic (<6.0) conditions of SSMP-1 are indeed measured accurately, this acidity is probably due to high content of organic acids. According to Heising et al. (1999), the Fe-oxidizing GSB do best with pH values of 6.8-7.0, as observed from the authors’ multiple treatments.

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While the reaction for photosynthetic use of sulfide as an electron donor is: 6CO2 + 12H2S + light à <C6H12O6> + 6H2O + 12S (1)

The reaction for use of Fe(II)2+ is: CO2 + 4Fe(II)2+ + 11H2O + light à <CH2O> + 4Fe(III)(OH) 3 + 8H+ (2)

The redox couple Fe(II)2+/Fe(III)(OH)3 has a midpoint redox potential of 0.15 V, such that P840, at 0.24 mV should be able to oxidize Fe(II)2+. This holds at neutral pH (Widdel et al., 1993). At acidic pH, the redox couple Fe2+/Fe3+ with midpoint potential 0.77 V renders conditions unfavorable for P840 to oxidize Fe2+, unless P840’s potential somehow shifts in tandem. I need time to read up more on the properties of P840 at low pH, and have not yet found any prior work on this in the literature. However, the fact that an iron-oxidizing purple non-sulfur bacteria was found at this site indicates that the photosynthetic oxidation of ferrous iron must be possible, at least sometimes at this site. Apparently, R. palustris Strain TIE-1 was sampled at a time after heavy rain when the road by the puddle was flooded, so the pH was probably closer to neutral.

Role  of  Light   The irradiance measured at School Street Marsh probably provides close to the higher fluxes received at this site throughout the year. Conditions on the day of measurements had patchy, diffuse clouds, and moments of clear sky in the early afternoon, contributing to occasionally high direct beam radiation and enhanced scattering of diffuse radiation into the tree canopy covering the School Street Marsh Puddle. The spectra available are biased toward the red and near-infrared wavelengths required by GSBs. The PAR (400-840 nm) fluxes of 0.03 – 0.14 µmol/m2/s without an overlying biofilm, and 0.03-0.04 µmol/m2/s underneath a brown biofilm are about 2 orders of magnitude less than that recommended by Heising et al. (1999) for enrichment (15-80 µmol-photons/m2/s, but also an order of magnitude larger than the radiation at the hydrothermal vent (Beatty et al., 2005). So, at this site at the sediment surface, light is not necessarily limiting.

Incubation  Reactions   The lack of appearance of iron oxidation within the given time frame does not yet imply there are no iron-oxidizing GSBs in the inoculants, because they could emerge still months later. However, the gradual blackening of the precipitate in some of the incubations is a sign that a sulfate reducer has produced sulfide, which then could possibly give: FeCO3 + H2S --> FeS + H2CO3

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The carbonic acid could further acidify the medium, rendering it even less favorable for GSB iron oxidation. I did not have MOPS buffer in the medium, since it was not called for in the enrichment of Heising et al. (1999).

Conclusions   This project was an example of putting the cart before the horse: chemical characterization of the field sites was actually done after the enrichments were started, due to anxiousness to start the enrichments early for a limited time frame, not knowing earlier what field equipment was available, and trial and error with different ways of measuring pH and trying to do so on fresh samples. I assumed the site should be conducive to utilization of iron as an electron donor for anoxygenic photosynthesis since the purple non-sulfur bacterium that does this had already been found here. Evidently conditions can change. If I were to do this project over again, and had more time, field site characterization would come first. Although the School Street Marsh Puddle is highly acidic, further upstream where there is more regular flow of water and less allocthonous inputs of plant matter, the water quality could still be iron enriched but with less acidic pH. This could explain the known presence of iron-oxidizing purple non-sulfur bacteria. Further characterization of the site could include fluorescence in situ hybridization (FISH) on water samples with the 16S rRNA probe GSB-532 (Overmann and Tuschak, 1997; Tuschak et al., 1999) to look for presence of GSBs. It would also be worth building a clone library for the site. The enrichment medium could be improved by adding MOPS buffer, and an inhibitor for sulfate reducers such as sodium molybdate (Bernhard Schink, personal communication) or others in the literature. I would also do a number of replicates (bottles were a scarce resource during the project period). Otherwise, isolation of a new species or strain of Fe-oxidizing green sulfur bacteria still seems promising, given sufficient time with the right inoculant. Next it would be more interesting to learn the mechanisms of anoxygenic photoautotrophic iron oxidation through more thorough review of the literature, and learn something about the evolutionary pathway(s) for this function.

