MICROBIAL COMMUNITIES:From Life Apart to Life Together

AMERICAN ACADEMY OF MICROBIOLOGY

Copyright © 2003American Academy of Microbiology1752 N Street, NWWashington, DC 20052http://www.asmusa.org

This report is based on a colloquium, “Microbial Com-munities: Advantages of Multicellular Cooperation,”sponsored by the American Academy of Microbiologyheld May 3-5, 2002, in Tucson, Arizona.

The American Academy of Microbiology is the honorificleadership group of the American Society for Micro-biology. The mission of the American Academy ofMicrobiology is to recognize scientific excellence andfoster knowledge and understanding in the microbio-logical sciences.

The American Academy of Microbiology is grateful forthe generous support of the following organizations:

National Science FoundationU.S. Department of EnergyNIDCR, National Institutes of HealthAurora Biosciences CorporationMicrobia, Inc.Centers for Disease Control

and Prevention Foundation

The opinions expressed in this report are those solely ofthe colloquium participants and may not necessarilyreflect the official positions of our sponsors or theAmerican Society for Microbiology.

MICROBIAL COMMUNITIES:From Life Apart to Life Together

AMERICAN ACADEMY OF MICROBIOLOGY

Merry R. Buckley

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Board of Governors,American Academy of Microbiology

Eugene W. Nester, Ph.D. (Chair) University of Washington

Kenneth I. Berns, M.D., Ph.D. Mount Sinai Medical Center, New York

James E. Dahlberg, Ph.D. University of Wisconsin, Madison

Arnold L. Demain, Ph.D. Drew University

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

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

Abraham L. Sonenshein, Ph.D. Tufts University Medical Center

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

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

Colloquium Steering Committee

J. Willliam Costerton, Ph.D. (Co- Chair) Montana State University

E. Peter Greenberg, Ph.D. (Co-Chair) University of Iowa

Anne K. Camper, Ph.D. Montana State University

Clay Fuqua, Ph.D. Indiana University

Paul E. Kolenbrander, Ph.D. National Institutes of Health

Roberto G. Kolter, Ph.D. Harvard Medical School

James A. Shapiro, Ph.D. University of Chicago

Carol A. Colgan American Academy of Microbiology

Colloquium Participants

Douglas H. Bartlett, Ph.D. Scripps Institute of Oceanography

Anne K. Camper, Ph.D. Montana State University

J. Willliam Costerton, Ph.D. Montana State University

Rodney Donlan, Ph.D. Centers for Disease Control and Prevention

Gary M. Dunny, Ph.D. University of Minnesota

Clay Fuqua, Ph.D. Indiana University

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

Jo Handelsman, Ph.D.University of Wisconsin, Madison

Caroline S. Harwood, Ph.D. University of Iowa

Barbara H. Iglewski, Ph.D. University of Rochester Medical School

Howard F. Jenkinson, Ph.D. University of Bristol Dental School, UK

Heidi B. Kaplan, Ph.D. University of Texas Medical School

Paul E. Kolenbrander, Ph.D. National Institutes of Health

Roberto G. Kolter, Ph.D. Harvard Medical School

Howard K. Kuramitsu, Ph.D. State University of New York, Buffalo

Jared R. Leadbetter, Ph.D. California Institute of Technology

Dennis Mangan, Ph.D.NIDCR, National Institutes of Health

Soeren A. Molin, Ph.D. Technical University of Denmark

Leland S. Pierson, Ph.D. University of Arizona

James A. Shapiro, Ph.D.University of Chicago

Joan Strassmann, Ph.D. Rice University

Graham C. Walker, Ph.D. Massachusetts Institute of Technology

Stephen C. Winans, Ph.D. Cornell University

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1.0 Executive Summary

A colloquium was convened in Tucson, Arizona, May 3-5, 2002, by the American Academy of Microbiology todiscuss issues surrounding microbial communities andtheir role in human health, industrial processes, andecological functions. Discussions centered around theimportance of microbial community functions to humanand environmental concerns, the status of currentresearch findings in the field, the technologies availablefor investigating communities, and educational and collaboration needs. The colloquium attendees agreedon a number of recommendations with respect toresearch, education, and collaboration.

The size of microbes belies the massive impacts theyhave across the globe. Some microbial communities areenormous. Their contributions are diverse, and theirimpacts can be felt on every scale—from subtle humaninfections to the treatment of chemical contaminationto the cycling of the elements that are most critical tomaintaining life on the planet.

Research has shed light on some of the phenomena sur-rounding microbial communities, providing clues abouttheir stability, development, and the mechanisms thatgovern the locations of individual members. However,much work lies ahead. The contribution of unculturablemicrobes to the functioning of many microbial communi-ties remains a vast, unanswered question. Antibioticresistance is often enhanced in microbial communitiesthat have organized into biofilms. The problems associ-ated with this resistance must be resolved. Moreover,the role of intercellular signaling and contact-dependentgene regulation is intriguing and should be investigatedfurther in order to better understand the ability of communities to act as an integrated unit. Developmentof a few appropriate model communities, which wouldhelp researchers to address these and other questions,is highly recommended.

Microbial communities operate on every scale and incountless different environments. As a result, researchand education efforts need to incorporate contributionsof scientists from many different scientific disciplines;from molecular biology to oceanography, the participa-tion of professionals in all relevant fields should be fostered and valued. Improving the public’s grasp ofthe issues surrounding microbial communities throughthe popular press, the Internet, and other outlets is also encouraged.

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2.0 Introduction

Right now, inside the water pipes in your home, thickaccumulations of organisms are living off the meagernutrients they glean from tap water. Though mostlyharmless to human health, these accumulations are dif-ficult to remove and nearly impossible to prevent, andthey foul waterlines and corrode countless miles ofwater pipes every year. After tap water leaves the faucetand drains down the sink, it follows the waste pipesfrom your home, under the streets to the treatmentfacility, and arrives at a biological reactor, seething andgurgling as it digests every manner of substance.Human and animal waste, soaps, food, solvents, andmore are degraded and mineralized by the organismsthat cling to the surfaces of the tiny reactor particlesdesigned to support them.

