3.1 Analysis of the growth of B. subtilis
The growth analysis revealed significant variations in behavior among the different culture media tested. It is important to highlight that the data collected during this analysis formed the foundation for assessing how well the microorganism adapted in the metal-based assays without any additional sources of nutrients. This information suggests that the microorganism's ability to thrive and reproduce varies depending on the specific conditions provided by the culture media. The results also hold significance for understanding how the microorganism responded to metal exposure in an environment where its nutritional resources are limited, which is a key aspect of the assays being conducted.
In Fig. 1, it is possible to observe that in TSB33, the growth of the microorganism was less evident in comparison to TSB100. This difference can be attributed to the reduced availability of nutrient sources in TSB33. Despite the disparity, both media exhibited an exponential growth phase at 4 h of cultivation.
Figure 1 The outcomes of bacterial growth in the standard TSB medium, with two different dilutions: TSB100 and TSB33. Different shapes indicate different sampling materials, with black circles for TSB100 and triangles for TSB33
For TSB100, the highest concentration of cell mass was reached at 72 h of culture, reaching 3 g/L. On the other hand, in the TSB33 culture, the maximum cell mass concentration was achieved between 15 h and 96 h of culture, with levels ranging from 0.6 to 0.7 g/L.
By the Student t test, when the biomass was compared, the weight in TSB33 was always significantly lower than it was in TSB100, except at 48 h and 72 h, when the growth of biomass was equal regardless of the concentration. These results indicate that the best methodology to evaluate the growth of the microorganism against metals is the determination by concentration - cell mass in g/L. Furthermore, they indicate that the assays can be conducted using metal by reducing the nutritional components of the culture medium by 67% (TSB33).
3.2 B. subtilis growth in the presence of each heavy metal
For Pb, the removal capability by the microorganism B. subtilis was evaluated in TSB33 and water, by the X-Ray Fluorescence analysis. The behavior of the microorganism regarding its growth is presented in Fig. 2.
Figure 2 Analysis of B. subtilis mass growth X (g/L) and the consumption of Pb (ppm) in TSB33 and water
The assay utilizing TSB33 and water started with 500 ppm of Pb and cell mass at a concentration of 0.31 g/L. For TSB33 - unfilled bars - in the first hour of cultivation, there was a 5-fold decrease in the concentration of Pb, from 500 ppm to 100 ppm. However, in the 4-h cultivation, there was a 3-fold increase in its concentration, from 100 to 350 ppm. During the first 18 h of culture, the concentration of Pb varied and eventually reached approximately 180 ppm. This variation in concentration coincided with the exponential growth phase of the B. subtilis, represented by unfilled diamonds.
After 24 h, the main differences regarding the growth of the microorganism and the consumption of Pb were observed because the cell mass in the culture increased by 17 times reaching 5.47 g/L and the concentration of Pb decreased by 8.3 times from the initial value, reaching the value of 60 ppm. In 48 h of cultivation, the cell mass increased by 56 times, reaching 17.42 g/L, and no Pb was found in the sample. However, in 144 h of the culture, the concentration value of Pb was approximately 100 ppm and the cell mass was similar to its initial phase (0.39 g/L).
For the water assay – represented by gray bars - the Pb concentration reduced from 500 ppm to 8 ppm (a 61-fold reduction), after 1 h of cultivation. Between 4 and 72 h of cultivation, the Pb concentration increased and oscillated between 20 and 40 ppm. At 120 and 144 h of cultivation, the Pb concentration increased to 20 and 30 ppm. The cell mass concentrations – represented by asterisks – were around 0.25 g/L, the highest value observed was 14 g/L and was not directly related to the low concentration of Pb. However, B. subtilis, for both analyses, grew from 8 h to 48 h, indicating actively multiplication with rapid growth of their population and a decrease in the concentration of Pb.
