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KC and the
Ground Sludge Band
After
10 years of hard work punctuated by serendipity, leaps of
faith, and intuition, an unsinkable team of scientists
devise an ingenious system to clean water contaminated
with carbon tetrachloride, a toxic
chemical.
By: Marina
Chicurel
An ominous toxic threat lurks beneath the ground at a
remote site in south-central Washington. Born in 1955, it is
a liquid plume of toxin spreading slowly underground.
Already it reaches down 18 stories into the soil and sprawls
across an area larger than 1,000 football fields.
The liquid is the waste product of the extraction process
used to recover plutonium for making nuclear weapons. From
1955 to 1973, government workers dumped more than 100,000
gallons of carbon tetrachloride, a toxic chemical, into
trenches and directly onto the soil at the U.S. Department
of Energy site in Hanford, Washington. The chemical seeped
into the ground and formed a huge plume that is slowly
creeping through the underground water.
A deceptively clear and sweet-smelling liquid, carbon
tetrachloride causes headaches, weakness, nausea and
vomiting. Even worse, it is a probable human carcinogen that
can seriously damage the liver and kidneys.
In an attempt to contain the Hanford disaster, in 1996,
the government set up a system of pumps that removes the
carbon tetrachloride from the water. But the system can
handle only a small fraction of the plume. As of now, there
is no way to fully restrain it, much less clean it up.
The carbon tetrachloride at Hanford is spreading through
an unpopulated area slowly enough that, for the moment,
there is no real danger of it affecting nearby communities.
The chemical can linger for thousands of years, however.
Sooner or later, if left undisturbed, it is bound to cause
serious injury to humans and damage to the environment.
The Hanford site is not the only underground aquifer
contaminated with carbon tetrachloride. Although the exact
extent of the problem remains unknown, aquifers in virtually
every community in the U.S. could be polluted. Carbon
tetrachloride is used only rarely these days because of its
known toxicity and because, like the better-known
chlorofluorocarbons used until recently in refrigerants, it
destroys the ozone layer. But in the past, it was produced
in large amounts to make insecticides, refrigerants,
dry-cleaning agents, and solvents for oils, varnishes and
resins.
The first steps toward a solution of this widespread
problem, however, are well under way. Craig Criddle, an
environmental engineer now at Stanford University, and his
collaborators at Michigan State University have brought
together two novel approaches: a bacterium that degrades
carbon tetrachloride and an ingenious system of wells that
exposes the contaminated water to the microbe. The system is
deceptively simple in appearance—a row of perforated
plastic pipes sunk 80 feet into the ground, connected to an
aboveground set of reservoirs and mixers. What’s impossible
to glean from viewing the apparatus, however, are the 10
years of hard work punctuated by serendipity, leaps of
faith, and intuition that went into its creation.
An abandoned bucket
In 1988, Craig Criddle, then a graduate student at
Stanford, was hired by professor of environmental
engineering Dunja Grbic-Galic to search for microorganisms
that could break down carbon tetrachloride. “I took the job
because the work was consistent with my research project,
because I needed the money, and because Dunja was a very
nice person to work with,” says Criddle.
Using pollution-eating microbes to clean up the
environment, also known as bioremediation, was just
beginning to gain momentum. Bioremediation promised to
overcome some of the problems faced by more traditional
cleaning methods. Bacteria are free, inexhaustible, and run
themselves, whereas traditional remedies can be expensive
and labor-intensive. For example, many traditional methods
use activated charcoal to trap contaminants. This process
cleans the water, but leaves contaminated charcoal behind.
Cleaning the charcoal is possible, but it is a costly
process that requires heating it above 15000 F.
Bioremediation promised to completely eliminate contaminants
in a single, cheap step.
Today, one form of bioremediation—in situ
bioremediation—is common. In this approach, the addition
of nutrients spurs bacteria, already present in the aquifer,
to degrade contaminants. In situ bioremediation has
efficiently cleaned up aquifers polluted with diesel fuel
and gasoline, for example.
A less common alternative, called bioaugmentation,
requires adding non-native microbes to aquifers. Criddle was
skeptical of bioaugmentation. He doubted that an added
microbe would be able to compete with other local bacteria
for space and food.
