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Journal of Soils and Sediments

, Volume 18, Issue 6, pp 2136–2147 | Cite as

Combined bioremediation of soil co-contaminated with cadmium and endosulfan by Pleurotus eryngii and Coprinus comatus

  • Ying Wang
  • BoWen Zhang
  • NanJun Chen
  • Can Wang
  • Su Feng
  • Heng Xu
Reclamation and Management of Polluted Soils: Options and Case Studies

Abstract

Purpose

The subjects of this study were to investigate the remediating potential of the co-cultivation of Pleurotus eryngii and Coprinus comatus on soil that is co-contaminated with heavy metal (cadmium (Cd)) and organic pollutant (endosulfan), and the effects of the co-cultivated mushrooms on soil biochemical indicators, such as laccase enzyme activity and bacterial counts.

Materials and methods

A pot experiment was conducted to investigate the combined bioremediation effects on co-contaminated soil. After the mature fruiting bodies were harvested from each pot, the biomass of mushrooms was recorded. In addition, bacterial counts and laccase enzyme activity in soil were determined. The content of Cd in mushrooms and soil was detected by the flame atomic absorption spectrometry (FAAS), and the variations of Cd fractions in soil were determined following the modified BCR sequential extraction procedure. Besides, the residual endosulfan in soil was detected by gas chromatography-mass spectrometry (GC-MS).

Results and discussion

The results indicated that co-cultivation of P. eryngii and C. comatus exerted the best remediation effect on the co-contaminated soil. The biomass of mushroom in the co-cultivated group (T group) was 1.57–13.20 and 19.75–56.64% higher than the group individually cultivated with P. eryngii (P group) or C. comatus (C group), respectively. The concentrations of Cd in the fruiting bodies of mushrooms were 1.83–3.06, 1.04–2.28, and 0.67–2.60 mg/kg in T, P, and C groups, respectively. Besides, the removal rates of endosulfan in all treatments exceeded 87%. The best bioremediation effect in T group might be caused by the mutual promotion of these two kinds of mushrooms.

Conclusions

The biomass of mushroom, laccase activity, bacterial counts, and Cd content in mushrooms were significantly enhanced, and the dissipation effect of endosulfan was slightly higher in the co-cultivated group than in the individually cultivated groups. In this study, the effect of co-cultivated macro fungi P. eryngii and C. comatus on the remediation of Cd and endosulfan co-contaminated soil was firstly reported, and the results are important for a better understanding of the co-remediation for co-contaminated soil.

Keywords

Cadmium Combined bioremediation Coprinus comatus Endosulfan Pleurotus eryngii 

1 Introduction

Heavy metals and persistent organic pollutant contaminations are major environmental concerns on a world scale, in particular with the rapid urbanization and industrialization in China (Sun et al. 2011). Cadmium (Cd) is a metal of great environmental concern in agricultural ecosystems because of its high toxicity to animal and human health and its strong mobility in soils (Perronnet et al. 2000). Endosulfan, which is highly toxic to fish, invertebrates, humans, and most animal species, provoking acute and chronic symptoms at relatively low exposure levels, is a broad-spectrum organochlorine insecticide that has been used extensively against insect pests that damage agricultural crops (Ulčnik et al. 2013). With the rapid development of urbanization, industrialization, and traditional use of pesticides during past decades, soils contaminated by Cd and organochlorine insecticides have been accelerated in China (Wang et al. 2012). Therefore, it has been very urgent to remediate the contaminated soils. However, remediation of soils co-contaminated by heavy metals and organic pollutants is more complex than by a single pollutant, and there are few appropriate technologies to decontaminate the co-contaminated soils because of the great differences of properties between heavy metals and organic pollutants (Liu et al. 2015). Most organic pollutants are hydrophobic, easily integrated with soil organic matters, and difficult to be removed, while heavy metals are mostly with high mobility, water/acid-soluble, and easily combined with soil Fe-Mn oxides and organic matters (Qi et al. 2006). Therefore, the development of alternative approaches for remediating heavy metal and organic pollutant co-contaminated soils is of significant environmental and commercial importance.

