Journal of Mi nerals & Materials Characteriza tion & Engineering, V ol. 10, No.9, pp.817-825, 2011
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817
Laboratory Studies on Phosphorus Removal from Nigeria’s Agbaja Iron Ore
by Bacillus Subtilis
C. N. Anyakwo1 and O.W. Obot2
1Department of Metallurgical and Materials Engineering, Federal University of Technology,
Owerri, Nigeria. Email: [e-mail]
2Department of Mechanical Engineering, Faculty of Engineering, University of Uyo, Nigeria.
ABSTRACT
The investigation into whether or not Bacillus subtilis can remove phosphorus from the
Nigerian Agbaja iron ore was carried out with careful monitoring of the population of the
removing agent as well as pH of the system. 1.00mm/0.50mm, 0.50mm/0.25mm, and
0.25mm/0.125mm ore fractions obtained from sieving of the crushed iron ore in Shital Test
kits, were used in sub-merged cultur e of nutrien t broth (NB) medium for 10 weeks. B. s ubtili s
which was part of the rich microflora found on the ore surface was cultivated in nutr ient-rich
media and later inoculated in sterilized 100ml of NB in 250ml conical flask and 1g of each of
the equally sterilized ore fractions was added. At weekly intervals, a set of samples was
removed, treated through series of chemical reactions to obtain ammonium phospho-
molybdate precipitate which was back-titrated with 0.1 N-HCl to determine the amount of
phosphorus left in samples and consequently, the amount removed. The laboratory
investigations found out that B. subtilis has the capability to remove phosphorus from the
Nigerian Agbaja iron ore, recording an impressive average of 65.73% P . Also found out was
the systematic reduction in bacterial cells count in colony forming unit per mililitre, the
initial load 3.4x105 cfu/ml increased to 4.8x107 cfu/ml from where it declined to 1.3x106
cfu/ml, which justified the pH trend observed during the process of cumulative phosphorus
removal. The reduction in microbial activity may be attributed to antimicrobial components
of the ore, pyrite, and other heavy metals which may have affected the phosphorus uptake
from ore.
1. INTRODUCTION
In 2007 the world iron ore production rose by 11% to 1.640 billion tons. It was expected that
in 2008 and 2009 the trend was likely to continue. According to the latest report released by

818 C. N. Anyakwo and O.W. Obot Vol.10, No.9
the United Nations Conference on Trade and Development (Unctad), it was found out that
world production of iron ore fell by 6.2% in 2009 to 1.52 billion tons [1].
Iron ore is one of the solid minerals found in the Federal Republic of Nigeria. 20 iron ore
deposits have been documented [2]. The Nigerian Agbaja iron ore is the largest of all the
deposits and estimated to be about 1.2 billion tons [3]. The principal constituent mineral is
goethite with minor hematite, maghematite, siderite, quartz, kaolinite and pyrite [2]. A recent
analysis of Agbaja iron ore using AAS (UniCam 939), calorimetry and titrimetry methods is
presented in Table 1.
Table 1 The Nigerian Agbaja Iron Ore Analysis
Sample Fe
% SiO2 % P2O5 % MgO % Cu2O % ZnO % LOI % S % MnO2 % Al2O3 %
Ore
sample 51.50 0.57 1.25 0.08 0.005 0.091 8.01 3.25 0.001 34.77
Agbaja iron ore which analyzed 43-51.50%F e is very rich in phosphorus (1.5-3.0%P2O5) and
the phosphorus is uniformly associated with the iron minerals [4, 5, 6, 7]. Phosphorus is a
deleterious inclusion that is responsible for steel brittleness causing it to fracture and snap at
very low stress values, also associated with phosphorus are the problems of strong primary
segregation during solidification of castings and the formation of high phosphorus brittle
streaks between metal grains thereby impeding plastic deformation. Thus, for high quality
steels, the phosphorus acceptable level is in the range 0.03-0.02 Wt% or even less [8].
Apart from the high phosphorus status, the Agbaja iron ore is also associated with
beneficiation difficulties [9, 10, 11] w hich, after many attempts by research ers, resulted in it s
abandonment on grounds of above technical reasons. In mineral processing the use of
microbes has gained significant popularity and the works of researchers in this area are well
documented. This evolving trend popularly called ‘‘biobeneficiation’’ and which has been
defined as – a biological process in which the microorganisms used produce as a consequence
of their metabolism, a ch emical by -produ ct (mineral acids, or ganic acid s, poly mers, enzy mes,
chelating agents, etc) is fast gaining a wider applicab ility [sic]. The by-product in turn attack
the gangue minerals contained in the ore, dissolving them and thus producing their selective
removal [12]. Using the bacterium Burkholderia caribensis isolated from a Brazilian high-
phosphorus iron ore [13], a team of researchers were able to mobilize between 5-20% of the
phosphorus originally contained in the ore in 21 days of treatment in shake-flask cultures.
Similarly in another work, Aspergillus niger also isolated from the same Brazilian high-
phosphorus ore was employed to remove phosphorus from the same ore and again in 21 days
between 13.8-33.2% was achieved [14]. In the beneficiation of iron ore slime Aspergillus
niger and Bacillus circulans were able to remove alumina with B. circulans and A. niger
showing 39 and 38 percents, respectively of alumina removal after 6 and 15 days,
respectively of in situ leaching at 10% pulp density. Also a culture filtrate leaching with A.
niger removed 20% alumina at 2% pulp density with 13 day old culture filtrate. B. circulans
was more efficient than A. niger for selective removal of alumina [15]. Earlier on A. niger

