BOARD OF GOVERNORS, AMERICAN ACADEMY OF MICROBIOLOGY

R. John Collier, Ph.D. (Chair) Harvard University Medical School

Kenneth I. Berns, M.D., Ph.D. University of Florida Genetics Institute

Arnold L. Demain, Ph.D. Drew University

E. Peter Greenberg, Ph.D. University of Washington

Carol A. Gross, Ph.D. University of California, San Francisco

J. Michael Miller, Ph.D. Centers for Disease Control and Prevention

Stephen A. Morse, Ph.D. Centers for Disease Control and Prevention

Harriet L. Robinson, Ph.D. Emory University

George F. Sprague, Jr., Ph.D. University of Oregon

David A. Stahl, Ph.D. University of Washington

Judy A. Wall, Ph.D. University of Missouri-Columbia

COLLOQUIUM STEERING COMMITTEE

Jennie Hunter-Cevera, Ph.D. (Co-Chair) University of Maryland Biotechnology Institute

David Karl, Ph.D. (Co-Chair) University of Hawaii-Honolulu

Russell Hill, Ph.D. Center for Marine Biotechnology University of Maryland Biotechnology Institute

Edward Ruby, Ph.D. University of Wisconsin-Madison

James Staley, Ph.D. University of Washington

Naomi Ward, Ph.D. The Institute for Genomic Research

Carol Colgan, Director American Academy of Microbiology

COLLOQUIUM PARTICIPANTS

Douglas Bartlett, Ph.D. University of California, San Diego

Craig Carlson, Ph.D. University of California, Santa Barbara

Colleen Cavanaugh, Ph.D. Harvard University

Feng Chen, Ph.D. Center of Marine Biotechnology University of Maryland Biotechnology Institute

Sallie Chisholm, Ph.D. Massachusetts Institute of Technology

Edward DeLong, Ph.D. Massachusetts Institute of Technology

Paul Falkowski, Ph.D. Institute of Marine and Coastal Sciences Rutgers University

Chris Francis, Ph.D. Stanford University

Jed Fuhrman, Ph.D. University of Southern California

Margo Haygood, Ph.D. Scripps Institute of Oceanography

John Heidelberg, Ph.D. The Institute for Genomic Research

Russell Hill, Ph.D. Center of Marine Biotechnology University of Maryland Biotechnology Institute

Jennie Hunter-Cevera, Ph.D. University of Maryland Biotechnology Institute

David Karl, Ph.D. University of Hawaii-Honolulu

Nancy Knowlton, Ph.D. Scripps Institute of Oceanography

Mary Ann Moran, Ph.D. University of Georgia

L. Nicholas Ornston, Ph.D. Yale University

Michael Rappé, Ph.D. Hawaii Institute of Marine Biology University of Hawaii

Margaret Riley, Ph.D. University of Massachusetts-Amherst

Forest Rohwer, Ph.D. San Diego State University

Eugene Rosenberg, Ph.D. Tel Aviv University, Israel

Edward Ruby, Ph.D. University of Wisconsin-Madison

Garriet Smith, Ph.D. University of South Carolina-Aiken

Mitchell Sogin, Ph.D. Marine Biological Laboratories Woods Hole, Massachusetts

James Staley, Ph.D. University of Washington

Curtis Suttle, Ph.D. University of British Columbia, Canada

Naomi Ward, Ph.D. The Institute for Genomic Research

John Waterbury, Ph.D. Wood Hole Oceanographic Institute

Jonathan Zehr, Ph.D. University of California, Santa Cruz

SPONSOR OBSERVER

Lita Proctor, Ph.D. Marine Microbiology Initiative The Gordon and Betty Moore Foundation

MARINE MICROBIAL HABITATS

Marine microbes continue to have a profound influence over the biosphere, but where, precisely, are marine microbes found? What are their various habitats like? How do you aseptically sample the habitats? These questions can be answered in a number of ways, based on the level of resolution that is of interest (see Table 1). The marine environment occurs on many scales, and there are many niche levels from which to approach a description of habitats. For example, a microbe floating in the middle of the Pacific Ocean could be described as “free-living,” but this is no more or less accurate than the descriptors “pelagic” (meaning “open water,” not sediment) or “within the Central Pacific gyre.”

TABLE 1

EXAMPLES OF HOW THE MARINE ENVIRONMENT MAY BE DIVIDED INTO DIFFERENT MICROBIAL HABITATS.

Perhaps the most important factor in defining marine microbial habitats is the distance over which these organisms interact with their environments. The habitat attributes that are apparent to the naked eye are usually less important to a marine microbe than the microscopic and submicroscopic facts, including concentrations of nutrients, the presence of gels and particulate matter, metal concentrations, light levels, pH, ultraviolet exposure and solar flux, temperature, oxygen saturation, and redox. Hence, the scale at which marine microbial habitats are most relevant is very small, but defining the boundaries of these habitats is difficult to accomplish in a controlled laboratory experiment and is even more difficult to define for a microbial cell embedded in the environment.

Microbial habitats in the oceans are influenced by an almost innumerable array of forces and factors, including salinity, currents, terrestrial inputs, and climate. Salinity is relatively constant in the open ocean, but is less stable in coastal areas. Ocean and seafloor currents have been shown to behave in ways other than previously thought, greatly affecting our understanding of transport processes in the deep sea. Terrestrial inputs create gradients of nutrients, pollutants, and other matter that affect habitats. Climate effects represent the largest scale of influence on microbial habitats. Temperature, precipitation, and wind (including windborne particulate matter) can each impact marine communities in a number of ways.

Importantly, marine microbes themselves exert influence on their habitats by consuming, producing, and sequestering a variety of compounds. Hence, in the oceans, gradients of materials important to micro- and macroorganisms alike are often controlled by processes carried out by microbes.

In the marine environment and elsewhere, interfaces tend to be hotspots of diversity and biological activity. Marine microbial habitats at interfaces include the air-water, water-sediment, water-ice, and host macroorganism-water interfaces. The sub-millimeter scale of physical and chemical variability in these habitats poses a serious challenge to studying interface habitats in detail.

