ASM-UnchartedReport-WEB.qxd

A REPORT FROM THE AMERICAN ACADEMY OF MICROBIOLOGY

T H E U N C H A RT E D M I C RO B I A L W O R L D :
m i c r o b e s a n d t h e i r a c t i v i t i e s i n t h e e n v i r o n m e n t

BY CAROLINE HARWOOD AND MERRY BUCKLEY

The Uncharted Microbial World: Microbes and Their Activities in the Environment

This report is based on a colloquium, sponsored by the American Academy of

Microbiology, convened February 9-11, 2007, in Seattle, Washington.

The American Academy of Microbiology is the honorific leadership group of the

American Society for Microbiology. The mission of the American Academy of

Microbiology is to recognize scientific excellence and foster knowledge and under-

standing in the microbiological sciences. The Academy strives to include

underrepresented scientists in all its activities.

The American Academy of Microbiology is grateful for the generosity of the follow-

ing organizations for support of this project:

■

Gordon and Betty Moore Foundation

■

Defense Advanced Research Projects Agency (DARPA)

■

U.S. Department of Energy

The opinions expressed in this report are those solely of the colloquium partici-

pants and do not necessarily reflect the official positions of our sponsors or the

American Society for Microbiology.

American Academy of Microbiology

1752 N Street, NW

Washington, DC 20036

http://www.asm.org

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

E. Peter Greenberg, Ph.D.

University of Washington

Carol A. Gross, Ph.D.

University of California, San Francisco

Lonnie O. Ingram, Ph.D.

University of Florida

J. Michael Miller, Ph.D.

Centers for Disease Control and Pre-
vention

Stephen A. Morse, Ph.D.

Centers for Disease Control and Pre-
vention

Edward G. Ruby, Ph.D.

University of Wisconsin, Madison

Patricia Spear, Ph.D.

Northwestern University

George F. Sprague, Jr., Ph.D.

University of Oregon

Judy A. Wall, Ph.D.

University of Missouri-Columbia

COLLOQUIUM STEERING

COMMITTEE

Caroline Harwood, Ph.D. (Chair)

University of Washington

Colleen Cavanaugh, Ph.D.

Harvard University

Nancy Freitag, Ph.D.

University of Illinois at Chicago Med-
ical School

Stephen Giovannoni, Ph.D.

Oregon State University

David Stahl, Ph.D.

University of Washington

Carol Colgan, Director

American Academy of Microbiology

COLLOQUIUM PARTICIPANTS

John Baross, Ph.D.

University of Washington

Douglas Berg, Ph.D.

Washington University School of Med-
icine, St. Louis

Kathryn Boor, Ph.D.

Cornell University

Daniel Buckley, Ph.D.

Cornell University

Donald Button, Ph.D.

University of Alaska-Fairbanks

Gerard Cangelosi, Ph.D.

Seattle Biomedical Research Institute

Colleen Cavanaugh, Ph.D.

Harvard University

Richard Darveau, Ph.D.

University of Washington

Timothy Ford, Ph.D.

Montana State University

Larry Forney, Ph.D.

University of Idaho

Nancy Freitag, Ph.D.

University of Illinois at Chicago Med-
ical School

E. Peter Greenberg, Ph.D.

University of Washington

Caroline Harwood, Ph.D.

University of Washington

The Uncharted Microbial World: Microbes and Their Activities in the Environment

A REPORT FROM THE AMERICAN ACADEMY OF MICROBIOLOGY

T H E U N C H A RT E D M I C RO B I A L W O R L D :
m i c r o b e s a n d t h e i r a c t i v i t i e s i n t h e e n v i r o n m e n t

The Uncharted Microbial World: Microbes and Their Activities in the Environment

iii

COLLOQUIUM PARTICIPANTS

(CONTINUED)

Roberto Kolter, Ph.D.

Harvard Medical School

J. Gijs Kuenen, Ph.D.

Delft University of Technology, The
Netherlands

Jared Leadbetter, Ph.D.

California Institute of Technology

Michael Madigan, Ph.D.

Southern Illinois University

Stanley Maloy, Ph.D.

San Diego State University

Douglas Nelson, Ph.D.

University of California, Davis

Oladele Ogunseitan, Ph.D., M.P.H.

University of California, Irvine

Martin Polz, Ph.D.

Massachusetts Institute of Technol-
ogy

Anna-Louise Reysenbach, Ph.D.

Portland State University

Amy Schaefer, Ph.D.

University of Washington

Bernhard Schink, Sc.D.

University of Konstanz, Germany

Thomas Schmidt, Ph.D.

Michigan State University

Holly Simon, Ph.D.

Oregon Health and Sciences Center

James Staley, Ph.D.

University of Washington

David Stahl, Ph.D.

University of Washington

Michael Wagner, Ph.D.

University of Vienna, Austria

Rachel Whitaker, Ph.D.

University of Illinois

Anne Michelle Wood, Ph.D.

University of Oregon

Karsten Zengler, Ph.D.

Marbis Corporation, Cardiff-by-the-
Sea, California

Stephen Zinder, Ph.D.

Cornell University

OTHERS

Millie Donlon, Ph.D.

DARPA (Sponsor Representative)

Merry Buckley, Ph.D.

Ithaca, New York (Science Writer)

Carol Colgan, Director

American Academy of Microbiology

Peggy McNult, Manager

American Academy of Microbiology

A REPORT FROM THE AMERICAN ACADEMY OF MICROBIOLOGY

T H E U N C H A RT E D M I C RO B I A L W O R L D :
m i c r o b e s a n d t h e i r a c t i v i t i e s i n t h e e n v i r o n m e n t

EXECUTIVE SUMMARY

Microbes are the foundation for all of life. From the air we breathe to the soil we

rely on for farming to the water we drink, everything humans need to survive is

intimately coupled with the activities of microbes. Major advances have been

made in the understanding of disease and the use of microorganisms in the indus-

trial production of drugs, food products and wastewater treatment. However, our

understanding of many complicated microbial environments (the gut and teeth),

soil fertility, and biogeochemical cycles of the elements is lagging behind due to

their enormous complexity. Inadequate technology and limited resources have

stymied many lines of investigation. Today, most environmental microorganisms

have yet to be isolated and identified, let alone rigorously studied.

The American Academy of Microbiology convened a colloquium in Seattle, Wash-

ington, in February 2007, to deliberate the way forward in the study of

microorganisms and microbial activities in the environment. Researchers in microbi-

ology, marine science, pathobiology, evolutionary biology, medicine, engineering,

and other fields discussed ways to build on and extend recent successes in micro-

biology. The participants made specific recommendations for targeting future

research, improving methodologies and techniques, and enhancing training and

collaboration in the field.

Microbiology has made a great deal of progress in the past 100 years, and the

useful applications for these new discoveries are numerous. Microorganisms and

microbial products are now used in industrial capacities ranging from bioremedia-

tion of toxic chemicals to probiotic therapies for humans and livestock. On the

medical front, studies of microbial communities have revealed, among other

things, new ways for controlling human pathogens. The immediate future for

research in this field is extremely promising. In order to optimize the effectiveness

of community research efforts in the future, scientists should include manageable

systems with features like clear physical boundaries, limited microbial diversity,

and manipulability with the goal of understanding fundamental principles that may

apply to more complex systems. A great deal of microbial genetic and phenotypic

diversity remains to be explored, and the commercial and medical potential locked

up in these unknowns should compel the field to move forward.

Future microbiology research will build on the successes of the past using new

techniques and approaches. Uncultivated microbes hold great promise for industry,

medicine, and the recycling of precious resources, and research and technology

must make inroads in overcoming the barriers that prevent their study. In many

cases, we will no longer be able to rely on isolated, pure cultures of microorgan-

isms, but must use communities of microorganisms, which presently are poorly

understood. Indeed, community-level studies can benefit from deconstructing

microbial communities and analyzing the component members separately, but this

is not feasible in every system. The effects of perturbation on microbial communi-

ties also require study. Humans rely on the services of microbes in innumerable

The Uncharted Microbial World: Microbes and Their Activities in the Environment

ways, but we have little or no predictive understanding of how microbial communi-

ties respond to disturbance.

Research must address current limitations in detecting microscale interactions

among microbes by enhancing current technologies and fostering new microscopic

tools, biosensors, and gas sensors for appropriate small scales. Genomics, which

has enabled great progress in microbiology research of individual species, must be

applied to communities of microorganisms. This will require improved methods of

DNA extraction and amplification from environmental samples and improved strate-

gies for DNA sequence assembly. In the future, genome sequencing efforts should

continue the exploration of evolutionarily diverse microbes, as well as help reveal the

mechanisms by which closely related microbes evolve.

Technological advances have spurred every great leap in microbial biology, and in

order to move forward, new methods for revealing the activities of microorgan-

isms must be continually developed. Today, researchers need access to better

techniques for enriching and isolating novel microorganisms, particularly

approaches that enable them to mimic the low nutrient conditions to which many

environmental microbes are adapted. Other outstanding needs include methods

for performing

in situ work and bioinformatics tools.

Finally, there are several ways that training and education in microbiology are fail-

ing to adequately prepare the next generation of scientists for the challenges

ahead. Training in some of the long-established disciplines, including enrichment

and isolation, physiology, enzymology, and biochemistry, needs to be revitalized.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

INTRODUCTION

Humans live in the midst of a seething, breathing microbial world. Microorgan-

isms populate every conceivable habitat, both familiar and exotic, from the surface

of human skin to rainforest soils to hydrothermal vents in the ocean floor. All around

us, microbes are exploiting locally available, favorable chemical reactions and living

off the energy released by these transformations. In doing so, microbes are the

engine behind global biogeochemical cycles that release and absorb oxygen and

greenhouse gases in the atmosphere, fix nitrogen for plant growth, and recycle

dead material into useful nutrients for new life. In short, microorganisms are the

keystone of global health, and make life as we know it possible.

