A Microbial Observatory Examining Microbial Abundance, Diversity, Associations and Activity at Seafloor Brine Seeps

This project is a collaborative effort between the Joye group, Dr. Ian MacDonald (Florida State University), Dr. Andreas Teske (University of North Carolina), Dr. Kai Uwe-Hinrichs (University of Bremen Germany), and Drs. Kirsten Habicht, Bo Thamdrup and Don Canfield (University of Southern Denmark). We are working at several brine seeps in the Gulf of Mexico, which are described below in more detail, and will be conducting research cruises to these sites in 2010, 2011 and 2012. The following group members are involved in this work:

** PhD and Post Doctoral Positions are available on this project contact mjoye (at) uga (dot) egu if you are interested in learning more**

Why study Seafloor Brines?

Fig. 1. Salt tectonics: Fluid flow along the faults associated with the salt diapir (hatched block, L image) result in surface features including mud volcanoes (middle image) and brine pools (R image). From MacDonald and Fisher (1996).

We propose to examine the microbiology of two types of brine seeps in the GoM. Brine pools occur where brine fills surface depressions creating “lake-like” features on the seafloor. Mud volcanoes occur where brine, oil, gas and fluidized mud are actively expelled, inducing large temperature fluctuations and seabed alterations.

Fig. 2. Side-scan sonar mosaics of the GC233 brine pool (BP, upper L) and the GB425 mud volcano (MV, upper R) and still pictures of the sites. Lower panels refer to areas in the white square (BP) and red square (MV). At the BP, a variety of macrofauna interact with the pool (lower L). At the MV, vigorous gas venting is common (lower R) and macrofauna are rare.

Figure 1A.

The chemical composition and salinity of brines is certain to influence the structure and activity of the resident microbial community. To date, most of what we know about the microbiology of seafloor brines comes from studies in the eastern Mediterranean (Med) and Red Seas. The Med brines derive from the dissolution of Messinian evaporates containing both halite (NaCl) and gypsum (CaSO4) and thus may have elevated concentrations of sulfate (up to 135 mM SO42-) and hydrogen sulfide (up to 10 mM). The temperature (T) of Med brines is slightly elevated above the bottom water (by ~3ºC) and the pH is ~6.6. Red Sea brines have a geothermal source and thus have lower sulfate (10 mM), higher T (23-67 ºC) and lower pH (5.5). The GoM brines share features with both Med and Red Sea brines. GoM brines are derived from dissolution of Jurassic halite and thus contain no sulfate. Some GoM brines (e.g. GC233 and GB425, see below) are derived from the deep subsurface, so their temperature is ~10ºC or more higher than bottom water. Other brines (e.g., GK2, see below) are derived from dissolution of salt structures near the sediment-water interface and thus have temperatures similar to the bottom water. The pH of the GoM brines is circumneutral (~7.2) and the salinity is about 150‰. The hydrogen sulfide concentration of GoM brines varies but is generally lower (<1 mM) than that observed in Med brines (up to 10 mM). A common feature of GoM and Med and Red sea brines is the presence of a 1 to 3 m thick chemocline separating the dense brine and the overlying seawater; this chemocline is a zone of elevated of microbial abundance and activity.

Seafloor brines are extreme environments, meaning the resident microbial populations possess unique physiological adaptations to osmotic and sometimes thermal stress. Despite the harsh nature of the habitat, microorganisms often attain markedly high levels of diversity and productivity by exploiting the resources available in brines. Microbiological investigations of Med and Red Sea brines showed diverse and metabolically active microbial communities that were distinct from the microbial communities in the overlying water column. In most Med brines, Bacteria were more abundant than Archaea. Med brines contained a variety of Bacteria, including representatives from the γ-, δ-, and ε- subdivisions of the Proteobacteria; Halobacteria; KB1, a new candidate division of the Euryarchaeota; MSBL-1 (Mediterranean Sea Brine Lakes group 1); and several new candidate divisions of Bacteria, including MSBL-2 through 6. Candidate division MSBL-2 is related to candidate division SB1, which was discovered along the chemocline of the Shaban Deep, Red Sea. Microorganisms related to the ANME-1 group of anaerobic methane oxidizers were found in Med brines and a diverse array of halophilic or halotolerant microorganisms were cultured from the chemocline and brine; two of these isolates were less than 92% similar to known microorganisms. In terms of microbial activity, substantial rates of sulfate reduction and methanogenesis were documented and rates of other processes (e.g. fermentation) were presumed to be substantial. The microbial diversity and assorted metabolic capabilities documented in Med and Red Sea brines illustrates that seafloor brines are a fruitful area for identifying new and unusual microorganisms.

