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The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New... Marine Systems. PLoS ONE 11(11): e0165635. doi:10.1371/journal.pone.0165635 Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the Editor: Zhe-Xue Quan, Fudan University, CHINA amount of methane (CH ) released into the environment. AMO acts as a sink of CH , con- 4 4 verting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)cli- Received: September 5, 2016 mate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO Accepted: October 15, 2016 events, especially at times when the role of methane in the carbon cycle was more pro- Published: November 8, 2016 nounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) Copyright:© 2016 Rush et al. This is an open lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34- access article distributed under the terms of the pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the Creative Commons Attribution License, which BHP compositions were examined for species of the recently described novel Type I permits unrestricted use, distribution, and reproduction in any medium, provided the original methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a author and source are credited. novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP Data Availability Statement: All relevant data are only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopen- within the paper and its Supporting Information tol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel struc- files. tures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC- Funding: The work was funded by a Starting Grant BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subse- (No. 258734) awarded to HMT for project quently, sediments and authigenic carbonates from methane-influenced marine environ- AMOPROX, from the European Research Council. ments were analysed. Most samples also did not contain significant amounts of KM and JCN thank Lundin Petroleum for financial support. The funder had no role in study design, aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 1 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Competing Interests: The authors have declared methanotophic bacteria. However, the BHP composition of the marine samples do point that no competing interests exist. toward the novel MC-BHPs components being potential new biomarkers for AMO. Introduction Methane (CH ) is a potent greenhouse gas, and its atmospheric concentration has tripled since pre-industrial times (e.g. [1,2]). Global oceans hold large subsurface reservoirs of CH in the form of gas hydrates. These stores are precariously dependent on temperature and pressure. A rapid destabilisation of gas hydrates has been proposed to have caused vast releases of marine CH in the past [3]. Increased input of CH into the atmosphere has been interpreted through 4 4 records of excursions of significantδ C depletion in the geological record, such as in the Palaeocene-Eocene Thermal Maximum (PETM) [4–7]. CH release into the atmosphere is regulated by methanotrophy, which converts CH into 4 4 CO , thereby playing a key role in the carbon biogeochemical cycle [8]. Although traditionally anaerobic archaea have been the most studied methanotrophs (e.g., AMNE-1 and ANME-2; cf. [9]), recent observations have highlighted the importance of bacteria performing aerobic CH oxidation (AMO) in marine, estuarine, and riverine fan environments (e.g., [10–14]). For example, pelagic AMO activity rose significantly after the Macondo oil well blowout in 2010 [15]. However, this activity was short-lived, highlighting the complexity of natural community interactions in response to increased CH [16]. It is thus important to recognise and trace methanotrophy during past extreme events in order to understand its potential to mitigate future CH release. AMO bacteria belong to two phyla, Proteobacteria and Verrucomicrobia. Most isolates of Verrucomicrobia are thermoacidophilic [17–20], and have been found primarily in acidic, geo- thermal environments [21]. Aerobic methanotrophic members of Proteobacteria belong to two distinct classes, separated based on their carbon assimilation pathways [8]. Type II methano- trophs, members of the Alphaproteobacteria, are associated with terrestrial settings ([8], and references therein), whereas Type I methanotrophs members of the Gammaproteobacteria are widespread in aquatic systems, although they are also found in terrestrial systems. Both Type I and Type II methanotrophs are known to synthesise bacteriohopanepolyol (BHP) lipids [22]. BHPs are precursors to hopanes, which are the most ubiquitous lipids in the geological record [23,24]. Therefore, being able to trace AMO using hopanoid biomarkers is of value to the study of the carbon cycle in the past. Previous work exploring hopanoids as biomarker lipids found that methylation at the C-3 3Me 3Me 3Me position (3-Me-BHPs; Fig 1, I , II , III ) was a possible indication of methanotrophic origin [25,26]. However, attributing C-3 methylation to AMO was challenged by the revelation that bacteria other than methanotrophs have the genes to methylate at C-3 [27]. C-3 methyla- tion is more likely a requirement for cell survival in late stationary phase [27]. Moreover, not all methanotrophic bacteria have the gene to methylate at this position, [27,28], nor are 3-Me BHP precursors found in all CH -influenced environments (Table 1). However, AMO bacteria are often considered to be the most likely source of 3-Me hopanoids in marine sediments due to their depleted carbon isotope signatures (e.g., [29]) and because they are frequently accom- panied by 4-methylated steroids, which are also known as biomarkers of methanotrophic bac- teria (e.g., [30,31]). In other studies, diplopterol and diploptene have been interpreted as biomarkers for methanotrophy, notably in anoxic environments [32–35]. However, neither diplopterol nor diploptene is source-specific to AMO [36], and these studies also relied on very PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 2 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems 3Me MC Fig 1. Chemical structures of bacteriohopanepolyol lipids. I, aminopentol; I , 3-methyl-aminopentol; I , methylcarbamate-aminopentol; II, 3Me MC 3Me MC aminotetrol; II , 3-methyl-aminotetrol; II , methylcarbamate-aminotetrol; III, aminotriol; III , 3-methyl-aminotriol; II , methylcarbamate-aminotriol. MC MC MC The proposed structure of methylcarbamate(MC)-aminopentol (I ), MC-aminotetrol (II ), and MC-aminotriol (III ) are tentatively based on mass spectral identification (S1 File). doi:10.1371/journal.pone.0165635.g001 negativeδ C lipid values (e.g., ca. -61‰ for diplopterol; -61 to -74‰ for diploptene) as an indication of CH being the carbon source for the organism producing these lipids. Yet, recent work has emphasised that not all AMO-derived carbon shows a depleted isotope signature, especially in terrestrial systems where Type II methanotrophs tend to dominate. For example, only limited depletion in C, with values between -25‰ and -40‰, have been reported for hopanoids with an inferred methanotrophic origin from modern peat bogs [37,38] as well as other ancient lignite deposits [39]. Also, BHPs in Congo deep sea fan sediments, originating from low-latitude wetlands [40], had higher C values than expected (i.e., C hopanol ~ -41‰; [14]). In marine CH seep carbonates from the Gulf of Mexico, BHPs, hopanoic acids, and 4-methylated sterols of aerobic methanotrophs were found with similarδ C values as the CH source [41]. These observations can be the result of dilution from other heterotrophic bac- terial sources that make it difficult to identify subordinate methanotroph contributions [37]. The metabolic pathways used by the AMO bacteria for CH assimilation can also have a pro- found effect on the level of isotopic depletion, with values for serine pathway methanotrophs (Type II) ranging from 12‰ depleted to 10‰ enriched relative to the CH substrate [42]. Fur- thermore, to analyseδ C of intact BHPs, these must first be converted into primary alcohols by periodic acid/sodium borohydride cleavage [43]. For example, theδ C value of 35-amino- bacteriohopane-30,31,32,33,34-pentol (aminopentol; Fig 1, I) is measured on the C -hopanol product, which includes all converted hexa-functionalised BHPs (i.e., not only aminopentol), as well as any free-hopanols that are present in a sample before BHP conversion. While the contamination of free-hopanols can be circumnavigated by column separations (e.g., [41]), measuring theδ C values of intact BHPs is not currently possible. Aminopentol is thought to be the most diagnostic BHP for AMO (see review in [14]). Ami- 3Me nopentol and its methylated and unsaturated homologues (i.e., I ,ΔI) have been found PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 3 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 1. Presence and absence of aminopentol and related methylated and unsaturated homologues in previously investigated environmental settings. aminopentol aminotetrol aminotriol 3Me 3Me 3Me Sample I ΔI I II II III III Reference Soils Pasture [manured] (UK) + - - + - + + [112] Pasture [not manured] (UK) - - - + - + + [112] Rice Paddy (Vietnam) + - - + - + - [112] Woodland (North East England) + - - + - + + [112] South West France + - - + - + - [116] Amazon + - - + - + - [109] Congo + + - + - + + [40] Lean Delta Peamafrost [ice complex] + - - + - + - [110] Human Sewage [treated] - - - + - + - [117] Forest, Grassland Soils (Alberta, Canada) - - - + - + - [118] Peat and Lignite River Tet Catchment (France) + - - + - + - [116] Moorhouse (UK) + - - + - + - [46] Misten Bog (Belgium) + - - + - + - [119] Bisendorfer Moor (Germany) + - - + - + + [111] The Cobham Lignite (UK) + - - + - + - [49] Geothermal Environments + Cyanobacterial mat (Surprise Valley, Nevada, USA) + - - + - + - [120] Silica Sinter (Orakie Korako, Taupo Volcanic Zone, New Zealand) + - + + - + - [113] Silica Sinter (Champagne Pool, Taupo Volcanic Zone, New Zealand) - - - - - + - [121] Orange mat (Yellowstone, USA) - - - - - + - [122] Other Microbial mats Mars Oasic (Antarctica) - - - - - + - [48] Hypolith (Devon Island, Arctic) - - - - - + - [48] Cyanobacterial Mat (Christmas Island, Kiribati) - - - - - + - [123] Lake Sediments Lake Windermere (UK) + - - + - + - Lake Cadagno (Switzerland) + - - + - + - [26] Lake Holzmaar (Germany) + - - + - + - [26] Lake Nkunga (Kenya) + - - + - + - [45] Priest Pot (England) + - - + - + - [26,45] La Piscina de Yuriria (Mexico) + - + + + + - [26,45] Laguna de Zempoala (Mexico) + - - + - + - [26] Loch Ness (UK) + - - + - + - [26,45] Lake Druzhby (Antarctica) + - - + - + - [26,45] Sombre Lake (Signy Island, Southern Atlantic Ocean) + - - + - + - [26] Heywood Lake (Signy Island, Southern Atlantic Ocean) + - - + - + - [26] Ace Lake [Freshwater unit] + - - + - + - [104] Ace Lake [meromictic unit] - - - + - + - [104] Ace Lake [marine unit] - - - - - + - [104] Wetlands Amazon + + - + - + - [109] Congo + + - + - + - [40] Stream/River/Estuary Sediments (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 4 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 1. (Continued ) aminopentol aminotetrol aminotriol 3Me 3Me 3Me Sample I ΔI I II II III III Reference Arctic Rivers (Indigirka, Kolyma, Lena, Ob, Yenisei, Yukon, Mackenzie) + - - + - + - [124] Glacial Out¯ow Stream (Svalbard) + - - + - + - [125] Kalix River [surface sediment transect] + - - + - + - [126] Kolyma River [surface sediment transect] + - - + - + - [127] Yenisei River [mouth, and surrounding area] + - - + - + - [128] Yangtze River [estuary, inner shelf] + - - + - + - [82,129] Congo River [estuary] + + - + - + - [14,40] Water Column Priest Pot [pond] (UK) + - - + - + - River Water (Panama) + - - + - + - [130] Yenisei River + - - + - + - [128] Black Sea [oxic/anoxic transition zone] + - + + - + - [12] Black Sea [oxic/anoxic transition zone] + - - + - + - [108] Gotland Deep, Baltic Sea + - - + - + - [107] Marine Water (off Panama coast) - - - - - + - [130] Pelagic [sub-oxic and anoxic] (Arabian Sea, Peru Margin, Cariaco Basin) - - - - - + - [90] Cariaco Basin - - - - - + - [131] California Current - - - - - + - [91] Modern/Recent Marine Sediments Gotland Deep, Baltic Sea + - - + - + - [107] Congo River Deep-Sea Fan + + - + - + - [14] Amazon Shelf and Fan + - - + - + - [109] East Siberian Arctic Shelf + - - + - + - [79,126] Black Sea - - - + - + - [88] Southwest African Coast - - - + - + (+) [89] Chukchi Sea - - - + - + - [132] Alaskan Beaufort Sea - - - - - + - [132] Baltic Sea - - - + - + - [133] Marine Carbonates Carbonate, Gulf of Mexico - - - + - + - [72] Authigenic carbonates, Gulf of Mexico - - - + - + - [41] Seep Carbonate, Arabian Sea - - - + - + - [106] Other samples Membrane Foulant [river water] + - - - - + - [134] Membrane Foulant [brackish water] + - - + - + - [134] Membrane Foulants [seawater] - - - - - + - [134] Mussel Gill Tissue - - - + - + - [103] +, detected in at least one of the samples; -, not detected in any of the samples (+) indicates a methylated aminotriol was detected but the position of methylation was not identi®ed ¶Talbot and Farrimond, unpublished data doi:10.1371/journal.pone.0165635.t001 almost exclusively in Type I aerobic methanotrophs [22,44–46]. Moreover, aminopentol (I) has been found in a wide range of environments, which indicates potential as a biomarker for AMO (Table 1). Additionally, 35-aminobacteriohopane-31,32,33,34-tetrol (aminotetrol, II) and 35-aminobacteriohopane-32,33,34-triol (aminotriol, III) are also synthesised by Type I PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 5 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems and Type II AMO bacteria. However, II and III are less source-specific as both are synthesised by some species of sulfate reducing bacteria (SRB) of the genus Desulfovibrio [47], and III is synthesised by many other aerobic bacteria ([48], and references therein). Aminopentol made up a very minor proportion of the BHP composition in a SRB culture (<0.1% of total BHPs in Desulfovibrio salexigens). The ratio of aminopentol to the more ubiq- uitous aminotriol was 1:1352 [47]. We can therefore discount SRB as the source of aminopen- tol in an environmental sample with a high ratio of aminopentol:aminotriol. A similar approach was recently used by [49] based on the ratio of aminotetrol:aminotriol which has been found in the range 1:20–100 in some species of Desulfovibrio SRB [47,50,51]. Interest- ingly, some of the species of SRB cultures analysed by Blumenberg et al. [47,50,51] also synthe- sised diplopterol and diploptene, which could explain the enhanced presence of these lipids in CH -influenced anoxic sites. Representatives from only a small number of Type I methanotroph genera have been tested for BHP production (e.g., [22,36,46,52,53]. Many more recently described genera, including novel genera from marine and other (hyper)saline environments, have yet to be explored (e.g., [54–56]). Moreover, relatively few studies of BHP distributions have targeted marine environ- ments (Table 1). It is important, therefore, to determine whether aminopentol, which is seen as a diagnostic marker for Type I methanotrophs, is present in methanotrophs isolated from marine and other saline environments, and whether we can find aminopentol in CH -influ- enced marine sediments. This knowledge will have implications for the use of aminopentol as a biomarker to trace AMO in modern and ancient marine environments. In this study, our goal was to develop an appropriate biomarker approach for AMO, which will allow high throughput analysis of sediment without the requirement for laborious chemical conversion steps prior to compound specific isotope analysis. To this end, we screened the BHP distributions of three genera of aerobic methanotrophs (i.e., Methylomicrobium, Methylomari- num and Methylomarinovum). Methylomarinum and Methylomarinovum have not previously been investigated for BHPs. Two species of Methylomicrobium have been described previously [52,53], however, we include an additional species Methylomicrobium kenyense. These data are combined with literature BHP distributions of other AMO genera, including the recently reported Type I genus, Methylobacter [28], in order to facilitate interpretation of BHP distribu- tions in six selected modern marine sediments from CH -influenced systems. Two marine sites not influenced by CH were also investigated as controls for background marine BHP signatures. Methods Methanotroph pure cultures Cultivation of Methylomarinum vadi IT-4. Previously described Methylomarinum vadi IT-4 was isolated from a microbial mat sample (in-situ temperature 30–40°C) collected at a shallow marine hydrothermal system (depth, ~23 m) in a coral reef off Taketomi Island, Oki- nawa, Japan [56,57]. Cultivation of this strain was performed at JAMSTEC, Japan, using MJmet medium at pH 6.6 at 37°C. A detailed site description and the enrichment and isolation procedures can be found in [57]. Cultivation of Methylomarinovum spp. Previously described Methylomarinovum caldi- curalii IT-9 was isolated from the hot vent fluid (52°C) collected at the main vent site (depth, 23 m) in the shallow marine hydrothermal system where Methylomarinum vadi IT-4 was iso- lated [55,57]. Methylomarinovum sp. IN45 is a new isolate from a deep-sea hydrothermal field in Okinawa Trough, Japan (H. Hirayama, pers. comm.). The cultivation of strains IT-9 and IN45 was performed at JAMSTEC, Japan, using MJmet medium at 45°C, and at pH 6.2 and 6.6, respectively. