Analysis of diffuse flow communities separated EPR from the Guaymas sites according to both Bray-Curtis similarity (**Figure 6**) and nonmetric dimensional scaling (NMDS) of the

nonparametric Theta – Yue and Clayton (θyc) similarity coefficients (Schloss and Handelsman, 2005; Schloss et al., 2009) (**Figure 7**). To explain the grouping of the samples along the axes, the correlation of the relative abundance of individual OTUs in the NMDS dataset was calculated. The vector values of the most abundant OTUs were overlaid on the NMDS plot (**Figure 7A**). The OTUs that most contributed to the spatial distribution of the samples were within the *Epsilonproteobacteria*. About half of these were within the *Sulfurovum* genera, the others did not classify at genera level. Three other OTUs within the *Sulfurovum* genera also were correlated but were left off the plot because of their similarity to the vectors of the other *Sulfurovum* genera (data not shown). A *Thermococcus* (OTU-29) that was found in very high abundance in three Guaymas samples (4477, 4479, 4481), and a *Planctomycetes* (OTU-2918 & 2438) and an unclassified bacteria (OTU-65) found in high abundance in Guaymas 4476, 4478, or 4480 also significantly contributed to the observed distribution (**Figure 7A**).

Out of 24 constituent geochemical features (**Table 2**), iron, cobalt and ammonium concentrations correlated best to the

macrofauna-associated; open symbols, not macrofauna-associated.

community similarity plot (*r >* 0*.*6, confirmed with function bioenv in *R* where *r* = 0*.*6) (**Figure 7B**). Ammonium was correlated more with the distribution of microbial communities in the Guaymas Basin samples, while iron and cobalt were better correlated with communities in the EPR samples. Also included on the plots were constituents whose r values were between 0.2 and 0.6 (**Figure 7B**). These included temperature, sulfide, nickel, and phosphate (between 0.2 and 0.4) and magnesium, potassium, lithium, barium, strontium, and nitrite (between 0.4 and 0.6).

#### **DISCUSSION**

Our experimental approach allowed us to deeply sample microbial diversity and community composition between two geochemically and geographically distinct hydrothermal vent diffuse flow environments. Using statistical subtraction of taxa found in surrounding deep-sea water, it was possible to directly compare vent-specific taxa and further partition this diversity by taxonomic domain. As with other microbial diversity studies of hydrothermal vent microbial communities (Huber et al., 2007, 2010), *Epsilonproteobacteria* dominated the vent specific taxa in most samples. Samples collected near macrofaunal communities had a higher abundance of bacteria, specifically *Epsilonproteobacteria*, than those collected near sediments or vents not populated by macrofauna. Unexpectedly, we found that some diffuse flow environments contained archaeal communities that were of higher diversity and evenness than co-existing bacterial communities. Additionally, one of the dominant taxa groups found in the off axis water, Thaumarcheaota (formerly marine Crenarchaeota) MGI (Delong, 1992; Brochier-Armanet et al., 2008), was differentially present between the two geographic locations. The second, SUP05, is found in many oxygen minimum zones, including hydrothermal vent plumes (Sunamura et al., 2004; Walsh et al., 2009). Nevertheless, distinct patterns in microbial community composition between sites were apparent after statistical subtraction of taxa present in background seawater. These patterns were correlated both to specific taxa (e.g., *Epsilonproteobacteria*, *Planctomycetes*) and to environmental factors (e.g., iron, ammonium).

#### **DISTINCTIONS IN MICROBIAL DIVERSITY BETWEEN DIFFUSE FLOW SAMPLES**

The diversity of hydrothermal vent microbial communities is extensive, especially when measured with HTS techniques (Huber et al., 2007, 2010). In this study, total microbial diversity in both locations was positively correlated with the prevalence of Bacteria, especially *Epsilonproteobacteria*, within the vent-specific component of the community. Additionally, epsilonproteobacterial abundance as measured by frequency analyses was also significantly correlated with overall microbial richness. Other studies have noted high levels of epsilonproteobacterial diversity in hydrothermal vent environments (Huber et al., 2007, 2010; Opatkiewicz et al., 2009).

Collection near macrofaunal communities was correlated with increased overall diversity but decreased phylum-level diversity. These communities were dominated by *Sulfurovum* spp. within the *Epsilonproteobacteria*. This class of Proteobacteria may be more diverse than other groups due to their genetic makeup, where much of the group lacks standard DNA repair gene pathways seen in other bacterial groups (Miller et al., 2007; Campbell et al., 2009). We also observed higher levels of archaeal than microbial diversity or evenness in three out of five of the macrofauna-associated sites. This finding contrasts with prior reports, where the diversity of archaeal populations was lower than bacterial diversity when different sets of primers and different library constructs are used (Huber et al., 2002, 2003, 2010; Opatkiewicz et al., 2009). The resolution afforded by using a single set of domain-independent primers, as well as sampling of macrofauna-associated and non-macrofauna sites, likely contributed to our discovery of high levels of archaeal diversity at the vent sites.

While diversity estimates were not significantly different for vent-specific microbial communities between geographic locations, which agrees with our Good's coverage estimates, richness estimates were significantly different, even when corrected for sequencing effort. Several location-specific factors may account for the decreased richness observed in the Guaymas samples. Historically, Guaymas Basin hydrothermal fluids are depleted in sulfides and enriched in ammonium, nickel, methane and hydrocarbons as compared to the EPR spreading center (Edmond et al., 1982; Von Damm et al., 1985). Extreme or disturbed conditions often result in less richness (Campbell et al., 2010; Fierer and Lennon, 2011), and these features of the Guaymas site may act to decrease richness. Additional properties not investigated here, such as diffuse flow rates or numbers of particles may also be different between the sites and affect microbial richness. Nevertheless, even at the gross level of richness estimates, there were clear biogeographic effects on deep-sea hydrothermal vent communities.

#### **MICROBIAL COMPOSITION ANALYSES OF DIFFUSE FLOW SAMPLES**

Off-axis microbial communities from each region, EPR and Guaymas, were significantly different from one another. Our analyses revealed that archaeal phylotypes, specifically within the MGI clade, were the most abundant phylotypes in the seawater surrounding hydrothermal vents and differentiated the two regions. The MGI clade has been reported to dominate seawater microbial communities adjacent to hydrothermal vents and plumes, in some cases up to 46% of the entire community (Huber et al., 2002; Takai et al., 2004; Dick and Tebo, 2010). The dominant MGI Thaumarchaeaota OTU at the EPR site grouped with other MGI species within the sub-tropical and equatorial deep water cluster; whereas the dominant MGI OTU at the Guaymas Basin grouped with North Atlantic clones within the *amo*A archaeal isolates cluster. It is likely that most MGI found in deep waters, including the major MGI at the basalt-dominated EPR are different than the MGI at Guaymas in terms of their metabolic properties (Agogue et al., 2008; Bouskill et al., 2012). Despite the low latitude location of the Guaymas site, the occurrence of a taxon related to *amo*A Archaea from North Atlantic suggests that the most abundant MGI species in Guaymas sedimentarydominated deep-sea water is an autotrophic archaeal ammonium oxidizer. The generally higher ammonium concentrations in the Guaymas samples support this hypothesis. Therefore, our data indicate that the dominant deep-sea taxa from the EPR are most likely heterotrophs or mixotrophs and the dominant taxa from Guaymas are most likely autotrophs. This could be driven mostly by environmental conditions, where Guaymas Basin sites have high levels of ammonium resulting from high temperature breakdown of photosynthetic organisms sinking from the productive surface waters of the Gulf of California (Von Damm et al., 1985). Recent metagenomic and metatranscriptomic studies of both background and plume waters indicated high levels of chemolithoautotrophic processes from Guaymas Basin environment, supporting this findings (Baker et al., 2012; Lesniewski et al., 2012).

After statistical subtraction of OTUs within background deep-sea water, detailed patterns in microbial community structure emerged between the samples from various diffuse flow environments. In general, bacterial species distributions between the EPR and Guaymas indicated a trend toward dominance of autotrophic-associated taxa (*Epsilonproteobacteria*) at the EPR sites and heterotrophicassociated taxa (*Planctomycetes, Alteromonas, Thermosipho, Thermococcus*) at the Guaymas sites. At some of the sites (4472, 4478, 4480), the *Epsilonproteobacteria* were dominated by OTUs related to the *Sulfurovum*, *Sulfurocurvum*, and *Sulfuromonas* genera. Members of these genera are autotrophs and generally mesophilic and microaerophilic, but may respire nitrate, and use various sulfur species as electron donors (Campbell et al., 2006). These sites generally had higher pH and lower sulfide concentrations than others, perhaps indicating that these bacterial groups were actively oxidizing the sulfide. The epsilonproteobacterial communities at the other EPR sites (4470, 4473, 4474) were more evenly distributed between OTUs within the *Sulfurocurvum*, *Sulfuromonas,* and *Sulfurovum* genera and *Arcobacter*/*Sulfurospirillum* genera. While isolates within the *Sulfurospirillum* are heterotrophic, members of the *Arcobacter* can be autotrophic as well, and both can use sulfur as an electron donor (Campbell et al., 2006). Two of the samples taken at the same site (4476 and 4478) had relatively high levels of OTUs within the *Nautiliaceae*. Members of this family are thermophilic anaerobic autotrophs who obtain energy from hydrogen and may respire nitrate (Alain et al., 2002; Voordeckers et al., 2005; Campbell et al., 2006, 2009). Differential dominance of epsilonproteobacterial genera at vents has been previously observed at the Axial and Mariana Arc seamounts (Huber et al., 2007, 2010; Opatkiewicz et al., 2009) but not in the EPR or Guaymas Basin spreading centers.

At most of the Guaymas sites, member genera within the *Planctomycetes* were particularly frequent in the vent-specific samples. The *Planctomycetes* have been described in multiple habitats and include some genera that perform anaerobic ammonium oxidation (Neef et al., 1998; Jetten et al., 2001; Chistoserdova et al., 2004). This is the first study to demonstrate that members of the *Planctomycetes* occur at high abundance (*>*5% in the vent-specific community) within hydrothermal vent environments, although they have been found in terrestrial thermal springs (Kanokratana et al., 2004; Elshahed et al., 2007). *Planctomycetes* are generally considered heterotrophic, and isolates from low temperature sulfide springs are able to reduce sulfur species, therefore this group may be important in the heterotrophic cycling of sulfur in marine hydrothermal environments as well (Elshahed et al., 2007).

The two Guaymas samples taken from the Southern Site (4479 and 4481), as well as one other Guaymas sample from Pagoda and one of the EPR samples (V-vent) had more than 25% archaeal phylotypes. The high percentage of Archaea in the Guaymas samples was driven by a single OTU belonging to the *Thermococcales* order. Members of the *Thermococcales*, frequently isolated from hydrothermal vents, are generally considered anaerobic heterotrophic hyperthermophiles (Holden et al., 2001; Slobodkin et al., 2001; Jolivet et al., 2004; Teske et al., 2009; Perevalova et al., 2011). V-vent contained a large percentage of members of the *Archaeoglobales* and unclassified Euryarchaeota, many of which may be heterotrophic (Kletzin et al., 2004; Rusch and Amend, 2008). The sulfate-reducing *Archaeloglobales, Theromococcales*, and other Euryarchaeota are most likely hyperthermophiles as well (Stetter, 1996) and thus, diffuse flow from these sites are enriched with microbes originating from very high temperature environments.

While we base our discussion on the known thermal and metabolic properties of cultured Bacteria and Archaea, we did find some interesting trends in the data that suggest that, after subtraction of off-axis microbes, some sites are dominated by thermophilic to hyperthermophilic heterotrophs and others by mesophilic autotrophs. This indicates that not all diffuse flow environments are equally represented by microbes across all thermal regimes. It could also be the result of a lack of complete diversity and compositional coverage of all the samples. However, Good's estimates of coverage were quite high, even with subtraction of OTUs specific to off-axis bottom water.

#### **CONCLUSIONS**

This study underscores the utility of using HTS techniques combined with simultaneous amplification of the 16S rRNA gene from both Bacteria and Archaea to statistically separate off axis from vent-specific taxa. We found that overall diversity was significantly lower in the sedimentary Guaymas vent environments than in the basaltic EPR deep-sea hydrothermal vent environments, which has implications for understanding environmental controls of microbial diversity in these extreme habitats (Huber et al., 2007; Quince et al., 2008). Our study also suggests that environment-specific factors such as proximity to macrofaunal communities can have a dramatic effect on microbial community richness, diversity and composition. Furthermore, we observed unique microbial sub-communities that were specialized to the

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diffuse flow environment and were different between spreading centers. Regrettably, we can only speculate as to the physiological features of member populations within diffuse flow communities as these observations are based on 16S rRNA gene homologies to known microbes. Deeper understanding of connections between phylogeny and physiology in these diffuse flow specialized microbial phyla can only come through genome level studies (Baker et al., 2012; Lesniewski et al., 2012).