In situ decontamination approaches such as phytoremediation have been proved to have great potential to reduce the content of pollutants in soil. For example, alfalfa, ryegrass, pole bean, and Ricinus communis are considered to have the ability to remediate contaminated soils with heavy metals and organic pollutants through degradation, assimilation, and metabolism (Huang et al. 2011; Souza et al. 2012; Chigbo et al. 2013). Previous studies have illustrated that many plants are effective in removing heavy metals and decomposing organic matters from soil. However, the growth speed of many plants is slow, and phytoremediation is costly. On the contrary, mushroom has a short growth cycle and a large yield. Meanwhile, mushroom shows great potential to tolerate and accumulate heavy metals such as Cd, copper, and mercury from soil because of its ability to produce intracellular and extracellular chelating compounds and antioxidant enzymes (Jiang et al. 2015b; Cen et al. 2012). Besides, mushroom has the capacity to degrade organics including polycyclic aromatic hydrocarbons (PAHs), phenols, dyes, etc., on account of its capacity to secrete ligninolytic enzymes, such as laccase (Akpinar and Urek 2014; Krastanov 2000; Zhu et al. 2013). Therefore, mushroom may be considered as a potential approach for the bioremediation of contaminated sites combined with heavy metals and organics.

Pleurotus eryngii is a sort of common white-rot fungus. In our previous experiments, we found that P. eryngii had the capacity to absorb heavy metal from contaminated soil (Tang et al. 2016). And P. eryngii is capable of secreting ligninolytic enzymes to degrade PAHs (Akpinar and Urek 2014; Hadibarata et al. 2014). Coprinus comatus is also a type of white-rot fungus that performs well in the absorption of heavy metals and generating laccase, which makes C. comatus specialized in accumulating heavy metals and decomposing organic pollutants (Xiang and Ding 2010; Cen et al. 2011; Wu 2015). Therefore, both P. eryngii and C. comatus have the potential to remediate heavy metal and organic contaminated soils. And there is a possibility to cultivate P. eryngii and C. comatus simultaneously owing to the similar cultivation condition they shared (Kang et al. 2000; Chaiyama et al. 2007). However, no information has been reported about the bioremediation of heavy metal and organic compound-contaminated soil with co-cultivated mushrooms.

Therefore, the objectives of this study were to investigate (1) the influence of Cd and endosulfan as co-contaminants on the growth of mushrooms, (2) the potential bioremediation ability of P. eryngii and C. comatus on co-contaminated soils, and (3) the effects of the co-cultivation with two different mushrooms on soil biochemical indicators, such as laccase enzyme activity and microbial counts.

2 Methods and materials

2.1 Chemicals

Endosulfan was purchased from XianNong Biological Technology Co., Ltd., China, with the effective composition content of 350 g/L. All chemicals and reagents utilized in this study were of analytical grade (Kelong Chemical Reagent Factory, Chengdu, China).

2.2 Experiment design and soil preparation

Forty-eight treatments were set in our study, including 12 blank control treatments cultivated without mushroom and with different levels of contamination (H1–H12), 24 control treatments cultivated with mycelia bags of P. eryngii or C. comatus and different levels of contamination (P1–P12, C1–C12), and 12 experiment treatments cultivated with combined mycelia bags of P. eryngii and C. comatus and different levels of contamination (T1–T12). Three replications were set in each treatment, and all treatments are shown in Table 1.
Table 1

The design and contamination content of all the treatments

Treatment

Mushroom

Concentration

Cd

Endosulfan

H1

None

10

0

H2

None

10

200

H3

None

10

400

H4

None

10

800

H5

None

30

0

H6

None

30

200

H7

None

30

400

H8

None

30

800

H9

None

0

0

H10

None

0

200

H11

None

0

400

H12

None

0

800

T1

P. eryngii and C. comatus

10

0

T2

P. eryngii and C. comatus

10

200

T3

P. eryngii and C. comatus

10

400

T4

P. eryngii and C. comatus

10

800

T5

P. eryngii and C. comatus

30

0

T6

P. eryngii and C. comatus

30

200

T7

P. eryngii and C. comatus

30

400

T8

P. eryngii and C. comatus

30

800

T9

P. eryngii and C. comatus

0

0

T10

P. eryngii and C. comatus

0

200

T11

P. eryngii and C. comatus

0

400

T12

P. eryngii and C. comatus

0

800

P1

P. eryngii

10

0

P2

P. eryngii

10

200

P3

P. eryngii

10

400

P4

P. eryngii

10

800

P5

P. eryngii

30

0

P6

P. eryngii

30

200

P7

P. eryngii

30

400

P8

P. eryngii

30

800

P9

P. eryngii

0

0

P10

P. eryngii

0

200

P11

P. eryngii

0

400

P12

P. eryngii

0

800

C1

C. comatus

10

0

C2

C. comatus

10

200

C3

C. comatus

10

400

C4

C. comatus

10

800

C5

C. comatus

30

0

C6

C. comatus

30

200

C7

C. comatus

30

400

C8

C. comatus

30

800

C9

C. comatus

0

0

C10

C. comatus

0

200

C11

C. comatus

0

400

C12

C. comatus

0

800

There were three replicates for each treatment

Soil samples used in this study were collected from Chengdu, Sichuan province of China. All of the soil samples were air-dried and milled to sieve through a 2-mm sieve to wipe out extraneous matters in soil, and stored for further experiments. The main properties of soil are given in Table 2.
Table 2

