Effect of Secondary Metabolites Present in Brassica nigra Root Exudates on Anthracene and Phenanthrene Degradation by Rhizosphere Microorganism

Abstract

Rhizoremediation is a strategy for recovering soils contaminated with polycyclic aromatic hydrocarbons (PAHs) based on the interaction of plants and microorganisms in soil pollutant removal. Secondary metabolites are components of root exudates of plants, which have been shown to have an effect on the microbial degradation of aromatic compounds in the rhizosphere. In this study, microorganisms capable of degrading anthracene and phenanthrene were isolated from the rhizosphere of Brassica nigra, and the effect of root exudates and plant secondary metabolites identified in the microbial degradation of PAHs was evaluated. The degradation percentages achieved when anthracene and phenanthrene were used as the sole source of carbon and energy were 35.1% ± 0.3% and 17.5% ± 5.1%, respectively. However, when glucose was added to the culture medium the percentage decreased to 24.8% ± 2.4% for anthracene and increased to 40.6% ± 3.6% for phenanthrene. In trials with root exudates, degradation percentages were 18.5% and 26.8% for anthracene and phenanthrene, respectively. In total, six secondary metabolites were identified in root exudates of B. nigra as follows: flavanone, flavone, Iso-flavanone, 7-hydroxyflavanone, 7-hydroxyflavone, and 6-hydroxyflavone. All had a negative effect on the degradation of anthracene except for 6-hydroxyflavone and 7-hydroxyflavanone. With phenanthrene, the most significant effect was obtained with glucose and 6-hydroxyflavone, which increased the degradation percentage to 51.8 ± 4.0. The results indicate that the composition of root exudates may affect microbial degradation and the effect of secondary metabolites, depending on PAH structure.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic compounds that reach the environment through natural processes (Muratova et al., 2015) or from industrial activities such as spillage of petroleum and incomplete combustion of fossil fuels (Retnam et al., 2013; Cobas et al., 2014). Because of their toxicity, and teratogenic and carcinogenic effects on humans and animals, 16 dangerous PAHs, including anthracene and phenanthrene, have been categorized as priority contaminants by USEPA (Shahsavari et al., 2015).

Soil is one of the environmental matrices most affected by contamination with PAHs due to the hydrophobicity and affinity of these compounds for organic matter (Gan et al., 2009). PAHs are recalcitrant and persist in soils for a long time (De Boer and Wagelmans, 2016). Some soil organisms can accumulate PAHs. This has serious consequences on the environment, since they are biomagnified through food chains, making their toxic impact affect different species, including animals and plants (Zelinkova and Wenzl, 2015). This contamination also causes economic loss, because contaminated soil cannot be used for agricultural processes. For this reason, the recovery of soils contaminated with PAHs is an issue of global concern. Currently there are physicochemical methods available for the removal of PAHs from soil. However, these are expensive and can cause alterations in the ecological dynamics of soil (Chen et al., 2015). Therefore, biological processes have become a promising tool for the recovery of contaminated soils, as they are highly efficient and more economical and environmentally friendly (Meena et al., 2016).

Phytoremediation is the use of plants to enhance biodegradation and pollutant removal (Dietz and Schnoor, 2001) and is considered a cost-effective and environmentally friendly form of remediation technology (Arthur et al., 2005). Several research works on phytoremediation of contaminated soil have concluded that this method is a promising tool for the cleaning of this. However, many key issues in this field need to be studied further, especially those related with the interactions between plants and soil microorganisms (Farraji et al., 2016). Rhizoremediation is a type of phytoremediation that uses the association between plants and rhizospheric microorganisms for degradation or removal of pollutants such as PAHs (Shahsavari et al., 2015). Previous studies have shown that various types of plants can stimulate and enhance microbial degradation of PAHs, especially in the rhizosphere (Shahsavari et al., 2013). This increase in the degradation of PAHs has been related to increasing density of microbes in a plant root zone (Olson et al., 2004). The accelerated biodegradation of PAHs in the root zone can also be attributed to the release of oxygen into the rhizosphere by plant roots (Macek et al., 2000). However, other studies have shown that root exudates have an effect on the microbial metabolism of PAHs. Wang et al. (2014) reported that low-molecular weight organic acids present in the root exudates of various plants enhanced PAH removal percentages. Joner et al. (2002) reported an increase in the degradation of three or five ring PAHs using artificial root exudates. Yoshitomi and Shann (2001) demonstrated that root exudates were responsible for enhancing the microbial mineralization of pyrene. However, Rentz et al. (2004) reported repression of phenanthrene degradation by Pseudomonas putida with root exudates of different plants.

