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Journal of Environmental Management 92 (2011) 2786e2793

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Journal of Environmental Management

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

Prediction of metal-adsorption behaviour in the remediation of watercontamination using indigenous microorganisms

E. Fosso-Kankeu a,*, A.F. Mulaba-Bafubiandi a, B.B. Mamba b, T.G. Barnard c

aMinerals Processing and Technology Research Centre, Department of Metallurgy, Faculty of Engineering and the Built Environment, University of Johannesburg, PO Box 526,Wits 2050, Johannesburg, South AfricabDepartment of Chemical Technology, Faculty of Science, University of Johannesburg, PO Box 17011, Doornfontein, Johannesburg, South AfricacWater and Health Research Centre, Faculty of Health Sciences, University of Johannesburg, PO Box 17011, Doornfontein 2028, Johannesburg, South Africa

a r t i c l e i n f o

Article history:Received 23 August 2010Received in revised form16 May 2011Accepted 12 June 2011Available online 6 July 2011

Keywords:Mine wastewaterHeavy metalsLight metalsBinding affinityBiosorbent behaviourAdsorption kinetics

* Corresponding author. Tel.: þ27115596529; fax: þE-mail address: elvisf@uj.ac.za (E. Fosso-Kankeu).

0301-4797/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jenvman.2011.06.025

a b s t r a c t

In recent years, the adsorption of heavy metal cations onto bacterial surfaces has been studied exten-sively. This paper reports the findings of a study conducted on the heavy metal ions found in mineeffluents from a mining plant where Co2þ and Ni2þ bearing minerals are processed. Heavy metal ions arereported to be occasionally present in these mine effluents, and the proposed microbial sorption tech-nique offers an acceptable solution for the removal of these heavy metals. The sorption affinity ofmicroorganisms for metal ions can be used to select a suitable microbial sorbent for any particularbioremediation process. Interactions of heavy metal ions (Co2þ and Ni2þ) and light metal ions (Mg2þ andCa2þ) with indigenous microbial cells (Brevundimonas spp., Bacillaceae bacteria and Pseudomonas aeru-ginosa) were investigated using the Langmuir adsorption isotherm, pseudo second-order reactionkinetics model and a binary-metal system. Equilibrium constants and adsorption capacities derived fromthese models allowed delineation of the effect of binding affinity and metal concentration ratios on theoverall adsorption behaviour of microbial sorbents, as well as prediction of performance in bioremedi-ation systems. Although microbial sorbents used in this study preferentially bind to heavy metal ions, itwas observed that higher concentrations (>90 mg/[) of light metal ions in multi-metal solutions inhibitthe adsorption of heavy metal ions to the bacterial cell wall. However, the microbial sorbents reducedNi2þ levels in the mine-water used (93e100% Ni2þ removal) to below the maximum acceptable limit of350 mg/[, established by the South African Bureau of Standards. Competition among metal ions forbinding sites on the biomaterial surface can occur during the bioremediation process, but microbialsorption affinity for heavy metal ions can enhance their remediation in dilute (<5 mg/[ heavy metal)wastewaters.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Water pollution by metal ions has emerged as one of the seriouschallenges currently being faced by water authorities in SouthAfrica. The increase in industrial and artisanal activities whichdischarge waste containing metal ions into the environment is oneof the causes leading to this situation. Operations such as mining,mineral processing and metal extraction (artisanal, small scale andindustrial), energy and fuel production, along with fertilizer andpesticide use contribute to the pollution of the environment withheavy and light metal ions (Volesky, 1990; Bishop, 2002). Althoughheavy metal ions are more toxic, the occurrence of higher

27115596329.

