Copper-induced adaptation, oxidative stress and its tolerance in Aspergillus niger UCP1261

Background: The effects of exposure to copper, during growth, on the production of biomass, total protein, catalase, glutathione-S transferase, glutathione peroxidase, peroxidase, polyphosphate, acid and alkaline phosphatases, ultrastructure and the ability to remove this metal from Aspergillus niger , obtained from caatinga soil, were evaluated. Results: Allparameterstestedwerein ﬂ uencedbytheconcentrationofmetalintheculturemedium.Thepresence of metal induced high levels of antioxidant enzymes, including lipid peroxidation, thereby revealing the appearance of an oxidative stress response. The variation in polyphosphate levels indicates the participation of the polymer in response to stress induced by copper. The activities of the phosphatases were positively in ﬂ uenced by growing them in the presence of copper. Ultrastructure changes in the cell surface, electron density, thickness, and septation were visualized by exposing cells to increasingly larger concentrations of metal. The isolate was able to remove the agent from the growth medium, while maintaining its physiological functions. The metal removed from the cultures exposed to 0.5 mM, 1 mM and 2 mM copper exhibited percentages of removal equivalent to 75.78%, 66.04% and 33.51%. Conclusions: The results indicate that the isolate was able to grow in high concentrations of copper, activates mechanisms for adaptation and tolerance in the presence of metal, and is highly ef ﬁ cient at removing the agent. Such data are fundamental if a better understanding is to be reached of the cellular and molecular abilities of native isolates, which can be used to develop bioprocesses in environmental and industrial areas.


Microorganism and culture conditions
The microorganism A. niger UCP/WFCC 1261, was isolated from caatinga soil of Serra Talhada, Pernambuco, Brazil, and belongs to the Culture Collection of the Catholic University of Pernambuco (UCP)/ World Federation for Culture Collection (WFCC), and was used to study growth at various concentrations of copper. The strain was maintained on Potato Dextrose Agar (PDA) at 5°C. For the production of the pre-inoculum, the strain A. niger was grown on Sabouraud Dextrose Agar (SDA) medium and incubated at 28°C for 7 d.

Metallic solution
To prepare the stock solution of 100 mM, Cu 2+ ions were prepared by dissolving 24.968 g of CuSO 4 ·5H 2 O in 1 L of distilled and deionized water. From the stock, a standard solution was prepared of working solutions of 0.5 mM, 1 mM and 2 mM of copper used in these experiments.

Determination of growth curve
Pre-inoculum corresponding to 5% of a spore suspension of 10 7 cells/ mL, obtained from cultures of A. niger were inoculated in 250 mL Erlenmeyer flasks containing 100 mL of Sabouraud (broth) medium added copper at levels of 0.5 mM, 1 mM and 2 mM at pH 5, and were incubated in a shaker at 150 rpm, at 28°C, for 15 d. Control samples were grown in the absence of metal. The mycelia collected during the cultivation ranges reported were subjected to freeze drying, and subsequently kept in a vacuum desiccator until constant weight. The average dry weight in triplicate was used to establish the graph corresponding to the growth curve.

Glucose consumption
Treated and control samples corresponding to the supernatants from cultures grown in Sabouraud broth medium, served to determine the consumption of glucose in the medium. The glucose was measured by the enzymatic colorimetric method (Test-Lab) [23]. A standard curve was prepared using glucose solution (0.5-5.0 g/mL), and the concentration of glucose was determined by reading the absorbance at 510 nm, using a digital spectrophotometer (Spectronic Genesys 2 Mod).

Determination of pH
The pH of the growth in the culture media was monitored by the mean of three measurements. Subsequently, a pH curve was established with these values.

