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JES-01323; No of Pages 12

Development of a novel myconanomining approach forthe recovery of agriculturally important elements fromjarosite waste

Ankita Bedi1,3, Braj Raj Singh1, Sunil K. Deshmukh1, Nisha Aggarwal2,Colin J. Barrow3, Alok Adholeya1,⁎

1. TERI–Deakin Nanobiotechnology Centre, Biotechnology and Management of Bioresources Division, The Energy and Resources Institute(TERI), New Delhi 110003, India. E-mail: [email protected]. Sri Aurobindo College, Department of Chemistry, University of Delhi, New Delhi 110007, India3. School of Life and Environmental Sciences, Deakin University, Victoria 3216, Australia

A R T I C L E I N F O

⁎ Corresponding author. E-mail: [email protected]

https://doi.org/10.1016/j.jes.2017.09.0171001-0742/© 2017 The Research Center for Ec

Please cite this article as: Bedi, A., et al.,important elements from jarosite waste,

A B S T R A C T

Article history:Received 21 March 2017Revised 15 September 2017Accepted 30 September 2017Available online xxxx

In this study, an ecofriendly and economically viablewastemanagement approach have beenattempted towards the biosynthesis of agriculturally important nanoparticles from jarositewaste. Aspergillus terreus strain J4 isolated from jarosite (waste from Debari Zinc Smelter,Udaipur, India), showed good leaching efficiency along with nanoparticles (NPs) formationunder ambient conditions. Fourier-transform infrared spectroscopy (FT-IR) and transmissionelectron microscopy (TEM) confirmed the formation of NPs. Energy dispersive X-rayspectroscopy (EDX analysis) showed strong signals for zinc, iron, calcium and magnesium,with these materials being leached out. TEM analysis and high resolution transmissionelectronmicroscopy (HRTEM) showed semi-quasi spherical particles havingaverage size of 10‐50 nm. Thus, a novel biomethodology was developed using fungal cell-free extract forbioleaching and subsequently nanoconversion of the waste materials into nanostructuredform. These biosynthesized nanoparticles were tested for their efficacy on seed emergenceactivity of wheat (Triticum aestivum) seeds and showed enhanced growth at concentration of20 ppm. These nanomaterials are expected to enhance plant growth properties and beingtargeted as additives in soil fertility and crop productivity enhancement.© 2017 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences.

Published by Elsevier B.V.

Keywords:BioleachingFourier transform infraredspectroscopy (FTIR)Transmission electronmicroscopy (TEM)JarositeNanoparticlesSeed-emergence activity

Introduction

Presently, an annual production of approximately 960 milliontonnes (MT) of solid waste as by-products of processes likeindustrial, mining, agricultural and municipal has beenreported in India. Out of this, around 4.5 MT are consideredto be hazardous in nature (Er. Nitisha Rathore and Er.Devendra, 2014). Jarosite is one such important solid waste

s.in (Alok Adholeya).

o-Environmental Science

Development of a novelJ. Environ. Sci. (2017), htt

material, which is generated during the hydrometallurgicalmetallic zinc extraction process of zinc industries. Currently,substantial quantity of jarosite waste is being generateduniversally and China, Canada, USA, Japan, Australia, Spain,Holland, France, Yugoslavia, Korea, Brazil, Mexico, Norway,Germany, Argentina, Belgium and India are top producers(Pappu et al., 2011). Approximately 2.5 MT of such zincresidues are being disposed of per annum globally (Asokan,

s, Chinese Academy of Sciences. Published by Elsevier B.V.

myconanomining approach for the recovery of agriculturallyps://doi.org/10.1016/j.jes.2017.09.017

2 J O U R N A L O F E N V I R O N M E N T A L S C I E N C E S X X ( 2 0 1 7 ) X X X – X X X

2007; Asokan et al., 2006). About 0.25 MT of jarosite is releasedin India per annum (Er. Nitisha Rathore and Er. Devendra,2014).

The generated jarosite waste is hazardous in nature due tothe presence of toxic heavy metals (Al, Cu, Cd, Cr, Pb etc.) andis posing severe hazards to the exposed abiotic and bioticcomponents of the ecosystem. In order to avoid environmen-tal problems caused by the leaching of heavy metals fromjarosite waste, researchers are developing methods/technol-ogies for its management (Katsioti et al., 2006; Vyas, 2011). Inthe last two decades, various jarosite waste managementstrategies have been developed by researchers for their safedisposal and application like the development of landfill,construction and ceramic materials. Each managementstrategy has its own advantages and disadvantages, andthese are non-renewable in nature and require large scaleset up (Acharya et al., 1992; Katsioti et al., 2006). The reportedmethod in this paper is an alternative ecofriendly biologicalapproach to the existing physico-chemical methods ofjarosite waste management.

