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Anne Lanham, Lillian Mwanri

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The Effect of Flavonoids on the Microbial Mineralisation of Polycyclic Aromatic Hydrocarbons in Soil

Aniefiok E. Ite*, Kirk T. Semple

Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, United Kingdom

Abstract: The effect of flavonoids (flavone, morin hydrate and 3-hydroxyflavone)on the microbial mineralisation of polycyclic aromatic hydrocarbons (PAHs) in soil slurry by the indigenous microbial communities has been investigated. The rates and extents of ¹⁴C–PAHs (¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene)mineralisation in artificially spiked soils were monitored in the absence and presence of flavonoids applied at three different concentrations (50, 100 and 200 µg kg⁻¹) either as single compounds or as a mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio). Respirometric and microbial assays were monitored in fresh (0 d) and pre–incubated (28 d) artificially spiked soils following amendment with flavonoids. The highest extents of¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrenemineralisation (P< 0.001) were obtained in fresh artificially spiked soils amended with 100 µg kg⁻¹3-hydroxyflavonecompared to those obtained in unamended soils. However, amendment of fresh artificially spiked soils with higher concentrations of morin hydrate (≥ 100 µg kg⁻¹) inhibited mineralisation of¹⁴C–pyrene by indigenous microbial communities.Apart from naphthalene and pyrene spiked soils amended with morin hydrate, pre–incubated artificially spiked soils amended with 100 µg kg⁻¹ flavonoids had the highest extents of microbial mineralisation of PAHs compared to those obtained in unamended soils. The results obtained showed enhanced microbial mineralisation of ¹⁴C–PAHs in fresh artificially spiked soils amended with 50 – 100 µg kg-1 flavonoidsand in addition, low molecular weight PAHs (containing < 4 aromatic rings) exhibited higher extents of microbial mineralisation compared to high molecular weight PAH.Depending on its (bio) available concentration and solubility in soil, flavonoids can either stimulate or inhibit mineralisation of PAHs by indigenous soil microbial communities. This study suggests that phytochemical–microbe interactions are essential drivers of ecosystem functions relevant for the biodegradation of petroleum–derived or organic contaminants in soils.

Keywords: microbial mineralisation, PAHs, flavone, morin hydrate, 3-hydroxyflavone, soil

Citation: Aniefiok E. Ite, Kirk T. Semple, The Effect of Flavonoids on the Microbial Mineralisation of Polycyclic Aromatic Hydrocarbons in Soil. International Journal of Environmental Bioremediation & Biodegradation, 3 (3). 66-78. January 2015. doi:10.12691/ijebb-3-3-1

1. Introduction

Petroleum hydrocarbon contamination is a widespread and well recognised environmental problem in most oil–producing countries, causing acute and diffuse contamination of soil and sediments, surface and groundwater, marine environment and terrestrial ecosystems [1,2]. Microbial transformation plays a major role in degradation of most petroleum–derived contaminants, such as benzene, toluene, ethylbenzene and xylenes (BTEX), aliphatic and polycyclic aromatic hydrocarbons [2]. The main pathway for polycyclic aromatic hydrocarbons (PAHs) removal from environmental compartments is considered to be microbial transformation and growth–linked mineralisation by indigenous microbial communities [2,3,4]. Over the last few decades, biological and/or microbial degradation of PAHs in soils and sediments has been studied extensively [5-13]. It is widely known that PAHs can be resistant to biodegradation and may persist in the natural environment because of their hydrophobic nature, resulting in low water solubility and a tendency to be adsorbed to the soil and sediment matrices [14,15,16]. Biodegradation of organic contaminants in soil can be limited by factors such as adsorption, mass transfer and bioavailability, while combined plant–microbial systems can lead to more efficient growth–linked mineralisation in the root zone or rhizosphere [17]. Several researcheshave demonstrated that interactionsbetween plants and microorganisms are essential for thebiologicaland/or microbial transformation of organic contaminants in soil [17-22].

