Fungal degradation of polycyclic aromatic hydrocarbons (PAHs) by Scopulariopsis brevicaulis and its application in bioremediation of PAH-contaminated soil

ABSTRACT

A fungal strain PZ-4 isolated from an aged polycyclic aromatic hydrocarbon (PAH)-contaminated soil was found to have the ability to degrade PAHs, which was identified as Scopulariopsis brevicaulis based on 18S rRNA gene sequence. PZ-4 was able to remove phenanthrene (60%), fluoranthene (62%), pyrene (64%) and benzo[a]pyrene (82%) in liquid medium after 30 days of incubation. Microcosms were set up to evaluate the bioremediation potential of PZ-4 in a PAH-contaminated soil. After incubation for 28 days, 77% of total PAHs were removed from the soil with the addition of the PZ-4 suspension, the highest PAHs removal occurred in phenanthrene (89%) and benzo[a]pyrene (75%). These results indicate that this fungal strain might be a promising candidate for bioremediation of PAH-contaminated soils. It is also the first description of soil bioremediation with S. brevicaulis.

KEYWORDS:

• Biodegradation
• bioremediation
• polycyclic aromatic hydrocarbons
• soil
• fungal

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment with potential mutagenicity and carcinogenicity, which are generated from natural combustion processes as well as from human activities (Cerniglia 1992). Some PAHs are acutely toxic, mutagenic and carcinogenic (Boonchan et al. 2000). The deleterious properties of PAHs have made their remediation a critical need.

Bioremediation is one of the promising technologies to reclaim PAH-contaminated sites due to its relatively low cost and limited impact on the environment (Liebeg & Cutright 1999). Many isolated bacterial and fungal species have been reported to be capable of biodegrading PAHs. As a main group of microorganisms, flamentous fungi (including white rot fungi (WRF)) are important in the detoxification and cleaning up of contaminated environment (Harms et al. 2011). Recent studies have reported several fungal species with the capacity to degrade a series of PAHs, such as naphthalene, phenanthrene, fluoranthene, chrysene, pyrene and benzo[a]pyrene (Kiehlmann et al. 1996; Saraswathy & Hallberg 2002; Mollea et al. 2005; Mineki et al. 2015). Compared to bacteria, there are advantages in using fungi for bioremediation as they possess extracellular enzymes and their mycelia provide deeper penetration and larger surface area for absorption in soil.

PAH biodegradation has been reported by using different species of WRF such as Phanerochaete chrysosporium, Pleurotus ostreatus, Bjerkandera adusta, Irpex lacteus, Trametes versicolor, etc. (Wang et al. 2008; Mir-Tutusaus et al. 2014). The PAH-transforming capability of WRF may be related either to their cytochrome P-450 enzymes, or to the extracellular ligninolytic enzymes, including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Rafin et al. 2000). Although WRF possess an efficient enzyme system, the depletion of PAHs by WRF in soil is slow because they are not well adapted to soil environmental conditions that could consequently induce a slow growth in soil (Tortella et al. 2005; Li et al. 2012). To overcome growth-related disadvantages of WRF, there is a need to isolate fast growing non-white rot fungal strains capable of degrading PAHs. It has been reported that some non-WRF, Fusarium solani (Rafin et al. 2000), Aspergillus niger (Wang et al. 2008) and Penicillium janthinellum (Gao et al. 2010), were found to have the ability to degrade or mineralize PAHs. However until now, the full potential of biodegradation by non-WRF has not been fully investigated for bioremediation purposes, although these microorganisms are also abundant in heavily contaminated sites (Romero et al. 2001).

In the present study, a non-white rot fungal strain PZ-4 was isolated from an aged PAH-contaminated soil, its degradation ability of PAHs, especially HMW-PAHs, was investigated in liquid medium. Furthermore, soil microcosms were also set up to test the potential of fungal remediation in a PAH-contaminated soil.

Material and methods

Soil

Soil used in this study was collected from the surface layer (0–15 cm) of an uncontaminated area in Wuxi, Jiangsu Province, China. The soil pH was 6.6, organic matter 19.4 g kg⁻¹, total nitrogen 0.9 g kg⁻¹, total phosphorus 0.4 g kg⁻¹, total potassium 14.4 g kg⁻¹ and CEC 21.2 cmol kg⁻¹. The soil was air-dried, homogenized, passed through a 2-mm sieve, and stored at 4°C until use.

