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Simultaneous bioaccumulation of multiple metals fromelectroplating effluent using Aspergillus lentulus

Abhishek Mishra, Anushree Malik*

Applied Microbiology Lab, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas,

New Delhi 110 016, India

a r t i c l e i n f o

Article history:

Received 27 March 2012

Received in revised form

13 June 2012

Accepted 21 June 2012

Available online 4 July 2012

Keywords:

Bioaccumulation

Multiple metal

Electroplating effluent

Aspergillus lentulus

* Corresponding author. Tel.: þ91 11 2659115E-mail addresses: anushree_malik@yaho

0043-1354/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.watres.2012.06.035

a b s t r a c t

Toxic impacts of heavy metals in the environment have lead to intensive research on

various methods of heavy metal remediation. However, in spite of abundant work on

heavy metals removal from simple synthetic solutions, a very few studies demonstrate the

potential of microbial strains for the treatment of industrial effluents containing mixtures

of metals. In the present study, the efficiency of an environmental isolate (Aspergillus len-

tulus FJ172995), for simultaneous removal of chromium, copper and lead from a small-scale

electroplating industry effluent was investigated. Initial studies with synthetic solutions

infer that A. lentulus has a remarkable tolerance against Cr, Cu, Pb and Ni. During its

growth, a significant bioaccumulation of individual metal was recorded. After 5 d of

growth, the removal of metals from synthetic solutions followed the trend Pb2þ

(100%) > Cr3þ (79%) > Cu2þ (78%), > Ni2þ (42%). When this strain was applied to the

treatment of multiple metal containing electroplating effluent (after pH adjustment), the

metal concentrations decreased by 71%, 56% and 100% for Cr, Cu and Pb, respectively

within 11 d. Based on our results, we propose that the simultaneous removal of hazardous

metals from industrial effluents can be accomplished using A. lentulus.

ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction the individual metals (Franklin et al., 2002). Microorganisms

The water pollution owing to hazardous heavy metals is one

of the most important environmental problems throughout

the world. Chromium, copper, lead and nickel, being

commonly employed in several industries are wide spread

contaminants of the environment (Donmez and Aksu, 1999).

These metals are frequently encountered together in indus-

trial wastewaters from electroplating, electronics wire

manufacturing, oil refineries and copper sulphate

manufacturing industries (Stoll and Duncan, 1996; Zakaria

et al., 2006). The remediation of such complex industrial

streams containing cocktail of metal mixtures is vital because

the mixture of metals are reported to be even more toxic than

8; fax: þ91 11 26591121.o.com, anushree@rdat.iitdier Ltd. All rights reserved

could be employed as potent bioremediators to deal with such

problem (Malik, 2004). In particular, the filamentous fungi

offer an efficient system due to large surface area and easy

solideliquid separation (Iskandar et al., 2011).

Ample literature is available concerning metal bio-

accumulation under single metal exposure (Malik, 2004).

However, such results cannot be extrapolated to the mixture

of metals because the combined effects of multiple metals on

the same microbial strain/consortium are usually different

from the additive effects of the individual metals involved

(Gikas, 2008). Some studies have dealt with metal removal

from binary mixtures such as Ni and Co (Gikas, 2008) and Cu

and Ni (Acikel and Alp, 2009). More recently, metal removal

.ac.in (A. Malik).

.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 84992

from growth media amended with multiple metal mixtures

containing Cd, Cu, Zn and Pb (Pan et al., 2009) or Cd, Cr, Pb and

Ni (Joshi et al., 2011) has been reported. These studies offer

interesting insights into the effect of certain metal combina-

tions. Nevertheless, these studieswere conducted in synthetic

mixtures.

Some strains have been tested for removal of a particular

metal from actual industrial effluents during their growth.

Removal of Cr from industrial effluent has been researched

using bacterial (Ganguli and Tripathi, 2002) and fungal agents

(Srivastava and Thakur, 2006; Sharma and Adholeya, 2011).

Removal of other metals such as Cu, Zn, Ni, Cd and Pb from

industrial effluents has also been attempted using bacterial,

yeast and algal biomass (Pumpel et al., 2003: Roy et al., 2008;

Mishra and Malik, in press). Nevertheless, majority of the

studies have targeted or achieved the remediation of a single

metal even though complex effluents were dealt with. This

happens because themicrobial strainsmay not be able to cope

with various other metals present in the effluent (Zakaria

et al., 2006). Recently, Shivakumar et al. (2011) have used

two indigenous fungal strains (Aspergillus niger and Aspergillus

flavus) to remove heavy metals from the paper mill effluent.

Here both the strains caused an impressive removal of Pb but

Zn, Cu, Cr and Ni were not very effectively removed. In view of

the above scenario, the existing gap on the multiple metal

removal from actual effluents/waste streams can be readily

identified. Such investigations are essential to develop a field

worthy bioremediation technology. In the present study, we

propose Aspergillus lentulus as an efficient remediator for the

simultaneous removal of Cr, Cu, Pb and Ni.

