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Biokemistri
Nigerian Society for Experimental Biology
ISSN: 0795-8080
Vol. 20, Num. 2, 2008, pp. 77-84
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Biokemistri, Vol. 20, No. 2, Dec, 2008, pp. 77-84
Soil bacterial
flora and enzymatic activities in zinc and lead contaminated soil
Victor Oluoha Nwaugo1*, Reginald Azuonye
Onyeagba1, Emmanuel Iroha Akubugwo2 and Osita Ugbogu1
Departments of1Microbiology and 2Biochemistry, Abia State University, Uturu, Nigeria
*To whom correspondence should
be addressed. E-mail: vonwaugo@yahoo.com, Tel: 234063494654
Received 28 November 2008
Code Number: bk08012
Abstract
Soil
bacterial flora and enzymatic activities in lead and zinc contaminated soil of
Ishiagu, Nigeria were investigated. The physicochemical properties measured
showed that the mining pit had acidic pH (5.6) which gradually increased till
7.5 in the control. Organic matter was only 2.57mg/g in the pit but gradually
reached 7.41mg/g in control. Pb concentration was higher at pit 360.52mg/g,
305.46mg/g at 5m away and lowest at control 36.16mg/g. Zn was 217.47mg/g at the
pit, 176.32mg/g at 5m, 106.18mg/g at 10m and only 40.67mg/g at control. This
showed a gradual fall away from the pit. Major organisms at the pit were Pseudomonas
and Bacillus species (30% each) and Mocrococcus and Chromobacter
species (20% each) E. coli, Salmonella and Lactobacillus
species, which occurred in the control soil, were absent in the pit soil but
occurred at various rates in other soil samples. Bacterial prevalence,
diversity, and bioload were all high in the control, followed by 100m away
while values decreased significantly towards the pit. Soil enzymatic activities
correlated negatively with heavy metal concentration. This showed that the
higher the heavy metal concentration the lower the enzymatic activities. Urease,
dehydrogenase activity, hydrogen peroxidase
and polyphenol oxidase were adversely affected but alkaline phosphatase did not
show any significant effect.
Keywords:Soil, Zinc, Lead, Bacteria,
Bioload, gradient
INTRODUCTION
Microbial flora in relation to soil bioload and contamination is a
significant component of the quality of the soil. The type of activities
prevalent in any given environment determines the type of contamination in that
area1-3. Soil and water bodies have been sinks for many hazardous
wastes, organic wastes sewage and several other waste types generated from
different human activities. Many of these wastes contain heavy metals which
contaminate the receiving sink.
The soil serves several human needs and several other natural
functions. Nature too, has placed several minerals (metallic and non metallic)
in the soil. To reach and obtain them requires extensive drilling or excavation4-8.
Most industrial wastes are often not well treated before disposal9-11.
The most commonly encountered heavy metals include Pb, Cd, Zn, Hg
and As12,13. Heavy metal toxicity represents an uncommon but
clinically significant medical hazard often unrecognized and inappropriately
treated. In nature, Pb and Zn are often found together13,14. Both
metals have found extensive use in mans activities. Waste resulting from their
mining and use liter many places.
In most cases, large ponds and heaps of wastes are left in the
trail of excavations for these elements. While rains wash the waste heaps into
the surrounding water bodies and farmlands, the ponds overflow their banks
resulting in pollution even outside the area of production15. The
above scenario is typical of the Pb or Zn mining fields of Ishiagu, Ebonyi State in Nigeria. Several reports have it that such pollution inhibits soil microbial
activities but information from the study area is lacking in spite of the
extensive mining activities there. This work then aims at establishing the level
of pollution, soil bacterial flora and enzymatic activities in Ishiagu in
relation to the soil health or quality.
MATERIALS
AND METHODS
Ishiagu, the study area lies in the northern part of Abia State, South East Nigeria where rock blasting and heavy metal (Pb or Zn) mining
activities are common. Two major heavy metal mining companies in the area
excavate for Pb and Zn and abandon the wastes generated in heaps leaving large
ponds in the typical Guinea Savannah climate farmland. Indigenes also scavenge
for the same minerals in the abandoned heaps.
Soil properties and heavy metal extraction
The climatic condition of the area is Guinea savannah and has
typical uniform sandy, loamy soil. The soil pH and temperature were determined
directly at the site using multipurpose tester (Jenway HANNA 1910 model). Soil
Organic matter content was determined using loss of ignition method as
described by Lee et al.2 involving the use of furnace (MAC
2000). The soil moisture was also determined by the drying to constant weight
method as in the publication of the American Public Health Association (APHA)16.