The biological approach based on interactions betweenfungi and metallic elements/compounds has been wellestablished, and the inherent ability of fungi to extract and/or bioaccumulate metallic elements/compounds is alreadyapplied in biotechnological processes such as bioleachingand bioremediation. As an outcome of research in thenanoparticles biosynthesis field, it has been reported thatfungi possess inherent capability to synthesize metallicnanostructured materials by the intra- or extra-cellular re-duction of metallic elements/compounds (Mishra and Rhee,

RAJASTHAN

a

Fig. 1 – (a) Jarosite sample collection site (24°35′58″N 73°49′8″E) (httas-collected.

Please cite this article as: Bedi, A., et al., Development of a novelimportant elements from jarosite waste, J. Environ. Sci. (2017), htt

2010; Ren et al., 2009; Santhiya and Ting, 2005). As a novelmethod, myconanomining (Fungi mediated bioleaching andconversion of bulk metallic elements/compounds into nano-structures) is considered safe and ecologically benign for theconversion of bulk inorganic (metal based) materials intonanostructured forms.

In thismyconanomining approach, theuse of fungal biomassaqueous extract containing secretome for bioleaching fromcollected jarosite waste materials and subsequent biosynthesisof nanoparticles (plant nutrients Fe and Zn) is a possibility thathas not been applied extensively. The use of myconanominingfor bioleaching and biosynthesis of metal nanoparticles fromjarosite waste can offer several advantages over other environ-mental biological process, such as: (i) more biomass production,(ii) fungal secretome contains large amounts of extracellularproteinswith diverse functions, (iii)more biosorption ofmetallicelements/compounds at low pH and (iv) high metal reducingactivity of secretome.

Therefore, considering the importance and future scopeof myconanomining approaches, the following objectiveshave been formulated for this study (i) Total metal analysisof jarosite waste using Atomic Absorption Spectroscopytechnique; (ii) Isolation and characterization of promisingfungal strain for myconanomining from jarosite wasteusing culture enrichment technique; (iii) Bioleaching andbiosynthesis of plant nanonutrients via myconanominingapproach using Aspergillus terreus strain J4 and (iv) in-vitroassessment as a nutrient use efficacy of biosynthesized metalnanoparticles on wheat using seed emergence promotingactivity.

MAP OF RAJASTHAN

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p://wikimapia.org/171241/HZL-Debari (03/12/2015)); (b) jarosite


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Fig. 2 – (a) EDX (Energy dispersive X-ray spectroscopy)spectrum and (b) XRD (X-ray diffraction) analysis of jarositewaste based on reference powder diffraction files (PDF)respectively.

Table 1 – Chemical analysis of the metal content injarosite.

Metal Concentration(mg/kg)

Concentration globally(million tonnes) ⁎

Zinc 33,504.55 ± 15.61 ~0.082Iron 37,912.79 ± 0.70 ~0.095Lead 14,162.74 ± 9.15 ~0.035Sulfur 10,042.31 ± 3.27 ~0.025Aluminium 5122.50 ± 7.11 ~0.012Copper 284.0427 ± 14.77 ~0.0005Silica 116.083 ± 5.01 ~0.0002

⁎ These concentrations may vary by 5%–10% depending on variousparameters like location, temperature, humidity, collection etc.

3J O U R N A L O F E N V I R O N M E N T A L S C I E N C E S X X ( 2 0 1 7 ) X X X – X X X

1. Materials and methods

1.1. Chemicals and medium

All chemicals (analytical grade) used in this study were fromFischer Scientific (Mumbai, India). Potato dextrose agar, Potatodextrose broth, Mycological peptone, and Agar Extra purewere purchased from HiMedia (Mumbai, India) sterilized byautoclaving at 120 °C for 15 min at 15 Pa before use. Wheat(Triticum aestivum) seeds (Raj 3765 variety from Rajasthan,India) were purchased and stored in a dry place.

1.2. Jarosite waste sample collection and their elemental,mineralogical and structural analysis

The jarosite waste was procured from the Debari Zinc smelterplant of Hindustan Zinc Limited (HZL) situated in Udaipur(24°35′58″N 73°49′8″E) (Fig. 1) and transported in sterile tubes.The HZL is India's only integrated Zinc Company, operatingfrom mine to finished Zn metal and its current productioncapacity of 88,000 t per annum of high grade zinc. This companysupplied around 73% of India's requirements. The collectedjarosite waste sample was air-dried at room temperature,homogenized, sieved through fine sieve, and stored in ambercolor containers under dark and dry conditions.

