TABLE OF CONTENTS

Cruise Location Map (Fig.1)................................................................................................ 3

Errata ................................................................................................................................... 4

NeMO '99 Scientific Party................................................................................................... 5

1 CRUISE OVERVIEW........................................................................................................6
Dives on Axial (Fig. 2)......................................................................................................... 6b

SE Caldera SRZ, Northern Vents and Dive Tracks (Fig. 3)................................................. 7

SE Caldera SRZ, Southern Vents and Dive Tracks (Fig. 4)................................................. 8

ASHES Vent Field, Vent Names and Locations (Fig. 5)...................................................... 9

2 DISCIPLINE SUMMARIES..............................................................................................10

2.1 VOLCANOLOGY...............................................................................................................10

2.2 CHEMISTRY...................................................................................................................... 11

2.2a Hydrothermal Fluid Sampling.............................................................................................. 11

2.2b OsmoSampler and OsmoAnalyzer Operations..................................................................... 12

2.2c Gas Sampling........................................................................................................................ 13

2.2c Studies of Dissolved Gases from Hydrothermal Vent Systems............................................ 14

2.3 MICROBIOLOGY..............................................................................................................14

2.3a Microbiological Sampling for Molecular Microbial Ecology Analysis............................... 14

2.3b Hydrothermal Fluid Microbiology....................................................................................... 16

2.3c Microbial Food Webs........................................................................................................... 17

2.4 MACROBIOLOGY............................................................................................................ 18

2.4a Biology of Low Temperature Sites....................................................................................... 18

2.5 HYDROTHERMAL DEPOSITS.......................................................................................20

2.6 ROCK SAMPLING AND PETROLOGIC STUDIES ....................................................20

3.0 Non-ROPOS OPERATIONS............................................................................................. 22

3.1 Mooring Deployments /CTD's /XRF Analysis.....................................................................22

3.2 NeMO'99 Website and Public Outreach.............................................................................. 22

4 NAVIGATION................................................................................................................... 23

4.1 Navigation Overview........................................................................................................... 23

4.2 Final Calibrated Transponder Positions............................................................................... 24

4.3 Vents and Markers Location Table...................................................................................... 24

5 NeMO OPERATIONS....................................................................................................... 27

5.1 1998 Dive Dates and Locations - ROPOS Dives R460 -R480......................................... 27

5.2 1999 Dive Dates, Locations and Tasks - ROPOS Dives R482 - R503............................ 28

5.3 Experiments Deployed and Recovered - 1998 and 1999................................................. 30

5.4 1999 ROPOS Samples - Dives R483 - R503..................................................................... 32

5.5 Dive Map Nomenclature..................................................................................................... 50

5.6 1999 ROPOS Dive Logs - Dives R483 - R503 ................................................................. 51

(dive maps inserted in dive log, where appropriate)

R483 Dive Log..................................................................................................................... 51

R484 Dive Log..................................................................................................................... 54

R485 Dive Log..................................................................................................................... 58

R486 Dive Log..................................................................................................................... 60

R487Dive Log..................................................................................................................... 61

R488 Dive Log..................................................................................................................... 63

R489 Dive Log ................................................................................................................. 78

R490 Dive Log ................................................................................................................. 79

R491 Dive Log..................................................................................................................... 80

R492 Dive Log..................................................................................................................... 96

R493 Dive Log..................................................................................................................... 108

R494 Dive Log..................................................................................................................... 112

R495 Dive Log..................................................................................................................... 130

R496 Dive Log..................................................................................................................... 149

R497 Dive Log..................................................................................................................... 155

R498 Dive Log..................................................................................................................... 165

R499 Dive Log..................................................................................................................... 168

R500 Dive Log..................................................................................................................... 171

R501 Dive Log..................................................................................................................... 179

R502 Dive Log..................................................................................................................... 192

R503 Dive Log..................................................................................................................... 203

Cover: Image of high resolution pencil beam bathymetry (Imagenex). Bathymetry grid cell size is 2 meters. Depths range from 1500 - 1575 meters. Orange/red colors shallowest, blues/purples deepest. Imagenex data were collected by the NOAA Vents Program using ROPOS during NeMO'98 and NeMO'99. Data were supplemented with additional bathymetry collected on the Cleft'99 cruise in September 1999. Bathymetry processing and cartography by William W. Chadwick Jr., with assistance from Robert W. Embley and Susan G. Merle.

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Errata

Dive Logs:

ROPOS dive logs were saved in .html or Excel .csv (comma delimited) files. The .html files were not an option for editing, as they can only be saved as text files, and lose all their rows and columns. The .csv files were a little easier to work with so they were the files edited for the dive logs. (As you may recall, we could not edit the logs at sea.) There was a problem with the .csv dive log files as well. Any double letters or double numbers were absent. I tried to find those errors and correct them, but be aware, especially regarding numbers, as those errors were particularly hard to spot.

Navigation in general:

1999 navigation not as robust as 1998. 1999 nav fixes were often to the west or southwest of '98 fixes. Unfortunately this is not always the case. The farther south we went, the worse the acoustic nav. That is because the southern transponder net could not be calibrated with the ASHES net. (See section 4 for more details.) Be aware of that and use the dive plots as a guide.

