Introduction

Continuous addition of heavy metals through industrialization and urbanization is now accelerating environmental pollution. Metal ions concentrate and accumulate in the food chain causing serious threat to ecosystem. Zinc (Zn) is one of the most important trace elements in living organisms and is essential for growth and development of microorganisms, plants, and animals. But obviously most essentials as well as nonessential metals showed signs of toxicity above a certain concentration. Obviously Zn at higher concentrations may become toxic and elevated levels have been widely reported to be a soil contaminant [1, 2]. So, Zn is considered as an important contender for remediation of soil or environment. Conventional methods for treating harmful metals were inefficient, costly and not eco-friendly, so a shove was felt for searching an alternative way for heavy metal treatment. Bioremediation, by means of biosorption (ability of biological materials to accumulate heavy metals from contaminated wastes) has been considered as a useful approach for remediation of heavy metals due to its low cost and high efficacy. Among different microbial masses, fungal biomass grasps distinct advantages due to its different inherent characteristics. Biosorption is based on passive (metabolism-independent) or active (metabolism-dependent) accumulation processes [3]. During non-metabolism dependent biosorption, metal uptake occurred by means of physicochemical interaction between the metal and the functional groups present on the microbial cell surface. This is based on physical adsorption which takes place with the help of van der Waals forces [4], ion exchange and chemical sorption, which is not dependent on cell metabolism, whereas in metabolism-dependent intracellular uptake metal ions are transported across the cell membrane [5–7]. Potential of filamentous fungi in bioremediation of heavy metals containing industrial effluents and waste waters has been increasingly reported from different parts of the world [8]. Among different filamentous fungi, various researchers highlighted the efficacy of Aspergillus group in heavy metal bioremediation.

Amylases (comprised of α-amylase, gluocoamylase and α-glucosidase) and Cellulase (comprised of an endo-1,4-β-glucanase [Carboxymethyl cellulose; CMCase], a 1,4-β-cellobiohydrolase [Exoglucanase] and a 1,4-β-glucosidase [Cellobiase]) are crucial for basic cell metabolism [9]. These industrially important enzymes played another role in lignocellulosic waste (produced by the agricultural industry, forestry stations, and municipal solid wastes [10]) degradation. Metal ions may inhibit enzyme reactions by complexing the substrate, reacting with the protein-active groups of enzymes and enzyme-substrate complex [11]. Previous researchers have reported that heavy metals can decrease the ability of fungal growth and production of extracellular enzyme activity [12] and can consequently affect the cycling of carbon and nutrients. Growth of fungi and the production of extracellular enzymes are associated with the carbon and nutrient cycles. Several studies have shown that certain metals like Fe, Hg, Ag, Cd, Pb inhibited the laccase activity [13, 14]. Therefore being a heavy metal Zn can hamper normal activities of amylase and cellulase enzymes of A. terreus.

It is well established that oxidant–antioxidant status of a cell or the system concerned is an important marker of stress induced effects [15]. Although under normal conditions of growth of an organism reactive oxygen species (ROS) are formed as a by-product of various metabolic pathways and there exists a balance between ROS generation and its scavenging activity. However, the equilibrium is disturbed when the body is under stress conditions. For further persistence antioxidative enzyme system is functionally motivated to prevent cellular damages by converting ROS to oxygen and water [16]. ROS is a known initiator of antioxidative defense system (ADS) [17]. To combat the damage caused by ROS, living organisms including fungi adopted some intracellular enzymatic systems (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase) as well as non-enzymatic antioxidants (ascorbic acid, glutathione, metallothioneins, carotenoids etc.). Metallothioneins (MTs; small sized (<7 KDa), cysteine riched protein having metal binding Cys-rich domains) are engaged in metal homeostasis and detoxification [13, 18].This protein showed high affinity towards zinc and possesses high antioxidant activity. There is lack of information about the behaviour of filamentous fungi exposed to heavy metal stress, whether various physiological changes occurred when the cells were exposed to different environmental stress.