After treatment, wastewater is released from the facilityinto a nearby watercourse, where communities ofmicroscopic organisms thrive in the water column, onthe surfaces of rocks, and on sediment particles,exploiting and exchanging the remaining nutrients fromthe effluent and from naturally occurring sources. Asthe water continues to flow downstream, it joins largerrivers and eventually meets the oceans, home to vastcommunities of suspended microorganisms thatprocess all the materials necessary for life. Unfath-omable quantities of carbon, oxygen, nitrogen, andother elements pass through the cells of the oceanplankton every day, continually contributing to globalatmospheric gases and buffering the chemistry of theworld’s seas.

The films on the insides of household water pipes, theroiling tank of bioreactor grains, the microscopic assem-blies of organisms that extract nutrients from streams,and the ocean plankton responsible for global cycling ofnutrients are all examples of microbial communities,groups of microorganisms that exist together, fre-quently organizing and functioning as a single unit.Their impacts extend beyond the home and beyondwater treatment facilities to impact almost every aspectof human and environmental health.

A colloquium was convened in Tucson, Arizona, May 3-5, 2002, to discuss microbial communities, theirimpacts on humans and the environment, and direc-tions for future efforts in the field. The core topic of thecolloquium, “microbial communities,” is a seeminglystraightforward term, but it is a source of some debate.In a general sense of the phrase, a microbial communityis any group of microorganisms that have different func-tions and activities from one another. A liberal definitionidentifies microbial communities simply as assem-blages of microorganisms, whether single species or

multiple species, existing in the same place. This description echoes the macroecological concept ofplant and animal communities—assemblages of singlespecies or multiple species of organisms. Scientiststend to agree that many, if not most, communities arecomposed of more than one species, but it remainsuseful to include single species assemblages within thedefinition of “community.” Another attribute of manymicrobial communities is interaction among the mem-ber organisms. Although microbial communities oftenexhibit collaborative activities and complex structure,these are not defining characteristics. The broadest definition, “microorganisms that exist in a definedplace,” is used in this report.

This report summarizes the discussions and recommen-dations of the colloquium participants. Discussionscentered around the roles of microbial communities inhuman health and in ecological and industrial pro-cesses, the current extent of our knowledge in the field,the techniques currently available for studying thesecommunities, and recommendations for future efforts inresearch, collaboration, and education. The recommen-dations included in this report have significance forresearchers investigating microbial communities andaligned topics, scientific funding agencies, the popularpress, and other interested groups.

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3.0 The Impacts of MicrobialCommunities on Humans and the Environment

The impacts of microbial communities can be felt in everyaspect of life and in every corner of the globe. From theevolution of higher organisms to the scourge of antibioticresistance to the degradation of hazardous wastes, micro-bial communities are capable of radical impacts on humanhealth and the health of our environment.

In perhaps what could be called the “original” exampleof microbial communities, the origins of eukaryotesmay be attributable to close associations between dif-ferent kinds of microbial cells. It is well accepted bymost evolutionary microbiologists that the eukaryoticcell arose from a close association between differenttypes of single-celled organisms, eventually leading tothe engulfment of one microbe by another, leading toendosymbiosis. According to this theory, endosymbioticrelationships arose between one type of single-celledhost organism and a series of other, different species oforganisms that imparted various capabilities. Mitochon-dria, which allow eukaryotes to generate chemicalenergy efficiently by using cross-membrane gradientswithin the cell to drive the energy production, arethought to have arisen in this way. There is also com-pelling evidence that the chloroplasts that allow plantsto utilize light energy arose from an associationbetween a eukaryote-like cell and a photosynthetic sym-biont that found hospitable conditions inside theeukaryote. Hence, microbial communities can be seenas the incubator in which the ancestors of all higher lifeon the planet were born.

Among non-scientists, the topic of microbes usuallygoes hand in hand with discussions of disease.Although disease-causing microbes constitute a tinyfraction of all microorganisms, the impacts of these badcharacters are impossible to ignore. Microbial communi-ties are responsible for a variety of common afflictions,but conditions caused by biofilms are often the most difficult to treat. Microbial communities exist in count-less different environments, including those that exist onsolid surfaces. In this type of habitat, a microbial com-munity is called a biofilm. It is important to note that, asassemblages of microbes on a surface, all biofilms aremicrobial communities, but not all microbial communi-ties are biofilms. Liquid and semi-solid environmentsalso play host to thriving microbial communities.

Diseases caused by biofilms include gingivitis, an infec-tion of the lining of the gums that turns aggressive anddestructive under the appropriate conditions. Microbialcommunities can be responsible for endocarditis, aninfection of the lining of the chambers of the heart, and

persistent otitis media, a common condition in whichthe middle ear becomes infected by a refractory biofilmcommunity. Biofilms can develop on almost any solidsurface that is implanted in the human body, posingconsiderable risk in any procedure that requires the useof catheters or device implants. The diseases for whichbiofilm microbial communities are responsible oftenexhibit heightened resistance to antimicrobial treatmentand to host defenses.

Microbial communities are also responsible for manyprocesses of industrial and environmental significance.Corrosion brought about by microbial communitiescosts industry billions of dollars every year. Especiallyimpacted are those applications in which water is incontinuous contact with a solid surface, as in coolingsystems or in marine trades. This power of microbialcommunities to degrade solid and dissolved sub-stances is harnessed in wastewater treatmentbioreactors, which break down sewage into carbondioxide, which is released, and methane, which can beharvested for producing energy. Microbial degradationis also put to use in the biodegradation of anthro-pogenic chemicals to harmless products via processesknown as bioremediation. Some substances, like PCBs,are not readily degraded by single microbial species,but instead require the coordinated action of multi-species communities to carry out the various steps ofthe process.

Microbial communities are also responsible for many ofthe processes carried out in the global cycling of water,oxygen, carbon, hydrogen, sulfur, and other critical sub-stances. Water and the elements that microbialcommunities interchange are fundamental to the con-tinued existence of life on earth. Microbial contributionsto the processes that move these substances throughthe biosphere are so great that they impact weatherpatterns and affect the processes that buffer and con-tribute to global warming.

