Heavy metal tolerance and adaptability assessment of indigenous filamentous fungi isolated from industrial wastewater and sludge samples

Accepted Manuscript Heavy metal tolerance and adaptability assessment of indigenous filamentous fungi isolated from industrial wastewater and sludge samples Pawan Kumar Rose, Rani Devi PII: DOI: Reference:

S2314-8535(18)30062-3 https://doi.org/10.1016/j.bjbas.2018.08.001 BJBAS 304

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Beni-Suef University Journal of Basic and Applied Sciences

Received Date: Revised Date: Accepted Date:

29 January 2018 29 June 2018 6 August 2018

Please cite this article as: P.K. Rose, R. Devi, Heavy metal tolerance and adaptability assessment of indigenous filamentous fungi isolated from industrial wastewater and sludge samples, Beni-Suef University Journal of Basic and Applied Sciences (2018), doi: https://doi.org/10.1016/j.bjbas.2018.08.001

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Heavy metal tolerance and adaptability assessment of indigenous filamentous fungi isolated from industrial wastewater and sludge samples

Heavy metal tolerance and adaptability assessment of indigenous filamentous fungi isolated from industrial wastewater and sludge samples

Pawan Kumar Rose a, * and Rani Devi a

a

Department of Energy and Environmental Sciences, Chaudhary Devi Lal University, Sirsa,

Haryana -125055, India

* Corresponding author: Department of Energy and Environmental Sciences, Chaudhary Devi Lal University, Sirsa, Haryana -125055, India E-mail address: [email protected]

Abstract A total of twenty-five isolates of fungi from the wastewater and sludge samples of a steel industry were screened for their resistance to Cu(II) and Ni(II). Three strains were finally 1

selected and identified as Aspergillus awamori, Aspergillus flavus and Aspergillus niger. The growth behavior and concurrent bioaccumulation of Cu(II) and Ni(II) by each fungus was investigated as a function of pH of the growth medium and concentration of Cu(II) and Ni(II). pH for maximum growth with concurrent bioaccumulation of Cu(II) and Ni(II) was observed, respectively as 4.0 and 5.0 for A. awamori, 5.0 and 5.0 for A. flavus and 4.0 and 5.0 for A. niger. The growth behavior with increasing heavy metals concentration was also examined as tolerance index. Synergistic and inhibitory effect on growth with lower and higher concentration of both heavy metals, respectively observed for each fungus. Our finding indicates that the resistant strains isolated from their contaminated habitat have better pertinence and application in heavy metals bioremediation.

Keywords: Aspergillus awamori; Aspergillus flavus; Aspergillus niger; Heavy metal; Tolerance index

1. Introduction Heavy metals are a kind of natural resource used in several industrial, agricultural and domestic applications. However, conjointly they act as a pollutant, which can be very toxic to many life forms and brings various environmental issues. Unlike organic pollutant, heavy metals are non-biodegradable, exhibit toxicity even at low concentration and persistent in nature (Fu and Wang, 2011; Rose and Devi, 2015). Heavy metals have the tendency to accumulate in the body of living organisms, through the bioaccumulation process. Further, biomagnification process elevates the concentration of heavy metals in the body to such a level that they begin to produce toxic effects, and many of them become carcinogenic. As heavy metals are non-biodegradable, highly toxic and stable in the environment, so their 2

removal becomes an absolute necessity. Bioremediation is an environmental sound technology that restrains the burden on natural resources as associated with conventional technologies. Bioremediation process has shown many advantages over other available conventional technologies in the removal of heavy metals laden wastewater. It is easy in operation, less expensive, feasible, produce less toxic sludge and has a short period of operation (Mungasavalli et al., 2007). It has shown selectivity for specific heavy metal and it removes the metal more efficiently even with its presence in low concentration with less impact on cleanup personnel (Singh et al., 2010; Xie et al., 2017). It permanently eliminates the pollutants and easily bind up with other existing physical and chemical methods (Xie et al., 2017). Mechanism of heavy metals bioremediation is defined by the life form of the microorganism. The non-viable (dead) form abstracts heavy metals by biosorption and the viable (live) form by bioaccumulation mechanism. The biosorption is a rapid, passive and metabolic energy-independent process, which ties the heavy metals on the surface of biosorbent . However, bioaccumulation is a slow, active and metabolic energy-dependent process primarily involves conveying of heavy metals across the membrane into the cell (Volesky, 1990; Uslu et al., 2003; Acıkel and Alp, 2009; Fomina and Gadd, 2014; Zotti et al., 2014). Bioaccumulation is an intracellular heavy metals accumulation process that involves a variety of physical, chemical and biological mechanisms. It includes a combination of surface reactions, intra- and extra-cellular precipitation and intra- and extracellular complexation reactions (Kadukova and Vircikova, 2005; Fomina and Gadd, 2014). Heavy metals removal by non-viable form shown distinct advantages over the viable form. Uptake of heavy metal by non-viable is free from the metal toxicity to living cell, requirement of expensive nutrients and growth media (Dhankhar and Hooda, 2011). They can be easily produced from simple fermentation process under aseptic environments and 3

