Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by Cladosporium sphaerospermum isolated from an aged PAH contaminated soil

Abstract

The ability of a Deuteromycete fungus, Cladosporium sphaerospermum, previously isolated from soil of an aged gas manufacturing plant, to degrade polycyclic aromatic hydrocarbons was investigated. This strain was able to degrade PAHs in non-sterile soils (average 23%), including high molecular weight PAHs, after 4 weeks of incubation. In a microcosm experiment, PAH depletion was clearly correlated to fungal establishment. In liquid culture, this strain degraded rapidly benzo(a)pyrene during its early exponential phase of growth (18% after 4 days of incubation). Among extracellular ligninolytic enzyme activities tested, only laccase activity was detected in liquid culture in the absence or in presence of benzo(a)pyrene. C. sphaerospermum might be a potential candidate for an effective bioremediation of aged PAH-contaminated soils.

Introduction

Sites in the vicinity of gas manufacturing plants have frequently been contaminated with compounds such as polycyclic aromatic hydrocarbons (PAHs), which are produced by incomplete combustion processes of organic carbon-based material[1]. PAHs are hydrophobic compounds and their persistence in the environment is chiefly due to their low water solubility. This class of compounds is of increasing interest because of their toxic, mutagenic and carcinogenic properties[2]. Consequently, the US Environmental Protection Agency has listed 16 PAHs as priority pollutants. Clean-up of former gas plant sites is therefore desirable in order to avoid public health hazards.

Although PAHs may undergo chemical oxidation, photolysis and volatilization, microbial degradation is the major process affecting PAH persistence in nature[3]. Bioremediation, expected to be an economic and efficient alternative method to other remediation processes such as chemical or physical ones, has been developed as a soil clean-up technique. However, the success of PAH bioremediation projects has been limited by the failure to remove high-molecular weight PAHs[4]. The recalcitrance of PAHs to microbial degradation has been related to their hydrophobic nature. These compounds are thus regularly bound to soil particles, resulting in low bioavailability to microorganisms [5,6]. This phenomenon is especially enhanced in aged contaminated soils, so many biodegradation studies have focused on isolating microorganisms and evaluating their degradative ability on high molecular weight compounds.

In soil habitats, filamentous fungi offer certain advantages over bacteria for bioremediation. The majority of studies have focused on white rot fungi, particularly Phanerochaete chrysosporium [7–10]. These fungi are able to degrade some five-ring PAHs and detoxify PAH-polluted soils and sediments through the production of extracellular lignin-degrading enzymes. Non-lignolytic fungi, such as Cunninghamella elegans [3,11] and Penicillium janthinellum [12] can also metabolize a variety of PAHs to polar metabolites. Moreover, numerous filamentous fungi are especially suited for terrestrial habitats and can reach xenobiotics even immobilized in micropores due to their multicellular mycelium [13,14].

In a search for indigenous soil filamentous fungi with potential to degrade PAHs with four or more rings, our laboratory has isolated a collection of telluric fungi (belonging to Mastigomycetes, Zygomycetes and Deuteromycetes) from gas manufacturing plant soils and found numerous isolates with high capacity to degrade PAHs[15]. The objectives of the present work were to evaluate the ability of one of previously screened isolates, Cladosporium sphaerospermum, to degrade PAHs in aged coal tar contaminated soils and in liquid culture.

