Thompson, Janelle R.

The Microbial Ecology of Yellowstone Hot Springs

How do we define the habitats characteristic of geothermal environments?

Habitats are structured by the environmental gradients that determine the ability of an organism to meet its basic needs.

-Chemical gradients: pH, redox potential, carbon and nutrients
-Light gradients: intensity, quality
-Moisture gradients: exposure to desiccation
-Thermal gradients: temperature tolerance
-Mechanical gradients: hydrodynamics (Re), frequency of physical disturbance
-Biological gradients: competition, predation, mutualism

The positioning of organisms along the environmental gradients that define their habitats can be described by a classical model of 'n'-dimensional niche-space, where n is the number of relevant gradients in the environment (Hutchinson, 1969). An organism's ecological niche is its mode(s) of survival. Ecological models suggest that only one organism ('the competitive dominant') will occupy a given niche in a given habitat, so, the more 'niches' possible in a habitat, the higher the potential diversity.

With the Niche space model in mind, we can observe the effects of temperature, pH and light as three variables that visibly delineate the distribution of species in Yellowstone geothermal environments.

Hyperthermophiles, organisms that grow at temperatures above 80C, thrive in the high temperature pools. Their biochemical adaptations to high temperature environments include thermostable enzymes and membranes and unique enzyme systems to prevent and repair heat-induced damage. These organisms obtain energy by oxidizing the reduced sulfides, hydrogen and metals present in the geothermal fluid (lithotrophy). For example:

H2 ---> H2O + 2e-
H2S ---> SO42- + 6e-
Fe(II) ---> Fe(III) + 1e-

The electrons liberated by oxidation of the reduced compounds are either routed through biochemical reactions generating energy, ultimately being placed on a terminal electron acceptor (like oxygen or nitrate), or they are used to reduce CO2 to the oxidation state of biomass (biomass is ~50% C by dry weight).

Sulfate and ferric iron, produced by sulfide- and iron-oxidizing microbes, both add acidity to an environment. Many hot spring environments are characterized by their low pH due in part to the activity of acid-producing hyperthermophilic microbes (sulfur and iron compounds are also oxidized abiotically). Many hot spring hyperthermophiles are also acidophilic (requiring acidic conditions to grow). As temperatures fall below 75C, the cyanobacteria become apparent, forming brightly colored mats. Cyanobacteria cannot survive in environments with pH's below ~4. However, other photosynthetic microbes, such as species of eukaryotic algae, can survive in extremely acidic environments.

Photosynthesis at Dragon's Mouth: Mud Volcano Area

The hot springs in the Mud Volcano area are rich in geothermal sulfides and reduced metals, evidenced by their rotten egg smell, and the cloudy gray water caused by suspended precipitates of FeS(s). Sulfide and metal oxidizing microbes create a low pH environment where cyanobacteria cannot survive. However, the dark-green eukaryotic algea (Cyanidium cladarium) can survive at these low pH's (to pH 0!)

In some cases we see a pink-tinted rock emerging from the FeS (s) ladden water. At the high-water level a band of dark-green algae is apparent. Beneath the high water level, photosynthesis should be inhibited by the limited availability of light. Atop the rock, where the environment might not be as acidic, a lighter green shade may indicate growth of cyanobacteria. The zonation of dark green algae in the splash-zone of the Dragon's Mouth was a large-scale feature spanning the perimeter of the spring.

In geothermal springs zonation of organisms can be observed along a horizontal temperature gradient from the hottest source waters through the cooler run-off streams.

Gradients in a Hot Spring: West Thumb Geyser basin

At the West Thumb geyser basin some of the patterns described above were evident. In this image the clear pool in the back is presumably the hottest; a suitable habitat for hyperthermophiles. As water temperatures cool, pigmented microbial mats are evident at the periphery of this pool, and in the shallower fore-ground pools. The run-off stream, also in the fore-ground (right by the yellow clump of flowers and the tall blade of grass), looks like it harbors a mixture of different microbial mat communities. There is an intense dark green community that seems to parallel the source flow of the run-off stream, while the orange community seems to exists in the more dispersed flow of the stream and at the periphery of the pools.

Microbial Mats

The brightly colored microbial mats are a conspicuous feature of many Yellowstone geothermal vents. These mats are composed of communities of microorganisms embedded within an extracellular matrix of polysaccharides and proteins. Light is a dominant vertical gradient in these microbial mat communities. Photosynthetic microbes (like plants) are specialized to thrive under differing light intensities and qualities. Deeper in the microbial mat, low light adapted species will thrive, while at the surface of the mat species adapted to high light conditions will predominate. Carotenoid pigments (like those present in carrots) protect the cells from harmful radiation (UV) during seasons with high light intensities. Carotenoid pigments, and other light-absorbing pigments like chlorophyll give mats their characteristic colors. When incident light intensities decrease seasonally, the mats may become more greenish as the photosynthetic cells reduce their protective carotenoid pigments and increase their concentrations of photosynthetic chlorophyll molecules.

More Microbial Mats at Hot Springs in the West Thumb Geyser basin

Many non-photosynthetic microbes are also pigmented (such as the pink filament forming Aquifex species) presumably for protection against light damage or by accumulation of colored metabolites. Non-photosynthetic microbes must gain their energy by consuming organic matter (heterotrophy) or inorganic matter (lithotrophy). The geothermal spring is a source of reduced sulfur, molecular hydrogen and reduced metals that may be "eaten" as fuel for lithotrophy. Lithotrophic and heterotrophic microbes occur both in the microbial mats, and throughout the water column.