Dive Plots:

R492 Missing acoustic nav at end of dive, after JD183 2130. We weren't getting any fixes, and there were no good fixes recorded in the nav logbook to supplement the acoustic fixes. The dive continued for a couple more hours, ending about 250 meters southeast of Bag City. Will fill in last two hours of nav with adjusted ship nav, eventually.

R494 Video went out at JD185 1810, therefore there was no bottom image from that time until ROPOS came up. 1810 official end of dive.

R495 There are gaps in the nav from JD186 1454 - 1640 and 2236 - JD187-0000. We were getting out of the range of the ASHES net transponders. The dive plots just show a straight line connecting the times. Acoustic nav ends on JD187 0115, but the dive continued until 0158. All the nav gaps will be filled with adjusted ship nav, eventually.

R501 No acoustic nav from JD191 2251 - JD192 0521. ROPOS on the deck or in the water column from 0230 - 0530. Need to fill in JD191 2251 - JD192 0230 with adjusted ship nav, eventually. The dive plot shows the ROPOS track from JD192 1411 - 1520, but ROPOS was transiting off the bottom at the time and there was no video.

R494 and R497 Plots state that the nav fixes for the ship are at the stern. That is not accurate. We presume the GPS fixes were referenced to the GPS antennae, about 37 meters fore of the stern. To get actual position at stern one would have to apply a 37 meter lay-back to the ship navigation. (To confuse the matter more: Navigation fixes transcribed in the nav logbook are positions at stern, because we put the pointer in that position on the nav screen and wrote down that fix. More information than you want to know?)

Scientific Participants, Title and Affiliation:

Name Title Affiliation

Robert Embley Chief Scientist/Geologist PMEL

Dave Butterfield Chemist JISAO/PMEL

Bill Chadwick Geologist CIMRS/OSU/PMEL

Kim Juniper Biologist UQ

Michael Perfit Geologist UF

Craig Moyer Microbiologist WWU

Steve Scott Geologist UT

Thomas Chapin Chemist MBARI

Leigh Evans Chemist CIMRS/OSU/PMEL

Jim Gendron Chemist PMEL

Susan Merle Navigation/Geologist CIMRS/OSU/PMEL

Julie Huber Microbiologist/Grad Student UW

Marlene le Bel Biologist/Student UQ

Catherine Charpentier Biologist/Grad Student UQ

Christian Levesque Biologist/Grad Student UQ

Sheryl Roadruck Microbiologist UW

Andy Graham Chemist UW

Jean Marcus Navigation/Biologist/Grad Student UV

Maia Tsurumi Biologist/Grad Student UV

Paul Johnson Navigation/Geologist U Hawaii

Karen Lynch Geologist WWU

John Chadwick Geologist/Grad Student UF

Kevin Roe Chemist JISAO/PMEL

Nicole Nasby NeMO Web/Grad Student OSU

Naaznin Pastakia Geologist/Grad Student UT

Greg Pillette Teacher Beaverton High School

Keith Shepherd ROPOS Team Leader CSSF

Bob Holland Engineer CSSF

Keith Tamburri Engineer CSSF

Kim Wallace Engineer CSSF

Mike Dempsey Engineer CSSF

Craig Elder Engineer CSSF

Ian Murdock Engineer CSSF

Participating Organizations:

PMEL Pacific Marine Environmental Laboratory

UW University of Washington

OSU Oregon State University

CIMRS Cooperative Institute for Marine Resources Studies (NOAA/OSU)

JISAO Joint Institute for the Study of Atmosphere and Oceans (NOAA/UW)

WWU Western Washington State University

CSSF Canadian Scientific Submersible Facility

UF University of Florida

UQ University of Quebec

UT University of Toronto

UV University of Victoria

MBARI Monterey Bay Aquarium Research Institute

1 OVERVIEW OF NeMO'99 - Bob Embley

During the NeMO99 cruise, ROPOS recovered more than 260 geologic, biologic and chemical samples, retrieved 29 experiments and and instruments, and deployed 25 experiments. ROPOS made 18 successful dives totaling 204 hours of bottom time in 21 days on site. Despite major problems with the hydraulic systems on the ROPOS, which caused us considerable downtime, the hardworking ROPOS team and the efforts of the science party were able to recover to make this year's cruise rival the success of NeMO98.

The chemical and biological effects of the 1998 diking event are still apparent on the eastern side of the caldera where the lava flow was erupted. Vigorous hydrothermal venting continues in many places (e.g. Marker 33, Cloud, and Magnesia), whereas there has been reduction or cessation in others. Although the amount of visually apparent microbial activity (such as the white floc in the water column) decreased, "snowblower" activity continued at some vents (e.g. Magnesia Vent) and hyperthermophilic microbes were again found in abundance at most of the diffuse (low temperature) sites, (whose temperature is well below their optimal growth rate). These observations show that there is still enhanced subsurface microbial growth more than a year and a half after the eruption. Of particular interest was the discovery of a hyperthemophile growing optimally at 102C.