2. Materials and methods

2.1. Microorganism and growth conditions

A fungal strain previously isolated from Vardhman textile

effluent, Baddi, Himachal Pradesh, India and identified as A.

lentulus FJ172995 was used (Sharma et al., 2009). Fungal strain

was cultured at 180 rpm and 30 �C using sterilized composite

broth media (pH 6.5 � 0.2) containing (g L�1) K2HPO4 (0.5),

MgSO4 (0.1), NH4NO3 (0.5), NaCl (1.0), yeast extract (5.0) and

glucose (10.0) (pH 6.5). Glucose was autoclaved separately and

later added to the media to avoid precipitation.

2.2. Determination of minimum inhibitoryconcentrations (MIC)

Tolerance to heavy metals was determined in terms of the

minimum inhibitory concentration (MIC) of metals for A. len-

tulus. Metal solutions were prepared by dissolving their

respective salts, chromium sulphate [Cr2(SO4)3$6H2O], copper

sulphate [CuSO4$5H2O], lead nitrate [Pb(NO3)2] and nickel

sulphate [NiSO4$6H2O] in double distilled water (DDW). Metal

solutions were sterilized by passing through Millipore syringe

filter of 0.22 mm pore size. In order to estimate the MIC,

composite broth medium was amended with respective

metals at different concentration, viz. Cr(III):

100e12,000 mg L�1; Cu(II): 75e1000 mg L�1; Pb(II):

100e5500 mg L�1 and Ni(II): 70e500 mg L�1. These flasks were

inoculated with spore suspension (106 spores ml�1) of A. len-

tulus and incubated at 30 �C and 180 rpm on an orbital shaker.

Growth was monitored after every 24 h for the next 5 d. MIC

was defined as the minimum inhibitory concentration of the

heavy metal that inhibited the growth of A. lentulus.

2.3. Effect of initial metal ion concentration and pH onbioaccumulation

In order to study the effect of metal ion concentration, steril-

ized composite broth medium was mixed with respective

heavy metal solution to get the desired concentrations (based

on MIC determination) of metal ions viz. Cr(III):

1000e5000 mg L�1; Cu(II): 75e800 mg L�1; Pb(II):

100e4000 mg L�1 and Ni(II): 70e210 mg L�1. The flasks were

inoculated and incubated as described in Section 2.2. Residual

metal concentration, pH and biomass were measured at the

end of the experiments. After 5 d of incubation, the contents of

the flasks were filtered and fungal pellets were washed with

DDW. The biomasswasmeasured after drying at 80 �C for 24 h.

Residualmetal ion concentration in filtratewas determined by

digestion with nitric acid and sulphuric acid (3:1 ratio). The

digested solution was filtered through Whatman filter no. 01.

The heavy metal concentration in filtrate was quantified by

Atomic Absorption Spectrophotometer (PerkinElmer AAna-

lyst200) using standard protocols (APHA, 1989). Oxidizing air/

acetylene flame was used and all measurements were carried

out in triplicate.

In order to investigate themorphological changes resulting

from the metal stress, SEM of the biomass grown in the

presence and absence of metal ions was done using SEM

(ZEISS EVO 50) under the following analytical condition:

EHT ¼ 20.00 kV, WD ¼ 9.5 mm, Signal A ¼ SE1. Samples for

SEM were prepared using standard protocol (Tyagi and Malik,

2010).

To study the influence of pH on the uptake of Cu(II) and

Ni(II), the initial pH of the brothmedium containing respective

metals [Cu(II): 80 mg L�1; Ni(II): 70 mg L�1] was adjusted to the

desired value (2e8) using sterile H2SO4 and NaOH solutions. A

parallel series of flasks (pH 2e8) was also run in order to

evaluate the effect of pH in absence of metal stress. The flasks

were inoculated, incubated and monitored as describe above.

2.4. Metal removal from electroplating effluent

The ability of A. lentulus to remove metals from electroplating

effluent was tested and multiple metal (Cr, Cu & Pb) removal

by its growing biomass was studied using filter-sterilised

effluent. Electroplating effluent was collected from an

industry located in Okhla Industrial Area, New Delhi, India.

The effluent was characterized by standard methods of waste

water analysis for the total dissolved solid (TDS), total sus-

pended solid (TSS), chemical oxygen demand (COD) and

selected metals (APHA, 1989). Batch studies on the metal

bioaccumulation were carried out after dilution of the effluent

with composite media in the ratio of 1:1 and 4:1 (efflu-

ent:media) as well as with nutrient supplemented effluents

(without dilution). For supplementation, two main compo-

nents of composite media (glucose and yeast extract) were

used in different combinations. The effluent samples were

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 8 4993

inoculated with spore suspension (5% v/v) and incubated at

180 rpm and 30 �C for 11 d. The experimental series was

repeated after pH adjustment (z7) of the effluent.