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concentrations of light metal ions in the water system is the sourceof aesthetic, operational and possible health problems (Nies, 1999;Roane and Pepper, 2000). Light metal ions could also negativelyaffect water bioremediation processes intended to remove heavymetal ions as these light metal ions tend to compete with the tar-geted toxic metal ions for binding sites on the biomaterial surface.Current environmental legislations require industrial and miningoperations to further process their effluents prior to release into theecosystem. Remediation of toxic metal ion contamination of watersources is therefore important in order to restore wastewater to itsdesired usability, as usable water is already scarce in South Africa(Van Vuuren, 2007). Metalemicrobe interactions have been wellstudied and have so far been considered for metal uptake fromsolutions (Brady and Duncan, 1994a; Liu et al., 2002b; Vijver et al.,2004; Hassan et al., 2008). The myriad of studies done on bio-sorption of metals using microorganisms have shown that for the

0 100 200 300 400 500 6000.0

2.5

5.0

7.5

10.0

C o 2 + 50m g/L

C o 2 + 100m g/L

C o 2 + 200m g/L

N i 2 + 50m g/L

N i 2+ 100m g/L

N i 2+ 200m g/L

Exposure time (min)

Metal io

n u

ptake m

g/g

Fig. 1. Uptake of heavy metal ions (Co2þ and Ni2þ) by the Bacillaceae bacteria asa function of time and initial concentration.

0 100 200 300 400 500 6000

1

2

3

4

5

M g 2+ 50m g/L

M g 2+ 100m g/L

M g 2+ 200m g/L

C a 2+ 50m g/L

C a 2+ 100m g/L

C a 2+ 200m g/L

Exposure time (min)

Metal io

n u

ptake m

g/g

Fig. 2. Uptake of light metal ions (Mg2þ and Ca2þ) by the Bacillaceae bacterium asa function of time and initial concentration.

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e2793 2787

same metal species various microorganisms exhibit differentsorption abilities (Ferraz and Teixeira, 1999; Wang and Chen, 2006;Cabuk et al., 2006; Choi et al., 2009), implying that microorganisms’adsorption affinity may vary depending on the specific metal ions.Owing to this principle of adsorption selectivity, investigation ofthe biosorption behaviour of a wide range of metal ions andmicroorganisms should allow for the identification of suitablemicrobial sorbents with a strong affinity for particular targetedmetal ions that can lead to effective bioremediation of contami-nated water.

Heavy metal ions are highly toxic to both eukaryotes andprokaryotes and their presence in the environment adverselyaffects the ecosystem. The remediation of heavy metal pollution inwater systems is therefore important, and the use of microorgan-isms as biosorbents could possibly enhance the rehabilitation andprotection of the environment.

This paper reports on work done as part of a larger projectfocussing on surface water pollution by mining effluents. Experi-ments were performed on a laboratory scale to evaluate theadsorption capabilities of three indigenous microorganisms iso-lated from Ni2þ-contaminated mining effluent for the removal oflight and heavy metal ions from synthetic and mine-water solu-tions. The impact of light metal ions on the biosorption of heavymetal ions is herein studied and theoretical predictions are madefor the improvement of bioremediation at an industrial level basedon kinetic and isothermal reaction analyses.

2. Methodology

2.1. Experimental solutions

2.1.1. Synthetic solutionAnhydrous crystals (analytical grade) of CoSO4, NiSO4, MgSO4

and CaSO4 were each dissolved in sterile distilled water to obtaina stock solution of 1000 mg/[. From this solution, aliquots of 5 m[,7.5 m[, 10 m[, 15 m[ and 20 m[ of the metal solution were mixedwith aqueous microbial solutions (0.3 g of biomass in steriledistilled water) to yield final concentrations of 50 mg/[, 75 mg/[,100 mg/[, 150 mg/[ and 200 mg/[, respectively, made up to a finalvolume of 100 m[.

2.1.2. Mine-water solutionMine-water samples were collected at a mining plant, located

near Machadodorp, Mpumalanga Province, South Africa, usingsterile plastic bottles. The water samples were preserved at around8 �C during transport to the laboratory. Physicochemical analyseswere performed within 15 h and the samples were used thefollowing day for metal-removal experiments.

