1 Introduction

One of the great problems for coal washery industries is the dumping of effluent released after washing of coal that contains impurities such as heavy metals (like nickel, lead, iron, chromium, manganese, copper, cobalt, aluminium, arsenic, cadmium, etc.), fine particles of coal and many other pollutants [1, 2]. Huge amount of water and different materials like detergents, coagulants, flocculant and surfactant are used during processing of coals in coal washery plants which ultimately produce a significant quantity of effluent [3]. Discharge of this coal washery effluent into the river and other wetlands causes adverse effects on the aquatic organisms as it contains many of the non-biodegradable elements that have deleterious impact on animal physiology [4].

To overcome the problem of CWE pollution, treatment of these waste waters before their discharge into the water bodies is very important. Many methods such as ion exchange method, electrochemical treatment, use of membrane technologies and chemical precipitation are being used at large scale in different industries to decontaminate the contaminated environment [5]. However, these methods are very costly, not suitable to the environment and not much effective [6]. Hence, it is very important to develop a fast, cost-effective and ecofriendly method for eliminating heavy metals from effluent such as CWE. Use of mushroom in remediation of toxic elements has been increased many folds in recent years. It is known to have high metal binding capacity due to its high percentage of cell wall [7] and can also detoxify or convert toxic substance to less toxic form [8]. These are the reasons why macrofungi are considered as a promising option for remediation process.

There are several in vitro studies in which mushrooms were used for remediation of heavy metals from the synthetic media [9,10,11]. But, in vivo use of mushroom for decontamination of natural waste water released from different industries is scanty. Recently, it has been tried to decontaminate the CWE using P. ostreatus in vitro condition and reported significant decrease in metals from CWE [2]. Therefore, this study is designed to examine the role of P. ostreatus in decontamination of metals from CWE in vivo condition. The source of studied CWE was a Coal Washery Plant of BCCL (Bharat Coking Coal Limited), Dhanbad, India.

For remediation purpose, content of various metals (Cr, Ni, Pb, Co, Mn, Zn and Cu) in the grown mushrooms and in their paddy substrate soaked with different percentages of CWE, i.e. 75% CWE, 50% CWE and 25% CWE, was examined. Adverse impact of CWE on mushroom health was also manifested by measuring antioxidant enzyme activities, lipid peroxidation (LPO) and metallothionein proteins concentration in the fruiting bodies.

Apart from remediation efficiency, mushrooms are also beneficial for hypercholesteraemic patients as it influences the lipid profile and also improves the antioxidant condition [12, 13]. They are also a good source of several elements (Cu, Zn, Mo, Se and Fe), and they are involved in many important biochemical [14]. Although metals are required in trace amount for normal metabolic process and growth of mushroom, their excess amount may lead to severe physiological problems resulting low-quality mushrooms with reduced nutritional values. Hence to know the effect of CWE on the nutritional content of the mushrooms, protein, lipid, ash and fibre contents were also analysed in the fruiting bodies grown on substrate soaked in different percentages of CWE.

2 Materials and methods

2.1 Maintenance of mushroom culture and collection of CWE

The culture of P. ostreatus was obtained from Directorate of Mushroom Research, Solan (Himachal Pradesh), India. Malt dextrose agar (MDA) medium (temperature 25 ± 2 °C and pH 6–6.5) was used as culture media. Culture was subcultured at every three weeks of interval. CWE was collected in five containers each having capacity of 100 L, from a coal washery plant of Bharat Coking Coal Limited (BCCL), Jharkhand, India.

2.2 Preparation of spawn

Spawn is the vegetative growth of fungus grown on cereal grains. Method of Singh et al. [15] was used to prepare the spawn. Washed wheat grains were half boiled and spread over a tilted platform to remove excess water, and then, buffers (CaCO3 and CaSO4 in 3:1 ratio) were mixed with them. Bottles were half filled with wheat grain and were plugged by cotton. These bottles were autoclaved at the temperature 121 °C and pressure 15 psi for 30 min and left for overnight. Then after inoculation of bottles was done by transferring inoculums of P. ostreatus from cultured plate and these filled bottles were incubated in incubation chamber at a temperature of 25 ± 2 °C.