2.5. Statistical analysis

All the studies were conducted in triplicates and the results

are presented as means of the replicates along with standard

deviation (represented as error bars). Data were analysed by

using SPSS (version 17) statistical software. The effects of

initial metal ion and initial pH on A. lentulus were examined

using one way ANOVA. Duncan multiple range test was used

to compare the significance of the differences among treat-

ments at P values of <0.05.

3. Results and discussion

3.1. Metal tolerance

The MIC of different metals for A. lentulus was recorded as

300 mg L�1 for Ni, 850 mg L�1 for Cu, 5000 mg L�1 for Pb and

more than 120,000 mg L�1 for Cr(III). Besides, our previous

study (Sharma et al., 2009) reported MIC of 550 mg L�1 for

Cr(VI). A relativelywide range ofMICs of Cr, Cu, Pb andNi have

been reported for various microbial strains. Certain fungal

strains isolated by Iskandar et al. (2011) had very high metal

tolerance. However,A. niger (Dursun et al., 2003a;Parameswari

et al., 2010), A. flavus (Liu et al., 2009) and Aspergillus sp.

(Ezzouhri et al., 2009) had comparatively lowerMICs for Cr, Cu,

Pb and Ni. Overall, higher tolerance for several metals as

recorded in the present case was not commonly observed. It is

possible that binding or chelating of metal ions by the

complex media components, such as yeast extract, may have

reduced the metal toxicity. Hence, A. lentulus seems to be

a better candidate for remedialmeasures as it couldwithstand

higher Cr, Cu, Pb and Ni levels. It is beneficial to utilize such

resistant strain, since the toxicity of multiple metals can

inhibit the survival as well as themetal sequestration capacity

of strain (Donmez and Aksu, 2001; Dursun et al., 2003b).

3.2. Toxicity response against various metals

The variations in MIC values suggest that the resistance level

against individual metals is different. This could be related to

varied toxicity responses of A. lentulus to different metals.

Metal specific changes in the growth pattern and morphology

were clearly observed. Although all the metals caused the

growth inhibition at respective concentrations, the response

of the organism in terms of pellet morphology was quite

different.While in presence of Cu(II), the diameter of the pellet

increased from 0.5 mm (control) to 4e6 mm (800 mg L�1 Cu(II)

concentration). Such an increment in the size was not

observed in the presence of Ni and Cr(III). Earlier, our results

have demonstrated that the enhancement in the pellet size

due to mycelia aggregation is a typical response by the fungus

to avoid metal toxicity against Cr(VI). The pellets those fail to

aggregate are unable to bear the metal toxicity resulting in

death (Sharma et al., 2009). Thus, it seems that changed

morphology supports higher MIC of toxic metals. On the other

hand, in the presence of lead ions, mycelium became

ruptured. It appeared like a ball with solid core and a few

filaments protruding out from the outer shell. This was

different from smooth surface of the pellets, as observed in

the presence of other metals.

The SEMmicrographs depicted a clear distinction between

the control (Fig. 1A) and metal stressed mycelia (Fig. 1BeE).

The long, ribbon like fungal hyphae in the control pellets were

uniformly shaped and loosely packed, resulting in small

pellets but with fluffy and porous structure (Fig. 1A). However,

in the presence of metals, mycelia appeared short, dense and

broken. In response to Ni(II), decreased mycelial length was

verymuch evident (Fig. 1E). Twisting and looping of individual

hyphae and the formation of tightly packed intertwined

hyphal strands was observed in response to Cr(III) and Cu(II)

stress (Fig. 1BeC). Further, the mycelia were more deformed

and had a higher tendency to aggregate in the presence of

Pb(II) ions (Fig. 1D). It seems to be a toxicity response of the

organism that might reduce the surface area exposed to toxic

metal. Lilly et al. (1992) also observed the twisting and looping

of individual hyphae and the formation of intertwined hyphal

strands in response to metal stress.

3.3. Effect of initial metal ion concentration on growthand metal ion bioaccumulation

The growth of A. lentulus was significantly influenced by the

initial metal ion concentration (Table 1). In case of the control,

the stationary growth phase was achieved after 3 d due to

exhaustion of glucose (data not shown). However, the growth

was delayed in the presence of metal ions resulting in an

extended lag phase. During this period, the fungus adapted to

metal ions. Lower biomass production was observed in the

presence of metals as compared to the control. Percent

reduction in biomass in the presence of Ni(II) ions was

increased from 19 to 76% (at 70e140 mg L�1), while in the

presence of Cu(II) ions it was increased from 16 to 77% (at

80e800 mg L�1) after 5 d of the growth. Cr(III) ions showed

least inhibition (35% at 5000 mg L�1) whereas in the presence

of 1000mg L�1 Pb(II) ions, 40% growth inhibitionwas observed.