CHANGE OVER TIME

Microbial habitats change over many time scales—diel (daily), seasonal, decadal, and longer. Many of the changes induced by human activities can impact marine microbial communities and, in turn, can impact the ways by which those communities modulate the environment and climate. Temporal changes in marine microbial habitats can be illustrated by describing three disparate habitats: the central Pacific gyre, the Chesapeake Bay, and hydrothermal vents. The central Pacific gyre is an open ocean habitat that changes on a diel basis, but it has also exhibited changes over decades as shifts occur between community domination by diatoms and by picoplankton. The Chesapeake Bay exhibits diel changes, marked seasonal changes, and profound decadal changes over the past couple of centuries as human activities have taken their toll. Hydrothermal vents exhibit both short and long periods of fluctuation, changing over the course of minutes, hours, and decades. This variability creates an ephemeral and unpredictable habitat for microorganisms.

In the coming years, if observed trends in greenhouse gas emissions continue, increasing concentrations of atmospheric carbon dioxide are expected to result in a pH decline of 0.3 in the oceans—a small number that signifies big changes. This would be an utterly radical transformation of the ocean habitat for microorganisms and macroorganisms alike. To illustrate, a similar pH shift in the acidity of human blood would result in acidosis and a painful death.

INTERACTIONS BETWEEN SEDIMENT-DWELLING AND PLANKTONIC MARINE MICROBIAL COMMUNITIES

In general, marine microbes in and near sediments interact with and intercept reductants diffusing from the sediments below and oxidants diffusing from the water column above. Conversely, planktonic microbes intercept carbon compounds from photosynthetic activities near the surface of the ocean and control the downward flux of nutrients to the sediments. The connections between subsurface and planktonic environments are probably greatest in zones where the sea floor is spreading. However, details of the interactions between the microbial communities of marine sediments and communities in the water column are not known—a clear gap in the current knowledge.

MARINE SEDIMENTS, BIOFILMS, AND EARLY LIFE

In some ways, the physical and chemical circumstances of marine sediments are thought to reflect those that nurtured the beginnings of life on this planet. Studying marine sediment and the life that exists there today could provide insight into early life:

• >> Redox coupling may have been important to fostering formation of the organic molecules that propagated early life. Marine sediments harbor marked layering of redox potentials that enable extensive redox coupling.
• >> Methane may have been one of the building blocks of early life, and there are numerous areas of the ocean floor where methane percolates up from the deep subsurface.
• >> Heat and pressure could have facilitated formation of early biological molecules, including amino acids. These conditions exist in parts of the sea floor that combine high depth with communication with the earth's hot core.
• >> Given the pivotal role of iron in the functioning of many enzymes, iron is thought to have been abundant where enzyme systems first evolved. Marine sediments are usually iron-rich environments.
• >> Before the dawn of oxygenic photosynthesis, the earth's atmosphere was anaerobic, a condition mirrored by sub-surface marine sediments.

It can be argued that shallow microbial mats and biofilms are the most appropriate systems for modeling early life, as the high metabolic diversity and spatial separation of metabolisms of these arrangements closely reflect fossilized examples of early microbial communities. Microbial communities that dwell in marine sediments can be construed as biofilm or mat communities, given that these communities dwell on the surfaces of sediment particles (like biofilms) and can form thick accumulations of interacting cells (like microbial mats).

COMMUNITY COMPOSITION

Although intuitively apparent, it can be difficult to reach agreement on the precise technical definition of a microbial community. It is largely agreed that microbial communities are groups of microorganisms that interact and, together, accomplish more than those same organisms would separately. Communities are also influenced by common factors and/or by each other. However, the question of whether chemical interdependence, a form of interaction, is required among members of a community is more controversial. A continuum of interactions, ranging from obligate to minimal, is thought to exist among members of microbial communities; strict interdependence is not necessarily a requirement for the designation “community.” Microbial communities, in which members interact, are distinct from microbial assemblages, in which members merely coexist.

PREDICTING THE COMPOSITION OF MARINE MICROBIAL COMMUNITIES

The particular physical and chemical conditions of a given marine habitat, including resource availability, select for distinct groups of microorganisms, and there is a certain amount of predictability in the character of the resulting community.

Mapping the microbial species onto the physical and chemical variability of marine habitats is becoming increasingly feasible in certain habitats and with certain well-described species. For example, the general distributions of two planktonic genera, Synechococcus and Prochlorococcus, are well-understood information that can be extrapolated to unknown planktonic communities. The presence of certain broadly-defined functional groups, such as nitrogen fixers or calcium carbonate producing microbes, can also be predicted.

Most marine microbial communities are not yet fully described, however, so it is difficult, if not impossible, to predict the species-level composition of a marine microbial community. Hence, it is often possible to predict the functions in place in a given marine microbial community, but it is seldom possible to predict the genera or species present.

The scale and level of resolution of the inquiry into a marine microbial community are important factors in predictability. Communities can be unpredictable on a small scale (1 meter) but predictable over larger scales (kilometers). The level of resolution often determines the results of a study on community diversity. Deep, exhaustive sampling can reveal much greater diversity and complexity than shallow sampling. Predictability in marine microbial communities that is based on 16S rRNA genes may not be corroborated by further work at the genome sequence level. Genome sequences usually reveal much greater and, currently, unpredictable diversity.

The biome concept may be useful in predicting the composition of microbial communities. By this reasoning, the energy inputs into an ecosystem are evaluated, and the role this energy plays in defining the attributes of the microbial community is examined.

Alternatively, a microbial community may be structured by an evolutionary history that prevents the spread of that community into new environments or, conversely, prevents the entry and establishment of new species into that community. If a community's history dictates its composition, it would be very difficult to predict the structure of the community from transitory features like nutrient status and temperature.