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 6 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Cultivation of Methylomicrobium spp. Methylomicrobium alcaliphilum and Methylomi- crobium kenyense were first isolated from highly alkaline soda lakes in Russia and Kenya, respectively [58]. Both M. alcaliphilum (DSM-number 19304) and M. kenyense (DSM-number 19305) are from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures). The cultivation of both strains was done at the Center for Applied Geosciences at the University of Tübingen, Germany, at pH 9.1 and at 28°C with a high salt NMS medium (1.5% NaCl). Marine sediment and carbonate samples Håkon Mosby Mud Volcano (HMMV). HMMV has been extensively studied for both aerobic and anaerobic methanotrophy [13,59]. The flow of CH in the center of the HMMV is restricted, and AMO was observed to be the most dominant process within the centre of the crater, performed by Type I methanotrophs [60]. Sediments from the HMMV were collected aboard the RV Polarstern (cruise ARK XXIV/2; 2009) and the RV Maria S Merian (cruise MSM 16/2; 2010) [61]. Barents Sea carbonate crust (BSCC). The Barents Sea is a well-studied area of active CH seepage. The seafloor geology is marked by pockmarks [62], gas hydrates, gas flares [63], and patches of carbonate crusts [64], indicating active CH seepage [10,64]. We analysed eight depths (each 2–3 cm thick) from a push core (P120020 PR4) of a cold seep carbonate crust sampled at Loppa High/Polheim Sub-Platform area in the SW Barents Sea (72° 34’02.07”N, 20° 52’05.96”E). The core penetrated to a depth of 19 cm below sea floor (cmbsf ). These samples were taken aboard the RV Fugro Meridian in September 2012 by Lundin Petroleum Norway. Amon Mud Volcano (AMV). The Amon mud volcano (AMV) is located in the Nile deep-sea fan, in the Eastern Basin of the Mediterranean Sea. Hydrocarbons, muds, and fluids are transported to the surface via one main feeding channel from the deep subsurface, creating a stark thermal gradient in the sediment. Oxidation of CH in the water column directly above 13 2 AMV has been attributed to AMO using C and H isotopic values [65]. Sediments from AMV were collected aboard the RV Meteor (cruise M70/2, BIONIL; 2006) and the RV Maria S Merian (cruise MSM 13/3; 2009) [66]. New Zealand Seeps (NZS). The Hikurangi continental margin, east of New Zealand’s North Island has been described as a biogeographically “new” cold seep province, characterised by endemic faunal communities [67]. Surface (0–2 cmbsf) and subsurface (10–12 cmbsf) sedi- ment samples from three New Zealand seep (NZS) sites were collected aboard the RV Sonne (cruise So-191; 2007) [68]. These were dominated by distinct biota: Frenulata (Omakere Ridge), Ampharetidae (Wairarapa Takahae), and sulfur-oxidising bacteria (Wairarapa Takahae). Gulfo Dulce (GD) surface sediments. A sill at 60 m water depth physically cuts off Golfo Dulce from the Pacific Ocean, which promotes anoxic conditions within the basin. Recently, GD was shown to contain authigenic carbonate formations at shallow (ca. 10 m) water depth [69]. GD sediments were collected along a transect from 10 to 140 m water depth in March 2008, as described in [70]. Gulf of Mexico (GoM) cold seeps. The Gulf of Mexico (GoM) holds an abundance of thermogenic gas. The venting of this gas from deep subsurface forms gas hydrates, free CH , which are the sources of energy for microbial chemosynthetic communities, and authigenic carbonates [71]. Pancost et al., [72] described the BHP composition of material from five car- bonate rock and nodule sites in GoM. Two of the carbonates were shown to contain low quan- tities of aminopentol. Here, we reinvestigated the BHP signatures of these sediments. GoM sediments. The GoM also houses the outflow of the Mississippi River Delta, trans- porting terrestrial material into the Gulf. Three GoM sites to the best of our knowledge not PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 7 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems influenced by CH (27°30'N, 87°20'W; 28°20'N, 89°38'W; 26°50'N, 92°40'W; two sediment depths at each site) were investigated in this study [73]. Peru Margin (PM). An intense upwelling regimes fertilises surface water productivity on the Peru Margin (PM). This lends to oxygen utilisation in the water column, causing the East- ern South Pacific Oxygen Minimum Zone (ESP OMZ) [74]. Three PM sediments were ana- lysed (10–15, 20–25 and 40–45 cmbsf ) from a core taken within the ESP OMZ, at 100 m water depth [73,75]. Lipid extraction Total lipid extraction. All freeze-dried bacterial cells and marine sediments, except the Barents Sea samples extracted at GFZ Potsdam, were extracted using a modified Bligh-Dyer method [76,77]. Briefly, freeze-dried material was extracted in 19 mL of a 10:5:4 (v:v:v) mixture of MeOH:chloroform:H O in a 50 mLTeflon tube. This mixture was sonicated for 15 min at 40°C, and centrifuged for 10 min. The supernatant was transferred to a second tube, and the residue re-extracted twice more. The chloroform in the supernatant was separated from the aqueous phase by adding water until the H O:MeOH ratio was 1:1 (v:v), and collected. This procedure was repeated for the subsequent extractions. The collected chloroform total lipid extract (TLE) was dried by rotary evaporation in a round-bottom flask. The extraction protocol at GFZ Potsdam was similar but used a mixture of MeOH:DCM:ammonium acetate buffer [78]. Solid Phase Extraction. In-house comparisons have shown that amino-BHPs are better detected after solid phase extraction (SPE). An aliquot of the TLE was separated over a 1 mg NH solid phase extraction cartridge, as described in [79]. Briefly, the aliquot was dissolved and loaded onto a hexane-rinsed cartridge using 200 μL chloroform. Six mL of a 98:2 (v:v) diethyl ether:acetic acid solution was eluted. Residual material was dissolved with 200 μL 2:1 (v:v) chloroform:MeOH and loaded onto the cartridge, followed by elution with 10 mL of MeOH. BHPs were isolated from the MeOH fraction. Lipid analyses BHP preparation and HPLC/APCI–MSn analyses. A known amount (ca. 5–10 μg/g dry sediment) of internal standard (5α-pregnane-3β,20β-diol) was added to SPE extracts of the TLE for BHP analysis. Samples were acetylated in 0.5 mL of a 1:1 (v:v) mixture of acetic anhy- dride and pyridine at 50°C for 1 h, then left to stand overnight at room temperature [80]. Sol- vent was dried under a stream of N on a 50°C heating block. BHP samples were dissolved in MeOH:propan-2-ol (3:2; v:v), and filtered on 0.2 μm PTFE filters. BHPs were analysed by high performance liquid chromatography coupled to positive ion atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS), using a data- dependent scan mode (2 events) on an HPLC system equipped with an ion trap MS, as described in [46,81]. Further structural information for novel BHPs was obtained by way of MS spectra. BHP concentrations were (semi) quantitatively estimated based on the response factor of authentic standards (M. Rohmer; Strasbourg, France and [46,77]) relative to the inter- nal standard. Results In this study we investigated the BHP distributions in species of three AMO marine genera, and of eight marine environments, six of which were CH -influenced. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 8 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Novel nitrogen-containing BHP components In addition to the ‘regular amino-BHPs’ (e.g., I, II, and III; Figs 1 and 2), a suite of novel com- pounds were found in the methanotrophs and screened marine samples. Identification of these compounds is described in detail in the Supplementary Information (S1 File). Briefly, these components were related to the 35-amino-BHPs but differ in their terminal groups at C-35, which are tentatively proposed to comprise a methylcarbamate rather than a simple amine on 2 3 the basis of interpretation of their APCI MS and MS spectra. In each case, the novel com- MC MC MC MC pounds (I , I ’, II , III ; Figs 1 and 2) elute after their ‘regular’ amino-BHP analogues (i.e., I, I’, II, III; Fig 2). This indicates that the tentatively-assigned terminal group structures are less polar than the regular terminal amines (after acetylation). The novel structures include: MC 35-methylcarbamate-bacteriohopane-32,33,34-triol (MC-triol herein; III ), 35-methylcarba- MC mate-bacteriohopane-31,32,33,34-tetrol (MC-tetrol herein; II ), 35-methylcarbamate-bac- MC MC MC teriohopane-30,31,32,33,34-pentol (MC-pentol herein; I ) and an isomer of I (I ’) akin Fig 2. Distribution of nitrogen-containing BHPs in novel Type I methanotroph cultures. Partial mass chromatograms (15±25 min) showing relative abundances (%) of BHPs (shaded peaks) in the acetylated total lipid extracts of (a) Methylomarinum vadi IT-4, (b) Methylomarinovum caldicuralii IT-9, (c) Methylomarinovum sp. IN45, (d) Methylomicrobium alcaliphilum, and (e) Methylomicrobium kenyense. doi:10.1371/journal.pone.0165635.g002 PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 9 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems to the early-eluting aminopentol isomer (I’), which was found, based on mass spectra, in a cul- ture of Methylovulum-like strain M200 [46]. Methanotroph BHP signatures Four previously untested methanotrophs isolated from marine or saline, alkaline lacustrine environments, belonging to the three genera Methylomarinum, Methylomarinovum, and Methylomicrobium, were analysed for their BHP composition. An additional species Methylo- microbium alcaliphilum, the partial BHP composition of which was recently reported in [53], is also shown here in full for comparison with Methylomicrobium kenyense. All of the metha- notroph cultures investigated only synthesised BHPs with a nitrogen atom at C-35 position (nitrogen-containing BHPs herein). The relative abundances of BHPs are indicated as the per- centage of total BHPs in acetylated extracts, and are presented in Fig 2. The low starting mass of some of the dry cell material led to uncertainty in the calculations of absolute concentrations, which are therefore not reported. BHP inventory of Methylomarinum vadi IT-4. The most abundant BHPs in Methylo- MC marinum vadi IT-4 were aminotriol (III), 22.3% and MC-triol (III ), 22.7% (Fig 2A). Ami- MC MC nopentol (I), MC-pentol (I ), aminotetrol (II), and MC-aminotetrol (II ) made up 15.4%, 14.0%, 11.1%, and 11.8% of total BHPs, respectively, with lower levels of the aminopentol iso- MC mers (I’ and I ’; Fig 2A). No C-3 methylated or unsaturated equivalents of aminotriol, ami- notetrol and aminopentol were found in Methylomarinum vadi IT-4. BHP inventory of Methylomarinovum spp. The most abundant BHP in Methylomarino- vum caldicuralii IT-9 was aminopentol (I), 37.4% (Fig 2B). However, Methylomarinovum sp. IN45 had only 5.1% aminopentol (Fig 2C). M. caldicuralii IT-9 had 30.8% MC-pentol, 14.4% aminotetrol, and 13.3% MC-tetrol, and lower abundances of aminopentol isomer, MC-pentol isomer, aminotriol, and MC-triol (Fig 2B). The most abundant BHP in Methylomarinovum sp. MC IN45 was MC-tetrol (II ), making up 51.1%. Methylomarinovum sp. IN45 also contained 12.7% aminotetrol, 15.6% MC-pentol, 12.1% MC-triol and lower levels of aminotriol, and the aminopentol isomers (Fig 2C). No C-3 methylated nor unsaturated equivalents of aminotriol, aminotetrol and aminopentol were present in either strains. BHP inventory of Methylomicrobium spp. The Methylomicrobium alcaliphilum and Methylomicrobium kenyense cultures did not contain aminopentol (I) above detection limit (Fig 2D and 2E) although M. kenyense was found to contain minor abundance of 3-Me-amino- 3Me pentol (I ; 1.0%; Fig 2E). The most abundant BHP in both Methylomicrobium cultures was aminotriol (III), making up ca. 65% of all BHPs in both species. The second most abundant 3Me BHP was 3-Me-aminotriol (III ) at 31.5% in M. alcaliphilum and slightly less in M. kenyense (23.9%). Both species also contained lower levels of aminotetrol (II) and 3-Me-aminotetrol 3Me 3Me (II ). M. kenyense also contained unsaturated compounds (ΔIII andΔIII ; Fig 2E). The only MC compound identified in either Methylomicrobium sp. was MC-triol and then only at low levels (<3%). Marine sediment and carbonate BHP signatures Eight marine settings were studied for their BHP signatures (Table 2). Six of these were known to be influenced by CH (i.e., HHMV, BSCC, AMV, NZS, GoM cold seeps, GD) and two were used as comparison background marine levels (i.e., GoM sediments, PM). Håkon Mosby Mud Volcano (HMMV). None of the HMMV sediment samples contained aminopentol (Table 2). The most abundant amino- and MC-BHPs in HMMV samples was CME either aminotriol (III) or MC-triol (III ), making up 22–52% and 32–62% of total nitrogen- containing BHPs, respectively. Unsaturated aminotriol, unsaturated MC-triol, aminotetrol, PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 10 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. Concentrations (μg/g sediment) of amino-BHPs in marine sediment samples. Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 Håkon Mosby Mud Volcano (HMMV) new mud ¯ow 0±1 PS74/2 169±1 bdl bdl bdl bdl 0.02 0.01 bdl 0.17 0.02 0.14 bdl 0.01 bdl 0.38 cm PUC3 229 aged ¯ow 0±1 cm ARK XXIV-2 bdl bdl bdl bdl 0.01 0.01 bdl 0.29 0.05 0.81 bdl 0.13 bdl 1.3 PS74 172±1 237 center 0±1 cm MSM16/2 847±1 bdl bdl bdl bdl 0.01 0.01 bdl 0.63 0.09 1.26 bdl 0.19 bdl 2.19 MUC 53 center 10±12 cm MSM16/2 HMMV bdl bdl bdl bdl 0.01 0.02 bdl 0.17 0.02 0.17 bdl 0.02 bdl 0.41 new mud ¯ow 0±1 MSM16/2 838±1 bdl bdl bdl bdl 0.02 0.01 bdl 1.07 0.09 0.82 bdl 0.06 bdl 2.07 cm MUC 33 newer mud ¯ow 0±1 MSM16/2 855±1 bdl bdl bdl bdl 0.01 0.01 bdl 1.1 0.13 1.24 bdl 0.15 bdl 2.64 cm MUC 73 newer mud ¯ow 10± MSM16/2 HMMV bdl bdl bdl bdl 0.02 0.01 bdl 0.17 0.02 0.11 bdl 0.01 bdl 0.33 12 cm 83 aged ¯ow 0±1 cm MSM16/2 823±1 bdl bdl bdl bdl 0.02 0.02 bdl 0.56 0.05 0.88 bdl 0.07 bdl 1.61 MUC 19 aged ¯ow 10±12 cm MSM16/2 HMMV bdl bdl bdl bdl 0.01 0.01 bdl 0.15 0.01 0.11 bdl 0.01 bdl 0.29 Barents Sea Carbonate Crusts (BSCC) depth pro®le 0±2 cmbsf bdl bdl bdl bdl 1.36 bdl bdl 4.45 0.12 bdl bdl bdl bdl 5.93 2±4 cmbsf bdl bdl bdl bdl 0.21 bdl bdl 0.93 0.06 bdl bdl bdl bdl 1.2 4±6 cmbsf bdl bdl bdl bdl 0.04 bdl bdl 0.27 0.03 0.02 bdl bdl bdl 0.36 6±8 cmbsf bdl bdl bdl bdl 0.03 bdl bdl 0.19 0.02 0.02 bdl bdl bdl 0.26 8±10 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.07 bdl 0.02 bdl bdl bdl 0.09 10±13 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.03 bdl 0.01 bdl bdl bdl 0.04 13±16 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.08 bdl bdl bdl bdl bdl 0.08 16±19 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.11 bdl bdl bdl bdl bdl 0.11 Amon Mud Volcano (AMV) central dome 0±1 cm M70/2a 760 bdl bdl bdl bdl 0.02 0.07 bdl 0.42 0.13 2.67 bdl 0.96 bdl 4.27 PUC33 24 sulfur band 0±1 cm M70/2a 765 0.18 bdl 0.12 bdl 0.38 0.27 bdl 1.24 0.81 1.33 bdl 1.02 bdl 5.35 PUC49+50 71 sulfur band 10±12 M70/2 81 0.36 0.04 0.11 bdl 0.3 0.11 bdl 1.07 1.09 0.48 bdl 0.5 bdl 4.06 cm sulfur band 0±1 cm M70/2a 790 0.36 bdl 0.11 bdl 1.49 0.49 bdl 3.82 1.29 1.49 bdl 0.61 bdl 9.67 PUC68 172 sulfur band, white MSM13/3 947±1 0.09 bdl 0.11 bdl 0.31 0.24 bdl 0.64 0.36 0.71 bdl 0.44 bdl 2.9 mat PUC31 73D sulfur band 0±1 cm MSM13/3 968±1 0.08 bdl 0.06 bdl 0.75 0.49 bdl 0.73 0.46 0.77 bdl 0.28 bdl 3.62 PUC15 122 bacterial mats 0±1 MSM13/3 929±1 0.05 bdl 0.01 bdl 1.13 0.17 bdl 1.11 0.43 0.5 bdl 0.16 bdl 3.56 cm PUC22 45D (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 11 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. (Continued ) Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 bacterial mats 10±12 MSM13/3 50D bdl bdl bdl bdl 0.01 bdl bdl 0.03 bdl 0.02 bdl bdl bdl 0.07 cm gassy centre, 10±12 MSM13/3 61D bdl bdl bdl bdl 0.01 bdl bdl 0.04 0.01 0.02 bdl 0.01 bdl 0.08 cm New Zealand Seeps (NZS) 0±2 cmbsf Frenulata 45 bdl bdl bdl bdl 0.09 0.06 bdl 0.21 0.03 0.29 bdl 0.05 bdl 0.73 10±12 cmbsf Frenulata 45 bdl bdl bdl bdl 0.13 0.03 bdl 0.26 0.04 0.15 bdl 0.02 bdl 0.63 0±2 cmbsf Ampharetidae 0.06 bdl bdl bdl 1.11 0.17 bdl 1.32 1.17 2.59 bdl 0.26 bdl 6.67 10±12 cmbsf Ampharetidae bdl bdl bdl bdl 0.01 0.02 bdl 0.08 0.02 0.13 bdl 0.02 bdl 0.28 0±2 cmbsf Sulfur-oxidising 0.02 bdl bdl bdl 0.66 0.32 bdl 2.24 0.23 2.13 bdl 0.21 bdl 5.8 bacteria 315 10±12 cmbsf Sulfur-oxidising bdl bdl bdl bdl 0.03 bdl bdl 0.14 0.02 0.1 bdl bdl bdl 0.28 bacteria 315 Golfo Dulce (GD) Surface Sediment 10 m water depth SG 1 bdl bdl bdl bdl bdl bdl bdl 0.01 bdl bdl bdl bdl bdl 0.01 24 m water depth SG 2 0.16 bdl bdl bdl 0.03 bdl bdl 0.36 bdl 0.02 bdl bdl bdl 0.57 77 m water depth SG 4 0.02 bdl bdl bdl 0.01 bdl bdl 0.15 0.03 0.01 bdl bdl bdl 0.23 90 m water depth SG 5 0.04 bdl bdl bdl 0.02 bdl bdl 0.17 0.05 0.01 bdl bdl bdl 0.29 110 m water depth SG 6 bdl bdl bdl bdl 0.01 bdl bdl 0.08 0.07 0.02 bdl bdl bdl 0.18 120 m water depth SG 7 0.04 bdl bdl bdl 0.02 bdl bdl 0.36 0.2 0.02 bdl bdl bdl 0.63 140 m water depth SG 8 0.01 bdl bdl bdl <0.01 bdl bdl 0.11 0.08 0.01 bdl bdl bdl 0.21 Gulf of Mexico (GoM) Cold Seeps GC 234 CNSTS bdl bdl bdl bdl 0.01 bdl bdl 0.09 bdl bdl bdl bdl bdl 0.1 234 4436 bdl bdl bdl bdl <0.01 bdl bdl 0.04 bdl bdl bdl bdl bdl 0.04 GC 234 tube worm bdl bdl bdl bdl <0.01 bdl bdl 0.02 bdl bdl bdl bdl bdl 0.02 roots 4435 GC 232 bdl bdl bdl bdl 0.01 bdl bdl 0.01 bdl bdl bdl bdl bdl 0.02 GC 185 bdl bdl bdl bdl 0.76 bdl bdl 0.2 bdl bdl bdl bdl bdl 0.95 GoM Sediments West Gulf WG2-099/6 bdl bdl bdl bdl <0.01 bdl bdl 0.01 bdl bdl bdl bdl bdl 0.01 26Ê50'N, 92Ê40'W WG2-099/9 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl <0.01 bdl bdl bdl 0.01 Eastern Gulf 88-C-1 DCS 128/ bdl bdl bdl bdl bdl bdl bdl 0.01 bdl 0.01 bdl bdl bdl 0.02 27Ê30'N, 87Ê20'W DCS 128/13 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl <0.01 bdl bdl bdl 0.01 DCS 128/21 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl 0.01 bdl bdl bdl 0.01 Central Gulf CGD-136/17 bdl bdl bdl bdl bdl bdl bdl 0.01 bdl 0.01 bdl bdl bdl 0.01 28Ê20'N, 89Ê38'W CGD-136/13 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl 0.01 bdl bdl bdl 0.01 Peru Sediment 10±15 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.09 bdl bdl bdl bdl bdl 0.09 20±25 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.02 bdl bdl bdl bdl bdl 0.02 (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 12 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. (Continued ) Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 40±45 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.09 bdl bdl bdl bdl bdl 0.09 base peak = [M + H±CH3COOH]+ bdl±below detection limit doi:10.1371/journal.pone.0165635.t002 and MC-tetrol all made up <10% of total nitrogen-containing BHPs. Some HMMV samples contained relatively high concentration of anhydrobacteriohopanetetrol (anhydro-BHT). Minor abundances of BHT and BHT isomer, BHT-cyclitol ether (BHT-CE), and BHT-glucos- amine (BHT-G) were detected in some samples (S1 Table). Barents Sea carbonate crust (BSCC). The 19 cm BSCC core contained a majority of ami- MC notriol (III; 72–100% of nitrogen-containing BHPs; Table 2). MC-triol (III ; 0–23%) peaked between 4 and 13 cm. Aminotetrol (II; 0–23%) was detected in the upper 8 cm. Minor contri- bution of unsaturated aminotriol (ΔIII; 0–7%) was detected in the upper sediments. Other BHPs detected included BHT, and low concentrations of anhydroBHT, BHT isomer, 2-methyl-BHT (2-Me-BHT), adenosylhopane, BHT-CE, and BHT-G (S1 Table). Amon Mud Volcano (AMV). The most abundant nitrogen-containing BHPs in sedi- ments from the Amon mud volcano were aminotriol (III; 9.8–48.9%) and MC-triol MC (III ;11.8–62.5%) (Table 2). Other nitrogen-containing BHPs in these sediments were ami- notetrol (II; 0.5–31.8%), unsaturated aminotriol (ΔIII; 3.0–27.0%) and unsaturated MC-triol MC MC (ΔIII ; 0–22.5%). Minor abundances of aminopentol (I; 0–8.9%), and MC-pentol (I ; 0–3.8%) were found in some AMV sediments. Other BHPs in the AMV sediments were BHT, anhydroBHT, 2-Me-BHT, 3-Me-BHT, BHT-CE, BHT-G, BHT isomer, and adenosylhopane (S1 Table). New Zealand Seeps (NZS). The most abundant nitrogen-containing BHP in the sedi- MC ments from NZS were aminotriol (III; 28.5–72.7%), MC-triol (III ; 13.2–46.4%) and amino- tetrol (II; 5.1–20.6%) (Table 2). Unsaturated aminotriol (ΔIII), unsaturated MC-triol MC MC (ΔIII ), and MC-tetrol (II ) all made up <10%. Two sediment samples contained amino- pentol, albeit in low abundance (<1% of nitrogen-containing BHPs). Both of these aminopen- tol positive samples also contained soil marker BHPs [77,82]. Other BHPs found in NZS samples were BHT, anhydroBHT, 2-methyl-BHT, 3-methyl-BHT, BHT isomer, adenosylho- pane, and BHT-CE (S1 Table). Gulfo Dulce (GD). The most abundant nitrogen-containing BHP in GD surface sedi- ments was aminotriol (III; 44.3–100%; Table 2). Unsaturated aminotriol (ΔIII) made up MC 0–41.3%. MC-triol (III ; 0–11.4%) and aminotetrol (II; 0–6.0%) were found in most samples. Aminopentol was found in all but two surface samples, one of which was the shallowest site. Aminopentol abundance decreased with increasing water depth with the highest abundance at 24 m water depth (27.6% of nitrogen-containing BHPs). BHT, anhydroBHT, BHT isomer, and 2-Me-BHT were also found in GD (S1 Table). Gulf of Mexico (GoM) cold seeps. GoM cold seeps only contained aminotriol (III; 20.5– 100%) and aminotetrol (II; 0–79.5%) nitrogen-containing BHPs (Table 2). Other BHPs included BHT, soil marker BHPs, 2-Me-BHT, BHT isomer, and BHT-CE (S1 Table). PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 13 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems GoM sediments. GoM sediments from near the outflow of Mississippi River Delta showed an abundance of aminotriol (III; 26.6–80.7% of total nitrogen-containing BHPs), MC-triol MC (III ; 0–73.4%) (Table 2). One sample from the Western Basin had 19.3% aminotetrol (II) relative to total BHPs; however, the concentration of aminotetrol was low (<0.01 μg/g sedi- ment). Other BHPs included BHT, BHT isomer, anhydroBHT, 2-methyl-BHT, BHT-CE, and soil marker BHPs (S1 Table). Peru Margin (PM). The only nitrogen-containing BHP detected in sediments from the PM was aminotriol (III). Other BHPs found at this site included BHT, BHT isomer, anhy- droBHT, 2-Me-BHT, adenosylhopane, and BHT-CE (S1 Table). Discussion BHP distributions in aerobic methanotrophs Previously reported BHP distributions in AMO bacteria. Traditionally, Type I and Type II AMO bacteria had been distinguished by their different BHP signatures (e.g. [52]; see also review in [14]). Prior to the investigation of BHPs in the Methylovulum-like strain M200 [46], most screened Type I methanotrophs synthesised a high percentage of aminopentol (I) and lower contributions from aminotetrol (II) and in some cases aminotriol (III), and clustered in the left-hand corner of the amino-BHP ternary plot (Fig 3A). In contrast, Type II methano- trophs did not contain aminopentol, had varying contributions from II and III, and clustered along the right-hand axis of Fig 3A. The high relative abundance of III observed in Methylovu- lum-like strain M200 was, therefore, originally seen as an outlier [46]. Similarly, [52] showed that a culture of Methylomicrobium album did not contain aminopentol. At the time this was Fig 3. Relative composition (%) of `regular' amino-BHPs. Distributions of aminotriol, aminotetrol and aminopentol, including their C-3 methylated homologues, where present, are shown for (a) methanotroph cultures including literature data (circles) and data from new cultures of Methylomicrobium, Methylomarinum, and Methylomarinovum (diamonds; this study) and (b) sediments and microbial mats from methane-rich marine settings (HMMV = Håkon Mosby mud volcano; BSCC = Barents Sea carbonate crust; AMV = Amon mud volcano; NZ = New Zealand; GD Golfo Dulce; GoM = Gulf of Mexico; PM = Peru Margin). Methanotroph literature data from [36,42,46,52,83]. Literature data from [36,42,83] was based on GC-MS hopanol quantifications after periodic acid treatment. Therefore, it is not possible to distinguish between amino- and non-amino-BHPs with the same number of functional groups. This is especially significant for the Type II aerobic methanotrophic bacteria that are known to synthesise bacteriohopanetetrol. For this reason, the axes of plot (a) are shown as functionality of the BHP-side chain. doi:10.1371/journal.pone.0165635.g003 PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 14 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems presumed to be a contaminated culture, however, [53] also did not report I synthesis in cul- tures of Methylomicrobium alcaliphilum. All of the recently analysed Methylobacter spp. [28] join the more typical Type I methanotrophs in the left-hand corner of the plot, however, Methylobacter sp. BB5.1 increased the spread of the cluster with almost 40% III content. Amino-BHP distributions in previously untested Type I AMO bacterial cultures. It was assumed that the screened species of AMO Type I bacteria investigated in this study would display similar BHP distributions as those of previously reported Type I bacteria. All three bac- terial genera screened do indeed only contain amino-BHPs (Fig 2). However, the relative distri- bution of specific nitrogen-containing BHPs varies between genera, as well as between species belonging to the same genus. To allow for a more accurate comparison with data from the liter- ature (Fig 3A, circles), only aminopentol, aminotetrol, aminotriol, and their methylated equiva- lents were considered when producing the ternary plot of ‘regular’ nitrogen-containing BHPs of the novel Type I cultures (Fig 3A, diamonds). Aminopentol is the most abundant BHP in the novel species Methylomarinovum cadicuralii IT-9, which is in agreement with literature BHP compositions of most other Type I methano- trophs (Fig 3A, circles), e.g., Methylococcus capsulatus [44,52], Methylomonas sp. [42,46,52], psychrotolerant isolate CEL 1923 [42], and Methylocaldum tepidum [22]. However, a species in the same genus (Methylomarinovum sp. IN45) has a much lower abundance of aminopentol (5.0% of total amino-BHPs; Fig 2C). Methylomarinum vadi IT-4 shows relatively high propor- tions of aminopentol, but it is not the most abundant BHP (Fig 2A). Moreover, in our screen- ing of two species of Methylomicrobium spp., aminopentol was not detected, similar to reported cultures of Methylomicrobium album and Methylomicrobium alcaliphilum [52,53]. Our results seem to confirm the near-absence of aminopentol in all screened Methylomicro- bium spp., which are the first Type I methanotrophs apparently unable to synthesise amino- pentol. However, changes in BHP composition can occur at different growth stages and under different conditions (e.g. [27,83,84]), so further studies would be required to fully confirm this. It appears that the BHP distributions of Methylomicrobium and Methylovulum, which do not synthesise high amounts of aminopentol, should also no longer be considered outliers given the low levels of aminopentol in M. vadi IT-4 and Methylomarinovum spp. (Fig 2). This sug- gests a greater variance in the BHPs of Type I methanotrophs than previously thought. Fur- thermore, as Methylomicrobium has been isolated from a diverse range of marine environments [85–87], the absence of aminopentol in this genus might greatly affect its appli- cation as a marine aerobic methanotrophy biomarker. This, however, does not invalidate the use of aminopentol as a biomarker for methanotrophy. There is also significant variation in the relative abundances of the other nitrogen-contain- ing BHPs in Type I methanotrophs. A suite of novel BHPs identified as methylcarbamate (MC) BHPs are detected in all three genera screened in this study (Fig 2). These have not been reported in previous studies. Therefore, data available at Newcastle University from the analy- ses of Methylococcus capsulatus (Talbot et al., unpublished), Methylovulum-like strain M200, Methylomonas methanica, Methylomonas-like strain M5 [46], and Methylobacter spp. [28] MC were re-examined. III was identified retrospectively in Methylomonas methanica, and all MC species of Methylobacter. III constituted up 9.8% of total amino-BHPs in Methylobacter sp. MC BB 5.1. II was present in low abundance (<0.2%) in two of the three species of Methylobac- ter (Methylobacter sp. BBA 5.1 and Methylobacter sp. BB 5.1), but absent in Methylobacter mar- MC inus A 45. I was absent in Methylococcus capsulatus and Methylovulum-like strain M200, MC but was present in all species of Methylobacter. I was also present in Methylomonas metha- nica but absent in a different species of this genus (i.e., Methylomonas-like strain M5; [46]). These results indicate that MC-BHPs are not universally present when their regular PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 15 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems homologues are detected, and may be species specific and/or dependent on variations in growth conditions such as pH. The two species of Methylomarinovum display significant variations in their BHP composi- tions (Fig 2B and 2C). Methylomarinovum sp. IN45 had a relatively low level of aminopentol. MC The most abundant BHP in Methylomarinovum sp. IN45 is II . Although in lower abun- MC MC dances, III and I are also higher in comparison to their ‘regular’ homologues in this spe- cies compared to M. caldicuralii IT-9 (Fig 2B and 2C). In contrast, the most abundant BHP in Methylomarinovum caldicuralii IT-9 is aminopentol, followed by almost equal amounts of MC I . The different relative BHP distributions between the Methylomarinovum spp. highlight that there can be significant variations within a genus. Methylomarinovum sp. IN45 was iso- lated from a deep-sea hydrothermal field and perhaps the high levels of methylcarbamate com- ponents observed are the result of a physiological adaptation to higher pressure in this environment. This may explain why the relative abundances of components in Methylomarino- vum caldicuralii IT-9, the same genus but isolated from a shallow submarine hydrothermal environment, are quite different. Perhaps the complex functionality of the terminal group of the methylcarbamate components is more effective at stabilising the cell membrane and decreasing fluidity under these conditions. 3Me 3-methylaminotriol (III ) was observed in both Methylomicrobium spp. (23.9–31.5% of total BHPs; Fig 2D and 2E) in agreement with a recent report in [53]. This compound was accompanied 3Me by low levels of 3-methylaminotetrol (II ) in both species and trace amounts of 3-methylamino- 3Me pentol in M. kenyense (I ). The absence of C-3 methylated structures in the previously investi- gated Methylomicrobium album strain BG8 [52] may appear inconsistent with the organisms investigated here; however, genomic investigations have revealed that M. album is separated from halo(alkali)philic representatives of the Methylomicrobium genus such as M. alcaliphium and M. kenyense [58], and perhaps specific environmental conditions influence the BHP composition of Methylomicrobium spp. as they seemingly do within the Methylomarinovum genus. 