Considering the exposed metal concentration, Ni(II) caused

a higher growth inhibition than Cu(II) followed by Pb(II) and

Cr(III). Similar results were also observed by Moore et al.

(2008), who had reported that Cu(II) and Ni(II) causes a signif-

icant inhibitory effect on the biomass production. Donmez

and Aksu (2001) have also reported the reduction in the

biomass growth rate both for adapted (from 3.84 d�1 to

1.42 d�1) and non-adapted (from 3.84 d�1 to 1.1 d�1) Candida sp.

in the presence of 578.7 mg L�1 Cu(II) ions. A more severe

reduction in the growth rate was observed for both the

adapted (from 2.4 to 0.3 d�1) and non-adapted (3.36e1.1 d�1)

Candida sp. in the presence of 63.6e375.8 mg L�1 Ni(II) ion.

Table 1 also shows the metal uptake capacities of A. len-

tulus as a function of the initial concentration of themetal ions

in themedium. Removal of Cu(II) andNi(II) ionswas enhanced

with the increasing initial metal ion concentration. The

maximum specific metal uptake was determined as

124.50 mg g�1 (at 800 mg L�1) for Cu(II) and 11.05 mg g�1 (at

140 mg L�1) for Ni(II). However, maximum specific Cr(III)

(331.48 mg g�1) and Pb(II) (1120.65 mg g�1) uptake was

Fig. 1 e Scanning Electronmicrographs of pellet of Aspergillus lentulus. (A) In absence of metal; (B) at 2000 mg LL1 of Cr(III); (C)

at 100 mg LL1 of Cu(II); (D) at 2000 mg LL1 of Pb(II) and (E) at 100 mg LL1 of Ni(II).

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 84994

observed at the initial metal concentration of 4000 mg L�1.

Maximum Cu, Ni and Pb uptake capacities of A. lentulus were

significantly higher than those reported for Cu in A. niger and

Rhizopus arrhizus (Dursun et al., 2003a); Cu and Pb in Fusarium

sp. and Penicillium (Pan et al., 2009) and Cu and Ni for

unidentified fungal strain (Moore et al., 2008). Either these

strains had lower uptake capacities or most of these could not

survive up to the concentration tested in the present study.

3.4. Effect of initial pH on growth and metal ionbioaccumulation

pH plays a very critical role in the microbial metal uptake by

influencing the metal speciation and solution chemistry as

well as surface properties of the microbial cell. Since prelim-

inary experiments revealed that pH did not significantly alter

the removal of Cr(III) and Pb(II), growth and metal uptake was

studied over a wide range of pH (pH 2e8) for Cu(II) and Ni(II)

ions only. The results (Table 2) demonstrate that the biomass

production is not so significantly affected by the extremes of

pH in the absence of metals. However, in the presence of

metals, lower pH (2 and 3) decreased the growth as well as

Cu(II) and Ni(II) bioaccumulation properties of A. lentulus.

Biomass production increased with pH up to 5.0 and 6.0 in the

presence of Cu(II) and Ni(II), respectively, while bio-

accumulated quantity of Cu(II) and Ni(II) increasedwith pH up

to 4.0 and 6.0, respectively. Earlier, Dursun et al. (2003b)

determined optimum pH values for the maximum Cu(II)

accumulation as 4.5 (R. arrhizus) and 5.0 (A. niger). Our results

corroborate with that of Dursun et al. (2003b) with respect to

the optimum pH for Cu (II) ion removal by A. niger.

3.5. Metal removal studies from electroplating effluent

Electroplating effluent used in the present study contained

significantly higher concentrations of metal ions such as

chromium, copper and lead. Other metals such as cobalt and

nickel were either not present or were below detection levels

(Table S1). No fungal growth and metal removal were

observed in the neat effluent inoculated with A. lentulus. The

Table 1 e Bioaccumulation of copper, nickel, chromium and lead from broth medium by A. lentulus (at 30 �C, 180 rpm and120 h).

Copper Nickel

Co

(mg L�1)Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

Co

(mg L�1)Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

75 4.86 58.80 12.09 78.40 70 4.55 23.58 5.18 33.69

80 4.73 62.00 13.12 77.50 75 4.59 31.60 6.89 42.13

400 3.79 218.30 57.59 54.58 140 2.39 26.45 11.05 18.89

800 1.27 158.30 124.50 19.79 210 1.37 14.11 10.28 6.72

Chromium(III) Lead

Co

(mg L�1)Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

Co

(mg L�1)Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

1000 4.64 793.77 170.96 79.38 500 4.67 355.2 76.06 71.04

2000 4.21 1190.67 282.89 59.53 1000 3.72 566.9 152.39 56.69

3000 4.17 1204.62 289.09 40.15 2000 1.84 1125.8 611.85 56.29

4000 4.04a 1339.51 331.48 33.49 3000 0.67 596.0 889.55 19.87

5000 4.03a 1302.30 323.23 26.05 4000 0.46 515.5 1120.65 12.89

xm: Dried biomass; Co: Initial metal ion concentrations; Cacc: Bioaccumulated metal ion concentrations after five days; qm: Specific metal ion

uptake determined as the amount of metal per unit of dry biomass.

