Interfacial effect of Stropharia rugoso-annulata in liquid medium: interaction of exudates and nickel-quintozene

Abstract

This work investigated the accumulation of nickel (Ni) and dissipation of quintozene (PCNB) by the mycelia of Stropharia rugoo-annulata (S. rugoo-annulata), together with the correlation between cell exudates and contaminants removal in liquid medium. Results showed that the removal rates of PCNB accounted for 20.75–55.26% and 42.39–90.92% of the initial concentration (125 mg kg⁻¹) in un-inoculated and inoculated media, respectively. Ni accumulation in mycelia of S. rugoo-annulata at the end of experiment was 81.09 mg kg⁻¹ when the initial concentration of Ni was 30 mg L⁻¹ in polluted media, among which the proportion of NaCl-extractable (56.34%) was dominant. These results showed that PCNB and Ni were remarkably removed by mycelia incubation. The concentrations of cell exudates (macromolecular substances, low-weight-molecular organic acids (LMWOAs), liglinolytic enzymes) were quite different in both polluted and natural media inoculated with S. rugoo-annulata, indicating that the production of exudates was closely related to PCNB and Ni. Besides, the results of scanning electron microscopy (SEM) and diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS) demonstrated that the pollutants influenced the surface phenotypic structure but not for basic cell structures, implying mycelia of S. rugoo-annulata could well tolerate the pollutants. Our results suggested the presence of S. rugoo-annulata was effective in promoting the bioremediation of Ni–PCNB and laid the foundation for a better understanding of the mechanism about contaminant removal by mushroom.

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1. Introduction

Several contaminates are widely persistent in soil and water as a result of human activities. Among these, heavy metals and organic pollutants are the two major chemical families that cause water and soil pollution.¹ Understanding the transport and fate of contaminants is of primary importance for pollutant management and treatment optimization. Ni is one of the eight common heavy metals and is of great toxicity to organisms,² as well as being the second excess metal in the soil of Chengdu, China. PCNB is often used as a fungicide and is persistent in both soil and water, which causes sensitization by skin contact and is very toxic to aquatic organisms.³ There are many sites co-contaminated with PCNB and Ni because of the traditional use of pesticide. Compared with individual pollutants, combinations in the form of metal–organic complexes are more complex and difficult to remediate.⁴

Mycoremediation is a cost effective and ecological method for organic pollutants dissipation,⁵ and mushroom as a representative has shown the potential to remove not only organic pollutants but also heavy metals.⁶ Mushroom is easy to cultivate, and has aggressive growth and biomass production,⁷ which overcome the disadvantage of phytoremediation and lay the basis for the pollutants removal. Furthermore, Agaricus bisporus, Trametes versicolor and Pleurotus ostreatus have been reported to remediate in the polluted sites both in situ and ex situ.⁸ S. rugoso-annulata, as one of the most efficient degraders for organics among decomposing fungi, has also been identified to degrade dyes, benzo(a)anthracene, benzo(a)pyrene and dibenzo(a,h)anthracene.^9,10 Nevertheless, there is lack of information on synchronous remediation of Ni–PCNB by S. rugoso-annulata.

Previous studies on bioremediation (phytoremediation and mycoremediation) for soil and water which contaminated with heavy metals and organics mainly focused on the accumulation of heavy metals and dissipation of organics.^2,4 However, little is known about remediation mechanism of pollutants and their surroundings, particularly for the cell exudates. Therefore, studying the correlation between cell exudates and contaminants removal is likewise critical and could lay the foundation of pollution control in co-contaminated conditions.

Cell exudates consist of different carboxylic acids, alcohols, carbohydrates, proteins, lipids and all kinds of enzymes,^8,11 which can influence the pH and biocompatibility of organic matters in rhizosphere sediment and play an important role in contaminates removal.¹² The combinations of carbohydrates and proteins have great effects on metal complexation, especially for the proportion of carbohydrates.¹³ LMWOAs exuded by mushroom varied with species, and have been shown to influence metal bioavailability,¹⁴ which are closely linked to metal migration and removal from contaminated sites.¹⁵ In addition, ligninolytic enzymes (laccase and manganese peroxidase (MnP)) from mushroom are conducive to the degradation of many organic pollutants including phenols, polychlorinated biphenyls, dyes etc.^16–18 Many researchers have reported that exudates were relative to metals uptake or organics degradation.^9,16 However, there is lack of systemically research on the relation between the contaminants removal and exudates. In addition, the effects of mixed pollutants of Ni and PCNB on cell exudates, and how exudates relate to pollutants removal, are unknown.

This paper attempts to clarify the correlation between mixed pollutants (Ni, PCNB) removal and exudates from S. rugoso-annulata. The aim is to detect the exudates variation of S. rugoso-annulata under the co-contamination surroundings, and finally find whether the S. rugoso-annulata has the potential to degrade PCNB and accumulate Ni synchronously in co-contaminated conditions.

2. Materials and methods

2.1 Experiment design

In this study, single PCNB (50, 75, 100, 125, 150, 200 mg L⁻¹) and Ni (10, 20, 30, 40, 50, 100 mg L⁻¹) treatments were set to screen the optimum addition level, respectively. Based on the preliminary experiment, the initial concentrations of PCNB and Ni were 125 and 30 mg L⁻¹ in the co-contamination experiment, respectively. The experimental designs are shown in Table 1, which revealed the CK were media polluted with PCNB and Ni without inoculation, T1 represented the media inoculated mycelia without any pollutants spiked, and T2 were treatments with both PCNB + Ni contamination and mycelia inoculation. All these groups were tested with three replications.