Basic properties of the untreated soil used for pot experiment before planting mushrooms

Properties

Values

pH

6.27 ± 0.04

Water holding capacity (%)

12.67 ± 0.29

Organic content (g/kg)

19.51 ± 0.32

CEC (cmol/kg)

10.43 ± 0.31

Total Cd (mg/kg)

Not detected

Results are expressed as means ± standard deviation (n = 3)

The concentration of trace elements in soil was an important indicator of environmental contamination (Narin et al. 1997; Soylak and Türkoglu 1999), and the level of Cd was selected on the basis of several literature studies of China and Europe (Li et al. 2015; Wang and Mulligan 2004; Chavez et al. 2016). Both heavy metal and organic contaminations used in this study were artificially added. Cd-contaminated soil was prepared by the addition of CdCl2·2.5H2O (0, 10, 30 mg/kg) into the stored soil, and marked as Cd0, Cd10, and Cd30 group, respectively. In order to make Cd uniformly distributed in soil, soil was equilibrated in a dark room for 2 months prior to the experiment. After that, Cd-contaminated soils were spiked with endosulfan (dissolved in acetone) at the concentrations of 0, 200, 400, and 800 mg/kg. Firstly, acetone-dissolved endosulfan was added into 25% of the prepared Cd-contaminated soil (6 kg), and the same amount of acetone was also added into the treatments without endosulfan. After acetone volatilization, the 25% of Cd-contaminated soil was mixed thoroughly with the rest of the 75%. Afterwards, co-contaminated soil was covered with aluminum foil and equilibrated in a dark room for 1 week.

2.3 Pot experiments

The pot experiment was carried out in a greenhouse to keep a constant temperature and to prevent direct sunlight. The experiment was conducted in plastic pots with 6 kg of the prepared soil. The mycelia bags were purchased from Shuangliu, Chengdu. In experiment treatments, the plastic pots were zoned into two regions, and the mycelia bags of both P. eryngii and C. comatus were cut into round pieces of about 0.25 kg each. Then, the round pieces of mycelia bags were spread on the prepared soil per 0.5 kg in two regions individually. After that, the mycelia bags were covered with the abovementioned soil for 3–4 cm. In the control treatments, 1 kg of round pieces of P. eryngii mycelia bags or C. comatus mycelia bags was cultivated in plastic pots through the same way as experiment treatments. In the blank control treatments, the same treatment was conducted as the control treatments, except cultivated with the same amount of sterilized mycelia bags. At the bottom of each pot, a plastic dish was set to collect leachate, and all of the pots were placed randomly in the greenhouse. During the cultivating period, deionized water was added through a watering can three times a week to compensate for water loss, and soil moisture content was contained at approximately 65% of the water holding capacity. About 2 months later, the mature fruiting bodies were harvested from each pot and washed with deionized water, then dried at 60 °C in an oven for 4 days. The dry mushrooms were powdered with a grinder after dry weight was recorded. In addition, soils from each pot were collected carefully, and dried under the natural condition for further experiments.

2.4 Heavy metal analysis

The concentration of Cd in mushrooms and soil was determined by the flame atomic absorption spectrometry (FAAS; Varian, SpectrAA 220FS). A powdered mushroom sample (0.2 g) was digested with the mixture of HNO3/HCl/HClO4 (3:2:3, v/v/v) in a microwave using medium high, medium, and low temperature each for 3 min. Metal speciation in soil was performed by the BCR procedure (Sungur et al. 2015). There were four chemical forms of Cd that could be sequentially extracted from soil: HOAc-extractable, reducible, oxidizable, and residual fractions.