2.2. Biomass preparation

Indigenous strains of Pseudomonas aeruginosa, Brevundimonasspp. and Bacillaceae bacteria isolated from waters around themining areas as reported previously (Fosso-Kankeu et al., 2009),were grown on nutrient agar as described by Fosso-Kankeu et al.(2010) and kept at 4 �C.

Single colonies of the microorganisms were inoculated into200 m[ of nutrient broth in sterile Erlenmeyer flasks, usinga procedure previously described by Fosso-Kankeu et al. (2010),and incubated overnight at 37 �C in a shaking incubator (150 rpm).Aliquots (30 m[) of the cultures were centrifuged at 8000 rpm for15 min to collect the cells. The recovered cell pellet (w0.1 g) waswashed several times with sterile distilled water to remove residualmedium and was suspended in appropriate volumes of steriledistilled water and used for the sorption experiments.

2.3. Metal biosorption experiment

Metal and cell solutions were mixed in sterile 250 m[ Erlen-meyer flasks to the specified concentrations as described in Section2.1.1; sterile distilled water was added and made up to a finalvolume of 100 m[. Optimal biosorption conditions were ensuredi.e., neutral pH and standard biomass concentration of 0.3 g/[ cells(wet cells) as described by Fosso-Kankeu et al. (2010). The mixturewas incubated at 37 �C in a shaking incubator (150 rpm) and 5m[ ofthe mixture was collected at time intervals of 20 min, 60 min,300 min and 480 min. The samples were centrifuged at 8000 rpmfor 15 min and the amount of residual metal in solution wasdetermined in the supernatant as described below.

2.4. Metal ions quantification and experimental procedure

The concentration of residual metal ions in the supernatant wasdetermined using an Inductively Coupled Plasma-Optical EmissionSpectrometer (ICP-OES). All experiments were done in triplicatewith an abiotic control (without microorganisms) that assisted inaccounting for metal loss due to precipitation. The error discrep-ancy between replicates was less than 10%. Average values of thetriplicate results obtained were used for plotting the graphs.

2.5. Theoretical studies

2.5.1. Isotherm and reaction kineticsVariation of adsorption capacity was studied over time with

various aqueous solution concentrations of metal ions; trends of

Ce (m g /L )

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

q e (mg/

g)

0

2

4

6

8

1 0

B . b a c te riu m - C o 2 +

B re v u n d im o n a s s p - C o2 +

P . a e ru g in o s a - C o2 +

Fig. 3. Langmuir isotherm plots for the biosorption of Co2þ by each microbial strain.

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e27932788

experimental data were evaluated based on the theoreticalmethods such as the Langmuir adsorption isotherm and pseudosecond-order reaction kinetics. The adsorption capacity at equi-librium was determined using the following equation:

qeðmg=gÞ ¼ ðC0 � CeÞVm

where:

qe is the adsorption capacity in mg/gC0 is the initial concentration of metal ions in solution (mg/[)Ce is the equilibrium of metal ions (mg/[)m is the biomass (g)V is the volume of the solution ([)

2.5.2. StatisticsThe adsorption capacity of microorganisms for heavy and light

metal ions was determined over time and was recorded ona Microsoft Excel spreadsheet. The sorption affinity of each

Ce 0 5 0 1 0 0

qe (m

g/g)

0

2

4

6

8

1 0

Fig. 4. Langmuir isotherm plots for the bioso

microorganism for heavy vs. light metal was estimated by deter-mining the differences in adsorption capacities between the twotypes of metal ions, using the Wilcoxon signed-rank test. Thedifference was deemed significant when the p-value was <0.05.

2.6. Metal tolerance

To determine the effect of the metal concentrations on themicroorganism’s viability after exposure to the prepared experi-mental solutions, aliquots of the mixture were plated onto nutrientagar and incubated at 37 �C overnight to determine the number ofsurviving cells after each time interval and metal concentrationcombination.