But before deciding which kind of bioremediation the team
would aim for, Criddle first had to determine whether a
suitable microbe even existed. He incubated bacteria from
several different aquifers into separate bottles with a food
solution and carbon tetrachloride. Just as he was finishing
setting up his rows of bug-filled bottles, Criddle paused.
“As an afterthought, I remembered some old aquifer material
sitting in a bucket at the back of a cooler,” he says. “I
figured I’d just give it a try for the fun of it.”
A couple of days later, Criddle went to measure the
levels of carbon tetrachloride in his bottles and found that
the contaminant had disappeared from the bottle containing
bacteria from the old bucket. Even more importantly, he
found no chloroform in the bottle.
Other scientists before Criddle had discovered bacteria
that break down carbon tetrachloride. For every molecule
these bacteria broke down, however, they produced a molecule
of chloroform—a probable carcinogen that can cause
breathing and heart problems. Criddle’s abandoned bucket
seemed to contain a bacterium that degraded carbon
tetrachloride without producing chloroform. In addition, it
seemed to be getting rid of nitrate, a harmful substance
that can cause methemoglobinemia—a disease, which infants
are particularly susceptible to, in which the blood fails to
carry its normal load of oxygen. Because nitrate is used as
a fertilizer and carbon tetrachloride was used in grain
silos as an insecticide, these chemicals are often found
together in aquifers near agricultural areas.
Criddle isolated the bacterium from the bottle and
eventually identified it. In honor of his wife, Karrie
Criddle, he dubbed it KC. Although he became more excited
about the project, Criddle remained cautious.
“At the time I thought that it was unlikely that we had
isolated anything unique,” he says. So he ordered previously
isolated strains of the same type of bacteria from the
largest repository of known microorganisms, the American
Type Culture Collection in Virginia. None of them could
perform KC’s trick of degrading carbon tetrachloride and
getting rid of nitrate without producing chloroform.
KC was one of a kind—an exceptional find. It provided
the first hope that an insidious and widespread pollutant
might be eventually conquered through bioremediation.
Criddle was delighted, but unaware of his discovery’s full
implications—as yet he could not imagine the role KC
would play in his future.
Catering to KC’s finicky tastes
Like a trainer who discovers a gifted athlete, Criddle
was eager to hone KC’s talents and test it in the field. The
next step appeared simple: KC’s performance had to be tested
in a real aquifer water sample. So Criddle added KC directly
to some carbon tetrachloride-contaminated water from a site
at Moffett Field near Mountain View, California.
KC disappointed Criddle by eating very little carbon
tetrachloride. For some reason, the bug could devour the
pollutant when growing in the original food mixture, but not
when placed in the contaminated water sample.
Divining KC’s finicky tastes proved a challenge. Criddle
spent months testing each component of his food solution. He
discovered that it wasn’t enough to have a solution that
merely allowed KC to grow. For example, iron stimulated KC’s
growth but inhibited its carbon tetrachloride-degrading
abilities. So for each component Criddle tested, he had to
measure both KC’s growth and its performance. He also found
out that he couldn’t just test each component individually,
because the combination of certain components had effects
that couldn’t be predicted from the individual tests alone.
He had to mix and match different components to tease apart
KC’s tastes. Finally, Criddle concluded that two conditions
had to be balanced carefully for KC to grow and transform
carbon tetrachloride: acidity and amounts of iron.
Criddle realized how lucky he had been when he performed
that first experiment. By chance, he had chosen the
abandoned bucket and used the right degree of acidity and
amounts of metals in preparing his feeding solution. What
was not chance or luck, however, were the years of hard work
that followed to understand just what had happened that
first time.
Soon after this discovery in 1990, Criddle graduated and
took a faculty position at Michigan State University in East
Lansing. Although enthusiastic about his own future, Criddle
did not regard KC’s as golden. As promising as KC had once
appeared, it now seemed too demanding to ever qualify for
practical use. “I doubted that it would ever be more than a
laboratory curiosity,” says Criddle.
A winding road to success
As the young head of a lab, Criddle had many affairs to
attend to, and although he maintained an academic interest
in KC, it was not a priority. For a year, the project
languished. Then Greg Tatara, a graduate student at the
time, and Michael Dybas, a postdoctoral fellow, joined
Criddle’s lab and enthusiastically took over the exploration
of KC’s predilections.
Tatara was attracted to the project because it involved a
recently discovered organism—KC’s lifestyle, genes, and
physiology were a mystery, uncharted territory that he could
adventurously explore.
