Mycoremediation potential of Coprinus comatus in soil co-contaminated with copper and naphthalene

Abstract

Experiments were conducted to investigate the effects of mycoremediation by Coprinus comatus (C. comatus) on the biochemical properties and lettuce growth in copper and naphthalene (Nap) co-contaminated soil. The results showed a significant enhancement of Nap dissipation upon incubation with C. comatus, and its removal ratios ranged from 96.00% to 97.16% with the level of contaminates, which were associated with the production of ligninolytic enzymes. The accumulation of copper in the body of C. comatus showed a positive correlation with the augment of metal loaded, and the proportion of acetic acid extractable copper in unplanted soils was larger than in soils with C. comatus. Lettuce grown in bioremediated soils showed a higher biomass and germination percentage and lower copper uptake than in non-bioremediated soils. These results, which suggest the accumulation of copper and degradation of Nap by C. comatus, provide a candidate for bioremediation in sites containing multiple pollutants.

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1. Introduction

Heavy metals and PAHs released into the environment seriously threaten natural ecosystems and public health due to their toxicity and bioaccumulation.^1–3 Due to long-term anthropogenic activities, the Cu concentration in soils with repeated application of Cu salts as fungicides can reach values 10–100-fold larger than in non-contaminated soils, and the excessive buildup of Cu in topsoil has affected plant communities and plant performances.^4–6 Naphthalene (Nap) has the lowest molecular weight among the sixteen PAHs listed as priority pollutants by the United State Environmental Protect Agency (USEPA), and a high concentration of Nap is commonly found in both aqueous and solid phases in the environment.^7–9 In multiple metal and organic polluted sites, the co-contamination of Cu and Nap often occurs in soil environments as a result of wastewater irrigation, solid waste disposal, and industrial activities.¹⁰ Moreover, the remediation of sites co-contaminated by metal and organic pollutants is a very complex problem, because the chemical processes and remediation technologies are different for each group of pollutants. Therefore, it is critical to develop a cost-effective and eco-friendly technology to remove heavy metals and PAHs from co-contaminated soils.

In recent years, phytoremediation has received considerable attention for the assimilation, metabolization, detoxification and degradation of metal and organic chemical contaminants.^11–13 However, there are many limitations to hypertoremediation. For example, hyperaccumulators are generally small and grow slowly, which make them difficult to accumulate a mass of pollutants.¹⁴ In addition, because of the lack of PAHs degradation capacity, the dissipation of pollutants by growing hyperaccumulators is enhanced very slightly.¹⁵

Compared with hyperaccumulators, mushroom, which has big biomass and grows quickly, has been cultivated all over the world.¹⁶ To date, there are numerous promising results that indicate mushroom has a high accumulation for heavy metals, including cadmium, lead, and copper.^14,17 Moreover, previous studies have illuminated that mushroom has the capacity to degrade organic compounds on account of its production of ligninolytic enzymes.^18,19 Therefore, mushroom possesses a more effective mechanism than plants to remediate heavy metal and organic co-contaminated soils.

Coprinus comatus is a white rot basidiomycete with a high content of proteins and has an excellent performance in producing ligninolytic enzymes.²⁰ However, little information is available on the effectiveness of mycoremediation concerning heavy metal and organic pollutants, particularly about the remediation by C. comatus for co-contaminated soils of heavy metals and organics. The aim of this study is to investigate the influence of co-contamination on the growth of C. comatus and the fate of pollutants in the soil and mushroom. After C. comatus was harvested, lettuce (Lactuca sativa L.) was used to test the effect of bioremediation because a large number of studies have indicated that massive plants could accumulate heavy metals and organics, and the toxicity of pollutants has a serious effect on the growth of plants.^21,22 Several researchers have demonstrated that lettuce growth is inhibited in soils contaminated with heavy metals.^23,24 Hence, the growth response and heavy metal accumulation of lettuce could be used to further evaluate the remediation performance.

