Journal of Minerals & Materials Characterization & Engineering, Vol. 9, No.12, pp.1131-1138, 2010
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1131
Phosphorus Removal Capability of Aspergillus Terreus and Bacillus Subtilis
from Nigeria’s Agbaja Iron Ore
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
Comparative phosphorus removal capability of a microbial fungus-Aspergillus terreus and a
bacterium-Bacillus subtilis from Nigeria’s Agbaja iron ore was investigated. Manual crushing
and sieving of the iron ore in Shital Test Kits produced 5 grain size distributions namely:
≥1.0mm, 1.0mm/0.50mm, 0.50mm/0.25mm, 0.25mm/0.125mm, 0.125mm/0.00mm, from which the
first and last grain sizes were excluded from the experiment on technical grounds. Then 1g of
each of the remaining 3 grain sizes was carefully weighed for each set of experiment. Culturing
and subsequent inoculation of the microbes (which are native to the iron ore) in conical flasks
containing sterilized, weighed iron ore samples in 100ml of equally sterilized malt extract and
nutrient broths were left to stand for 7 weeks in the laboratory. At weekly interval, the samples
were 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 research found out
Aspergillus terreus and Bacillus subtilis have separately and successfully removed phosphorus-
a deleterious non-metalic inclusion from Nigeria’s Agbaja iron ore, which previously defiled all
attempts at phosphorous removal and beneficiation techniques. However their ability at
phosphorus removal was promising and varied with the grain sizes of ore. Across all grain sizes
used, 58% and 66% phosphorus was removed in 7 weeks, respectively by the two microbes. It
was also obvious that the capacity of the microbes in removing phosphorus was seriously
hampered by a decline in activity as indicated by the reduction in microbial population in the
microcosm. For B. subtilis the density of viable cells increased from an initial load 3.4 x 105
cfu/ml, at the beginning of experiment, to 2.2 x 107 in 2 weeks but later declined to 0.9 x 106
cfu/ml at the end of 7 weeks. For A. terreus the initial biomass weight of 1g inoculated increased
to 1.993 g dry weight within 3 weeks but later decreased to 0.910g dry weight in the 7th week.
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. Our
findings have confirmed the stronger phosphorus removal capability of B. subtilis under

1132 C.N. Anyakwo and O.W. Obot Vol.9, No.12
submerged culture technique. It is however suggestive that A. terreus may perform optimally if
the submerged medium is constantly aerated.
Key words: Microbes, Culture, Crushing, Digestion, Broth, Furnace, Biodegradation
1. INTRODUCTION
The Nigeria’s Agbaja iron ore reserve having about 47.5%Fe [1] remains till date the largest
known in the country but had long been abandoned due to its very high phosphorus content
variously estimated at about 0.76–0.89Wt% [2, 6]. The mineralogy of Agbaja iron ore revealed
abundant goethite with minute magnetite. Hematite, pyrite, siderite and chlorite were also
identified [3]. A successful removal of phosphorus to a metallurgical acceptable level therefore,
from the over 1.2 billion tons ore reserve [4] can mean an economic boom for the country with
quite substantial multiplier effects in the areas of job creation and downstream sectors of iron
and steel industry.
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 0.010-0.020 Wt% or even less [5].
A flurry of research activities into the removal of phosphorus from the Nigeria’s Agbaja iron ore
commenced in the eighties. Researchers predominantly applied the conventional froth flotation
technique for the removal of phosphorus but failed because the phosphorus is not associated with
the gangue but is in bonding with the iron [6,7]. An evolving trend in mineral processing
currently gaining popularity is the use of microbes and the works of researchers in this regards
are well documented. Using the bacterium Burkholderia caribensis isolated from a Brazilian
high-phosphorus iron ore, Delvasto et al [8] 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 [9]. 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 [10]. Earlier on A. niger 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 [11]. In this work, a microbial leaching approach has been adopted for
comparative investigations of the phosphorus removal capabilities of Aspergillus terreus and

