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•  The Lost City Hydrothermal Field (LCHF) is located near the summit of the seafloor mountain Atlantis Massif, which is 15 km west of the Mid-Atlantic Ridge in the Atlantic Ocean.

•  LCHF is a collection of strikingly large carbonate chimneys (up to 60

m tall). They were formed by mixing of cold seawater with subsurface vent fluids, which are produced by a set of geochemical reactions known as ‘serpentinization’.

•  These anaerobic, exothermic reactions produce elevated levels of

H2, CH4, and other organic compounds. •  Also, during serpentinization, olivine (the primary mineral of the

Earth’s mantle) is hydrated and oxidized by seawater, producing the minerals serpentine, magnetite, and brucite.

Olivine + H2O + CO2 → Serpentine + Magnetite + Brucite + CH4 +

H2 + other organics

Background

•  During October-December of 2015, International Ocean Discovery Program (IODP) Expedition 357 to the Atlantis Massif: “Serpentinization and Life” recovered a total of 57 m of rock cores from 17 different drill holes at 9 sites (Fig. 2).

Proposed work

•  The experimental approaches: I have adapted and optimized DNA extraction and purification techniques that were previously developed for hydrothermal chimneys1,5, magnetic nanoparticles6, Mars analogue substrates7: 1.  pH modification of the DNA extraction buffer: using pH 10 instead

of 8 that was suggested by Lever, et al. (2015). 2.  Chemicals that prevent adsorption of DNA to minerals:

combination of dATP and sodium pyrophosphate.

3.  SCODA (synchronous coefficient of drag attenuation) technology: utilizing Aurora purification system (Boreal Genomics) to separate DNA molecules from minerals in an iterative process such that tiny quantities of DNA can be efficiently accumulated and concentrated in a single final preparation from multiple aliquots of large volumes of crushed rock.

Implications

References 1.  Brazelton, et al. (2006) Methane- and sulfur- metabolizing microbial communities

dominate the Lost City hydrothermal field ecosystem. App. and Environ. Microbiol. 72, 6257-6270.

2.  Schrenk, M.O., et al. (2004) Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge. Environ Microbiol. 6, 1086-1095.

3.  Schrenk, M.O., et al. (2013) Serpentinization, Carbon, and Deep Life, Rev. Mineral. Geochem. 75, 575-606.

4.  Saeki, K. & Sakai, M. (2009) The influence of soil organic matter on DNA adsorption on Andosol, Microbes Environ. 24, 175-179.

5.  Schrenk, M.O., et al. (2003) Incidence and diversity of microorganisms within the walls of an active black smoker hydrothermal chimney. Appl. Environ. Microbiol. 69, 3580-3592.

6.  Tanaka, T., et al. (2009) Contributions of Phosphate to DNA Adsorption/Desorption Behaviors on Aminosilane-Modified Magnetic Nanoparticles. Langmuir. 25, 2956-2961.

7.  Direito, S.O.L., et al. (2012) Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals. FEMS Microbiol Ecol. 81, 111–123.

8.  Schulte, M., et al. (2006) Serpentinization and its implications for life on early Earth and Mars. Astrobiology. 6, 364-376.

9.  Martin, W., et al. (2008) Hydrothermal vents and the origin of life. Nat. Rev. Microbiol., doi:10.1038/nrmicro1991, 1-10.

10. Mumma, M. J., et al. (2009) Strong release of Methane on Mars in northern summer 2003. Science. 323, 1041-1045.

11. Ehlmann, B. L., et al. (2010) Geologic setting of serpentine deposits on Mars. Geophys. Res. Lett. 37: doi:10.1029/2010GL042596.

12. Glein, C.R., et al. (2015) The pH of Enceladus’ ocean. Geoch. Cosmochimi. Acta. 162, 202–219.

13.  Neish, C., et al. (2012) A new approach for DNA detection in Mars analogue soils using SCODA. AbSciCon. JHU/APL.

14.  Carr, C. E., et al. (2013) Life Detection with the Enceladus Orbiting Sequencer. Aerospace conference. IEEE. 1-12.

15.  Hsu, H., et al. (2015) Ongoing hydrothermal activities within Enceladus. Nature. 519, 207-210.

•  Previous environmental DNA sequencing studies on the water and rock samples from LCHF chimneys1,2 showed that the anaerobic chemolithoautotrophs use H2 and CH4 gas produced by serpentinization as their energy sources (Fig. 1).

•  Anoxic interior parts of the carbonate chimneys are inhabited by

methanogenic and methanotrophic archaea (Methanosarcinales), while the exterior parts are dominated by sulfur-metabolizing bacteria (Fig. 1).