2. Materials and methods

2.1 Soil preparation

Soil samples used in this study were collected from the campus area in Sichuan University, Chengdu, China, with pH 7.12, 1.68% organic matter, and originally free of Nap and 26 mg Cu kg⁻¹. Soil samples were air dried and sieved through a 2 mm mesh, carefully weighed and then spiked with heavy metal and organic pollutants. The levels (mg kg⁻¹) of Cu and Nap added to the soil were T0 (Cu0 + Nap0), T1 (Nap250), T2 (Nap500), T3 (Cu100), T4 (Cu200), T5 (Cu300), T6 (Cu100 + Nap250), T7 (Cu100 + Nap500), T8 (Cu200 + Nap250), T9 (Cu200 + Nap500), T10 (Cu300 + Nap250), T11 (Cu300 + Nap500), which included planted and unplanted groups with three replicates. Briefly, the bulk soil was first mixed with Nap dissolved in acetone. Then, the solutions of Cu (as CuCl₂) with different concentrations were added to the Nap-spiked soils. After the evaporation of acetone, the spiked soils were sieved again through a 2 mm mesh and packed into pots (2 kg dry weigh soil per pot), then covered with aluminum foil, and equilibrated in a dark room for two months prior to the experiment.

2.2 Pot experiments

This experiment was carried out in clean plastic pots (height 9 cm and diameter 12 cm) containing 2 kg of the abovementioned contaminated soil and 0.1 kg of a mycelia bag of C. comatus, which was purchased from Shuangliu, Chengdu, China. In three replicates, the soil was wetted with deionized water three times a week to approximately 65% soil field water capacity. At the bottom of each pot, there was a plastic dish to collect any potential leachate. After approximately 60 days, the mature fruiting bodies were harvested from the pots, washed with deionized water and dried for 4 days at 60 °C in an oven.

After C. comatus was harvested, the soil from each pot was collected carefully, air-dried and then sieved through a 3 mm mesh again for the lettuce experiment. Each pot was sowed with thirty seeds of lettuce, and wetted with deionized water every day. After 30 days, lettuce was harvested, washed with deionized water and dried for 2 days at 60 °C in an oven.

2.3 Soil analysis

Soil samples of cropped and uncropped C. comatus were collected at harvest, oven dried at 80 °C for three days and the BCR sequential extraction procedure was applied for metal speciation according to Quevauviller et al. with some modifications.²⁵ Briefly, 1.0 g of soil was shaken at 25 °C, 250 rpm for 16 h with 40 mL of 0.11 M CH₃COOH, then centrifuged for 5 min at 8000 rpm and the supernatant was collected for assay in the acetic acid extraction state. Combined with the oxidation state, the abovementioned residue was shaken at 25 °C, 250 rpm for 16 h with a 40 mL mixture of 0.5 M NH₂OH·HCl and 0.05 M HNO₃, then centrifuged for 5 min at 8000 rpm and the supernatant was collected for assay. For organic combination of state, the abovementioned residue was added to 10 mL 30% H₂O₂ (pH = 2.5), placed in a bath at 85 °C for about 1 h until the volume of liquid was less than 3 mL, then the residue was extracted with 10 mL 30% H₂O₂ and when the volume of liquid was less than 1 mL, 50 mL 1.0 M CH₃COONH₄ (pH = 2) was finally added and centrifuged for assay. For the residual fraction, the abovementioned residual soil was digested with a mixture of 6 mL HNO₃, 4 mL HClO₄, and 3 mL HF using a microwave digestion method to extract the residual fraction. All fractions of Cu in the samples were determined by flame atomic absorption spectrometry (AAS; VARIAN, SpecterAA-220Fs).

Extraction of Nap from the soil was performed according to the method described by Huang et al. with some modifications.²⁶ Concentrations of Nap were determined by HPLC with a UV-vis detector, operating at a wavelength of 254 nm and a reverse phase 5 μm C-18 column (250 × 4.6 mm). The mobile phase used was acetonitrile–water (90 : 10, v/v) at a flow rate of 0.6 mL min⁻¹.