Exudates have a complex composition and each component may have a different effect on the metabolism of rhizospheric microorganisms (Long et al., 2010; Martin et al., 2014; Wang et al., 2014). Although there are many studies on the effect of root exudates on the biodegradation of PAHs, little is known about the effect on microbial activity of each of the mixture components, such as carbohydrates, carboxylates, amino acids, or plant secondary metabolites (PSMs). PSMs are especially interesting in relation to plant rhizospheric microorganisms, as it has been reported that some can act as a growth substrate or as inducers of metabolic pathways for the degradation of aromatic compounds, including PAHs.

Although in recent years the amount of research related to rhizoremediation has increased, very little is yet known about the effect of PSM on the degradation of PAHs by soil microorganisms. The composition of plant exudates varies, so not all plants can be useful in this process. Therefore, it is necessary to identify the PSM present in plant root exudates and understand how they influence PAH degradation by rhizospheric microorganisms. The objective of this study was to identify secondary metabolites from root exudates of Brassica nigra (mustard) and evaluate their effect on anthracene and phenanthrene biodegradation by a microorganism isolated from the rhizosphere.

Materials and Methods

Soil

The soil used in this study was taken from the B horizon of garden soil (sandy clay loam). The nitrogen and phosphorus concentrations were 1,713 and 124 mg/Kg, respectively, and the pH was 7.23. The soil was air-dried and passed through a 2-mm sieve before being used for experiments. It was then artificially contaminated with a mix of anthracene and phenanthrene (50 mg/Kg) dissolved in acetone (Anthracene purity >96%; Alfa Aesar, MA and phenanthrene purity >98%; Sigma-Aldrich, MO). The soil was thoroughly mixed to ensure homogeneous distribution of the compounds, and the solvent was allowed to evaporate for 12 h. After the addition of the PAHs, the soil was kept for 6 months in a closed vessel protected from light and maintained at room temperature. Subsequently, the concentration of the PAHs was tested, and variations were not detected (US-EPA, 2007). B. nigra seeds were certified and purchased from a local market. The treated soil was distributed in 5 plastic pots (500 g of soil per pot), and 20 seeds of B. nigra were sown. Control pots without plants were used to determine PAH loss by abiotic processes. The plants were grown for 6 months at a temperature between 22°C and 26°C and relative humidity of 60%, with a 16-h photoperiod (100 μmol m² s⁻¹).

Experimental designs

The experiment was performed in two phases; isolation of rhizosphere microorganisms able to degrade anthracene and phenanthrene, followed by identification of PSM in root exudates of B. nigra and evaluation of their effect on microbial degradation of PAHs.

Bacterial isolation from rhizospheric soils

Soil samples were collected from the rhizosphere of plants grown in anthracene and phenanthrene contaminated soil. The collected rhizospheric soils were mixed together to form a homogenized composite sample. Bacterial quantification was carried out by serial dilution method using agar Luria-Bertani (LB). Ten gram of the sample was diluted with 90 mL of buffer phosphate and shaken for 3 h at 30°C and 120 rpm. Then, 100 μL of diluted extracts was plated on a petri dish containing LB agar and incubated at 30°C for 48 h.

