Mycoremediation of heavy metal (Cd and Cr)–polluted soilthrough indigenous metallotolerant fungal isolates

Ibrar Khan & Maryam Aftab & SajidUllah Shakir &

Madiha Ali & Sadia Qayyum & Mujadda Ur Rehman &

Kashif Syed Haleem & Isfahan Touseef

Received: 5 December 2018 /Accepted: 15 August 2019# Springer Nature Switzerland AG 2019

Abstract Remediation of heavy metals, other than mi-crobial bioleaching method, is expensive and unsuitablefor large contaminated areas. The current study wasaimed to isolate, identify, and test the potential of indig-enous fungal strains for heavy metal removal fromcontaminated soil. A total of three metallotolerant fun-gal strains, i.e., Aspergillus niger (M1DGR),Aspergillusfumigatus (M3Ai), and Penicillium rubens (M2Aii),were isolated and identified by phenotyping andgenotyping from heavy metal–contaminated soil ofHattar Industrial Estate, Pakistan. A. niger was foundto be the most successful strain for the removal of heavymetals from the contaminated soil with maximum bio-accumulation efficiency of 98% (Cd) and 43% (Cr). Incontrast, A. fumigatus showed comparatively low butstill considerable bioleaching potential, i.e., 79% and69% for Cd and Cr removal, respectively. Maximummetal uptake efficiency, i.e., 0.580 mg g−1 and0.152 mg g−1 by A. niger strain was noticed for Cdand Cr with Czapek yeast extract (CYE) and Sabourauddextrose broth (SDB) media, respectively. A. fumigatus(M3Ai) exhibited the maximum bioleaching capacity

(0.40 mg g−1) for Cr with CYE medium. The resultsreveal that A. niger M1DGR and A. fumigatus M3Aicould be used to develop new strategies to remediate soilcontaminated with heavy metals (Cd and Cr) througheither in situ or ex situ mycoremediation.

Keywords Heavymetal .Hattar IndustrialEstate (HIE) .

Mycoremediation . Indigenousmicrobes

Introduction

In modern era, the soil environment is being continu-ously polluted with heavy metals due to rapid industri-alization, mining, and other technological related ad-vancements. As a result, heavy metal pollution hasbecome a serious issue and a matter of high consider-ation throughout the world (Iram et al. 2013;Ayangbenro and Babalola 2017). Interestingly, theseheavy metals also perform some important biologicalfunctions both in animals and in plants because theirred-ox properties and chemical coordination gave theman extra benefit to maintain the control mechanisms likehomeostasis, degradation, transport, and binding to thetarget cell (Singh et al. 2011; Xavier et al. 2019).

Although essential heavy metals including iron, zinc,and copper are required by a living organism in traceamounts, their concentration above a specific level istoxic and can be harmful to living organisms (Varol andSünbül 2018). Some heavy metals such as cadmium,chromium, and arsenic are so harmful that they maycause cancer and can occupy a single or multiple classes

Environ Monit Assess (2019) 191:585 https://doi.org/10.1007/s10661-019-7769-5

I. Khan (*) :M. U. RehmanDepartment ofMicrobiology, Abbottabad University of Science &Technology, Abbottabad 22010, Pakistane-mail: abrar@aust.edu.pk

M. Aftab : S. Shakir :M.Ali : S. Qayyum :K. S. Haleem :I. Touseef (*)Department of Microbiology, Hazara University,Mansehra 21300, Pakistane-mail: isfahan@hu.edu.pk

of carcinogens designated by the International Agencyfor Research on Cancer (IARC) and the US Environ-mental Protection Agency (USEPA) (Tchounwou et al.2012; Rahman and Singh 2019). On the other hand,various metals like Fe, Zn, Ni, and Cu are essentialmetals for growth and other functions; however, if theirlevel increases from a minimum acceptable range, theymay also become dangerous to animals and plants(Qayyum et al. 2016a).

