Acidithiobacillus caldus, Leptospirillum spp, Ferroplasma spp and Sulphobacillus
spp. for use in cobalt and copper removal from water.
N.P. Dlamini1,2, A. F. Mulaba – Bafubiandi,1 B.B. Mamba2
1Minerals
Processing and Technology Research Group, Department of Extraction
Metallurgy, Faculty of Engineering and the Built Environment, University of
Johannesburg, PO Box 526, Wits 2050, South Africa.
2Department
of Chemical Technology, Faculty of Science, University of
Johannesburg, P.O Box 17011, Johannesburg 2028, South Africa
Tel (011)5596215
Fax (011)5596194
*Email amulaba@uj.ac.za
Abstract
Bacteria from the genus Acidithiobacillus, Leptospirillum, Ferroplasma,
Sulphobacillus and Bacillus are often associated with water remediation
(Rzhepishevska, 2008). In the present study a consortium of Acidithiobacillus caldus,
Leptospirillum spp., Ferroplasma spp and Sulphobacillus spp was cultured and used
to remove copper and cobalt from aqueous solution. Acidithiobacillus caldus,
Leptospirillum spp, Ferroplasma spp and Sulphobacillus spp reclaim both copper and
cobalt from their sulphate synthetic solutions. The bacteria have successfully removed
up to 54 % of copper from 0.07M and 39 % from 0.66M copper sulphate solutions.
They also removed up to 23 % and 21 % of cobalt from 0.07M and 0.66M cobalt,
respectively from cobalt sulphate solutions.
Introduction
Recent progress in the use of microorganisms for industrial applications promotes not
only the bacterial leaching in mineral bearing ores but also the microbial treatment of
metal contaminated water (Brierley, 1982). Water contamination is a very serious
problem which is increasing in magnitude. For example, with respect to ground water
contamination, about 10 % of the population in South Africa uses ground water for
drinking and a large number of industries and agricultural systems depend on clean
and safe groundwater supplies (Botha, 2006). Contamination of ground water can
come from garbage dumps, industrial waste dumps, mine dumps, septic tanks and
many more(Vullo et al, 2008). Considerable effort is being expended to remediate this
problem. Contamination of groundwater resources by heavy metals in South Africa is
also on the increase, because of the large and small scale mining activities taking
place in the country.( Mail & Guardian online newspaper, SA may face watercontamination crisis Feb 03 2008 10:38)
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Heavy metals which contaminate water bodies include copper, cobalt, iron,
manganese, chromium, nickel, gold, lead and many other metals. Although most of
these metals have vital human bodily functions, their ingestion in high quantities can
result in serious health implications which may be fatal. It is therefore evident that
concentrations of these metals in drinking water have to be closely monitored. Table
1 shows the allowable concentrations of copper and cobalt in drinking water
according to the South African National Standards (SANS), 2006
Determinant
Copper
Cobalt
unit
µg
µg
Recommended limit
< 500
< 1000
Table 1: showing the allowable concentrations of copper and cobalt in drinking water
according to SANS (dwarf, 2005, Botha 2006)
Most commonly reported mechanisms for metal removal from solution are adsorption
(biosorption, chemisorption or specific adsorption) and precipitation in the form of
hydroxides (Fe3+, Cr3+, and Al3+), carbonates (Fe2+, Mn2+) or sulphides (Pb2+, Co2+,
Cd2+, Cu2+, Ni2+, Fe2+, Zn2+) (Bojinova et al., 2006). While these methods have been
successful in removing metal species from water, the challenge for research is the use
of novel techniques that are less costly, more efficient and environmentally friendly
for example convectional methods can be used for high grade ores effectively while
for low grade ores these methods can be expensive. With respect to the environment,
the removal of metals from solutions emanating from metallurgical and mining
operations would serve to protect ground and surface water, especially when taking
into consideration repercussions from acid mine drainage. The use of microorganisms for the removal of metals from metallurgical aqueous solutions has to the
best of our knowledge not been reported largely. Recently a report has been published
where bacterial species such as Pseudonomonas species have demonstrated capacity
to remove significant amounts of heavy metals by biosorption (Donalkova et al.,
2005; Vullo et al., 2008), In the present study, copper and cobalt removal has been
performed using micro-organisms to ascertain their metal removal efficiency. What is
unique about the study is that the microorganisms will be utilised in removing metal
species not just from water but also from aqueous metallurgical solutions and a real
leached ore where the possibility of interference by other metal ions present in such
solutions may be possible. In investigating the efficiency of the use of microorganisms for the removal of cobalt and copper from metallurgical solutions, the
preliminary results to be presented in this study have emanated from the removal of
these metals from synthetic aqueous sulphate solutions.
EXPERIMENTAL
Methods
A mixed strain culture of bacteria was sourced from Mintek-Randburg, South Africa.
