The Adsorption And Rematining Of Bacillus Thuringinesis Toxin In Minerals And Soil Colloids

The wide-spread cultivation of transgenic plants would pose a risk to natural and agricultural ecosystems, especially the transgenic Bacillus thuringinesis (Bt) plants, for they release Bt toxin to the soil ecosystem by root exudates, plant biomass and pollen. This study investigated the adsorption and desorption of Bt toxin by montmorillinite, kaolinite, goethite, silicon dioxide, red soil (Ultisol), latosol (Oxisol), yellow brown soil (Alfisol) and yellow cinnamon soil (Alfisol). The kinetic and thermodynamic parameters were analyzed to understand the adsorption mechanisms, while infrared spectra, circular dichroism (CD) spectra and fluorescence spectra were used to investigate the secondary structure changes of Bt toxin during the adsorption and desorption process. The remaining of Bt toxin in soils with or without microorganisms were also studied. This investigation helps to evaluate the behavior and fate of Bt toxins in the soil ecosystem. The main results are described below.1) The isotherm adsorption curves were L-type, and the adsorption data fitted well with both Langmuir and Freundlich isotherm models, but the Freundlich equation was more suitable. The adsorption was much easier at low temperature than that at high temperature at the initial Bt toxin concentration varying from 0 to 1 000 mg L^-1. The negative values of the standard free energy (Δ_rG_m^θ) indicated that the adsorption of Bt toxin by minerals and soil colloids were spontaneous. The standard enthalpy changes (Δ_rH_m^θ) of the toxin adsorbed by montmorillinite was positive while that by others were negative, suggesting that the adsorption by montmorillinite was endothermic and by others were exothermic. The standard enthalpy changes (Δ_rH_m^θ) were all less than 40 kJ mol^-1, showing that the adsorption was physicosorption.2) The Bt toxin could be adsorbed easily by minerals and soil colloids, and the adsorption amount in the first 0.25 h was the 75%-80% of the equilibrium adsorption amount. The adsorption kinetic of Bt toxin can be described by the intra-particle diffusion model, however, the value of the contant C was not 0. This phenomenon showed that the speed of intra-particle diffusion was not the only dominant factor of the adsorption. Besides, the adsorption dynamic of Bt toxin consisted with the pseudo-first-order, pseudo-second-order, and Elovich equations in which the pseudo-second-order had the best fitting results, and it indicated that the adsorption is mainly controlled by the surface adsorption process. 3) The adsorption amount of the toxin were greatest at pH 6 by goethite, montmorillonite, kaolinite, yellow brown soil, and yellow cinnamon soil, while the maximum adsorption by silicon dioxide, red soil, and latosol presented at pH 7. The ]overall trend was the adsorption capacity of the toxin decreased with the pH increase, which is attributed to the isoelectric point (IEP) of Bt toxin and the point of zero charge (PZC) of minerals and soils.4) The adsorption of Bt toxin by minerals and soil colloids were affected by low-molecular-weight organic acid ligands and inorganic salts. Low concentrations (< 10 mmol L^-1) of organic acid ligands (acetate, oxalate, citrate) inhibited toxin adsorption, whereas high concentrations promoted adsorption, and the inorganic salts had the opposite effects. The effect degree of the different organic acid ligands was oxalate > citrate > citrate, while H₂PO₄^- > NO₃^- and NH₄⁺ > K⁺ for inorganic ions.5) The adsorbed Bt toxin was hardly desorbed. The desorption rate of the toxin by 0.1 mmol L^-1 NaCl and phosphate was less than 5.3% and 13.1%, respectively, which indicated that a small proportion of the toxin adsorbed by minerals and soil colloids via electrostatic forces and ligand exchange. The desorption rates of Bt toxin were increased obviously when the organic acid ligands were present, the results suggested that organic acid ligands can markedly loose the bond of Bt toxin to minerals and soil colloids, and oxalate had the most significant effects among the tested organic acid ligands.6) Two fluorescence peaks of Bt toxin presented at 338 ran of tryptophan residues and 314.5 nm of the tyrosine residues by the excitation at 282 nm. The tryptophan peak of the toxin desorbed from kaolinite, montmorillonite and soil colloids were red-shifted 5 to 9.5 nm, and their tyrosine peak remained at 314.5 nm without shift. However, the two peaks of the toxin desorbed from goethite and silicon dioxide didn't shift obviously. The red-shift of the fluorescence peaks suggested that the polarity of the microenvironment of the toxin increased during the adsorption and desorption. The CD spectra showed that theα-helix andβ-sheet content of the toxin after desorption from minerals and soil colloids increased while P-turn and random coil content decreased in comparison to native toxin, which indicated that the ordered structure (α-helix +β-sheet) of the toxin increased in a certain extent during the adsorption and desorption. The special IR spectra of amideâ… (1653 cm^-1) and amideâ…¡(1543 cm^-1) of the adsorbed toxin did not shifted obviously in comparision to the native toxin, and the peaks at 1055, 1240 and 2362 cm^-1 were disappeared, these results indicated that the radicle of C-N played a key role in the adsorption of Bt toxin by minerals and soil colloids with no obvious changes in the secondary structure of Bt toxin after adsorption.7) At 25℃, the remaining amount of the free Bt toxin (without soils) when the initial concentration was 0.3μg g^-1 decreased as the cultivation time increase, and the remaining amount was less than 55% of the initial content after cultivating 10 days. The results indicated that the free toxin could degrade rapidly, and the degradation of the toxin with microorganisms (especially extracted from the soil planting non transgenic Bt plants) was more than that without microorganisms. However, as the cultivation time increase, the remaining of Bt toxin in soils (latosol, yellow brown soil and yellow cinnamon soil) increased firstly (1-2 days) and then gradually decreased. The half-life (DT₅₀) of Bt toxin in soil tested was about 10 days, and the effects of macroorganisms on the degradation of Bt toxin in soils were not obvious. The degradation rates of Bt toxin in yellow brown soil and yellow cinnamon soil were lower that that in latosol at cultivation time varying from 10 to 40 days, with no obvious differences of degradation rate in 3 tested soils at the first cultivation days (less than 10 days).