Plants employ several mechanisms to remediate soils and/or ecosystem contaminated with petroleum–derived or organic contaminants. Phytoremediation studies have demonstrated enhanced biodegradation of organic contaminants in the rhizosphere of certain plants [23,24,25]. For example, Yoshitomi and Shann [24] attributed enhanced polycyclic aromatic hydrocarbons (PAHs) degradation in the rhizosphere to the stimulation effect of plant root exudates. Some studies have shown the positive effect of root exudates, including phenolic compounds, terpenes and flavonoids, on microbial activity in soil and on biodegradation of polychlorinated biphenyls(PCBs) [26,27,28]. It is widely known that roots secrete an enormous number of chemical compounds (phytochemicals) into the root zone orrhizosphere where the secreted chemicals mediate multi-partite interactions and alter their immediate environments around plant roots [29,30]. Plant secreted chemical compounds, including salicylic acid, monoterpenes and flavonoids, may facilitate transformation of organic contaminants with similar structures [18]; induce genes encoding enzymes involved in the degradation process, increase contaminant bioavailability, and/or increase in microbial activity and selective enrichment in the numbers of microbial communities that are capable of degrading specific contaminants[17,19,20]. In a study using a 'rhizosphere metabolomic' approach, Narasimhan et al. [31] identified a large majority of aromatic acids or phenylpropanoids, including flavonoids, in plant exudates. Flavonoids, which are common to vascular plants other than legumes [32], are known components of plant root exudates [33], plant tissue and plant based products.

Flavonoids, being O-containing heterocyclic compounds, belong toa diverse group of polyphenolic secondary metabolic compounds which has a general C₆–C₃–C₆ carbon framework (Figure 1). Flavonoids are produced by the phenylpropanoid biosynthetic pathway in plants and accumulate inthe vacuoles for release in exudates [32]. Based on the biosynthetic pathways, flavonoidscan be divided into about30 classes depending on the structure of the propane fragment (–C₃–) [34]. From further elucidation of the degree of oxidation of the C₃ unit, flavonoids could also be classified into six major subclasses: chalcones, flavones, flavonols, flavandiols, anthocyanins and proanthocyanidins, and a seventh subclass called aurones. In few studies [35,36], it has been demonstrated that some flavonoids can promote movement of microbial communities towards plant and stimulate microbial activities in rhizosphere. In a review, Cescoet al. [33] summarized the types and concentrations of flavonoids in root exudates and most of these were from plants grown in solution. Flavonoid concentrations vary widely and are dependent on plant growth conditions, sampling techniques, nutrient supply and plant species [33]. However, the fate of flavonoids in the rhizosphere depends on the physical–chemical properties of the soil and the presence of active microbial communities. Depending on their modifications, flavonoids persistence in the rhizosphere soils varies and can be < 72 h, depending on the chemical structure of the flavonoid [37]. Indigenous soil microbial communities undoubtedly influence the quality and quantity of flavonoids, both through modification of root exudation patterns and microbial catabolism of exudates [38,39]. Therefore, plant–secreted flavonoids may influence the population structure of rhizosphere associated microbial communities, stimulate the overall population size and activity of indigenous soil microbial communities that have the capability to degrade specific organic contaminants.

Figure 1. Chemical structures of some of the major groups of flavonoids

The hypotheses for this study were: (i) the presence of flavonoids above a threshold concentration in contaminated soil may stimulate microbial activity and subsequent prolific microbial population growth, and (ii) the addition of appropriate concentration of flavonoids or a mixture of flavonoidsmay increase bioavailability of organic contaminant, intrinsic microbial catabolic activity and enhance microbial mineralisationof PAHs in soil by active indigenous microbial communities. To address these hypotheses, the following aims were considered: (i) assessment of the microbial mineralisation of ¹⁴C–PAHs(¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene) in soils, (ii) monitoring of microbial mineralisation of PAHs in artificially spiked soils in the absence and presence of flavonoids at three different concentrations (50, 100 and 200 µg kg⁻¹) either as single compounds or as a mixture of flavonoids(flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio) and (iii) monitoring of PAHs (naphthalene, phenanthrene and pyrene) mineralisation by indigenous microbial communities and enumeration of microbial cell numbersin fresh (0 d) and pre–incubated (28 d) artificially spiked soils following flavonoids amendment.

2. Materials and Methods

2.1. Materials

Naphthalene (>96%); [UL–¹⁴C] naphthalene (specific activity = 2–10 mCi mmol⁻¹, radiochemical purity > 95%), phenanthrene (>96%); [9–¹⁴C] phenanthrene (specific activity = 50 mCi mmol⁻¹, radiochemical purity 99.6%), and pyrene (>98%); [4,5,9,10-¹⁴C]pyrene (specific activity = 7.5 mCi mmol⁻¹, radiochemical purity 98.6%)were obtained from American Radiolabeled Chemicals (ARC) UK. Sigma–Aldrich UK suppliedflavone (≥98%), morin hydrate (>95%) and 3-hydroxyflavone (≥98%).Fisher Scientific UK supplied the nutrient agar, sodium hydroxide (NaOH) used for the CO₂ traps and the mineral basal salts (MBS) solution reagents. Ringer’s solution pellets and plate count agar (PCA) powder were obtained from Oxoid Ltd, UK. Fisher Scientific UK supplied toluene and acetone used forvarious experimental procedures. Schott Duran® bottles (250 ml) with TeflonTM lined screw caps were supplied by Schott UK and the metal fittings used to make the respirometers were supplied by RS Components Ltd, UK. The 7 ml glass scintillation vials and Goldstar liquid scintillation cocktail were supplied by Meridian UK.