In this context the present paper highlighted about Zn uptake and removal efficacies of a Zn tolerant species, A. terreus. It has evaluated whether Zn exposure causes any alteration in metabolic enzyme (CMCase and α-amylase) activity, and whether antioxidant enzymes (superoxide mutase; SOD and catalase; CAT) and metallothionein protein participated in protection of A. terreus against Zn stress.

Material and Methods

Isolation, Identification and Culture of Fungi

Aspergillus terreus was isolated from soil of garbage dump site of Dhapa, Kolkata by standard plating methods in potato dextrose media. The species was purified by streaking repeatedly on the same medium and the fungus was identified microscopically, followed by standard fungal identification key [19].The isolated strain was cultured in potato dextrose broth with the following composition: peeled potato (400 g), dextrose (25 g), dissolved in 1 l of distilled water. Final pH was around 7. The medium was autoclaved at 121 °C for 20 min. Cultures were maintained in agar slants (potato dextrose broth plus 20 g/l agar).

Assessment of Metal Tolerance

Assessment of metal (Zn) tolerance (in terms of determination of minimum inhibitory concentration; MIC) of A. terreus was done after isolation from soil. Different concentrations of Zn solutions were added separately to PDA medium and inoculated with 100 μl of spore suspension to determine MIC. ZnSO4, 7 H2O (Merck) salt was used for running all experiments.

Estimation of Zn Uptake and Removal Potential of A. terreus from Zn Supplemented Media

One 8 mm disk of fungal colony from petri plates of A. terreus was transferred to different Zn enriched liquid media with a sterilized cork borer and allowed for growth in an orbital shaker at 100 rpm for 8 days with incubation at 29 °C. Uptake of Zn (mg/g of tissue) and removal percentage were carried out following the method of Srivastava and Thakur [20] using atomic absorption spectrophotometer (FI-HG-AAS Perkin Elmer Analyst 400).

Analysis of Functional Groups Responsible for Metal Adsorption by Fourier Transform Infrared Spectroscopic (FTIR) Study

The functional groups (present in fungal cell wall) involved in metal adsorption are studied through FTIR. Samples were prepared considering the methods of Xu et al. [21].The lyophilized fungal mycelium (lyophilized at −85 °C under high vacuum condition using freeze dryer; Virtis EL-65, New York) was mixed with oven dried (100 °C) potassium bromide (KBr) in the ratio of 1:100. FTIR spectrum of samples was recorded in JASCO-6300 FTIR instrument with a diffuse reflectance mode (DRS8000) attachment.

Estimation of Extracellular Enzyme Assay

Enzyme Extract

After 8 days of shaking incubation the biomass part and some portion of broth were separated for determining uptake and removal (%) potential. Remaining broth was centrifuged at 12,860×g for 15 min to avoid any solid debris. The supernatant was used for enzyme assay. CMCase activity and α-amylase activity were studied according to the method of Denison and Koehn [22] and Bernfield [23] respectively.

CMCase [EC 3.2.1.4] activity was calculated with the help of glucose standard curve (1 Unit/ml = amount of enzyme which releases 1 µmol glucose under assay condition) and α-amylase [EC 3.2.1.1] activity was calculated with the help of maltose standard curve (1 Unit/ml = amount of enzyme which releases 1 µmol maltose under assay condition) using a UV–Vis spectrophotometer, (Perkin Elmer Lambda-25) keeping a blank (without sample).

Sample Preparation for Scanning Electron Microscope (SEM)

Samples were prepared according to the methods of Kaminskyj and Dahms [24] and Mishra and Malik [25]. Eight days old fungal mycelium were cut into small pieces(maximum 1 mm to 2 mm in diameter) and fixed in 2.5 % glutaraldehyde (Sigma) solution for 1–2 h, after that the samples were washed in buffer(0.1 M phosphate buffer, pH 7.4) for 1–2 min and then dehydrated in series of ethanol water solution (30, 50, 70, 90 and 100 % ethanol; 10–15 min in each) and finally kept in ethyl acetate (100 %).The samples were critical point dried under a CO2 atmosphere for 20 min. Mounting was done on aluminium stubs and gold coated with IB2 ION coater. Coated samples were viewed at 25 kV with scanning electron microscopy (HITACHI S-530).