After 3–4 days of inoculation, the fungal mycelium started to spread on the grains in the form of white net web-like mycelia. After 18–21 days, white mycelial growth was observed in entire bottles.

2.3 Preparation of paddy straw substrate and spawning

Before preparation of substrate, dilution of the CWE was done with distilled water (DW) in three combinations, i.e. 75% CWE + 25% DW, 50% CWE + 50% DW and 25% CWE + 75% DW. Raw effluent (100% CWE) was also kept along with diluted CWE. Substrate was prepared by dipping the 2 kg small piece of paddy straw in 8 L of distilled water (taken as control), pure and diluted CWE separately and kept at RT for overnight. Next morning, spawning of substrate was carried out, i.e. 30 g spawn grains were mixed with each 1000 g wet substrate which was further tightly packed in polythene bags. In this way, six bags of each CWE-soaked group were prepared along with distilled water-soaked group (control). In last at the bottoms of each bag (1 × 1 cm), 12 holes were made to remove extra moisture and entry of air. All the spawned bags were kept in mushroom house for 45 days on metal rack for growth analysis of mushroom. After full growth of fruiting bodies, they were collected from each group on 35th day and 42nd day for study of various metal concentration and nutritional parameters. Fruiting bodies are full grown mushrooms that comprise of stem, cap and gills which are what eaten by people (Fig. 1). Substrates were also collected along with fruiting bodies from each group for metal analysis.

Fig. 1
figure 1

Growth of fungal mycelia on substrate soaked with different percentages of CWE. a Growth of fungal mycelia on substrate soaked with 100% CWE. b Growth of fungal mycelia on substrate soaked with 75% CWE. c Growth of fungal mycelia on substrate soaked with 50% CWE. d Growth of fungal mycelia on substrate soaked with 25% CWE. e Growth of fruiting bodies on substrate soaked with 75% CWE. f Growth of fruiting bodies on substrate soaked with 50% CWE. g Growth of fruiting bodies on substrate soaked with 25% CWE

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2.4 Growth analysis of fungus grown on paddy substrate

The growth of fungus can be divided into mycelial stage, pinhead stage, early fruit body stage and mature fruit body stage. The day when fungus was spawned, mycelial growth was not seen which was denoted by a negative sign (−). We observed the growth of fungus after 7-day interval and measured its mycelial growth in terms of positive sign (+). Each positive sign (+) represents the 10% mycelial growth, and it was maximum (100%) in control (DW-soaked substrate) group after 21 days of spawning, then after pinhead stage started to appear followed by early fruit body and mature fruit body stages (Table 1).

Table 1 Growth of fungal mycelia grown on paddy straw soaked with different percentages of CWE
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2.5 Heavy metal analysis in fruiting bodies and paddy substrate

Five gram of fruiting bodies from each group were dried in Petri dish in an oven at 120 °C till there was no further weight loss. Dried fruiting bodies and substrate were kept into digestion flasks having a mixture of nitric acid and perchloric acid (4:1 v/v). Then after digestion flask was heated on a hot plate at 120 °C so that all materials get dissolved in acids. After complete digestion, double-distilled water was added to sample to make the volume up to 25 ml. Concentration of metals (Mn, Ni, Zn, Cu, Pb, Co and Cr) was analysed using inductively coupled plasma mass spectrophotometry (ICP-MS).

2.6 Analysis of antioxidant enzyme activities in fruiting bodies

For the antioxidant enzymes assay, homogenate of fungal tissue was prepared in ice cold 0.1 M phosphate buffer saline (pH 7.0). Centrifugation was done at 2500 rpm for 10 min, at 4 °C followed by second centrifugation at 12,000g for 20 min at 4 °C. The supernatants were then kept at − 80 °C for enzyme estimation. Then after total protein in the supernatant was measured using Lowry et al. [16]. Method of Das et al. [17] was used for analysis of SOD activity (Unit/mg protein) in fungal tissue using reaction cocktail containing phosphate buffer, α-methionine, Triton X-100, HAC and EDTA and Griess reagent. For CAT assay (nmole/mg/sec), Aebi [18] method was used. Its assay mixture had phosphate buffer, H2O2 and enzyme extract. LPO in fungal tissue (nmoles TBARS/mg protein) was analysed by method given by Ohkawa et al. [19] using reaction cocktail (SDS, acetic acid, thiobarbituric acid and butylated hydroxyl toluene).

