Another option is to use bacteria. For the past 25 years, Sallie (Penny) Chisholm, the Lee and Geraldine Martin Professor of Environmental Studies, has been studying Prochlorococcus, an ocean-dwelling bacterium that she calls "a pretty spectacular organism." Of all organisms that perform photosynthesis, this single-celled bacterium is both the most abundant and the smallest—less than 1 micron in diameter. It accounts for fully 10 percent of all photosynthesis on Earth and forms the base of the ocean food chain. It also has the smallest genome of any known photosynthetic cell. "Three billion years of evolution has streamlined its genome, and it now contains the least amount of information that can make biomass from solar energy and carbon dioxide," says Chisholm, who has a joint appointment in civil and environmental engineering (CEE) and biology. "It makes sense that we try to understand it—inspired by its simplicity—and see if we can use this understanding to help us design microorganisms that efficiently produce biofuels directly from sunlight."
In 2010, Chisholm's much-studied bacterium delivered a surprise: As it grows, it naturally releases small, spherical, membrane-bound vesicles containing fatty oils related to those that make algae so appealing. This was a serendipitous discovery. In 2008, Chisholm's group needed some images of Prochlorococcus for a publication. Using a scanning electron microscope, then-graduate-student Anne Thompson PhD '10 took the images—and they showed small spheres near the surfaces of the Prochlorococcus cells. The spheres remained a mystery to the ocean biologists until 2010, when Steven Biller joined Chisholm's group as a postdoctoral associate in CEE. Based on his work with soil bacteria, he proposed—and subsequently confirmed—that the spheres are lipid-bound vesicles.
That finding is remarkable for two reasons. While many species are known to release vesicles, the behavior has never before been observed in a marine organism—and it could significantly change today's understanding of marine ecosystems, including their influence on the global carbon cycle. "Prochlorococcus is making organic carbon from sunlight and then packaging it up and releasing it into the seawater around it," says Chisholm. "What we need to figure out now is, Why and how? And what role do these vesicles play in ocean food webs and the ocean carbon cycle?"
Equally surprising, this is the first observation of vesicle
release in an organism that performs photosynthesis. The implications
for industrial use—including biofuels production—are significant. Given
just sunlight, carbon dioxide, and water, Prochlorococcus would
continually release lipid-containing vesicles, which could be collected
without disturbing the growing bacteria. "With algae, retrieving the
lipids requires destroying one batch of cells and starting with a new
batch," says Biller. "With Prochlorococcus, it could be a 'continuous
culture.'"
Technical challenges, new insights
Chisholm stresses that such commercial applications are "way down the
road." For now, research in her lab focuses on developing a fundamental
understanding of the newly observed behavior. For example, how often
does a Prochlorococcus cell release vesicles? How many does it release?
And what's inside them?
To answer those questions, Biller overcame a series of technical
challenges. First he developed improved methods of culturing large
quantities of Prochlorococcus cells. Then he designed techniques for
filtering off the vesicles and concentrating and purifying them—while
keeping them intact. But his biggest problem was how to count the
individual vesicles. Standard methods of counting particles don't
provide sufficient resolution to look at the vesicles, which are less
than 100 nm in diameter. After some trial and error, Biller was
successful in adapting recent advances in nanoparticle analysis
techniques to studying these tiny bacterially derived structures.
Using his new approaches, he determined that vesicles are present in
large concentrations in growing cultures. Indeed, they outnumber the
Prochlorococcus cells themselves—in some cases by a factor of 10. They
are generated by strains of Prochlorococcus that grow in bright light
(such as near the ocean surface) as well as in dimmer light (typical of
the deep ocean). Vesicles appear to be produced continually during some
phases of cell growth, and they are stable under laboratory conditions:
Over the course of two weeks, the size and concentration of vesicles in a
laboratory culture remained essentially unchanged. Finally, the
vesicles contain not only lipids but also DNA, RNA, and a diverse set of
proteins.
Unfortunately, the lipids in the vesicles from Prochlorococcus
are not the optimal kind for making biofuels, notes Biller. "But because
of its simple genome, it's a good model for us to use in exploring the
mechanisms that control the formation and extrusion of vesicles and
determine their content," he says. "Once we understand how it works,
that mechanism could eventually be utilized in more robust and
fast-growing organisms, and the contents of the vesicles could be
manipulated."