Table 1 The design of all the treatments^a

	CK	T1	T2
a +: the ingredient(s) was/were contained. −: the ingredient(s) was/were not contained.
S. rugoso-annulata	−	+	+
Ni + PCNB	+	−	+

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Potato culture medium was used for the S. rugoso-annulata cultivation in present work.¹⁹ Strain of S. rugoso-annulata was purchased from Sichuan Academy of Agricultural Sciences (China), and then was propagated in a culture dish (9 cm diameter).

0.5 mL of acetone filtrate containing 0.025 g mL⁻¹ PCNB, which dissolved by acetone (≥99%) in sterile test tubes and filtered through 0.22 μm filter membrane to get rid of germs, was added to flask. After the solvent was evaporated, 100 mL of medium per flask containing 30 g mL⁻¹ Ni²⁺ (NiCl₂·6H₂O) was added to PCNB-spiked medium. Finally, the initial concentrations of PCNB and Ni in medium were 125 mg L⁻¹ and 30 mg L⁻¹, respectively. The flasks were inoculated with 5 pieces (1 cm diameter) of S. rugoso-annulata and shaken at 27 °C for 15 days, then the growth and pH were measured. At the end of each growth phase the exuded material in the culture was obtained by tangential filtration through 0.65 mm hollow fiber polysulfone cartridges.¹³ The mycelia were washed with deionized water, dried at 60 °C for 3 days in oven and dry weights were recorded. The pH of medium was determined by a pH meter (METTLER TOLEDE S210) after standing for 10 min. The filtrate was stored at 4 °C for exudation analysis within 24 h.

2.2 Chemical characterization of the S. rugoso-annulata exudates

Total dissolved proteins, exo-polysaccharides and lipids were determined in previously filtered samples. Proteins determination was based on the Coomassie brilliant blue reagent with crystalline bovine albumin as standard and determined at 595 nm.²⁰ Total dissolved carbohydrates were based on the phenol-sulfuric acid reaction with glucose as standard and quantified at 487 nm.²¹ Lipids were measured by Rose-Gottlieb method.²¹

Laccase and MnP activity in exudates were analyzed through spectrophotometry.^22,23 Laccase was determined with 2,2-azino-di-[3-ethyl-benzothiazolin-sulphonate] (ABTS) as a substrate. One activity unit of laccase was defined as the amount of enzyme that oxidised 1 μmol ABTS min⁻¹. MnP determination was based on the oxidation Mn²⁺ to Mn³⁺. One unit of the enzyme activity of MnP was defined as 0.1 of ΔOD_{240 nm} increase min⁻¹.

LMWOAs (including oxalic acid, malic acid, lactic acid, citric acid, succinic acid and vitamin C) were determined by high performance liquid chromatography (HPLC, Shimadzu CBM-20A, Japan) according to Chen et al.²⁴ All samples were prepared with Milli-Q water (Milipore RiOs-DI, France) and filtered through 0.45 μm membranes.

2.3 Heavy metals analysis

The accumulation of Ni in hypha was determined by flameless atomic absorption spectrophotometry (FAAS, VARIAN, SpectrAA-220Fs) according to Xu et al.²⁵ The dry hypha (0.3 g) was digested for 40 min with a microwave in a mixture of concentrated nitric acid (HNO₃), 30% hydrogen peroxide (H₂O₂) and perchloric acid (HClO₄) (5 : 4 : 3, v/v). The digestion procedure was assessed by analyzing tea certified reference material (GBW 07605, GSV-4) and soil (GSBZ 50014, ESS-4), most of the determined recovery values for Ni were above 95%.

Metal speciation in hypha was performed by the method of Lu et al.²⁶ with some modifications. 0.5 g of fresh hyphae was extracted by 50 mL of the extraction solutions listed below in order and shaken for 22 h at 25 °C. The homogenate was centrifuged at 5000 rpm for 10 min and the first supernatant solution was transferred in a flask bottle. The sedimentation was re-suspended twice in extraction solution and shaken for 2 h at 25 °C, centrifuged at 5000 rpm for 10 min, then pooled the supernatant of the three suspending. Each of the pooled supernatant solution was evaporated on an electric-plate at 70 °C to constant weight, then digested for 40 min with a microwave in a mixture of HNO₃, 30% H₂O₂ and HClO₄ (5 : 4 : 3, v/v).

(1) 80% ethanol, extracting ethanol-soluble protein-bound metal ions, (2) deionized water, extracting water-soluble metal of organic acid complexes, (3) 1 M sodium chloride (NaCl), extracting pectates and protein integrated metal, (4) 2% acetic acid (HAc), extracting sparingly soluble Ni phosphate, and (5) 0.6 M hydrochloride (HCl), extracting insoluble metal oxalates. The residual metal in mycelia was measured as the procedure of the total metal determination, and the metal concentration in digestion solution was determined by FAAS.

2.4 PCNB analysis

PCNB concentration in medium was analyzed through gas chromatography-mass spectrometer (GC-MS). 10 mL of filtrate was extracted with 50 mL of mixed extraction agent containing acetone and petroleum ether (1 : 4) in a shake flask for 30 min, then in ultrasonic for 20 min. Centrifuging the extracts and collecting organic layer, then the extracts were added with 0.2 g of anhydrous sodium sulfate to remove any moisture and concentrated in a rotary evaporator, finally the contents were transferred to a sample vial and re-suspended in 1.5 mL n-hexane and 1 μL sample was analyzed by Agilent 6890 N GC-MS. A capillary column with a temperature gradient from 120 °C to 280 °C at a rate of 10 min⁻¹ was used to separate the compounds. The final hold time was 5 min, and detector temperature was 250 °C. PCNB recovery was measured by adding a known concentration of standard (50 mg kg⁻¹) to uncontaminated medium, and the recoveries of PCNB from spiked samples were above 85%.