2.4 Analysis of mushroom

C. comatus was harvested once their fruit bodies unfolded and washed with deionized water three times. Fresh samples (0.5 g) were quickly frozen in liquid nitrogen and grinded using a pre-cooled mortar and pestle, and then extracted in 5 mL of 200 mM phosphate buffer (pH 7.8) at 4 °C. The homogenate was centrifuged at 5500 rpm for 30 min and the obtained supernatant was used to measure the soluble protein and ligninolytic enzymes. The soluble protein content in C. comatus was measured using bovine serum albumin as the standard protein.²⁷ Laccase activity was measured as described by Palmieri et al. with one unit of laccase activity defined as the amount of enzyme that catalyzed the oxidation of 3-ethylbenzothiazolone-6-sulfonic acid (ABTS) at 30 °C in 1 min.²⁸ Manganese peroxidase (MnP) activity was measured according to Lopez et al. with one unit of enzyme activity defined as the amount of enzyme that can produce 1 μM Mn³⁺ from the oxidation of Mn²⁺ per minute.²⁹ Lignin peroxidase (LiP) activity was measured as described by Tien et al. with one unit of LiP defined as 1 μM of veratryl alcohol (VA) oxidized to veratraldehyde per minute.³⁰ After the fruit bodies of C. comatus were oven-dried, the mushroom powder samples (0.1 g) were digested with a mixture of 3 mL HNO₃, 1 mL 30% H₂O₂ and 1 mL HF in a microwave and then diluted to 10 mL with deionized water. Finally, the concentration of Cu in C. comatus was determined by FAAS.

2.5 Analysis of lettuce

Germination percentage was recorded two weeks after the lettuce seeds were sowed and no seeds sprouted afterward. After about 30 days, the lettuce was harvested, washed with deionized water three times, and dried at 65 °C for two days to determine the dry weight and the content of heavy metal. The concentration of Cu in lettuce was measured as the same as the determination of Cu in mushroom.

2.6 Date analysis

Translation factors (TF) values of metal from soils to mushrooms were calculated according to the following formula:

The percentage of TCP removal from soils was calculated as follows:

All treatments were replicated three times in this experiment. Treatment means were evaluated using variance and Tukey's test (p < 0.05). Statistical analysis was carried out using SPSS 18.0.

3. Results and discussion

3.1 Mushroom growth

The growth of C. comatus was significantly affected by Cu, PAHs and their interactions. As shown in Table 1, the addition of Cu could facilitate the growth of C. comatus under a low level (T3), which results in an increase at the rate of 18.25% when compared with the control group (T0). However, C. comatus showed visual signs of toxicity in response to single Nap contamination and to mixed contaminants, and total biomass significantly decreased by 19.24% and 22.86% in the high Cu treatments (T4 and T5). Furthermore, it was observed that an addition of Nap further decreased the biomass of C. comatus in Cu treatments compared to the Cu treatment alone. The highest decrease occurred in the 200 and 300 mg Cu kg⁻¹ samples with Nap compared the samples without Nap, whereas no significant difference was detected in the other treatments.

Table 1 Biomass (dry weight) of C. comatus grown contaminated soils for 60 days^a

Treatment	Biomass (g per pot)
	Total	Cap	Stipe
a Results are expressed as means ± SD (n = 3). Data within columns with different letters indicate a significant difference (Tukey HSD p < 0.05).
T0	12.16 ± 1.810c	6.42 ± 0.184f	5.74 ± 1.626e
T1	8.99 ± 4.172ab	4.48 ± 1.365ab	4.50 ± 2.807c
T2	8.50 ± 5.105a	4.24 ± 1.478a	4.26 ± 3.627bc
T3	14.38 ± 3.543d	6.96 ± 2.107g	7.42 ± 1.435f
T4	9.25 ± 0.127ab	5.08 ± 0.085cde	4.17 ± 0.042abc
T5	9.05 ± 4.130ab	5.38 ± 2.510e	3.66 ± 1.619a
T6	9.82 ± 1.336b	4.76 ± 0.205bcd	5.07 ± 1.541d
T7	9.02 ± 2.058ab	4.62 ± 1.775abc	4.40 ± 0.283c
T8	8.83 ± 1.471ab	4.50 ± 0.495ab	4.33 ± 0.976c
T9	9.38 ± 3.988ab	5.22 ± 2.411de	4.16 ± 1.577abc
T10	8.51 ± 2.857a	4.78 ± 0.488bcd	3.73 ± 2.369ab
T11	8.51 ± 1.577a	4.14 ± 1.892a	4.37 ± 0.764c