Colonization of the new vents continued. Tubeworms had arrived at Marker 33 and were present in greater densities at sites that only contained small patches in 1998 (e.g., Nascent Vent). Analyses of the water samples, biological grabs, and video observation should tell us much about the evolution of the chemical and biological systems on the summit of Axial.

Mapping of the new lava flow and associated hydrothermal system continued in 1999. We located the (probable) southern end of the lava flow about 6 kilometers south of the caldera along a spectacular fissure system that was followed for hundreds of meters. The eruption was entirely contained in the 1 to 2 meter wide fissure until it overflowed its fissure and erupted in the large mound of lava that was found in 1998 at 4552'N. Farther north towards the caldera, the trace of the 1998 eruption became more confused as the slightly older eruptions mixed with the 1998 lava. Several new vents (Bag City, Crevice, Coquille) were found south of Marker 113 (the southernmost point investigated in 1998). The vent fluid sampler recovered 113 water and filter samples from all the (still active) major vents.

The ASHES vent field was only visited on two dives in 1999 because of the early downtime of ROPOS, and part of one dive was again spent at the CASM vent field in the northern part of the caldera.

Active hydrothermal venting was only found on the summit of Axial, probably where the underlying zone of molten lava permanently resides. The summit area contains hydrothermal systems formed during several different volcanic or tectonic (earthquake generated) events, providing a natural laboratory for studying colonization and evolution of these unique chemosynthetic ecosystems.

2 DISCIPLINE SUMMARIES

2.1 VOLCANOLOGY - Robert W. Embley and William W. Chadwick

Volcanology studies during NeMO'99 focused on, 1) continuing our instrumental monitoring efforts, 2) expanding our Imagenex sonar bathymetry of the 1998 eruption site, and 3) extending our mapping of the 1998 lava flow south to the area of the southern SeaBeam depth anomaly at 45 52' (confirmed as a new lava flow during dive R465 last year).

The extensometers that were recovered from Axial's north rift zone during NeMO'98 yielded an interesting data set. The instruments had been in place during the 1998 eruption and recorded a 4 cm contraction at the time of the eruption. This represents deflation of the entire summit area of Axial as magma intruded into the south rift zone, and is consistent with the rumbleometer instruments that recorded dramatic subsidence in Axial caldera. The extensometer and rumbleometer data can be modeled together to estimate the depth of the magma reservoir beneath the caldera and the volume of magma removed during the eruption/intrusion. These results were published in the December 1999 issue of Geophysical Research Letters. After the data was downloaded from the extensometers in 1998, they were re-deployed in the same area, and we recovered these again in 1999. Unfortunately, all 4 instruments failed during the 1999 deployment due to connector leaks and failure of components on the circuit boards. The status of these instruments for future deployments is currently being assessed.

The high-resolution bathymetry that can be collected with the Imagenex sonar was one of the highlights of NeMO'98. We were able to double the area of Imagenex coverage in 1999, mainly to the west of the area mapped in 1998. This helped define the western contact of the 1998 lava flow, and also showed many drain-out channels in the older lava to the west, suggesting that the previous eruptions from this area were more voluminous that the 1998 eruption. The Imagenex map fills a critical observational gap between multibeam bathymetry and bottom observations, and helps us understand what we see from ROPOS. It helps us map out the 1998 lava flow in unprecedented detail, including collapse areas and flow morphologies, puts the locations of vents sites and samples in a meaningful spatial context, and helps us interpret the sequence of events during the eruption. This is the first high-resolution map of a mid-ocean ridge eruption site and we will continue to expand the Imagenex coverage in future years.

During NeMO'98, our geologic mapping efforts were concentrated in the 3 km north of Marker 113. However, we know that the 1998 eruption extended at least 7 km south of Marker 113, so during NeMO'99, we extended this mapping to the south. Observations during dive R495 showed that even the large pillow mound that erupted at 45 52' was fed from a narrow fissure. Within a few hundred meters south of the pillow mound, 1998 lava can be seen filling, but not overflowing, a fissure that is 1-1.5 m wide. This extraordinary observation is essentially the surface outcrop of the 1998 dike that fed the eruption at this location, and the width of the fissure we saw is the minimum amount of seafloor spreading that occurred during this event. Between the pillow mound at 45 52' and Marker 113, the story is more complicated because there are other very young lava flows that are difficult to distinguish from the 1998 lava, except that locally they host clearly pre-1998 vent communities (mainly large, dead tubeworms). We were able to map out the distribution of lavas of at least 4 different ages during dives R492, R493, R494, R495, and R501. However, these dives only covered limited parts of this large area, and additional mapping will be necessary in future years to complete this mapping effort. In collaboration with M. Perfit and his students at the University of Florida, a careful collection of basalt samples was made during these mapping dives to determine if there are distinctive chemical differences between the 1998 lava flow and the surrounding older lavas. These results might help in the mapping effort and have implications for the evolution Axial's magmatic system.

