Iron oxides from the caldera of Axial Volcano, a site of hydrothermal vent activity along the Juan de Fuca Ridge, were found to consist predominantly of microbial structures in hydrated whole mounts examined using an environmental scanning electron microscope. Novel observations were made of the iron oxides revealing the spatial relationships of the bacteria within to be more consistent with microbial mats than mineral precipitates. The bacterial structures are attributed to the sheaths of Leptothrix ochracea, the stalks of Gallionella ferruginea, and the filaments of a novel iron oxidizing PV-1 strain, based on the distinctive morphological characteristics of these three bacteria. Energy dispersive X-ray spectroscopy revealed the presence and distribution of Fe, Si, and Cl on the bacterial sheaths, stalks and filaments. The iron oxides were identified by X-ray diffraction to be two-line ferrihydrite, a poorly ordered iron oxyhydroxide. Adsorption of Si in particular to two-line ferrihydrite likely contributes to its stability on the seafloor, and might also be a preservation mechanism creating microfossils of the bacterial structures encrusted with ferrihydrite. Presumptive evidence of the sub-seafloor presence of L. ochracea, G. ferruginea and PV-1 at Axial Volcano was obtained from the presence of these bacteria on a trap that had been placed within an active vent, and also in a vent fluid sample. If indeed these bacteria are present in the sub-seafloor, it may be an indication that the surface expression of iron oxide deposits at Axial Volcano is minimal in comparison to what exists beneath the seafloor.
Iron redox gradients in advective aqueous systems can provide ideal habitats for aerobic and neutrophilic iron oxidizing bacteria such as Leptothrix ochracea and Gallionella ferruginea[1–5]. In these environments, the sheaths and stalks of these bacteria typically become encrusted with poorly ordered iron oxide precipitates. Although a degree of uncertainty still exists with regards to the metabolic utility of Fe(II) oxidation by L. ochracea[2,6], Hallbeck and Pederson  have demonstrated that G. ferruginea relies on Fe(II) oxidation as an electron donor for autotrophic growth. Subsequent hydrolysis of Fe(III) produced by the bacteria results commonly in the precipitation of poorly ordered ferric oxides on the helical stalk of G. ferruginea and the sheath of L. ochracea.
The ability of bacteria to influence mineral precipitation is being increasingly recognized as an important process in environmental geochemistry [8–13]. With respect to iron oxide formation at neutral pH, iron oxidizing bacteria play at least two roles. First, they have been shown to increase the rate of iron oxidation by up to four orders of magnitude over the rate of strictly inorganic oxidation [4,14]. Second, they inherently lower the degree of supersaturation required for iron oxide precipitation by behaving as geochemically reactive solids for heterogeneous surface nucleation .
The main objective of this study was to investigate microbial contributions to the fabric and the hydrated structure of iron oxides from Axial Volcano, a site of active hydrothermal venting situated along the Juan de Fuca Ridge in the north-east Pacific Ocean. Specifically, environmental scanning electron microscopy (ESEM) was used to examine hydrated whole mounts of chemically unaltered specimens, which has proven to be invaluable in such geomicrobiological studies . The advantage of ESEM over traditional scanning and transmission electron microscopy (SEM and TEM) is that SEM and TEM require a substantial amount of sample preparation, potentially altering the architecture and composition of specimens, whereas ESEM does not.
The combination of ESEM's imaging and energy dispersive X-ray spectroscopy (EDS) capabilities can provide both composition and element distribution information of the iron oxide precipitates. Iron, oxygen and silicon are the major elements that have been detected in mid-ocean ridge (MOR) iron oxide deposits [17–21], however, the distribution of Si is debatable as siliceous diatoms are commonly present in MOR iron oxides. To our knowledge, this architectural and compositional distribution ESEM investigation is the first to be performed on MOR iron oxides.
A secondary objective of this study was to investigate the occurrence of iron oxidizing bacteria in the sub-seafloor of Axial Volcano. The formation of low temperature vents involves the mixing of hot Fe(II)-rich hydrothermal fluids (i.e. >350°C, reduced and acidic) with cold oxygenated ocean water (i.e. circumneutral, 4°C) that has percolated down through faults and fissures in the seafloor . Mixing below the seafloor creates mildly reducing microaerophilic conditions that are ideal for bacterial iron oxidation. The capture and retention of iron oxidizing bacteria on a trap that had been placed in an active vent, and the presence of these bacteria in a vent fluid sample were used to test for their presence in the sub-seafloor at Axial Volcano.