Antioxidative Enzyme Assay

Enzyme Extract

Mycelium of tested fungus was washed with cold water and homogenized with 0.015 M potassium phosphate buffer (pH 7.8) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF) that inhibits proteases, and is frozen in liquid nitrogen. It is then centrifuged in 25,720×g for 20 min at 4 °C. The supernatant was taken as enzyme extract.

Super Oxide Dismutase (SOD) Assay [EC: 1.15.1.1]

Estimation of SOD was followed by Paoletti and Mocali [26] based on inhibition of NADH oxidation upon the addition of enzyme.

Catalase Assay (CAT) [EC:1.11.1.6]

Estimation of CAT was followed by Aebi [27] by measuring the rate of H2O2 degradation.

Estimation of Total Metallothionein in Spectrophotometric Method

Quantification of total metallothionein was estimated according to the method of Viarengo et al. [28] and modified according to Pal et al. [29]. The metallothionein concentration was estimated considering reduced glutathione (GSH) standard curve.

Statistical Analysis

Statistical analyses were based on the mean values performed on 3–6 replicates for each set of experiments. Students t test was analyzed to determine the level of significant differences between control and treated samples. The difference is considered to be significant in case of p < 0.05. A Pearson correlation test was used to obtain correlation coefficients between sets of data.

Results and Discussion

Serial dilution technique was used to isolate fungal strains from 225 ppm Zn enriched soil samples of Dhapa, Kolkata. Appeared colonies in PDA media (Potato Dextrose Agar; pH 7) were further purified on the same media by streaking method and all strains were maintained in slant of PDA at 29 °C. Four different strains were isolated and tested for their Zn tolerance assessment. Among all isolated strains A. terreus (MIC of A. terreus for Zn is 9100 ppm) was selected for further experiments for its potential zinc tolerance.

Atomic absorbtion spectroscopic (AAS) data revealed that Zn accumulation and subsequent removal efficacy of A. terreus was found to be decreased with increasing Zn concentration (from 100 ppm to 9000 ppm). Results shown in Fig. 1 illustrated maximum removal (75.3 %) and uptake (18.3 mg/g of tissue) of Zn by A. terreus from 100 ppm Zn enriched growth medium. Further increase in Zn concentration caused decrease in uptake potential (9.65, 7.62 and 5.87 mg/g of tissue from 7000, 8000 and 9000 ppm of Zn enriched media respectively). From 9000 ppm Zn enriched medium A. terreus could remove almost 35 % Zn. Zn accumulation and subsequent removal efficacies indicated A. terreus as a potential Zn tolerant fungal strain.

Fig. 1
figure 1

Percent of removal (%) and uptake of zinc (mg/g of tissue) by Aspergillus terreus from different zinc enriched growth media. (Within each group values are significantly different at ≤0.05; error bars are standard deviations)

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It is well established that microorganisms are able to grow in heavy metal enriched environment with a significant metal uptake capacity [20]. Fungi belonging Rhizopus and Aspergillus have already been reported with potential heavy metal removal efficacies from contaminated aqueous solutions [29, 30]. Reports regarding potential of A. niger for removing heavy metals including Zn has been cited by a host of earlier researchers. Akthar and Mohan [31] reported that killed mycelium of A. niger could remove copper and zinc from contaminated lake waters. Price et al. [32] has shown potential of A. niger to remove 91 % of Cu and 70 % of Zn from a treated swine effluent. The potential of living A. niger to remove cadmium and zinc from aqueous solution is also reported by Liu et al. [18]. Metals at elevated levels may block the functional groups of biologically important molecules (enzymes and transport systems for essential nutrients and ions), displace and/or substitute the essential metal ions and disrupt cellular and organellar membrane integrity [33]. This might be the origin behind decreasing Zn removal potential of A. terreus at higher concentrations, as documented in the present results and also reported by previous researchers [33, 34].