#### **ACKNOWLEDGMENTS**

We thank the captain and crew of the R/V Atlantis and especially the pilots and technicians of the DSV Alvin for their essential roles in the collection of specimens used in this study. We would also like to acknowledge the support from JCVI Joint Technology Center for their assistance with next-gen sequencing. This research was supported by grants from the National Science Foundation to K. Eric Wommack, Shannon J. Williamson and S. Craig Cary (MCB-0731916), to S. Craig Cary (OCE-0550491) and to Barbara J. Campbell (OCE-0825468). Computational infrastructure support from the University of Delaware Center for Bioinformatics and Computational Biology Core Facility and Delaware Biotechnology Institute was made possible through funding from Delaware INBRE (National Center for Research Resources 2 P20 RR016472-09; National Institute of General Medical Sciences 8 P20 GM103446-12); and Delaware EPSCoR (National Science Foundation EPS-081425). The New Zealand Marsden Fund provided financial support for S. Craig Cary (UOW0802), and Charles K. Lee (UOW1003).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 February 2013; accepted: 17 June 2013; published online: 24 July 2013.*

*Citation: Campbell BJ, Polson SW, Zeigler Allen L, Williamson SJ, Lee CK, Wommack KE and Cary SC (2013) Diffuse flow environments within basalt- and sediment-based hydrothermal vent ecosystems harbor specialized microbial communities. Front. Microbiol. 4:182. doi: 10.3389/ fmicb.2013.00182*

*This article was submitted to Frontiers in Extreme Microbiology, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Campbell, Polson, Zeigler Allen, Williamson, Lee, Wommack and Cary. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

### The microbiology of deep-sea hydrothermal vent plumes: ecological and biogeographic linkages to seafloor and water column habitats

### *Gregory J. Dick1,2,3\*, Karthik Anantharaman1, Brett J. Baker1, Meng Li 1, Daniel C. Reed1 and Cody S. Sheik1*

1 Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, MI, USA

2 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA

3 Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

#### *Edited by:*

Andreas Teske, University of North Carolina at Chapel Hill, USA

#### *Reviewed by:*

James F. Holden, University of Massachusetts Amherst, USA Victoria Jean Bertics, Harvard University, USA

#### *\*Correspondence:*

Gregory J. Dick, Department of Earth and Environmental Sciences, University of Michigan, 2534 CC Little Building, Ann Arbor, MI 48109-1005, USA. e-mail<EMAIL_ADDRESS>
Hydrothermal plumes are an important yet understudied component of deep-sea vent microbial ecosystems. The significance of plume microbial processes can be appreciated from three perspectives: (1) mediation of plume biogeochemistry, (2) dispersal of seafloor hydrothermal vent microbes between vents sites, (3) as natural laboratories for understanding the ecology, physiology, and function of microbial groups that are distributed throughout the pelagic deep sea. Plume microbiology has been largely neglected in recent years, especially relative to the extensive research conducted on seafloor and subseafloor systems. Rapidly advancing technologies for investigating microbial communities provide new motivation and opportunities to characterize this important microbial habitat. Here we briefly highlight microbial contributions to plume and broader ocean (bio)geochemistry and review recent work to illustrate the ecological and biogeographic linkages between plumes, seafloor vent habitats, and other marine habitats such as oxygen minimum zones (OMZs), cold seeps, and oil spills. 16S rRNA gene surveys and metagenomic/-transcriptomic data from plumes point to dominant microbial populations, genes, and functions that are also operative in OMZs (SUP05, ammonia-oxidizing Archaea, and SAR324 Deltaproteobacteria) and hydrocarbon-rich environments (methanotrophs). Plume microbial communities are distinct from those on the seafloor or in the subsurface but contain some signatures of these habitats, consistent with the notion that plumes are potential vectors for dispersal of microorganisms between seafloor vent sites. Finally, we put forward three pressing questions for the future of deep-sea hydrothermal plume research and consider interactions between vents and oceans on global scales.

**Keywords: chemosynthesis, chemoautotroph, deep-sea, biogeochemistry, hydrothermal, vent, biogeography**

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#### **INTRODUCTION**

Deep-sea hydrothermal plumes occur where seafloor vents inject hydrothermal fluids replete with potential microbial energy sources such as H2S, Fe, Mn, CH4, and H2 into the deep oceans. These hot, chemically reduced fluids rapidly mix with cold, oxidizing seawater, forming hydrothermal plumes that rise hundreds of meters off the seafloor and disperse hundreds of kilometers away from their source. Because of their extensive spatial coverage and easily detectable hydrothermal signals (Fe, Mn, turbidity, Helium-3), plumes played an important role in the history of deep-sea hydrothermal vent research (Lupton and Craig, 1981) and continue to be utilized for discovery of new seafloor hydrothermal systems (German et al., 2010). Hydrothermal plumes are highly variable in terms of scale and chemical and physical properties, and can be detected by a variety of methods (chemical, physical, optical), thus the definition of a plume depends on the parameter being measured (Lupton, 1995). For many of the geochemical and microbiological processes of interest here, "plume" often refers to hydrothermal fluid that has been heavily diluted by seawater (e.g., ∼1:10,000), but some work has addressed microbial processes in

the rising portion of the plume where hydrothermal constituents are more concentrated.

Hydrothermal plumes are found at vents sites distributed globally along the mid-ocean ridge system (**Figure 1**) at a frequency correlated to seafloor spreading rate (Beaulieu et al., 2012). Deepsea vent systems continue to be discovered at a rapid pace; over 500 vent fields are now known, nearly double the number known before the year 2000 (Beaulieu et al., 2012). Yet much of the midocean ridge system remains unexplored, especially at ultra-slow spreading ridges, which have only recently been recognized to host hydrothermal activity (German et al., 2010) and are particularly abundant in the Arctic and Southern oceans. Hydrothermal venting in shallow waters is also widespread (Prol-Ledesma et al., 2005), but here wefocus only on deep-sea systems. Given the global distribution and extent of hydrothermal venting, it is clear that deep-sea vents exert significant influence on the chemistry of the global oceans (Elderfield and Schultz, 1996). A recent modeling study of hydrothermal contributions to the marine iron inventory (Tagliabue et al., 2010) highlights the global impacts of vents (**Figure 1**).

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#### **MICROBIAL MEDIATION OF PLUME BIOGEOCHEMISTRY: TRACE ELEMENTS, PHOSPHORUS AND CARBON**

The impact of deep-sea hydrothermal vents on ocean chemistry involves several processes that are influenced by microbial activities in hydrothermal plumes. First, vents are thought to be a significant source of Fe and Mn to the oceans because their concentrations in hydrothermal fluids are up to 106 times that of background seawater (Elderfield and Schultz, 1996; Tagliabue et al., 2010; Sander and Koschinsky, 2011). The oceanic fate of these metals is influenced by scavenging and oxidation, which are promoted by microorganisms (Cowen and Bruland, 1985; Cowen et al., 1986; Mandernack and Tebo, 1993; Dick et al., 2009), and by binding with organic matter, which is presumably derived from microbial activity (Bennett et al., 2008; Toner et al., 2009; Breier et al., 2012; Holden et al., 2012). Second, the iron and manganese oxides produced by microbial oxidation are extraordinarily reactive (Goldberg, 1954; Tebo et al., 2004) and thus remove phosphorous and trace elements (rare earth elements, potassium, vanadium, arsenic, chromium, uranium) from seawater via scavenging and co-precipitation reactions (Feely et al., 1998; German and Von Damm, 2004). Because of the rapid mixing of seawater with hydrothermal fluids and the large volumes of plumes, the entire volume of the global oceans cycles through hydrothermal plumes and is scavenged of reactive elements on relatively short times scales (2.4 × 105 y; Kadko, 1993). Thus plumes essentially act as a filter for the global oceans, scavenging them of phosphorous, rare earth elements, and trace metals, and acting as a chemical sink for these elements (Kadko, 1993). As these Fe and Mn oxides and their scavenged elements are deposited to the seafloor, they form metalliferous sediments that potentially preserve a record of seawater nutrient status and chemistry that is valuable from paleoceanographic perspectives (Feely et al., 1998). Similarly, banded iron formations, which are likely sourced from

hydrothermal activity, provide a Precambrian record of ocean chemistry (Konhauser et al., 2009; Planavsky et al., 2011). Thus, microbially mediated metal oxide formation and the properties of the resulting biogenic minerals in plumes influence scavenging reactions and outcomes in terms of ocean chemistry, and understanding these processes is critical for interpretation of the sedimentary record for paleoceanographic purposes.

The third process by which plume microorganisms mediate broader ocean biogeochemistry is chemosynthetic fixation of carbon. Chemosynthetic activity at vents was recognized upon the initial discovery of deep-sea hydrothermal vent ecosystems (Jannasch and Wirsen, 1979) and in early plume studies (Winn et al., 1986) yet the magnitude of chemosynthesis in plumes remains poorly constrained. Based on extrapolation of data from the Southern East Pacific Rise to the global oceans, Maruyama et al. (1998) estimated that net primary production in plumes represents 0.1–1% of total marine photosynthetic net primary production. Because only a small fraction of surface organic carbon reaches the deep oceans, the hydrothermal contribution could represent up to 25% of the global deep ocean organic carbon inventory (Maruyama et al.,1998). Thermodynamic models support the idea that plumes are a significant source of chemosynthetically derived organic carbon to the deep oceans (McCollum, 2000), and several observational studies confirm an important contribution of plumes to deep-sea organic carbon on regional scales (De Angelis et al., 1993; Cowen and German, 2003; Lam et al., 2004, Lam et al., 2008). Indeed, plumes are enriched with organic carbon, some of which is labile, and are responsible for dispersing it kilometers away from vent sites (Roth and Dymond, 1989; Cowen et al., 2001; Shackelford and Cowen, 2006; Lam et al., 2008; Bennett et al., 2011a,b). More recently, transcriptomic evidence confirms that autotrophy is a prevalent process in plumes (Baker et al., 2012; Lesniewski et al., 2012; Anantharaman et al., 2013).

Two recent developments provide fresh motivation for reexamining the global impact of hydrothermal vents on the chemistry and biology of the oceans. First, hydrothermal activity along the mid-ocean ridges is more common than previously recognized, especially at slow-spreading systems (German et al., 2010; Beaulieu et al., 2012). Slow-spreading ridges represent a large but poorly explored portion of the global mid-ocean system and host high-energy, H2-rich systems that are potential hotspots for chemosynthesis (Amend et al., 2011). Second, the discovery of cryptic biogeochemical cycling of sulfur (Canfield et al., 2010) and widespread chemolithoautotrophy in the broader pelagic oceans (Aristegui et al., 2009; Swan et al., 2011) suggests that current models underestimate chemosynthesis and raise questions regarding ecological connections between plume and other pelagic environments, which we consider below.

#### **MICROBIAL COMMUNITIES IN DEEP-SEA HYDROTHERMAL PLUMES**

Despite the well-recognized importance of microorganisms in the biogeochemistry of hydrothermal plumes, few studies have characterized microbial communities that inhabit them. Thus the physical source, taxonomic composition, and ecological nature of these organisms remain poorly understood. Potential sources of microbes for deep-sea hydrothermal plumes can be divided into three broad categories: (i) seafloor (or sub-seafloor) communities, (ii) background deep seawater communities, or (iii) growth *within* the plume (**Figure 2**). The traditional view is that plume microbes are likely sourcedfrom highly productive biological communities that inhabit vent chimneys and surrounding areas (Winn et al., 1986). Such sources could also include bottom water that is heavily influenced by low-temperature diffuse flow (Kadko et al., 1990), which may be responsible for geochemical flux comparable to focused hydrothermal venting and contains microbes from the subsurface biosphere (Wankel et al., 2011; Akerman et al., submitted). Indeed, tracer studies indicate that diffuse flow and larvae of vent fauna can be entrained into plumes (Jackson et al., 2010). Alternatively, plume microbes could be derived primarily from ambient background seawater, which seems feasible given that plumes are a mix of >99% seawater and ∼0.01% hydrothermal fluid (Lupton et al., 1985) and that benthic and pelagic habitats differ greatly (Zinger et al., 2011). Regardless of the original source of microbes, it is likely that plume communities are dynamic in time and space. Shifts in microbial community structure are to be expected as the geochemical environment of hydrothermal fluids evolves with plume age (i.e., become more dilute and oxidized). Consistent with this notion are observations of progressive removal of electron donors (Kadko et al., 1990) and morphological evidence of changes in microbial communities with plumes age (Cowen and Li, 1991). However, only recently has plume microbial diversity been sampled and analyzed with molecular tools in a spatially resolved manner.

Many studies have reported elevated microbial biomass and activity in plumes relative to background, suggesting that plume microbes are distinct from those in the ambient water column (Winn et al., 1986; Naganuma et al., 1989; Juniper et al., 1998; Maruyama et al., 1998; O'Brien et al., 1998; Lam et al., 2004, Lam et al., 2008; Dick et al., 2009). In one of the first applications of

molecular tools to deep-sea hydrothermal plumes, Sunamura et al. (2004) showed that the Suiyo Seamount hydrothermal plume is dominated by just two phylotypes, one group of *Gammaproteobacteria* and one group of *Epsilonproteobacteria*. Interestingly, both of these phylotypes were most closely related (at the time) to symbionts of hydrothermal vent animals – the *Gammaproteobacteria* to bivalve gill symbionts and the *Epsilonproteobacteria* to ectosymbionts of the tubeworm *Riftia pachyptila* and shrimp *Rimicaris exoculata* (Sunamura et al., 2004). The *Gammaproteobacteria*, designated "SUP05," were also found to dominate low-temperature diffuse flow emanating from a bivalve-colonized mound (99% of cells; Sunamura et al., 2004), raising the possibility that microbial communities in the subsurface or animal symbioses are sources of plume microbes (see further discussion of links between plumes and symbionts below). Indeed, SUP05-like sequences have also been retrieved from diffuse hydrothermal fluids on the seafloor at the Juan de Fuca ridge (Huber et al., 2003; Bourbonnais et al., 2012; Anderson et al., 2013). However, recent rRNA gene surveys show that SUP05 are also widely distributed in pelagic environments, raising the question of whether the connection between plumes and subsurface is physical (i.e., transport only) or ecological (i.e., actively operating in similar niches in both environments) in nature. An extreme example of the physical transport of seafloor and/or subsurface material to the water column is "snowblower" vents that discharge elemental sulfur and bacterial filaments (Haymon et al., 1993; Crowell et al., 2008). Thermophilic microbes derived from the subsurface have also been observed in eruptive event plumes (Summit and Baross, 1998). *Epsilonproteobacteria* are also commonly encountered in vent seafloor environments (Nakagawa et al., 2005; Campbell et al., 2006; Zinger et al., 2011), again highlighting potential connections between the seafloor and plumes. Clear signals of *Epsilonproteobacteria* and other seafloor hydrothermal microbes have been observed in plumes at the Mid-Cayman Rise (German et al., 2010), in the Iheya hydrothermal field (Nakagawa et al., 2005), the Logatchev hydrothermal plume (Perner et al., 2013), and in descending particles from plumes at the East Pacific Rise, which are genetically distinct from surrounding seawater (Sylvan et al., 2012). Recent studies employing fine-scale phylogenetic approaches and coupled DNA and RNA approaches hold great promise for elucidating the niche space and distribution of sulfur-oxidizing *Gammaproteobacteria* and *Epsilonproteobacteria* in subsurface, seafloor, plume, and background environments. Anderson et al. (2013) noted partitioning of distinct clades of sulfur-oxidizing *Gammaproteobacteria* in vent environments (SUP05 in plumes and animal symbioses) versus others [Arctic96BD-19 in background and oxygen minimum zones (OMZs)] and noted that sulfide concentration likely controls the balance of *Gammaproteobacteria* versus *Epsilonproteobacteria*. Consistent with that view, coupled RNA and DNA analyses revealed showed that *Epsilonproteobacteria* are more active in the reducing environment of the subseafloor, whereas *Gammaproteobacteria* are more active at the seafloor where mixing with seawater is more prevalent (Akerman et al., submitted).