2.7. Effect of metal ions on cell morphology

Microbial cells incubated overnight in a binary-metal solution ofNi2þ plus Co2þ (50mg/[ each) and the control (not exposed tometalions) were centrifuged and the resulting pellet was washed withsterile distilled water, harvested and placed on a copper grid, then

(m g /L )1 5 0 2 0 0 2 5 0

B . b a c te r iu m - N i2 +

B re v u n d im o n a s s p - N i2 +

P . a e ru g in o s a - N i2 +

rption of Ni2þ by each microbial strain.

C e (m g /L )

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

q e (mg/

g)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

B . b a c te riu m - M g 2 +

B re v u n d im o n a s s p - M g 2 +

P . a e ru g in o s a - M g 2 +

Fig. 5. Langmuir isotherm plots for the biosorption of Mg2þ by each microbial strain.

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e2793 2789

dried for 1 h in an oven. The grid with dried cells was mounted onthe specimen aluminium stub using double-sided carbon tape.Scanning electron microscopy (SEM) was performed using a lowvacuum mode SEM machine at 5.00 kV and 25 000 magnitude.

3. Results and discussions

3.1. Bio-uptake of heavy metals from synthetic solutions

Cobalt and nickel bearing minerals are mined and processed inSouth Africa and these metal ions have been reported to be occa-sionally present in discharged mine-water hence these two heavymetal ions were considered in this study. The removal of theseheavy metal ions from synthetic solutions using indigenousmicroorganisms such as Bacillaceae bacteria, Brevundimonas spp.and P. aeruginosa was investigated and analyzed as a function oftime and initial metal concentrations. The adsorption uptakecapacity (qe mg/g) was found to increase with time when using theBacillaceae bacteria and P. aeruginosa as biosorbents. The trend of

Ce 0 50 100

qe (m

g/g)

0

1

2

3

4

5

Fig. 6. Langmuir isotherm plots for the bioso

metal removal by Brevundimonas spp. was inconsistent and low;this could be explained by the breakage of bonds linking the metalto the external cell surface, or alternatively metal excretion fromthe cells via efflux transport, can result in a decrease in the metalremoval rate (Fosso-Kankeu et al., 2009). Overall, the adsorptioncapacity of the Bacillaceae bacteria surpassed the performance ofother bacteria tested (Fig. 1).

3.2. Bio-uptake of light metals from synthetic solutions

Light metal ions often occur in surface water at high concen-trations (Fosso-Kankeu et al., 2009) and thus may affect theremoval of heavy metal ions (Co2þ and Ni2þ). To confirm thisexperimentally, Ca2þ and Mg2þ, also divalent metal ions, wereselected and their adsorption within a period of time by the samemicroorganisms was studied. Removal of these metals as a functionof time and initial concentration showed a relative increase ofremoval rate (qe mg/g) with time; however, P. aeruginosa and Bre-vundimonas spp. did not remove the metals consistently, as metal

(m g /L )150 20 0 250

B . bac te riu m - C a 2+

B revund im onas sp - C a 2+

P . ae ru g inosa - C a 2+

rption of Ca2þ by each microbial strain.

Table 1Parameters derived from Langmuir plots illustrating adsorption affinity ofP. aeruginosa for metal ions.

Parameters Metal ions

Co Ni Mg Ca

K ([/mg) 0.092 0.1057 0.0152 0.0734qm (mg/g) 6.2 6.63 2.42 3.026R2 0.996 0.9965 0.9235 0.9885

Table 3Statistical values of the comparison of adsorption capacity of heavy and light metalions.