Extraction and determination of total protein
Samples of A. niger mycelium, collected at intervals of 3, 6, 9, 12 and 15 d of cultivation were collected and washed in phosphate buffered saline, pH 7.2 three times to remove waste. Samples of 10 mg of mycelium were lyophilized, and subjected to the extraction process using urea buffer/trichloroacetic acid. Initially, the samples were incubated in 0.5 mL of 10% trichloroacetic acid for 5 min at room temperature, washed three times with 90% acetone and allowed to air dry. Subsequently, the samples were incubated in 0.2 mL buffer containing 1% of SDS, 9 M of urea, 25 mM of Tris-HCl, pH 6.8, 1 mM of EDTA and 0.7 M of mercaptoethanol. The sample was stirred, boiled for 2 min, and stirred and boiled again for another minute. The samples were submitted to quantification by the Biuret method [24], which is based on the principle that the copper ions in an alkaline medium (the Biuret reaction) will interact with the peptide bonds of the protein, forming a purple color, which has absorbance to 545 nm and is proportional to the protein concentration in the sample.

Preparation of samples for determination of oxidative enzymes
Samples of 1 g of mycelium, collected at intervals of 3, 6, 9, 12 and 15 d were collected by filtration, washed with deionized water and mixed with a solution containing potassium chloride 1.15% phenyl methyl sulfonyl fluoride (protease inhibitor) at a concentration of 100 mM in isopropanol. The homogenized samples were centrifuged for 10 min at 3000 rpm in a refrigerated centrifuge. The supernatants were collected and used to determine the enzymatic activities antioxidants and lipid peroxidation.

Catalase (Cat)
The high speed of reaction of this enzyme associated with lower "affinity" enables its activity with high concentrations of H 2 O 2 (10 mM) to be determined. This was done using the rate of consumption of H 2 O 2 in the first minute of the reaction, 240 nm (ε = 40 M -1 cm -1 ) [25]. The disappearance of hydrogen peroxide without the presence of the sample is discounted. The enzyme assay was performed in 50 mM of 40-second potassium phosphate buffer (KPi); 0.5 mM of EDTA, pH 7, containing 0.012% of Triton X-100. 10 mM of H 2 O 2 is used as the substrate initiator. The absorbance baseline is deduced from the readings of the reaction in the absence of the test sample. The data are expressed as IU/mg protein. Proteins were measured by the Biuret method [24].

Glutathione-S-transferase (GST)
Conjugation of GSH with the substrate chlorodinitrobenzene (CDNB) catalyzed by GST produces a compound which can be detected at 340 nm (ε = 9600 M -1 cm -1 ). Enzyme activity is proportional to the speed of production of the conjugate compound [26]. This activity is a discounted reaction baseline reading obtained from the reaction between GSH and CDNB assay without the presence of the sample. The 5 min enzyme assay was performed in 100 mM of KPi, 1 mM of EDTA, pH 7, containing 1 mM of GSH. The substrate, 1 mM of CDNB, was used as the initiator. The baseline absorbance was reduced from the readings of the reaction in the absence of the test sample. The data is expressed as IU/mg protein. Proteins were measured by the Biuret method [24].

Glutathione peroxidase (GPx)
The enzyme, using GSH to degrade an organic peroxide such as t-butyl peroxide (t-BOOH), or cumene, generates oxidized glutathione (GSSG), which in turn is reduced by glutathione reductase, was added to the medium reaction with consumption of NADPH (ε = 6220 M -1 cm -1 ). The consumption of NADPH is monitored spectrophotometrically at 340 nm as glutathione reductase [27] is determined. This is a discounted rate of consumption of NADPH. The consumption of basal is obtained by reading the enzymatic assay without the presence of the substrate (peroxide). The enzyme assay is performed for 5 min in KPi, 50 mM of ethylenediamine tetraacetic acid, 0.5 mM of EDTA, pH 7, containing 0.2 mM of NADPH, and 1 mM of GSH, 0.2 U/mL of GR purified from yeast. 5-10 min of incubation is needed with the reagents (except initiator substrate) to activate the enzyme. 1 mM of CuOOH (cumene hydroperoxide) is used as substrate initiator. The data are expressed as IU/mg protein. Proteins were measured by the Biuret method [24].