conveniently immobilized with good feasibility of biomass regeneration and heavy metals recovery by desorption process (Ahluwalia and Goyal, 2007; Singh et al., 2010). However, viable form removes heavy metals simultaneously with the growth of its biomass during culturing process and separate biomass production process (e.g., cultivation, harvesting, drying, processing and storage) are not demanded (Gonen and Aksu, 2008). Though, this system (using viable biomass) has many limitations for heavy metals accumulation. The viable biomass requires necessary nutrients, growth media and growth of biomass is also suppressed due to high concentration of heavy metals (most significant), salts and extreme pH (Donmez and Aksu, 1999; Uslu et al., 2003; Gonen and Aksu, 2008). Isolation and screening of heavy metals resistant microorganisms can overcome these limitations as they have the ability to survive in higher heavy metals concentration and potential for heavy metals accumulation (Gonen and Aksu, 2008). Identification of indigenous microbial species that are adapted to heavy metals contaminated environment has the ability for long-term application in wastewater treatment. These species can remarkably contribute towards the development of economical and efficient bioremediation processes (Singh et al., 2016). Application of fungi in the bioremediation is known as mycoremediation and term was coined by Paul Stamets. Fungi is a better choice as bioremediater due to their considerable tolerance toward toxic heavy metals, immense growth rate and admirable metalbinding properties. They can easily and economical culture in mass quantities because to their short multiplication cycle and easily modified morphologically and genetically (Fu and Wang, 2011; Dhankhar and Hooda, 2011). The viable fungus cells have usually high intracellular uptake values and lesser sensitivity towards extreme fluctuation in pH, temperature, aeration and nutrition (Anand et al., 2006). The heavy metals bioremediation potential of different species of fungi was reported from various parts around the world (Ahluwalia and Goyal, 2007; Wang and Chen, 2009; Dhankhar and Hooda, 2011; Abbas et al., 2014). 4

However, potential of filamentous fungi habitat at contaminated sites in India have not been well explored (Singh et al., 2010). There are confined studies in our country on the systematic isolation of filamentous fungi from the heavy metals contaminated site from their diversity. Followed by the screening of heavy metals resistant/tolerant strains among the isolates are obtained from the site. Furher the assessment of growth behavior and bioaccumulation potential of fungal mycelia in heavy metals containing medium, tolerance behavior and heavy metals sorption capacity study. Considering these aspects the present study was undertaken for: 1) isolation and identification of heavy metals resistant filamentous fungi habiting wastewater and sludge of a steel industry, 2) assessing the effects of heavy metals on the mycelia growth and bioaccumulation potential of isolated filamentous fungi as a function of initial pH and initial heavy metals concentration in a conventional batch system, 3) evaluating the tolerance behavior of isolated filamentous fungi for heavy metals in terms of tolerance index.

2. Materials and Methods 2.1. Sample collection and analysis The wastewater and sludge samples were collected from a steel industry located near the Rohtak city of Haryana, India. Rohtak is a southerneast district of Haryana state having a semi-arid condition with great variation in temperature in a year. The wastewater samples were collected in a pre-cleaned, sterilized, polyethylene bottle of two liter capacity and sludge sample in plastic zipper bags. The samples were carried to the laboratory in ice bags (4oC). The pH of the wastewater sample was measured at the location using handy pH meter (Eutech pHScan 2). The physico-chemical characteristics of wastewater were analyzed using standard APHA methods (APHA, 2012). 5

