Nigerian Journal of Microbiology, 2009, Vol.

, 23(1);1830 - 1838

Assessment of Physicochemical and Biological Indices of Fluvial Deposits in Abandoned Mine

Pits in Ishiagu, South Eastern Nigeria

*Nwaugo, V.O.1, 2Onwuchekwa.I.S., 3Ogbonna, C. and 1Onyeagba, R.A

1
Environmental Microbiology Unit, Abia State University, Uturu
2
Hydrology and hydrobiology Unit, Abia State Univeristy, Uturu
3
Environmental Resources Management, Abia State University, Uturu
Abstract
The physicochemical and microbiological indices of heavy metal mine pit soil
were assessed using standard methods. PO4, SO4, NO3 and TOC were higher in alluvial
deposits of abandoned mine pits than active mine pit soil and control soil. Soil from the
alluvial deposits had high silts and clay contents while active pit soil had more sand particles.
Pb and Zn concentrations were statistically (P=0.05) higher in the active pit soil and above
the acceptable limits in all soil samples. Cd, Cu, and Fe were within acceptable levels. Total
heterotrophic, cellulolytic, nitrifying and phosphate solubilizing bacterial counts were higher
in the abandoned mine alluvial deposits. Iron oxidizing and sulphur oxidizing bacterial counts
were higher in the acidic active mine soil but sulphur reducing bacteria were more prevalent
in the neutral alluvial deposit soils of the abandoned pits. Most adversely affected bacterial
group was the nitrifying group, and the least was the total heterotrophic bacterial group.. All
soil enzymes whose activities were determined followed the trend of microbial loads ie
showing more activities in mine alluvial soil than the active mine pit soil. These enzymes
were dehydrogenase, which was the most adversely affected, urease, glucosidase, acid and
alkaline phosphatase.

Keywords; mine tailing, properties, bacteria, metals, adverse.

Corresponding author: Dr. V.O. Nwaugo
E-mail: vonwaugo@yahoo.com Tel; 2348063493654

Introduction cases, such soils contain silt and clay

The soil is very important to human
existence for various reasons especially
agriculture. However, the soil has been
subjected to several abuses including
dumping of wastes and other
contaminating activities (Weillinga et al,
1999, Nwaugo et al, 2006, 2007a). The
soil also contains several minerals and
metals, which are only mined either by
drilling or excavation. The excavation
leaves large ditches and ponds on its
trail. When these ditches are water or
sand filled, they serve other purposes for
man (Nwaugo et al, 2006). According to
Weillinga et al., (1999) and Cloassens et
al., (2005), alluvial deposits of most
mine pits contain soil slightly different
from the surroundings, as it contains
mainly washed down particles. In most
particles with less of sand and high
organic matter content (Prescott et al
,2003).
The complex geochemical nature of
the environment portrays equally
complex microbial flora. In the same
vein microorganisms mediating the
biogeochemical transformations
involving recycling of nutrients and
energy, are also complex. Lee et al
(2002). Aon and Colaroneri (2001), and
Pascual et al., (2000) agreed that the
complexity of the microbial population
gives rise to a complex array of enzymes
catalyzing different transformation
processes in alluvial soil of mine pits.
Generally, this type of alluvial soil
litter all excavated mine areas in
Nigeria, especially in the rural areas.