Dybas’s original plan was to isolate the genes that
underlie KC’s carbon tetrachloride-eating abilities. The
genes, he thought, would provide them with a powerful handle
to begin solving the problem of carbon tetrachloride
pollution. Dybas planned to search for KC mutants that were
unable to degrade carbon tetrachloride. By comparing the
genes of those mutants to normal KC genes, he hoped to
identify the carbon tetrachloride-degrading genes.
But when Dybas and Tatara arrived in the lab, they
couldn’t get normal KC to degrade carbon tetrachloride
consistently. “Out of 50 batches, only five would work,”
says Tatara. KC’s fastidious nature was rearing its ugly
head again.
Criddle began to worry once more about KC’s ability to
successfully compete with other local bacteria for food and
space. But this didn’t slow down Dybas. “Mike was just a
crazy man,” says Criddle. “One of the great things about
having new people in the lab is their ignorance of the
‘impossible.’”
Joining forces, Dybas and Tatara set out to more
carefully determine what KC needed to degrade carbon
tetrachloride efficiently. They soon discovered KC secreted
molecules that could degrade carbon tetrachloride on their
own, even when there were no bacteria present. This
suggested they might be able to use these simple molecules
and avoid dealing with the complicated KC.
Unfortunately, the molecules were devilishly sensitive to
acidity. Dybas spent no less than six months testing the
ways in which tiny differences in acidity affected the
molecules’ activity. With unflinching persistence he found
out exactly how to coax those molecules into doing their
very best job.
And then something terrible happened. Without any obvious
explanation, the molecules stopped working. Even when Dybas
adjusted the conditions precisely, he could not wring any
activity out of them. “The last time I had activity was the
last day of George Bush’s presidency,” says Dybas. “Since
Bill Clinton’s been in office I’ve never been able to get
it. I can’t directly correlate that with him, but I have my
suspicions.”
Dybas was disappointed, but not defeated. He decided to
set the molecules aside and go back to working with the
whole bacterium. He repeated the experiment Criddle had
tried three years earlier, testing KC’s ability to degrade
carbon tetrachloride in an aquifer sample of contaminated
water. Dybas now knew more than Criddle had known then,
however.
Dybas understood, better than anyone else, that KC needed
very particular conditions to do its job. So instead of just
dumping KC into the water as Criddle had initially done, he
first adjusted the acidity of the sample and added a food
supplement for KC to munch on. Finally, KC came through. It
passed its carbon tetrachloride-eating test with flying
colors.
This was the first experiment to show that KC didn’t need
a carefully prepared lab solution to survive and destroy
carbon tetrachloride; with a little help, it could work its
magic in real aquifer water. Since there were other,
naturally occurring bacteria in the water, it was also the
first indication that KC could effectively compete with
other bacteria for space and food. The researchers stood on
the threshold of developing a new approach to environmental
restoration. All they needed now was a site where they could
challenge their wonder-microbe in a real-world setting.
KC moves out of the lab and into the real world
At about the same time that Dybas’s experiments were
meeting with success, a student in one of Criddle’s classes
provided just what the team needed. Tim Mayotte had worked
for six years as the head hydrogeologist on a carbon
tetrachloride-contaminated aquifer in the village of
Schoolcraft in southwest Michigan. When he learned of
Criddle’s carbon tetrachloride-decontaminating bugs, he told
Criddle there might be an opportunity for testing KC.
But before dumping KC into the Schoolcraft aquifer, the
team had to do more work. What if KC itself was toxic? They
decided to have KC’s metabolic waste products tested for
cancer-causing compounds. The results came back negative.
The team also tested the effects of KC on the germination of
corn seedlings, since corn is grown in a field close to
Schoolcraft. KC seemed to allow germination just fine.
Finally, since the groundwater flows into a lake, they
tested KC’s effects on fish. Dybas even set up an aquarium
in his office where fish swam in a soup of KC. The fish
seemed fine. Although none of these tests proved KC is
harmless, they at least suggested the bacterium would not
cause an environmental disaster at Schoolcraft.
Besides testing KC’s environmental friendliness, the team
had to resolve another issue before injecting KC into the
Schoolcraft aquifer. Criddle and his co-workers had done all
their tests in bottles with watery solutions. The
Schoolcraft aquifer is primarily made up of sand saturated
with water that moves slowly through it—more like slush
than a water solution. Would the notoriously discriminating
KC cooperate under these conditions?
