Experimental Technology and Management

在环境生物学实验教学过程中，如何使学生掌握检测重金属生物有效性的方法至关重要。该教学设计利用全细胞生物传感器zntA对重金属的特异性响应机制，设计了利用该菌株定量检测水体和土壤中铅(Pb)生物有效性浓度的实验。实验包括培养基制备、基因工程菌培养、重金属Pb暴露实验及菌株生物发光信号检测等环节。结果表明，随着Pb浓度的增加，zntA的生物发光信号逐渐增强，zntA对Pb的检测限为4μg·L^–1。基于zntA的生物发光信号值的强弱，成功定量了水体和土壤中生物有效态Pb的浓度。该实验教学设计为环境科学相关课程提供了创新性教学案例，具有较高的推广价值。

[Objective] Heavy metal(HM) pollution has posed an increasingly serious threat to the environment and human health. Therefore, accurately assessing the bioavailability of HMs is crucial for their environmental risk assessment(ERA). Traditional detection methods employed for ERA, such as chemical extraction, cannot reflect the actual interactions between HMs and organisms. However, plant-and animal-based biological detection methods suffer from several limitations, including time-consuming procedures and complex operational requirements. Therefore, developing a rapid, efficient, and operationally simple ERA technique for HMs is of great significance. This laboratory course aimed to utilize the whole-cell bioreporter “zntA” for designing and implementing a novel, experimental, teaching method for the quantitative detection of lead(Pb) bioavailability in water and soil, providing a case for innovative environmental science education. [Methods] This experiment employed a Type I whole-cell bioreporter, zntA, to detect Pb bioavailability through a specific response mechanism. The experimental design included strain isolation and cultivation, HM exposure assays, and bioluminescence detection. The bacterial strain was cultured in a lysogeny broth medium, harvested via centrifugation, and subsequently resuspended in an optimized minimal medium to obtain the bacterial suspension. A standard curve was plotted using standard Pb solutions with a 0–0.4 mg·L^-1 gradient. Water samples were filtered through a 0.45 μm membrane, while soil samples were prepared as slurries at 0.78–25 mg·mL^-1. These samples were then mixed with the bacterial suspension, and the bioluminescence kinetics were recorded for 7 h with a microplate reader. To address the issue of optical signal interference caused by soil particles, a light-blocking calibration model was established to compensate for signal attenuation induced by soil particulates, thereby enhancing the detection accuracy of the bioreporter for soils. [Results] Per the results, zntA exhibited a dose-dependent response to Pb, with bioluminescence peaking at 140 min. The limit of detection was 4 μg·L^-1, much lower than the drinking water guideline of 10 μg·L^-1 set by the WHO. An analysis of the samples from natural water bodies revealed Pb bioavailability of 0.21(W1) and 0.18 mg·L^-1(W2). For soil samples, the optical interference caused by soil particles was corrected by measuring light transmittance and optical density. A mathematical model correlating slurry concentration with optical signal obstruction was established, offering a calibration method for complex matrices. After calibration, the bioavailable Pb concentration in the soil was detected to be 0.69 mg·kg^-1. These results show that excessive soil slurry concentrations caused signal suppression due to toxic effects. [Conclusions] This experiment successfully applied whole-cell bioreporter technology to environmental biology teaching, demonstrating its advantages of speed and sensitivity over conventional methods. By optimizing the experimental procedures and introducing a light-blocking calibration model, interference from soil matrices was effectively addressed, providing a reliable solution for detecting HM bioavailability. The experimental design not only enhanced the practical and research skills of students but also served as a model for disseminating environmental monitoring technology-related.