Materials and methods

Chemicals, media and soils

Benzo(a)pyrene (BaP) and veratryl alcohol were purchased from Acros Organic (Noisy le Grand, France). A 16 EPA-PAH kit was purchased from Restek (Evry, France). Dichloromethane (DCM), N′, N′-dimethylformamide (DMF), acetone and other solvents and chemicals, except when specified otherwise, were obtained in the highest purity available from Merck (Darmstadt, Germany). Vanillylacetone was purchased from Interchim (Montluçon, France) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) from Sigma (Steinheim, Germany). Malt yeast extract agar (MYEA) medium was prepared as follows: 2.0% malt extract, 0.2% yeast extract and 1.5% agar in distilled water. The composition of the BSM–glycerol medium (BSM–Gly) was (per liter of distilled water): 10 g glycerol; 2 g KH₂PO₄; 0.14 g CaCl₂.2H₂O; 0.07 g MgSO₄.7H₂O; 2.5 mg thiamine hypochloride; 7.5 ml Tween 80; 1.84 g D-diammonium tartrate; 1 g yeast extract; and trace-element solution contained (per liter): 70 mg FeSO₄.7H₂O; 46.2 mg ZnSO₄.7H₂O; 35 mg MnSO₄.H₂O; 7 mg CuSO₄.5H₂O. This culture medium was adjusted to pH 6.5. Soils samples, provided by Rhodia (France), were from two different PAH contaminated soils at aged gas manufacturing plant sites in France. Soils were denoted as ROG and PER and contained approximately 839.7 and 1098.9 mg total PAH/kg of soil, respectively (Table 1). These soils were classified as slimy-clayey sand using particle size distribution test[16]. Soil samples were stored aerobically at 4 °C in the dark until use, approximately 2 months after collection.

Microorganism and preparation of fungal inocula

This study was carried out using C. sphaerospermum that has been isolated previously from ROG soil collected from a gas plant site[15]. Cultures were maintained on MYEA slants at 18 °C and subcultured every 3 months.

An inoculum was prepared from a suspension of spores by washing a one-week-old culture of C. sphaerospermum (grown on MYEA in Petri dishes) with 4 ml of sterile deionized water. The fragments of mycelium were removed from the spore suspension by filtration through sterile glass wool. The concentration of spores per volume of the suspension was estimated using a Malassez haemocytometer (Fisher Scientific Labosi, Elancourt, France). For soil experiments, mycelial inoculum was prepared as described previously[15]. Before inoculation into the soil, the spore suspension was transferred to a solution of glucose-sucrose (providing a final concentration of glucose and sucrose of 5 mg each/g of soil) for 36 h at 25 °C in order to induce spore germination and hyphal elongation. Germinated spores were introduced into soil (at a final concentration of 10⁴ spores/g of soil). Killed fungal inoculum was also prepared by adding HgCl₂ (0.7 g l⁻¹) to the 36 h cultures.

PAH degradation in soil

To assess the ability of C. sphaerospermum to degrade PAH, two sets of experiments were conducted, that is culture tubes and microcosms.

(i) Culture tubes (25 ml) containing 7 g of air-dried soil (passed through 2 mm sieve prior to use) were inoculated with the mycelial inoculum to give an initial concentration of 10⁴ spores/g of soil for each soil treatment (either ROG or PER). Biotic controls were non-sterile soil samples containing the same glucose-sucrose solution in order to detect the biodegradation due to indigenous microorganisms. Similarly, HgCl₂-killed mycelium controls were set up in order to assess PAH adsorption on fungal hyphae. Initial PAH concentrations were determined three times before each inoculation treatment. Three replicates were run for each treatment. All soil samples were moistened to about 70% of their water holding capacity. After inoculation, the tubes were closed with sterile cotton plugs and incubated in the dark at room temperature for one month before PAH extraction.

(ii) In a microcosm study, each microcosm was setup in a plastic tank (35 × 25 × 20 cm) closed with aluminum foil, and contained 5 kg of soil from the PER site. Air-dried soil was passed through a 10 mm sieve prior to use. Each microcosm was inoculated with mycelial inoculum in a final concentration of 10⁴ spores/g of soil. The control microcosm (biotic degradation) was not inoculated but modified with the same solution of glucose–sucrose. Water content was adjusted to about 70% of the water holding capacity and the moisture content of each microcosm was maintained throughout the experiment. All treatments were conducted in duplicate at room temperature over a period of 60 days. Triplicate soil samples from each microcosm were collected after 0, 30, and 60 days in order to analyze the fungal populations and PAH degradation as described below.