3Me 3Me No C-3 methylated equivalents of aminotriol (III ), aminotetrol (II ), nor aminopentol 3Me (I ) were present in Methylomarinum vadi IT-4 or the Methylomarinovum spp., adding to examples of Type I methanotroph species that contain amino-BHPs, but not their C-3 methyl- ated equivalents (e.g., see review in [14]). The most abundant BHPs in Methylomarinum vadi MC IT-4 were aminotriol (III) and MC-triol (III ), which were present in equal amounts (Fig MC MC 2A). Similar amounts of II and II , and I and I were also observed in this culture. A high proportion of III is unusual for a Type I methanotroph, but has been observed before in the Methylovulum-like strain M200. (Fig 4A; [46]). The new data reiterate that aminopentol is not always the most abundant BHP in Type I methanotrophs, nor necessarily the most appropriate biomarker for AMO. BHPs in marine environments Lack of BHP diversity in marine environments. The screened marine sediments and authigenic carbonates do not show large diversity in their BHP signatures (S1 Table). The lim- ited BHP distributions are also comparable to other reported marine sediment BHP signatures, all dominated by BHT and BHT isomer, from a number of locations including the Black Sea [88], the Benguela upwelling system [89], and the Arabian Sea [90]. More recently a similar pattern was also seen in water column samples from the California Current system, where the wide diversity observed in the gene responsible for hopane cyclisation (squalene-hopene cyclase) was not reflected by distinct BHP fingerprints related to this potential range of source- organisms [91]. However, genetic information is quickly lost, and we must strive to find lipid biomarkers to trace particular metabolisms in the geological record. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 16 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Fig 4. Relative composition (%) of methylcarbamate(MC)- BHPs. Distributions of MC-triol, MC-tetrol and MC-pentol are shown for (a) methanotroph Type I cultures, Methylobacter, Methylomicrobium, Methylomarinum, and Methylomarinovum, and (b) marine sediments (HMMV = Håkon Mosby mud volcano; BSCC = Barents Sea carbonate crust; AMV = Amon mud volcano; NZ = New Zealand; GD Golfo Dulce; GoM = Gulf of Mexico). Methylobacter data from [28]. doi:10.1371/journal.pone.0165635.g004 Non-nitrogen-containing BHP concentrations in the screened sediments do not show remarkable signatures (S1 Table). BHT and anhydro-BHT, thought to be a degradation prod- uct of BHT and other composite BHPs such as BHT cyclitol ether [92], were found at all sites. The presence of soil marker BHPs at some sites, particularly NZS, indicates that these sedi- ments could be influenced by terrestrial input of organic matter (e.g., [77,82,93]). However, as adenosylhopane is an intermediate in the biosynthesis of all other side-chain extended BHPs [94], other sources cannot be entirely excluded. BHT isomer, a biomarker for anaerobic ammo- nium oxidation [70], was found in high concentrations in GD (previously reported in [70]), as well as in the PM sediments, which underlie the Peruvian OMZ, where anammox is known to be an important process [95], and where BHT isomer has previously been reported from the water column within the OMZ [90]. The most abundant of the three regular amino-BHPs in the CH -influenced marine sediments was aminotriol, which is not source-specific (e.g., [48]). Aminopentol in marine sediments. Although aminopentol was found in significant abundance in some of the reported and screened Type I methanotroph cultures (Fig 2), it was not found to be abundant in most of the CH -influenced marine sites in this study (Table 2). In fact, it was only detected in AMV, GD surface sediments, and two NZS samples (Fig 3B; Table 2). The discrepancy between the distinct amino-BHP signatures of isolated Type I AMO bacteria and signatures of CH -influenced marine sites is highlighted in the ternary plots of the relative composition of aminopentol, aminotetrol, and aminotriol (Fig 3A cf. Fig 3B). These differences could be due to the particular methanotrophic bacterial community responsible for methanotrophy in the CH -influenced marine sediments. Ruff et al. [96] found that diversity in the global CH seep microbiome was controlled by environmental factors such as PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 17 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems temperature and electron acceptor availability. Considering their findings, it is possible that the environmental conditions in most marine CH -influenced sediments favour specific methano- troph communities. For example Methylomicrobium spp., found in saline environments [85– 87] and saline, highly alkaline environments [58,97], and which do not produce aminopentol in significant amounts (Fig 2), could be present. However, the absence of C-3 methylated com- pounds is confounding for a Methylomicrobium source, pointing towards other methanotrophs that do not synthesise aminopentol. Yan et al. [98] found that 85% of the operational taxo- nomic units (OTUs) from the same sites as our GoM cold seeps did not group with known sequences of a subunit of particulate methane monooxygenase (pmoA). This would suggest the presence of novel methanotrophic species in GoM. In the same way, significant pmoA diversity has been observed in sediments from the North American margin [99], a shallow CH seep [100], a marine estuary [101], and hydrocarbon seeps [102]. pmoA OTUs from the NZS sedi- ments grouped with methanotrophic endosymbionts [68], including Bathymodiolus spp., which have been shown to contain neither aminopentol nor methylated BHPs [103]. Neverthe- less, the absence of methylated amino-BHPs in the screened marine sediments (Table 2) may suggest Methylomicrobium album, or a related species that also does not synthesise methylated amino-BHPs, being the dominant methanotroph in CH -influenced marine environments. These are not the first reports of marine CH -influenced environments not containing ami- nopentol (Table 1). For example, using methods targeting the functional gene pmoA, which is produced by most methanotrophs, Type I methanotrophs were detected in all three units of Ace Lake sediments. However, aminopentol was only detected in sediments deposited under freshwater conditions (unit III) despite the fact that the modern meromictic water column, containing relict seawater left behind after the sea level fell around 9000 years ago, hosts the Type I methanotroph Methylosphaera hansonii [104]. No aminopentol was detected in the methanotrophic symbionts in the gill tissue of a cold-seep mussel, despite other lipid-based evi- dence suggesting the presence of a Type I methanotroph [103,105]. Similarly, CH seep car- bonates from Alaminos Canyon, northern Gulf of Mexico [41] and the Northern Arabian Sea [106] were found to lack aminopentol. Conversely, aminopentol was detected in the water col- umn of the Baltic Sea with supporting evidence for the presence of Type I methanotrophs from C-depleted PLFAs [107]. Aminopentol was also detected in the water column of the Black Sea in the oxic-anoxic water transition, but not in the underlying sediment [12,88,108]. The presence of aminopentol in sediments from the AMV, located on the Nile deep-sea fan, in the Eastern Basin of the Mediterranean Sea (Table 2) may be explained by Nile River outflow carrying terrestrial wetland methanotrophy signatures into the Mediterranean, as seen in the Amazon and Congo River fans [14,109]. This would appear to indicate that aminopentol is still an excellent biomarker for terrestrial AMO. The near-absence of soil-marker BHPs in AMV (Table 2) may still point towards in-situ marine production of aminopentol. However, the rela- tive abundance of soil-markers in terrestrial settings has recently been found to be strongly influenced by environmental factors; higher temperatures and low pH (in peatlands) can both strongly reduce the relative proportion of soil marker BHPs as a proportion of total BHPs [40,110,111]. Aminopentol was found in NZS sediments that also contained soil marker BHPs (Table 2). Therefore, aminopentol in sediments from NZS may have originated from terrestrial sources. Aminopentol in GD surface sediments may be the result of a distinct AMO commu- nity living in the specific environment prone to carbonate formation in GD. Unfortunately, samples were not properly preserved to be able to determine AMO diversity using genetic- based analyses of the pmoA gene in these sediments. The cumulative results of the studied marine sites do, however, indicate that an absence of aminopentol is not necessarily evidence for the absence of methanotrophs or aerobic methane oxidation. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 18 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Alternative BHP biomarkers for AMO and implications for the marine sedimentary record of methanotrophy Regular amino-BHPs. Screened Type I methanotrophs also produced varying amounts, depending on the genera, of aminotetrol (II) and aminotriol (III) (Fig 3A), both of which were found in CH -influenced marine sediments (Fig 3B; Table 2). However, these two amino- BHPs are less source-specific to methanotrophic bacteria than aminopentol, and do not make 3Me ideal biomarker lipids for methanotrophy. Given that 3-Me-aminotriol (III ) made a signifi- cant contribution to the amino-BHP abundance in screened cultures of Methylomicrobium spp. (23.9 and 31.5% of total amino-BHPs; Fig 2D and 2E) and 9.8% in Methylobacter sp. 3Me BB5.1 [28], it was expected that III would be an important amino-BHP in CH -influenced 3Me marine sediments. However, III was not found in any of the screened sediments (Table 2). Methylomicrobium alcaliphilum and Methylomicrobium kenyense are adapted to high alkalin- 3Me ity, but not necessarily to high salinity [58]. This distinct lack of III in marine sediment samples would seem to indicate that the Methylomicrobium species we investigated are not the primary source of amino-BHPs in CH -influenced marine environments. 3Me III has only occasionally been reported from environmental samples including some soils [82,112] and most recently in a peat core from Germany [111], but only at very low levels 3Me (Table 1). Other C-3 methylated amino-BHPs are even less common (Table 1). I was first reported from a neo-volcanic, eutrophic and saline lake sediment (La Piscina de Yuriria, Mexico; [45]), and subsequently from a geothermal silica sinter (Opaheke Pool hot spring, 3Me New Zealand; [113]). The pentafunctionalised II was also present in the Mexican lake sedi- ment. Both of these compounds were reported in one study on the Black Sea water column [12], but were absent at another site [108]. The apparent discrepancy between the very limited occurrence of C-3 methylated BHPs (as measured using the periodic acid cleavage technique which converts polyfunctionalised BHPs into GC-amenable primary alcohols; e.g., [36,75]) and their wider occurrence in the form of 3-Me hopanes in ancient rocks and oils was first identified in [114]. These authors found 3-Me-BHPs to be abundant only in a very limited number of settings, under quite specific conditions (i.e., some alkaline lakes). The occurrence of 3-Me-hopanes in marine authigenic carbonates [31,115], which form under highly alkaline conditions are also consistent with a Methylomicrobium source ([58], and references therein). It was further suggested that 3-Me-BHPs and hexafunctionalized BHPs appear to have differ- ent sources (possibly, but not necessarily restricted to, only Type I methanotrophs; [114]). Cul- ture studies (on the moderately thermophilic Type I methanotroph Methylococcus capsulatus) have shown that production of C-3 methylated compounds may be related to growth stage. Higher relative proportions of methylated BHPs replaced the non-methylated equivalents dur- ing stationary phase growth [83], and appear to be necessary for maintaining intra-cellular membrane structures [27]. These important physiological roles for methylated BHPs are at odds with the very sparse occurrence of these compounds in modern settings (Table 1), and clearly our understanding of the factors controlling their biosynthesis and subsequent preser- vation in sediments is still limited, hampering interpretation of certain BHP signatures. Methylcarbamate-BHPs. Most of the marine sediments influenced by CH contained at least MC-triol, albeit at relatively low abundances (Fig 4B; Table 2). The fact that the MC-BHPs were found in all strains of methanotrophs analysed, though not all components in the suite were present in every strain, shows the biomarker potential of these BHPs for AMO (Fig 3A). MC MC-tetrol (II ) was the most abundant component in Methylomarinovum sp. IN45, and MC-BHPs were found in higher abundance than the ‘regular’ 35-amino-BHP homologues, which may allow this particular hydrothermal vent species to be identified in environmental CME settings. Unsaturated MC-triol (ΔIII ) was found in high abundance in AMV, HMMV, and PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 19 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems NZS, but was not found in any of the methanotroph cultures. This is possibly because the BHP signatures in most CH -influenced marine sediments are sourced from AMO bacteria that have no cultured relatives or at least none which have been tested for BHP production. Given the small diversity in BHPs found in marine sediments and the need for an AMO bio- marker, there appear to be few BHPs that meet the criteria of being source-specific and abun- dant. This has significant implications for the development of a proxy using aminopentol to trace AMO in marine settings. Applying MC-BHPs combined with the traditional suite of amino-BHPs (e.g. aminopentol, aminotetrol, and aminotriol) seems to be the most appropriate biomarker course for AMO. Conclusions Isolated methanotrophs from previously unexamined genera and species displayed marked dif- ferences in their relative abundances of amino-bacteriohopanepolyols (BHPs). Aminopentol (I) was the most abundant BHP in Methylomarinovum caldicuralii IT-9, which fits with the typical BHP signature of known Type I methanotrophs. However, the BHP signatures of Methylomarinovum sp. IN45 and Methylomarinum vadi IT-4 both did not show aminopentol as the most abundant BHP. Moreover, neither of the Methylomicrobium spp. contained amino- pentol and only one contained a low level of 3-methyl-aminopentol showing that not all Type I methanotrophs synthesise aminopentol, agreeing with previous environmental studies. Con- sidering Methylomicrobium can be prevalent in marine environments, this has implications for the use of aminopentol as a biomarker for marine methanotrophy. A suite of components related to amino-BHPs, but with methylcarbamate (MC) terminal groups, were detected for the first time, and were present in all Type I methanotroph strains tested. Marine sediments influenced by CH did not contain significant amount of aminopentol, but did contain MC-BHPs. This study highlights the relatively low BHP diversity within marine sediments, and indicates that the combined use of MC-BHPs and amino-BHPs might be preferential to trace aerobic methane oxidation (AMO) in marine settings. Supporting Information S1 File. Identification of novel BHP compounds in methanotroph cultures. (DOCX) S1 Table. Concentrations of other BHPs in marine sediment samples. Concentration (μg/g sediment) of other BHPs in marine sediments samples presented in this study. (XLSX) Acknowledgments We are deeply thankful to A. Boetius for supplying the sediments from HMMV, AMV, and NZS. Additional information about these sites can be found on the Pangaea database (http:// www.pangaea.de/).The PM sediments analysed in this study were originally provided by T. Eglinton and J. Repeta. R.D. Pancost is thanked for use of GoM cold seep material. The GoM sediments used in this study were provided by R. Sassen and S. Sweet of the Geochemical & Environmental Research Group (GERG) at Texas A&M University, U.S.A. We thank the cap- tain and the crew of the RV Kaiyo, the operation team of the ROV Hyper-Dolphin, and the shipboard scientific party for the invaluable help in collecting deep-sea samples. We also thank M. Abe and K. 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Biofouling 27: 21±32. doi: 10.1080/08927014.2010.536614 PMID: 21104476 PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 27 / 27 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems

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Abstract

