Pesticide removal by the bioaugmentation process in secondary treated wastewater

Pentachlorophenol (PCP), extensively used as a herbicide, insecticide, fungicide, wood preservative, resin, lubricant, and dye intermediate, has been commonly found in groundwater, sediment, and surface soils (Lemke et al. 2021; Mundeja et al. 2021). The World Health Organization (1996) reports that the concentration of PCP in industrial wastewater ranged between 2 and 50 mg L⁻¹, the concentration in rivers is 10.5 mg L⁻¹, and the concentration in water samples is 0.01 mg L⁻¹. Since PCP is a toxic substance with high mutagenicity and carcinogenicity, researchers have considered removing it from industrial wastewater (Seyedi et al. 2019). PCP can be degraded in the environment by chemical (Asgari et al. 2021), microbiological (Werheni Ammeri et al. 2021a, 2021b, 2022a, 2022b, 2022c, 2022d), and photochemical processes (Cabezuelo et al. 2021). Many strains of bacteria and fungi, such as Sphingomonas chlorophene (Yang et al. 2006), Acinetobacter ISTPCP-3 (Sharma et al. 2009), and Sphingobium chlorophenolicum ATCC 39723 (Dams et al. 2011), have been shown to have PCP degradation capabilities. The transformation of PCP in an aqueous solution led to the release of chlorine, its quantification showed the PCP degradation efficiency (Asgari et al. 2021). Members of the genus Pseudomonas showed remarkable metabolic and physiological versatility, allowing the colonization of various terrestrial, and aquatic habitats and tolerance against many xenobiotics (Hosu et al. 2021), and are of great interest because of their increasing potential in biotechnological (Hassen et al. 2021; Raio & Glimcher 2021). Iron is an essential nutrient for most living organism to sustain their growth, although it is mostly unavailable in the environment. Researchers report that the iron cycle can be combined with the carbon cycle, the fate of heavy metals, and the conversion of nitrogen and organic pollutants (Li et al. 2012; Yu et al. 2013). Recently, it was observed that PCP can be chemically dechlorinated by the high reaction activity of adsorbed Fe (II) produced from the dissimilatory iron reduction process mediated by iron-reducing microorganisms (Wang et al. 2017). Siderophores, produced by a different class of microorganisms (bacteria, fungi, etc.), are small molecules with a high affinity for Fe (III) and can be produced in iron-limited conditions (Neilands 1981). Therefore, many strategies have been developed by organisms such as fungi, plants, or bacteria to have access to this element essential for their growth (David et al. 2019). Several Pseudomonas species have the capacity to supply and excrete, below iron-restricting conditions, soluble yellow-green fluorescence pigments (Bultreys et al. 2003) named pyoverdines (PVD) or pseudobactins, which act as siderophores for those bacteria (Meyer 2000). These molecules are idea to be related to pathogenesis (Fuchs et al. 2001). Bacteria can come to be tolerant to oxidative pressure with the aid of using secreting extracellular polymeric substances and forming biofilm (Rovida et al. 2021). The bacterial flair of colonizing a poisonous biotic floor boomed with biofilm development. The environmental pressure situations may want to affect biofilm production (Poole 2014) and represent a unique mode of increase that lets for survival in adverse environments (Elias & Banin 2014). Meliani & Ben Soltane (2014) stated that Pseudomonas isolates broaden an essential biofilm mass development, to defend cells from adverse environments. The microorganisms that make up biofilms may be used of polluting substances as a supply of carbon and energy (Cohen et al. 2002; Petrova & Sauer 2012).

The aim of this study was to check the capability of the Plant Growth-Promoting Rhizobacteria (PGPR) strain Ps. putida AE015451 to tolerate and remove the pesticide PCP in liquid MSM or treated secondary wastewater (TSWW). We follow the effect of the PCP during the pyoverdine secretion (PVD) by the Ps. putida AE015451. The biofilm formation capacity of this strain was studied under stressed conditions following PCP and Fe addition. The effect of PCP–Fe interaction on the production of PVD and biofilm development by Ps. putida AE015451 was also observed.

The novelty in our work is the study of the capacity of the bacterial strain Ps. putida AE015451 in the elimination of PCP in secondary wastewater. Also, the effect of Fe addition in the improvement of PCP biodegradation. Thus, the effect of Fe–PCP interaction in the production of pyoverdine-type siderophore. The main study's findings showed us how effective the Pseudomonas bacteria are in removing harmful pesticides.