Evolution, disease, corrosion, degradation, bioremedia-tion, and global cycling are only a few of the manythousands of ways that microbial communities impactour lives. Microbes are ubiquitous across the surface ofthe earth, and every day they are found to affect humanhealth and the environment in new and unforeseenways. As with other basic research, a detailed under-standing of microbial communities will provide insightsthat can drive practical applications. Research intomicrobial communities will enable development of newtechniques for controlling microbial growth, generatingnew natural products including novel pharmaceuticals,improving the safety of medical products and proce-dures, and optimizing the environmental processes thataffect global ecological health. Further insights intothese impacts are gained as scientists use traditional

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and cutting-edge techniques to tease apart the storiesbehind microbially mediated processes and the commu-nities that drive them.

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4.0 Research on MicrobialCommunities: Early Progress

Microbial communities can be found in every corner ofthe globe, from the permafrost soils of the arctic circleto termite guts in sub-Saharan Africa, and on everyscale, from microscopic films on implanted medicaldevices to the oceans’ planktonic communities thatbreathe the world’s gases and cycle its nutrients.Despite this, microbes at the community level are onlybeginning to be understood by researchers. Inroadshave been made in grasping the processes at work incommunity stability, development, structure, and otherareas, and advances continue as researchers investi-gate these ubiquitous and diverse assemblages.

Fitting Microbial Ecology Into Macroecology ParadigmsA communication gap has developed between microbi-ologists and ecologists, occasionally growing so widethat some in ecology have claimed that microbial ecol-ogy falls short of “true” ecology. However, microbialcommunities have been found to adhere to the samebasic rules of ecology that macro-communities follow,including the maintenance of different levels of organi-zation, temporal progression, and the existence climaxcommunities. However, there are characteristics inher-ent to microbial communities that are due to the smalldistances and microenvironments relevant to microbes.

Another issue is the concern that microbes may engagein horizontal genetic exchange within communities.These attributes set microbial communities apart fromcommunities of metazoan organisms and require thatgeneral ecological principles be adapted to the particularidioms of microbial ecology.

Resilient and Stable One of the more fascinating attributes of microbial com-munities is the paired properties of resiliency andstability they exhibit. Microbial communities are capableof recovering from and adapting to radical habitat alter-ations by altering community physiology andcomposition. In this way, they are able to maintain greatstability in structure and function over time. Environ-mental alterations bring about community change byimpacting individual components of the community, butthe results are manifested at the level of the wholecommunity. It is tempting for microbiologists to attrib-ute community stability to genetic diversity andfunctional redundancy, but experimental work has notyet proven this principle to hold true in every situation.

Microbial communities share the ability to maintain biological stability, known as homeostasis, with other

biological and ecological entities. Microbes within communities are often buffered from changing environ-mental conditions, but when exposed to conditions thatexceed this adaptive tolerance, a microbial communitywill destabilize. These conditions, which can includenutrient starvation, viral infection, and other environ-mental insults, can be viewed as catastrophes for thecommunity. Human activities, in particular, can beresponsible for destabilization of communities. Forexample, supplying a diet of grain to cattle has beenshown to lead to a lower rumen pH, which alters thegut community and fosters the growth of pathogenicstrains like Escherichia coli 0157:H7. Oftentimes, a radi-cally different microbial community may take the placeof the former under the new conditions.

Development FROM EARLY AGGREGATES TO MATURE COMMUNITIESMicrobial communities are diverse, and they may beformed by a variety of different “developmental” pro-cesses. Initial aggregates that colonize a surface, forinstance, may originate from pre-formed aggregates orsingle cells, and can develop into mature communitiesthrough a number of routes, including the recruitmentof planktonic members, clonal growth, and surfacemotility. A progression of colonizers eventually results ina complex, mature community that does not necessarilyresemble the original colonizers. Often, the structuringof aggregates will be dictated by opportunities for co-metabolism. In the case of dental plaque, for exam-ple, the aggregated cells of the mature community arethe product of, but substantially different from, the initial aggregates. Progression and change are notroutes followed by all microbial communities, however, and none of the rules of community development are universally applicable.

DEVELOPMENTAL PROGRAMMINGAmong eukaryotes, development refers to the specificseries of changes an organism undergoes during thepassage from the embryonic state to maturity. Theseare irreversible processes that take the organism froma lower to a higher state of organization, changing itsshape and structure. However, microbes remain highlymutable, even when fully “mature.” This metabolicplasticity can make it difficult to determine where phys-iological adaptation to the immediate surroundingsends and true development to a final, programmedstate begins. As a result, microbial community develop-ment cannot always be directly equated witheukaryotic development.

When the plasticity of many species at many stages ofdevelopment is combined in a microbial community,studying development becomes complicated, and thedistinctions between programming and synergistic

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community development may be irrelevant. However,there are clear examples of traditional developmentalprogramming influencing single species communities.Fruiting body formation among the myxobacteria is anexcellent example. Initiation of fruiting body formationoccurs under nutrient limitation and results in develop-ment of complex, programmed multicellular structuresthat contain differentiated, stress resistant spores. This process is mediated, at least in part, via exchangeof a series of diffusible signals and cell-contact depend-ent recognition that drive the direction and rate of development.

Among multispecies communities the clear examplesare not numerous, but they do exist. The “genus”Chlorochromatium is actually a paired community thatacts as a single organism. One partner is a phototrophthat can respond to light, and the other member is aheterotroph that provides motility for swimming to thatlight. Clearly, formation of this two-member communityis developmentally programmed, since the memberspecies are inherently designated to achieve this rela-tionship successfully, and they develop to fulfill it. Thereare other places where one might look for these interac-tions, including oral communities, bioreactor flocs, andtermite guts. There is evidence that other microbialcommunities form synergistically, relying on multipleinteractions between individuals to dictate the eventualarchitecture of the community.

Another issue of community development and program-ming is reversibility. Development of microbialcommunities is much more plastic than classic meta-zoan development. Microbial development cannot beconfined to those processes that are irreversible. How-ever, there are examples of effectively irreversiblemicrobial development. For example, among certain filamentous cyanobacteria, every tenth cell evolves intoa heterocyst that fixes nitrogen, and loses the ability todedifferentiate, or revert to a normal cell structure. Suchexamples are limited, and most microbial systemsretain the ability to revert to autonomous units undercertain environmental conditions.