Certain tight associations, like the one between the bacterium Vibrio angulara and the alga Ulva, can allow researchers to use the presence of one species to predict the presence of a partner species. Recent studies have shown Ulva propagules will not establish themselves on a surface in the absence of chemical signals from their biofilm partner, V. angulara. Hence, if Ulva is detected in a biofilm, it can be assumed that V. angulara, too, is present.

In seeking to better predict the structure of marine microbial communities, there is a need to know more about possible keystone species—organisms that may be present at low or high numbers but perform indispensable functions for the community.

MICROBIAL DISTRIBUTION IN THE OCEANS

The issue of whether marine microbial species are either cosmopolitan, or are more provincial and limited to certain geographical areas, was raised decades ago. The question endures today because of the bearing it has on the conservation of bio-diversity. If marine microbes are not cosmopolitan, then does the international scientific community need to act to preserve the microbial diversity harbored in endangered habitats?

Current evidence indicates that most marine microbes are not cosmopolitan, but, instead, are restricted to specific habitat types or geographic locations. However, there are a few examples of truly cosmopolitan organisms, including the deep-sea marine group I archaea. As new, higher resolution technologies become available, further research may show other microorganisms to be more widespread than previously thought.

Free-living marine microbes may be more cosmopolitan than symbionts, biofilm-associated microbes, and others. Extinction is a real possibility for symbiotic microbes since they are dependent on the survival of their host and many, many species of marine macroorganisms are currently endangered.

It is essential to note that the number of representatives of a given group is not necessarily linked to the importance of that group in the functioning of the community. Common organisms may not play a critical role in the dynamics of a given community despite their numbers, and organisms that only muster 0.1% prevalence, like nitrogen fixers, can be of pivotal importance. Consequently, it is not known whether cosmopolitan microorganisms like Synechococcus are common because they are essential to their communities or because they are weedy individualists that can survive in a wide spectrum of environments.

One observation that appears to support the idea that marine microbes are largely cosmopolitan is the establishment of novel microbial communities in the wake of a disturbance. Often, the new communities are dominated by microbial types that were previously present in low or undetectable numbers.

The long-standing question of how to taxonomically divide microbes creates some confusion in discussions about cosmopolitan distributions. A working definition of an appropriate taxonomic unit is needed to address research in microbial distribution in the oceans and elsewhere.

Millimeter-scale analyses of marine habitats may help to reveal the distribution variability of marine microbes over very small spatial scales.

OUTSTANDING QUESTIONS ABOUT MARINE MICROBIAL DISTRIBUTION

A number of outstanding questions about marine microbial distributions remain to be addressed. These include:

• >> What is the ecological relevance of the functional gene diversity observed in marine microbial communities? Does the diversity have an impact on ecosystem function or long-term stability?
• >> Are changes in microbial communities predictive of changes in environmental function?
• >> The long-term variability of marine microbial communities is poorly understood. Can intermittent sampling, like taking community snapshots, address this question or is continuous sampling necessary to evaluate community composition shifts?
• >> Are there keystone species in marine microbial communities that are critical to a given function? How can scientists identify those organisms?

FORCES AT WORK IN MAINTAINING STEADY STATE IN MARINE MICROBIAL COMMUNITIES

Many marine surface waters maintain steady populations of approximately 10⁶ bacteria and 10⁷ viruses per milliliter, a condition that may be determined by nutrient limitations and predation controls enforced by protists and viruses. It is thought that, in some cases, microbial population numbers are kept at about an order of magnitude below the carrying capacity of the habitat—presumably by predation. It is thought that viruses may coexist with their hosts, helping to structure communities and diversity, but some studies have shown that the removal of viruses from a system has no effect on bacterial abundance and community structure. Protists, on the other hand, are thought to play a more antagonistic, less discriminating role in steady state maintenance of bacterial cell abundances by consuming many different types of bacterial prey.

MACROECOLOGICAL THEORY AND OCEAN MICROBES

It only became feasible to test ecological theory as it applies to marine microbial communities within the last 10 years, after basic questions about the abundance and species distribution of marine microorganisms had been answered. In general, ecological concepts like predation, competition, and diversity appear to be applicable to marine microbial communities, but particular problems related to scale, food webs, the species concept, and the traditional focus on biochemical and geochemical sciences within marine science preclude broad application of ecological theory to these communities.

The small scales relevant to microbes are often not addressed adequately by traditional macroecology. Also, the structure of food webs in microbial systems is likely to take on a very different form than those described by traditional macroecology.

The species concept poses a big stumbling block for applying ecological theory in marine microbial systems. The grouping of organisms into clusters of species is the basis of most ecological theory, but most microorganisms do not fall neatly into species categories because of their ability to reproduce asexually. It may be that genes or genomes are more appropriate taxonomic units than species for modeling in microbial systems.

To a certain extent, the biogeochemical and geoscience focus of marine science has prevented robust integration of ecological and evolutionary theory into marine microbiology. Rigorous inclusion of ecological science within classical oceanographic science is needed to satisfactorily address contemporary challenges in marine microbiology. This will require extensive dialogue between theoretical ecologists, evolutionary scientists, physical and chemical oceanographers, and marine microbiologists.

With man-made pressures on the world's natural systems growing annually, a thorough grasp of the ecology of all environments, the oceans included, will be necessary to mitigate damages and manage our natural resources for the benefit of future generations. Because of their short generation times and relatively simple physiology, microorganisms have been and will continue to be powerful model systems for testing ecological theories.

FACTORS THAT IMPACT MARINE MICROBIAL DIVERSITY

The list of factors that impact marine microbial diversity is not a short one. Nearly every measurable physical, chemical, and biotic variable in the marine environment has been found to increase, decrease, or otherwise alter microbial diversity. See Table 2 for a list of several of the more important factors.

TABLE 2

FACTORS THAT IMPACT MARINE MICROBIAL DIVERSITY.

For the most part, the extent to which each of these influences actually operates in the environment and the contexts in which they are important remain to be determined.

