lable at ScienceDirect

International Biodeterioration & Biodegradation 65 (2011) 309e317

Contents lists avai

International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Detoxification and accumulation of chromium from tannery effluent and spentchrome effluent by Paecilomyces lilacinus fungi

Seema Sharma a,b, Alok Adholeya a,*

aBiotechnology and Management of Bioresources Division, The Energy and Resources Institute, DS Block, India Habitat Centre, Lodhi Road, New Delhi 110003, IndiabCentre for Bioresources and Biotechnology, Teri University, DS Block, India Habitat Centre, Lodhi Road, New Delhi 110003, India

a r t i c l e i n f o

Article history:Received 26 August 2010Received in revised form1 December 2010Accepted 1 December 2010Available online 30 December 2010

Keywords:Paecilomyces lilacinusChromiumCane sugarTannery effluentSpent chrome effluent

* Corresponding author. Tel.: þ91 11 2468210024682145.

E-mail address: aloka@teri.res.in (A. Adholeya).

0964-8305/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.ibiod.2010.12.003

a b s t r a c t

The tannery industry process involves chromium (Cr) salts as a main constituent of the process. The Crrecovery is a part of the process where other salts are used to achieve separation and recovery for usingCr back in the process. The process steps may contain both forms of Cr [Cr(VI): hexavalent and Cr(III): trivalent]. The recovery of Cr from tannery industry effluent through biological systems is muchneeded. The diverse physicochemical characteristics of these effluents may limit the growth of micro-organisms and hence the limitation towards possible practical application of microorganisms in realindustrial effluent conditions. The present study attempted the ability of the Cr-resistant fungus Paeci-lomyces lilacinus [isolated through an enrichment culture technique at 25 000 mg l�1 of Cr(III)] to growand remove Cr [Cr(VI) and Cr(III)] from two physicochemically different undiluted tannery industryeffluents (tannery effluent and spent chrome effluent) in the presence of cane sugar as a carbon source.Such attempts are made keeping in view the potential integration of biological processes in the overall Crremoval and recovery processes to improve its efficiency and environmental sustainability. The fungushas broad pH tolerance range and can reduce Cr(VI) both in acidic (pH 5.5) and alkaline (pH 8.0)conditions. The fungus showed the ability to remove Cr(VI) (1.24 mg l�1) and total Cr (7.91 mg l�1) fromtannery effluent below the detection level within 18 h and 36 h of incubation, respectively, and ability toaccumulate 189.13 mg Cr g�1 of dry biomass within 600 h of incubation from spent chrome effluent[containing 3731.4 mg l�1 of initial Cr(III) concentration].

At 200 mg l�1 of Cr(VI) in growth media, with 100% detoxification and with only 10.54% of total Craccumulation in the biomass, P. lilacinus showed Cr(VI) reduction as a major mechanism of Cr(VI)detoxification. The time-course study revealed the log phase of the growth for the maximum specificreduction of Cr(VI) and stationary phase of the growth for its maximum specific accumulation of both theforms of Cr [Cr(III) and Cr(VI)] in its biomass. In growth media at 50 mg l�1 and 200 mg l�1 of Cr(VI),P. lilacinus showed 100% reduction within 36 h and 120 h of incubation, respectively. The high degree ofpositive correlation and statistically high degree of relationship (r2 ¼ 0.941) between the fungal growthand % Cr(VI) reduction by the fungus support the role of metabolically active cellular growth in Cr(VI)reduction by the fungus. Results indicate that expanded solid (sludge) retention times (SRTs) (stationaryphase) can be recommended for the removal of Cr(III) through accumulation. In case of Cr(VI), reductionneeds a priority; therefore, a non-expanded SRT is recommended for designing a continuous-flowcompletely stirred bioreactor so that a log phase of cellular growth can be maintained during thereduction process. This study reveals the strong potential of P. lilacinus fungi for the removal of Cr fromtannery effluent and spent chrome effluent.

� 2010 Elsevier Ltd. All rights reserved.

x2631, 2628; fax: þ91 11

All rights reserved.

1. Introduction

Chromium (Cr) compounds are used in industries for chromeplating, wood preservation, textile dyeing and pigmentation,manufacturing pulp and paper, and tanning. The wastewaterresulting from these industrial processes contains high concen-tration of Cr, which contaminates the natural environment,

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317310

affecting human health (McLean and Beveridge, 2001;Congeevaram et al., 2007). The tannery industry, whichcommonly uses basic Cr(III) sulphate [Cr(H2O)5(OH)SO4] fortanning process, is a major cause for high influx of Cr to thebiosphere, accounting for 40% of the total industrial use (Barnhart,1997). In nature, Cr exists in two stable forms Cr(III) and Cr(VI).Characteristics like higher solubility in water, rapid permeabilitythrough biological membranes, and subsequent interaction withintracellular proteins and nucleic acid makes Cr(VI) comparativelymore toxic than Cr(III) (Congeevaram et al., 2007). Due to thedifference in the toxic nature of both the forms of Cr, the dischargeof Cr(VI) into surface water is regulated to below 0.05 mg l�1 by theUS EPA, while the total Cr [including Cr(III), Cr(VI) and its otherforms] is regulated to below 2 mg l�1 (Baral and Engelken, 2002).

The conventional physical and chemical methods used forremoval of heavy metals from the effluent, such as precipitationwith carbonates, sulphides and hydroxide, adsorption on activatedcarbon, use of ion-exchange resins and membrane-separationprocesses, are responsible for generation of pollution and are notcost-effective (Volesky and Holzen, 1995; Kratochvil et al., 1998;Camargo et al., 2003). An alternative to these methods is theremoval of heavy metal contaminants by microorganisms. Themetal removal ability of microorganisms, including bacteria(Cheung and Gu, 2005; Thacker et al., 2007), microalgae (Kratochvilet al., 1998;Matsunaga et al., 1999; Gupta et al., 2001a, b; Gupta andRastogi, 2008) and fungi (Tobin and Roux, 1998; Srivastava andThakur, 2006), has been studied extensively. Fungi, in general, arewell-known for their ability to biosorb and bioaccumulate metals(Pillichshammer et al., 1995; Dursun et al., 2003; Nouri et al., 2005;Park et al., 2005) and have also been reported to be involved inreduction (biotransformation) of Cr(VI) to Cr (III) form (Pal, 1997;Gouda, 2000; Acevedo-Aguilar et al., 2006; Morales and Cristiani,2008). The common Cr(VI) detoxification mechanisms reported inCr-resistant microorganisms are periplasmic biosorption, intracel-lular bioaccumulation and biotransformation through direct enzy-matic reaction (Lovley, 1993; Lee et al., 2000; Valls et al., 2000) orindirectly with metabolites (Camargo et al., 2003). In Cr(VI)-resis-tant filamentous fungi, such as Aspergillus (Gouda, 2000; Acevedo-Aguilar et al., 2006), Penicillium (Acevedo-Aguilar et al., 2006),Trichoderma (Morales and Cristiani, 2008) and Phanerochaete (Pal,1997), the Cr(VI) detoxification through transformation of Cr(VI)to Cr(III) form was observed due to cellular metabolism processesbased on the reducing power of carbon sources.