Despite their undeniable roles in the processes and conditions that sustain life,

science is only beginning to understand microorganisms. Many microbes and

microbial systems have been isolated and studied in the laboratory, and new tech-

niques are constantly being devised for studying microbes without cultivation, but

the vast majority of microbial life has resisted or escaped these efforts. This part

of the microbial world largely remains a mystery to science. Microbes are of funda-

mental importance to every form of life on earth, but in spite of major recent

advances we lack the technological tools and resources to explore them fully.

Ignorance of the microbial world and of the specific roles microorganisms play in

the biogeochemical cycles that sustain our planet is a massive waste of the enor-

mous potential for microorganisms to benefit humankind. Through fossil fuel

burning, pollution, and resource exploitation, humans have impacted the very

ecosystems that provide the essential elements for our continued existence.

Researchers currently lack an understanding of the normal, baseline state of many

microbial systems, and they know even less about how microorganisms will be

affected by drastic environmental changes, whether microbes will amplify or

dampen these effects in their habitats and what those effects signify for human

beings. For example, it is not known how climate change will impact carbon

cycling by microorganisms, an important factor in predicting the future concentra-

tions of carbon dioxide in the atmosphere. Also, clearing forested land for farming

increases the emission of nitrous oxide (a significant greenhouse gas) from soil

and diminishes the soil’s ability to absorb methane (another important greenhouse

gas), but it is not known why this happens or whether it can be prevented. Micro-

bial systems are robust but can be pushed to the limit, and science needs to find

ways microbial consortia may eventually fail in their roles in maintaining ecosystem

integrity. These scientific challenges must be met with a resolve befitting the real

urgency of the environmental problems we face.

In addition to their roles in the global biogeochemical cycles, microorganisms

perform innumerable other functions related to ecosystem health and human

health. The influence of microorganisms can be felt in the structure of food webs,

bioenergy production, waste management and treatment, food production, and

symbiotic nitrogen fixation for plants, to name a few examples. As human popula-

The Uncharted Microbial World: Microbes and Their Activities in the Environment

tions climb, reliance on microorganisms to perform these functions and others for

maintaining human and ecological health will grow too, and the stakes could not

be higher.

Research must continue to explore the microbial world in order to enable accu-

rate predictions and effective manipulations of environmental systems. This

report suggests avenues to explore for accelerating the pace of discovery in

microbial biology.

ESTIMATES OF THE NUMBER OF MICROORGANISMS ON
THE PLANET

On a global scale, one estimate places the total number of microbial cells at

and the number of viral particles at 10

(Whitman, 1998), but these large

numbers are associated with uncertainty, particularly with respect to inaccessible

environments like deep sediments, which are little explored and poorly charac-

terized as to their microbial inhabitants. Moreover, estimates of the total number

of microbes on the planet are less instructive than estimates of total microbial

biomass or biomass turnover, which are not available.

In individual microbial systems, it can be useful to derive estimates of the num-

ber of participating microbial cells, since fluctuations in cell density can be an

indication of perturbation. However, for many health applications and most environ-

mental systems, baseline shifts can go undetected because information about cell

density in the undisturbed state is lacking.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

ESTIMATING THE TAXONOMIC
DIVERSITY OF MICROORGANISMS

Estimates of the total number of distinct

microbial types (also called taxonomic diver-

sity) vary widely, but, judging from the

known microbial species, this diversity is

extremely large. To estimate the total number

of prokaryotic species on Earth requires spec-

ulation on the average volume of material

that would have to be sampled to encounter

a unique species. A simple calculation illus-

trates this:

In the example presented here, we limit our-

selves to estimating the prokaryotic species

present in the top 1 km of the Earth’s crust.

Based on the known dimensions of the Earth,

this is estimated to be 5.1 X 10

If each 1 m

of soil (one million grams) con-

tains only one unique species (by the 70%

DNA-DNA hybridization measure defining a

species), there are ~10

prokaryotic species in

the top 1 km of Earth’s crust.

A more conservative estimate assumes there is

only 1 unique species in each km

(one billion

or one quadrillion, 10

, grams), in which

case there are “only” ~10

prokaryotic species

on Earth.

Incredibly, this conservative estimate yields a

total number of species that is only two orders

of magnitude larger than some estimates of

the species diversity found in a single gram of

soil (Gans et. al., 2002), but still an order of

magnitude more than the estimated number of

insect species (30 million; Erwin, 1982), he

most diverse class of macroorganisms known.

The small toy boat represents the Known Biological

Diversity, while the great, complex ship represents the

number of Unknown Microbial Species.

THE CURRENT LANDSCAPE

Technological advances and creative, collaborative research have enabled a

great wave of discoveries in microbiology in the past 100 years, including novel

organisms and communities for use by industry and agriculture, as well as

treatments and preventatives for human infectious diseases. There are several

characteristics to target when selecting microbial communities for future

research, including clearly delineated physical characteristics, reproducibility,

and replicability.

METHODOLOGY FOR DISCOVERING NOVEL MICROBES

The methods used for discovering new microorganisms can be roughly divided

into two broad categories:

molecular techniques

and

cultivation- or

enrichment-dependent

approaches. (In this context “molecular” connotes the

use of genetic material for analysis.) There is a degree of overlap between these

two approaches, and they do not capture all of the methods available to

researchers in microbial biology. Also, molecular methods are often combined with

cultivation-dependent approaches in a single investigation. Most microbiological

research tools fit nicely into either the “molecular” or “cultivation” categories,

however, and they summarize a fundamental decision that must be made when

planning an investigation into microbial systems: to cultivate or not to cultivate?

Molecular techniques

There are two types of information that can be recovered by analyzing the gene

content of environmental DNA: phylogenetic or functional. Phylogentically informa-

tive genes (e.g., 16S rRNA) have been used to survey diversity, whereas genes

encoding specific functions (nitrogen fixation, nitrification, denitrification, etc.) have

been used to evaluate potential processes. In one molecular approach that is com-

mon today, researchers extract DNA from environmental samples, copy the

ribosomal RNA (rRNA) genes in the sample using the polymerase chain reaction

(PCR), clone the rRNA genes in a fast-growing organism, sequence the genes, and

analyze the phylogenetic relationship of those genes to each other and to genes

from known organisms. In another approach, referred to as metagenomics,

researchers eliminate the PCR and clone and sequence large segments of DNA

directly from environmental samples. This eliminates the restrictions and biases

associated with PCR.

DNA hybridization approaches, including microarray technology, are used to

detect organisms for which the target genes are already known. This type of molec-

ular approach can provide “barcodes” or “fingerprints” of individual microbes or

communities of microbes. These identification techniques enable researchers to

track changes in community composition and relative abundance over time or

between various experimental treatments. Researchers may also use these

The Uncharted Microbial World: Microbes and Their Activities in the Environment

approaches to predict or identify which barcodes represent microbes that should be

targeted for cultivation.

A drawback to applying molecular techniques is that it is not always possible to

predict the physiology of a microorganism from its phylogenetic relationship to

other organisms. Also, similarity at the genetic level does not always translate into

similarity at the functional level, since extremely subtle variations in gene sequence

can have drastic effects on the function of a gene. Most molecular methods only

examine a single gene or a subset of genes and, therefore, neglect the vast major-

ity of the genetic information that dictates function, and many novel organisms bear

little genetic resemblance to known organisms in any case. Members of the bacter-

ial genus

Dehalococcoides, for example, catalyze anaerobic dehalogenation of

several very toxic chemicals, and they constitute a novel phylum level group of

organisms. Phylogenetic analysis of the organism placed it far from any reference

organisms, so the physiology of

Dehalococcoides was only understood after it was

cultivated in the laboratory. However, recent methodological advances now enable

microbiologists to investigate with molecular tools important aspects of the physiol-

ogy of uncultured microorganisms in their natural habitat on a single cell level. For

example, these approaches, which include the combination of FISH (fluorescence

situ hybridization) and microautoradiography, FISH and Raman spectroscopy, and

FISH and secondary ion mass spectrometry, provide insights into which substrates

are assimilated by uncultured microorganisms under different environmental condi-

tions and thus also provide important guidance for subsequent cultivation efforts

(Wagner et al, 2006).

The development of molecular techniques has contributed to:

■

Our current perception of microbial diversity. For example, the number of recog-

nized bacterial phyla increased from 12 in 1887 to more than 80 in 2004. Of these

80 bacterial phyla, more than 50 have no cultured representative.

■

An appreciation that many uncultivated groups of bacteria are present in high

numbers in some environments, e.g., the discovery that crenarchaeota repre-

sent one of the ocean’s most abundant microbial cell types.

■

The use of metagenomics to harvest genes from uncultured microorganisms

and use them (via heterologous expression) for production of biotechnologi-

cally important products, such as antibiotics and enzymes.

■

The identification of the microorganisms important for chemical processes,

e.g., the discovery of microbes that catalyze nitrogen and phosphorous

removal in wastewater treatment plants and which are thus essential to pre-

vent eutrophication. These key players in the world’s largest biotechnological

process are mostly uncultured (

Nitrospira, Accumulibacter), and since we can

now detect them by molecular tools, the conditions in treatment plants can be

optimized for their activities.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

Cultivation and Enrichment

The alternative to molecular methods—cultivation and enrichment—has limitations.

