Journal of Minerals and Materials Characterization and Engineering, 2012, 11, 848-852
Published Online August 2012 (http://www.SciRP.org/journal/jmmce)
Comparative Phosphorus Removal Capabilities of
Eurotium herbarorium and Clostridium Species on
Nigeria’s Agbaja Iron Ore
Obotowo William Obot1, Charles Nwachukwu Anyakwo2
1Department of Me ch an ical Engin e er in g, Faculty of Engineering, University of Uyo, Uyo, Nigeria
2Department of Metallurgical and Materials Engineering, Federal University of Technology, Owerri, Nigeria
Received July 1, 2012; revised July 31, 2012; accepted August 15, 2012
ABSTRACT
A study of phosphorus removal capabilities of Eurotium herbarorium and Clostridium species from Nigeria’s Agbaja
iron ore was carried out. Iron ore sample was crushed, sieved to obtain 0.50 mm/0.25 mm particle size distribution and
cultured with mineral oil medium to facilitate microbial growth. Fungi and bacteria that concurrently grew were sub-
cultured in Sabouard dextrose agar and nutrient agar solutions that support fungal and bacterial growth, respectively,
and characterized using standard procedures. Ore was exposed to these microbes to effect phosphorus removal in stan-
dard media and later analyzed at weekly interval using the standard volumetric ammonium phospho-molybdate method.
The fermentation broth media were analyzed for iron, copper, cadmium, zinc, nickel, manganese and lead using the
atomic absorption spectrophotometer. The microorganisms markedly removed phosphorus from the ore with 61.48%
and 69.20%, respectively. For the fungus pH remained in the acidic region and basic for the bacterium. Trace elements
analyses of the initial and final ore-co ntaining media recorded marked reduction in the concen tration of these elements.
A plausible explanation that is supported by literature is that the microorganisms accumulated them. This probably ac-
counts for the drastic decrease in fungal biomass and bacterial density with the concomitant decrease in phosphorus
removal observed towards the end.
Keywords: Microbes; Culture; Biodegradation; Trace; Metal; Biomass
1. Introduction
The Nigeria’s Agbaja iron ore reserve with its rich iron
content 47.5% - 51.50% Fe [1] is the country’s largest
but had long been abandoned due to its high phosphor-
ru s status which researchers variously estimated at about
0.76 - 0.89 wt% [2,3]. The mineralogy of Agbaja iron ore
revealed abundant goethite with minute magnetite and
some hematite, pyrite, siderite and chlorite also identi-
fied Uwadiale [4]. A successful removal of phosphorus
to a metallurgical acceptable level, therefore, from the
over 1.2 billion tonnes of ore reserve Uwadiale [5] can
mean an economic boom for the country with quite sub-
stantial multiplier effects in the areas of job and wealth
creation for the downstream sectors of iron and steel in-
dustry.
The negative effects of phosphorus in high quality
steels namely: the effect of steel brittleness coupled with
the effect of strong primary segregation during solidifi-
cation of castings and the formation of high phosphorus
brittle streaks between metal gr ains which impede plastic
deformation are undesirable and therefore should be
minimized as much as possible. Thus, for high quality
steels, the phosphorus acceptable level is in the range of
0.020 - 0.030 wt % or e ven less Kudrin [6] .
A flurry of research activities into the removal of
phosphorus from the Nigeria’s Agbaja iron ore com-
menced in the eighties. Researchers predominantly ap-
plied 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 [3,7]. An evolving trend in mineral processing
currently gaining popularity is the use of microbes and
the works of researchers in this regards are well docu-
mented [8-10 ].
The current work th erefore intends to adopt the micro-
bial degradation approach for comparative investigations
of the phosphorus removal capabilities of Eurotium her-
barorium and Clostridium species on Nigeria’s Agbaja
iron ore. This approach is cheap, environmental friendly
and has a potential for easy incorporation into existing
iron and steel making technologies.

O. W. OBOT, C. N. ANYAKWO 849
2. Materials and Methods
The major raw material, iron ore, was obtained from
Agbaja in Lokoja area of Kogi State of Nigeria. A sizable
piece of the iron ore was crushed in the laboratory using
hammer and anvil and then a standard laboratory Shital
Test Kits was employed to sieve 0.50 mm/0.25 mm par-
ticle size distribution. This was an alyzed for composition.
