Biosorption of metals from gold mine wastewaters Biosorption of metals from gold mine wastewaters by by Penicillium Simplicissimum Penicillium Simplicissimum immobilised on immobilised on

zeolitezeolite

E.N. Bakatula, E.M. Cukrowksa, I.M. Weiersbye, C.J. Straker, E.N. Bakatula, E.M. Cukrowksa, I.M. Weiersbye, C.J. Straker, H. TutuH. Tutu

University of the Witwatersrand, Johannesburg, South AfricaUniversity of the Witwatersrand, Johannesburg, South Africa

More than 50 000 tons of gold mined leaving behind more than 244 mine tailings dumps. More than 35 000 tons of gold still remain in

deep resources

4 - 6 billion tons of mine waste

~30 million tons of sulphur (Witkowski and Weiersbye, 1998)

~430 000 tons of low-grade uranium (Winde et al., 2004)

Over 70 minerals have been identified in the primary ores: quartz, pyrite, pyrrhotite, galena, arsenopyrite, gersdofite, sphalerite, urananite…

Rising tide of acid mine water threatens Rising tide of acid mine water threatens JohannesburgJohannesburg

““The water is currently around 600 m below the The water is currently around 600 m below the city’s surface but is rising at a rate of between city’s surface but is rising at a rate of between 0.4 and 0.9 m per day”0.4 and 0.9 m per day”

- Telegraph, 6 Sep 2010- Telegraph, 6 Sep 2010

Acid mine drainageAcid mine drainage

2FeS2FeS22 + 7O + 7O22 + 2H + 2H22O => 2FeSOO => 2FeSO44 + 2H + 2H22SOSO44

4FeSO4FeSO44 + O + O22 + 4H + 4H22O => 2FeO => 2Fe22OO33 + 4H + 4H22SOSO44

NeutralisationNeutralisationCaCOCaCO33 + H + H22SOSO44 => CaSO => CaSO44 + H + H22O + COO + CO22

• Cyanidation of gold-bearing ores (Elsner’s equation):Cyanidation of gold-bearing ores (Elsner’s equation):

4Au + 8CN4Au + 8CN- - + O+ O2 2 + 2H+ 2H22O O =>=> 4Au(CN) 4Au(CN)--2 2 + 4OH+ 4OH--

Gold extractionGold extraction

Water from slimes dam collected in water retain reservoirs

(Courtesy: Prof. T.S. McCarthy)(Courtesy: Prof. T.S. McCarthy)

Broken down pumps result in Broken down pumps result in leakage of contaminated water leakage of contaminated water to natural water bodiesto natural water bodies

Elevated elements include S, Mg, U, Fe, Cu, Mn, Zn, Pb, Ni, As, Cr, U (Coetzee et al., 2003; Naicker et al., 2005; Tutu et al., 2008).

To underlying aquiferTo underlying aquifer

Seepage and surface flowSeepage and surface flow

EvaporationEvaporation

“Reactive transport modelling”

Leached solution + Solid minerals → Predicted solution

+ “New” contact solution “New” predicted solution

Barriers for containment of toxic elements

Precipitation barrierPrecipitation barrier

e.g. liming to precipitate elements, pH dependent

Evaporation barrierEvaporation barrier

e.g. evaporation of salt-laden shallow groundwater

Redox barrierRedox barrier

e.g. precipitating elements using redox differentials

Adsorption barrierAdsorption barriere.g. can be natural or engineered e.g. reactive barriers

Biosorptive properties of fungi: Penicillium simp.

Biosorption - property of biomaterials as bacteria, yeast, fungi, agriculture wastes, etc. to bind and to concentrate metals from aqueous solutions by active (metabolically) and passive modes (physico-chemical pathways)

Metal sorption and accumulation depends on diverse factors, such as pH, temperature, organic matter, ionic speciation and the presence of other ions in solution which may be in competition, etc.

Many potential binding sites are present in fungal cell walls, including chitin, amino, carboxyl, phosphate, sulfhydryl and other functional groups, which may act individually or synergistically to bind cations.

Zeolite structure is mainly composed of 3 components: alumino-silicate framework (with a repeating pore network), exchange cations and water within the pores.

Both Si/Al ratio and the cation contents determine the properties of most zeolites.

The general formula is: (M2+, M2+)O. Al2O3. gSiO2.

zH2O They have a net negative charge due to isomorphous replacement of Si4+ by Al3+

and this negative charge is balanced by the extra-framework cations (Na+, K+, Ca2+

and Mg2+).