Researchers have estimated that less than 1% of the microorganisms observed by

microscopy in soil yield to conventional cultivation efforts (Torsvik and Øvreås, 2002),

and once isolated, a researcher can never be certain that an organism’s behavior

under controlled conditions mirrors its behavior in the environment. Furthermore, cul-

tivation-based approaches are generally not suitable to determine microbial

community structure and dynamics over time and often lead to the isolation of micro-

bial weeds, which are well adapted to the conditions offered in the laboratory but not

necessarily important in the environment under investigation. Despite these draw-

backs, the value of cultivation-dependent approaches for making discoveries in

microbiology is undeniable. They have formed the historical foundation of all microbi-

ology, and today studying microbes in the laboratory is still pivotal to making many

important discoveries. Even the interpretation of data acquired independently of culti-

vation, like metagenomics data in which the genomes of a community of organisms

are pooled and studied as a unit, relies on information gathered from cultured and

characterized organisms.

The possible inspirations for initiating an enrichment experiment include the

need to acquire a disease-causing organism for study, biogeochemical observa-

tions that provoke inquiries into microbial activities, a need to acquire specific

metabolisms for commercial purposes, or a need to acquire microbes with specific

properties for use in agriculture.

Researchers usually use one or more “knowns” to tailor enrichments for an organ-

ism of interest. Using an understanding of the environmental conditions favored by an

organism, together with molecular (genetic clues about the microbe’s abilities) or

physical information (e.g., whether the organism is more likely to thrive on surfaces or

in liquid) as a guide for designing culture conditions, is a powerful approach. For

example, if researchers know that a certain microbial transformation is thermodynami-

cally possible, they can recreate those conditions in the laboratory to single out the

organisms that carry out that process. Enrichment and cultivation approaches like

these are increasingly being supplemented with environmental observations made

using techniques that do not rely on cultivation, leading to ever more targeted isola-

tion strategies. Sulfate-reducing bacteria, for example, were first isolated years ago,

but new molecular techniques have helped identify novel, numerically-dominant types

of sulfate reducers that have subsequently been targeted by enrichment experiments.

In some cases, biogeochemical observations have led to very targeted cultiva-

tion of previously unknown organisms, including iron-reducing bacteria and a

defined anaerobic methane-oxidizing consortium of microbes. Some types of bac-

teria were predicted by thermodynamic calculations to exist, and then the

respective conditions in laboratory bioreactors were established that led to their

subsequent isolation.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

Cultivation has allowed researchers to discern some of the fundamental principles

that govern biology and enabled basic discoveries about microorganisms

themselves. Discoveries using cultivation and enrichment have contributed to:

■

Discerning the unifying biological principles of life.

Studies of isolated

microbes have revealed a great deal about biochemistry, genetics, bioenerget-

ics, evolution, ecology, and population biology that can be applied to the whole

of biology. Also, molecular biological reagents derived from cultivated bacteria

(Taq polymerase, restriction enzymes, etc.) have enabled genetic studies of

animals and plants.

■

Identifying sources and mechanisms of biogeochemical processes.

Selective

enrichment of microorganisms has enabled researchers to understand the

processes that make life on earth possible—the biogeochemical cycles of the ele-

ments. This is especially true of the nitrogen cycle, which tends to be driven by

specialized groups of microbes that mediate each different step in the transforma-

tion of ammonia to nitrogen gas back to ammonia.

■

Defining the limits and nature of life.

Work with microbial extremophiles

(which live in habitats in which most higher organisms cannot) and bacteria

with small genomes has illuminated the limits of life—the chemical, physical,

and genetic constraints beyond which life (as we know it) is not possible.

■

Understanding the mechanisms of disease and pathogenesis.

Thanks to

work with cultured organisms, we now know that microbes and microbial

communities are not only the cause of infectious disease, they are also essen-

tial for normal immune development and defense against pathogens. Other

suspected links between certain diseases (possibly including heart disease,

inflammatory bowel syndrome, and others) and bacteria and bacterial commu-

nities have yet to be confirmed. Enrichment and isolation will be important in

these investigations.

■

Basic discoveries about microorganisms themselves.

Microbiological dis-

covery has traditionally been driven by the identification of new capabilities in

organisms that are newly cultivated. For example, the isolation of bacteria that

can completely degrade environmental pollutants made untenable the widely

held view that many of these substances were completely recalcitrant to

biodegradation. Also, our understanding of mechanisms of antibiotic resistance

derives from studies of antibiotic resistant isolates of bacteria and fungi. The

novel and striking features of microorganisms that have been discovered by

studying isolates can be divided among morphology, physiology, abundance,

and phylogeny.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

Specific discoveries that have arisen from cultivation and enrichment include, but

are not limited to:

■

Proteorhodopsin-containing bacteria.

Proteorhodopsin, discovered as late

as 2000 and found in numerous species of marine bacteria, probably plays a

significant role in energy cycling the biosphere.

■

Methods to genetically engineer crops

come from basic studies of the Ti

plasmid from the plant pathogen

Agrobacterium tumefaciens.

■

The nanoarchaea.

■

Autotrophic ammonia oxidizing crenarchaea.

■

Aerobic anoxygenic phototrophs.

■

Anaerobic methane-oxidizing denitrifiers.

■

Probiotic organisms.

■

Interactions in biofilms.

Communities of microbes that form biofilms have

been approached through an understanding of model organisms, which allow

discovery of the mechanisms of intercellular communication and cooperation.

■

Genome sequencing.

Pure cultures have enabled the first genomes to be

sequenced, leading to the discovery of evolutionary mechanisms in microbes.

■

Modern day analogues of ancient metabolisms.

Researchers have been able

to reconstruct ancient biogeochemical cycles and interpret Earth’s history using

microbes that mirror early life forms.

■

New pathogens.

The discovery of novel pathogens has led to new treat-

ments and new thinking about the epidemiology of certain diseases, such as

AIDS,

Legionella pneumophila (the causative agent of Legionnaires’ disease),

and

Helicobacter-induced stomach ulcers.

COMMERCIAL PROGRESS ENABLED BY STUDY OF
MICROORGANISMS

Microorganisms have great commercial significance, and the wealth of bacteria,

viruses, archaea, and microscopic eukaryotes that have yet to be cultivated and

understood pose a tantalizing untapped resource for industry. There are two general

approaches for adapting uncultivated microbes and microbial communities for com-

mercial purposes: using an understanding of community function to identify useful

community components, and bioprospecting.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

An understanding of community functions and the chemical language of microor-

ganisms can identify new targets for commercial applications. Quorum sensing, for

example, was discovered by studies carried out at the community level. Discoveries

in this area may lead to more refined methods of controlling biofilms that contribute

to biofouling or antibiotic resistance.

Bioprospecting (also sometimes referred to as “biodiversity prospecting”)

seeks to capture useful aspects of the world’s biological diversity in order to apply

them in industrial endeavors, medicine, or other areas. It is most reasonable to

carry out bioprospecting efforts in communities where the desired activities have

been demonstrated or are probably occurring. For example, to find compounds

involved in microbe-microbe interactions, research should focus on biofilm com-

munities and other settings where multiple species of microbes live in close

proximity. Bioprospecting for DNA from microbial communities in specific environ-

ments with desirable properties (e.g., high or low temperatures, or a certain level

of acidity) has proven valuable for selectively plucking out genes that encode use-

ful enzymes. Certain antimicrobials and anticancer drugs (including bryostatin)

have been isolated through bioprospecting in the microbial communities in and

around marine invertebrates.

Commercial applications for microbes and microbial products include:

■

Bioremediation and bioaugmentation.

Microbes have been put to use degrad-

ing organic chemicals through direct metabolism (in which the microbe uses the

material for food or energy) and through co-metabolism (through which the

microbe apparently gains nothing). They have also been used to carry out chemi-

cal transformations of inorganic materials in order to make those products less

mobile or bioavailable in the environment. Applications include both

in situ treat-

ment (at the site of contamination) and treatment of waste streams in

manufacturing settings.

■

Aids in mining operations.

Bacteria are used in microbial enriched oil recov-

ery and to extract precious materials from ore.

■

Probiotics.

Numerous probiotics products (consumables containing microorgan-

isms that are thought to offer health benefits) are available to consumers today.

Probiotics are occasionally used in medical settings as well; patients are some-

times administered a collection of probiotic microorganisms to head off

colonization by

Clostridium difficile after a dose of broad-spectrum antimicrobials.

■

Manufacture of biofuel and other energy products.

Bacteria are used to

digest corn and sugarcane in the manufacture of ethanol, and researchers are

exploring their use in transforming chemical energy into electrical energy in

microbial fuel cells.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

■

Agricultural applications.

Bacteria are used to digest grasses and other fodder

to make silage, a feed material that can be stored for use during winter months

when pastures are not available. Also, legume seeds, such as beans and peas,

are often coated with nitrogen-fixing bacteria prior to planting to ensure the

plants develop the proper nitrogen-fixing communities. A gene that encodes

the insecticidal delta-endotoxin of

Bacillus thuringiensis (a bacterium commonly

called Bt) has been inserted into certain crops to improve insect resistance, and

the bacteria themselves are sometimes sprinkled on crops to limit infestations.

Although the neurotoxins produced by

Clostridium botulinum have been a per-

sistent problem in the food canning industry, the botulinum toxin is used in the

medical and cosmetic (Botox) industries. Finally, the bacterial compound mon-

ensin is used to increase digestion efficiency in dairy cattle.

■

Food manufacture.

Microorganisms are put to work in food manufacture in

many different capacities, including fermentation processes and flavor

enhancement. Microbes are also significant in terms of food spoilage and

food safety. There have been enormous and frequent food recalls due to

microbial contamination.

■

Industrial applications.

Heat stable enzymes isolated from thermophilic bac-

teria, like Taq, lipase, esterases and others, have proven extremely useful in

biotechnology.

■

Wastewater treatment.