2.5 Phenotypic changes

To evaluate the phenotypic and structure or biochemical changes of hypha induced during pollutants exposure, SEM equipped with a Princeton Gamma-Tech energy dispersive spectroscopy (EDS) microanalysis system and DRIFTS were used. For SEM, regular cross sections of mycelia samples were generated using a surgical blade and the pieces were coated with 2–3 nm of gold prior to analysis. Samples were analyzed using an accelerating voltage of 20 kV at 250–400 magnification and a working distance of 14 mm. For DRIFTS, 5% mixture of freeze-dried hyphae were prepared by grinding and thoroughly mixing with potassium bromide (KBr). Infrared spectra were recorded using a Thermo Scientific Nicolet 6700 FTIR equipped with a smart collector diffuse reflectance accessory and mercury cadmium telluride (MCT/A) detector. Each samples spectra were at an average of 254 spectra collected with 4 cm⁻¹ resolutions.

2.6 Statistics

Mean and standard deviation values of three replicates were calculated in present experiment. Statistical analyses were performed using statistical product and service solutions (SPSS) for Windows, Version 21.0, and means were compared using least significant differences (LSD) calculated at a significance level of P < 0.01. All figures were performed using Origin V8.5 software.

3. Results and discussions

3.1 Screening of co-contaminants level

Growth parameter is an important indicator of stress response for mushroom under environmental stress,²⁷ thus the biomass of S. rugoso-annulata was used to screen the pollutants level under different initial concentrations of Ni and PCNB (Fig. 1). The results showed that the Ni treatments had a stronger toxicity to the S. rugoso-annulata compared with the PCNB treatments, and the growth of S. rugoso-annulata was significantly inhibited when the concentration of Ni reached to 30 mg L⁻¹. For PCNB treatments, higher concentrations of PCNB enhanced the biomass of mycelia. And the 125 mg L⁻¹ of PCNB immensely promoted the increase of biomass compared to mycelia grown in the medium without any spiked, which was in line with the study that organics contributed to facilitate the growth of plants in certain concentration.²⁸ Therefore, given to the combined effects of PCNB and Ni, 125 mg L⁻¹ PCNB and 30 mg L⁻¹ Ni were selected as initial concentrations in the co-contaminated experiment.

Fig. 1 Biomass of S. rugoso-annulata grown in media spiked with different initial concentrations of Ni and PCNB. Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

3.2 Biomass of S. rugoso-annulata

To evaluate the tolerance of mycelia for the initial concentrations of PCNB (125 mg L⁻¹) and Ni (30 mg L⁻¹) chosen in preliminary experiment, the biomass of S. rugoso-annulata was recorded (Fig. 2). Compared to media without any pollutants spiked, the growth of mycelia in polluted media were slightly inhibited in the first 11 days (Fig. 2). Moreover, the biomass of S. rugoso-annulata in polluted medium was 106.22% of control in natural medium at the end of experiment, and increased with the increment of the incubation time. These results suggested the initial concentrations of PCNB and Ni chosen in preliminary experiment exhibited no visible toxic symptom on the growth of mycelia, which further indicated the S. rugoso-annulata could well tolerate the co-contamination of PCNB and Ni.

Fig. 2 Biomass of S. rugoso-annulata grown in media spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹). Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

Although it was proved that heavy metals could damage the growth and metabolism of mushroom,^29,30 the PCNB facilitated the growth of the S. rugoso-annulata as shown in Fig. 2, which contributed to enhance the tolerance of mycelia for pollutants. Besides, the toxicity of the co-contamination of PCNB and Ni might obtain a certain degree of alleviation through a number ways depending on biodegradation, bioaccumulation, chelation or precipitation of mycelia.²⁶ Therefore, these results demonstrated the mycelia of S. rugoso-annulata may possess a great potential to tolerate the co-contamination of Ni and PCNB, and hence, it is a feasible choice for remediation.

3.3 PH change in medium

The pH in all inoculated groups decreased compared with un-inoculated groups (Fig. 3), which might attribute to the secretion of cell exudates that could help to reduce the pH in media, such as organic acids.¹² Besides, the pH values in inoculated media without pollutants were lower than that in inoculated media with pollutants except for the last incubation time, suggesting the pollutants probably influenced the production of exudates. Moreover, the gradually decline trend in inoculated groups was perhaps attributable to the secretion of exudates, whose carboxyl, sulphonic and amide groups contributed to the increase of hydrogenion (H⁺) in solution.³¹ The decrease of pH could result in metal activation,³² and it was reported that the mobilization of metals significantly increased with the decrease of pH in the rhizosphere.³³ Thus the decrease of pH might greatly promote the uptake of Ni by the mycelia of S. rugoso-annulata.

Fig. 3 pH values of all media in inoculation and un-inoculation medium spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹). Error bars represent the standard deviation of three samples. CK: control, without any spike. T: treatment, with initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹).