The cyanobacteria live at the surface of the microbial mats in zones of high light intensity. Cyanobacteria are photosynthetic microbes that produced oxygen. The most thermophilic cyanobacter (Synechococcus lividus) survives at temperatures up to 75°C. Eukaryotic algae have a more restricted temperature tolerance than the cyanobacteria (to 56°C), and can coexist with cyanobacteria in the high-light intensity zones. Green and purple sulfur bacteria, which are also photosynthetic, but have a more primitive photosynthesis mechanism than cyanobacteria, are generally adapted to growth under lower light conditions and tend to be distributed beneath the zone of cyanobacterial growth (although some yellowstone microbial mats are composed predominantly of Chloroflexus species- a green sulfur bacterium). Bacteria feeding on exuded dissolved organic matter and dead biomass (heterotrophs) consume oxygen produced by cyanobacterial photosynthesis. When the oxygen has been depleted, other organisms use alternative electron acceptors such as nitrate, sulfate or CO2 to fuel the degradation of organic carbon (denitrifiers, sulfate reducers and methanogens, respectively).

Encrustation and preservation (lamination) of the mat by minerals precipitating out of the overflowing water competes with microbial degradation of the mat material. Laminated microbial mats are thought to be the precursors of ancient stomatolites. New layers of microbial mats continually grow over the encrusted layers, forming laminations characteristic of fossilized mats. Micro-environments around microbes (e.g. pH, and redox potentials) can influence carbonate, metal, and silicate deposition, contributing to the mineral composition of sedimentary rocks. For example, the oxygen produced by photosynthesis can lead to encrustation of cyanobacteria with precipitated ferric iron in iron-rich vent fluid.

Microbial Mat ecosystems

The warm environments influenced by geothermal springs are colonized year-round by a variety of species including mosses, grasses, insects and flowering plants. Much of the vegetation adapts to the season, growing close to the warm ground during harsh winter conditions and growing taller during the summer.

A typical food chain connecting the microbial mat to the larger geothermal vent ecosystem is depicted below.

Notes on Biotechnology

The enzymes produced by "extremophiles" (the thermophiles, hyperthermophiles, and acidophiles we've been discussing) are a major interest for biotechnological development. These enzymes catalyze biochemical reactions at high temperatures and/or low pH, are more stable than enzymes from mesophiles (prolonged shelf life), and are of use in the PCR (polymerase chain reaction), laundry soaps, other "industrial" applications

The recent Diversa Corporation - Yellowstone National Park agreement now makes Yellowstone a partial beneficiary of any future profits gained from exploitation of the parks microbiological resources.

How do we study microorganisms from natural environments?

> Traditional: Enrichment culturing
An attempt to grow target organisms by reconstructing elements of their natural environment in the lab while excluding environmental factors that could allow non-target organisms to grow.

Problems: Observed cultured diversity rarely reflects distribution of organisms in situ.
-Numerically dominant organisms in enrichment cultures are often "lab weeds"
-Limited culturing technology, often don't know how to best culture an organism until it has been studied in the lab.

Benefits: Once an organism can be cultured, its physiology can be investigated, providing information about its ecological role in its natural habitat. Hyperthermophiles were discovered by enrichment culture of hot spring waters and their subsequent cultivation enabled their ecological role in high temperature environments to be studied.

> Modern: Combine Molecular Microbial Ecology with culture-based methods
Use the DNA sequence of the ribosomal subunit genes (and other genes) to estimate the evolutionary distance between organisms.

- On-line databases now contain tens of thousands of these sequences.
- Use DNA sequence information to design fluorescent probes to signal the presence of target groups of organisms in situ (Fluorescent in situ hybridization - FISH).
- Use the polymerase chain reaction (PCR) to synthesize copies of a target gene from the gene sequences of organisms present in the environment. With sufficient DNA the variation in sequences can be analyzed to infer what types of organisms were present in the environmental sample.

Problems: Biases in the analytical methods do exists, but they are not well characterized

-Variable DNA extraction efficiencies for different organisms in environment
-Variable PCR amplification efficiencies for different DNA sequences
-Artificial genetic diversity introduced by analytical methods

Benefits: The application of molecular biology to study microorganisms in situ has led to an explosion of information about microbial diversity that would have been impossible by the current culturing methodologies. Many studies combine culturing and molecular work.

Advances in Molecular Microbial Ecology:

The ability to identify organisms based on their DNA sequences has led to many significant advances in the exploration of microbial diversity.

- The recognition of the 3 Domains of life: Bacteria, Archaea, and Eucarya from evolutionary trees generated from ribosomal DNA sequence divergence.
- The recognition of the staggering amount of uncharacterized microbial diversity (less than 1% species characterized to date).
- In Obsidian pool, Yellowstone, a "Kingdom" of Archaea (Korarchaeota) was discovered by PCR based methods, similar organisms have since been detected in other hot spring environments.
- A hyperthermophilic organism, evolved in a geothermal habitat, has been championed as the "common ancestor" of the 3 domains of life. The deepest branching sequences (most primitive living organisms) within the Bacteria and the Archaea are thermophiles.
The geothermal features of Yellowstone national park have been studied by microbiologists for decades, and the application of DNA sequence information to these studies has rapidly accelerated the pace of discovery. Yellowstone's hot springs have wide-ranging biological significance, from the unique diversity of organisms harbored there-in which expand our views of where (and how) life can survive, to the potential utility of these new-found microbial resources to industry and medicine. Preservation of these resources is a primary objective of park managers, and continued stewardship will ensure that they remain intact for observation by future generations of tourists and scientists.

On-line references:

Life at High Temperatures
Thomas D. Brock
http://www.bact.wisc.edu/Bact303/b1

Microbiology in Yellowstone National Park
David M. Ward
http://www.wfed.org/resources/reports/article5.htm

Hydrothermal Systems: Doorways to Early Biosphere Evolution
Jack D. Farmer
http://www.geosociety.org/pubs/gsatoday/gsat0007.htm