Biosorption of metals from gold mine wastewaters
by Penicillium Simplicissimum immobilised on
zeolite
E.N. Bakatula, E.M. Cukrowksa, I.M. Weiersbye, C.J. Straker, H. Tutu
University of the Witwatersrand, Johannesburg, South Africa
Over 70 minerals have been identified in the
primary ores: quartz, pyrite, pyrrhotite, galena,
arsenopyrite, gersdofite, sphalerite, urananite…
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)
Rising tide of acid mine water threatens
Johannesburg
“The water is currently around 600 m below the
city’s surface but is rising at a rate of between
0.4 and 0.9 m per day”
- Telegraph, 6 Sep 2010
Gold extraction
• Cyanidation of gold-bearing ores (Elsner’s equation):
4Au + 8CN- + O2 + 2H2O => 4Au(CN)-2 + 4OHAcid mine drainage
2FeS2 + 7O2 + 2H2O => 2FeSO4 + 2H2SO4
4FeSO4 + O2 + 4H2O => 2Fe2O3 + 4H2SO4
Neutralisation
CaCO3 + H2SO4 => CaSO4 + H2O + CO2
Water from slimes
dam collected in water
retain reservoirs
(Courtesy: Prof. T.S. McCarthy)
Broken down pumps result in
leakage of contaminated water
to 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).
Evaporation
Seepage and surface flow
“Reactive transport
modelling”
To underlying aquifer
Leached solution + Solid minerals → Predicted solution
+ “New” contact solution
“New” predicted solution
Barriers for containment of toxic elements
Precipitation barrier
e.g. liming to precipitate elements, pH dependent
Evaporation barrier
e.g. evaporation of salt-laden shallow groundwater
Redox barrier
e.g. precipitating elements using redox differentials
Adsorption barrier
e.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.
Structure of zeolite
Zeolite structure is mainly composed of 3 components: alumino-silicate framework (with a
repeating pore network), exchange cations and water within the pores.
The general formula is: (M2+, M2+)O. Al2O3. gSiO2. zH2O
M+ = Na + or K + ; M2+ = Mg2 +, Ca2 + or Fe2 +
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+).
 Both Si/Al ratio and the cation
contents determine the properties
of most zeolites.
Two main mechanisms are attributed to heavy metal
removal by natural zeolites:
(i) ion exchange and (ii) adsorption
Ion 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
Growth of penic.simp. after 5 days
Light Microscopy (100X)
Penicillium strains are halotolerants (able to grow in presence or absence of
salt).
 Batch and column sorption experiments were done
in living as well as inactive biomass for Co, Cu, Fe,
Hg, Cr, Ni, U and Zn metals (single and multicomponent solutions).
 The concentrations of metals remaining in solution
were determined using ICP-OES.
RESULTS AND DISCUSSION
Zeolite – Composition and characteristics
XRF characterisation of zeolite
Constituent
Value (%)
Surface area: 69 m2/g
Si O2
40.6
Al2O3
32.92
Average pore volume : 0.002 cm3/g
Fe2O3
0.01
Average pore diameter: 150 Å
FeO
0.08
CEC: 61.06 meq/100 g
MnO
0.01
MgO
0.06
CaO
0.03
Na2O
19.92
K2O
0.25
TiO2
0.02
P2O5
0.01
H2O
6.1
Growth curves for Penicillium simp.
100
600
500
pH 2
60
pH 3
pH 4
pH 5
40
pH 6
Harvest (mg)
Harvest (mg)
80
pH3
300
200
100
0
0
5
10
15
Day
Penicillium simp.
20
25
pH4
pH5
20
0
pH2
400
pH6
0
5
10
15
20
25
Day
Zeolite-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 biomass
CEC
C
H
N
S
meq/100 g
%
%
%
%
ZeoliteFungi
82.50
0.388
2.295
0.254
0.102
Natural
zeolite
61.06
0.219
2.209
n.d
n.d
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.
 Characterization 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.
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.
Adsorption studies…..
Effect of contact time
(Zeolite-Living fungi)
Kinetics
25
25
Cu
Cu
20
Cr
U
15
Fe
Ni
10
Cr
Qe (mg/g)
Qe (mg/g)
20
Equilibrium
U
15
Fe
Ni
10
Hg
Hg
Co
5
Co
5
Zn
Zn
0
0
0
50
100
150
200
Time (min)
Single component syst: Ci = 100 mg/L
pH 3 (2.5 g in 500 mL)
0
50
100
150
200
Time (min)
Multi component syst: Ci = 100 mg/L
pH 3 (2.5 g in 500 mL)
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
60
60
50
Cu
50
Cu
Cr
U
Fe
30
Ni
Hg
20
Cr
40
Qe (mg/g)
Qe (mg/g)
40
U
Fe
30
Ni
Hg
20
Co
10
Zn
0
Co
Zn
10
0
0
50
100
150
200
Time (min)
Single component syst: Ci = 100 mg/L
pH 3 (2.5 g in 500 mL)
0
50
100
150
200
Time (min)
Multi components syst: Ci = 100 mg/L
pH 3 (2.5 g in 500 mL)
Inactive microbial biomass frequently exhibits a higher affinity for metal
ions than viable cells, probably due to the absence of competing protons
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.