Means within a column followed by same superscript letter are not significantly different (P < 0.05).

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 8 4995

toxicity could possibly be attributed to the high concentration

of free metal ions in the absence of strong binding ligands

present in the growth media. Therefore, anticipating the

nutrient deficiency or toxic effects of free metals ions on A.

lentulus, the effluent was either diluted with media (1:1 and

4:1; effluent: composite media) or supplemented with nutri-

ents (glucose and/or yeast extract).

3.5.1. Effect of various treatments on metal removalTable 3 shows the changes in metal concentrations in various

treatments. The dilution of effluents effectively reduced the

initial metal load, thereby alleviating the toxicity and

favouring better growth (3e4 g L�1 biomass) as compared to

the undiluted effluents (0e1.5 g L�1 biomass). In the case of Cr,

a gradual decline in metal concentration was observed till

11th d (in diluted effluents). In the case of undiluted and

supplemented effluents, major fraction of Cr removal

occurred in the first 5 d beyond which it remained almost

constant. Among the various supplementations, the best

performance (132.64 mg L�1 Cr removal) was obtained with

0.5 g L�1 glucose and 5 g L�1 yeast extract combination fol-

lowed by that (122.20 mg L�1) in effluent supplemented with

5 g L�1 glucose and 5 g L�1 yeast extract. Supplementations

with either glucose or yeast extract resulted in very poor Cr

removal. Almost similar results were obtained with respect to

Cu removal (Table 3). In contrast to this, complete Pb removal

was observed (Table 3) in all the nutrient supplementations

(except 5 g of glucose) and dilution series. Nevertheless, Pb

was not removed in the control and neat effluent, where the

growth of A. lentulus was not favoured. Overall, the absolute

metal removal as well as specific metal uptake per unit

biomass was higher in the supplemented effluents as

compared to the diluted ones.

3.5.2. Effect of pH on metal removal from effluentsThe pH of the electroplating effluent was 2.35. The effect of pH

neutralization (z7) on the metal removal in supplemented

and diluted effluent series is shown in Table 3. After the pH

adjustment, the growth of biomass was significantly

enhanced, especially in the effluents supplemented with

combination of glucose and yeast extract. With nutrient

supplementation (0.5 g L�1 glucose and 5 g L�1 yeast extract)

and pH adjustment, better removal of Cr (71%) and Cu (57%)

was noticed compared to 53% (Cr) and 37% (Cu) removal in the

effluent without pH neutralization. In the case of Pb, metal

removal remained unaffected by pH. Significant enhancement

in Cr and Cu removal was also noticed in the diluted effluents

with pH adjustment, whereas the lead removal was only

slightly affected. Above results deduce that the pH plays an

important role in enhancing the removal of certain metals

from industrial effluent.

Overall, these results indicate that A. lentulus can accom-

plish simultaneous removal of multiple metals with dilution

and nutrient supplementation of the electroplating effluent.

Other studies in the literature have reported substantial

decline in the metal uptake from the nutrient supplemented

tannery effluents byA. niger (Srivastava and Thakur, 2006) and

Pseudomonas aeruginosa (Ganguli and Tripathi, 1999) as

compared to synthetic solution. Nevertheless, in the present

study, the metal uptake from nutrient supplemented indus-

trial effluents by A. lentulus is not significantly curtailed

compared to the bioaccumulation of the metals from metal

amended growth media. Our previous studies have estab-

lished the removal of Cr from small-scale electroplating

industry and CETP effluents (Sharma et al., 2011). Neverthe-

less, these effluents were dominated by Cr contamination and

were devoid of other metals. The present study makes

another very important contribution to the research by

demonstrating simultaneous removal ofmultiplemetals from

complex electroplating effluents bearing a mixture of Cr, Cu &

Pb. The investigations reported in the literature often target

remediation of a single metal for example Cr from tannery

(Srivastava and Thakur, 2006; Benazir et al., 2010; Ganguli and

Tripathi, 1999) or electroplating effluents (Ganguli and

Table 2 e Effect of pH on fungal growth andmetal accumulation at 80mg LL1 Cu(II) & 70mg LL1 Ni(II) (at 30 �C, 180 rpm and120 h).

