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Searching for EET-microbes from the crustal deep biosphere of North Pond, Mid-Atlantic Ridge, using cathodic poised potential experiments - Manchester Geobiology Research in Progress meeting
- 1. Searching for EET-microbes from the crustal deep biosphere of North Pond, Mid-Atlantic Ridge, using cathodic poised potential experiments Dr. Rose Jones (rjones@bigelow.org), Dr. Beth Orcutt
- 3. Andy Fisher (2006). Hydrogeology Journal
- 4. Tremblay and Zhang, 2015. Frontiers in Microbiology Microbes get energy from solids Mineral Substrate
- 5. Cool, oxic crust Solid Fe Cool, oxic crust Aqueous Fe
- 6. Electron donor Electron acceptor e-e-Fe2+ (aq) + 0.5 O2 (g) + 2.5 H2O (l) Fe3+(OH-) 3 (s) + 2 H+ (aq)
- 7. + - Working Counter Ag/AgCl reference electrode Semi-permeable membrane Potentiostat Fixed voltage, variable current Cells Sample e- Electrode
- 8. Can marine deep subsurface lithotrophic microbes use solid substrates?
- 9. Field Site: North Pond Edwards et al (2011).
- 10. Edwards et al (2011).
- 11. Ocean Crust Wellhead FLOCS CORKs Fluid flow Edwards et al (2011).
- 13. Ship- board Storage 1 year 4C T0 DNA Sequencing SEM Imaging T end electrode DNA SequencingElectrode Enrichment 1 2 3 4 5 -0.2 V vs. SHE DNA Sequencing
- 14. No electricity + inoculum -0.2 V vs SHE + inoculum -0.2 V vs SHE - inoculum = No cells
- 16. -0.2 V Vs. Standard Hydrogen Electrode (SHE) Shipboard
- 17. -0.2 V Vs. Standard Hydrogen Electrode (SHE) Activity over time U1382A 151 mbsf Control (blue) Basalt chips (Red i, Orange ii, yellow iii)
- 18. -0.2 V Vs. Standard Hydrogen Electrode (SHE) Activity over time U1382A 151 mbsf Control (blue) Basalt(Red) Glass wool (Green) Pyrhottite (Purple)
- 19. Activity over time U1382A 151 mbsf Wellhead samples Control (blue) Basalt(Red) Glass wool (Green) Pyrhottite (Purple)
- 20. U1382A 151 mbsf GW = Glass Wool B=Basalt P = Pyrrotite FC = Fluid control OC = Offline control Relative Abundance (%)
- 21. Conclusions Microbes in marine subsurface basalt can use solid substrates as electron donor We can use bioelectrochemistry to enrich for rock-hosted iron- oxidizing microbes
- 23. 1 2 3 4 5 Treatment Voltage Sample 1 Offline - Mix 2 Fluid -0.4 V - 3 Working -0.4 V Glass Wool 4 Working -0.4 V Basalt 5 Working -0.4 V Pyrrotote
- 26. Indum Tin Oxide Carbon Cloth
Editor's Notes
- I’m Dr. Rose Jones. I’ve spent the last two and a half years as a postdoc at Bigelow Laboratory of Ocean Science in Maine, on the very top of the Northeast coast of the USA. The project I’ve been working on involves methods testing bioelectrochemical techniques as a way to search for microbes that use solid inorganic sources of energy – EET (Extracellular Electron Transport) such as iron from the deep subseafloor in particular. I’ll first go into a bit about why we might be interested in the deep subsurface, go into a bit of theory of using electricity to search for microbes in this environment, then how it worked out in practice.
- We’re used to thinking about the Earth like this: But, it can also look like this <animation>. If 70% of the world is covered by ocean, then 70% is sea-bed. And yet, only 5% of this area is mapped, never mind understood, in any great detail. Doesn’t stop at the sea bed skin – it extends down into the crust.