Peroxidase
The presence of peroxidase activity was determined using pyrogallol and hydrogen peroxide as substrates [28]. Samples (0.1 mL for each activity measured) were subjected to reaction with 0.5 ml of H 2 O 2 (5 mmoL L -1 ), and 1.0 mL of pyrogallol (12.69 mmoL L -1 ), in 1.4 mL of sodium phosphate buffer 0.1 moL L -1 , pH 6. The reading activity was conducted at 420 nm after 1 min of reaction. The enzymatic activity was measured in enzyme units mg -1 protein. The proteins were measured by the Biuret method [24].

Lipid peroxidation
Lipid peroxidation was estimated by the TBARS [29], method with some modifications. The method forms a pinkish-red pigment comprising two molecules of thiobarbituric acid (TBA) and malondialdehyde (MDA). An aliquot (100 μL) of sample was added to 1 mL of solution containing 400 μL of acetic acid buffer, 1.3 M, 400 μL of TBA at 0.8% and 200 μL of sodium dodecyl sulfate (SDS) at 8.1%. This was mixed and incubated to 95°C for 60 min. The reaction of TBA with MDA produces a chromophore which can be measured photometrically at 532 nm. Data were expressed as nmol/mg protein. Proteins were measured by the Biuret method [24].

Extraction and polyphosphate determination
The polyphosphate was extracted and assayed according to the method described by McGrath and Quinn [30]. Samples of 10 mg of mycelium (dry weight) obtained from the incubation medium during the cultivation of the fungus at intervals of 3, 6, 9 12 and 15 d were collected and washed twice in 1.5 M NaCl solution containing 0.01 M EDTA and 1 mM NaF. They were then placed in wash solution and sonicated on ice for 2 min 12 times with intervals of 1 min at 16 KHz. The resulting extract was centrifuged at 12000 × g for 10 min at 4°C to remove the cell debris. To determine the total cell content of the polyphosphate, 100 μL of concentrated HCl was added to 0.5 mL of cell extract and heated at 100°C for 45 min. The phosphate released was determined using the colorimetric method Subbarow [31], which is based on the reaction of inorganic phosphorus with ammonium molybdate in acidic media, resulting in a complex phosphomolybdate blue color, quantified spectrophotometrically at 600 nm, and the intensity of which is directly proportional to the concentration of phosphorus. A standard curve was prepared, for which a solution of phosphorus (0.5-5.0 mg/dL) was used. Data were collected in a digital spectrophotometer, Spectronic, Genesys model 2. The concentration of polyphosphate was expressed in milligrams per deciliter (mg/dL) of phosphorous and given as means of triplicates. A non-hydrolyzed sample was used as a control to determine the level of free phosphate in the cell. The overall polyphosphate is expressed in milligrams per gram of biomass.

Activity of acid and alkaline phosphatase
To determine the enzymatic activity of acid and alkaline phosphatase in culture medium, aliquots of the supernatant fluid and metabolic biomass were collected at intervals of 3, 6, 9, 12, and 15 d during cultivation. Samples of 36 mg of mycelium (wet weight) were collected at intervals of 3, 6, 9, 12, and 15 d of cultivation, washed with deionized water and incubated in 3 mL of extraction solution, consisting of 0.02 M of sodium acetate buffer, pH 4.5 for acid phosphatase, and 50 mM of alkaline phosphatase Tris-HCl, pH 7.5, containing 5% of glycerol. The samples were macerated by mortar and pestle for 5 min, and then homogenized for 2 min on ice. The resulting extract was centrifuged at 12000 × g for 10 min at 4°C to remove cellular debris. To determine enzyme activity, the Lab-Test Kit was used and cell extracts were collected and subjected to spectrophotometric determination using Lab-Test Kits. These are based on the enzymatic hydrolysis of monophosphate thymolphthalein releasing thymolphthalein released in its blue at different pH values. The resulting color of the reaction is directly proportional to the enzyme concentration and is measured at 590 nm. The values of the concentrations were given in International Units (IU), which represent the amount of enzyme that catalyzes the splitting of 1 μmol substrate/ minute/liter of sample, according to Joh et al. [32].

