**4. Discussion**

**3.3. Combined effects of cinnamic and vanillic acids and pH on cell count of the isolate 13M**

This phase focused on the evaluation of pH, cinnamic and vanillic acids (combined through a 2k experimental design) on the viability of the isolate 13M. Figure 9 shows the evolution of cell count in some selected combinations of the design; when MSM medium was adjusted to pH 7.0 in presence of 2 g/l of the phenols (combination D) the viable count (7 log cfu/ml) was drastically reduced to ca. 4.73 log cfu/ml. On the other hand, an alkaline pH played a protective role, as the viable count was not reduced both in absence and with phenol added (combinations

**Figure 9.** Viable count of the isolate 13M in some selected combinations of 2k design (see Table 1).

protective role toward viable count.

13M after 12 days in MSM. Ns, not significant. R2

Finally, Figure 10 shows the increase/decrease of cell count after 12 days; a positive value indicates a significant increase in the viable count, while a negative value indicates a death kinetic. These values were used as input data to run a multiple regression procedure and pinpoint the weight of each factor of the design; the results of this statistical analysis are listed in Table 4. The individual effects of phenols were not significant, while their interactive term played a negative role, i.e., it was the leading factor of death kinetic; on the other hand, the statistical analysis pinpointed a positive mathematical effect of pH, thus confirming its

> **Terms Statistical effect** pH 3.46 Cinnamic acid ns Vanillic acid ns pH/Cinnamic acid ns pH/Vanillic acid ns Cinnamic acid/vanillic acid -3.06

> > R2 ad 0.776

**Table 4.** Standardized effects of vanillic and cinnamic acids and pH on the reduction of the viable count of the isolate

ad, determination coefficient corrected for multiple regression.

E and H).

94 Wastewater Treatment Engineering

The disposal of wastewaters represents a major problem. Namely, OMWs have the highest polluting rate within the food industry, especially for the high concentration of phenolic compounds. An increased interest in environmental issues has favored the introduction of new technologies as alternative ways to traditional methods. A promising approach is represented by bioremediation, which reduces the pollution load of various by-products using the phenoldegrading ability of some microorganisms. In a previous research, we selected some promising yeasts (*Pichia holstii* and *Candida boidinii*) able to reduce phenol content in OMW [26, 28]. Hereby, we evaluated bacterial ability to grow in a phenol-enriched medium; after isolation and phenotyping, we preliminary identified the most promising strains as *Bacillus* and *Pseudomonas* spp. These bacteria are normal constituents of wastewater microflora [29, 30]. After the screening with vanillic and cinnamic acids (chosen as representative of the most important phenolic moieties: coumaric and hydroxybenzoic compounds), we focused on viability in the presence of these compounds, as well as in a caffeic acid, rutin, oleuropein, and tyrosol enriched medium. Caffeic acid and rutin are representative of secondary phenolic compounds, while oleuropein is a high molecular weight phenolic glycoside responsible for the bitter taste in olive fruits. Oleuropein is present in olive mill wastewater as a result of debittering treatments and the extraction process of table and olive oil, respectively. Tyrosol is a product of hydrolysis of oleuropein. We used higher concentrations (1 g/l, 2 g/l, and 3 g/l) than those generally present in OMWs to evaluate bacterial viability in extreme conditions.

In some samples, phenols exerted a bactericidal action, due probably to their ability to form hydrogen bonds with proteins and/or enter cells. The resistant microorganisms generally convert phenols into carbolic acids; these latter compounds are transported through a transmembrane shuttle system into the cytoplasm. Carbolic acid is converted to catechol, and after three enzymatic steps, oxaloacetate is formed; the final step is the conversion of oxalacetate to acetaldehyde and pyruvate. The enzymes involved are oxygenase, hydroxylase, peroxidase, tyrosinase, and peroxidase [31, 32]. These products, finally, follow the main metabolic pathway up to their complete mineralization by mitochondrial chain [33, 34]. The oxalacetate can also be used in other cellular activities. The isolates 13M, 44M, and 6P strains were catalase positive and this trait is an important requirement as it is related to phenol degradation [28].

The isolates showed a good metabolic capacity towards simple phenolic compounds, as they were generally able to survive, with some exceptions to this generalized statement. In addition, some isolates (e.g., 13M) significantly reduced the concentration of some compounds in the broth and these are desired traits to select a promising microorganism acting as a bioreme‐ diation tool [35].

Concerning the second step of the research (combined effects of phenols and pH), the most important result was the effect of alkaline pH, as it seemed to exert a protective role on cell viability, thus we could suggest that phenolic metabolism at basic pHs is favored because the enzymes might have an optimal pH of 9. In these conditions a high presence of hydroxyl ions is ensured and it is very important as they represent a fundamental substrate used in the first step of the catabolic pathway of phenol to obtain catechol. These assumptions, however, require a confirmation. Finally, the protective effect of alkaline pH suggests the potential use of these isolates for the bioremediation of alkaline washing water of table olives.