PCP (MW = 266,337 > 99% purity) was purchased from Sigma-Aldrich (USA) and high-performance liquid chromatography (HPLC) grade solvents were purchased from Merck, Germany. All chemicals used for the culture media preparation and other reagents used were purchased from Sigma-Aldrich or Fluka.

Wastewater with no detectable PCP was sampled in April 2021 at the level of Gabes El Hamma wastewater treatment plant in the arid region of southern Tunisia. The sewage treatment plant has a secondary wastewater treatment system (STWW) with activated sludge. STWW samples were stored at 4 °C to determine key physical and chemical properties: cation exchange capacity (CEC) and pH, organic carbon, chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), nitrate, chloride, total nitrogen, and total carbon (Brook et al. 1971; Werheni Ammeri et al. 2023).

The Ps. putida strain AE015451 used in this work was isolated from an industrial wastewater plant and analyzed by partial 16S rRNA gene sequencing by Mehri et al. (2011). The strain was selected by testing its resistance and ability to eliminate PCP in MSM at a concentration of 30–300 mg L⁻¹ for 168 h (Mundeja et al. 2021). The composition of MSM was in mg L⁻¹: KH₂PO₄, 800; Na₂HPO₄, 800; MgSO₄·7H₂O, 200; CaCl₂·2H₂O, 10; NH₄Cl, 500; and 1 mL of trace metal solution comprising in (mg L⁻¹) FeSO₄·7H₂O, 5; ZnSO₄·H₂O, 4; MnSO₄·4H₂O, 0.2; NiCl·6H₂O, 0.1; H₃BO₃, 0.1; CoCl₂·6H₂O, 0.5; ZnCl₂, 0.25; EDTA, 2.5 with a pH 6.3. PCP concentration was added to the medium after autoclaving. The flasks were incubated at 30 °C under constant shaking at 160 rpm min⁻¹ using an incubator shaker (ZHWY-2102 P). All the treatments were carried out in triplicate.

The preparation of Ps. putida AE015451 inoculum for the bioaugmentation process study was made into the nutrient broth for 24 h at 30 °C. The obtained inoculum was centrifuged for 10 min at 12,000 rpm and 4 °C. The test of PCP removal capacity was described by Werheni Ammeri et al. (2021) and was released in MSM and sterile STWW. The choice of MSM liquid medium is specific to Pseudomonas bacteria according to the literature (Karn et al. 2010a; Werheni Ammeri et al. 2017, 2021a, 2021b) and in addition contains only PCP as a carbon source. The STWW was sterile after three autoclaves at 120 °C for 15 min. A variation in different parameters has been considered: variation of inoculum volume (0.5, 1, and 2 mL), PCP (30, 50, 70, 100, 200, and 300 mg L⁻¹), iron concentration (5, 10, and 20 mg L⁻¹), and pH (4, 5, 6.3, 7, or 8). The experiment was performed in Erlenmeyer flasks containing 100 mL of MSM or sterile STWW. Samples for measurement were taken to the initial time of incubation (T0) and after 168 h of incubation (TF). The experiment was carried out in triplicates.

The microbial biomass values of the Ps. putida AE015451 strain used in this study were measured by a spectrometer (UV–Vis Dual BEAM UVS-2700) at 600 nm and at 24 h intervals to measure the OD of the samples. The bacterial biomass was estimated according to the formula, Bacterial biomass = OD/k, with k the number of bacteria counted on MSM agar in 1 mL of inoculum (Cabezuelo et al. 2021).

The study of PCP removal was quantified by HPLC analysis (Rao et al. 2017; Werheni Ammeri et al. 2022b). The PCP was extracted by methanol solution, and a volume of 1 mL of the different treatments taken at 24 h incubation intervals. The prepared suspension was vortexed for 5 min and could stand at 20 °C for 10 min. After, the sample was vortexed for 5 min and centrifuged at 8,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm sterile filter. The filtrate was analyzed by a Perkin Elmer Series YL9100 HPLC as reported by Karn et al. (2010a). All analyzes were carried out in triplicates.

The chloride content in MSM and STWW for different treatments in the bioaugmentation process was determined in a neutral medium using a silver nitrate titration method in the presence of potassium chromate described by Werheni Ammeri et al. (2021a).

