3.1. Impact of the Surface Roughness on Bacteria Adhesion
As mentioned in the introduction section, it is well known that both surface topography and its chemistry play an important role in the adhesion process of microorganisms. For that reason, two gold substrates with different surface roughness were selected for experiments, i.e., the commercially available gold layer on glass (Au200) and the electrochemically synthesized gold layer on the copper substrate (Au(E)). Firstly, the surface morphology and crystallinity of both substrates were examined by using AFM and XRD, respectively. The obtained results are shown in Figure 2
AFM images revealed that the differences in the surface morphology of materials are significant. The Au(E) plates have a heterogeneous topography with micro- and submicrometric scale domains (Figure 2
b). On the other hand, Au200 samples can be described as smooth and with a relatively repeatable nanoscale surface morphology (Figure 2
a). Moreover, the distinction between smooth and rough surfaces is also visible in the roughness parameter (Ra
), i.e., 2.4 and 7.0 nm for Au200 and Au(E), respectively.
To get an insight into the nature of the gold coverage, XRD measurements were adapted. As can be seen from Figure 2
c, the Au200 samples revealed sharp reflexes of gold (at 38° and 82° for (111) and (222) planes, respectively), which indicate that gold crystals are surface-oriented with the same crystallographic plane. This may contribute to the surface smoothness visible in the AFM image. When it comes to the Au(E) sample, the diffractogram shows reflexes for both copper and gold; however, the shape of the signals from Au indicates its polycrystalline nature. Therefore, during electrodeposition, Au crystals grow in different directions at various speeds, which results in higher surface roughness, as shown in the AFM image.
The gold substrates were further tested in regard to the adhesion of bacteria cells. As mentioned earlier, three Lactobacillus spp.
were chosen for these experiments, i.e., Lactobacillus rhamnosus
GG, Lactobacillus plantarum
299v, and Lactobacillus
acidophilus. The SEM images of examined surfaces after 48 h cultivation are shown in Figure 3
As presented above, for both tested materials, the growth of L. rhamnosus
GG on gold substrates is significant. After adhesion, microorganisms grew, spread, and proliferated on all available areas, forming a lace-like network, contrary to our previous work, where such a structure was observed only for modified electrodes [18
]. It might be related to the different composition of the gold-rich electrolyte used for the deposition process or extended cultivation time. However, the difference between the smooth and rough Au is visible in the SEM images since the structure of the biofilm is denser on the polycrystalline surface. Also, slightly higher mortality was observed on the Au200 substrate, which will be described later. Such behavior is not typical for the growth of other bacteria strains on nanorough gold surfaces. As shown by Nguyen et al. [37
], nanoscaled topography significantly reduces the adhesion of Pseudomonas aeruginosa
(also a rod-shaped bacteria) when compared to the smooth gold substrate. Moreover, when it comes to other Lactobacillus
strains, the surface coverage on the Au(E) substrate is significantly different (Figure 3
c,d). Not only the coverage of the surface was much smaller, but also the percentage of the damaged bacteria cells increased in the order L. rhamnosus
GG < L. plantarum
< L. acidophilus
(see in the next section).
In order to gain more quantitative insight into the surface coverage, the Gram method was employed to determine the amount of bacteria on the surface for selected systems and confirm whether the distribution of microorganisms in the macro scale is uniform. The samples were prepared in the same way as those for SEM imaging, which enabled the direct comparison of the results. The obtained data are shown in Figure 4
and Figure S1
The obtained results proved that in the case of the L. rhamnosus
GG strain, an almost ideal surface coverage is obtained for the Au(E) substrate, while for Au200, the measured absorbance, and therefore, the coverage, is significantly lower (Figure 4
and Figure S1
). These results confirm that in the case of L. rhamnosus
GG, the surface nanoroughness enhances the colonization of gold. When it comes to other Lactobacilli strains, the surface coverage is almost negligible, especially for Lactobacillus acidophilus
, where the absorbance is at the control level. Such results may be explained by the presence of the pili in the cell wall of Lactobacillus rhamnosus
GG, which are responsible for the adhesion process [38
]. Such adhesive proteins were not determined for the other two strains. The fact that Lactobacillus rhamnosus
GG is not adhering using its entire surface but only with nano-objects scattered over the cell surface, allows us to conclude that this process will be critically sensitive to the specific surface of the substrate. This could explain why, for rough surfaces, we have observed more bacteria. Another aspect that could explain it is the characteristic 3D lace multi-layer structure created by bacteria on the surface. Such an arrangement provides more points of contact with the surface, which will significantly favor rougher surfaces.