3.4 Chemical characterization of the S. rugoso-annulata exudates

3.4.1 Macromolecular substances in exudates. Fig. 4 shows macromolecular substances of exudates excreted by mycelia in media with or without Ni (30 mg kg⁻¹) and PCNB (125 mg kg⁻¹). Macromolecular substances (protein, polysaccharides and lipid) were detected during the assay to investigate their effects and relations with the pollutants removal. Protein, polysaccharides were both highest on the 9th day, accounting for 213.96% and 490.93% of control, respectively (Fig. 4). Fig. 4A shows the protein contents firstly increased in the first 9th days, then decreased at the anaphase of growth period in polluted media. The enhancement of protein contents might be due to the inducement of Ni since it was reported that heavy metals could promote the secretion of exudates.³⁴ The decline in protein contents probably ascribed to the damage produced by Ni with the increase of enrichment amounts in mycelia,³⁵ and consequently resulting in the reduction of the protein contents. Lipid contents were relative low in both natural and polluted media, but the contents of lipid in polluted media were higher compared to natural media in later culture periods (Fig. 4B), indicating the pollutants also could slightly promote the lipids secretion by mycelia. Fig. 4C shows the polysaccharides contents in polluted media were higher than that of in natural media apart from on the 3rd day, indicating the contaminants had a vast promotion on the secretion of polysaccharides. Similar to the trend of protein contents, polysaccharides likewise increased then decreased in the whole incubation time, showing the mycelia would secret more polysaccharides and proteins under the inducement and stimulation of pollutants.³⁵ Nevertheless, Ni produced harmful effect on the growth of mycelia with the increase of accumulation amounts in mycelia, thus affecting the secretion of proteins and polysaccharides.^35,36 Klai Nouha also observed the same tendency of protein and polysaccharide contents in wastewater contaminated with heavy metals,³⁷ which was akin to our study.

Fig. 4 Concentrations of macromolecular substances of exudates in inoculated media spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹). (A) Protein, (B) lipid, (C) polysaccharide. Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

The macromolecular substances have many sites for adsorption of metals, such as aromatics, aliphatics in proteins, and hydrophobic regions in carbohydrate,³⁸ and it could also adsorb phenanthrene,³⁶ benzen,³⁸ and dye,³⁴ which might attribute to the hydrophobic regions in extracellar exudates.^34,36 Polysaccharides were reported to complexation with heavy metals,³⁹ and Goodwin et al. demonstrated the affinity of polysaccharides to cadmium (Cd) and copper (Cu).⁴⁰ Besides, Fukushi et al. expounded the important role of proteins in fixing of metals ions.⁴¹ For lipids, G. Guibaud found there were no correlations between lipids, nucleic acids and uronic acids contents and their affinity for metals,⁴² and therefore lipids probably hadn't significant influence on metal complexation. These results suggested that the proteins and polysaccharides excreted by mycelia of S. rugoso-annulata probably acted as an important response to environmental stress, which contributed to the removal of PCNB and Ni, and laid the basis for better understanding the bioremediation mechanism of pollutants by mushroom.

3.4.2 LMWOAs. LMWOAs were proved to be closely related to the mechanism of bioremediation of heavy metal and organic compounds,³³ and therefore, the LMWOAs were chosen to evaluate the removal of PCNB and the transportation of Ni. Oxalic, vitamin C, malic, succinic, lactic, succinic and citric acids were detected in both natural and polluted media (Fig. 5). However, the concentrations of the six LMWOAs varied significantly along with the incubation time. In polluted medium, the highest contents of oxalic, lactic, malic, succinic and citric acids reached up to 0.85, 0.79, 0.62, 0.62 and 0.48 mg mL⁻¹, respectively, and the concentration of vitamin C were low in both natural and polluted media (Fig. 5). These results showed S. rugoso-annulata could secret relative high concentrations of LMWOAs even in the co-contamination environment, attesting its potential to remove pollutants in complex environment. Compared to natural media, concentrations of LMWOAs in polluted media were lower in the early culture time but were higher in the middle of culture time (7th and 9th days) except for vitamin C. Besides, Hou et al. also found the contents of the water-soluble organic acids (citric acid, succinic acid and glutaric acid) increased in pyrene and lead co-contaminated soil.³¹ These results together suggested the pollutants might inhibit the mycelia in the early culture stage but promote in the middle stage.¹¹ Furthermore, it was interesting to note that the secretion of citric and malic acids were closely linked. Once the citric acids were excreted less, more malic acids would be secreted. Similar to our study, Lu et al. also found this tendency between citric and malic acids when investigating the effect of LMWOAs secreted by mangrove in the presence of Cd.⁴³ These phenomena might indicate the citric and malic acids might act together under environmental stress.

Fig. 5 Concentrations of LMWOAs of exudates in inoculated media spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹). (a) Oxalic acid, (b) lactic acid, (c) vitamin C, (d) citric acid, (e) malic acid, (f) succinic acid. Error bars represent the standard deviation of three samples.

It has been widely reported that LMWOAs might be related to the tolerance and transportation of heavy metals in plants.¹⁵ Besides, previous studies reported that the organic acids secreted from plant were in response to high concentration of metal as a detoxifying mechanism.^44,45 The LMWOAs could directly activate metals, and thus making metal available for mushroom uptake.³² In addition, metals bioavailable are also greatly influenced by the pH in their surroundings,⁶ and pH of exudates solution was reported to connect with the LMWOAs secreted by the root of plant.³³ What's more, the LMWOAs could not only affect pH and the form of heavy metals,³³ but also promote biocompatibility, absorbability and biodegradability of polycyclic aromatic hydrocarbons (PAHs).¹¹ Citric acids and other organic acids can significantly increase the accumulation of manganese (Mn), zinc (Zn), Cu, lead (Pb), and Cd.^13,46 Succinic and lactic acids were positively related to the removal percentage of PAHs.^11,15 Wherefore, it can be easily speculated that the LMWOAs secreted by S. rugoso-annulata probably were significantly conductive to the removal of contaminants and were closely related to the mechanism of contaminants removal by mushroom.