2.5 Endosulfan analysis

The amount of endosulfan residual in soil was analyzed using a gas chromatograph-mass spectrometer (GC-MS; Shimadzu, GCMS-QP2010). Five grams of air-dried soil sample was extracted with 25 mL of n-hexane and 5 mL of 0.05 M H2SO4 in shake flasks for 30 min, then heated under ultrasonic bath for an hour at 40 °C. Finally, the above extracts were centrifuged, concentrated in a rotary evaporator, and transferred into a 1.5-mL sample vial. To separate the compounds, a Hewlett Packard-5 capillary column was used with a temperature gradient from 100 to 25 °C at a rate of 15 °C/min. The initial and final hold time was 2 and 5 min, respectively, and the detector temperature was 260 °C. The recovery of endosulfan was measured by adding a known concentration of the standard (250 mg/kg) into uncontaminated soil, and the recovery of endosulfan from spiked soils was above 89.67%.

2.6 Soil bacterial counts

Bacterial counts were conducted on Luria-Bertani agar through the plate spread method; 5 g of soil samples was diluted with aqueous serially and spread on the agar plate, and the total number of colony-forming units (CFU) was counted until incubation for 3–5 days at 34 °C in the dark.

2.7 Assay of laccase activity in soil

The extraction of laccase enzymes was in a modified method of Baldrian (Baldrian and Wiesche 2000). In brief, 1 g of soil sample was mixed with 5 mL phosphate buffer (0.05 M, pH 7.0) in a flask and incubated on ice for 1 h, shaking the flask occasionally. The suspension was centrifuged and collected for estimation of enzyme activities immediately. Laccase activity was measured by monitoring the oxidation of 3-ethylbenzothiazolone-6-sulfonic acid (ABTS). Ten milliliters of malonate-sodium buffer and 10 mL of ABTS were mixed up, putting the mixture into water bath at 30 °C. The laccase activity was determined spectrophotometrically at 420 nm with the reaction mixture that contained 1 mL of buffer, ABTS mixture, and 30 μL of the clean suspension; then, the absorbance was recorded every 30 s for three times, and the absorbency variation was calculated by taking the average value. The activity of laccase was defined as the amount of enzyme that catalyzed 1 μmol of substrate per minute per milliliter (Zhang et al. 2010).

2.8 Data analysis

The bioconcentration factor (BCF) of metal in mushrooms that came from soil was calculated according to the following equation:
$$ \mathrm{BCF}=\frac{\mathrm{Metal}\ \mathrm{concentration}\ \mathrm{in}\ \mathrm{mushroom}}{\mathrm{Metal}\ \mathrm{concentation}\ \mathrm{in}\ \mathrm{soil}} $$
The endosulfan removal rate from soil was calculated as the following:
$$ \begin{array}{l}\mathrm{Endosulfan}\ \mathrm{removal}\ \mathrm{rate}\ \left(\%\right)=\\ {}\frac{\mathrm{Initial}\ \mathrm{concentration}\ \mathrm{of}\ \mathrm{endosulfan}\ \mathrm{in}\ \mathrm{soil}-\mathrm{Concentration}\ \mathrm{of}\ \mathrm{endosulfan}\ \mathrm{in}\ \mathrm{soil}\ \mathrm{after}\ \mathrm{harvest}}{\mathrm{Initial}\ \mathrm{concentration}\ \mathrm{of}\ \mathrm{endosulfan}\ \mathrm{in}\ \mathrm{soil}}\\ {}\times 100\%\end{array} $$

Mean and standard deviation values of three replicates were calculated. Statistical significance was evaluated using SPSS package (version 21.0) with ANOVA, and means were compared using least significant differences (LSDs) calculated at a significance level of P = 0.01. All statistics were performed using the Origin 8 (USA).

3 Results and discussion

3.1 Growth response

With the same amount of mycelia bags in each pot, the biomass of mushrooms in the experiment group (T group) was 1.57–13.20% (except for T2, T6, and T7 treatments) and 19.75–56.64% higher than the group cultivating with P. eryngii (P group) or C. comatus (C group) individually (Fig. 1). In addition, with different concentrations of Cd and endosulfan, the biomass of mushrooms in P group was 12.51–132.22% higher than in C group. As a result, it was apparent that Cd had greater inhibiting effects on the biomass of mushrooms than endosulfan, both in the control and in the experiment treatments. Moreover, the inhibiting effects on mushroom biomass were enhanced with the increased concentration of endosulfan. However, the inhibition caused by Cd could be alleviated by endosulfan at some certain concentrations. When the concentration of endosulfan was 400 mg/kg, it was obvious that the biomass of mushrooms both in the control and in the experiment treatments increased compared to the treatments only spiked with Cd. On the contrary, the toxicity of Cd to mushroom enhanced with the addition of a high concentration (800 mg/kg) of endosulfan.
Fig. 1