Microbial  Diversity  2014  Thoughts   This project provided me experience with using environmental microsensors (and their shortcomings), SEM, HPLC for pigments, the various means of extracting pigments, using an anoxic chamber, in general getting to know what utensils are out there, and especially with making media for culturing microbes. I learned that formulating the growth medium is a mix of both science and a lot of experience, with some leeway in what is necessary or influential to organisms. I also poured agar plates in anticipation of doing more streaking, and prepared media for shake tubes, which unfortunately did not get used due to the slow growth of these organisms and the intense technique required for making shake tubes.

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Making the medium involved a complicated series of steps for anoxic mixing and inoculation. As notes to myself, I would put the DCMU directly in the stock medium and not try to mix in a syringe per bottle in the anoxic chamber. Probably an autoclaved bottle could be brought into the anoxic chamber to mix the FeCl2 stock, DCMU, and, as necessary, salinity augmentation, to avoid contamination and the laborious process of mixing in syringes. Syringe bottles and tubes probably should be acid washed. For future students of the course, I would recommend not picking a slow-growing organism for your individual project (I was forewarned, but plowed ahead anyway, since prospects seemed promising).

Acknowledgments  Being able to take this legendary course was a real privilege. I would like to thank the course instructors, Dianne Newman for cheerfully showing me how to streak plates, and Jared Leadbetter for his stern admonishments about good microscope and medium habits. I want especially to thank Kurt Hanselmann for his tireless patient guidance in using the spectrometers, SEM, and microscopes; Arpita Bose for taking direct interest in my project and her helpful advice on the enrichment medium and various things to try and look for; Verena Salman for doing the microsensor profiles for me and advice on caring for water samples; the anoxic chamber buddies – Ben, Marton, Jake, Bing – for coordinating on the airlock; and the TA’s and everyone in Microbial Diversity 2014 in general for their camaraderie and help. I am grateful to Dan Repeta at WHOI and his lab members, René Boiteau and Serena Dao, for generously allowing me to use their HPLC. Thanks are also expressed for support for my taking the course provided by MBL sponsors, the NASA Goddard Space Flight Center Training Office, and Michael New of the NASA Astrobiology Institute through his support of the NAI Team, The Virtual Planetary Laboratory (PI Victoria S. Meadows).

Appendix   Enrichment Medium for Fe(II)-oxidizing green sulfur bacteria: The enrichment medium was prepared as follows from the stock solutions listed below, and the final medium molarities are given in the tables for each solution. To 0.8 L of MilliQ water, the following were added aerobically: 10 ml of S1 Freshwater solution, 25 ml of S2 100 mM KH2PO4, 6.5 ml of S3 1.0 M NH4Cl, and 1 ml of S4 1.0 M Na2SO4. The pH was adjusted to 6.8 with droplets of concentrated HCl. The desired molarities for KH2PO4, NH4Cl, and Na2SO4 follow that suggested by Heising et al. (1999). The volume was brought to 0.93 L with MilliQ water. This was autoclaved for 45 minutes, then cooled under continuous stream of 80% N2/20% CO2. In an anoxic chamber, to the cooled mixture was added aseptically: 30 ml of S5 1.0 M NaOH, 1 ml of S6 vitamin stock solution, and 1 ml of S7 trace minerals stock solution. This made the base solution for enrichment, without the iron source or inhibitor. The Fe(II) source and DCMU were added later to the base solution in incubation bottles. A batch of 40 ml serum bottles were allowed to equilibrate, opened, in an anoxic