Isolation of bacteria degrading PAHs was performed by the selective enrichment technique (Hilyard et al., 2008) to obtain the most efficient microorganisms. Ten gram of composite sample was added to 100 mL of minimal salt medium (MSM) (Zeinali et al., 2007), which contained 1 mL of anthracene and phenanthrene mixture (100 mg/L) as the sole carbon and energy source for microorganisms. At weekly intervals sample from the primary enrichment was transferred to 100 mL of fresh MSM containing the same carbon source at double concentration. PAH utilization in cultures was evidenced through observation of a decrease in anthracene and phenanthrene crystals, medium color changes, and an increase in bacterial biomass. After five transfers, 100 μL of sample was removed and spread onto MSM agar plates prepared with Noble agar (Difco, NJ). Anthracene and phenanthrene were provided as the sole carbon source by placing crystals in the Petri dish lid. The inoculated plates were incubated at 30°C for 72 h. After incubation, the plates were observed for bacterial growth, and strains were purified by streak cultures several times. To select the strains with the greatest degradation capacity, a bacterial suspension was prepared in MSM and added in portions of 5 mL to tubes containing 125, 250, 500, 1,000, and 2,000 mg/L of anthracene or phenanthrene and incubated at 30°C and 120 rpm for 72 h. Turbidity was then measured in each of the tubes, and the microorganisms that grew at the highest concentration of PAHs were selected for subsequent assays. The isolates were also evaluated for plant growth promoting properties like phosphate solubilization (Gupta et al., 2012), indole acetic acid (IAA) production (Sachdev et al., 2009), and nitrogen fixation.

Identification of isolates

Isolated microorganisms were initially identified based on morphological and biochemical characteristics, including Gram and spore staining, oxidase test, mannitol, TSI, Simmons citrate, urease, indole, and motility. Molecular identification was done by 16S rRNA gene sequencing, and polymerase chain reaction (PCR) was carried out using F27 and R1492 as primers (Yao et al., 2013). The final volume of reaction was 50 μL, which contained 1 × MgCl₂ 2.5 mM, dNTPs 0.2 mM, primers 0.25 μM, Taq polymerase (Bioline, MA) 0.1 U, and 10 ng/μL of ADN. PCR product was analyzed in 1% agarose gel and then purified using Invisorb Fragment CleanUp Kit (Invitek GmbH, Berlin, Germany) and sequenced by Macrogen. The sequences obtained were edited using Genius^® software (Randall et al., 2009), and the presence of sequence chimeras was assessed by CHIMERA CHECK software. Edited sequences were compared with the 16S rRNA gene through the BLAST database at GenBank.

Obtaining root exudates

Root exudates were collected using the solution culture method. The seeds were first soaked by shaking in 70% ethanol for 10 min and rinsed with deionized water. They were then sterilized by soaking in 2% hypochloric solution for 10 min and rinsing with deionized sterile water for 5 min. Groups of thirty seeds were transferred aseptically to glass flasks containing 100 mL of one-tenth strength Hoagland's solution. Flasks were covered at the bottom and sides to the level of the growth medium. The plants were grown for 8 weeks in a climate-controlled chamber under a fluorescent light with a 16-h light/8-h dark cycle at 26°C and relative humidity of 60%. The Hoagland's solution was replaced whenever necessary to complete the initial volume. Root exudates were collected under sterile conditions, filtered through 0.45 μm Whatman^® (Sigma-Aldrich), and stored at 4°C.

Identification of PSM from root exudates

The analysis was performed by UHPLC-MS/MS chromatography, using an UltiMate 3000 RS UHPLC system (Thermo Scientific, MA). Separation of the compounds was performed using a Hypersil GOLD aQ C18 column (100 × 2.1 mm, 1.9 micron particle size) from the same manufacturer. Detection of the compounds was performed using a high resolution mass spectrometer (Orbitrap Thermo Scientific Q Exactive quadrupole). An electrospray ionization source was used in positive mode. For the acquisition and processing of data, Xcalibur 2.2 software with Qual Browser (Thermo Scientific) was used.

PAH degradation

Before calculating anthracene and phenanthrene biodegradation, it was assessed whether the M2.7 strain was able to use root exudates and identified secondary metabolites as a carbon source. While root exudates were capable of supporting bacterial growth, secondary metabolites did not support the growth of microorganisms. For this reason, sodium acetate and glucose were used as a carbon source during evaluation of the effect of PSMs on the degradation of PAHs.