2.7 Analysis of metallothionein concentration in fruiting bodies

Viarengo et al. [20] method was used to estimate total metallothionein concentration. Ellman’s reagent was used for its assay. Detail of the procedure has been described by Vaseem et al. [2].

2.8 Analysis of nutritional content in fruiting bodies

2.8.1 Total protein

Mushroom was homogenised in phosphate buffer and centrifuged at 4000 rpm. Resulting precipitate was dissolved in 0.5 N NaOH and centrifuged at 7000 rpm, and obtained clear solution was used for estimation of protein concentration using method of Lowry et al. [16].

2.8.2 Total lipid

Estimation of total lipid was done by the method of Folch et al. [21] by extracting lipid in extraction solvent (chloroform:methanol 2:1 v/v).

2.8.3 Crude fibre

Crude fibre was determined by method of Raghuramulu et al. [22].

2.8.4 Total ash

For determination of ash content, mushroom sample was heated in a muffle furnace (in atmospheric conditions) for about 5–6 h at 600 °C and sample was then cooled in a desiccator and weighed.

Then, total ash was calculated as (Raghuramulu et al. [22]):

Ash content (g/100 g sample) = weight of ash × 100/weight of sample taken.

2.9 Statistical analysis

Data are presented as mean ± standard deviation (n = 3). One-way analysis of variance (ANOVA) followed by the Duncan's Multiple Range Test (DMRT) was used to find out significant difference between the two mean values. Differences were considered significant at the level of p < 0.05. In tables and figures, alphabets denote the result of DMRT. Different alphabets show significant difference (p < 0.05) in the different values of means.

3 Results and discussion

3.1 Physicochemical characteristics of CWE

The effluent was found to be highly contaminated with many heavy metals which were present above their permissible limits (Mn: 14.63 mg/l, Ni: 1.789 mg/l, Zn: 4.458 mg/l, Cu: 2.337 mg/l, Pb: 45.355 mg/l, Co: 3.514 mg/l, Cr: 1.123 mg/l) [2]. Other physicochemical parameters of the CWE were as follows: total dissolved solid (mg/l): 739 ± 169.44, total suspended solids (mg/l): 4624.333 ± 323.62, turbidity (NTU): 27.667 ± 2.517, electrical conductivity (µS/cm): 1.063 ± 0.118, BOD (mg/l): 65.821 ± 5.43, hardness (mg/l): 413 ± 7.211, phosphate (mg/l): 35.78 ± 3.324, nitrate (mg/l): 28.46 ± 4.98 [2].

3.2 Effect of different percentages of CWE on growth of fungus

Growth of fungal mycelia on the substrate soaked with different percentages of CWE is shown in Table 1 and Fig. 1. The mycelia grown on different percentages of CWE have shown different levels of growth stages. From Table 1 and Fig. 1A, it can be clearly seen that fungal mycelia could not grow on the substrate soaked with 100% (raw) effluent. High concentration of different toxic metals in the raw effluent might have caused severe effects on the growth of mycelia and did not allow them to survive (Fig. 1A). The fruiting bodies grown on the substrate containing 50% CWE (Fig. 1F) and 25% CWE (Fig. 1G) showed good and continuous growth in comparison with the mycelia grown on 75% CWE (Fig. 1E) (growing at a slower speed and developed pinhead stage later). The dilution of CWE caused decrease in concentration of different metals in the CWE which led to the normal growth of the mycelia in diluted effluents like 75%, 50% and 25% effluent and made mycelia efficient for metal accumulation from the CWE and supported in its normal growth. Higher content of metals in the 75% CWE in comparison with the 50% and 25% diluted effluent might be the cause of late growth of mycelia and distorted fruiting bodies in 75% diluted CWE. Maximum growth was observed in control group, and pinhead stage and fruiting bodies growth was seen first in control than other groups.