Vol.9, No.12 Phosphorus Removal Capability 1133
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
Bacillus subtilis on Nigeria’s Agbaja iron ore. This approach is economical, environmental
friendly and has an uncommon potential for easy incorporation into existing iron and steel
making technologies.
2. MATERIALS AND METHODS
The main material for this research, iron ore, was obtained from Agbaja area near Lokoja in Kogi
State of Nigeria. A sizable piece of the iron ore was crushed in the laboratory and a standard
laboratory Shital Test Kits mesh was employed to sieve the grains obtained into ≥1.00mm,
1.00mm/0.50mm, 0.50mm/0.25mm, 0.25mm/0.125mm and 0.125mm/0.00mm grain size
distributions. The first and last grain sizes were not used because of handling(large pieces and
dust, respectively). The microbes for the experiment were those native to the iron ore
environment and were obtained from the ore samples. In order to generate sufficient amount of
microbes necessary to cause degradation of phosphorus in the iron ore, the microbes were
initially cultured in mineral oil medium (MOM).
10g of each of the grain sizes of iron ore was serially diluted up to 10-6(6 times by 10 folds
dilution). Each final dilution was taken to seed sterile petri dishes and about 20ml of MOM at 45
oC was added to each seeded petri dish, swirled and allowed to set and thereafter incubated for
14 days. Some petri dishes were incubated aerobically and while others anaerobically. Bacteria
and fungi colonies were counted and recorded in colony forming unit per milli-litre(cfu/ml).
The fungi colonies were sub-cultured into Sabouard dextrose agar (SDA) and the bacterial
colonies into Nutrient agar (NA). The growth colonies were characterized and identified using
standard manuals for fungal and bacterial identification [12,13]. The isolates were Aspergillus
terreus and Bacillus subtilis.
In order to allow biodegradation of phosphorus in iron ore using the test organisms to take place,
100ml of malt extract broth (MEB) was prepared and dispensed into 250ml conical flasks. Also,
100ml of nutrient broth (NB) was prepared and similarly dispensed into 250ml conical flasks.
The conical flasks were sterilized using autoclave at 121 OC at 10 psi. On cooling, 1g of an
already sterilized iron ore from the sample bottles was accurately weighed and mixed with each
of the 100ml MEB and NB media earlier dispensed into the 250ml flasks. A loopful of A.
terreus spores from the culture in petri dishes was used to inoculate each of the MEB, and 1ml of
the B. subtilis broth culture was used to inoculate each of the NB aseptically. Some conical
flasks of each of the media were not inoculated with the test organisms, and they served as the
control.
Finally, a batch of 12 conical flasks representing each of the 3 mesh sizes from both MEB and
NB media and their controls were removed for phosphorus analysis periodically beginning from
week 1 to the 7th, when the stock was exhausted. The samples were treated through series of
standard chemical reactions [14] to obtain the data presented in Figs.1–5.

1134 C.N. Anyakwo and O.W. Obot Vol.9, No.12
3. RESULTS AND DISCUSSION
A comparison of the capabilities of A. terreus and B. subtilis at removing phosphorus from
Nigeria’s Agbaja iron ore in 1.0mm/0.50mm grain size for the period of 7 weeks is presented in
Fig.1. In the first instance, B. subtilis (a faster self-generating microbe) removed more
phosphorus from Week 1 till about Week 5 and thereafter slowed down from Week 6 to Week 7.
During this time its population degenerated from 1.4 x 107 cfu/ml to 0.9 x 106 cfu/ml. A. terreus,
a slower self-generating fungus population rose from 0.692g dry weight in Week 1 to a peak of
2.786g dry weight in Week 3 and quickly dropped to 0.963g dry weight in Week 7 averaging out
in stagnation. One possible explanation for this pattern is that the microbes having actively
degraded phosphorus in the ore samples from the 1st week were also actively dying of toxicity
due to biodegradation wastes and this resulted in their diminished population in the microcosm.
In Fig.2 for instance, the potential of B. subtilis to remove phosphorus from 0.50mm/0.25mm-
sized Nigerian Agbaja iron ore was higher than that of A. terreus and dominated effectively till
midway of Weeks 3 and 4. This period witnessed a population of 1.92 x 107 – 5.6 x 106 cfu/ml
which continued to diminish to 1.0x106 cfu/ml at Week 7. During this period, the microbial
count for A. terreus grew from 0.726 – 1.964 g dry weight and then went on to remove more
phosphorus till Week 7.
0.00
0.20
0.40
0.60
0.80
1.00
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Wt.%PhosphorusOreContent
DurationofPhosphorusRemoval,Weeks
Figure1ComparisonofPhosphorusRemoval
CapabilitiesofAspergillusterreus andBacillus subtilis in
1.0mm/0.50mm‐sizedNigerianAgbajaIronOrefor 7
Weeks
asp.T
bac.S