The cell surfaces of microorganisms are negatively chargedowing to the presence of various anionic structures. This givesmicroorganisms an ability to bind metal cation (Chen and Hao,1998). Metal removal through biosorption is the process in whichlive/dead microbial biomass or adsorbents developed from bio-logical and industrial waste materials (Gupta et al., 1997; Srivastavaet al., 1997; Gupta et al., 2001a, b; Gupta et al., 2009) is simply usedas an adsorbent (Volesky, 2001), whereas the bioaccumulationprocess involves using growing biomass in the removal or bindingof metal ions (Dursun et al., 2003). Recent reports employinggrowing cultures of bacteria, fungi and marine microalgae indicatethat intracellular metal levels are often higher than the biosorbedones (Kapoor et al., 1999; Matsunaga et al., 1999; Perez-Rama et al.,2001; Kader et al., 2007). Moreover, on one hand, the biosorptionmethods are often sensitive to ambient conditions, such as pH,ionic strength and the presence of organic or inorganic ligands,while, on the other hand, it lacks specificity in metal binding(Baudet et al., 1988; Kumar et al., 2008). Application of active andgrowing cells might be a better option due to their ability ofself-replenishment, continuous metabolic uptake of metals afterphysical adsorption, and the potential for optimization throughdevelopment of resistant species and cell surface modification

(Wilde and Benemann, 1993; Sandau et al., 1996; Malik, 2004).Apart from this, using growing cultures in bioremoval could avoidthe need for a separate biomass-production process, for example,cultivation, harvesting, drying, processing and storage prior to theuse.

Though it is a well-known fact that biological treatment couldsignificantly reduce the costs for chemicals and energy comparedwith conventional physical and chemical treatment, there are stillsome problems in applying this to the real wastewater treatmentprocesses due to the death of microbial cells in high concentrationsof Cr, low reducing and accumulation rate compared with the highgeneration rate of wastewaters, continuous supplies of expensivenutrients or chemicals and difficulty in the separation of cells aftertreatment (Park et al., 2005; Katarzyna, 2007; Vijayaraghavan andYeoung-Sang, 2008). These existing application gaps were thereason behind isolating a Cr-resistant fungus, identified as Paeci-lomyces lilacinus, from the tannery sludge and evaluating thepotential of growing fungal cells for Cr(VI) detoxification and Cr(III)accumulation at higher concentrations both in growthmedia and intannery industry effluent. The physicochemical characteristics,such as pH, Cr concentration, chemical oxygen demand (COD) andbiological oxygen demand (BOD) of discharged tannery industryeffluent, vary from industry to industry (Tobin and Roux, 1998;Nouri et al., 2005; Prigione et al., 2009; Ramteke et al., 2010),which may inhibit the growth of microorganisms and the overallCr-removal efficiency. Therefore, the ability of microorganisms tosurvive and remove Cr from physicochemically diverse tanneryindustry effluent is warranted. Considering this, the potential ofP. lilacinus in case of real industrial effluent was evaluated bystudying the ability of the fungus to remove Cr from two physico-chemically different tannery industry effluents (spent chromeeffluent and tannery effluent). The spent chrome effluent is theeffluent generated during the process of chrome tanning of hidesand skins, whereas tannery effluent is the final composite effluentdischarged from the leather industry to common effluent-treat-ment plants (CETPs) for final treatment. Further, the effect ofgrowth phase on Cr(VI) detoxification and Cr(III) accumulation wasstudied to generate some basic data, which will be important indesigning solid (sludge) retention time for a continuous-flowcompletely stirred (CFCS) bioreactor in future, which is a generalreactor type for wastewater treatment plants for efficient Cr(VI)detoxification and Cr(III) accumulation by fungi.

2. Materials and methods

2.1. Isolation and characterization of fungi

The Cr-resistant fungi were isolated from tannery sludge [con-taining 25 000 mg l�1 of Cr(III)] through an enrichment culturetechnique. The sludge sample was collected from a tannery wastedisposal site in Kanpur, India. For isolation of fungi, 1 g ofcontaminated sludge sample was inoculated in a 500-ml Erlen-meyer flask containing 100 ml of potato dextrose broth (PDB)media with initial Cr(III) concentration of 5000 mg l�1 [sourceCr2(SO4)3, CDH Lab Chemicals, India]. The flask was incubated ina shaking incubator (Innova 4080, New Brunswick Scientific, USA)at 30 �C,100 rpm in dark conditions. Startingwith 5000mg l�1 of Cr(III), the successive Cr(III) concentrations used for culture enrich-ment were 10 000 mg l�1, 15 000 mg l�1, 20 000 mg l�1 and25 000 mg l�1 in PDB media. The cultures were enriched bysuccessively transferring the 2% v/v of culture aliquot from lower tothe higher concentrations after every 360 h of incubation. Aftercompletion of incubation at final concentration of Cr(III)(25 000mg l�1), a Cr-resistant fungus was isolated from broth usinga serial dilution and plating technique on potato dextrose agar

Table 1physicochemical characteristic of spent chrome effluent and tannery effluent.

Parameter Spent chrome effluent Tannery effluent

EC (ms cm�1) 22.64 18.03pH 3.5 8Total Cr (mg l�1) 3731.4 � 91 7.91 � 0.64Cr(VI) (mg l�1) NDa 1.24 � 0.32Total P (mg l�1) 824.46 � 46.2 772.50 � 55.5Total Kjeldahl N (mg l�1) 1200 � 118.24 818 � 92.6COD (mg l�1) 1250 � 96.5 520 � 22.4BOD (mg l�1) 880 � 32.4 325 � 18.6

Mean � SD.a ND: not detectable.

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317 311

(PDA) plates amended with 25 000 mg l�1 of Cr(III). The isolate wasidentified as P. lilacinus at the Indian Type Culture Collection atIndian Agriculture Research Institute (IARI), NewDelhi, India, basedon their morphological structures, such as colour, diameter of themycelia and microscopic observation of spore formation. Theisolate was further verified through molecular characterizationbased on the internal transcribed spacer (ITS) sequence of nuclearribosomal DNAs.

Pure culture of the isolated fungal strain was grown in PDB at30 �C in a shaking incubator (100 rpm) for 72 h in dark condition.After incubation culture was centrifuged in a sterile centrifuge tubeat 5000g for 10 min at room temperature to get the biomass in theform of a pellet. The pellet was washed thrice with sterile distilledwater to make the biomass free from media components and wasfurther used for DNA extraction. Genomic DNAwas extracted usingDNeasy plant mini kit, according to the manufacturer’s instructions(QIAGEN, USA). The ITS region of rDNAwas amplified by polymerasechain reaction (PCR) using ITS1 [5-TCCGTAGGTGAACCTGCGG-3] andITS4 [5-TCCTCCGCTTATTGATATGC-3] primers (White et al., 1990).PCR amplification was performed in a total volume of 25 ml ina thermocycler (Applied Biosystems 2400, USA). Each PCR mixturecontained 1 ml of template DNA (46 mg ml�1), 2.5 ml of 10� Taq DNApolymerase buffer (Invitrogen, USA), 0.75 ml of 50 mM MgCl2 solu-tion, 0.5 ml of each deoxynucleoside triphosphate (10 mM), 0.125 mlof Taq DNA polymerase (5 Uml�1), 18.125 ml of deionised water, and1 ml of each primer (10 mM). Amplification was performed for 35cycles under the following conditions: after 5 min of initial dena-turation at 94 �C, each cycle consisting of denaturation at 94 �C for1 min, primer annealing at 55 �C for 1 min and primer extension at72 �C for 2.5 min, followed by a 10-min final extension step at 72 �Cin the last cycle. Negative control was carried out without templateto ensure there was no contamination. PCR product was purifiedusing a QIAquick spin column (QIAGEN, USA) according to themanufacturer’s instructions. Purified PCR product was subjected toelectrophoresis in 1% (w/v) agarose gels and visualised afterethidium bromide staining with a Tris-acetate- EDTA (TAE) buffersystem to verify that the DNA had not been degraded. The purifiedPCR product was sequenced by Lab India Instruments, Pvt. Ltd(Gurgaon, India) on an automated multicapillary DNA sequencer,ABI Prism 3130 � l Genetic Analyser (Applied Biosystems, USA),using the Big Dye Terminator v3.1 Ready Reaction Cycle SequencingKit (Applied Biosystems). The recovered ITS gene sequences werecompared with known sequences from the United States NationalCenter for Biotechnology Information (NCBI) database by using theBasic Local Alignment Search Tool (BLAST) algorithm to identify themost similar sequence.