The biomass (g/pot) of P. eryngii and C. comatus growing in soil with different concentrations (mg/kg) of Cd and endosulfan. Error bars represent the standard deviation of three sampled pots. Different uppercase letters indicate significant (P < 0.01) difference among treatments with the same mushroom-planting pattern and different pollutant concentrations. Different lowercase letters denote significant (P < 0.05) differences among treatments with different mushroom-planting patterns and the same pollutant concentration

The present study illustrated that the composition and concentration of contaminations had a direct effect on the biomass of mushrooms, and Cd showed greater toxicity than endosulfan, especially at a high concentration of Cd (30 mg/kg). Similar to our research, Batty et al. showed that plants grown in soil containing pyrene had greater growth rates than that grown in zinc-contaminated soil (Batty and Anslow 2008). Lu et al. also suggested that the addition of pyrene increased the plant biomass in Cu-contaminated and Cd-contaminated treatments, and the biomass was even slightly higher than the group without any pollutant (Lu et al. 2014). Besides, other chlorinated organic compounds like pentachlorophenol could also alleviate the inhibition on plant growth caused by heavy metals (Qi et al. 2006).

Shown from previous and our studies, mushrooms could grow without pronounced toxicity stress under the heavy metal and organic contaminated conditions. During the mushroom growth process, endosulfan could alleviate the toxicity caused by Cd at relatively low concentration, but at high concentration, inhibition also existed to the biomass of mushroom. Moreover, combined cultivating of P. eryngii and C. comatus resulted in higher biomass, testifying that co-cultivation of P. eryngii and C. comatus had better ability to tolerate the toxicities of Cd and endosulfan than individual cultivation, which indicated that they had potential to co-remediate the co-contaminated soil.

3.2 Soil bacterial counts

The bacterial counts in all treatments are shown in Fig. 2. At the same concentration of pollutants, the bacterial counts in T group were higher than in P and C groups. Such as T8 (30 mg/kg of Cd, 800 mg/kg of endosulfan) treatment, the bacterial numbers were 3.38 times that of P8 and 71.51% higher than C8. Between planted and unplanted groups (H group), the bacterial counts were significantly different. Especially in T group, the bacterial counts were 23.53–123.68 times that of H group. Zhang et al. reported that bacteria were the primary components and were important indicators of soil fertility (Zhang et al. 2004). Mushroom could produce intracellular and extracellular chelating compounds during the growth process, such as thiol (SH) functional groups containing compounds and antioxidant enzymes that had the ability to chelate with heavy metals and scavenge reactive oxygen species (ROS), thereby alleviating the toxicities of heavy metal and organic to bacteria and mushrooms (Gadd 1994; Bai et al. 2003). The chemical compounds such as organic acids, polysaccharides, and proteins produced by mushrooms also played important roles in heavy metal chelation as well as energy and nutrition supplies for bacteria (Baldrian 2003; Gadd 1994; Martino et al. 2002). In addition, different mushrooms were reported to have the capacity to secrete various kinds and amounts of extracellular compounds in the contaminated soils (Roy et al. 2000). Thus, in the co-cultivated group, there perhaps existed more species and amounts of extracellular compounds than in the individually cultivated groups, which consequently resulted in higher bacterial counts in soil.
Fig. 2

Bacterial counts in soil with different concentrations of Cd and endosulfan (mg/kg). Error bars represent the standard deviation of three sampled pots. Different lowercase letters denote significant (P < 0.01) differences among treatments with different mushroom-planting patterns and the same pollutant concentration