The elemental, mineralogical and morphological charac-teristics of jarosite waste material were determined by atomicabsorption spectroscopy (AAS) and transmission electronmicroscopy (TEM). For the elemental composition of jarositewaste, ~0.5 g of finely powdered sample was refluxed withHNO3 for acid-digestion using Method 3050 B (Arsenic et al.,1996). The digested sample was later cooled, filtered anddiluted (100–1000 times) for chemical analysis by using atomicabsorption spectrometer (iCE 3500, Thermo Fischer Scientific,USA). Mineralogical composition was determined by powderX-ray diffraction. The X-ray diffraction (XRD) patterns ofpowder sample was recorded on MiniFlex™ II benchtop XRDsystem (MiniFlex™ II, Rigaku Corporation, Japan) operating at40 kV and a current of 30 mAwith Cu Kα radiation (λ = 1.54 Å).The diffracted intensities were recorded from 20 to 80°2θ angles. Themorphology (microstructure) and total elemen-tal composition of jarosite waste sample was analyzed usingTEM (TECNAI G2 T20 TWIN, FEI, Netherlands) equipped withenergy dispersive X-ray spectrometer (EDS) (LN2 detector,EDAX Inc., Netherlands). Ten milligrams of sample was dis-solved in 1-mL MilliQ (MQ) water and sonicated for 5 min fordisintegration of the microparticles. The sonicated sample(~10 μL) was drop casted on a carbon-coated nickel grid and airdried under dark conditions. The prepared grid was analyzed atan accelerated voltage of 200 kV and the TEM micrographs andEDX spectrum images were obtained.

1.3. Isolation and screening of bioleachor fungal strains fromjarosite waste

Various fungal strains were isolated from jarosite usingculture enrichment technique. Around 20 g of jarosite wassuspended in 100 mL of mycological peptone. After 48 hr ofincubation (140 r/min, 30 ± 2 °C), 100 μL of sample was plated


on potato dextrose agar (PDA) plates. The concentration of Znwas successively increased in the suspension from 10,000 to40,000 ppm using ZnSO4·7H2O (Qualigens, Mumbai, India), byfilter sterilization using 0.22-μm mdi membrane technologiessterile syringe filters (Advanced Microdevices Pvt. Ltd.,Ambala Cantt., India). The standard spread plate techniquewas used to isolate the filamentous fungi on PDA plates afternecessary dilution (up to 10−4) in three replicates (Ilyas et al.,2013; Joshi et al., 2011). The colonies obtained were picked and



subcultured on PDA plates for further purification. Thesefungal isolates obtained were then subjected to 40,000-ppmZn to check for metal tolerance. Isolates thus obtained, werefurther purified on fresh PDA plates and later screened forbioleaching and nanoparticles biosynthesis efficiency.

1.4. Assessment of growth kinetics of promising bioleachorfungal strain J4

Among the 5 fungal isolates obtained, the fungal strain J4was identified as a potential bioleachor of zinc (Zn) element/compound from jarosite waste. The isolated strain J4 waspreserved using 50% glycerol in sterile Milli Q and stored at−80 °C until further used.

Ergosterol is the major sterol present in the cell membranesof filamentous fungi, andmonitoring its level is a usefulmethodfor estimating growth kinetics (Axelsson et al., 1995; Klamer andBaath, 2004; Steudler andBley, 2015). Fiftymilligramsof strain J4fungal mycelium was taken at regular time interval of 24 hr(starting after 48 hr). The mycelium was ground using a motorandpestlewith liquidnitrogen, followedby theaddition of 1 mLof absolute ethanol. This mixture was agitated for 30 sec, keptin ice for 1 hr and then centrifuged for 5 min at 14,000 r/min.The supernatant was collected, and a pellet was suspended in1 mL of absolute ethanol and treated once again as describedabove. The two supernatants were pooled together, filteredusing 0.22-mm nitrocellulose filters (Millipore, Darmstadt,Germany), and the filtrate was analyzed for Ergosterol usingthe protocol of Lindblom (Mille-Lindblom et al., 2004) with some

a

c

Fig. 3 – Transmission electron microscopy (TEM) of jarosite as su(d) 100 nm.