This exploits the natural capability of microorganisms

to degrade and recycle the essential elements on Earth. Millions of tons of

organic and inorganic waste are treated annually, and more and more of the

energy contained in this waste is recovered as biogas (methane). Important

advances have also been made in recycling of sulfur and heavy metals.

■

Others.

Microbes are used in the manufacture of biodegradable plastics,

green chemistry applications, and bacterial ice nucleation proteins are used in

snow manufacture.

PROGRESS IN UNDERSTANDING PATHOGENESIS AND
DISEASE ENABLED BY STUDY OF COMMUNITIES OF
MICROORGANISMS

Through studying human-associated microorganisms in sick and healthy individu-

als, scientists have discovered that microbial communities can define not only

human disease, but also human health. Subtle perturbations in the microbial com-

munities that exist on or in our bodies can give rise to illness or even death. Oral

health, in particular, relies on microbial community interactions; fluctuations in the

numbers of various microorganisms in the human mouth can bring on conditions

ranging from bad breath to periodontal disease and has also been associated with

systemic problems, such as arthritis and cardiovascular disease. In the gut, micro-

The Uncharted Microbial World: Microbes and Their Activities in the Environment

bial communities are now known to be necessary for the production and absorption

of certain nutrients, and their development is intricately linked with the develop-

ment of proper immune response and gut blood vessels. Studying microbial

communities has also illuminated the mechanisms by which opportunistic human

pathogens take hold. Scientists now know that vaginosis, for example, is not solely

due to invasion by a foreign pathogen, but often results from an imbalance in the

various members of the steady state (“normal”) vaginal microbial community that

allows an opportunist to grow unchecked. The use (and overuse) of antibiotics has

precipitated many of these insights into human health by triggering a change or

collapse in the steady state communities that exist in and on the human body.

Research into microbial communities in the environment has yielded insights and

tools for managing human health and disease. Scientists are continually discovering

new antibiotics, anti-inflammatories, and other bioactive compounds in the natural

communities associated with marine invertebrates and other niches. Community-

level studies have also revealed the dynamics of antibiotic resistance genes in

human-associated and environmental communities and how antibiotic resistance

genes and infections are spread. Studies of

Vibrio cholerae in its natural surround-

ings have revealed that the pathogen exists in communities on the surface of

comparatively large copepods. These creatures, along with their bacterial passen-

gers, are effectively removed by filtering contaminated water through several layers

of cloth, rendering the water safe to drink (Colwell et al., 2003).

Current work with human-associated microbial communities could lead to even

greater advancements in medicine. Researchers have identified a tentative associa-

tion between the composition of gut microbial communities and obesity. Further

studies may illuminate the interactions that bring about this correlation, possibly

allowing doctors to prescribe community-altering therapies for overweight patients.

Other experimental work has successfully fashioned a strain of the cavity-causing

bacterium

Streptococcus mutans that does not produce acid, a deficiency that

makes the strain unable to degrade teeth. Researchers are currently investigating

whether inoculating the mouth with acid deficient

S. mutans can force native, acid-

producing strains to extinction and lower the incidence of dental caries.

Scientists have learned that the health of animals and plants is also linked to the

state of their respective microbial communities. In cattle, rapid changes in diet

from grass to grain unbalance the microbial community of the rumen, leading to

poor production efficiency, rumen acidosis, and potentially death. A slow transition

allows the microbiota to adapt and preserves the health of the animal. Black band

disease in corals is caused by a consortium of microorganisms and may require

the contributions of polymer degraders, fermentative organisms, and sulfidogens.

Conditions like rumen acidosis and black band disease can only be understood by

studying entire communities of organisms.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

WELL-DEFINED MICROBIAL SYSTEMS AND COMMUNITIES
FOR STUDY

The work of “defining” certain microbial communities has been ongoing for sev-

eral decades, but progress toward this goal can be measured in different ways.

There are two levels in defining a community: achieving a process level

understanding (knowing what the community does) and achieving a compositional

understanding (knowing which species make up the community).

The oral microbiota is somewhat defined, in terms of both its processes and its

composition. The successional processes of these communities, whereby differ-

ent species of microorganisms come and go in the wake of disturbance, are well

characterized and reasonably well understood. Relationships between community

composition in the mouth and the oral health of the patient are also well estab-

lished, but gum disease does not appear to fit the paradigm established by Koch,

who introduced a set of postulates meant to test the causative relationship

between a microorganism and a disease. Rather than following a one-microbe-one-

disease trajectory, research shows that gum disease is the result of complex

multispecies community interactions (Jenkinson and Lamont, 2005). Some organ-

isms are associated with health, while others are associated with disease.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

KOCH’S POSTULATES

Three rules for experimental proof of the pathogenicity of an organism

were presented in 1883 by the German bacteriologist, Robert Koch. A

fourth was appended by E.F. Smith in 1905. Briefly, these rules state:

The suspected causal organism must be constantly associated with

the disease.

The suspected causal organism must be isolated from an infected

plant and grown in pure culture.

When a healthy susceptible host is inoculated with the pathogen from

pure culture, symptoms of the original disease must develop.

The same pathogen must be re-isolated from plants infected under

experimental conditions.

These rules of proof are often referred to as “Koch’s Postulates.”

Other microbial communities that could be labeled “well-defined,” in terms of

their composition and the processes they carry out, include the communities that

inhabit the rumen of cattle, anaerobic sludge granules and other waste water treat-

ment communities, deep subsurface sediment communities, the symbiotic

community of medicinal leeches, and the communities in certain extreme environ-

ments, such as acid mine drainage pools at Iron Mountain in California (Tyson et al.,

2004). The communities important for certain food products (including yogurt, pick-

les, sauerkraut, and cheese) are well-defined, but the role of microbial communities

in permitting pathogens to survive or coexist with natural communities in food is

not understood. Although the processes and compositions of each of these com-

munities have been studied extensively, less is known about the effects of

perturbation (including the addition of antibiotics and other micropollutants, for

example) on these communities.

Obtaining a robust, precise, high-resolution community profile remains a

challenge even in the simplest of communities. An understanding of the

processes—and which organisms are carrying them out—is more meaningful and

important than an exhaustive census of each and every organism in a community.

Phages (viruses that infect bacteria or archaea) probably play a profound role in

the organization and functions of microbial communities, but their diversity and

host range are seldom evaluated in community-level studies, and science knows

relatively little about them.

Selecting “easy” communities for study

No single microbial community will meet all of the criteria for an “easy” system

to study, but the following is a list of guidelines for targeting manageable commu-

nities for these investigations. (Not all criteria are equally important for different

types of studies, and discovery-driven studies, which should not necessarily aim to

meet these criteria, maintain an important place in research.) Communities

targeted for complete characterization should be:

■

Clearly delineated by physical boundaries.

The boundaries of a microbial

community should be easily delineated so that researchers can determine with

precision which microorganisms are part of the community and which are not.

The human digestive tract is one example of a clearly bounded community.

■

Reproducible.

Research data on a particular microbial community should be

reproducible among different laboratories.

■

Replicable.

Microbial systems for which replicates can be obtained (including

termite guts or the oral cavity) are preferable to those which cannot (including

unique geological features, like Lake Michigan, for example).

■

Manipulable.

For research purposes, the ideal microbial community is capable

of being altered for experimental purposes.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

■

Accessible.

Microbial communities targeted for study should be physically

accessible for sampling purposes and technically accessible for measuring

inputs and outputs of the system.

■

Scaled appropriately.

The physiochemical that a particular community of

microbes interacts with can vary in size, depending on the type of community. It

is helpful if this environment is sufficiently large to be amenable to

measurement and characterization.

■

Growing and active.

The activities and fluxes of active microbial communities are,

in general, easier to measure than they are in less active communities.

■

The key fluxes into and out of the system should be known.

Substantive

and credible background information is helpful.

■

Manageably diverse.

This is technology-dependent.

■

Stable over reasonably long time frames.

■

Genetically tractable.

WHISTLEBLOWERS: MICROBES AS SIGNALS OF
ENVIRONMENTAL DEGRADATION

Microbes and microbial communities are intimately linked with their environ-

ments, and in many cases, ecological disturbances are reflected in changes in the

abundance or behavior of these organisms before detection of other outward

signs. Toxic algal blooms, for example, can result from nutrient pollution in coastal

marine environments. Under the right conditions, these blooms may also signify

the presence of

V. cholerae (the bacterium that causes cholera) in coastal waters.

Pathogen-related illnesses in wildlife can also signify ecological disturbance; black

band disease and bleaching (by Vibrio species) in coral, for example, has been

linked to increases in water temperature resulting from global climate change.

Malodorous products from sulfur reducing bacteria can signify tainted water or

even spoiled food products and generally serve as a good indicator of “things and

places to avoid.” The presence of genes for degrading human-made chemicals

could potentially be used to identify areas of chemical contamination. Fecal col-

iform bacteria and other organisms found in the human gut are routinely used to

identify sewage contamination in water. Lichens, which are extremely sensitive to

atmospheric sulfur dioxide, can be used to detect elevated levels of this

substance in areas of concern. The pollution-sensitive bacterium

Thioploca gradu-

ally disappeared from Lake Erie waters as that lake became more and more

eutrophied (Larkin and Strohl, 1983), serving as an indicator of adverse conditions.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

UNEXPLORED ACTIVITIES ENCODED BY MICROBIAL GENES

Despite the extensive amount of work done to characterize microbes and their

communities, many unknowns remain at the genetic level. Today, as much as a third

of the genetic content of even the most thoroughly studied organisms remains to be

annotated, so although the sequences of many genes (in many organisms) are

known, the functions and significance of these genes remain unresolved.

Judging from what is already known of the functional diversity of the microbial

world, the potential locked up in poorly understood genes is considerable. These

“unknown” genes could reveal useful processes for industry, bring to light

processes that science has not yet conceived of, fill gaps in our understanding of

metabolic pathways, be responsible for disease processes not yet understood, and

unearth previously unknown functions of microorganisms in the environment.