Climate change, which will be felt by marine microbial communities as changes in ocean temperatures, will undoubtedly alter the diversity of communities in unforeseen ways. Climate change should be considered a major top-down controller of microbial communities.

Pollution, including nitrogen inputs due to anthropogenic nitrogen fixation, also impact marine microbial diversity (see section on Humans and Marine Microbes). Anthropogenic nitrogen inputs to the oceans now comprise about half the total nitrogen inputs to the oceans, a circumstance that has resulted in vast dead zones in coastal areas and an increased incidence of harmful algal blooms.

INTERACTIONS BETWEEN MARINE MICROBES AND MARINE MACROORGANISMS

Most marine ecosystems are fueled by the regeneration of nutrients—processes mediated by marine microorganisms. This is the most fundamental service provided by marine microbes; they are responsible for the cycles that sustain all living things in the oceans.

Microbes also play more direct roles in the health of corals and other marine organisms. For example, corals die when the bacteria that live on their surfaces are removed, although the mechanism behind this observation is not known. Also, bacteria associated with squid eggs have been shown to protect the eggs from fungal infection. Biofilm bacteria are known to broadcast attraction cues that affect the settlement of invertebrate larvae in those biofilms.

SYMBIOSES WITH INVERTEBRATES

A number of marine invertebrates, including species of corals, sponges, squids, shipworms, and others, are associated with unique species of bacterial and/or archaeal symbionts. Symbiosis between a microbe and a marine invertebrate affords the microbe shelter, nutrients, and possibly a route for reproduction and dispersal, and offers the host a variety of benefits. In some cases, one invertebrate species may be host to many, possibly hundreds, of unique microbial species, a detail that has important implications for marine microbial diversity considering the fact that about 1,000 coral species and more than 5,000 sponge species populate the oceans. The diversity of marine symbionts is an understudied field.

The diversity of marine microbial symbionts and the closeness of symbiotic relationships may be determined by the mode of transmission employed by the invertebrate hosts. Transmission is accomplished either by “horizontal” or “vertical” means. Studies have found that horizontally-transmitted symbionts, which are dispersed in the environment and picked up by invertebrates, are more taxonomically diverse than vertically-transmitted symbionts with respect to the internally transcribed spacer (ITS) regions of their ribosomal DNA. (The ITS region is variable in length and sequence and can be used to identify and establish relatedness between microorganisms.) The reason for this difference may be the fact that vertical transmission, passing symbionts from generation to generation through the gametes, more easily allows the transmission of identical clonal organisms between members of an invertebrate species. The full genome sequences of both vertically- and horizontally-transmitted marine symbionts are currently being studied and will probably shed more light on the diversity of these organisms.

Vertical transmission is likely to lead to a close symbiosis between the microbe and its host, since inheriting a symbiont from one's forbearers affords few opportunities for host- and symbiont-switching. In horizontal transmission, the host is more likely to acquire symbionts appropriate for the particular location where the host settles down.

It is possible that in the early stages of a new symbiotic relationship, horizontal transmission of a symbiont can be used effectively, but as the relationship becomes tighter and more necessary to the survival to the microbe and its host, transmission must shift to the vertical mode, via the gametes. Symbionts have been known to dispose of those parts of their genomes that are redundant within the protective confines of the host. Giving up part of its genome can render a microbe less fit for survival outside the host, making vertical transmission necessary for the survival of the symbiont.

In studying marine symbionts in situ, it can be difficult to separate the symbionts from the pathogens and transient populations of microbes that may also be found in and around an invertebrate. Phylotype-specific probes that preferentially detect microbes with high concentrations of RNA compared to those with lesser concentrations can be used for this purpose.

MICROBIAL EFFECTS ON THE ECOLOGY AND LIFE HISTORY OF MARINE INVERTEBRATES

Microbial symbionts can have profound effects on their hosts—effects that have consequences for the ecology and life history of these invertebrates. For example, local microbial diversity has been shown to impact the suite of symbionts harbored by a widespread species of mussel. Studies of the ITS region of the ribosomal DNA of mussel symbionts and their environments have shown that one species of mussel has different populations of bacteria in different geographic areas, a reflection of the bacterial populations available for colonization.

In more rigid, obligatory symbioses, like that between the hydrothermal vent tube worm Riftia and its symbionts, the presence of free-living symbionts is necessary for an invertebrate to colonize a given area, posing a limitation on the habitats available to that organism. In another example of a tight relationship (as described previously), the bacterium Vibrio angulara is required for the algae Ulva to establish itself on a surface.

There is some evidence that the algae to blame for harmful algal blooms (which have caused as much as $1 billion damage over the past decade) require not only the appropriate nutrients in order to proliferate into a bloom, but they are also reliant on certain bacterial associates. In other words, if the bacterium is present, an algae bloom is possible and if the bacterium is missing then a bloom may not happen. The mechanism behind this phenomenon is not yet known.

MARINE VIRUSES

At first glance, the numbers of viruses found in marine waters appears to be exceedingly high. Locations studied to date have revealed viral counts on the order of 10,000,000 viruses per milliliter of water. However, these numbers are less surprising when one considers the number of prospective microbial hosts available to those viruses. Approximately 1,000,000 microbes are found in a milliliter of seawater, making the ratio of viruses to hosts roughly 10:1, a reasonable proportion for ensuring that a virus meets up with prospective hosts often enough to propagate itself before is disintegrates. The half life of viruses averages between two to four days, so a virus population must be sufficiently large to ensure that at least some of its members meet up with an appropriate host during that window of time. Viruses that infect rare hosts may need to live for longer periods of time to survive the interval between infection events.

Marine viruses likely play a number of important roles in the ecology of marine microbes. Obviously, viruses act as predators, causing the mortality of marine microbes. Viruses also help to maintain the high levels of microbial genetic diversity observed in marine ecosystems because hosts are known to manipulate their genomes to evade diseases. By moving DNA between the host cells, viruses act as agents of sex, shuffling genetic information around the community and providing new and surprising combinations of genes in their hosts.