Our previous work documented microbial diversity and activity in sediments influenced by brine and/or oil and gas seepage. We determined patterns of microbial abundance and activity across a 2m chemocline separating the overlying seawater and underlying brine at a mud volcano and a brine pool (Joye et al. 2009 Nature Geoscience).

Fig. 3. Geochemical parameters and microbial activity a brine pool (A, B) and a mud volcano (C, D); hashed line denotes the seawater-brine interface. Note different scales for methanogenesis (MOG) rates. Rates in µmol or nmol per liter per day. Ac_MOG=acetoclastic MOG; H_MOG=hydrogenotrophic MOG.

The elevated H2 and acetate concentrations together with acetate-δ13C data implied homoacetogenesis was more important in the brine pool than in the mud volcano. Rates of sulfate reduction were 10 times higher in the brine pool than in the mud volcano while rates of methanogenesis were almost 1000 times higher in the mud volcano. No AOM was detected, possibly because of the high H2 concentrations. Data describing the identity and diversity of the microorganisms in the brines is, at this time, limited to the sulfate reducers. The surface layers of brine pool and mud volcano harbor sulfate-reducing bacterial populations that oxidize acetate and aromatic compounds (Desulfosarcinales, Desulfobacterium). The brine pool contained sulfide- and hydrogen-oxidizing e-Proteobacteria as well, consistent with observed higher hydrogen levels and with sulfide production via sulfate reduction (Fig. 3). In contrast, the mud volcano contained fermentative d-Proteobacteria of the family Syntrophaceae that grow syntrophically with hydrogen-consuming methanogens; this is consistent with lower hydrogen concentrations and higher rates of hydrogenotrophic methanogenesis observed there (Fig. 3). Analysis of dissimilatory sulfite reductase genes is in progress.

This work generated a variety of intriguing questions, such as: What novel microorganisms inhabit these systems? Why does H2 accumulate to such high concentrations in the brine pool? Why are sulfate reduction rates lower in the mud volcano? Why are methanogenesis rates lower in the brine pool? How important are homoacetogenesis and sulfur disproportionation in these brines? We will address these and other questions to understand the relationship between brine chemistry/dynamics and microbial community composition and activity during this Microbial Observatory project.

We will apply a suite of microbiological, biogeochemical, isotopic, molecular, and physiological techniques to document the activity and distribution of microorganisms at brine seeps. Our overall goal is to identify and analyze the microbial populations inhabiting brine seeps characterized by different fluid flow rates and chemical regimes. Our work has six main objectives:

1. Quantify the abundance, diversity and activity of microorganisms mediating carbon and sulfur transformations in brine seep habitats;
2. Quantify the relationship between microbial diversity and activity and environmental gradients;
3. Identify the environmental controls on microbial activity and distributions;
4. Identify metabolically active microorganisms;
5. Elucidate carbon and sulfur flow in the microbial food web using stable isotope studies of biomarkers, DNA and RNA;
6. Isolate and characterize microorganisms from brine seep habitats.

The nuts and bolts of the Brine Microbial Observatory Project

We will carry out research cruises and laboratory studies to examine microbial diversity and activity at seafloor brine seeps. We will address the following hypothesis and questions at four sites over a 5-year research program (2009-2014): We hypothesize that differences in fluid flow generate variability in brine chemical composition, which strongly influences microbial community composition and activity within and between sites.

This hypothesis implies that identifying and quantifying the physical and chemical constraints and their effects on microbial community composition and activity is essential for developing an ecosystem-level understanding of brine-impacted cold seeps, which are globally distributed marine habitats with the unique role of re-injecting fossil carbon into the living biosphere.