Since the functions of many of the genes in well-known microbial species are not

known, many of the current predictions about microbial metabolism may be

incomplete, and serious limits are placed on the ability to carry out genomic and

metagenomic studies. Every new gene that is characterized has multiplicative

value in science’s ability to identify similar, related genes in other organisms.

Functional studies of genes in the past 20 years show that, although general cate-

gories of function can often be assigned, specific functions of genes are often not

predictable from their sequences, so care must be taken in extrapolating the func-

tions of unknown genes from the gene sequences of known proteins.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

RESEARCH ISSUES

The future of research in microbial biology is inspiring. Research concerns

include the large fraction of microbial life that remains uncultivated, accurate con-

struction and deconstruction of microbial communities for study in the laboratory,

predicting the effects and outcomes of disturbance in microbial communities, find-

ing the functions of unknown microbial genes, spatial scale in community

interactions, co-evolution of microbial species, the role of genomics in microbiol-

ogy research, and the ways in which the principles of population biology and

evolution can be applied in microbiology.

UNCULTIVATED MICROORGANISMS: FUTURE BENEFITS FOR
THE PLANET AND COMMERCIAL PURPOSES

Microbiologists estimate that over 90% of bacteria are not captured by the cur-

rent collection of cultivation techniques, a deficit that leaves the vast majority of

the microbial world largely hidden from the eyes of science (Staley and Konopka,

1985). These uncultivated microorganisms could potentially be useful for increas-

ing the sustainability of human activities on this planet and for a number of

commercial purposes.

There are great hopes for microbes to repair the damage of human activities on

the health of ecosystems. Microbes may eventually be isolated for degrading and

detoxifying recalcitrant wastes that cannot currently be treated, for example, or the

powers of uncultivated nitrifiers in the soil could be directed to produce more fertile

farmland without the use of chemical fertilizers or other nitrogen-rich amendments.

Other uncultivated microorganisms may eventually be induced to impact the atmos-

phere and climate in some beneficial way. Oxygenic phototrophs in the oceans, for

example, could be encouraged to generate more oxygen, or aerobic methane oxi-

dizers in soils could be induced to consume a greater share of the atmospheric

methane than they already bear. In all probability, science is still not aware of all the

ways in which microorganisms could be useful for managing greenhouse gases.

In the future, the most significant commercial feature of uncultivated microbes

may be their novel metabolites, including antimicrobials and other bioactive com-

pounds. Genome studies of cultivated bacteria hint at the wealth of compounds

waiting to be discovered.

Myxococcus xanthus alone appears to have 50-60 path-

ways for the synthesis of small molecules, most of which have yet to be

characterized in any way.

Streptomyces avermitilis makes several useful

compounds that we know about, but it is suspected of making 30 other small mol-

ecules that may be valuable.

As demand for novel compounds grows, it will become increasingly important to

develop innovative techniques for accessing them. Isolating DNA from environmen-

tal samples and then inducing another microorganism to express those genes has

The Uncharted Microbial World: Microbes and Their Activities in the Environment

been a successful approach in the past, but it remains impossible in most cases to

isolate and express entire pathways, due to a lack of suitable expression hosts. It

may be important to develop more and better hosts for expression of uncultivated

microbial genes in the future.

Targeting uncultivated microorganisms and their animal hosts in the search for

bioactive compounds is often rewarding, since symbionts and hosts often develop

chemical defenses to prevent other microbes from establishing spurious

infections. It is important to note that useful compounds that may eventually come

from uncultivated microorganisms are not limited to antibiotics; microorganisms

produce an incredibly diverse array of bioactive compounds. The availability of

appropriate screening methods to test the activities of these substances currently

limits the ability to find and use them.

Novel enzymes, which may eventually be identified and from DNA extracted

from uncultivated microorganisms in the environment, also present a great poten-

tial resource for commercial use. The symbionts of termite guts, for example, are

thought to be species-specific, and each symbiotic community produces a vast

number of biocatalysts for wood degradation—a process that is critical for any

number of industrial processes, including ethanol manufacturing. Considering that

there are over 2,600 different species of termites, the commercial resource these

communities represent is impressive. A number of enzymes from other unculti-

vated microorganisms are already on the market, including enzymes that can

tolerate detergents and heat.

An understanding of microbial ecology/diversity should enable more effective devel-

opment of both probiotics and prebiotics. (Prebiotics are defined as nondigestible

food ingredients that may beneficially affect the host by selectively stimulating the

growth and/or the activity of a limited number of bacteria in the colon.)

Barriers to cultivation

The ability to cultivate the majority of microorganisms found in the environment

is likely impeded by difficulties in replicating the conditions the microorganisms

prefer, genetic and ecological barriers, overgrowth by nontarget organisms, and

basic human error, including researcher impatience. Many bacteria, archaea,

viruses, and microscopic eukaryotes prosper only in exceedingly narrow sets of

conditions, and replicating these microbial environments in controlled laboratory

settings is very difficult. The presence of trace contaminants, incorrect concentra-

tions of nutrients, the absence of signal molecules, failure to recreate

microbe-microbe or microbe-host interactions, and failure to provide the right nutri-

ents can all work against the researcher trying to isolate a particular strain.

Growing microorganisms may also change the chemical conditions of the growth

media, thereby inhibiting their own growth. The unknown presence of lysogenic

phage, which selectively destroys certain species of bacteria and archaea, may

also impede cultivation.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

Genetic and ecological barriers to cultivation include the dependence of

microorganisms on symbiotic or mutualistic settings that cannot realistically be

recreated in the laboratory. Cell density-dependent microbial growth can also pre-

vent researchers from collecting sufficient organisms for study. Batch culture

methods, which dominate the established methods for cultivation, can impair cul-

tivation by preventing the isolation of organisms that are not adapted to grow to

high densities. Still other organisms require a minimum inoculum size that cannot

be achieved with today’s methods. It is also possible that some organisms have

complex life strategies that make growth in simple laboratory-devised habitats

impossible. In these microbes, reproduction may be triggered by seasonal or envi-

ronmental cues that researchers do not (or cannot) replicate.

Oftentimes, the organism of interest does not have time to establish itself under

the culture conditions before the flask is taken over by other, faster growing organ-

isms. Mutations can also arise that change the strain of interest in favor of faster

growth and greater fitness in the culture environment. For example, under the

selective pressures of cultivation, dellovibrios that are largely host dependent can

acquire the ability to grow independently or other talents that are completely dif-

ferent from the

in situ organism.

Human error, including failure to wait a sufficient length of time for cultures to grow

and a lack of imagination and creativity in designing cultivation conditions, probably

prevents successful cultivation in some cases. Moreover, although the ability to study

cultivated microorganisms has enabled many of the most important advances in the

field, a bias in funding agencies in favor of molecular, culture-independent techniques

may ultimately discourage cultivation efforts.

It may not be possible to separate microorganisms that have co-evolved to fit

one another’s functions. Symbionts and pathogens, for example, have developed

smaller, sparer genomes as their relationships with other organisms allowed them

to do so, and they may not be able to survive outside of these carefully balanced

arrangements. Microbiology needs to move beyond its dependence on pure cul-

tures of organisms and appreciate the value of defined but mixed communities.

IS THE WHOLE A SUM OF ITS PARTS? DECONSTRUCTING
AND MODIFYING MICROBIAL COMMUNITIES

In general, it will be difficult to isolate and identify every individual component of

a natural microbial system. However, research on an intact microbial community

can get extremely complicated if the community or the analysis is not distilled to

target the process or organisms of greatest interest. Community deconstruction,

including studies of waste water systems, pulp mill systems, and artificial rumens,

has successfully illuminated community processes and functions of a number of

systems. There is unsurpassed clarity in the interpretation of results from studies

of pure cultures; using a co-culture of two organisms squares the complexity of a

The Uncharted Microbial World: Microbes and Their Activities in the Environment

pure culture, and a culture of three organisms cubes the complexity. Results from

pure culture studies can lead back to the intact community by generating hypothe-

ses that are testable on

in situ processes.

Although there are significant technical difficulties in dismantling communities, it

is possible, and there are far more tools available for these projects now than ever

before, including single cell optical tweezers, improved FISH and flow cytometry

techniques, and other single cell technologies.

The availability of long-term microbiological study sites is important for studies

that seek to deconstruct natural microbial communities. Model systems in which a

researcher can start simply and add additional complexity one step at a time are

also helpful. Suitable model systems that have been or could be used include:

■

waste water treatment communities,

■

rumen communities,

■

some chemostat communities,

■

winogradsky columns, and

■

phytoplankton communities.

Ideally, microbiologists would develop a set of rules (or “Koch’s postulates for

communities”) for testing success in deconstructing communities. Possible tenets

of this system could include:

Gain an understanding of the environment, including chemical processes and

members,

Acquire the isolates that carry out these processes, and

Return to the environment and demonstrate that the selected organisms are

responsible for the processes of interest.

A deconstructed community that is subsequently reconstructed from constituent

key players should retain the key characteristics and process rates of the original, and

there is probably redundancy in the microorganisms that can be targeted to accomplish

this. Success in deconstructing a microbial community can often be measured by

reproducibility and observation of the predicted results.

Although deconstruction can offer valuable insights into microbial communities,

researchers need to bear in mind that, even in laboratory cultures, populations of

microorganisms in the environment are never genetically identical. Even single

nucleotide polymorphisms, in which one base out of thousands differs from that of

The Uncharted Microbial World: Microbes and Their Activities in the Environment

the most closely related organism, can have a huge impact on the characteristics of

a microbe, so subtle genetic differences among members of a microbial population

can be meaningful to community function. When isolating an organism from a com-

munity, researchers generally whittle a population down to a single ribotype (genetic

type), but it is not known how well this organism represents the rest of the popula-

tion. Moreover, there is a chance that the process of dissecting a community will

introduce genetic changes to a given member (see

Barriers to Cultivation

, above)

by spontaneous mutation or gene/plasmid loss.

