PHE (C₁₄H₁₀, purity 98%) was purchased from Sigma-Aldrich (Madrid, Spain) and radiolabeled compound ¹⁴C-PHE (36 mCi⋅mmol⁻¹, purity 99.9%, and radiochemical purity 100%) was acquired from the Institute of Isotopes (Budapest, Hungary). HPBCD was obtained from CycloLab Cyclodextrin Research and Development Laboratory Ltd. (Budapest, Hungary).

Mineral Salt Medium (MSM) composition (g L⁻¹): 4.0 Na₂HPO₄; 2.0 KH₂PO₄; 0.8 MgSO₄; 0.8 NH₄SO₄. The micronutrients solution (SNs) was composed of (mg L^-1): 12.5 NiCl₂ 6H₂O; 25.0 SnCl₂ 2H₂O; 12.5 ZnSO₄ 7H₂O; 12.5 Al₂(SO₄)₃ 18H₂O; 75.0 MnCl₂ 4H₂O; 12.5 CoCl₂ 2H₂O; 37.5 FeSO₄ 7H₂O; 10.0 CaSO₄ 2H₂O; 3.75 KBr; 3.75 KCl; 2.50 LiCl (Fenlon et al., 2011). The mixture of MSM and SNs (50:1) was named NS in the study.

Five soils (PLD, LL, ALC, CR, R) with diverse physicochemical properties were collected from different points in the South of Spain. PLD soil is an agricultural soil located in Los Palacios y Villafranca—Seville (37°10′20.0″N 5°55′21.9″W), where wheat, cereals, and vineyard crops are mainly cultivated. The agricultural soil LL from Vejer de la Frontera—Cádiz (36°17′52.6″N 5°52′45.2″W) is devoted to intensive agriculture of carrot, cotton, leek, etc. ALC soil was taken from Alcornocales Natural Park (36°20′54″N 5°36′14″O) and is characterized by high organic matter (OM) content. CR soil is an agricultural soil from an area of olives within the experimental farm La HAMPA (IRNAS-CSIC) (37°17′28.3″N, 6°3′55.4″W). R soil, collected from a palm trees zone in Conil de la Frontera—Cádiz (36°18′32.4″N, 6°08′58.6″W), has been managed with a huge number of insecticides. The soil samples collection was conducted from the superficial horizon (0–20 cm), air-dried for 24 h at room temperature, and sieved (2 mm). Table 1S shows the physicochemical properties of the studied soils and their textural classification. The pH was determined in a proportion of 1:2.5 soil/water extract. The particle size distribution was evaluated using Bouyoucos densimeter; the calcination or muffling method was used to estimate the OM content of the soil weight loss on ignition (LOI) or calcination, the quantification of OM was determined by K₂Cr₂O₇ oxidation, and the manometric method was used to measure the total carbonate content.

The PHE-degrading microorganism selected, S. indicatrix CPHE1, corresponds to S. maltophilia CPHE1 (NCBI number: MT138842) published by Lara-Moreno et al. (2021). Subsequent studies indicated greater phylogenetic closeness with the species S. indicatrix rather than S. maltophilia (Lara-Moreno et al., 2023). CPHE1 strain was isolated in our lab from a highly contaminated industrial soil as reported by Lara-Moreno et al. (2021). The sequencing data of S. indicatrix CPHE1 were deposited in the National Center for Biotechnology Information (NCBI), under the BioProject ID PRJNA868539. The whole genome shotgun generated has been deposited in DDBJ/ENA/GenBank under the accession number JANQDV000000000. The version described in this paper is version JANQDV010000000. The raw data was deposited in the Sequence Read Archive (SRA) under the accession number SRR21098193 (Lara-Moreno et al., 2023).

Lara-Moreno et al. (2023) provided relevant information about the genome. The genome of S. indicatrix CPHE1 deposited in NCBI (JANQDV000000000) (Lara-Moreno et al., 2023) consists of 163 contigs, with a size of 4.553.664 bp, 66.1% of Guanine-Cytosine (G-C) content, and 4137 genes described.