The geologic results from this project also aid in putting into context the remarkable changes observed in the chemical and biologic systems at Axial Volcano since the 1998 eruption. The mapping provides the geologic context for the along-strike variations in the chemical and biologic characteristics of the hydrothermal system. For example, we were able to compare the general pattern of hydrothermal flow from the new eruption with that of the pre-eruptive system (mapped by previous camera tows and dives), suggesting that the conduit system was not dramatically changed by the dike injection and eruption. The north-south pattern appears to be similar, but there was apparently a small East-West displacement between the pre- and post-eruptive hydrothermal systems. The center of maximum hydrothermal flow appears to have remained in roughly the same place. This is also apparently where the maximum diversity in the microbial population is. The "snow blower" vents occur primarily at the northern portion of the system, where the lowest flow rates are located.

2.2 CHEMISTRY

2.2a Hydrothermal Fluid Sampling - David Butterfield

The main goals for fluid sampling for this cruise were 1) re-sample vent fluids from sites sampled last year and expand the sampling to cover a broader range of the eruption area in the SE caldera and rift zone, 2) re-sample diffuse, warm vents and hot vents in the ASHES field, and 3) sample vent fluids from CASM. The vent fluid studies are intended to use chemistry to understand the connections between volcanic activity, hydrothermal heat loss, and microbial activity in hydrothermal systems. Fluid chemistry is also an important measure of habitat conditions for studies of vent fauna colonization and species distribution.

Very few studies to date have focused intensively on the variation in diffuse fluid chemistry within a region. This work may yield new insights into the variability of sub-seafloor processes in a hydrothermal system.

Hot Fluid Sampler

We used the Hot Fluid Sampler (HFS) for the second year during this cruise, after several improvements were made over the proto-type model of last year. The sampler has a lower profile in front of the ROV so that visibility and ability to sample are not severely impaired. The plumbing layout was improved to allow more filters on the instrument. This year we were configured to take 6 piston samples (3 modified with high-vacuum valves for gas sampling), 8 collapsible bag samples with optional in-line filters, and 10 additional filters without water collection. One of the bag samplers was used as a flushing line for two titanium gas-tight samplers so that fluids of known temperature could be sampled by different types of samplers for comparison. On one dive, we can collect 10 discrete water samples for fluid chemistry, 5 water samples for gas analysis, and up to 17 filters for a variety of analytical purposes.

Several types of filters were used. Membrane filters of .45 or .2 micron pore size were used to collect particulate material for: xrf chemical analysis and SEM, particulate elemental sulfur, particulate organic carbon, lipids, and fluorescent in-situ hybridization (FISH) analysis. In addition, "Sterivex" high through-put cartridge filters were used to collect particles for DNA extraction. All of the analytical work done on the filters is designed to help us understand the chemical and microbiological variation among different types of vents. The in-situ filtration capability of the fluid sampler is an important advance over all previous vent fluid sampling instrumentation in that it allows us to concentrate particulate material from large volumes of water for multiple analyses at a given vent site, and it also allows us to separate the dissolved components from the particulate components at the time of venting, thereby eliminating uncertainty about post-sampling particle formation within the water sample containers. In many instances, we collect both filtered and unfiltered water from the same site for comparison. Overall, we have an excellent set of samples for fluid chemistry, gas chemistry, particle chemistry, and microbiology.

On-board fluid analysis:

Kevin Roe was the principal fluid analyst on this cruise, and carried out analysis for pH, alkalinity, hydrogen sulfide, dissolved silica, and ammonia. Most of our chemical analysis occurs on shore. We measured refractive index of the hotter vent fluids to estimate their salinity or chlorinity. Dave Butterfield analyzed particulate elemental sulfur by colorimetry on a subset of samples.

We were fortunate to have Andy Graham on board. Andy received cuts of most of the fluid samples for methane and hydrogen analysis by gas chromatography. In many cases, vent fluid samples arrived on deck containing a gas phase and a liquid phase. When this occurred we measured the volume of the liquid and gas portions and Andy analyzed both for gas content. In this way, the fluid sampler appears to be superior to the traditional titanium major samplers because it has much better gas retention capability, and the samplers are transparent, allowing us to separate the gas portion from the liquid. We cannot rule out some gas loss through the check valves, but comparison of the gas results from different samplers will help to evaluate this.

Initial results:

HFS was deployed on 2 short aborted dives and 3 complete dives in the 1998 eruption area, plus one complete dive at ASHES. HFS proved to be quite efficient. For example, on the first HFS dive, we had only 30 minutes of bottom time at marker 33 and took 5 samples (2 water chemistry, one gas chemistry, and two filters). For the entire cruise, we collected 43 vent fluid samples for water chemistry with HFS, 13 gas piston samples, and over 50 filters. In addition, titanium gas-tight samples were collected on many dives (see Leigh Evans report) and these will be analyzed for major element chemistry.

We have not yet processed the raw data from the last two dives, but the initial results appear similar to last year. Relative to the CoAxial eruption area, where there were large changes in composition and a decay in heat output one year after the eruption, we saw little change on the seafloor in the eruption area. Diffuse venting continued in many areas. The maximum measured temperature at marker 33 is higher than last year (78 versus 55 degrees). Looking at the ratio of hydrogen sulfide to silica (the ratio is only slightly sensitive to the amount of entrained seawater during sampling) it appears that the samples from throughout the eruption area this year are just slightly lower in sulfide relative to silica. Methane appears to be slightly higher this year than last year. We cannot say anything about the changes in salinity until we do high-precision analysis on shore.