2.2. Soil Sampling and Analysis

A pristine agricultural soil (DystricCambisol) was collected from the A horizon (5 – 20 cm depth) from Myerscough Agricultural College, Lancashire, UK. Prior to spiking, the soil was air–dried for 24 h and subsequently homogenised by passing through a 2 mm mesh sieveto remove stones and residual plant materials [21]. The field moisture content was determined in triplicate by oven drying at 105 °C for 24 h [40]. Soil texture was determined using sedimentation, 40 g air dried soil and thesoil pH was determined using a calibrated pH meter (Model 657R-00), ratio 10 g soil:25 ml dH₂O. The total extractable organic carbon content was determined using loss on ignition method (450°C for 24 h) and a Carlo Erba CHNS–OEA 1108 CN–Elemental analyzer was used to determine the total carbon and nitrogen contents. In addition, the phosphate content was determined by acid digestion with HNO₃ and a phosphate reducing agent (neutralized with NaOH) was used to develop the characteristic blue colour for spectrometric determination at 882 nm (Cecil CE 1011 UV Spectrometer). All measurements were triplicate determinations.

2.3. Soil Spiking with Target Polycyclic Aromatic Hydrocarbons (PAHs)

Prior to spiking, soilsamples were rehydrated with deionised water to 70% soil water–holding capacity. Samples of the soil (240 g wet weight) were then spiked with ¹²C–labelled naphthalene, phenanthrene and pyrene using acetone as the carrier solvent to give a final ¹²C–hydrocarbon concentration of 20 mg kg⁻¹ (dry weight). Each soils piked mixture was then blended following the method developed by Doick et al. [41]. In addition, controlsconsisting of rehydrated soil (120 g wet weight) only were produced as analytical blanks. The artificially spiked soils and controls were stored in amber glass jars (in triplicates) with loosely fitted Teflon–linedTM screw caps to allow ambient oxygen exchange. The samples of artificially spiked soil and controls were incubated in darkness at 21.5 ± 0.5°C and 45% relative humidity for 0 to 28 d. The pre–incubated artificially spiked soils were sampled at two time intervals (0and 28 d) for both respirometric assays and microbial analysis.

2.4. Mineralisation of ¹⁴C–PAHsin Soil

The extents of mineralisation of 14C–PAHs (naphthalene, phenanthrene and pyrene) in the artificially spiked soils were measured (in triplicate) through the evolution of ¹⁴CO₂ producedusing respirometric assays [42]. The mineralisation assay was performed in respirometers which consist of modified 250 ml Schott Duran® bottles containing 10 g ± 0.1 g soil (wet weight) and 30 ml autoclaved minimal basal salt solution [41]. Each of the respirometers was spiked with naphthalene, phenanthrene and pyrene standards prepared in toluene to deliver ¹²C–PAHs (naphthalene, phenanthrene and pyrene) concentration of 10 mg kg⁻¹ soil dry weight with an associated ¹⁴C–activity of 83 Bq g⁻¹ soil dry weight. A 7 ml scintillation vial containing 1 ml NaOH (1 M) solution was suspended from the lid of each respirometer to trap ¹⁴CO₂ that evolved as a result of microbial mineralisation of¹⁴C–PAHs. In addition, unamended respirometers were prepared as outlined above, with 10± 0.1 g naphthalene, phenanthrene and pyrene spiked soils (wet weight) and 30 ml of autoclaved MBS solution.

To investigate techniques to optimise the microbial mineralisation of target organic contaminants, respirometers were also prepared as above with the following treatments: (i) fresh artificially spiked soils (0 d) amended with 50, 100 or 200 µg kg⁻¹flavonoids (flavone, morin hydrate and 3-hydroxyflavone) and a mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio) using toluene as the carrier solvent, (ii) pre-incubated artificially spiked soils(28 d) amended with 50, 100 or 200 µg kg⁻¹flavonoids (flavone, morin hydrate and 3-hydroxyflavone) and a mixture of flavonoids prior to mineralisation assays, and (iii) artificially spiked soils with no flavonoids amendment were used as controls to assess any increase in rates and extents of microbial mineralisation of the target organic contaminants.