modification. Analysis was carried out using high performanceliquid chromatography (HPLC, CBM-20A, Shimadzu, Japan)equipped with a quaternary pump (LC - 20AT, Shimadzu,Japan), solvent degasser system (DGU - 20 A5, Shimadzu,Japan), autosampler (SIL – 20A, Shimadzu, Japan) and diodearray detector (SPDM-20A, Shimadzu, Japan). Inbuilt software(LC solution, Shimadzu, Japan) was used to control theHPLC pump and acquire data from the diode array. A C18Phenomenex column (Gemini-NX 250 mm × 4.6 mm × 5 μmparticle diameter) was used for the analysis. A series ofergosterol standards of varying range 10–50 ppmwere preparedin ethanol. The standard peak was obtained with a UV detectorset at 282 nm and a runtime of 20 min. The mobile phase wasmethanol (97%) and water (3%) at a flow rate of 1 mL/min andthe injected sample volume was 50 μL.

1.5. Taxonomic characterization of fungal strain J4

1.5.1. Morphological characterizationThe fungal strain J4 was presumptively identified on the basisof monographs (Nyongesa et al., 2015; Samson et al., 2011)and macro as well as micro morphological features. Themorphological features of strain J4 were observed usingscanning electron microscopy (SEM) (Carl Zeiss, Oberkochen,Germany). The strain J4 was subcultured on PDA andincubated at 25 °C for 4–5 days. After incubation, fungaldiscs were taken and then immersed in fixative solution(modified Karnovsky's fixative containing 2.5% glutaralde-hyde, 2.5% paraformaldehyde, 0.05-mol/L cacodilate buffer

b

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ch at different scale of (a) 1 μm, (b) 0.5 μm, (c) 0.2 μm and



and 0.001-mol/L CaCl2) at pH 7.2. Cacodilate buffer was thenused to wash the discs (thrice for 10 min each), followed bypost-fixation in 1% osmium tetraoxide solution and water for1 hr (Silva et al., 2011). The samples were then washed withsterile MQ water three times and subjected to dehydration inacetone (25%, 50%, 75%, 90% and 100%) for 10 min, followed bycritical point drying (CPD) (Emitech K850, Berkshire, UK). Thesample was later assembled on double-sided carbon tapeplaced on aluminium stubs and coated with gold–palladiumin a sputter coater (Quorum Technologies SC7620, Berkshire,UK) and viewed in SEM at an accelerating voltage of 10 kV.

1.5.2. Molecular characterizationFor molecular identification of the fungal strain J4, totalgenomic DNA was extracted from ~50 mg of mycelia usingthe DNeasy Plant MiniKit 50 (Qiagen, Germany) according tothe manufacturer's instructions. Internal transcribed spacer(ITS) region (ITS 1–5.8S–ITS 2) of rDNA was amplified usingprimer set: ITS1;5′-TCCGTAGGTGAACCTGCGG-3′ and ITS4;5′-TCCTCCGCTTATTGATATGC-3′ as described by White andco-workers (White et al., 1990). The polymerase chain reaction

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Fig. 4 – (a) Fungal isolates (coding J1–J5) on potato dextrose agarefficiency from Jarosite.


(PCR) amplification was performed in a Veriti 96-well ThermalCycler (Applied Biosystems, Massachusetts, USA). Negativecontrol reaction without any template DNA was carried outsimultaneously to confirm the absence of any contamination.The PCR product was analyzed on 1.2% agarose gel containingethidium bromide (10 mg/mL) (Sigma-Aldrich, St. Louis, USA)and amplicons were extracted and purified using gel extrac-tion kit (Qiagen, Germany). The purified PCR product wasligated using 2X rapid ligation buffer. The master mix (10 μL)was prepared as follows: 5 μL of 2X rapid ligation buffer, 1 μLof pGEM® - T Easy Vector (50 ng) (Promega, USA), 2 μL of PCRProduct and T4 DNA ligase (3 Weiss units/mL). The finalvolume was made up using deionized water; 0.5 mL tubesknown to have low DNA-binding capacity were used. Thereactions were mixed by pipetting and incubated overnight at4 °C to allow the ligation reaction to stabilize and increase thenumber of transformants. The next day, transformation wasconfirmed using the competent cells (Escherichia coli DH5-α).The final products were analyzed on 1% agarose. The streakedcolonies corresponding to samples showing bright bandsin the gel were then inoculated in 5-mL Luria broth (LB)

J3 J4 J5

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Fig. 5 – (a) pH of MilliQ with washed biomass recorded atvarious time intervals to confirm the release of organic acids;(b) Growth kinetics study of Aspergillus terreus strain J4 byErgosterol assay.