Marine Systems. PLoS ONE 11(11): e0165635. doi:10.1371/journal.pone.0165635 Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the Editor: Zhe-Xue Quan, Fudan University, CHINA amount of methane (CH ) released into the environment. AMO acts as a sink of CH , con- 4 4 verting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)cli- Received: September 5, 2016 mate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO Accepted: October 15, 2016 events, especially at times when the role of methane in the carbon cycle was more pro- Published: November 8, 2016 nounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) Copyright:© 2016 Rush et al. This is an open lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34- access article distributed under the terms of the pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the Creative Commons Attribution License, which BHP compositions were examined for species of the recently described novel Type I permits unrestricted use, distribution, and reproduction in any medium, provided the original methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a author and source are credited. novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP Data Availability Statement: All relevant data are only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopen- within the paper and its Supporting Information tol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel struc- files. tures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC- Funding: The work was funded by a Starting Grant BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subse- (No. 258734) awarded to HMT for project quently, sediments and authigenic carbonates from methane-influenced marine environ- AMOPROX, from the European Research Council. ments were analysed. Most samples also did not contain significant amounts of KM and JCN thank Lundin Petroleum for financial support. The funder had no role in study design, aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 1 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Competing Interests: The authors have declared methanotophic bacteria. However, the BHP composition of the marine samples do point that no competing interests exist. toward the novel MC-BHPs components being potential new biomarkers for AMO. Introduction Methane (CH ) is a potent greenhouse gas, and its atmospheric concentration has tripled since pre-industrial times (e.g. [1,2]). Global oceans hold large subsurface reservoirs of CH in the form of gas hydrates. These stores are precariously dependent on temperature and pressure. A rapid destabilisation of gas hydrates has been proposed to have caused vast releases of marine CH in the past [3]. Increased input of CH into the atmosphere has been interpreted through 4 4 records of excursions of significantδ C depletion in the geological record, such as in the Palaeocene-Eocene Thermal Maximum (PETM) [4–7]. CH release into the atmosphere is regulated by methanotrophy, which converts CH into 4 4 CO , thereby playing a key role in the carbon biogeochemical cycle [8]. Although traditionally anaerobic archaea have been the most studied methanotrophs (e.g., AMNE-1 and ANME-2; cf. [9]), recent observations have highlighted the importance of bacteria performing aerobic CH oxidation (AMO) in marine, estuarine, and riverine fan environments (e.g., [10–14]). For example, pelagic AMO activity rose significantly after the Macondo oil well blowout in 2010 [15]. However, this activity was short-lived, highlighting the complexity of natural community interactions in response to increased CH [16]. It is thus important to recognise and trace methanotrophy during past extreme events in order to understand its potential to mitigate future CH release. AMO bacteria belong to two phyla, Proteobacteria and Verrucomicrobia. Most isolates of Verrucomicrobia are thermoacidophilic [17–20], and have been found primarily in acidic, geo- thermal environments [21]. Aerobic methanotrophic members of Proteobacteria belong to two distinct classes, separated based on their carbon assimilation pathways [8]. Type II methano- trophs, members of the Alphaproteobacteria, are associated with terrestrial settings ([8], and references therein), whereas Type I methanotrophs members of the Gammaproteobacteria are widespread in aquatic systems, although they are also found in terrestrial systems. Both Type I and Type II methanotrophs are known to synthesise bacteriohopanepolyol (BHP) lipids [22]. BHPs are precursors to hopanes, which are the most ubiquitous lipids in the geological record [23,24]. Therefore, being able to trace AMO using hopanoid biomarkers is of value to the study of the carbon cycle in the past. Previous work exploring hopanoids as biomarker lipids found that methylation at the C-3 3Me 3Me 3Me position (3-Me-BHPs; Fig 1, I , II , III ) was a possible indication of methanotrophic origin [25,26]. However, attributing C-3 methylation to AMO was challenged by the revelation that bacteria other than methanotrophs have the genes to methylate at C-3 [27]. C-3 methyla- tion is more likely a requirement for cell survival in late stationary phase [27]. Moreover, not all methanotrophic bacteria have the gene to methylate at this position, [27,28], nor are 3-Me BHP precursors found in all CH -influenced environments (Table 1). However, AMO bacteria are often considered to be the most likely source of 3-Me hopanoids in marine sediments due to their depleted carbon isotope signatures (e.g., [29]) and because they are frequently accom- panied by 4-methylated steroids, which are also known as biomarkers of methanotrophic bac- teria (e.g., [30,31]). In other studies, diplopterol and diploptene have been interpreted as biomarkers for methanotrophy, notably in anoxic environments [32–35]. However, neither diplopterol nor diploptene is source-specific to AMO [36], and these studies also relied on very PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 2 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems 3Me MC Fig 1. Chemical structures of bacteriohopanepolyol lipids. I, aminopentol; I , 3-methyl-aminopentol; I , methylcarbamate-aminopentol; II, 3Me MC 3Me MC aminotetrol; II , 3-methyl-aminotetrol; II , methylcarbamate-aminotetrol; III, aminotriol; III , 3-methyl-aminotriol; II , methylcarbamate-aminotriol. MC MC MC The proposed structure of methylcarbamate(MC)-aminopentol (I ), MC-aminotetrol (II ), and MC-aminotriol (III ) are tentatively based on mass spectral identification (S1 File). doi:10.1371/journal.pone.0165635.g001 negativeδ C lipid values (e.g., ca. -61‰ for diplopterol; -61 to -74‰ for diploptene) as an indication of CH being the carbon source for the organism producing these lipids. Yet, recent work has emphasised that not all AMO-derived carbon shows a depleted isotope signature, especially in terrestrial systems where Type II methanotrophs tend to dominate. For example, only limited depletion in C, with values between -25‰ and -40‰, have been reported for hopanoids with an inferred methanotrophic origin from modern peat bogs [37,38] as well as other ancient lignite deposits [39]. Also, BHPs in Congo deep sea fan sediments, originating from low-latitude wetlands [40], had higher C values than expected (i.e., C hopanol ~ -41‰; [14]). In marine CH seep carbonates from the Gulf of Mexico, BHPs, hopanoic acids, and 4-methylated sterols of aerobic methanotrophs were found with similarδ C values as the CH source [41]. These observations can be the result of dilution from other heterotrophic bac- terial sources that make it difficult to identify subordinate methanotroph contributions [37]. The metabolic pathways used by the AMO bacteria for CH assimilation can also have a pro- found effect on the level of isotopic depletion, with values for serine pathway methanotrophs (Type II) ranging from 12‰ depleted to 10‰ enriched relative to the CH substrate [42]. Fur- thermore, to analyseδ C of intact BHPs, these must first be converted into primary alcohols by periodic acid/sodium borohydride cleavage [43]. For example, theδ C value of 35-amino- bacteriohopane-30,31,32,33,34-pentol (aminopentol; Fig 1, I) is measured on the C -hopanol product, which includes all converted hexa-functionalised BHPs (i.e., not only aminopentol), as well as any free-hopanols that are present in a sample before BHP conversion. While the contamination of free-hopanols can be circumnavigated by column separations (e.g., [41]), measuring theδ C values of intact BHPs is not currently possible. Aminopentol is thought to be the most diagnostic BHP for AMO (see review in [14]). Ami- 3Me nopentol and its methylated and unsaturated homologues (i.e., I ,ΔI) have been found PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 3 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 1. Presence and absence of aminopentol and related methylated and unsaturated homologues in previously investigated environmental settings. aminopentol aminotetrol aminotriol 3Me 3Me 3Me Sample I ΔI I II II III III Reference Soils Pasture [manured] (UK) + - - + - + + [112] Pasture [not manured] (UK) - - - + - + + [112] Rice Paddy (Vietnam) + - - + - + - [112] Woodland (North East England) + - - + - + + [112] South West France + - - + - + - [116] Amazon + - - + - + - [109] Congo + + - + - + + [40] Lean Delta Peamafrost [ice complex] + - - + - + - [110] Human Sewage [treated] - - - + - + - [117] Forest, Grassland Soils (Alberta, Canada) - - - + - + - [118] Peat and Lignite River Tet Catchment (France) + - - + - + - [116] Moorhouse (UK) + - - + - + - [46] Misten Bog (Belgium) + - - + - + - [119] Bisendorfer Moor (Germany) + - - + - + + [111] The Cobham Lignite (UK) + - - + - + - [49] Geothermal Environments + Cyanobacterial mat (Surprise Valley, Nevada, USA) + - - + - + - [120] Silica Sinter (Orakie Korako, Taupo Volcanic Zone, New Zealand) + - + + - + - [113] Silica Sinter (Champagne Pool, Taupo Volcanic Zone, New Zealand) - - - - - + - [121] Orange mat (Yellowstone, USA) - - - - - + - [122] Other Microbial mats Mars Oasic (Antarctica) - - - - - + - [48] Hypolith (Devon Island, Arctic) - - - - - + - [48] Cyanobacterial Mat (Christmas Island, Kiribati) - - - - - + - [123] Lake Sediments Lake Windermere (UK) + - - + - + - Lake Cadagno (Switzerland) + - - + - + - [26] Lake Holzmaar (Germany) + - - + - + - [26] Lake Nkunga (Kenya) + - - + - + - [45] Priest Pot (England) + - - + - + - [26,45] La Piscina de Yuriria (Mexico) + - + + + + - [26,45] Laguna de Zempoala (Mexico) + - - + - + - [26] Loch Ness (UK) + - - + - + - [26,45] Lake Druzhby (Antarctica) + - - + - + - [26,45] Sombre Lake (Signy Island, Southern Atlantic Ocean) + - - + - + - [26] Heywood Lake (Signy Island, Southern Atlantic Ocean) + - - + - + - [26] Ace Lake [Freshwater unit] + - - + - + - [104] Ace Lake [meromictic unit] - - - + - + - [104] Ace Lake [marine unit] - - - - - + - [104] Wetlands Amazon + + - + - + - [109] Congo + + - + - + - [40] Stream/River/Estuary Sediments (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 4 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 1. (Continued ) aminopentol aminotetrol aminotriol 3Me 3Me 3Me Sample I ΔI I II II III III Reference Arctic Rivers (Indigirka, Kolyma, Lena, Ob, Yenisei, Yukon, Mackenzie) + - - + - + - [124] Glacial Out¯ow Stream (Svalbard) + - - + - + - [125] Kalix River [surface sediment transect] + - - + - + - [126] Kolyma River [surface sediment transect] + - - + - + - [127] Yenisei River [mouth, and surrounding area] + - - + - + - [128] Yangtze River [estuary, inner shelf] + - - + - + - [82,129] Congo River [estuary] + + - + - + - [14,40] Water Column Priest Pot [pond] (UK) + - - + - + - River Water (Panama) + - - + - + - [130] Yenisei River + - - + - + - [128] Black Sea [oxic/anoxic transition zone] + - + + - + - [12] Black Sea [oxic/anoxic transition zone] + - - + - + - [108] Gotland Deep, Baltic Sea + - - + - + - [107] Marine Water (off Panama coast) - - - - - + - [130] Pelagic [sub-oxic and anoxic] (Arabian Sea, Peru Margin, Cariaco Basin) - - - - - + - [90] Cariaco Basin - - - - - + - [131] California Current - - - - - + - [91] Modern/Recent Marine Sediments Gotland Deep, Baltic Sea + - - + - + - [107] Congo River Deep-Sea Fan + + - + - + - [14] Amazon Shelf and Fan + - - + - + - [109] East Siberian Arctic Shelf + - - + - + - [79,126] Black Sea - - - + - + - [88] Southwest African Coast - - - + - + (+) [89] Chukchi Sea - - - + - + - [132] Alaskan Beaufort Sea - - - - - + - [132] Baltic Sea - - - + - + - [133] Marine Carbonates Carbonate, Gulf of Mexico - - - + - + - [72] Authigenic carbonates, Gulf of Mexico - - - + - + - [41] Seep Carbonate, Arabian Sea - - - + - + - [106] Other samples Membrane Foulant [river water] + - - - - + - [134] Membrane Foulant [brackish water] + - - + - + - [134] Membrane Foulants [seawater] - - - - - + - [134] Mussel Gill Tissue - - - + - + - [103] +, detected in at least one of the samples; -, not detected in any of the samples (+) indicates a methylated aminotriol was detected but the position of methylation was not identi®ed ¶Talbot and Farrimond, unpublished data doi:10.1371/journal.pone.0165635.t001 almost exclusively in Type I aerobic methanotrophs [22,44–46]. Moreover, aminopentol (I) has been found in a wide range of environments, which indicates potential as a biomarker for AMO (Table 1). Additionally, 35-aminobacteriohopane-31,32,33,34-tetrol (aminotetrol, II) and 35-aminobacteriohopane-32,33,34-triol (aminotriol, III) are also synthesised by Type I PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 5 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems and Type II AMO bacteria. However, II and III are less source-specific as both are synthesised by some species of sulfate reducing bacteria (SRB) of the genus Desulfovibrio [47], and III is synthesised by many other aerobic bacteria ([48], and references therein). Aminopentol made up a very minor proportion of the BHP composition in a SRB culture (<0.1% of total BHPs in Desulfovibrio salexigens). The ratio of aminopentol to the more ubiq- uitous aminotriol was 1:1352 [47]. We can therefore discount SRB as the source of aminopen- tol in an environmental sample with a high ratio of aminopentol:aminotriol. A similar approach was recently used by [49] based on the ratio of aminotetrol:aminotriol which has been found in the range 1:20–100 in some species of Desulfovibrio SRB [47,50,51]. Interest- ingly, some of the species of SRB cultures analysed by Blumenberg et al. [47,50,51] also synthe- sised diplopterol and diploptene, which could explain the enhanced presence of these lipids in CH -influenced anoxic sites. Representatives from only a small number of Type I methanotroph genera have been tested for BHP production (e.g., [22,36,46,52,53]. Many more recently described genera, including novel genera from marine and other (hyper)saline environments, have yet to be explored (e.g., [54–56]). Moreover, relatively few studies of BHP distributions have targeted marine environ- ments (Table 1). It is important, therefore, to determine whether aminopentol, which is seen as a diagnostic marker for Type I methanotrophs, is present in methanotrophs isolated from marine and other saline environments, and whether we can find aminopentol in CH -influ- enced marine sediments. This knowledge will have implications for the use of aminopentol as a biomarker to trace AMO in modern and ancient marine environments. In this study, our goal was to develop an appropriate biomarker approach for AMO, which will allow high throughput analysis of sediment without the requirement for laborious chemical conversion steps prior to compound specific isotope analysis. To this end, we screened the BHP distributions of three genera of aerobic methanotrophs (i.e., Methylomicrobium, Methylomari- num and Methylomarinovum). Methylomarinum and Methylomarinovum have not previously been investigated for BHPs. Two species of Methylomicrobium have been described previously [52,53], however, we include an additional species Methylomicrobium kenyense. These data are combined with literature BHP distributions of other AMO genera, including the recently reported Type I genus, Methylobacter [28], in order to facilitate interpretation of BHP distribu- tions in six selected modern marine sediments from CH -influenced systems. Two marine sites not influenced by CH were also investigated as controls for background marine BHP signatures. Methods Methanotroph pure cultures Cultivation of Methylomarinum vadi IT-4. Previously described Methylomarinum vadi IT-4 was isolated from a microbial mat sample (in-situ temperature 30–40°C) collected at a shallow marine hydrothermal system (depth, ~23 m) in a coral reef off Taketomi Island, Oki- nawa, Japan [56,57]. Cultivation of this strain was performed at JAMSTEC, Japan, using MJmet medium at pH 6.6 at 37°C. A detailed site description and the enrichment and isolation procedures can be found in [57]. Cultivation of Methylomarinovum spp. Previously described Methylomarinovum caldi- curalii IT-9 was isolated from the hot vent fluid (52°C) collected at the main vent site (depth, 23 m) in the shallow marine hydrothermal system where Methylomarinum vadi IT-4 was iso- lated [55,57]. Methylomarinovum sp. IN45 is a new isolate from a deep-sea hydrothermal field in Okinawa Trough, Japan (H. Hirayama, pers. comm.). The cultivation of strains IT-9 and IN45 was performed at JAMSTEC, Japan, using MJmet medium at 45°C, and at pH 6.2 and 6.6, respectively. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 6 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Cultivation of Methylomicrobium spp. Methylomicrobium alcaliphilum and Methylomi- crobium kenyense were first isolated from highly alkaline soda lakes in Russia and Kenya, respectively [58]. Both M. alcaliphilum (DSM-number 19304) and M. kenyense (DSM-number 19305) are from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures). The cultivation of both strains was done at the Center for Applied Geosciences at the University of Tübingen, Germany, at pH 9.1 and at 28°C with a high salt NMS medium (1.5% NaCl). Marine sediment and carbonate samples Håkon Mosby Mud Volcano (HMMV). HMMV has been extensively studied for both aerobic and anaerobic methanotrophy [13,59]. The flow of CH in the center of the HMMV is restricted, and AMO was observed to be the most dominant process within the centre of the crater, performed by Type I methanotrophs [60]. Sediments from the HMMV were collected aboard the RV Polarstern (cruise ARK XXIV/2; 2009) and the RV Maria S Merian (cruise MSM 16/2; 2010) [61]. Barents Sea carbonate crust (BSCC). The Barents Sea is a well-studied area of active CH seepage. The seafloor geology is marked by pockmarks [62], gas hydrates, gas flares [63], and patches of carbonate crusts [64], indicating active CH seepage [10,64]. We analysed eight depths (each 2–3 cm thick) from a push core (P120020 PR4) of a cold seep carbonate crust sampled at Loppa High/Polheim Sub-Platform area in the SW Barents Sea (72° 34’02.07”N, 20° 52’05.96”E). The core penetrated to a depth of 19 cm below sea floor (cmbsf ). These samples were taken aboard the RV Fugro Meridian in September 2012 by Lundin Petroleum Norway. Amon Mud Volcano (AMV). The Amon mud volcano (AMV) is located in the Nile deep-sea fan, in the Eastern Basin of the Mediterranean Sea. Hydrocarbons, muds, and fluids are transported to the surface via one main feeding channel from the deep subsurface, creating a stark thermal gradient in the sediment. Oxidation of CH in the water column directly above 13 2 AMV has been attributed to AMO using C and H isotopic values [65]. Sediments from AMV were collected aboard the RV Meteor (cruise M70/2, BIONIL; 2006) and the RV Maria S Merian (cruise MSM 13/3; 2009) [66]. New Zealand Seeps (NZS). The Hikurangi continental margin, east of New Zealand’s North Island has been described as a biogeographically “new” cold seep province, characterised by endemic faunal communities [67]. Surface (0–2 cmbsf) and subsurface (10–12 cmbsf) sedi- ment samples from three New Zealand seep (NZS) sites were collected aboard the RV Sonne (cruise So-191; 2007) [68]. These were dominated by distinct biota: Frenulata (Omakere Ridge), Ampharetidae (Wairarapa Takahae), and sulfur-oxidising bacteria (Wairarapa Takahae). Gulfo Dulce (GD) surface sediments. A sill at 60 m water depth physically cuts off Golfo Dulce from the Pacific Ocean, which promotes anoxic conditions within the basin. Recently, GD was shown to contain authigenic carbonate formations at shallow (ca. 10 m) water depth [69]. GD sediments were collected along a transect from 10 to 140 m water depth in March 2008, as described in [70]. Gulf of Mexico (GoM) cold seeps. The Gulf of Mexico (GoM) holds an abundance of thermogenic gas. The venting of this gas from deep subsurface forms gas hydrates, free CH , which are the sources of energy for microbial chemosynthetic communities, and authigenic carbonates [71]. Pancost et al., [72] described the BHP composition of material from five car- bonate rock and nodule sites in GoM. Two of the carbonates were shown to contain low quan- tities of aminopentol. Here, we reinvestigated the BHP signatures of these sediments. GoM sediments. The GoM also houses the outflow of the Mississippi River Delta, trans- porting terrestrial material into the Gulf. Three GoM sites to the best of our knowledge not PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 7 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems influenced by CH (27°30'N, 87°20'W; 28°20'N, 89°38'W; 26°50'N, 92°40'W; two sediment depths at each site) were investigated in this study [73]. Peru Margin (PM). An intense upwelling regimes fertilises surface water productivity on the Peru Margin (PM). This lends to oxygen utilisation in the water column, causing the East- ern South Pacific Oxygen Minimum Zone (ESP OMZ) [74]. Three PM sediments were ana- lysed (10–15, 20–25 and 40–45 cmbsf ) from a core taken within the ESP OMZ, at 100 m water depth [73,75]. Lipid extraction Total lipid extraction. All freeze-dried bacterial cells and marine sediments, except the Barents Sea samples extracted at GFZ Potsdam, were extracted using a modified Bligh-Dyer method [76,77]. Briefly, freeze-dried material was extracted in 19 mL of a 10:5:4 (v:v:v) mixture of MeOH:chloroform:H O in a 50 mLTeflon tube. This mixture was sonicated for 15 min at 40°C, and centrifuged for 10 min. The supernatant was transferred to a second tube, and the residue re-extracted twice more. The chloroform in the supernatant was separated from the aqueous phase by adding water until the H O:MeOH ratio was 1:1 (v:v), and collected. This procedure was repeated for the subsequent extractions. The collected chloroform total lipid extract (TLE) was dried by rotary evaporation in a round-bottom flask. The extraction protocol at GFZ Potsdam was similar but used a mixture of MeOH:DCM:ammonium acetate buffer [78]. Solid Phase Extraction. In-house comparisons have shown that amino-BHPs are better detected after solid phase extraction (SPE). An aliquot of the TLE was separated over a 1 mg NH solid phase extraction cartridge, as described in [79]. Briefly, the aliquot was dissolved and loaded onto a hexane-rinsed cartridge using 200 μL chloroform. Six mL of a 98:2 (v:v) diethyl ether:acetic acid solution was eluted. Residual material was dissolved with 200 μL 2:1 (v:v) chloroform:MeOH and loaded onto the cartridge, followed by elution with 10 mL of MeOH. BHPs were isolated from the MeOH fraction. Lipid analyses BHP preparation and HPLC/APCI–MSn analyses. A known amount (ca. 5–10 μg/g dry sediment) of internal standard (5α-pregnane-3β,20β-diol) was added to SPE extracts of the TLE for BHP analysis. Samples were acetylated in 0.5 mL of a 1:1 (v:v) mixture of acetic anhy- dride and pyridine at 50°C for 1 h, then left to stand overnight at room temperature [80]. Sol- vent was dried under a stream of N on a 50°C heating block. BHP samples were dissolved in MeOH:propan-2-ol (3:2; v:v), and filtered on 0.2 μm PTFE filters. BHPs were analysed by high performance liquid chromatography coupled to positive ion atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS), using a data- dependent scan mode (2 events) on an HPLC system equipped with an ion trap MS, as described in [46,81]. Further structural information for novel BHPs was obtained by way of MS spectra. BHP concentrations were (semi) quantitatively estimated based on the response factor of authentic standards (M. Rohmer; Strasbourg, France and [46,77]) relative to the inter- nal standard. Results In this study we investigated the BHP distributions in species of three AMO marine genera, and of eight marine environments, six of which were CH -influenced. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 8 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Novel nitrogen-containing BHP components In addition to the ‘regular amino-BHPs’ (e.g., I, II, and III; Figs 1 and 2), a suite of novel com- pounds were found in the methanotrophs and screened marine samples. Identification of these compounds is described in detail in the Supplementary Information (S1 File). Briefly, these components were related to the 35-amino-BHPs but differ in their terminal groups at C-35, which are tentatively proposed to comprise a methylcarbamate rather than a simple amine on 2 3 the basis of interpretation of their APCI MS and MS spectra. In each case, the novel com- MC MC MC MC pounds (I , I ’, II , III ; Figs 1 and 2) elute after their ‘regular’ amino-BHP analogues (i.e., I, I’, II, III; Fig 2). This indicates that the tentatively-assigned terminal group structures are less polar than the regular terminal amines (after acetylation). The novel structures include: MC 35-methylcarbamate-bacteriohopane-32,33,34-triol (MC-triol herein; III ), 35-methylcarba- MC mate-bacteriohopane-31,32,33,34-tetrol (MC-tetrol herein; II ), 35-methylcarbamate-bac- MC MC MC teriohopane-30,31,32,33,34-pentol (MC-pentol herein; I ) and an isomer of I (I ’) akin Fig 2. Distribution of nitrogen-containing BHPs in novel Type I methanotroph cultures. Partial mass chromatograms (15±25 min) showing relative abundances (%) of BHPs (shaded peaks) in the acetylated total lipid extracts of (a) Methylomarinum vadi IT-4, (b) Methylomarinovum caldicuralii IT-9, (c) Methylomarinovum sp. IN45, (d) Methylomicrobium alcaliphilum, and (e) Methylomicrobium kenyense. doi:10.1371/journal.pone.0165635.g002 PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 9 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems to the early-eluting aminopentol isomer (I’), which was found, based on mass spectra, in a cul- ture of Methylovulum-like strain M200 [46]. Methanotroph BHP signatures Four previously untested methanotrophs isolated from marine or saline, alkaline lacustrine environments, belonging to the three genera Methylomarinum, Methylomarinovum, and Methylomicrobium, were analysed for their BHP composition. An additional species Methylo- microbium alcaliphilum, the partial BHP composition of which was recently reported in [53], is also shown here in full for comparison with Methylomicrobium kenyense. All of the metha- notroph cultures investigated only synthesised BHPs with a nitrogen atom at C-35 position (nitrogen-containing BHPs herein). The relative abundances of BHPs are indicated as the per- centage of total BHPs in acetylated extracts, and are presented in Fig 2. The low starting mass of some of the dry cell material led to uncertainty in the calculations of absolute concentrations, which are therefore not reported. BHP inventory of Methylomarinum vadi IT-4. The most abundant BHPs in Methylo- MC marinum vadi IT-4 were aminotriol (III), 22.3% and MC-triol (III ), 22.7% (Fig 2A). Ami- MC MC nopentol (I), MC-pentol (I ), aminotetrol (II), and MC-aminotetrol (II ) made up 15.4%, 14.0%, 11.1%, and 11.8% of total BHPs, respectively, with lower levels of the aminopentol iso- MC mers (I’ and I ’; Fig 2A). No C-3 methylated or unsaturated equivalents of aminotriol, ami- notetrol and aminopentol were found in Methylomarinum vadi IT-4. BHP inventory of Methylomarinovum spp. The most abundant BHP in Methylomarino- vum caldicuralii IT-9 was aminopentol (I), 37.4% (Fig 2B). However, Methylomarinovum sp. IN45 had only 5.1% aminopentol (Fig 2C). M. caldicuralii IT-9 had 30.8% MC-pentol, 14.4% aminotetrol, and 13.3% MC-tetrol, and lower abundances of aminopentol isomer, MC-pentol isomer, aminotriol, and MC-triol (Fig 2B). The most abundant BHP in Methylomarinovum sp. MC IN45 was MC-tetrol (II ), making up 51.1%. Methylomarinovum sp. IN45 also contained 12.7% aminotetrol, 15.6% MC-pentol, 12.1% MC-triol and lower levels of aminotriol, and the aminopentol isomers (Fig 2C). No C-3 methylated nor unsaturated equivalents of aminotriol, aminotetrol and aminopentol were present in either strains. BHP inventory of Methylomicrobium spp. The Methylomicrobium alcaliphilum and Methylomicrobium kenyense cultures did not contain aminopentol (I) above detection limit (Fig 2D and 2E) although M. kenyense was found to contain minor abundance of 3-Me-amino- 3Me pentol (I ; 1.0%; Fig 2E). The most abundant BHP in both Methylomicrobium cultures was aminotriol (III), making up ca. 65% of all BHPs in both species. The second most abundant 3Me BHP was 3-Me-aminotriol (III ) at 31.5% in M. alcaliphilum and slightly less in M. kenyense (23.9%). Both species also contained lower levels of aminotetrol (II) and 3-Me-aminotetrol 3Me 3Me (II ). M. kenyense also contained unsaturated compounds (ΔIII andΔIII ; Fig 2E). The only MC compound identified in either Methylomicrobium sp. was MC-triol and then only at low levels (<3%). Marine sediment and carbonate BHP signatures Eight marine settings were studied for their BHP signatures (Table 2). Six of these were known to be influenced by CH (i.e., HHMV, BSCC, AMV, NZS, GoM cold seeps, GD) and two were used as comparison background marine levels (i.e., GoM sediments, PM). Håkon Mosby Mud Volcano (HMMV). None of the HMMV sediment samples contained aminopentol (Table 2). The most abundant amino- and MC-BHPs in HMMV samples was CME either aminotriol (III) or MC-triol (III ), making up 22–52% and 32–62% of total nitrogen- containing BHPs, respectively. Unsaturated aminotriol, unsaturated MC-triol, aminotetrol, PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 10 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. Concentrations (μg/g sediment) of amino-BHPs in marine sediment samples. Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 Håkon Mosby Mud Volcano (HMMV) new mud ¯ow 0±1 PS74/2 169±1 bdl bdl bdl bdl 0.02 0.01 bdl 0.17 0.02 0.14 bdl 0.01 bdl 0.38 cm PUC3 229 aged ¯ow 0±1 cm ARK XXIV-2 bdl bdl bdl bdl 0.01 0.01 bdl 0.29 0.05 0.81 bdl 0.13 bdl 1.3 PS74 172±1 237 center 0±1 cm MSM16/2 847±1 bdl bdl bdl bdl 0.01 0.01 bdl 0.63 0.09 1.26 bdl 0.19 bdl 2.19 MUC 53 center 10±12 cm MSM16/2 HMMV bdl bdl bdl bdl 0.01 0.02 bdl 0.17 0.02 0.17 bdl 0.02 bdl 0.41 new mud ¯ow 0±1 MSM16/2 838±1 bdl bdl bdl bdl 0.02 0.01 bdl 1.07 0.09 0.82 bdl 0.06 bdl 2.07 cm MUC 33 newer mud ¯ow 0±1 MSM16/2 855±1 bdl bdl bdl bdl 0.01 0.01 bdl 1.1 0.13 1.24 bdl 0.15 bdl 2.64 cm MUC 73 newer mud ¯ow 10± MSM16/2 HMMV bdl bdl bdl bdl 0.02 0.01 bdl 0.17 0.02 0.11 bdl 0.01 bdl 0.33 12 cm 83 aged ¯ow 0±1 cm MSM16/2 823±1 bdl bdl bdl bdl 0.02 0.02 bdl 0.56 0.05 0.88 bdl 0.07 bdl 1.61 MUC 19 aged ¯ow 10±12 cm MSM16/2 HMMV bdl bdl bdl bdl 0.01 0.01 bdl 0.15 0.01 0.11 bdl 0.01 bdl 0.29 Barents Sea Carbonate Crusts (BSCC) depth pro®le 0±2 cmbsf bdl bdl bdl bdl 1.36 bdl bdl 4.45 0.12 bdl bdl bdl bdl 5.93 2±4 cmbsf bdl bdl bdl bdl 0.21 bdl bdl 0.93 0.06 bdl bdl bdl bdl 1.2 4±6 cmbsf bdl bdl bdl bdl 0.04 bdl bdl 0.27 0.03 0.02 bdl bdl bdl 0.36 6±8 cmbsf bdl bdl bdl bdl 0.03 bdl bdl 0.19 0.02 0.02 bdl bdl bdl 0.26 8±10 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.07 bdl 0.02 bdl bdl bdl 0.09 10±13 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.03 bdl 0.01 bdl bdl bdl 0.04 13±16 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.08 bdl bdl bdl bdl bdl 0.08 16±19 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.11 bdl bdl bdl bdl bdl 0.11 Amon Mud Volcano (AMV) central dome 0±1 cm M70/2a 760 bdl bdl bdl bdl 0.02 0.07 bdl 0.42 0.13 2.67 bdl 0.96 bdl 4.27 PUC33 24 sulfur band 0±1 cm M70/2a 765 0.18 bdl 0.12 bdl 0.38 0.27 bdl 1.24 0.81 1.33 bdl 1.02 bdl 5.35 PUC49+50 71 sulfur band 10±12 M70/2 81 0.36 0.04 0.11 bdl 0.3 0.11 bdl 1.07 1.09 0.48 bdl 0.5 bdl 4.06 cm sulfur band 0±1 cm M70/2a 790 0.36 bdl 0.11 bdl 1.49 0.49 bdl 3.82 1.29 1.49 bdl 0.61 bdl 9.67 PUC68 172 sulfur band, white MSM13/3 947±1 0.09 bdl 0.11 bdl 0.31 0.24 bdl 0.64 0.36 0.71 bdl 0.44 bdl 2.9 mat PUC31 73D sulfur band 0±1 cm MSM13/3 968±1 0.08 bdl 0.06 bdl 0.75 0.49 bdl 0.73 0.46 0.77 bdl 0.28 bdl 3.62 PUC15 122 bacterial mats 0±1 MSM13/3 929±1 0.05 bdl 0.01 bdl 1.13 0.17 bdl 1.11 0.43 0.5 bdl 0.16 bdl 3.56 cm PUC22 45D (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 11 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. (Continued ) Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 bacterial mats 10±12 MSM13/3 50D bdl bdl bdl bdl 0.01 bdl bdl 0.03 bdl 0.02 bdl bdl bdl 0.07 cm gassy centre, 10±12 MSM13/3 61D bdl bdl bdl bdl 0.01 bdl bdl 0.04 0.01 0.02 bdl 0.01 bdl 0.08 cm New Zealand Seeps (NZS) 0±2 cmbsf Frenulata 45 bdl bdl bdl bdl 0.09 0.06 bdl 0.21 0.03 0.29 bdl 0.05 bdl 0.73 10±12 cmbsf Frenulata 45 bdl bdl bdl bdl 0.13 0.03 bdl 0.26 0.04 0.15 bdl 0.02 bdl 0.63 0±2 cmbsf Ampharetidae 0.06 bdl bdl bdl 1.11 0.17 bdl 1.32 1.17 2.59 bdl 0.26 bdl 6.67 10±12 cmbsf Ampharetidae bdl bdl bdl bdl 0.01 0.02 bdl 0.08 0.02 0.13 bdl 0.02 bdl 0.28 0±2 cmbsf Sulfur-oxidising 0.02 bdl bdl bdl 0.66 0.32 bdl 2.24 0.23 2.13 bdl 0.21 bdl 5.8 bacteria 315 10±12 cmbsf Sulfur-oxidising bdl bdl bdl bdl 0.03 bdl bdl 0.14 0.02 0.1 bdl bdl bdl 0.28 bacteria 315 Golfo Dulce (GD) Surface Sediment 10 m water depth SG 1 bdl bdl bdl bdl bdl bdl bdl 0.01 bdl bdl bdl bdl bdl 0.01 24 m water depth SG 2 0.16 bdl bdl bdl 0.03 bdl bdl 0.36 bdl 0.02 bdl bdl bdl 0.57 77 m water depth SG 4 0.02 bdl bdl bdl 0.01 bdl bdl 0.15 0.03 0.01 bdl bdl bdl 0.23 90 m water depth SG 5 0.04 bdl bdl bdl 0.02 bdl bdl 0.17 0.05 0.01 bdl bdl bdl 0.29 110 m water depth SG 6 bdl bdl bdl bdl 0.01 bdl bdl 0.08 0.07 0.02 bdl bdl bdl 0.18 120 m water depth SG 7 0.04 bdl bdl bdl 0.02 bdl bdl 0.36 0.2 0.02 bdl bdl bdl 0.63 140 m water depth SG 8 0.01 bdl bdl bdl <0.01 bdl bdl 0.11 0.08 0.01 bdl bdl bdl 0.21 Gulf of Mexico (GoM) Cold Seeps GC 234 CNSTS bdl bdl bdl bdl 0.01 bdl bdl 0.09 bdl bdl bdl bdl bdl 0.1 234 4436 bdl bdl bdl bdl <0.01 bdl bdl 0.04 bdl bdl bdl bdl bdl 0.04 GC 234 tube worm bdl bdl bdl bdl <0.01 bdl bdl 0.02 bdl bdl bdl bdl bdl 0.02 roots 4435 GC 232 bdl bdl bdl bdl 0.01 bdl bdl 0.01 bdl bdl bdl bdl bdl 0.02 GC 185 bdl bdl bdl bdl 0.76 bdl bdl 0.2 bdl bdl bdl bdl bdl 0.95 GoM Sediments West Gulf WG2-099/6 bdl bdl bdl bdl <0.01 bdl bdl 0.01 bdl bdl bdl bdl bdl 0.01 26Ê50'N, 92Ê40'W WG2-099/9 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl <0.01 bdl bdl bdl 0.01 Eastern Gulf 88-C-1 DCS 128/ bdl bdl bdl bdl bdl bdl bdl 0.01 bdl 0.01 bdl bdl bdl 0.02 27Ê30'N, 87Ê20'W DCS 128/13 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl <0.01 bdl bdl bdl 0.01 DCS 128/21 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl 0.01 bdl bdl bdl 0.01 Central Gulf CGD-136/17 bdl bdl bdl bdl bdl bdl bdl 0.01 bdl 0.01 bdl bdl bdl 0.01 28Ê20'N, 89Ê38'W CGD-136/13 bdl bdl bdl bdl bdl bdl bdl <0.01 bdl 0.01 bdl bdl bdl 0.01 Peru Sediment 10±15 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.09 bdl bdl bdl bdl bdl 0.09 20±25 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.02 bdl bdl bdl bdl bdl 0.02 (Continued ) PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 12 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Table 2. (Continued ) Amino-BHPs MC 3Me MC 3Me MC 3Me MC 3Me I ΔI I I II II II III ΔIII III III ΔIII ΔIII Total nitrogen- containing BHPs (ug/g sediment) Base Peak 830 828 846 844 772 788 786 714 712 730 728 728 726 40±45 cmbsf bdl bdl bdl bdl bdl bdl bdl 0.09 bdl bdl bdl bdl bdl 0.09 base peak = [M + H±CH3COOH]+ bdl±below detection limit doi:10.1371/journal.pone.0165635.t002 and MC-tetrol all made up <10% of total nitrogen-containing BHPs. Some HMMV samples contained relatively high concentration of anhydrobacteriohopanetetrol (anhydro-BHT). Minor abundances of BHT and BHT isomer, BHT-cyclitol ether (BHT-CE), and BHT-glucos- amine (BHT-G) were detected in some samples (S1 Table). Barents Sea carbonate crust (BSCC). The 19 cm BSCC core contained a majority of ami- MC notriol (III; 72–100% of nitrogen-containing BHPs; Table 2). MC-triol (III ; 0–23%) peaked between 4 and 13 cm. Aminotetrol (II; 0–23%) was detected in the upper 8 cm. Minor contri- bution of unsaturated aminotriol (ΔIII; 0–7%) was detected in the upper sediments. Other BHPs detected included BHT, and low concentrations of anhydroBHT, BHT isomer, 2-methyl-BHT (2-Me-BHT), adenosylhopane, BHT-CE, and BHT-G (S1 Table). Amon Mud Volcano (AMV). The most abundant nitrogen-containing BHPs in sedi- ments from the Amon mud volcano were aminotriol (III; 9.8–48.9%) and MC-triol MC (III ;11.8–62.5%) (Table 2). Other nitrogen-containing BHPs in these sediments were ami- notetrol (II; 0.5–31.8%), unsaturated aminotriol (ΔIII; 3.0–27.0%) and unsaturated MC-triol MC MC (ΔIII ; 0–22.5%). Minor abundances of aminopentol (I; 0–8.9%), and MC-pentol (I ; 0–3.8%) were found in some AMV sediments. Other BHPs in the AMV sediments were BHT, anhydroBHT, 2-Me-BHT, 3-Me-BHT, BHT-CE, BHT-G, BHT isomer, and adenosylhopane (S1 Table). New Zealand Seeps (NZS). The most abundant nitrogen-containing BHP in the sedi- MC ments from NZS were aminotriol (III; 28.5–72.7%), MC-triol (III ; 13.2–46.4%) and amino- tetrol (II; 5.1–20.6%) (Table 2). Unsaturated aminotriol (ΔIII), unsaturated MC-triol MC MC (ΔIII ), and MC-tetrol (II ) all made up <10%. Two sediment samples contained amino- pentol, albeit in low abundance (<1% of nitrogen-containing BHPs). Both of these aminopen- tol positive samples also contained soil marker BHPs [77,82]. Other BHPs found in NZS samples were BHT, anhydroBHT, 2-methyl-BHT, 3-methyl-BHT, BHT isomer, adenosylho- pane, and BHT-CE (S1 Table). Gulfo Dulce (GD). The most abundant nitrogen-containing BHP in GD surface sedi- ments was aminotriol (III; 44.3–100%; Table 2). Unsaturated aminotriol (ΔIII) made up MC 0–41.3%. MC-triol (III ; 0–11.4%) and aminotetrol (II; 0–6.0%) were found in most samples. Aminopentol was found in all but two surface samples, one of which was the shallowest site. Aminopentol abundance decreased with increasing water depth with the highest abundance at 24 m water depth (27.6% of nitrogen-containing BHPs). BHT, anhydroBHT, BHT isomer, and 2-Me-BHT were also found in GD (S1 Table). Gulf of Mexico (GoM) cold seeps. GoM cold seeps only contained aminotriol (III; 20.5– 100%) and aminotetrol (II; 0–79.5%) nitrogen-containing BHPs (Table 2). Other BHPs included BHT, soil marker BHPs, 2-Me-BHT, BHT isomer, and BHT-CE (S1 Table). PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 13 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems GoM sediments. GoM sediments from near the outflow of Mississippi River Delta showed an abundance of aminotriol (III; 26.6–80.7% of total nitrogen-containing BHPs), MC-triol MC (III ; 0–73.4%) (Table 2). One sample from the Western Basin had 19.3% aminotetrol (II) relative to total BHPs; however, the concentration of aminotetrol was low (<0.01 μg/g sedi- ment). Other BHPs included BHT, BHT isomer, anhydroBHT, 2-methyl-BHT, BHT-CE, and soil marker BHPs (S1 Table). Peru Margin (PM). The only nitrogen-containing BHP detected in sediments from the PM was aminotriol (III). Other BHPs found at this site included BHT, BHT isomer, anhy- droBHT, 2-Me-BHT, adenosylhopane, and BHT-CE (S1 Table). Discussion BHP distributions in aerobic methanotrophs Previously reported BHP distributions in AMO bacteria. Traditionally, Type I and Type II AMO bacteria had been distinguished by their different BHP signatures (e.g. [52]; see also review in [14]). Prior to the investigation of BHPs in the Methylovulum-like strain M200 [46], most screened Type I methanotrophs synthesised a high percentage of aminopentol (I) and lower contributions from aminotetrol (II) and in some cases aminotriol (III), and clustered in the left-hand corner of the amino-BHP ternary plot (Fig 3A). In contrast, Type II methano- trophs did not contain aminopentol, had varying contributions from II and III, and clustered along the right-hand axis of Fig 3A. The high relative abundance of III observed in Methylovu- lum-like strain M200 was, therefore, originally seen as an outlier [46]. Similarly, [52] showed that a culture of Methylomicrobium album did not contain aminopentol. At the time this was Fig 3. Relative composition (%) of `regular' amino-BHPs. Distributions of aminotriol, aminotetrol and aminopentol, including their C-3 methylated homologues, where present, are shown for (a) methanotroph cultures including literature data (circles) and data from new cultures of Methylomicrobium, Methylomarinum, and Methylomarinovum (diamonds; this study) and (b) sediments and microbial mats from methane-rich marine settings (HMMV = Håkon Mosby mud volcano; BSCC = Barents Sea carbonate crust; AMV = Amon mud volcano; NZ = New Zealand; GD Golfo Dulce; GoM = Gulf of Mexico; PM = Peru Margin). Methanotroph literature data from [36,42,46,52,83]. Literature data from [36,42,83] was based on GC-MS hopanol quantifications after periodic acid treatment. Therefore, it is not possible to distinguish between amino- and non-amino-BHPs with the same number of functional groups. This is especially significant for the Type II aerobic methanotrophic bacteria that are known to synthesise bacteriohopanetetrol. For this reason, the axes of plot (a) are shown as functionality of the BHP-side chain. doi:10.1371/journal.pone.0165635.g003 PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 14 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems presumed to be a contaminated culture, however, [53] also did not report I synthesis in cul- tures of Methylomicrobium alcaliphilum. All of the recently analysed Methylobacter spp. [28] join the more typical Type I methanotrophs in the left-hand corner of the plot, however, Methylobacter sp. BB5.1 increased the spread of the cluster with almost 40% III content. Amino-BHP distributions in previously untested Type I AMO bacterial cultures. It was assumed that the screened species of AMO Type I bacteria investigated in this study would display similar BHP distributions as those of previously reported Type I bacteria. All three bac- terial genera screened do indeed only contain amino-BHPs (Fig 2). However, the relative distri- bution of specific nitrogen-containing BHPs varies between genera, as well as between species belonging to the same genus. To allow for a more accurate comparison with data from the liter- ature (Fig 3A, circles), only aminopentol, aminotetrol, aminotriol, and their methylated equiva- lents were considered when producing the ternary plot of ‘regular’ nitrogen-containing BHPs of the novel Type I cultures (Fig 3A, diamonds). Aminopentol is the most abundant BHP in the novel species Methylomarinovum cadicuralii IT-9, which is in agreement with literature BHP compositions of most other Type I methano- trophs (Fig 3A, circles), e.g., Methylococcus capsulatus [44,52], Methylomonas sp. [42,46,52], psychrotolerant isolate CEL 1923 [42], and Methylocaldum tepidum [22]. However, a species in the same genus (Methylomarinovum sp. IN45) has a much lower abundance of aminopentol (5.0% of total amino-BHPs; Fig 2C). Methylomarinum vadi IT-4 shows relatively high propor- tions of aminopentol, but it is not the most abundant BHP (Fig 2A). Moreover, in our screen- ing of two species of Methylomicrobium spp., aminopentol was not detected, similar to reported cultures of Methylomicrobium album and Methylomicrobium alcaliphilum [52,53]. Our results seem to confirm the near-absence of aminopentol in all screened Methylomicro- bium spp., which are the first Type I methanotrophs apparently unable to synthesise amino- pentol. However, changes in BHP composition can occur at different growth stages and under different conditions (e.g. [27,83,84]), so further studies would be required to fully confirm this. It appears that the BHP distributions of Methylomicrobium and Methylovulum, which do not synthesise high amounts of aminopentol, should also no longer be considered outliers given the low levels of aminopentol in M. vadi IT-4 and Methylomarinovum spp. (Fig 2). This sug- gests a greater variance in the BHPs of Type I methanotrophs than previously thought. Fur- thermore, as Methylomicrobium has been isolated from a diverse range of marine environments [85–87], the absence of aminopentol in this genus might greatly affect its appli- cation as a marine aerobic methanotrophy biomarker. This, however, does not invalidate the use of aminopentol as a biomarker for methanotrophy. There is also significant variation in the relative abundances of the other nitrogen-contain- ing BHPs in Type I methanotrophs. A suite of novel BHPs identified as methylcarbamate (MC) BHPs are detected in all three genera screened in this study (Fig 2). These have not been reported in previous studies. Therefore, data available at Newcastle University from the analy- ses of Methylococcus capsulatus (Talbot et al., unpublished), Methylovulum-like strain M200, Methylomonas methanica, Methylomonas-like strain M5 [46], and Methylobacter spp. [28] MC were re-examined. III was identified retrospectively in Methylomonas methanica, and all MC species of Methylobacter. III constituted up 9.8% of total amino-BHPs in Methylobacter sp. MC BB 5.1. II was present in low abundance (<0.2%) in two of the three species of Methylobac- ter (Methylobacter sp. BBA 5.1 and Methylobacter sp. BB 5.1), but absent in Methylobacter mar- MC inus A 45. I was absent in Methylococcus capsulatus and Methylovulum-like strain M200, MC but was present in all species of Methylobacter. I was also present in Methylomonas metha- nica but absent in a different species of this genus (i.e., Methylomonas-like strain M5; [46]). These results indicate that MC-BHPs are not universally present when their regular PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 15 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems homologues are detected, and may be species specific and/or dependent on variations in growth conditions such as pH. The two species of Methylomarinovum display significant variations in their BHP composi- tions (Fig 2B and 2C). Methylomarinovum sp. IN45 had a relatively low level of aminopentol. MC The most abundant BHP in Methylomarinovum sp. IN45 is II . Although in lower abun- MC MC dances, III and I are also higher in comparison to their ‘regular’ homologues in this spe- cies compared to M. caldicuralii IT-9 (Fig 2B and 2C). In contrast, the most abundant BHP in Methylomarinovum caldicuralii IT-9 is aminopentol, followed by almost equal amounts of MC I . The different relative BHP distributions between the Methylomarinovum spp. highlight that there can be significant variations within a genus. Methylomarinovum sp. IN45 was iso- lated from a deep-sea hydrothermal field and perhaps the high levels of methylcarbamate com- ponents observed are the result of a physiological adaptation to higher pressure in this environment. This may explain why the relative abundances of components in Methylomarino- vum caldicuralii IT-9, the same genus but isolated from a shallow submarine hydrothermal environment, are quite different. Perhaps the complex functionality of the terminal group of the methylcarbamate components is more effective at stabilising the cell membrane and decreasing fluidity under these conditions. 3Me 3-methylaminotriol (III ) was observed in both Methylomicrobium spp. (23.9–31.5% of total BHPs; Fig 2D and 2E) in agreement with a recent report in [53]. This compound was accompanied 3Me by low levels of 3-methylaminotetrol (II ) in both species and trace amounts of 3-methylamino- 3Me pentol in M. kenyense (I ). The absence of C-3 methylated structures in the previously investi- gated Methylomicrobium album strain BG8 [52] may appear inconsistent with the organisms investigated here; however, genomic investigations have revealed that M. album is separated from halo(alkali)philic representatives of the Methylomicrobium genus such as M. alcaliphium and M. kenyense [58], and perhaps specific environmental conditions influence the BHP composition of Methylomicrobium spp. as they seemingly do within the Methylomarinovum genus. 