2Materials and methods
2.1Site description and sample collection
Axial Volcano is a large MOR axis volcano located at the center of the Juan de Fuca Ridge (lat. 46°N, long. 130°W) in approximately 1500 m of water in the north-east Pacific Ocean . Hydrothermal venting in the area results from ongoing MOR spreading of the Juan de Fuca plate. Sampling was performed at both active and inactive hydrothermal vent sites during the New Millennium Observatory (NeMO) expedition in June 2001 aboard NOAA's Ronald H. Brown with the Canadian ROPOS remotely operated vehicle (ROV). The iron oxide deposits sampled included mound-like (∼0.1–0.5 m high) accretions to widespread blanket-type deposits from six different sites throughout the caldera of Axial Volcano. Another site sampled in the proximity of low temperature hydrothermal venting was South Cleft (44°38.53N, 130°11.19W), south of the Axial Volcano caldera, along the Juan de Fuca ridge. A limited amount of sediment was recovered from this site due to the time constraints of the cruise, allowing for a microscopic analysis only. Collection of iron oxide samples was accomplished using a suction sampler on the ROPOS ROV.
In an effort to evaluate the sub-seafloor occurrence of iron oxidizing bacteria, two vent sampling techniques were employed. The first method utilized a bacterial trap that had been placed in an active, low temperature (10°C) vent for 1 year situated in such a way that it was completely surrounded by vent fluids. It was assumed that hydrothermal fluids surrounded the trap for the duration of the deployment as venting was still active upon its recovery. The 202-μm nylon mesh (Nytex) on the trap was noted to have developed a light coating of iron oxide precipitate. After recovery, the mesh was cut into several small fragments (∼3 cm2), and fixed with 3% glutaraldehyde in a 1.5-ml sterile nalgene capsule. The second sampling technique involved recovery of low temperature hydrothermal fluid directly using a hydrothermal fluid and particulate sampler from a site of active venting in the proximity of iron oxides. Vent fluid was retained after a steady temperature on the intake and outtake probes was reached to ensure that ambient water was not sampled.
The samples were initially processed at sea by transferring collected iron oxides to 1-l autoclaved and acid washed nalgene bottles. Then, approximately 1 ml of wet iron oxide sediment was dispensed into autoclaved and acid washed 1.5-ml nalgene centrifuge capsules, fixed with 3% (v/v) glutaraldehyde and stored at 4°C until examined by ESEM.
For each sample, approximately 0.5 ml of wet sediment was transferred directly from the centrifuge capsule using a 1.5-ml glass Pasteur pipette onto a 1-cm-diameter stainless steel slug. For the bacterial trap specimens, the 3-cm2 section of the iron oxide encrusted mesh was cut into a 1-cm2 section and placed on the metal slug. To look for bacteria in the vent fluid sample, particulate matter was first concentrated by centrifugation and then pipetted in a small drop of water onto the metal slug.
A Philips XL30 ESEM operating at 20 keV was used to examine the samples. The temperature controlled Peltier sample stage was set to 3°C, and the vapor pressure set to 5.4 Torr to give a relative sample surface humidity of 95%. This level of humidity was chosen as it revealed on average the most detail with the greatest amount of sample hydration, allowing the constituents of the iron oxides to remain in their native state. In a few instances, where greater detail of bacterial structures was desired, the vapor pressure was lowered to remove slightly more water and expose more of the individual bacteria.
A Princeton-GammaTech energy dispersive X-ray spectrometer was used to assess the elemental composition of the iron oxide precipitates, and to generate element distribution maps of the samples. Analyses were performed at an accelerating voltage of 20 keV for 100 s, live time.
2.4X-ray diffraction (XRD)
The mineralogy of iron oxides present at Axial Volcano was assessed by XRD. To remove residual seawater sodium chloride, the samples were centrifuged for 5 min at 3000×g and rinsed with 18 MΩ ultrapure water. This cycle was repeated four times. After drying overnight in an oven set at 60°C, samples were thoroughly crushed using a mortar and pestle. Approximately 0.2 g of powdered iron oxide was mixed with 1 ml of acetone in a plastic weighing dish and spread evenly over a glass slide. A Philips XRD system with a PW1830 HT generator, PW1050 goniometer and PW3710 electronics control was run at 40 kV and 40 mA with a lower pulse height distribution (PHD) of 32 and an upper PHD of 80 to aid in reduction of secondary iron fluorescence. Samples were analyzed using Cu K-α radiation, a step size of 0.020 at a rate of 0.85 s/step from a 2θ of 4° to 90°.