The microbes for the experiment were those native to the
iron ore environment and were obtained from the crushed
ore sample. In order to generate sufficient amount of
microbes necessary to cause degradation of phosphorus
in the ore, the microbes were initially cultured in mineral
oil medium (MOM). 10 g of the particle size of ore was
serially diluted up to 10–6 (6 times by 10 folds dilution).
The final dilution was taken to seed sterile petri dishes
and about 20 ml of MOM at 45˚C was added to each
seeded petri d ish, swirled and allowed to set and thereaf-
ter incubated for 14 days. Some petri dishes were incu-
bated aerobically and while others anaerobically. Bacte-
ria and fungi colonies were counted and recorded in col-
ony forming unit per milli-litre (cfu/ml). The fungi colo-
nies 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
[11,12]. The isolates with stronger survival chances were
Eurotium herbarorium and Clostridium species.
In order to allow biodegradation of phosphorus in the
iron ore using the test organisms to take place, malt ex-
tract broth (MEB) and nutrient broth (NB) were prepared
to standard and 100 ml of each dispensed into 250 ml
conical flasks. The conical flasks were sterilized by auto-
claving at 121˚C at 15 psi for 15 minutes. On cooling, 1
g of an already sterilized ore was mixed with each of the
100 ml MEB and NB media. 1 g wet weight of Eurotium
herbarorium spores from the culture in petri dishes was
used to inoculate each of the MEB, and 1ml of the Clos-
tridium species 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, some
were inoculated with test organisms but without ore
samples and they served as the control.
Finally, a batch of conical fl asks represent i ng bot h M EB
and NB media were removed for phosphorus analysis
weekly for 10 weeks. The treated ore samples were digested
through the standard volumetric ammonium phospho-
molybdate method [13] and the precipitate back titrated
with 0.1 N-HCl solutions using phenolphthalein as indi-
cator. The fermentation broth media were analyzed for
iron (Fe), copper (Cu), cadmium (Cd), zinc (Zn), nickel
(Ni), manganese (Mn) and lead (Pb) using UNICAM
solaar 969 atomic absorption spectrophotometer (AAS).
The pH of the fermentation broth media was monitored
using pH meter (EIL 70 20, Kent Industrial Measurement
Ltd.). The population of the microbes was carefully
monitored also within the period by harvesting the fungal
mycelia in each MEB flask and drying on filter paper in
the oven at 50˚C for 12 hours. Each dried mass was re-
corded. Bacterial count in colony forming unit per mili-
litre involved culturing 1 ml of fermented NB in fresh
nutrient agar to allow the growth of Clostridium species
which was estimated using the colony counter. The data
obtained from the experiment are presented in Figures
1-8.
3. Results and Discussion
The result of iron ore co mpositional analysis is presen ted
in Table 1. LOI (Lo ss on ign ition) was obtained at 939˚C.
The analysis of Fe, Mg, Cu, Zn and Mn was carried out
using AAS (Unicam 939). P was analyzed by colorimetry
using ammonium vanadate. S was analyzed by Eschka
method. Al was analyzed by Titrimetry while SiO2 by
colorimetry using ammonium molybdate.
Table 1. Result of iron ore compositional analysis.
Mineral Fe(T) SiO2 P
2O5 MgO Cu2O ZnO LOI S MnO2 Al2O3
Fe2O3 51.50 0.57 1.25 0.08 0.005 0.091 8.01 3.25 0.001 34.77
(a) (b)
Figure 1. (a) MEB + Eurotium herbarorium and NB + Clostridium species Initial Analytical Results without Nigeria’s Agbaja
iron ore 0.50/0.25 mm samples; (b) MEB + Eurotium herbarorium and NB + Clostridium species initial analytical results
without Nigeria’s Agbaja iron ore 0.50/0.25 mm samples.