The optimum pH (range 2.5e9.5) for the growth of the funguswas studied in 50-ml PDB in a 250-ml Erlenmeyer flask [pH of themedia was adjusted with predetermined amounts of filter-steri-lised (0.22-mm membrane filter, Millipore, USA) 1 M HCl or 1 MNaOH] inoculated with three circular mycelial agar discs of 7 mmdiameter (the colonies were cut from the edges of the activemycelial colony growing on a PDA plate). The inoculated flaskswereincubated in a shaking incubator (100 rpm) at 30 �C for 120 h indark condition and growth of the fungus was studied in terms ofdry biomass.

2.2. Media preparation

The Cr [Cr(VI) and Cr(III)] removal studies were carried out ingrowth medium, spent chrome effluent and tannery effluent. Thegrowth medium used was PDB (HiMedia, India) with differentconcentrations of Cr [Cr(VI) and Cr(III)]. Different concentrations ofCr-containing growth media were prepared by mixing appropriateamount of Cr [Cr(VI) and Cr(III)] stock solutions and growth media

(Camargo et al., 2003). Stock Cr(VI) solution (source K2Cr2O7,Qualigens, India) of 5000 mg l�1 and Cr(III) solution of35 000 mg l�1 were prepared by dissolving a measured amount ofK2Cr2O7 and Cr2(SO4)3, respectively, in distilled water and werefilter-sterilised with a 0.22-mm-membrane filter. The tanneryeffluent and spent chrome effluent (Table 1) used in this studywereabsolute and filter-sterilised. The tannery effluent was collectedfrom an effluent collection pond of a CETP and the spent chromeeffluent was collected from a functional leather factory, Jajmau,Kanpur, the leather hub city of India, located in the state of UttarPradesh. The tannery effluent was alkaline in nature and containingboth the forms of Cr [Cr(VI) and Cr(III)], whereas the spent chromeeffluent was acidic in nature and containing only Cr(III).

2.3. Inoculum preparation and growth conditions

Throughout this study, all experiments were set up in a 250-mlErlenmeyer flask containing 50 ml media/effluent. The inoculumsize used was 0.4 mg (dry weight) per ml. The cultures wereincubated in a shaking incubator (100 rpm) at 30 �C in darkcondition. The inoculumwas prepared in 200-ml PDB in a 1000-mlErlenmeyer flask inoculated with 12 circular mycelia agar discs of7 mm diameter. The flask was incubated in a shaking incubator(100 rpm) at 30 �C for 72 h in dark conditions. After incubation,fungal cells were homogenised using a magnetic stirrer (Spinit,Multilab, India) and aliquots of equal volume of homogenisedfungal cells were centrifuged (5000g for 10 min) in a sterilecentrifuge tube to get the equal biomass in the form of pellet. Thepellet was washed thrice with sterile distilled water to removemedia components and was used as an inoculum in suspendedform in the small aliquot of the same media/effluent inwhich it hasto inoculate. In time-course experiments, for every specified periodof incubation, separate sets of flasks were maintained with thesame inoculum size and growth conditions.

2.4. Cr(VI) reduction and accumulation study in growth media

The Cr(VI) reduction and accumulation ability of P. lilacinus wasexamined in growth media (pH 5.5) amended with three differentconcentrations of Cr(VI) (200 mg l�1, 500 mg l�1 and 1000 mg l�1).Mediawere inoculated with fungal biomass and were incubated for120 h. After incubation, cultures were withdrawn and centrifuged,and the supernatant and pellets were analysed for residual Cr(VI) inthe media and Cr accumulation by the biomass, respectively. Theaccumulation by the fungus was examined to detect any loss of Cr(VI) due to biotransformation of Cr(VI) to Cr(III) and/or accumula-tion by microbial biomass. The biomass-free controls (abioticcontrol) were used to detect any possible abiotic Cr(VI) reductionbrought about by media components.

1 Calculated as: Cr accumulation by fungi in mg g�1 � total fungal biomass in g.2 Calculated as: [net Cr (mg) accumulated by fungi/net Cr (mg) in 50 ml spent

chrome effluent] �100.

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317312

2.5. Kinetics of fungal growth, Cr(VI) reduction and accumulationin growth media

To gather some information regarding the effect of growthphase on Cr(VI) reduction and accumulation by the fungus, thetime-course study of fungal growth, Cr(VI) reduction and accu-mulationwas carried out in growth media (pH 5.5) at 200 mg l�1 ofCr(VI) concentration. Samples were read at a regular interval afterevery 24 h of incubation till the Cr(VI) concentration reaches belowthe detection level. After every incubation period, cultures werewithdrawn and centrifuged, and the supernatants were analysedfor residual Cr(VI) in the media and subsequently pellets wereanalysed for fungal growth and Cr accumulation by the biomass.

2.6. Cr(III) accumulation from growth media

The accumulation ability of P. lilacinus in case of Cr(III) wasexamined in growth media (pH 3.5) amended with three differentconcentrations (10 000mg l�1, 15 000 mg l�1 and 20 000 mg l�1) ofCr(III) after 600 h of incubation. After incubation, cultures werewithdrawn and centrifuged, and pellets were analysed for Cr(III)accumulation by fungal biomass.

2.7. Kinetics of fungal growth and Cr(III) accumulation in growthmedia

To gather some information regarding the effect of growthphase on Cr(III) accumulation by the fungus, the time-course studyof Cr(III) accumulation and fungal growthwas carried out in growthmedia (pH 3.5) at 10 000 mg l�1 of Cr(III) concentration. Thesamples for growth and accumulation were read at a regularinterval after every 120 h of incubation till the growth of the fungusreaches the stationary and decline phase. After every incubationperiod, cultures were withdrawn and centrifuged, and pellets wereanalysed for fungal growth and Cr(III) accumulation.

2.8. Selection of carbon source and effect of pH on Cr(VI) reduction

As the ultimate target for removal of Cr using the fungus wouldbe the effluents coming out of the industries, therefore, the selec-tion of carbon source to enhance the growth of fungus was carriedout directly in filter-sterilised tannery effluent amended with 2%(w/v) of four different carbon sources (dextrose, sucrose, jaggerypowder and cane sugar). The growth medium was used as a posi-tive control and tannery effluent without any carbon source wasused as a negative control of the experiment. The cultures wereincubated for 120 h and the growth of the fungus was compared.

Taking into the consideration the alkaline (pH 8) nature oftannery effluent, a comparative Cr(VI) reduction study was con-ducted in growth media at pH 8 and pH 5.5 to find out any signif-icant difference in the reduction efficiency of the fungus at50 mg l�1 of Cr(VI) concentration. The biomass-free control at pH 8was used to detect any possible abiotic Cr(VI) reduction broughtabout by increase in pH of growth media. The samples for residualCr(VI) in growth media were analysed after every 6 h of incubation.

2.9. Cr removal from tannery effluent

Considering the carbon source as an important electron donor ina redox reaction of Cr(VI) to Cr(III) form, the effect of selectedcarbon source (2% w/v of cane sugar) on Cr(VI) (1.24 mg l�1) andtotal Cr (7.91 mg l�1) removal by the fungus was studied in filter-sterilised tannery effluent. The treatment without carbon sourcewas used as a control. The samples for residual Cr(VI) and total Crwere analysed after every 6 h of incubation. The biomass-free

controls in tannery effluent (with and without carbon source) wereused to detect any possible abiotic Cr(VI) reduction brought aboutby the carbon source added in the effluent or by any other chemicalcomponents present in the effluent and to detect any change intotal Cr concentration with time.