- The subsurface isn’t a solid block. It’s full of cracks and flow paths, which are full of fluid. (Think about groundwater coming into a basement). If you think about hydrothermal plumes, with the clouds of super-heated water coming out, that water has to come from somewhere. Fluid volume in the oceanic crust is estimated at ∼2% of the total ocean volume. This fluid is ocean water that gets changed as it passes along the cracks, with it’s water chemistry changing as it goes. We also know there are microbial cells in these subsurface cracks that are distinct from deep ocean microbial communities. What we don’t really understand yet is how they can be surviving in such an extremely nutrient and energy poor environment: The marine subsurface is mainly basalt – a low silica mineral high in manganese and iron and is very low in organic carbon. At ocean water pH, iron and other dissolved minerals may also present in fluids in very low concentrations.
- There are however, certain chemolithotrophic microbes with mechanisms that allow them to give and take electrons for energy directly from solid minerals. Either via electron shuttle molecules or direct contact of conductive pili. By conductive, I mean that electrons and therefore electricity can pass relatively freely along it. These are being ID’d in more environments all the time – wastewater, estuarine sediment etc. These microbes included some that can use iron in a solid mineral as an energy source, including one from a hydrothermal vent. But, no-one had looked for them in the deep oxic crustal subsurface.
- Thermodynamically speaking, there is a case for this direct-electron-transfer of iron coupled to oxygen as a potential ecological niche. This is the total Gibbs free energy of all potential metabolically relevant redox reactions in at cool oxic crust, normalized by the concentration of each present, and adjusted for environmental conditions like temperature. When we consider only the aqueous iron and then when we take that still in solid form in the basalt, We can see that there is more energy when you adjust to include energy from soild Fe/O – in black, as opposed to aqueous Fe only – grey. Values taken from our field site, so this is particularly relevant.
- Which means a lot of potential space for this redox reaction to happen in. We normally think of iron oxidation as a two-step process, where iron abiotically reacts, releasing it into solution. Microbes then catalyze aqueous iron oxidation. The electron moves from electron donor to acceptor, diverted along the way into a cell’s machinery for conversion to ATP and so on. However, extracellular electron transport capable microbes don’t need to wait for iron to abiotically react. They take the electron direct from the solid phase.
- So, for microbes that can interact with a solid phase for energy, can do this <>, instead of the chemical redox reaction. In this growth setup, the oxidation and reduction halves of a reaction are separated. Ion flow between electrodes completes the circuit. Using the potentiostat, we can measure the rate of flow of electrons along the wire (the current), and set the energy level (the voltage) of the system - essentially controlling which redox reactions will happen or not. We can therefore change the energy at which we feed the electrons to vary the community that will grow on the electrode, essentially fooling the microbe into thinking we are giving it a rock, when we are growing it on a surface of our choosing.
- This site is in around 4400m water.
- It’s a pool of sediment collected in amongst the peaks of the mid-atlantic ridge. In 2011, holes were drilled into the crust, then capped the boreholes to stop surface water entering the hole.
- Long strings of instruments and porus cases of sterile rock chips were suspended within the holes. The idea being that rock-hosted microbes in the crust would settle on the rocks, which would make it easier for us to collect – like putting food in a humane trap to catch mice. In 2014, wellhead experiments were installed with similar cases of sterile rock chips, bathed in fluids coming up from within the boreholes. Some of the sites had multiple levels of fluid, isolated from each other by packers with the assumption that deeper fluids might carry different microbial communities to higher ones.
- In November 2017, we went back to North Pond on R/V Atlantis, and retrieved the CORK strings using the ROV Jason.
- I took rock crush from the CORK strings, stored them at 4C for ~1 year. DNA was then collected from the rocks at the beginning of each experiment – T0 to account for storage effects, then they were added as inoculum to electrochemical cells and subjected to a voltage of -0.2V vs. SHE for 1-2 weeks. DNA was then taken at the end of the experiment to see how the community responded, and images taken of the community on the electrode pieces.
- When we look at the glass electrodes under light microscope, we can see a clear difference in morphology between no-voltage control and those with electrons passing through.