3.4.3 Ligninolytic enzymes system. Previous researches have summarized that the ligninolytic enzymes secreted by mushroom played an important role in the degradation process of diverse organics.^16–18 With respect to ligniolytic enzymes activity, only laccase and MnP were detected in both polluted and natural media inoculated with mycelia (Fig. 6). Laccase in polluted media showed highest activity on the 9th day, approximately ten times higher than that of control (Fig. 6A). Moreover, laccase activities in trails with pollutants was higher than in trails without pollutants except for on the 3rd day, which probably ascribed to the stimulation and inducement of contaminants in polluted media.^6,8 In polluted media, the activity of laccase increased in the first 9th days and then decreased but increased at last, which was in agreement with the report of HA Akdoganthe et al. who studied the biodegradation of fluorene by P. ostreatus.⁴⁷ MnP in media decontaminated with Ni and PCNB presented higher activities than in natural media during the whole culture period (Fig. 6B), and MnP activity distinctly increased by 589.80% on day 11th compared to natural medium. What's more, MnP activities changed with the similar tendency as laccase did in polluted media, indicating a positive co-effect of high levels of Ni and PCNB on MnP and laccase activities in the initial growth stage. The falling of laccase and MnP activities might be owing to the less inducement effect as well as more serious biological toxic effect of pollutants, which the pollutants in medium were relative lower than the initial concentrations at the end of experiment and produced more damage to the mycelia for the accumulation effect.⁴⁵ Besides, Zhou et al. also discovered similar phenomenon in studying the influence of Cu and trichlorophenol on MnP and laccase activities.⁴⁵

Fig. 6 Concentrations of ligninolytic enzymes of exudates in inoculated media spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹). (A) Laccase, (B) MnP. Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

There are many reports about the enzymatic degradation of xenobiotic compounds by ligninolytic enzymes system.^47–49 Laccase was reported to be capable of translating, degrading organic compounds and complexing heavy metals,³² it has been documented to catalyze the oxidation of a wide varieties of organics with phenolic structures, such as PAHs, polychlorinated biphenyls, synthetic dyes, pesticides, and iron–cyanide complexes.⁵⁰ Hence the exudation of laccase would result in a better degradation of PCNB. Acevedo et al. reported the five rings PAHs were greatly removed with the enhancement of MnP produced by Anthracophyllum discolor,⁴⁹ therefore MnP probably likewise contributed to the degradation of PCNB. Although lignin enzymes (LiP) was included in ligninolytic enzymes system, the LiP might be too low to detect in present study. Wesenberg et al. who studied the white-rot fungi and their enzymes for the treatment of industrial dye effluence, demonstrated that the shaking culture condition was more suitable for the exudation of laccase but not for the production of LiP,⁵¹ and thus probably resulting in the un-detection of LiP in present study. Even so, our results still indicated the pollutants could efficiently promote the secretion of ligninolytic enzymes (laccase and MnP), and resulting in a better degradation of PCNB. Besides, it could be inferred that the exudation of ligninolytic enzymes might closely connect with the removal of pollutants by mushroom.

3.5 Metal content and speciation in hyphae of S. rugoso-annulata

The accumulation of Ni in the mycelia was 81.09 mg kg⁻¹ as the initial concentration of Ni was 30 mg L⁻¹ in polluted medium at the end of experiment. However, the enrichment of Ni was reported only 3.16 mg kg⁻¹ in Agrocybe aegerita inoculated with bacteria in Cd–Ni (100 mg L⁻¹) contaminated soil, and reached to 7 mg kg⁻¹ in the Ni hyper-accumulator associated with organic acids when the initial concentration of Ni was 25 mg kg⁻¹.^52,53 Thus our results revealed that the mycelia of S. rugoso-annulata could well bio-accumulate Ni in co-contamination medium. Besides, the metal speciation was assayed by using the sequential extraction method (Fig. 7). The percentages of heavy metals in different fractions from high to low were: NaCl-extractable (56.43%) > ethanol-extractable (23.52%) > water-extractable (11.94%) > HAc-extractable (5.13%) > HCl-extractable (1.73%) > residual (1.67%). Bioavailable fractions including NaCl-extractable, ethanol-extractable and water-extractable were dominant, which were conductive to Ni translocation from medium to mycelia,¹⁵ and therefore promoting the removal of Ni. The residual was the lowest proportion in mycelia of S. rugoso-annulata, which might ascribe to the activation of LWMOAs.³²

Fig. 7 Metal speciation in mycelia grown in media spiked with initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹) after 15 day incubation. Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

Ni uptake by the mycelia of S. rugoso-annulata was not only dependent on its total concentration, but also its chemical forms.⁵⁴ On one hand, The LMWOAs secreted by mycelia could influence the forms of heavy metals by acidizing insoluble heavy metal fraction, chelating and reducing heavy metal⁵⁵ as well as influenced the pH,³³ contributing to enhance the uptake and simultaneously alleviate the detrimental effect of Ni. Hou et al. reported that adding citric acid increased the mobile forms of Pb.³¹ Other studies also found that Ni uptake was enhanced via interaction with citric acid.⁵³ In addition, the pH values of inoculated media were relative low (Fig. 3), which were conductive to the mobilization of Ni, and thus further promoting the Ni uptake by mycelia. On the other hand, the extracellular macromolecular substances have many adsorption sites for metals, such as carboxyl, phosphoryl, sulfhydryl, phenolic and hydroxyl groups.³⁸ These sites offered cation exchange potential and hence complexed with Ni,³⁹ which were helpful for alleviating the toxicity of Ni to mycelia. Moreover, Guibaud discussed the correlations existing between the parameters of complexation with Cd, Pb and Cu and the composition in proteins and polysaccharides, and found that the coefficients were relatively high,⁴² clarifying the important effects of macromolecular substances in fixing heavy metals and enhancing the tolerance of mycelia for metals. Accordingly, the LMWOAs and macromolecular substances secreted by S. rugoso-annulata might promote the enrichment of Ni and alleviate the toxicity of Ni, collaboratively leading to the removal of Ni in polluted media.