Further, to confirm the significant role of cane sugar in Cr(VI)reduction by the fungus at higher C(VI) concentration, a compara-tive reduction study was conducted at 50mg l�1 in tannery effluentwith and without cane sugar (2% w/v). The concentration of Cr(VI)in tannery effluent was increased by adding measured volume offilter-sterilised stock solution of Cr(VI). The samples for residual Cr(VI) were analysed after every 6 h of incubation.

2.10. Cr removal from spent chrome effluent

To find out the ability of fungus to remove Cr(III) from spentchrome effluent, Cr(III) accumulation by P. lilacinus was studied infilter-sterilised spent chrome effluent [containing 3731.4 mg l�1 ofCr(III)] amended with cane sugar (2% w/v). Spent chrome effluentwithout cane sugar was used as a control of the experiment. Thebiomass was analysed for Cr(III) accumulation after 600 h ofincubation and further net Cr accumulation by fungal biomass1 and% Cr removal by fungal biomass2 from spent chrome effluent werecalculated.

2.11. Analytical methods

Samples were centrifuged at 5000g for 10 min at roomtemperature (30 �C), and supernatants and pellets were separated.The supernatants were analysed for residual Cr(VI) with a UVeVi-sible Spectrophotometer (UV-2450, SHIMADZU, USA) at 540 nmusing diphenylcarbazide (Greenberg et al., 1981). The Cr(VI)concentrations in tannery effluent and spent chrome effluent wereanalysed following the same standard protocol (Greenberg et al.,1981) at 540 nm using diphenylcarbazide. For studying Cr accu-mulation by fungal biomass, pellets were washed thrice with steriledistilled water dried at 80 �C till constant weight and acid-digestedwith 5 ml of HNO3 and 1 ml of HF in closed Teflon vessels at 170 �C(Kalra et al., 1989), using amicrowave accelerated reaction system 5(MARS5) (CEM Corporation, USA). Mycelial dry weight of the trip-licate sample was determined before digestion. For analysing totalCr in effluent (tannery effluent and spent chrome effluent),measured volumes of effluents were acid-digested followinga standard protocol (USEPA 3015A, 1998) using a MARS5. Thedigested samples of fungal biomass and effluent were analysed fortotal Cr metal. The analysis was carried out by TJA Solutions atomicabsorption spectrophotometer (AAS), Model SOLAAR M5 Series,graphite furnace equipped with FS 95 auto sampler (TJA Solutions,UK). The concentration of Cr(III) was calculated as total CreCr(VI).The electrical conductivity (EC), pH, COD, BOD, total nitrogen andtotal phosphorus of tannery effluent and spent chrome effluentwere analysed using standard methods (APHA, 1995). For evalua-tion of fungal growth, biomass was collected by centrifugation(5000g for 10 min), washed thrice with sterile distilled water anddried at 80 �C till constant weight and values recorded.

2.12. Statistical analysis

Triplicates were set up for each parameter tested. A completelyrandomised design was used and sampling was random. The

Table 2Cr(VI) reduction and accumulation by fungi from growth media after 120 h ofincubation.

Cr(VI) concentration ingrowth media (mg l�1)

200 500 1000

Reduction (%) 100 � 0.00 48 � 1.35 16 � 1.85Accumulation (mg g�1) 8.61 � 0.22 3.16 � 0.36 0.004 � 0.00

Mean � SD

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317 313

correlation and linear regression analysis were carried out usingMS-Excel. Error bars represent standard deviations (SD).

3. Results

3.1. Isolation and characterisation of fungi

Through a serial dilution and plating technique, the majority ofcolonies (seven out of nine colonies) obtained on PDA plates[amended with 25 000 mg l�1 of Cr(III)] were of identicalmorphology (Fig. 1) and were identified as P. lilacinus. The fungusP. lilacinus could grow at a pH range of 2.5e9.5, optimum pH being5.5 for growth. Analysis of the ITS-rDNA sequence (529 bp) revealedthat the isolated fungus was closely related to P. lilacinus (NCBISequence Viewer, accession number GQ229080.1) with 99%similarity.

3.2. Cr(VI) reduction and accumulation study in growth media

The Cr(VI) reduction and accumulation ability of fungus is pre-sented in Table 2. After 120 h of incubation period, the fungusshowed the ability to reduce Cr(VI) at all the three concentrationstested. In biomass-free controls, no measurable changes in Cr(VI)concentrations were detected after 120 h of incubation. In terms ofCr(VI) accumulation, the fungus showed the ability to accumulateCr in its biomass, at 200 mg l�1 and 500 mg l�1 of Cr(VI) but at1000 mg l�1, the fungus showed almost negligible amount ofaccumulation in the biomass. In general, it was observed that withincrease in Cr(VI) concentration in the media, there was decrease inreduction and accumulation ability of fungi.

3.3. Kinetics of fungal growth, Cr(VI) reduction and accumulationin growth media

From the time-course data analysis, it was observed that thespecific Cr(VI) reduction [calculated as Cr(VI) reduction/drybiomass] by the fungus was increased when cells were in activephase of the growth (log phase) (Fig. 2a) between 48 h and 96 h ofincubation and the specific Cr(VI) accumulation [calculated as Cr(VI) accumulation/dry biomass] by the fungus was increased when

Fig. 1. Fungal colonies on a PDA plate amended with 25000 mg l�1 of Cr(III) after serialdilution and plating technique. The dark greeneblack colour of media is due toCr2(SO4)3 added as a source of Cr(III).

cells were in stationary phase (Fig. 2b) of the growth between 96 hand 120 h of incubation. The fungus showed 100% detoxification of200 mg l�1 of Cr(VI) in 50 ml of growth media [i.e. net detoxifica-tion of 10 mg of Cr(VI) in 50 ml media]. Out of 10 mg of total Cr(VI)in growth media, the fungus was able to accumulate in the biomassonly 1.05 mg of Cr(VI) (calculated as Cr accumulation by fungi inmg g�1 � total fungal biomass in g) i.e., 10.54% of the total Cr(VI) in50 ml of growth media. These observations and calculationsconfirmed that P. lilacinus has the ability to reduce Cr(VI) and alsothe capability to accumulate Cr(VI) in the biomass as a mechanismof Cr(VI) resistance, but the major mechanism of Cr(VI) detoxifi-cation observed was reduction of Cr(VI) to Cr(III) form. Due to thetoxic nature of Cr(VI), removal through reductionwill always be thepriority; therefore, a non-expanded solid (sludge) retention times(SRT) (to maintain log phase of the growth) may be recommendedusing the fungal isolate in removal of Cr(VI) from industrialwastewater.

To find out the extent of interdependency and relationshipbetween the fungal growth and Cr(VI) reduction, and between thefungal growth and Cr(VI) accumulation, linear regression equations

Fig. 2. a. Kinetic study of fungal growth and % Cr(VI) reduction by P. lilacinus. b. Kineticstudy of fungal growth and Cr(VI) accumulation by P. lilacinus.

Fig. 3. Kinetic study of fungal growth and Cr(III) accumulation by P. lilacinus.

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317314

were estimated between the variables. The estimated regressionequation (y ¼ 895.9 x � 13.05) between fungal growth (x) and % Cr(VI) reduction (y) by the fungus showed very high degree of posi-tive correlation (0.97) and statistically very high degree of rela-tionship (r2 ¼ 0.941) between the variables. This indicates that Cr(VI) reduction by the fungus was significantly (P � 0.01) dependenton the growth of the fungus, whereas the estimated regressionequation (y¼ 63.72 x� 1.119) between fungal growth (x) and Cr(VI)accumulation (y) by the fungus showed non-significant correlation(0.84) and relationship (r2 ¼ 0.702) between the variables andindicates that Cr(VI) accumulation by the fungus was not depen-dent on the growth of the fungus. The results obtained throughregression analysis are in concurrence with results obtainedthrough time-course data analysis. The results support the role ofmetabolically active fungal cells in Cr(VI) reduction.