1830
Nwaugo et al., 2009
The quality of and microbial activities in
such soil remains largely unexplained in
Nigeria. Such heavy metal mining
activities (excavation) have been on for
several years (over thirty-five years)
unabated in Ishiagu, South eastern,
Nigeria, This study was therefore
designed to investigate the
physicochemical properties of the
alluvial deposits in abandoned and
active Pb and Zn mine pits including the
microbial profile and enzymatic
activities in Ishiagu. This will ascertain
the suitability of such soil for
agricultural purposes.
Materials and methods
The study area is Ishiagu, a rural
Pb/Zn mining community of Ebonyi
State, South Eastern Nigeria. It is
located between latitudes 7o30’ and
7o37’E and longitudes 5o52’ and 6o00’
N(Fig. 1). The study area lies in the
typical Guinea Savanna area of Nigeria
characterized by tall grasses and a few
trees with a mean annual rainfall
between 1750 – 2000mm. The people
are mainly farmers.
Sampling
Four of the abandoned pit alluvial
soil and two recent/active mining pits
located in the same area were randomly
sampled. The control was taken from
another unexcavated natural area, a good
distance(500m) away from the mining
sties. At each sampling site, three
samples collected according to Dick et
al., (1990), were pooled to give one
main sample. While microbiological and
enzymatic activities were analyzed
within 2 - 3 hours of collection, the
samples for physicochemical parameters
were stored at 40C until required for
analysis within 1-3 days of collection.
Physicochemical parameters
The pH and temperature were
determined using Jenway Multipurpose
Tester (HANNA 1910 model). The
phosphate (PO4), and Sulphate (SO4)
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Nig.J.Microbiol., 23(1);1830 - 1838
were determined by the methods stated
in UNEP(2004) analytical methods. The
organic carbon content was determined
by the loss of ignition method using
MAC 2000 furnace while the moisture
content was determined gravimetrically
after drying at 105oC till constant weight
was obtained. The heavy metal contents
of the various soil samples were
determined using HACH/DO/2010
Atomic Absorption spectrophotometer
after digestion.
Microbial diversity
The diluted (ten-fold serial dilution)
samples were spread plated on various
specific culture media for the desired
bacterial group and incubated
appropriately for 24-72 hours. The
bacterial groups and the media used
include phosphate solubilizing bacteria
(PSB) with phosphate solubilizing
bacteria medium (US patent, 2003),
nitrifying bacteria (NB) with modified
mineral salt agar and cellulose Agar for
cellulolytic bacteria (CB). Postgate
medium (modified) was used for
sulphate reducing bacteria. Sulphur
oxidizing bacteria and iron oxidizing
bacteria were cultured as described by
Harrison (1984). Sulphate oxidizing
bacteria was estimated using
Thiosulphate medium B as described by
Chessbrough (2003)
Soil enzymatic activities
The activities of five enzymes
were evaluated. These include
dehydrogenase produced by all
microorganisms and indirectly related to
organic carbon content, urease,
phosphatases (acid and alkaline)
important in nitrogen and phosphorus
recycling respectively. The activities of
glucosidase known to influence carbon
utilization was also assessed.
Dehydrogenase was analyzed
according to Li et al., (2005) involving
the use of triphenyl tetrazolum chloride
(TTC) amended soil with formation of
 Nwaugo et al., 2009
triphenyl formazon (TPF) read at
485nM. Urease activity was determined
colorimetrically as described by
Kandeler and Gerder (1988) with urea
amended soil. Alkaline and acid
phosphatases were investigated
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Nig.J.Microbiol., 23(1);1830 - 1838
according to Tabatabai (1997) at pH of
11 and 5.5 respectively using p-
nitrophenyl phosphate as substrate and
formation of p-nitrophenol. Glucosidase
activity was estimated as described by
Eivazi and Tabatabai (1988).
Nwaugo et al, 2009
Results
Physicochemical parameters