Viruses can also play a role in the ecology of host cells by lysogenic conversion, a phenomenon in which a phage changes the phenotype of a host cell by either introducing genetic material into the host's genome or by other means. It has been found, for example, that marine viruses can carry the genes necessary for photosynthesis, and that these genes are regularly transferred between host cells of Prochlorococcus. This temporary storage in viruses and the efficient shuffling of the genes among Prochlorococcus species probably has had a profound impact on the evolution of these photosynthesis genes and on the ecology of Prochlorococcus.

The diversity of marine viruses is not limited to bacteriophages (viruses that infect and lyse bacteria). Many other forms of viruses, including DNA and RNA viruses with a wide variety of different sizes, host ranges, and biological properties, remain almost entirely uncharacterized. Marine viruses represent a new, unexplored world of diversity.

THE ABUNDANCE AND DISTRIBUTION OF BACTERIAL AND VIRAL PATHOGENS

In the marine environment, as elsewhere, the distribution of a bacterial or viral pathogen is directly determined by the virulence of the pathogen and the number of susceptible hosts available. This balance between hosts and pathogens generates and maintains the diversity of both groups. However, this delicate relationship breaks down in some instances. For example:

• >> Lateral gene transfer between bacteria (carried out by viruses) and lysogenic conversion may be important mediators of change from non-pathogenic to pathogenic states in some bacteria.
• >> Opportunistic pathogens like Vibrio cholerae do not comply with the rules of host availability since they can exist outside the host.
• >> Climate-related factors, including temperature, have been shown to trigger a pathogenic state in certain opportunistic pathogens like Vibrio shiloi and coral symbiotic algae called zoozanthellae.
• >> Human activities and chemical pollution can also influence the abundance of pathogens by stressing and destabilizing microbial communities.

SCALE OF MICROBIAL PROCESSES IN THE SEA

Microbial interactions and processes have implications over a very wide range of scales in the oceans, from the nanometer scale (0.0000000001 meter) to the kilometer scale (1,000 meters) and greater. The global outcome of microbial metabolic processes is the integration of interactions on very small scales. In designing studies of marine microbial communities, it is important to remember the importance of microscales. They should not be overlooked.

GLOBAL CYCLES OF BIOELEMENTS

The cycles of nitrogen, oxygen, carbon, sulfur, phosphorous, iron, and other bioelements that sustain life on this planet are driven, in part, by the microorganisms in the oceans. Microbes are capable of using every natural compound on the planet and most of the human-made compounds as well. It is this metabolic flexibility that secures microbes' importance in the cycling of the bioelements. Microorganisms control the rate-limiting steps of the cycles that no other organisms can execute. Microbes also strengthen the feedback systems that increase the stability of the cycles of the bioelements. The details of many key processes in the bioelemental cycles remain unknown, and it is largely unknown which microbes are the largest contributors to these cycles, or if it is even possible for one species to be dominant.

Although the exact contributions of marine microbes to the biogeochemical cycles is uncertain, because of their metabolic capabilities and their sheer numbers, marine microorganisms are thought to be major players in every cycle relevant to life. It is estimated that if the oceans were emptied of microbes, the carbon dioxide in earth's atmosphere would increase sevenfold. Moreover, half of the microbially-mediated nitrogen fixation occurs in the oceans, and nitrification and denitrification, two key processes that set the pace of the nitrogen cycle, are carried out by microbes. Marine microbes may also play a role in cloud formation by cycling compounds such as dimethylsulfide into the atmosphere. A constant efflux of methane from the surface of the oceans has been detected, and although the process is not entirely understood, it is undoubtedly microbially-mediated.

As more and more of the key players in the cycles of bioelements are cultivated and studied in the laboratory, and as metagenomic studies continue to contribute to the mapping of metabolic processes onto ocean depths and provinces, scientists are coming to a better, more sophisticated understanding of how elemental cycling is carried out in the oceans.

UNIQUE METABOLIC CAPABILITIES OF MARINE MICROBES

Marine microorganisms possess a number of metabolic capabilities that cannot be found in terrestrial microorganisms. As in other ecosystems, the geochemical habitat drives the evolution of different metabolic capabilities in the marine environment. For example, cold environments near the arctic drive different adaptations than high temperature, high pressure habitats near hydrothermal vents. Methane seeps are another example of a uniquely marine environment that has motivated novel metabolic capabilities. In methane seeps, high sulfate concentrations combine with high methane concentrations to favor the anaerobic oxidation of methane, a metabolism found only in the oceans.

The light-driven proton pump proteorhodopsin, which is found only in marine bacteria, is thought to play an important role in the energy balance of the biosphere because of its ability to efficiently generate energy from light. The use of sodium-dependent transporters is also limited to marine microorganisms.

Marine symbioses, including the symbioses between macroorganisms and bioluminescent bacteria and between shipworms and nitrogen-fixing cellulolytic bacteria, have given rise to many unique metabolic activities.

ADAPTING TO EXTREME ENVIRONMENTS

The oceans are host to many different kinds of extreme environments, and marine microbes have found numerous ways to thrive in those places by either changing their biochemistry to cope with the conditions or by creating barriers to keep the harsh conditions out of their cells.

In cold environments, psychrophilic bacteria cope with the conditions by maintaining flexible membranes and by circulating natural antifreeze compounds throughout the cell. In extremely hot areas of the oceans, hyperthermophilic microbes use subtle changes in their proteins to maximize the number of stabilizing salt bridges and ion pairings that keep enzymes together in the heat. Reverse gyrase, which allows DNA to maintain its structure during replication at high temperatures, is another unique enzyme used by hyperthermophiles.

In areas of high salt concentrations, many halophiles have adapted by altering their proteins, making them acidic, and hence, resistant to the protein-scattering effects of high salt concentrations. Other halophiles circulate osmolytes, small organic molecules that prevent water loss to the highly salty surroundings outside the cell.