M+ = Na + or K + ; M2+ = Mg2 +, Ca2 + or Fe2 +

Structure of zeoliteStructure of zeolite

Two main mechanisms are attributed to heavy metal removal by natural zeolites: (i) ion exchange and (ii) adsorptionIon exchange properties of zeolites are due to the weakly bonded extra-framework cations which are mobile and easily exchanged with solution cations.

It is a good adsorbent and ion-exchange agent which is determined by its unique structure and large specific surface area. Thus, it has found wide applications in wastewater treatment research.

The zeolite was selected due to its capacity for immobilising micro-organisms and to its large surface area.

EXPERIMENTAL WORK

Penicillium simpliccissimum was maintained on the following solid media: 40 g L-1 Potato Dextrose Agar (PDA) and 50 g L-1 Malt Extract Agar (MEA).

For experimental purpose, cultures were grown at 25oC in liquid medium at pH 2 to 7, comprising the following: (NH4)2SO4, KCl, MgSO4.7H2O, EDTA-Fe, ZnSO4.7H2O, MnSO4.H2O, CaCl2.2H2O, K2HPO4, yeast, glucose in 1 L of sterilised deionised water.

1 g of zeolite was added to the medium, the mixture was inoculated after autoclaving. The immobilized biomass was separated from the broth by filtration and washed with deionised water.

Penicillium simp. strains

Light Microscopy (100X)Growth of penic.simp. after 5 days

Penicillium strains are halotolerants (able to grow in presence or absence of salt).

Batch and column sorption experiments were done Batch and column sorption experiments were done in living as well as inactive biomass for Co, Cu, Fe, in living as well as inactive biomass for Co, Cu, Fe, Hg, Cr, Ni, U and Zn metals (single and multi- Hg, Cr, Ni, U and Zn metals (single and multi- component solutions).component solutions).

The concentrations of metals remaining in solution The concentrations of metals remaining in solution were determined using ICP-OESwere determined using ICP-OES..

RESULTS AND DISCUSSIONRESULTS AND DISCUSSION

Zeolite – Composition and characteristics Zeolite – Composition and characteristics

XRF characterisation of zeolite

Constituent Value (%)

Si O2 40.6

Al2O3 32.92

Fe2O3 0.01

FeO 0.08

MnO 0.01

MgO 0.06

CaO 0.03

Na2O 19.92

K2O 0.25

TiO2 0.02

P2O5 0.01

H2O 6.1

Surface area: 69 m2/g

Average pore volume : 0.002 cm3/g

Average pore diameter: 150 Å

CEC: 61.06 meq/100 g

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Harves

t (m

g)

Day

pH 2

pH 3

pH 4

pH 5

pH 6

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500

600

0 5 10 15 20 25Harve

st (m

g)

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pH2

pH3

pH4

pH5

pH6

Penicillium simp. Zeolite-Penicillium simp.

Growth curves for Growth curves for Penicillium simp.Penicillium simp.

The growth of fungus showed ~ 10-fold increase in biomass when immobilized on zeolite (600 mg/g at pH 4).

Elemental analysis of the biomassElemental analysis of the biomass

CEC C H N S

meq/100 g % % % %

Zeolite-Fungi

82.50 0.388 2.295 0.254 0.102

Natural zeolite

61.06 0.219 2.209 n.d n.d

Characterization of the biomassCharacterization of the biomass

SEM analysis revealed that the biofilm covered uniformly the zeolite surface.

Infrared spectra of the biomass pointed to more compounds released after 10 days of inoculation and confirmed the presence of functional groups which include: hydroxyl, carbonyl, carboxyl, amine, imidazole, phosphate groups.

The % of C was high in the biomass; these results confirm the presence of organic compounds released by the fungi as revealed by with the IR spectra.

Adsorption studies

Kinetic models and sorption isotherms

Mathematical models (Pseudo 1st and 2nd order and Intraparticle diffusion models) were employed for the prediction and comparison of the binding capacity and to design the sorption process.

The ‘isotherm’, a curve describing the retention of a substance on a solid at various concentrations, is a major tool to describe and predict the mobility of this substance in the environment. Langmuir and Freundlich isotherms are the most commonly used.

Models have an important role in technology transfer from a laboratory scale to industrial scale.

Effect of contact time (Zeolite-Living fungi) Kinetics

Adsorption studies…..