In microcosm experiments, soil colonization by autochthonous fungi and by C. sphaerospermum was estimated using colony forming unit (CFU) count. Three soil samples of 5 g were taken from each treatment on every sampling day (0, 30 and 60 days), added to 100 ml sterile deionized water in a 250 ml Erlenmeyer and shaken for 30 min at room temperature. The solid particles were allowed to settle for 30 min. Serial dilutions were performed and 100 μl of each dilution were spread homogeneously on the surface of an agar plate (containing 15 ml MYEA supplemented with a solution of sterilized chloramphenicol (0.02%)). Three replicates per dilution were prepared. Plates were incubated at 25 °C for several days prior to CFU determinations. Identification of C. sphaerospermum was performed by macro- and microscopic observations.

PAH extraction from soil samples

Optimal extraction conditions were determined after preliminary assays. After incubation, soils were oven-dried (30 °C, overnight), passed through a 0.12 mm sieve and further extracted. Samples (5 g) were extracted in a Soxhlet apparatus for 16 h with dichloromethane. The extracts were concentrated and analyzed by gas chromatography (GC) using a model Auto-System GC (Perkin–Elmer Co, Inc.) equipped with a flame ionisation detector[15]. The percentage of PAH depletion (%D) was given by the formula: %D = 100 [(M_HgCl2−M_T)/M_I], in which M_HgCl2 was the quantity of PAH obtained in HgCl₂-killed mycelium control, M_T was the quantity of PAH obtained in each treatment and M_I was the initial PAH quantity present in soil.

BaP depletion in liquid cultures

Cultures for pure-culture assays were established as follows: BaP (0.252 g l⁻¹ resulting in 10⁻³ mol l⁻¹) dissolved in 1 ml acetone was added to 250 ml empty Erlenmeyer flasks. After total evaporation of the organic solvent, the BSM–Gly medium (50 ml per flask) was added. The flasks were sterilized at 121 °C for 20 min. Inoculation was performed by adding a spore suspension to reach a fungal concentration of 10⁵ spores/ml. To detect abiotic BaP degradation, flasks without fungi were prepared and processed analogously. In order to detect adsorbed BaP on fungal hyphae (extraction controls), flasks containing 50 ml BSM–Gly, but no BaP, were inoculated with C. sphaerospermum. Every 48 h, the obtained mycelia were suspended in 50 ml BSM–Gly with BaP (0.252 g l⁻¹ resulting in 10⁻³ mol l⁻¹) and stirred for 4 h on a reciprocating shaker (Laboshake, Gerhardt, France, 90 min⁻¹) at 4 °C. These treatments allowed us to determine the adsorption processes on hyphae, used in further calculations of BaP degradation. All treatments were incubated at 25 °C with a 12 h photoperiod for 10 days on a reciprocating shaker (Laboshake, 90 min⁻¹). Triplicates were used to determine fungal biomass, enzyme activity and BaP depletion every 48 h.

Fungal growth was estimated by biomass produced on alternating days over a total period of 10 days. Mycelium was filtered through pre-dried cellulose filters in a vacuum filtration apparatus. Filters were lyophilised and biomass dry weight was measured.

BaP extraction and analytical procedure

The culture filtrate was extracted twice with 100 ml of DCM/ethyl acetate (50:50, v/v) and once with 100 ml of DCM. The organic and aqueous phases, containing mycelium, were separated. Mycelium was filtered, washed with 50 ml of acetone and lyophilized. Lyophilized mycelium was extracted for 16 h in a Soxhlet apparatus with DCM in order to remove adsorbed and non-metabolized BaP on hyphae. The aqueous phase was extracted three times with 50 ml DCM. Organic fractions obtained from the mycelium and from the aqueous phase were pooled with the previously obtained organic fraction and concentrated in 100 ml of DCM. BaP concentrations were determined using HPLC Waters 2690 system fitted with a Waters Symmetry^R, C18, 5 μm, 100 Å column and a Waters 996 Photo Diode Array Detector. The separation was achieved with a 5 min linear gradient of acetonitrile/water (60:40 to 100:0, v/v) at a solvent flow rate of 0.3 ml min⁻¹ and ending with acetonitrile/water (100:0, v/v) during 50 min. Concentrations were determined by UV absorbance at 254 nm.