Control Copper Nickel

pH Xm

(g L�1)Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

Xm

(g L�1)Cacc

(mg L�1)qm

(mg g�1)Uptakeyield (%)

2 4.71 1.71 2.9 1.70 3.63 2.11a 2.58 1.22 3.69

3 5.76 3.465 11.69 3.37 14.61 2.13a 11.29 5.31 16.13

4 5.99a 4.206 63.87 15.19 79.84 2.50 21.46 8.57 30.66

5 6.15 4.888 61.77 12.64 77.21 4.26 28.61 6.72 40.87

6 6.22 4.241a 52.82 12.45 66.03 5.59 28.97 5.19 41.39

7 5.98a 4.262a b 50.01 11.73 62.51 4.86 26.82 5.52 38.31

8 5.86 4.27b 42.41 9.93 53.01 4.61 14.79 3.21 21.13

xm: Dried biomass; Co: Initial metal ion concentrations; Cacc: Bioaccumulated metal ion concentrations after five days; qm: Specific metal ion

uptake determined as the amount of metal per unit of dry biomass.

Means within a column followed by same superscript letter are not significantly different (P < 0.05).

Table 3e Biomass production and removal ofmetals from electroplating effluentwithout andwith pH adjustmentz7 (after11 days at 30 �C, 180 rpm).

Experimentalset up

Conditions Xm

(g L�1)Copper Chromium Lead

qm(mg g�1)

Metalremoval (%)

qm(mg g�1)

Metalremoval (%)

qm(mg g�1)

Metalremoval (%)

Control Without pH adjustment 0.00 0.00 0.00 0.00 0.00 0.00 0.00

With pH adjustment z7 0.00 0.00 0.30 0.00 0.00 0.00 4.44

Neat Without pH adjustment 0.00 0.00 1.19 0.00 2.47 0.00 6.67

With pH adjustment z7 0.12 3.33 0.59 173.67 11.09 8.17 10.89

5Y Without pH adjustment 0.14 14.29 2.96 169.43 12.62 64.29 100.00

With pH adjustment z7 0.42 15.24 9.48 89.81 20.06 21.43 100.00

5G Without pH adjustment 0.00 0.00 1.93 0.00 3.64 0.00 65.56

With pH adjustment z7 0.18 12.78 3.41 150.22 14.38 33.67 67.33

5G þ 5Y Without pH adjustment 1.52 16.64 37.48 56.42 45.62 5.57 94.00

With pH adjustment z7 2.76 13.70 56.00 44.43 65.23 3.26 100.00

0.5G þ 5Y Without pH adjustment 1.38 18.12 37.04 71.80 52.70 6.52 100.00

With pH adjustment z7 2.22 17.25 56.74 59.95 70.79 4.05 100.00

04:01 Without pH adjustment 3.14 7.07 41.11 13.83 28.88 2.29 100.00

With pH adjustment z7 3.76 8.91 62.04 28.49 71.22 1.91 100.00

01:01 Without pH adjustment 4.04 4.91 58.81 13.89 59.70 1.11 100.00

With pH adjustment z7 4.48 4.94 65.63 18.78 89.49 1.00 100.00

5Y, 5.0 g L�1 yeast extract; 5G, 5.0 g L�1 glucose; 5G and 5Y, 5.0 g L�1 glucose and 5.0 g L�1 yeast extract; 0.5G and 5Y, 0.5 g L�1 glucose and 5.0 g L�1

yeast extract; 01:01, Effluent: Media; 04:01, Effluent: Media.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 84996

Tripathi, 1999, 2002) and that of Cu from electroplating

effluent (Sze et al., 1996). Some studies have reported simul-

taneous removal of metals on pre-cultivated biomass by bio-

sorption method (Miretzky et al., 2006; Vilar et al., 2009;

Machado et al., 2010). However, studies depicting uptake of

multiple metals (Cu, Cr, Cd, Ni and Zn) during the growth of

strains in effluents have been rarely accomplished (Stoll and

Duncan, 1996; Zakaria et al., 2006). Zakaria et al. (2006) have

employed Acinetobacter sp. for the treatment of multiple

metals containing electroplating effluent. Although it could

take up Cr(VI) with 94e96% efficiency but the othermetals (Pb,

As, Hg, Cu, Fe, Ni & Cd) could not be removed. Such strains are

hence not suitable for the treatment of multiple metal con-

taining waste water. In view of the above, the capability of A.

lentulus to simultaneously remove multiple metals from the

waste water is very promising. To the best of our knowledge

this is the first study to demonstrate efficient and

simultaneous removal of the metals from electroplating

effluent in bioaccumulation mode.