3.3 Efficiency of P. ostreatus in remediation of heavy metals from CWE

Remediation efficiency of Pleurotus for heavy metals from CWE was investigated from 0 day (spawn condition) to 42nd day (fully grown fruiting bodies) of exposure on the substrate soaked with different percentages of CWE. The data from 100% CWE grown fungus are not included in the results because the mycelia could not grow on it.

Figure 2A shows bioremediation of different metals like Ni, Zn, Cu, Co, Cr, Mn and Pb from the substrate soaked with 75% CWE. From Fig. 2A, it can be clearly seen that significant (p < 0.05) amount of all the metals have been remediated from the substrate from 0 day to 35th day to 42nd day. The percentage decrease in metal concentration in 75% CWE was as follows Ni: 87.76%, Zn: 91.54%, Cu: 100%, Co: 62.36%, Cr: 66.39%, Mn: 86.95% and Pb: 82.38%.

Fig. 2
figure 2

a Metals concentration in substrate soaked with 75% CWE. b Metals concentration in fruiting bodies grown on substrate soaked with 75% CWE

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Figure 2B shows concentration of metals accumulated in the fungus fruiting bodies after bioremediation of substrate soaked with 75% CWE. From the figure, it can be clearly seen that concentration of all the metals significantly increased in fruiting bodies from 0 day to 42nd day. The final concentration of metals in the fruiting body after mycoremediation was as follows (mg/kg): Mn: 8.56, Ni: 1.12, Zn: 2.762, Cu: 0.959, Pb: 27.023, Co: 1.482, Cr: 2.261 (Table 3).

In the 50% and 25% CWE containing substrates, significant decrease in different metals was observed from 0 day to 42nd day (Figs. 3a and 4a, respectively). In 50% CWE, the percentage decrease in different metals was as follows: Ni: 98.36%, Zn: 89.52%, Cu: 100%, Co: 89.98%, Cr: 88.80%, Mn: 72.89% and Pb: 78.18%, while in case of 25% CWE, the decrease was as follows: Ni: 100%, Zn: 99.04%, Cu: 100%, Co: 100%, Cr: 100%, Mn: 84.66% and Pb: 67.92%. Percentage decrease in concentration of most of the metals was maximum in the substrate soaked with 25% CWE in comparison with the 75% and 50% CWE. This shows the maximum bioremediation efficiency of Pleurotus in 25% CWE. Decreased metal concentration caused less accumulation of metals in the fruiting bodies in diluted effluent that has direct effect on its growth. Percentage decrease in metals from all the diluted effluents is shown in Table 2. Significant difference in the decrease in most of metals was also observed in all the diluted effluents (p < 0.05). Many researches have been performed that reported capability of metal build-up of an organism depends upon its environmental metal concentration [23,24,25,26]. Milovanovic et al. [27] reported that P. ostreatus mycelium had good growth and biomass yield in low concentration of metal, while its growth and biomass yield reduced in higher concentration of metals. In this way, result of present study shows that metal concentration in the environment decides an organisms’ ability of metal accumulation.

Fig. 3
figure 3

a Metals concentration in substrate soaked with 50% CWE. b Metals concentration in fruiting bodies grown on substrate soaked with 50% CWE

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Fig. 4
figure 4

a Metals concentration in substrate soaked with 25% CWE. b Metals concentration in fruiting bodies grown on substrate soaked with 25% CWE

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Table 2 Percentage decrease in the concentration of metals from the substrate soaked with different percentages of CWE after bioremediation using P. ostreatus on 42nd day
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Low concentration of metals in diluted effluents has less toxic effects on the fruiting bodies and makes the fruiting bodies capable for removing significant amount of metals from CWE. This can be demonstrated by the significant accumulation (p < 0.05) of different metals in mushrooms grown on substrate soaked with 75%, 50% and 25% CWE from 0 day to 42nd day (Figs. 2b, 3b and 4b). In control groups, there were no significant changes observed in metals concentration in fruiting bodies as well as in substrate.

