Final Report Summary - SUBSTRATE USE (Linking substrate consumption to consumer identity in carbon-cycling microbes inhabiting anoxic marine sediments) The seafloor consisting of marine sediment and the underlying Earth’s crust is the largest carbon sink on Earth, and exerts an important control on ocean chemistry, atmospheric gas composition, and Earth’s climate (Hedges and Keil 1995). To a large extent the fate of carbon in the seafloor is controlled by microbes, which use carbon compounds as energy sources for growth and survival. Our understanding of the metabolic activities of these microbial communities remains rudimentary (e.g. Parkes et al. 2000, D’Hondt et al. 2002, Biddle et al. 2006, Inagaki et al. 2006, Lipp et al. 2008). Several guilds of microbes that differ in metabolism carry out remineralization under anaerobic conditions in sediments (e.g. Froelich et al. 1979, Canfield et al. 1993). The two quantitatively most important ones in marine sediments are sulfate reducers and methane producers (Jørgensen, 1982). Based on porewater concentration profiles of sulfate and methane the activities of both groups are spatially separated. According to the traditional view sulfate reducers outcompete methanogens for shared substrates in the presence of sulfate due to higher Gibbs free energy yields of sulfate reduction. Methanogens are thought to become dominant deeper in sediments, where sulfate is absent. Acetogenesis, a metabolism that involves the reductive synthesis of acetate (Drake et al. 2006), is believed to be absent due to lower free energy yields from shared substrates compared to sulfate reduction or methanogenesis.Project SUBSTRATE USE investigated the following research questions:1. Populations of sulfate reducers, methanogens, and acetogens are present in both sulfate reducing and methanogenic seafloor environments.2. Growth of sulfate reducers, methanogens, and acetogens can be selected for and predicted based on media composition. 3. Substrate specialists will end up dominating in single-substrate media, whereas substrate generalists will end up dominating in mixed-substrate media. I address these separately in the following sections:1. The examination of sulfate reducing, methanogenic, and acetogenic microbial populations in anaerobic seafloor-environments.In this first part of the project, I was able to demonstrate that the traditional view of spatial separation of sulfate-reducing and methanogenic microbial communities does not apply to oceanic crustal environments. Instead both groups overlap heavily in range, perhaps due to differences in substrate use. Combined isotopic analyses on C- and S- isotopic compositions indicate that both sulfate reducers and methanogens are metabolically active. Using laboratory-based stable isotope probing experiments, I was able to demonstrate that both groups of organisms to be alive and active. This was the first study to ever demonstrate the presence and viability of these microbes in the oceanic crust, and was published in Science earlier this year (Lever et al. 2013).I also supervised two students on projects on the distributions of methane producers and acetogens. My PhD student Xihan Chen (2011-present) examined the distributions of methane producing and anaerobic methane oxidizing Archaea in relation to sulfate and methane concentrations in coastal sediments of Aarhus Bay. This work is close to completion by now, but not published yet. Xihan’s preliminary data show (1) that methane producing Archaea are indeed present in sulfate-containing sediment, thus arguing against the dogma that communities of methane-producing Archaea are competitively excluded by sulfate reducing bacteria in sulfate-rich sediment, and (2) a distinct zonation of methanogenic Archaea in response to the presence/absence of sulfate, with known methylotrophic methanogens dominating methane-producing communities in sulfate zones, while hydrogenotrophic methanogens and to a lesser extent aceticlastic methanogens dominate methane zones.The other student was a visiting Masters student, Laura Piepgras, from the University of Bremen, who visited from April to May 2012 and examined the distribution of acetogenic and other C-fixing microbes in subseafloor sediments of Aarhus Bay and the Bering Sea, as well as hydrothermal surface sediment of the Guaymas Basin using PCR primers I had designed. These PCR primers amplify a key gene of C-fixation (acsB) that is found in all acetogenic microbes. Laura was able to detect phylogenetically diverse communities of microorganisms that are likely to be acetogenic bacteria, based on DNA sequence similarity to cultured acetogen strains.2. Growth of sulfate reducers, methanogens, and acetogens can be selected for and predicted based on media composition. Under supervision by myself, and fellow postdoctoral scientist, Dr. Kasper Kjeldsen, our PhD student Hyunsoo Na conducted flow-through, batch and stable-isotope labelling experiments in which she incubated marine sediments from Aarhus Bay with the key energy substrate acetate to examine the role of sulfate reducers, methanogens, and (reverse) acetogens in the cycling of this compound. Her data, which will be published this year, indicate sulfate reducers of the family Desulfobacteraceae to be the main consumers of acetate in the presence of sulfate. In the absence of sulfate, acetate is taken up by uncultured bacterial groups – not as expected by methanogens. Using sediments from the Guaymas Basin and Aarhus Bay, I have also initiated incubation experiments with stable-isotope (13C) labelled methylated substrates (formate, acetate, methanol, dimethylbenzoic acid). The main aim of these experiments is to determine which microbes are responsible for