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The present study clearly demonstrates that heavy metal contaminants and PAHs have a direct effect on biomass production, and Nap shows stronger toxicity than Cu. Similar to our study, Chigbo et al. suggested that pyrene has stronger inhibition on Brassica juncea than Cu, and the increased growth in low Cu concentration could be related to the effects Cu has on various macronutrient contents (N, P, K, Na, Mg).³¹ Zhang et al. showed that the interaction of Cd and PAHs caused stronger inhibition on Juncus subsecundus than Cd or PAHs alone.³² Our results, however, are different from the report by Zhang et al., which showed that pyrene did not alleviate the toxicity of Cd to Z. mays.¹⁵ These results suggest that the growth response to the joint toxicity of metal and organic contaminants depend on certain factors, including species and characteristics of the pollutants.

Although growth was inhibited under some treatments of Cu and Nap, C. comatus displayed excellent tolerance to toxicity stress and confirmed its potential ability to remediate metal and PAHs co-contaminated soils.

3.2 Mushroom soluble protein content and enzyme activities

The soluble protein content and enzyme activities in mushroom were measured after 60 days of incubation. The soluble protein content in C. comatus decreased from 27.52 to 73.39% for Cu (T3–T5) and from 29.81 to 45.11% for Nap (T1–T2) compared to the control (Fig. 1), which showed that contaminants with Cu and Nap could effectively induce the protein content in the bodies of C. comatus. Moreover, it was obvious that the co-effects of Cu and Nap led to an induction of protein content in C. comatus. When 300 mg Cu kg⁻¹ was mixed with 500 mg Nap kg⁻¹ (T11), the protein decrease reached maximum, about 645.79% lower than the control.

Fig. 1 Concentration of soluble protein and activities of laccase, LiP and MnP in C. comatus exposed to different treatments of Cu and Nap. Different letters represent significant differences between the sampling at least p < 0.05.

The laccase and LiP activities (Fig. 1) in C. comatus displayed a significant increase under the joint stress of Cu and Nap in comparison with the control. In the same concentration of Nap, laccase and LiP activities tended to increase with the level of Nap increasing from 0 to 500 mg kg⁻¹ in the soil. The maximum laccase and LiP activities were observed in T11 and T9, which were 316.09% and 240.49% higher than the control, respectively.

MnP activity was more complex than laccase activity and LiP activity (Fig. 1), and it reached the maximum in 250 mg Nap kg⁻¹ mixed with 200 mg Cu kg⁻¹ (T8), which is about 232.15% higher than control. As the figure shows, the activity of MnP increased in the soil spiked with 250 mg Nap kg⁻¹ as compared with the soil spiked with Cu of 0, 100, 200 and 300 mg kg⁻¹ alone, particularly in 200 mg Cu kg⁻¹. In the same concentration of Nap, however, MnP activity showed no significant difference (p < 0.01) at 100 and 300 mg Cu kg⁻¹ compared to the control.

Heavy metal and PAHs are known to increase the activities of ligninolytic enzymes (laccase, MnP, and LiP), which can partly reduce toxicity stress and degrade organic compounds.^33,34 In Pleurotus ostreatus, the addition of Cu (0.5–5 mM) or Cd (1–5 mM) not only induces laccase by the expression of laccase genes, but also positively affects the activity and stability of the enzyme.³⁵ These results presented in our study prove that the secretion of ligninolytic enzymes in C. comatus could be enhanced in co-contaminants, which attests the potential removal of PAHs.