2.2b OsmoSampler and OsmoAnalyzer Operations - Thomas Chapin

Changes in the chemical composition of hydrothermal effluent after a tectonic-volcanic event have been documented (e.g., Baker et al., 1987, 1998; Butterfield and Massoth, 1994; Von Damm et al, 1995; Massoth et al., 1995; Massoth et al., in press; Wheat et al., to be submitted) and a conceptual model has been developed that theorizes the chemical evolution of venting fluids (Butterfield et al., 1997). However, the timing of these changes is uncertain. To date observations of temporal variability in the chemical composition of hydrothermal fluids has relied on repeated submersible operations and the collection of discrete samples. While this technique provides some temporal constraints, a continuous water sampler or analyzer allows one to collect more samples with limited need for costly submersible operations. Our goal for this cruise was to deploy two short-term (two weeks) and two long-term (one year) continuous sampling systems to provide temporal constraints for observing hourly to daily and weekly to monthly chemical cycles in the hydrothermal effluent. Data from these samplers and their comparison to samples collected using traditional discrete sampling techniques will allow us to determine the temporal scale of chemical change in the hydrothermal effluent as the hydrothermal system evolves and may provide constraints for understanding the physical and chemical conditions at depth and the path for fluid circulation.

Two sampling systems were deployed, OsmoSamplers and OsmoAnalyzers. OsmoSamplers are continuous water samplers that use the osmotic pressure that is created across a semi-permeable membrane by solutions of differing salinity (Theeuwes and Yum, 1976; Jannasch et al., submitted). This pressure drives water across the membrane at a speed that is dependent on the surface area of the membrane, type of membrane, salt gradient, and temperature. An excess of salt is maintained on one side of the membrane, thus only temperature affects the flow of water in the sampler. Pumps in an OsmoSampler are used to continuously draw sample through a small bore (0.8 mm id) tubing that is attached to a 40-cm-long T-handle. An additional pump was used to add acid to the sample stream in most of the OsmoSamplers. A 1.5-m-long section of tubing separates the sample intake from the pump to allow the pump to be placed in an area void of hydrothermal influence and thus minimizes temperature (pump rate) fluctuations. A temperature recorder with a resolution of 0.0018°C is attached to the T-handle to monitor the same water that is being collected by the OsmoSampler. Chemical data are obtained by retrieving the sampler, cutting the sample tubing into sections, extracting the seawater, and analyzing the seawater for chemical species of interest. Time-stamps for individual samples are determined assuming a uniform temperature at the pump that translates into a uniform rate of pumping.

OsmoAnalyzers, in contrast to OsmoSamplers, use osmotic pumps to deliver reagents into a sample stream for in situ analysis (Jannasch et al 1994). An iron OsmoAnalyzer was deployed at Marker 33 to continuously measure Fe at 15-minute intervals over the next 6-9 months.

Two long-term acid addition OsmoSamplers, deployed from the NeMO 1998 September cruise were recovered on this cruise. These samplers, one at Marker 33 and one at Milky vent, continuously collected sample for 9 months providing 163 0.5-mL samples. Analysis for major elements and trace metals will be performed later in our laboratory. Milky vent started at 9.5 C but dropped to 3.6 C by the time the OsmoSampler was recovered. There was no visible floc venting from Milky and the low temperatures at the end of the deployment indicate that the diffuse vent had died out. Marker 33, on the other hand, continued to vent hydrothermal fluids up to 80 C and was quite vigorous. The NeMO-98 long term acid addition OsmoSamplers deployments appear to have been a success and will provide one of the first long-term continuous records of the chemical signature of hydrothermal fluids.

During NeMO-99, four long-term OsmoSamplers were deployed. Three OsmoSamplers were deployed at the Marker 33, in the hottest section with temperatures up to 80 C. The OsmoSamplers consisted of one regular acid addition, one bio-OsmoSampler which has a sodium azide biocide to prevent bacterial growth, and a long term Cu-OsmoSampler which will collect samples for gas analysis. Another long-term acid addition OsmoSampler was deployed in 70 C water at Magnesia vent, just north of Milky vent. Tremendous clouds of white flocculent material were coming out of Magnesia and it looked like a snowstorm.

A 2-week record high temperature record from the Hell vent was recovered. Unfortunately the deployment of a long-term acid addition OsmoSampler and temperature probe was not successful.

References:

Baker, E. T., G. J. Massoth, and R. A. Feely. 1987. Cataclysmic hydrothermal venting on the Juan de Fuca Ridge. Nature, 329, 149-151.

Baker, E. T., G. J. Massoth, R. A. Feely, G. A. Cannon, and R. E. Thomson. 1998. The rise and fall of the CoAxial hydrothermal site, 1993-1996. J. Geophys. Res., 103, 9791-9806.

Butterfield, D.A., and G. J. Massoth. 1994. Geochemistry of north Cleft segment vent fluids: Temporal changes in chlorinity and their possible relation to recent volcanism. J. Geophys. Res., 99, 4951-4968.