In contrast with the hypothesis that seafloor environments are the major source of plume biota, microbial communities in hydrothermal plumes at Guaymas Basin in the Gulf of California are distinct from those of the underlying seafloor habitats such

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**communities in background seawater, vent chimneys, near-vent animal symbioses, subsurface environments, near-bottom waters, and recirculation of aged plumes.** Microbial growth within the plume also shapes the plume community, including utilization of hydrothermally sourced organic carbon produced chemosynthetically or hydrothermally. Hydrothermal plumes contain distinct regions (e.g., the rising plume and neutrally buoyant plume) with steep gradients of physical and chemical properties that likely hold distinct microbial communities.

as hydrothermal sediments or chimneys (Dick and Tebo, 2010). Rather, Guaymas Basin plume communities closely resemble those from background seawater samples taken just above the plume or in the neighboring Carmen Basin, which is 100 km away and does not host hydrothermal activity. Metagenomic and metatranscriptomic data further reinforces the plume-water column connection, showing that the metabolically active microbes in Guaymas plumes are pelagic rather than benthic in nature, and suggesting an ecological boundary between seafloor and plume (Lesniewski et al., 2012). These studies interpreted the above-plume and Carmen Basin samples as true background communities, and this is supported by the absence of detectable physical and chemical tracers of hydrothermal activity in those samples. However, another possibility is that this deep seawater surrounding Guaymas Basin is impacted by microbes that are exported from the highly productive chemoautotrophic plumes. Processes that could facilitate export include ascending and descending particles and migratory zooplankton (Cowen et al., 2001), buoyant transparent exopolymeric substances (Shackelford and Cowen, 2006; Prieto and Cowen, 2007), and large scale advection such as mesoscale eddies (Adams et al., 2011). Regional influence of vents on deepsea microbial communities may be particularly important in the Gulf of California, where restricted basins could limit dispersal of plumes and mixing with true non-hydrothermally-impacted seawater. We will re-visit the potential impact of hydrothermal plumes on the broader deep oceans below.

#### **ECOLOGICAL AND BIOGEOGRAPHIC LINKAGES BETWEEN HYDROTHERMAL PLUMES AND OTHER MARINE HABITATS**

There is growing recognition that chemical species that fuel microbial growth in hydrothermal plumes (H2, various sulfur species, ammonium, and iron) also support microbial growth in

marine environments well beyond hydrothermal systems, including OMZs, oils spills, and cold seeps (Paull et al., 1984; Lösekann et al., 2007; Tavormina et al., 2008, 2010; Redmond et al., 2010), whale falls (Baco and Smith, 2003; Tringe et al., 2005; Goffredi and Orphan, 2010), and within microenvironments of organic-rich particles in the oxic water column (Karl et al., 1984; **Figure 3**). Indeed, recent molecular surveys show that microorganisms that are abundant in deep-sea hydrothermal plumes are also abundant in these other marine habitats. SUP05 and another group of uncultivated putative sulfur-oxidizing bacteria, SAR324 *Deltaproteobacteria*, are abundant in OMZs, where they play important roles in linking the sulfur and nitrogen cycles (Lavik et al., 2009; Walsh et al., 2009; Canfield et al., 2010; Stewart et al., 2012; Wright et al., 2012). These two groups have also been identified in the dark pelagic oceans (e.g., Swan et al., 2011; Ghiglione et al., 2012). In addition, methanotrophic populations and functional genes for methane oxidation (particulate methane monooxygenase; *pMMO*) that are prevalent in the Guaymas Basin hydrothermal plume are closely related to those in plumes of the Deepwater Horizon oil spill (Lesniewski et al., 2012; Li et al., unpublished). Cultures also support connections between plumes and other marine environments; close relatives of the Mn(II) oxidizing alphaproteobacterium SI85-9A1 (>99% 16S rRNA gene sequence identity), which was originally isolated from the Saanich Inlet oxic/anoxic interface (Dick et al., 2008), have been isolated from the surface of *Alvinella pompejana* tubeworms at 9◦N East Pacific Rise (Anderson et al., 2009), and from plumes of the Lau Basin. Finally, *Halomonas* and *Marinobacter* species detected in hydrothermal plumes are present throughout the oceans (Kaye and Baross, 2000, 2004; Kaye et al., 2011). Hence, plume ecological niches and the microorganisms that fill them appear to be widespread in the oceans, and understanding their distribution is paramount for understanding the dispersal of plume microorganisms, the "inoculation" of plumes with microbes from background seawater, and ultimately for understanding the distribution and abundance of chemoautotrophy throughout the global oceans. Below we focus on comparisons of plumes to OMZs and seafloor environments.

#### **CONNECTIONS TO OXYGEN MINIMUM ZONES**

Oxygen minimum zones are widely distributed in the oceans and are expanding due to anthropogenic global change (Wyrtki, 1962; Stramma et al., 2008; Wright et al., 2012). Reduced O2 concentrations favor alternative terminal electron acceptors, the products of which drive chemoautotrophic metabolisms. Hence, microbial metabolisms that take advantage of redox gradients mediate OMZ biogeochemistry and contribute significantly to the global cycling of nitrogen and greenhouse gasses (Wright et al., 2012). OMZs in which the concentration of O2 falls below 20 μM, a threshold below which some anaerobic metabolisms operate (Kalvelage et al.,2011; Ulloa et al.,2012), overlap geographically with the midocean ridge system, especially in the Eastern Pacific (**Figure 1**). Although OMZs and deep-sea hydrothermal vent plumes are typically separated vertically by 1500 m or more, communication

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**marine habitats that are sources of electron donors for chemosynthetic growth and support microbial communities similar to those found in deep-sea hydrothermal plumes.** Electron donors for

sea vents and as the products of anaerobic microbial respiration in low-O2 environments. OMZ and redox schematics after Wright et al. (2012).

between these habitats likely occurs via sinking particles. Whereas nitrogen cycling was classically thought to dominate the microbial ecology and metabolism of OMZs, cryptic sulfur cycling has also been recently demonstrated in the Eastern Tropical South Pacific OMZ (Canfield et al., 2010). Such cryptic sulfur cycling occurs when sulfide produced by sulfate reduction is rapidly oxidized back to sulfate. Though easily overlooked by chemical methods, numerous reports of abundant sulfur-oxidizing autotrophs in OMZs (Stevens and Ulloa, 2008; Lavik et al., 2009; Walsh et al., 2009) and even in oxic waters (Swan et al., 2011) suggest that this process may be widespread in the broader oceans.

To evaluate potential connections between the microbial ecology of deep-sea hydrothermal plumes and other marine environments, we compared the most abundant microbial groups across a variety of pelagic and benthic habitats including both oxic and anoxic as well as hydrothermal and non-hydrothermal environments (**Figure 4**). Although there are obviously a variety of environmental and biogeographic factors that shape microbial community structure in these diverse habitats, there are some striking similarities in terms of the organisms that dominate. SUP05 are among the most abundant microbial groups in the Guaymas Basin and Suiyo Seamount hydrothermal plumes, Saanich Inlet and ETSP OMZs, and in coastal waters of the Benguela upwelling zone off Namibia (Lavik et al., 2009). SUP05 are also found at appreciable abundance in the OMZ of Guaymas Basin (Anantharaman et al., 2013) and in the Black Sea redoxcline (Fuchsman et al., 2011). The SAR324 group of *Deltaproteobacteria* (also known as the Marine Group B), which has recently been implicated in hydrocarbon and sulfur metabolism and autotrophy (Swan et al., 2011; Li et al., unpublished; Sheik et al., unpublished), is also abundant in widespread environments (**Figure 4**). It should be noted there is considerable diversity within these important groups, which likely reflects specific ecological adaptations. For example, there are clades of SAR324 that appear to be specific to Saanich Inlet (Wright et al., 2012), and there is plasticity of electron donors and acceptors within the SUP05 (Anantharaman et al., 2013). Enigmatic groups such as SAR406, SAR86, and Arctic97B-4 also exhibit similar distributions to those of SAR324 and SUP05 and further highlight the links between deep-ocean and pelagic environments.

#### **PLUMES AS DISPERSAL VECTORS FOR VENT MICROBES, SYMBIONTS, AND LARVAE**

Hydrothermal plumes entrain both focused and diffuse hydrothermal fluids and thus are potential dispersants of vent-associated organisms (Jackson et al., 2010). Indeed, vent-associated larvae have been observed in plumes (Mullineaux et al., 1995), where they are likely transported great distances to colonize vent sites (Mullineaux et al., 2010). As discussed previously (Dick and Tebo, 2010; Lesniewski et al., 2012), the microbial community of the Guaymas Basin hydrothermal plume is clearly distinct from seafloor communities (**Figure 4**). Despite this ecological boundary between hydrothermal seafloor and plume habitats, hydrothermal plumes could play a key role in the dispersal of seafloor

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**FIGURE 4 | Microbial community structure and abundance of key populations in plumes and other marine habitats.** 16S rRNA gene clone and pyrosequencing reads were downloaded from Genbank or VAMPS (vamps.mbl.edu) repositories and classified to the Silva v. 111 database (Quast et al., 2013) using the Mothur software package (Schloss et al., 2009).  \*Primer sets used were bacterial specific and did not allow for comparison of Archaea.

hydrothermal organisms such as hydrothermal sediment and chimney-associated microbes. Consistent with this notion, deep sequencing of 16S rRNA genes from Guaymas Basin (Anantharaman et al., 2013) revealed the presence, albeit at low abundance, of candidate division bacteria that are common in seafloor hydrothermal environments (**Figure 4**). To our knowledge, this represents the first evidence of seafloor microbes in the rare portion of the plume microbial community at a chronic vent site [they have been detected previously in eruptive event plumes (Summit and Baross, 1998)], and suggests that plumes could indeed be an important mechanism of dispersal of deep-sea vent organisms.

Potential connections between free-living microbes in plumes and symbionts of hydrothermal vent animals have long been recognized, but views on the nature of this relationship are evolving. The similarity of free-living SUP05 in plumes to symbionts was noted with the original discovery of SUP05 (Sunamura et al., 2004). More recently, plume SUP05 werefound to have and express genes for H2 oxidation (Anantharaman et al., 2013) that are highly similar to those of bathymodiolin mussel symbionts at deep-sea vents (Petersen et al., 2011), and symbiont-like methanotrophs were detected in the Guaymas Basin hydrothermal plume (Li et al., unpublished). The traditional view of these microbial symbionts is that they evolved from free-living ancestors to form a few distinct symbiont clades. However, recent analysis shows that symbionts are phylogenetically interspersed with free-living forms of the bacteria, suggesting numerous evolutionary transitions between symbiotic and free-living forms (Petersen et al., 2012). Furthermore, horizontal gene transfer has been implicated as a significant mechanism of metabolic evolution of the symbionts (Kleiner et al., 2012). Bathymodiolin symbionts are thought to be transmitted horizontally and acquired by each generation from the environment (Petersen et al., 2012), and the relatively low diversity of symbionts within animal populations suggests that the animals carefully select symbionts from the pool of diversity present within free-living communities. However, the mechanisms by which this selection takes place, the degree to which symbionts and free-living populations are genetically and ecologically distinct, and the time scales over which transitions between free-living and symbiotic lifestyles occur all remain intriguing frontiers for understanding the relationship between animal symbionts and free-living plume microorganisms.

#### **THE INFLUENCE OF BENTHIC AND PELAGIC HABITATS ON PLUME MICROBIAL COMMUNITIES**

What controls the relative contribution of benthic versus pelagic microbes to plume microbial communities? Although the limited data on microbial communities in plumes provides few answers, there are some preliminary clues. Both physical and biological factors likely play a role in determining the balance of seafloor versus water column microbes in plumes. Physical factors include (i) the fluid flux and entrainment rate of the rising plume, (ii) the properties and habitability of the near-vent environment that could potentially be entrained, such as temperature and material properties (e.g., hard substrate versus easily transported sediments or biological material such as biofilms or dense animal communities), (iii) bathymetry of the seafloor surrounding the vent environment [e.g., bathymetric highs lead to rapid dilution and dispersal whereas restricted volumes such as Guaymas Basin or the Suiyo Seamount caldera tend to accumulate hydrothermal chemistry and biota (German andVon Damm, 2004)], and (iv) the bioenergetic potential of plume geochemistry (i.e., high concentration of energy-rich electron donors is more likely to support a seafloor-derived community in the plume). Biological factors that influence the balance of seafloor versus water column microbes likely hinge on the properties of the water mass in which venting takes place, including the cell density and the structure of the community with regard to metabolic potential. Denser microbial communities and those that hold a large portion of organisms able to utilize inorganic electron donors for lithotrophic growth will promote a greater water column contribution to plume microbial communities. Finally, the microbial growth response to temperature likely influences the degree to which seafloor microbes are metabolically active in hydrothermal plumes. Thermophiles or hyperthermophiles are unlikely to be metabolically active in cold hydrothermal plumes, whereas mesophiles or psychrophiles from lower-temperature seafloor habitats may indeed remain active at plume temperatures.