2.2. Reagents and glassware The chemicals and reagents were utilized throughout in the present study were of analytical reagent grade. A stock solution of Cu(II) and Ni(II) (1,000 mg/L) was prepared by dissolving salts of copper sulfate (CuSO4.5H2O) and nickel sulfate (NiSO4.6H2O), respectively in double distilled water. The desired concentrations for experiments were obtained by the dilution of stock solutions. All the aqueous solutions were prepared using double distilled water. Borosil glasswares were cleaned properly with labolene, acid (10% nitric acid), tap and double distilled water, dried and sterilized in a hot air oven at 180oC for 4 h successively prior to using for the experimental purpose. The concentration of heavy metals was measured by atomic absorption spectrophotometer (LabIndia AA-7000). 2.3. Media used Three different types of media were used in the present study for the isolation of filamentous fungi from wastewater and sludge samples. i) Potato Dextrose Agar (PDA) with following composition (g/L): potato infusion 300.0, dextrose 20.0, agar 15.0, pH 5.6; ii) Czapek Yeast Extract Agar (CYEA) with following composition (g/L): sucrose 30.0, yeast extract 5.0, K2HPO4 1.0, NaNO3 0.3, KCl 0.05, MgSO4.7H2O 0.05, FeSO4.7H2O 0.001, ZnSO4 0.001, CuSO4 0.0005, agar 15.0) and iii) Martin Agar (MA) with following composition (g/L): dextrose 10.0, peptone 0.5, MgSO4.7H2O 0.5, KH2PO4 1.0, rose bengal dye 0.03, streptomycin 0.033, agar 15.0, pH 5.0. PDA and CYEA were procured from Hi-Media Lab. Pvt. Ltd., Mumbai, India and MA was prepared as per the given composition by Martin (1950). PDA and CYEA media were modified by addition of rose bengal dye (0.03 g/L). Rose bengal dye suppresses the development of bacteria (Ottow and Glathe, 1968) and recommended in standard methods for the enumeration of yeasts and moulds from water (APHA, 1998). Modified CYEA media was used for fungus screening and MIC experiments. Rest of the experiments were performed using Czapek Yeast Extract (CYE) broth, which has 6

similar composition as CYEA but without agar. CYE broth was also supplemented with rose bengal dye. 2.4. Isolation of filamentous fungi Serial dilution and standard spread plate techniques were employed for filamentous fungi isolation from both wastewater and sludge samples. Aliquots of 1 ml from different dilutions were spread on the modified PDA, modified CYEA and MA media plates (Triplicates). The inoculated plates were incubated for 72 h or more at 30°C, developed heterogeneous colonies were picked on the basis of frequency of occurrence for purification. Pure cultures of isolates were obtained by streaking repeatedly colonies on modified CYEA media plates. Pure isolates culture were maintained at 4oC and monthly subcultured on modified CYEA slants served as stock cultures. 2.5. Screening and identification of heavy metals resistant filamentous fungi Purified fungal isolates were screened based on their resistant to copper as Cu(II) and nickel as Ni(II) using two successive steps. In primary screening step (qualitative), one loopful of fungal growth was inoculated on the sterilized modified CYEA media supplemented with 100 mg/L concentration of heavy metal (individually) by streaking method. Visible growth after a period of 72 h or more of incubation at 30oC were observed and further screened, based on growth rates (kd) experiment that was the secondary screening step (quantitative). The fungal mycelia disk (8 mm) was aseptically inoculated on the center of sterilized modified CYEA media plates supplemented with heavy metals (100 mg/L, individually). Plates were incubated at 30oC and mycelia growth (diameter) was measured every day using a ruler. The fungal strains demonstrated maximum mycelia growth in the minimum time period (greater value of kd) were subjected to identification and evaluation of growth behavior, bioaccumulation potential and tolerance behavior. Finally selected fungal strains were identified and characterized on the basis of their macroscopic features on culture media and 7

microscopic features under the compound microscope by preparing slides with lactophenolcotton blue staining dye. The initial identity of isolates was done using taxonomic guides and standard procedures (Barnett and Hunter, 1998). The growth rate (kd) of individual fungus was measured by following equation. kd = D/T, Where, D: Average diameter of the fungal colony, mm (excludes diameter of inoculum, 8 mm), T: time period in hours (h). 2.6. Minimum inhibitory concentration assay Minimum inhibitory concentration (MIC) is the minimum heavy metal concentration that entirely inhibits the noticeable growth of microorganisms. The Cu(II) and Ni(II) tolerance was determined as the MIC against isolated fungal strains. Modified CYEA media plates inoculated by spreading 100 µl volume of fungal spore suspension. A circular trough was made in solid media and filled with a sterilized solution of heavy metals (dip method) of desire concentration ranging from 100 mg/L-6000 mg/L. The inoculated plates were incubated at 30oC for 5 days, and growth was recorded every day. 2.7. Growth and bioaccumulation studies The effect of initial pH on the fungal growth and bioaccumulation potential was carried out in 250 ml Erlenmeyer flask with 100 ml working volume of modified CYE broth media containing 100 mg/L of heavy metal concentration (individually). The pH of the sterilized (121°C for 15 min at 15 psi) accumulation medium was adjusted to the desired value (3.5, 4.0, 4.5, 5.0, 5.5, 6.0) using sterile H2SO4 (0.1N) and NaOH (0.1N) solutions. Afterward, the accumulation medium was inoculated with 8 mm disc of fungus mycelium with spore from the 7 days old seeded modified CYEA plates, pre-inoculated at 30oC of respective fungal strain under sterile environment. After 72 h of incubation at 30 oC on an orbital shaker at 150 8