Respirometers containing only 10 ± 0.1 g rehydrated soil (wet weight) and 30 ml of autoclaved MBS solution were also prepared as analytical blanks. During the 14 d mineralisation assay sampling period, respirometers were placed securely on an orbital shaker (SANYO Gallenkamp) at 21C and shaken at 100 rpm to agitate and ensure adequate mixing of the soil slurry. The ¹⁴CO₂ traps were replaced every 24 h. Scintillation fluid (5 ml) was added to each spent ¹⁴CO₂ trap and stored in darkness overnight to reduce the effects of chemilumiescence [21]. The trapped ¹⁴C-activity was counted using a Packard Canberra Tri-Carb 2300TR liquid scintillation counter and quantified using standard counting protocols with an automatic quench correction [43].The lag phases, rates and extents of ¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene mineralisation in the soil slurries were calculated based on the percentage of trapped ¹⁴CO2 over the total pool of ¹⁴C–labelled carbon.

2.5. Enumeration of Total Heterotrophic and PAH–degrading Bacteria

The number of total heterotrophic bacteria (THB) and indigenous naphthalene, phenanthrene and pyrene–degrading bacteria were evaluated following standard aseptic plate count techniques [44]. In brief, 1 ± 0.1 g soil was extracted with 10 ml quarter–strength sterile Ringer’s solution following proper mixing and 0.1 ml extracts were then serially diluted with Ringer’s solution. Serial dilutions of bacterial suspension (0.01 ml) were inoculated onto plate count agar for THB and agar–agar plates amended with 0.1% naphthalene, phenanthrene and pyrene as the sole carbon source for naphthalene, phenanthrene and pyrene–degrading bacteria. The inoculated plates were incubated at 25 ± 0.5 °C and the cell number of THB was counted after 48 h and > 7 d for naphthalene, phenanthrene and pyrene–degrading bacteria. The microbial cell number was expressed as colony–forming units per gram soil dry weight (CFU g–1).

2.6. Statistical Analysis of Data

Statistical analysis of the results after blank correctionwas performed at various time intervals and statistically verified using t–tests after normality and equal variance tests (Tukey test, P ≤ 0.05) using statistical software –SigmaStat®, Version 3.5 (Systat Software Inc., USA). PAHsmicrobial mineralisation profiles in artificially spiked soils in the absence and presence of flavonoidsare presented using graphing software package – SigmaPlot®, Version 12.5 (Systat Software Inc., USA).

Table 1. Physicochemical and microbial characteristics of Myerscough soil. Values are the mean (n = 3) ± standard errors of the mean (SEM)

3. Results

3.1. Mineralisation of ¹⁴C–PAHs in Soil

The physicochemical and microbiological properties of the soil are presented in Table 1. The ability of the indigenous soil microbial communities to mineralise ¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene were measured in fresh artificially spiked soils (Figure 2 – Figure 4 [A, B & C] and Table 2) and pre–incubated artificially spiked soils (Figure 2 – Figure 4 [D, E & F] and Table 3) in the absence and presence of flavonoids.The lag phases (time taken to achieve 5% mineralisation) in fresh artificially spiked soils significantly (P < 0.001) varied between each of the target organic contaminants (51.93 ± 1.27 to 64.44 ± 1.93 h in systems with naphthalene; 112.17 ± 0.92 to 167.28 ± 0.25 h in systems with phenanthrene and 140.43 ± 3.49 to 201.57 ± 3.16 h in systems with pyrene; Table 2). In systems containing pyrene, amendment with 100 μg kg⁻¹ morin hydrate resulted in statistically (P < 0.05) longer lag phases compared to the unamended systems (control) and fresh artificially spiked soils amended with 50 μg kg⁻¹ morin hydrate. Apart from artificially spiked soils amended with 200 μg kg−1 3–hydroxyflavone, flavonoids amendment did not affect lag phases (P > 0.05) in systems containing naphthalene. However, shorter lag phases were often measured in fresh artificially spiked soilsamended with 3–hydroxyflavone and the mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio). After 28 dpre–incubation, there were significant (P < 0.001) reductions in lag phases in artificially spiked soils containing naphthalene (4.55 ± 0.02 to 7.13 ± 0.01 h) and phenanthrene (4.48 ± 0.02 to 6.55 ± 0.12 h) compared to systems containingpyrene (13.29 ± 0.83 to 28.34 ± 3.16 h)as presented in Table 3. The pre–incubated artificially spiked soils amended with 50, 100 and 200 μg kg⁻¹ flavonoidsexhibited similar (P> 0.05)lag phases in systems containing naphthalene and phenanthrene. Although there were no statistical reductions of lag phases in soils in the absence and presence of flavonoids amendment (P > 0.05), longer lag phases were measured in fresh artificially spiked soils amended with morin hydrate. After 28 d pre–exposure, lag phases were significantly (P < 0.001) shorter in pre–incubated artificially spiked soils (Table 3) compared to those measured in fresh artificially spiked soils (Table 2).