3.3 Cu accumulation and translocation in mushroom

Metal accumulation and translocation in the fruiting bodies of C. comatus were significantly influenced by the concentration of Cu, PAHs, and their interactions (Fig. 2). The Cu concentration in the cap and stipe of C. comatus tended to increase with increasing amounts of Cu in the soil, which was 8.21–103.7 mg kg⁻¹ and 5.58–65.2 mg kg⁻¹ across all the treatments, respectively. Comparison of Cu-alone and Cu–Nap contamination indicated that the addition of Nap could increase the accumulation of Cu in the cap (except in T8) and in the stipe (except in T8 and T10). Especially in T11, the accumulation of Cu in the cap and stipe reached maximum, which was about 128.50% and 116.34% higher, respectively, than in T5. Some previous reports also have shown that the interaction between metals and PAHs could influence metal uptake and accumulation in co-contaminated soil. Increased Zn concentrations were found in the shoots of Indian mustard (Brassica juncea) grown in soils contaminated with a mixture of pyrene and Zn.³⁶ The PAHs increased Cu uptake by a salt marsh plant (Halimione portulacoides) in elutriate, but not in the presence of sediments.³⁷ However, Lin et al. found that the ability of Cu phytoextraction of Zea mays L. is inhibited under Cu-Pyr co-contaminated soil.²¹ Chen et al. observed a slight decrease in the accumulation of Cu in Lolium perenne in Cu–2,4-dichlorophenol co-contaminated soil.³⁸ Furthermore, it was also observed that the accumulation of Cu was higher in the cap than that in the stipe, which agrees with these reports.^17,39,40 In the absence of Nap, the TF value results first significantly increased in 100 mg Cu kg⁻¹ (T3), then decreased in 200 and 300 mg Cu kg⁻¹ (T4 and T5), which is about 133.57%, 100.95% and 102.14% higher than the control, respectively. In soil co-contaminated with Cu and Nap, however, Nap influenced the Cu concentration and accumulation, which depends on the various levels of Cu treatment. For example, in 100 and 200 mg Cu kg⁻¹ soil, the TF values first decreased in 250 mg Nap kg⁻¹, then increased in 500 mg Nap kg⁻¹ and even reached 2.07 in T7. However, in high doses of Cu (300 mg Cu kg⁻¹), the TF values first significantly increased in 250 mg Nap kg⁻¹ and then decreased in 500 mg Nap kg⁻¹. Specifically, in lower Cu-polluted soil, high Nap increases the translocation of Cu, and in highly Cu-polluted soil, low Nap decreases the translocation of Cu. Our results could be explained from the results by Alkio et al. wherein PAHs may passively penetrate stipe cell membranes without any carrier, which can therefore facilitate the penetration of metals or metal complexes into the cell, increasing the metal in the cap.⁴¹ Moreover, due to the complex interactions of PAHs and metals in soil, the translocation efficiency of Cu is influenced by different concentrations in varying degrees.

Fig. 2 Cu concentration (column) in C. comatus and TF values (line) in treatments with different concentrations of Cu and Nap. Different letters represent significant differences between the sampling at least p < 0.05.

3.4 Cu speciation in soil

For phytoremediation or mycoremediation, Cu must be bioavailable, which suggests that metal accumulation in mushroom is dependent on not only their total concentration, but also their chemical forms.⁴² To study the distribution of the different forms of Cu in soil, four chemical fractions of Cu in planted and unplanted soils were determined using the BCR method and the concentrations obtained are shown in Table 2. On the one hand, it was observed that the HOAc extractable Cu decreased and the immobilized metals were transformed mainly into oxidizable forms in the planted soil after 60 days culture compared with the unplanted soil with Cu added treatments. The proportion of HOAc extractable Cu in the planted soil decreased by 3.08–20.04% and oxidizable Cu increased by 19.26–107.18% relative to the unplanted soil (Fig. 3). A possible explanation for this could be that the exchangeable form of Cu in the planted soil was the predominant species for Cu uptake by mushroom, which is consistent with the result of Cu accumulation in C. comatus (Fig. 2). Hence, C. comatus can significantly decrease the concentration of active and bioavailable heavy metals by their uptake and thus accelerate the stability process. On the other hand, the proportion of reducible and residual Cu either remains stable or changes only slightly. It may be assumed that the short incubation time did not lead to any marked change in the reducible and residual portion of the heavy metal.⁴³

Table 2 Concentrations of different species of Cu in planted and unplanted C. comatus soils^a

