Development of a new biological-based method for the detection of heavy metals in water sources

The contamination of water sources by heavy metals represents a critical issue with serious implications for both the environment and human health. Effective monitoring of these pollutants is essential to mitigate human exposure to toxic metals and protect ecosystems. However, the methods currently available for detecting heavy metals present several limitations, such as high operational costs, methodological complexity, and prolonged analysis times. Consequently, there is an increasing need to develop alternative approaches capable of overcoming these limitations, providing more rapid, cost-effective, and user-friendly solutions for the monitoring of heavy metal contamination. This thesis aimed at developing an innovative, colorimetric biological-based method for the rapid detection of heavy metals in potentially contaminated water sources. The development of this method relied on the use of the microorganism B. subtilis. Indeed, studies have shown that the enzyme α-amylase, expressed by B. subtilis, is inhibited by heavy metals, including cadmium, lead and mercury. This inhibition has been exploited to develop a variant of the MBS method suitable for detecting the potential presence of heavy metals in artificially and naturally contaminated water samples. Indeed, the MBS method, by measuring the catalytic activity of oxidoreductase enzymes involved in the main metabolic pathways of bacteria, allows an unequivocable correlation between the observed enzymatic activity and the number of viable bacteria present in the sample. This is made possible by the presence of a redox indicator in the growth medium which in condition of hypoxia, induced artificially by limiting external oxygen supply (i.e., by applying vaseline oil at the air-sample interface), causes a color change of the solution at a rate inversely proportional to the bacterial concentration. Indeed, once the internal available oxygen is depleted, bacterial metabolic activity will result in the reduction of the redox indicator, responsible for color change of the growth medium. The variant of the MBS method developed within this thesis has been based on the principle that in the presence of heavy metals the time required for the color change should be slow down, due to a decrease in bacterial growth caused by diminished α-amylase activity. Indeed, since α-amylase catalyzes the hydrolyses of internal α-1,4-glycosidic linkages in starch in low molecular weight products, such as glucose, maltose and maltotriose units, in the presence of starch as the sole carbon source in the growth media, microorganisms’ growth depends on α-amylase activity. The results obtained showed that the developed method is effective in detecting the presence of heavy metals in artificially contaminated water samples, showing limits of detection (LOD) of 5 µg/L for cadmium, 10 µg/L for lead, and 6 µg/L for mercury. Interestingly, these concentration values correspond to the legal thresholds for cadmium and lead in potable water established by Legislative Decree 31/2001 and align with the guideline value for mercury recommended by the World Health Organization (WHO). However, it is important to highlight that the developed method is not intended to provide precise quantitative measurements of heavy metal concentrations. Rather, it was designed as an early warning heavy metal detection system. Consequently, the method aims to provide an indication of the potential contamination, rather than quantifying the levels of contaminants. The selectivity of the method was assessed toward the metals most found in natural waters such as, manganese, copper, and nickel. The results showed that the responses of these metals, at concentrations up to the legal limit established for irrigation water, do not overlap with the responses induced by cadmium, mercury, and lead, demonstrating that the presence of these metals in water sources, at the analyzed concentrations, does not interfere with the detection of the heavy metals investigated in this study. The results obtained in terms of the rapidity of the analytical response highlighted that heavy metal contamination can be detected within a time range of 3.5 to 9 hours, and a color change response within and no later than 3.5 hours can be considered indicative of the absence of the considered heavy metals. In conclusion, the results obtained showed that the method developed within this thesis project presents some distinctive features that can offer perspectives and implications in the field of developing cost-effective, portable, and user-friendly devices that would facilitate on-site analysis and allow even non-expert personnel to perform rapid analyses. However, before reaching this final goal, studies on naturally contaminated water sources, especially those derived from areas known to be potentially impacted by heavy metal contamination, are needed to confirm the effectiveness of the method in more complex matrices. In this context, studies are underway, and the preliminary results are promising even if still too preliminary to be discussed in this work.