Klebsiella planticola: The GM bacteria that almost...

From a post by Dr Robert Mann, on the New Zealand GE list 8 February 2001

The story really shows the awesome power of genetic engineering, the multidisciplinary nature of the review it requires, and the folly of releasing GM microbes before very extensive contained studies.

This is a very good example of how slight changes in a highly evolved bacterium can greatly change its ecological significance.

Klebsiella planticola, a common soil bacterium, was genetically engineered by a German research institute to make ethanol for industrial purposes.

The inventors had planned a recycling system: farmers would give them agricultural slash, which would be used for the bacterial fermentation; the resulting ethanol would be separated out, and the sludge could be given back to the farmers to spread on their fields as fertilizer.

It all sounded very good for the environment, but how much soil ecology impinged on the planning is unclear.

Dr Elaine Ingham of Oregon State University and her graduate student M.T. Holmes discovered to their alarm that soils containing the engineered organism killed wheat seedlings, most likely through alcohol production in the root system, which kills roots at very low concentrations. Mycorrhizal fungi were also killed.

Had the engineered sludges been returned to farmers, it would have drastically degraded their soil, rendering them unable to grow many or all plants.

Since K. planticola is a ubiquitous organism, found in the root systems of plants all over the world, the GM mutant could have spread and made ALL soil unable to support crops!

Microorganisms are easily spread on surfaces of insects, on the feet of birds, on people's feet, etc; this engineered bacterium could have spread world-wide quite rapidly.


Dr Ingham describes what happened:

Good Intentions and Engineering Organisms that Kill Wheat

by Elaine Ingham, Oregon State University

A genetically engineered Klebsiella-planticola had devastating
effects on wheat plants while in the same kind of units, same incubator,
the parent bacteria did not result in the death of the wheat plants.

Consider that the parent species of bacteria grows in the root systems of
every plant that has been assessed for Klebsiella's presence. The
bacterium also grows on and decomposes plant litter material. It is a very
common soil organism. It is a fairly aggressive soil organism that lives
on exudates produced by the roots of every plant that grows in soil.
This bacterium was chosen for those very reasons to be engineered:
aggressive growth on plant residues.

Field burning of plant residues to prevent disease is a serious cause of
air pollution throughout the US. In Oregon, people have been killed
because the cloud from burning fields drifted across the highways and
caused massive multi-car crashes. A different way was needed to get
rid of crop residues. If we had an organism that could decompose the
plant material and produce alcohol from it; then we'd have a win-win
situation. A sellable product and get rid of plant residues without
burning. We could add it to gasoline. We could cook with it. We could
drink grass wine-although whether that would taste very good is
anyone's guess. Regardless, there are many uses for alcohol.

So, genes were taken out of another bacterium, and put into
Klebsiella-planticola in the right place to result in alcohol
production. Once that was done, the plan was to rake the plant residue
from the fields, gather it into containers, and allow it to be
decomposed by Klebsiella-planticola. But, Klebsiella would produce
alcohol, which it normally does not do. The alcohol production would
be performed in a bucket in the barn. But what would you do with the
sludge left at the bottom of the bucket once the plant material was
decomposed? Think about a wine barrel or beer barrel after the wine
or beer has been produced? There is a good thick layer of sludge left
at the bottom. After Klebsiella-planticola has decomposed plant
material, the sludge left at the bottom would be high in nitrogen and
phosphorus and sulfur and magnesium and calcium-all of those
materials that make a perfectly wonderful fertilizer. This material
could be spread as a fertilizer then, and there wouldn't be a waste
product in this system at all. A win-win-win situation.

But my colleagues and I asked the question: What is the effect of the
sludge when put on fields? Would it contain live Klebsiella-planticola
engineered to produce alcohol? Yes, it would. Once the sludge was spread
it onto fields in the form of fertilizer, would the
Klebsiella-planticola get into root systems? Would it have an effect
on ecological balance; on the biological integrity of the ecosystem; or on
the agricultural soil that the fertilizer would be spread on?