4. Conclusion

The aim of the present study was to examine the bio-

accumulation characteristics of A. lentulus for the simulta-

neous removal of metal ions from industrial effluents. A.

lentulus harbours significant tolerance against Cr, Cu, Pb and

Ni, as depicted by the high MIC values. Morphological obser-

vations and SEM indicated that the varied toxicity responses

were evoked against different metals. Consequently, the

growth, specific metal uptake capacities and metal removal

yields also varied with themetal. While Ni exerted the highest

toxicity and growth inhibition, the maximum metal uptake

was observed for Pb among the tested metals. Generally, the

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 8 4997

metal uptake was affected by pH and initial metal ion

concentration. Further, simultaneous removal of Cr (71%), Cu

(56%) and Pb (100%) was accomplished from nutrient supple-

mented electroplating effluent. The ability ofA. lentulus for the

simultaneously removal of various hazardous metals such as

Cr, Cu and Pb is remarkable and bears high potential for

industrial applications.

Acknowledgement

The Ministry of Environment and Forests and Indian Council

of Agricultural Research Govt. of India, are gratefully

acknowledged for providing the fund. The authors wish to

thank Mr. Sabal Singh and Dr. H. K. Malik (IIT Delhi, India) for

kind technical support in experimental work and critical

review of the manuscript, respectively.

Appendix A. Supplementary material

Supplementary data associated with this article can be found,

in the online version, at http://dx.doi.org/10.1016/j.watres.

2012.06.035.

r e f e r e n c e s

Acikel, U., Alp, T., 2009. A study on the inhibition kinetics ofbioaccumulation of Cu(II) and Ni(II) ions using Rhizopusdelemar. Journal of Hazardous Materials 168 (2e3), 1449e1458.

APHA, 1989. Standard Methods for the Examination of Water andWastewater 17th. American Public Health Association-American Water Works Association-Water Pollution ControlFederation, Washington, D.C.

Benazir, J.F., Suganthi, R., Rajvel, D., Pooja, M.P.,Mathithumilan, B., 2010. Bioremediation of chromium intannery effluent by microbial consortia. African Journal ofBiotechnology 9 (21), 3140e3143.

Donmez, G., Aksu, Z., 1999. The effect of copper(II) ions on growthand bioaccumulation properties of some yeasts. ProcessBiochemistry 35 (1e2), 35e142.

Donmez, G., Aksu, Z., 2001. Bioaccumulation of copper(II) andnickel(II) by the non-adapted and adapted growing Candida sp.Water Research 35 (6), 1425e1434.

Dursun, A.Y., Uslu, G., Tepe, O., Cuci, Y., Ekiz, H.I.A., 2003a. Acomparative investigation on the bioaccumulation of heavymetal ions by growing Rhizopus arrhizus and Aspergillus niger.Biochemical Engineering Journal 15 (2), 87e92.

Dursun, A.Y., Ulsu, G., Cuci, Y., Aksu, Z., 2003b. Bioaccumulationof copper(II), lead(II) and chromium(VI) by growing Aspergillusniger. Process Biochemistry 38 (12), 1647e1651.

Ezzouhri, L., Castro, E., Moya, M., Espinola, F., Lairini, K., 2009.Heavy metal tolerance of filamentous fungi isolated frompolluted sites in Tangier, Morocco. African Journal ofMicrobiology Research 3 (2), 35e48.

Franklin, N.M., Stauber, J.L., Lim, R.P., Petocz, P., 2002. Toxicity ofmetal mixtures to a tropical freshwater alga (Chlorella sp.): theeffect of interactions between copper, cadmium, and zinc onmetal cell binding and uptake. Environmental Toxicology andChemistry 21 (11), 2412e2422.

Ganguli, A., Tripathi, A.K., 1999. Survival and chromate reducingability of Pseudomonas aeruginosa in industrial effluents. Lettersin Applied Microbiology 28 (1), 76e80.

Ganguli, A., Tripathi, A.K., 2002. Bioremediation of toxicchromium from electroplating effluents by chromate reducingPseudomonas aeruginosa A2Chr in two bioreactors. AppliedMicrobiology and Biotechnology 58 (3), 416e420.

Gikas, P., 2008. Single and combined effects of nickel (Ni(II)) andcobalt (Co(II)) ions on activated sludge and on other aerobicmicroorganisms: a review. Journal of Hazardous Materials 159(2e3), 187e203.

Iskandar, N.L., Zainudin, N.A.I.M., Tan, S.G., 2011. Tolerance andbiosorption of copper (Cu) and lead (Pb) by filamentous fungiisolated from a freshwater ecosystem. Journal ofEnvironmental Sciences 23 (5), 824e830.

Joshi, P.K., Swarup, A., Maheshwari, S., Kumar, R., Singh, N., 2011.Bioremediation of heavy metals in liquid media through fungiisolated from contaminated sources. Indian Journal ofMicrobiology 51 (4), 482e487.

Lilly, W.W., Wallweber, G.J., Lukefahr, T.A., 1992. Cadmiumabsorption and its effect on growth and mycelial morphologyof the basidiomycete fungus, Schizophyllum commune.Microbios 72, 227e237.