Results obtained in the various alluvial soil
samples had similar values but different from
those from active mining pit soil. Values from the
control were found to be close to those from the
alluvial soil in the abandoned pits.
Table 1 shows the physicochemical parameters
of the soil samples analyzed. Abandoned pit
alluvial soil had pH values 6.96 – 7.14 while the
active pits had 5.5 – 5.7 and control 6.52. The
values range of PO4 was 5.82 – 6.0mg/g, 2.24 –
2.48mg/g and 4.91mg/g for pit alluvial soil, active
pit soil and control soil respectively. This same
trend was also observed in SO4, NO3, TOC and
moisture content (Table 1) respectively.
Values obtained for Cu, Cd and Fe were low and
non-significant (P = 0.05). On the other
hand,values for Pb and Zn were significantly (P =
0.05) high and exceeded the safety levels as
directed by CEC (1986). This was especially so in
the active pit soils while the abandoned alluvial
pit soils were within the upper acceptable limits
(Table 1).
Microbial diversity
All bacterial groups determined were present in
all soil samples analyzed but at different rates.
Similar trends were observed in all the groups
except the SRB and IOB which were highest in
the active pit soil. In all the other bacterial groups,
highest biological loads were observed in alluvial
soil of the abandoned pits, followed by the control
while the least were in the active mining pit soil
samples (Table 2). Nitrifying bacteria (2.7 x 103 –
9 x 101 cfu/g) were the most adversely affected
followed by the PSB (2.4 x 103 – 1.10 x102cfu/g).
Cellulolytic bacteria had a range of 2.83 x 103 –
1.2 x 102 cfu/g. The least affected bacteria were
the total heterotrophic group with a range of 6.7 x
106 – 4.3 x 104 cfu/g.
Analysis of pH and microbial counts showed
that acidic pH favoured the proliferation of IOB
and SOB more than SRB while alkaline pH had
the opposite effects. Similarly there were
suppressive effects of the heavy metals (Pb and
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Nig.J.Microbiol., 23(1);1830 - 1838
Zn) on all the bacterial groups determined as soil
with high metallic concentrations had low
microbial counts and vice versa (Table 2).
Analysis of the bacterial groups showed that the
nitrifying, cellulolytic and phosphate solubilizling
bacteria were not significantly different in
prevalence in the various soil samples.
Again,values ofs total heterotrophic bacteria were
also significantly higher than the values of the
other groups of bacteria determined (Table 2).
Soil enzymatic activities
Soil enzymatic activities were observed to
follow the pattern of total heterotrophic bacteria.
The highest activities were in the alluvial soil of
the abandoned pits, followed by the control soil
before the active mining pit soil (Table 3).
The most adversely affected enzyme was
dehydrogenase which had 7.82 – 8.31 mg/g-16h-1
in abandoned pit alluvial soil and only 0.59 – 0.92
mg/g-16h-1 in the active mining pit soil (only
11.07% of the highest activity). Urease had 4.25 –
1.03 mg/gh-3-1 activities with lowest value in the
active pit soil and highest in the alluvial soil. The
active mine soil had only 25.85 – 24.21% of the
highest activities in the abandoned pit alluvial
soil.
The phosphatases – acid and alkaline did
not show as much differences in activities as the
other enzymes. The difference between the
highest values (2.27 µ-mol-p-nitrophenol in
alluvial pit soil) and the lowest (1.41 µ-mol-p-
nitrophenol in recent or active pit soil) was only
59.49% while that of alkaline phosphatase was
55.46% (2.47 – 1.37). This shows that the
environmental factors did not have very
significant effect on the phosphatases compared
to the other enzymes.
Observations in the glucosidase activity also
showed similar trends as other enzymes. The
lowest activity was in the soil from active mining
pit (1.7 mg/g-16h-1) and the highest (6.7mg/g-6h-1)
in the tailing soil. Values for the control are
shown on Table 2.
 Nwaugo et al, 2009 Nig.J.Microbiol., 23(1);1830 - 1838