One of the experiments that Michael Holmes did for his Ph.D. work was to
bring typical agricultural soil into the lab, sieve it so it was nice and
uniform, and place it in small containers. We tested it to make sure it
had not lost any of the typical soil organisms, and indeed, we found
a very typical soil food web present in the soil. We divided up the
soil into pint-size Mason jars, added a sterile wheat seedling in
every jar, and made certain that each jar was the same as all the

Into a third of the jars we just added water. Into another third of the
jars, the not-engineered Klebsiella-planticola, the parent organism,
was added. Into a final third of the jars, the genetically engineered
microorganism was added.

The wheat plants grew quite well in the Mason jars in the laboratory
incubator, until about a week after we started the experiment. We came
into the laboratory one morning, opened up the incubator and went,
"Oh my God, some of the plants are dead. What's gone wrong? What did
we do wrong?" We started removing the Mason jars from the incubator.
When we were done splitting up the Mason jars, we found that every
one of the genetically engineered plants in the Mason jars was dead.
Wheat with the parent bacterium, the normal bacterium, was alive and
growing well. Wheat plants in the water-only treatment were alive and
growing well.

From that experiment, we might suspect that there's a problem with this
genetically engineered microorganism. The logical extrapolation from this
experiment is to suggest that it is possible to make a genetically
engineered microorganism that would kill all terrestrial plants. Since
Klebsiella-planticola is in the root system of all terrestrial plants,
presumably all terrestrial plants would be at risk.

So what does Klebsiella-planticola do in root systems? The parent
bacterium makes a slime layer that helps it stick to the plant's roots.
The engineered bacterium makes about 17 parts per million alcohol.
What is the level of alcohol that is toxic to roots? About one part
per million. The engineered bacterium makes the plants drunk, and
kills them.

But I am not trying to say that all genetically engineered organisms are
technological terrors. What I am saying is that we have to test each and
every genetically engineered organism and make sure that it really does
not have unexpected, unpredicted effects.

They have to be tested in something that approximates a real world
situation. I've worked with folks in the Environmental Protection Agency
(EPA) and I know the tests the EPA performs on organisms. They often begin
their tests with "sterile soil." But if it's sterile, then it's not really
soil. Soil implies living organisms present. If you use "sterile soil" and
add a genetically engineered organism to that sterile material, are you
likely to see the effects of that organism on the way nutrients are
cycled, or on the other organisms in that system? No, you're not
likely to. So it's probably no surprise that no ecological effects
are found when they test genetically engineered organisms in sterile
soil. They really need to put together testing systems, which assess
the effects of the test organism on all of the organisms present in

What do we mean, organism-wise, when we talk about soil? Agricultural
soil should have 600 million bacteria in a teaspoon. There should be
approximately three miles of fungal hyphae in a teaspoon of soil. There
should be 10,000 protozoa and 20 to 30 beneficial nematodes in a teaspoon
of soil. No root-feeding nematodes. If there are root feeding
nematodes, that's an indicator of a sick soil.

There should be roughly 200,000 microarthropods in a square meter of soil
to a 10-inch depth. All these organisms should be there in a healthy soil.
If those conditions are present in an agricultural soil, there will be
adequate disease suppression so that it is not necessary to apply
fungicides, bactericides, or nematicides. There should be 40 to 80% of the
root system of the plants colonized by mycorrhizal fungi, which will
protect those roots against disease.

What happens when you apply the most fungicides and pesticides to soil?
In every single case where we have looked at foodweb effects of
pesticides, there are non-target organism effects, and usually very
detrimental effects. The sets of beneficial organisms that suppress
disease are reduced. Organisms that cycle nitrogen from
plant-not-available forms into plant-available forms are killed.
Organisms that retain nitrogen, phosphorus, sulfur, magnesium,
calcium, etc. are killed. Organisms that retain nutrients in the soil
are killed. Once retention is destroyed, where do those nutrients go?
They end up in our drinking water; or end up in our ground water. You
and I as taxpayers have to pay in order to clean up that water so we
can drink it.

Wouldn't it be much wiser to keep those organisms present in the soil so
those nutrients would be retained and become available to the next crop of
plants instead of ending up in our drinking water where we have to pay in
order to have clean drinking water? How do you do that? You get the
organisms back into the soil. If you grow the proper number and types of
bacteria, fungi, protozoa, nematodes and microarthropods, mycorrhizal
fungi in the root systems of the plants, you can do away with
pesticides. It's been done. We can reduce significantly the amount of
fertilizer that goes into that soil. In experiments that have been
done all over the country, all over the world, inorganic fertilizer
inputs have been reduced, or are not added at all, without reduction
in plant growth. Where green manure or legumes are not available,
approximately 40 pounds of nitrogen fertilizer, once every four
years, are still necessary.