(HiMedia, Mumbai, India) for plasmid isolation. LB wasincubated at 37 °C, 180 r/min overnight. Before sending thesample for sequencing, plasmid was extracted using geneaidhigh speed plasmid mini kit (New Taipei City, Taiwan).Nucleotides similarity analysis was carried out by nucleotidebasic local alignment search tool (BLASTN) against thesequences available in GenBank database (Altschul et al.,1990), and dataset files were prepared. The sequences weresubmitted in GenBank database under accession no. KX953882.Construction of Phylogenetic tree of strain J4 was performedusing an interactive software Phylogeny.fr (http://phylogeny.lirmm.fr/phylo_cgi/simple_phylogeny.cgi) following standardparameters.

1.6. Bioleaching and biosynthesis of metal nanoparticles fromjarosite waste using promising fungal strain J4

The strain J4 was inoculated in 100 mL of potato dextrose broth(PDB) in a 250-mL Erlenmeyer flask and incubated at 28 °C for96 hr on a rotary shaker at 140 r/min. The obtained biomasswasthen harvested by centrifugation (Microcentrifuge, HeraeusBiofuge-stratos, USA) followed by 3 subsequent washingswith sterile MQ. Twenty grams of harvested biomass wasre-suspended in 100 mL of autoclaved MQ and subjected torotary shaking at 140 r/min, 28 °C for 96 hr, and pH of thesecretome was recorded at regular time intervals up to 144 hr.After the re-suspension, biomass was filtered using Whatmannfilter paper No.1, and the secretome (extracellular proteins andorganic acids) was collected. The obtained secretome ~100 mLwas mixed with 10 g of jarosite waste sample for bioleachingof metals elements/compounds and incubated at 28 ± 1 °C(140 r/min) for 24 hr on a rotary shaker at 140 r/min. Thebioleachate from the reaction mixture was collected into newflask and stirred at 35 ± 2 °C for 3 hr for the complete nucleationof metals with strain J4 secretome. The biosynthesized metalnanoparticles (denoted as ‘bMNPs’) were harvested usingcentrifugation at 5000 r/min for 10 min followed by washingthree times with sterile MQ water. The dried bMNPs samplepowder was stored in amber color vials at room temperatureunder dry and dark conditions until used for furthercharacterization.

1.7. Characterization of biosynthesized metals nanoparticles(bMNPs)

TEM and EDAX of bMNPs were carried out as described inSection 1.2. FTIR (Fourier-transform infrared spectroscopy)spectra of jarosite as control and bMNPs sample wererecorded using a Nicolet 6700 FT-IR (Nicolet 6700, ThermoFischer Scientific, USA). This spectrum helps in identificationof the capping agents responsible for biosynthesized nano-particles stabilization. The spectrum was obtained by anaverage scan of 64 in the range 400–4000/cm. Zeta potentialwas recorded to further confirm the presence of surface-bound proteins on nanoparticles indicated by the negativecharge. Fifty microliters of sample was taken for measure-ment of zeta potential. The sample was then transferred to apolycarbonate zeta cell (having gold plated electrode) and thepotential was measured at 25 °C using Zeta Sizer (Nano ZS90,Malvern, UK).


1.8. in-vitro assessment of bMNPs as plant nanonutrients

Wheat (Triticum aestivum) seeds (Raj 3765 variety fromRajasthan, India) were purchased locally and kept in dryplace. For in-vitro experiment, different dosages of Zn i.e. 10 to50 ppm were prepared. Wheat seeds were surface-sterilizedfirst by washing with 0.01% HgCl2 twice for 2 min eachfollowed by sterile distilled water three times. Seeds weredipped in different dosages of 10–50 ppm and then sonicatedfor 30 sec followed by shaking for about 2 hr. Untreated seeds(C) as absolute control and seeds treated with bulk ZnSO4

(40 ppm) as positive control were taken. Treated seeds wereplaced on 0.8% water agar plates (6 seeds each plate). Eachreaction was carried out in 3 replicates. The plates were sealedwith parafilm and kept in the plant growth room (25 °C).Following 3 to 7 days, seed germination in each treatmentwas recorded in percentage. Seeds that had coleoptile longerthan 2 mm were considered germinated; and the others wereconsidered non-germinated. Seedling vigor index (SVI) wasmeasured from the root and shoot length of geminated seedsusing the formula given below (Abdul-Baki and Anderson,1973; Jayarambabu et al., 2014).