•  However, the chimneys are continuously exposed to oxidized

seawater, so they may not be accurate representatives of the deep, subsurface habitats.

DepartmentofBiology,UniversityofUtah

ShahrzadMotamediandWilliamJ.Brazelton

Explora?onofnovelsubsurfacemicrobialcommuni?eswithinseafloormantlerocks

Figure 2: Schematic of putative biogeochemical processes in low-temperature, actively serpentinizing systems (LCHF)3. There is not a clear image of what is going on in serpentinite rocks in marine subsurface environment, where exposure to oxidized seawater is limited.

Figure 3: International Ocean Drilling Program Expedition to the Atlantis Massif: October-December 2015. A) The BGS Rockdrill (RD2). B) MARUM-MeBo70 drill. C) Expedition 357 drill sitesare shown in black circle. The yellow star shows the “Lost City”.

C

•  The hypotheses: 1.  Novel microbial communities live within the serpentinite

subsurface of the LCHF. 2.  Variations of microbial diversity among drill holes are correlated

with the concentrations of H2 and CH4 in the holes. 3.  The microbial communities of serpentinite rocks mostly comprise

anaerobic archaea and bacteria, while gabbroic and basaltic rocks host mostly aerobic bacteria.

•  The objectives: 1.  Extract DNA from rock samples for environmental 16S rRNA gene

sequencing. 2.  Use two different analytical approaches to demonstrate the

uniqueness of the serpentinite-inhabiting microbes.

•  The challenges: 1.  Low-biomass samples: The total amount of biomass in deep-sea

oceanic crust can be exceedingly low, challenging our ability to even detect life, not to mention our ability to sequence DNA from such samples.

2.  DNA adsorption to the minerals (Fig. 4): many of the minerals that

make up serpentinite rocks reduce the yield of DNA extractions, prevent effective DNA purification, and act as inhibitors to DNA sequencing reactions.

A B

Figure 4: Mechanisms and sites of DNA adsorption on soil particles4. A) The DNA molecules associate with the surface of negatively charged materials such as phyllosilicates (including serpentine, clay minerals, etc.) via a bridging of cations. B) Phosphate groups on the edge of the DNA molecule may bind via a ligand exchange reaction directly to OH groups of some minerals (e.g. Al or Fe) on the edge of phyllosilicates.

Figure 5: Boreal Genomics’ Aurora instrument (left) and reusable cartridge (right). Images from www.borealgenomics.com

•  Lost City has been studied as a model for the origin and early evolution of life on Earth1,8,9.

•  Lost City is also similar to other potential sites in our solar system for sustaining life: 1.  The presence of methane in the atmosphere of Mars could be

the result of serpentinization in the Martian subsurface, either today or in the past10,11.

2.  The Cassini orbiter confirmed that one of Saturn’s moons,

Enceladus, has a liquid water ocean under its frozen surface with alkaline pH (11-12), which is consistent with serpentinization12

(Fig. 7).

•  This study will provide the first census of diversity of microbes living in a marine subsurface serpentinite habitat.

•  SCODA and NASA missions: 1.  The technology was used for DNA detection in Mars analog soils

from Atacama desert, Chile13. 2.  Potential use during collection of ice grains from Enceladus, by

concentrating the possible nucleic acids and long charged polymers14.

1b.

Figure 7. Hydrothermal systems on Enceladus. The fluid pH and reaction temperature is pretty similar to LCHF on Earth15.

Olivine Serpentine

??

M0076(AM-11)

30°06'

30°08'

30°10'

30°12'

M0074 (AM-09)

M0070 (AM-07)

M0075(AM-03)

M0073(AM-05)

M0071(AM-04) M0068

(AM-02)

M0069 (AM-06)M0072 (AM-01)

2500 2000 1500 1000Depth (mbsl)

42°10' 42°08' 42°06' 42°04'42°12'W

N

U1309A−EU1309FU1309G−H

U1310

U1311

MeBoRD2

Expedition 304/305

Expedition 357

2 km

AM-08

AM-10Planned

Lost City

A

B

C

S. 1 S. 2 S. 3

OTU 1 0.2% 0.01% 0.9%

OTU 2 0.03% 0.5% 2.5%

OTU 3 6% 3.3% 0.09%

OTU 4 0.6% 0.7% 0.6%

16S rRNA amplicons

Microbial Community

Pre-Clustering

OTUs Abundance Table

Figure 6: Processing sequences. Each dot in red circles on the plot above shows the microbial community of each drill hole. The distances between the dots on the plot

show the dissimilarities between the microbial communities.

Multivariate plot