rpm, the fungal biomass was harvested by filtration technique using whatman filter paper no. 1. The fungal growth at different pH in the influence of heavy metals was measured by taking constant weight of dry biomass of mycelium (g/L) at 80oC. The residual heavy metals concentration in broth medium was measured after digestion with nitric acid and sulphuric acid (3:1 ratio) by atomic absorption spectrophotometer using standard protocols (APHA, 2012). The metal uptake capacity was determined as the amount of heavy metal accumulated per unit of dry weight of cells at the end of microbial growth (mg/g). 2.8. Effect of heavy metal concentration on fungal growth The effect of heavy metal on fungal growth was mapped by tolerance index. The tolerance index (TI) is a ratio of fungal growth under the stress of heavy metal to control (without heavy metal) for the same period of time (Valix et al., 2001; Valix and Loon, 2003). Experiments were performed with 100 ml working volume of sterilized modified CYE broth supplemented with two different concentrations (100 mg/L and 200 mg/L) of Cu(II) and Ni(II) (individually). pH of the accumulation medium was set to optimum value acquired from earlier growth experiment using sterile H2SO4 (0.1N) and NaOH (0.1N) solutions. The flasks were inoculated with fungal spores and incubated at 30oC on an orbital rotary shaker at 150 rpm with constant shaking. The flasks were removed from shaker at predetermined time intervals (24, 48, 72, and finally at the completion of the experiment 96 h) and analyzed for the fungal biomass. An another set with similar conditions, except heavy metal presence named as control was also performed. 3. Results and Discussion 3.1. Physio-chemical analysis of samples The wastewater was colorless but turbid. The observed pH of 3.08 indicated acidic nature of wastewater. The concentration of copper, nickel, lead, and total chromium was 35.08 mg/L, 9

64.22 mg/L, 2.94 mg/L and 112.34 mg/L in wastewater sample and 19.34 mg/L, 60.86 mg/L, 1.56 mg/L and 89.23 mg/L in sludge sample, respectively. 3.2. Isolation of filamentous fungi In present study, a total of twenty-five isolates; twelve from wastewater and thirteen from sludge samples of fungi were isolated. Maximum diversity of fungi were observed on modified CYEA media plates followed by PDA and MA (data not shown). 3.3. Screening and identification of heavy metals resistant filamentous fungi 3.3.1. Qualitative screening The screening of Cu(II) and Ni(II) resistant filamentous fungi were performed with twentyfive isolated fungal strains. Screening was based on the growing ability of isolates on solid modified CYEA media supplemented with 100 mg/L concentration of heavy metals., despite the fact the metal speciation and solubility are affected by agar (Zapotoczny et al., 2007). Preliminary qualitative screening data (Table 1) revealed that a total of eleven and nine isolates (assigned with arabic numbers) showed visible growth on Cu(II) and Ni(II) supplemented agar plates, respectively. The growth was represented as positive results (+) in various degrees like very good visible growth (++) and visible growth (+). The isolates assigned with number 2, 5, 6, 8, 9, 11 from wastewater sample and 3, 7, 8 from sludge sample showed positive results (growth) for both heavy metals in comparison to the rest of isolates (Table 1). The isolates exhibited growth inhibition may attribute to the metal toxicity on growing cell. Reduction in fungal growth at higher heavy metals concentration may be due to the increment in duration of lag phase against control (without heavy metal) (Iram et al., 2012) or various biological factors (Dwivedi et al., 2012). Our results of qualitative screening corroborate with that of Dwivedi et al. (2012) and Abd El Hameed et al. (2015). 3.3.2. Quantitative screening