Figure 2. Development of microbial mineralisation of ¹⁴C–naphthalene in fresh artificially spiked soils amended with flavonoids at concentrations of (A) 50 µg kg -1 , (B) 100 µg kg ⁻¹ and (C) 200 µg kg ⁻¹ and pre–incubated artificially spiked soils amended with flavonoids at concentrations of (D) 50 µg kg ⁻¹, (E) 100 µg kg ⁻¹ and (F) 200 µg kg ⁻¹. Error bars are the standard error of mean (SEM) and data are presentated as means (n = 3)

Figure 3. Development of microbial mineralisation of ¹⁴C–phenanthrene in fresh artificially spiked soils amended with flavonoids at concentrations of (A) 50 µg kg -1 , (B) 100 µg kg ⁻¹ and (C) 200 µg kg ⁻¹ and pre–incubated artificially spiked soils amended with flavonoids at concentrations of (D) 50 µg kg ⁻¹, (E) 100 µg kg ⁻¹ and (F) 200 µg kg ⁻¹. Error bars are the standard error of mean (SEM) and data are presentated as means (n = 3)

The rates of ¹⁴C–PAHs mineralisation in fresh artificially spiked soils in the absence and presence of flavonoids amendment varied from 0.44 ¹⁴CO₂–0.97 ¹⁴CO₂% h⁻¹ in systems with naphthalene; 0.24 ¹⁴CO₂– 0.84 ¹⁴CO2 % h⁻¹ in systems with phenanthrene; and 0.19 ¹⁴CO₂– 0.19 ¹⁴CO2 % h⁻¹ in systems with pyrene (Table 2). Apart from¹⁴C–phenanthrene, the rates of ¹⁴C–naphthalene and ¹⁴C–pyrene mineralisation were not significantly (P > 0.05) enhanced in soils amended with morin hydrate compared to controls. The fastest rates of ¹⁴C–PAHs mineralisation were measured in fresh artificially spiked soils amended with 50 – 100 µg kg⁻¹flavone, 3–hydroxyflavone and the mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio) as presented in Table 2. However, fresh artificially spiked soils amended with 50 – 200μg kg⁻¹morin hydrate generally exhibited the lowest rates of ¹⁴C–PAHs mineralisation (P < 0.05) compared to other flavonoids amendment and unamended conditions. After 28 dpre–exposure, therates of mineralisation in pre–incubated artificially spiked soilsin the absence and presence of flavonoids amendment varied from 0.73 ¹⁴CO₂– 1.10 ¹⁴CO2 % h⁻¹ in systems with naphthalene; 0.76 ¹⁴CO₂– 1.14 ¹⁴CO2 % h−1 in systems with phenanthrene; and 0.18 ¹⁴CO₂– 0.38 ¹⁴CO2 % h⁻¹ in systems with pyrene (Table 3). The maximum rates of ¹⁴C–PAHs mineralisation in pre–incubated artificially spiked soils (Table 3) were significantly (P< 0.001) higherthan those measured in fresh artificially spiked soils (Table 2). With exception of morin hydrate, flavonoids amendment revealed subtle differences in the rates of PAHs mineralisation between various treatments (Table 3). The rates of ¹⁴C–naphthalene and ¹⁴C–phenanthrene mineralisation in soils were significantly (P < 0.001) higher compared to pre–incubated artificially spiked soilscontaining ¹⁴C–pyrene.

Figure 4. Development of microbial mineralisation of ¹⁴C– pyrene in fresh artificially spiked soils amended with flavonoids at concentrations of (A) 50 µg kg -1 , (B) 100 µg kg ⁻¹ and (C) 200 µg kg ⁻¹ and pre–incubated artificially spiked soils amended with flavonoids at concentrations of (D) 50 µg kg ⁻¹, (E) 100 µg kg ⁻¹ and (F) 200 µg kg ⁻¹. Error bars are the standard error of mean (SEM) and data are presentated as means (n = 3)