#### **METABOLIC AND FUNCTIONAL LINKAGES BETWEEN PLUMES AND OTHER MARINE HABITATS**

Recent reports of genomic and metabolic plasticity within microbial groups such as SUP05 (Anantharaman et al., 2013) underscore the need to use caution when inferring microbial metabolism and function from 16S rRNA genes of plume populations, even at fine phylogenetic scales. On the other hand, the emergence of random shotgun metagenomic and metatranscriptomic approaches, driven by rapidly increasing throughput and decreasing costs of DNA sequencing, provides new opportunities to directly assess microbial metabolism and its effect on biogeochemistry in deepsea hydrothermal plumes. Lesniewski et al. (2012) used a parallel metagenomic and metatranscriptomic approach to show that ammonium, methane, and sulfur are the primary energy sources in the Guaymas Basin hydrothermal plume. Genomes and transcriptomes of abundant microbial groups were subsequently reconstructed to reveal the genetic potential and expression of specific microbial populations (Baker et al., 2012; Anantharaman et al., 2013; Li et al., unpublished; Sheik et al., unpublished). Comparison of these large 'omics datasets to those from other marine environments provides a view of the dynamics of both community-wide functions and specific microbial groups across distinct settings, and potentially provides powerful insights into the factors that govern microbial and ecosystem functions in the deep sea.

We analyzed the abundance of key functional genes in shotgun sequencing datasets from recent studies of the Guaymas Basin hydrothermal plume (Lesniewski et al., 2012), the Deepwater Horizon oil spill (Goldstamp Gm00382, IMG TaxonID<PHONE_NUMBER>), the Eastern Tropical Pacific OMZ (Stewart et al., 2012), and the North Pacific subtropical gyre (DeLong et al., 2006; **Figure 5**). This data shows that functional genes for oxidation of nitrogen, sulfur, hydrogen, and hydrocarbons that are highly expressed in the Guaymas Basin hydrothermal plume are also widely present and expressed in these other disparate marine habitats (**Figure 5**). Genes for ammonia oxidation are abundant

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**FIGURE 5 | Abundance of functional genes and transcripts for oxidation of selected nitrogen, sulfur, hydrogen, and hydrocarbon species in the following samples and studies: Eastern Tropical South Pacific OMZ (Stewart et al., 2012), Hawaii Ocean Time Series (DeLong et al., 2006), Guaymas Basin plumes and background (Lesniewski et al., 2012), and Deepwater Horizon oil spill (Goldstamp Gm00382, IMG TaxonID<PHONE_NUMBER>).** Metagenomic sequences from these studies were used as queries with BLASTX against databases containing the following sequences: AMO – ammonia monooxygenase subunit A (from both ammonia-oxidizing archaea and bacteria); Nxr – nitrite oxidoreductase subunit A; DsrA – dissimilatory sulfite reductase subunit A; SoxA – sulfur oxidation protein subunit A; HupL and HydB – group 1 membrane-bound Ni,Fe hydrogenase, large subunit; pMMO – particulate methane monooxygenase subunit A; pEMO – putative particulate ethane monooxygenase subunit A, includes sequences from Methylococcaceae

bacterium species ET-HIRO (AB453962 and AB453963) and ET-SHO (AB453960 and AB453961), and environmental sequences (Redmond et al., 2010; Li et al., unpublished; SAR324\_pHMO: putative C2–C4 alkane oxidizing monooxygenase subunit A, contains sequences from SAR324\_J09 (Swan et al., 2011) and Guaymas Basin SAR324 (Li et al., unpublished; Sheik et al., unpublished). BLASTX bit scores >50 were considered positive matches. Abundance of sequence reads recruiting to each functional gene is shown as a percentage of total (putative) mRNA-containing cDNAs. rRNA were identified using Ribopicker [Version 0.4.3 (Schmieder et al., 2012)] with the comprehensive Ribopicker database "rrnadb" and removed from all datasets. The absence of data for samples simply indicates that it was not identified at the sequencing depth and does not necessarily imply the absence of genes/transcripts. Note that due to novelty of sequences, in some cases the pathway and substrate specificity are uncertain.

and highly expressed in nearly all datasets analyzed here except for surface waters of the ALOHA station, consistent with the widespread abundance of ammonia-oxidizing Archaea (Karner et al., 2001; Francis et al., 2005), specific populations of which are stimulated in the ammonium-rich hydrothermal plumes of Guaymas Basin (Baker et al., 2012). NXR (nitrite oxidoreductase) genes for nitrite oxidation (Lucker et al., 2010; Baker et al., 2013) were recovered from many of the datasets, especially OMZs. However, this result should be interpreted with caution given the novelty of NXR genes; it is difficult to distinguish the forms utilized by nitrite-oxidizing bacteria versus anaerobic ammonia oxidation (anammox) bacteria (Strous et al., 2006). Thus, we suspect that a significant fraction of NXR hits in the OMZ are from genes involved in anammox. Sulfur oxidation systems (*sox* and *dsr* genes) are most prevalent in the Guaymas Basin hydrothermal plume and in the OMZ cores, but they are also detectable in Guaymas Basin background and in some samples in the oxic water column at HOT (Hawaii Ocean Time Series). These sulfur oxidation genes are present in all metatranscriptomes of the Guaymas Basin, but only in a subset of those from the Gulf of Mexico, HOT, and ETSP OMZ. These results, especially expression of sulfur oxidation genes in oxic waters (HOT), are consistent with widespread cryptic geochemical cycling of sulfur (Canfield et al., 2010), perhaps in association with organic-rich particles (Karl et al., 1984; Wright et al., 2012). H2 oxidation genes are less widely distributed, but are most prevalent in ultramafic vent chimney samples (Brazelton et al., 2012) and in the Guaymas Basin hydrothermal plume, consistent with H2 being sourced primarily from hydrothermal fluids and being rapidly utilized in plumes (Kadko et al., 1990). However, H2 oxidation genes were also detected in some OMZ, ALOHA, and background Guaymas samples (Anantharaman et al., 2013), consistent with H2 production in association with organic-rich particles in the oxic water column also being a substantial source of H2 as predicted decades ago (Karl et al., 1984).

Utilization of methane and other hydrocarbons as an energy source represents another potential connection between hydrothermal plumes and other marine microbial habitats. The capability of naturally occurring marine microbes to consume hydrocarbons has recently been highlighted by the Deepwater Horizon disaster (Mason et al., 2012). While the connections need further investigation, natural sources of hydrocarbons in the deep sea (Jorgensen and Boetius, 2007) such as hydrocarbon seeps, hydrothermal vents, or *in situ* water column production (Karl et al., 2008) may prime deep ocean microbial communities for hydrocarbon degradation. Guaymas Basin hydrothermal fluids have unusually high concentrations of methane due to interaction with sediments that overlay the ridge. High methane concentrations are also found at the Endeavour Segment of the Juan de Fuca ridge (De Angelis et al., 1993), and ultramafic systems on the Mid-Atlantic Ridge (Charlou et al., 1998) and the Mid-Cayman Rise (German et al., 2010). The imprint of methane is clearly apparent in microbial communities of the Guaymas Basin hydrothermal plume; genes for pMMO are among the most abundant mRNAs represented in the metatranscriptome (Lesniewski et al., 2012). Recent work shows that there is extensive diversity of pMMOs and related Cu membrane monooxygenases at Guaymas, and that they affiliate phylogenetically with enzymes involved in C2–C4 alkane oxidation (Li et al., unpublished). We refer to these methane, ethane, and butane monooxygenases collectively as particulate hydrocarbon monooxygenases (pHMOs). pHMOs are also present in the metatranscriptome of the Deepwater Horizon oil spill (Mason et al.,2012; Li et al., unpublished) and in afew samples from station ALOHA (**Figure 5**). pHMOs were only detected at low levels in a few of the shotgun sequencing datasets, but they have been reported to be abundant in OMZs off Costa Rica (Tavormina et al., 2013).

#### **COMPARATIVE METATRANSCRIPTOMICS OF POPULATIONS IN PLUME AND BACKGROUND: SUP05 AND Thaumarchaeota**

One of the remarkable conclusions emerging from comparison of different interfacial redox environments in the oceans is that the same microbial groups often dominate disparate environmental settings. For example, despite marked environmental differences between deep-sea hydrothermal plumes and OMZs (e.g., depth, temperature, pressure, nutrient availability, water mass history, quantity, and quality of DOC and POC), they share several of the most abundant microbial populations, including SUP05, SAR324, and*Thaumarchaea* (**Figure 4**). The nature of these two oxic/anoxic interfaces is quite different: in plumes, reduced chemicals are injected into an oxic water column, whereas in OMZs, reduced chemical species are produced through anaerobic microbial respiration of organic carbon. However, many of the microbial players appear to be the same, indicating that these organisms thrive at redox interfaces regardless of differences in other environmental parameters described above.

Recent metatranscriptomic studies (Baker et al., 2012; Lesniewski et al., 2012; Stewart et al., 2012; Anantharaman et al., 2013) permit detailed views of the gene expression patterns of these microbial groups and their roles in biogeochemistry. Comparison of transcripts from OMZ and Guaymas Basin plume/background samples recruited to SUP05 and *Thaumarchaea* genomes shows that the overall patterns of transcript abundance are quite similar between the two environments (**Figure 6**). Indeed, the differences between samples *within* each environment (plume versus background at Guaymas, different depths of the OMZ) appear to be as significant if not greater than differences between environments. For *Thaumarchaea*, acquisition of ammonium and oxidation of ammonia dominate the metatranscriptome in both environments. Conserved hypothetical proteins of unknown function are similarly highly expressed in both environments, highlighting large gaps in our knowledge of what are likely critical functions for these organisms. In addition to obtaining ammonia directly from the environment, uptake of urea also appears to be an important source of ammonia for both populations (**Figure 6**). Utilization of urea has now been noted for *Thaumarchaeota* in diverse settings such as sponge symbionts (Hallam et al., 2006), soil (Tourna et al., 2011), polar waters (Alonso-Saez et al., 2012), and surface waters of the Gulf of Maine (Tully et al., 2012).

Like *Thaumarchaea*, transcript abundance profiles of SUP05 show striking similarities in the genes that are most highly expressed in plumes and OMZs (**Figure 6B**). Many of the highly expressed genes are involved in cell maintenance and growth (e.g., translation and DNA replication), which is expected in any

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environment. However, similarly high transcript abundance of other genes likely reflects common interactions between SUP05 and the two different environments. Ammonium transporters were the most highly expressed genes in all OMZ and plume samples except the 200 m depth of the OMZ. A predicted ABC (ATP-binding cassette) transporter for amino acids is expressed in all samples, but it appears to be most abundant in the deep sea.

SUP05 potentially plays an important role in linking the sulfur and nitrogen cycles by coupling the oxidation of reduced sulfur species to the reduction of nitrate or nitrite (Walsh et al., 2009; Canfield et al., 2010). Perhaps the most intriguing difference between plume and OMZ transcript profiles is that the

*soxA* gene for sulfur oxidation is highly expressed in plumes but not in deep background waters or the OMZ. This suggests a difference in the form of sulfur used by SUP05 in plumes versus OMZs; abundant *soxA* transcripts point to thiosulfate oxidation in plumes, whereas free sulfide may be the preferred substrate in OMZs, as hypothesized by Walsh et al. (2009). Additional differences between populations of SUP05 include diversity in terms of electron donors and acceptors. SUP05 at the Saanich Inlet oxic/anoxic interface were described as anaerobes (Walsh et al., 2009), but SUP05-related symbionts appear to be aerobic (Kuwahara et al., 2007; Newton et al., 2007), and those in the Guaymas Basin plume are primarily aerobic (Anantharaman et al., 2013). Guaymas Basin SUP05 also have genes for H2 oxidation that do

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not appear to be present in other populations (Anantharaman et al., 2013).

#### **THREE BIG QUESTIONS FOR THE FUTURE OF DEEP-SEA HYDROTHERMAL PLUME MICROBIOLOGY**

The recent explosion of new data on deep-sea microbial communities has revealed connections between deep-sea hydrothermal plumes and other marine habitats. These insights raise intriguing new questions regarding the roles of deep-sea hydrothermal systems in the chemistry and biology of the broader oceans. At the same time, revolutionary technological advances provide new opportunities to address these questions. One exciting future goal is to define how the interplay between microbiology and geochemistry in hydrothermal plumes extends to global-scale interactions

between mid-ocean ridges and deep-sea microbiology. The global mid-ocean ridge hosts geochemically diverse hydrothermal systems. This geochemical diversity is tied to geological differences in the underlying host rock of hydrothermal systems, which shapes the quantity and types of metabolic energy available for chemoautotrophic growth in hydrothermal plumes (Amend et al., 2011). Indeed, vents show the highest variability in biodiversity of all marine systems (Zinger et al., 2011). Although microbial growth in plumes is predicted to be significant for carbon budgets in the deep oceans (McCollum, 2000), several first-order questions remain poorly constrained and need to be addressed.

#### **WHAT SHAPES THE STRUCTURE OF MICROBIAL COMMUNITIES IN DEEP-SEA HYDROTHERMAL PLUMES?**

As we have seen, hydrothermal plume communities are composed of microbes from both seafloor and pelagic environments, and while data is scarce, the balance between these two sources appears to be variable and depends on both physical and biological factors. In addition to external contributions, chemoautotrophic growth *within* plumes likely contributes to the plume microbial community. Hence, the structure of communities in deep-sea hydrothermal plumes is shaped by both the geochemistry of the seafloor hydrothermal system (which shapes seafloor microbial communities and the energy available for growth in plumes) and the composition of communities in the surrounding water column. Microbial biogeography in the deep oceans is largely controlled by deep-sea circulation (Ghiglione et al., 2012) and hydrography (Galand et al., 2010), thus if the contributions from surrounding seawater are significant, then the geographic location of vents along the global deep conveyor belt of circulation could be an important determinant of plume community composition. Determining the relative importance of vent geochemistry and geography in shaping the community structure of plumes should be possible by tracking microbial diversity along local gradients of vent geochemistry in locations such as the Eastern Lau Spreading Center (Tivey et al., 2012). Expanded sampling and characterization of microbial communities in geographically widespread plumes in the broader deep sea will also be critical to evaluate biogeographic characteristics of deep-sea hydrothermal plumes.