3.4. Cr(III) accumulation from growth media

The Cr(III) accumulation ability of the fungus from growthmedia is presented in Table 3. The fungus showed the ability toaccumulate Cr(III) at all the three concentrations tested after 600 hof incubation. In general, it was observed that with increase in Cr(III) concentration in growth media, there was decrease in accu-mulation ability of fungi.

3.5. Kinetics of fungal growth and Cr(III) accumulation in growthmedia

In a time-course study at 10 000 mg l�1 of Cr(III) in growthmedia (Fig. 3), the fungus was able to accumulate 69.78 mg Cr g�1

of dry biomass within 600 h of incubation. The fungus was able togrow till 480 h of incubation, and after that, the growth becomesstationary. The maximum specific Cr(III) accumulation [calculatedas: Cr(III) accumulation/dry biomass] by the fungus was observedwhen cells were in stationary phase of the growth between 480 hand 600 h of incubation. Therefore, expanded SRT (stationaryphase) may be recommended using fungal isolate in removal of Cr(III) from industrial wastewater.

3.6. Selection of carbon source and effect of pH on Cr(VI) reduction

In a comparative growth study, the highest growth of funguswas observed in case of PDB (0.243 g dry weight), which was thepositive control, and the lowest growth was observed in case oftannery effluent without any carbon source (0.063 g dry weight),which was the negative control of the experiment. Out of fourdifferent carbon sources tested, the most preferred carbon sourceby the fungus was cane sugar (0.216 g dry weight). The growth ofthe fungus in case of sucrose, jaggery powder and dextrose was ofthe order of 0.186 g > 0.136 g > 0.13 g dry weight, respectively.

In reduction study at two different pH conditions in growthmedia at 50 mg l�1 (Fig. 4), the fungus showed Cr(VI) reductionbelow the detection level within 36 h and 42 h of incubation inacidic (pH 5.5) and alkaline (pH 8) conditions, respectively. Theresults demonstrate high reduction efficiency of the fungus inacidic condition at its optimum pH (5.5) for the growth and effi-ciency decreases at alkaline pH condition. In biomass-free control

Table 3Cr(III) accumulation by fungi from growth media after 600 h of incubation.

Cr(III) concentration ingrowth media (mg l�1)

10 000 15 000 20 000

Accumulation (mg g�1) 69.78 � 1.88 57.9 � 2.81 37.8 � 3.95

Mean � SD.

at pH 8, no measurable changes in Cr(VI) concentrations weredetected throughout the experiment.

3.7. Cr removal from tannery effluent

It was observed that in presence of cane sugar, the Cr(VI) andtotal Cr removal by the fungus was increased in tannery effluent(Fig. 4). The fungus showed removal of 1.24 mg l�1 of Cr(VI) belowthe detection limit within 18 h of incubation in the presence of canesugar. In the absence of cane sugar, it takes 24 h to remove1.24 mg l�1 of Cr(VI) below the detection limit. In terms of total Crremoval, in the presence of cane sugar, the fungus showed 96.84%of removal within 30 h of incubation and after 36 h of incubation,the concentration reaches to below the detection level. In theabsence of cane sugar, the fungus showed 47.5% and 49.6% of totalCr removal within 30 h and 36 h of incubation, respectively, andafter that Cr removal activity became constant throughout theexperiment. In biomass-free controls, no measurable changes in Cr(VI) and total Cr concentrations were detected throughout theexperiment. The observations indicate an important role of carbon

Fig. 4. Comparative Cr(VI) reduction in acidic and alkaline pH conditions in growthmedia and in tannery effluent (TE) with and without cane sugar at 50 mg l�1 of Cr(VI)concentration (dotted lines). Comparative Cr(VI) (1.24 mg l�1) and total Cr (7.91 mg l�1)removal by P. lilacinus from tannery effluent with and without cane sugar (bold lines).

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317 315

source in Cr(VI) and total Cr removal by P. lilacinus from tanneryeffluent.

In case of tannery effluent at 50 mg l�1 of Cr(VI) and in presenceof cane sugar (Fig. 4), 95.5% and 100% reduction was observedwithin 42 h and 48 h of incubation, respectively, by the fungus. Inthe absence of cane sugar, the fungus showed only 4.1%(2.05mg l�1) reduction of Cr(VI) within 24 h of incubation and afterthat activity became constant throughout the experiment. Theresults reveal a significant role of carbon source in Cr(VI) reductionby the fungus. The observations suggest that limited availablenutrients present in effluent limit the ability of fungus to grow andreduce Cr(VI) in tannery effluent. The addition of cane sugar asa carbon source not only supports the growth, but also enhancesthe Cr(VI) reduction ability of the fungus. The results indicate thatin case of P. lilacinus, a carbon source is required to provide thereducing power needed to decrease Cr(VI) in tannery effluent.

3.8. Cr removal from spent chrome effluent

The chromium removal ability of the fungus from spent chromeeffluent is presented in Table 4. The fungus showed tremendousability to grow and accumulate Cr from spent chrome effluent withand without cane sugar. It showed significant amount of Cr(III)removal out of 186.57 mg of Cr(III) in 50 ml of spent chromeeffluent after 600 h of incubation. The Cr removal ability of thefungus increased 8.27 times that of the control treatment in pres-ence of cane sugar.

4. Discussion

The Cr-resistant fungus, P. lilacinus, isolated [at 25 000 mg l�1 ofCr(III) in growth media] in this research study showed the detoxi-fication of Cr(VI) and accumulation of Cr(III) in its biomass whilegrowing in Cr-containing media/effluent (tannery effluent andspent chrome effluent). The fungus showed broad pH tolerancerange (2.5e9.5), optimum pH being 5.5 for growth and preferredcane sugar (out of four different carbon sources) for the highestgrowth in tannery effluent. It showed the ability to biotransform(reduce) Cr(VI) to Cr(III) form and also the capability to accumulateCr(VI) in the biomass as a mechanism of Cr(VI) detoxification,which is different from the mechanism reported in case of other Cr(VI)-resistant fungi, such as Trichoderma (Morales and Cristiani,2008), Aspergillus (Gouda, 2000; Acevedo-Aguilar et al., 2006),Penicillium (Acevedo-Aguilar et al., 2006) and Phanerochaete (Pal,1997). In all the above-mentioned fungi, biotransformation of Cr(VI) to Cr(III) form has been reported as a sole mechanism of Cr(VI)detoxification. With 100% detoxification and with only 10.54% oftotal Cr accumulation in the biomass [at 200 mg l�1 of Cr(VI)],P. lilacinus showed biotransformation as a major mechanism of Cr(VI) detoxification.

In growth media at 50 mg l�1 and 200 mg l�1 of Cr(VI), P. lila-cinus showed 100% detoxification within 36 h and 120 h of incu-bation, respectively. The highest concentration of Cr(VI)(1000 mg l�1), which allows the growth and Cr(VI) reductionactivity by P. lilacinus, was much higher than the commonlyreported Cr(VI)-resistant bacteria (Humphries and Macaskie, 2002;

Table 4Cr removal by fungi from spent chrome effluent after 600 h of incubation.

Treatments Cr accumulation by fungi (mg g�1) Total fungal dry bioma

With canesugar

189.13 � 20 0.236 � 0.013

Control 62.84 � 3.64 0.086 � 0.005

Mean � SD.