The extents of ¹⁴C–PAHs mineralisation in fresh artificially spiked soilsin the absence and presence of flavonoids amendmentranged from 27.30 ± 1.98 to 46.30 ± 1.27 % in systems with naphthalene; 28.10 ± 0.33 to 48.73 ± 1.35 % in systems with phenanthrene; and 19.01 ± 1.64 to 30.88 ± 1.26 % in systems with pyrene (Figure 2 – Figure 4 [A, B and C] and Table 2). Fresh artificially spiked soils amended with 100 µg kg⁻¹3–hydroxyflavonehad the highest extents of ¹⁴C–naphthalene (46.30 ± 1.27), ¹⁴C–phenanthrene (48.73 ± 1.35) and ¹⁴C–pyrene (30.88 ± 1.26) mineralisation compared to umamended soils (controls).Apart from artificially spiked soilsamended with morin hydrate, there were no significant (P> 0.05) differences in the extents of ¹⁴C–naphthalene and¹⁴C–phenanthrene mineralisation in fresh artificially spiked soils amended with flavonoids (Table 2). However, the extents of¹⁴C–pyrene mineralisationin fresh artificially spiked soilswere significantly (P< 0.05) inhibited following amendment with > 100 µg kg⁻¹morin hydrate. After 28 dpre–exposure, extents of ¹⁴C–PAH smineralisation in pre-incubated artificially spiked soils in the absence and presence of flavonoids ranged from 36.30 ± 1.37 to 50.59 ± 1.07 % in systems with naphthalene; 42.86 ± 0.79 % to 55.48 ± 0.32 in systems with phenanthrene; and 29.96 ± 1.64 to 44.30 ± 0.88 % in systems with pyrene (Figure 2 – Figure 4 [D, E and F]; Table 3). There were significant (P< 0.001) increases in the extents of¹⁴C–naphthalene (4.55 ± 0.02 % to 7.13 ± 0.01 %) and ¹⁴C–phenanthrene (4.48 ± 0.02 % to 6.55 ± 0.12 %) mineralisation compared to extent of ¹⁴C–pyrene (13.29 ± 0.83 % to 28.34 ± 3.16 %) mineralisation in pre-incubated artificially spiked soils in the absence and presence of flavonoids (Table 3). Apart from the pre-incubated artificially spiked soils containing ¹⁴C–phenanthrene amended with 100 µg kg⁻¹ morin hydrate, there was no statistical significant (P > 0.05) enhancement in the extents of ¹⁴C–naphthalene and ¹⁴C–pyrenemineralisation following morin hydrate amendments. Although soil–contaminant pre–exposure significantly (P< 0.05) increased the extents of ¹⁴C–PAH mineralisation in soils, there were no significant (P > 0.05) differences in the extents of ¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene mineralisation in artificially spiked soils amended with flavonoids. The highest extents of ¹⁴C–PAHS mineralisation were measured in pre–incubated artificially spiked soils amended with 50 – 100 µg kg-1 flavone, 3–hydroxyflavone and the mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio). Overall, the extents of mineralisation increased in the order of naphthalene ≥ phenanthrene > pyrene and pre-incubated artificially spiked soils exhibited higher extents of ¹⁴C–PAH mineralisationcompared to freshartificially spiked soils.

Table 2. Microbial Mineralisation of ¹⁴C–PAHs (¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene) in fresh artificially spiked soils(0 d) amended with flavonoids at concentrations of 50 µg kg ⁻¹, 100 µg kg ⁻¹and 200 µg kg ⁻¹. Values are the mean (n=3) ± standard error of the mean (SEM)

Table 3. Microbial Mineralisation of ¹⁴C–PAHs (¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene) in pre–incubated artificially spiked soils (28 d) amended with flavonoids at concentrations of 50 µg kg ⁻¹, 100 µg kg ⁻¹ and 200 µg kg ⁻¹. Values are the mean (n=3) ± standard error of the mean (SEM)

Table 4. Microbial cell numbers of total heterotrophic and PAHs degrading microbial communities after mineralisation assays in fresh (0 d) artificially spiked and pre–incubated (28 d) artificially spiked soils amended with flavonoids at concentrations of 50 µg kg ⁻¹, 100 µg kg ⁻¹ and 200 µg kg ⁻¹. Values are the mean (n=3) ± standard error of the mean (SEM)