Considering the results presented above, it might be stated that the colonization of the metallic surfaces by microorganisms, especially gold, depends not only on the surface properties of the material (roughness in particular), but also on the individual properties of bacteria species, including the structure of the cell wall.
3.2. Dextran Derivatives as Additional Surface Modifying Agents
A negatively charged gold surface may affect the adhesion of bacteria cells because their biological membranes also have a negative potential. This effect can be eliminated by using polycationic films deposited by using the layer-by-layer technique. It is a well-developed and straightforward method that allows covering a negatively charged surface with a molecular layer of the cationic polymer [40
]. This leads to a change of the surface charge and, in consequence, may influence its adhesive properties. Three cationic dextran derivatives differing in a molar mass confirmed and characterized by gel permeation chromatography (GPC) method (detailed data are shown in the Supplementary Material, Figure S2
) were selected for this study. In our earlier publications [31
], we have discussed the effect of positive charge and molecular weight of polysaccharides on their biological activity and toxicity. Due to the electrostatic mechanism of nanocoating formation on the surface of gold, we imposed the synthesis conditions that the polymer charge should be fixed while the molar mass will be a variable. Such an approach is also justified by the fact that the charge will affect the toxicity, which may distort the obtained results [32
]. Therefore, the zeta potential of the respective solutions was measured to confirm that we have obtained polymers with the desired charge. The results are shown in Table 1
The obtained zeta potential values allow us to state that the assumed goal has been achieved, and the charges of obtained polymers differ insignificantly and these differences can be regarded as negligible. The effectiveness of Au(E) substrates coverage by polymer coatings was confirmed by XPS (Figure 5
), IR, and AFM (Figures S3 and S4 in the Supplementary Material
An overview of the survey spectra for the produced samples is shown in Figure 5
a. A detailed examination of the XPS spectra in relation to the unmodified samples (Figure 5
b) showed that characteristic carbon C1s peaks are well visible for samples with dextran derivatives. In particular, typical C–C/C–H (285.0 eV) and C–O/C–OH (286.5 eV) bonds can be observed for these materials (Figure 5
c). A peak corresponding to the caroxyl groups observed at 288.3 eV results from oxidation of carbon impurities at the surface. Moreover, the presence of a Cl 2p peak at 199.6 eV, which is a part of the polymer matrix, was found (Figure 5
d). The presence of Cl in the spectra comes from the modification procedure, where glycidyltrimethylammonium chloride (GTMAC) was used in the synthesis of cationic derivatives of dextran. Although no peaks from N1s (at around 400 eV) were present, the Cl 2p spectra demonstrate that the modification was performed successfully. Also, peaks from phosphorus, sodium, and potassium were present due to the preparation procedure (see paragraphs in Section 2.2
, Section 2.2
, Section 2.3
). Additionally, for all the prepared samples, the characteristic XPS gold (Au4f ~83.0 eV) and copper (Cu2p ~932.0 eV) peaks related to the presence of the Au coating on the Cu substrate were also visible (Figure 5
The presence of the dextran derivative monolayer on the Au(E) substrate was also confirmed by the IR spectra (Figure S3
). Similarly to the previously reported data [18
], the characteristic peaks for dextran derivatives were observed for the polymers with higher molecular mass. In particular, the O-H, C-H, and C-C stretching bands were observed at 3394 cm−1
, 2941 cm−1
, and 1272 cm−1
, respectively. Moreover, the CH3
-N bond was also visible at 1462 cm−1
, ascribed to the C-H bending vibrations.
