Comparative Phosphorus Removal Capabilities of Eurotium Herbarorium and Clostridium Species on Nigeria’S Agbaja Iron Ore

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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

Email: [email protected], [email protected]

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

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





final

initial

%100



(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 .

—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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