There are an array of techniques, e.g., filtration,chemical precipitation, reverse osmosis, membranetechnology, oxidation and reduction, ion exchange,and electrochemical treatment, for the removal of heavymetals from a contaminated environment. However,these techniques have some serious demerits associatedwith them. The most important one is their inability toremove heavy metals found at lower concentration (≤100 mg/L) (Dixit et al. 2015). Additionally, these tech-niques are not only expensive and do not require energysources but their application may also alter the proper-ties of the soil, but the heavy metal contaminants maynot be removed completely. Furthermore, the pollutantmay also be displaced to other sites in the environmentwhere they can accumulate and may cause the sameissue (Rohwerder et al. 2003; Qayyum et al. 2016c;Sajjad et al. 2019).

Alternatively, bioleaching is a biological process inwhich microorganisms (fungi, algae, and bacteria) uptakeheavy metals from the environment (Das et al. 2008;Chatterjee et al. 2019). Among all bioleaching microbes,fungi are considered the most suitable candidates for bio-remediation due to their high tolerance to heavymetals anda higher surface-to-volume ratio (Nath et al. 2018; Lima deSilva et al. 2012). The main advantage to use an indige-nous fungal strain is that they are adapted not only to thepresence of contaminants but also to the environmentalcondition of the site. Thus, there is a need to develop newstrategies to utilize indigenous fungal strains for heavymetal removal from contaminated soil.

The Hattar Industrial Estate is situated in KhyberPakhtunkhwa (KPK) Province of Pakistan. A large num-ber of chemical units, textile industries, heavy electricalplants, paper-manufacturing industries, food processingmills, steel mills, vegetable oil–processing plants, andleather-manufacturing units exist in this industrial estate.During industrial production, the leaking in the productionprocess and stacking behavior results in soil contaminationwith heavy metals. In addition, wastewater containingthese metals contaminates the nearby streams and runoff

to Tarbela Dam, Topi Tehsil, Khyber Pakhtunkhwa, Paki-stan (Naidu et al. 2012). The selected industrial area hasneither been explored previously for the determination ofcadmium and chromium concentrations in the soil envi-ronment nor has its indigenous fungal community beenidentified and tested for its potential to remove toxic heavymetals from contaminated soil. Therefore, this study pro-vides first-ever insight into the indigenous metal-resistantfungal strains having application for effective removal ofcadmium and chromium from contaminated soil and otherenvironments.

Materials and methods

Sample collection

The soil samples were collected in sterile shopping bagsfrom top soil/surface soil (0–15 cm) from different sitesof National Radio Telecommunication Corporation(NRTC) high-tech industry, Hattar Industrial Estate,KPK, Pakistan, using the protocol reported earlier(Qayyum et al. 2016b). The collected samples wereair-dried for 2 weeks and were ground using the labo-ratory facilities of the Department of Microbiology,Hazara University Mansehra in strict adherence toprotocols.

Determination of heavy metal (Cd and Cr)concentration

Atomic absorption spectrophotometry (AAS) for totalheavy metal concentration analysis (Chowdhury et al.2017) was performed at Biological Sciences Laboratory,Life Sciences Department, COMSAT Institute of Infor-mation Technology (CIIT), Abbottabad, Pakistan.

Samples were prepared by acid digestion method asmentioned by Page et al. (1982). Briefly, 0.5 g of eachsoil sample was weighed and added into a 250-mLErlenmeyer flask containing 5 mL nitric acid (HNO3,67%). After overnight incubation, 2 mL perchloric acid(HCLO4) was added and heated at 60 °C in a fume hoodfor overnight or until it turned cloudy. The mixture wasallowed to cool and subsequently filtered withWhatmanfilter paper supplied by Sigma-Aldrich (Saeed et al.2011). The suspensions were diluted to obtain a rangeof concentrations, i.e., 10−1 to 10−4 mL. The concentra-tion of heavy metals was determined from the finaldilution using atomic absorption spectrophotometry