Table 1: Physicochemical parameters of the various alluvial deposit soil and active mine pit soil’

PARAMETER MTS 1 MTS 2 MTS 3 MTS 4 AMS 1 AMS2 Control

Soil
a a a b c c d
Clay % 13+01.00 13+02.00 14+02.00 15+02.00 6+0.3.00 4+0.06 09+1.00
Silt % 35+03.00a 34+02.00a 35+02.00a 37+03.02b 15+0.01c 18+1.01c 28+0.02d
Sand % 52+02.00a 53+03.00a 51+02.00a 48+02.00b 79+0.03c 78+0.02c 63+0.02d

Colour Dark Dark Dark Dark Brown Brown Brown

pH 7.14+02a 6.76+0.3b 7.24+02a 6.98+0.3b 5.6+0.2c 5.5+0.3c 6.5.2+0.2d
Temp oc 29.7+03a 29.8+02a 29.5+0.2a 29.4+0.2a 28.2+04b 28.4+0.3b 28.9+0.3b
a a a a c c d
PQ4 mg/g 5.97+0.11 5.64+0.3 5.82+0.10 6.01+0.13 2.42+0.3 2.24+0.2 4.91+0.11
a b b c d d e
SO4 mg/g 42.35+0.21 46.39+0.24 44.78+0.29 53.27+0.21 14.36+0.26 16.75+0.27 37.64+0.12
a a b b c c d
NO3 mg/g 4.36+0.11 4.24+0.17 4.74+0.14 4.82+0.13 0.82+0.18 0.91+0.16 2.87+0.12
TOC mg/g 1.02+0.2a 1.27+0.2b 1.30+0.3b 1.47+0.3b 0.32+0.4c O0.39+0.4c 0.92+0.2d
a b b a c c d
Moisture % 4.41+2.1 3.97+2.6 3.68+2.5 4.01+2.2 1.21+0.4 1.33+0.6 2.67+1.3
a a a b c c d
NH4 mg/g 0.41+0.1 0.45+0.1 0.47+0.1 0.64+0.1 0.12+0.03 0.11+0.6 0.29+0.1
a b c d a a
Cd mg/g 0.06+0.1 0.05+0.1 0.03+0.01 0.04 +0.1 0.06+0 0.06+0.2 0.01+.00d
Cr mg/g 0.04+0.0a 0.04+.00a 0.02+0.1b 0.03+00b 0.04+0.01a 0.04+0.00a 0.01+ 00c
a a a a b a c
Pb mg/g 100.81+3.1 104.62+5.1 06.46+3.1 102.12+3.1 191.49+5.1 186.42+32 40.41+22
a a a a b b c
Zn mg/g 42.41+1.3 39.21+1.6 38.51+2.0 40.11+1.9 109.41+3.7 112.64+40 26.79+2.1
a a a a b b
Fe mg/g 21.36+1.3 26.71+1.6 22.31+2.0 23.16+2.1 36.7+3.6 34.39+4.1 18.66+1.3c
MTS = Mine tailing soil AMS =Active mine soil
Values followed by the same alphabets are not significantly different but those followed by different alphabets
are significantly different

Table 2: Bacterial diversity in bioloads (cfu/g) of the various soil samples from abandoned alluvial and active
mine soils examined.

Parameter MTS 1 MTS 2 MTS 3 MTS 4 AMS 1 AMS 2 Control

Soil
NB 2.7X103 2.4x102 2.1X102 2.5X103 0.4X101 0.6X101 1.2X103
4 4 4 4 2 2 4
CE 2.5 x10 2.6+10 2.6 X10 2.9 X10 1.2 X10 1.4 X10 1.2 X10
3
PSB 2.1X10 2.0+103 2.4 X102 2.2 X103 1.4 X102 1.2 X102 2.8 X102
SRB 3.9X102 4.6X102 4.1 X102 4.5 X102 1.6 X102 1.6 X102 2.2 X102
3 2 3 3 4 4 2
IOB 6.7X10 6.1X10 6.4 X10 6.8 X10 2.4 X10 2.4 X10 3.1 X10
3 3 3 2 3 3 3
SOB 1.7X10 1.2X10 1.0 X10 1.1 X10 1.9 X10 1.9 X10 1.1 X10
THB 6.6X107 6.3X107 6.1 X102 6.7 X107 4.3 X104 4.7 X104 4.4 X106