Treatment	HOAc soluble-Cu (mg kg^−1)		Reducible-Cu (mg kg^−1)		Oxidizable-Cu (mg kg^−1)		Residual-Cu (mg kg^−1)
	Planted	Unplanted	Planted	Unplanted	Planted	Unplanted	Planted	Unplanted
a Data within columns with different letters indicate a significant difference (Tukey HSD p < 0.05).
T0	0.08a	0.00a	5.48a	5.92a	5.22a	5.06a	8.65a	7.20a
T1	0.96a	0.76a	4.42a	6.16a	4.34a	4.07a	8.68a	7.66a
T2	1.48a	1.20a	3.92a	4.40a	4.34a	4.78a	9.15ab	8.24a
T3	31.16b	48.04c	59.56c	56.08b	14.24b	11.82b	10.62bc	11.29bc
T4	38.36b	50.80c	64.36c	57.60b	15.03b	12.37b	12.56de	11.16bc
T5	32.40b	34.84b	31.92b	52.40b	14.75b	13.09b	18.28f	11.22bc
T6	54.96c	64.48d	88.88e	83.24c	22.11c	15.02c	13.13de	12.11bcd
T7	97.64d	99.16e	99.60f	105.96d	26.24def	16.44cd	13.93e	12.34bcd
T8	119.04ef	100.08e	78.40d	104.76d	23.60cd	17.93d	13.94e	13.05d
T9	123.96f	151.28f	102.47f	129.96e	24.86de	16.78cd	13.18de	12.68cd
T10	112.76e	140.64f	105.92f	135.31e	28.16f	16.20cd	11.97cd	16.54e
T11	126.39f	155.70f	104.32f	130.45e	26.83ef	15.24c	12.04cde	15.72e

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Fig. 3 Cu speciation in soil with different concentrations of Cu and Nap.

3.5 Removal of Nap in soil

The concentrations of Nap in the soil after about 60 days are shown in Fig. 4. The residual concentrations of Nap in C. comatus planted soil were significantly lower than in the unplanted soil. In 250 and 500 mg Nap kg⁻¹ soil, the residual concentrations of Nap in the C. comatus-planted soils were 7.01–8.12 and 18.07–20.12 mg Nap kg⁻¹, which are about 27.06–33.28% and 28.76–30.10% lower than in the unplanted soils, respectively. Furthermore, the removal ratios were elevated in the planted soil, and the maximum of removal ratio (97.20%) was observed in T7 compared with the treatments (93.41–94.3%) without the incubation of C. comatus (Fig. 4). These results indicate that the removal of Nap is clearly enhanced by planting mushroom. The effect of heavy metals on the dissipation of PAHs may be positive or negative, while the presence of Cu showed no-significant effect on the removal of Nap in this study.

Fig. 4 Residual concentration (column) and removal rate (line) of Nap in soil with different concentrations of Cu and Nap. Different letters represent significant differences between the sampling at least p < 0.05.

The fates of PAHs in spiked soils mainly include volatilization, leaching, plant uptake, biodegradation, photo-degradation, and other abiotic losses.⁴⁴ Volatilization, photo-degradation, and microbial activity are most possibly related to the removal of Nap in unplanted soil, and the enhanced removal of Nap in planted soil can be attributed to the phenomena of mushroom uptake and biodegradation. Previous studies reported the removal pathway of PAHs in plants such as Tall fescue, Tagetes patula, Rumex crispus.^45–47 The presence of C. comatus could cause the production of ligninolytic enzymes (Fig. 1) and thus lead to the degradation of Nap.

3.6 Lettuce growth response

Plant growth in response to pollutants is sensitive. The growth response of lettuce and Cu uptake by lettuce are shown in Table 3. The biomass of lettuce was unaffected by residual Nap because Nap in soils is very rare after remediation. However, the biomass of lettuce gradually decreased with increasing concentrations of Cu, which agrees with the previous reports.^23,48 Compared with the non-remedied soils, there was a significant increase of biomass in soils after growing C. comatus and the maximum biomass was observed in T10, which is about 313.64% higher than in the non-remedied soil. In addition, the trend of germination percentage (Table 3) of lettuce was similar to the biomass and the maximum germination percentage in the remedied soil was observed in T11, which is about 262.47% higher than in the non-remedied soil. Moreover, there was a rather straightforward comparison of Cu accumulation in lettuce between the non-remedied and remedied soil (Table 3). Planting mushroom significantly decreased Cu accumulation in lettuce and the maximum decrease was 67.58% in T5, which is consistent with the result of HOAc extractable Cu in soils (Fig. 3). The abovementioned results suggest that incubation with C. comatus could facilitate growth, induce the Cu accumulation of lettuce and further confirm the beneficial remediation effect of mushroom in Cu and Nap co-polluted soil.