the turnover of these compounds in marine sediments. These experiments still await completion, because establishing a sensitive method of stable-isotope probing, including a suitable ultra-centrifugation method that ensures clear 12C- and 13C-DNA-peak separation required months of work. I anticipate finishing the analyses on these experiments this year and hopefully publishing the results next year.3. Substrate specialists will end up dominating in single-substrate media, whereas substrate generalists will end up dominating in mixed-substrate media. Most of the second half of 2011, I spent on modelling competitive outcomes in single- versus mixed-substrate sediment samples. This study, which was published in Frontiers in Microbiology (Lever 2012) came to the clear conclusion that the existence of acetogens in marine sediments – despite the lower energy yields of acetogenesis compared to sulfate reduction and methanogenesis from shared substrates – can be explained by the ability of acetogens to access larger substrate spectra and thereby pool energy from substrates not utilized by sulfate reducers and methanogens, as well as the fact that all substrates shared by these three metabolic groups except H2, CO, and formate, are occur at sufficiently high porewater concentrations so that sulfate reduction, methanogenesis, and acetogenesis from these substrates are all energy-yielding. This article received considerable attention by fellow scientists in the field, as a result of which Prof. Aharon Oren from Hebrew University in Jerusalem wrote a commentary article on my article (Oren 2012). I presented these results at several international conferences, including a workshop on minimum energy requirements of life held in Aarhus in May 2012. After this workshop I was asked to first-author a review on minimum energy requirements of life along with co-authors, which is currently under revision with FEMS Microbiology Reviews (Lever et al., under revision). Due to my expertise in the application of functional genes to study microbes in the subseafloor environment, moreover, I was asked by FEMS Microbiology Ecology to submit a review on past functional gene studies surveys in the marine subseafloor. This study, which I wrote and was accepted by the journal in 2012, appeared in the print version of the journal earlier this year (Lever 2013). Highlights of my research:Together with an international team of scientists, I was able to for the first time conclusively demonstrate the presence of alive and active microbial populations in subseafloor basalt (Lever et al. 2013). Using energetic modelling, I for the first time provided a theoretical framework for why acetogens can coexist with metabolic guilds that have higher energy yields of metabolic reactions, such as sulfate reducers and methanogens (Lever 2012). In recognition of my expertise in the use of functional genes to link microbial identity to microbial metabolism, I was asked to write a comprehensive review on our knowledge on functional genes and microbial metabolic processes in subseafloor environments (Lever 2013). Analyses of several experiments involving additions of stable isotope labelled energy substrates to sediment incubations are still ongoing and will result in several further publications within the next 2 years.REFERENCES Biddle JF, Lipp JS, Lever MA, Lloyd KG, Sørensen KB, Anderson R, Fredricks HF, Elvert M, Kelly TJ, Schrag DP, Sogin ML, Brenchley JE, Teske A, House CH, Hinrichs K-U (2006) Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc Nat Acad USA 103:3846-3851.Canfield DE, Jørgensen BB, Fossing H, Glud R, Gundersen J, Ramsing NB, Thamdrup B, Hansen JW, Nielsen LP, Hall PO (1993) Pathways of organic carbon oxidation in three continental margin sediments. Mar Geol 113:27-40. Drake HL, Küsel K, Matthies C (2006) Acetogenic Prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, eds. The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community. New York: Springer 2:354-420.Froelich PN, Klinkhammer GP, Bender ML, Luedtke NA, Heath GR, Cullen D, Dauphin P, Hammond D, Hartman B, Maynard V (1979) Early oxidation of organic matter in pelagic sediments of the eastern equatorial Atlantic: suboxic diagenesis. Geochim Cosmochim Acta 43:1075-1090.Hedges JI, Keil RG (1995) Sedimentary organic matter preservation: an assessment and speculative synthesis. Mar Chem 4:81-115.Inagaki F, Nunoura T, Nakagawa S, Teske A, Lever M, Lauer A, Suzuki M, Takai K, Delwiche M, Colwell FS, Nealson KH, Horikoshi K, D’Hondt S, Jørgensen BB (2006) Biogeographical distribution and diversity of microbes in methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin. Proc Nat Acad Sci USA 103:2815-2820.Jørgensen BB (1982) Mineralization of organic matter in the sea bed - the role of sulphate reduction. Nature 296:643-645. Lever MA (2012) Acetogenesis in the energy-starved deep biosphere – a paradox? Frontiers in Microbiology 2:1-18.Lever MA (2013) Functional Gene Surveys from Ocean Drilling Expeditions – A Review and Perspective, FEMS Microbiol Ecol. 84:1-23.Lever MA, 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 MA, Rogers K, Lloyd KG, Overmann JO, Schink B, Thauer RK, Hoehler TM, Jørgensen BB. Microbial life under extreme energy limitation: a synthesis of laboratory-based experiments and in-situ evidence from the deep biosphere, FEMS Microbiology Reviews, under revision.Oren A (2012) There must be an acetogen somewhere. Commentary on “Lever MA. Acetogenesis in the energy-starved deep biosphere – a paradox?” Frontiers in Microbiol 3:1-2.Parkes RJ, Cragg BA, Wellsbury (2000) Recent studies on bacterial populations and processes in subseafloor sediments: a review. 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