Subsurface microbial physiology

Marine subsurface sediments are densely populated diverse microbial communities. These microbes live by degrading dead organic matter sedimenting from the overlying water column. Because the degradability of the buried organic matter decreases rapidly with sediment depth subsurface sediments are extremely energy-limited (http://geomicrobiology.au.dk/research/slowlife). Still the microorganisms manage to harvest sufficient energy for sustaining high population sizes and thus maintain the biological activities, which are key for the cycling of carbon, sulfur and other essential elements in the marine subsurface.

Our agenda

The Center for Geomicrobiology studies the physiology of subsurface microbial populations in order to understand how their metabolic activities are controlled and are coupled to the cycling of elements. Furthermore, our studies aim to identify the traits that allow buried microorganisms to subsist under extreme energy limitation. Our research on the physiology of marine subsurface microbes is a combination of analytical studies on environmental samples, isotopic tracer experiments, microbial cultivation, and modeling.

Analytical studies

To examine in situ activity of microbes, we measure concentrations and isotopic compositions of key metabolites in environmental samples. Metabolites analyzed include O2, nitrate, Fe(II), sulfate, methane, ethane and propane, dissolved inorganic carbon, in addition to volatile fatty acids, H2, carbon monoxide and methanol. To complement these geochemical data, we study the phylogenetic composition and concentrations of diagnostic genes (DNA) and gene transcripts (mRNA) that allow us to link microbes to microbially-driven processes. We combine analysis of targeted key diagnostic genes using PCR, and broad-based sequencing of all genes in the sediment using metagenomics and metatranscriptomics.

Isotopic tracer experiments

By adding radioactive tracers (e.g. 35S or 14CO2) to sediments and measuring their conversion to metabolic end products, we obtain estimates of metabolic rates of microbial processes, such as sulfate reduction and methanogenesis. In addition, we study microbial substrate use via stable isotope probing (SIP). In this method, isotopically labeled microbial substrates are added to samples at environmentally relevant concentrations. Energy production pathways and substrate assimilation are then examined by monitoring the incorporation of isotopic label into metabolic end products, biomass of single cells and bulk nucleic acids (DNA or RNA). Isotopic compositions of metabolic end products and single cells are measured by isotope ratio mass spectroscopy and NANO-SIMS. Isotopically labeled nucleic acids are extracted and microbes that have assimilated added substrates identified via DNA sequencing followed by phylogenetic analyses.

Microbial cultivation

Cultivation and isolation remain the only means by which the physiology of any given microbial strain can be thoroughly characterized. In cultivating subseafloor microbes, we are particularly interested in organisms adapted to conditions in deeply buried sediments. This implies slow growth, low maintenance energy and adaptation to low substrate concentrations. A key question is whether or not unique guilds of subseafloor microbes exist that are physiologically adapted to the low-energy conditions that prevail in the subsurface. Our efforts to address this question include investigating the kinetics of substrate uptake and turnover in pure cultures of known microbial species, for example sulfate-reducing bacteria.

Modeling of activity

We model net reaction rates of microbial metabolic pathways, e.g. oxic respiration, sulfate reduction or methane oxidation, based on porewater concentration profiles of electron acceptors and electron donors. In addition, using thermodynamic calculations, we determine the in situ energy yields of metabolic reactions in the subsurface. The combination of modeling with quantitative data on cell abundances is then used to investigate in situ energy availability to microbes in the subsurface.

Metabolic potential

Fluorescence-activated cell sorting (FACS) is our tool of choice for isolating microorganisms from marine sediments. We are currently developing methods to isolate specific target cells for genomics analyses using FACS.

In addition to our own efforts, we maintain a collaboration with the Single Cell Genomics Center at the Bigelow Laboratory for Ocean Sciences, USA. Together with this partner, we routinely isolate single microbial cells from marine sediments using FACS and amplify their genomic material. Subsequent genome reconstruction enables us to deduce the metabolic potential of the isolated cells.

 


KEY PUBLICATIONS FROM CfG

Jørgensen, BB, Marshall, IPG. (2016) "Slow Microbial Life in the Seabed". Annual Review of Marine Science 8: 311-332.

Na, H., Lever, M. A., Kjeldsen, K. U., Schulz, F., Jørgensen, B. B., (2015) "Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate (13) C-acetate in coastal marine sediment". Environmental Microbiology Reports 7: 614-22.

Ozuolmez, D., Na, H., Lever, M. A., Kjeldsen, K. U., Jørgensen, B. B., Plugge, C. M. (2015) "Methanogenic archaea and sulfate reducing bacteria co-cultured on acetate". Frontiers in Microbiology, 6.

Glombitza, C., Jaussi, M., Røy, H., Seidenkrantz, M., Lomstein, B. A., Jørgensen, B. B., 24.8.2015, "Formate, acetate, and propionate as substrates for sulfate reduction in sub-arctic sediments of Southwest Greenland". Frontiers in Microbiology, vol. 6, nr. AUG

Lever M. A, Rogers KL, Lloyd KG et al. (2015) "Life under extreme energy limitation". FEMS Microbiology Reviews 39(5): 688-728.

Lever M. A., Rouxel OJ, Alt J, Shimizu N, Ono S, Coggon RM, Shanks WC, Lapham L,

Elvert M, Prieto-Mollar X, Hinrichs KU, Inagaki F, Teske AP. (2013) "Evidence for microbial carbon and sulfur cycling in deeply buried ridge flank basalt". Science 339: 1305-1308.

Lever M. A. (2012) "Acetogenesis in the energy-starved deep biosphere – a paradox?". Frontiers in Microbiology 2: 1-18.

Røy H. (2014) "Experimental assessment of community metabolism in the subsurface". Kallmeyer, J., Wagner, D. (eds.), Microbial Life of the Deep Biosphere, Walter de Gruyter, p. 303-317

Wasmund K, Schreiber L, Lloyd KG, Petersen DG, Schramm A, Stepanauskas R, Jorgensen B. B., Adrian L. (2014) "Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia, phylum Chloroflexi". ISME J 8:383-397.

Lloyd KG, Schreiber L, Petersen DG, Kjeldsen KU, Lever MA, Steen AD, Stepanauskas R, Richter M, Kleindienst S, Lenk S, Schramm A, Jorgensen BB. (2013) "Predominant archaea in marine sediments degrade detrital proteins". Nature 496:215-218.

Tarpgaard IH, Røy H, Jørgensen BB (2011) "Concurrent low- and high-affinity sulfate               reduction kinetics in marine sediment". Geochimica et Cosmochimica Acta 75:2997-3010.