Fieldwork expands the options
Based on their laboratory data, the researchers estimated that
Prochlorococcus worldwide could release on the order of 1,027 vesicles
per day—a significant contribution to the marine ecosystem. But many
factors could influence vesicle production in the wild, so the team
decided to take direct measurements. They collected hundreds of liters
of seawater in two locations: the nutrient-rich coastal waters of
Vineyard Sound in Massachusetts and the nutrient-sparse waters of the
Sargasso Sea near Bermuda. They used their laboratory techniques—scaled
up to handle larger volumes of water—to test the samples on board
research vessels. As with their lab cultures, they found numerous
vesicles in the samples from both types of ocean environments. And their
analyses showed that the vesicles contained DNA from many kinds of
bacteria—not just Prochlorococcus.
That finding potentially extends vesicle production to organisms that
are ubiquitous in ocean systems extending from pole to pole. "This adds
a whole new dimension to marine microbial ecosystems that we hadn't
realized was there," says Biller. "And while Prochlorococcus was our
entry point into this concept for biofuels production, it looks like
there may be applications to many other organisms."
Vesicles as nutrients for other bacteria
These curves show the growth of a marine heterotroph—a
nonphotosynthetic organism—in three laboratory cultures. One culture
includes a mixture of organic carbon compounds ("+organic carbon mix");
another includes only added Prochlorococcus vesicles ("+vesicles"); and
the last has no source of fixed carbon ("control"). Optical density, or
OD, is a common measure used for estimating cell concentrations in
liquid cultures. The data show that the vesicles alone provide enough
nourishment for the cells to increase in number over 50 hours.
Prochlorococcus thus appears to facilitate the growth of
heterotrophs—and in return, the heterotrophs may protect Prochlorococcus
by neutralizing toxic compounds that would harm it.
Wasteful behavior?
An intriguing question is why Prochlorococcus would make and release
vesicles. Jettisoning their hard-earned organic carbon seems
inconsistent with the need for this streamlined organism to make
efficient use of scarce resources. What function could the vesicles
serve? Biller and Chisholm don't have an answer to that question, but
they've come up with several hypotheses—ideas with potential impacts on
both understanding marine ecosystems and developing commercial-scale
biofuels systems.
In working with Prochlorococcus, Chisholm and her colleagues have
found that the bacterium is "happier" in the company of
heterotrophs—organisms that can't synthesize their own food and need a
source of organic carbon
to grow. "We went through heroic efforts to separate the
Prochlorococcus and their heterotrophic friends in seawater samples,"
says Chisholm. "Then we realized that when we grow them together, the
cultures grow faster and are more stable." In a series of experiments,
Biller showed that the newly identified lipid vesicles can serve as
nutrients for the heterotrophs.
What does Prochlorococcus get in return? It is not fully understood,
but others have shown that in the process of becoming streamlined,
Prochlorococcus lost certain enzymes that other species use to
neutralize toxic oxygen compounds produced during metabolism. The
heterotrophs can perform that detoxification task, taking care of the
problem for Prochlorococcus.
Another hypothesis is that the vesicles help protect Prochlorococcus
from phage, viruses that infect bacteria. The surface of a vesicle
contains material from the outer membrane of its parent cell, including
protein receptors that phage use to identify their "prey." The vesicles
therefore may serve as a decoy—"much as a fighter jet trying to evade an
incoming missile may throw out chaff so that the missile goes after the
chaff instead of the jet," says Biller. To test that idea, Biller mixed
purified Prochlorococcus vesicles with a phage known to infect the
Prochlorococcus source of the vesicles. Electron micrographs revealed
many phage attached to vesicles. Moreover, their shortened tails suggest
that they have injected their DNA into the vesicles, thereby becoming
inactive.
A final hypothesis is that the vesicles assist in the exchange of
genetic material between individual bacteria—a phenomenon known to occur
in some bacteria as a means of developing genetic diversity and sharing
useful genes. "We know that bacteria are swapping genes among
themselves at surprisingly high frequencies—maybe by using phage or
direct cell-to-cell contact," says Biller. "But it wasn't clear that
those mechanisms alone could explain the apparent rates at which genes
are moving around. This is one possibility of another way that DNA might
be exchanged in these communities."
Benefits of multiple-scale study
The researchers' latest results confirm the validity of Chisholm's
decades-long approach to studying Prochlorococcus. "Our studies of this
bacterium have ranged in scale from genes to cells to populations and
then to the community they're embedded in and up to the global scale,"
she says. That approach, called integrative systems biology, has obvious
benefits for understanding global ecosystems and—in the longer term—for
developing practical systems involving mass cultures that are
fast-growing, stable, and productive. Says Chisholm, "Studying model
systems such as Prochlorococcus in an expansive sense—from the phage
that infect them to the other microbes that they grow with in
nature—will ultimately have relevance for any kind of large-scale
production of biomass for biofuels and other types of high-energy
compounds."
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