Butterfield, D. A., I. R. Jonasson, G. J. Massoth, R. A. Feely, K. K. Roe, R. E. Embley, J. F. Holden, R. E. McDuff, M. D. Lilley, and J. R. Delaney. 1997. Seafloor eruptions and evolution of hydrothermal fluid chemistry. Phil. Trans. R. Soc. Lond. A, 355, 369-386.

Jannasch, H. W., K. S. Johnson and C. M. Sakamoto. 1994. Submersible, osmotically pumped analyzers for continuous determination of nitrate in situ. Anal. Chem. 66, 3352-3361.

Jannasch, H. W., C. G. Wheat, M. Kastner, and D. Stakes. 1998. Long-term in situ osmotically pumped water samplers. Deep Sea Res., submitted.

Massoth, G. J., E. T. Baker, R. A. Feely, D. A. Butterfield, R. E. Embley, J. E. Lupton, R. E. Thomson, and G. A. Cannon. 1995. Observations of manganese and iron at the CoAxial seafloor eruption site, Juan de Fuca Ridge. Geophys. Res. Lett., 22, 151-154.

Massoth, G. J., E. T. Baker, R. A. Feely, J. E. Lupton, R. W. Collier, J. F. Gendron, K. K. Roe, S. M. Maenner, and J. A. Resing. 1998. Manganese and iron in hydrothermal plumes resulting from the 1996 Gorda Ridge Event. Deep Sea Res., in press.

Theeuwes, F., and S. I. Yum. 1976. Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations. Ann. Biomed. Eng., 4, 343-353.

Von Damm, K. L., S. E. Oosting, R. Kozlowski, L. G. Buttermore, D. C. Colodner, H. N. Edmonds, J. M. Edmond, and J. M. Grebmeier. 1995. Evolution of East Pacific Rise hydrothermal fluids following an oceanic eruption. Nature, 375, 47-50.

Wheat, C. G., H. W. Jannasch, F. J. Sansone, J. N. Plant, and C. L. Moyer. 1998. Hydrothermal Fluids From Loihi Seamount After the 1996 Event: A Year of Change Monitored With a Continuous Water Sampler. Earth Planet. Sci. Lett., to be submitted.

2.2c Gas Sampling - Lee Evans

The primary goal of gas sampling during the NeMO '99 expedition was direct sampling of vent fluids by way of Titanium Gastight bottles and modified pistons from the PMEL Hot Fluid Sampler (HFS). Approximately 30 useful samples were gathered and their available gas contents extracted and sealed in glass ampoules for chemical analysis. Analyses include helium isotopes, hydrogen and methane.

As with 1998's samples, the geographic coverage of sampling included the east side of the caldera along the region of the 1998 lava flow, Ashes vent field on the west side and Casm vent field to the north. Time series measurements will be possible at about 5 vent sites. The coverage of diffuse vent samples was extended southward on the east side in the direction of the vestige of the eruptive fissure.

This year's method modifications present a significant improvement as compared with those used to gather samples in 1998. At least some of 1998's collection were a bit more dilute than what is desirable. Both the plumbing scheme for titanium gastight bottles and the sample integrity of the gas piston samplers (HFS) were improved.

2.2d Studies of Dissolved Gases from Hydrothermal Vent Systems - Andy Graham

The main focus of our lab is the study of dissolved gases from hydrothermal vent systems. For this cruise I brought a gas chromatograph on board and analyzed fluid samples from the hot fluid sampler, suction sampler and a niskin bottle mounted on ROPOS. The main gases that I analyzed were dissolved hydrogen and dissolved methane. These gases are important in the hydrothermal vent community because certain microbes can oxidize these gases and use them as an energy source. Over 50 samples were analyzed ranging from 300 ^oC Inferno vent to 4 ^oC Magnesia vent. From an initial glance the data vent such as Marker 33 and Virgin Mound still appear to contain high concentrations of both hydrogen and methane. Further analysis and a comparison to last year's data will occur.

2.3 MICROBIOLOGY

2.3a Microbiological Sampling for Molecular Microbial Ecology Analysis

Western Washington University, Biology Department: Craig L. Moyer & Karen Lynch.

Introduction

One of the greatest challenges in microbial ecology is the accurate identification and description of microbial populations within their respective communities. This information is central to determining the extent of global microbial diversity, which remains the least understood of all the biological size classes. To address this challenge, molecular biological techniques using small-subunit ribosomal RNA (SSU rRNA) gene sequences have been applied to describe the structure and diversity of different microbial communities. The current endeavor is to examine specific habitats with known biogeochemical characteristics (e.g., S, Fe, Mn) to learn more about the dominant microorganisms residing therein. The focus of this study at Axial Volcano is to estimate the microbial community structure and diversity to assess the degree of commonality and uniqueness among local hydrothermal vent habitats, (i.e., vent-associated sediments, free-living microbial mats, microbes associated with subsurface floc-ejecta), and to also compare these results with distal hydrothermal vent habitats. This study will also allow for the enhanced development of a comprehensive global perspective regarding the diversity of deep-sea microbial communities.

