2.3 MICROBIOLOGY

2.3a Microbiological Sampling for Molecular Microbial Ecology Analysis. Western Washington University, Biology Dept.- Craig L. Moyer & Jeff Engebretson.

Introduction

A challenge to microbial ecologists is the accurate description and identification of microbial populations within their respective communities. Because hydrothermal vent communities are so obviously dependent on their respective microbial populations, understanding the abundance and diversity in the vent environment is very important for integrated biological studies. Furthermore, scientists have recently hypothesized that if life were to exist on other global systems it would likely be analogous to hydrothermal vent systems here on earth. Perhaps modern communities at hydrothermal vents are evolved directly from the first microbial communities on early earth. To address the 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 assess the microbial community structure at local hydrothermal vent habitats (i.e., vent-associated sediments, free-living microbial mats, microbes associated with subsurface floc-ejecta). Succession in the microbial community will also be analyzed with the data collected via Nemo'98, '99 and '00. 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 study of naturally-occurring microbial populations. The majority (typically >90-99%) of microbes in nature have not yet been cultivated using traditional techniques. A further exacerbation is that "weedy" or opportunistic microorganisms preferentially grow in the relatively nutrient-rich media used for isolations. Consequently, it is very unlikely that collections of microbial isolates are representative of in situ diversity and community structure. 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. These lessons can then be used to focus selective enrichment culture techniques toward related, previously-identified 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). The focus of our study will be to analyze the SSU rRNA gene. Each SSU rRNA gene contains regions which are highly conserved (the same from one organism to another) and regions that are variable. The variable regions will allow us to not only discern one organism from another but also to identify how closely related they are. 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). The phylotypes at Axial volcano will be analyzed with other microorganisms in an evolutionary context by the use of an online database called the Ribosomal Database Project (RDP) at NSF's Center for Microbial Ecology at Michigan State University. The RDP currently contains over 15,000 partial and whole SSU rRNA sequences.



Experimental Design and Methods

Shipboard Processing and Storage of Samples

Microbial sampling occurred by three approaches. First, a suction device was used to obtain free-living and microbial mats from the surface of various hydrothermal vent habitats. The samples were contained in a sample bottle by the use of 202 m nylon mesh. During NeMO 2000, suction samples were obtained from Marker 33, Cloud vent, Bag City, Marker 113, Hillock/Phoenix vent, Hell vent and an Fe-hydride site at the base of the west wall of the ASHES vent field.

Second, the deployment and recovery of microbial traps at diffuse-flow vents. Microbial traps were constructed using glass wool as a substrate for microbial growth placed inside a cluster of three 3" sections of 4" o.d. plexiglass tubing, surrounded top and bottom by a 202 m nylon mesh (Nytex) to exclude macrofauna and meiofauna grazing. These were deployed with the idea of achieving a time-series on both a short-term (days) and long-term (annual) scale. The objective was successfully achieved with long-term recoveries made at Marker #33, Gollum, Ropos and Mushroom vents. Short-term recoveries were also made from Marker 33 and Cloud vent. Traps deployed for long-term recovery were at Marker #33, Gollum and Ropos vents. Overall, Marker 33 and Cloud vent are the sites where we will be getting thorough time-series data as both short- and long-term recoveries were made at these sites from NeMO'98 through NeMO'00.

A third method of collection was with the use of an osmotic pump which was deployed at Marker #33 during NeMO '99. A small diameter (1.2 mm) capillary was used to continuously collect free-floating bacteria for approximately one year. Preliminary results show that approximately 0.8 mls of diffuse-flow vent fluid were collected per day.

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 subsamples were also I) cryo-preserved (again using liquid nitrogen quick-freezing) with 40% glycerol, and (ii) aliquots were stored at 4 C, both for enrichment culture and selection. Another series of subsamples were fixed with 2.5% EM grade glutaraldehyde for examination with SEM and epifluorescence microscopy.