There are alternatives to community deconstruction for pursuing an understanding of

microbial communities. In the “encapsulation approach,” individual cells can be encap-

sulated within small agarose beads, one cell per bead, then a meta-community can be

assembled by combining certain beads and incubating under conditions like those

found

in situ. After clonal replication within the beads, individual beads may be

removed for characterization. This approach permits cross-talk among the different

members of the community during incubation and allows the isolation of single cells,

but alters the physical and spatial interactions of the community.

Another alternative to deconstruction is to selectively remove (or “knock out”) an

individual member of a community using antibiotics, phage, antibodies, filtration by

size, or another tool, and then observe community function to determine the role of

that member. Metagenomics, proteomics, or transcriptomics approaches may be

used to reveal genetic or protein components of the community. Flow cytometry can

also be used in these efforts as an analytical tool or as a cell sorting technique to

physically separate individual components.

A microbial community does not necessarily need to be disassembled in order to

learn about it. Microbiologists could take a cue from ecology research by manipu-

lating individual parameters in a system and observing the effect on community

structure.

Constructing artificial communities

Constructing artificial microbial communities can be revealing. In general, the basic

properties and individual processes of a community can be recompiled relatively easily

by drawing representatives from different categories of metabolisms. Phenomena and

processes that can be studied in artificially constructed systems include:

■

Host-symbiont and host-pathogen interactions,

■

Metabolic interactions (including syntrophy),

■

Predator-prey interactions,

■

Evolution (either experimentally or

in silico using computer models),

■

Quorum sensing,

The Uncharted Microbial World: Microbes and Their Activities in the Environment

■

Dehalogenation,

■

Gene exchange, and

■

Biofilm formation.

Gnotobiotic animals, which are born and raised in a sterile environment, and gnoto-

biotic mice in particular, area good platform on which to build complex microbial

systems one step at a time.

Bacteroides’ metabolic interaction, for example, has

been studied in the mouse gut, where they stimulate complex carbohydrate produc-

tion and then metabolize these sugars.

Identifying communities that impact human health and environmental safety

that should be studied at the community level

There are some questions about microbial communities that currently cannot be

answered using a reductionist approach that treats the whole as a sum of its

parts. Studies of

V. cholerae within the context of its microbial community, for

example, led to the discovery that the bacterium tends to adhere to the surface of

much larger zooplankton, making the bacterium easy to filter out using a folded

piece of fabric. This simple treatment, which has saved countless lives since it

was recommended, may not have been considered without information gathered

in community-level studies.

Among the microbial communities that impact environmental integrity and

human health, it is important to identify those that are most in need of study as

intact entities. Communities can be identified and prioritized by evaluating them

according to ecological criteria (Is it widespread? Does it perform a keystone func-

tion? Does it perform a quantitatively important function?) or according to the

degree to which they impact human health and well being. Epidemiologists and

experts in exposure assessment can also play an important role here.

Almost every agent of infectious disease needs to be studied at the community

level, since pathogens either arise from or pass through environmental communi-

ties during the infection process. Virulence (the degree to which a pathogen is able

to cause disease) is partly determined by community interactions, and in most

cases it is not known which factors enhance or inhibit virulence. It is particularly

critical to understand community dynamics in cases where natural communities

serve as reservoirs of disease, thereby maintaining a baseline level of infection in

humans despite treatment and eradication efforts. Communities that expose

humans to toxic microbial products also require study. Some examples of

pathogen-laden communities that should be studied more thoroughly include com-

munities in coastal environments (which have become breeding grounds for

pathogens), the communities of the human gut and mouth, and the communities

that thrive in moist air conditioning ducts and water lines in homes.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

The impact of microbial communities on chemical contaminants in the environ-

ment is another area where study is needed. Some compounds are degraded or

detoxified only by communities of organisms working together, while other com-

pounds resist degradation or are made more toxic by microbial communities.

Research needs to address these issues and determine which compounds are

broken down and which are made more toxic and how microbial communities

accomplish these transformations.

Researchers need to study drug effectiveness on pathogens within the context

of their communities in the human body. Chemicals that are effective on

pathogens in a Petri plate in the laboratory are not always effective when used to

treat an infection. It is also important to consider microbial communities present in

agricultural animals as these are often the source of food contamination and reser-

voirs of human disease.

Examples of directing and modifying natural communities

A microbial community may be designed and constructed piece by piece to carry

out a specific process, but it is also possible to alter or direct an intact community to

achieve the same goal. There are a number of examples in which this type of man-

agement has been accomplished successfully. Very good examples are various civil

and industrial wastewater treatment plants where a complex but selected microbial

community is doing its daily or seasonal job of sewage or industrial waste degrada-

tion. It is possible, for instance, to stimulate methanotrophic activity in a community

in order to boost the co-metabolism of trichloroethylene, and

Dehalococcoides bacte-

ria can be stimulated or added to a community to boost anaerobic dehalogenation of

tetrachloroethylene. Other examples of microbial community alteration include:

■

The addition of

probiotic microorganisms

to the microbial consortia of the

gut in humans and animals, which are commonly administered to aid digestion

or fight pathogens,

■

The addition of

Geobacter species

to stimulate microbial uranium precipitation

in ground water systems,

■

Coating legume seeds with

Rhizobium bacteria

to prompt colonization of the

plant and improve plant productivity, and

■

Use of

Bacillus cereus

for biocontrol of fungal infections in plants.

In the future, microbial community manipulation may successfully replace acid-

forming species of

Streptococcus mutans in the mouth with strains that are less

prone to forming dental caries. Phage control of bacteria is another promising area

of research. The FDA recently approved the use of a Listeria phage as a food

safety additive (Bren, 2007).

The Uncharted Microbial World: Microbes and Their Activities in the Environment

MICROBIAL COMMUNITY STABILITY

The stability of a microbial community, that is, its ability to maintain species com-

position and processes within certain bounds, mostly relies on three basic features:

the variability of the environment, the diversity of the community, and the interac-

tions among community members.

Environmental variation has a profound influence on gene expression, species

composition, and the relative abundance of members within microbial communi-

ties. High microbial community diversity, specifically functional diversity and

redundancy, is critical to overcoming perturbation. There are many settings, includ-

ing sewage treatment communities, the human microflora (including the gut,

mouth, and vagina), bioremediation, and agriculture, in which a better knowledge

of stability-diversity connections is needed. The presence of cells in resting phase

may also boost a community’s stability. Since macromolecule synthesis, transport,

and metabolism, in general, are greatly slowed in resting microbes, slow-growing

and non-growing organisms may not have to copy with chemical contaminants to

the degree actively-growing members do. Systematic studies of the connection

between diversity and stability are few and far between, and much of what is

known on this topic comes from anecdotal evidence. It is not known whether key-

stone groups (without which the community could not recover) exist, for example.

This situation may change as new high throughput methods for monitoring com-

munity composition, like DNA microarrays, become available.

Predicting the outcome of disturbance

Very little is currently known about the specific outcomes of disturbance in

microbial communities, even in communities that are directly related to human

health and well being, like wastewater treatment and the human gut. In Los Ange-

les, for example, a seemingly innocuous ban on metals dumping in municipal

sewers changed the microbial system drastically, encouraging the unpredicted

growth of

Thiobacillus denitrificans, which in turn promoted widespread corrosion

of sewer pipes. Mathematical models for predicting the results of community-level

disturbance are very limited in number and scope.

Research needs to address this deficit in the predictability of microbial systems

by evaluating model systems at “baseline” or preperturbation state and then

directly measuring the effects of perturbation in these systems.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

UNLOCKING THE FUNCTIONS OF UNKNOWN
MICROBIAL GENES

A great deal of uncertainty remains in drawing the link between a nucleotide

sequence in the genome of a microorganism and the processes that organism car-

ries out in its environment. The difficulty of this problem can be illustrated by past

experience with a thoroughly studied model organism; the genome of

Escherichia

coli K12, for which mutants have been created for many open reading frames,

maintains a large fraction of unknown genes. Apparently, all the standard tools have

been applied to studying this organism, but little is currently understood.

There has obviously been some progress in identifying gene functions, however,

and several different approaches to the problem that have met with success can

provide guidance for making future discoveries. These approaches include:

■

Relate gene expression patterns to environmental (chemical/physical/biotic)

parameters like nutrient limitation. Other, more qualitative factors, like where

the gene is expressed within the cell or microcolony or consortium, can also

point to a gene’s function.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

PREDICTING RESPONSES FROM AN UNCHARACTERIZED
COMMUNITY: WHAT COMES OUT OF THE BLACK BOX?

In certain settings, the composition and processes of a particular commu-

nity do not need to be understood in detail in order to predict community

response to change, since experience and the laws of thermodynamics are

sufficiently instructive. Wastewater treatment and many food fermentation

processes are examples of longstanding successes in this capacity. How-

ever, there is now concern that existing wastewater treatment systems are

not effectively removing pharmaceuticals (e.g., birth control, antibiotics)

and food additives. These are seriously impacting natural systems to which

the treated water is released. Thus, it is essential that established micro-

bially-based treatment processes be revisited to ensure that they retain

function as the composition of waste streams change with changes materi-

als discharged by both households and industry. In most cases, however, it

is not acceptable to treat microbial communities as black boxes with

unknown contents and predictable outputs. A novel synthetic chemical, for

example, may be introduced to soil or water, but without understanding

soil and water microbial communities, their metabolic resources, and the

processes they carry out is not possible. Research should continue to

investigate the composition and activities of microbial communities in

order to predict the reactions of communities to disturbance.

