# Evaluation of _Aspergillus flavus_ Growth on Weathered HDPE Plastics Contaminated with Diesel Fuel
## 3 Results
### Fungal Growth Dynamics on Plastic Substrates
#### 3.3.6 Temporal Evolution of Mycelial Growth and Halo Formation  
The temporal analysis of _Aspergillus flavus_ growth on different plastic matrices revealed distinct patterns based on substrate and incubation temperature. Overall, a progressive increase in total colony and halo areas was observed over the 17-day incubation period, although with notable differences among treatments.  
At 30 \({}^{\circ}\)C (see Figure 1), the gray plastic matrix contaminated with weathered diesel demonstrated the most pronounced and sustained progression over time, both in radial mycelial expansion and in the formation of diffusible extracellular metabolites. Colony areas increased in a near-linear fashion from 10 mm\({}^{2}\) on day 0 to 355.8 mm\({}^{2}\) by day 17, while halo areas reached up to 2823.4 mm\({}^{2}\) during the same period. This pattern reflects highly favorable conditions for fungal growth and metabolism, with relatively low measurement errors across time points, suggesting robust reproducibility among replicates.  
Figure 1: Temporal evolution of fungal colony (**left**) and halo areas (**right**) at 30 \({}^{\circ}\)C.  
At 25 \({}^{\circ}\)C (see Figure 2), the same substrate exhibited effective growth, albeit with a slight reduction in expansion rate, particularly during the final days of the experiment. Colony areas reached 190 mm\({}^{2}\) and halos 1437.97 mm\({}^{2}\), indicating a deceleration compared to the values observed at 30 \({}^{\circ}\)C. This behavior suggests that, although the temperature still supports significant activity, the metabolic performance of the fungus is partially constrained.  
In contrast, the white plastic matrices contaminated with non-weathered diesel exhibited the lowest fungal development under all conditions, with slower growth and greater data dispersion, as reflected by the standard errors (Table 5). At 20 \({}^{\circ}\)C and 25 \({}^{\circ}\)C (see Figures 2 and 3), increases in area were low and erratic, with colony sizes barely exceeding 45 mm\({}^{2}\) and halos showing high variability and little sustained expression over time. This dynamic supports the hypothesis that both the lack of contaminant weathering and the nature of the white polymer (possibly containing inhibitory additives) negatively impacted the colonization and metabolism of _A. flavus_.  
In turn, the uncontaminated gray plastic (control) exhibited progressive and more stable growth, particularly in the formation of halos. At 20 \({}^{\circ}\)C and 25 \({}^{\circ}\)C (see Figures 2 and 3), these reached total areas of 1987.5 mm\({}^{2}\) and 1538.96 mm\({}^{2}\), respectively, while colony areas approached 100 mm\({}^{2}\), with lower relative variability. This suggests that although the absence of diesel does not stimulate fungal metabolism as a carbon source, the plastic substrate alone does not significantly inhibit its growth.  
Figure 2: Temporal evolution of fungal colony (**left**) and halo areas (**right**) at 25 \({}^{\circ}\)C.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Temperature (\({}^{\circ}\)C)** & **Matrix Type** & **CV (\%)** \\ \hline
20 & Gray + Diesel & 29.8 \\ \hline
20 & White + Diesel & 50.2 \\ \hline
20 & Control (Gray) & 34.2 \\ \hline
25 & Gray + Diesel & 33.8 \\ \hline
25 & White + Diesel & 55.1 \\ \hline
25 & Control (Gray) & 24.0 \\ \hline
30 & Gray + Diesel & 35.2 \\ \hline
30 & White + Diesel & 58.3 \\ \hline
30 & Control (Gray) & 40.4 \\ \hline \hline \end{tabular}
\end{table}
Table 5: Coefficients of variation (CV%) by plastic matrix and temperature.  
Overall, these results highlight that the fungal growth of _A. flavus_ is influenced not only by the availability of contaminants as a substrate but also by factors such as the prior weathering of diesel, incubation temperature, and the composition of the base polymer. The incubation temperature of 30 \({}^{\circ}\)C was clearly the most favorable, particularly when combined with weathered gray plastic matrices, thereby establishing a useful framework for future applied studies on fungal-based biocleaning.