Biosorbents Comparedmetals

All Ci considered Order of absorption

Bacillaceae bacteria Co2þ vs. Mg2þ P < 0.001 Co2þ > Mg2þ

Co2þ vs. Ca2þ P < 0.001 Co2þ > Ca2þ

Ni2þ vs. Mg2þ P < 0.001 Ni2þ > Mg2þ

Ni2þ vs. Ca2þ P < 0.001 Ni2þ > Ca2þ

Brevundimonas spp. Co2þ vs. Mg2þ P ¼ 0.701 Co2þ w Mg2þ

Co2þ vs. Ca2þ P ¼ 0.368 Co2þ w Ca2þ

Ni2þ vs. Mg2þ P ¼ 0.216 Ni2þ w Mg2þ

Ni2þ vs. Ca2þ P ¼ 0.475 Ni2þ w Ca2þ

P. aeruginosa Co2þ vs. Mg2þ P < 0.001 Co2þ > Mg2þ

Co2þ vs. Ca2þ P < 0.001 Co2þ > Ca2þ

Ni2þ vs. Mg2þ P < 0.001 Ni2þ > Mg2þ

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e27932790

release in solution was followed by re-uptake, a phenomenon thatFosso-Kankeu et al. (2009) previously ascribed to physiologicalneeds following starvation of microorganisms in aqueous solution.The relative increase in metal ion removal was well demonstratedin the case of metal removal by the Bacillaceae bacteria (Fig. 2).

Ni2þ vs. Ca2þ P < 0.001 Ni2þ > Ca2þ

All microorganisms Co2þ vs. Mg2þ P < 0.001 Co2þ > Mg2þ

Co2þ vs. Ca2þ P < 0.001 Co2þ > Ca2þ

Ni2þ vs. Mg2þ P < 0.001 Ni2þ > Mg2þ

Ni2þ vs. Ca2þ P < 0.001 Ni2þ > Ca2þ

3.3. Comparative bio-uptake of metals from mixed syntheticsolutions

In the presence of all four metal ions (Co2þ, Ni2þ, Mg2þ andCa2þ) in solution, while keeping the biomass constant, the limita-tion in terms of the surface space available on the binding sitespossibly led to a competition among metal ions for interactionwithfunctional groups on the biosorbent.

3.3.1. Affinity of microorganisms for metalsUptake of metal ions by microorganisms requires physical

interaction between the two, hence the possibility that the removalrate of specific metal ions varies from one microorganism toanother depending of the degree of affinity. The ability of a micro-organism to adsorb metal ions indicates greater sorption affinity,and this ability can be determined using the Langmuir adsorptionisotherm model. This model illustrates a non-linear and saturatedadsorption process that can be explained by the followingequation:

qe ¼ ðqmkCeÞ=ð1þ kCeÞwhere:

qe is the amount of metal adsorbed onto the unit amount of thebiomass (mg/g)Ce is the equilibrium concentration (mg/[)qm is a complete monolayer (mg/g)k is the adsorption equilibrium constant related to the strengthof the binding site

Among the results shown in Figs. 3e6 only the P. aeruginosametal ion adsorption plots exhibit a trend much closer to a satu-rated monolayer isotherm when considering all four metals, i.e.,Co2þ, Ni2þ, Mg2þ and Ca2þ, and these are therefore suitable for theapplication of the Langmuir adsorption isotherm model in thecurrent study. In a multi-metal solution, the sorption affinity ofBacillaceae bacterium for metal ions tends to be affected and metalattachment on the binding site could become unstable, possiblyleading to incomplete saturation of the cell surface; in the case of

Table 2Pseudo second-order reaction kinetic parameters for the adsorption of Co2þ, Ni2þ, Mg2þ

Biosorbents Cobalt Nickel

R2 qe mg/g K2 g/mg min R2 qe mg/g K2 g/mg

B. bacteria 0.9966 1.44 0.0329 0.9992 1.9135 0.047Brevundimonas 0.3508 na 0.021 0.8734 0.1075 naP. aeruginosa 0.9991 0.9543 0.0813 0.9998 1.2885 0.1144

na: not available.

Brevundimonas spp., inconsistency of metal binding to cell surfacewas noted.