- This is them under light microscope. These guys are consistently ultrasmall coccoids, averaging between 1003 00 nm in diameter. This is pretty representative of the elecrode communities.
- This plot shows the change in current over time, normalized by electrode area. See that the current – the electron flow increasingly changes over time, separating from the abiotic control and the fluid community. There are therefore microbes from the deep subsurface taking up electrons from a solid substrate. Values are – because ‘-’ here is electrical i.e. direction of electron flow, not mathematical – (Carbon cloth electrode = underestimate)
- This plot also shows current over time, normalized by electrode area and volume of sample added. From downhole rocks at U1382A (~151 mbsf).
- This plot also shows a change in current over time indicating growth, with glass wool substrate having the most response. This is probably because this was used as a packing material at the end of rock crush columns so any biomass washed through would collect here. Biomass was too low for sequencing here. <Additional notes: These wellhead samples came from from rocks bathed in bottom fluid but in cases on the surface next to the boreholes, but allowing for more material. This meant that there was more starting material and we could get DNA off of the electrodes. The curves are less dramatic however. It’s definitely clear that the NP13 from U1383C shallow have some response. All of which makes me suspect that we’re seeing activity on the electrodes, rather than true growth. Which isn’t entirely unexpected due to the very limited medium available and the relatively short incubation time for am inorganic-eating microbe growing at 4C. It also made me suspect they were mixotrophs – capable of using inorganics for energy but requiring organic carbon to grow, or perhaps using this metabolism as a last-resort>
- When we look at the wellhead samples - which had more starting sample mass to work with - we get this, which shows that very little interaction with the electrodes is going on.
- Sequencing seems to confirm this, as there isn’t as clear of a difference between controls and Echem. Therefore, wellhead samples appear to have less EET microbes in the community.
- The next steps with this work is to dig further into the identity of the EET microbes and work out how they relate to the fresh communities. <Further things to explore with this approach includes adding carbon to encourage growth of rock-eating heterotrophs, further shipboard experiments with freshly collected samples, and searching for microbes that use solid minerals as electron acceptors – those that breathe rock rather than eat it. Incubating for more than two weeks – particularly with extremely nutrient poor and cold environments like this might also be useful>
- Each experiment had:
- This was set up on ship.
- And in the lab. Both set-ups were done in at 4C, as this approximates the temperatures in situ at North Pond – approaching that fo ambient deep seawater.
- The electrodes I used were: ITO = Conductive coating on glass = like smartphone screens. Lab CC = Shipboard. Much higher restance – i.e. much harder for electrons to flow through – like fighting through heavy traffic vs. 1 am in winter.
- Representative cyclic voltammetry (CV) scans of indium tin oxide (ITO) electrodes incubated with various mineral substrates indicate alteration products formed as a result of incubation at -0.2V vs SHE. Here, showing data from Hole U1383C shallow wellhead samples (NP13 experiment, Tables 1 and 2); see Supplemental Figure 2 and BCO-DMO dataset for data from other experiments. CV data plotted as current normalized to ITO surface area and data from abiotic controls, and additionally normalized against CV scans at the start of the incubation. Top) CV sweep of NP13 Glass wool shows peaks at -0.8, -0.5 and 0.2 V vs Ag/AgCl on both the incubated ITO electrode at end of experiment (dark blue) and on a fresh ITO electrode inserted into incubated media (light blue), indicating a biological interaction at equivalent to H2/O, Fe2/O2 and an as yet unknown mechanism. Bottom) NP13 Basalt. Dark brown, CV scan of incubated ITO electrode at end of experiment; Light brown, fresh ITO electrode inserted into incubated media. CV sweep shows peaks at -0.8, -0.5 and 0.2 V vs Ag/AgCl on both the incubated ITO electrode at end of experiment (dark blue) and on a fresh ITO electrode inserted into incubated media (light blue), indicating a biological interaction at equivalent to H2/O, Fe2/O2 and an as yet unknown mechanism. Note, data from Pyrrhotite incubation not shown because the signal is indistinguishable from noise.