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Time (min)Qe (m

g/g)

CuCrUFeNiHgCoZn

Single component syst: Ci = 100 mg/L pH 3 (2.5 g in 500 mL)

Multi component syst: Ci = 100 mg/L pH 3 (2.5 g in 500 mL)

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Time (min)

Qe

(mg/

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CuCrUFeNiHgCoZn

Kinetics

Equilibrium

The biosorption was fast (10 minutes) and the kinetics includes 2 phases:

(1) associated with the external cell surface and (2) intra-cellular accumulation/ reaction depending on the cellular metabolism.

Effect of contact time (Zeolite-Inactive fungi)

Kinetics

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Time (min)

Qe (m

g/g)

CuCrUFeNiHgCoZn

Multi components syst: Ci = 100 mg/L pH 3 (2.5 g in 500 mL)

Single component syst: Ci = 100 mg/L pH 3 (2.5 g in 500 mL)

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Time (min)

Qe

(mg/

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CuCrUFeNiHgCoZn

Inactive microbial biomass frequently exhibits a higher affinity for metal Inactive microbial biomass frequently exhibits a higher affinity for metal ions than viable cells, probably due to the absence of competing protons ions than viable cells, probably due to the absence of competing protons produced during metabolism.produced during metabolism.

Kinetics of metal ion sorption governs the rate, which determines the residence time and it is one of the important characteristics defining the efficiency of an adsorbent.

Metal ions

Pseudo first-order parameters

Pseudo second-order parameters

Intraparticle diffusionparameters

qe K1 /[min-1] R2 qe K2/[g mg-1 min-1]

R2 Kid/[mg g min]

C R2

Cu2+ 0.022 0.075 0.715 0.168 0.062 0.999 0.005 0.028 0.829

Co2+ 0.014 0.072 0.618 0.182 0.0574 0.998 0.006 0.031 0.928

Cr3+ 0.034 0.065 0.794 0.206 0.0505 1.000 0.007 0.034 0.842

Fe2+ 0.012 0.011 0.466 0.193 0.054 1.000 0.006 0.032 0.936

Hg2+ 0.072 0.029 0.751 0.0305 0.526 0.992 0.002 0.004 0.865

Ni2+ 0.002 0.02 0.507 0.183 0.057 1.000 0.006 0.031 0.928

UO22+ 0.033 0.019 0.846 0.0174 0.425 0.773 0.007 0.002 0.982

Zn2+ 0.005 0.014 0.625 0.164 0.063 0.999 0.005 0.028 0.927

The pseudo 2nd order model (dqt / dt = k2 (qe – qt)2 fits better the biosorption kinetics.

Kinetic models (Zeolite – Inactive fungi)

The film diffusion coefficient was in the range of 2.03 x 10The film diffusion coefficient was in the range of 2.03 x 10-6-6 and 3 and 3 x 10x 10-7-7 cm cm22/s for the metals studied./s for the metals studied.

The pore diffusion coefficient was between 3.55 x 10The pore diffusion coefficient was between 3.55 x 10-7-7 and 0.52 x and 0.52 x 1010-7-7 cm cm22/s./s.

The metal diffusion through the film is the rate limiting step. The metal diffusion through the film is the rate limiting step. According to Michelson: According to Michelson: DDff = 10 = 10-6-6 - 10 - 10-8-8 cm cm22/s /s andand D Dpp = 10 = 10-11-11 - - 10-10-

1313 cm cm22/s/s..

The film and pore diffusion equationsThe film and pore diffusion equations ((DDff = 0.23 r = 0.23 r00 δ q δ qee / t / t½½ andand DDpp = = 0.03 r0.03 r00

22 / t / t½½ ) ) were used to check whetherwere used to check whether the the diffusion step controlled diffusion step controlled ion exchangeion exchange or not or not ..

DDff == the film diffusion coefficient (cm the film diffusion coefficient (cm22/s),/s), DDpp = = the pore the pore diffusion coefficient (cmdiffusion coefficient (cm22/s)/s), , rr00 = = the radius of zeolite , δthe radius of zeolite , δ = = the the film thickness (0,001 cm, assuming film thickness (0,001 cm, assuming the the geometry of the geometry of the spherical particles) and spherical particles) and tt½½ is the half time for the ion-is the half time for the ion-exchange process (min). exchange process (min).

Effect of pH (Zeolite-Living fungi)

IsothermsLangmuir and Freundlich isotherms were used to fit the experimental data.