The following specific research goals will be examined to address the primary hypothesis and research objectives (noted in parentheses):

1. Spatio-temporal Variability: Document differences in microbial populations and activity between brine pool and mud volcano sites and between years within a given site (objectives 1, 2).
2. Chemical Composition: Determine how brine chemical composition influences microbial abundance, community composition and activity (objectives 2, 3).
3. Functional and Phylogenetic Diversity: Quantify the microbial metabolisms and key functional genes found across habitats and evaluate how metabolic diversity relates to overall phylogenetic diversity as determined by 16S rDNA and rRNA analysis (objectives 1, 4).
4. Regulation of Microbial Activity: Elucidate the factors – community composition, physiological constraints (e.g., limitation by nutrients or bioactive trace metals, inhibition by salt, or competition for substrates), or environmental factors – that regulate the distribution and activity of microorganisms in seafloor brines (objective 3).
5. Flow of Energy and Carbon: Determine which chemicals serve as primary sources of metabolic energy and cell carbon to support microbial growth (objective 5).
6. Regulation of Sulfur Isotopes: Quantify sulfur isotopic fractionation during sulfate reduction to understand the sulfur isotope composition of sulfate and reduced sulfur (objective 5).
7. Novel Microorganisms: Isolate and characterize brine microorganisms (objective 6).

Research Cruises

Fig 4. Gulf of Mexico overview with general bathymetry and stations (top) and a detailed station map (bottom) [mud volcano=red; brine pool=blue; exploratory sites=yellow]. Maps made via GeoMapApp.

Two additional sites representing ‘end member’ brines, discovered during JASON ops in 2007, will be sampled. Site 1 (“Hot Site”) is a small mud volcano at a depth of 1000m. The central brine pool was highly active, discharging copious gas and fluidized mud. The fluid salinity was approximately 210‰ and the temperature was elevated substantially above the bottom water. Site 2 (“Red Crater”) is an ~1-km wide crater at a depth of 2200m. The edges of the crater were noted by dark-stained depressions filled with reducing brines. The central crater was filled with reddish-colored brine (salinity ~39‰). We suspect that site 1 represents the freshly formed mud volcano since the salinity approaches that expected for halite saturation. Site 2 appears to have undergone dilution with seawater over extended time given the low salinity. At these sites, we will do CTD casts through the brine and collect bottle (or 1 brine-trapper) samples from the core brine and chemocline to evaluate rates of key microbially-mediated processes and to generate some preliminary data on microbial community composition.

We will sample these sites during cruises using the R/V Altantis and manned submersible ALVIN in 2010 and 2011. Cruises will consist of sampling each site and recovering and deploying monitoring equipment. For the four main sites, a minimum of two dives per site is required to accomplish site photo-documentation, brine sampling using the “brine trappers”, temperature-salinity profiling using a CTD, and deployment of monitoring equipment. Two sets of brine-trapper samples will be collected at three sites, while three sets of brine-trapper samples will be collected at the other site (the intensively sampled site) to provide a measure of spatial heterogeneity and obtain material for laboratory experiments.