Let's talk about why today's conventional agricultural systems require
such massive inputs of pesticides and fertilizers. When a healthy soil is
first plowed out of native grassland, for example, the disease-suppressive
bacteria and fungi, protozoa and nematodes are present. For the first 5 to
15 years after plowing native grassland you don't have to use any
pesticides. No fertilizers are required because there is natural nutrient
cycling, natural nitrogen retention, and disease suppression. As you plow
that soil, you start to kill the beneficial organisms, you lose the
organic matter, and you lose the food to feed the beneficial
organisms. After about 10 to 15 years, if you're not adding back
adequate plant residue to feed those organisms, you lose them, and
start having significant disease problems. Then you either leave that
land and farm elsewhere, or in the US, we used fertilizers to keep
yields high. As more and more of the organisms were killed by the
salt effect of the fertilizers, and the constant plowing mined out
more and more of the organic matter, starving the beneficial
organisms to death, disease became a serious problem. And we started using
more and more pesticide to knock the disease back.

In California, around 1955, those disease problems became so severe that
they thought they would lose agricultural production. So the University of
California came up with a better way to kill those disease-causing
organisms. It's called methyl bromide. This chemical kills disease-causing
organisms-but it also kills everything else. There is very little natural
disease suppression going on in agricultural soils in California.

How many organisms are left in strawberry fields that have been
methyl-bromided 2 to 3 times a year for the last 14 years? There are no
microarthropods left. There are no beneficial nematodes left; only root
feeding nematodes. And there is nobody to control root-feeding nematodes
in those soils. How many protozoa are left in that soil? None. You
cannot cycle nutrients. There is nobody home to make nitrogen
plant-available. So what do you have to do? You have to add
fertilizer. We force ourselves to have to add fertilizer. We have no
other choice if you're going to grow those plants in those soils.

How many fungi do you have left in that soil? No beneficial fungi-they're
all disease-causing. How many bacteria are left? All are gone, except for
100 per gram of soil. We should have 600 million per teaspoon in that
soil; we have 100 left. There is nothing left to retain nitrogen in
those soils, nothing. So you apply fertilizer. What happens to the
fertilizer? Whatever fertilizer contacts the roots of the plants is
indeed taken up; the rest of it flushes through the soil into the
ground water, into the river. Take Santa Maria River in California as
an example. This land has had methyl bromide applied 2 to 3 times a
year for the last 14 years or more. Fertilizer is applied as
sidedress when strawberries are planted. About two weeks later, the
river goes up to around 150 parts per million nitrates. What is the
toxic level for nitrate for humans? Ten parts per million nitrates is
what the EPA tells us. It used to be three parts million but that
evel was increased. Can you drink that water in the river in the Santa
Maria valley? Not unless you'd want to die. You would destroy your kidneys
pretty fast if you drank that water. It is high in nitrate. It is so toxic
that you can't even put that water back on the plants. The high nitrate
burns the plants.

We have a simple solution for this problem. Get the right kind of
organisms, the right numbers of organisms, back in the soil and let them
start performing their functions again. Put food for the organisms back
into the soil; put the organisms back into the soil. It's that
simple. Send us your soil samples and we can tell you whether you
have that food web in your soil.

How are you going to fix that set of organisms it if you don't have a
healthy foodweb? We can help you with that question. We can indeed move
towards that time when we really don't need pesticides anymore; where you
only apply fertilizer once every four years and in very small amounts. We
can move to a sustainable agriculture. It takes time and effort, but it is

This article is adapted from the presentation the author gave on July
18, 1998 at the First Grassroots Gathering on Biodevastation: Genetic

See also: Holmes, M.T., Ingham, E.R., Doyle, J.D., & Hendricks, C.W.
(1998). Effects of Klebsiella-planticola SDF20 on soil biota and
wheat growth in sandy soil. Applied Soil Ecology, 326, 1-12.




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