3.6 Removal of PCNB

PCNB was greatly removed both in un-inoculated and inoculated media at the end of experiment, accounting for 55.26% and 90.92% of the initial concentration (125 mg L⁻¹), respectively (Fig. 8). The higher remove rates of PCNB in inoculated media showed the mycelia of S. rugoso-annulata could well degrade the PCNB. Compared to other reports, the removal rate of PCNB by pcnb-21 isolated from PCNB polluted soil was about 89.7% in liquid medium as the initial concentration was 100 mg kg⁻¹, and the degradation rates of PCNB was 66.26–77.68% of initial concentration (10 mg kg⁻¹) after alfalfa growth in the soils for 20 days.^56,57 These results confirmed that the beneficial effect of S. rugoso-annulata in removing PCNB. Besides, the removal rates of PCNB were highly significant difference (P < 0.01) between inoculated and un-inoculated groups (Fig. 8), demonstrating that the presence of S. rugoso-annulata significantly facilitated the removal of PCNB, which was consistent with the results of previous researches studying the bioremediation of heavy metal and organics co-contamination.^29,32 Moreover, it was worth noting that the removal rates gradually increased in inoculated media. And the drastic reduction in PCNB mainly occurred in 9–11th days, which was exactly the period of the maximum concentration of exudates (Fig. 4 and 6), manifesting that exudates secreted by S. rugoso-annulata were tightly linked with the removal of PCNB. Researchers also found that the exudates of plant had the positive effects on organic pollutants biodegradation,^33,44 and Joner et al. reported that adding artificial exudates could increase five- and six-ring PAHs desorption in soil, and thus promoting the removal of PAHs.⁵⁸ Furthermore, Table 2 shows that PCNB residues in inoculated media were lower than that in un-inoculated medium, indicating the degradation of PCNB was remarkably enhanced by S. rugoso-annulata compared to the natural dissipation. These results in the present study coupled with other works showed that the exudates secreted by mycelia of S. rugoso-annulata significantly promoted the removal of PCNB.

Fig. 8 Removal rates of PCNB in media spiked with initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹) with different culture time. Error bars represent the standard deviation of three samples. Columns denoted by different letters indicated significant (P < 0.01) differences among different treatments.

Table 2 Residual concentrations of PCNB in un-inoculation and inoculation media^a

Incubation time (days)	PCNB content (mg L^−1)
	Un-inoculation	Inoculation
a Values in each row followed with different letters indicated significant (P < 0.01) difference between un-inoculation and inoculation media. Values represent means ± standard deviation.
3	99.06 ± 3.21^a	72.01 ± 2.14^c
5	85.52 ± 1.67^b	41.50 ± 2.96^e
7	72.44 ± 1.14^c	37.48 ± 1.63^e
9	60.19 ± 5.04^d	30.28 ± 0.67^f
11	59.30 ± 0.73^d	17.76 ± 0.90^g
13	57.82 ± 2.26^d	14.11 ± 0.41^g
15	55.92 ± 2.76^d	11.35 ± 1.06^g

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The enhanced dissipation of PCNB in polluted medium by the mycelia of S. rugoso-annulata might be due to the reasons as follows: (a) mycelia uptake. Previous studies have reported that mushrooms have the ability to tolerate and accumulate organic pollutants,^5–7 even explored the pathway that the persistent organic pollutants were mineralized in plant, such as Tall fescue²⁶ and Tagetes patula.¹ (b) The effects of exudates. Mushroom would secret lots of metabolites, such as macromolecular substances, LMWOAs and ligninolytic enzymes.^13,28 These exudates could remove the organics by a micro-interfacial process,⁵⁹ including complexation, transformation and biodegradation. Firstly, the carbohydrates and proteins played an important but indirect role in the PCNB removal process. They had great effects on Ni complexation,¹³ which contributed to the decrease the toxicity of Ni, thus promoting the growth of mycelia and enhancing the uptake and biodegradation of PCNB. Secondly, the LMWOAs could promote biocompatibility, absorbability and biodegradability of PAHs,¹¹ which were beneficial to translocate of organics and finally led to the biodegradation of PCNB. Besides, LMWOAs could directly promote PAHs elimination through abiotic oxidation, which could rapidly degrade a variety of organic compounds. For example, the process that carboxylic acid produces peroxy acid could cause stress responses of mushroom, and leading to the emission of free hydroxyl radicals (–OH).³³ Thirdly, the ligninolytic enzymes secreted by S. rugoso-annulata also had the ability to transform and degrade PCNB. Laccase was reported to transform and degrade trichlorophenol and chlorinated phenols,⁵⁰ and MnP could degrade phenolic compounds and hormones.⁶⁰ (c) Hydrophobic interactions. Some of the exudates containing hydrophobic regions, probably being helpful for the sorption of PCNB via hydrophobic interactions.³⁸ These above results suggested that degradation of PCNB was remarkably promoted by the presence of S. rugoso-annulata, and PCNB removal was probably inextricably correlated to the cell exudates.

3.7 Hypha changes induced by pollutants

Fig. 9 shows the changes in surface structure and phenotypic of hyphae cultured in natural and polluted medium. The results of SEM elaborated that the surface of hyphae incubated in natural medium were mesh-like, smooth and clear (Fig. 9a). Besides, the hyphae possessed a larger specific surface area, which probably was conducive to the adsorption of pollutants in liquid medium. Nevertheless, the mycelia of S. rugoso-annulata became loose, shriveled and wrinkled when prolonged to expose in polluted medium (Fig. 9b), demonstrating that the pollutants might impair the surface structure of mycelia in the long run. What's more, phenotypic images of mycelia balls also showed that the hyphae incubated in natural medium were more plump and the pileups were bigger than those in contaminated medium (Fig. 9A and B), which corresponded well with the results of SEM.