*Values reported are mean values of three times estimation results

NB = Nitrifying bacteria, CB = Coliform bacteria, PSB = Phophate solubilization bacteria
,SRB = Sulphur reducing bacteria, SOB = Sulphur oxidizing bacteria, IOB = Iron oxidizing bacteria
THB = Total heterotrophic bacteria

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Nwaugo et al, 2009 Nig.J.Microbiol., 23(1);1830 - 1838

Table 3: Enzymatic activities in the various soil samples examined

Enzyme MTS 1 MTS 2 MTS 3 MTS 4 AMS 1 AMS 2 Control soil

Dehydrogenase 8.21+0.6a 7.91+0.4a 7.82+0.4a 8.10+0.3a 0.82+0.8b 0.91+0.8b 5.3+0.6 c
(mg/g-gh-1)

Urease 4.25+0.4a 4.42+0.3a 4.52+0.3a 4.36+0.3a 1.03+0.1b 1.10+0.1b 2.77+0.2c

(mg/g-1 3h-1)

Acid phosphatase 2.12+0.5a 2.41+0.4a 2.37+0.4 a 2.41+0.4a 1.41+0.1b 1.46+0.1b 1.94+0.2c

umol-p-nitrophenol

Alkaline 2.47+0.4a 2.44+0.4a 2.49+0.4a 2.46+0.4a 1.42+0.02b 1.38+0.03b 1.87+0.2c

phosphatase
u-mol-p-nitrophenol

Glucosidase mg/g- 6.7+0.4a 6.4+0.4a 6.4+0.4a 6.6+0. 4a 1.7+0.3 b 1.0+0.3b 3.2+0.1c

6h-1
Values followed by the same alphabets are not significantly different
Values followed by different alphabets are significantly different

Baath et al., (2005), and Schoenhaltz et

Discussions and conclusions
The washed down soil in
abandoned mine pits have been known to
be very complex and different from the
surrounding soil (Weillinga et al, 1999,
Nwaugo et al, 2007b). This was evident
in this study. Physicochemical parameters
determined in this study, showed
significant differences in values according
to the soil type analyzed. PO4, SO4, NO3,
TOC and moisture content were
significantly more in the alluvial soil in
the abandoned mine pits. Statistical
differences were equally evident in soil
particle sizes investigated, as silt and clay
particles were more available in the
alluvial soil while the active pit soil had
mainly sandy particles. This might have
resulted from the action of rain water run-
off which could carry smaller particles
of soil, and clay with less sand into the
abandoned pits. These particles had
higher PO4, SO4, NO3 and TOC content
than the sandy soil of the active mining
pit where excavation was still on.
Nwaugo et al., (2007b) had reported a
similar observation. These observations
equally agree with Cloasson et al., (2005),
al., (2000) who had earlier reported that
the washing down of particles into a pit,
brings NO3, PO4, SO4 and organic carbon
into such pit. Again, excavation activities
in mines, bring up sand particles to the
surface. These could therefore account
for the poor soil quality of the active mine
pit soil and the opposite for the alluvial
soil of the abandoned mine pits as
observed in this study.
Further observations indicated that
soil moisture contents were higher in the
abandoned mine alluvial soil than other
soil samples. This observation could be
attributed to high silt and clay content of
the alluvial soil which could retain more
water than the sandy soil. The pH
showed neutral values in the abandoned
pits following accumulation of materials
from various sources that could act as
buffering materials thereby reducing the
acidity. The active mine soil samples
were still fresh and acidic due to the mine
effluent water. Several researchers have
reported acidic pH in active mines of
heavy metals (Weillinga et al, 1999,
Pascul et al, 2000).