Kinetic models (Zeolite – Inactive fungi)
Metal
ions
Cu2+
Co2+
Cr3+
Fe2+
Hg2+
Ni2+
UO22+
Zn2+
Pseudo first-order
parameters
Pseudo second-order
parameters
Intraparticle diffusion
parameters
qe
K1 /[min-1]
R2
qe
K2/[g mg-1
min-1]
R2
Kid/[mg g
min]
C
R2
0.022
0.075
0.715
0.168
0.062
0.999
0.005
0.028
0.829
0.014
0.072
0.618
0.182
0.0574
0.998
0.006
0.031
0.928
0.034
0.065
0.794
0.206
0.0505
1.000
0.007
0.034
0.842
0.012
0.011
0.466
0.193
0.054
1.000
0.006
0.032
0.936
0.072
0.029
0.751
0.0305
0.526
0.992
0.002
0.004
0.865
0.002
0.02
0.507
0.183
0.057
1.000
0.006
0.031
0.928
0.033
0.019
0.846
0.0174
0.425
0.773
0.007
0.002
0.982
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.
The film and pore diffusion equations (Df = 0.23 r0 δ qe / t½ and Dp =
0.03 r02 / t½ ) were used to check whether the diffusion step controlled
ion exchange or not .
Df = the film diffusion coefficient (cm2/s), Dp = the pore
diffusion coefficient (cm2/s), r0 = the radius of zeolite , δ = the
film thickness (0,001 cm, assuming the geometry of the
spherical particles) and t½ is the half time for the ionexchange process (min).
The film diffusion coefficient was in the range of 2.03 x 10-6 and 3
x 10-7 cm2/s for the metals studied.
The pore diffusion coefficient was between 3.55 x 10-7 and 0.52 x
10-7 cm2/s.
The metal diffusion through the film is the rate limiting step.
According to Michelson: Df = 10-6 - 10-8 cm2/s and Dp = 10-11 10-13 cm2/s.
Effect of pH
(Zeolite-Living fungi)
Isotherms
Langmuir and Freundlich isotherms
were used to fit the experimental data.
30
30
25
Cu
Cu
25
Cr
Qe(mg/g)
20
U
Fe
15
Ni
10
Hg
Co
5
Zn
0
2
3
4
5
pH
6
7
8
Single component syst: Ci = 500 mg/L
(1 g in 100 mL)
Qe (mg/g)
Cr
U
20
Fe
15
Ni
Hg
10
Co
5
Zn
0
2
3
4
5
6
7
8
pH
Multi-component syst: Ci = 500 mg/L
(1 g in 100 mL)
An increase of AC was observed for U at pH 5 which is close to its hydrolysis
pH.
N
[ i2+ ]TO T =
8 5 .1 9 mM
1
L og C onc .
-1
N i2+
H
N iO
( H )2 (c )
+
-3
NO
i H
-5
+
N i2O H
-7
-9
2
OH
3
4
pH
5
6
Nickel species distribution using Medusa software
3+

Effect of pH
(Zeolite-Inactive fungi)
Isotherms
60
60
Cu
50
Cr
U
40
Fe
30
Ni
20
Hg
10
Co
Qe (mg/g)
Qe (mg/g)
50
Cu
Cr
U
40
Fe
30
Ni
20
Hg
Co
10
Zn
Zn
0
0
2
3
4
5
6
7
8
pH
Single component syst: Ci = 500 mg/L
(1 g in 100 mL)
2
3
4
5
6
7
8
pH
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+ .
Qe (mg/g)
Effect of pH (Natural zeolite)
12
Cu
10
Cr
8
U
6
Fe
Ni
4
Hg
2
Co
0
2
3
4
5
6
7
8
Zn
pH
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. )
25
Qe (mg/g)
Cr
U
15
Fe
10
Ni
Qe (mg/g)
Cu
20
25
Cu
20
Cr
U
15
Hg
5
Co
Co
0
Zn
Zn
20
0
20
30
40
50
60
17.49
-80.57
-6.424
109.9
-75.84
-27.91
-120.4
-113.2
∆ Ho
kJ/mol
104.4
-481.0
-38.32
651.1
-452.4
-166.6
-718.9
-675.7
40
50
60
70
Temperature (oC)
Multicomponent syst: Ci = 100 mg/L, pH = 3
(0.5 g in 100 mL)
Single component syst: Ci = 100 mg/L, pH = 3
Ea
kJ/mol
30
70
Temperature (o C)
Cu
Cr
U
Fe
Ni
Hg
Co
Zn
Ni
10
Hg
5
Fe
∆ Go
kJ/mol
25oC
-17.66
-6.232
-1.513
-25.96
0.986
-5.860
-5.689
-7.373
40oC
-18.09
-12.78
-1.833
-22.24
-5.200
-6.896
-12.75
-19.47
60oC
-19.29
-16.58
-2.484
-16.89
-7.767
-9.978
-20.64
-21.72
Physisorption: 5 ≤ Ea ≤ 40 kJ mol-1
Chemisorption: 40 ≤ Ea ≤ 800 kJ mol-1.