Single component syst: Ci = 500 mg/L Multi-component syst: Ci = 500 mg/L

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2 3 4 5 6 7 8pH

Qe (m

g/g)

CuCrUFeNi HgCoZn

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2 3 4 5 6 7 8pH

Qe(

mg/

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Cu

CrU

Fe

Ni Hg

CoZn

An increase of AC was observed for U at pH 5 which is close to its hydrolysis pH.

(1 g in 100 mL) (1 g in 100 mL)

Nickel species distribution using Medusa software

2 3 4 5 6- 9

- 7

- 5

- 3

- 1

1L

og C

onc.

p H

H +N i 2 +

N i 2 O H 3 +

N i O H +

O H

N i ( O H ) 2 ( c )

[ N i 2 + ] T O T = 8 5 . 1 9 m M

Effect of pH (Zeolite-Inactive fungi) Isotherms

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Qe (m

g/g)

pH

Cu

Cr

U

Fe

Ni

Hg

Co

Zn0

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2 3 4 5 6 7 8

Qe (m

g/g)

pH

Cu

Cr

U

Fe

Ni

Hg

Co

Zn

Single component syst: Ci = 500 mg/L (1 g in 100 mL)

Multi-component syst: Ci = 500 mg/L (1 g in 100 mL)

The presence of Fe2+ and Zn2+ was found to influence uranium uptake in the multi-component system. AC was constant ( 40-50 mg/g) at the pH range 2 - 7 for Cu2+, Fe2+, Hg2+, Co2+ , Zn2+ .

Effect of pH (Natural zeolite)

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Qe (m

g/g)

pH

Cu

Cr

U

Fe

Ni

Hg

Co

Zn

Natural zeolite, single component syst. Ci = 500 mg/L (1 g in 100 mL)

Competition between cations and protons for binding sites means that sorption of metals like Cu, Cr, Ni, Co and Zn is often reduced at low pH values.

Effect of Temperature (Zeolite-Living fungi)

Thermodynamics (Ea, ΔGo and ΔHocalculated from the experimental data. )

Single component syst: Ci = 100 mg/L, pH = 3 Multicomponent syst: Ci = 100 mg/L, pH = 3 (0.5 g in 100 mL)

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Temperature (oC)

Qe

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CuCrUFeNiHgCoZn

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Qe

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Cu

Cr

U

Fe

Ni

Hg

Co

Zn

  Ea ∆ Ho   ∆ Go    kJ/mol kJ/mol   kJ/mol  

      25oC 40oC 60oCCu 17.49 104.4 -17.66 -18.09 -19.29Cr -80.57 -481.0 -6.232 -12.78 -16.58U -6.424 -38.32 -1.513 -1.833 -2.484Fe 109.9 651.1 -25.96 -22.24 -16.89Ni -75.84 -452.4 0.986 -5.200 -7.767Hg -27.91 -166.6 -5.860 -6.896 -9.978Co -120.4 -718.9 -5.689 -12.75 -20.64Zn -113.2 -675.7 -7.373 -19.47 -21.72

Physisorption: 5 ≤ Ea ≤ 40Physisorption: 5 ≤ Ea ≤ 40 kJ mol kJ mol-1-1 Chemisorption: 40 ≤ Ea ≤ 800Chemisorption: 40 ≤ Ea ≤ 800 kJ mol kJ mol-1-1..

ΔΔG = -RTln KG = -RTln Kdd

KKdd = q = qee/C/Cee

Effect of Temperature (Zeolite - Inactive fungi)

Thermodynamics (Ea, ΔG and ΔH

calculated from the experimental data)

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Qe

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CuCrUFeNiHgCoZn

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Qe

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CuCrUFe

NiHgCoZn

Single component syst: Ci = 100 mg/L pH=3 (0.5 g in 100 mL)

Multi-component syst: Ci = 100 mg/L pH = 3 (0.5 g in 100 mL)

  Ea ∆ H   ∆ G    kJ/mol kJ/mol   kJ/mol  

      25oC 40oC 60oCCu -1.338 -7.986 -14.04 -20.37 -23.08Cr 19.80 21.71 -25.75 -27.29 -29.04U 12.16 -254.4 1.483 -1.964 -2.735Fe 10.35 493.2 -21.28 -22.31 -24.65Ni -64.18 118.2 -16.86 -16.30 -16.97Hg -42.62 -383.0 0.460 -3.593 -7.007Co 82.64 61.80 -19.51 -19.81 -21.11Zn 3.637 72.56 -19.02 -20.22 -21.34

Effect of initial concentration (Multi-component system)

Zeolite-Inactive fungi, pH= 3 (1 g in 50 mL)

Zeolite-Living fungi, pH= 3 (1 g in 50 mL)

The metal uptake was constant for all the metals studied except for Ni, Hg and U.