Scanning electron microscopy
A scanning electron microscope was used to analyze the mycelium surface, of control and exposed cells, to different concentrations of copper, after 3 and 15 d of contact. The mycelium was collected after centrifugation and filtration, washed twice with phosphate buffered saline, pH 7.2, and fixed with 0.1 M of cacodylate buffer containing 2.5% of glutaraldehyde, pH 7.2. After 3 h, the mycelium was washed twice with 0.1 M of cacodylate buffer, pH 7.2. Samples were post-fixed with 0.1 M of cacodylate buffer containing 0.05% of malachite green for two hours in the dark. This was followed by washing in 0.1 M of cacodylate buffer, pH 7.2, 2.5% of glutaraldehyde, pH 7.2. The mycelium was dehydrated in ethanol (v/v): 50%, 70%, 90% and 100%. The mycelia were then mounted on metal supports, observed and photographed in a LV5.600 JEOL scanning microscope operating at 20 keV.

Determination of copper removal
To determine copper removal from the culture medium, supernatant samples withdrawn at intervals of 3, 6, 9, 12 and 15 d were subjected to an atomic absorption spectrophotometer, model spectrophotometer (GBC 932 AA). All experiments were performed in triplicate. The biomass biosorption and the removal efficiency were evaluated. The assay was performed in triplicate. The sorption efficiency (%) was calculated according to the expressions: where C o and C e are the initial and equilibrium concentrations (mg L -1 ).

Statistical analysis
To evaluate the influence of copper on growth, antioxidant enzyme activity, lipid peroxidation, polyphosphate, acid and alkaline phosphatase, and removal of copper, the data were submitted to variance analysis using Statistica 7.0 software. The means between treatments were compared by Tukey's t-test at 5% probability.

Effects of copper on the growth profile
Initially, the behavior of the strain was evaluated on the characteristics of growth by increasing the concentrations of copper in the medium. The growth profile of A. niger at time intervals in cultures in the absence and presence of copper at concentrations of 0.5 mM, 1 mM and 2 mM, was associated with a corresponding glucose consumption. Fig. 1a and Fig. 1b, present the data obtained for isolated growth in the presence of copper and glucose consumption.
Approximately 58.45% of glucose was consumed in the indicated time intervals of the culture control during the first 3 d of cultivation and the cultures were treated with 0.5 mM, 1 mM and 2 mM copper respectively and 63.1%, 50.4% and 23.6% were consumed. Based on the growth curve, it appears that after 15 d of cultivation, although glucose consumption had occurred, all cultures kept growing even after the carbon source in the presence of the metal was exhausted (Fig. 1a). This represents the tolerance of A. niger to the presence of copper.
It was found that the length of time and concentration had an influence on the biomass of A. niger. Treatments with 0.5 mM and 1 mM copper did not differ statistically from the control culture and after 15 d of culture, an increase in the biomass concentration of 1 mM with 1.7 g/L was observed as was a control sample with 1.3 g/L. However, a significant reduction was observed in the biomass concentration of 2 mM copper, with 1.0 g/L being obtained (Fig. 1b).
Assessing the potential of growth of A. niger in the presence of copper, it was found that the fungus is able to grow at concentrations of up to 300 mg L -1 in the culture medium [33]. In this study, the growth of A. niger, accompanied by the production of biomass revealed that copper at concentrations of 0.5 mM, 1 mM and 2 mM has a positive effect on this variable. However, higher copper concentrations increased the lag phase and decreased the carbon source consumption. These data confirmed the information regarding the effects of copper on the growth of A. niger.