The culture contained four types of bacterial strains namely Acidithiobcillus caldus,
Leptospirillum spp, Ferroplasma spp and Sulphobacillus spp. This was then grown in
a culture media known as Postgate’s medium C (Postgate, 1984). This medium
contained in (g/L) 1.0 (NH4)2SO4, 0.1 KCl, 0.5 K2HPO4, 0.5 MgSO4, 8.5 elemental
Sulphur, 7.5 FeSO4.7H2O. That is, 20ml of culture (mixed strain sample) was mixed
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with 100ml of nutrient medium and thoroughly shaken to mix in a 1000ml volumetric
flask as illustrated in figure 3 below. This was kept at constant stirring using a
magnetic stirrer and the temperature was maintained at 36oC and the pH constantly
monitored to be between 1.5 and 2.0. 20ml of the mixed strain bacteria were
inoculated into Cobalt and Copper sulphate solutions and extraction was allowed to
take place at 36oC. Analysis was done using the atomic absorption spectrophotometer
(AAS) before and after removal.
Identification and distribution Analysis
The microorganisms were viewed under the fluorescent microscope to verify if the
mixed strain culture really contained the microorganisms that were thought to be
present. This was done by diluting the bacteria with nutrient medium (1: 90) and
increasing the magnification of the microscope to view closely single bacterial
cells.To observe their distribution and to note the most abundant species, the culture
medium was diluted with the nutrient medium 5X (1ml bacteria: 20ml nutrient
media), 10X (1ml bacteria: 40ml nutrient media) and 20X (1ml bacteria: 80ml
nutrient media) for clarity under the microscope since it was dense i.e. the populations
were high.
Slurry Analysis
The mixed strain also had slurry at the bottom of the culture flask. To ensure that the
composition of the slurry did not alter the results, this was studied using the scanning
electron microscope (SEM). The slurry was dried and then coated with gold before
analysis. SEM images and composition of slurry were obtained.
Copper and Cobalt removal
Copper and cobalt sulphate solutions (0.07M, 0.33M and 0.66M) were prepared in the
laboratory. The mixed strain bacteria (20 mL) were then inoculated into the prepared
cobalt and copper sulphate solutions and the removal of the metals from solutions was
allowed to take place at room temperature. Analysis of the solutions after Co(II) and
Cu(II) removal was done using the Atomic Absorption Spectrophotometer after 1, 3,7
and 12 days respectively.
RESULTS AND DISCUSSION
Identification and distribution Analysis
The fluorescence microscopy results could only identify four out of the five bacterial
strains believed to be in the mixed culture and these are Acidithiobcillus caldus,
Leptospirillum spp, Ferroplasma spp and Sulphobacillus spp as shown below.
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IDENTIFIED
SHAPE
MICROORGANISM
Sulphobacillus spp
Leptospirillum spp
Ferroplasma spp
Acidithiobacillus
caldus
SIZE
rod
GRAM
positive and
GRAM
negative
Negative
0.3-0.8 µm
wide and0.62.0 µm long
spiral
0.3-0.5 µm
Negative
wide and 0.93.0 µm long
pleomorphic Varying width Negative
and 0.4-0.9 µm
long
rod
extremely short Positive
AUTOTROPHIC OR
CHEMOLITROPHIC
Strictly
chemolithotrophic and
stricly autotrophic.
Strictly
chemolithotrophic and
strictly autotrophic.
Strictly
chemolithotrophic and
strictly autotrophic.
Partially
chemolithotrophic
Table 2: Characteristics of identified microorganisms
Figure 1: Micrograph of the identified microorganisms (10X magnification;1cm=80
µm.)
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Distribution Analysis
Figure 2: Micrograph of the identified microorganisms (5X magnification; 1cm=200
µm.)
This micrograph shows a 5X dilution i.e. the bacterial strains suspended in five times
more of the required amount of nutrient medium. In this micrograph the bacteria are
clumped together and it is not easy to identify the various strains present. The scale of
this micrograph is 1:200µm.
Figure 3: Figure 2: Micrograph of the identified microorganisms (10X
magnification; 1cm=80 µm.)
This micrograph shows a 10X dilution i.e. the bacterial strains suspended in ten times
more of the required amount of nutrient medium. In this micrograph the bacteria are
quite manageable with a few clusters though. The scale of this micrograph is1: 80 µm.
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Figure 4: Micrograph of the identified microorganisms (20X magnification; 1cm=35
µm.)
This micrograph shows a 20X dilution i.e. the bacterial strains suspended in twenty
times more of the required amount of nutrient medium. In this micrograph the bacteria
are perfectly spaced with none adhearing to the other to an extent that makes
identification difficult. The scale of this micrograph is 1: 35µm.
From the micrographs above it is evident that the most abundant of all four microbes
is Ferroplasma spp which are mostly pleomorphic cells and Sulphobacillus spp which
are rod shaped a few Leptospirillum spp and Acidithiobacillus caldus were also
observed.