O. W. OBOT, C. N. ANYAKWO
850
Figure 1(a) presents the result of initial analyses of
MEB + Eurotium herbarorium and NB + Clostridium
species without the iron ore samples. It shows the con-
centration of the trace metals in the broth cultures in the
presence of the microorganisms. The MEB does not
contain Zn, Ni, MN and Pb but has some amounts of Fe,
Cu and Cd ions. On the other hand, NB contains Fe, Cu,
Cd and Zn but no Ni, Mn and Pb ions. Figure 1(b) is a
scale up modification of Figure 1(a) without values for
Fe ions concentration to give better visualization of
amounts of the other very insignificant trace metals.
Figures 2(a) and (b) present the initial analytical re-
sults of MEB + Eurotium herbarorium and NB + Clos-
tridium species cultures with the ore sample. Figure 2(b)
is a scale up modification of Figure 2(a) without values
for Fe concentration, again for visualization effect. Com-
paring Figures 1 and 2 shows that the introduction of
iron ore sample into both MEB and NB cultures, resulted
in increase in the trace metals concentration. For MEB
inoculated with Eurotium herbarorium, Zn, Mn and Pb
ions increased 31%, 317% and 105%, respectively. NB
inoculated with Clostridium species during the same in-
terval recorded 5.5% and 424% increase for Ni and Pb,
respectively. The trace metals increase is expected with
the introduction of the ore as this has increased whatev er
concentration of trace metals that came with the MEB
medium. However, it is observed that in the MEB culture,
the concentration of Fe and Cd decreased by 11% and
80% respectively. It is possible that the decrease in trace
metals is due to bio-accumulation by the microbes.
(a)
(b)
Figure 2. (a) Concentration of trace metals in broth media
during phosphorus removal from Nigeria’s Agbaja iron ore
by Eurotium herbarorium and Clostridiumn species for 10
weeks; (b) Initial concentration of trace metals in broth
media during phosphorus removal from Nigeria’s Agbaja
iron ore by Eurotium herbarorium and Clostridium species.
Comparing Figures 2 and 3 shows trace metals sharp
decrease. It is possible that in the MEB culture Eurotium
herbarorium accumulated 100% of Cu, Cd and Pb; 68%
Fe, 49% Zn and 89% Mn ions at the end of 10 weeks
experimentation. During the same period Clostridium
species in NB culture possibly also accumulated 100%
Fe and Cd, also 60% Zn and 79% Pb. In NB culture 87%
Cu, 92% Ni and 38% Mn ions were released back. It is
confirmed in literature that the microorganisms can ac-
cumulate trace metals under favourable conditions and
also release same back to the environment if the condi-
tions change. It is equally known that the over-accumu-
lation of trace metals disrupt the normal functioning cells
due to toxicity [14]. The reduction in cells population
observed in Figure 5, especially towards the end of ex-
perimentation with its concomitant reduction in phos-
phorus removal capability of the microbes about the
same period, may be facts to show why the stagnation
noticed from the 7th week till the end of the experiment
occurred. It is most probable that the microbes having
over-accumulated trace metals especially Pb, simply died
and this could have aff ected this route of processing iron
ore to reduce phosphorus.
The removal of phosphorus in the ore by Eurotium
herbarorium and Clostridium species for 10 weeks is
presented in Figure 4. During this period, the initial
phosphorus con tent in the ore 0 .89 wt% reduced to 0.339
wt% and 0.271 wt%, respectively.
Figure 3. Concentration of trace metals in broth media
during phosphorus removal from Nigeria’s Agbaja iron ore
by Eurotium herbarorium and Clostridium species at end of
10 weeks.
Figure 4. Variation of wt% P content during phosphorus
removal by Eurotium herbarorium and Clostridium species
from Nigeria’s Agbaja iron ore 0.50/0.25 mm for 10 weeks.

O. W. OBOT, C. N. ANYAKWO 851
The variation of the biomass weight of Eurotium her-
barorium in MEB and the log of the density of Clostrid-
ium species in NB submerged media bearing the iron ore
with time is shown in Figur e 5.
initia
%P
%P
The variation of the pH of the MEB and NB cultures
with time during the experimentation period is shown in
Figure 6.
