Bacterial size and morphology
SEM microphotographs of attached bacteria allowed the statistical evaluation of bacterial sizes. It could be observed that in the presence of Al2O3-NP the attached bacteria were significantly smaller than those of the control (average lengths of 0.8µm and 1.4µm, respectively). The frequency distribution of the lengths (Fig. 2C) showed the marked influence of the environment both, in the bacterial size and morphology, the smaller sizes correspond to coccoid shapes. Conversely, in the presence of Zn-NP the bacterial size was larger, like that of the control. Thus, aluminium ions release by the Al2O3-NP may be a toxic agent for bacteria causing morphological and size changes in response to the stress condition. Consequently, considering that complexation of the cations may reduce the toxic effect and that the components of humic acids (HA, frequently present in natural aqueous environments) may function as chelator agents, we decided to add HA to the Al2O3-NP suspension and to investigate their influence in the physicochemical and biological response.
Adsorption of humic acids. Figure S2 shows that the E465/E665 ratio in the liquid filtrate of a Al2O3-NP suspension containing HA is close to 3, indicating that the high molecular weight aromatic structures remain in the liquid filtrate and the low molecular weight aromatic compounds containing carboxylic and/or carbonyl groups are adsorbed on the Al2O3-NP surface. DLS results (Table S1) showed that the distribution of the diameters of the Al2O3-NP aggregates changes if HA is present.
FTIR-ATR results presented in Figure S3 complement this information (see SI for details). There, the spectra corresponding to HA, Al2O3-NP and the solid (HA-Al2O3-NP(s), Figure S3 left) and liquid phases (HA-Al2O3-NP(l), right) obtained by centrifugating the HA-Al2O3-NP suspensions are shown. Interestingly, the ATR-IR spectrum of HA-Al2O3-NP(s) showed the appearance of new important peaks at 3350 and 1620 cm− 1, characteristic of HO− and COO− vibrations, respectively, which were not observed for Al2O3-NP. The observed shift in the bands due to carboxylic groups on HA- Al2O3-NP (s) seems to support the formation of Al-carboxylic complexes on the Al2O3−NP surface, in line with E465/E665 results and literature reports . On the other hand, in case of the liquid filtrate HA-Al2O3-NP(l) the shift of the band at 1586 cm− 1 to 1637 cm− 1 can be attributed to the carboxylate groups with covalent coordinated bonds formed when HA and Al(III) are present. Additionally, a shift from the 1090 cm− 1 (HA) to 1114 and 1124 cm− 1 for HA-Al2(SO4)3 and HA-Al2O3-NP(l), respectively, also indicates the formation of coordination complexes between Al(III) and the organic aliphatic molecules with -OH groups.
Release of Al(III) ions. To determine the release of Al(III) by suspended Al2O3-NP, the concentration of Al-containing species in the liquid phase was evaluated. To that purpose, Al2O3-NP(l) was analysed by ICP. The results showed that the Al-containing species released by Al2O3-NP in SIM suspensions was 0.993 ppm, while in the presence of HA (HA-Al2O3-NP(l)), the NP were able to release 2.77 ppm. Thus, these results are in accordance with FTIR-ATR findings supporting that the presence of HA favours the release of less toxic Al-carboxilic complexes.
Addition of HA-Al2O3-NP to the culture medium. The addition of HA-Al2O3-NP suspension to the bacterial culture medium resulted in the increase of the number of attached bacteria in relation to the control. SEM observations revealed that 3D colonies with EPS strings were formed and, consequently, HA addition probably led to the reduction of the stress by the chelation of Al-containing ions.
In HA-Al2O3-NP-containing medium isolated bacteria did not form a network. The average size was slightly larger with less coccoid shape bacteria than in the case of Al2O3-NP without HA but significantly smaller that the control (Fig. 1D, Fig. 2C).
As expected, the complexation of the Al ions by HA reduced the toxicity caused by these ions, however, the wellbeing growth condition not be achieved since their size is smaller.
In agreement, EDS analysis after the biofilm formation showed a decrease of Al/C ratio from 0.088 in presence of Al2O3-NP to 0.070 in presence of HA-Al2O3-NP, revealing a decrease of the relative Al content on the surface after the addition of these HA-containing NP due to the consequent complexation process.
Al2O3-NP toxicity and bacterial adaptive behaviour.
Pseudomonas species display different strategies to survive at high levels of Al ions. ,. One of them is the generation of complexation agents where, among ligands of Al ions, citrate is a very good one . Cellular changes are displayed and lead to a metabolic shift in order to convert malate to citrate to overcome the stress produced by high concentrations of Al ions . It should also be considered that the resistance of P. aeruginosa biofilms decrease with time and they may be eradicated at similar ions concentrations than planktonic cells after 1 day exposure .
Results reported here show that in the case of P. aeruginosa transformations other than chemical can be produced since morphological and size changes of cells and architectural reforms of the biofilm take place. In fact, Fig. 2C shows bacterial size distributions for each condition and a sharp maximum close to 0.8 µm (corresponding to ca. 70% of the cells) can be seen for Al2O3-NP suspension while broad peaks in the 0.6–1.2 µm and in the 1.0-1.6 µm range are depicted for the HA-Al2O3-NP and HA-control conditions, highlighting the influence of the medium composition on the bacterial size distribution. Analogously, it has been reported that under stress conditions the morphology of these cells change to U shape and finally to coccoid bacteria probably by modifications in cell wall crosslink or metabolic activity. Morphological changes, motility and surface properties changes were also observed for media with sub-MIC antibiotics . Atypical results are shown here for Al2O3-NP since they show that the original 3D aggregates disappear and a 2D network is formed with nanotubes as connectors of small coccoid bacteria.
Nanotubes are intercellular connections between neighbouring bacteria when the surface density of some bacteria is low. It has been hypothesized that these nanotubes enable bacteria scavenge and deliver molecules inside them and represent an important form of bacterial communication in nature, providing a network for exchange of cellular molecules within and between species . It was found that nanotubes mediate the transfer of cytoplasmic molecules between adjacent cells ,. Moreover, they may also enable transiently acquired nonhereditary resistance to antibiotics. Besides, the transference of plasmids also occurred granting hereditary features to recipient cells .
On the other hand, eDNA that may be produced through explosive cell lysis events could facilitate the survival in non-nutrient stress condition . It was hypothesized that in unfavourable conditions weak bacterial cells die, and the survival cells live on their expense (strategy known as “bust-and-boom”) . The detailed observation of Fig. 1B (left, red arrow) reveals that the direction of the nanotubes of several bacteria is focused on a particular cell that may act as a nutrient donor. Thus, bust-and-boom strategy may also be used by P. aeruginosa in the networks.
Notwithstanding that there is a persistent tendency in environmental literature to link toxicity with EPS formation our results show that, under the in vitro conditions analysed, EPS is not the way that P. aeruginosa use to adapt to the metal toxicity.