Selective enrichment culture has severe limitations as an approach to the cultivation of naturally-occurring microorganisms. The majority (typically >90-99%) of microbes in nature have not yet been cultivated using traditional techniques. Consequently, it is very unlikely that collections of microbial isolates are representative of in situ diversity and community structure. Furthermore, because relatively nutrient-rich media are generally used for isolations, "weedy" or opportunistic microorganisms may be selected rather than those dominant in the natural community. The approach, herein, is to ascertain a microbial community's primary members through molecular (i.e., cell component) means and then to attempt to further characterize their respective phylogeny or natural history. Obtaining a better representation of microbial community structure and diversity is crucial to aspects of microbial ecology where Bacteria and Archaea interact with one another and with their environment, e.g., global biogeochemical cycling of matter, succession and disturbance responses, predator-prey relationships, and trophic-level interactions. These lessons can then be used to focus enrichment culture techniques towards ecologically significant taxa. This approach has been successfully used to isolate the dominant iron-oxidizer bacterial taxon found within the microbial community at hydrothermal systems located at Loihi Seamount, North Gorda Ridge, and other habitats (Emerson and Moyer, 1997; unpublished results).

Cell component analyses provide a culture-independent means of investigating microorganisms as they occur at hydrothermal vent systems (Moyer et al., 1994;1995; 1998). While several types of cell components have been analyzed, the SSU rRNA molecule offers an amount and type of information that makes it one of the best culture-independent descriptors or biomarkers of microorganisms. In recent years a detailed theory of evolutionary relationships among the domains Bacteria, Archaea and Eucarya has emerged from comparisons of SSU rRNA "signature" sequences. For example, each SSU rRNA gene contains highly conserved regions found among all living organisms as well as diagnostic variable regions unique to particular organisms or closely related groups. Additionally, each SSU rRNA gene contains about 1,500 nucleotides of sequence information that can be obtained and utilized to differentiate among closely-related and distantly-related groups of microorganisms. This type of molecular approach allows the autecology of microorganisms to be studied whether or not they can be been cultivated (Moyer et al., 1996). In addition, the phylogenetically described taxa or "phylotypes" can be placed in a synecology context through the examination of SSU rRNA clone libraries generated from a microbial community and habitat diversity can be analyzed through rarefaction (Moyer et al., 1998). These features make SSU rRNAs particularly useful for studies of molecular microbial ecology, where a broad and unknown range diversity of microorganisms is likely to exist. Currently, over 10,000 SSU rRNA sequences from both cultured isolates and environmental phylotypes have been made available for study through the Ribosomal Database Project at NSF's Center for Microbial Ecology at Michigan State University.

Experimental Design and Methods

Shipboard Processing and Storage of Samples

A dual approach was used for microbial sampling. First, a "slurp" gun suction device was be used in combination with a rotating rosette of sample bottles to "vacuum" and capture free-living microbial mats from the surface of various hydrothermal vent habitats. Slurp gun samples were successfully obtained from the East-Side of Axial at (1) Marker #33 Vent, (2) Markers N6 & N4 aka Cloud Vent, and (3) Magnesia aka Whiteout Vent. We also began to investigate the phenomena of the "bag creature" this year with slurp samples collected on the East side of Axial at both Axial Gardens (Marker #113) and from a new site entitled Joystick Vent (Marker #42). This is a characteristic jelly-like residue, which looks to be composed of complex polysaccharide globules that form in and around low temperature diffuse flowing vents in conjunction with microbial mats. No suction samples used for microbiology were obtained from the vicinity of the ASHES vent area this year.

Second, the deployment and recovery of microbial traps using glass wool as a substrate for microbial growth. Microbial traps were constructed using a cluster of three 3" sections of 4"o.d. plexiglass tubing, surrounded top and bottom by a 202 µm nylon mesh (Nytex) to exclude macrofauna and meiofauna grazing. These were placed directly into diffuse vents and were used to collect colonizing microorganisms in an effort to examine community succession. These were deployed with the idea of attempting a time-series with both short-term (days) and long-term (annual) time scales. This objective was successfully achieved with long-term recoveries from last year's NeMO98 made at both Marker #33 and Cloud Vent (Marker N4), short-term recoveries from deployments made this year were also made at Marker #33, and Cloud Vent (Marker N4) on the East-side of Axial Volcano. New long-term deployments again were made at both of these two East-side sites. Unfortunately, only a single successful recovery from the ASHES Vent Field was made. This occurred at Gollum Vent, where two long-term deployed microbial traps were heroically recovered (in spite of the onset of foul weather conditions) and a fresh trap was deployed. Short-term recoveries from the ASHES area remains illusive, but may again be attempted next year, in addition to attempting continued long-term recoveries from each of the following locations where microbial traps have been previously deployed: Gollum, ROPOS, Hillock, Mushroom Vents.

Microbial samples collected were each independently processed. Microbial biomass preservation was achieved by quick-freezing in liquid nitrogen and storing on dry ice until return to the laboratory. These samples will be used for the direct extraction of nucleic acids. A series of sub-samples were also (i) cryo-preserved (again using liquid nitrogen quick-freezing) with 40% glycerol, and (ii) aliquots were stored at 4C, both for enrichment culture selection. Another series of sub-samples was fixed with 2.5% EM grade glutaraldehyde for examination with SEM and epifluorescence microscopy.