Genes potentially associated with PHE biodegradation were bioinformatically predicted in S. indicatrix CPHE1 genome by two diverse strategies: (i) searching for specific annotation of gene products by RAST server and subsequently clustering them into phylogenetic trees using reference sequences; a bibliographic and database search of enzymes of other bacteria has been carried out to identify reference sequences. Data obtained allowed the improvement of the genome automatic annotation of the CPHE1 strain genes. Similarities between the whole set of selected enzymes have been analyzed by Clustal Omega obtaining a phylogenetic analysis. (ii) Analyzing the sequence homology with respect to reference sequences of bacterial strains able to biodegrade PHE (Cébron et al., 2008; Izmalkova et al., 2013) or bacteria frequently detected in the water systems by other studies (Stingley et al., 2004; Elufisan et al., 2020).

Considering the first approach, Supplementary Figure S1 shows the distribution of the annotated genes in different subsystems of S. indicatrix CPHE1 genome. Particular attention was focused on the subsystem of genes that participate in the aromatic metabolism compounds (boxed in red); these annotated genes could take part in the central catabolism of the metabolites formed during the degradation of aromatic compounds. Beta-ketoadipate pathway is an aromatic compound degradation route widely distributed in soil bacteria and fungi. On the one hand, catechol 1,2-dioxygenase converts catechol generated from aromatic hydrocarbons into intermediates of the tricarboxylic acid cycle (TAC). On the other hand, protocatechuic acid is turned into intermediates of the TAC catalyzed by protocatechuate 3,4-dioxygenase. Both enzymes act by cleaving aromatic ring. Catechol-1,2-dioxygenase, has been studied in species belonging to Pseudomonas genus due to its ability to cleave the aromatic ring of catechol molecule (Rodríguez-Salazar et al., 2020). Other annotated genes in the S. indicatrix CPHE1 genome could encode enzymes that participate in the salicylate and gentisate pathway, compounds that have been identified as intermediaries of PAH catabolism (Grund et al., 1992). The salicylic acid pathway has been described for several PHE-degrading bacterial genera or other PAHs, such as Pseudomonas (Lin et al., 2014), Streptomyces (Ishiyama et al., 2004), or Stenotrophomonas (Huang et al., 2013). Several authors have described monooxygenases (Medić et al., 2020) and dioxygenases (Moody et al., 2005; Gao et al., 2013; Kumari et al., 2017) as enzymes responsible for catalyzing the initial reaction of PAHs degradation pathway to produce hydrodiol or dihydrodiol.

For this reason, we initially focused on the study of genes annotated as monooxygenases in the genome of S. indicatrix CPHE1. A phylogenetic analysis was carried out using the proteins reported in Table 1 (Figure 1). The phylogenetic tree demonstrated no similarities between the annotated CPHE1 sequences and the reference monooxygenase (AlkB, UniProtKB: A4XPE8), described in Pseudomonas aeruginosa P6 as responsible for petroleum hydrocarbon degradation (Wang et al., 2019; Medić et al., 2020). Since the other reference sequences were not involved in PAH degradation pathways, it has been ruled out that monooxygenases encode the first step in the PHE degradation.

A similar approach was then undertaken for another enzyme class since the initial reaction of PAH biodegradation in S. indicatrix CPHE1 could be catalyzed by a multicomponent system of a dioxygenase, producing dihydrodiol (Kumari et al., 2017). This hypothesis is supported by two fundamental facts; first, the presence of similar dioxygenases has been detected in several PAHs degrading bacteria such as Pseudomonas (Prabhu and Phale, 2003; Cébron et al., 2008), Mycobacterium (Stingley et al., 2004; Moody et al., 2005), and Stenotrophomonas (Kumari et al., 2017). Second, the metabolite dihydroxyphenanthrene was identified during the PHE biodegradation of S. indicatrix CPHE1 (Lara-Moreno et al., 2021). These data would suggest the intervention of dioxygenase in PHE biodegradation.