1835
 Nwaugo et al, 2009
The heavy metals concentration
showed a remarkable trend as only Pb and
Zn had concentrations above acceptable
limits following the standards of CEC
directive, (CEC Directive, 1986, Van-Rensburg
et al, 1998). Values for Cu, Cd, and Fe were
within acceptable limits, though Fe had higher
values in the active mining pits with its reddish
brown colour. The high Pb and Zn values could
be attributed to water from the mining pits.
These are the metals mined in the area, so parts
of the ore could dissolve in the available soil
water. Brenar et al., (1995), and Fortin et al.,
(1995) had reported that the concentration of
any particular heavy metal in soil is a reflection
of the metal mined in that area, which agrees
well with this study.
Observations in the microbial
diversity and structure showed a higher
microbial prevalence in the alluvial soil of
the abandoned pits. This could be
attributed to its rich microbial nutrients
availability and complexity. The
availability of SO4, PO4, NO3 and
metabolizable organic matter in the soil
indicated that nutrients were available to
support the proliferation of
microorganisms. However, the only
difference was the concentrations coupled
with the other accompanying
environmental factors.
The most sensitive bacterial group
was the nitrifying bacteria while the least
was the total heterotrophic bacteria. The
THB group is a very complex group
comprising of all forms of viable bacterial
species in the soil. It is not specialized,
hence is the sum total of all available
viable bacteria. The nitrifying bacteria are
very sensitive to environmental changes,
especially heavy metals and oxygen
tension. Mining is known to greatly alter
these factors (Lee et al, 2002, Pascul et al,
1996) . The conditions in the active pit
were neutralized in the mine alluvial soil
hence the increase in microbial load. The
same trend was observed in the
cellulolytic and phosphate solubilizing
1836
Nig.J.Microbiol., 23(1);1830 - 1838
bacteria. Much of the materials washed
into the abandoned mine were plants and
other organic materials which contained
high cellulose and phosphate. Thes served
as substrates for the cellulolytic and
phosphate solubilizing bacteria. Kahn, and
Scullion (2000) had a similar observation.
While IOB and SOB were more
prevalent in the acidic soil of the active
mines, SRB were higher in the more
neutral abandoned mine alluvial soil.
This observation agrees with Cloassen et
al., (2005), Fortin et al., (1995) and
Weillinga et al., (1999). This observation
tends to agree that microbial diversity is
complex but depends on the interactions
of several environmental factors.
The observations of more microbial
loads in the alluvial soil of the abandoned
pits indicated the high availability of
utilizable substrates and condusive
environmental factors. Konopka et al.,
(1998), and Prescott et al., (2003) have
stated that micro-organisms proliferate in
any habitat with enough nutrients and
conducive environment. This could be the
case in the abandoned mine alluvial soil
with enough microbial nutrients (NO3,
PO4, SO4 and TOC) and good
environmental conditions (more neutral
pH, temperature and less heavy metal
concentrations).
Dehydrogenase, produced by all
forms of microorganisms (Lee et al,
2002) was the most adversely affected
enzyme. This correlated positively with
the microbial loads. Active mine pit soil
had the least microbial loads except in the
case of IOB and SOB bacteria.
Summation of the microbial loads showed
that the alluvial soil of the abandoned pits
had higher loads than the control and the
active mine pit in that order. This then
follows that the enzymatic activities are
dependent on the producing
microorganisms i.e. low microbial loads
will result in low enzymatic activities and
high load result in higher activities. The
high cellulose/carbohydrate components
 Nwaugo et al., 2009
in the abandoned mine pit alluvial soil
provided substrate for the glucosidase
while the protein served as substrate for
urease. The phosphorus-containing
compounds, which were high in the
abandoned mine alluvial soil served as
substrates for the phosphatases. This then
supported the high enzymatic activities in
such soil over the other soil types with
less utilizable materials (substrates).
Generally enzyme activities values
obtained in this study fall within those
reported by Li et al., (2005), Kuppeman
and Carreiro (1997) and Nwaugo et al.,
(2007ab).
The results showed the alluvial soil
of the abandoned mine pits as being more
complex in microbial diversity and
nutrients. This also indicated more
complex biogeochemical transformations
in such soil with its complex array of
microorganisms and enzymes.
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