We will use temperature-salinity profiles, temperature-salinity loggers, and time-lapse digital video to document spatial and temporal variability of fluid flow at seafloor brine seeps. Modeling of temperature and salinity profiles will provide estimates of fluid flow velocities. The aim of this work is to document the variability in and magnitude of fluid flux at the different sites, not to constrain fluid flow in an absolute sense; though the latter is a worthy goal, it is well beyond the primary scope of this proposal, which is microbiology. We are prepared to modify our plans to take advantage of opportunities to sample episodic or extreme discharge events at mud volcanoes. At the four main sites, the following tasks will be completed during two ROV dives (three dives at the intensively sampled site): 1. The site will be surveyed and photo-documented using down-looking digital mosaic camera (detailed in section VII-5). 2. Brine samples will be collected using the brine trapper; additionally, a deep (>5m) brine sample will be collected using a gas-tight fluid sampler lowered into the brine using a winch. The brine-trapper consists of an outer anodized aluminum tube (the interior is Teflon coated) pierced by a regularly spaced windows and a series of inner Delrin® plugs connected by a rod. The ends of each plug are fitted with an O-ring bore seal so that the spaces between the plugs form a series of internal compartments. An hydraulic actuator moves the plug and rod assembly up or down in the tube. The compartments are open to the outer environment when aligned with the windows and closed when the plugs are aligned with the windows. Each sampling compartment is fitted with a high-pressure valve and sampling port. The trapper is mounted horizontally on the ROV and deployed by rotating it downward. Sampling compartments are set ~20 cm apart throughout the length to capture the transition from the overlying seawater through the chemocline to the underlying brine. The two brine trappers will be mounted ~2m apart and deployed on the same dive to collect replicate profiles from a given location. Using both trappers assures that we will have ample material for microbiological and biogeochemical analyses and for laboratory experiments (enrichment, isolation, etc.; see below). 3. A SeaBird SBE53-MP CTD (modified for hypersaline conditions) will be lowered into the brine using a ROV-mounted winch to characterize the thermal and salinity structure; at least 4 CTD profiles will be obtained to characterize spatial variability. 4. Monitoring equipment, i.e. a rotary time-lapse camera system and T-S sensor strings, will be deployed at positions in (sensors) or at the edge of (camera) the brines to document the T-S regime (sensors) and discharge of turbid fluid and gas bubbles (camera) over a year. Cameras and temperature-salinity loggers will be deployed at two sites per year, generating annual records for each of the four primary sampling sites during the field program.

A fluid profile from the overlying seawater into the brine will be obtained by lowering the brine trapper vertically, maintaining the upper three chambers in the overlying seawater and immersing the remainder are in the brine. The seawater-brine interface is identified visually by noting the change in refractive index. The brine trapper is positioned and then sampling occurs (the chambers are opened, then closed). Using ALVIN’s dynamic positioning system, we can place the two brine trappers at comparable locations with respect to the brine-seawater interface, assuring that the contents are replicates of one other. Upon return to the surface, the brine trappers are transferred to a cold van, where they are sampled. The volume of gas in each chamber will be quantified by connecting a 20L Tedlar bag containing distilled water (and no gas phase) to the sampling port. As the pressure valve is opened, venting gas is introduced into the top of the bag while water is displaced through a one-way valve at the bottom; the volume of displaced water is recorded as a proxy for the gas volume. Three 20 mL samples of the venting gas will be collected into an evacuated serum vial for determination of concentrations of C1-C5 alkanes, carbon dioxide, hydrogen, and hydrogen sulfide (methods in the following section). The gas volume and concentration of individual components will be used to calculate in situ gas concentrations. After degassing, fluid samples from replicate chambers (the comparable depth from each trapper) will be transferred to argon-purged sterile bottles, and aliquots will be dispensed for various analyses (see below).

How we will do this

During cruises, a suite of core experiments will be carried out and environmental data will be obtained at each study site. More detailed sampling and experimentation will be carried out at one of the study sites each year so that detailed data will be generated for each of the four main study sites over the 4-yr field program. This work focuses on the microbial communities mediating carbon and sulfur transformations in anoxic brines, the anoxic chemocline (seawater-brine transition), and the oxic overlying seawater. These brines are laden with ammonium (up to 15 mM) but are nitrate (NO3-) and oxygen (O2) free, making it unlikely that denitrification, dissimilatory nitrate reduction to ammonium, anammox, nitrification or nitrogen fixation are important in the brines. However, [some of] these processes may be important where brine-derived NH4 is mixed with oxygen- and NO3--rich bottom waters. We will attempt to leverage funding from other agencies for N cycling work in the overlying bottom waters.