For this reason, nine dioxygenases annotated in the genome of S. indicatrix CPHE1 have been exhaustively analyzed and compared with similar dioxygenases of bacterial genomes deposited in several databases. All dioxygenases have been compared by multiple alignments with reference proteins (Table 2), including two dioxygenases known for their involvement in naphthalene biodegradation (NAHAc of P. putida G7 and NIDA of the only naphthalene dioxygenase described in Stenotrophomonas) (Cébron et al., 2008; Kumari et al., 2017). As shown in the tree in Figure 2, the enzyme DO2 is the closest phylogenetically to the reference proteins (NAHAc and NIDA). However, annotated dioxygenases have been considered for the subsequent RT-PCR expression experiments since there are no conclusive results to exclude any of them as putative responsible for deoxygenation.

After the analysis of the possible dioxygenases, a thorough analysis of the annotated dehydrogenases of S. indicatrix CPHE1 was performed, since the dihydrodiol dehydrogenase enzyme catalyzes the second step of the PHE biodegradation route described by Stingley et al. (2004). This enzyme removes two hydrogen atoms from the molecule, oxidizing it, the electrons and protons from the hydrogen atoms are captured by the oxidized form of the coenzyme, which is reduced. Among 119 CPHE1 proteins annotated as dehydrogenases, nine were selected based on their function in relation to the biodegradation of PAHs (Table 3). These amino acid (aa) sequences were analyzed in a phylogenetic tree using diverse dehydrogenase references, including dehydrogenases known as catalysts of the second step of the PAHs biodegradation pathway (NahB, BphB, and NidD). Two different trees were obtained based on the considered aa references (Figure 3); the first tree (Figure 3A) comprises dihydrodiol naphthalene dehydrogenase (NahB) described for P. stutzeri AN10, naphthalene dehydrogenase (NahB) of P. putida AK5, and cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase (BphB) of P. putida 9816-4. Figure 3B includes naphthalene dehydrogenase (NidD) described in Stenotrophomonas sp. IITR87 which resulted in phylogenetically close to five (out of nine) CPHE1 dehydrogenases.

Although PABA DH (betaine aldehyde dehydrogenase) and SUCB (oxoglutarate dehydrogenase dihydrolipoamide succinyltransferase component) were the closest CPHE1 sequences to the respective reference proteins (for each tree), it should be noted that reference proteins belong to different bacterial genera, consistent in a wide protein sequences variability (Keshavarz-Tohid et al., 2019).

Studies of gene expression have been carried out by RT-PCR experiments to know which annotated dioxygenase of S. indicatrix CPHE1 was expressed throughout the PHE biodegradation process after inoculating CPHE1 strain.

Validation of the samples is reported in Supplementary Figure S2 showing the PCR amplification of 16S rDNA gene using genomic DNA (positive control) from S. indicatrix CPHE1, cDNA obtained from RNA extracted from CPHE1 strain without contact with PHE (t₀) and RNA samples taken at different times from the PHE biodegradation assay (t₁, t₃, t₇, t₁₄, and t₂₁).

Several authors have used the semi-quantitative RT-PCR to confirm the involvement of genes in the biodegradation pathway of various pollutants. For example, Thanh et al. (2019) observed the expression of dbfA1A2RBC genes that share 99%–100% identity with Paenibacillus sp. YK5 genes (putatively involved in dibenzofuran degradation). The induction of four of these genes was observed after treatment with dibenzofuran through RT-PCR. Di Canito et al. (2018) studied the role of Rhodococcus opacus R7 oxygenases in the o-xylene degradation. The important involvement of akb genes was demonstrated using RNA obtained from R7 cells in the presence of o-xylene by RT-PCR. The result showed that the transcription was induced by the contaminant. In another study, Churchill et al. (2008) checked the ability of Mycobacterium sp. CH-2 to mineralize PHE, pyrene, and FLT. Primers were designed based on the dioxygenases nidAB and pdoA2B2 and an alkane monooxygenase. RT-PCR analysis indicated that alkane monooxygenase is constitutively expressed, however, nidAB, and pdoA2B2 were expressed only in the presence of PAHs.

