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Binding of Bt protein to soil particles

Binding sites and soil horizons

It is a cool, sunny July morning on the maize trial field. Scientists from the University of Göttingen are digging up a soil profile. The University of Göttingen’s Institute of Applied Biotechnology in the Tropics (IBT) is part of the maize research group. It is responsible for characterising the soil on the trial fields. Its scientists are also investigating in the laboratory how much Bt protein the soil can bind and whether the Bt protein can move to lower soil layers.

Dr Jürgen Niemeyer (above) and Dr Christian Ahl (below) digging up a soil profile. (video clip in german language)

First the topsoil is removed and then the first subsoil horizon.

A soil profile on the maize trial field. The topsoil is the darkest because it contains the highest proportion of organic matter. The paler subsoil horizons contain larger proportions of clay and iron oxides.

If a soil and water mixture (left) is left for a few hours or days, the large particles, e.g. grains of sand, fall to the bottom first, while the smaller, lighter particles, e.g. clay particles, remain suspended in the water for longer. Since the Bt proteins primarily bind to the clay particles, the clay and water mixture (right) is siphoned off. The clay particles are freeze-dried and used for further research.

Dr Sibylle Pagel-Wieder prepares the sorption measurements.

Defined amounts of clay and Bt protein are mixed with water and agitated for half an hour (left). The samples are then centrifuged. This causes the soil particles to settle (right). The remaining solution is analysed further.

Results of the ELISA colour test, which measures the amount of Bt protein using a marked antibody. The first three columns on the left show the initial Bt solution. The columns on the right show the solution that remained after agitating for 30 minutes with clay. Since the clay particles have bound some of the Bt protein, the protein concentration in the later solutions is lower and the blue colouration is paler.

A whole arsenal of shovels, spades, bore sticks and rules are lined up at the edge of the field. Soil is being dug out of an area measuring one metre by one metre. After a quarter of an hour the pile of earth is already quite big. The top 30 centimetres have been taken out and suddenly the soil looks much lighter. The field consists of several layers, or ‘horizons’. These have different structures and therefore different chemical and physical characteristics. Jürgen Niemeyer and Christian Ahl gradually expose three different horizons; by the time they have finished, the hole is almost a metre deep. The different layers are removed separately and will later be shovelled back in the correct sequence.

Before the first crop of maize was sown in 2008 in the first year of the three-year trial, soil profiles were dug up at three sites on the trial field and soil samples were taken at 40 different sites on each of the 40 plots. The soil samples are measured for important parameters, including pH value, grain size distribution and humus and nitrogen content. The results of the soil characterisation are made available to the other members of the research group to enable them to determine whether differences between the different maize variants could also be due to different soil structures on the different plots. However, the tests carried out to date have revealed that the soil on the trial field is very homogenous.

Detecting bound proteins indirectly

In the afternoon, Sibylle Pagel-Wieder begins analysing the soil samples in the IBT institute’s laboratory. The work surface is covered with large glass bottles filled with a brownish soil and water mixture. What looks to the layman like mud is actually a sedimentation experiment. The different components of a soil horizon are separated by leaving the mixture to stand so that the larger, heavier particles sink to the bottom.

The IBT scientists are primarily interested in whether Bt proteins travel to lower soil layers. Whether a soluble substance like a protein is carried down to lower soil layers when it rains depends partly on how strongly it is bound (sorbed) by the upper soil layers, which would then hold it back. This in turn depends on the chemical and physical characteristics of the soil and of the protein. The IBT team is investigating which soil and protein characteristics have the greatest influence on the sorption of various Bt proteins.

The research is difficult because sorbed proteins cannot be detected directly. If standard procedures for obtaining proteins are used on soil samples, the only proteins obtained are those that are bound only lightly, if at all, to soil particles. To detect sorbed proteins, one would first have to separate them from the soil particles. However, this is not really possible without changing the proteins chemically or destroying them.

Sibylle Pagel-Wieder therefore uses an indirect method of measurement. After a number of stages in which she obtains the clay particles, to which most of the proteins bind, she mixes a precise weight of clay with water and a known amount of Bt protein, centrifuges them to separate the clay particles off after half an hour and measures the amount of Bt protein left in the liquid. From this she calculates the amount of bound protein and the ratio of free to bound protein. This experiment is conducted with various protein concentrations. Sibylle Pagel-Wieder has succeeded in refining the measurement method to such an extent that it can be used on protein concentrations as low as those found in the field.

At the same time, the team examines the samples to measure various parameters, such as electrical charge and the total surface of the clay particles. This data is correlated with the sorption measurements.

Bt proteins in the soil: different behaviour

The first Bt protein to be examined by the IBT institute was Cry1Ab, which targets the European corn borer. These studies are complete. The binding of Cry1Ab to soil particles depends largely on the electrical charge and the total surface area of the soil particles and on the proportion of organic carbon. For Cry3Bb1, which primarily protects Bt maize plants against the Western corn rootworm, all the measurements are complete, but analysis of the data will not be finished until the end of 2009. However, there are already indications that Cry3Bb1 behaves very differently. Cry1A.105 and Cry2Ab2 are also being studied within the new maize research group.

Earlier research conducted by the IBT institute showed that less than 10 per cent of the Bt protein is desorbed again, i.e. once Bt protein is bound to soil particles, it usually remains bound. It is known that sorbed Bt proteins cannot be broken down by bacteria, but their insecticidal effect remains intact. IBT measurements have shown that the ratio of unbound to bound Bt protein is about 1:200, i.e. if 0.2 nanograms of Bt protein per gram of soil is found in the soil samples from the trial field, there will be an estimated 40 nanograms of sorbed Bt protein per gram of soil. These amounts are still a long way below the lethal dose (LD50 ) for the target organisms, let alone non-target organisms. The results on the behaviour of Bt proteins in the soil are also valid in principle for Bt proteins that are sprayed e.g. by organic farmers.