The marine environment presents an unparalleled opportunity for studying life in extreme environments. Nowhere else on earth do so many different environmental conditions exist in such compact areas. The oceans are home to extremely high pressures and to the highest temperatures of any aquatic system. Deep sea brines, which have high concentrations of both salts and heavy metals, are singular habitats where life thrives despite the difficulties. Because of these diverse habitats, the oceans are unique with respect to the challenges posed to life and with respect to the wealth of biological diversity that has arisen to cope with those challenges.

IMPACTS OF HUMAN-INDUCED CHANGE ON THE CYCLING OF ELEMENTS IN THE OCEANS

Since the industrial revolution, vast quantities of many important bioelements have been taken from relatively inert or inaccessible sources and released into the planet's air, soil, and water. Inputs to the environment on this scale directly and indirectly impact the oceans, and atmospheric inputs of greenhouse gases have induced rapid climate change. As world populations climb and resource exploitation soars, environmental inputs and climate change are accelerating at an alarming rate. Some significant alterations in the microbial communities of the oceans, and, therefore, in the global cycling of the bioelements, are anticipated in the wake of these escalating changes.

The impacts of global temperature changes resulting from climate changes will be felt by microbial enzyme systems. Different enzyme systems have different temperature optima, so microecological processes in a given area may be depressed or stimulated depending on the nature of the local temperature change (up or down) and the optima of the enzyme systems involved. These changes are not predictable, since the optimum temperature for enzymes are not easy to decipher from an organism's habitat.

In some areas of the world, climate change has resulted in a decrease in rainfall. In Saharan Africa, a decline in rainfall combined with human activities has led to desertification of many areas and encouraged an increase in wind-born dust, resulting in an increase in the deposition of African dust in the oceans. The metals carried in this dust, including iron, are likely to impact marine microbial communities and the cycles they carry out that sustain the biosphere. (As a side note, in China's Gobi Desert, irrigation and other land use practices have actually decreased the size of selected deserts. This has led to a decrease in dust deposition, and could cause iron limitation in the marine waters where dust and the iron it carries is otherwise deposited.)

Human inputs of nitrogen to the oceans, which comprise roughly half of the total nitrogen inputs, are impacting the global cycling of nitrogen in both known and unknown ways. For example, nitrogen pollution has had an impact on nitrous oxide cycling in coastal systems, apparently resulting in a net increase in nitrous oxide production. Nitrogen-burdened, oxygen-depleted coastal regions account for only 2% of the ocean surface area, but these regions contribute about 20% of the nitrous oxide the oceans release to the atmosphere. The concentration of nitrous oxide in the atmosphere is increasing at an accelerating rate, and it is not clear how this trajectory will change with global climate change. It is likely that escalating nitrogen inputs will continue to perturb and transform the nitrogen cycle and that nitrous oxide emissions from the oceans will continue to climb.

Because of their rapid growth rates and metabolic flexibility, marine microbes could possibly serve as a buffer that helps dampen large scale changes resulting from human-induced modifications of the oceans. There is a great deal of uncertainty about how well this dampening effect works in practice, however. It may be that marine microbes have a certain buffering capacity, but when it is exceeded conditions will decline precipitously.

THE CONSEQUENCES OF GLOBAL CLIMATE CHANGE AND HUMAN INPUTS OF BIOELEMENTS TO THE OCEANS ARE POTENTIALLY DISASTROUS.

The consequences of global climate change and human inputs of bioelements to the oceans are potentially disastrous. Temperature increases can have an indirect effect on the acidity of ocean water, and thus, on the marine habitat. The solubility of carbon dioxide increases with temperature, and dissolved carbon dioxide combines with water to form carbonic acid (H₂CO₃) which subsequently dissociates into H⁺ and HCO³⁻. These materials lower the pH of the water. The concentration of carbon dioxide from human sources is also on the rise. Hence, a decrease in the pH of seawater is expected to echo the global warming trend and ongoing increases in carbon emissions. Ocean acidification will have a direct impact on corals. They are expected to grow more slowly and become more fragile under these circumstances, decreasing the ability of coral reefs to act as protective island barriers even as storm intensity increases because of climate change.

Climate change may also bring about big changes in the stratification of the oceans, reducing mixing and reducing the output of marine fisheries.

HUMAN IMPACTS ON MARINE MICROBIAL DIVERSITY

Over six billion people now populate the world, and the impacts of our activities are felt in every corner of the globe, including the oceans. The biodiversity of marine microbes is likely to be affected by human activities on both global and local scales.

On the global scale, all organisms in the oceans, including microbes, will respond to the chief feature of global climate change: temperature changes. Elevated temperatures have been found to bring about increases in the numbers of certain waterborne pathogens. Higher temperatures can also trigger the activity of certain microbial genes, including virulence genes that can lead environmental pathogens to cause disease in humans, corals, and other macroorganisms.

Temperature changes in the oceans may also disturb the delicate balance between the numbers of bacteria and phages. The checks and balances system between viruses and their bacterial hosts has recently been recognized as a determinant of bacterial abundance and diversity and of the efficiency of carbon cycling in a given microbial community. It is possible that climate change may not have drastic outward effects on the balance between bacteria and phage, however, given the flexibility of the system and the adaptability of microorganisms.

On a local level, the ozone hole over the Antarctic, a consequence of human releases of ozone-depleting chemicals, is probably having effects on microbial populations there. The gap in the ozone layer allows high levels of ultraviolet rays to reach the surface of the ocean, and is likely to increase microbial mutation rates and change microbial community composition in Antarctic waters.

Local impacts also include the release of exotic ballast water. The practice of ballast water dumping is known to release both invasive macroorganisms and potentially harmful microorganisms in coastal waters.

Nutrient loading, whether from fish farming activities or from runoff, has had massive impacts on coastal environments. Fish farms have proven responsible for eutrophication (additions of high concentrations of nutrients) of surrounding waters, resulting in large changes in the local microbial communities. Fish farming activities and runoff together have resulted in an increase in the frequency of coastal phytoplankton blooms, including harmful algal blooms that cost fishermen and governments millions of dollars every year.