Effect of copper on total protein and lipid peroxidation (TBARS)
The data obtained from analyzing the TBARS in response to the presence and absence of copper in the cultivation time intervals are shown in Fig. 2a and Fig. 2b.
Treatment with copper in diverse concentrations induced the appearance of a distinct protein profile for all the samples tested (Fig. 2a).
The results showed that despite the changes in total protein content in the samples treated with copper, no significant differences were observed between treatments (P N 0.05). However, the content of total protein indicates that a significant effect on protein content has been found between cultivation times. There was an increase over the control which might demonstrate the inductive effect of copper content on protein isolated from A. niger.
Many microorganisms synthesize proteins, and are able to sequester metal ions, especially Cu, Pb and Cd, found at high concentrations in polluted soils. Metal transport proteins may be involved in metal tolerance either by extruding toxic metal ions from the cytosol out of the cell or by allowing metal sequestration into intracellular compartments [10,34].
In this study, increasing total protein content in response to copper (0.5, 1 and 2 mM) concentration and the protein levels can support the synthesis of key proteins involved in detoxification system A. niger against excess copper indicating that it is able to neutralize the toxic effect of heavy metals.
Throughout the experimental period, it was found that the control culture exhibited lower levels of lipid peroxidation compared to treated cultures (Fig. 2b). Treatments with 0.5 mM, 1 mM and 2 mM of copper induced an increase in the peak levels for TBARS according to time intervals. Treatment with 2 mM copper induced the highest TBARS difference from the average, relative to treatment with 0.5 mM and 1 mM copper. Note that the TBARS levels among the treated samples varied according to the culture and differed significantly 5% from the control sample over time.
These data reveal that oxidative stress resulting, among others, in membrane lipid peroxidation is involved in the mechanisms of the toxicity of copper toxicity and suggests that this fungus exhibits (an ability to copper), to some extent, with the increased level of lipid peroxides. Curvularia lunata in the mycelia exposed to Ni 2 , the levels of TBARS (lipid peroxidation products) increased and ranged between 156 and 823% over the control [35].