Vol.10, No.9 Labortory Studies on Phosphorus Removal 819
isolated from Nigeria’s Agbaja iron ore was used to mobilize phosphorus from the same ore
and in 49 days of leaching, 81%, 63% and 68% efficiencies for Mesh 5, Mesh 100 and Mesh
250 grain sizes, respectively, were achieved [16]. The objective of the present study
therefore, is aimed at using B. subtilis, a bacterium, to biobeneficiate the Nigerian Agbaja
iron ore to a metallurgical acceptable level. Biobeneficiation is an alternative, ecologically
friendlier approach, to the traditional physical and chemical methods of dephosphorisation.
2. MATERIALS AND METHODS
The iron ore, Plate 2.1, was collected from Agbaja, an indigenous community near Lokoja,
Kogi State of Nigeria. Standard glass wares, heavy equipment, chemicals and disposables
used in the phosphorus removal process were sourced locally.
Plate 2.1 Agbaja iron ore
1kg of the iron ore was manually crushed in the laboratory using hammer and anvil to
generate tiny particles which were screened using Shital Test sieves into 1.00/0.50mm,
0.50/0.25mm and 0.25/0.125mm particle size fractions, Plates 2.2.
(a) (b) (c)
Plate 2.2 Ore Sample Particle Size Fractions
(a) 1.00/0.50mm, (b) 0.50/0.25mm, (c) 0.25/0.125mm
10 g of each of the iron ore particle size fractions was weighed using Adventurer-AR3130
(with readability 001g) and placed in 90 ml of sterile water in 250 ml conical flask and then
serially diluted to 10-6. Using a pipette 1 ml from each final dilution was taken to seed 10
sterile Petri dishes and about 20 ml of mineral oil medium, the recipe of which is given in
Table 2.1., at 45 OC was added to each seeded Pet ri dish, swirled and allowed to stand for 14
days.

820 C. N. Anyakwo and O.W. Obot Vol.10, No.9
Two batches of 5 Petri dishes each were allowed to stand aerobically and anaerobically. At
the end of this duration, growth colony of microbes was observed.
Table 2 Recipe for Mineral Oil Medium
Reagent Quantity,
()
Sodium Chloride 1.20
Iron Sulphate 1.02
Magnesium Sulphate 0.05
Potassium di-hydrogen
Phosphate 0.25
Di-potassium
hydrogen Phosphate 1.25
Agar-agar 6.00
Water 1 litre
Bacterial colonies were sub-cultured in nutrient agar and incubated for 24 hours 28±2 OC.
Using the standard manual for bacterial identification [17], the developed growth colonies
were characterized and identified as Bacillus subtilis. These microbes were preserved for
subsequent use in the experiment.
19.85g of NB powder was mixed with 2.4 litres of distilled water in a sizeable container to
produce NB medium. 100ml of this was dispensed into 250ml conical flasks. The conical
flasks with content were sterilized using autoclave at 121 OC at 15 psi for 15 minutes. On
cooling, 1g of the already sterilized set of samples was accurately weighed and mixed with
each of the 100ml NB medium earlier dispensed into the 250ml flasks. 33 flasks were
prepared. 1ml of the B. subtilis broth culture was used to inoculate each of the flasks
aseptically. 3 conical flasks w ere not inoculated with the test organisms, and they served as
the control.
Finally, a batch of 3 conical flasks representing each of the 3 particle size fractions was
removed for phosphorus analysis weekly. The pH and cells count of the microbes w er e noted
on weekly basis as well. The samples were treated through series of standard chemical
reactions [18] to obtain ammonium phospho-molybdate precipitate which was back-titrated
with 0.1 N-HCl to determine the amount of phosphorus left in samples and consequently, the
amount removed. The data obtained from above procedure were organized and presented in
Figures 1-5.

Vol.10, No.9 Labortory Studies on Phosphorus Removal 821
3. RESULTS AND DISCUSSION
The result of phosphorus removal by B. subtilis in nutrient broth medium using the ore
particle size fractions 1.00/0.50mm, 0.50/0.25mm and 0.25/0.125mm for 10 weeks is
presented in Figure 1. From the initial ore concentration of 0.89 wt. % the phosphorus
content was lowered to 0.368 wt. %, 0.258 Wt. % and 0.289 wt. %, respectively for the
particle size fractions by Week 10. The microbes removed phosphorus rapidly from Week 1
to Week 7 and afterwards phosphorus removal was observed to have stagnated from Week 8
to Week 10.