3.2. Numbers of Total Heterotrophic and PAH–degrading Bacteria

Amendment of artificially spiked soils with flavonoids greatly stimulated the growth of total heterotrophic bacteria and PAH–degrading microbial communities compared to the unamended soils (controls) as presented in Table 4. The numbers of total indigenous microbial communities in fresh artificially spiked soils amended with flavonoids ranged from 10⁵ – 107 colony forming units per gram soil dry weight (CFU g⁻¹) for heterotrophic bacteria and 10⁶ – 10⁹ CFU g⁻¹ for PAH–degrading microbial communities. The microbial cell number of heterotrophic bacteria and PAH–degrading microbial communitieswere in the range of 106 CFU g⁻¹ in artificially spiked soils in the absence of flavonoids amendment. In artificially spiked soils in the presence of flavonoids amendment, the numbers of CFU g⁻¹ of total heterotrophic bacteria and PAH–degrading microbial communities greatly increased after 28 d pre–incubation compared to those obtained in the fresh artificially spiked soils (Table 4). The numbers of naphthalene and phenanthrene degraders were significantly (P < 0.05) increased in the pre–incubated artificially spiked soilscompared to the number of pyrene degrader sin fresh artificially spiked soils. The greatest numbers of PAH–degrading microbial communities in the range of 10⁹ CFU g⁻¹ were measured in systems amended with 50 – 100 µg kg⁻¹3-hydroxyflavone, flavone and the mixture of flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio)following 28 d soil–contaminant pre–exposure. In direct comparison, there were significant (P < 0.001) increases in the numbers of PAH–degrading microbial communitiesin artificially spikedsoils amended with 100 µg kg⁻¹ 3-hydroxyflavone compared to unamended soils. In this study, the highest numbers of PAH–degrading microbial communities were measured in artificially spiked soils amended with 100 µg kg⁻¹ 3-hydroxyflavone.

4. Discussion

The extents of ¹⁴C–PAH mineralisation in fresh artificially spiked soils indicate that the indigenous microbial communities were capable of degrading naphthalene, phenanthrene and pyrene. PAHs are environmental contaminants (of either natural or anthropogenic origin) that are ubiquitously detectable in soils from both non-polluted and polluted areas [45]. Although PAHs biodegradation is feasible in previously uncontaminated soils, soil–contaminants pre–exposure of the indigenous microbial communities has been linked to enhanced microbial activity and growth–linked mineralisation of PAHs in soils [11,16,21,46]. The sigmoidal nature of ¹⁴C–naphthalene, ¹⁴C–phenanthrene and ¹⁴C–pyrene mineralisation curves observed in fresh and pre–incubated artificially spiked soils show that microbial acclimation is required prior to enhanced rates of mineralisation [47]. In this study, shorter lag phases and enhanced microbial activities can be attributed to increase in microbial populations, induction of catabolic enzymes and microbial adaptation following 28 d pre–incubationof the PAHs spiked soils [13]. From the results obtained, naphthalene exhibited the shortest (P < 0.001) lag phases compared to phenanthrene and pyrene under the same treatment conditions. The observed rapidmicrobial mineralisationin fresh artificially spiked soils could be attributed to selective enrichment in the numbers of microbial community members that have the ability to degrade naphthalene[8]. However, microbial degradation for different petroleum–derived contaminants are known to vary as a result of physicochemical and biological properties of the soil [48].

Enhanced mineralisation of PAHs observed in artificially spiked soils amended with 50 – 100 µg kg⁻¹ flavone, 3-hydroxyflavone and a mixture of the three flavonoids (flavone, morin hydrate and 3-hydroxyflavone at a 1:1:1 ratio) can be attributed to selective increase in numbers of PAH–degrading populations and stimulation of appropriate enzymatic pathways [49,50]. Several flavonoids released from plants can regulate activities of rhizosphere associated microbes at micromolar and nanomolar concentrations [36]. According to Reilley et al. [9], the addition of organic acids, typically found in root exudates, enhanced the rates of pyrene mineralisation in rhizosphere soil. Siciliano and Germida [51] have shown enhanced losses of PAHs in planted soils and suggested that the mechanism by which this occurs is via plant root exudates stimulating the microbial communities involved in the dissipation of aromatic compounds. It is known that the degradation of flavonoids by rhizospheric bacteria leads to the formation of intermediates, including resorcinol, phloroglucinolphenylacetic acid, substituted cinnamic acids and protocatechuic acid [52]. Although these organic compounds are likely to be mineralised through the β-ketoadipate pathway [53], protocatechuate is an intermediate in the degradation of PAHs in some microorganisms [54]. In a study, Da Silva et al.[55] reported that phenolic-rich root extracts obviously stimulated phenanthrene mineralisation as well as total oxygenase activity. Leigh et al.[56] demonstrated that seasonal fine root death releases several flavones which act as substrates for polychlorinated biphenyl (PCB) degrading bacteria. The release of organic compounds, such as flavonoids, into the root zone or rhizosphere increases the concentration of available organic carbon and also stimulates the activity of soil heterotrophic bacteria, which are largely prevailing over soil autotrophic microorganisms [57].