INTEGRATION OF SIGNALS DURING DEVELOPMENTDifficulties also arise when considering whether microbial communities integrate signals to build com-munities. Integration, the merging of multiple signals into a smaller set of coherent signals that driveorganization of a community, probably does not play arole in mixed-species microbial community develop-ment, as there is no central processing function withincommunities. “Integration” at the community level is notnecessarily required for ordered community develop-ment. Rather, the responses of individual cells to theirenvironment, including their neighbors, is combinatorial

within the community, and manifests itself as the overallphenotype of that community. In this way, each organ-ism responds or integrates independently and it is notnecessary to postulate integration across species.

How microbes in communities react to signals will beilluminated by further studies of phenomenon like quorum sensing, a type of signal production governinggene expression. Quorum sensing has been observedin more than 30 species of gram-negative bacteria anda number of gram-positive species. Having been foundin pure culture, as well as in intact microbial communi-ties, quorum sensing runs the gamut of microbialsettings. In quorum sensing, the chemical signal, an N-acyl-homoserine lactone, for example, can be usedas a measure of the population density in a given envi-ronment. When density reaches a critical level, thesignal accumulates and this increased signal concen-tration tells the bacteria that they have reached aquorum. In turn, they respond coordinately in someprescribed fashion. For example, quorum sensing in apathogen may turn on synthesis of factors that are onlyuseful when present in a large amount, such as anantibiotic or a virulence factor. Hence, the gene productof interest is not produced when the microbe firstinvades the host, but it is “turned on” all at once inresponse to the appropriate signal. In some cases, ithas been shown that this type of signaling is necessaryfor biofilm development, directly connecting this signal-ing process to community development.

MICROBES RESPOND TO THE PRESENCE OF OTHERSResearch has uncovered many examples of microbesresponding to the presence of other species. This is acritical characteristic of some communities, whichapparently rely on these interactions to develop anappropriate structure or to optimize metabolicprocesses. The microbes in dental biofilms, for exam-ple, show a stepwise aggregation that can only be byinteractions among the various members of the com-munity. Bioreactor flocs, the engineered assemblagesof microbes that accomplish a given metabolic process,also exhibit interactions that determine the relative posi-tioning of the members.

Another example of interspecies communication is syn-trophic hydrogen transfer, which is carried out in mixedcommunities. Interspecies hydrogen transfer allowsmicrobial communities to carry out process that wouldbe energetically impossible without the contributions ofeach species. In these cases, one syntrophic partnercarries out a process that under normal conditions isenergetically unfavorable: the production of hydrogen.The other microbial partner consumes hydrogen,thereby allowing the hydrogen-producer to continuewith the otherwise unfavorable process. In these situa-

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tions, cells of the two species come into close contactwith one another, allowing for the efficient transfer ofhydrogen, little accumulation of this valuable substance,and minimal loss to diffusion.

Mechanisms for Positioning CellsWithin many kinds of microbial communities, secondarystructure can buffer sensitive members from environ-mental fluxes for sensitive members, maximize theability of two or more species to carry out coupled func-tions, and create micro-environments conducive toexotic, localized forms of metabolism. For some com-munities, this structural organization is apparentlygoverned on a community-wide scale, via mechanismslike quorum sensing, in which a signal from every mem-ber of the community contributes to the architecture ofthe community. In other communities, complex configu-rations can come about in seemingly uncontrolled waysthat are actually closely guided by pair-wise interac-tions. For example, the communities harbored in termiteguts that allow the insects to digest cellulose are highlystructured, but it is thought that this organization resultsfrom small-scale interactions, rather than from an over-all architectural plan.

In order to achieve the appropriate secondary structurefor a community, cells must position themselves, mov-ing within the community to the appropriate locations. Itis likely that many different mechanisms control this cellular positioning, but most communities apparentlyemploy chemical gradients to dictate the appropriatemovements. An example of gradients in action is themigration of cells within microbial mat communities.Microbial mats are characterized by steep gradients of sulfide and oxygen. Sulfide-oxidizing bacteria, likeBeggiatoa, position themselves along this gradient tooptimize their ability to make energy from transferringelectrons from sulfide to oxygen. At night, when oxygenis limiting in the mat, Beggiatoa glide close to the sur-face, where atmospheric oxygen is available. During theday, when oxygen is produced by photosynthetic mem-bers, oxygen is present deeper in the mat, andBeggiatoa glide downwards, where oxygen and sulfideconcentrations are optimal.

Scales of distanceThe distances between cells in a microbial communitycan be critical to community functions, and distancesranging from the length of the entire community downto the length of an individual cell can play a role. Manycommunities require that very short distances be main-tained between cells. Myxococcus, for example,requires “head-to-head” contact between cells in orderto initiate fruiting body development. Likewise, the

collaborative interspecies hydrogen transfer betweenmethanogens and ethanol fermenters requiresextremely close contact, as do all known communitiesthat carry out interspecies hydrogen transfer.

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5.0 Technologies Available forStudying Microbial Communities

There are many techniques available for the study ofmicrobial communities. Conversely, there are also manyopportunities to develop new methods. Althoughnumerous biological, chemical and physical techniquesmay be brought to bear on the study of multicellularmicrobiology (see list below), two general approacheshave revolutionized the study: advanced imaging tech-nology and molecular probing. Advanced imagingtechniques apply an analysis, which can be automated,to an image or many images of a microbial community,allowing measurements of any of a number of differentparameters, including the genetic and phenotypic diver-sity of the community, the physical distribution ofdifferent members within the community, or the viabilityand metabolic activity of the community and its mem-bers. Molecular probing entails the use of a nucleic acidor immunological component to label and identify thegenetic or metabolic contributions of different membersof a community.