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with standard curve obtained through PerkinElmer pureatomic spectroscopy standards, i.e., cadmium, 1000 μg/mL in 2% HNO3 (CAS# Cd 7440-43-9); copper,1000 μg/mL in 2% HNO3 (CAS# Cu 7440-50-8); lead,1000 μg/mL in 2% HNO3 (CAS# Pb 7439-92-1); andmercury, 1000 μg/mL in 10% HNO3 (CAS# Hg 7439-97-6) (AAnalyst™ 700 atomic absorption spectrome-ter—PerkinElmer).

Isolation and identification of metallotolerantindigenous fungi

Isolation and colony morphology of indigenous fungalstrains from metal-contaminated soil samples having po-tent uptake efficiency were carried out by using sabourauddextrose agar (SDA) (peptone 10 gL−1, agar 15 gL−1, anddextrose 40 gL−1), potato dextrose agar (PDA) (potatoextract 4 gL−1, dextrose 20 gL−1, and agar 15 gL−1), andmalt extract agar (MEA) (malt extract 20 gL−1, peptone6 gL−1, agar 15 gL−1, and dextrose 20 gL−1) mediaautoclaved at 121 °C for 15 min and supplemented with0.05% chloramphenicol. The cell morphology was ob-served under a fluorescence microscope (OlympusCKX41-A32PH) (Khan et al. 2017a). Images were record-ed using the cellSens Standard software. Furthermore, theisolated mold colonies were re-purified, were preserved onPDA slants, and were stored at − 80 °C for further use(Khan et al. 2014, 2017a).

The total genomic DNAs of the fungal isolates wereisolated and purified using the method described by Al-Samarrai and Schmid (2000). Nuclear ribosomal internaltranscribed spacer (ITS) was amplified using ITS: 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′ primers (Qayyum et al.2016b; Pedersen et al. 1997). The sequence obtained wasaligned using BLASTanalysis network service of NationalCenter for Biotechnology Information (NCBI) for compar-ison with currently available sequences. The sequenceswhich showed over 98% similarities with currently acces-sible sequences were regarded as the same species. Fur-thermore, multiple alignments were performed usingClustal X 1.83 and MEGA 4.0 for the construction ofphylogenetic tree (Khan et al. 2017b; Tamura et al. 2013).

Bioleaching capacity analysis of indigenous fungalstrains

Three types of media, i.e., yeast peptone glucose (YPG),czapek yeast extract (CYE), and sabouraud dextrose

broth (SDB), were used to check the percentage effi-ciency and heavy metal uptake capacity of resistantindigenous fungal isolates by using a two-stepbioleaching process.

Optimization with YPG medium

Each 250-mL flask containing 49 mL pre-sterilizedYPG medium (1% yeast extract, 2% peptone, and 2%glucose) was autoclaved at 121 °C for 15 min. Total1 mL of spore suspension of each strain was inoculatedin pre-sterilized 49 mL of YPG flask and incubated at30 °C. After 72 h of incubation, 2.5 g of sterilizedcontaminated soil sample was added to the respectiveflask.

Bioleaching experiment was performed by furtherincubating the mixture in a shaking incubator at130 rpm and 30 °C for 72 h. The flask containingYPG broth with 2.5 g of sterilized heavy metal–contaminated soil sample served as control.

Optimization with CYE medium

Each 250-mL flask containing 49 mL of CYE medium(yeast extract 5 mg L−1, sucrose 30 mg L−1, sodiumnitrate 0.3 mg L−1, dipotassium hydrogen phosphate1 mg L−1, magnesium sulfate 0.05 mg L−1, zinc sulfate0.001 mg L−1, ferrous sulfate 0.001 mg L−1, coppersulfate 0.0005 mg L−1 , potassium chlor ide0.05 mg L−1, and agar 15 mg L−1) was autoclaved at121 °C for 15 min. Total 1 mL of spore suspension ofeach strain was inoculated in pre-sterilized 49-mL CYEflask and incubated at 30 °C. After 72 h of incubation,2.5 g of sterilized contaminated soil sample was addedto the respective flask.