#### **DO HYDROTHERMAL PLUMES INFLUENCE THE DIVERSITY OF DEEP-SEA MICROBIAL COMMUNITIES ON A GLOBAL SCALE?**

So far we have focused on the contribution of background deepsea microbes to plumes, but the interaction between communities in these two environments may in fact be bi-directional. Cell count data and thermodynamic modeling indicate that deep-sea hydrothermal plumes are productive environments compared to surrounding deep waters, and several studies have suggested that vents influence deep-sea microbial communities in their vicinity (Moyer et al., 1998; Takai et al., 2004). Active venting occurs in every ocean basin at over 1000 vent fields globally (Beaulieu et al., 2012), and surface-generated mesoscale eddies potentially transport hydrothermal plumes long distances (Adams et al., 2011). Although dispersion of plumes may be largely constrained by the ridge axis (Speer et al., 2003) or confined to a narrow corridor 10 km to either side of the ridge axis (German and Von Damm, 2004), deep-sea microbial communities are relatively stable (Ghiglione et al., 2012), so microbes exported from plumes could persist for long periods of time. Clearly there is potential, but to what extent does chemoautotrophic growth in plumes influence the broader deep oceans? The answer is poorly constrained, but given recent insights into cryptic chemoautotrophy throughout the oceans, the nature of potential influence takes on new meaning. Rather than being unique oases of chemoautotrophy in the oceans, perhaps vents should be viewed as one of many potential sources of electron donors that collectively maintain widespread chemoautotrophy in the oceans. Advances in thermodynamic-bioenergetic modeling of plumes on a global scale, incorporation of this data into ecological models, and evaluation of the results with experiments and observations provides great promise for addressing this question. Finally, even if the answer is that plumes do not influence broader deep-sea microbial communities, the value of deep-sea hydrothermal plumes as natural laboratories to examine the response of deep-sea microorganisms to diverse geochemical perturbations should not be ignored.

#### **HOW DOES MICROBIAL ACTIVITY IN DEEP-SEA HYDROTHERMAL PLUMES IMPACT CARBON BUDGETS OF THE DEEP OCEANS?**

The deep oceans hold the largest reservoir of rapidly exchangeable inorganic carbon on the Earth's surface. Microbial autotrophy potentially converts dissolved inorganic carbon into the organic phase within the biota. Subsequent grazing and viral predation transfer this carbon through the food chain or into the dissolved organic carbon pool. Although plume autotrophy likely has a negligible impact on the dissolved inorganic carbon inventories over global and short-term scales, its impact on organic carbon in the deep sea, especially on regional spatial scales, is potentially significant. In particular, the possibility that microbial autotrophy, heterotrophy, and/or lysis in hydrothermal plumes contribute to a significant conversion of inorganic matter to refractory organic matter, and thus sequestration of carbon as a component of the "microbial carbon pump" (Jiao et al., 2010), has not been explored in any detail. Improved measurements of plume microbial carbon fixation rates and studies of the fate of that carbon are required to evaluate this possibility. Another important but understudied aspect of microorganisms in deep-sea hydrothermal plumes is their role in modulating the flux of hydrocarbons from the seafloor through the water column and into the atmosphere (Orcutt et al., 2011). Future investigation on the inter relationship between plume microbial activity and hydrocarbon degradation will provide a better understanding of the impact of hydrothermal plume microbial activity on deep ocean carbon cycling.

Although this review has focused on hydrothermal plumes from high-temperature venting along the mid-ocean ridges, it should also be noted that low-temperature venting, including ridge-flank circulation, also likely contributes significantly to the processes described here. In fact, some estimates suggest that low-temperature ridge flank systems drive fluxes of fluid, heat, and solutes that are larger than those from high-temperature hydrothermal systems (Wheat and Mottl, 2000; Fisher and Harris, 2010). The microbial biogeochemistry of such systems is poorly known, but (McCarthy et al., 2011) showed that chemosynthesis

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by crustal microbial communities is a major source of dissolved organic carbon in ridge-flank and on-axis hydrothermal fluids sampled from the Juan de Fuca Ridge. Thus ridge-flank systems likely amplify the contributions of hydrothermal/subsurface circulation to the biology and geochemistry of the oceans and strengthen ecological and biogeographic connections between these systems.

#### **REFERENCES**

### **ACKNOWLEDGMENTS**

We are grateful to Anna-Louise Reysenbach for providing Lau Basin seafloor microbial data and to Alessandro Tagliabue for providing modeled iron distributions. This project is funded by the Gordon and Betty Moore Foundation through Grant GBMF 2609 to Dr. Gregory J. Dick and by National Science Foundation grants OCE 1029242 and OCE 1038006 to Dr. Gregory J. Dick.

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G. J. (2012). The metatranscriptome of a deep-sea hydrothermal plume is dominated by water column methanotrophs and lithotrophs. *ISME J.* 6, 2257–2268.

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in a ridge-flank hydrothermal system. *Nat. Geosci.* 4, 32–36.

from Irina II in the Logatchev field. *Environ. Microbiol.* 15, 1551–1560.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 February 2013; paper pending published: 08 March 2013; accepted:*

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*30 April 2013; published online: 21 May 2013.*

*Citation: Dick GJ, Anantharaman K, Baker BJ, Li M, Reed DC and Sheik CS (2013) The microbiology of deep-sea hydrothermal vent plumes: ecological and biogeographic linkages to seafloor and water column habitats. Front. Microbiol. 4:124. doi: 10.3389/fmicb.2013.00124*

*This article was submitted to Frontiers in Extreme Microbiology, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Dick, Anantharaman, Baker, Li, Reed and Sheik. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

### Diversity and phylogenetic analyses of bacteria from a shallow-water hydrothermal vent in Milos island (Greece)

#### *Donato Giovannelli1,2,3\*, Giuseppe d'Errico3, Elena Manini 3, Michail Yakimov4 and Costantino Vetriani 1,2\**

1 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA

2 Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA

3 Institute for Marine Science - ISMAR, National Research Council of Italy - CNR, Ancona, Italy

4 Institute Coastal Marine Environment - IAMC, National Research Council of Italy - CNR, Messina, Italy

#### *Edited by:*

Anna-Louise Reysenbach, Portland State University, USA

#### *Reviewed by:*

Karine Alain, Centre National de la Recherche Scientifique, France Casey R. J. Hubert, Newcastle University, UK

#### *\*Correspondence:*

Donato Giovannelli, Institute for Marine Science - ISMAR, National Research Council of Italy - CNR, Largo Fiera della Pesca 60125, Ancona, Italy e-mail<EMAIL_ADDRESS>Costantino Vetriani, Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901, USA e-mail<EMAIL_ADDRESS>
Studies of shallow-water hydrothermal vents have been lagging behind their deep-sea counterparts. Hence, the importance of these systems and their contribution to the local and regional diversity and biogeochemistry is unclear. This study analyzes the bacterial community along a transect at the shallow-water hydrothermal vent system of Milos island, Greece. The abundance and biomass of the prokaryotic community is comparable to areas not affected by hydrothermal activity and was, on average, 1*.*34 × 108 cells g−1. The abundance, biomass and diversity of the prokaryotic community increased with the distance from the center of the vent and appeared to be controlled by the temperature gradient rather than the trophic conditions. The retrieved 16S rRNA gene fragments matched sequences from a variety of geothermal environments, although the average similarity was low (94%), revealing previously undiscovered taxa. Epsilonproteobacteria constituted the majority of the population along the transect, with an average contribution to the total diversity of 60%. The larger cluster of 16S rRNA gene sequences was related to chemolithoautotrophic Sulfurovum spp., an Epsilonproteobacterium so far detected only at deep-sea hydrothermal vents. The presence of previously unknown lineages of Epsilonproteobacteria could be related to the abundance of organic matter in these systems, which may support alternative metabolic strategies to chemolithoautotrophy. The relative contribution of Gammaproteobacteria to the Milos microbial community increased along the transect as the distance from the center of the vent increased. Further attempts to isolate key species from these ecosystems will be critical to shed light on their evolution and ecology.

**Keywords: bacteria,** *Epsilonproteobacteria***, shallow-water hydrothermal vent, Milos, geothermal**

#### **INTRODUCTION**

Microbes are one of the most abundant life forms on Earth, they are ubiquitous, possess a great metabolic plasticity and drive major biogeochemical cycles (Staley and Reysenbach, 2002). Studies carried out in geothermal and extreme environments have shown that life on Earth is far more diverse, widespread, and resistant to extreme conditions than previously thought. Despite the crucial roles of prokaryotes in extreme ecosystems, our understanding of their diversity and ecological relevance in these environments is limited.

Deep-sea hydrothermal vent ecosystems are largely based on chemolithoautotrophic primary production (Jannasch, 1985; Bach et al., 2006; Nakagawa and Takai, 2008). Photoautotrophic contribution to these ecosystems is limited to the sinking of low-quality particulate material from the photic zone (Comita et al., 1984). In contrast, shallow-water hydrothermal vent systems (located at depth *<*200 m) are largely influenced by photosynthesis (Tarasov et al., 2005). In these environments, chemolithoautotrophy and photoautotrophy occur simultaneously and spatial separation is often influenced by

steep thermal and geochemical gradients (Wenzhöfer et al., 2000).

Shallow-water hydrothermal vents are widespread ecosystems that have been previously understudied compared to their deep-sea counterparts (InterRidge vents database, http://www*.* interridge*.*org/), despite the fact that these systems were known long before the discovery of the deep-sea vents on the Galapagos Rift in 1977 (Lonsdale, 1977). Because of their proximity to the surface, shallow-water hydrothermal systems are influenced both by geothermally generated reducing power and by light, and can be described as "high energy" environments, where microbial metabolism is fueled by different energy sources (Baross and Hoffman, 1985). According to Dando et al. (1995, 2000), the emission of carbon dioxide from the Milos venting area alone (35 km2) could account for 10% of the carbon dioxide emission caused by the venting associated with the Mid-Oceanic Ridges. It has been suggested that the conditions found in shallow-water hydrothermal systems could resemble those in which life originated and where metabolic divergence begun (Nisbet and Fowler, 1996; Nisbet and Sleep, 2001; Martin et al., 2008).

Understanding the microbiology of shallow-water hydrothermal vents is necessary to evaluate how microorganisms influence biogeochemical cycles. The geology and chemistry of the hydrothermal system located in Paleochori Bay, a sandy bay off the South East coast of the island of Milos, was previously investigated by Dando et al. (2000) and Valsami-Jones et al. (2005). In Paleochori Bay, the vents are located in shallow waters, with temperatures ranging from 25 to 119◦C (Botz et al., 1996) and extensive gas and fluid seepage. Early microbiological studies based on fingerprinting approaches showed the presence of bacteria associated to the *Cytophaga-Flavobacteria-Bacteroides* as well as *Arcobacter* spp. (*Epsilonproteobacteria*) and *Thiomicrospira* spp. (*Gammaproteobacteria*) (Brinkhoff et al., 1999; Sievert et al., 1999a, 2000a,b).

In this study, we carried out an environmental survey of a shallow-water hydrothermal vent located in Paleochori bay, Milos island, Greece. The site was sampled to investigate the structure and diversity of the bacterial community along a 1.5 m transect starting at the center of one of the vents.

#### **MATERIALS AND METHODS**

#### **SITE DESCRIPTION AND SAMPLE COLLECTION**

The Milos hydrothermal system is one of the largest in the Mediterranean Sea. It is part of the Hellenic Arc, whose eastern section reaches the Turkish coast and the island of Kos, and Methana to the west. Extensive submarine venting occurs offshore, from the intertidal zone to depths of more than 100 m, with an approximate extension of 34 km2 of seabed (Dando et al., 2000).

Inside the Paleochori bay (**Figure 1**), the venting area is characterized by strong degassing activities coupled with fluid seepage (Valsami-Jones et al., 2005). The entire shallow venting area is surrounded by patches of the seagrass *Posidonia oceanica,* and the vents occur as areas of high temperature and degassing on the sandy bottom (referred to hereafter as the center of the vent) that gradually decrease to ambient conditions as the distance from the vent increases. Steep thermal and redox gradients occur vertically, with temperature increasing with the depth of the sediment, while more gradual temperature and redox changes occur horizontally as the distance from the center of the vent increases (Sievert et al., 1999a, 2000a; Dando et al., 2000; Wenzhöfer et al., 2000). Temperatures of up to 119◦C at a vent site in 10 m water depth have been reported (Botz et al., 1996). Venting fluids are enriched with freshwater with varying salinity. The composition of CO2 released with the fluids ranged between 54.9 and 91.9%, while the concentrations of H2S, CH4 and H2 were ≤8.1, ≤9.7, and ≤3%, respectively (Botz et al., 1996; Dando et al., 2000). In addition, the hydrothermal fluids have been shown to contain elevated concentrations of reduced inorganic chemicals, such as NH+ 4 (up to 1 mM), H2S (up to 1 mM) and Mn2+ (up to 0.4 mM)

(Fitzsimons et al., 1997). Arsenic and sulfur precipitates are common in proximity to vent orifices, and dense brines can be found in sediment depressions (Dando et al., 2000).

SCUBA divers collected sediment samples using push-cores during the MAMBA cruise in 2010. Starting from the center of a vent located at a depth of 12 m (designated as MV1; 36◦ 40.351 N, 24◦ 31.108 E), a horizontal transect consisting of four stations located 50 cm apart from each other was sampled (MV1, 0 cm distance from the center of the vent; MV2, 50 cm distance; MV3, 100 cm distance; MV4, 150 cm distance; **Figure 1** and **Table 1**). Additionally, a single station was sampled at ca. 50 cm from the center of the vent in a yellow sediment patch (F12). The transect was characterized by a 20◦C thermal gradient (45 and 25◦C at stations MV1 and MV4, respectively) and differences in sediment color were observed. Station MV4 appeared only marginally influenced by hydrothermal activity as the temperature of its surface sediments was close to ambient and *P. oceanica* was observed in close proximity (**Table 1**).