Large-scale harvesting of the marine macrofauna by humans has changed the food webs of the oceans and is likely to be affecting marine equilibria and microbial diversity, driving the numbers of some species up and others down. In Peru in the 1970s, for example, over-fishing led to large phytoplankton blooms and a succession of bacteria that created large zones of depleted oxygen, called “dead zones,” in coastal waters. In this case, human activities led directly to a fundamental and devastating change in the microbial ecosystem. The exact nature of the effects of food web changes resulting from over-fishing is little understood and requires further study.

CRITICAL MICROBIALLY-MEDIATED EQUILIBRIA THAT IMPACT ENVIRONMENTAL AND HUMAN HEALTH

While it is true that marine microbial systems drive the biogeo-chemical cycles that make life possible on this planet, marine microbes also have more direct, immediate effects on human health and the well-being of the ocean ecosystem. Many of these direct effects are the result of fragile microbial equilibria, balancing points between opposing trends that could lead to serious repercussions for humans and our environment.

The equilibrium between bacteria and viruses in the oceans is an example of the kind of critical balancing act microbes perform every day. Changes in water temperature and ultraviolet radiation (UV), two factors known to be impacted by human activities, are known to disturb the relative numbers of bacteria and viruses in the oceans, with possibly disastrous results for human health. The virulence of a virus that preys on the bacterium responsible for cholera (Vibrio cholerae), for example, is affected by subtle changes in temperature. Hence, coastal temperature is a key determinant of the burden of cholera in coastal waters—an important matter to humans who live near those areas. Studies indicate UV can convert viruses between active and dormant forms, so atmospheric changes that increase the amount of light in these wavelengths that reaches the oceans can upset the balance between viruses and their hosts, possibly leading to uncontrolled epidemics in fish, invertebrates, or humans.

Microbial communities in the oceans also maintain the balances that can keep harmful algal blooms in check. Algal blooms can poison humans and wildlife that consume shellfish tainted by the algae. It is now known that nutrient pollution can disturb coastal marine microbes, triggering these blooms.

Symbiotic equilibria between marine microbes and their hosts could also be upset by human activities. Increases in water temperature, for example, compel corals to drive out their bacterial symbionts. Marine microbes mediate the nutrient ratios in seawater, and releases of nutrients from runoff, wastewater treatment facilities, or other sources that upset those ratios can seriously unbalance ecosystem health.

Marine systems are highly connected with one another—more so than terrestrial systems. As a result, altering microbially-mediated equilibria in one part of the ocean will often have impacts on adjacent areas and far-flung regions. The coupling between the sediment (called the benthic zone) and water (called the pelagic zone) in coastal areas is particularly tight and changing one of those components will inevitably affect the other. Similarly, the deep ocean collects material from the upper ocean—the two are somewhat separate but inextricably linked.

APPLYING MARINE MICROBES USING BIOTECHNOLOGY

The great metabolic diversity of marine microbes makes the oceans a rich source of biological material for biotechnology applications. A variety of secondary metabolites, enzymes, polymers, and metabolic processes can be found only in the microbes that inhabit the oceans, and it is likely that many more useful products will be found as research continues. The biotechnology potential of these organisms is a major driving force behind the push to characterize marine microbial diversity.

Different marine habitats will yield microbes suited to different applications. Exploring hot environments, for example, will unearth thermophilic bacteria that produce thermostable polymerases that could be used in industrial applications.

Collections of libraries and culture collections of marine microorganisms would be very useful for harvesting biotechnological products from marine microbes. Basic research needs to provide the fundamental libraries, tools, and meta-data that can be linked and passed on to biotechnological and biomedical interests for devising applications for marine microbes and their products. Innovative ways of linking research to real-world applications are needed.

USING MARINE MICROBES TO AMELIORATE ENVIRONMENTAL DETERIORATION

The metabolic diversity of marine microorganisms not only makes them useful in biotechnology applications, it makes them versatile tools for addressing environmental problems. One prime environmental application for these organisms is bioremediation—the treatment of chemical contamination using microorganisms. The cleanup of hydrocarbons, specifically petroleum products, is an especially pressing matter in the oceans, where accidents aboard oil tankers can release thousands of gallons of oil in a single incident. In some cases, fertilizing the indigenous microbial communities on the affected beaches with nitrogen and phosphorus can speed the degradation of the spilled oil, but bioremediation options in open water are limited because of difficulties in delivering sufficient nutrients to sustain biodegradation.

It is now known that the actions of an entire microbial community are necessary to break down complex organic matter, including petroleum. A super-organism that could carry out the entire process was sought, but was never isolated or designed.

A cluster of genes that carry out the degradation of chlorinated biphenyls, called the BPH cluster, have been isolated and are now being applied in treating dredged contaminants from the Hudson River estuary.

Today, most research in bioremediation in the marine environment is focused on the organisms already present in the affected areas. For a number of reasons, few efforts are underway to develop engineered microorganisms to address problems of chemical contamination. There are particular problems with respect to releasing microbes for bioremediation into aquatic environments; confining the organisms to the site of concern, for example, would be difficult.

Finally, there is a possibility that marine viruses can be used to control harmful algal blooms in a sort of “viral therapy.” Viruses may be isolated from the algal species responsible for blooms, then engineered or otherwise enhanced in the laboratory, and released in a bloom. This approach holds great potential for controlling diseases in aquaculture settings.

GENOMICS AND MARINE MICROBES

Genomics, the study of all or part of the genome of an organism to answer questions about that organism, describes a relatively new and powerful set of research techniques that can be applied in marine microbiology. Genomics will allow researchers to uncover previously unknown abilities and metabolisms and define the interrelationships that hold marine microbial communities together, revolutionizing our understanding of the field in the coming years.