SVI ¼ Average root length in cmþ Average shoot length in cmð Þ�Germination percentage



1.8.1. Statistical analysisThe data presented is expressed in terms of mean ± standarddeviation (SD) of three replicates. Raw data here was analyzedusing IBM SPSS Statistics 19. One-way analysis of variancewith a Duncan's post-hoc test of significance at p ≤ 0.05 wasused to study the effect of different dosages of bMNPs ongermination and growth characteristics of wheat seedlings.

2. Results and discussion

2.1. Characterization of jarosite waste

The elemental and morphological characteristics of collectedjarosite waste were determined by AAS and TEM techniques,respectively. The metal content of jarosite waste of HZLdetermined by AAS is shown in Table 1. It was observed to

a b

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Fig. 6 – Colony morphology on (a) solid media PDA (potato dextroScanning electron microscopy images showing structural morphstrain J4; and (e) Phylogenetic tree based on the internal transcristrain J4 in comparison with the related species sequences.


mainly contain zinc (~34,000 ppm), iron (~38,000 ppm), sulfur(~11,000 ppm) and lead (~14,000 ppm), along with trace ele-ments like copper and aluminium. The results are presented asmean ± SD of samples setup in triplicates. Heterogeneity wasobserved when the data obtained was compared with otherreports (Acharya et al., 1992; Ilyas et al., 2013; Pappu et al., 2011).These differences may be due to the origin of substrate andvarious physical parameters like temperature and climate ofthe sample collection site. Jarosite is essentially a hydroussulfate of chemical formula KFe3+3(OH)6(SO4)2 released as aby-product of zinc purification and refining.

The presence of iron, sulfur, potassium and zinc in jarositewas determined by EDX spectroscopy (Fig. 2a). These resultswere in correspondence to earlier reports indicating higherconcentration of these elements (Asokan et al., 2006). XRDpattern of as received jarosite waste sample is shown inFig. 2b. The obtained XRD pattern was analyzed to confirm the

d

se agar); (b) liquid media PDB (potato dextrose broth);ology of fungal conidiophore (c) and spores (d) of A. terreusbed spacer region nucleotide sequence of culture A. terreus


Fig. 8 – Comparative graph of FTIR (Fourier transforminfrared spectroscopy) spectrum of jarosite as control andbiosynthesized metal nanoparticles (bMNPs) indicating thevariation in the frequency and intensities which may be dueto the conformational changes.


presence of minerals and data revealed that themajor intensepeaks are assigned to jarosite (JCPDS 26-1014) and gypsum(JCPDS 21-0816) minerals. The difficulties were faced duringthe minor minerals phase (quartz and franklinite) determina-tion from XRD data, because of strong overlap with peaks ofmajor minerals (jarosite and gypsum) (Mehra et al., 2016).However, other minerals (Barite and galaxite) have beenreported from the jarosite waste but were not detected inthe collected waste sample. The TEM analysis indicates thatjarosite waste sample has aggregated crystalline structureswith irregular shaped amorphous particles with size range~100 to 500 nm (Fig. 3).

2.2. Isolation and screening of fungal strains with bioleachingproperties from jarosite waste

The percentage of bioleaching and nanoparticle biosynthesisefficacy (%) from jarosite, after 96 hr of reaction, of all the fivefungal isolates, is shown in Fig. 4. The results are presented asmean ± SD of samples setup in triplicates. Under equivalentexperimental conditions, different isolated fungal cultureshave shown varying percentage of leaching of zinc and iron,along-with nanoparticles biosynthesis from jarosite. Thevariation in the inherent capability of leaching and nanopar-ticle biosynthesis of isolated fungal culturesmay be attributedto the exogenously released different organic acids, such asgluconic, citric, oxalic acid, and enzymes such as reductases,that impact nanoparticles formation (Brisson et al., 2016;Duran et al., 2005; Seh-Bardan et al., 2012). It was observedthat A. terreus strain J4 showed the highest leaching efficiencyof 67.1% as compared to other isolates.

A drop in pH from 7.05 to 5.21 after 96 hr of reaction timewas observed, which remained almost constant thereafter asseen in Fig. 5a. The results were presented as mean ± SD ofsamples set up in triplicates. These were in correlation withthe previous reports which stated that leaching efficiency offungus could be attributed to the organic acids released byfungal biomass. Oxalic acid and citric acid have been reported

ba

Fig. 7 – Biosynthesized nanoparticles from Jarosite-colorchanged from (a) white yellow to (b) brick red.


to be involved in the leaching process (Ambreen et al., 2002;Aung and Ting, 2005; Brandl et al., 2001; Ren et al., 2009;Santhiya and Ting, 2005). Fig. 5b shows the growth kineticstudy of A. terreus strain J4 by ergosterol assay. Five-day oldculture was used for further studies.