Cores were retrieved on board, sediment horizons separated (0–1, 3–5, and 10–15 cm) and processed according to the analytical procedure described in following paragraphs.

#### **ORGANIC MATTER COMPOSITION**

Aliquots of sediments were immediately frozen at −20◦C for determination of organic matter quantity and composition. Total protein concentrations were determined on sediment sub-samples according to Hartree (1972). Total carbohydrates were analyzed according to Gerchakov and Hatcher (1972) and expressed as glucose equivalents. Total lipids were extracted from the sediment by direct elution with chloroform:methanol (1:1 v/v) according to Bligh and Dyer (1959) and then determined according to Marsh and Weinstein (1966). All readings were performed spectrophotometrically. Carbohydrate, protein and lipid concentrations were converted into carbon equivalents using the conversion factors 0.40 and 0.49 and 0.75 mgC mg−1, respectively, and normalized to sediment dry weight (Fabiano et al., 1995). Biopolymeric organic carbon was calculated as the sum of the C equivalents of protein, lipid and carbohydrate.

Chlorophyll-a and phaeopigments were extracted from sediment sub-samples according to Plante-Cuny (1974). Briefly, a few mg of MgCO3 were added to 1 g of wet sediment to avoid chlorophyll-a degradation. Samples were supplemented with 90% acetone, sonicated and incubated in the dark at 4◦C for 12 h. Following incubation, the samples were centrifuged to remove the sediment and the concentration of the pigments in the supernatant were determined spectrofluorimetrically (ex. 430 nm, em. 665 nm) before and after acidification with HCl 0.1 N. Concentrations were calculated against a standard curve and normalized to sediment dry weight. Total phytopigments (CPE) were obtained from the sum of chlorophyll-a and phaeopigments.

#### **PROKARYOTIC ABUNDANCE AND BIOMASS**

Total prokaryotic abundance was determined by direct counts after staining with acridine orange (Danovaro et al., 2002). Briefly, 0.5 g of each sample was supplemented with 5 mM tetrasodium pyrophosphate and incubated for 15 min before sonication. The samples were then stained with 0.025% (wt/vol) acridine orange and filtered on 0.2 mm pore-size Nucleopore black polycarbonate filters, under low vacuum (*<*100 mm Hg). The filters were analyzed as described by Fry (1990), using epifluorescence microscopy (Zeiss Axioskop 2; 1000 × magnification). The total prokaryotic abundance was normalized to sediment dry weight after desiccation.

Prokaryotic biovolume was estimated using the image analysis software ImageJ (Schneider et al., 2012). Average carbon content was assumed to be 310 fg C µm3 (Fry, 1990). The prokaryotic biomass (PBM) was normalized to sediment dry weight.

**Table 1 | Sampled stations, temperature, organic matter content and main characteristic of the area.**

Means are reposted with standard deviation. BPC, Biopolymeric organic carbon; CPE, total phytopigments.

#### **GENOMIC DNA EXTRACTION AND PCR AMPLIFICATION**

Genomic DNA was extracted from sediment biomass by the phenol:chloroform method (Maniatis, 1989). Briefly, aliquots of frozen sediments (ca. 0.5 g) were resuspended in extraction buffer (100 mM Tris-HCL, 100 mM EDTA, 1.5 M NaCl pH 8.0), supplemented with 10 mg ml−1 lysozyme and incubated for 30 at 37◦C. Subsequently, 20% SDS was added and each sample was incubated with agitation for 1 h at 60◦C. Sediments were removed by centrifugation (5 min at 14,000 × g) and supernatants were collected and extracted with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) followed by one extraction with chloroform:isolamyl alcohol (24:1). DNA was then precipitated overnight with 0.1 volumes of sodium acetate and 0.7 volumes isopropanol, washed with 70% ice-cold ethanol, resuspended in PCR grade water and visualized on 1% agarose gel.

The bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal bacterial primer 8F (5 - AGA GTT TGA TCC TGG CTC AG-3 ) and 1517R (5 -ACG GCT ACC TTG TTA CGA CTT-3 ) (Weisburg et al., 1991). Aliquots of 5µl of PCR products were visualized by staining with ethidium bromide on 1.5% agarose gel.

#### **DENATURING GRADIENT GEL ELECTROPHORESIS**

A preliminary assessment of the diversity of the sediment bacterial communities was carried out by Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the bacterial 16S rRNA gene. The full-length 16S rRNA gene was obtained as described above, gel-purified and used as a template for nested PCR to amplify the V3 region using the GC-clamp primer 338F-(GC) (5 -CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG-3 ) and 519R (5 - GWA TTA CCG CGG CKG CTG-3 ). DGGE was performed with a D Gene system (Bio-Rad Laboratories, Hercules, CA). PCR products (15 ml) were applied directly onto 6% (wt/vol) polyacrylamide gels in 1 × TAE (0.04 M Tris base, 0.02 M sodium acetate, 1 mM EDTA pH 7.4), with denaturant gradient from 40 to 60% (where 100% denaturant contains 7 M urea and 40% formamide). Electrophoresis was performed at a constant voltage of 45 V for 14 h. After electrophoresis, the gels were incubated for 15 min in 0.5 mg l−1 ethidium bromide, rinsed for 10 min in distilled water, and photographed with a UV Foto Analyst system (Fotodyne, Inc., Hartland, WI).

#### **LIBRARY CONSTRUCTION AND RFLP ANALYSIS**

Amplified 16S rRNA gene fragments were excised from agarose gels, purified, and cloned into the pCR2-TOPO vector using the TOPO-TA Cloning Kit (Invitrogen, Inc., Carlsbad, California) following the manufacturer's instructions. The resulting ligation products were used to transform competent *Escherichia coli* TOP10 cells. Recombinant *E. coli* clones were grown on Luria-Broth media supplemented with 100 µg ml−1ampicillin. Sixty to ninety clones for each library were randomly chosen and analyzed for insert-containing plasmids by direct PCR followed by gel electrophoresis of the amplified products. Insert 16S rRNA gene fragments were digested with HaeIII and MspI (Promega, Inc., Madison, Wis.) restriction endonucleases for 3 h at 37◦C and

subjected to Restriction Fragment Length Polymorphism (RFLP) analysis on 3% low-melting agarose gel. Clones were grouped into operational taxonomic units (OTUs) based on their RFLP profiles, and sequences (about 900 bases) were obtained from representative clones of each OTU.

#### **STATISTICAL AND PHYLOGENETIC ANALYSES**

Analysis of variance (ANOVA) using the statistical R-Software (R-Cran project, http://cran*.*r-project*.*org/) was carried out to identify significantly different samples, which were subjected to the Tukey HSD *post-hoc* test. Where ANOVA assumptions were rejected, a more conservative level of *p* was chosen (Underwood, 1991). DGGE profiles were analyzed with ImageJ (Schneider et al., 2012) and R-software for cluster analysis to identify significant differences in the composition of bacterial communities. Briefly, the position of each DGGE band was recorded using the ImageJ Gel plugin and the resulting matrix was fed to R for the determination of the distance matrix based on Jaccard dissimilarity and used for cluster analysis.

Sequences obtained from libraries were manually checked for quality, primers were removed and the resulting sequences were aligned using ClustalW (Larkin et al., 2007) and SeaView (Gouy et al., 2010). The software Bellerophon (Huber et al., 2004) was used to identify chimeric sequences, which were removed from the dataset (2.2 % of total sequences were identified as chimeric). Neighbor-Joining trees were constructed using the Jukes-Cantor correction and tree topologies were tested using 1000 bootstraps replications (Perriere and Gouy, 1996). Sequences of cultured bacteria retrieved as top blast hits against our sequences were included as references in the alignment. Phylogenetic designation of the sequences to a specific group was obtained by integrating blastn and EzTaxon results with phylogenetic analyses (Chun et al., 2007). Chao1 non-parametric diversity estimator and rarefaction curves were computed using Rarefaction software (http://www2*.*biology*.*ualberta*.*ca/jbrzusto/ rarefact*.*php).

The sequences from this study are available through GenBank under accession numbers from KC463698 to KC463741.

#### **RESULTS**

#### **SEDIMENTARY ORGANIC MATTER CONTENT**

Biopolymeric organic carbon (BPC) in the surface sediments decreased along the transect from the center of the vent (MV1) toward the background station (MV4) with values ranging from 0*.*45 ± 0*.*03 to 0*.*23 ± 0*.*08 mg C g−1 at station MV1 and station MV4, respectively (**Table 1**). At station F12, BPC was highest (1*.*51 ± 0*.*08 mg C g−1). The observed horizontal gradients were significant (ANOVA *p <* 0*.*001), and a BPC minimum at a depth of 3–5 cm was observed at all stations. Proteins dominated the carbon pool at all stations with an average contribution to BPC of 55%, followed by lipids and carbohydrates. Protein contribution to the carbon pool decreased along the transect from MV1 to MV4 (**Table 1**). In contrast, total phytopigment (CPE) concentration increased along the transect from MV1 to MV4 (ANOVA *p <* 0*.*001). Chlorophyll-a concentrations were extremely low, and phaeopigments were the most abundant class. The quantity and composition of the organic matter of the yellow surface sediments at station F12 differed from that of the other stations with proteins contributing nearly 95% of the BPC (**Table 1**).

#### **PROKARYOTIC ABUNDANCE AND BIOMASS**

In general, total prokaryotic abundance decreased as the depth of the sediment increased at all stations, despite their proximity to vent orifice (**Figure 2A**; ANOVA *p <* 0*.*001). Values ranged from 0*.*8 ± 0*.*4 × 108 to 2*.*7 ± 1*.*1 × 108 cells g−1 in the surficial sediments for MV1 and MV4, respectively, indicating higher abundances in the surface sediment layers and an increasing trend along the transect from MV1 to MV4. These trends were statistically significant (ANOVA *p <* 0*.*001). Prokaryotic biomass in surficial sediments ranged from 1*.*61 ± 1*.*06 to 11*.*95 ± 6*.*3µg C g−1 in station MV2 and MV4, respectively (**Figure 2B**). Again, a trend of decreasing biomass with increasing sediment depth and increasing biomass along the transect toward the periphery of the vent was observed (**Figure 2B**; ANOVA *p <* 0*.*01). Prokaryotic abundance in station F12 was comparable to station MV1, while prokaryotic biomass was significantly higher. The decreasing trend along the vertical sediment profile was more evident at stations located further away from the center of the vent. Inter-replicate variability was also higher at those stations.

#### **PROKARYOTIC DIVERSITY**

DGGE was used to investigate the diversity of the sediment bacteria from the 0–1, 5–10 and 10–15 cm layers along the transect stations, and that from the 0–1 cm layer of station F12 (**Figure 3**). Similar DGGE profiles were obtained from stations MV1, MV3 and MV4 and F12 (data were not obtained for station MV2). Cluster analysis based on the DGGE profiles indicated that the bacterial communities from the same sediment layers tend to group together (**Figure 3**). A relevant group included communities from the 0–1 cm sediments from the transect stations and from F12, while the 3–5 and 10–15 cm layers from MV1 and MV4

formed a second group (**Figure 3**). The bacterial communities from the 0–1 cm layer of stations MV1 (center of the vent) and MV4 (periphery of the vent) were selected to construct 16S rRNA gene libraries.

Both the MV1 and MV4 libraries were dominated by sequences that could be assigned to the *Epsilonproteobacteria* (60 and 59% for MV1 and MV4, respectively; **Figure 4**). Other common phyla in both libraries were sequences belonging to the CFB (20 and 6% for MV1 and MV4, respectively; **Figure 4**) and *Gammaproteobacteria* (7 and 20% for MV1 and MV4, respectively; **Figure 4**). The number of gammaproteobacterial clones increased at the periphery of the vent and the ratio of *Epsilon*to *Gammaproteobacteria* was 8.2 and 2.9 at stations MV1 and MV4, respectively. Sequences related to the *Deltaproteobacteria*, Planctomycetes, *Actinobacteria* and *Ignavibacteria* were also found. Four percent of the sequences retrieved from station MV1 could not be assigned to any known lineage (**Figure 4**).

The sequences were further analyzed by aligning them against closest cultured relatives and a neighbor-joining tree was constructed (**Figure 5**). Both libraries are well represented in the tree, with numerous sequences clustering together despite their different origins. A large number of sequences obtained from the libraries were related to *Sulfurovum lithotrophicum* (Inagaki et al., 2004), and clustered around this sequence on the tree (average similarity of 93%; **Figure 5** and **Table 2**). The *Sulfurovum*related sequences were organized in two discrete clusters, each containing clones from both libraries, and represented 44% of the clones in each library. The same clusters had best hits in the non-redundant database to sequences identified during environmental surveys in a variety of submarine geothermal

**FIGURE 4 | Bacterial community structure at station MV1 and MV4 surface sediments (0–1 cm) as obtained by the analysis of the sequences.** Groups are shown at the phylum level based on the results of BLAST and position on the phylogenetic tree. CFB, Cytophaga-Flavobacteria-Bacteroides group.

environments such as the Logatchev field on the Mid-Atlantic Ridge (Nakagawa et al., 2005; Hügler et al., 2010), Vailulu seamount in the Samoa and submarine volcanoes on the Kermadec Arc (Hodges and Olson, 2009).

A second major group of epsilonproteobacterial sequences was related to *Sulfurospirillum* spp. (90.8% average similarity), and clustered outside of the *Sulfurospirillum* group, constituting 13.6 and 5.8% of the two libraries respectively (**Figure 5**

**Table 2 | Sequenced clones, top blast hits to the non-redundant database (closet relative) and best hit among cultured bacteria (closest cultured relative).**

(Continued)

#### **Table 2 | Continued**

and **Table 2**). Clones closely related to the *Epsilonproteobacterium Sulfurimonas authotrophica* were found exclusively in library MV1 (2.5% of the library sequences), while sequences related to *Sulfurimonas paralvinellae* constituted 8.8% of the MV4 library (93% similarity).