The results of the CPHE1 dioxygenase gene expression are shown in Figure 4 obtained with specific primers designed on CPHE1 genes reported in Table 2. At the top of the figure are presented the results of the PCR when the 16S rRNA gene was amplified under non-limiting conditions, which serves as a reference gene. Signal intensity quantification (ImageJ.JS) of PCR results indicated that the cDNA of the t₀, t₁, t₃, and t₂₁ samples are the least concentrated, followed by the t₁₄ sample. The t₇ sample was twice more concentrated. These data will allow for assessing the expression levels of the rest of the studied genes.

A previous study carried out by Lara-Moreno et al. (2023) showed that the gene annotated as cysteine dioxygenase (cysDO) was expressed in the presence of PHE from the third day of treatment. The KEGG database indicates that the cysDO enzyme is involved in different biodegradation pathways of aromatic compounds, including naphthalene (M00534), salicylic acid (M00638) and phthalic acid (M00623). In addition, Wang et al. (2017) studied the participation of cysDO enzyme in the degradation of aromatic compounds. However, in the currently work, gene expression study has been expanded, all dioxygenases annotated in the genome of CPHE1 strain were analyzed with the aim to know their involvement in PHE biodegradation pathway. The electrophoresis gel obtained is shown in Figure 4. Gene expression of 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (ardDO), gamma-butyrobetaine dioxygenase (bbhDO) or putative dioxygenase (DO2) was not detected. In the case of tryptophan-2,3-dioxygenase (tphDO), homogentisate dioxygenase (hmgDO), 4-hydroxyphenylpyruvate dioxygenase (hppDO), and nitropropan dioxygenase (npDO) genes, a repression of expression was observed in the presence of PHE. And in case of 3-hydroxyanthranilate 3,4-dioxygenase a constituent expression was detected. It concludes that cysDO was the only one expressed in the presence of PHE. These results suggest the involvement of a deoxygenation in the PHE degradation pathway when S. indicatrix CPHE1 was inoculated. In addition, as indicated above, the presence of dihydrodiol metabolites was detected after inoculating CPHE1 strain (Lara-Moreno et al., 2021). These results suggest the involvement of a dioxygenation in the PHE degradation pathway when S. indicatrix CPHE1 was inoculated.

Beside the dioxygenase gene expression, the expression of other genes involved in PHE biodegradation route was investigated relying on the second bioinformatic approach based on the comparison of S. indicatrix CPHE1 genes with sequences belonging to bacterial strains known for their involvement in PHAs biodegradation.

Indeed, Stingley et al. (2004) described the most common PHE biodegradation pathways for Mycobacterium vanbaalenii strain PYR-1. These genes were compared to the only two genomic studies (Kumari et al., 2017; Elufisan et al., 2020) that have been published on PHE degradation route in Stenotrophomonas genus. For instance, the genes involved in this degradation pathway showed at least 97% identity compared with Stenotrophomonas IITR87 genome (Kumari et al., 2017).

Thus, CPHE1 genes showing a high identity with respect to the genes described in the M. vanbaalenii PYR-1 genome were taken into consideration as references for the gene expression studies in S. indicatrix CPHE1 (Table 4). In addition, genes described in other bacterial species, reported as PAH-degrading, were included in Table 4 as references, accordingly to the hypothesized metabolic pathway.