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A total of nine fungal isolates out of twenty-five screened from the primary qualitative experiments were subjected to growth rates (kd) experiment. Among these nine isolates, four shown higher values of kd (data not shown) in following order: 11 > 7 > 3 > 6 (initially assigned with a number). These four fungal isolates were identified as Aspergillus awamori (number 11 from wastewater sample), Aspergillus flavus (number 7 from sludge sample), Aspergillus niger (number 3 from sludge sample), Penicillium sp. (number 6 from wastewater sample). Congeevaram et al. (2007), Zafar et al. (2007) and Ezzouhri et al. (2009) screened heavy metals resistant filamentous fungi and observed that the most tolerant fungi belonged to genera Aspergillus and Penicillium. Congeevaram et al. (2007) reported that species of Aspergillus are efficient reducer of heavy metals. Dwivedi et al. (2012) isolates and identified various Ni(II) tolerant fungal species as Aspergillus niger (Ni19, Ni27, Ni33) and Aspergillus flavus (Ni35, Ni36). Moreover, Iram et al. (2012) isolates fifty-four fungal strains from the industrial effluent irrigated agricultural field of Faisalabad (Chakera Chack) and among these, thirteen were identified as Aspergillus niger and twenty-two as Aspergillus flavus. The concurrent appearance of different Aspergillus species may be due to either they are adapted to surrounding conditions or produced large numbers of readily airborne spores even from a small parental biomass. From screening data, A.awamori, A. flavus and A. niger were identified as better fungal strains in term of growth on heavy metals (100 mg/L) supplemented media. Therefor, these three fungal strains were selected for subsequent studies. 3.4. Minimum inhibitory concentration The MIC of Cu(II) and Ni(II) for studied fungal strains is demonstrated in Table 2. The selected strains shows greater tolerance for Cu(II) in comparison to Ni(II). The MIC were in the range of 4.0-5.5 mg/ml for Cu(II) and 2.5-5.0 mg/ml for Ni(II) (Table 2). Cu(II) tolerance was exhibited in order of A. awamori > A. niger > A. flavus and for Ni(II) the order was as A. 11

awamori ≈ A. flavus > A. niger. Among three strains, A. awamori demonstrated greater tolerance for both heavy metals, which is also reflected in Figure 1 and Figure 3. The value of MIC counsel that the level of tolerance against any individual heavy metal was reliant on the type of strains. Three strains of an equivalent genus (Aspergillus) exhibited a marked distinction in heavy metal tolerance level may be attributable to different strategies or mechanisms of tolerance shown by diverse fungi. 3.5. Effect of pH on fungal growth and bioaccumulation The pH of the accumulation media is a significant factor for the fungal growth and bioaccumulation of the heavy metals as it influences the microbial surface properties, solution chemistry and metal speciation. In order to determine the optimum pH for effective growth and bioaccumulation of heavy metals by filamentous fungi, batch mode experiments were performed at initial pH values (3.5, 4.0, 4.5, 5.0, 5.5, 6.0) and heavy metals concentration (100 mg/L). The comparison of growth and heavy metals uptake by A. awamori, A. flavus and A. niger at different initial pH value is displayed in Figure 1. The maximum growth (dry weight, measured in g/L) and Cu(II) bioaccumulation quantity (measured in mg/g) was observed at pH 4.0 for A. awamori (12.30 g/L and 7.13 mg/g) and A. niger (9.46 g/L and 7.02 mg/g) and at pH 5.0 for A. flavus (10.41 g/L and 6.99 mg/g). For Ni(II) containing culture media, the maximum growth and bioaccumulation quantity were observed at pH 5.0 for A. awamori (10.19 g/L and 6.78 mg/g), A. flavus (12.31 g/L and 7.64 mg/g) and A. niger (9.50 g/L and 6.53 mg/g). The growth of A. awamori, A. flavus and A. niger and bioaccumulation of Cu(II) and Ni(II) was decreased below and above these optimum pH values. The results showed that the A. awamori exhibited higher growth in Cu(II) containing media in comparison to A. flavus and A. niger and later two presented almost similar growth. Growth in Ni(II) containing media appeared in the following pattern: A. flavus > A. awamori 12

