**3.2. Viability and phenol reduction by some selected isolates**

Three isolates (6P, 13M, and 44M; see Table 3 for their phenotypic traits) were selected and used as targets to assess their viability in the presence of various phenolic compounds (caffeic, cinnamic and vanillic acids, oleuropein, and rutin and tyrosol) at different concentrations (1 g/l, 2 g/l, and 3 g/l); moreover, we focused also on microorganism ability to reduce phenol content. Figure 3 reports the viability of the isolate 6P in the presence of caffeic acid; the initial cell number was 7 log cfu/ml. Then, it underwent a strong reduction within 5 days (ca. 1.5 log cfu/ml at 1 g/l and 2 log cfu/ ml at 3 g/l); in the last days of storage we found a tailing effect, with a residual cell count of 5 log cfu/ml. Similar results were found in the presence of cinnamic acid, tyrosol, rutin, and oleuropein (data not shown).

Vanillic acid at 2 g/l reduced the viable count by 3 log cfu/ml in 5 days with a final tailing effect and a residual cell count of 3–4 log cfu/ml. The lowest concentration (1 g/l) resulted in a slower death kinetic, with a similar residual viable count (Figure 4).


The screening was also performed after isolate growing in MSM broth with low amounts of phenols; this step could be referred as an induction phase, aimed at inducing the resistance to phenols. Figure 2 shows the results with 1 g/l of vanillic acid. There were 32 out of 119 isolates that acquired the ability to grow in MSM with vanillic acid; at the lowest concentration (0.5 g/ l) 49 strains were able to grow. The same protocol was also used for cinnamic acid, but only a single isolate was able to grow after induction both at 0.5 g/l and 1 g/l (the isolate 26M).

(a)

(b) Figure 3: Viability of the isolate 6P in MSM+caffeic acid (mean values ± standard deviation). \*, viable

**Figure 2.** Screening of the isolates on MSM with vanillic acid (0.5 g/l and 1.0 g/l) after the induction. The numbers on

Three isolates (6P, 13M, and 44M; see Table 3 for their phenotypic traits) were selected and used as targets to assess their viability in the presence of various phenolic compounds (caffeic, cinnamic and vanillic acids, oleuropein, and rutin and tyrosol) at different concentrations (1 g/l, 2 g/l, and 3 g/l); moreover, we focused also on microorganism ability to reduce phenol content. Figure 3 reports the viability of the isolate 6P in the presence of caffeic acid; the initial cell number was 7 log cfu/ml. Then, it underwent a strong reduction within 5 days (ca. 1.5 log cfu/ml at 1 g/l and 2 log cfu/ ml at 3 g/l); in the last days of storage we found a tailing effect, with a residual cell count of 5 log cfu/ml. Similar results were found in the presence of cinnamic

Vanillic acid at 2 g/l reduced the viable count by 3 log cfu/ml in 5 days with a final tailing effect and a residual cell count of 3–4 log cfu/ml. The lowest concentration (1 g/l) resulted in a slower

count in MSM+caffeic acid are significantly different from control.

the pictures indicate if the targets are able or not able to grow.

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acid, tyrosol, rutin, and oleuropein (data not shown).

death kinetic, with a similar residual viable count (Figure 4).

**3.2. Viability and phenol reduction by some selected isolates**

**Table 3.** Phenotyping of the isolates selected for the second step of the research. F, metabolism under aerobic and anaerobic conditions.

**Figure 3.** Viability of the isolate 6P in MSM+caffeic acid (mean values ± standard deviation). \*, viable count in MSM +caffeic acid are significantly different from control.

**Figure 4.** Viability of the isolate 6P in MSM+vanillic acid (mean values ± standard deviation). Letters indicate signifi‐ cant differences (one-way ANOVA and Tukey's test; P<0.05).

Phenols did not affect the viability of the strain 44M and the viable count was at 6–7 log/ml for the entire running time (data not shown). Some interesting results were found for the isolate 13M. Tyrosol at 3 g/l reduced the viable count by 3 log cfu/ml within 5 days, thereafter cell number increased up to 6–7 log cfu/ml (Figure 5); this trend could be the result of a kind of induction and adaptive evolution with phenols added. Caffeic acid at 1 g/l and 2 g/l caused a slight viability loss (1–2 log cfu/ml), while cell number was below the detection limit after 25 days at the highest concentration (3 g/l), thus suggesting a possible dose-dependent bacteri‐ cidal effect (Figure 6). The other phenols did not affect the viable count (data not shown).

**Figure 5.** Viability of the isolate 13M in MSM+tyrosol (mean values ± standard deviation). \*, significantly different from control (t-student test, P<0.05).

**Figure 6.** Viability of the isolate 13M in MSM+caffeic acid (mean values ± standard deviation).

We focused also on phenol content; Figure 7 shows the removal of vanillic acid by the isolate 13M and 44M (the initial content of the compound was 1 g/l). Both the strains were able to reduce its concentration in the broth, although the isolate 13M showed higher removal efficiency (ca. 18% after 33 days). The isolate 44M was also able to reduce cinnamic and caffeic acids by 24%–27% after 33 days (Figure 8). Removal was always found at 1g/l of phenols; higher initial amounts completely depleted bioremediation, probably due to a possible saturating action on the membrane.

Phenols did not affect the viability of the strain 44M and the viable count was at 6–7 log/ml for the entire running time (data not shown). Some interesting results were found for the isolate 13M. Tyrosol at 3 g/l reduced the viable count by 3 log cfu/ml within 5 days, thereafter cell number increased up to 6–7 log cfu/ml (Figure 5); this trend could be the result of a kind of induction and adaptive evolution with phenols added. Caffeic acid at 1 g/l and 2 g/l caused a slight viability loss (1–2 log cfu/ml), while cell number was below the detection limit after 25 days at the highest concentration (3 g/l), thus suggesting a possible dose-dependent bacteri‐ cidal effect (Figure 6). The other phenols did not affect the viable count (data not shown).

**Figure 5.** Viability of the isolate 13M in MSM+tyrosol (mean values ± standard deviation). \*, significantly different

**Figure 6.** Viability of the isolate 13M in MSM+caffeic acid (mean values ± standard deviation).

We focused also on phenol content; Figure 7 shows the removal of vanillic acid by the isolate 13M and 44M (the initial content of the compound was 1 g/l). Both the strains were able to

from control (t-student test, P<0.05).

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**Figure 7.** Removal of vanillic acid (initial content, 1 g/l) in MSM inoculated with the isolates 13M and 44 M; data are reported as absorbance fall. Mean values ± standard deviation.

**Figure 8.** Removal of cinnamic and caffeic acids (initial content, 1 g/l) in MSM inoculated with the isolate 44 M; data are reported as absorbance fall. Mean values ± standard deviation.