Fig. 9 SEM and hypha balls images of S. rugoso-annulata after 15 day incubation. (A) and (a) hypha grown in the medium contaminated with Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹), (B) and (b) hypha grown in the medium without pollutants.

In order to characterize the functional group types revealed on mycelia, the infrared spectrum analyses of mycelia grown in natural medium and polluted medium were carried out (Fig. 10). In polluted medium, the broad bio-sorption peak around 3396.53 cm⁻¹ and 2924.04 cm⁻¹ were indicative of –OH, amino (–NH₂), and methylene (–CH₂) groups stretching of carbohydrate and protein, respectively. The bio-sorption band of 1652 cm⁻¹ was protein amide I band, which was generated by the stretching vibration of amide (–CONH) in protein amide I. In addition, the bio-sorption band at 1076.41 cm⁻¹ and 1039.61 cm⁻¹ might be the cyano (–C–N) and carbonyl(–C=O) stretching vibration the of phosphate and polysaccharide.⁶¹ Compared with mycelia growing in natural medium, the slight displacement and decrease of vibration intensity in polluted medium at 3391.32, 2924.04, 1652.40, 1076.41 and 1049.35 cm⁻¹ might imply that the –OH, phosphate, –C=O and –CONH were involved in adsorption and complexation process of Ni. Overall, the absorption of infrared spectral and shape of peak for mycelia were consistent in natural medium and polluted medium, suggesting the adsorption of Ni hadn't changed the basic cell structure of mycelia, which explained its big biomass in medium contaminated with Ni and PCNB, further indicating the mycelia of S. rugoso-annulata possessed huge potential to remove PCNB and Ni from the co-contamination surroundings.

Fig. 10 DRIFTS of mycelia grown in media spiked with or without initial concentrations of Ni (30 mg L⁻¹) and PCNB (125 mg L⁻¹).

4. Conclusion

The present work identified the presence of S. rugoso-annulata was very effective in promoting the bioremediation of liquid medium contaminated with Ni and PCNB. And the accordance between the tendency of PCNB removal rates and exudates production expounded the secretion of exudates was closely related to the degradation of PCNB. Furthermore, the results of SEM and DRIFTS implied the mycelia could well tolerate the co-contamination of Ni and PCNB. Therefore, our results showed the mycelia of S. rugoso-annulata were a suitable candidate for in situ or ex situ mycoremediation of the PCNB and Ni co-contaminated sites, particularly for remediation of water contaminated with PCNB–Ni. Besides, the mycelia of S. rugoso-annulata may be application technically feasible for environmental remediation purposes because of the aggressive growth and biomass production along with their efficacy in remediation for PCNB and Ni. These results were also very important for a better understanding of the pollutants removal mechanism, especially for the interaction of exudates with contaminants, and laying the basis for pollution control in co-contaminated surroundings.

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.