Activity of antioxidant enzymes
The results presented in Fig. 3a, Fig. 3b, Fig. 3c, and Fig. 3d reveal the activity of antioxidant enzymescatalase, glutathione S transferase, glutathione peroxidase, and peroxidase, in response to the concentration of copper (0.5, 1 and 2 mM).
These results showed that the time and concentration of copper in catalase activity had a significant effect (P b 0.05). At all-time intervals, the catalase activity remains higher in samples treated with copper (Fig. 3a).
It appears that enzyme activity increases with the length of cultivation and that within 3 d of cultivation, catalase activity increased 50% in the samples treated with 0.5 mM and 1 mM copper respectively 52.6 UI/mg and 61.8 UI/mg compared to control with 26 UI/mg. Furthermore, the presence of copper at concentrations of 0.5 mM and 1 mM led to a progressive increase in enzymatic activity compared to the concentration of 2 mM with 40.46 UI/mg. The highest level of CAT was obtained at the end of the experiment after 15 d in culture with 110.26 UI/mg, 119.26 UI/mg and 90.68 UI/mg treatments respectively 0.5 mM, 1 mM and 2 mM compared to control with 64.4 UI/mg.
Results on the effect of exposure concentrations of copper and the interaction of time and copper on peroxidase activity were similar to those for catalase activity (Fig. 3b). It is observed that the presence of Results are means (μ) ± standard deviations (σ) for thee replicates. * discriminates significant differences (P b 0,5) from control using Tukey test. Fig. 3. Biological responses of A. niger apices exposed to Cu (a, b, c, and d) for the activity of the enzymes catalase, peroxidase, glutathione S-transferase (GST) and glutathione peroxidase (GPx) respectively, in the culture medium within 15 d. Results are means (μ) ± standard deviations (σ) for thee replicates. * discriminate significant differences (P b 0.5) from control using Tukey test. metal in the culture medium resulted in increased activity compared to control culture (P b 0.05). Peroxidase activity in time intervals of cultivation among treated and control samples shows that a progressive increase in the activity of the enzyme was obtained at the end of the experiment after 15 d in culture. This increased 60% and demonstrated that time was a significant factor in the enzyme expression. The treatment with 0.5 and 1 mM copper with respectively 0.02262 UI/mg and 0.02546 UI/mg compared to control with 0.0164 UI/mg, and 2 mM 0.01852 UI/mg had the highest level of peroxidase activity.
On determining the activity of glutathione S transferase (GST), the positive effect of exposure to copper compared to control culture and the culture time was observed. The enzymatic response was dependent on the concentration of copper used (Fig. 3c). The data on the levels of glutathione S-transferase activity were increasing the time intervals between a 5% of significance. The results differing among the concentrations used achieved after 15 d of cultivation levels of 0.358 IU/mg 0.445 IU/mg, 0.5 IU/mg and 0.5434 IU/mg respectively stop control, 0.5 mM, 1.0 mM and 2 mM, thus verifying the inductive effect of copper on the activity of the enzyme.
The activity of glutathione peroxidase (GPx) in response to the presence and absence of copper in the time intervals in treated and control samples is shown in Fig. 3d. The glutathione peroxidase activity shows a progressive increase in the activity of the enzyme for treatment and control samples in the time intervals. It is observed that the presence of metal in the culture medium resulted in increased activity compared to control culture (P b 0.05). The treatment with 1 mM of copper had the highest level of GPx activity with 0.03758 UI/mg compared to control with 0.02066 UI/mg. Cellular responses related to the adaptation process when the occurrence of oxidative stress includes increased activity of antioxidant enzymes and/or increasing the concentration of non-enzymatic antioxidant components [36].
Enzymes like CAT, which detoxifies hydrogen peroxide and GPx, which catalyzes the reduction of hydrogen peroxide and other peroxides, are essential to maintain cellular redox balance, and are activated during stress induced by heavy metals [11,37]. Additionally, glutathione S-transferase is the enzyme responsible for the conjugation of glutathione to electrophilic xenobiotics, thereby reducing its toxicity. This makes them more hydrophilic, thus enabling the conveyor system to eliminate these conjugates into the extracellular environment. They are metabolized via the mercapturic acid pathway [38].
In the present study it was demonstrated that the isolate of A. niger, obtained from the soil of caatinga, exhibited catalase, glutathione S-transferase, glutathione peroxidase, and peroxidase activities. These enzyme activities increased during exposure to copper. This result possibly indicates an induction mechanism of H 2 O 2 formation. The increase in enzyme activities is a mechanism for metal detoxification via degradation of hydrogen peroxide, thus confirming the data in the literature.
A. niger has an activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione reductase in response to oxidative stress induced by the presence of hydrogen peroxide. The existence and activity of antioxidant enzyme catalase and superoxide dismutase have also been reported in the protection of the A. niger conidiophores subjected to thermal stress [39,40]. Several species of fungi under stress induced by copper have been shown to have activation of enzymes, such as catalase. An increase in peroxidase activity and a reduction in glutathione reductase have also been reported [41,42].

Effects of copper on polyphosphate behavior
The behavior of polyphosphate isolated from A. niger over time for the control samples and treated with copper sulfate is shown in Fig. 4.
The highest total polyphosphate was obtained at 3 d of cultivation for control cultures and those treated with 2 mM copper, with values of 0.66 mmoL L -1 and 0.86 mmoL L -1 , respectively. For treatment with 0.5 mM and 1 mM of copper, polyphosphate content of 0.54 mmoL L -1 , and 0.61 mmoL L -1 was obtained, respectively. The content of the polymer decreased over cultivation time. For the treatment with 2 mM, a decrease of 73% in the polymer was found after 15 d. The results revealed, for the treated and control cultures, that the cultivation time and metal concentration were important factors for the polymer behavior in A. niger, and showed significant variations at the level of 5%.
The data indicate the potential for polyphosphate accumulation by the fungus A. niger, as well as its degradation related to tolerance/survival in the presence of coper ions.
The detoxification of heavy metals, including copper, is also reported as dependent on the metabolism of polyphosphate. The polymer is associated with the potential of tolerance to heavy metals, as well as resistance to stress induced by heavy metals in various bacteria, yeasts and filamentous fungi [43,44,45,46,47,48].
The metabolism of polyphosphate was investigated in Trichoderma harzianum showed that the presence of cadmium induced a reduction in polyphosphate content related to the concentration used [49].
The presence of heavy metals, cells can activate two mechanisms: the increase in or accumulation of degradation of the polymer and the start of the synthesis cycle for the release of phosphorus sequestering and chelating of metal ions by reducing intracellular flow and consequently their toxicity [50,51,52].
In this study, we observed variations in the content of polyphosphate between control cultures and those exposed to copper. This variation was related to the length of cultivation, the growth phase, and the concentration of metal. Thus, reducing the polymer content in the cultures of A. niger exposed to metal revealed the reduction of the polymer, indicating its use to maintain cell viability, which supports the functions of the metal in the maintenance of cell metabolism. Additionally, the data obtained for control cultures also demonstrate the use of metal at different stages of growth. These data are the first reports of the behavior of polymer A. niger.