Table 3 Biomass (dry weight), germination percentage, and Cu concentration of lettuce fed in non-remedied and remedied soils^a

Treatment	Biomass (g)		Germination percentage (%)		Cu concentration in lettuce (mg kg^−1)
	Non-remedied	Remedied	Non-remedied	Remedied	Non-remedied	Remedied
a Results are expressed as means ± SD (n = 3). Date within columns with different letters indicate a significant difference (Tukey HSD p < 0.05).
T0	0.71 ± 0.03fg	0.78 ± 0.04de	95.00 ± 12.0h	93.33 ± 10.6cd	10.20 ± 1.2ab	9.68 ± 0.6a
T1	0.76 ± 0.05g	0.70 ± 0.02bc	93.33 ± 8.5h	90.00 ± 9.8bc	9.73 ± 0.8a	9.27 ± 0.6a
T2	0.70 ± 0.03ef	0.72 ± 0.03bc	95.00 ± 11.5h	91.67 ± 9.8bc	12.47 ± 1.3b	10.89 ± 1.2ab
T3	0.61 ± 0.02de	0.73 ± 0.03cd	83.33 ± 9.8fg	95.00 ± 11.5d	42.07 ± 3.7d	15.34 ± 1.4de
T4	0.67 ± 0.03ef	0.80 ± 0.05e	86.67 ± 10.6g	90.00 ± 10.5bc	33.69 ± 2.8c	12.13 ± 1.0bc
T5	0.62 ± 0.04def	0.78 ± 0.04de	80.00 ± 8.5f	91.67 ± 11.5bc	40.12 ± 3.9d	13.65 ± 1.4cd
T6	0.56 ± 0.03cd	0.69 ± 0.02bc	66.67 ± 7.4d	88.33 ± 8.5bc	46.22 ± 4.1ef	14.27 ± 1.4cde
T7	0.50 ± 0.02c	0.80 ± 0.02e	73.33 ± 8.0e	93.33 ± 12.0cd	45.83 ± 3.9ef	13.25 ± 1.0bc
T8	0.48 ± 0.01c	0.78 ± 0.03de	70.00 ± 7.2de	86.67 ± 9.2b	45.67 ± 4.3e	15.02 ± 1.6de
T9	0.34 ± 0.02b	0.67 ± 0.01b	46.67 ± 5.88c	66.67 ± 7.8a	48.39 ± 4.7f	15.69 ± 1.3de
T10	0.22 ± 0.01a	0.69 ± 0.02bc	33.33 ± 4.2b	66.67 ± 8.6a	47.40 ± 4.6ef	16.38 ± 1.6e
T11	0.25 ± 0.01a	0.62 ± 0.02a	26.67 ± 2.3a	70.00 ± 7.2a	48.30 ± 4.6f	15.72 ± 1.4de

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4. Conclusions

The conclusions based on this experiment are as follows: (1) C. comatus is tolerant to all concentrations of co-contamination and shows potential ability to remove heavy metal from co-contaminated soil (7.03–84.45 mg kg⁻¹ for Cu). (2) Planting C. comatus facilitates the removal of Nap, and the removal ratios are over 96.0%. (3) The presence of C. comatus decreases HOAc extractable Cu (3.08–20.04%) in soil. (4) The activities of ligninolytic enzymes significantly increase when C. comatus is exposed to Cu and Nap pollutants, which could be beneficial for defense against Cu and Nap toxicity stress. (5) The effect of remediation with C. comatus enhances the biomass and germination percentage of lettuce and significantly decreases the accumulation of Cu. These findings, therefore, provide evidence for the potential mushroom remediation of Cu and Nap co-contaminated soil with C. comatus.

Acknowledgements

This study was financially supported by the NSFC (No. 41171253, No. J1103518) and the National High Technology Research and Development Program of China (No. 2013AA06A210). The authors wish to thank Professor Guanglei Cheng and Dong Yu from Sichuan University for their technical assistance.

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