What is more, the AFM measurements showed that due to the small amount of the deposited polymer, the surface is slightly smothered when compared to the uncoated Au(E) substrate, but still rougher than the pristine Au200 substrate (Figure S4
Furthermore, the biocompatibility of the modified gold surfaces was assessed using the same Lactobacilli
strains as for the non-modified samples. The SEM microphotographs of Au(E) substrates covered with polymer layers after 48 h of cultivation are shown in Figure 6
Based on the SEM microphotographs, it may be stated that for Lactobacillus rhamnosus
GG strain cultured on all types of modified Au(E) samples (Figure 6
a,d,g), no significant differences were observed when compared to the non-modified ones (see Figure 3
b). Bacteria were spread on the whole surface, and a well-developed bacterial network was present. No negative impact of the polymeric layer on the bacteria was observed, which is in good agreement with our previous work [18
]. In the case of Lactobacillus plantarum
299v, a visible improvement in the surface coverage was observed for all types of modifiers (Figure 6
b,e,h). On the other hand, for Lactobacillus acidophilus,
a decrease (Figure 6
c,i) or no change (in the case of Dex40) (Figure 6
f) in the surface colonization was observed.
The presented results showed that by modifying the metallic surface with even a monolayer of biocompatible substance, an improvement in the bacteria adhesion is evident. However, it should be underlined that this dependency is not unambiguous for all strains, so the reasons behind such different behaviors should be further examined.
For all tested surfaces variants for the Lactobacillus rhamnosus GG strain, an additional observation is that we are not dealing with a system as stable as a classic biofilm, and bacteria can be detached in both cases (Au(E) and Au 200) with the help of a cell scraper. The surfaces covered with bacteria can be removed and re-immersed in the liquid without any detachment, but other operations, such as heavy rinsing, affect the stability of both systems in a similarly negative way.
This poor grip to the surface is due to the fact that only a few bacteria growing in the lowest layers of the 3D structure are bound to the substrate, and most of the cells are only bound to each other. It can be considered as a disadvantage, but this strain does not occur usually in nature in the form of a biofilm and cannot exist as a colony of bacteria; therefore, this may be the only form in which they can appear in a more massive cluster. Additionally, thanks to such a 3D lace multi-layer structure, bacteria have large access to nutrients, and even those individuals furthest from the surface can live without limitations.
3.3. The Influence of the Surface on the Welfare of Microorganisms
Based on the SEM measurements, one can only assess the degree of coverage of the surfaces by microorganisms. To assess their welfare, experiments on the non-fixed bacteria cells were conducted. All of the examined surfaces were used for fluorescent live/dead staining to establish the percentage of damaged bacteria cells on each surface and for all bacteria strains (Figure 7
As already described for the uncovered gold layers, the number of damaged bacteria cells varied between strains; however, the tendency for smooth and rough gold was the same, i.e., the highest percentage of damaged bacteria cells was observed for Lactobacillus acidophilus
. When the samples were modified with cationic dextran derivatives, the situation seems to be more complex. For Lactobacillus rhamnosus GG
, cells’ viability was slightly improved or remained at a similar level for the nanorough Au(E) samples. However, it should be pointed out that significantly higher surface coverage was observed on the dextran-modified than unmodified samples, which can be seen on microphotographs from the fluorescent and scanning electron microscopes (Figure 8
). On the other hand, when the Au200 substrate was modified, an increase in the percentage of damaged bacteria cells was observed. Interestingly, similar tendencies were also observed for the other two tested strains on both gold surfaces, which was also confirmed microscopically (see Figures S5 and S6 in the Supplementary Materials
). Such results may indicate that the enhancement of the adherence and proliferation of the bacteria cells due to the presence of dextran derivatives may also be species dependent.
Another indicator for the well-being of the examined bacteria on the tested substrates is the amount of produced lactic acid, a metabolite of all three tested bacteria strains. It should be mentioned that in the experimental setup, bacteria live both on the gold surface and in the surrounding medium (due to the detachment of some of the bacteria during cultivation). Therefore, their metabolic products are present in the medium used for the determination of LA. Obviously, such an approach is not ideal, but it gave us an insight into the biological activity of the entire system. The results are shown in Table 2
, and the calibration curve is shown in Figure S7
As shown, for L. acidophilus strain, the measured concentrations are at the limit of detection, so it is hard to clearly confirm the bacterial activity/presence on the surface. On the other hand, L. rhamnosus and L. plantarum generated a significant amount of LA that further proves that live bacteria are present and maintain their metabolic activity. What is more, the results obtained for LA production also confirmed that the proposed modifications are the most beneficial for Lactobacillus rhamnosus GG, whereas, for other strains, the results are inconclusive.
Nonetheless, this demonstrates that the tested substrates are not toxic towards the Lactobacilli strains; however, when designing a biosurface for specific applications, many factors that could affect its performance should be assessed.