Deficient iron Casamino-acid liquid culture medium (CAA) is used in this study and comprises 5 g of casamino-acid, 1.18 g of K₂HPO₄ and 0.25 g of MgSO₄ in 1 L of deionized water. The sterile medium CAA was inoculated with the strain Ps. putida AE015451 and incubated for 48 h at 25 °C as described by Mehri et al. (2011). Different treatments were performed in this experiment depending on the volume inoculum variation, the concentration of PCP (30, 50, and 100 mg L⁻¹) and at different pH values varying between 5 and 8. The amount of pyoverdine extracted from the culture medium was spectrophotometry determined at 400 nm according to the method described by Navazio (2005) and David et al. (2020).

This biofilm formation protocol was adapted from methods used by Merritt et al. (2003) and Malcova et al. (2008). Bacterial strains were incubated in heart-brain broth (BHI: MAST DM106) and MSM with 0.25% glucose for 24 h at 30 °C (Werheni Ammeri et al. 2022d). The growing bacterial culture was transferred to a 96-well microtiter plate. Microtiter plate tests were performed in triplicate, in triplicate per well/treatment (PCP or no and Fe) along with negative controls. Calculate OD by subtracting the mean of 3-fold OD from ODC (control). Absorbance was measured at 550 nm using an automated system (UV–Vis Dual BEAM UVS-2700). Strains were classified as low, medium, or high biofilm producers based on OD (Mehri et al. 2014; Werheni Ammeri et al. 2021a, 2021b).

Statistical analysis was performed using the SPSS 21.0 software. Data collected, PCP removal %, chloride and bacterial biomass content of MSM and STWW samples, were subjected to analysis of variance (ANOVA), and the means were separated by the Duncan test at P ≤ 0.05. Differences between the two sampling times were compared by an independent-sample t-test (two-tailed). All data were presented as mean ± SD (n = 3). The relationship between the parameters was carried out by the Minitab software to generate the specific response and the contour plot.

The results obtained from the characterization of the STWW sample used in this study are presented in Table 1. The STWW sample has a basic pH of 8.7 and a high COD and BOD₅ of 632.0 and 380 mg L⁻¹, respectively. According to the COD/BOD₅ ratio lower than 2, the studied effluent is easily biodegradable (Płuciennik-Koropczuk & Myszograj 2019). However, the COD recorded for our sample is lower than textile wastewater according to the work of Yakamercan & Aygün (2020). Thus, the chloride level in STWW is 14.6 g L⁻¹. The nitrogen value obtained from the STWW showed a value of 48.0 mg L⁻¹. The STWW showed a higher chloride value as compared with a preliminary study conducted Werheni Ammeri et al. (2021). The growth of Ps. putida in the STWW sample was rather difficult.

In this study, the selection of a strain of Ps. putida AE015451 species was based on its ability to eliminate PCP molecules (Werheni Ammeri et al. 2022a, 2022b, 2022c, 2022d). As reported by Lee et al. (2011), the strain Pseudomonas sp. (Bu34) can remove the rate of 4,000 mg L⁻¹ PCP. Also, Karn et al. (2010a) proved that Ps. stutzeri CL7 can grow up in the PCP contaminated medium (600 mg L⁻¹). In our previous study Werheni Ammeri et al. (2017), we demonstrated that Ps. fuorescens can tolerate and remove up to 250 mg L⁻¹ of PCP in the MSM medium after 96 h of incubation at controlled conditions.

The chloride content in the control sample (MSM or STWW + Ps. putida AE015451) significantly increased at TF compared to T0 (Table 2). Different experimental conditions were studied such as PCP, Fe content, inoculum volume, and pH of the matrix. Karn et al. (2010a) proved that the PCP used and the discharge of chloride with inside the medium was essential because of the PCP mineralization.