For microbial degradation studies, 50 mL sterile amber glass flasks were used containing 10 mL of MSM. The flasks were supplemented with 50 mg/L of anthracene or phenanthrene and inoculated with the selected strain at OD_600nm = 1. To evaluate the effect of the extra carbon source on PAH degradation, 30 mM of glucose or sodium acetate was added to MSM when necessary. Similar experiments with uninoculated flasks served as control. The cultures were incubated at 30°C and 120 rpm for 48 h. All assays were made in triplicate. After the incubation time, PAHs were extracted and quantified by HPLC.

Degradation of PAHs in root exudates

For these assays root exudates were used as the culture medium and extra carbon source. Overnight culture of the selected strain was centrifuged at 5,000 rpm for 10 min and washed twice in MSM. Bacterial suspension at OD₆₀₀ nm of 1 was added to amber sterile glass tubes containing 10 mL of filter-sterilized root exudates amended with anthracene or phenanthrene at 50 mg/L. The cultures were incubated for 48 h with shaking at 30°C. All assays were made in triplicate. After the incubation time, PAHs were extracted and quantified by HPLC.

Effect of PSM on degradation of PAHs

Selected strain was grown overnight at 30°C in LB medium. The bacterial cultures were centrifuged at 5,000 rpm for 10 min and washed twice in MSM. The suspensions were adjusted to OD₆₀₀ nm of 1 with MSM amended with the secondary metabolites identified in root exudates (Table 1) at a concentration of 30 mM. Bacterial suspension measuring 10 mL was added to amber sterile glass tubes containing 30 mM of glucose or sodium acetate and anthracene or phenanthrene at 50 mg/L dissolved in acetone, and the solvent was allowed to evaporate. The tubes were incubated for 48 h at 30°C and 120 rpm. Similar experiments were carried out without microorganisms as controls. After incubation time the PAHs were extracted and quantified by HPLC.

Table 1.

Secondary Metabolites Identified in Root Exudates of Brassica nigra

Metabolite	Chemical structure
Flavanone
Flavone
Iso-flavanone
7-hydroxyflavanone
7-hydroxyflavone
6-hydroxyflavone

Extraction, analysis, and quantification of PAHs

Before the extraction of the PAHs, the cultures were centrifuged at 7,000 rpm for 10 min to remove bacterial cells. For extraction, 10 mL of dichloromethane was added to the supernatant, stirred at 180 rpm for 30 min, and then centrifuged at 5,000 rpm for 10 min. The extract was reduced to a volume of 500 μL and then reconstituted with acetonitrile. The recovery percentage with this method was 98%. The residual anthracene and phenanthrene were analyzed by reverse phase HPLC (Agilent 1100 HPLC) with a diode array detector (DAD Agilent G1315B DAD) with a Phenomenex^® Luna C-18 column (125 × 4.5 mm × 5 μm 100 Å). The mobile phase consisted of acetonitrile water (75:25, v/v), with an isocratic flow of 0.76 mL/min and an injection volume of 2 μL. The column temperature was 18°C. The DAD was set at a wavelength of 230 nm. The concentration of each PAH was calculated according to a calibration curve constructed from a standard composed of a mix of the two PAHs, with which calibration standards of 50, 100, 150, 200, and 250 μg/L were prepared in acetonitrile as solvent. The identification of each PAH was based on peak and retention time. The solvent curves showed a correlation coefficient R² ≥ 0.997 and it was therefore established that the obtained areas were directly proportional to the concentration of the compound.

Statistical analysis

Data were analyzed using the software R, under a fixed-effects design and comparison of means, taking into account four variables.