Microbial communities operate on many differentscales, and techniques applied to the study of a particu-lar community should be tailored accordingly. From thethin oral biofilms that form on the surfaces of teeth tothe vast, continuous communities of cyanobacteria inthe open ocean, microbial communities run the gamutin size and impact, and the analytical methods used incharacterizing them should be applied with an eye tothe scale in which the investigator is interested. Manytools are available for these analyses, ranging frommicroscopic techniques to satellite imagery, andresearchers need to maintain a focus on the scale ofthe question at hand when selecting among them.12

TABLE 1ANALYTICAL TECHNIQUES FOR THE STUDY OF MICROBIAL COMMUNITIES

In situ Techniques and Microscopy/StainingNon destructive EM and AFMSEMCSLMStarFISH – combination labeling and FISHViability staining and Metabolic activity

Molecular and Genetic TechniquesMutant ScreensReporter GenesGenomics, Proteomics, MetabalomicsAntibody Libraries16S-rRNA Gene TechniquesTRFLP and DGGEQ-PCRMetagenomics

Spectroscopic techniquesIRMRNMRFTIR

Microprobes

Physical techniques

Natural isotope techniques

In Situ Techniques for Examining Microbial CommunitiesTwo distinct types of in situ techniques are available:nondestructive real-time monitoring and destructivetechniques that kill the bacteria while retaining the origi-nal structure of the community. To study microscopicbiofilm communities, both types of methods are heavilydependent on confocal scanning laser microscopyimaging techniques. In destructive techniques, cellsfrom any microscopic microbial community can be fixedand embedded in resin prior to staining and imaging. Innon-destructive techniques, the community must begrown in a flow cell that allows microscopy of the livingcommunity from early colonization through to theachievement of a climax community. Among nonde-structive in situ techniques for studying microbialcommunities, optical fiber imaging holds particularpromise for the field. Advanced fiber optical imagingsystems are being developed that can provide a confo-cal image, allowing a three-dimensional view of thecommunity. Advances are also being made in the reso-lution and depth that can be achieved by the technique.

Both nondestructive real-time monitoring and destruc-tive techniques are critically important to the study ofmicrobial communities. However, there is a tendencyfor these approaches to be descriptive or qualitativeand poorly controlled. Efforts are being made todevelop more rigorous techniques that allow quantita-tive measurements and appropriate controls whileretaining the structure and viability of the communityunder study. It must also be kept in mind that in situtechniques like these are a complement to invasive,reductionist approaches, not a replacement for them.Destructive techniques that dissect the community areof equal importance and need to be incorporated intothe experimental design.

Molecular TechniquesThe use of molecular techniques has radically changedthe practice of microbiology research, allowing fine-scale analyses of microbial communities that werenever before possible. Within the past 20-30 years,microbiologists have implemented these powerfulgenetic and biochemical techniques, shedding light onthe development of microbial communities, the relation-ships within communities, and the contributions ofindividual populations. Some specific issues and cau-tions associated with the use of individual moleculartechniques are discussed below.

REPORTER CONSTRUCTS AND EXPRESSED SEQUENCE TAGSReporter constructs, particularly those coupled with fluorescence microscopy, play an important role instudying microbial communities. A reporter is an exoge-nous gene that has been placed within the genome of a

microbe to signal the activity of another, endogenousgene of interest. The reporter gene is transcribed andtranslated into a protein when the gene of interest isactivated by the host, and it demonstrates a visible orotherwise easily measurable signal. In this way, theexpression and activity of a gene that belongs to thehost can be determined by measuring these parametersin the reporter protein. For example, the green fluores-cent protein (GFP) reporter can be placed in the vicinityof the promoter of a gene that is expressed in the pres-ence of a heavy metal. Under the microscope, cells thatproduce the protein will fluoresce, highlighting thoseareas where the heavy metal is present. New reporterswith more specificity and applicability to a wider rangeof target functions are needed in the field.

Another tool in the arsenal of molecular techniques isthe molecular probe, a DNA or RNA sequence fusedwith a fluorescent molecule that is complementary tothe mRNA sequence of a protein of interest. If the geneis transcribed by a member of the community, themolecular probe adheres to the mRNA and fluoresces,identifying the location and relative level of transcrip-tion. Reporters and tags allow measurements of geneexpression and activity under given conditions, provid-ing information about the activity and functions of themember of interest.

GENOMICS AND PROTEOMICSGenomics is a rapidly evolving field, and the definitionof the term “genomics” is debatable. For the purposesof microbial community analysis, genomics may bedefined as an array of analytical techniques that exploitthe genome, that is, the entire genetic complement ofan organism, to determine some attribute of that organ-ism. In other words, genomic techniques utilize theentire genome or genome fragments of an organism todetermine the activity or role that organism plays in thecommunity. Generally speaking, genomic techniquesare separated from other genetic techniques by focus-ing on aspects of part or all of the genome, rather thanaspects of individual genes.

Although it is a relatively new window on the microbialworld, genomics is already used to great advantage instudying microbial communities. DNA microarrays areoften used to evaluate gene expression in individualspecies or mixed communities. A microarray is a devicethat has been spotted with DNA representing the genesfrom the microbe or community of interest. In the lab,the microbe is cultivated under any of a number of dif-ferent conditions, for example, in the presence ofoxygen or in the absence of oxygen. The mRNA isextracted from these cultures and added to the microar-ray device to determine which genes are “turned on” or“turned off” under different conditions. The locations onthe array that give off a binding signal indicate which

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genes were expressed in the culture. This enables theresearcher to identify those genes that are necessaryfor survival under particular conditions or to identify thegenes that merit further study.

Though interesting information may be derived fromwhole genome analysis of almost any microbial com-munity, it is recommended that the communitieschosen for these efforts should be carefully selected sothat they are highly representative and have the poten-tial to inform other areas of microbiology and ecology. It is also recommended that genomics be applied atvery small scales in order to assess heterogeneitywithin communities. Oftentimes, the scale of experi-mental design is inappropriately large, which may leadto overgeneralizations about microscale communityinteractions and processes.

In some cases, the genome of a microbial communitycan be treated as a single entity, and work is currentlyunderway to sequence the genomes of entire commu-nities. These metagenomic approaches, which treat thegenetic content of communities as one large sample ofgenes and DNA (or Environmental-DNA, e-DNA), arepermitting an understanding of the genetic potentialwithin microbial communities without having to knowdetailed information regarding the individual membersof that community.

