2 Methodology

3 Results

What are Archaea up to in marine sediments? Stable isotope incubation experi-ments reveal low activity of sedimentary “Thaumarchaeota”.

Sabine K. LENGGER1,*, Yvonne A. LIPSEWERS1, Henk DE HAAS2, Jaap S. SINNINGHE DAMSTÉ1 and Stefan SCHOUTEN1

1. Marine Organic Biogeochemistry, Royal NIOZ Netherlands Institute for Sea Research, P. O. Box 59, 1790 AB Den Burg, Texel, The Netherlands.2. Marine Geology, Royal NIOZ Netherlands Institute for Sea Research, Texel, The Netherlands..

* Present address: Petroleum and Environmental Geochemistry Group, University of Plymouth, Plymouth PL4 8AA, UK. e-mail: sabine.lengger@plymouth.ac.uk

1 IntroductionArchaea are small unicellular organisms, much like bacteria, but fundamentally and evolutionary different (in fact, more different from bacteria than humans are from slime molds; Fig. 1). Thaumarchaeota are amongst the most abundant mi-croorganisms in aquatic environments. The membrane lipids of their cells are preserved well and thus widely used as mo-lecular fossils. However, their metabolism in marine sedi-ments is still debated. While in cultures and mesocosms, pe-lagic and sedimentary Thaumarchaeota have been shown to be autotrophic ammonia oxidizers (Wuchter et al., 2003), i.e. building biomass from inorganic carbon and gaining energy from the oxidation of ammonia, there is some evidence for heterotrophic metabolisms in Thaumarchaeota, i.e. building biomass from organic carbon and gaining energy from oxi-dation of organic compounds. They were shown to grow on the substrates pyruvate (Tourna et al., 2011), amino acids (Ouverney and Fuhrman, 2000) and glucose (Takano et al., 2010).

In this study, we used different substrates for incubation of marine sediment cores from the Iceland Shelf, which con-tained heavier (but not radioactive, thus stable) carbon iso-topes - 13Carbon instead of 12Carbon. As only 1% of the carbon in the natural environment is 13C, we could then look for enrichment of this 13C in specific lipids produced by bac-teria and eukaryotes vs. Archaea.

90°

Air in (pipette)

Drilled holes 2.5 mm

filled with silicon

Stopper

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Yellow tape

Distance 1 cm

Airpump

Filter 0.2 mm

Water bath for optimum temperature (4°C)

Acyclic biphytane

Bicyclic biphytane

Tricyclic biphytane

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REFERENCES: Ouverney, C. C., Fuhrman, J. A.: Marine Planktonic Archaea Take Up Amino Acids, Appl. Environ. Microbiol., 66, 4829-4833, 2000. Wuchter, C., Schouten, S., Boschker, H. T. S., and Sinninghe Damsté, J. S.: Bicarbonate uptake by marine Crenarchaeota, FEMS Microbiol. Lett., 219, 203-207, 2003. Takano, Y., Chi karaishi, Y., Ogawa, N. O., Nomaki, H., Morono, Y., Inagaki, F., Kitazato, H., Hinrichs, K.-U., and Ohkouchi, N.: Sedimentary membrane lipids recycled by deep-sea benthic archaea, Nature Geoscience, 3, 858-861, 2010. Tourna, M., Stieglmeier, M., Spang, A., Könneke, M., Schintlmeister, A., Urich, T., Engel, M., Schloter, M., Wagner, M., Richter, A., and Schleper, C.: Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil, Proc. Natl. Acd. Sci. USA, 108, 8420-8425, 2011.

ACKNOWLEDGEMENTS: The authors would like to thank the Master and crew of the R/V Pelagia and the participants of the Long Chain Diols Cruise 64PE341, in particular M. Baas, S. Rampen, M. Besseling, L. Handley and W. Lenthe, as well as the NIOZ technical department (Y. Witte, J. Schilling). We would also like to thank L. Moodley, H.T.S. Boschker, L. Pozzato, J. Middelburg, G. Duineveld and M. Lavaleye for helpful advice in planning the experiments and K. Koho / Utrecht University and L. Stal and N. Bale for providing equipment. We are grateful for analytical assistance to J. van Ooijen, S. Crayford, A. Mets, M. Verweij and J. Osse-baar. S.K.L. was funded by a studentship from the Darwin Center for Biogeosciences to S. S. This is a publication of the Darwin Center for Biogeosciences. NIOZ is part of the Netherlands Organization for scientific research (NWO).

AA +

Glu +

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+Pyr+

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-Pyr

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Dissolved ‚CO2

‚ (from respiration)

12C13C

13CAdded substrate containing 99%

Natural substrate containing 99%

HCO3-Amino acid (AA)

Pyruvate (Pyr)Glucose (Glu)

Available carbon enriched in

Bacteria and Eukaryotes

Archaea and Thaumarchaeota

Make archaeal lipids(Biphytanes)

Make bacterial / eukaryotic lipids (fatty acids)

13C-labeled substrates were added to freshly recovered sediment cores from the Iceland shelf. The cores were incu-bated for 4-6 days. Then, the sediment was extracted and the specific archaeal and bacterial lipids were analyzed on their 13C content, as well as the CO2 dis-solved in the water (Fig. 2). An increased 13C content compared to cores incubated without the enriched substrates would indicate that they were growing as they then took up the added substrates, rich in 13C.

ARCHAEA

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BACTERIA Thaumarchaeota

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Incubation

Figure 2. Incubation scheme.

The added 13Carbon was respired as seen by the increase in 13C in the dissolved inorganic carbon to up to 1800 ‰ (Fig. 3, difference between incubation and background is plotted). Bacteria / Eukaryotes clearly incorporated the 13Carbon into their lipids (100 ‰; Fig. 4). However, the values of archaeal lipids did not change upon incubation (Fig. 5).

Figure 3. Difference between 13C values of dissolved inorganic carbon in the incubated cores from the background incubations (∆δ13C).

Figure 4. Difference between 13C values of fatty acids found in the incubated cores from the ones found in the background incubations (∆δ13C), illustrating the uptake of 13C into C16 and C18 fatty acids at 0-1 cm sediment depth.

Figure 5. Difference between 13C values of biphytanes found in the incubated cores from the ones found in the background incubations (∆δ13C), illustrating the lack of uptake of 13C into biphytanes at 0-1, 1-2 and 6-7 cm sediment depth.

Acyclic biphytane

Tricyclic biphytane

C16 fatty acid

C18 fatty acid

4 Conclusions

There was no substan-tial incorporation of the 13C from the sub-strates into the archae-al lipids, in contrast to the bacterial lipids and the respired CO

2 which

showed an increase in δ13C. It is thus possible that archaea, are not taking up the substrates used. However, as they were varied, it is more likely that benthic Thaumarchaeota are growing only slowly. Turnover times and generation times were esti-mated to be at least several years.

How ∆δ13C is calculated

The carbon isotope ratio 13R is the fraction of 13C compared to 12C : 13R = 13C/12C . The δ notation uses this ratio and compares it to a standard. A common standard for 13C is Vienna Pee-Dee Belemnite.

δ13C

Sample = (13R

Sample/13R

VPDB - 1 )x 1000 ‰

The ∆δ13C here is calculated by subtracting the background (cores

where no labeled substrates where added) from the incubations with the different labeled substrates.

Figure 1. The tree of life.

HCO3

-

HCO3-Amino acid (AA)

Pyruvate (Pyr)Glucose (Glu)

Figure 6. The incubations team.