Bioleaching experiment was performed by furtherincubating the mixture in a shaking incubator at130 rpm and 30 °C for 72 h. The flask containingCYE broth with 2.5 g of sterilized contaminated soilsample served as control.

Optimization with SDB medium

Each 250-mL flask containing 49 mL of SDB medium(1% peptone and 2% dextrose) was autoclaved at121 °C for 15 min. Total 1 mL of the spore suspensionof each strain was inoculated in pre-sterilized 49-mLSDB flask and incubated at 30 °C temperature. After

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72 h of incubation, 2.5 g of sterilized contaminated soilsample added to the respective flask.

Bioleaching experiment was performed by furtherincubating the mixture in a shaking incubator at130 rpm and 30 °C for 72 h. The flask containingSDB broth with 2.5 g of sterilized contaminated soilsample served as control. All the experiments were runin triplicate.

Heavy metal uptake by fungal isolates

Three different media (YPG, SDB, and CM) containingheavy metals were used to evaluate the heavy metal’suptake ability of each metallotolerant fungal isolates.All the experiments were performed in triplicate. Brief-ly, each strain was separately used to inoculate one ofthe abovementioned media containing specific heavymetal. After incubation for 72 h, media in flask weretransferred into 50-mL falcon tubes and centrifuged at4000 rpm for 15 min. The supernatant was stored at −4 °C for estimation of heavy metal contents throughAAS. The harvested fungal biomass obtained wasrinsed 3–4 times with double distilled water and driedin a hot air oven (80 °C for 18 h) and weighed. Theuptake of heavy metal by fungal biomass was calculatedusing the following equation.

Qe ¼ Ci−C f� �

VM

where Q is the concentration (mg g−1) of heavy metalacquired by fungal biomass, ci being the initial concen-tration of heavy metal (mg L−1), cf the final concentra-tion of heavy metal (mg L−1), V (L) is the volume ofaqueous medium, and M is the dry weight (g) of fungalbiomass (Faryal et al. 2007).

Statistical analysis

To compare the difference of methods, one-wayANOVA at p < 0.05 level of significance was appliedby using LSD (least significant difference) and Tukey’stest by using GraphPad Prism software. Analysis usingeach method was performed in triplicate.

Results

Soil characterization and its heavy metal concentrationanalysis

Soil characterization is the most important component todetermine the level and nature of contamination in soil(Qayyum et al. 2016d; Gonzalez et al. 2019). Soil sam-ple (S3) revealed a heavy metal contamination of0.0586 mg L−1 and 0.3565 mg L−1 for Cd and Cr,respectively (Table 1).

Isolation and identification of fungal isolates

A total of 3metallotolerant indigenous fungal isolates wereisolated from the soil samples (n= 4) collected fromHattarIndustrial Estate. The isolates were identified on the basisof their morphological (Fig. 1) and molecular characteris-tics using ITS sequences as mentioned in the “Materialsand methods” section.

The results of morphological analysis were further sup-ported by the topology of the phylogram. The phylogramconfirmed that the morphological characterization of fun-gal strains isolated in this study was correct. The identifiedstrains were designated as M1 DGR (Aspergillus niger),M3 Ai (Aspergillus fumigatus), and M2 Aii (Penicilliumrubens) (Fig. 2).

Bioleaching capacity analysis

Microorganisms have the ability to remove toxic heavymetals more efficiently at a low cost. Microbial remediationhas the advantage of removing a large amount of heavymetals. The microbe-associated technologies provide a re-placement of the conventional method for the complete orpartial removal of heavy metals as well as its improvement.