Seeing from Fig. 2, the bacterial counts were influenced by the concentration of Cd. Compared to the treatments without pollutant, the bacterial counts individually decreased 38.31, 32.30, 46.90, and 62.74% in T, P, C, and H groups when the concentration of Cd increased to 30 mg/kg. However, the bacterial counts displayed diverse changes along with the concentration of endosulfan. With a low concentration (200 mg/kg) of endosulfan, the bacterial counts were higher than in the treatments without endosulfan, particularly as the concentration of Cd was low. It had been reported that bacteria in soil had the capacity to absorb organic pyrene as substrate for microbial growth and thus enhanced the soil microbial biomass carbon (Lu et al. 2013). In the current study, the low concentration of endosulfan might be used as energy and substance resources to promote the activities of bacteria. But in the treatments with a high concentration (800 mg/kg) of endosulfan, the toxicity of Cd was enhanced. Compared to the treatments with low concentration (10 mg/kg) of Cd but without endosulfan (T1, P1, C1, H1), the addition of a high concentration (800 mg/kg) of endosulfan resulted in 30.83, 32.66, 16.32, and 4.74% decrease of bacterial counts in T, P, C, and H groups, respectively. Similar results were reported by Gogolev and Wilke (1997) in which the toxicity of heavy metals (Cd, Cu, Zn) increased with the addition of fluoranthene (Gogolev and Wilke 1997). Sikkema et al. (1994) explained that lipophilic compounds such as PAHs had the narcotic mode of toxic action and could interact with the lipophilic components of cytoplasmic membranes of bacteria, thus affecting the permeability and structure of bacteria (Sikkema et al. 1994). Therefore, with the addition of PAHs, heavy metal could penetrate into bacteria more easily. In our experiment, the high concentration of endosulfan, which also had a lipophilic property and probably destroyed the structure of bacterial cells, consequently resulted in a bacterial count decrease.

3.3 Total metal in mushroom and metal speciation in soil

3.3.1 Total metal in mushroom

After harvesting, the content of Cd in mushroom was measured and the data are shown in Fig. 3. Compared to P and C groups (1.04–2.28 and 0.67–2.60 mg/kg), the concentration of Cd was higher in T group (1.83–3.06 mg/kg). It could be observed that the content of Cd in mushroom was enhanced with the increase of Cd concentration in soil. Meanwhile, the content of Cd in mushroom was also affected by the concentration of endosulfan. As shown in the results, when the concentration of endosulfan was 400 mg/kg, the content of Cd in mushrooms decreased 2.25, 12.12, and 50.11% and 18.17, 40.84, and 10.22% in T3, P3, and C3 (with 10 mg/kg of Cd) and T7, P7, and C7 (with 30 mg/kg of Cd), respectively. However, in the condition of 800 mg/kg endosulfan, the content of Cd in mushroom presented an increased tendency, which corresponded to the mushroom growth response.
Fig. 3

Heavy metal content in P. eryngii and C. comatus with different concentrations of Cd and endosulfan (mg/kg). Error bars represent the standard deviation of three sampled pots. Different uppercase letters indicate significant (P < 0.05) differences among treatments with the same mushroom-planting pattern and different pollutant concentrations. Different lowercase letters denote significant (P < 0.05) differences among treatments with different mushroom-planting patterns and the same pollutant concentration

In our experiments, there perhaps existed some interactions between P. eryngii and C. comatus in the co-cultivated group to help with the mushroom growth processes and tolerate the toxicity caused by Cd and endosulfan.

3.3.2 Metal speciation in soil

There were all possible chemical forms (speciation) of heavy metal in solid, liquid, or biotic phases (Krishnamurti and Naidu 2000), and the bioavailability of heavy metal for organisms was directly determined by the speciation distribution. Figure 4 showed that the percentages of residual and HOAc-extractable Cd in T, P, and C groups were 3.19–4.60, 2.09–4.29, and 2.47–4.03% and 38.63–71.89, 22.06–47.63, and 21.20–56.94%, respectively. Thus, it could be summed up that the amounts of residual and HOAc-extractable Cd in soil increased with the co-cultivation of P. eryngii and C. comatus.
Fig. 4

Metal speciation in soil with different concentrations of heavy metal and endosulfan (mg/kg)