The variation of the pH of the medium with time during the experimentation period is shown
in Figure 2. The pH increased from the initial 6.91 to 8.19 by Week 2 and thereafter
remained in the basic re gion till Week 10. It therefore remained in the basic region for most
of the experimental period.

0.000
0.200
0.400
0.600
0.800
1.000
012345678910
Wt. % Phosphorus Content in Ore
Time (week)
Figure 1Curve of Weight Percent
Phosphorus Content vs Time for Nigeria's Agbaja
Iron Ore during Phosphorus Removal by Bacillus
subtilisfor 10 weeks
1.00/0.50mm
0.50/0.25mm
0.25/0.125mm
0.00
2.00
4.00
6.00
8.00
10.00
012345678910
Substrates pH
Time (week)
Figure 2Curve of Substrates pH vs Time
during Phosphorus Removal fr om N igeria's
Agbaja Iron Ore by Bacillus subtilisfor 10 weeks
1.00/0.50mm
0.50/0.25mm
0.25/0.125mm

822 C. N. Anyakwo and O.W. Obot Vol.10, No.9
The log of the growth of B. subtilis in the nutrient broth medium bearing the
iron ore during phosphorus removal for 10 weeks is presented in Figure 3.

Figures 4(a) and (b) present the variation of the iron ore phosphorus content, the substrates
pH and the log of density during phosphorus removal from Agbaja ore 1.00/0.50mm and
0.50/0.25mm, respectively for 10 weeks by B. subtilis. It is observed that a rapid phosphorus
removal occurred in Week 1 lowering its content from an initial 0.89 wt. % to 0.65 wt. %.
The same period experienced a rapid cells multiplication from the initial log of cells density
5.531 cfu/ml to 7.322 cfu/ml also, an increased pH from 6.89 to 8.16. A steady phosphorus
removal starting from Week 2 till the end of Week 7 was observed and thereafter it stagnated
till the end of the experiment in Week 10. This period of an intensive phosphorus removal
was also coincident with the time of maximum cells growth.

0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
012345678910
Log of Density (cfu/ml)
Time (Week)
Figure 3 Growth of Bacillus subtil isin NB medium
during Phosphorus Removal from Nigeria's
Agbaja Iron Ore for 10 weeks
0
0.2
0.4
0.6
0.8
1
012345678910
pHx10 ; Log of Den sity (cfu/ml)x10;
Wt. % P
Time (Week)
Figure 4(a) Variation of Iron Ore Phosphorus
Content, Substrates pH and Log of Density during
Phosphorus Removal from Nigeria's Agbaja Iron
Ore 1.00/0.50mm by B. subtilisfor 10 Weeks
%P
pH
Log of Density

Vol.10, No.9 Labortory Studies on Phosphorus Removal 823


Figure 5 presents the percent phosphorus removed by B. subtilis in 10 weeks. The percent
phosphorus removed from the ore was calculated using Equation 1.
%  % %

%
100……………………………………1
% – the initial weight % phosphorus content in ore
%
 – the final weight % phosphorus in treated ore.

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
012345678910
pHx10 ; L og o f D en sity(cf u /ml) x1 0 ;
Wt. % P
Time (Week)
Figure 4(b) Variation of Iron Ore Phosphorus
Content, Substrates pH and Log of Density during
Phosphorus Removal from Nigeria's Agbaja Iron
Ore 0.50/0.25mm by B. subtilisfor 10 Weeks
%P
pH
Log of Density
0
10
20
30
40
50
60
70
80
012345678910
% Phosphorus Removed
Time (Week)
Figure 5Curve of Percent Phosphorus
Removed vs Time by Bacillus subtilisfor 10 Weeks
1.00/0.50mm
0.50/0.25mm
0.25/0.125mm

824 C. N. Anyakwo and O.W. Obot Vol.10, No.9
4. CONCLUSION
Bacillus subtilis has the capability to successfully remove phosphorus from Nigeria’s Agbaja
iron ore. The result of this research shows that an average of 65.73%P removal across the 3
particle size fractions was achieved. The highest removal of 71.01%P was achieved for
0.50/0.25mm.
ACKNOWLEDGEMENT
Particularly deserving great thanks are the staff and Heads of Departments of Microbiology
and Chemical Engineering of University of Uyo, Akwa Ibom State. Few names will not fail
to be mentioned and I wish to thank my Supervisor, Engr. (Dr.) C. N. Anyakwo who, actually
not only conceived the work and provided the experimental strategies stage by stage, but also,
brought his knowledge and expertise to bear on the entire work. Engr. Peter Asangausung,
the Senior Technologist in-charge of the Chemical Engineering Laboratory where the bulk of
this work was done, for his resourcefulness, keen interest and constant prayers; Dr. A. O. Ano
of the Nigerian Root Crops Research Institute, Umuahia, Abia state, whose elastic patience
limit was over stretched many times in an unprecedented manner by my over-keeping
borrowed equipment worth millions of naira in order to complete this research.
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