Stasinakis et al., 2004; Viamajala et al., 2004; Cheung and Gu, 2005;Thacker et al., 2007) and fungi (Pal, 1997; Gouda, 2000; Acevedo-Aguilar et al., 2006; Morales and Cristiani, 2008). Nonetheless, itis important to consider that the microbial chromate-resistanceand chromateereduction parameters are correlated with mediumcomposition and cell density (Mergeay, 1995; Wang, 2000). In caseof trivalent Cr in growth media, P. lilacinus showed the accumula-tion of 69.78 mg Cr g�1 of dry biomass, 57.9 mg Cr g�1 of drybiomass and 37.8 mg Cr g�1 of dry biomass, respectively, at10 000 mg l�1, 15 000 mg l�1 and 20 000 mg l�1 of Cr(III) within600 h of incubation. In this study, the highest concentration of Cr(III), which allows the growth and Cr(III) accumulation by P. lilaci-nus, was much higher than the all reported Cr(III)-resistantmicroorganisms in existing literature (Pillichshammer et al., 1995;Zetic et al., 2001; Nouri et al., 2005; Onyancha et al., 2008). Ingeneral, the higher tolerance level of P. lilacinus in case of Cr(III)may be due to the fact that Cr(VI) is 100 times more toxic than Cr(III) (Beszedits, 1988).

The fungus P. lilacinus showed the ability to grow in vitro intotwo physicochemically different tannery industry effluents(tannery effluent and spent chrome effluent) without any amend-ment, and demonstrated a real adaptation to these polluted envi-ronments. Besides having ability to reduce and accumulate Cr athigher concentrations in growth media, the fungus showed theability to perform the desired activity in absolute industrial effluentat its natural pH conditions, which is the major limiting factor tilldate (Tobin and Roux, 1998; Malik, 2004; Nouri et al., 2005;Srivastava and Thakur, 2006). Various fungi (Acevedo-Aguilaret al., 2006; Srivastava and Thakur, 2006; Fukuda et al., 2008;Morales and Cristiani, 2008; Coreno-Alonso et al., 2009), bacteria(Camargo et al., 2003; Pal and Paul, 2004; Thacker et al., 2007;Quintelas et al., 2008) and adsorbents developed from biological(Gupta et al., 2001a,b; Gupta and Rastogi, 2008, 2009) and indus-trial waste materials (Srivastava et al., 1996; Gupta et al., 1999; Aliand Gupta, 2007; Gupta et al., 2010) have been reported forremoval of Cr [Cr(III) and Cr(VI)], but all reported studies are insynthetic media or in diluted industrial effluent with modifiednutrient and pH conditions. However, Prigione et al. (2009) havereported the removal of 40% of Cr within 24 h from conventionallytreated (chemical physical and biological treatments) tanneryeffluent [containing 0.45 mg l�1 of Cr(III)] using inactivated fungal(Cunninghamella elegans) biomass. In our study, P. lilacinus showedthe detoxification of 1.24 mg l�1 of Cr(VI) below the detection levelwithin 18 h of incubation and removal of total Cr (7.91 mg l�1)below the detection level within 36 h of incubation from tanneryeffluent in the presence of 2% w/v of cane sugar. Even though theexperimental conditions and approach of the above-mentionedstudy cannot be compared with the present study, but still in termsof Cr removal efficiency, P. lilacinus showed higher removal effi-ciency than the above-mentioned inactivated fungal biomass(C. elegans) from tannery effluent. In a separate study, Acevedo-Aguilar et al. (2006) have reported 96% reduction of 50 mg l�1 ofchromate by a Penicillium sp. after 72 h in an electroplating liquidwaste diluted with nutrient media. In our study, in case of Cr(VI)concentration increased upto 50 mg l�1 in tannery effluent, P. lila-cinus showed 100% detoxification within 48 h of incubation in

ss (g) Net Cr accumulation by fungi (mg) Cr removal from spentchrome effluent (%)

44.93 � 7.28 24 � 3.9

5.42 � 0.47 2.9 � 0.25

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317316

presence of 2% w/v of cane sugar. In absence of cane sugar, thefungus showed only 4.1% of Cr(VI) detoxification within 24 h ofincubation and after that activity becomes constant. This studyreveals the role of carbon source in enhanced fungal growth, Cr(VI)and total Cr removal from tannery effluent by P. lilacinus. The time-course study at 200 mg l�1 of Cr(VI) in growth media reveals themaximum specific Cr(VI) reduction by the fungus when cells werein maximum metabolically active phase (log phase) of the growth.This study clearly indicates that the Cr(VI) reduction in tanneryeffluent by the fungus results from cellular metabolism processesbased on the reducing power of the carbon source. Similar obser-vations have been reported in case of Aspergillus sp., Penicillium sp.and Trichoderma inhamatum (Acevedo-Aguilar et al., 2006; Moralesand Cristiani, 2008). There was a high degree of positive correlationand statistically high degree of relationship (r2 ¼ 0.941) obtainedbetween Cr(VI) reduction and growth of the fungi. The findingsupports the role of metabolically active cellular growth in Cr(VI)reduction by fungi. In a comparative Cr(VI) reduction study in acidic(pH 5.5) and alkaline (pH 8) conditions in growth media at50 mg l�1, P. lilacinus showed higher reduction efficiency in acidiccondition at its optimum pH for the growth. The difference inreduction efficiency may be explained by the fact that Cr(VI)reduction needs metabolically active cellular growth and highermetabolically active cellular growth will be obtained at optimumpH for the growth; hence, the highest Cr(VI) reduction will beobtained at optimum pH for the growth of the fungus. Similarobservations have been reported in case of Bacillus and Arthrobacterbacteria (Camargo et al., 2003).

In case of trivalent Cr, Pillichshammer et al. (1995) have reportedthe ability of Mucor hiemalis for uptake of 21.4 mg Cr g�1 of dryweight from 1 mM of Cr concentration. Zetic et al. (2001) havereported about Saccharomyces cerevisiae to accumulate 30e45 mgCr g�1 of cells. In another study, Nouri et al. (2005) have reportedabout Aspergillus sp. to remove 97.6% of Cr with an uptake ability of83.7 mg Cr g�1 from tannery effluent containing 240 mg l�1 of Cr(III). This study reports the ability of P. lilacinus to accumulate189.13 mg Cr g�1 of dry biomass from spent chrome effluentamended with 2% (w/v) of cane sugar within 600 h of incubation,which is the highest ever reported Cr accumulation by any micro-organism from real industrial effluent. The time-course study at10 000 mg l�1 of Cr(III) in growth media reveals the maximumspecific bioaccumulation of Cr(III) by the fungus when cells were instationary phase of the growth.

In this study, P. lilacinus showed higher Cr removal efficiency intannery effluent than the spent chrome effluent. This difference inremoval efficiency may be explained by the fact that tanneryeffluent supports the fast growth of the fungus and therefore thehigher Cr removal efficiency, whereas in spent chrome effluent, thegrowth rate of the fungus decreases and hence the Cr removalefficiency. It might be possible that higher COD and BOD in spentchrome effluent, which are indicators of higher organic mattercontent and higher oxygen demand in the effluent, suppresses thegrowth and the overall Cr removal efficiency of the funguscompared to the tannery effluent. However, the specific ability ofP. lilacinus to adapt and remove Cr from two physicochemicallydifferent tannery industry effluent (tannery effluent and spentchrome effluent) makes this fungus very suitable for practicalapplication in both small-scale and large-scale industrial effluent aswell as in CETPs.