3Me 3Me No C-3 methylated equivalents of aminotriol (III ), aminotetrol (II ), nor aminopentol 3Me (I ) were present in Methylomarinum vadi IT-4 or the Methylomarinovum spp., adding to examples of Type I methanotroph species that contain amino-BHPs, but not their C-3 methyl- ated equivalents (e.g., see review in [14]). The most abundant BHPs in Methylomarinum vadi MC IT-4 were aminotriol (III) and MC-triol (III ), which were present in equal amounts (Fig MC MC 2A). Similar amounts of II and II , and I and I were also observed in this culture. A high proportion of III is unusual for a Type I methanotroph, but has been observed before in the Methylovulum-like strain M200. (Fig 4A; [46]). The new data reiterate that aminopentol is not always the most abundant BHP in Type I methanotrophs, nor necessarily the most appropriate biomarker for AMO. BHPs in marine environments Lack of BHP diversity in marine environments. The screened marine sediments and authigenic carbonates do not show large diversity in their BHP signatures (S1 Table). The lim- ited BHP distributions are also comparable to other reported marine sediment BHP signatures, all dominated by BHT and BHT isomer, from a number of locations including the Black Sea [88], the Benguela upwelling system [89], and the Arabian Sea [90]. More recently a similar pattern was also seen in water column samples from the California Current system, where the wide diversity observed in the gene responsible for hopane cyclisation (squalene-hopene cyclase) was not reflected by distinct BHP fingerprints related to this potential range of source- organisms [91]. However, genetic information is quickly lost, and we must strive to find lipid biomarkers to trace particular metabolisms in the geological record. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 16 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Fig 4. Relative composition (%) of methylcarbamate(MC)- BHPs. Distributions of MC-triol, MC-tetrol and MC-pentol are shown for (a) methanotroph Type I cultures, Methylobacter, Methylomicrobium, Methylomarinum, and Methylomarinovum, and (b) marine sediments (HMMV = Håkon Mosby mud volcano; BSCC = Barents Sea carbonate crust; AMV = Amon mud volcano; NZ = New Zealand; GD Golfo Dulce; GoM = Gulf of Mexico). Methylobacter data from [28]. doi:10.1371/journal.pone.0165635.g004 Non-nitrogen-containing BHP concentrations in the screened sediments do not show remarkable signatures (S1 Table). BHT and anhydro-BHT, thought to be a degradation prod- uct of BHT and other composite BHPs such as BHT cyclitol ether [92], were found at all sites. The presence of soil marker BHPs at some sites, particularly NZS, indicates that these sedi- ments could be influenced by terrestrial input of organic matter (e.g., [77,82,93]). However, as adenosylhopane is an intermediate in the biosynthesis of all other side-chain extended BHPs [94], other sources cannot be entirely excluded. BHT isomer, a biomarker for anaerobic ammo- nium oxidation [70], was found in high concentrations in GD (previously reported in [70]), as well as in the PM sediments, which underlie the Peruvian OMZ, where anammox is known to be an important process [95], and where BHT isomer has previously been reported from the water column within the OMZ [90]. The most abundant of the three regular amino-BHPs in the CH -influenced marine sediments was aminotriol, which is not source-specific (e.g., [48]). Aminopentol in marine sediments. Although aminopentol was found in significant abundance in some of the reported and screened Type I methanotroph cultures (Fig 2), it was not found to be abundant in most of the CH -influenced marine sites in this study (Table 2). In fact, it was only detected in AMV, GD surface sediments, and two NZS samples (Fig 3B; Table 2). The discrepancy between the distinct amino-BHP signatures of isolated Type I AMO bacteria and signatures of CH -influenced marine sites is highlighted in the ternary plots of the relative composition of aminopentol, aminotetrol, and aminotriol (Fig 3A cf. Fig 3B). These differences could be due to the particular methanotrophic bacterial community responsible for methanotrophy in the CH -influenced marine sediments. Ruff et al. [96] found that diversity in the global CH seep microbiome was controlled by environmental factors such as PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 17 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems temperature and electron acceptor availability. Considering their findings, it is possible that the environmental conditions in most marine CH -influenced sediments favour specific methano- troph communities. For example Methylomicrobium spp., found in saline environments [85– 87] and saline, highly alkaline environments [58,97], and which do not produce aminopentol in significant amounts (Fig 2), could be present. However, the absence of C-3 methylated com- pounds is confounding for a Methylomicrobium source, pointing towards other methanotrophs that do not synthesise aminopentol. Yan et al. [98] found that 85% of the operational taxo- nomic units (OTUs) from the same sites as our GoM cold seeps did not group with known sequences of a subunit of particulate methane monooxygenase (pmoA). This would suggest the presence of novel methanotrophic species in GoM. In the same way, significant pmoA diversity has been observed in sediments from the North American margin [99], a shallow CH seep [100], a marine estuary [101], and hydrocarbon seeps [102]. pmoA OTUs from the NZS sedi- ments grouped with methanotrophic endosymbionts [68], including Bathymodiolus spp., which have been shown to contain neither aminopentol nor methylated BHPs [103]. Neverthe- less, the absence of methylated amino-BHPs in the screened marine sediments (Table 2) may suggest Methylomicrobium album, or a related species that also does not synthesise methylated amino-BHPs, being the dominant methanotroph in CH -influenced marine environments. These are not the first reports of marine CH -influenced environments not containing ami- nopentol (Table 1). For example, using methods targeting the functional gene pmoA, which is produced by most methanotrophs, Type I methanotrophs were detected in all three units of Ace Lake sediments. However, aminopentol was only detected in sediments deposited under freshwater conditions (unit III) despite the fact that the modern meromictic water column, containing relict seawater left behind after the sea level fell around 9000 years ago, hosts the Type I methanotroph Methylosphaera hansonii [104]. No aminopentol was detected in the methanotrophic symbionts in the gill tissue of a cold-seep mussel, despite other lipid-based evi- dence suggesting the presence of a Type I methanotroph [103,105]. Similarly, CH seep car- bonates from Alaminos Canyon, northern Gulf of Mexico [41] and the Northern Arabian Sea [106] were found to lack aminopentol. Conversely, aminopentol was detected in the water col- umn of the Baltic Sea with supporting evidence for the presence of Type I methanotrophs from C-depleted PLFAs [107]. Aminopentol was also detected in the water column of the Black Sea in the oxic-anoxic water transition, but not in the underlying sediment [12,88,108]. The presence of aminopentol in sediments from the AMV, located on the Nile deep-sea fan, in the Eastern Basin of the Mediterranean Sea (Table 2) may be explained by Nile River outflow carrying terrestrial wetland methanotrophy signatures into the Mediterranean, as seen in the Amazon and Congo River fans [14,109]. This would appear to indicate that aminopentol is still an excellent biomarker for terrestrial AMO. The near-absence of soil-marker BHPs in AMV (Table 2) may still point towards in-situ marine production of aminopentol. However, the rela- tive abundance of soil-markers in terrestrial settings has recently been found to be strongly influenced by environmental factors; higher temperatures and low pH (in peatlands) can both strongly reduce the relative proportion of soil marker BHPs as a proportion of total BHPs [40,110,111]. Aminopentol was found in NZS sediments that also contained soil marker BHPs (Table 2). Therefore, aminopentol in sediments from NZS may have originated from terrestrial sources. Aminopentol in GD surface sediments may be the result of a distinct AMO commu- nity living in the specific environment prone to carbonate formation in GD. Unfortunately, samples were not properly preserved to be able to determine AMO diversity using genetic- based analyses of the pmoA gene in these sediments. The cumulative results of the studied marine sites do, however, indicate that an absence of aminopentol is not necessarily evidence for the absence of methanotrophs or aerobic methane oxidation. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 18 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Alternative BHP biomarkers for AMO and implications for the marine sedimentary record of methanotrophy Regular amino-BHPs. Screened Type I methanotrophs also produced varying amounts, depending on the genera, of aminotetrol (II) and aminotriol (III) (Fig 3A), both of which were found in CH -influenced marine sediments (Fig 3B; Table 2). However, these two amino- BHPs are less source-specific to methanotrophic bacteria than aminopentol, and do not make 3Me ideal biomarker lipids for methanotrophy. Given that 3-Me-aminotriol (III ) made a signifi- cant contribution to the amino-BHP abundance in screened cultures of Methylomicrobium spp. (23.9 and 31.5% of total amino-BHPs; Fig 2D and 2E) and 9.8% in Methylobacter sp. 3Me BB5.1 [28], it was expected that III would be an important amino-BHP in CH -influenced 3Me marine sediments. However, III was not found in any of the screened sediments (Table 2). Methylomicrobium alcaliphilum and Methylomicrobium kenyense are adapted to high alkalin- 3Me ity, but not necessarily to high salinity [58]. This distinct lack of III in marine sediment samples would seem to indicate that the Methylomicrobium species we investigated are not the primary source of amino-BHPs in CH -influenced marine environments. 3Me III has only occasionally been reported from environmental samples including some soils [82,112] and most recently in a peat core from Germany [111], but only at very low levels 3Me (Table 1). Other C-3 methylated amino-BHPs are even less common (Table 1). I was first reported from a neo-volcanic, eutrophic and saline lake sediment (La Piscina de Yuriria, Mexico; [45]), and subsequently from a geothermal silica sinter (Opaheke Pool hot spring, 3Me New Zealand; [113]). The pentafunctionalised II was also present in the Mexican lake sedi- ment. Both of these compounds were reported in one study on the Black Sea water column [12], but were absent at another site [108]. The apparent discrepancy between the very limited occurrence of C-3 methylated BHPs (as measured using the periodic acid cleavage technique which converts polyfunctionalised BHPs into GC-amenable primary alcohols; e.g., [36,75]) and their wider occurrence in the form of 3-Me hopanes in ancient rocks and oils was first identified in [114]. These authors found 3-Me-BHPs to be abundant only in a very limited number of settings, under quite specific conditions (i.e., some alkaline lakes). The occurrence of 3-Me-hopanes in marine authigenic carbonates [31,115], which form under highly alkaline conditions are also consistent with a Methylomicrobium source ([58], and references therein). It was further suggested that 3-Me-BHPs and hexafunctionalized BHPs appear to have differ- ent sources (possibly, but not necessarily restricted to, only Type I methanotrophs; [114]). Cul- ture studies (on the moderately thermophilic Type I methanotroph Methylococcus capsulatus) have shown that production of C-3 methylated compounds may be related to growth stage. Higher relative proportions of methylated BHPs replaced the non-methylated equivalents dur- ing stationary phase growth [83], and appear to be necessary for maintaining intra-cellular membrane structures [27]. These important physiological roles for methylated BHPs are at odds with the very sparse occurrence of these compounds in modern settings (Table 1), and clearly our understanding of the factors controlling their biosynthesis and subsequent preser- vation in sediments is still limited, hampering interpretation of certain BHP signatures. Methylcarbamate-BHPs. Most of the marine sediments influenced by CH contained at least MC-triol, albeit at relatively low abundances (Fig 4B; Table 2). The fact that the MC-BHPs were found in all strains of methanotrophs analysed, though not all components in the suite were present in every strain, shows the biomarker potential of these BHPs for AMO (Fig 3A). MC MC-tetrol (II ) was the most abundant component in Methylomarinovum sp. IN45, and MC-BHPs were found in higher abundance than the ‘regular’ 35-amino-BHP homologues, which may allow this particular hydrothermal vent species to be identified in environmental CME settings. Unsaturated MC-triol (ΔIII ) was found in high abundance in AMV, HMMV, and PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 19 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems NZS, but was not found in any of the methanotroph cultures. This is possibly because the BHP signatures in most CH -influenced marine sediments are sourced from AMO bacteria that have no cultured relatives or at least none which have been tested for BHP production. Given the small diversity in BHPs found in marine sediments and the need for an AMO bio- marker, there appear to be few BHPs that meet the criteria of being source-specific and abun- dant. This has significant implications for the development of a proxy using aminopentol to trace AMO in marine settings. Applying MC-BHPs combined with the traditional suite of amino-BHPs (e.g. aminopentol, aminotetrol, and aminotriol) seems to be the most appropriate biomarker course for AMO. Conclusions Isolated methanotrophs from previously unexamined genera and species displayed marked dif- ferences in their relative abundances of amino-bacteriohopanepolyols (BHPs). Aminopentol (I) was the most abundant BHP in Methylomarinovum caldicuralii IT-9, which fits with the typical BHP signature of known Type I methanotrophs. However, the BHP signatures of Methylomarinovum sp. IN45 and Methylomarinum vadi IT-4 both did not show aminopentol as the most abundant BHP. Moreover, neither of the Methylomicrobium spp. contained amino- pentol and only one contained a low level of 3-methyl-aminopentol showing that not all Type I methanotrophs synthesise aminopentol, agreeing with previous environmental studies. Con- sidering Methylomicrobium can be prevalent in marine environments, this has implications for the use of aminopentol as a biomarker for marine methanotrophy. A suite of components related to amino-BHPs, but with methylcarbamate (MC) terminal groups, were detected for the first time, and were present in all Type I methanotroph strains tested. Marine sediments influenced by CH did not contain significant amount of aminopentol, but did contain MC-BHPs. This study highlights the relatively low BHP diversity within marine sediments, and indicates that the combined use of MC-BHPs and amino-BHPs might be preferential to trace aerobic methane oxidation (AMO) in marine settings. Supporting Information S1 File. Identification of novel BHP compounds in methanotroph cultures. (DOCX) S1 Table. Concentrations of other BHPs in marine sediment samples. Concentration (μg/g sediment) of other BHPs in marine sediments samples presented in this study. (XLSX) Acknowledgments We are deeply thankful to A. Boetius for supplying the sediments from HMMV, AMV, and NZS. Additional information about these sites can be found on the Pangaea database (http:// www.pangaea.de/).The PM sediments analysed in this study were originally provided by T. Eglinton and J. Repeta. R.D. Pancost is thanked for use of GoM cold seep material. The GoM sediments used in this study were provided by R. Sassen and S. Sweet of the Geochemical & Environmental Research Group (GERG) at Texas A&M University, U.S.A. We thank the cap- tain and the crew of the RV Kaiyo, the operation team of the ROV Hyper-Dolphin, and the shipboard scientific party for the invaluable help in collecting deep-sea samples. We also thank M. Abe and K. Tanaka for their assistance in cultivation. PLOS ONE | DOI:10.1371/journal.pone.0165635 November 8, 2016 20 / 27 Biomarkers of Aerobic Methanotrophy in Marine Systems Author Contributions Conceptualization: KAO DR HMT. Formal analysis: DR KAO HMT. Investigation: KAO DR FRS. Methodology: KAO DR HMT. Resources: DB AK HH JP SWP JCN KM MK. Writing – original draft: DR KO HMT. Writing – review & editing: DB AK HH JP SWP JCN KM MK. References 1. Lelieveld J, Crutzen PJ, Dentener FJ (1998) Changing concentration, lifetime and climate forcing of atmospheric methane. Tellus B 50: 128±150. 2. IPCC (2007) Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL (Eds.)]. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA. 3. 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