The percentage of BaP depletion was given by the formula: [(m_EC−m_T)/m_EC] ∗ 100, in which m_EC was the quantity of BaP recovered in extraction controls (conducted to detect adsorbed BaP on fungal hyphae) and m_T was the quantity of BaP recovered in each treatment[17].

Enzyme assays

Enzyme assays were conducted in 1 ml reaction mixtures at 30 °C using the extracellular medium of fungal BSM–Gly cultures as an enzyme source. Laccase activity was measured by monitoring the oxidation of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm[18]. Specific activity was determined using ε= 36,000 M⁻¹ cm⁻¹. LiP activity was determined by the veratryl alcohol oxidation assay[19]. MnP was assayed according to the method of Paszczynski et al.[20] with vanillylacetone as a substrate.

Results

PAH biodegradation in soils by C. sphaerospermum

The first experiment was conducted in culture tubes containing 7 g of soil. C. sphaerospermum was tested for its PAH degradation capacity in two different soils under non-sterile conditions: either ROG soil (the native soil) or PER soil (in which the strain is exogenous). These soils had similar PAH contamination, with high amounts of high molecular weight PAHs (Table 1). After 4 weeks of incubation with only autochthonous microbial population, up to 13% loss of PAH occurred in PER soil, in contrast to low disappearance (1%) in ROG soil (Table 2).

Inoculation of polluted soils with C. sphaerospermum led to a total PAH loss of 26% in ROG soil and 21% in PER soil. In comparison to the biotic control, the total PAH loss was significantly greater in both soils inoculated with C. sphaerospermum after 4 weeks of incubation. Inoculation with Cladosporium led to a significant improvement in the depletion of in particular five- and six-ring PAH in both soils (Table 2).

PAH degradation in PER soil in microcosm

PAH degradation by C. sphaerospermum was further examined in microcosms containing PER soil. We chose this soil to study not only the capacity of this fungus to degrade PAH but also to examine the potential of this isolate to be maintained in other than native soils. In this experiment, soil colonization and PAH depletion (Table 3) were examined.

The autochthonous fungi, detected in both treatments (controls or inoculated ones), remained at a constant level (above 5 × 10³ CFU/g of soil) throughout the experiment (data not shown). C. sphaerospermum, which was not initially present in PER soil, was never detected in control treatments. After C. sphaerospermum was introduced into soil (10⁴ CFU/g of soil), it colonized the soil and reached an average density of 5 × 10³ CFU/g of soil after 4 weeks of incubation. The C. sphaerospermum population remained constant during the 8 weeks of experiment.

After 4 weeks of incubation, growth of C. sphaerospermum in PER soil allowed a significantly higher PAH depletion (11%) than biotic control (6%). After 8 weeks incubation, no significant difference between biotic control and inoculated treatment was observed. 16% and 5% of high molecular weight PAHs were depleted in treatments inoculated with C. sphaerospermum and in biotic controls, respectively.