Fungal heavy metal removal efficiency

All tested fungal isolates revealed high metal-resistantabilities. Two strains were highly resistant to cadmium(Cd) and two strains were highly resistant to chromium(Cr) (Table 2). All isolates (Aspergillus fumigatus, Pen-icillium rubens, and Aspergillus niger) showed higherefficiency for Cd removal with 79%, 98%, and 98% inSDB medium. While the lowest efficiency, i.e., 76%,75%, and 79%, was observed with CYE medium.

On the other hand, for removal of Cr, Aspergillus nigerandAspergillus fumigatus strains showed higher efficiency

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for clearance of Cr (43% and 69%, respectively) in SDBmedium. However, the lowest efficiency, i.e., 35% (Asper-gillus niger) and 48% (Aspergillus fumigatus), was ob-served in CYE medium.

Heavy metal uptake by fungal isolates

Bioremediation is an advanced and promising technol-ogy for recovery and removal of heavy metals in pol-luted water and lands. Microorganisms have adoptedmechanisms for their survival in a heavy metal–polluted environment. The heavy metal uptake efficien-cy for all three indigenous fungal isolates from heavymetal–contaminated soil sample was also analyzed ondifferent media (SDB, CYE, and YPG) using formulagiven in the “Materials and methods” section.

Uptake of Cd (mg g−1) by indigenous fungalisolates The maximum (0.465 mg g−1) uptake efficien-cy for Cd removal from heavy metal–contaminated soilwas noticed for Aspergillus fumigatus M3Ai straingrown on SDB medium while the lowest uptake

efficiency of this strain was 0.450 mg g−1 with CYEmedium (Fig. 3). Similarly, the highest uptake efficiencyof Penicillium rubens M2Aiii strain was observed as0.575 mg g−1 with SDB medium and the lowest uptakeefficiency was 0.441 mg g−1 with CYE medium. Fur-thermore, the maximum uptake efficiency for Cd re-moval for Aspergillus niger M1DGR strain was ob-served as 0.580 mg g−1 with CYE medium and thelowest uptake efficiency was 0.464 mg g−1 with YPGmedium.

Uptake of Cr (mg g−1) by indigenous fungal isolates Themaximum uptake efficiency (0.152 mg g−1) for Cr re-moval from heavy metal–contaminated soil was noticedfor Aspergillus niger M1DGR strain when grown inSDB medium (Fig. 4). On the other hand, the lowestefficiency (0.130 mg g−1) was observed by the samestrain with CYE and YPG media. Likewise, the highestuptake efficiency of Aspergillus fumigatus M3Ai strainwas observed as 0.40 mg g−1 with CYEmedium and thelowest uptake efficiency of this strain was 0.07 mg g−1

with SDB medium.

Table 1 Characteristics of soil sample and concentration of its heavy metals

Soil code Industrial site pH value Electrical conductivity Moisture content Heavy metal Heavy metal concentration

S3 NRTC 6.2 2.2 ms cm−1 2.5% Cd 0.586 ± 0.025 mg L−1

NRTC 5.5 1.7 ms cm−1 3.1% Cr 0.3565 ± 0.031 mg L−1

Fig. 1 The colony morphology of the strains Aspergillus niger (a), Aspergillus fumigatus (c), and Penicillium rubens (e) on PDA medium.b, d, and f Mycelium and chain of spores of A. niger, A. fumigatus, and P. rubens, respectively

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Discussion

Environmental pollution is the contamination of soil,water, and air with pollutants such as heavy metals. Asa consequence of industrial progress and urbanization,heavy metal pollution has become one of the mostserious environmental problems worldwide during re-cent years (Sharma et al. 2007; Fu and Wang 2011).

