A 94 C geothermal pool, with a level-maintaining siphon, near Gerlach, Nevada. Sediment from the floor of this pool was enriched on pulverized miscanthus at 90 C and subsequently transferred to filter paper in order to isolate microbes able to subsist on cellulose alone. Photo: Joel Graham, University of Maryland |
Bioprospectors from the University
of California, Berkeley,
and the University of Maryland School of Medicine have found a microbe in a Nevada hot spring that
happily eats plant material—cellulose—at temperatures near the boiling point of
water.
In fact, the microbe’s cellulose-digesting enzyme, called a cellulase, is
most active at a record 109 C (228 F), significantly above the 100 C (212 F)
boiling point of water.
This so-called hyperthermophilic microbe, discovered in a 95 C (203 F)
geothermal pool, is only the second member of the ancient group Archaea known
to grow by digesting cellulose above 80 C. And the microbe’s cellulase is the
most heat tolerant enzyme found in any cellulose-digesting microbe, including
bacteria.
“These are the most thermophilic Archaea discovered that will grow on
cellulose and the most thermophilic cellulase in any organism,” says coauthor
Douglas S. Clark, UC Berkeley professor of chemical and biomolecular
engineering. “We were surprised to find this bug in our first sample.”
Clark and coworkers at UC Berkeley are teaming with colleagues, led by Frank
T. Robb, at the University of Maryland (U-Md) School of Medicine in Baltimore,
to analyze microbes scooped from hot springs and other extreme environments
around the United States in search of new enzymes that can be used in extreme
industrial processes, including the production of biofuels from hard-to-digest
plant fiber. Their team is supported by a grant from the Energy Biosciences
Institute (EBI), a public-private collaboration that includes UC Berkeley, in
which bioscience and biological techniques are being applied to help solve the
global energy challenge.
“Our hope is that this example and examples from other organisms found in
extreme environments—such as high-temperature, highly alkaline or acidic, or
high salt environments—can provide cellulases that will show improved function
under conditions typically found in industrial applications, including the
production of biofuels,” Clark says.
Clark, Robb, and their colleagues, including UC Berkeley professor Harvey W.
Blanch and postdoctoral researcher Melinda E. Clark, and U-Md postdoctoral
researcher Joel E. Graham, will publish their results online in Nature
Communications.
Many industrial processes employ natural enzymes, some of them isolated from
organisms that live in extreme environments, such as hot springs. The enzyme used in the popular
polymerase chain reaction to amplify DNA originally came from a thermophilic
organism found in a geyser in Yellowstone
National Park.
But many of these enzymes are not optimized for industrial processes, Clark said. For example, a fungal enzyme is currently
used to break down tough plant cellulose into its constituent sugars so that
the sugars can be fermented by yeast into alcohol. But the enzyme’s preferred
temperature is about 50 C (122 F), and it is not stable at the higher
temperatures desirable to prevent other microbes from contaminating the
reaction.
Hence the need to look in extreme environments for better enzymes, he said.
“This discovery is interesting because it helps define the range of natural
conditions under which cellulolytic organisms exist and how prevalent these
bugs are in the natural world,” Clark says. “It indicates that there are a lot of potentially useful cellulases in places
we haven’t looked yet.”
Robb and his colleagues collected sediment and water samples from the 95 C
(203 F) Great Boiling Springs near the town of Gerlach
in northern Nevada
and grew microbes on pulverized Miscanthus
gigas, a common biofuel feedstock, to isolate those that could grow with
plant fiber as their only source of carbon.
After further growth on microcrystalline cellulose, the U-Md and UC Berkeley
labs worked together to sequence the community of surviving microbes to obtain
a metagenome, which indicated that three different species of Archaea were able
to utilize cellulose as food. Using genetic techniques, they plucked out the
specific genes involved in cellulose degradation, and linked the most active
high-temperature cellulase, dubbed EBI-244, to the most abundant of the three
Archaea.
Based on the structure of the enzyme, “this could represent a new type of
cellulase or a very unusual member of a previously known family,” Clark says.
The enzyme is so stable that it works in hot solutions approaching
conditions that could be used to pretreat feedstocks like Miscanthus to break
down the lignocelluloses and liberate cellulose. This suggests that cellulases
may someday be used in the same reaction vessel in which feedstocks are
pretreated.
The newly discovered hyperthermophilic cellulase may actually work at too
high a temperature for some processes, Clark
said. By collecting more hyperthermophilic cellulases, protein engineers may be
able to create a version of the enzyme optimized to work at a lower
temperature, but with the robust structural stability of the wild microbe.
“We might even find a cellulase that could be used as-is,” he says, “but at
least they will give us information to engineer new cellulases, and a better
understanding of the diversity of nature.”