Results of their expression in the presence of PHE are shown in Figure 5. The expression of the homologous gene encoding aldolase hydratase (phdG), responsible for transforming 2-hysroxybenzo(h)chromene-2-carboxylic acid to 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid, was induced after 7 days in contact with the contaminant, suggesting its involvement in the PHE degradation pathway carried out by S. indicatrix CPHE1. Usually, in the majority of publications, the gene that encodes the α subunit hydroxylating dioxygenase is identified as a functional marker gene. However, it has been observed that due to low phylogenetic resolution and the lack of specificity, it is derived by erroneous estimation of bacteria that degrade PAHs (Liang et al., 2019). For this reason, Liang et al. (2019) proposed the hydratase-aldolase gene as a biomarker for bacteria involved in PAHs degradation. Therefore, this gene could represent a powerful biomarker to explore the potential of PAH-degrading bacteria in ecosystems, which would have a high impact on the bioremediation of contaminated soils with PAHs. Levieux et al. (2018) and Story et al. (2001) demonstrated that bifunctional enzymes hydratase-aldolase are present in bacterial strains which can use PAHs as the only carbon and energy source, indicating that despite catalyzing an important step in the PAHs degradation, their mechanisms are not very well established.

With the aim of deepening the study of the genes responsible for the degradation of PHE, Stenotrophomonas sp. Pemsol genes were also considered, since Pemsol strain can degrade anthraquinone, naphthalene, PHE, phenanthridine, and xylene (Elufisan et al., 2020). Five genes from CPHE1 were selected due to their high homology with Pemsol genes involved in PHAs biodegradation pathway (Table 5). Figure 6 shows the gene expression at different times: the gene encoding catechol-2,3 dioxygenase was constitutively expressed while the expression of biphenyl-2,3-diol 1,2-dioxygenase (bphC) was induced after 7 days of treatment, the correspondent gene product catalyzes the transformation of 3,4-hydroxyphenanthrene to 2-acid hydroxycenzochromene-2-carboxylic. bphC gene has been also studied in Sphingomonas, and Sphingobium genera which are able to metabolize PHE via both meta- and ortho-cleavege pathways (Waigi et al., 2015; Macchi et al., 2019). Moreover, Yun et al. (2014) concluded that PHE induced a strong regulation of bphC gene, which is involved in the attack to cleavage the aromatic ring in the lower catabolic route of PAHs in Novosphingobium pentaromativoransus species.

All these results together revealed that PHE induces the expression of specific genes (cysDO, phdG, and bphC) in S. indicatrix CPHE1 degradation pathway. Figure 7 summarizes the metabolic steps and the time of activation of expressed genes. The gene annotated as cysDO was induced after 3 days of inoculation, coinciding with the point where PHE biodegradation begins to be significant (Lara-Moreno et al., 2021), so this dioxygenase could be responsible for catalyzing the first step of the PHE biodegradation pathway. In addition, the induction of bphC and phdG genes by the presence of PHE from respectively 14 days and 21 days indicates that they could act in intermediate steps as extradiol dioxygenase responsible for the first aromatic ring cleavage and hydratase aldolase responsible for transforming 2-hysroxybenzo(h)chromene-2-carboxylic acid to 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid, respectively.

PHE abiotic dissipation was assessed in the five selected soils, adding HgCl₂ solution to kill the soil endogenous microbiota. The results concluded that abiotic dissipation was not observed in this study (data not shown). Figure 8 and Table 6 show the PHE mineralization curves and kinetic parameters obtained after the application of the different treatments in the investigated soils. An extension of mineralization of 3.6%, 1.7%, 2.3%, 8.7%, and 1.5% after 120 days for PLD, LL, ALC, CR and R soils was respectively observed when the contaminated soils are not subjected to any bioremediation treatment, with DT₅₀ values in the range 1133–6343 days (3–17 years). These results evidenced the need to resort to assisted natural attenuation using biostimulation and bioaugmentation.