Liu, Y.G., Fan, T., Zhou, N., He, Y.C., Min, Z.Y., Wu, S.D., 2009.Isolation, identification and its bioaccumulationcharacteristics of a fungus strain with resistance to Cu2þ andZn2þ. Journal of Central South University (Science andTechnology) 40, 60e66.

Machado, M.D., Soares, H.M.V.M., Soares, E.V., 2010. Removal ofchromium, copper, and nickel from an electroplating effluentusing a flocculent brewer’s yeast strain of Saccharomycescerevisiae. Water Air & Soil Pollution 212 (1e4), 199e204.

Malik, A., 2004. Metal bioremediation through growing cells.Environment International 30 (2), 261e278.

Miretzky, P., Saralegui, A., Cirelli, A.F., 2006. Simultaneous heavymetal removal mechanism by dead macrophytes.Chemosphere 62 (2), 247e254.

Mishra, A., Malik, A. Recent advances in microbial metalbioaccumulation. Critical Reviews in Environmental Scienceand Technology, in press.

Moore, B.A., Duncan, J.R., Burgess, J.E., 2008. Fungalbioaccumulation of copper, nickel, gold and platinum.Minerals Engineering 21 (1), 55e60.

Pan, R., Cao, L., Zhang, R., 2009. Combined effects of Cu, Cd, Pb,and Zn on the growth and uptake of consortium of Cu-resistant Penicillium sp. A1 and Cd-resistant Fusarium sp. A19.Journal of Hazardous Materials 171 (1e3), 761e766.

Parameswari, E., Lakshmanan, E., Thilagavathi, A., 2010.Biosorption and metal tolerance potential of filamentous fungiisolated from metal polluted ecosystem. Electronic Journal ofEnvironmental, Agricultural and Food Chemistry 9 (4),664e671.

Pumpel, T., Macaskie, L.E., Finlay, J.A., Diels, L., Tsezos, M., 2003.Nickel removal from nickel plating waste water usinga biologically active moving-bed sand filter. Biometals 16 (4),567e581.

Roy, S., Ghosh, A.N., Thakur, A.R., 2008. Uptake of Pb(2þ) bya cyanobacterium belonging to the genus Synechocystis,isolated from East Kolkata Wetlands. Biometals 21 (5),515e524.

Sharma, S., Adholeya, A., 2011. Detoxification and accumulationof chromium from tannery effluent and spent chrome effluentby Paecilomyces lilacinus fungi. International Biodeterioration &Biodegradation 65 (2), 309e317.

Sharma, S., Malik, A., Satya, S., 2009. Application of responsesurface methodology (RSM) for optimization of nutrientsupplementation for Cr (VI) removal by Aspergillus lentulusAML05. Journal of Hazardous Materials 164 (2e3), 1198e1204.

wat e r r e s e a r c h 4 6 ( 2 0 1 2 ) 4 9 9 1e4 9 9 84998

Sharma, S., Malik, A., Satya, S., Mishra, A., 2011. Development ofa biological system employing Aspergillus lentulus for Crremoval from a small-scale electroplating industry effluent.Asia-Pacific Journal of Chemical Engineering 6 (1), 55e63.

Shivakumar, C.K., Thippeswamy, B., Krishnappa, M.,Ananthamurthy, K.S., 2011. Heavy metals accumulationpotency of Aspergillus niger and Aspergillus flavus indigenous topaper mill effluent. The Bioscan 6 (4), 691e696.

Srivastava, S., Thakur, I.S., 2006. Isolation and process parameteroptimization of Aspergillus sp. for removal of chromium fromtannery effluent. Bioresource Technology 97 (10), 1167e1173.

Stoll, A., Duncan, J.R., 1996. Enhanced heavy metal removal fromwaste water by viable, glucose pretreated Saccharomycescerevisiae cells. Biotechnology Letters 18 (10), 1209e1212.

Sze, K.F., Lu, Y.J., Wong, P.K., 1996. Removal and recovery ofcopper ion (Cu2þ) from electroplating effluent by a bioreactor

containing magnetite-immobilized cells of Pseudomonasputida 5X. Resources, Conservation and Recycling 18 (1e4),175e193.

Tyagi, A.K., Malik, A., 2010. Liquid and vapour-phase antifungalactivities of selected essential oils against Candida albicans:microscopic observations and chemical characterization. BMCComplement. Alternative Medicine, 10e65.

Vilar, V.J.P., Martins, R.J.E., Botelho, C.M.S., Boaventura, R.A.R.,2009. Removal of Cu and Cr from an industrial effluent usinga packed-bed column with algae Gelidium-derived material.Hydrometallurgy 96 (1e2), 42e46.

Zakaria, Z.A., Zakaria, Z., Surif, S., Ahmad, W.A., 2006.Bioremediation of Cr(VI)-containing ElectroplatingWastewater Using Acinetobacter Sp.. International Conferenceon Environment (ICENV) 13e15 November 2006, Penang,Malaysia.