Unlike organic pollutants, heavy metals are non-biodegradable and accumulate in living organisms viathe food chain, and some of these metals are extremelytoxic in relatively low concentrations. Due to their non-biodegradable and persistent nature, the industries usingheavy metals in their production activities face the is-sues of removing these pollutants (Fazli et al. 2015;Sawyerr et al. 2019). Their accumulation in the

Fig. 2 The phylogenetic tree of the fungal isolates and other fungal species relatives based on a neighbor-joining analysis of ITS sequences,with the Fusarium nisikadoi NR137623 as an outgroup (Khan et al. 2019, under review)

Table 2 Removal efficiency of heavy metals by fungal isolates

Soilcode

Heavy metal Initial heavy metalconcentration

Fungal isolates Mediaused

Final concentration of metal(mg L−1)

Removalefficiency

S3 Cadmium(Cd)

0.586 mg L−1 Aspergillus fumigatus(M3Ai)

CYE 0.136 ± 0.01 76%

SDB 0.121 ± 0.02 79%

YPG 0.126 ± 0.01 78%

Penicillium rubens(M2Aiii)

CYE 0.145 ± 0.03 75%

SDB 0.011 ± 0.04 98%

YPG 0.068 ± 0.02 88%

Aspergillus niger(M1DGR)

CYE 0.009 ± 0.05 79%

SDB 0.006 ± 0.02 98%

YPG 0.122 ± 0.04 96%

Chromium(Cr)

0.3565 mg L−1 Aspergillus niger(M1DGR)

CYE 0.201 ± 0.01 35%

SDB 0.200 ± 0.03 43%

YPG 0.23 ± 0.02 43%

Aspergillus fumigatus(M3Ai)

CYE 0.185 ± 0.03 48%

SDB 0.107 ± 0.02 69%

YPG 0.118 ± 0.01 66%

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environment is more worrying for the population ofdeveloping countries which mostly rely on industriesand further seeking to increase their number (Shannonet al. 2010). Hence, the contamination of soil environ-ment with heavy metals is a great concern due to the factthat it consequently affects the health of animals andplants (Mishra et al. 2019).

Prior to the application of biological processes forthe removal of heavy metals from the environment,conventional methods like filtration, chemical oxida-tion, reverse osmosis, and precipitation methods areused. These techniques had some demerits, for in-stance, if the concentration of heavy metals wasbelow 100 mg/L, then it cannot be removed by thesetechniques. Similarly, these techniques were

expensive, were difficult to operate, and producesome secondary contaminants (Ahmad et al. 2019).Keeping in mind the demerits of these methods,biological removal of heavy metals can be efficient,easy, cost-effective, and environmentally friendly.Fungi, yeast, bacteria, and Actinomycetes are thebest known bioaccumulating agents (Mudhoo et al.2012; Yang et al. 2019). Among fungi, Penicillium,Aspergillus, and Fusarium species can be consideredas the best bioaccumulators (Iram et al. 2013). Sim-ilarly, some bacteria like Bacillus spp., Pseudomonasspp., Enterobacter spp., and Aeromonas spp. have anability to resist heavy metals and uptake them toremove or detoxify (Mathivanan and Rajaram 2014;Iram et al. 2013; Babu and Pathak 2019).

Fig. 3 Uptake of Cd (mg g−1) byindigenous fungal isolates inCYE, SDB, and YPG media.Values are presented as mean ±SEM (n = 3). Bars with asterisksdiffer significantly, *p < 0.05;**p < 0.01; ***p < 0.001;****p < 0.0001; ns, non-significant

Fig. 4 Uptake of Cr (mg g−1) byindigenous fungal isolates inCYE, SDB, and YPG media.Values are presented as mean ±SEM (n = 3). Bars with asterisksdiffer significantly, *p < 0.05;**p < 0.01; ***p < 0.001;****p < 0.0001; ns, non-significant

Environ Monit Assess (2019) 191:585 Page 7 of 11 585

In economic development of Pakistan, Hattar Indus-trial Estate plays a vital role since it houses a number ofmedium- and large-scale industries (Rasheed et al.2013). Due to heavy industrialization, the accumulationof toxic heavy metals such as Cd and Cr is very likely.Therefore, keeping in mind the toxic effects of theseheavy metals to both animal and human health, thisstudy was conducted to evaluate the presence and con-centrations of cadmium and chromium in this industrialsoil and to isolate the fungi responsible for their removal(Table 1).