Chloride content was determined for the different PCP concentrations (30, 50, 70, 100, 200, and 300 mg L⁻¹). Different behaviors of the chloride content were observed in MSM or STWW samples (Table 2). A significant decrease in chloride content at T0 and TF was observed by increasing PCP concentration, from 1.98 ± 0.03 g L⁻¹ of the 50 mg L⁻¹ to 1.33 ± 0.04 g L⁻¹ of the 100 mg L⁻¹ PCP contaminated MSM samples of T0 time and from 4.20 ± 0.03 to 1.60 ± 1.03 g L⁻¹ of the 30 and 200 mg L⁻¹ PCP contaminated MSM samples of TF time (Table 2). In addition, a significant increase in the chloride content was observed from T0 to TF in the 30–100 mg L⁻¹ PCP contaminated MSM samples, by increasing the PCP to 200 and 300 mg L⁻¹ not significantly changes from T0 and TF, were observed (Table 2). Contrariwise, in the STWW sample by increasing the PCP concentration, a significant increase of the chloride content was observed by reaching 8.33 ± 2.04 and 14.20 g L⁻¹ at T0 and TF in the 300 and 200 mg L⁻¹ of PCP (Table 2). Furthermore, a significant increase from T0 to TF in the different PCP contaminated STWW samples was observed (Table 2). Using PCP as the sole carbon source led to a release of chlorides in the medium. These results agreed with the study of Werheni Ammeri et al. (2021), in which Ps. putida strain can remove PCP molecule 800 mg L⁻¹ and release proportional rates of chloride.

The effect of iron rate variations (5, 10, and 20 mg L⁻¹) on the chloride content in MSM and STWW supplemented with PCP 30 mg L⁻¹ is represented in Table 2. In both liquids tested, the release of chloride decreased by increasing Fe concentration and this result could be explained by the decrease of PCP degradation. A significant increase in chloride concentration from T0 to TF was observed (Table 2). Removal of PCP by Ps. putida AE015451 in the presence of an increasing concentration of iron (5, 10, and 20 mg L⁻¹) corresponds to a decrease in chloride content in the MSM and STWW media. According to the results in both liquid media, the chloride level appeared proportional to the increase of Fe concentrations 5, 10, and 20 mg L⁻¹. When the experiment ended, the chloride level at these latter concentrations of Fe was 2.35, 2.30, and 1.95 g L⁻¹ in MSM and 3.8, 3.0, and 3.0 g L⁻¹ in STWW. These results could be explained by the ability of Ps. putida AE015451 to degrade PCP even at a high rate (300 mg L⁻¹). Similarly, in STWW, PCP appeared proportional to the release of chloride, but this release was greater by using a FeSO₄ rate of 5 mg L⁻¹.

Thus, PCP being chelated and the ionic transfer was enhanced by iron in the biotope, which facilitates the development of bacteria (Chen et al. 2011; Lin et al. 2014). Thus, since iron is an essential element for the metabolism of aerobic microorganisms, bacteria can also assimilate iron and grow (Beasley et al. 2019). Besides, the Fe addition changes the pH to acid (Martin et al. 2008).

The effect of inoculum volume (0.5, 1, and 2 mL) in the presence of 30 mg L⁻¹ of PCP contaminated MSM or STWW samples is reported in Table 2. In MSM at T0 by increasing the inoculum volume, a significant decrease to 0.37 ± 0.04 g L⁻¹ of the chloride content was observed. At TF, the chloride content significantly increased compared with T0 in all the treatment and within the TF time a significant decrease from 6.20 ± 0.03 to 4.60 ± 0.04 g L⁻¹ was observed by increasing the inoculum volume from 0.5 to 2 mL. In the STWW sample at T0, no significant change was observed in the chloride content (Table 2), and at TF, in which no further changes were observed compared with T0, the greater value of the chloride content was registered in the sample with 1 mL of inoculum (Table 2).

The pH of the medium is typically regarded as a crucial factor in PCP elimination (Karn et al. 2010a, 2010b). In this study, the variation of the pH between 4, 5, 6.3, 7, and 8 in 30 mg L⁻¹ of PCP contaminated MSM and STWW samples is reported in Table 2. The increase of the pH in the MSM sample allowed a significant increase of the chloride content in both T0 and TF (Table 2). But in STWW samples, the chloride content showed a decrease from 2.01 ± 0.66 to 1.04 ± 0.99 g L⁻¹ and from 3.80 ± 0.33 to 2.40 ± 0.44 g L⁻¹ in T0 and TF, respectively (Table 2). In both MSM and STWW samples, a chloride content increase in T0 to TF was observed. The pH of the media played an important role in the PCP mineralization by microbial biomass, as reported by Cabezuelo et al. (2021) and Karn et al. (2010a, 2010b). Thus, the pH ranging between 6.5 and 8 enhanced the PCP biodegradation by augmenting its solubility and availability in the medium (Hechmi et al. 2013).