Previous researches reported that mushrooms had the capacity to secrete organic acids, such as citric, ketoglutaric, malic, succinic, and fumaric acids, during the growth processes (Yoshida et al. 1986; Ribeiro et al. 2008). The low molecular weight organic acids (LMWOAs) had the ability to alter soil pH and consequently affect the bioavailability of heavy metals. The study by Najafi and Jalali indicated that the increased concentration of LMWOAs decreased the pH of soil and thus promoted Cd removal from soils (Najafi and Jalali 2015). Figure 4 presents that the percentage of HOAc-extractable Cd in T group was 43.67–119.14 and 19.56–88.63% higher than in P and C groups under the same pollutant condition, which might be caused by the different amounts of LMWOAs produced during the growth processes of P. eryngii and C. comatus. Researchers reported that the bioavailability of heavy metals to organisms in soil enhanced with the increase of HOAc-extractable percentage (Ying et al. 2007; Chen et al. 2010). Therefore, the mobility of Cd in soil could be accelerated by the increased percentage of HOAc-extractable Cd, and consequently induced the higher content of Cd in mushroom. The bioavailability of Cd and other heavy metals was also governed by soil microorganisms and microbial-mediated progresses. Soil microorganisms could alter the metal bioavailability by releasing specific compounds to form complexes with Cd or by changing the pH of soil (Vig et al. 2003). From the result, the Cd speciation distributions were in accordance with the number of bacteria in different treatments, illustrating that microorganisms played an important role in altering the speciation of Cd in this study. Moreover, endosulfan played an important part in the speciation distributions of Cd. When the endosulfan concentration increased from 0 to 400 mg/kg, the percentage of HOAc-extractable Cd decreased to about 5.27–31.62, 10.28–19.3, and 3.72–29.23% in T, P, and C groups, respectively, which was coincident with the heavy metal accumulation in mushrooms, indicating that organic pollutant had the ability to alleviate the toxicity to mushrooms, and probably through forming metal-organic complexes. On the contrary, as the concentration of endosulfan was 800 mg/kg, the percentage of HOAc-extractable Cd increased compared with the lower concentration of organic, which meant that a high concentration of endosulfan had the capacity to promote Cd removal from soil. Jiang et al. reported that the addition of phenathrene enhanced the accumulation of Cd in Pleurotus cornucopiae in general (Jiang et al. 2015a). Similarly, Wang et al. also illustrated that the presence of PAHs decreased the stimulatory quality of Cd on the biomass of plant (Sedum alfredii) and the Cd phytoextraction efficiency (Wang et al. 2012).

In addition, as shown in Fig. 5, the BCF of Cd in T, P, and C groups were 9.16–24.30, 4.70–13.91, and 7.17–15.51%, respectively. And higher BCF in T group demonstrated that Cd could be removed more efficiently with the co-cultivation of P. eryngii and C. comatus under the same pollutant condition.
Fig. 5

Bioconcentration factor (BCF) in treatments with different concentrations of Cd and endosulfan (mg/kg). Error bars represent the standard deviation of three sampled pots. Different letters indicate significant (P < 0.05) differences among treatments with the same mushroom-planting pattern and different pollutant concentrations

3.4 Laccase activity

In all treatments, laccase was only detected in the groups cultivating with mushrooms. Figure 6 shows the laccase activity in different treatments. Comparing the laccase activity among T, C, and P groups with the same pollutant, in T group, laccase activity presented 16.89–81.90 and 21.14–116.02% higher than in P and C groups, respectively. Moreover, in all groups, laccase activity presented downtrends as the concentration of Cd increased, which might be caused by the enhanced toxicity to mushroom. Besides, laccase activity was influenced by the addition of endosulfan. Higher activity was detected in the treatments with endosulfan, especially as the concentrations of endosulfan were 200 and 400 mg/kg. Nevertheless, the activity of laccase exhibited downtrends when endosulfan concentration was 800 mg/kg, which was also in conformance with the growth response of mushroom (Fig. 1), and providing further evidences that laccase activity could be affected by the change of growth condition of mushrooms. Therefore, endosulfan had the capacity to simulate the activity of laccase, and different concentrations of endosulfan had different impacts on laccase activity. In the research of Forootanfar et al. (2012), the activity of laccase was simulated by organic pollutants, like guaiacol, xylidine, and veratryl alcohol at the concentration of 250 μM, but decreased below or higher than the critical concentration (Forootanfar et al. 2012).
Fig. 6

Laccase activity in soil with different concentrations of Cd and endosulfan (mg/kg). Error bars represent the standard deviation of three sampled pots. Different letters indicate significant (P < 0.05) differences among treatments with the same mushroom-planting pattern and different pollutant concentrations

More importantly, laccase secreted from fungi had the capacity to transform xenobiotic compounds and catalyze the oxidation process of organic pollutants, including PAHs, polychlorinated biphenyls, and pesticides (Baldrian 2008; Sinsabaugh 2010). In this study, the activity of laccase secreted from mushrooms cultivated in the co-contaminated soil illustrated that both P. eryngii and C. comatus had the potential to resist the toxicities of soil pollutants and remediate endosulfan-polluted soil.

