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"The termite is a remarkable machine," said Dr. Raymond L. Orbach,
Under Secretary for Science, U.S. Department of Energy, whose program
supports DOE JGI. "Termites can digest a frightening amount of wood in
a very short time, as anyone who has had termites in their house is
painfully aware. Instead of using harsh chemicals or excess heat to do
so, termites employ an array of specialized microbes in their hindguts
to break down the cell walls of plant material and catalyze the
digestion process. Industrial-scale DNA sequencing by DOE JGI was key
to identifying the genetic structures that comprise the tools that
termites use. Our task now is to discover the metabolic pathways
generated by these structures to figure out how nature digests plant
materials. We can then synthesize the novel enzymes discovered through
this project to accelerate the delivery of the next generation of
cellulosic biofuels."
While termites have been the subject of keen scientific study for more
than a century, the precise identity and role of the microbes from
their digestive tract remained a mystery. With this new work, the
symbiotic orchestration of these compartmentalized, complex microbial
communities required for wood digestion is now coming to light.
Like cows, termites have a series of stomachs, each harboring a
distinct community of microbes under precisely defined conditions.
These bugs within bugs are tasked with particular steps along the
conversion pathway of woody polymers to sugars that can then be
fermented into fuels such as ethanol. The mandibles of the insect
chomp the wood into bits, but the real work is conducted in the dark
recesses of the belly, where the enzymatic juices exuded by microbes
attack and deconstruct the cellulose and hemicellulose, which, along
with lignin, are the basic building blocks of wood.
The tiny insects that gave up their stomach contents to advance the
frontiers of science were isolated on a safari into the rainforest of
Costa Rica, the world's geographic hotbed of biodiversity for termites,
by co-author Jared Leadbetter of Cal Tech, first author Falk Warnecke
of DOE JGI's Microbial Ecology Program, and members of Verenium and
INBio. Traipsing through the jungle, the team came upon a massive,
tumor-like nest of termites clinging to an otherwise nondescript tree.
With a flick of a machete, the contents of this dense network of
tunnels forged from wood waste were revealed, along with a frenzy of
higher termites from the genus Nasutitermes, which are only about the
size of the date imprinted on a penny.
Foregoing the funnel-headed "soldiers," the project focused on the
larger "workers," with bulbous heads and inflated bellies. In the
laboratory of INBio, researchers armed with fine forceps and needles
painstakingly extracted the contents of the workers' third paunch or
hindgut, referred to as P3, a distended kink in the convoluted
plumbing system of the termite. Each sample was barely visible to the
naked eye, and care was taken not to contaminate it with material from
neighboring stomachs. Contents from 165 specimens were purified,
yielding only a few valuable drops - a veritable microbial mosh pit -
that was sent on ice to Verenium for DNA extraction and preparation,
then on to DOE JGI's Walnut Creek (CA) Production Genomics Facility
for sequencing.
From the sample, about 71 million letters of fragmented genetic code
were elaborated and computationally reassembled, like putting Humpty
Dumpty back together again, to tease out the identities of the
microbial players in the mixture and the metabolic profile of the
enzymes that they produce. From this reconstructed liquid puzzle
emerged the identities of a dozen different phyla - broad groupings of
microbial life forms.
"Our analysis revealed that the hindgut is dominated by two major
bacterial lineages, treponemes and fibrobacters," said co-author Phil
Hugenholtz, DOE JGI's Microbial Ecology Program head. "Treponemes have
long been recognized in the termite gut due to their distinctive
cork-screw shape, but fibrobacters were an exciting new find, because
they have relatives in the cow rumen known to degrade cellulose. We
could directly link the termite fibrobacters and treponemes to enzymes
capable of breaking down wood. However, fibrobacters are specialists
in this regard and don't appear to participate in sugar fermentation,
leaving that to the treponemes. This project has really given me a new
appreciation for the lowly termite, a mobile miniature bioreactor."
In the termite P3 compartment alone, more than 500 genes related to
the enzymatic deconstruction of cellulose and hemicellulose were
identified by Hugenholtz and colleagues. This dataset has since been
uploaded by DOE JGI onto its metagenome data management and analysis
system, IMG/M for public access and further analysis.
"Adapting these findings for an industrial-scale system is far from
easy," said Eddy Rubin, JGI Director. "Termites can efficiently
convert milligrams of lignocellulose into fermentable sugars in their
tiny bioreactor hindguts. Scaling up this process so that biomass
factories can produce biofuels more efficiently and economically is
another story. To get there, we must define the set of genes with key
functional attributes for the breakdown of cellulose, and this study
represents an essential step along that path."
Nature paper first author Falk Warnecke is a postdoctoral fellow in
the Hugenholtz lab. Other DOE JGI authors include Natalia Ivanova,
Rotem Sorek, Susannah Tringe, Hector Garcia Martin, Victor Kunin,
Daniel Dalevi, Julita Madejska, Edward Kirton, Darren Platt, Ernest
Szeto, Asaf Salamov, Kerrie Barry, Natalia Mikhailova, Nikos Kyrpides,
and Director Rubin.
These findings follow on the heels of the announcement by DOE
Secretary Samuel Bodman in June that DOE will invest up to $375
million in three new Bioenergy Research Centers to accelerate basic
research in the development of cellulosic ethanol and other biofuels.
DOE JGI will conduct genome sequencing in support of these centers.
The termite hindgut whodunit builds upon DOE JGI's pioneering "metagenomic"
research, where genetic material is isolated, identified, and
characterized directly from environmental samples, providing a profile
of a particular (often extreme) ecological niche. Published
investigations by DOE JGI include glimpses into such diverse slices of
the biosphere as acid mine drainage, a gutless worm, farm soil,
submerged whalebones, and sewage sludge.
Currently among the scores of projects in the sequencing queue at DOE
JGI are metagenomes from contents of the Tammar wallaby forestomach,
the Asian longhorned beetle gut, and other exotic species that promise
to be treasure troves of enzymes involved in cellulose deconstruction.
These targets were submitted through DOE JGI's Community Sequencing
Program (CSP), which provides the scientific community with access to
high-throughput sequencing for projects of relevance to DOE missions.
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