The linear form (Ce/qe¼ 1/kqmþ Ce/qm) of the Langmuir isothermmodel allows the derivation of the equilibrium constant from theplot (Ce/qe vs. Ce). Values of the equilibrium constant (k) foradsorption of Co2þ, Ni2þ, Mg2þ and Ca2þ by P. aeruginosa are showninTable 1. The k value has been reported to positively correlate to thestrength of the binding sites (Gabr et al., 2008; Baysal et al., 2009),therefore the sorption affinity of any particular microorganism fora metal ion can be illustrated by an increase in the equilibriumconstant. Obtained values of k for heavy metals (Co2þ, 0.092; Ni2þ,0.1057) were higher than the values for light metals (Mg2þ, 0.0152and Ca2þ, 0.0734), implying that P. aeruginosa had more affinity forCo2þ and Ni2þ than for Mg2þ and Ca2þ. The overall order ofadsorption affinity (Ni2þ> Co2þ> Ca2þ>Mg2þ) shows that bindingsites onmicrobial cell membranes preferentially attach onemetal infavour of another; this variation in the interaction has been ascribedto the difference inmetal:ionic ratio (Mattuschka and Straube,1993;Chong and Volesky, 1995; Sag et al., 2002) and the difference inelectronegativity of the metal ions (Allen and Brown, 1995; Stummand Morgan, 1996). The electronegativity order (Ni2þ >

Co2þ > Mg2þ > Ca2þ) as established by Pauling (1932) can explainthe affinity of microbial sorbent for heavy metal ions, but thedifference in adsorption affinity between light metal ions could alsodepend on the ion-exchange principle wherebyMg2þ is more easilyreleased from cells than Ca2þ in exchange for heavy metal ionadsorption (Brady and Duncan, 1994b).

The determination of the biosorption rate of a metal ion ona given biosorbent is very important for the prediction of bio-sorption capacity and can therefore provide information needed forthe design and construction of a bioremediation plant. The pseudosecond-order kinetic model widely used (Choi et al., 2009; Yahayaet al., 2009; Baysal et al., 2009) was considered in this study todetermine the adsorption behaviour of microorganisms over

and Ca2þ on microbial sorbents.

Magnesium Calcium

min R2 qe mg/g K2 g/mg min R2 qe mg/g K2 g/mg min

0.9853 0.7518 0.2253 0.9955 0.7685 0.20050.8108 0.0407 1.0344 0.8434 0.0392 na0.9937 0.6454 0.2607 0.9136 0.1832 0.2364

R atio (N i2+ concentra tion /100 m g L -1)

0 .0 0.2 0.4 0.6 0.8 1.0

Abso

rptio

n ca

paci

ty q

e (m

g/g)

0

1

2

3

4

5

N i2+ - 20 m in N i2+ - 60 m in N i2+ - 300 m in M g 2+ - 20 m in M g 2+ - 60 m in M g 2+ - 300 m in

Fig. 7. Competitive effect of Mg2þ on the adsorption of Ni2þ by P. aeruginosa.

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e2793 2791

a period of time. Important parameters of this kinetic model arepresented in the following equation:

t=qt ¼ 1=k2q2e þ t=qe

where:

k2 is the equilibrium rate constant of pseudo second-orderadsorption (g/mg min)qe is the equilibrium adsorption amount (mg/g)qt is the adsorption amount at time t (mg/g)

The plot of t/qt vs. t shows that metal-adsorption follows thepseudo second-order reaction kinetic model only when the bio-sorbents are the Bacillaceae bacteria and P. aeruginosa, but not withBrevundimonas spp. This trend is well demonstrated in the case ofthe adsorption of heavy metal ions which gives correlation coeffi-cient (R2) values >0.9966 (Table 2).

