How Microscopy Techniques Can Contribute to Understand Bacterial Responses to Environmental Pollutants?

FERNANDEZ M.; AGOSTINI E.; MORALES G. M.; GONZÁLEZ P. S.

La Plata

Workshop; Imaging Techniques for Biotechnology and Biomedical Applications -Workshop-; 2016

Several industrial operations released highly dangerous inorganic and organic compounds such as Cr(VI) and phenol into the environment. Both compounds are listed as priority pollutants by the US Environmental Protection Agency (EPA) due to their toxicity and persistence. The great ability of indigenous microorganisms for biotransformation of toxic compounds is of considerable interest for the emerging area of environmental bioremediation. For this reason, we isolated a bacterial strain from tannery sediments, which was identified as Acinetobacter guillouiae SFC 500-1A able to simultaneously remove high Cr(VI) and phenol concentrations. The mechanisms involved in Cr(VI) removal as well as phenol biodegradation were previously studied (Ontañon et al., 2015). Bacterial cells are known to adapt to toxic heavy metals including Cr(VI), by changing their morphology, however, the effects of organic C D compounds such as phenol, on bacterial morphology are poorly studied. In this work we studied the effect of Cr(VI) and/or phenol on A. guillouiae SFC 500-1A analyzing morphological changes. Cell length, width and height were determined, as well as surface characteristics and ultrastructural changes. These parameters were 6 µm evaluated using various analytical techniques like optic microscopy (OM), atomic force microscopy of TY medium supplemented with Cr(VI) 25 mg/L, phenol 300 mg/L or both contaminants simultaneously, were inoculated (10% V/V) with a bacterial culture grown overnight, until late exponential phase. Then, they were incubated at 28±2 °C and 200 rpm, during 16 h. The effect of Cr(VI), phenol and both contaminants on cell morphology and surface topology of A. guillouiae SFC 500-1A was examined by AFM. Bacterial cells growing in control condition showed morphological characteristics of Gramnegative cocci or coccobacilli (Figure 1 A). Frequency graphs showed that most of the analyzed cells reached length ranges between 0.6 and 2.4 µm, width between 0.60 and 1.35 µm and height between 0.20 and 0.36 µm. When bacteria were treated with Cr(VI) 25 mg/L, cells increased their size (length, width and height) (Figure 1 B). Most of cells had a length between 0.9 and 1.8 µm, however, cells with a length up to 3.6 µm were also observed. Besides, cell surface was analyzed in different growth condition. Cell surface was smooth and no significant changes were observed in control conditions in the cell surface as well as after treatments with Cr(VI), which would be beneficial for a bacterium with potential for Cr(VI) remediation. When A. guillouiae SFC 500-1A was exposed to phenol 300 mg/L, cells tended to form aggregates and increased in size compared to control (Figure 1 C). The majority of cells had a length in the range of 0.9 and 2.7 µm, a cell width from 0.9 to 1.05 µm, and a significant increase was observed in cell height, which varied between 0.32 and 0.48 µm in most of the cells. Also, an effect of phenol on cell surface was observed, for instance, the presence of waves compared to cells growing in control conditions. Finally, AFM showed that bacteria simultaneously exposed to Cr(VI) 25 mg/L and phenol 300 mg/L were remarkably deformed and adhered together respect to control cells, growing without contaminants (Figure 1 D). Important differences in length, width and height were observed respect to control cells. Most of the cells had a length in a frequency range between 1.2 and 1.8 µm, while width and height ranged between 1.05-1.20 µm and 0.28-0.40 µm, respectively. In addition, the cell surface was also roughened (with presence of waves) (Figure 1 D), similarly to that observed when bacteria grew only in presence of phenol. The characteristics observed, could reflected extracellular polysaccharides, since the production of them was increased in these treatments (data not shown). On the other hand, TEM gives the possibility to study the cell physiology and especially changes in cell ultrastructure as a result of exposure to contaminants. The most significant changes were observed when bacteria grow in Cr(VI) 25mg/L. Cells of A. guillouiae SFC 500-1A showed considerable morphological changes, such as the increment in size and a reduced dark cytoplasmic space or increased periplasmic space by detachment of the inner membrane. A large number of electron dense precipitates, were observed. Possibly, the electron-opaque particles are attributable to chromium precipitations. These observed inclusions were unevenly distributed in the cytoplasm, as well associated with the periplasm and outer membrane and even localized extracellularly. Moreover, it is important to note that exposure of this strain to one or both contaminants did not produce cellular lysis as was also reported by others authors (Batool et al., 2014).