> A. niger. The growth and bioaccumulated quantity for A. awamori favour Cu(II) than Ni(II), pattern was reverse for the A. flavus and no significant differences were noticed for A. niger. No experiment was conducted at higher pH (pH > 6.0) because copper and nickel precipitate as Cu(OH)2 and Ni(OH)2, respectively, due to the high concentration of hydroxyl ions. The results illustrated in Figure 1, revealed that the pH playes an important role in enhancing the growth of fungal biomass and bioaccumulation of heavy metals. The optimum range of pH for maximum growth and bioaccumulation of Cu(II) and Ni(II) by of A. awamori, A. flavus and A. niger studied in the present study with much reported literature has shown similarity (Table 3). 3.6. Effect of heavy metal concentration on fungal growth The effect of different concentration of heavy metals on fungal growth was expressed in terms of tolerance index (TI). TI values greater than one indicates resistant or tolerant nature of fungi, and TI values lower than one reflects susceptible or non-tolerant nature of fungi toward a specific heavy metal and its concentration. Valix et al. (2001) and Valix and Loon (2003) proposed a five-phase growth behavior of fungi under the exposure of heavy metal as shown in Figure 2. (a) Lag phase: No growth or very little growth occurred. (b) Rapid or first accelerated growth phase: very fast initial growth. (c) Retarded or death phase: Decline in growth. (d) Similar or stationary phase: No difference in fungi growth with and without heavy metals. (e) Enhanced or second accelerated growth phase: growth often exceeds the control. A good adaptive tolerance behavior of fungi against a particular heavy metal is measured as higher TI values at similar or stationary phase (d) (TI >1), which is related to the high initial fungal growth rate at rapid phase (b) and relatively low fungal death rate at retarded or death

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phase (c). The growth behavior of A. awamori, A. flavus and A. niger at 100 mg/L and 200 mg/L concentration of Cu(II) and Ni(II) is presented in Figure 3. The trend demonstrated in Figure 3 (A), suggests that the A. awamori has better tolerance for Cu(II) in following order: Cu(II), 100 mg/L > Cu(II), 200 mg/L followed by Ni(II), 100 mg/L. Figure 3A, also suggests that A. awamori does not fully adapt to Ni(II) at 200 mg/L concentration as TI values in similar or stationary growth phase (d) remaines below one and growth pattern cannot follow the five-phase growth characteristic. All the three species of Aspergillus shows higher growth at both concentrations of Cu(II) than the control indicates its utilization as nutrient because copper is an essential metal for diverse biological actions such as a co-factor in different enzymatic processes. According to Srivastava and Thakur (2006), Aspergillus sp. can accumulate toxic heavy metals more than the nutritional need. The growth in terms of dry weight (g/L) of A. awamori at lower concentration (100 mg/L) of Cu(II) was observed 5.82 g/L (TI=1.79) at 24 h of the incubation period than at higher concentration (200 mg/L) which was 4.04 g/L (TI=1.24). A. flavus and A. niger displayed almost similar value of TI at 24 h of incubation for both Cu(II) concentration. However, both of them were beneath of A. awamori (Figure 3). The TI value declined at the end of rapid phase (b) with the rise of Cu(II) concentration (100 mg/L to 200 mg/L) may be either lower concentration utilize as nutrient which in return doesn’t introduce any stress during the uptake of heavy metal and/or development of some tolerance mechanism. Although higher concentration of the same heavy metal brings toxicity and/or some kind of stress besides the nutriental need, which restrains the growth during rapid phase (b) as observed from Figure 3. A. flavus exhibited a better tolerance for Ni(II) at lower concnetration in comparison with Cu(II) at both concentrations. However, the adaptive behavior was akin and difference was not significant (Figure 3B). In Figure 3, the growth curve of A. flavus and A. niger with Cu(II) at both concentrations are nearly alike, indicated similar adaptive behavior of both 14

fungus with Cu(II). However, exceptional adaptive behavior of A. niger for Ni(II) at the both concentrations indicated that the fungus was not fully adapted as the value of TI remained below one throughout the studied period of time and growth pattern unmatched with fivephase growth characteristics. The higher toxicity of nickel for A. niger was already illuminated (Avakyan, 1974). None of the studied fungal strains demonstrated tolerance toward high concentration of Ni(II). It didn't mean that these fungi were not able to tolerate high heavy metal concentration. A detailed study at every step should be lessened (Anahida et al., 2011). Anahida et al. (2011) observed a weaker tolerance for Ni(II) by Aspergillus niger and Aspergillus foetidus. All the metals, whether essential and nonessential at certain levels or concentrations causes toxicity to the microorganism. The extend of metal toxicity is a function of isolate type and its location of isolation. Every isolates behave differently against different heavy metals. Some isolates are highly tolerant, while others are sensitive and unable to survive even at very low concentration of heavy metals (Munoz et al., 2012). In present study, nickel appear as more toxic heavy metal to tested fungi. Usually, the sites aren’t contaminated by a single heavy metal and this non-uniformity of pollution affect the selection of isolates, which is controlled either by the most toxic heavy metal or by synergistic action of different heavy metals (Baldrian and Gabriel, 2002). Fungi belongs to same genus as in present study not bounded to exhibit similar tolerance for a specific heavy metal. All three studied fungi displayed a better tolerance to Cu(II) in comparison to Ni(II). Table 4 data indicates the existence of different tolerance strategies or resistance mechanism shown by each fungi, e.g. Aspergillus niger resists copper by involving copper metallothionein synthesis active process (Kermasha et al., 1993). Our results are in confirmation of the findings of other researchers (Valix et al., 2001; Valix and Loon, 2003; Ezzouhri et al., 2009; Shivakumar et al., 2011; Anahida et al., 2011; Munoz et al., 2012; Iram 15