Despite their power, genomics approaches cannot be the only tools used to address marine microbial biology. Traditional laboratory approaches in genetics, biochemistry, physiology, and ecology are still necessary to validate the biology. Functional studies will often be necessary to follow up on genomics studies that uncover unknown genes, for example. Conserved hypothetical genes pose another problem; it is not often possible to discern protein function from the sequence of a hypothetical gene alone. Better motif recognition tools could help improve the ability to discern function from sequence. Microarray studies could also help by identifying where and under what conditions the gene of interest is expressed. The integration of genomics, transcriptomics, proteomics, with ecological, biophysical, and chemical techniques will be necessary to delineate fundamental physiological processes, understand their regulation, and determine how they relate to biogeochemical cycles.

METAGENOMICS

Genomic sequencing of single species has made great contributions to our understanding of marine microbial systems, and metagenomics (in which the genomes of a group of organisms are lumped and studied together) approaches will extend that knowledge and add to our understanding of marine microbial processes.

Metagenomics confers the potential to map the metabolisms of microbes in space and time. It may even be possible to identify differences in metabolic potential between different ocean regions using metagenomics approaches. Archiving of genomic and metagenomic samples will be necessary to provide baseline data against which microbial community structures can be assessed over time. Moreover, genomic and metagenomic information provide the fundamental for microarray and proteomics-based technologies that have shown the promise for unveiling complex microbial functions. Community-based proteomics have been applied to investigate the major expressed proteins from estuarine bacterioplankton.

Protein expression of Chesapeake Bay picoplankton community shown by 2-D gel analysis (Courtesy of Feng Chen). The gel was stained with SYPRO Ruby. The IP ranged from 5 to 8, and the molecular mass from 10–100Kda.

To make the best progress in the field, it will be critical to tie metagenomics studies to the existing knowledgebase in biological oceanography. One way to accomplish this is to locate large scale studies at existing study sites, like the ocean time series stations Hawaiian Ocean Time-series (HOT) and the Bermuda Atlantic Time-series Study (BATS), in order to have a rich context of existing data available for understanding new metagenomics data.

It will be important to make metagenomic data and other genomics datasets accessible to other researchers and as easy to use as possible.

16S DEPENDENT TECHNIQUES

The level of resolution of techniques that rely on the 16S ribosomal RNA gene is another pressing issue in research on marine microbes. Ribosomal RNA gene sequences are often used to identify microorganisms and 16S group probes are used assess the diversity of groups of microbes. New information has brought up questions about whether 16S genes offer sufficient resolution to inform researchers about the distribution of different microbes. It may be that 16S genes are simply too highly conserved across related strains of bacteria and archaea to be useful in many analyses and that studies that rely on denaturing gradient gel electrophoresis (DGGE) or other 16S-dependent methods are seriously underestimating microbial diversity.

Greater resolution of microbial species can now be achieved using other tools, including the internal transcribed spacer (ITS) region between the 16S and 23S genes, and even measures such as genome size. Studies using these methods have shown that 16S genes can miss out on a lot of genetic diversity, confirming that the definition of a microbial species is indeed a complicated issue.

RESEARCH FUNDING

There are a number of urgent questions in marine microbial ecology, like how climate change will affect marine microbes and biogeochemical cycling that can only be addressed by large national initiatives. More interagency programs are needed to target these types of projects. There is also a need for centers of excellence in marine microbiology to focus the efforts of the community on the big questions of the day.

NEW COLLABORATIONS

Penetrating questions in marine microbiology rarely stay within the confines of one person's expertise—they require the input of many varied perspectives. Collaboration across international boundaries and across disciplines needs to be encouraged.

TEACHING AND EDUCATIONAL OPPORTUNITIES

There are a number of innovative ways to convey marine microbiology to the undergraduate students, graduate students, and postdoctoral associates who will be the next generation of scientists.

Training in marine microbiology should be multidisciplinary, exposing students to such diverse disciplines as molecular biology, laboratory microbiology, ecological theory, oceanography, and bioinformatics. The challenge here lies in expressing all the basic subjects in these fields to the students in a coherent manner.

A semester at sea is a valuable part of training in marine microbiology. It may be advisable to coordinate with the University-National Oceanographic Laboratory System (UNOLS) to get more students involved in projects onboard research vessels.

Summer internships or “lab swap” arrangements in which a student spends a semester in a new lab would foster interdisciplinary interactions among graduate students, undergraduates, and postdoctoral associates. Arrangements like this would inevitably lead to productive interactions between the student's mentors, as well. “Research experience for undergraduates” programs would also help to improve the quality of undergraduate training in marine microbiology.

Postdoctoral programs that train individuals in cross-disciplinary fields, like bioinformatics, should be targeted with a slant on marine microbiology.

There is a need for a good textbook in marine microbiology. A book compiled with the input of many experts that integrates information across the disciplines of microbiology, ecology, evolution, and oceanography, would probably be most welcome by instructors.

It is important to train marine biologists and marine ecologists in the basics of microbiology and molecular techniques during undergraduate and graduate training. In order to imprint upon these students the importance of microbial systems, it is critical to present microbiology in a way that makes a big impact, conveying the importance of microbiology to our understanding of the oceans, rather than detailed concepts. One way to accomplish this is by appointing effective guest lecturers.

COMMUNICATING THE ISSUES

The scientific community needs to take a more active role in conveying the issues surrounding marine microbes to the public and to policy makers. A number of different approaches can be used in educating nonscientists on the importance of microscopic creatures (including the microbes that live in the oceans) to our everyday lives.

Video is a very appealing way to reach the public, particularly younger audiences. A film festival for graduate students, to be held annually at the meeting of an invested scientific society, would be an excellent way to foster projects of this kind and to highlight the research of graduate students. The film festival sponsored by the European Life Scientist Organization would be a good model on which to base a new film festival endeavor.

Video games and children's books with microbiology themes are other ways to reach young audiences.

Television shows with forensic science themes have boosted the interest of students and the public in forensics education. Perhaps television shows with microbiology themes could be used to stimulate interest in this field.

To reach policymakers, it may be a good idea for marine micro-biologists to participate in the existing program of short courses for congressional interns. Topics specific to marine science could be presented to these fast-track nascent politicians.