Acid and alkaline phosphatase behavior
The results obtained for the intracellular acid and alkaline phosphatases detected in treated and control samples are presented in Fig. 5a, Fig. 5b.
High levels of acid phosphatase activity of control and treated samples were evaluated in relation to the culture period. Variance analysis showed significant variations for the cultivation time. The results obtained for alkaline phosphatase activity also showed different values among samples during the time intervals. After 6 d of cultivation, increased levels of the enzyme were detected in the cultures treated with 0.5 mM, 1 mM and 2 mM of copper, respectively, which corresponded to 2.48 IU/L, 2.45 IU/L and 2.29 IU/L, an increase of 25% compared to the control with 1.69 IU/L. At intervals of 9 and 12 d phosphatase activity varied for samples treated with 0.5 mM, 1 mM and 2 mM copper, and maintained a behavior similar to the control culture. At 15 d of cultivation, values were very similar between the treated and control sample. As demonstrated by the variance analysis at the 5% level, there was no significant effect on the enzyme activity related to copper concentrations. However, it was identified that cultures treated with copper had a slight increase in phosphatase activity, which was proportional to the concentration, thus showing an inductive effect of exposure to copper.
The results obtained for alkaline phosphatase activity also demonstrated a significant effect of culture time on the expression of enzymatic activity.
The activity of the phosphatase also induced copper in A. niger, was described by Tsekova and Todorova [33]. Where observed the increased activity of the phosphatase with exposure to copper. Acid phosphatase activity in Yarrowia lipolytica increased with increasing concentrations of Cu 2+ in the medium. In addition, the content of phosphate involving cellular polyphosphate was reduced by the addition of Cu 2+ [53].
The microorganisms to reduce the concentration of free heavy metal, may sequester the form of phosphate salts. The hydrolysis of organic phosphorus acid phosphatase and by a concurrent precipitation of heavy metals on the surface of the cell help to protect and enables these support microorganisms to tolerate higher doses of heavy metals [2].

Scanning electron microscopy
Ultrastructural analysis showed changes in the density of mycelia, the electron density pattern, the texture of the cell surface and the thickness of hyphae in treated samples compared to control culture. The intensity variations are related to the concentration of the metal to which cells were exposed (Fig. 6).
Heavy metals are reported to induce structural changes and the effects associated with the type of metal and its concentration. Basically, exposure to metals and their subsequent connection modify the properties of the structure, such as the texture of electron density [41,45,47]. The data obtained in this study confirm the effects of copper in ultrastructure A. niger, isolated from soil of the caatinga.
The reports about the tolerance of microorganisms to copper display data indicated that within the same genus, species and strains react differently to different heavy metals and their concentrations.  Additionally, the sensitivity is also related to the stage of cell growth. Thus, these studies do not enable a concentration limit to be determined. For example, while other microorganisms can only tolerate up to 6000 mg/kg of metal, showing metal resistance mechanisms, adsorption and intracellular compartmentalization [54].
In this study, data on the ability of removing copper revealed that the strain was able to remove a high percentage of the metal, when exposed to copper during growth and has metabolically active cells, suggesting the involvement of cumulative processes and binding to the cell surface, related to the presence of chitin and chitosan in its walls.