et al., 2012) that the isolates response to heavy metals is a function of type of isolate under study, its place of isolation, metal type and its concentration in the medium. The present study was carried out to explore the capability of Aspergillus awamori, Aspergillus flavus and Aspergillus niger isolated from the contaminated site for the removal of Cu(II) and Ni(II) as a function of pH and initial metal concentrations. pH is a most significant parameter in any bioremediation process. As per the available literature, bioaccumulation capabilities of A. awamori and A. flavus have never been explored for the removal of Cu(II) and Ni(II) at different pH conditions. To be a novel isolates for the bioremediation process, fungi should be metal tolerant as well as the fungal isolates must have the capability for the metal biosorption. A high TI values by A. awamori and A. flavus for both the metals indicated that these species were significantly tolerant to both metals at given metal concentrations as well as the fungal species have shown effective metals uptake. The present research work provides some necessary information about these fungal strains and confirms their bioaccumulation ability. The information provided in the present investigation may be helpful for further refining of the bioremediation processes, especially dealing with the copper and nickel contaminated sites.

4. Conclusion The present study demonstrated that the heavy metals resistance/tolerance of fungal isolates appeared to be correlated with the site of isolation. A positive cumulative trends between fungal biomass and metal accumulation at a specific pH confirmed that the influence of pH on the bioaccumulation was attributed to organism specific physiology. In the present study the fungal growth had shown synergistic and inhibitory association with lower and higher 16

concentration, respectively for both heavy metals. Our finding confirmed that the isolates response to heavy metals is depended on indigenous isolates tested, its site of isolation, metal under consideration and its concentration in the medium.

Compliance with ethical standards Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of Interest: The authors declare that they have no conflict of interest.

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Table Legends Table 1 Screening of fungal isolates for heavy metal resistance. Heavy

Wastewater sample

metal

Isolates assigned number

type

Ni(II)

1

2

3

4

5

6

7

8

9

10

11

12

-

++

-

-

+

++

-

+

+

-

++

-

23

Cu(II)

-

++

-

+

+

++

-

+

+

+

++

-

Sludge sample Isolates assigned number 1

2

3

4

5

6

7

8

9

10

11

12

13

Ni(II)

-

-

++

-

-

-

++

+

-

-

-

-

-

Cu(II)

-

-

++

-

-

++

+

-

-

-

-

-

- : no visible growth, + : visible growth, ++ : very good visible growth

Table 2 Minimum inhibitory concentration of fungal isolates. Isolates

MIC (mg/ml) Cu(II)

Ni(II)

Aspergillus awamori

5.0 < MIC < 5.5

4.5 < MIC < 5.0

Aspergillus flavus

4.0 < MIC < 4.5

4.5 < MIC < 5.0

Aspergillus niger

4.5 < MIC < 5.0

2.5 < MIC < 2.75

24

Table 3 Comparison of other fungal strains from selected literature with the present work. Fungal strain

Source

Dm, (g/L)

qm, (mg/g)

Reference

Rhizopus arrhizus Aspergillus niger Rhizopus arrhizus

Procured from US Department of Cu(II), 50 mg/L Agriculture Culture Collection Cu(II), 50 mg/L Procured from US Department of Cu(II), 50 mg/L Agriculture Culture Collection Procured from US Department of Cu(II), Agriculture Culture Collection

4.5 5.0 4.5

1.53 1.45 1.53

7.32 6.92 7.32

Dursun et al., 2003a

5.0

2.2

11.3

Dursun 2003b

et

al.,

Rhizopus delemar

Procured from US Department of Cu(II) and Ni(II), Agriculture Culture Collection 100 mg/L