Proteomic techniques focus interest on proteins, usingthe entire complement of proteins produced or on asubset of the proteome to determine the activity or con-tribution of an organism to the community. Proteomicscould potentially have an important place in microbialcommunity analysis, but the technical issues involved inidentifying proteins from multiple microbes within amixed population remain daunting.

The development of new “-omics,” techniques that takea step back from the reductionist approaches of tradi-tional molecular techniques to analyze parameters thatare a function of whole-organism or community-widecharacteristics, may be on the horizon. Metabalomics,for example, is already an accepted suite of techniques.The comprehensive study of the sugars and polysac-charides that make up exterior of cells—e.g.,glycomics—is a particularly important area to develop inthe study of microbial communities, which often rely onthese substances for community adherence and struc-ture. Community analysis is wide open to suchlarge-scale analysis techniques, but, again, the scale ofsampling must always be carefully considered.

MICROSAMPLINGMicrosampling, which can be used to extract samplesas small as a single cell from a community, is a power-ful way to analyze activities within communities. Laser

capture microscopy, optical tweezers, and advancedcell sorting approaches allow access to single cells orsmall patches of cells, enabling the analysis of environ-ments, gradients, and physiology on microscopicscales. Further development of these techniques willgreatly advance our ability to dissect the activities andcomposition of biofilms over very limited spatial scales.

INTEGRATING DIFFERENT TYPES OF MEASUREMENTSIn order to obtain a comprehensive picture of microbialcommunities, there is a need to integrate multiple levelsof analysis. It has been shown repeatedly that the mostpowerful studies are those that combine many tech-nologies to tackle a single question. For example,studies that sequence and analyze community riboso-mal RNA and investigate the physiology of individualspecies provide a simultaneous view of communitydiversity and activity. The use of multiple technologiesin microbial community analysis is critical to gaining anaccurate, integrated view of the many facets theseassemblages present.

AVERAGING DATA FROM COMMUNITIESAnalytical techniques that provide an average picture ofa microbial community are essential for establishingbaseline activity, but they may mask significant micro-heterogeneity. In the analysis of microbial communities,it is critical to integrate the results of studies on multiplescales of resolution to derive meaningful conclusions.For example, some community-wide measurementswould identify the pH in a dental plaque community asbeing fairly constant, but studies of micro-heterogeneityhave shown that areas of extremely low pH (which maybe the initiation sites of dental caries) exist. Therefore,techniques that average measurements over an entirecommunity should be complemented with approachesthat investigate gradients of gene expression and activ-ity over very short distances. In some situations, thevarious measurements of community activity, gradients,species distribution, and other parameters may cancelout over the scale of the entire community, but the finedetails are nonetheless important to a thorough under-standing of community dynamics.

Technologies Needed to Advance the FieldThere are many areas of microbial communitiesresearch that require development of new analyticalmethods. For example, new staining and imagingapproaches, coupled with improved confocal micro-scopes that allow more rapid data collection, wouldprovide clearer views of the physical structures ofmicrobial communities. The lack of methods for analy-sis of cell surface structures, such as matrix materialwithin biofilms, is severely limiting for the field andpresents an area that should be developed. Moreover,tools for rapid diversity analyses, like microarrays that

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allow assessments of phylogenetic composition withincommunities, are needed.

Improvements of existing analytical methods are alsonecessary. For example, enhancement of noninvasive,microscale sampling and physical measurementsshould be explored. In research of microbial biofilms,the shortage of available technologies often limits therate of advancement in the field, and more focus onmethods and technology development for this area inparticular is warranted.

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TABLE 2WIDER APPLICATION OF TECHNOLOGIES TO BENEFITMICROBIAL COMMUNITIES RESEARCH

Single Cell Technologies

Additional Staining \ Imaging approaches

Noninvasive Microscale Physical Measurement (NMR)

Rapid Detection of Initial Colonization – Blood Chemistry – Probes

Tools to Examine Cell Surfaces – Carbohydrate Chemistry/Structure Within Communities

Rapid Diversity Analysis

Microflow Cell Tools

Confocal Laser Microscopy: in situ, multiphoton, better image analysis capabilities

Atomic Force Microscopes

Non-destructive Electron Microscopy

Better Image Analysis to Quantify and Characterize Community Structure

Novel Microbial Cultivation Techniques

6.0 Outstanding Knowledge Gaps

The current understanding of microbial communities,their structure, development, composition, and diver-sity, is at best incomplete and imperfect. Recognizedknowledge gaps include the contributions of organismsthat cannot be studied in culture, the mechanismsbehind enhanced antimicrobial resistance in communi-ties, and the significance of contact-dependent generegulation among the diversity of microbial communi-ties. In addition, it should be noted that relevant modelsystems for addressing these knowledge gaps are few.Model systems should be developed further in order toenable researchers to extrapolate a thorough under-standing of a limited number of systems to the largerdiversity of microbial communities. Microbes play cru-cial roles in human health and in ecological andindustrial processes. Improving our understanding ofthe known knowledge deficiencies will not onlyenlighten the management and use of communities inthese roles, but will also reveal new arenas for researchand implementation of microbial communities.

What are the Contributions of Not Yet Cultivated Microorganisms?The issue of uncultivated organisms rings loudly in theears of all microbial ecologists, particularly those con-cerned with the interactions of cells in communities. Ithas been estimated that as many as 99% of all micro-bial species have not yet been grown in culture, andmany may, in fact, be resistant to cultivation by availabletechniques. As a result, there are limited opportunitiesfor studying the genetic, biochemical, and metaboliccapacities of the vast majority of single-celled organ-isms. This discrepancy—that most of the microbes inthe world cannot be studied in fine detail in the lab—isat the root of what may be an immense gap in ourunderstanding of the microbial world.

Oral biofilms offer a good demonstration of this gap inthe current knowledge. When viewed with a micro-scope, it is clear that half of the organisms in dentalplaque are spirochetes, with distinct spiral-shaped mor-phology and corkscrew motility, but most of theidentified cell types have not yet been grown in the lab.Many such uncultivated organisms are apparently activein the community, and they likely play a role in oralhealth. In many communities, including the oral flora, itis not known whether active but uncultivated microbescontribute a large extent or a small percentage to com-munity activity, and it remains a source of active debateand investigation.