The inhibition of pyrene mineralisation and decreases in number of PAH–degrading microbial communities following amendment of artificially spiked soils with higher concentrations of morin hydrate (≥ 100 µg kg⁻¹) could be attributed to formation of toxic intermediates/metabolites and/or preferential utilisation of the soluble flavonoids. However, interpretation of mineralisation kinetics may be hampered when compounds with high toxicity to bacteria are involved as mineralisation rates may be reduced as a result of toxic inhibition [58]. In a further possible explanation, the solubility of flavonoids in solvents depends on their existing forms and chemical structures. Flavone is insoluble in water [59] and 3-hydroxyflavone is insoluble in water at room temperature [60]. However, morin hydrate is soluble in water (0.25 mg ml⁻¹, 20 °C; 0.94 mg ml⁻¹,100 °C) and it is also soluble in methanol (50 mg ml⁻¹) [61]. Qiuet al.[62] reported that the addition of flavone and morin at concentrations greater than 10 mumol l⁻¹ hindered ¹⁴C-B[a]P mineralisation in root zone soil slurries. Flavonoids and other phenolics have been found to inhibit a range of root pathogens and pests, ranging from bacteria to fungi and insects [63]. This has been attributed to their role as antimicrobial toxins [64] and anti- or pro-oxidants [65]. In addition, metabolism of flavonoids by rhizosphere associated microbial communities could alter the relative abundance of some indigenous microbial species, activity of microbial populations and contaminant availability in the soil [39]. This study showed that the rates and extents of ¹⁴C–PAHs mineralisation in soils were influenced by the available concentrations of flavonoids.

In the pre–incubated artificially spiked soils, enhanced rates and extents of ¹⁴C–PAHs mineralisation could be attributed to acclimatisation of microbial communitiesand subsequent increase in the numbers of microorganisms capable of degrading PAHs. Furthermore, soil chemical changes related to the presence of flavonoids and products of their microbial turnover could affect microbial activity, population diversity, availability of nutrientsand solubility of contaminants in the soil slurries. Rhodes et al.[66] found that pre–incubated spiked woodland soils exhibited a higher level of [UL-¹⁴C] 2,4-dichlorophenol degradation, which was subsequently enhanced by the addition of monoterpenes (P < 0.001), with the VOC mix and α–pinene amendments showing increased [UL-¹⁴C] 2,4-dichlorophenol catabolism. The apparent high levels of PAHs mineralisation in the pre–incubated soils could be attributed to selective enrichment and relative abundance of indigenous PAH–degrading microbial species compared to fresh artificially spiked soils. The results of this study suggested that contaminated soils amended with appropriate concentrations of flavonoids can promote growth of indigenous microbial communities capable of degrading PAHs and subsequently stimulate growth–linked mineralisation of ¹⁴C–PAHs in soils. Overall, the results of this study agree with the findings of Johnson et al. [10] and there is an indirect evidence for soil microbial response to plant–secreted chemicals during the biodegradation of petroleum contaminated soil.

5. Conclusion

The results obtained in this study have shown that amendments of contaminated soils with some flavonoids at appropriate concentrations (50 – 100 µg kg⁻¹) enhancedthe catabolic capabilities of indigenous soil microbial communities and subsequentgrowth–linked mineralisation of ¹⁴C–PAHs.In this study, soils amended with 100 µg kg⁻¹ 3–hydroxyflavone exhibited the highest extents of ¹⁴C–PAH mineralisation, while amendment with morin hydrate at higher concentrations (≥ 100 µg kg⁻¹) inhibited ¹⁴C–pyrene mineralisation. This study provide further understanding into the role of flavonoids on the development of microbial mineralisation of ¹⁴C–PAHsin fresh and pre–incubated artificially spiked soils.Depending on its concentrations, addition of flavonoids and other secondary compounds may either stimulate or inhibit microbial degradation of PAHs in soil.In practice, the effects of single compounds or mixed secondary plant metabolites on microbial mineralisation may likely become complicated due to apparent solubility and bioavailability of mixed contaminants in soils.Therefore, researchers should be cautious in attempts to manipulate flavonoids rhizosphere signals during phytoremediation processes due to the overlapping functions of many flavonoids as either stimulators or inhibitors of microbial activity. Further research is recommended in order to better understand the diversity and functions of phytochemicals in bioremediation of petroleum–contaminated soils.

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