■

Construct a strain with a knockout mutation for a known gene, then examine

changes in the relative amounts of transcribed genes to infer which of the

unknown genes are associated with the known, interrupted gene.

■

Study adaptive radiation within a clade to understand what functions have

been selected.

■

Create a metabolic model of an organism, then create targeted knockouts of

unknown genes and reexamine metabolic fluxes within the cell.

■

Use transposon mutagenesis to create libraries of mutants, then expose the

mutants to selective regimes to determine via microarray hybridization which

mutants are selected against.

■

Use protein structure modeling to predict functions.

SPATIAL SCALE OF MICROBIAL COMMUNITY FUNCTIONS
AND INTERACTIONS

When designing an investigation of a microbial community, it is important to

determine the spatial scales on which the community and the system operates,

since sampling and other factors rely directly on the scaling of the system. For

example, in biofilms and sediments the use of microelectrodes has revealed that

steep gradients of oxygen or sulfide can exist over a few millimeters or less.

Unfortunately, there is no stock set of rules to follow for determining the scales

relevant in all community-oriented research, but there are some general guidelines

that can be tailored on a case-by-case basis. They include:

■

Identify the spatial constraints

on the community by making chemical and

physical measurements. Do multiscale sampling and determine the scale at

which the desired property or process is relevant. However, due to the small

scales involved and the limitations of the current generation of technology,

researchers often have limited ability to determine the relevant parameters at

the right scales.

■

Measure the inputs and outputs

of the system and determine the scaling.

Technology also limits the ability to get this done in some settings.

■

Identify how rates and fluxes change with scale

to see if change with

scale is predictable or if relationships are scale-dependent. For example, in a

small, well-stirred fermentor, the mixing time of a growth limiting nutrient

may be ignored relative to its rate of biological consumption, whereas in a

large fermentor, the mixing time may become the rate-limiting factor and/or

lead to strong fluctuations of this nutrient in the growth vessel. In such

cases, the proper engineering approach is to perform mathematical modeling

The Uncharted Microbial World: Microbes and Their Activities in the Environment

and experimental verification to identify the critical time constants (bottle

necks) of the process.

■

Use mathematical modeling

to predict scales when possible.

Microscale interactions among species are critical in almost all microbial

systems, and they play roles in many different phenomena related to microbial

communities, including competition for nutrients, symbiotic relationships, quorum

sensing, gene transfer (especially by conjugation), interspecies hydrogen transfer,

viral infection, predation, cross feeding, antibiotic production, and density-depend-

ent growth. Bulk measurements will often miss microscale activities and

underestimate the rates of many microbial transformations. For example, cluster-

ing around particulate nutrient sources in the oceans, where nutrient

concentrations are relatively high, can lead to a greater growth rate than would be

estimated from measurements of the bulk solution. Modeling suggests that if

these interactions are taken into account, the overall carbon turnover of the

oceans would have to be scaled up by a factor of two or three.

In systems in which the black box approach (which treats a microbial system as

a simple unit with inputs and outputs) works well at answering the relevant ques-

tions, as it does with issues of substrate conversion rates in a bioreactor; for

example, microscale interactions may be neglected.

Tools for studying microscale interactions include reporter genes (which are

extremely useful for studying biofilms), fluorescence

in situ hybridization (FISH)

coupled with digital image analysis, FISH-microautoradiography and FISH-Raman

spectroscopy to reveal cross feeding, fluorescent quenching techniques (which

can be used to study physical interactions), microelectrodes, microoptodes, single

cell reporter tools (e.g., green fluorescent protein system and carbon source

reporters), chemical-specific dyes, and secondary ion mass spectroscopy. More

work is needed to enhance these technologies. Further work is also needed to

miniaturize scanning electron microscopy and other microscopic tools, develop

biosensors, microsensors, and gas sensors, and to generally improve the ability to

make

in situ environmental measurements at appropriate scales.

CO-EVOLUTION IN INTERACTING MICROBIAL SPECIES

Microorganisms are constantly co-evolving to improve their standing in the con-

text of competitive and symbiotic relationships with other microbes. Arms races

with antibiotics can drive competing microbes to create new weapons and, in turn,

create new defenses. Another form of antagonism—phage-host interactions—

determines the success or failure of certain genotypes by influencing host

population size and other factors. Succession in oral biofilms is partly determined

by ligand-receptor binding that determines physical assembly of different species.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

Evolved changes in microorganisms can reverberate through a microbial community

and cause the adaptive landscape to shift for other organisms. There may be synergis-

tic interactions in which an organism has reached a fitness peak and any changes the

organism makes will decrease the fitness of the interacting partners. Involvement in an

interaction like this may also decrease the ability of an organism to act as a generalist

within the community, since any new adaptation moves it out of sync with a partner

that it needs to survive.

MICROBIAL COMMUNITIES IN AGRICULTURAL SYSTEMS—
MANAGING HUMAN IMPACTS

Intensive agricultural practices are known to have serious ecological impacts, and

microbial communities are usually the first to feel these effects. The concern here

lies not necessarily in the impacts to the microorganisms themselves, but in the

implications these effects hold for livestock health, human health, and the environ-

ment. Examples of the impacts of agriculture on microbial communities include the

fostering of antibiotic resistant bacteria in livestock-associated consortia, which can

eventually lead to untreatable infections in animals as well as humans. Pathogen

loads, including human pathogens, can be drastically elevated in lakes and soils near

industrial farms. Nutrient pollution in water bodies and groundwater down gradient

of farms can cause eutrophication—blooms of toxic dinoflagellates and toxic

cyanobacteria.

By developing more microbial sentinels to display specific stress responses

indicative of system disturbance, monitoring strategies could be devised to make

use of impacts to microbial communities and head off consequent impacts to

animals, humans, and the environment.

GENOMICS AND ENVIRONMENTAL MICROORGANISMS

The ability to sequence and interrogate entire microbial genomes has revolution-

ized microbiology. Genome sequencing, on its own, is important for examining the

evolution of strains over relatively short periods; researchers can use sequencing to

follow genetic adaptations over time in a single strain. The tools of genomics, which

study an organism’s genome for information regarding the organism’s activities, are

superb hypothesis generators.

Comparative genomics

, for example, allows estima-

tion of the mechanisms and rates of genome diversification, adaptation, and

speciation. Metagenomics, in which groups of genomes from diverse organisms are

studied as a whole, has revealed many new details, including the importance of

transposons in bacteriophages.

Environmental genomics

can expose the differen-

tial distribution of genes and metabolic types in different types of environments.

Although the cost of sequencing genomes is coming down, in the future,

researchers must continue to be discerning about which microorganisms they select

The Uncharted Microbial World: Microbes and Their Activities in the Environment

for sequencing. Among the genome sequences currently available in the public

domain, there is a marked overrepresentation by human pathogens, and there are

many underrepresented groups that remain to be characterized. It is important to

sequence clusters of closely related organisms to reveal the eco-physiological prop-

erties that maintain diversity within a family or species, as well as more divergent

organisms. Viruses are agents of gene transfer in microbial communities and also

are important targets for sequencing. It is important to ground predictions arising

from genomics with experiments in the laboratory wherever possible.

The pace of genome sequencing is brisk, but the current lack of resources in bioin-

formatics and data analysis (which are used to make sense of raw sequence data)

pose a serious limitation on progress in this field. More work is particularly needed in

functional genomics, which is used to link sequences with specific functions.

APPLYING THE PRINCIPLES OF POPULATION BIOLOGY AND
EVOLUTION TO THE STUDY OF MICROBIAL COMMUNITIES

Although microbes share many characteristics with larger organisms, science

has historically dealt with them very differently, excluding microbes and their com-

munities from the scrutiny afforded in the endeavors of population biology,

ecology, and evolution studies. Scientists now recognize that it is useful and

appropriate to apply the principles learned in these fields to the study of microor-

ganisms, since a deep understanding of ecology, natural history, and physiology is

perhaps the most important foundation for advancing understanding of gene func-

tion. Some of the fundamental themes of microbiology that should be addressed

using these principles include:

■

The definition of a microbial species.

Questions about the nature of the

species concept as it applies to microbes, how species are formed, and how to

define diversity should all be dealt with.

■

The origins of novel phenotypes and how they are maintained.

Research

should address the how genes are carried into a community, the nature of

genetic drift in microorganisms, the environmental parameters that control the

selection of phenotypes, and whether or not there are measurable rates of

gene transfer in nature.

■

The biogeography of microorganisms.

It is clear that as far as principle

types of metabolism is concerned the old axiom “everything is everywhere” is

undoubtedly true, but it is unclear whether any given microbial

type/(sub)species can be found wherever one looks. When it comes to the

fine detail of niche differentiation it may well be that specific variants of

species/families operate on a more localized basis (Foti et al., FEMS Microbial

Ecology 56, 95-101 (2006)).

The Uncharted Microbial World: Microbes and Their Activities in the Environment

■

Improving the predictive capacity with respect to the activities of micro-

bial communities.

Ecology, which focuses on developing predictive

hypotheses, has a great deal to offer in the study of microorganisms.

■

Resolution and precision in community analysis.

The practice of blind test-

ing in data collection and in data analysis is standard in ecology research, but it

has eluded microbiology, a shortfall that probably has an impact on standardi-

zation of methods.

METHODOLOGIES AND TECHNIQUES

Microbiology is both enabled and limited by technology. Although the technology

and methodology support for environmental microbiology has progressed quickly

in recent years, the challenges that lie ahead cannot be met without further

progress in cultivation methods, functional genomics, and other techniques.