2.3. Morphological and molecular characterization ofbioleachor fungal strain J4

Preliminary identification of fungal isolate was carried out onthe basis ofmorphological characteristicswhichwere similar tothose described by Thom and Church (1918). Fig. 6a–d showsisolated fungi maintained on solid media (PDA), liquid media(PDB), SEM images of fungal conidiophore and spores respec-tively. Indeed, the authentic taxonomic characterization of thestrain J4 was carried out by comparing the nucleotide sequenceobtained using BLAST of National Centre for BiotechnologyInformation (NCBI) followed by phylogenetic tree using Maxi-mum Likelihood Method (Edgar, 2004; Guindon et al., 2010)(Fig. 6e). BlastN sequence alignment analysis revealed ∼99%sequence homology of sequenced amplicon with the strains ofAspergillus terreus. The phylogenetic analysis revealed the closetaxonomic homology of strain J4 with the Aspergillus terreusstrain LCF17 (Accession No.FJ867934.1).

Zeta potential (mV)

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Fig. 9 – Zeta potential of nanoparticles biosynthesized fromjarosite using cell-free extract of A. terreus strain J4.


Table 2 –Measurement growth characteristics of wheatseedlings at different concentration of jarosite nanoparticles(with respect to Zn) within 7 days.

Treatments Germination (%) Seedling vigor index

Untreated (C) 88.87 ± 0.64ab 1716.23 ± 0.12ab

Bulk ZnSO4 91.8 ± 0.47ab 1651.29 ± 0.24ab

10 ppm 88.87 ± 0.64ab 1996.72 ± 0.05ab

20 ppm 100.0 ± 0.01a 2390.0 ± 0.75a

30 ppm 77.77 ± 0.60b 1602.35 ± 0.68bc

40 ppm 72.22 ± 0.60b 1300.07 ± 0.26c

50 ppm 72.21 ± 0.60b 1404.82 ± 0.77c

Values with same letters are not statistically different. Data isrepresented as mean ± SD of 3 replicates.


2.4. Characterization of biosynthesized nanoparticles (NPs)

The color of NPs changed fromyellow to brick red indicating thebiosynthesis. Fig. 7 shows the dried bMNPs from jarosite.

The samples kept in −80 °C were analyzed to identify themolecules that may be responsible for the observed bioleachingactivity by the fungal cell-free extract. Fig. 8 displays the FTIRspectrum showing an absorption peak at ~1621 and ~1425 cm−1

corresponding to the amide I and N\H vibration of NH+4

functional group of jarosite (Adamou et al., 2016) and~3408 cm−1 the\OHstretch (Becheri et al., 2008). The absorptionbands at ~1196, ~1078, and ~631 cm−1 correspond to thevibration mode of SO4

2− (Liu et al., 2007; Mymrin et al., 2005;Tsakiridis et al., 2005). It has been observed that there is variationin frequency and intensity of the bMNPs when compared withjarosite as control. This could be due to the conformationalchanges that occurred during the nucleation of metals. Thus,presence of these functional groups indicates that proteins forma capping layer on the nanoparticles, preventing them fromagglomeration and thus stabilizing them in a solution. Fig. 9shows the negatively charged zeta potential of −10.2 mV for the

a

c

Fig. 10 – Transmission electron micrograph of (a) bMNPs of an avresolution Transmission electron microscopic image at scale of 1nanoparticles and (c) EDX spectrum.


biosynthesized nanoparticles which were obtained usingcell-free extract of A. terreus strain J4 which further indicatesprotein-capping of the biosynthesized nanoparticles, whichcontributes to particle stability.