Figure 7 presents the percent phosphorus reduced by
Eurotium herbarorium and Clostridium species in 10
weeks. The percent phosphorus reduced from the ore was
calculated using Equation (1):
initial
%
PP


final
initial
%%
%100
PP
 (1)
Figure 5. Growth of Enrotitum herharorium and Clostridium
species during phosphorus removal from Nigeria’s Agbaja
iron ore 0.50/0.25 mm for 10 weeks.
Figure 6. Curve of subsr ates pH vs time during phosphorus
removal from Nigeria’s Agbaja iron ore 0.50/0.25 mm by
Eurotiu m herbar orium and Clostridium species for 10 weeks.
Figure 7. Curve of % phosphorus removed vs time for Ni-
geria’s Agbaja iron ore by Eurotium herbarorium and Clos-
tridium sp e cies for 10 weeks .
l
—The final wt% phosphorus in treated ore.
—The initial wt% phosphorus content in ore
final
Figure 8(a) shows that in the first week, an initial
rapid drop in biomass weight that also corresponded to a
rapid drop in ore’s phosphorus concentration occurred.
Thereafter for a period of two weeks a marked increase
in biomass weight that peaked at the third week took
place while phosphorus intake by the fungus continued to
take place. Beyond this point the biomass weight began
to decrease rapidly while phosphorus intake also contin-
ued. It is however seen that when the biomass weight
dropped to 1 g dry weight, phosphorus removal attained
its lowest value and below this value no further phos-
phorus removal effectively continued. The highest per-
cent cumulative removal for Eurotium herbarorium was
61.48%. Removal occurred in an increasing acidity cul-
ture attaining a pH value 2.30 in 10 weeks.
Figure 8(b) shows on the other hand, a gradual phos-
phorus removal in the ore occurring throughout the pe-
riod thus lowering its content from an initial value of
0.890 wt% to 0.271 wt%. The same period experienced
rapid cells multiplication from an initial log of cfu/ml
5.531 to a climax 7.92 2 in the 4th week and then a grad-
ual reduction in cells population. During this period the
substrates pH value increased from 7.13 to 9.61. The
(a)
(b)
Figure 8. (a) Variation of iron ore phosphorus content, sub-
strates pH and biomass weight during phosphorus removal
from Nigeria’s Agbaja iron ore 0.50/0.25 mm by Eurotium
herbarorium for 10 weeks; (b) Variation of iron ore phos-
phorus content, substrates pH and log of density during
phosphorus removal from Nigeria’s Agbaja iron ore 0.50/
0.25 mm by Clostridium species for 10 weeks.

O. W. OBOT, C. N. ANYAKWO
852
period of a steady phosphorus removal was also coinci-
dent with the time of maximum cells growth. The highest
phosphorus cumulative removal attained was 69.20%.
4. Conclusion and Recommendation
The comparative phosphorus removal capability of Eu-
rotium herbarorium and Clostridium species on Nige-
ria’s Agbaja iron ore was investigated with 61.48% and
69.20% cumulative removal respectively attained in 10
weeks. The initial rapid removal capacity shown in the
fermentation broths till about the 6th and 7th weeks, re-
spectively for the two organisms apparently could not be
sustained due to over-accumulation of trace metals and
other toxic waste products of biodegradation and this
probably adversely affected the further phosphorus re-
moval. It is recommended that further studies on the
chemistry of metabolic broth wastes be considered as this
may reveal the interacting parameters which can be ad-
vantageously harnessed for a continuous bio-degradation
that is possible with microorganisms.
5. Acknowledgements
We thank all those who in one way or the other sup-
ported this work especially, the staff of the Departments
of Microbiology and Chemical Eng ineering of Univ ersity
of Uyo, also the staff of the Ministry of Science and
Technology, Akwa Ibom State. Deserving special appre-
ciation is Dr. A. O. Ano of the Nigerian Root Crops Re-
search Institute, Umuahia, Abia state, for lending equip-
ment worth millions of naira free of charge in order to
complete this research. And finally, Engr. Peter Asan-
gausung, the Senior Technologist in-charge of the
Chemical Engineering Laboratory where the bulk of this
work was done, we thank for his resourcefulness and
demonstrated concern.
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