A number of explanations have been proposed for theapparent unculturability of many microbial species.

First, it is highly likely that many microbes are, in fact,culturable through the use of current technology, butthey resist cultivation because the precise growth conditions have not been made available. Some recentsuccesses bear this theory out. In the case of Campy-lobacter, it was only lately understood that micro-aerophilic conditions are needed for its growth. Arecent study evaluated the percentage of clinical vaginalswabs from which Staphylococcus aureus can begrown, and showed that 90% of swabs were reportedback as negative for this species. However, whengrowth plates were incubated for a longer period thanthat originally dictated by the clinical protocol, theorganism was found to have developed from many pre-viously “negative” samples; this almost universalpresence of this potential pathogen was confirmed in aseparate study by the use of FISH probes and PCR. Inthis case, the culture conditions were defined by clini-cians’ need to get fast results, but they limited thevalidity of the final result.

Another cause for poor culturability may be that criticalelements present within the microbial community envi-ronment are lacking in traditional cultivation techniques.Current cultivation techniques will need improvement ifinvestigators hope to capture the organisms thatrequire the nutritional, chemical, or physical support ofthe surrounding microbial community.

It is important to distinguish between metabolicallyactive microbes that cannot be cultivated due to ourlimited knowledge of their physical and nutritionalrequirements and those organisms that are quiescent ordormant and cannot be revived in culture. Clearly,microbes in the first category are expected to haveenormous impacts on the activity of their respectivemicrobial communities. Microbes that fall in the secondcategory may also impact the community, as they maycontribute to the structure of biofilms, for example, butdo not deplete resources. Dead cells may, in fact,release nutrients into the community that support thegrowth of other members.

More research is needed to investigate the contributionof non-cultivatable cells within microbial communities.Fortunately, we now have some tools that allow for thistype of research. The application of meta-genomicsapproaches, in which DNA is extracted from a totalsample without separating out the individual organismsand the resulting library of genetic sequences is ana-lyzed, is proving fruitful, and driving new questions forresearch. For example, research has shown that a meta-genomic library of soil microorganisms that is probedfor 16S ribosomal RNA (rRNA) sequences may containup to 25-30% Acidobacter sequences. Only three mem-bers of this genus have been cultured, leavinginvestigators with many questions (and few answers)

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regarding the metabolic role of these organisms in soil.An important caveat when interpreting non-cultivationbased discoveries like this is the all too frequent discon-nect between genetic fingerprints and metaboliccapabilities. The genetic sequences of marker loci likethe 16S rRNA gene are only hints about the phenotypiccharacteristics of a given organism. Nevertheless, meta-genomics and other molecular techniques will allowinvestigators to make major inroads into the world ofviable, but not yet cultivated, microbes.

For many years, microbiologists relied solely on tradi-tional culture-based approaches, in which only thosemicroorganisms that could be grown in a lab were usedto extrapolate to the whole of microbial diversity and microbial communities. Over-reliance on thesetechniques has likely introduced a number of misunder-standings with regard to the in situ activity ofmicroorganisms. The use of cultivation-based tech-niques in modern-day research should be examinedcarefully, and extraneous experimentation that wouldlead only to conclusions relevant to the bench-topbehavior of pure-culture microorganisms should be lim-ited. The focus of current and future research should beon the wider world of microbial diversity, and should notbe limited to those few non-representative organismsthat are easily maintained in the laboratory environment.

Enhanced Resistance to Antimicrobials in Microbial Communities Biofilm microbial communities often exhibit heightenedresistance to antimicrobial treatment as compared withtheir free-living counterparts. Antimicrobial resistance inbiofilms and other microbial assemblages is a criticallyimportant issue and has extensive practical implicationsfor medicine, industry, and the environment. It is widelyagreed that the field merits a great deal more experi-mental work.

Who is a member of the community and who isn’t? One point that is so fundamental to the topic of micro-bial communities that is often overlooked is thequestion of how to define the limits of a microbial com-munity. In the environment, functionally definedmicrobial communities exist in continuity with oneanother, and the distinctions between them blur. It isdifficult to delimit these communities in almost anyhabitat, separating the cells and species that are mem-bers from those that are not. Resolving this issueshould be a key theme for discussions among investiga-tors, and an attempt should be made to come to someagreement on what defines a member of a given micro-bial community.

What are the right model systems for community interactions? A major missing component in microbial communityresearch is the development of one or a few goodmodel systems to determine the fundamental mecha-nisms at work in communities. An ideal model systemwould be comprised of well-characterized individualmembers that together accomplish functions that arebeyond the capabilities of the individual species. Whilethe study of this model system should not precludeinvestigating the diversity of microbial communities,some significant effort should be directed towardsdeveloping one or more systems. Initially, work shouldfocus on characterizing communities with only twomember species. Chlorochromatium is an excellent can-didate for such a model. Once inroads have been madeinto understanding two-member communities, furtherresearch can branch into investigating multi-speciescommunities. Multispecies candidates include dentalcommunities, microbial mats, termite or gypsy mothgut communities, and the communities that inhabit therumens of cows.

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7.0 Education,Training,and Collaboration

The Role of Multidisciplinary CollaborationMicrobial communities are complex and phenomenarelated to their development, function, dynamics, andimpacts are not limited to the domain of any single tra-ditional scientific discipline. Hence, research intomicrobial communities requires the intellectual andtechnical skills of professionals from many differentfields, including microbiology, ecology, medicine, popu-lation biology, environmental chemistry, molecularbiology, biochemistry, soil science, plant science,hydrology, geology, engineering, and others. Because of the breadth of skills required in this field, the development of multidisciplinary collaborations isstrongly recommended.

Table 3 identifies pairs of traditional disciplines that areparticularly well suited to collaborative efforts and areasof research that are best suited to these types of inves-tigations. Interest in collaboration depends to a largeextent on attracting diverse researchers to this arenaand may be accomplished through seminar or sympo-sium sponsorship at large scientific meetings, such asthe meetings of the American Society for Microbiology(ASM). Recruitment may also be accomplished by indi-vidual scientists, who, already engaged in research onmicrobial communities, recognize persons outside thefield who may contribute and propose collaborations.However, successful collaborations can only be estab-lished if financial resources are available to supportthem. Scientists need to encourage funding agenciesand corporations to both recognize the need for multi-disciplinary work and to encourage joint projects byfunding specific grants and research.