4.0

1.89

-

Açıkel 2009

and

Alp,

Aspergillus lentulus FJ172995

Isolated from textile industry wastewater

Cu(II), 80 mg/L Ni(II), 70 mg/L

5.0 6.0

4.88 5.59

12.64 5.19

Mishra and Malik, 2012

Aspergillus niger

Isolated from electroplating Ni(II), 50 mg/L industry wastewater

5.0

5.09

-

Kumar et al., 2012

Aspergillus awamori

Isolated from steel industry wastewater

Cu(II), 100 mg/L Ni(II), 100 mg/L

4.0 5.0

12.30 10.19

7.13 6.78

Present study

Aspergillus flavus

Isolated from steel industry sludge Cu(II), 100 mg/L Ni(II), 100 mg/L

5.0 5.0

10.41 12.31

6.99 7.64

Present study

Aspergillus niger

Isolated from steel industry sludge Cu(II), 100 mg/L Ni(II), 100 mg/L

4.0 5.0

9.46 9.50

7.02 6.53

Present study

Aspergillus niger

Heavy metal type Optimum and concentration pH

Dm (g/L) = dried weight (g) of fungus biomass per litre (L). qm (mg/g) = maximum uptake capacity of heavy metal (mg) per (g) of dried fungus biomass. 25

Uslu et al., 2003

Table 4 Tolerance and adaptability assessment of fungal strains. Fungal strain

Heavy metal Cu(II)

Aspergillus awamori

100 mg/L 3 2 1

Aspergillus flavus

2 1 1

Aspergillus niger

2 1 1

Criteria Growth rate (dry weight, g/L) at end of 24 h of incubation TI value at similar phase (d) Five-phase growth characteristics

6

Ni(II) 200 mg/L 2 4 1 1

100 mg/L 3 5 1 1

200 mg/L 0 0 0 0

5

0 0 0

0

0

0 0 0

0

4

2 0 1

3

3 1 1

4

2 1 1

4

0 0 0

Level Below the control Equal to the control Above the control Well above the control Below one Almost equal to one Above one Not-follow Follow

Assigned value 0 1 2 3 0 1 2 0 1

Comment: The fungal strain with highest number display better tolerance and adaptability towards that heavy metal and concentration and conditions are reverse for lowest number.

26

Figure Legends Figure 1. The effect of initial pH on the maximum fungal growth and heavy metals uptake capacity of (A) A. awamori, (B) A. flavus, and (C) A. niger. Dm: dried weight (g) of fungal biomass per litre (L) with ─●─ Cu(II) and ─■─ Ni(II); qm: maximum uptake capacity of ─○─ Cu(II) and ─□─ Ni(II) (mg) per (g) of dried fungal biomass.

Figure 2. Five-phase growth behaviour of fungi in the presence of heavy metal (Valix et al., 2001; Valix and Loon, 2003).

Figure 3.

Tolerance behaviour of (A) A. awamori, (B) A. flavus, and (C) A. niger with

─●─ Cu(II), 100 mg/L; ─○─ Cu(II), 200 mg/L; ─■─ Ni(II), 100 mg/L; ─□─ Ni(II), 200 mg/L.

27

Figure 1.

A

12

8.0

13 12

7.5

8.0

13

8

6.0

7

7.0

10 9

6.5

8

6.0

5.5

3.5

4.0

4.5

5.0

pH

5.5

6.0

5.0

5.5

6 5

7.5

7.0

10 9

6.5

8

6.0

7

7

6

Dm (g/L)

6.5

8.0

11

qm (mg/g)

9

Dm (g/L)

7.0

10

C

12

7.5

11

qm (mg/g)

Dm (g/L)

11

5

B

3.5

4.0

4.5

5.0

5.5

6.0

5.0

5.5

6 5

3.5

4.0

4.5

5.0

pH

pH

28

5.5

6.0

5.0

qm (mg/g)

13

Figure 2.

b

c

e

d

Tolerance index

a

Time (day)

29

Figure 3.

2.00

A

1.75

Tolerance index

Tolerance index

1.50 1.25 1.00 0.75

2.00

B

1.75

1.75

1.50

1.50

Tolerance index

2.00

1.25 1.00 0.75

1.25 1.00 0.75

0.50

0.50

0.50

0.25

0.25

0.25

0.00

0

1

2

Time (day)

3

4

0.00

0

1

2

3

Time (day)

30

4

C

0.00

0

1

2

Time (day)

3

4

31