Figure 2 shows the FTIR spectra of A. terreus before and after Zn exposure. Data analysis showed involvement of hydroxyl group (–OH), asymmetric and symmetric stretching vibration of CH2 group, amide group (–NH), SO3 groups, stretching vibration of C–O, C–N groups, carboxylate group (–COO) and carbonyl group (–CO) in Zn adsorption by A. terreus [21]. Significant shifting of peaks at regions 3380, 2925, 1746,1247, 1151 and 1079 cm−1 were noted in A. terreus biomass grown in Zn enriched media as compared to the control group having no metal supplementation. One new peak at 1411 cm−1 region was found in the group grown in Zn supplemented media. Similar shifting in peaks in fungal biomass treated with heavy metals have been reported earlier by a host of researchers [21, 35]. These observations indicated the involvement of the related functional groups in the Zn biosorption process on fungal (A. terreus) cell surface.

Fig. 2
figure 2

FTIR spectra of A. terreus biomass. a Control b Zn loaded biomass

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Figure 3 represents the change in CMCase and α-amylase activities of Zn exposed A. terreus with respect to control (without Zn stress) counterparts. A. terreus grown in Zn supplemented medium showed decrease in activities of both the enzymes. The intensity of the effect in Zn-induced stress is more on α-amylase activity than that on CMCase activity. While A. terreus grown in 100 ppm Zn supplemented medium showed 7.9 % decline in CMCase activity (Fig. 2), the same group showed 15 % decline in α-amylase activity (Fig. 3) when compared to that of the normal control counterparts. Enzyme activities were gradually decreased with increasing Zn concentrations (1000–9000 ppm) and maximum decline (79 % decline in α-amylase activity and 64 % in CMCase activity) was noticed when A. terreus was grown in 9000 ppm Zn enriched medium. This ensures the role of Zn in distortion of enzyme functioning in the concerned fungi which is possibly due to association of Zn with transcriptional as well as translational pathways as postulated by Baldrian et al. [36, 37]. Pearson correlation matrix showed a positive correlation between CMCase and α-amylase activities (Table 1a).

Fig. 3
figure 3

Change in CMCase and α-amylase activity (IU/ml) of zinc exposed Aspergillus terreus with respect to control (unexposed). (Each value is significantly different at p ≤ 0.05 from control; error bars are standard deviation)

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Table 1 Values of correlation matrix (Pearson)
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In parallel to the present observation Huang et al. [38] found inhibition of CMCase activity and cellulose degradation capacity of Phanerochaete chrysosporium under Pb stress. Cd, Cu, Pb, Mn and Zn also affected cellulase and xylanase activity of P. ostreatus cultivated in lignocellulosic residues [36].

Assessment of morphological changes in response to Zn exposure in A. terreus was performed by scanning electron microscopy (SEM). Fig. 4a represents fungal hyphae without zinc exposure; the hyphae were cylindrical, septate, normal in shape while after being exposed to zinc the hyphal structures were deformed with corrugation, flocculation and became narrower (Fig. 4b). Various groups of researchers reported structural deformities in metal exposed hyphal structures [21].