The addition of inorganic nutrients (NS in Figure 8 and treatment B in Table 6) produced a slight activation of the indigenous microbiota of LL, ALC, and CR soils, and therefore PHE mineralization slightly increased (after 120 days 13.1, 12.3, and 17.3%, respectively). On the contrary, its effect was much greater in the cases of PLD and R soils, in which 53.2% and 42.6% of PHE mineralization were achieved, respectively, decreasing DT₅₀ values from 3243 days to only 61.1 d for PLD soil, and from 6343 days to 335 days for R soil. To confirm the presence of PHE-degrading bacterial strains in the studied soils, an enumeration of colony forming units per Gram of soil (CFU g⁻¹ soil) in presence of PHE as the only carbon source was carried out (Supplementary Table S3). The CFU counts were in line with the observed mineralization results when the soil microbiota was stimulated using NS. The microbiota of PLD and R soils turn out to be the most active in PHE mineralization, containing the highest number of CFU g⁻¹ (1.5 × 10⁷ and 8.6 × 10⁷, respectively) of PHE degrading bacteria; CFU g⁻¹ for LL and CR soils were an order of magnitude less than in PLD and R soils, likewise a lower extent of mineralization. ALC soil only reached 12.3% of mineralization and its microbiota was the least rich in potential PHE degraders (1.9 × 10⁵ CFU g⁻¹).

OM influences the control of PAH bioavailability in soils (Abdel-Shafy and Mansour, 2016). It has been demonstrated that the interaction between PAHs and the soil increases to longer aging time, causing a decrease in the bioavailability of the pollutants (Ehlers and Loibner, 2006). To increase their bioavailability, extractants such as cyclodextrins have been used. In a previously published work, Morillo et al. (2012) confirmed the formation of inclusion complexes between HPBCD and PHE which caused an increase in its water solubility (376 times higher in the presence of 100 mM HPBCD). HPBCD is considered suitable for application to contaminated soils since the addition of HPBCD to these soils would cause an increase in the concentration of PHE in the soil solution making it more bioavailable (Duán et al., 2021; Lara-Moreno et al., 2022b). Figure 8 (Treatment C) shows an increase in the percentage of mineralization with respect to treatment with only NS (Treatment B) in all cases except in PLD soil. In ALC, CR, and R soils the extent of mineralization reached up to 58.4%, 87.3%, and 61.3%, respectively (Table 6). In addition, the kinetic processes were accelerated, showing a decrease of DT₅₀ from 2307, 573 and 335 days to 61.6, 26.4, and 12.1 days after treatment, respectively.

Gao et al. (2014) also evaluated the effect of OM on the biodegradation process and the effect of HPBCD to improve PHE bioavailability. The study concluded that there is a significant inverse relationship between the PHE adsorption capacity to the soil and the extracted fraction with HPBCD and PHE biodegradable fraction. Morillo et al. (2014) performed fluorene (FLU) and fluoranthene (FLT) adsorption-desorption in different soils in the presence of HPBCD. The results confirmed that FLU is more resistant to extraction in soils since its initial adsorption to minerals and OM of the soil is greater due to its smaller size in the case of FLT.

PHE extraction experiments from soils have been carried out to know the effect produced by NS solution in comparison to HPBCD on the PHE bioavailability (Figure 9). The OM content is very different in the studied soils. This difference influenced the PHE amounts present in the soil solution when NS was added: 9.6% (CR), 10% (LL), 9.2% (PLD), 2% (R), 0.4% (ALC) (listed in increased order of OM content). PHE concentration in the soil solution was higher in all soils when HPBCD was added: 18.8% (CR), 20.4% (LL), 19.2% (PLD), 3.9% (R), 1.2% (ALC), and the observed increase was due to the formation of complexes between HPBCD, and PAHs as previously observed by Morillo et al. (2014). The results confirmed the inverse relationship between the soil OM content (Supplementary Table S1) and the PHE extracted. However, the determined PHE fraction in solution in the presence of NS or HPBCD was not directly correlated with the observed PHE biodegradation curves and the extent of extraction reached for the different soils, concluding that, PHE degradation depends on other factors apart from the fact that HPBCD is able to increase PHE bioavailability.