1. Microbiology and molecular ecology

The goals of this component are to document patterns of microbial abundance, diversity and associations, to characterize and compare/contrast the microbial communities over depth and between sites, and to isolate and characterize novel microorganisms. These goals are related to objectives 1, 2, 4, and 6. Detailed phylogenetic analyses cannot be completed on every sample given the large number of samples this work will generate (a minimum of ~32 brine samples will be collected at each site [2 sets of replicate brine trappers, volumes combined, and 2 deep brine bottle samples], or ~130 samples total for the four main sites). For comparisons between sites, we will carry out detailed phylogenetic characterization of the microbial community in the overlying water, in the middle of the chemocline, and in the deep brine. Cell counts and CARD-FISH enumerations will be carried out more frequently. We will use DGGE as a survey tool to identify discontinuities in microbial community structure along chemical and depth gradients to pinpoint representative samples for diversity analysis using clone libraries and pyrosequencing. Culture work will carried out using chemocline and deep brine samples.

2. Microbial activity

Quantifying rates of microbial activity is necessary to fulfill objectives 1, 2 and 3. We will determine rates of C and S cycling in samples by assaying a core suite of activities at each site in the complete brine trapper profile samples (SR, AOM, methanogenesis from CO2 and acetate, homoacetogenesis) complemented by other assays carried out at fewer depths (acetate oxidation, fermentation, methanogenesis from methylated amines and methanol). Rate measurements will be conducted on sub-samples from each depth of the profile collections as well as in deep brine samples (n=3 per depth plus killed controls). Activity of SR, AOM, homoacetogenesis and methanogenesis at simulated in situ pressure will be documented at 4 depths (top, middle and bottom of chemocline, and deep brine) at the intensively sampled site each year.

3. Linking phylogeny and activity

We will use three approaches to link microbial phylogeny with patterns of activity; a secondary goal is to constrain carbon flow. This work will be done at the intensively sampled site each year and addresses objectives 4 and 5.

4. Biogeochemistry

Determining the biogeochemical signature of the brine seeps will help us understand the controls on microbial distributions and activity and will help identify feedbacks between microorganisms and the geochemical environment and vice versa. Stable C and S bulk isotopic measurements combined with compound specific isotope data will help constrain C and S flow. This work is required to address objectives 2, 3 and 4.

5. Habitat characterization: imaging and instrumentation

Environmental characterization is required to address objectives 2 and 3. The PIs have access to a variety of specialized sampling and monitoring equipment, much of it developed by members of our own research team, including:

1. Time-lapse cameras for temporal documentation of bottom conditions and communities,
2. Salinity and Temperature loggers for recording in situ S and T over 12 months,
3. Brine trapper for obtaining vertically stratified samples through the upper 2 to 4 m of brine.

Instrument details are provided in the facilities statements. The salinity-temperature loggers, CTD profiling and brine trappers were described in section VI. Quantitative and documentary imaging will be carried out using two distinct camera systems. At each site, the ROV will survey the perimeter of each pool while recording down-looking digital images with the JASON mosaic camera. A series of small markers (floats on short lines) will be deployed to mark the edges of the pools and to facilitate repetitive sampling. Uniform scale mosaics of the pool edges will document the stability of the margin and changes in frequency and character of the habitat. A rotary time-lapse camera system will be deployed at a position where it can survey the discharge from the pool and regions of the pool edge colonized by bacterial mats and/or chemosynthetic macrofauna (e.g. Bathymodiolus mussels). These devices consist of a digital camera and light inside a tempered glass tube. The camera rotates 36º between shots to collect a 360° cylindrical panorama of the seep environment over a radius of approximately 3m. In total, this represents an area of ~28 m2 and volume of about ~43 m3 for each panorama. In the yearlong intervals between cruises, the cameras will be left in place to record time-lapse imagery for ~9 month periods (constrained by battery life). These long- term deployments will monitor brine dynamics over annual cycles (Vardaro et al. 2006). The separate images comprising a 360° panorama will be merged into Quick Time Virtual Reality (QTVR) to offer a unique educational perspective of the brine seep environment. For an example of a QTVR panorama from the deep sea collected with one of the rotary cameras, see: http://sci.tamucc.edu/news/2007-07/DeepSlope/movie.html

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We thank the National Science Foundation (NSF) for funding this research.

**Disclaimer** The content of this page is based in part on work supported by the NSF. Any opinions, findings, and conclusions or recommendations expressed here are those of the author (Mandy Joye) and do not necessarily reflect the views of the NSF.