The bacterial biomass in the control sample MSM or STWW + Ps. putida AE015451 showed an increase between T0 and TF (Table 3). Also, different experimental conditions were examined such as PCP, Fe content, bacterial inoculum volume, and pH of the medium.

Table 3 presents the results about the effect of PCP content variations of 30, 50, 100, 200, and 300 mg L⁻¹ on bacterial biomass (BBM) developed in the MSM medium and STWW at T0 and after 7 days (TF) of incubation at 30 °C. In MSM and STWW samples at T0, no significant changes were observed in BBM (Table 3), while, in MSM, at TF, a significant increase of the BBM was observed in all the PCP contaminated MSM (Table 3). Within TF time, the BBM value decreased by increasing the PCP concentration and went from 8.64 ± 0.98 log₁₀ CFU mL⁻¹ of 30 mg L⁻¹ to 4.20 ± 1.10 log₁₀ CFU mL⁻¹ of 300 mg L⁻¹ PCP contaminated MSM medium (Table 3). In STWW samples at TF, no further increase in terms of BBM was observed compared to T0 (Table 3). Contrariwise to what happens in the MSM medium at TF in the STWW sample, the BBM significantly increased from 7.85 ± 0.15 log₁₀ CFU mL⁻¹ of 30 mg L⁻¹ to 8.55 ± 0.33 log₁₀ CFU mL⁻¹ of 300 mg L⁻¹ PCP contaminated samples (Table 3).

Based on these results, this pesticide acts directly on the Ps. putida AE015451 growth; thus, bioaugmentation efficiency decreases. Therefore, the biodegradation of PCP decreases with its increasing rates (Werheni Ammeri et al. 2016) especially in the PCP contaminated MSM medium. In addition, the PCP has a negative effect on bacteria growth (Urrutia et al. 2013; Gałązka et al. 2018).

Table 3 shows the effect of the different rates of iron in MSM and TSWW on BBM at a PCP rate of 30 mg L⁻¹. Adding the different concentrations of iron (FeSO₄) at 5, 10, and 20 mg L⁻¹ led to a different behavior in MSM and STWW medium after 7 days (Table 3). At T0 in the MSM medium, a significant slight decrease of the BBM value was observed by increasing the Fe concentration. In addition, no significant changes were observed at TF compared to T0 (Table 3). At TF, the BBM did not significantly change by increasing the Fe amount (5, 10, and 20 mg L⁻¹) (Table 3). In STWW samples, the BBM value was 7.90 log₁₀ CFU mL⁻¹ on average at T0 and no significant changes were observed compared to TF (Table 3). Only the sample treated with Fe at 20 mg L⁻¹ significantly increased at TF to 8.61 ± 0.15 log₁₀ CFU mL⁻¹. These results are the consequence of the iron being in solution that can be easily assimilated at this step, and that helps the bacteria's resistance to PCP toxicity and contributes to its effective removal from the media.

In sterile STWW, increasing the rate of Fe leads to a better bacterial development, and the strain of Ps. putida by producing pyoverdine enhanced the effectiveness of PCP-polluted sites' bioremediation. This pyoverdine production is known as largely been affected by the Fe availability in the medium. The addition of Fe in the medium regulates the siderophore production and the PCP transformation throughout bacterial activity. In addition, the chloride content is untimely related to PCP degradation.

According to literature, the majority of the iron available in the medium and in the bacterial cell would be bound to siderophores, which are small peptides capable of forming complexes – siderophores Fe³⁺ – or iron chelators. These siderophores are a form of iron transit and hogging and a means of internalizing iron into the cell, which is necessary for its functioning; therefore, siderophores are well regarded as an important form of antagonism and rivalry between organisms living in the environment (Vaulont & Schalk 2015). Among the molecules synthesized by bacterial microorganisms and especially of the genus Pseudomonas, pyoverdines, fluorescent pigments, are fluorescent siderophores and an antibiotic oligopeptide produced in particular by Pseudomonas aeruginosa and Pseudomonas fluorescens, and are well considered as virulence and invasion factors of Pseudomonas (Mehri et al. 2011, 2012). So, bacteria can assimilate iron as an essential element for nutrient metabolism under aerobic condition (Beasley et al. 2019). In overall, iron is not soluble in liquid media which makes difficult its assimilation. As reported by Chen et al. (2011), iron can be chelated by PCP in the biological environment and become available for the metabolism and development of bacteria. The work of Chen et al. (2018) showed an interaction between PCP and Fe during degradation experiments related to some radical cations.