Laboratory Processing and Molecular Biology Analyses

Initially, all samples will be examined by epifluorescence microscopy 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 DNA-analysis 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 polymerase chain reaction 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 from the total genomic DNA with the use of oligonucleotide primers that are universally conserved among all eubacteria. Representative amplification products are then cloned in order to construct a clone library of the community's SSU rDNA genes. Clone libraries will then be examined through the use of Amplified Ribosomal DNA Restriction Analysis (ARDRA) and by using rarefaction as a metric for organismal diversity (Moyer et al., 1998). This approach, using tetrameric restriction endonucleases, has been show to detect >99% of the taxa (i.e. phylotypes) present within a model data set with maximized diversity (Moyer et al., 1996). SSU rDNA sequences will also be subjected to phylogenetic analyses (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 of bacterial SSU rRNA genes: efficacy 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 - Mausmi Mehta and Julie Huber, University of Washington

Because so little is known about the subsurface biosphere, scientists use a variety of "windows" to peer into this world, such as looking for evidence of microbial activity in deep sea basalts or examining diversity in hot springs on land. Our work at Axial uses diffuse fluids as a window into the subsurface and combines molecular, culture, and microscopic techniques to characterize and quantify microorganisms in diffuse fluids and examine their link to fluid chemistry in the subsurface biosphere at Axial Seamount over both time and space. This research will allow us to better understand the interactions between microbes and hydrothermal fluids, as well as examine the possibility of subsurface biotopes and how these biotopes may be related to one another, if at all, at Axial Seamount.

Using the hot fluid sampler, we have collected over 15 samples of diffuse fluids from the southeast rift zone and the ASHES vent field, as well as some higher temperature fluids from ASHES. We attempted to re-sample vents we have previously sampled in 1998 and 1999 to continue our time series study of changes in the microbial community and chemistry of fluids at Axial after the diking-eruptive event in January 1998. The diffuse fluids were used for culturing mesophiles, thermophiles, and hyperthermophiles on board in a variety of media, mostly anaerobic. The fluids were also preserved for microscopic counts on land, and we once again performed a number of quantitative enrichments (MPNs, Most-Probable Number technique) to monitor the presence of hyperthermophiles at certain vents over time. Preliminary results indicate there are still high numbers of anaerobic, heterotrophic, sulfur-using, hyperthermophiles at Marker 33, Marshmallow, and Cloud N6 vents, as well as significant numbers of hyperthermophilic methanogens at these vents and Gollum vent. The high numbers of cultures obtained from Marshmallow is especially surprising due to the high temperature of the fluid, with a maximum measured temperature of 150 C.

With the hot fluid sampler, we were also able to obtain a variety of in-situ filtered fluid samples for DNA extraction to look at microbial diversity, FISH (Fluorescent In-Situ Hybridization) to quantify and track certain microbes within and between vents, and lipid analysis to quantify and determine the physiological state of microbes. These filtered samples will be used in the time series study, as well as some new projects we are starting at Axial Seamount. One new aspect of our work is the search for nitrogen-fixing microorganisms that could be providing a source of fixed organic nitrogen to the rest of the vent community. The nitrogen cycle at hydrothermal vents has not been thoroughly investigated, but nitrogen isotope data indicate that nitrogen-fixation could be occurring at hydrothermal vents; and the fact that several thermophilic methanogens that have been isolated from vents possess the nitrogen fixation gene suggests that nitrogen fixation may be an important process here. One of the techniques that are being used in this search is an in-situ nitrogen fixation assay that involves using acetylene instead of nitrogen gas as the substrate for nitrogenase - the enzyme responsible for fixing nitrogen. Another method is to amplify and sequence the nitrogenase gene from the filtered fluid samples in order to get an estimate of the importance and diversity of nitrogen-fixing microorganisms at hydrothermal vents. These new methods, combined with our time series work, will allow us to better understand the microbial ecology of hydrothermal fluids.

Due to its variety of hydrothermal fluids and our ability to sample it over time, Axial provides an excellent window into the subsurface environment. By using a combination of molecular and culture methods to examine the unique microorganisms living in the subsurface, as well as working with geologists and chemists, we hope to better constrain the subsurface environment at Axial Seamount.