The iron oxides recovered from the sample sites at Axial Volcano and South Cleft were orange in color and poorly consolidated as they easily disaggregated when touched by ROPOS’ robotic arm during sampling. They appeared homogeneous in composition and lacked any notable textural differences. The vent fluid was transparent without obvious amounts of particulate matter, even after centrifugation.
The ESEM examination of the iron oxide deposits from all of the sample sites revealed an overwhelming presence of distinct bacterial forms as seen in Fig. 1a. The rod-like structure shown in Figs. 1b and 2a resembles the sheath of L. ochracea[2,6], while the distinctive helical structure in Figs. 1b and 2 resembles the stalk of G. ferruginea[7,24]. The presence of these iron oxidizing bacteria is also consistent with their physiological requirements being satisfied by the aqueous chemical conditions present at sites of low temperature hydrothermal venting . The filamentous, non-helical structures in Figs. 1b and 2 are similar to a novel iron oxidizing PV-1 strain that has been documented by Emerson and Moyer  in iron oxides from the Loihi Seamount, also a site of low temperature hydrothermal venting. This strain appeared similar to G. ferruginea, however, it was found to be a more similar phylogenetically to other neutrophilic obligate iron oxidizing bacteria such as ES-1 and ES-2 .
Some differences were noted among the various iron oxides in terms of their microbial architecture. Those from Fe-City (Fig. 1) were collected from a site of active low temperature venting, and represent a nascent deposit with very little detritus present. The iron oxides from ‘Wall of Caldera’ (Fig. 2) were not adjacent or even in the proximity of active venting and thus represent an older deposit. The older iron oxides contained bacterial sheaths, stalks and filaments that were more heavily encrusted with iron oxide precipitates than the newer deposits (i.e. Fe-City). Additionally, the longer exposure time experienced by the older iron oxides resulted in the accumulation of detrital particulate matter such as siliceous diatoms (Fig. 2b).
Major elements detected by EDS on the surface of the iron oxide encrusted bacterial sheaths, stalks and filaments included Fe, Si, and Cl. The detection of Fe is consistent with the presence of iron oxides and Cl is from seawater. The distribution maps of Fe, Si, and Cl of a sample of bacteriogenic iron oxide can be found in Fig. 3.
The iron oxide's XRD traces were characterized by two broad peaks at 2.6 Å and 1.5 Å for all of the samples analyzed (Fig. 4). These two peaks correspond to two-line ferrihydrite, a poorly ordered iron oxyhydroxide [26,27].
Bacterial structures resembling L. ochracea, G. ferruginea and PV-1 were present on the mesh of the bacterial trap. Some mineral particulate matter was also present that was likely captured from the vent fluid during the year long deployment of the trap. A sheath-like structure was observed in the hydrothermal vent fluid sample whose morphology was consistent with the sheath of L. ochracea.
4.1Fabric and hydrated structure
Traditional electron microscopic techniques (i.e. SEM and TEM) applied to the study of iron oxides require considerable sample preparation that drastically alters the natural hydrated state of specimens. Conversely, ESEM examination does not require dehydration and permits inspection of samples in their fully hydrated native state. While the presence of bacteria in MOR iron oxides has been previously documented [20,28], their wet physicochemical and spatial relationships have not. The most striking ESEM observation of the Axial Volcano samples was that the bacterial-like structures in some of the deposits were so prolific that their fabric appeared similar to the microbial fabric of the dense Beggiatoa mats from the Guyamas Basin . Traditionally these types of iron oxide deposits have been described as inorganic precipitates, however, it may be that a more accurate description of these deposits is microbial mats encrusted with poorly ordered iron oxides, or even iron oxide microbial mats.
The detection of Si on the two-line ferrihydrite encrusted bacterial surfaces, in addition to the presence of siliceous diatoms, is an indication that Si in these iron oxides is composed of both adsorbed and diatomaceous Si. The adsorption of Si in association with bacteria has been proposed to be a result of electropositive amine groups present on bacterial surfaces that may act to bind silicates at near neutral pH . However, it has also been noted that the total number of amine groups present cannot account for the amount of Si precipitation observed . An alternative mechanism to account for the sorption of Si to bacterial surfaces is a metal cation bridge provided by Fe, which is also more consistent with the observations of this study. Once Fe has been adsorbed to the bacterial surface, it is then available to bind aqueous Si. Furthermore, previous studies have noted that elevated concentrations of Fe sorption to the bacterial surface are associated with elevated concentrations of Si sorption [13,31]. It is then more likely that bacterial surfaces play an indirect role in Si sorption, and that this process occurs as a consequence of metal sorption.