Fig. 4
figure 4

Scanning electron microscopy (SEM) of Aspergillus terreus mycelium (a) control (b) after zinc exposure. (Samples viewed at 25 kV energy in microscope with ×2000 magnification)

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Zinc stress caused significant elevation in SOD and CAT activities in zinc exposed A. terreus with respect to their control counterparts. The increase in activities was directly proportional to the zinc concentrations. Stress enzyme activities showed just reverse to the metabolic enzyme activities. CAT enzyme played more potential role in scavenging activity than SOD in the present results when the fungal (A. terreus) strain exposed to same concentrations of zinc (Fig. 5). A. terreus showed 1.8 times more SOD activity and 4.08 times more CAT activity when the growth medium was enriched with 9000 ppm Zn, while at lower zinc concentration (100 ppm in growth media) 1.18 times enhanced SOD activity and 1.34 times more CAT activities were noted (Fig. 5). This might occur as SOD requires copper and zinc for its activity. Copper ions play functional role in the reaction by undergoing alternate oxidation whereas zinc ions seem to stabilize the enzyme. A. terreus showed 38–70 % augmentation in SOD activity and 70 % -2.5-fold augmentations in CAT activity when grown in 1000–8000 ppm of Zn. Participation of SODs in heavy metal toxicity has been reported by a group of researchers in plants, animals and microorganisms [14, 39], but reports about filamentous fungi is limited. However, SODs of filamentous fungi were reviewed by Frealle et al. [40] and some research on their response to metal and oxidative stress has been carried out [41], while CAT activity in fungi exposed to heavy metal stress is very rare. The present results are in tune with the findings of Vallino et al. [17], who reported 2.54-fold more SOD activity in ericoid mycorrhizal fungus Oidiodendron maius under Zn stress. Abhrasev et al. [42], observed enhanced level of SOD and CAT activities against thermal stress in A. niger. Pearson correlation matrix showed a positive correlation between SOD and CAT activities (Table 1b).

Fig. 5
figure 5

Effect of zinc stress on SOD and CAT activity of Aspergillus terreus [A. terreus grown in absence of zinc stress was taken as control. Both enzyme activities against zinc stress are significantly different (p ≤ 0.05) from control one]

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Spectrophotometric data of metallothionein protein revealed significantly higher amount of metallothionein in Zn induced A. terreus than that of unexposed counterparts (Table 2). Upregulation of metallothionein protein in Zn exposed A. terreus indicated that this small protein is responsible for heavy metal tolerance and subsequently allowed the fungi to survive in high Zn concentration (9000 ppm of Zn) as these small proteins are associated with metal homeostasis and oxidative stress along with numerous cellular functions [43]. The present results are in tune with the findings of Pal et al. [29], who found upregulation of metallothionein protein in Cd exposed A. niger with respect to wild type. Kameo et al. [44] accounted induction of metallothioneins in Beauveria bassiana treated with Cd and Cu. Similarly in Glomus intraradics, GintMT1 (metallothionein gene) was responsible for Cu stress [45]. In fungi this protein is primarily engrossed in metal toxicity and in stress response [46]. Results reflect role of MT in inducing heavy metal tolerance of the fungi allowing the fungi to survive in higher Zn enriched growth media.

Table 2 Quantitative estimation of metallothionein protein (µmol/mg of protein) in Aspergillus terreus exposed to different concentrations of Zn
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Data of the present study showing enhancement of anti-oxidative enzymes as well as non-enzymatic antioxidants in the tested fungal strain treated with Zn re-establish the toxic effect of heavy metals in producing cellular perturbations through production of ROS as has been cited by a host of earlier researchers [47, 48]. Response of antioxidant proteins and enzymes towards stress not only portrays immediate sensitivity or tolerance of A. terreus towards Zn but also gives an indication towards strategic development of adaptive response.

Conclusion

Based on the observations, it can be assumed that A. terreus is a Zn tolerant strain which can remove significantly higher amount of Zn from different Zn treated growth media. Even from extremely elevated concentrations of Zn enriched growth media (7000–9000 ppm) it showed significantly higher removal efficiency. Therefore the strain can be used for the decontamination of soil and effluent polluted by higher amount of Zn. Heavy metal stress like Zn, caused decrease in CMCase and α-amylase activities, the enzymes responsible for growth and nutrient assimilation in fungi. The detoxification of Zn by A. terreus is mediated by antioxidative enzyme system (SOD and CAT) as well as metallothionein protein which took part to sequester zinc inside the cell.