Education and TrainingIn addition to multidisciplinary research efforts, thecomplexity of microbial communities necessitates mul-tidisciplinary education and training. In the future, morestudents at the undergraduate and graduate levelsshould be exposed to the “communities view” of micro-bial biology. Moreover, students should receiveintegrated training in order to develop experience inmultiple relevant disciplines.

Small meetings, such as ASM and Gordon conferences,have advanced the field of microbial communitiesresearch and education and their continued support isencouraged. Furthermore, short courses for graduatestudents and early faculty that incorporate the study ofmicrobial communities have proven useful.

International CollaborationInternational collaboration between scientists active inthe field of microbial communities research is highlydesirable. Scientific research should have no borders,and international collaborations should be fostered, par-ticularly in the study of microbial communities, whichholds great significance the vitality of the planet and allthe world’s citizens.

There is a need for more direct mechanisms ofexchange among international laboratories, such asexchange programs for graduate students and postdoc-toral fellows and increased support for visitinginternational professorships. European efforts of thistype, which include money for travel and meetings

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TABLE 3COLLABORATIVE OPPORTUNITIES IN MICROBIAL COMMUNITIES RESEARCH

Potentially useful interdisciplinary collaborations(some of which are already being funded to someextent) include:

modeling and experimental research programsenvironmental scientists and clinical scientistsecologists and molecular biologistsevolutionary biologists and microbiologists

Research areas that are particularly appropriate forcollaborative work include:

context of structured communitiesdrinking water biofilmscorrelating the introduction of bioterrorism agentswith microbial communitiesremediation following a bioterrorism incidentprevention and reduction of bioterrorism agentsbeneficial applications of biofilms

among collaborating scientists, could serve as exam-ples. Institutions that might sponsor such internationalcollaborations include the Fogarty Center at the NationalInstitutes of Health, the World Health Organization(WHO), and the Gates Foundation. Biofilm workshopsthroughout the world sponsored by the National Sci-ence Foundation have been successful in advancinginternational research on the topic, and more ventureslike them should be encouraged.

Communicating the Importance and Practical Benefits of Research on Microbial Communities to the Public Scientists need to communicate to the public the crucialrole of microbial communities in everyday life. From themajor biogeochemical cycles to medical implications tothe possibilities for new commercial applications, micro-bial communities impact our lives in innumerable ways,and a greater awareness of this fact among the lay public would foster a greater ability to enhance the well-being of humans and the environment.

Importantly, these functions and potential applicationsare accomplished by microbes about which we knowlittle, that have never been cultivated, and that operatein nature not as single species or individuals of aspecies, but in aggregates or communities. Withgreater recognition of the importance of microbial com-munities will come greater support in the public sectorfor the research that fills these knowledge gaps andimproves the human condition.

In certain areas, improved understanding of microbialcommunities can directly benefit the public. For exam-ple, in the medical profession, disease is nowrecognized as a perturbation of the natural state, andmicrobial communities frequently play a role in main-taining that state. A grasp of this role can guide patientsand consumers in preventing harmful perturbations intheir own bodies, directed by the insight that maintain-ing a healthy flora is more attractive than medicines toamend perturbations.

In addition to receiving information about benefits tohealth and the environment, the public should be madeaware that microbial communities comprise a vast,untapped commercial resource. The potential applica-tions of microbial community functions are numerousand are limited only by the imaginations of scientistsand engineers. Possible uses of microbial communitiesinclude large-scale applications for pollution reductionand water and sludge treatment. One can envision anext technological revolution after the electronic soft-ware revolution—a “bioware” revolution with microbialcommunity products effectively reducing biofouling andcorrosion, drug discovery, and application of mixed

communities in industrial fermentation processes.Microbial processes can be exploited to their full poten-tial if scientists can identify the basic mechanisms ofcommunity establishment, function, and maintenance.

Improved public communication of current understand-ing of microbial communities can be carried out bynon-academic science advisory groups, academicresearchers, and government agencies. Preparing anddisseminating documents designed for public informa-tion purposes, such as this colloquium report, arecrucial to this effort. The Internet is a particularly power-ful tool for disseminating information. Individualresearchers could make information available to anextremely large audience by posting websites on theirown work or on the general topic of communities.These websites should be written in lay language, withvisual aids and interactive elements.

Educational materials, like television programs and arti-cles in popular magazines, can also be used to educatethe public. When targeting young audiences, microbiol-ogy education needs to incorporate up-to-date subjectmatter regarding microbial communities so that youngstudents will be familiar with these ideas as they aretrained. In school, science laboratory exercises shouldinclude investigations of microbial communities.

Scientific societies are perfectly positioned to supporteducation and communication activities, and theyshould be encouraged to continue to hold meetings onmicrobial communities. Both symposia at national sci-entific meetings, which communicate to fellowscientists, and meetings targeted to the public, whichcan attract journalists from the popular media, are help-ful in educating a wide audience.

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8.0 Summary of Recommendations

Recommendations for Research• Determining the contributions of organisms that can-

not be cultured in the lab to the development,structure, and function of microbial communitiesshould be an overriding theme for future research inthis field.

• Understanding the reasons for enhanced antibioticresistance of microbial communities is pivotal tomanaging persistent microbial community infections.

• Development of suitable model systems for thestudy of microbial communities would prove profitable to the field by enabling a thorough under-standing of the underlying order and processes atwork in these complex and dynamic systems.

Recommendations for Education and Collaboration• The phenomena relevant to microbial communities

research are not the exclusive realm of any single sci-entific discipline. Multidisciplinary collaborationamong scientists from many fields is most conduciveto making important contributions in the areas wherethe current knowledge is weakest.

• International collaborations are critical if the strengthsof research programs in the various nations involvedin the field are to be used to their full potential.

• The public is largely unaware of the innumerableimpacts that microbial communities have on dailylife. Improving the education of the public throughthe use of publications, television, and the Internet isencouraged so that the public can come to recognizethe importance of continued research in the field.

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