5. Conclusion

The results of this study showed that P. lilacinus has uniqueproperties towards adapting steep pH variation as well as efficientaccumulation and reduction mechanisms in higher and lower

concentration of Cr salts drawn from spent chrome effluent andtannery effluent. However, further experiments need to be carriedout with the objective of optimising the conditions, which wouldallow a more efficient removal of Cr from industrial effluent.Certainly, a possible application of the method on an industrialscale will be preceded by a phase of the optimisation of parametersand more generally, the reduction of operating costs. In this study,the time-course data of Cr metal removal and fungal growthprovided basic information for SRT design, which is an importantparameter in designing CFCS bioreactors for the removal of metalfrom industrial effluents (Congeevaram et al., 2007). According tothe kinetic data, expanded SRT (stationary phase) is recommendedwhile using P. lilacinus for the removal of trivalent Cr throughaccumulation. When using the fungus for detoxification of Cr(VI),however, a non-expanded SRT is recommended for the designing ofthe CFCS bioreactor, so that a log phase of cellular growth could bekept in the treatment system. Further desorption studies to recoverCr metal from the fungal biomass can be carried out as a finalapproach, along with the management of heavy-metal-ladenfungal biomass as an environment-friendly green method fordisposal.

Acknowledgments

This study was supported by the Department of Biotechnology.The authors wish to thank Dr R K Pachauri, Director-General, TheEnergy and Resources Institute, India, for providing infrastructuralfacilities. The assistance of Mr U Gangi Reddy for the analysis ofsamples is duly acknowledged.

References

Acevedo-Aguilar, F.J., Espino-Saldana, A.E., Leon-Rodriguez, I.L., Rivera-Cano, MaE.,Avila-Rodriguez, M., Wrobel, K., Wrobel, K., Lappe, P., Ulloa, M., Gutierrez-Corona, J.F., 2006. Hexavalent chromium removal in vitro and from industrialwastes, using chromate-resistant strains of filamentous fungi indigenous tocontaminated wastes. Canadian Journal of Microbiology 52, 809e815.

Ali, I., Gupta, V.K., 2007. Advances in water treatment by adsorption technology.Nature Protocols 1, 2661e2667.

American Public Health Association, 1995. Standard methods for the examination ofwater and wastewater, 19th ed. APHA, Washington, D.C.

Baral, A., Engelken, R.D., 2002. Chromium-based regulations and greening in metalfinishing industries in the USA. Environmental Science and Policy 5, 121e133.

Barnhart, J., 1997. Chromium chemistry and implications for environmental fate andtoxicity. Journal of Soil Contamination 6, 561e568.

Baudet, C., Sprott, G.D., Patel, G.B., 1988. Adsorption and nickel uptake in Meth-anothrix concilii. Archives of Microbiology 150, 338e342.

Beszedits, S., 1988. Chromium removal from industrial wastewaters. In: Nriagu, J.O.,Nieboer, E. (Eds.), Chromium in the natural and human environments. JohnWiley, New York, pp. 232e263.

Camargo, F.A.O., Okeke, B.C., Bento, F.M., Frankenberger, W.T., 2003. In vitroreduction of hexavalent chromium by a cell-free extracts of Bacillus sp. ES 29stimulated by Cu2þ. Applied Microbiology and Biotechnology 62, 569e573.

Chen, J.M., Hao, O.J., 1998. Microbial chromium (VI) reduction. Critical Reviews inEnvironmental Science and Technology 28, 219e225.

Cheung, K.H., Gu, J.D., 2005. Chromate reduction by Bacillus megaterium TKW3isolated from marine sediments. World Journal of Microbiology and Biotech-nology 21, 213e219.

Congeevaram, S., Dhanarani, S., Park, J., Dexilin, M., Thamaraiselvi, K., 2007. Bio-sorption of chromium and nickel by heavy metal resistant fungal and bacterialisolates. Journal of Hazardous Materials 146, 270e277.

Coreno-Alonso, A., Acevedo-Aguilar, F.J., Reyna-Lopez, G.E., Tomasini, A.,Fernandez, F.J., Wrobel, K., Wrobel, K., Gutiérrez-Corona, J.F., 2009. Cr(VI)reduction by an Aspergillus tubingensis strain: role of carboxylic acids andimplications for natural attenuation and biotreatment of Cr(VI) contamination.Chemosphere 76, 43e47.

Dursun, A.Y., Uslu, G., Tepe, O., Cuci, Y., Ekiz, H.I.A., 2003. A comparative investi-gation on the bioaccumulation of heavy metal ions by growing Rhizopusarrhizus and Aspergillus niger. Biochemical Engineering Journal 15, 87e92.

Fukuda, T., Ishino, Y., Ogawa, A., Tsutsumi, K., Morita, H., 2008. Cr(VI) reduction fromcontaminated soils by Aspergillus sp. N2 and Pencillium sp. N3 isolated fromchromium deposits. Journal of General and Applied Microbiology 54, 295e303.

Gouda, M.K., 2000. Studies on chromate reduction by three Aspergillus species.Fresenius Environmental Bulletin 9, 799e808.

S. Sharma, A. Adholeya / International Biodeterioration & Biodegradation 65 (2011) 309e317 317

Greenberg, A.D., Connors, J.J., Jenkins, D., Franson, M.A., 1981. Standard methods forthe examination of water and wastewater, 15th ed. American Public HealthAssociation, Washington, D.C.

Gupta, V.K., Carrott, P.J.M., Carrott, M.M.L.R., Suhas, 2009. Low cost adsorbents:growing approach to wastewater treatment e a review. Critical Reviews inEnvironmental Science and Technology 39, 783e842.

Gupta, V.K., Gupta, M., Sharma, S., 2001a. Process development for the removal oflead and chromium from aqueous solutions using red mud-an aluminumindustry waste. Water Research 35, 1125e1134.

Gupta, V.K., Mohan, D., Sharma, S., Park, K.T., 1999. Removal of chromium (VI) fromelectroplating industry wastewater using Bagasse fly ash e a sugar industrywaste material. The Environmentalist 19, 129e136.

Gupta, V.K., Rastogi, A., 2008. Sorption and desorption studies of chromium (VI)from nonviable cyanobacterium Nostoc muscorum biomass. Journal ofHazardous Materials 154, 347e354.

Gupta, V.K., Rastogi, A., 2009. Biosorption of hexavalent chromium by raw and acid-treated green alga Oedogonium hatei from aqueous solutions. Journal ofHazardous Materials 163, 396e402.

Gupta, V.K., Rastogi, A., Nayak, A., 2010. Adsorption studies on the removal ofhexavalent chromium from aqueous solution using a low cost fertilizer industrywaste material. Journal of Colloid and Interface Science 342, 135e141.

Gupta, V.K., Srivastava,A.K., Jain,N., 2001b. Biosorption of chromium(VI) fromaqueoussolutions by green algae Spirogyra species. Water Research 35, 4079e4085.

Gupta, V.K., Srivastava, S.K., Mohan, D., Sharma, S., 1997. Design parameters forfixed bed reactors of activated carbon developed from fertilizer wastematerial for the removal of some heavy metal ions. Waste Management 17,517e522.

Humphries, A.C., Macaskie, L.E., 2002. Reduction of Cr (VI) by Desulfovibrio vulgarisand Microbacterium sp. Biotechnology Letters 24, 1261e1267.

Kader, J., Sannasi, P., Othman, O., Ismail, l.B.S., Salmijah, 1.S., 2007. Removal of Cr(VI) from aqueous solutions by growing and non-growing populations ofenvironmental bacterial consortia. Global Journal of Environmental Research1, 12e17.

Kalra, Y.P., Maynard, D.G., Radford, F.G., 1989. Microwave digestion of tree foliage formulti element analysis. Canadian Journal of Research 19, 981e985.

Kapoor, A., Viraraghavan, T., Roy, C.D., 1999. Removal of heavy metals using thefungus Aspergillus niger. Bioresource Technology 70, 95e104.