Much to its delight, the team rapidly found out that KC
was capable of attaching to the sand, growing on its
surface, and breaking down carbon tetrachloride. Criddle and
his group set up a small field experiment. They drilled a
well and fitted it with a four-inch diameter PVC pipe ending
in a strainer-like screen that allows water and bacteria
through, but keeps the sand out. They then injected KC and
its feeding solution into the pipe and let it seep through
the screen and into the surrounding ground. The group also
drilled several other holes around this injection well to
draw out water samples for monitoring KC’s whereabouts and
accomplishments.
KC did not disappoint the team on its field debut. It
quickly started feasting on the carbon tetrachloride. But
after a few months, chloroform started to show up in the
monitoring wells. Like a schoolyard bully, the natural
population of bacteria in the ground was eating KC’s food
and converting carbon tetrachloride into chloroform.
Criddle’s initial concern about KC’s inability to compete
well with other bacteria was playing out.
To solve the problem and increase the size of the
operation at the same time, the team would have to figure
out how to distribute enough KC across a larger area, and
how to keep it well-fed so it wouldn’t be squeezed out by
neighboring bugs.
“We really had our back against the wall when we wanted
to scale up,” said Criddle. “We realized that it was a huge
challenge.”
A new idea
Just in time, geologist David Hyndman and civil engineer
David Wiggert, both of Michigan State University, breathed
new life into the project. Brainstorming with Criddle’s
group, they envisioned a curtain of bacteria intercepting
the plume and destroying it at its leading edge. Wiggert
suggested drilling a row of closely spaced wells to inject
KC and its feeding solution into the aquifer. The idea was
unprecedented.
For months, Hyndman ran computer simulations to determine
if and how this idea would work. Based on his calculations,
the scientists sketched out a working model. They would
drill a row of 15 one-inch-diameter wells, spaced about 3
feet apart, with strainer-like screens along most of their
lengths. Into each well, they would inject KC, letting the
bacteria seep out into the aquifer and attach to nearby
grains of sand. The researchers would use the slotted screen
pipes to periodically supplement KC’s diet with feeding
solution. To make sure the entire biocurtain was well-fed,
they would recirculate the feeding solution by using
alternate wells to inject and extract the solution in the
aquifer.
Trap-and-Treat
The key to this system is that it’s passive. Within the
aquifer, groundwater moves like a slow-flowing river at six
inches a day. Because it sticks to the sand, the carbon
tetrachloride contaminating the water moves even more slowly—about
two inches a day. Most bioaugmentation engineers
seeking to flush the contaminant out view its stickiness as
a problem that interferes with their goal. But the approach
of Criddle’s team actually takes advantage of the
stickiness.
The group’s system uses the aquifer’s natural water flow
and the carbon tetrachloride-trapping properties of the sand
to accumulate carbon tetrachloride for the bacteria to munch
on leisurely. Spacing the wells 3 feet apart creates a
uniformly dense curtain of KC, enabling it, through sheer
numbers, to outcompete the natural bacterial
inhabitants. Within a day or two, KC can break down most of
the carbon tetrachloride immediately surrounding it. During
the next six days, water is left to naturally drift through
the KC curtain. This stage requires no pumping. As the
contaminated water flows by, the cleaned sand acts as a
brake on the sticky carbon tetrachloride. At the end of the
six days, a pump delivers fresh food and the cycle
repeats.
This trap-and-treat technology is not speedy, but it is
very cheap and potentially very effective. Conventional
techniques for treating contaminated water are often limited
by the cost of running pumps continuously as well as the
expense of the labor to maintain them. In addition,
conventional techniques are not very effective at cleaning
sticky contaminants like carbon tetrachoride, which can’t be
easily flushed out with water. Hyndman and Wiggert’s
calculations suggested that by properly spacing the wells,
they could get away with only six hours of pumping a week,
at the paltry rate of 40 gallons per minute—only about
eight times faster than the rate at which a garden hose
fills a bucket.