Sequences related to the *Gammaproteobacteria* constituted the second largest group in the MV4 library and were mainly related to *Pseudomonas* spp. and members of the *Thioalophilus*/*Thioprofundum* cluster (98.5 and 91.7% average similarity, respectively) constituting 20.6% of the clones sequenced from this library. In the library from MV1, the *Gammaproteobacteria*-related sequences were related to *Thioalkalivibrio denitrificans* and *Thiothrix eikelboomii* (average similarity 90%), constituting only 6.2% of the library. The remaining gammaproteobacterial clones were associated with *Acinetobacter sp.* FR-W5Bb (98% similarity, 1.2% of the library) and a cluster that included clones MV1.01-C9 and MV1.01-F10, related to *Thioalkalivibrio denitrificans* (90% average similarity, 3.7% of the library, **Figure 5** and **Table 2**).

Sequences related to the *Flavobacteria*-*Bacteroides-Cytophaga* cluster were the second major group in library MV1, with an average similarity of 90% to *Cytophaga* sp. BHI60-95B (constituting 16% of the clones in the library). Sequences related to the same species were also found in library MV4 (5.9% of the library). Unique sequences related to the *Flavobacterium Gaetbulibacter jejuensis* and *Polaribacter sp.* j2-11 were present at station MV1 (93 and 91% similarity, respectively). *Deltaproteobacteria* were represented in library MV4 by sequences related to *Geothermobacter ehrlichii* (87.5% similarity, 5.8% of the clones in the library). MV1 sequences related to the *Deltaproteobacteria* represented 5% of the clones in this library and were associated to *Eubacterium* sp. OS (84% similarity).

Clones related to the *Ignavibacterium album* (87.7% similarity on average) were retrieved from both libraries, MV1 and MV4.

Despite the lower number of clone sequenced at station MV4, computed Chao1 diversity estimate indicated that diversity at MV4 was higher than MV1 (Chao1, 63*.*7 ± 22 and 28*.*8 ± 4*.*2 respectively). This can be also inferred from the slope visible on the rarefaction curves computed for both libraries (**Figure 6**).

#### **DISCUSSION**

Shallow-water hydrothermal vents are distributed worldwide and, while understudied relative to their deep-sea counterparts, they represent unique ecosystems where primary productivity is supported both by chemosynthesis and photosynthesis (Tarasov et al., 2005). In this study, we investigated the abundance, biomass, community structure and diversity of the prokaryotic community at a shallow-water hydrothermal vent in Paleochori Bay, Milos island, Greece (**Figure 1**).

Prokaryotic abundance was as high as 2*.*7 ± 1*.*1 × 108 cells g−1, with average values comparable to those reported in previous studies of the same area and other shallow-water hydrothermal systems, as well as at deep-sea vents (**Figure 2A**; Sievert et al., 1999b, 2000a,b; Nakagawa et al., 2005; Manini et al., 2008; Williamson et al., 2008; Maugeri et al., 2009, 2010). Prokaryotic abundance decreased with depth in the sediments at all stations, a general trend previously reported for shallow-water marine sediments (Molari et al., 2012). In contrast, prokaryotic abundance increased toward the periphery of the vent (**Figure 2A**). Prokaryotic biomass followed similar spatial patterns, with higher values at the surface (0–1 cm) and at station MV4 (**Figure 2B**). A previous report of the Milos vents indicated that deeper sediments tend to be hotter (Sievert et al., 2000b), while we

observed that surface sediments along the transect became gradually cooler at the periphery of the vent (**Table 1**). Hence, it appears that temperature and prokaryotic biomass are inversely correlated (**Figure 2**, Pearson moment correlation *r* = 0*.*617, *p <* 0*.*05).

Biopolymeric organic carbon (BPC), as well as proteins, decreased along the transect from the center (MV1) to the periphery of the vent (MV4), while carbohydrates and lipids remained fairly stable (**Table 1**). In contrast, phytopigments (CPE) followed an opposite pattern, increasing along the transect from MV1 to MV4 (**Table 1**). The highest concentrations of proteins and lowest concentration of CPE were measured at station F12 (yellow sediments, temperature 34◦C; **Table 1**). The increasing concentration of CPE as the sediment temperature decreases implies an increase of phototrophic organisms in the lower temperature regions of the vent system, probably due by the presence of previously described diatoms and cyanobacteria mats in the outer rings of similar vents (Thiermann et al., 1997). This is consistent with the general notion that photoautotrophs are less tolerant to elevated temperatures than chemoautotrophic or chemoheterotrophic microorganisms (Madigan et al., 2012). These findings suggest a gradual enrichment of phototrophic organisms along the decreasing thermal gradient. While BPC and proteins decreased along the transect from MV1 to MV4, prokaryotic abundance and biomass increased (**Table 1** and **Figures 2A,B**) and no significant correlation was found among the two parameters (Pearson moment correlation *r* = −0*.*214, *p >* 0*.*05). Since the transect was relatively short and all sampling stations were at the same depth, it is safe to assume that the input of organic matter from the water column and lateral advection was constant for all stations. We hypothesize that the decreasing prokaryotic biomass in the hottest section of the vent may lead to a decrease in carbon consumption, which is reflected in the higher concentration of measurable proteins and BPC. This hypothesis implies that temperature, rather than trophic resources, mainly controls the distribution of prokaryotes in this system.

Bacterial diversity, investigated by DGGE profiles, showed that the main DGGE banding pattern was highly conserved, while the highest number of unique bands was obtained from the 3– 5 and 10–15 cm depth profiles at station MV1 (**Figure 3**). In total, 30 unique bands with distinct electrophoretic mobility were obtained. As expected, the DGGE analysis highlights the presence of distinct bacterial populations in surface and deeper sediments, which likely reflect different thermal and redox regimes (**Figure 3**). Similar findings have been reported for a variety of environments, including shallow-water and deep-sea hydrothermal vents (Moyer et al., 1995; Sievert et al., 1999b, 2000a; Manini et al., 2008).

Previous studies of the Paleochori Bay vents based on DGGE and fingerprinting analyses reported a population dominated by the *Cytophaga-Flavobacteria-Bacteroides* cluster, *Gammaproteobacteria* of the genus *Thiomicrospira* and *Epsilonproteobacteria* of the genus *Arcobacter* (Brinkhoff et al., 1999; Sievert et al., 1999a, 2000a). Here, we integrated fingerprinting, sequencing and phylogenetic analyses to assess the bacterial diversity of the shallow-water vent ecosystems of Milos island. Our survey of the bacterial 16S rRNA gene sequences of the surface sediments of stations MV1 and MV4 showed that *Epsilonproteobacteria* were the most represented class in both libraries. *Epsilonproteobacteria* are well adapted to sulfidic conditions and are commonly detected in environmental surveys of geothermal environments, as well as isolated as pure cultures from deep-sea hydrothermal vents (Campbell et al., 2006; Sievert and Vetriani, 2012). In contrast, *Gammaproteobacteria* constituted only 7 and 20% of the MV1 and MV4 clone libraries, respectively. It is worth noting that the relative abundance of gammaproteobacterial clones increased from the center to the periphery of the vent (**Figure 4**). The *Epsilon-* to *Gammaproteobacteria* ratio decreased accordingly, from 8.8 at station MV1 to 2.9 at station MV4. Overall, the expected diversity, calculated as Chao1 and rarefaction analyses (**Figure 6**), appeared to be higher at MV4. Sievert et al. (1999a) reached a similar conclusion, as they found that the microbial diversity based on DGGE profiles was higher at the periphery of the vent. Despite this, the beta-diversity within the transect appeared to be relatively low as cloned sequences retrieved from both MV1 and MV4 were closely related (**Figure 5**).

Phylogenetic analysis showed a large number of the sequences retrieved from both libraries were placed in the *Sulfurovum* cluster, although the average similarity of the clones generated in this study to the type strain, *Sulfurovum lithotrophicum*, was only 93% (**Figure 5**). *S. lithotrophicum* is a sulfur-oxidizing *Epsilonproteobacterium* isolated from deep-sea hydrothermal sediments of the Okinawa trough (Inagaki et al., 2004) and it has since been identified in deep-sea vent communities worldwide (Campbell et al., 2006; Huber et al., 2007; Tokuda et al., 2007; Huber et al., 2010). The identification of *Sulfurovum*related clones in the Milos vents extended the distribution of this group of *Epsilonproteobacteria* to shallow-water hydrothermal systems. Currently, all the cultured *Epsilonproteobacteria* isolated from geothermal environments are chemolithoauthotrophs, although some of these bacteria have the ability to use one-carbon compounds (and in some rare cases complex organic carbon compounds; Campbell et al., 2006; Sievert and Vetriani, 2012). Given the phylogenetic distance between the *Epsilonproteobacteria* identified in this study and both cultured and uncultured relatives, it is possible that the shallow-water hydrothermal vents of Milos harbor members of the *Epsilonproteobacteria* with novel metabolic characteristics. One interesting hypothesis is that such *Epsilonproteobacteria* might be mixotrophic or facultatively heterotrophic. Herrmann et al. (2010) and Hubert et al. (2012) formulated similar hypotheses after detecting *Epsilonproteobacteria* in a benzene-degrading enrichment culture and in discharge waters collected from an oil sands reservoir, respectively. An effort to use alternative strategies to culture and isolate key members of the epsiloproteobacterial community is needed to understand their physiology and metabolism, and ultimately elucidate their role in these ecosystems.

A large number of the sequences retrieved in this study have low similarity to currently cultured bacterial strains (on average 90.1% similarity), while only four sequences (two for each library, 12.5% of the total investigated sequences) had similarities over 97%, a value considered as cut-off for closely related species/strains (Rossello-Mora and Amann, 2001). The nonredundant database indicated that the closest relatives to the Milos sequences were clones retrieved in the course of microbial diversity surveys of deep-sea hydrothermal vents (Lopez-Garcia et al., 2003; Nakagawa et al., 2005; Hügler et al., 2010), coldseeps (Wegener et al., 2008) and seamounts (Hodges and Olson, 2009). However, the average similarity with those sequences was rather low (93.6 and 96.7% for MV1 and MV4, respectively). This was particularly true for station MV1, where the average similarity between cultured and uncultured relatives was comparable (**Table 2**). This suggests that the Milos shallow-water hydrothermal vents harbor previously undiscovered taxa, and raises questions about the physiology and metabolism of the *Bacteria* at this site. Aside from temperature, the quantity and quality of organic matter found at the Milos vent (**Table 1**) are likely a key factor in selecting for heterotrophic or facultative heterotrophic bacteria. This observation is supported by the abundance of members of the *Cytophaga-Flavobacteria-Bacteroides* (CFB) cluster in the libraries (**Figure 4**). Since all cultured members of the CFB are heterotrophs, it is reasonable to speculate that the Milos CFB have a similar metabolism.

None of the 16S rRNA gene sequences identified in this study were related to the newly described genus *Galenea,* isolated from the same sediment samples (Giovannelli et al., 2012). In contrast to previous studies, none of the gammaproteobacterial 16S rRNA gene sequences were related to the genus *Thiomicrospira*, and none of the epsilonproteobacterial sequences were related to the genus *Arcobacter*, both of which were previously reported to be abundant in the Milos vents (Brinkhoff et al., 1999; Sievert et al., 1999b, 2000a). This suggests that spatial and/or temporal differences in the composition of the microbial communities of these vents could be enormous. Shallow-water hydrothermal vents are characterized by fluctuating conditions with elevated temporal and spatial variability of oxygen, salinity, composition of fluids, and venting regimes (Wenzhöfer et al., 2000), and they are furthermore affected by weather conditions, swell, tides and currents. Such variability creates microniches and high spatial and temporal heterogeneity, possibly leading to an increase of the overall community variability and gamma-diversity.

In conclusion, we showed that prokaryotic abundances at the shallow-water vents of Milos island are comparable to those reported in other shallow-water and deep-sea hydrothermal systems. Temperature appears to be the main driving factor in controlling prokaryotic distribution in proximity of the vent and photoautotrophy seems to increase in the lower temperature regions of the vent system. For the first time, *Sulfurovum*-related sequences were found in shallow-water hydrothermal sediments, which underscores their possible relevance in the microbial communities of both shallow-water and deep-sea hydrothermal vents. The Milos shallow-water hydrothermal vent investigated in this study harbors previously undescribed and unexpected diversity, as most of the sequences retrieved had a very low similarity to previously reported ones. This may be due to the abundance of organic matter in these systems, which may support *Epsilonproteobacteria* with novel metabolic characteristics. Further attempts to isolate key species in those ecosystems will be important to shed light on their ecology and evolution and to better understand these environments.