Copper removal
The results revealed that A. niger sample was able to remove the metal from culture supernatant. Additionally, the removal depended on the initial concentration used. Fig. 7 shows the residual copper in the medium.
Cells exposed to 0.5 mM and 1 mM of copper showed higher removal percentages compared to cultures treated with 2 mM. At 3 d of cultivation, the corresponding percentages of removal were 31.76%, 21.39% and 12.72% for cultures exposed to 0.5 mM, 1 mM and 2 mM of copper, respectively. At the end of 15 d of cultivation, the cultures exposed to 0.5 mM, 1 mM and 2 mM of copper exhibited percentages of removal equivalent to 75.78%, 66.04% and 33.51%. The percentage removal was used to perform the variance analysis relating the time of cultivation and the concentration of copper. The data express a significance level of 5%.
In this study, the data show that the strain was able to show growth even with the reduction of biomass, in concentrations of up to 2 mM of copper. This finding was confirmed by the expression of adaptive responses and tolerance that allowed cell survival.
The genus Aspergillus fungi is considered to be a fungus which has one of the greatest potential biotechnological and industrial uses nowadays due to its potential for producing numerous metabolites [15,55,56].
Microbial cells are used in bioremediation and biotransformation processes of textile dyes, oil and oil products, pesticides and heavy metals [2,56,57]. Thus, the biomass of fungi is an excellent sorbent material for metal ions [2,48].
Data in the literature show that microorganisms exhibit high potential for the removal and mobilization of copper. This removal can be performed efficiently by bacteria such as Pseudomonas, Bacillus, Staphylococcus, and yeasts such as Candida, Saccharomyces, Kluyveromyces, Schizosaccharomyces and filamentous fungi, such as Aspergillus, Mucor, Rhizopus, Trichoderma and Cunninghamella [2]. Therefore, we evaluated the potential for copper removal in A. niger isolate from soil of the caatinga. The data presented show that the isolate removed copper in stages during growth, such that by the end of the experiment, the percentage removed was 75.78%, 66.04% and 33.51% at concentrations of 0.5 mM, 1 mM and 2 mM, thus indicating there were greater efficiencies at lower metal content.
The species A. niger has been shown to be effective in the biotransformation of pesticides [57]. Additionally, many strains of A. niger have been extensively tested for their ability to leach and remediate heavy metals, lead, cadmium, nickel, zinc, copper, based on their capacity for physiological adaptation and tolerance resistance [58,59,60,61,62,63].
The cell walls of fungi act as a first barrier restricting the internalization of solutes. Chitin, chitosan, glycoproteins and melanin, the main components of cell walls of fungi, confer protection against metal ions [2]. The metals bound to the walls act like other materials used in ion exchange, adsorption, complexation, precipitation and crystallization [2]. Thus, it has been reported that yeast tolerant metals exhibit a higher binding potential of metals in relation to a wall of lesser tolerance. Some studies have shown that the walls of fungi can retain from 37% to 77% of copper [64].

Conclusions
It was found that there was influence of copper on the growth of the A. niger isolate UCP/WFCC 126, used in this study and a significant decrease in biomass concentration of 2 mM was observed. The isolate showed changes in the protein profiles directly related to the concentration of copper, and the microorganism was able to keep the content of proteins in the presence of copper in the medium which may suggest a possible detoxification engine due to exposure to this metal. The lipid peroxidation was observed as a result of exposure to the copper. The isolate exhibited activity of catalase, glutathione S-transferase, glutathione peroxidase, and peroxidase. These activities in cells exposed to copper were increased, revealing the activation of antioxidant response against stress induced by metal. The behavior of the polyphosphate had changed, indicating the role of polymer molecular responses of tolerance to stress induced by copper. Modifications of the fine structure, such as increased electron density, septation and shortening of the hyphae were observed. The intensity of the changes was proportional to the concentration of the copper. The isolate was able to remove the metal from the cultivation medium and has the potential for higher remediation. These data indicate the physiological and biotechnological potentials of the isolate, which should be evaluated more specifically for the development of bioprocesses.