Benzo(a)pyrene degradation in liquid cultures

BaP degradation by C. sphaerospermum was then investigated in liquid cultures with 10 days of incubation. We conducted this kinetic experiment to determine the time course for BaP degradation and the involvement of extracellular oxidative enzymes (Fig. 1) in PAH metabolism. In the absence or presence of BaP, C. sphaerospermum showed a similar growth pattern, with a short initial lag period followed by an exponential growth phase. After 6 days of incubation, fungal biomass had reached the highest level (112 mg dry weight per 50 ml of culture). The depletion of BaP occurred rapidly during the lag and early exponential growth phases and was about 18% within 4 days of incubation. The time course for extracellular laccase activity in a liquid medium is also shown in Fig. 1. In absence of BaP, laccase activity was detected between the 6th and 8th day and decreased thereafter. In comparison, cultures grown in presence of BaP showed a peak of laccase activity detected on the 8th day. Neither lignin peroxidase nor manganese peroxidase were detected in either treatment throughout the experiment.

Discussion

This study shows that C. sphaerospermum, a Deuteromycete fungus, recently isolated from aged gas manufacturing plant soil[15], is able to enhance PAH biodegradation in soils. We used non-sterile soil in order to reproduce the conditions prevailing during bioremediation. Under these conditions, an increased depletion of mainly high molecular weight PAHs (with five- and six-rings) was observed.

The success of bioaugmentation was demonstrated in the native soil of C. sphaerospermum and also in another aged contaminated soil (PER soil). Although the indigenous microbiota showed some degradative capacity on all 16 EPA-PAHs (13%) in PER soil, the inoculation of C. sphaerospermum significantly stimulated PAH degradation, especially high molecular weight PAHs. This shows that C. sphaerospermum can survive in non-native soil and compete with indigenous microbial populations. Bioaugmentation with PAH-contaminated soil has been monitored in other studies showing opposing results. In some examples, bioaugmentation has shown limited success for various reasons including die-off of laboratory-adapted strains, limited substrate availability, the inability of the inoculum to compete with the indigenous microbial populations or the absence of enzymes involved in PAH degradation [21,22]. Others have reported on significantly improved degradation of high molecular weight PAHs after bioaugmentation in PAH-contaminated soil [23–25], similarly as in our experiment. Bioaugmentation is especially promising for sites containing high PAH concentrations and/or a significant proportion of high molecular weight PAHs, as found in aged contaminated soils. In such soils, longer periods of contact of persistent organic pollutants (POP) with soil constituents remove POP from solution by adsorption to soil constituents, resulting in decreased bioavailability[26]. This phenomenon is especially important for high molecular weight PAHs characterized by low water solubility[3]. The mycelial growth of fungi, during soil colonization, might therefore penetrate contaminated soil to reach PAHs and subsequently enhance the contact between insoluble compounds and fungal walls[13]. This could be a real advantage for remediation of aged contaminated soils.

The first sets of experiments, conducted on small amounts of soil, do not serve as models for a three-dimensional environment composed of a solid matrix. As several biotic and abiotic factors affect the degradation of chemicals in the environment[27], more realistically scaled evaluations have to be compared to other methods. For this reason, a long-term study in microcosms containing PER soil was conducted. In both treatments (biotic control and inoculated treatment), lower PAH degradation rate was obtained in microcosms, even after eight weeks of incubation. This result could not be explained by a pitfall of fungal establishment in soils. Indeed, the assessment of fungal populations shows that C. sphaerospermum survived in PER soil microcosms even in the presence of indigenous microorganisms. Although degradation levels can vary significantly depending on the scale of the experiment, this study shows again the PAH-degrading capacity of C. sphaerospermum in soils, especially on five- and six-ring PAHs.

Among Cladosporium species known to degrade hydrocarbons, Cladosporium (Amorphotheca) resinae has received much attention in the last three decades. It was named the ‘kerosene' or ‘creosote' fungus because of its occurrence in aviation fuel causing damage by clogging filters and corroding pumps and tanks [28,29]. In other investigations, strains of Cladosporium herbarum, isolated from contaminated sites, were also previously found to be able to degrade PAH in liquid medium [30–32]. Few studies reported on Cladosporium sphaerosperum degradation This species was first isolated from a biofilter that had been used to remove toluene from waste gases[33] and was also demonstrated to use toluene, ethylbenzene and styrene as the sole source of carbon and energy[34]. Recently, Giraud et al.[35] reported depletion values of about 8.3% and 40.1% for anthracene and fluoranthene, respectively, in liquid synthetic culture medium inoculated with C. sphaerospermum. The ability of C. sphaerospermum to degrade high molecular weight PAHs suggests potential use for bioaugmentation of PAH-contaminated soil, and this isolate was deposited at the CNCM (Collection Nationale de Cultures de Microorganismes – Institut Pasteur, Number I-2255) for potential use for bioremediation processes[36].