In the present study, a total of three fungal strainswere isolated from heavy metal–contaminated soilsamples. Phenotyping characteristics like hyphaestructures of the isolates on different media wereperformed for initial identification (Fig. 1). The iso-lates were analyzed on the basis of their molecularcharacteristics by using ITS sequences, which con-firmed that the fungal strains isolated and used inthis study were M1 DGR (Aspergillus niger), M3 Ai(Aspergillus fumigatus), and M2 Aii (Penicilliumrubens) (Fig. 2). In 2016, Qayyum and co-workersisolated six fungal isolates including Rhizomucorpusillus, Aspergillus flavus, Aspergillus terreus, As-pergillus tubengensis, and Neosartorya hiratsukae,showing maximum tolerance to heavy metals(Qayyum et al. 2016b). According to another studyconducted in 2016 by Ahirwar and team, five bac-terial isolates including Proteus vulgaris, Bacilluscereus, Bacillus decolorationis, and Pseudomonasfluorescens were isolated from heavy metal–contaminated soil (Ahirwar et al. 2016).

Bioleaching is a biological process in which micro-organisms (fungi, algae, and bacteria) uptake heavymetals from the environment. Fungi have the ability touptake both essential and non-essential heavy metals(Veglio and Beolchini 1997; Fernández et al. 2018;Chatterjee et al. 2019). In the current study, one indig-enous fungal isolate (Aspergillus niger) was found to bethe most successful candidate for the removal of twoheavy metals (Cd and Cr) from contaminated soil. Thisfungus showed the best bioleaching potential, i.e. 98%and 43%, for Cd and Cr, when optimized in Sabourauddextrose broth (SDB) followed by yeast peptone glu-cose (YPG) and Czapek yeast extract (CYE) media,respectively (Table 2). Similarly, Penicillium rubenswas found the second best cadmium bioaccumulatorwith 98% bioleaching potential when optimized onSDB medium. Aspergillus fumigatus strain showed

comparatively low bioleaching potential for Cd and Crremoval with a cumulative bioleaching ability of 79%and 69% on SDB medium (Table 2). Zili and teamisolated five fungal species, i.e., Metarhiziumanisopliae, Saccharomyces cerevisiae, Fusariumoxysporum, and two species of Penicillium from agri-culture soil of China in 2016 and achieved maximumresult for their bioleaching ability against cadmium andother heavy metals (Zili et al. 2016).

All the fungal isolates were highly resistant to testedheavy metals (cadmium and chromium) and showedsignificant capacity to bioaccumulate them from the soilenvironment. Quantitatively, 0.580 mg g−1 cadmiumwas bioleached on CYE medium by Aspergillus nigerwhich is the maximum concentration of cadmiumbioleached by any strain in the present study (Fig. 3).Similarly, effective bioleaching potential for Cr, i.e.,0.152 mg g−1, was observed for the same strain whenoptimized on SDB medium (Fig. 4). Fungal isolate A.fumigatus (M3Ai) was considered as the strain withminimum bioleaching capacity for Cd showing 0.450,0.460, and 0.465 mg g−1 on CYE, SDB, and YPG,respectively. On the other hand, the same strain showedmaximum bioleaching capacity for Cr, i.e., 0.40 mg g−1

with CYE medium.The fungal isolate P. rubens also showed significant

bioleaching potential for Cd. Its bioleaching ability wasmaximum (0.575 mg g−1) on SDB medium, while onYPG medium, its ability was noted as 0.518 mg g−1. Incontrast to its bioleaching capacity on SDB and YPG,this fungus was not so much effective for bioleaching ofcadmium on CYE, i.e., only 0.441 mg g−1 concentrationof cadmium was bioleached. Heterotrophic fungi suchas Mucor sp., Aspergillus sp., Penicillium sp., andYarrowta sp. can remove both soluble and insolublemetal species from solution and are able to leach metalcations from solid waste (Rather et al. 2018).