After a thorough analysis of the CPHE1 strain genome and the degrading genes expression, the ability of S. indicatrix CPHE1 to remove PHE was used to improve the bioremediation with a bioaugmentation strategy. When S. indicatrix CPHE1 was inoculated in PLD, LL, ALC, and CR soils, improvements in the global extent of PHE mineralization were observed (59.9%, 33.5%, 65.3%, 75%, respectively), and in the mineralization kinetic parameters, reducing the values of DT₅₀ (31.5, 499, 36.3, and 17.2 days, respectively). The lowest values of DT₅₀ in these 4 soils were obtained when S. indicatrix CPHE1 was inoculated as compared to NS or HPBCD application. Shon et al. (2020) observed that Sphingopyxis soli KIT-001 showed interactions between the bacterial membrane and PAHs exerting a strong influence on biological processes such as metabolic activity and substrate absorption due to changes in membrane lipids, improving the PHE degradation efficiency.

On the contrary, in the case of contaminated R soil inoculated with S. indicatrix CPHE1, a reduction in the extent of mineralization (29.3%) was observed after 120 days, with respect to the degrading capacity shown by the soil microbiota stimulated with NS (42.6%) or the use of HPBCD (61.3%). This fact has been widely observed in the literature because of the competition between the degrading endogenous and exogenous bacteria, which indicates that the presence of S. Indicatrix CPHE1 would have partially disabled PHE degradation carried out by the soil endogenous microbiota, and vice versa (Cycoń et al., 2017; Nwankwegu et al., 2022). The presence of a high density of potential PHE-degrading bacteria in R soil (8.6 × 10⁷ CFU g⁻¹, Supplementary Table S3) would support this statement.

The combined application of S. indicatrix CPHE1 and HPBCD was also tested. The results showed that this co-application clearly increased the extent of PHE mineralization only in soil ALC (with the highest OM content, 13.9%) but the increase was not significant in LL and CR soils, and even a negative effect on the mineralization rate was observed in PLD and R soils in relation to other treatments. Besides, and not less important, DT₅₀ values were higher for all soils when S. indicatrix CPHE1 and HPBCD were co-applicated, indicating that both treatments should not be added together in these soils.

Previously, Stroud et al. (2009) also observed that HPBCD interfered with the microbial mineralization of PHE and hexadecane resulting in lower mineralization extents, but these authors gave no explanation for this fact. Equally, Villaverde et al. (2019) observed that pyrene mineralization in three soils was not increased or even reduced when using HPBCD together with the exogenous bacterial strains Achromobacter xylosoxidans 2BC8 and S. maltophilia JR62. The possibility that the increased concentration of PHE in soil solution due to HPBCD addition could be toxic for S. indicatrix CPHE1 is discarded because Lara-Moreno et al. (2021) demonstrated its capacity to degrade and mineralize 10 mg L⁻¹ PHE in aqueous solution, and its concentration in the soil solution in the present studies is substantially lower. However, since HPBCD could be biodegraded by soil microorganisms (Fenyvesi et al., 2005), the preference of S. indicatrix CPHE1 for HPBCD as a carbon and energy source instead of PHE could slow down the mineralization process (Supplementary Table S4).

Each of the studied soils showed different results after the application of the different treatments, so it is considered important to select a bioremediation strategy based on each specific soil and pollutant properties. LL, PLD, ALC, and CR soils required S. indicatrix CPHE1 inoculation to improve mineralization kinetics, although in the case of CR soil a higher biodegradation profile was observed only applying HPBCD. The most effective strategy for R soil turned out to be the use of HPBCD, which makes more bioavailable PHE in the soil through the formation of an inclusion complex, since its endogenous microbiota showed to be especially active as PHE-degrader.