For these metals, the uptake decreases until an initial concentration of 200 mg/ L for Ni and Hg; 400 mg/ L for U, most probably because of the xenobiotic effect.

Ni 2.48165 9.9944 24.98135 HgU 2.48805 10 25 Co

Hg 2.49045 9.9925 24.9926 Zn

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Cr

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Hg

Co

Zn

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Zn

Desorption studiesDesorption studies

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Des

orpt

ion

%

[HNO3] M

Cu

Cr

U

Fe

Hg

Ni

Zn

Co

Zeolite- Inactive fungi, Ci = 100 mg/L

The metal loaded in the biomass can potentially be desorbed in order to regenerate the biosorbent and to reclaim valuable metals.

This biosorbent was used 5 times without any loss of its adsorption properties.

Application on mine effluent

Sampling site

SW1 SW2 SW3 SW4 Pit waterpH 3.8 7.2 4.1 5.6 3.2SO4

2-

(mg/L)383.6 19.8 819.4 653.6 1669

Ci Cf % Ci Cf % Ci Cf % Ci Cf % Ci Cf %

Fe 6.10 0.018 99.7 4.50 0.004 99.9 5.80 0.006 99.9 0.60 < DL 99.8

Ni 6.00 0.012 99.8 1.60 0.040 97.5 1.80 0.010 99.4 4.70 0.005 99.9 10.70 0.010 99.9

Zn 4.30 0.004 99.9 1.60 0.002 99.9 1.70 0.005 99.7 14.80 0.015 99.9

Cr 0.30 0.005 98.3 0.04 < DL 97.5

Hg 0.30 < DL 99

U 0.20 < DL 100

After treatment with the zeolite-fungi, the quality of the effluent complied with the discharge standards for industrial wastewater.

Discharge standards for industrial wastewater (SAWQG)pH 6 – 9Fe 0.3 mg L-1

Ni 0.05 mg L-1

Zn 5 mg L-1

Cr 0.01 mg L-1

Hg 0.002 mg L-1

Ci = initial metal concentration (mg L-1 ) ; Cf = final conc. % = Removal % (1 g in 50 mL)

ConclusionsConclusions

The biosorbent displayed good adsorption of toxic metals even at low pH The biosorbent displayed good adsorption of toxic metals even at low pH values, making it an ideal sorbent for metals in effluents. Biosorption was values, making it an ideal sorbent for metals in effluents. Biosorption was described to be easy, safe, rapid, inexpensive and can be used described to be easy, safe, rapid, inexpensive and can be used to recover to recover heavy metals at very low concentration. heavy metals at very low concentration.

Non viable microbial biomass frequently exhibits a higher affinity for metal Non viable microbial biomass frequently exhibits a higher affinity for metal ions than viable cells, probably due to the absence of competing protons ions than viable cells, probably due to the absence of competing protons produced during metabolism. produced during metabolism.

Accumulation of metals from solutions by fungi can be divided into three Accumulation of metals from solutions by fungi can be divided into three categories:categories: (1) biosorption of metal ions on the surface of fungi.(1) biosorption of metal ions on the surface of fungi. (2) intracellular uptake of metal ions.(2) intracellular uptake of metal ions. (3) chemical transformation of metal ions by fungi (3) chemical transformation of metal ions by fungi

In the environment, In the environment, Penicillium simplPenicillium simpl. can grow in the silica matrix (tailings . can grow in the silica matrix (tailings dams) at low pH and adsorption of toxic metals occurs at that pH.dams) at low pH and adsorption of toxic metals occurs at that pH.

OutlookOutlook

Application to the recovery of metal complexes Application to the recovery of metal complexes (e.g. cyanide complexes) and metalloids (e.g. As)(e.g. cyanide complexes) and metalloids (e.g. As)

Exploring cheaper sources of silica e.g. fly ash, Exploring cheaper sources of silica e.g. fly ash, zeolites, liquid glasszeolites, liquid glass

AcknowledgementsAcknowledgements

• The Carnegie Corporation

• The National Research Foundation

• The Friedel Sellschop Foundation

• AngloGold Ashanti