Typically iron oxides that are poorly ordered (i.e. two-line ferrihydrite) will precipitate in association with bacteria [11,20]. Subsequent mineral ordering processes of the poorly ordered mineral will lead to increases in crystallinity forming different iron oxide phases such as hematite and goethite [26,27]. The comparison of the iron oxide's XRD traces from the different sample sites at Axial Volcano suggests that bacteriogenic two-line ferrihydrite is a stable phase of iron oxide in this environment. The XRD trace of a mineral will change as crystallinity changes. In the case of two-line ferrihydrite, the two broad lines will become larger, more defined peaks, and new lines will develop that represent developing crystal faces within the mineral. The XRD trace from ‘Fe-City’, where fresh iron oxides were forming around an active vent, was very similar to the XRD trace of the ‘Old Flow’, where it appeared that venting had not been active for some time suggesting that the iron oxides at this site were relatively older. The similarity of two-line ferrihydrite's XRD traces from this study may be an indication that it is a stable phase of iron oxide on the seafloor assuming local geological conditions (i.e. volcanism) remain constant.
The adsorption of Si in particular to the iron oxide encrusted bacterial surfaces may be a mechanism to account for the observed stability of two-line ferrihydrite on the seafloor. It has been documented that the adsorption of Si to poorly ordered iron oxides inhibits subsequent conversion to more crystalline iron oxides such as hematite and goethite [17,27,32]. These iron oxides are termed stable since over shorter time periods than a year (i.e. months), synthetic and/or inorganically precipitated two-line ferrihydrite will convert to more crystalline forms [27,32]. Conversion occurs to a more ordered structure in non-silicified iron oxides by evolving adsorbed water at elevated temperatures. As the water is evolved, small particles can then agglomerate and increase the ordering of the mineral, resulting in increased crystallinity. For silicified iron oxides, adsorbed water is replaced by adsorbed Si in the form of Fe–O–Si bonds. Elevated temperatures will have a reduced effect in evolving adsorbed species such as Si, thereby inhibiting the ordering of two-line ferrihydrite . In addition to silicification stabilizing two-line ferrihydrite, the process may also preserve the morphology of bacterial sheaths and stalks creating bacterial fossils that may survive anywhere from years to geological eras [33,34].
The detection of bacterial-like structures in the bacterial trap and hydrothermal vent fluid may be evidence that L. ochracea, G. ferruginea and PV-1 are present in low temperature mixing zones in the sub-seafloor at Axial Volcano. The exposure of the trap to ambient bottom waters did present the risk that the constituents of the trap were not all from the vent fluid, however, it still provided an opportunity to obtain bacteria from the hydrothermal fluids in an otherwise logistically difficult sample site (i.e. at a depth of 1500 m). Microaerophilic conditions are generated below the seafloor when oxygenated bottom waters encounter hydrothermal fluids that are acidic, reduced and enriched with metals such as Fe(II) . The prevailing microaerophilic conditions in the sub-seafloor can likely sustain low rates of inorganic Fe(II) oxidation, which is permissive to bacterially mediated Fe(II) oxidation and provides an ideal habitat for these types of bacteria .
If iron oxidizing bacteria are prolific in low temperature mixing zones beneath the seafloor, then it is reasonable to suggest that iron oxides will also be abundant beneath the seafloor due to the ability of bacteria to promote iron oxide precipitation [5,8,9,34]. This may be an indication that the sub-seafloor extent of iron oxide deposits is orders of magnitude greater than its surface expression. Consequently, the abundance of iron oxide deposits along MORs may be drastically underestimated along with the role these deposits play in the cycling of elements throughout the planet's oceans.
This research was funded by a Natural Science and Engineering Research Council of Canada-Collaborative Research Opportunities grant and a Hugh E. McKinstry student research grant from the Society of Economic Geologists. The authors of this paper wish to thank Dr. S. Douglas from N.A.S.A.'s Jet Propulsion Laboratory (JPL) in Pasadena, CA, USA for the use of JPL's high resolution ESEM. The bacterial trap used for this study was courtesy of C.L. Moyer and the vent fluid sample was from D.A. Butterfield. We also wish to thank the two anonymous reviewers for their constructive comments on the manuscript and all of the NeMO scientists and crew aboard NOAA's Ronald H. Brown in the summer of 2001.