Isolation and identification of fungus

Strain PZ-4 was isolated from an aged PAH-contaminated soil by two-month enrichment using mineral salt medium (MSM) with addition of pyrene to a final concentration of 50 mg L⁻¹. The PAH-contaminated soil was collected from the surface layer (0–15 cm) of an industrial plant in Wuxi, Jiangsu Province, China (Mao et al. 2012). The MSM was composed of (g L⁻¹): K₂HPO₄ 6.0, KH₂PO₄ 5.0, Na₂SO₄ 1.0, KCl 1.0, MgSO₄·7H₂O 0.2 and yeast extract 0.05. The fungus was maintained on Potato Dextrose Agar plates at 4°C. It was cultured in liquid medium at 28°C for four days before used. The liquid media included (g L⁻¹) glucose 20, KH₂PO₄ 2, MgSO₄·7H₂O 0.5, CaCl₂ 0.1 and yeast extract 0.2.

Total genomic DNA of PZ-4 was extracted using the fungal DNA Kit SK1375 (Sangon, Shanghai, China) according to the manufacturer's instructions. 18S rRNA gene fragment was amplified by PCR using the set of primers NS1 (5′-GTAGTCATATGCTTGTCTC-3′) and NS6 (5′-GCATCACAGACCTGTTATTGCCTC-3′). The PCR conditions were as follows: initial denaturation at 94°C for 5 min, 35 cycles at 94°C for 30 s, at 55°C for 35 s, and at 72°C for 1 min, followed by a final extension at 72°C for 8 min. The PCR products were purified by SK1131 Kit (Sangon) and sequenced by Shanghai Sangon Ltd. The sequences were aligned then using the BLAST program at NCBI and deposited at GenBank with the accession number (KC218924).

Degradation of PAHs in liquid culture

The degradation experiment was conducted in 250-mL Erlenmeyer flasks containing 44 mL of sterile liquid medium. Five milliliter of PZ-4 culture and 1 mL of stock solution of individual PAH (phenanthrene, fluoranthene, pyrene and benzo[a]pyrene) dissolved in dimethylformamyde (DMF) were added aseptically into the flasks. The final concentration of each PAH was 100 mg L⁻¹ for phenanthrene, fluoranthene and pyrene, except for benzo[a]pyrene which was supplied at 20 mg L⁻¹. The control experiments (CK) were performed with autoclaved PZ-4 cells (0.3 g L⁻¹). The incubation was carried out at 28°C statically in a constant temperature incubator for 30 days. All the experiments were performed in triplicate, and the results were expressed as mean values.

PAHs in liquid culture was extracted three times by CH₂Cl₂ (1:0.5, v/v) in a separating funnel on a shaker. The organic extract was dried over anhydrous Na₂SO₄, frozen and quantitatively analyzed by high performance liquid chromatography (HPLC). The efficiency of PAHs extraction from the liquid media was 85–115%. The biomass of strain PZ-4 was carried by weighing the total cells after drying at 60°C for 6 h in the oven.

Bioremediation of PAH-contaminated soil

The bioremediation potential of PZ-4 was evaluated using microcosms. Each microcosm comprised 1.0 kg non-sterile soil (dry weight). The soil was laid out thinly and the PAHs dissolved in acetone were added to the soil using a pipette at a rate of 200 mL of solution per kg of soil. Acetone was utilized as the carrier solvent as it solubilizes the PAHs and is easily evaporated. The soil was turned repeatedly during the additions and then left for 3 days to ensure evaporation of the acetone. The final concentration of phenanthrene, fluoranthene and pyrene in soil was 10 mg kg⁻¹, and the concentration of benzo[a]pyrene was 4 mg kg⁻¹ soil. For bioaugmented microcosms, soils were inoculated with 200 mL of fungal suspensions. Control microcosms (CK) were set up with the same treatment as described above except addition of sterile liquid medium. The water holding capacity of microcosms was adjusted to 60%. All microcosms were run in triplicates and incubated at room temperature for 28 days in darkness.

The microbial populations (bacteria and fungi) in soil samples were determined using the dilution plate method (Shen & Chen 2007). The soil samples (10 g) were diluted to 10⁻⁸ in sterile deionized water. Soil suspension (0.2 mL) was dispensed into a Petri plate and 15 mL of molten agar medium (Sinopharm, Shanghai, China) was added and incubated at 28°C for 72 h. The population of bacteria was determined by plating with nutrient agar while rose bengal agar was used for estimating fungi numbers. Both plate counts were carried out in triplicates per dilutions.

