Microbiology as a modern scientific discipline may be said to
have begun in 1859, when Louis Pasteur performed a set of elegant experiments that finally
laid to rest the "theory" of spontaneous generation. For almost a century and a
half, most of what we know about microbes comes from studying them in the laboratory.
"Yet fewer than one to five percent of the microbes we find in nature will grow in
the lab," says Tamas Torok of the Life Sciences Division. "The first challenge
is to find out who's out there." Torok is a member of the Center for Environmental
Biotechnology, a group of scientists from different divisions and disciplines headed by
Jennie Hunter-Cevera of Earth Sciences.
The probable reason why so few microbes can be grown artificially, says Torok, has to
do with the difficulty of simulating natural conditions. In nature, a gram of soil may
contain as many as 10 billion individual single-celled organisms, representing some 10,000
different species. "For a culture growing on nutrient in a dish, metabolism is simple
and food is plentiful," Torok says. "In nature, microbial metabolism is complex
and lean."
As a result of Mother Nature's leanness, it may take a population of microbes in the
deep subsurface of the Earth as long as a thousand years to produce a new generation; in
the lab scientists are used to seeing new generations in anywhere from a few weeks to a
few minutes.
"Microbes need the right mix of nutrients and the right surfaces to grow on,"
adds Stanley Goldman, Torok's colleague. "In nature they're usually interdependent
with other types, and they can be sensitive to change. Some microbes have been found as
deep as three kilometers, using hydrogen as a source of energy. In the laboratory we can
only grow them as a consortium."
Goldman and Torok have been working to survey and characterize the complex microbial
communities found in damaged environments, including closed military bases contaminated by
spilled fuel and other chemicals, watersheds polluted by agricultural run-off, and sites
used for high-level radioactive waste. Their goal is to understand how these intricate,
invisible communities behave and whether some of the microbes in them can help remediate
their toxic surroundings.
The potential uses of such information are manifold. Through oxidation or reduction,
some microbes can change metals from toxic to less toxic, or from mobile to less mobile.
Others can metabolize toxics such as toluene and trichloroethylene.
A vast majority of the genomes in the world are microbial, and probably most of the
world's actual biomass too. Many useful new enzymes and new antibiotics are waiting to be
discovered in nature, a goal that is already being pursued by commercial companies such as
Roche, Novo Nordisk, and the Diversa Corporation of San Diego, which has offered to return
royalties to the owner of the place where useful microorganisms are found -- for example,
to the National Park Service for interesting creatures recovered from the hot springs and
geysers of Yellowstone National Park.
Goldman and Torok use several methods of identifying microbes found in nature,
including gene sequencing and biomarker analysis. To secure large numbers of organisms,
the researchers, joined by Jennie Hunter-Cevera, have also developed techniques for
imitating nature in the lab -- among them, growing cultures with highly restricted
nutrients in cool darkness and on samples of rock or soil from the collection site.
In gene sequencing, very mixed natural populations are first "lysed," or
broken open. Genes called ssrRNAs are amplified, cloned, and then automatically sequenced.
These genes code for the nucleic acid component of the ribosome's small subunit.
(Ribosomes are tiny two-part functional units in the cells of all living creatures which
read messenger-RNA sequences and assemble proteins.) Although DNA from many different
kinds of bacteria (prokaryotes) and fungi, algae, and protozoa (eukaryotes) may be present
together in the broken cell mixture, their ssrRNA sequences, which are highly conserved,
can be readily distinguished by small differences -- differences which can also reveal
something of the evolutionary relationships among the organisms.
"It's important to remember that we're identifying phylotypes here, not
species," Torok says, emphasizing that "we can answer the question, `Who's out
there?' in terms of phylogenetic trees -- evolutionary relationships -- not in terms of
specific genetic identities."
"For identifying natural populations, ssrRNA sequencing is not a panacea,"
says Torok. "We miss some organisms because they don't all respond to our methods of
extraction from the soil. The cell walls of some are a lot tougher than others -- they
don't break -- so their DNA isn't well represented after extraction. And amplification
with PCR depends on using primers based on known organisms; we may be missing some that
are completely unrelated."
"Nevertheless," Goldman says, "we've already got a much better
real-world view of who's out there."
Goldman and Torok also perform biomarker analysis of fatty acids from the cell
membranes of the creatures in the same environmental samples and compare the results with
those from gene sequencing. The goal is to devise a range of identification methods, a net
with a finer mesh.
Says Goldman, "We're developing isolation techniques and novel probes, such as
fluorescent tags, to rapidly identify organisms in unexplored settings. We're also working
on microarray techniques to survey the relative abundance of different individual DNA
sequences in complex samples from entire communities."
Torok and Goldman's study of communities of microorganisms is a prime example of the
Center for Environmental Biotechnology's determination to build bridges among many
different scientists, disciplines, and institutions. It is a key research program that
promises to produce benefits for the Department of Energy and other federal agencies, both
within the state of California and across the nation, in both the public and private
sectors.
The Center for Environmental Biotechnology's web page can be found at at http://www-esd.lbl.gov/CEB/ceb.html.