Results and Discussion

Isolation and selection of bacteria

In total, 13 bacteria able to use anthracene and phenanthrene as sole carbon source were isolated from mustard rhizosphere soil. The isolates were sorted into three groups as follows: M2.1–M2.7, M3.1–M3.4, and M4.1–M4.2. Strain M2.7 was selected for further degradation studies because it exhibited the highest anthracene and phenanthrene degrading abilities of the six isolates. The success of rhizoremediation is based on plant–microbe interactions, which makes it especially useful for the identification of the different mechanisms of interactions between these organisms that can improve the removal of contaminants (Afzal et al., 2011). This is because microorganisms with properties promoting plant growth can contribute to the plant more easily tolerating stress conditions to which it is subjected in PAH contaminated soil, and compounds released into the rhizosphere by plant roots can stimulate microbial metabolism for PAH degradation. The data available in the literature does not make it possible to establish a relationship between the degradation capacity of PAHs by soil bacteria and plant growth promoting properties. However, Golubev et al. (2011) reported significant correlations between root-zone IAA content and rhizosphere effects for phenanthrene degraders. Given that rhizoremediation is based on the interaction between plants and microorganisms for the removal of soil pollutants (Kuiper et al., 2004), certain plant growth promoting properties were evaluated in all the isolates. The M2.7 strain was the only isolate that possessed some of these properties such as phosphorus solubilization, IAA production, and nitrogen fixation. For this reason, it was also selected for the subsequent tests.

Bacteria identification

The M2.7 strain was classified as Gram negative rods, oxidase and catalase positive negative. The analysis further showed that the 16S rRNA sequence of M2.7 had 99% identity to the 16S rRNA sequence of Erwinia sp. (JF494827.1) and Pantoea sp. (FJ593733.1). These genera are closely related phylogenetically, and some authors have reported that a single gene is not enough to obtain a reliable species identification (Zhang and Qiu, 2015), so identification at species level is difficult. These bacteria are commonly isolated from plants and natural environments (Brady et al., 2008). Some Erwinia species have been reported as plant pathogens, but strains of Pantoea with some plant growth promoting properties have been reported, and their effect on increased degradation of PAHs has been proven using plants cultured in soil inoculated with Pantoea sp. (Afzal et al., 2011).

Identification of secondary metabolites in exudates

The compounds exuded by plant roots are diverse and depend on environmental factors and the growth stage of the plant (Martin et al., 2014). Exudates may be composed of organic acids, polysaccharides, amino acids, fatty acids, sterols, vitamins, and enzymes (Walker et al., 2003). This study focused on identifying secondary metabolites because their effect on microbial community structure and rhizosphere metabolic activity has been widely reported (Singer et al., 2003; Luo et al., 2007; Jha et al., 2015). After analysis of the root exudates by UHPLC-MS/MS, six PSMs were identified as shown in Table 1. The structure of the compounds identified corresponds to phenolic compounds, which represent a very diverse group of PSM and seem crucial for the accelerated degradation of aromatic compounds by stimulating the bacteria PAHs catabolic pathway in the rhizosphere (Musilova et al., 2016). They have been reported as one of the most common components in root exudates produced by different plants used in phytoremediation processes. B. nigra has a great ability to release phenolic compound-rich root exudates and has been reported in phytoremediation research. For this reason it was selected for this study.

PAH degradation

Numerous bacteria from soil that degrade and utilize PAHs as their sole carbon source have been found (Hilyard et al., 2008; Regonne et al., 2013). However, there is not much knowledge about the effect of plants, especially root exudates, on the ability of rhizospheric bacteria to degrade PAHs. To evaluate this effect on the selected strain, its ability to both degrade PAHs and use them as carbon source was initially assessed. The effect of additional carbon sources on PAH degradation was also evaluated using compounds of sodium acetate and glucose, which are easily used by microorganisms. The degradation percentage was calculated through the difference in the concentration of PAHs between treatments with M2.7 strain and controls. The results are shown in Table 2. In the initial degradation assays without additional carbon sources, degradation percentages of 35.1% and 17.5% were obtained for anthracene and phenanthrene, respectively. Although both PAHs contain three fused aromatic rings, differences between anthracene and phenanthrene degradation by a microorganism have been reported (Seo et al., 2009). These differences have been linked to the compounds themselves, the metabolic pathway used by the microorganism, and environmental conditions.

Table 2.