3.5 Dissipation of endosulfan

The dissipation experiment results (Fig. 7) showed that the endosulfan in soil was dissipated both in planted and unplanted treatments in a great measure. The removal rates of endosulfan were 90.67–99.38, 88.99–99.20, 88.47–99.31, and 87.09–97.65% in T, P, C, and H groups, respectively. Among the treatments with the same concentration of pollutants, no significant difference of endosulfan residual was found in most of the planted groups. On the contrary, there was a significant (P < 0.05) difference among the planted and unplanted groups. More concretely, the residual of endosulfan in H4 (10 mg/kg of Cd and 800 mg/kg of endosulfan, without mushroom) was about 41.10, 16.34, and 31.27% higher than T4 (with co-cultivation of P. eryngii and C. comatus), P4 (with cultivation of P. eryngii), and C4 (with cultivation of C. comatus), respectively. The residual of endosulfan in soil was influenced by the presence of Cd. Generally, there was no obvious difference when the concentration of Cd was 10 mg/kg in comparison to the treatments without Cd. However, as the concentration of Cd increased to 30 mg/kg, the dissipation of endosulfan displayed an inhibiting effect in all treatments, which was due to a higher toxicity in soil caused by Cd. As the concentration of Cd increased to 30 mg/kg, biomass of mushrooms, bacterial counts, and laccase activity in soil all displayed declining trends. Besides, it was noteworthy that co-degradation of endosulfan by P. eryngii and C. comatus was more effective than individual degradation by P. eryngii or C. comatus, even though the advantage of co-degradation was not significant.
Fig. 7

The residual endosulfan in soil with different concentrations of Cd and endosulfan (mg/kg). Error bars represent the standard deviation of three sampled pots. Different letters denote significant (P < 0.05) differences among treatments with different mushroom-planting patterns and the same pollutant concentration

Kathpal et al. reported that the dissipation of total endosulfan residues occurred to an extent of 92–97% in the first 4-week period and by about 99% in 238 days in two distinct phases: in first-order kinetics through volatilization, hydrolysis, and metabolization, and in photochemical and microbial degradation (Kathpal et al. 1997). In the studies of Kamei et al., the dissipation of endosulfan was more effective in the presence of mushrooms. They demonstrated that the white-rot fungi Trametes hirsute had the capacity to degrade 90% of endosulfan after 14-day incubation (Kamei et al. 2011). Moreover, white-rot fungi had the capacity to degrade xenobiotics due to the capacity to produce extracellular laccases, oxidases, and hydrolases. In the culture media, fungi grown heterogeneously and interacted with endosulfan in physical and biochemical ways either occluded the compound in the mycelium or through the extracellular enzymes to degrade it (Rivero et al. 2012). Xu et al. demonstrated that combined plant cultivation was effective in PAH contamination remediation; planting with maize and ryegrass significantly enhanced the rate and extent of phenanthrene degradation. After 60 days of incubation, the concentration of phenanthrene was reduced from 52.53 to 4.50, 4.15, and 1.00 mg/kg in soil planted with ryegrass alone, maize alone, and a combination of ryegrass and maize, respectively (Xu et al. 2006). In addition, some bacteria in soil were able to use endosulfan as sulfur source and carry necessary enzymes to degrade the organic pollutant (Hussain et al. 2008). In the research of Wang et al., the microbial population in soil corresponds with PAH residue, and treatments with higher microbial population had higher PAH degradation effects and lower PAH residue (Wang et al. 2016).

In this study, the dissipation of endosulfan was complex, including physical, chemical, and biological processes. From the results above, both laccase activity and soil bacterial counts were enhanced with the cultivation of mushroom, which could lead to a strengthened effect on the dissipation of endosulfan. Hence, the co-degradation was a feasible biomethod to remediate the organic polluted soil and probably became more effective with some modification.

4 Conclusions

The growth response of P. eryngii and C. comatus in co-cultivated group and the interaction of Cd and endosulfan on their fates and soil biochemical properties in Cd and endosulfan co-contaminated soil were investigated in this study. Co-contamination level had an obvious influence on the biomass of mushroom and soil biochemical properties. The biomass of mushroom, laccase activity, bacterial counts in soil, and Cd content in mushroom were significantly enhanced in the co-cultivated group in comparison to the individually cultivated groups. The dissipation of endosulfan was evidently promoted by the presence of mushroom, and the co-cultivated group showed slightly higher dissipation effect than individually cultivated groups. The present study firstly reported the remediation effect by co-cultivated macro fungi P. eryngii and C. comatus on Cd and endosulfan co-contaminated soil, and the results are important for a better understanding of co-remediation for co-contaminated soil.

Notes

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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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Key Laboratory of Bio-Resources and Eco-Environment (Ministry of Education), College of Life SciencesSichuan UniversityChengduChina

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