Vol.9, No.12 Phosphorus Removal Capability 1135
1135
In Fig.3, A. terreus in Week 1 removed more phosphorus than B. subtilis and this trend was
sustained till start of Week 3. The microbial count of this fungus over this period rose from
0.686g dry weight to 1.993g dry weight while that of B. subtilis dropped 2.10x107 cfu/ml to
6.2x106 cfu/ml. Between Week 3 and Week 4 the overall performance of both microbes seems
to have stagnated during which time their average count stood at 1.755g dry weight and 5.0 x 106
cfu/ml, respectively. From end of Week 4 the population of the bacterium dropped seriously
from 3.7 x 106 cfu/ml to 1.0 x 106 cfu/ml in Week 7 while that of fungus increased initially from
1.516g dry weight to 1.168g dry weight in Week 5 and then nose-dived to 0.910g dry weight in
Week 7.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
1234567
Wt.%PhosphorusOreConte n t
DurationofPhosphorusRemoval,Weeks
Figure2 ComparisonofPhosphorusRemova l 
CapabilitiesofAspergillusterreus andBacillussubtilis
from0.50mm/0.25mm‐sizedNigerianAgbajaIronOre
fo r 7Weeks
asp.T
bac.S
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
1234567
Wt.%PhosphorusOreContent
DurationofPhosphorusRemoval,Weeks
Figure3 ComparisonofPhosphorusRemoval 
CapabilitiesofAspergillusterreus andBacillussubtilis
from0.25mm/0.125mm‐sizedNigerianAgbajaIronOre
for7Weeks
asp.T
bac.S

1136 C.N. Anyakwo and O.W. Obot Vol.9, No.12
The views separately presented in Figs.1-3 are better evaluated in Fig.4 where the three averages
are brought together for comparison. From Week 1 to midway of Weeks 4 and 5, Bacillus
subtilis removed more phosphorus than A. terreus. This clearly shows that while about 64% of
the duration of experimentation was dominated by B. subtilis in removing phosphorus, A. terreus
only dominated a mere 36%.
The average percentage of phosphorus removed by A. terreus and B. subtilis can still better be
evaluated when presented as in Fig.5. In the first week, 24% and 19% removal was achieved,
respectively by both microbes. At the end of the experiment duration, 58% and 66%
respectively, were recorded for the microbes with B. subtilis out-performing the other. Although
both microorganisms actively removed phosphorus from the ore samples even, when ore
contained pyrite[15] being an effective antimicrobial substance. It is also necessary to note that
the metabolic activity of A. terreus an obligate aerobe may have been affected by the excessively
moistened or reduced substrate of the submerged culture technique. Under this condition active
contact with the settled ore samples at the bottom of the flask may be hindered despite their
ability to produce hyphae. On the other hand, B. subtilis generally a micro-aerophilic bacterium
whose metabolic activities are less affected by the submerged culture technique employed in this
research performed better. Although a technique of periodic manual-shaking of the conical
flasks and content was adopted as a means of ensuring a more intimate interaction in the system,
it probably did not do much in altering the above observed microbial characteristics.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
1234567
Wt.%PhosphorusOreContent
DurationofPhosphorusRemoval,Weeks
Figure4ComparisonofAve rage Phosphorus
Removal byAspergillusterreus andBacillussubtilis from
NigerianAgbajaIronOrein7WeeksAcrossallGrain
Sizes
asp.T
bac.S

Vol.9, No.12 Phosphorus Removal Capability 1137
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4. CONCLUSION
Aspergillus terreus and Bacillus subtilis both have the capability to successfully remove
phosphorus from Nigeria’s Agbaja iron ore. The result of this research shows that 58 and 66
percents removal, respectively, were achieved in 7 weeks of experimentation when 1g of ore
samples were subjected in 100ml of media and inoculated with the microbes. A better
phosphorus removal capability demonstrated by Bacillus subtilis is natural because they are not
affected by the reduced nature of the test substrate. It is suggested that Aspergillus terreus may
perform optimally if the submerged medium is constantly aerated. However, these findings
necessitate further investigation.
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.
0
10
20
30
40
50
60
70
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%PhosphorusRemovalbyMicrobes
DurationofPhosphorusRemoval,Weeks
Figure5AveragePercen tagePhosphorusRemo val
byAspergillusterreus andBacillussubtilisfromNigerian
AgbajaIronOrefor7WeeksAcrossallGrainSizes
asp.T
bac.S

1138 C.N. Anyakwo and O.W. Obot Vol.9, No.12
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