ΔG = -RTln Kd
Kd = qe/Ce
Thermodynamics (Ea, ΔG and ΔH
Effect of Temperature
(Zeolite - Inactive fungi)
calculated from the experimental data)
30
30
Cr
U
20
Fe
15
Ni
Hg
10
Qe (mg/g)
Qe (mg/g)
Cu
Cu
25
25
Cr
20
U
Fe
15
Ni
10
Hg
Co
5
Zn
Co
5
Zn
0
0
20
30
40
50
60
70
20
Single component syst: Ci = 100 mg/L
pH=3 (0.5 g in 100 mL)
Cu
Cr
U
Fe
Ni
Hg
Co
Zn
-1.338
19.80
12.16
10.35
-64.18
-42.62
82.64
3.637
∆H
kJ/mol
-7.986
21.71
-254.4
493.2
118.2
-383.0
61.80
72.56
40
50
60
Temperature (oC)
Temperature (o C)
Ea
kJ/mol
30
25oC
-14.04
-25.75
1.483
-21.28
-16.86
0.460
-19.51
-19.02
Multi-component syst: Ci = 100 mg/L
pH = 3 (0.5 g in 100 mL)
∆G
kJ/mol
40oC
-20.37
-27.29
-1.964
-22.31
-16.30
-3.593
-19.81
-20.22
60oC
-23.08
-29.04
-2.735
-24.65
-16.97
-7.007
-21.11
-21.34
70
Effect of initial concentration (Multi-component system)
Ni
U
Hg
30
25
2.48165
2.48805
2.49045
9.9944 24.98135
10
25
9.9925 24.9926
Hg
Co
Zn
60
Cu
50
Cu
Cr
U
Fe
15
Ni
Hg
10
U
40
Qe (mg/g)
20
Qe (mg/g)
Cr
Fe
30
Ni
Hg
20
Co
Zn
Co
10
Zn
5
0
0
0
0
100
200
300
400
500
600
200
400
600
Concentration (mg/L)
Concentration (mg/L)
Zeolite-Living fungi, pH= 3
(1 g in 50 mL)
Zeolite-Inactive 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.
Desorption %
Desorption studies
120
Cu
100
Cr
80
U
Fe
60
Hg
40
Ni
20
Zn
0
Co
0
2
4
6
8
[HNO 3] M
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
3.8
383.6
pH
SO42(mg/L)
SW2
7.2
19.8
Ci
Cf
%
Fe
6.10
0.018
99.7
Ni
6.00
0.012
99.8
Zn
4.30
0.004
99.9
Ci
Pit water
3.2
1669
Cf
%
Ci
Cf
%
Ci
Cf
%
4.50
0.004
99.9
5.80
0.006
99.9
0.60
< DL
99.8
1.80
0.010
99.4
4.70
0.005
99.9
10.70
0.010
99.9
1.60
0.002
99.9
1.70
0.005
99.7
14.80
0.015
99.9
0.30
0.005
98.3
0.04
< DL
97.5
Hg
0.30
< DL
99
U
0.20
< DL
100
Cr
0.040
%
SW4
5.6
653.6
Ci
1.60
Cf
SW3
4.1
819.4
97.5
Ci = initial metal concentration (mg L-1 ) ; Cf = final conc. % = Removal % (1 g in 50 mL)
Discharge standards for industrial
wastewater (SAWQG)
pH
6–9
Fe
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
After treatment with the zeolite-fungi, the
quality of the effluent complied with the
discharge standards for industrial wastewater.
Conclusions
The biosorbent displayed good adsorption of toxic metals even at low pH
values, making it an ideal sorbent for metals in effluents. Biosorption was
described to be easy, safe, rapid, inexpensive and can be used to recover
heavy metals at very low concentration.
Non viable microbial biomass frequently exhibits a higher affinity for metal
ions than viable cells, probably due to the absence of competing protons
produced during metabolism.
Accumulation of metals from solutions by fungi can be divided into three
categories:
(1) biosorption of metal ions on the surface of fungi.
(2) intracellular uptake of metal ions.
(3) chemical transformation of metal ions by fungi
In the environment, Penicillium simpl. can grow in the silica matrix (tailings
dams) at low pH and adsorption of toxic metals occurs at that pH.
Outlook
 Application to the recovery of metal complexes
(e.g. cyanide complexes) and metalloids (e.g. As)
 Exploring cheaper sources of silica e.g. fly ash,
zeolites, liquid glass
Acknowledgements
• The Carnegie Corporation
• The National Research Foundation
• The Friedel Sellschop Foundation
• AngloGold Ashanti