Effect of Carbon Source on Degradation of Polycyclic Aromatic Hydrocarbons

	Anthracene	Phenanthrene
Control	35.1 ± 0.3	17.5 ± 5.1
Sodium acetate	35.3 ± 5.4	37.1 ± 4.0
Glucose	24.8 ± 2.4	40.6 ± 3.6

Glucose and sodium acetate were used to investigate the effect of an additional carbon source on PAH degradation. This was because some authors have reported repression of PAH degradation by some components of plant root extracts and exudates, including those that can be used as carbon sources (Rentz et al., 2005; Phillips et al., 2008). The degradation percentage for phenanthrene increased to 40.6% with the addition of glucose and to 37.1% with sodium acetate. For anthracene degradation the percentage with glucose was 24.8% and with sodium acetate 35.3%. These results show that the presence of an external carbon source affects the metabolism of PAHs in the M2.7 strain in different ways. Carbon sources may increase microbial biomass and thereby increase degradation, which would explain the results for phenanthrene using glucose and sodium acetate. However, other authors have reported that carbon sources such as glucose are metabolized more easily by microorganisms, which inhibits the degradation of toxic compounds like PAHs. Although we cannot exclude the possibility that glucose may exert catabolite repression similar to that observed for other compounds and strains, many other factors may have been responsible for the decrease in the degradation percentage of anthracene when glucose was added. The biochemical pathways for the bacterial degradation of PAHs such as anthracene and phenanthrene have been well investigated. Of these, the bacterial naphthalene dioxygenase system is particularly useful for oxidizing bi- and tricyclic PAH substrates such as phenanthrene and anthracene (Peng et al., 2008). Although the metabolic pathway used by the microorganism to degrade PAHs is not evaluated in this study, we cannot exclude the possibility that the M2.7 strain uses a different group of dioxygenase enzymes to degrade anthracene to that used for phenanthrene. This could explain why the metabolism of anthracene is affected by other carbon sources.

Effect of root exudates on degradation of PAHs

The degradation percentages of anthracene and phenanthrene when root exudates were used as culture medium are shown in Fig. 1. The degradation percentages obtained were 18.5% and 26.8% for anthracene and phenanthrene, respectively. Comparing these results with those obtained in the previous assay of degradation, a significant difference (p ≤ 0.05) was observed for both PAHs. In this assay, the degradation percentages were higher for phenanthrene, suggesting that the presence of other compounds, including carbon sources, had different effects on the metabolism of the two PAHs for the M2.7 strain. These results corresponded with the results obtained in the previous trial, where degradation of anthracene decreased in the presence of glucose. Rentz et al. (2004) report that root extracts of different plants repressed the expression of phenanthrene degradation for P. putida. However, in this study an apparent degradation repression was observed for anthracene. This suggests that the effect of root exudates on microbial degradation of PAHs depends on the species of microorganism or the metabolic pathway used for degradation. We cannot exclude the possibility that this effect is related to the composition of root exudates. This is because Rentz et al. (2004) evaluated the effect of root extracts and exudates of other plants aside from B. nigra and it is well known that the composition of exudates and extracts depends on the type of plant. In the analysis of mustard root exudates, it was demonstrated that these contain various phenolic compounds, including flavonoids, which have been related to increased microbial metabolism of aromatic compounds, including PAHs and polychlorinated biphenyls.

FIG. 1.

Effect of root exudates on degradation of PAHs. PAHs, polycyclic aromatic hydrocarbons.

Effect of PSM on PAH degradation

Secondary metabolites present in root exudates of plants are a very large group of compounds, covering different structures (Singer et al., 2003). Phenolic compounds, including flavonoids, are a group that has drawn attention in recent research on rhizoremediation processes due to the fact that they can induce catabolic pathways of aromatic compounds in different microorganisms (Pham et al., 2012; Musilova et al., 2016). Six PSMs, classified as flavonoids, were identified in this study, and their effect on anthracene and phenanthrene degradation by the M2.7 strain was evaluated. The results of these tests are shown in Table 3. When sodium acetate and PSM were used, significant differences (p ≤ 0.05) were observed in the degradation percentage of anthracene compared to the rates obtained without PSM. In the tests with sodium acetate plus PSM, degradation rates decreased in most trials, especially with flavanone, with which the rate decreased to 14.1%. The only PSM that had no effect on the degradation of anthracene was 7-hydroxyflavanone, with which the degradation percentage was 39.5%, similar to that obtained without this PSM (35.1%). No statistically significant differences were observed between degradation rates obtained in tests using glucose plus PSM and previous tests without PSM, except for the rate obtained with 6-hydroxyflavone which was 41.8%.