The final concentration of metals in the fruiting bodies grown on substrate soaked with 75%, 50% and 25% CWE is shown in Table 3. From Table 3, it can be clearly demonstrated that all the metals (except Pb) in fruiting bodies grown on different percentages of effluent were well below than their permissible limits suggested by various international agencies for food materials. Significant difference was also observed in most of the metals in all the diluted effluents (p < 0.05). Many researchers have also reported accumulation of various metals in the fruiting bodies of natural growing mushrooms and found their concentration below the level suggested by various agencies [28, 29].

Table 3 Final concentration of metals in the fruiting bodies grown on substrate soaked with different percentages of effluent on last day of bioremediation (42nd day)
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3.4 Antioxidant enzyme activities in fruiting bodies

There was significant alteration observed in antioxidant enzymes activities in fruiting bodies grown on different percentages of CWE (Table 4). Activity of SOD, catalase and LPO in 50% and 25% CWE increased from 0 day to 42nd day, while catalase activity did not show any significant difference in 75% CWE from 35th day to 42nd day. Antioxidant enzymes are capable of protecting the organisms by stabilising and deactivating the reactive oxygen species (one of the major causes of oxidative stress) produced my metals and other toxicants [30, 31]. Increased activities of antioxidant enzyme in 50% and 25% effluent grown mushrooms might be for protection of fungus from stress of heavy metals. Many researchers have also reported increased antioxidant enzymes activity in macrofungi exposed to metals [32,33,34]. High amount of metals in the 75% might have responsible interruption in the antioxidant systems, and fungus did not withstand the damage caused by accumulated metals.

Table 4 Antioxidant enzyme activities in fruiting bodies grown on substrate soaked with different percentages of effluent on different days of remediation
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3.5 Metallothionein concentration in fruiting bodies

Metallothioneins (cysteine-rich proteins) have high capacity to bind with metals. In this way, they protect the cells from adverse effect of metals [35]. In present study, increase in metallothionein concentration might be resulted to save the mushrooms from the metals accumulated in fungal mycelium grown on CWE [36]. Figure 5 denotes concentration of metallothionein protein in the fruiting bodies cultivated on substrate soaked with different percentages of CWE on 42nd day of experiment. It illustrates that metallothionein concentration was found to be significantly higher (p < 0.05) in the fruiting bodies grown on substrate containing CWE than the fruiting bodies of control group. Increase in metallothionein concentration is due to accumulation of various metals in fruiting bodies.

Fig. 5
figure 5

Metallothionein concentration in fruiting bodies grown on substrate soaked with different percentages of CWE

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3.6 Nutritional content in the mushrooms

Mushrooms are highly nutritious having high protein content, fibre, sugar, minerals and high quantity of amino acids like phenylalanine, threonine and tyrosine [37, 38]. As they are good source of various nutrients, it is also very important to analyse different nutritional contents of mushrooms cultivated on CWE. Hence, the effect of coal washery effluent was accessed by studying different nutritional contents of the mushroom fruiting bodies like total protein, total lipid, ash and fibre content (Table 5). The protein, lipids and fibre contents in the mushrooms grown on all percentage of CWE were found to be lower than those of control ones except in 25% CWE grown mushrooms in which protein content was higher in comparison with the control. The decreased protein, lipids and fibre content might be due to toxic effect rendered by heavy metals present in the CWE on the mushroom’s physiology. Increased concentration of ash in all the fruiting bodies cultivated on substrate with CWE in comparison with control (Table 5) might be the result of accumulated metals in the fruiting bodies grown on CWE.

Table 5 Nutritional contents of fruiting bodies grown on substrate soaked with different percentages of CWE
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4 Conclusion

Mycoremediation of coal washery effluent using a macrofungi Pleurotus ostreatus was found to be highly significant resulting decrease in concentration of metals from CWE. Metal accumulation was also observed in the fruiting bodies grown on different percentages of the CWE having metal concentration below than their permissible level suggested by different international agencies for food materials. Alteration in antioxidant enzyme activities and concentration of metallothionein proteins in fruiting bodies were also observed showing protective mechanism of mushroom against toxic effect of heavy metals present in effluent. The decreased nutritional content of the fruiting bodies grown on CWE in comparison with control ones was also the evidence of CWE toxicity.