R a tio (N i2 + c o n c e

0 .0 0 .2 0 .4

Abso

rptio

n ca

paci

ty q

e (m

g/g)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0

3 .5

N i2 + - 2 0 m in N i2 + - 6 0 m in

N i2 + - 3 0 0 m in C a 2 + - 2 0 m iC a 2 + - 6 0 m

C a 2 + - 3 0 0 m in

Fig. 8. Competitive effect of Ca2þ on the

The calculated values of the equilibrium adsorption amountwere very close to the experimental values when the Bacillaceaebacteria and P. aeruginosa were used as biosorbents. These valueswere comparatively higher for heavy metal ions than for their lightmetal ion counterparts, showing greater adsorption capability ofthe heavymetal ions by the two bacterial strains; similar to findingsreported by Wang and Chen (2006). Considering the solutionscontaining one biosorbent or three biosorbents, the statistical testscomparing adsorption of heavy and light metal ions, further con-firmed (Table 3) that heavy metal ion adsorption capacity (mg/g)was significantly higher than light metal ion adsorption capacity,except in cases where Brevundimonas spp. was used as a biosorbent.

3.3.2. Adsorption behaviour in binary-metal ion systemsTo simulate the behaviour of the most appropriate biosorbent

(P. aeruginosa) in a bioremediation plant, potential concentrationranges of metal ions in mine-water were prepared; solution ratios

n tra tio n /1 0 0 m g L-1

)

0 .6 0 .8 1 .0

n in

adsorption of Ni2þ by P. aeruginosa.

0 100 200 300 400 500 600-25

0

25

50

75

100

Ni2+

Mg 2+

Ca 2+

Exposure time (min)

Percen

tag

e u

ptake (%

)

Fig. 9. Remediation of Ni2þ from mining water by Bacillaceae bacteria in the presenceof high concentrations of light metal ions (Mg2þ and Ca2þ).

0 100 200 300 400 500 6000

50

100

150

Ni 2+

Mg2+

Ca 2+

Exposure time (min)

Percen

tag

e u

ptake (%

)

Fig. 11. Remediation of Ni2þ frommining water by P. aeruginosa in the presence of highconcentrations of light metal ions (Mg2þ and Ca2þ).

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e27932792

(1:10; 3:10; 5:0; 7:10; 9:10) of nickel to light metal ions (Mg2þ orCa2þ) were prepared up to a total binary-metal concentration of100mg/[. This was done to determine the effect of lower and higherconcentrations of light metal ions on Ni2þ adsorption byP. aeruginosa. Co2þ was not found in mine-water, therefore it wasnot considered for the study of the binary-metal system intended topredict bioremediation of heavy metal pollution of mine-water.Results (Figs. 7 and 8) show that an increase in Ni2þ adsorptioncapacity positively correlated with an increase of nickel to lightmetal ratio, but in Ni2þeCa2þ binary solution system the adsorptioncapacity at a ratio of 0.9 was lower than at a ratio of 0.7, possibly dueto microbial inhibition. It was observed that at a nickel to lightmetal (Mg2þ or Ca2þ) ratio of 0.1 P. aeruginosa did not adsorb Ni2þ tothe best of its ability; this observation could be ascribed to the factthat higher concentrations of Mg2þ ions easily occupied the bindingsites; however, in the range of nickel to light metal (Mg2þ or Ca2þ)ratio of 0.3e0.9, Ni2þ adsorption capacity was equal to or greaterthan light metal ion adsorption capacity, further confirming theaffinity of P. aeruginosa for Ni2þ. Considering the adsorptioncapacity of P. aeruginosa for Ni2þ in Ni2þeMg2þ and Ni2þeCa2þ

binary solution systems it can be predicted that in similar binary-metal solutions this protocol would be effective only in the reme-diation of dilute solutions.

In the next section, the above laboratory protocol, optimized fora synthetic solution, was used for the bioremediation of nickelcontaminated mine-water and prediction of biosorption capacity ofmicrobial biosorbents examined.

0 100 200 300 400 500 600-10

0

10

20

30

40

Ni 2+

Mg2+

Ca2+

Exposure time (min)

Percen

tag

e u

ptake (%

)

Fig. 10. Remediation of Ni2þ frommining water by Brevundimonas spp. in the presenceof high concentrations of light metal ions (Mg2þ and Ca2þ).

