## 3 Results
### Alternaria sp. Was Able to Grow with Extra-Heavy Crude Oil as the Sole Carbon Source  
The five fungal strains were assayed for growth at 0.5% of three different crude oils: light, heavy, or extra-heavy. All five strains were able to grow in light crude oil as the sole source of carbon. However, the heavier the crude oil was, the fewer strains were able to metabolize it. While four strains (_Alternaria_ sp., _Penicillium_ sp. 1, _Penicillium_ sp. 2, and _Stemplylium_ sp.) were able to grow in HCO, only one, _Alternaria_ sp., was able to grow in EHCO (Table 1). Under light microscopy, _Alternaria_ sp., _Penicillium_ sp. 1, and _Penicillium_ sp. 2 hyphae were observed strongly adhered to the surface of the HCO (Figure 4A-C). In contrast, _Stemplylium_ sp. presented hyphal growth that did not adhere to the oil (Figure 4D). Regardless of how attached the hyphae were to the crude oil, the main purpose of this experiment was to confirm the presence of mycelium, and this was successfully achieved, as shown in the images obtained. When grown in EHCO, hyphae and conidia of _Alternaria_ sp. also adhered to the sticky oil drops. By DIC microscopy, what appeared to be newly formed conidia at the tips of the hyphae were observed at the petroleum surface (Figure 5). By SEM, a dense mass of mycelium and conidia were observed as well growing at the surface of the petroleum (Figure 6). Figure 4: Differential interference contrast microscopy of fungal mycelia grown in Czapek minimal medium containing 0.5% _w/v_ of heavy crude oil (16–20\({}^{\circ}\) API). (**A**) _Alternaria_ sp. conidia (arrowheads) and mycelium (arrows) adhered to the oil after six days of incubation. (**B**) _Penicillium_ sp. 1 hyphae (arrows) and conidia growth (arrowheads) after 24 days of incubation; salts from the Czapek minimal medium (small arrows) can be observed. (**C**) _Penicillium_ sp. 2 mycelium (arrows) and conidia (arrowheads) embedded in salt (small arrows) and petroleum after 24 days of incubation. (**D**) _Stemplylium_ sp. hyphae (arrows) with no attachment to petroleum after 27 days of incubation. Scale bar = 25 μm. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{4}{c}{**Crude Oil Density**} \\ \cline{2-4}
**Identified Isolate** & **Light** & **Heavy** & **Extra-Heavy** \\  & **40\({}^{\circ}\) API** & **16–20\({}^{\circ}\) API** & **7–10\({}^{\circ}\) API** \\ \hline _Alternaria_ sp. & + & + & + \\ _Cladosporium_ & + & \(-\) & \(-\) \\ _halotolerans_ & & \(-\) & \(-\) \\ _Penicillium_ sp. 1 & + & + & \(-\) \\ _Penicillium_ sp. 2 & + & + & \(-\) \\ _Stemplylium_ sp. & + & + & \(-\) \\ \hline \hline \end{tabular} \(+\) growth, \(-\) no growth.  
\end{table} 
Table 1: Growth of selected fungi in crude oils of different densities. The culture was carried out in 50 mL of modified Czapek minimal medium with 0.5% _w/v_ of crude oil as the sole carbon source. For both _Pencilillium_ spp., the maximum average amount of protein (40.5 \(\pm\) 1.2 mg/L for _Pencilillium_ 1 and 46.0 \(\pm\) 13.7 mg/L for _Pencilillium_ 2) was obtained for the cultures amended with 0.75% \(\
icefrac{{v}}{{v}}\)/\(\
icefrac{{v}}{{v}}\) light crude oil (Figure S1). The protein concentration in _Pencilillium_  
Figure 5: Differential interference contrast microscopy of _Alternaria_ sp. grown in Czapek minimal medium containing 0.5% \(\
icefrac{{w}}{{v}}\) of extra-heavy crude oil (7–10\({}^{\circ}\) API). (**A**) Hyphae (arrow) and septate conidia (arrowhead) emerging from a dense mass of petroleum. (**B**) Mycelium (arrow) and conidia (arrowhead) with Czapek minimal medium salts and petroleum. Scale bar = 25 \(\upmu\)m.  
Figure 6: Scanning electron microscopy of _Alternaria_ sp. grown in Czapek minimal medium containing 0.5% \(\
icefrac{{w}}{{v}}\) of extra-heavy crude oil (7–10\({}^{\circ}\) API). (**A**) Hyphae (small arrows), conidia (arrowheads), and smooth petroleum surface (arrows). Scale bar = 100 \(\upmu\)m. (**B**) Mycelium (small arrows) and conidia (arrowheads). Scale bar = 200 \(\upmu\)m. (**C**) Hyphae (small arrows) and conidia (arrowheads).