**3. Results**

(0.5 g/l), MgSO4\*7H2O (0.2 g/l), CaCl2 (0.025 g/l), FeCl2 (0.005 g/l), Agar (12 g/l), cinnamic (C9H8O2) or vanillic acids (C8H8O4) (0.5 or 1 g/l; Sigma-Aldrich, Milan, Italy). After streaking the isolates onto the surface of this modified medium, the plates were incubated at 30°C for mesophilic bacteria and *Bacillus* and at 25°C for *Pseudomonadaceae* for 72 h. MSM without phenolic compounds was used as control. For a second assay, the isolates were preliminary grown in MSM broth added of 0.025 g/l and 0.05 g/l of cinnamic and vanillic acids and incubated for 24 h; thereafter they were streaked onto the surface of MSM with phenols, as

This step was performed on three selected strains (6P, 13M, and 44M); they were grown in TS broth incubated at 25°C (strain 6P) or at 30°C (strains 13M and 44M) for 48 h. Each culture was centrifuged at 4,000 rpm for 10 min and the pellet was re-suspended in sterile saline solution (0.9% NaCl); ca. 6–7 log cfu/ml were inoculated in MSM medium (1, 2, and 3 g/l), added with phenols (cinnamic acid; vanillic acid; caffeic acid-C9H9O4; rutin hydrate-C27H30O16 x H2O; tyrosol-C8H10O2; oleuropein-C25H32O13; phenols were purchased from Sigma-Aldrich). MSM

The samples were stored at 25°C–30°C for 33 days and periodically analyzed to evaluate the viable count on TSA and the content of phenols through the Folin-Ciocalteau method [27]. The analyses were performed in duplicate and the results analyzed through one-way Analysis of Variance (one-way ANOVA), using Tukey's test as the post-hoc comparison test, or t-student test for paired comparisons. The statistical analysis was performed using the software Statistica

**2.4. Combined effects of pH, cinnamic, and vanillic acids on the viable count of the strain**

The strain 13M was used as target; it was inoculated into MSM broth to 6–7 log cfu/ml. The amounts of cinnamic and vanillic acids and pH varied according to a 2k experimental (Table 1). The samples were stored at 30°C and periodically analyzed to evaluate the viable count and

**Combinations pH Cinnamic acid (g/l) Vanillic acid (g/l)** A 7 0 0 B 7 0 2 C 7 2 0 D 7 2 2 E 9 0 0 F 9 0 2 G 9 2 0 H 9 2 2

**2.3. Effect of phenolic compounds on the viable cell count**

without phenols was used as control.

phenol content (up to 12 days).

**Table 1.** Combinations of 2k design.

for Windows version 10.0 (Statsoft, Tulsa, OK, USA).

reported above.

88 Wastewater Treatment Engineering

**13M**