Fungal survival in heavy metal–contaminated envi-ronments mainly depends on intrinsic structural andbiochemical properties, genetical and/or physiologicaladaptation, morphological changes, environmentalmodification of heavy metal, its toxicity, and availability(Tiwari and Lata 2018; Gadd 1994). Biological mecha-nisms associated with fungal existence include extracel-lular precipitation, crystallization, transformation, andcomplexation of metal species by using mechanismssuch as reduction, oxidation, dealkylation, methylation,biosorption to cell walls, extracellular polysaccharideand pigments, impermeability or decreased transport,

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efflux, intracellular compartmentation, and sequestra-tion (Rose and Devi 2018; Jacob et al. 2018; Gadd1994). A particular organism may indirectly and/or di-rectly rely on several survival stratagems. Moreover,suitable treatment of laden microbial biomass can en-able recovery of valuable metals for recycling or furtherrestraint of highly toxic species. Though basic featuresof metal accumulation are common to most microbialgroups, fungi possess a number of traits which revealtheir physiological and morphological diversity (Daviset al. 2003; Atakan et al. 2018). The majority of fungiexhibit biochemical and structural differentiation withthe filamentous growth form supporting proficient col-onization of substrates. Therefore, due to diversity inresistance capability, different microbial cells may havedifferent heavy metal removal or uptake capacities(Atakan et al. 2018; Gadd 1994).

Goyal et al. (2003) investigated the biosorption ofCr(VI) and Fe(III) by Streptococcus equisimilis, S.cerevisiae, and Aspergillus niger. In 2005, Sen andco-workers did observational studies on biosorptionof Cr(VI) by using F. solani isolated from soil underdifferent growth conditions i.e. resting cells, growingcells, as well as non-living biomass. Significant Cr(VI) removal was observed using growing cells inbatch and continuous modes of operations and usingnon-living biomass in a batch bioreactor. They con-ducted the study to evaluate the potential of theresting cells of the Fusarium solani for Cr(VI) re-moval from aqueous solution with an aim to developa suitable operational strategy for treatment ofCr(VI)-contaminated wastewaters (Sen and Ghosh2011). Several researchers suggested the use of As-pergillus niger, Aspergillus sp., Penicillium sp., andFusarium sp. to remove the heavy metals Cr, Zn, Ni,Pd, and Cd (Jobby et al. 2018; Malik 2004; Iram et al.2009; Volesky and Holan 1995). In a recent studyconducted in 2017, six bacterial isolates includingChryseobacterium indoltheticum, Cupriavidusoxalaticus, Pseudomonas helmanticensis, Bacillusmycoides, Bacillus almalaya, and Acinetobactertjernbergiae showed higher tolerance to Cd, Pb, Cr,and Zn (Jiang et al. 2017). Thus, the use of microbialbiomass may therefore be considered as remedy forthe removal of toxic substances from the environ-ment. Not only being cost-effective, the indigenousmicroorganisms isolated also detoxify the contami-nated site itself, to exercise their natural power andremedy the situation.

Conclusion

Industrialization is the best known cause for heavymetalpollution of the soil and bioleaching is the most effi-cient, cost-effective, and environmentally friendlymeth-od. Fungi could be the most suitable bioaccumulatingagents for the removal of cadmium and chromium fromcontaminated soil. The current findings revealed that themycoremediation process using indigenous fungal iso-lates could be effective for leaching of heavy metalsfrom the industrial soil polluted by heavy metals anddemonstrated to be a promising technology towards abiological refinement of soils.

Funding information This work was financially supported bythe Higher Education Commission, Government of Pakistan underSRGP Program (no. 21-1259/SRGP/R&D/HEC/2017).

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