Fig. 10a shows TEM micrographs of bMNPs. After a reactionof 96 hr, the bioleachate contains semi-quasi spherical particles

Element Weight %C(K) 47.23

O(K) 12.25

Na(K) 3.79

Mg(K) 1.02

Al(K) 0.73

Si(K) 2.63

Cl(K) 1.13

Ca(K) 0.55

Fe(K) 5.96

Cu(K) 19.54

Zn(K) 5.12

b

erage size of 10 nm by fungal cell-free extract; (b) High0 nm showing fringes indicating crystalline structure of


0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

)mc(

ht gneL

Treatments

Root length (cm)

Shoot height (cm)

ba a

b bb

d

ba

c cd cd

b

cd

Fig. 11 – Root length and shoot height of respective treatments following 7 days. Values with same letters are not statisticallydifferent. Data is represented as mean ± SD of 3 replicates.


of approximately 10–50 nm with an average size range of 15 ±5 nm. The lattice structure of particles can be observed in highresolution transmission electron microscopy (HRTEM) micro-graphs, indicating their crystalline structure (Fig. 10b). Fig. 10cdisplays an EDX spectrum of bio-synthesized NPs. The spec-trumwas recorded from one of the densely populated NPs areaand confirmed the leaching of zinc and other metals by thefungal cell-filtrate, showing a strong signal of zinc and oxygenalong-with calcium and magnesium. The sharp optical absorp-tion peak in the range of 5–10 keV signified the presence of zincand 0–1 keV confirmed the presence of oxygen in the NPs. TheP, S, Cl, and K signals were probably due to X-ray emission frombiological macromolecules (carbohydrates/proteins/enzymesetc.) present in the cell wall of fungal mycelium. The strongpeaks of Ni and C are due to the carbon-coated nickel grid. Theusual carbon-coated copper gridwas not used so as tominimizethe interference from the grid, as jarosite already has copperpresent in it.

2.5. Effect of bMNPs on seed germination and root andshoot development

In-vitro results revealed that the increase in percentage ofseed germination as compared to control was observed ontreatment with as synthesized bMNPs (with respect to Zn)(Table 2). Seed germination is the beginning of a physiologicalprocess that needswater imbibition (Wierzbicka andObidzinska,1998). The increase in percentage of seed germination wasobserved on treatment with biosynthesized nanoparticles.Statistically significant effect on germination was recorded in20 ppm (100%) followed by 82.22% in case of 10 ppm treatedseeds as well as control which decreased with higher concentra-tions (30 ppm - 77.78%, 40 ppm - 72.22% and 50 ppm - 72.22%).This indicated that bMNPs did not have an adverse effect onthe wheat seed germination. Treatment of 20 ppm showedsignificant effect with the highest SVI of 2390 as comparedto other treatments. These results are related to the reportswhere it has been reported that nano-ZnO promotes seedgermination at lower concentrations in plants like Oryza sativaL. (Boonyanitipong et al., 2011), Vigna radiata L. (Jayarambabu


et al., 2014) etc. The increase in seed germination depends onthe adsorption, uptake, and penetration of biosynthesizednanoparticles. There are reports which indicate penetration inthe seed by nanoparticles (Khodakovskaya et al., 2009). Thisreport suggested multi-walled carbon nanotubes (MWCNTs)could penetrate in tomato seed and enhance germinationby increasing water uptake. Khot and co-workers have alsoreported that this enhanced germination could also be due tothe photo-generation of active oxygen like superoxide andhydroxide anions that increases the seed stress resistancecapacity and also capsule penetration of oxygen required forfaster germination process (Khot et al., 2012).

Length of root and shoot were recorded in cm after 7 daysand similar trends were observed, that is, lengths were foundto be inversely proportional to the concentration. At lowerconcentrations of 10 and 20 ppm, nanoparticles significantlyenhanced the growth of seedlings. However, there was nosignificant effect observed at higher concentrations (Fig. 11).These results are corroborated by earlier reports where thisreduction in the growth measurements has been recordeddue to toxicity of nanoparticles at higher concentrations(Boonyanitipong et al., 2011; Lee et al., 2008; Zhu et al., 2008).

3. Conclusion

The present study indicates the bioleaching potential ofA. terreus strain J4 from jarosite. The NPs synthesized rangefrom 10 to 50 nmwith an average size range of 15 ± 5 nm. Theyshowed goodeffect on seedling's growth at low concentrationof20 ppm as compared to higher concentrations and control. Tothe best of our knowledge, this is the first study of its kind. Thisfinding can be further used synthesizing green protein-cappedmetal nanoparticles with potential cost efficacy.

Acknowledgements

We duly acknowledge Ms. Deeprajni for HPLC, Ms. ShikhaSherawat for SEM, Mr. Ranjit and Mr. Palak Agarwal for AAS



and Mr. Chandrakant Tripathi and Mr. Aditya Gaur for TEManalysis. Dr. Sashidhar Burla and Ms. Richa Chaturvedi arealso acknowledged for assisting in molecular work. Thefellowship provided by Deakin University, Australia andinfrastructure support extended by The Energy and ResourcesInstitute, India is also duly acknowledged.

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