The mechanisms of tolerance, accumulation and adsorption of Cd by rice roots endophyte Bacillus sp. RE35

The cadmium (Cd) contamination in agricultural land has been attracting much attention worldwide due to its potential health risks. Rice grown in the polluted field may pose a serious threat to human health through dietary intake because its high Cd enrichment ability leads to Cd accumulation in the grain. It has been shown that plant endophytes have the ability to immobilize Cd, but there are few studies reporting the characteristics of rice endophytes with high Cd tolerance, and their Cd-immobilizing mechanisms under different concentrations of Cd need further studies. In this article, a liquid adsorption test was conducted to investigate the Cd tolerance and immobilizing mechanisms of the rice root endophyte Bacillus sp. RE35 that was previously screened for high Cd tolerance. The results showed that the maximum Cd tolerance concentration of strain RE35 could reach 600 mg/L, the maximum adsorption capacity was 28.49 mg/g, and the removal rates of 1 and 5 mg/L Cd2+ were 88.37% and 80.40%, respectively. The stain can tolerate such high concentrations of Cd equivalent to the stains isolated from Cd-contaminated soil. The strain RE35 showed distinct Cd accumulation and adsorption mechanisms at different Cd concentrations. When Cd concentration was lower than 20 mg/L, Cd2+ removal by strain RE35 was dominated by intracellular accumulation. The production of siderophores, as a possible transport carrier of Cd, was greatly reduced under Cd treatment, indicating that siderophores did not have significant contribution to the intracellular accumulation of Cd by the strain RE35. The determination of ATPase activities showed that they increased significantly with the increase of Cd concentrations, and it was speculated that the intracellular transport of Cd by strain RE35 may be related to ATP-dependent metal transporters. The distribution of Cd inside strain RE35 cells was investigated by high-resolution transmission electron microscopy and energy dispersive spectroscopy, and the results revealed that Cd was uniformly distributed in the cytoplasm of the cells. When the concentration of Cd was higher than 20 mg/L, the intracellular Cd accumulation of strain RE35 reached saturation, whereas the extracellular adsorption amount rose significantly with increasing Cd concentrations and gradually exceeded the intracellular accumulation. The production of protein component in the extracellular polymeric substances (EPS) by strain RE35 was positively related to the amount of Cd extracellular adsorption. The results of scanning electron microscopy and energy dispersive spectroscopy showed that more EPS appeared on the cell surface with the increase of Cd concentrations, and Cd adsorption on the cell surface was detected at the Cd concentration of 70 mg/L as well. The characterization of chemical groups on the cell surface by X-ray photoelectron spectroscopy and Fourier transform infrared absorption spectroscopy showed that the -NH2, amide I (C=O) and amide II (-NH, -CN) groups of extracellular proteins were involved in the Cd extracellular adsorption by the strain RE35. The rice root endophyte Bacillus sp. RE35 exhibited relatively high resistance and removal capacity of Cd2+. When the Cd concentration was lower than 20 mg/L, strain RE35 removed Cd2+ in solution mainly by intracellular accumulation, and when the Cd concentration increased, it was mainly through extracellular adsorption. The intracellular accumulation of Cd by strain RE35 might be related to ATP-dependent metal transporters, and at higher Cd concentrations, the strain could effectively adsorb Cd extracellularly through the secretion of EPS in addition to accumulating intracellularly. The amino and amide groups of proteins in EPS were closely related to the extracellular adsorption of Cd. This study elucidated the Cd accumulation and adsorption mechanisms of strain RE35 with high Cd tolerance under different Cd concentrations, providing valuable information on a strain resource for the development of endophyte microbial agents in the future.