#### **REFERENCES**

submarine hydrothermal areas around the island of Milos, Hellenic Volcanic Arc. *Cont. Shelf Res.* 15, 913–929. doi: 10.1016/0278-4343 (95)80002-U

#### **ACKNOWLEDGMENTS**

The authors wish to thank the Captain and Crew of R/V Urania for their assistance in sampling operation. This work has been supported by the InterRidge Student and Postdoctoral Fellowships 2011 to Donato Giovannelli and NSF grants MCB 08-43678 and OCE 11-24141 to Costantino Vetriani.

a linear photometric response. *Anal. Biochem.* 48, 422–427. doi: 10.1016/0003-2697(72)90094-2

determination of lipids. *J. Lipid Res.* 7, 574–576.

alloproteins and photosynthesis begin around hydrothermal vents? *Geol. Soc. Lond. Spec. Publ.* 118, 239–251. doi: 10.1144/GSL.SP. 19<IP_ADDRESS>

dance of Archaea and Bacteria along a thermal gradient of a shallowwater hydrothermal vent quantified by rRNA slot-blot hybridization. *Microbiology* 146, 1287–1293.

communities mediating the anaerobic oxidation of methane. *Environ. Microbiol.* 10, 2287–2298. doi: 10.1111/j.1462-2920.2008.01653.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 January 2013; accepted: 18 June 2013; published online: 08 July 2013.*

*Citation: Giovannelli D, d'Errico G, Manini E, Yakimov M and Vetriani C (2013) Diversity and phylogenetic analyses of bacteria from a shallowwater hydrothermal vent in Milos island (Greece). Front. Microbiol. 4:184. doi: 10.3389/fmicb.2013.00184*

*This article was submitted to Frontiers in Extreme Microbiology, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Giovannelli, d'Errico, Manini, Yakimov and Vetriani. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

### Biogeochemical implications of the ubiquitous colonization of marine habitats and redox gradients by *Marinobacter* species

#### *Kim M. Handley1,2\* and Jonathan R. Lloyd3*

1 Searle Chemistry Laboratory, Computation Institute, University of Chicago, Chicago, IL, USA

2 Computing, Environment and Life Sciences, Argonne National Laboratory, Chicago, IL, USA

3 School of Earth, Atmospheric, and Environmental Sciences, University of Manchester, Manchester, UK

#### *Edited by:*

Andreas Teske, University of North Carolina at Chapel Hill, USA

#### *Reviewed by:*

Purificacion Lopez-Garcia, Centre National de la Recherche Scientifique, France Juergen Wiegel, University of Georgia, USA

#### *\*Correspondence:*

Kim M. Handley, Searle Chemistry Laboratory, Computation Institute, University of Chicago, 5735 South Ellis Avenue, Chicago, IL 60637, USA.

e-mail<EMAIL_ADDRESS>
The Marinobacter genus comprises widespread marine bacteria, found in localities as diverse as the deep ocean, coastal seawater and sediment, hydrothermal settings, oceanic basalt, sea-ice, sand, solar salterns, and oil fields. Terrestrial sources include saline soil and wine-barrel-decalcification wastewater. The genus was designated in 1992 for the Gram-negative, hydrocarbon-degrading bacterium Marinobacter hydrocarbonoclasticus. Since then, a further 31 type strains have been designated. Nonetheless, the metabolic range of many Marinobacter species remains largely unexplored. Most species have been classified as aerobic heterotrophs, and assessed for limited anaerobic pathways (fermentation or nitrate reduction), whereas studies of low-temperature hydrothermal sediments, basalt at oceanic spreading centers, and phytoplankton have identified species that possess a respiratory repertoire with significant biogeochemical implications. Notable physiological traits include nitrate-dependent Fe(II)-oxidation, arsenic and fumarate redox cycling, and Mn(II) oxidation. There is also evidence for Fe(III) reduction, and metal(loid) detoxification. Considering the ubiquity and metabolic capabilities of the genus, Marinobacter species may perform an important and underestimated role in the biogeochemical cycling of organics and metals in varied marine habitats, and spanning aerobic-to-anoxic redox gradients.

**Keywords:** *Marinobacter***, marine, hydrothermal, biogeochemical cycling, hydrocarbon, iron, arsenic, opportunistic**

#### **INTRODUCTION**

Marine habitats are host to a diverse range of substrates and physicochemical regimes. Among these, hydrothermal features attract particular interest owing to ore-grade concentrations of metals, physicochemical extremes, and the presence of chemolithoautotrophic macrofauna and microbiota. Bacteria and Archaea occupying marine habitats have a substantial physical presence. There are an estimated 3*.*9 × 1030 prokaryotic cells in the open ocean and unconsolidated marine sediments, comprising ∼3*.*1 × 1011 tonnes of carbon (Whitman et al., 1998). This is slightly more than the estimated carbon content of terrestrial prokaryotes, and just under half of that in all plant life. Prokaryotes can contribute significantly to marine ecosystem functioning and biogeochemical cycles (Jørgensen, 2006), owing to their prevalence and enormous capacity for transforming their environments through metabolism of organic and inorganic matter (Gadd, 2010). Yet much of the marine microbial biomass remains unexplored, and there is still much to learn about heterotrophic and autotrophic bacterial functioning in the ocean (e.g., Moran et al., 2004; Emerson et al., 2010; Holden et al., 2012).

*Marinobacter* is a heterotrophic, and in some instances mixotrophic (Dhillon et al., 2005; Handley et al., 2009a,b), metabolically flexible genus found in an exceptionally wide range of marine and saline terrestrial settings, including various low-temperature hydrothermal environments (**Table 1**). The genus comprises Gram-negative *Gammaproteobacteria* within the *Alteromonadales* order. All known species are motile with polar flagella (excluding *M. goseongensis*, Roh et al., 2008), slightly to moderately halophilic (cf. DasSarma and Arora, 2012), aerobic heterotrophs (**Table 2**). However, few are confirmed strict aerobes, and several are facultative anaerobes (**Table 1**). All are rod-shaped, with the exception of the ellipsoidal *M. Segnicrescens* (Guo et al., 2007), and most are neutrophilic, except the slightly alkaliphilic *M. alkaliphilus*, which grows optimally at pH 8.5–9.0 (Takai et al., 2005; also see Al-Awadhi et al., 2007; **Table 2**). Although most species are mesophilic, many are psychrotolerant (also known as psychrotrophic) and capable of growth down to ∼4◦C (**Table 2**; Moyer and Morita, 2007). A couple of other species are either psychrophilic (with a growth optimum near 15◦C) or thermotolerant (with growth up to 50◦C and an optimum of 45◦C). This phenotypic versatility contributes to the ubiquity of this genus, and its ability to occupy diverse physicochemical regimes.

#### *MARINOBACTER HYDROCARBONOCLASTICUS***, DENITRIFICATION AND HYDROCARBONS**

The genus was created for *M. hydrocarbonoclasticus*, which was isolated from hydrocarbon-polluted sediment, collected from the

(Continued)

#### **Table 1 | Continued**

aValidly published species names as of April 2013.

bCarbohydrates used by species are glucose, glycerol, fructose, maltose, mannitol, sucrose, cellobiose, galactose, dextrin, sorbital, trehalose, xylose, ribose, sorbose, erythritol, inositol, dulcitol, arabinose, and N-acetyl-D-glucosamine.

c–g 87–94%, 89–97%, 96–99%, 99%, 96–98% 16S rRNA gene sequence similarity to Marinobacter species, respectively.

 The species has an obligate requirement for sodium. It grows readily on complex organic media containing yeast extract and peptone, and aerobically on a range of organic acids (acetate, butyrate, caproate, fumarate, adipate, lactate, citrate), and the amino acids L-glutamate, L-glutamine, and L-proline. Under anaerobic conditions *M. hydrocarbonoclasticus* can perform denitrification using a membrane-bound respiratory NarGHI complex to reduce nitrate (Correia et al., 2008). The nitrite formed is reduced to N2 *via* nitrite reductase cytochrome cd1 (Besson et al., 1995), nitric oxide reductase NorBC (EMBL-EBI ABM20188.1, ABM20189.1), and nitrous oxide reductase (Prudêncio et al., 2000). *M. hydrocarbonoclasticus* is most notable, however, for its ability to aerobically degrade liquid and solid, aliphatic (pristane, heneicosane, eicosane, hexadecane, tetradecane) and aromatic (phenanthrene, phenyldecane) hydrocarbons. It uses each hydrocarbon as a sole energy source, and produces large quantities of bioemulsifier. Bioemulsifiers (biosurfactants), are thought to aid in bacterial adhesion to hydrophobic surfaces, water-immiscible material breakdown, and competitor inhibition, and are attracting increasing interest for various industrial applications (Banat et al., 2000; Nerurkar et al., 2009; Williams, 2009; Soberón-Chávez and Maier, 2011).

Of the 34 species named since the genus was created, several exhibit hydrocarbonoclastic activity, while others remain untested (**Table 1**). Additional hydrocarbons utilized by *Marinobacter* species include squalene, which is metabolized under denitrifying conditions (Rontani et al., 2003), polycyclic aromatic hydrocarbons (PAHs) (Cui et al., 2008), hexane, heptane, petroleum ether (Shivaji et al., 2005; Antunes et al., 2007), *n*-pentadecane, *n*-tridecane, *n*-undecane, *n*-decane, *n*-nonane, butane, and kerosene (Takai et al., 2005).

This hydrocarbonoclastic capacity in *Marinobacter* has attracted attention owing to the potential for these bacteria to remediate crude oil contamination in environments as diverse as the Arabian Gulf (Al-Awadhi et al., 2007) and Artic sea ice (Gerdes et al., 2005). Nitrate reduction by *Marinobacter* species has also been exploited for potential use in oilfield maintenance. Dunsmore et al. (2006) showed reduction of added nitrate prevented deleterious growth of sulfate-reducing bacteria in produced water from a North Sea oilfield oil reservoir, controlling microbial souring reactions. The beneficial reduction of nitrate was largely attributed to indigenous *Marinobacter* species.

#### **Table 2 |** *Marinobacter* **species attributes.**

aGC contents range from 54.0–63.5% (average, 57.6%), using the Márquez and Ventosa (2005) value for hydrocarbonoclasticus.

bAll species are motile, excluding M. goseongensis. M. lutaoensis also has bipolar pili. The number of flagella on M. guineae cells is unknown.

cUnsheathed flagellum.

Abbreviations: Haloph, halophile; Mesoph, mesophile; temp, temperature; resp, respiratory; Y, yes; N, no; –, unknown; s-mod, slightly-moderately; psyt, psychrotolerant; psyph, psychrophile; therm, thermotolerant.

1–32References: refer to *Table 1*.

#### **EXPANDED FUNCTIONAL TRAITS OF THE GENUS**

Following the characterization of *M. hydrocarbonoclasticus*, the functional range of the genus has been further expanded to include (non-exhaustively) fermentation; the ability to respire at least 19 different carbohydrates (**Table 1**) and several extra amino (e.g., L-alanine, D-glutamate, L-phenylalanine; Antunes et al., 2007 and Green et al., 2006) and organic acids [e.g., malonate, formate, pyruvate, alpha-ketoglutarate; Kim et al. (2006) and Kharroub et al. (2011)]; degradation of the isoprenoid ketone 6,10,14-trimethylpentadecan-2-one (Rontani et al., 1997); growth on ethanol (Gu et al., 2007; Huo et al., 2008), phenol (Liebgott et al., 2006), and various Tweens (e.g., Takai et al., 2005; Green et al., 2006) following enzymatic evidence in *M. hydrocarbonoclasticus*; utilization of fumarate as an electron acceptor (Takai et al., 2005; Handley et al., 2009a); and oxidation/reduction of arsenic, iron or manganese (Handley et al., 2009a,b; Wang et al., 2012).

As for *M. hydrocarbonoclasticus*, all subsequently described species are able to grow aerobically on complex organic matter, and oxidize organic acids. Many, but not all are enzymatically able to reduce nitrate (**Table 1**). Lack of fermentation by *M. hydrocarbonoclasticus* was initially proposed as a distinctive feature of the genus; however, a number of subsequently isolated type strains exhibit both fermentative and respiratory metabolisms, owing to their ability to ferment glucose (**Table 1**), lactate (Handley et al., 2009a) and other substrates (Lee et al., 2012). Evaluation of recently available genome sequences also suggests certain *Marinobacter* species may exhibit enzymatic resistance to arsenic and heavy metals (e.g., Wang et al., 2012).

#### **BIOGEOGRAPHY AND PHYLOGENY**

*Marinobacter* colonize diverse saline habitats, e.g., sea ice and hydrothermal sediments, facilitated by psychrophilic to thermotolerant physiologies, and an ability to metabolize an array of (in)organic compounds under aerobic or anaerobic conditions. However, evaluation of phylogenetic trees, constructed using 16S rRNA gene sequences, suggest the genus is monophyletic, forming a single clade distinct from other closely related epsilonproteobacterial lineages (**Figure 1**).

Despite the physiological versatility of the genus, and the ability of some strains to grow (non-optimally) without salt (e.g., Huu et al., 1999; Sigalevich et al., 2000; Liebgott et al., 2006), *Marinobacter* appear to be geographically restricted to marine or terrestrial environments rich in sodium salts. This observation is consistent with the hypothesis that microorganisms exhibit non-random biogeographical differentiation and distribution, due in part to environmental selection (Martiny et al., 2006).

In marine environments, dispersal does not appear to be a limiting factor for *Marinobacter*. Strains have been isolated from, and phylogenetically detected in, oceans (Pacific, Atlantic, Indian, Arctic, and Antarctic) and seas, spanning the globe from pole to equator (**Table 1**; Kaye et al., 2011). They display both attached and planktonic lifestyles, and the distribution of the genus extends from deep-ocean (hydrothermal) benthic sediment and exposed basalt to surface water, or coastal (hydrothermal) sediment, hot spring water and sand (**Table 1**; **Figure 1**).

In many instances, terrestrial isolation sources can be clearly linked to the ocean (e.g., coastal solar salterns and hot springs, and polluted soil from a coastal oil field; **Table 1**). Isolation of species from terrestrial sources, up to 50–60 km inland, implies a greater degree of terrestrial dispersal (**Table 1**; **Figure 1** and Table S2 in Kaye et al., 2011). However, there is insufficient information regarding the nature of terrestrial isolation sources, and too few isolate and phylogenetic data, to judge how well-dispersed this genus is on land, or whether terrestrial sources are strictly independent from marine influences.

#### **LIFESYTLES**

In many respects *Marinobacter* species are generalists like their marine and terrestrial *Alteromonadales* cousins in the *Shewanella* genus. *Shewanella* species are respiratory generalists (e.g., Heidelberg et al., 2002), and at least one species (*S. baltica*) has been described as "very close to the ultimate [marine] *r*-strategist," starkly contrasting with genomically streamlined K-strategist (or oligotrophic) marine bacteria like *Prochlorococcus* (Caro-Quintero et al., 2011). In the presence of surplus organic carbon, *Marinobacter* can grow rapidly, out-competing other bacteria in enrichment cultures (e.g., Handley et al., 2010). This *r*-strategist (or copiotrophic) behavior renders them weed-like and relatively easy to cultivate, even compared with other heterotrophic marine bacteria (Kaye and Baross, 2000). *Marinobacter* can also excel under aerobic-to-anaerobic conditions with no added substrate, while in the presence of Fe(II) (Edwards et al., 2003; Handley et al., 2013a).