PAHs extraction and analysis

Five grams of frozen dried soil samples (in triplicates) were extracted with 60 mL dichloromethane in a Soxhlet apparatus for 24 h. Extracts were concentrated using a rotary evaporator and purified with column chromatography filled with activated silica gel before analysis by HPLC (Ping et al. 2007).

Analysis of PAHs were performed with a Shimadzu Class-VP HPLC system (Shimadzu, Japan), with a fluorescence detector (RF-10AXL). A reversed phase column C18 (VP-ODS 150 × 4.6 mm i.d., particle size 5 μm), using a mobile phase of water and acetonitrile mixture (4:6, v/v) at a constant solvent flow rate of 0.5 mL min⁻¹, was used to separate PAHs. The excitation and emission wavelengths for individual PAHs were set separately (Mao et al. 2012).

Data analysis

All the data obtained in the study were subjected to statistical analysis of one-way ANOVA, and post hoc Tukey's test with SPSS Version 13.0.

Results and discussion

Identification of the fungal strain PZ-4

In recent years, bioremediation through microorganisms represents the main route for the recovery of PAH-contaminated areas (Wu et al. 2009). Several PAH-degrading fungal strains had been isolated from an aged PAH-contaminated soil by the continuous transferring using MSM with pyrene as the sole carbon source and energy (Lisowska & Dlugonski 2003). The strain PZ-4 was selected among other strains for its relatively higher degradation ability of pyrene. The partial 18S rRNA gene sequence was used to BLAST search against GenBank databases. The result showed that the 18S rRNA sequence of PZ-4 was 99% identical to that of Scopulariopsis brevicaulis (accession no. KC218924). The morphological characteristics of strain PZ-4 including its spores were compared with those of the known species of fungi (Wei 1979) and it was strongly suggested that strain PZ-4 belongs to the S. brevicaulis.

Degradation of PAHs in liquid medium

Biodegradation trials at laboratory scale using a liquid medium are normally the first approach to establishing the ability of microorganisms to degrade specific pollutants. The degradation of individual PAHs (phenanthrene, pyrene, fluoranthene and benzo[a]pyrene) by PZ-4 in liquid medium after 30 days of incubation are shown in Figure 1. An initial decrease in concentration of PAHs was observed during the first 5 days for phenanthrene, pyrene, and benzo[a]pyrene. A sharp decrease in all PAHs concentrations was observed between 15 and 20 days, particularly for benzo[a]pyrene and pyrene, and between 20 and 25 days for phenanthrene, in treatments inoculated with PZ-4, compared to the control (non-inoculated) (Figure 1). After 30 days of incubation, the removal efficiency of PAHs in liquid was in the following order: benzo[a]pyrene had the highest removal (82.1%) followed by pyrene (64.3%), fluoranthene (61.9%) and phenanthrene (60.0%).

Figure 1. Concentration of PAHs in liquid medium (mg L⁻¹). Each point represents the average value of triplicate samples.

The fungal growth in liquid medium during the degradation of PAHs is shown in Figure 2. The growth rate of PZ-4 in the treatment with pyrene was higher than others in the first 25 days. A sharp growth increase occurred during the 15 and 25 days, caused the highest growth of PZ-4 in the treatment of pyrene and phenanthrene at day 25. The growth rate of PZ-4 in fluoranthene and benzo[a]pyrene increased gradually, which was lower than that in pyrene and phenanthrene.

Figure 2. The growth weight of PZ-4 in liquid medium with the addition of PAHs (g L⁻¹). Each point represents the average value of triplicate samples.

The degradation observed in this study should be from a cometabolic process, since PZ-4 cannot grow with PAHs as sole carbon and energy source in MSM (data not shown). Microorganisms can degrade PAHs through mineralization or cometablism (Cerniglia 1992). Many studies have reported on microorganisms that are capable of degrading LMW-PAHs as sole carbon and energy sources for growth, but the degraders of HMW-PAHs are relatively rare (Nam et al. 2001). HMW-PAHs are usually more persistent in soil because of their low bioavailability, due to their strong sorption onto soil organic matter (Li et al. 2008). Recently, the ability of a number of fungal isolates to degrade HMW-PAHs has been shown to mainly occur cometabolically with an alternative carbon source (Tekere et al. 2005; Hwang et al. 2007), only a few isolates have been reported to solely degrade HMW-PAHs as sole carbon and energy sources (Wu et al. 2009). The appropriate cosubstrates may induce the secretion of catabolic enzymes and thus promote the degradation of HMW-PAHs (Acevedo et al. 2011). For example, Hadibarata and Kristanti (2012) found that benzo[a]pyrene was more efficiently degraded in glucose-supplemented medium than in other cosubstrates.