Since the use of this microorganism represents a potential tool for soil bioremediation, we investigated its ability to degrade benzo(a)pyrene, a high molecular weight PAH in liquid medium, in another experiment. In the pure-culture conditions, the degradation of BaP occurred rapidly to a maximum of 18% after 4 days of incubation. In comparison with degradation obtained with white rot fungi (such as Pleurotus ostreatus), which usually started in the stationary growth phase, i.e. between the 10th and 15th days of culturing[37], this fungus degraded BaP during lag and early exponential phases. Such a rapid BaP disappearance until day 4 and a similar BaP removal, expressed as quantity per flask (about 1–2 mg per flask), were also observed with other fungi, such as Marasmiellus troyanus and Pleurotus sapidus [38]. Moreover, neither lignin peroxidases nor manganese peroxidases were detected in liquid culture throughout the experimentation. Laccase activity was detected on the 6th day and the 8th day in absence or in presence of BaP, respectively. Thus, a clear correlation between laccase activity and BaP disappearance in fungal cultures was not observed, as reported for the white rot fungus Pleurotus ostreatus [39]. Nevertheless, we cannot exclude the possible role of laccases from C. sphaerospermum, involving an indirect mechanism, as described for white rot fungi [40–42]. This metabolic pathway involves the participation of an oxidation mediator – a cooxidant – which could catalyze the initial oxidation of benzo(a)pyrene. In this case, this mediator could be produced during the exponential growth phase of C. sphaerospermum before the detection of laccase activity in liquid medium.

In previous studies, conducted with another Deuteromycete telluric fungus, Fusarium solani, we observed a similar rapid degradation of BaP during the exponential growth phase[43]. We suggested that BaP degradation occurred during spore germination, a physiological phase characterized by an important consumption of fungal lipids[44]. The lipid metabolism produces hydrogen peroxide (H₂O₂) enhancement. Consequently, this could induce formation of reactive oxygen species (ROS), such as hydroxyl radicals (^●OH)[45], which might be the agents that initiate BaP oxidation[17]. In this study, we hypothesize that H₂O₂ production and extracellular ROS, acting as small non-enzymatic agents, could play an essential part in PAH biodegradation by C. sphaerospermum. Cladosporium species are ubiquitous fungi, reported on leaf litter, on phylloplane, on senescent or dead materials and also as soil inhabitants. This saprophytic fungus significantly contributes to the degradation of dead plant remains. Little is known about the decay mechanism. Presumably, ROS might be the agents that initiate decay of plant cell walls in these different ecological situations. This increase could be a defense mechanism occurring during the hyphae growth phase, which allows the resources colonization. As this free-radical mechanism is highly non-specific and non-stereoselective, it might also contribute to PAH degradation in contaminated soils.

Further studies will be conducted to confirm the relative importance of these mechanisms involved in PAH degradation by C. sphaerospermum. Studies on the physiological status of this Deuteromycete soil fungus during BaP degradation are undergone, in order to optimize the potential of this strain for the degradation of xenobiotics.

Acknowledgements

Financial support for this research was provided by Rhodia (Lyon). The authors wish to express their gratitude to Dr. Dominique Petre and Dr. Frédéric Baud-Grasset for their expert assistance and helpful criticisms. The authors thank Rhodia for providing the PAH contaminated soil samples and soil data. The authors also thank S. Masset and A. Gay for help in English revision of this article.

References