Figure 3 Analysis of B. subtilis mass growth X (g/L) and the consumption of Cd and Cu (ppm) in water
The degradation of Cu and Cd by the microorganism B. subtilis was carried out exclusively in water due to the previous observations of Pb. The assay in water, containing 100 ppm of Cu, is represented by the black bars in Fig. 3. The finding shows, in the first hour of culture, the Cu value reduced by 8 times, from 100 to 13 ppm, remaining at this level until the period of 8 h. Within this period, the microorganism, represented by black circles, was in exponential phase growing from 1.46 g/L to 15.95 g/L. There was a reduction in cell mass between 8 and 48 h. Then, there was a new increase in cell mass, which remained until 120 h. The concentration of Cu varied between 12.5 ppm and 16.5 ppm throughout the process.
Also, the assay in water containing 100 ppm of Cd is represented by the gray bars in Fig. 3. Results showed, in the first hour of cultivation, Cd levels reduced by almost 4 times, going from 100 ppm to 27 ppm. In this period, the microorganism, represented by unfilled circles, was in exponential phase growing from 0.12 g/L to 15.5 g/L. In 2.5 h, the increase of cell mass was 5.7 g/L and reduction of Cd concentration was from 100 ppm to 27 ppm. In the period between 4 h and 144 h, the cell mass concentration varied between 11 g/L and 15.5 g/L. At 8 h, the greatest increase in cell mass was observed, reaching 15.5 g/L, and it was also possible to observe a 3.6-fold reduction of Cd concentration, from 100 ppm to 28 ppm. At 72 h, there was a 10-fold reduction in Cd concentration, from 100 ppm to 10 ppm. At 96 h, the cell mass was reduced to 11.4 g/L with an increase in Cd concentration to 21 ppm. The best results in relation to the growth of the microorganism and the consumption of Cd were obtained at 120 h with an increase of cell mass of 14.5 g/L, and a reduction of Cd concentration from 100 ppm to 9.8 ppm. At 144 h, there was an increase of 14.8 g/L and a reduction of Cd concentration from 100 ppm to 8.6 ppm.
3.3 Challenge analysis: B. subtilis growth in the mixture of heavy metals
Idealizing a system where the metals would be together, the analysis of the removal of 100 ppm of Pb, Cu, and Cd, in water by B. subtilis was performed and the behavior of the microorganism with respect to its growth can be observed in Fig. 4.
We can observe that B. subtilis, represented by the line, presented the beginning of the exponential phase after 1 h of contact with the metals. However, Cd (gray bars) and Pb (black bars) were reduced by approximately 50% in the first 10 min. In this assay, through ICP-MS, it was not possible to identify what happened to Cu because there was an increase in its concentration. Zhao et al. (2020) indicated that the presence of Fe and Cd ions inevitably affected the efficiency of Cu removal. In our experiment, the presence of Cd and Pb may have also affected the efficiency of Cu removal in a negative way.
Figure 4 Analysis of B. subtilis growth in the mixture of heavy metals and respective removal efficiencies in water
3.4 Removal efficiency in the presence of each heavy metal
In Fig. 5, at the 96th h of the process, Pb removal efficiency reached 100% whereas Cu reached 89% and Cd, 72%. The removal of Cd in the 1st h of contact with the cellular biomass was 73.7%. However, at the 144th h of process, Cd removal efficiency was 91.41%. The highest efficiency of Cd removal occurred at the 120th h and 144th h, reaching 91% and 92.3%, respectively.
Figure 5 Removal efficiency in the presence of Pb, Cu and Cd, separately, utilizing B. subtilis in water
3.5 Scanning electron microscope (SEM)
We only analyzed by this technique cell samples of the microorganism in contact with Pb because this metal presented the best removal efficiency. This efficiency varied at different times, reaching almost 100% of removal at certain times.
Figure 6 showed, qualitatively, the viability of the microorganism in water contaminated with Pb. In Fig. 6A, it is possible to observe no sign of change in the surface morphology of the bacterial cells, even in an enhanced image. Moreover, the presence of chemical elements and metal degradation in the bacterial cell mass was confirmed with the support of the Energy Dispersive Spectroscopy (EDS) analysis. Through EDS (Fig. 6B), it was possible to verify the spectrum of chemical elements in the cell mass and that there was adsorption of the Pb by B. subtilis.
Figure 6 Scanning Electron Microscope image of B. subtilis in the Pb sample in water (a) and EDS spectrum for the analyzed area, with the distribution of the elements (b)