Katarzyna, C., 2007. Bioaccumulation of Cr(III) ions by blue-green alga Spirulina sp.part I. A comparison with biosorption. American Journal of Agriculture andBiological Science 2, 218e223.

Kratochvil, D., Pimentel, P., Volesky, B., 1998. Removal of trivalent and hexavalentchromium by seaweed biosorbent. Environmental Science and Technology 32,2693e2698.

Kumar, P., Prasad, B., Mishra, I.M., Chand, S., 2008. Decolorization and COD reduc-tion of dyeing wastewater from a cotton textile mill using thermolysis andcoagulation. Journal of Hazardous Material 153, 635e645.

Lee, D.C., Park, C.J., Yang, J.E., Jeong, Y.H., Rhee, H.I., 2000. Screening of hexavalentchromium biosorbent from marine algae. Applied Microbiology and Biotech-nology 54, 445e448.

Lovley, D.R., 1993. Dissimilatory metal reduction. Annual Review of Microbiology 47,263e290.

McLean, J., Beveridge, T.J., 2001. Chromate reduction by a Pseudomonad isolatedfrom a site contaminated with chromate copper arsenate. Applied and Envi-ronmental Microbiology 67, 1076e1084.

Malik, A., 2004. Metal bioremediation through growing cells. Environment Inter-national 30, 261e278.

Matsunaga, T., Takeyama, H., Nakao, T., Yamazawa, A., 1999. Screening of microalgaefor bioremediation of cadmium polluted seawater. Journal of Biotechnology 70,33e38.

Mergeay, M., 1995. Heavy metal resistances in microbial ecosystems. In:Akkermans, A.D.L., van Elsas, J.D., De Bruijn, F.J. (Eds.), Molecular microbialecology manual. Kluwer Academic Press, Dordrecht, Netherlands, pp. 1e17.

Morales-Barrera, L., Cristiani-Urbina, E., 2008. Hexavalent chromium removal bya Trichoderma inhamatum fungal strain isolated from tannery effluent. WaterAir and Soil Pollution 187, 327e336.

Nouri, S.M., Nasseri, S., Mazaheri, A.M., Yaghmaian, K., 2005. Chromium bioremovalfrom tannery industries effluent by Aspergillus oryzae. Iran Journal of Envi-ronmental Health Science and Engineering 2, 273e279.

Onyancha, D., Ward, M., Catherine, N.J., Peter, O., Joseph, C., 2008. Studies ofchromium removal from tannery waste waters by algae biosorbents, Spirogyra

condensate and Rhizoclonium hieroglyphicum. Journal of Hazardous Materials158, 605e614.

Pal, N., 1997. Reduction of hexavalent chromium to trivalent chromium by Pha-nerochaete chrysosporium. In: Alleman, B.C., Leeson, A. (Eds.), In situ and on-sitebioremediation, Vol. 1. Batelle Press, Ohio, pp. 511e517.

Pal, A., Paul, A.K., 2004. Aerobic chromate reduction by chromium-resistant bacteriaisolated from serpentine soil. Microbiological Research 159, 347e354.

Park, D., Yun, Y.S., Park, J.M., 2005. Use of dead fungal biomass for the detoxificationof hexavalent chromium: screening and kinetics. Process Biochemistry 40,2559e2565.

Perez-Rama, M., Herrero, L.C., Abalde, A.J., Torres, E., 2001. Class III metallothioneinsin response to cadmium toxicity in the marine microalga Tetraselmis suecica(Kylin). Environmental Toxicology and Chemistry 20, 2061e2066.

Pillichshammer, M., Pumple, T., Eller, K., Klima, J., Schinner, F., 1995. Biosorption ofchromium to fungi. Biometals 8, 117e121.

Prigione, V., Zerlottin, M., Refosco, D., Tigini, V., Anastasi, A., Varese, G.C., 2009.Chromium removal from a real tanning effluent by autochthonous andallochthonous fungi. Bioresource Technology 100, 2770e2776.

Quintelas, C., Fernandes, B., Castro, J., Figueiredo, H., Tavares, T., 2008. Biosorption ofCr(VI) by a Bacillus coagulans biofilm supported on granular activated carbon(GAC). Chemical Engineering Journal 136, 195e203.

Ramteke, P.W., Awasthi, S., Srinath, T., Joseph, B., 2010. Efficiency assessment ofcommon effluent treatment plant (CETP) treating tannery effluents. Environ-mental Monitoring and Assessment 169, 125e131.

Sandau, E., Sandau, P., Pulz, O., 1996. Heavy metal sorption by microalgae. ActaBiotechnology 16, 227e235.

Srivastava, S., Thakur, I.S., 2006. Isolation and process parameter optimization ofAspergillus sp. for removal of chromium from tannery effluent. BioresourceTechnology 97, 1167e1173.

Srivastava, S.K., Gupta, V.K., Mohan, D., 1996. Parameters for the removal of lead andchromium from wastewater using activated carbon developed from fertilizerwaste material. Environmental Modeling and Assessment 1, 281e290.

Srivastava, S.K., Gupta, V.K., Mohan, D., 1997. Removal of lead and chromium byactivated slag - a blast-furnace waste. Journal of Environmental Engineering(ASCE) 123, 461e468.

Stasinakis, A.S., Thomaidis, N.S., Mamais, D., Lekkas, T.D., 2004. Investigation of Cr(VI) reduction in continuous-flow activated sludge systems. Chemosphere 57,1069e1077.

Thacker, U., Parikh, R., Shouche, Y., Datta, M., 2007. Reduction of chromate by cell-free extract of Brucella sp. isolated from Cr(VI) contaminated sites. BioresourceTechnology 98, 1541e1547.

Tobin, J.M., Roux, J.C., 1998. Mucor biosorbent for chromium removal from tanningeffluent. Water Research 32, 1407e1416.

United States Environmental Protection Agency, 1998. Method 3015A, revision 1:Microwave assisted acid digestion of aqueous samples and extracts. SW-846.EPA, Washington, D.C.

Valls, M., Atrian, S., de Lorenzo, V., Fernandez, L.A., 2000. Engineering a mousemetallothionein on the cell surface of Ralstonia eutropha CH34 for immobili-zation of heavy metals in soil. Nature Biotechnology 18, 661e665.

Viamajala, S., Peyton, B.M., Sani, R.K., Apel, W.A., Petersen, J.N., 2004. Toxic effects ofchromium (VI) on anaerobic and aerobic growth of Shewanella oneidensis MR-1.Biotechnology Progress 20, 87e95.

Vijayaraghavan, K., Yeoung-Sang, Y., 2008. Bacterial biosorbents and biosorption.Biotechnology Advances 26, 266e291.

Volesky, B., 2001. Detoxification of metal-bearing effluents: biosorption for the nextcentury. Hydrometallurgy 59, 203e216.

Volesky, B., Holzen, Z.R., 1995. Biosorption of heavy metals. Biotechnology Progress11, 235e250.

Wang, Y., 2000. Microbial reduction of chromate. In: Lovley, D.R. (Ed.), Environ-mental microbe e metal interactions. American Society for Microbiology Press,Washington, pp. 225e235.

White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. Amplification and direct sequencingof fungal ribosomal RNA genes for phylogenetics. In: lnnis, M.A.D.H.,Gelfand, J.J., Sninsky, J., White, T.J. (Eds.), PCR protocols: A guide to methods andapplications. Academic Press, San Diego, pp. 315e322.

Wilde, E.W., Benemann, J.R., 1993. Bioremoval of heavy metals by the use ofmicroalgae. Biotechnology Advances 11, 781e812.

Zetic, V.G., Thomas, V.S., Grba, S., Lutilsky, L., Kozlek, D., 2001. Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeastbiomass. Journal of Biosciences 26, 217e223.