Criddle found it very hard to imagine that such a low
level of pumping could clean an entire aquifer. “Even now
when I say it to people, I’m amazed,” says Criddle. “It
shocked me.” Criddle admits he was nervous about investing
money and effort into this next step. But the Michigan
Department of Environmental Quality, looking for ways to
reduce their costs for cleaning up aquifers, decided to fund
the team. “It was a big risk,” says Criddle. “I guess our
funding agency had faith in us.”
During the summer and fall of 1997, Criddle and his team
worked with three private companies to build a full-scale
system. Workers drilled 15 bore holes reaching eight stories
below the ground, and fitted each with a one-inch diameter
PVC pipe. Except for the uppermost 30 feet, the rest of the
pipes’ lengths were riddled with slots to let KC and its
food out into the aquifer. They also installed over 100
other wells to monitor KC and its decontaminating
progress.
Viewed from across the field that overlies the aquifer,
the installation resembles a 50-foot-long diamondback snake.
Each square of the diamond is a wooden trapdoor that, when
opened, reveals the upper portion of a pair of wells. Each
well is connected by underground tubing to the feeding vats,
KC reservoirs, and mixing system which are housed in a small
building nearby.
On January 7, 1998 the team injected KC into the system
along with “Mike’s recipe for KC à la carte,” as
Criddle calls the feeding solution developed by Dybas. At
first, samples from the monitoring wells didn’t show much of
a drop in carbon tetrachloride concentrations. But by then,
Criddle and his group had learned patience. After two
months, carbon tetrachloride levels began to drop
significantly and they have continued to drop steadily ever
since. Criddle says the system now degrades more than 95
percent of the carbon tetrachloride contained in the 2,500
gallons that drift through the biocurtain every day.
“There were a lot of challenges. There were a lot of
quick decisions,” says Dybas. “And moments of panic and
moments of victory.”
Today, Dybas is the project manager of the KC
decontaminating team at Michigan State, and Tatara works as
a consultant and a key member of the team. “Most people
don’t get to see their Ph.D. work go into the field,” says
Tatara. “It was a once-in-a-lifetime opportunity.” Criddle
returned to Stanford where he is now an associate professor
in the department of civil and environmental
engineering.
What lies ahead
An important test of the biocurtain system hinges on its
evaluation by other experts in the field. But the
preliminary results are encouraging. The time it will take
his system to clean the entire aquifer depends on how many
KC biocurtains are set up. Criddle calculates that with the
single biocurtain they have now, it would take approximately
25 years and cost $1.5 million. Working at an equivalent
rate, pump-and-treat—an approach that requires continuous
pumping and multiple steps to destroy carbon tetrachloride—would
cost about $6 million. Criddle says the group is
now working on ways of automating the system to further
reduce costs. “Although I started this journey as a
bioaugmentation skeptic,” says Criddle. “I now find myself a
believer.”
But more significant than its low cost is its potential
for improvement. Lycely Sepulveda-Torres, a graduate student
of Criddle, and Ron Crawford and his coworkers at the
University of Idaho, have recently cloned the genes that
endow KC with its carbon tetrachloride-eating capability,
and are now attempting to put them into other organisms. If
successful, they may be able to engineer less-demanding and
faster-growing bacteria that produce more carbon
tetrachloride-degrading molecules.
Encouraged by all this, Criddle is preparing to use the
trap-and-treat method to tackle other contaminants such as
chromium, tetrachloroethylene, and trichloroethylene. Other
researchers have already found bacteria that degrade some of
these contaminants, so setting up these new decontaminating
systems may be straightforward. In other cases, he will have
to search for entirely new strains of bacteria, but his
experience with KC will no doubt serve as a valuable
guide.
The problem of carbon tetrachloride contamination,
however, is far from solved. The monstrous plume at the
Hanford site remains out of control. It is so vast that,
right now, neither the new KC biocurtain nor conventional
pump-and-treat systems stand a chance of making a
significant dent in it.
Nevertheless, in looking to the future, it is worth
remembering the unpredictable path that research often
follows. “The story of KC shows that the microbial world is
full of unexpected surprises,” says Criddle. “And that
serendipity can play a big role, maybe bigger than we care
to admit.”
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- BIO
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- WRITER
Marina Chicurel
- B. Sc., basic medical
research, Universidad Nacional Autonoma de Mexico; Ph.D., neurobiology,
Harvard University.
Internship: New Scientist (Latin America correspondent).
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Text ©
1999 Marina Chicurel
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