Bioremediation of PAH-contaminated soil

Microorganisms play an important role in the degradation of PAHs in terrestrial and aquatic ecosystems (Vinas et al. 2005). Bioaugmentation is the most often used technique to increase the biodegradative capabilities of the indigenous microbial populations. In this study, the strain PZ-4 was used in the bioremediation of PAH-contaminated soil in laboratory-scale test. Plate counts of the microbial populations of bacteria and fungi in soil during the bioremediation process are shown in Figure 3. The number of fungi in control soil remained constant during 0–21 days, but it increased by threefold at day 28. This shows that PZ-4 can survive in non-native soil and compete with indigenous microbial populations. In contrast to control, much higher microbial counts were observed when stain PZ-4 was added in soil at day 0, and the highest microbial population (7.4 × 10⁸ CFU g⁻¹ soil) was detected at day 28, indicating a successful colonization of the inoculum. The number of bacteria increased significantly after inoculation in both of the treatments, but there was no significant difference between them after day 7, showing that the addition of MSM stimulated the growing of soil bacteria.

Figure 3. Plate counts of the microbial populations of fungi and bacteria in soil during the bioremediation process. Each point represents the average value of triplicate samples. CFU, colony-forming units

The concentrations of PAHs in the soil with and without bioaugmentation of PZ-4 are presented in Figure 4. In the bioaugmented soils, phenanthrene was rapidly removed from soil, about 80% of it was removed in the first 14 days, followed by a slower decrease. The removal of fluoranthene, pyrene and benzo[a]pyrene in the bioaugmented soils was relatively slower compared to phenanthrene. The PAHs concentration in bioaugmentated soils was significantly lower than that in control treatments from 14 to 28 days (p < .05).

Figure 4. Concentration of PAHs in PAH-contaminated soil (mg kg⁻¹ soil). Each point represents the average value of triplicate samples.

The degradation rate of phenanthrene was the highest, 89.1% of which was removed from the soil. Compared with control, significant degradation was observed in both total and individual PAHs (p < .01) in bioaugmented microcosms.

The bioaugmentation studies of PAH-contaminated soils have been conducted by enrichment with allochthonous fungi and white rot fungi (Wu et al. 2008). Yateem et al. (1998) have reported a TPH removal of 81.8% when a soil polluted with 3.1% of TPH was biotreated by bioaugmentation with white rot fungi within 12 months. Acevedo et al. found that (2011) Anthracophyllum discolor was able to remove phenanthrene (62%), anthracene (73%), fluoranthene (54%), pyrene (60%) and benzo(a)pyrene (75%) from soil after 60 days of incubation.

It was generally accepted that the biodegradation of LMW-PAHs occurred much more rapidly and extensively than that of the HMW-PAHs (Nam et al. 2001). However, high degradation of the latter would have been obtained in some conditions. Potin et al. (2004) found that some fungi isolates, Coniothyrium sp. and Fusarium sp. preferentially degraded HMW-PAHs than LMW-PAHs. In this study, the highest degradation percentage was obtained for benzo[a]pyrene in the liquid culture, and the removal efficiency of benzo[a]pyrene was also higher than that of pyrene and fluoranthene in contaminated soil. These results may be caused by the lowest initial concentration of benzo[a]pyrene (only about 25% of other PAHs) applied in this study. It showed that the initial amount of the pollutants can significantly influence the level of degradation. Although it has been reported that a series of fungal species could oxidize and degrade PAHs, to the best of our knowledge, this is the first report on degradation of PAHs by S. brevicaulis in detail. Further studies will be conducted to the metabolism pathway of PAHs by this fungal strain in liquid culture and PAH-contaminated soils.

Acknowledgments

We especially thank Dr Yucheng Wu from Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, for his helpful discussions. We also thank Professor Shixue Yin from Yangzhou University, College of Environmental Science and Engineering, Yangzhou, China, for language revision of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was funded by grants from the National Natural Science Foundation of China [41201315] and the Knowledge Innovation Program of the Chinese Academy of Sciences [KSCX2-EW-G-16-04].