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(3% H2) chamber, then plugged with blue butyl rubber stoppers and crimped shut. These were autoclaved for 45 minutes at 120 ºC, then brought back into the anoxic chamber for injection of enrichment medium. I used 20 ml syringes and 25g surgical needles aseptically (ethanol as a disinfectant) to inject 20 ml of base medium into each bottle. A 1 ml syringe and needle was used to inject 0.6 ml of S8 Ferric chloride stock solution into each bottle to achieve 8 mM Fe(II)Cl2 (so the fluid total volume came to 20.6 ml instead of 20 ml). This produced a gray precipitate suspended in a fine layer at the bottom of the bottle. For DCMU, because it has a low solubility, I used a sterile pipette tip to wipe one small dab of DCMU powder inside a syringe with 25g needle inserted into the bottle stopper, then used the syringe plunger to withdraw fluid from the bottle to rinse the DCMU into the bottle, repeating an additional time. Arpita Bose deemed 1.5 g/L of DCMU sufficient for inhibiting Photosystem II, and the amount introduced into the serum bottles should achieve around this concentration. For the Salt Pond and Trunk River sites, to augment the salinity of the stock medium, 4.4 ml of S10 was injected into each serum bottle, bringing the total volume to 25 ml and final salinity to 24%o. The serum bottles were then ready for inoculation. (NOTES: Didn’t add vitamins until ready to make incubations, because vitamins cannot be autoclaved. You need to have an autoclaved bottle in which to mix everything, in the anoxic chamber. Why not just mix everything in an empty, equilibrated, autoclaved big bottle? Probably can add DCMU to the base solution, rather than having to do each serum bottle individually). S1. Stock 100X Freshwater (“FW”) Base (per liter) (MD2014 stock) This stock solution was prepared aerobically. Component Amount

(g/L) Formula Wt. (g/mol)

100X Conc. (mM)

Vol. added per L

Final Conc. (mM)

NaCl 100 g 58.44 1711 mM 10 ml 17.11 mM MgCl2⋅6H2O 40 g 203.30 197 mM 1.97 mM CaCl2⋅2H2O 10 g 147.02 68 mM 0.68 mM KCl 50 g 74.56 671 mM 6.71 mM S2. Stock 100 mM KH2PO4 pH 7.2 (per liter) (MD2014 stock – ##VERIFY KH2PO4/K2HPO4 formula##) This stock solution was prepared aerobically. Component Amount

(g/L) Formula Wt. (g/mol)

100X Conc. (mM)

Vol. added per L

Final Conc. (mM)

KH2PO4 ? 135.901 100 25.5 ml 2.55 S3. Stock 1000X NH4Cl (MD2014 stock) This stock solution was prepared aerobically. Component Amount

(g/L) Formula Wt. (g/mol)

1000X Conc. (mM)

Vol. added per L

Final Conc. (mM)

NH4Cl 5.35 53.4913 1000 6.5 ml 6.5

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S4. Stock 1000X 1.0 M Na2SO4 (MD2014 stock) This stock solution was prepared aerobically. Component Amount

(g/0.1 L) Formula Wt. (g/mol)

1000X Conc. (mM)

Vol. added per L

Final Conc. (mM)

Na2SO4 14.2 g 142.04 1000 mM 1 ml 1 mM S5. Stock 300X 1.0 mM Sodium bicarbonate (NaHCO3) (add 30 ml for total 1 L) 2.16 g (30 mmol) of NaHCO3 was dissolved to 30 mL of MilliQ water. This stock solution was prepared aerobically in a 125 mL serum bottle in 30 mL of MilliQ water, then autoclaved sealed with plugged with blue butyl rubbers stoppers for 45 minutes. The solution was cooled under continuous stream of 80% N2/20% CO2, with surgical needle in-flow and out-flow for ~ 30 minutes. Component Amount

(g/0.03 L) Formula Wt. (g/mol)

Conc. (mM)

Vol. added per L

Final Conc. (mM)

NaHCO3 2.16 g 71.9958 1000 mM 30 ml 30 mM S6. Stock 1000X 13-Vitamin Solution (MD2014 stock). This stock solution was prepared aerobically.

Component Amount (g/0.1 L)

1000X Conc. (mM)

Vol. added per L

Final Conc. (mM)

10 mM MOPS, pH 7.2 1000 ml 10 mM 1 ml 10 µM Riboflavin 100 mg 0.1 mg/ml 0.1 µg/ml Biotin 30 mg 0.03 mg/ml 0.03 µg/ml Thiamine HCl 100 mg 0.1 mg/ml 0.1 µg/ml L-Ascorbic acid 100 mg 0.1 mg/ml 0.1 µg/ml d-Ca-pantothenate 100 mg 0.1 mg/ml 0.1 µg/ml Folic acid 100 mg 0.1 mg/ml 0.1 µg/ml Nicotinic acid 100 mg 0.1 mg/ml 0.1 µg/ml 4-aminobenzoic acid 100 mg 0.1 mg/ml 0.1 µg/ml pyridoxine HCl 100 mg 0.1 mg/ml 0.1 µg/ml Lipoic acid 100 mg 0.1 mg/ml 0.1 µg/ml NAD 100 mg 0.1 mg/ml 0.1 µg/ml Thiamine pyrophosphate 100 mg 0.1 mg/ml 0.1 µg/ml Cyanocobalamin (Vitamin B12)