References

1. Y. Sun, Q. Zhou, Y. Xu, W. Lin and X. Liang, J. Hazard. Mater., 2011, 186, 2075–2082.
2. J. W. Moore and S. Ramamoorthy, Heavy metals in natural water, Springer, New York, 1984.
3. T. L. Metcalfe, P. J. Dillon and C. D. Metcalfe, Chemosphere, 2008, 71, 1957–1962.
4. Y. Wang, F. Ling, L. Li, T. Luan and N. F. Y. Tam, Chemosphere, 2014, 99, 152–159.
5. C. O. Adenipekun and Y. Lawal, Nat. Sci., 2011, 9, 125–131.
6. R. Chen, Z. Zhou, Y. Liu, J. Jiang, Q. Li, H. Song, D. Pei and H. Xu, J. Soils Sediments, 2015, 15, 1083–1093.
7. P. G. Miles and S. T. Chang, Mushroom Biology Concise Basics & Current Developments, 1997.
8. C. O. Adenipekun and R. Lawal, Biotechnol. Mol. Biol. Rev., 2012, 7, 62–68.
9. M. Łebkowska and M. Załęskaradziwiłł, Desalin. Water Treat., 2014, 52(19–21), 3708–3713.
10. A. W. Taylor and P. E. Stamets, Implementing Fungal Cultivation in Biofiltration Systems – The Past, Present, and Future of Mycofiltration, ed. K. M. Wilkinson and D. L. Haase, National nursery proceedings, 2013, pp. 23–28.
11. Y. Wang, F. Ling, L. Li, T. Luan and N. F. Y. Tam, Chemosphere, 2013, 99, 152–159.
12. L. Landi, F. Valori, J. Ascher, G. Renella, L. Falchini and P. Nannipieri, Soil Biol. Biochem., 2006, 38, 509–516.
13. A. E. Tonietto, A. T. Lombardi, A. A. H. Vieira, C. C. Parrish and R. B. Choueri, Water Res., 2014, 49, 381–390.
14. W. H. O. Ernst, Appl. Geochem., 1996, 11, 163–167.
15. A. Sungur, M. Soylak and H. Ozcan, Chem. Speciation Bioavailability, 2014, 26, 219–230.
16. Y. S. Keum and Q. X. Li, Chemosphere, 2004, 56, 23–30.
17. G. Q. Zhang, Y. F. Wang, X. Q. Zhang, T. B. Ng and H. X. Wang, J. Cell. Biochem., 2010, 45, 627–633.
18. D. Daâssi, S. Rodríguez-Couto, M. Nasri and T. Mechichi, Int. Biodeterior. Biodegrad., 2014, 90, 71–78.
19. M. G. Kichenko, Gigiena I Sanitaria, 1950, 45–46.
20. M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
21. M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, Anal. Chem., 2002, 28, 350–356.
22. F. Xu, H. Chen and Z. Li, Bioresour. Technol., 2001, 80, 149–151.
23. K. Murugesan, Y. M. Kim, J. R. Jeon and Y. S. Chang, J. Hazard. Mater., 2009, 168, 523–529.
24. L. Chen, S. Luo, X. Li, Y. Wan, J. Chen and C. Liu, Soil Biol. Biochem., 2014, 68, 300–308.
25. H. Xu, J. Dai, J. Ji, F. Cen and Z. Yang, Environ. Eng. Manage. J., 2011, 10, 665–671.
26. M. Lu, Z. Z. Zhang, J. X. Wang, M. Zhang, Y. X. Xu and X. J. Wu, Environ. Sci. Technol., 2014, 48, 1158–1165.
27. X. B. Jing, N. He, Y. Zhang, Y. R. Cao and H. Xu, Can. J. Microbiol., 2012, 58, 45–53.
28. S. Lu, X. Yan, X. Liao, W. Yi, Z. Chong and L. Tao, Environ. Pollut., 2011, 159, 3398–3405.
29. Z. Bai, L. M. Harvey and B. Mcneil, Crit. Rev. Biotechnol., 2003, 23, 267–302.
30. D. Damodaran, K. Vidya Shetty and B. Raj Mohan, Ecotoxicol. Environ. Saf., 2014, 104, 414–422.
31. Y. Hou, X. Liu, X. Zhang, X. Chen and K. Tao, Int. J. Environ. Sci. Technol., 2016, 13, 887–896.
32. H. Liu, S. Guo, J. Kai, J. Hou, X. Han and H. Xu, J. Hazard. Mater., 2015, 294, 121–127.
33. G. Huang, G. Guo, S. Yao, N. Zhang and H. Hu, Int. J. Phytorem., 2015, 18, 33–40.
34. G. P. Sheng, M. L. Zhang and H. Q. Yu, Colloids Surf., B, 2008, 62, 83–90.
35. S. Clemens, Biochimie, 2006, 88, 1707–1719.
36. A. Liu, I. S. Ahn, C. Mansfield, L. W. Lion, M. L. Shuler and W. C. Ghiorse, Water Res., 2001, 35, 835–843.
37. K. Nouha, R. S. Kumar and R. D. Tyagi, Bioresour. Technol., 2016, 212, 120–129.
38. R. Späth, H. C. Flemming and S. Wuertz, Water Sci. Technol., 1998, 37, 207–210.
39. T. T. More, J. S. S. Yadav, S. Yan, R. D. Tyagi and R. Y. Surampalli, J. Environ. Manage., 2014, 144, 1–25.
40. J. A. S. Goodwin and C. F. Forster, Water Res., 1985, 19, 527–533.
41. K. Fukushi, S. Kato, T. Antsuki and T. Omura, Water Sci. Technol., 2001, 44, 453–459.
42. G. Guibaud, N. Tixier, A. Bouju and M. Baudu, Chemosphere, 2003, 52, 1701–1710.
43. H. Lu, C. Yan and J. Liu, Environ. Exp. Bot., 2007, 61, 159–166.
44. S. Meier, M. Alvear, F. Borie, P. Aguilera, R. Ginocchio and P. Cornejo, Ecotoxicol. Environ. Saf., 2012, 75, 8–15.
45. D. L. Jones and L. V. Kochian, Plant Soil, 1996, 182, 221–228.
46. U. Najeeb, L. Xu, S. Ali, G. Jilani, H. J. Gong, W. Q. Shen and W. J. Zhou, J. Hazard. Mater., 2009, 170, 1156–1163.
47. H. A. Akdogan and N. K. Pazarlioglu, Process Biochem., 2011, 46, 834–839.
48. Z. Zhou, Y. Chen, X. Liu, K. Zhang and H. Xu, RSC Adv., 2015, 5, 42768–42776.
49. F. Acevedo, L. Pizzul, M. D. P. Castillo, R. Cuevas and M. C. Diez, J. Hazard. Mater., 2011, 185, 212–219.
50. H. Forootanfar, M. M. Movahednia, S. Yaghmaei, M. Tabatabaei-Sameni, H. Rastegar, A. Sadighi and M. A. Faramarzi, J. Hazard. Mater., 2012, 209–210, 199–203.
51. D. Wesenberg, I. Kyriakides and S. N. Agathos, Biotechnol. Adv., 2003, 22, 161–187.
52. X. Li, S. Dong, Y. Yuan, W. Shi, M. Wu and H. Xu, RSC Adv., 2016, 6, 65816–65824.
53. R. Boominathan and P. M. Doran, J. Biotechnol., 2003, 101, 131–146.
54. X. Zhang, F. Lin, Y. Jiang, W. Ke and X. L. Feng, Environ. Monit. Assess., 2009, 155, 205–213.
55. K. R. Kim, G. Owens and R. Naidu, Pedosphere, 2010, 20, 494–504.
56. R. Li, J. W. Zheng, B. Ni, K. Chen, X. J. Yang, S. P. Li and J. D. Jiang, Pedosphere, 2011, 21, 31–36.
57. Y. Y. Li and H. Yang, J. Environ. Manage., 2013, 119, 143–150.
58. E. J. Joner, S. C. Corgié, N. Amellal and C. Leyval, Soil Biol. Biochem., 2002, 34, 859–864.
59. Y. Z. Wang and Q. X. Zhou, Chin. J. Ecol., 2012, 31, 1034–1042.
60. P. J. Kersten, B. Kalyanaraman, K. E. Hammel, B. Reinhammar and T. K. Kirk, Biochem. J., 1990, 268, 475–480.
61. P. R. Griffiths, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, Academic Press, 1991, p. 121.

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