NEEDED METHODS FOR ISOLATION AND ENRICHMENT

Researchers need access to better cultivation technologies in order to

adequately explore the world of uncultivated organisms that hold promise for med-

icine, industry, and the environment. Microbiology requires more methods that

allow researchers to mimic the conditions microorganisms encounter in their natu-

ral habitats, particularly the conditions in low nutrient environments and in nutrient

The Uncharted Microbial World: Microbes and Their Activities in the Environment

SPECIFIC TOPICS IN MICROBIOLOGY TO ADDRESS
USING PRINCIPLES FROM POPULATION BIOLOGY,
ECOLOGY, AND EVOLUTION

More specific topics that could be addressed using principles from

population biology, ecology, and evolution include:

■

The mechanisms of community assembly and interaction,

■

How predation dictates structure in microbial communities,

■

Host switching by bacteria and viruses (e.g., SARS),

■

Antibiotic resistance, including gain and loss of resistance from

populations,

■

Emerging infectious diseases,

■

Co-evolution of hosts and parasites,

■

The effect of different kinds of selection on microbes,

■

Development of new vaccines (the phenomenon of herd immunity

needs to be understood), and

■

The environmental manifestations of organismal interactions.

and oxygen gradients that form at interfaces and surfaces. In the environment,

microorganisms probably rely heavily on the activities of other species, but these

biotic services are often neglected in laboratory culture conditions. Cultivation

techniques that provide these partners for the organisms of interest are needed.

situ culture technologies, like nutrient-permeable bags, encourage the replication

of the organism of interest in a growth chamber inserted into a microbial system.

More and better refined technologies like this are needed.

Flow cytometry, which enables researchers to separate cells either on the basis of

their intrinsic properties, like cell shape, or on the basis of fluorescent tags, can be

extremely useful in selective cultivation, but continued improvements are needed.

For example, it would be extremely helpful if cells could be sorted after FISH identifi-

cation and if this FISH staining would not kill the cells (as current protocols do) and,

thus, the sorted cells could be used for cultivation. There is a single paper which

postulates that FISH staining of living cells might be possible (Nucleic Acids Res.

2005 33:4978-86). Laser capture microscopy with optical tweezers is a promising

technique for aiding cultivation, too, but here again, improvements are needed. In an

ideal situation, scientists would be able to use lasers to capture organisms sharing

the same general habitat, sequence those organisms, perform the bioinformatics

research to reconstruct their metabolisms, then sample environmental parameters to

ultimately decide how to cultivate the organisms in the laboratory.

Finally, research has long neglected the value of cultivating consortia of microor-

ganisms, partly because of a lack of suitable techniques. New methods are

needed that make it easier to exploit meta-interactions and grow microbial consor-

tia in the laboratory.

NEEDS FOR

IN SITU WORK

Oftentimes in microbiology, it is best to study the organisms of interest within

the context of their habitats,

in situ. This type of work can be difficult, and

researchers often find their investigations are limited by technology. More sensi-

tive methods for making

in situ measurements are needed, as are improvements

in the ability to make real time measurements of environmental conditions.

Research would benefit from miniaturized instrumentation for making real-time,

long-term

in situ measurements of cellular responses. Researchers also need bet-

ter tools for monitoring substrate consumption and compound excretion patterns,

gene expression, and the proteome in the environment.

BIOINFORMATICS NEEDS

Bioinformatics, which applies mathematics and computer science to problems

in biology, has opened up grand new possibilities for exploring the microbial

world, but more bioinformatics tools are needed to facilitate further progress and

The Uncharted Microbial World: Microbes and Their Activities in the Environment

there are a number of specific areas where improvements can be made.

Research needs include:

■

Methods for rapidly identifying nonfunctional genes (“junk DNA”),

■

New algorithms for assembling genomes from short sequence reads and from

metagenomic data,

■

Comparative bioinformatics approaches to identifying metabolic modules

that might allow inference of novel function of specific genes that are part

of that module.

OTHER NEEDED TECHNOLOGIES AND METHODS

Other key developments in technology and methodology to enable future

research on microorganisms and their activities in the environment include:

■

Improved

high throughput molecular and proteomic techniques

for moni-

toring community compositions.

■

Better

single cell genome replication techniques

that give higher coverage

and fewer errors. Current rolling circle genome replication technologies oper-

ate at low temperatures, a situation that encourages non-specific binding and

imperfect replication. Thermophilic phages should be mined for more suit-

able polymerases.

■

“Live” stains

that can identify uncultured cells without killing them.

■

Improvements in

autonomous underwater vehicles

to enable aseptic water

sampling.

■

New

genetic systems

, particularly environmentally important but so far under-

studied groups.

■

Better

models for protein prediction

from gene sequences.

■

Development of

ordered mutant libraries

of more organisms.

■

Better

protein crystallization methods

and methods of predicting and solving

crystal structures so that the functional details of more proteins can be elucidated.

■

remote sensing techniques

for monitoring community structure.

■

Refined

techniques

for measuring the function of uncultured microbial cells at

the single cell level.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

EDUCATION, TRAINING, AND COMMUNICATIONS ISSUES

Although the challenges that lie ahead in microbiology are compelling, it is

important to remember that discovery relies on the work of individuals. Emphasis

should be placed on effective training and fostering productive collaborations.

CURRENT GAPS IN TRAINING

The future of microbiology relies on the training and education going on today, but

there are a number of identifiable gaps in training that must be addressed. Current

training programs in microbial science do not place enough emphasis on critical

thinking and hypothesis or question building, and observational skills are being lost.

This sort of preparation may have to begin in K-12 education, where students could

be more effectively introduced to the excitement of natural discovery.

Although microbiology training seems to be keeping up with advances in tech-

nology, it is falling short when it comes to some of the more long-established

disciplines, including physiology, enzymology, and biochemistry, and long-estab-

lished techniques like culturing. Other weak areas in training include

bioinformatics, small molecule structural identification, biophysics, and chemistry.

Quantitative analysis, including mathematical modeling and complex statistics is

also not taught to a sufficient extent.

The failure of graduate programs to provide sufficient training in physiology is

particularly troubling, since a detailed knowledge of physiology is needed to inter-

pret the vast amounts of genomics data that are currently being generated by high

throughput techniques. Fellowships that emphasize physiology and improved

microbial physiology textbooks are sorely needed.

Some specific recommended changes for education and training that would help

to keep microbial science vital include:

■

Fellowships and travel grants for encouraging cross disciplinary interactions,

■

Web-based courses and reading lists for students and other scientists,

■

Intensive short courses in microbiology (including microbial diversity courses)

which offer students good exposure to the field and networking possibilities,

■

Revitalize devoted microbial science departments in colleges and universities as

they have often of late succumbed to consolidation with genetics and molecular

biology departments, a development that has weakened collaborative ties

between microbiologists. Re-equip many outdated college and university labora-

tories with new tools for microscopy and molecular analyses,

The Uncharted Microbial World: Microbes and Their Activities in the Environment

■

Make evolution and ecology standard components of microbiology training, since

almost all questions in microbiology must be viewed in these contexts, and

■

Develop new and better textbooks for microbial physiology, microbial diversity and

systematics, bioinformatics, and microbial ecology.

RECOMMENDED COLLABORATIONS

Collaborations among professionals from diverse areas of expertise are a hallmark

of successful microbiology research. Collaborations provide fresh perspectives on

study topics and help inspire dialog and creativity. There are a number of ways to

improve the state of collaborative interaction in microbial science. Microbiologists

should make connections with experts on particular environments in order to specifi-

cally tailor microbial cultivation conditions. Additional collaborations among

environmental microbiologists and infectious disease specialists would be helpful.

Engineers and applied physicists, too, should be involved in microbiology research to

help design improved instrumentation. Long-term ecological research stations can

provide good platforms for collaboration, provided they are associated with some

sort of central institution that can foster these interactions.

In general, the field needs more nucleation points, more common ground where

professionals can talk with each other. Proximity is essential for collaborative

research; sharing space in a laboratory is one way to promote collaboration. Train-

ing courses should also involve students from diverse academic backgrounds.

Unfortunately, the administrative structure of academic departments sometimes

inhibits interdisciplinary research. International collaboration is also difficult, since

strict customs security measures have made moving microbiological samples across

borders incredibly difficult. Exchange visits have also become more difficult for pro-

fessionals from certain countries.

Collaborations with industry can be fruitful. Industry may also be interested in

supporting graduate students conducting research in areas that interest the private

sector but funding agencies avoid.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

RECOMMENDATIONS

When selecting microorganisms for genome sequencing, it is important to

sequence clusters of closely related organisms as well as more divergent organ-

isms. Viruses, too, should be targeted by sequencing efforts, since they serve as

important agents of gene transfer in microbial communities. Technical barriers to

community sequencing projects should be lowered.

Microbiology also needs to move beyond its dependence on pure cultures of

organisms and appreciate the value of defined but mixed communities of microbes.

It may not always be possible to separate microorganisms that have coevolved to

fit one another’s functions and isolate them in pure cultures.

Research should test the responses of microbial communities to perturbation by

evaluating model systems at “baseline” or preperturbation state, and then directly

measuring the effects of perturbation in these systems. Very little is currently

known about the specific outcomes of disturbance in microbial communities, even

those that are directly related to human health and well-being.

Current technologies for making measurements at the microscale require

enhancement. Work is also needed to miniaturize scanning electron microscopy

and other microscopic tools, develop biosensors, and to generally improve the

ability to make

in situ environmental measurements.

Microbiology requires more methods that allow researchers to mimic the con-

ditions microorganisms encounter in their natural habitats, particularly the

conditions in low nutrient environments and in nutrient and oxygen gradients

that form at surfaces.

The Uncharted Microbial World: Microbes and Their Activities in the Environment

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