The result of inoculum volume variations of 0.5, 1, and 2 mL of the Ps. putida AE015451, at a PCP rate of 30 mg L⁻¹ in the MSM medium and sterile STWW, is reported in Table 3. The variation of the inoculum volume directly affected the BBM content in the MSM medium. Both at T0 and TF and by increasing the inoculum, the BBM showed a significant increase (Table 3). Besides, by comparing the two incubation periods, T0 and TF, the BBM showed a significant increase following the application of 0.5 and 1 mL of inoculum (Table 3). Contrariwise in the case of the STWW experiment, no significant changes in BBM are observed in both T0 with on average 7.87 log₁₀ CFU mL⁻¹, and TF with on average 8.25 log₁₀ CFU mL⁻¹ (Table 3).

Table 3 shows the effect of 4, 5, 6.3, 7, and 8 pH variations on the BBM content in MSM and STWW, respectively, at a PCP rate of 30 mg L⁻¹ at T0 and after 7 days. In the STWW medium, no significant changes in BBM content are registered at T0 and TF (Table 3). As for the MSM and at T0, a significant increase of BBM is registered, from 3.66 ± 0.92 log₁₀ CFU mL⁻¹ at pH 4 to 7.91 ± 1.03 log₁₀ CFU mL⁻¹ at pH 8 (Table 3). Equally and at TF, the BBM showed an increase in the MSM at pH 5 and 6.3 with values of 8.70 ± 0.92 and 9.24 ± 0.35 log₁₀ CFU mL⁻¹, respectively (Table 3). By increasing the pH to 7 and 8, the BBM showed a significant decrease as compared to the case of T0. Thus, the pH showed an influence on the microbial growth and the removal and biotransformation process of PCP. These results obtained could be explained by the importance of the pH in protecting the bacteria from stresses, as reported by Zarkan et al. (2019).

When 1 and 2 mL of inoculum are introduced in the CAA without PCP and at pH 5, the PVD content is revealed as important with 6.88 and 6.39 × 10⁻⁸ mol mL⁻¹, respectively (Figure 2(a)). Besides, the PVD content showed a decrease if the PCP increased from 30 to 50 mg L⁻¹. As well, no significant changes in PVD production at 30 mg L⁻¹ of PCP following the inoculum volume increase, whereas the PVD showed an increase after the inoculum increase, especially to 1 mL and with 5.38 × 10⁻⁸ μmol mL⁻¹ (Figure 2(a)). The addition of iron caused a net decrease in PVD production (CAA + Fe treatment; Figure 2(a)). The addition of Fe in CAA + PCP 30 and especially at 50 mg L⁻¹ showed a net development of the pyoverdine production. In the case of CAA + Fe + PCP 30 mg L⁻¹, a significant decrease in PVD is observed because of the Fe increase. In addition, the PVD production appeared not related to the inoculum volume, since at 5 and 10 mg L⁻¹ of Fe + 50 mg L⁻¹ PCP no significant changes are observed after the inoculum volume increase. Contrariwise, in the CAA + Fe + PCP 50 mg L⁻¹, a different pattern of behavior is seen. The PVD production reached the value of the control without PCP after adding 1 mL of inoculum showing 7.20 × 10⁻⁸ μmol mL⁻¹ (Figure 2(a)). These samples are characterized by a black precipitate at the bottom of the reaction tube.

The PVD production at pH 6.3 is also monitored and the result is reported in Figure 2(b). In the control experiment of CAA without PCP, the better PVD production was observed by adding 1 mL of the inoculum. In the experiment of CAA + PCP 30 mg L⁻¹, the PVD production reached the value registered for the control experiment without PCP after adding 1 or 2 mL of inoculum, with 7.32 × 10⁻⁸ μmol mL⁻¹ (Figure 2(b)). The addition of Fe at 5, 10, and 20 mg L⁻¹ allowed a reduction of the PVD production, but in the experiment inoculated with 1 mL of Ps. putida, we registered the larger value within this treatment with 4.85, 3.87, and 1.80 × 10⁻⁸ μmol mL⁻¹ in CAA + 5, 10, or 20 mg L⁻¹ of Fe, respectively (Figure 2(b)). Furthermore, CAA + Fe + PCP 30 mg L⁻¹ showed the larger value of PVD production (6.87 × 10⁻⁸ μmol mL⁻¹) as compared to the control without PCP, and with 5 mg L⁻¹ of Fe. In CAA + Fe + PCP 50 mg L⁻¹, no further changes compared to the control are observed about the PVD production (Figure 2(b)).