Table 3.

Effect of Plant Secondary Metabolites on Polycyclic Aromatic Hydrocarbon Degradation

	Anthracene		Phenanthrene
	Sodium acetate	Glucose	Sodium acetate	Glucose
Flavanone	14.1 ± 3.2	25.0 ± 4.8	36.5 ± 2.2	45.1 ± 3.2
Flavone	25.3 ± 0.8	31.8 ± 0.3	37.3 ± 3.4	34.4 ± 3.3
Iso-flavanone	19.9 ± 1.4	26.7 ± 1.6	30.7 ± 2.9	45.9 ± 1.9
7-hydroxyflavanone	39.5 ± 2.3	30.8 ± 2.1	23.3 ± 3.3	48.7 ± 2.1
6-hydroxyflavone	19.0 ± 4.4	41.8 ± 2.3	28.3 ± 3.8	51.8 ± 4.0
7-hydroxyflavone	24.6 ± 3.2	25.7 ± 0.9	33.1 ± 2.3	50.3 ± 1.8

Degradation rates obtained for phenanthrene with PSM and sodium acetate showed no significant difference to those obtained without PSM except in the case of 7-hydroxyflavanone, for which the degradation percentage decreased to 23.3%. In trials with glucose and PSM, the highest degradation percentages were obtained with 6- and 7-hydroxyflavone with 51.8% and 50.3%, respectively. For both anthracene and phenanthrene, a positive effect on degradation using glucose plus 6-hydroxyflavone was observed, with degradation percentages of 41.8% and 51.8% being obtained for anthracene and phenanthrene, respectively. These were higher than those obtained without the addition of this PSM.

These results suggest that the composition of the root exudates can influence the microbial metabolism associated with PAH degradation and this may decrease depending on the carbon sources available and the secondary metabolites in root exudates (Musilova et al., 2016). This composition is determined by environmental factors and the physiological state of the plant (Jones et al., 2009), so the influence will not be similar in different soil types and with different plants. The effect of root exudates will also depend on the rhizosphere microbial community and its metabolic capabilities. As suggested by the results obtained in this study, catabolic pathways respond differently to the presence of secondary metabolites. Some authors have reported that PSM can be metabolized by bacteria (Donnelly et al., 1994), increasing the number of microorganisms, which would increase degradation of pollutant compounds, including PAHs. However, the M2.7 strain did not have the ability to grow using any of the metabolites as a carbon source, suggesting that the effect on degradation is due to other interactions in the metabolic pathway. Understanding these interactions between plant and soil microorganisms is essential to avoid the catabolite repression of microorganisms and to enhance PAH degradation, to optimize phytoremediation processes.

Conclusions

In this study we have examined the effect of PSM on the PAH degradation for rhizobacteria. Our results indicate that some PSMs produced by B. nigra can influence anthracene and phenanthrene degradation by isolated bacteria. The results showed that the M2.7 strain had the ability to grow and degrade anthracene and phenanthrene when root exudates of B. nigra were used as culture medium. However, the degradation rates were different from those obtained with minimal medium, indicating that other components within the exudates were involved in the degradation of PAHs. The use of certain PSMs such as 6-hydroxyflavone with glucose increased degradation of both PAHs, but most others had a negative effect, reducing the rate of degradation. Interactions between different carbon sources and PSMs show that components of exudates can positively or negatively influence the microbial degradation of PAHs. This work highlights the need for obtaining better insights about the PSMs released by plants and about how soil microorganisms interact with PSMs. Different genera of bacteria have been related with PAHs rhizoremediation processes; therefore further insight about how these bacteria interact with PSMs is required to improve the rhizoremediation process.

Footnotes

Acknowledgment

The authors thank Colciencias (Grant No. 111557635893) for funding the project.

Author Disclosure Statement

No competing financial interests exist.

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