Slurry analysis
X 300 Magnification
X2 300 Magnification
Figure 5: SEM images of slurry
The slurry SEM images show some crevices which probably are due to burrowing of
the microorganisms.
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Composition of slurry
Figure 6: SEM result for slurry composition
From the SEM analysis it appears that the slurry is made up of clayey material. This
indicates the living milieu of the above bacterium. The EDX results indicate that A
contained 68% silicon, 26 % aluminum 8% potassium and some traces of iron and
magnesium, B on the other hand had 82 % silicon 9% aluminum and traces of Iron,
magnesium and molybdenum. It did not contain any copper or cobalt thus is wouldn’t
alter the concentrations thereof.
Copper and cobalt removal Results
The kinetics of Cu and Co removal is expressed in Figure 7 and Figure 8 below.
Concentration (M)
0.07
0.33
0.66
% removal
after 1 day
Cu
Co
18
16
20
13
16
11
% removal
after 3 days
Cu
Co
34
21
30
18
32
18
% removal
after 7 days
Cu
Co
54
23
43
21
38
21
% removal
after 12 days
Cu
Co
54
23
44
20
39
22
Table 3: Percentage of Cu and Co removal
As illustrated in Table 3 above, the table removal is highest with the low concentrated
solutions for both copper and cobalt. This can be shown graphically as shown in the
figures below.
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Figure 7: Graph showing Cu % removal using mixed strains at room temperature
Figure 8: Graph showing Co % removal using mixed strains at room temperature
Kinetics
Removal rate =
∆x
∆T
=
∆%
∆days
After day 1 for 0.07M Cu = 18% /day
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Concentration (M)
0.07
0.33
0.66
Removal rate
after 1 day
Cu
Co
18
16
20
13
16
11
Removal rate
after 3 days
Cu
Co
8 2.5
5 2.5
16 3.5
Removal rate
after 7 days
Cu
Co
5
2
3.25 0.75
2 0.75
Removal rate
after 12 days
Cu
Co
0
0
0.2 -0.2
0.2
0.2
Table 4: removal rates of Cu and Co in %/day
As presented in table 3, Cu removal is higher (54%) for lower concentrations
(0.07M). Cobalt also shows higher removal (23 %) in the low concentration (0.07M).
Cobalt in general has shown less removal by the mixed strain of Acidithiobcillus
caldus, Leptospirillum spp, Ferroplasma spp and Sulphobacillus spp. There is need to
mix the Cu and Co as it appears in the natural to observe the effect each one has on
the other. This is also confirmed by the removal rates as shown in Table 4. This table
shows that after a day the removal rate is high and it increases until a certain point
where it gradually decreases and then eventually stops.
From the obtained results above one can conclude that the mixed strain bacteria can
extract both copper and cobalt metals. This trend continues until after 7days and the
extraction rate becomes almost insignificant or drops. This can be explained using the
bacterial growth curve shown in below;
Figure
9:
Bacterial
growth
factories.com/content/4/1/13)
curve
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(http//www.microbial
cell
The curve illustrates the fact that the transfer of bacteria from one medium to another,
where there exist chemical differences between the two media, typically results in a
lag in cell division. This lag in division is associated with a physiological adaptation
to the new environment, by the cells, prior to their resumption of division. Lag phase
is followed by log phase during which binary fission occurs. Stationary phase is a
steady-state equilibrium where the rate of cell growth (division) is exactly balanced
by the rate of cell death. Stationary phase, in a standard bacterial growth curve, is
followed by a “die-off” of cells. Cell death in bacteria cultures means that the cells are
unable to resume division following their transfer to new environments. Typically this
die-off occurs exponentially, i.e., such that cell number graphed against time, using a
semi-log scale for cell number, results in a straight line. This can be interpreted as
follows; after a day, the microorganisms were still in the log phase and cell division
was rapid while after 7 days the bacterial cells were passing the stationary phase into
the decline phase, resulting in a few being able to absorb the metals thus the
stagnancy or drop in the rate at which the metals are removed.
CONCLUSIONS
From the preliminary results obtained thus far, it can be concluded that
microorganisms tend to remove or extract more metal ions at low concentrations and
shorter periods. . From the results generated thus far, it is evident that the mixed strain
of Acidithiobcillus caldus, Leptospirillum spp, Ferroplasma spp and Sulphobacillus
spp reclaim both copper and cobalt from their sulphate synthetic solutions. The
bacteria have successfully removed up to 54 % of copper from 0.07M and 39% from a
0.66M copper sulphate solutions. They also removed up to 23% and 22% from 0.07M
and 0.66M cobalt, respectively from cobalt sulphate solutions.
ACKNOWLEDGEMENTS
The authors wish to thank Mintek (South Africa) for providing the bacteria. Funding
from the National Research Foundation (NRF) and the University of Johannesburg is
gratefully acknowledged.
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