At pH 8, the better PVD production is achieved in the CAA supplemented with 30 mg L⁻¹ of PCP, with 6.98 × 10⁻⁸ μmol mL⁻¹, and in the CAA + Fe 10 mg L⁻¹ + PCP 30 mg L⁻¹ with 6.89 × 10⁻⁸ μmol mL⁻¹ (Figure 2(c)). The control experiment without PCP registered a decrease in PVD production as compared to the one recorded at pH 5 and 6.3 (Figure 2(a)–2(c)). Similar to the experiment at pH 5, the addition of Fe at 5, 10, and 20 mg L⁻¹ resulted in a decrease in the PVD production. No further changes registered in PVD production in the CAA + Fe (5, 10, and 20 mg L⁻¹) + PCP at 30 and 50 mg L⁻¹ (Figure 2(c)).

In contrast to the results obtained with PCP, the iron addition in the CAA does not affect the production of PVD. These most recent results can be attributed to the PVD's positive effect, which was carried out after PCP adaptation or neutralization by adsorption, conditioning the bacteria's tolerance to the PCP toxic effect.

This result followed Werheni Ammeri et al. (2016) and Karan et al. (2010) findings that showed Ps. putida to be more active at acidic pH for PCP removal. So, the PVD production was promoted in CAA and at pH 5. This result did not agree with the one of Mehri et al. (2011), who registered the best PVD production at pH 6.3.

Our research supports Saygin & Baysal (2020)'s findings that PCP and its different residual metabolites should encourage the creation of microbial biofilm to protect against harmful substances. Many researches have advocated a near affiliation among hydrocarbon removal and microbial biofilm development (Verhagen et al. 2011; Dasgupta et al. 2013; Meliani & Ben Soltane 2014). Besides, the study of Amaya-Chavez et al. (2006) and Saraswathy et al. (2001) confirmed that the microbial biofilm development represented a behavioral reaction to survival and success while microbial cells have been exposed in situ to diverse environmental constraints. To conclude, our main results showed that Pseudomonas and its siderophore producing pyoverdine contribute to the PCP removal in the presence of iron.

The effects of two independent variables such as PCP and Fe rates showed the maximum responses to 200 mg L⁻¹ PCP and 20 mg L⁻¹ Fe (Figure 4(a) and 4(b)).

The bioaugmentation of a secondary wastewater treatment process adopted in this study for PCP removal appeared to be an efficient alternative, less expensive, more extensive and more environmentally friendly tool as compared to the conventional physico-chemical methods. This bio-process showed high PCP removal for Ps. putida AE015451 and at 100 mg L⁻¹. After 72 h of incubation in STWW, this selected Pseudomonas strain removed about 90% of PCP. So, this strain of Ps. putida AE015451 could play an important role in the bioaugmentation process associated with its PGPR properties. Thus, it can help macrophytes to tolerate the toxicity of some compounds such as PCP and other insecticides, and enhance their decontamination properties by secreting PVD. The effect of adding iron as FeSO₄ showed this latter could react with PCP, forming neutral Fe–PCP complexes, which contribute to the development and growth of bacteria, and directly the pollution reduction. The ability of bacteria to form condensed biofilm enhances their degradative capacity and resistance to xenobiotic compounds such as PCP used as a carbon source. Our study demonstrates the effectiveness of PCP bio-removal by adding bacteria that can use the pollutant as a nutrient source. Further research is needed to extend these experiments to wastewater to develop bacterial weathering-based bioremediation processes.

The authors want to thank the University of Tunis El Manar for granting a Ph.D. Studentship to Ms Rim Werheni. So, this research was partly funded by the Tunisian Ministry of Higher Education and Scientific Research in the frame of the program contract 2019–2020 (CERTE).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.