3.4. Bioremediation of mining water

Studies on the adsorption behaviour of the microbial sorbentsusing synthetic solutions of metal ions demonstratedmore efficientremoval of heavy metal ions. These sorbents were then used tosequestrate Ni2þ from mine-water also containing Mg2þ and Ca2þ.Results (Figs. 9e12) show that the Bacillaceae bacteria were able toreduce Ni2þ to below the recommended concentration of 350 mg/[(SABS, 2005) while P. aeruginosa totally removed Ni2þ from thesolution. However, Brevundimonas spp. and the mixture of micro-organisms could only remove less than 50% of Ni2þ from mine-water. Overall the removal of Mg2þ and Ca2þ was almost negligibleand did not totally inhibit Ni2þ adsorption by P. aeruginosa.

3.5. Metal tolerance

Heavy and light metal ions impact differently on microorgan-isms. While heavy metal ions are mostly toxic for microorganisms,most light metal ions (trace elements) constitute nutrients requiredby microorganisms for their physiological functions (Sadler andTrudinger, 1967; Nies, 1999; Roane and Pepper, 2000). The mech-anism of metal absorption and adsorption depends on the micro-bial surface area available, physiology and the functional groups onthe binding site (Wang and Chen, 2006). Some metal ions (espe-cially heavy metals) affect the structure of the cell membrane, thesize of microbial cells as well as their metabolism. Microorganismtolerance to initial concentrations of metals at different timeintervals was studied by counting the colonies on the agar medium.It was found that heavy metal ions were more lethal to

0 100 200 300 400 500 6000

10

20

30

40

50

Ni 2+

Mg 2+

Ca 2+

Exposure time (min)

Percen

tag

e u

ptake (%

)

Fig. 12. Remediation of Ni2þ frommining water by the combined strains in presence ofhigh concentrations of light metal ions (Mg2þ and Ca2þ).

Fig. 13. Cell morphology of Bacilliaceae bacteria under scanning electron microscopy (25 000 mag) before (A) and after (B) exposure to Ni2þ and Co2þ.

E. Fosso-Kankeu et al. / Journal of Environmental Management 92 (2011) 2786e2793 2793

microorganisms after a period of exposure of 5 h. This effectincreases with an increase in metal initial concentrations. Higherconcentrations of light metal ions proved to be toxic also tomicroorganisms. P. aeruginosa and the Bacillaceae bacteria showedmore resistance to metal ions than Brevundimonas spp. Observationof the cell morphology of the Bacillaceae bacteria with scanningelectron microscopy showed (Fig. 13A and B) a reduction in the cellsurface areas in the presence of Ni2þ and Co2þ compared to thecontrol (not exposed to metal ions) cells. This implies that higherconcentrations of metal ions could affect their uptake by bacteriathrough inhibition that reduces surface binding size.

4. Conclusion

Investigation of the adsorption of heavy metal ions (Co2þ andNi2þ) in the presence of light metal ions (Mg2þ and Ca2þ) usingmicrobial biosorbents has allowed to determine the adsorptionbehaviour of the Bacillaceae bacteria and P. aeruginosa; this findingcan be used to facilitate prediction analyses. It was also found that,although the biosorbents preferentially bind to targeted heavymetals during remediation, in the presence of higher concentra-tions of light metal ions, competition for binding sites occurs andtherefore negatively affects the process. The use of microorganismsto sequestrate metal species in metallurgical solutions can bea viable alternative method for the bioremediation of heavy metalion contamination of source waters.

Acknowledgement

Funding for this project received from the National ResearchFoundation (NRF-under the profile MULI-I) and the University ofJohannesburg (Faculty of Engineering and the Built Environment) isgratefully acknowledged. Mine-water used in this study is fromNkomati Nickel Mines (South Africa). The authors are grateful toMintek (South Africa) for their contribution to the SEM study.

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