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

We also obtained a number of discrete filtered fluid samples for molecular analysis using the hot fluid sampler. These filtered samples will be used in total community DNA analysis to determine microbial diversity and phylogeny, FISH (Fluorescence In-Situ Hybridization) to quantify and track certain microbes, and lipid analysis to quantify and determine the physiological state of microbes. We will also perform epifluorescent counts on all preserved fluid samples for microbial enumeration.

This combination of culturing, microscopic, and molecular techniques will help us determine how any changes in the fluid chemistry over the past year may be reflected in the microbial community structure. Additionally, we will continue to explore the microbial ecology of new diffuse sites and high temperature microbes found here at Axial Seamount.

2.3c Microbial Food Webs - UQAM Disciplinary Summary, Kim Juniper

Emphasis this year was placed on the consolidation and expansion of a study of the dynamics of microbial food webs. The overall goal of the study is to understand how the structure of vent food webs add structure and complexity as new vents and vent fields are colonized by increasing numbers of species. Particularly relevant are questions of the importance of vestimentiferan tube worms as a keystone species that create habitat for other organisms, and the relative importance of free-living and symbiotic microbial production as food sources for animals. Suites of faunal samples were collected from new vents in the East Rift Zone as well as from mature and senescent vent sites on adjacent older lava flows. Tissue samples from these organisms will be analyzed for stable carbon and nitrogen isotopes and for lipid biomarkers. Samples of microbial mat, biofilms and particulate organic material collected from each site will be similarly analyzed to permit matching of deposit and suspension feeding animals to their food sources, as well as the identification of trophic levels. The microbial and particulate samples will also be analyzed for ATP and total lipid content, as estimates of microbial biomass available for consumer organisms. Samples were also preserved for a molecular study of the diversity of microorganisms that constitute the animal food supply.

Samples collected came almost entirely from basalt-hosted diffuse vents, with the exception of samples from T&S vent at CASM and 2 collections from the sulfide worm habitat at ASHES. Planned collections of sulfide edifice tube worm communities in the ASHES field were missed because of the weather-related early termination of the dive program.

A vent-field scale study of the development of food webs was initiated at the Cloud site in the East Rift Zone. This study will examine spatial relationships between geological features, the location and intensity of venting and the colonization of individual vents, much as we have previously done for sulfide edifices. This is very much a mapping based project that seeks to discover spatial and temporal patterns that lead to the development of testable hypotheses. Video imagery and photographs collected during two dives are being used to develop a map of the distribution of individual organisms in relation to geological features and venting. This map will be incorporated into a small-scale GIS later this summer, to facilitate quantification of organism distribution and habitat. A short Imagenex survey was also perform during the video transects. We will attempt to integrate the resulting data into the map, despite problems with imprecision of navigation along and between transects. Mapping will be repeated in subsequent years to document changes in venting and community composition.