Laboratory Processing and Molecular Biological Analysis

Initially, all samples will be examined by epifluorescence microscopy in an effort to ascertain biomass estimates and examine morphological diversity. A subset of these will also be examined through SEM and an analysis of extractable lipids, which provides an estimate of microbial biomass and initial clues into community structure. The overall molecular biological strategy used will be essentially that of Moyer et al. (1994, 1995; 1998) with a few technical and logistical improvements. The first step will be the efficient and direct extraction of high molecular weight nucleic acids from quick-frozen samples. This will be followed by PCR amplification of SSU rDNAs using previously defined conditions to maximize the equal representation from each population contained within a respective community. The concept is to proportionally amplify or make several copies using the total genomic DNA from a natural community serving as the template for oligonucleotide primers that are complementary to universally conserved SSU rDNA sequence positions. Representative SSU rDNA amplification products are cloned generating a clone library. Clone libraries will then examined through the use of Amplified Ribosomal DNA Restriction Analysis or ARDRA and by using rarefaction as a metric for organismal diversity (Moyer et al., 1998). This approach, using tetrameric restriction enzymes, has been shown to detect >99% of the taxa (i.e., phylotypes) present within a model dataset with maximized diversity (Moyer et al., 1996). SSU rDNA sequences will also be subjected to phylogenetic analysis (using distance matrix and maximum likelihood algorithms) to estimate the affiliated ancestral lineage for each dominant community member thereby yielding clues as to their respective evolutionary history and potential physiology.

References:

Emerson, D., and C. L. Moyer. 1997. Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH. Appl. Environ. Microbiol. 63:4784-4792.

Moyer, C. L., F. C. Dobbs, and D. M. Karl. 1994. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl. Environ. Microbiol. 60:871-879.

Moyer, C. L., F. C. Dobbs, and D. M. Karl. 1995. Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl. Environ. Microbiol. 61:1555-1562.

Moyer, C. L., J. M. Tiedje, F. C. Dobbs, and D. M. Karl. 1996. A computer-simulated restriction fragment length polymorphism analysis of bacterial SSU rRNA genes: effacacy of selected tetrameric restriction enzymes. Appl. Environ. Microbiol. 62:2501-2507.

Moyer, C. L., J. M. Tiedje, F. C. Dobbs, and D. M. Karl. 1998. Diversity of deep-sea hydrothermal vent Archaea. Deep-Sea Res. II. 45:303-317.

2.3b Hydrothermal Fluid Microbiology - Julie Huber and Sheryl BoltonWe returned to sea this summer to continue our research on microbial communities in diffuse fluids on Axial Seamount. Along with Dave Butterfield, we are trying to quantify the diversity of microbes and their metabolisms in relation to the chemistry of the diffuse fluids over time.

We have successfully cultured mesophiles, thermophiles, and hyperthermophiles from all diffuse fluids collected during this cruise. We have over 100 positive enrichments (which require confirmation on land) at temperatures ranging from 23 °C to 110 °C in a wide variety of media, mostly anaerobic. One very exciting find was the culturing of a microbe at 110 °C from Marker 33. This hyperthermophile (or group of hyperthermophiles) was successfully transferred six times while at sea. Currently, the highest known upper temperature limit for growth of a living organism is 113 °C by a sulfur-dependent hyperthermophilic Archaea called Pyrolobus. Our microbe, growing anaerobically with elemental sulfur, was isolated from ~70 °C fluid at Marker 33. We also found that it is producing large amounts of hydrogen and some carbon dioxide, as analyzed on the gas chromatograph by Andy Graham. The fact that we have found a microbe (or group of microbes) growing at such a high temperature, yet isolated from fluids much below its temperature of growth, strongly suggests that there is a hotter subsurface environment that these microorganisms are growing and thriving in. By studying this exciting microbe, we hope to learn more about the metabolic wonders of life at high temperature and the limits on life.

Additionally, quantitative enrichments (MPNs, Most-Probable-Number technique) were performed at a variety of temperatures from several sites. The table below contains the 95% confidence interval for the abundance of microorganisms that grow in the given media, given in microbes/liter. These data are preliminary and must be confirmed by microscopy on land.

Sample Site	Incubation Temp (°C)	Fluid Temp (°C)	Media Type	Microbes/L
Marker 33	90	78.0	Anaerobic, high organics	3000-96,000
Marker 33	90	78.0	Anaerobic, low organics	600-8800
Magnesia	55	5.6	Anaerobic, low organics	80-2400
Bag City	90	23.4	Anaerobic, high organics	300-7600
Bag City	90	23.4	Anaerobic, low organics	80-2400
15m N of Nascent	90	25.8	Anaerobic, high organics	60-880
Cloud N6	23	20.0	Aerobic, low organics	>48,000
Gollum	90	22.3	Anaerobic, high organics	In Progress
Marshmallow	90	71.8	Anaerobic, low organics	In Progress

5.4 1999 ROPOS Samples - Dives R483 - R503