10 mg 0.01 mg/ml 0.01 µg/ml

S7. 1000x HCl-Dissolved Trace Elements Stock Solution (per liter) (MD2014 stock) This stock solution was prepared aerobically. Component Amount

(g/L) Formula Wt. (g/mol)

1000X Conc (mM)

Vol. added per L (ml)

Final Conc. (mM)

20 mM HCl 1.7 ml na 20 mM 1 mL 20 µM

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conc. HCl FeSO4⋅7H2O 2100 mg 278.01 7.5 mM 7.5 µM H3BO3 30 mg 61.83 0.48 mM 0.48 µM MnCl2⋅4H2O 100 mg 197.91 0.5 mM 0.5 µM CoCl2⋅6H2O 190 mg 237.93 6.8 mM 6.8 µM NiCl2⋅6H2O 24 mg 237.69 1.0 mM 1.0 µM CuCl2⋅2H2O 2 mg 170.48 12 µM 12 nM ZnSO4⋅7H2O 144 mg 287.56 0.5 mM 0.5 µM Na2MoO4⋅2H2O 36 mg 241.95 0.15 mM 0.15 µM NaVO3 3 mg 121.93 25 µM 25 nM Na2WO4⋅2H2O 3 mg 329.85 9 µM 9 nM Na2SeO3⋅5H2O 6 mg 263.01 23 µM 23 nM S8. Ferrous chloride (FeCl2) stock solution This stock solution was prepared in an anoxic chamber. Two 40 mL serum bottles were prepared of 30 ml fluid each. 1.6 g (8 mmol) of FeCl2⋅4H20 was dissolved in 30 ml of MilliQ water, then filtered through a syringe and 20 µm filter via hypodermic needle into a plugged, autoclaved 40 ml serum bottle. Component Amount

(g/30 ml) Formula Wt. (g/mol)

30X Conc. (mM)

Vol. added per L (ml)

Final Conc. (mM)

FeCl2⋅4H2O 1.6 g 198.8118 0.2667 M 30 ml 8 mM S9. Other ingredients added (see description above): DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), Formula Weight 233.09 g/mol. S10. Salt Pond/Trunk River 24%o salinity augmentation Component Amount

(g/100 ml) Formula Wt. (g/mol)

Stock Conc. (M)

Vol. added per 20.6 (ml)

Final Conc. (mM)

NaCl 22.634 58.44 3.8731 M 4.4 ml 1.937 mM MgCl2 0.8755 203.3 0.15 M 0.33 mM References Beatty, J.T. et al., 2005. An obligately photosynthetic bacterial anaerobe from a deep-sea

hydrothermal vent. PNAS, 102(26): 9306-9310. Blankenship, R.E., 2002. Molecular Mechanisms of Photosynthesis. Blackwell Science,

321 pp. Hegler, F., Posth, N.R., Jiang, J. and Kappler, A., 2008. Physiology of phototrophic

iron(II)-oxidizing bacteria: implications for modern and ancient environments. FEMS Microbiology Ecology, 66(2): 250-260.

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Heising, S., Richter, L., Ludwig, W. and Schink, B., 1999. Chlorobium ferrooxidans sp. nov., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with "Geospirillum" sp. strain. Archives of Microbiology, 172: 116-124.

Jiao, Y.Y.Q., Kappler, A., Croal, L.R. and Newman, D.K., 2005. Isolation and characterization of a genetically tractable photo autotrophic Fe(II)-oxidizing bacterium, Rhodopseudomonas palustris strain TIE-1. Applied and Environmental Microbiology, 71(8): 4487-4496.

Overmann, J. and Tuschak, C., 1997. Phylogeny and molecular fingerprinting of green sulfur bacteria. Archives of Microbiology, 167(5): 302-309.

Tuschak, C., Glaeser, J. and Overmann, J., 1999. Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe. Archives of Microbiology, 171(4): 265-272.

Widdel, F. et al., 1993. Ferrous iron oxidation by anoxygenic phototrophic bacteria. Nature, 362: 834-836.