Soil heavy metals concentrations were estimated using the Atomic
Absorption Spectrophotometric (AAS) method after acid digestion as described in
APHA16 with HACH/D2/2010 spectophotometer.
Soil
enzymatic activities
The enzymes analysed in this work include dehydrogenase activity (EC 1.1.1.1) which
is the reduction of tetrazolium chloride (TTC) to triphenyl formazon (TPF),urease, hydrogen peroxidase, poly- phenol oxidase, acid and
alkaline phosphatases activities. The dehydrogenase activity was determined as described by Cassida et al17
and modified by Li et al18. 5.0g of soil was mixed in 10ml
0.25% aqueous triphenytetrazolium chloride (TTS). This was incubated in sealed
tubes at 30°C for 6 hours. The absorbance at 485nm of the methanol
extracts of the triphenylformazon (TDF) formed was measured using methanol as
blank. The result was expressed asTPF-1 dry soil 6h
The urease activity was estimated using the colourimetric method
based on NH3-N formation in the Urea-amended soil sample. The soil
was incubated at 37°C for 24 hours and the result expressed as mg NH3-N
g-1dry soil 24h19,20.
Soil hydrogen peroxidase activity was determined by the KMnO4
titration method. The result was expressed as The result was expressed as mLg-1 dry soil soil21. The polyphenol oxidase
activity was estimated by the colorimetric method as described in Ma et al22
and Li et al18 modified from Tabataabai and Bremearr23.
This was based on the purpurogallin formation in the pyrogallic
acid-supplemented soil samples. The amended soil was incubated at 30°C
for 3 hours and the result expressed as mg purpuragallin g-1dry soil 3h1.
The activities of both acid and alkaline phosphotases were determined
using the methods of Tabatabai (24 which involved the use of
Nitrophenyl/phosphate with CaCl2 and NaOH added to stop the
reactions and reading the result at 410nm.
Microbiological
analysis
Prevalence of soil bacterial species was determined using the
culture technique. Ten soil samples from each sampling point were collected at
one-week intervals and cultured on Tryptone soil Agar, McConkey Agar, and Mineral salt Agar. The
frequency of each organism was taken as occurrence or prevalence. Different bacterial
groups were also investigated using various culture media including Pb and Zn amended ones at concentration of 3mg/ml of the Pb and Zn10. The groups were Total Heterotrophic
Bacteria (THB), Coliform Bacteria (CB), Nitrifying Bacteria (NB), Pb-resistant
bacteria (PRB) and Zn-resistant Bacteria (ZRB). The bioloads of these organisms
were determined after ten-fold serial dilution of 1.0g of fresh soil sample as
described by Chessbrough25.
RESULTS
The results obtained in the physicochemical parameters and the
heavy metal concentration assessments are shown in Table 1. There was a general
gradient in all the parameters-either increasing or decreasing with distance
away from the excavated pit. The pH of the pit soil was weakly acidic (5.6) but
gradually changed to neural in 100m and control soil (7.0 and 7.5)
respectively. The organic matter that was only 2.57 in pit soil rose to 7.41 in
control. However soil moisture gradually decreased away from the water filed
pit but no significant change was observed in temperature (Table 1).
Concentrations of Pb and Zn followed the same pattern and were
quite significant (p = 0.05) highest at the pit lowest and further away.
Pb had 360.52 at the pit, 305.46 at 5m away while 100m and control had 112.53
and 36.16 respectively. Zn had 217.47 at the pit, 176.32 at 5m away and 40.67
at control (Table 1) with 51.77 and 41.67 for 100m and control soil
respectively. Pb and Zn values correlated negatively with pH and organic matter
content but positively with moisture content.
The frequency (occurrence) of each bacterial species observed in
the work is shown in Table 2. All the organisms had their lowest prevalence in
the pit soil but increased gradually till 100m which had no significant
statistical difference with control (p = 0.05). Bacillus and Pseudomonas
species were the most prevalent in the heavy metal source (pit) while E-coli,
Staphylococcus and Lactobacillus were found in 100m and control
samples (Table 2).
Table 3 shows the bioload of each of the groups of bacteria
estimated in relation to the effects of the heavy metals in soil. The bacterial
groups had their lowest bioload in pit, followed by the 5m away soil while the
highest bioloads were in the 100m and control soil samples.
The most affected was the nitrifying bacteria with only 1.7x101
of the pit and 2.8x104 and 3.1x104 in 100m and control
respectively. The least affected bacterial group was the total heterotrophic
bacteria
TABLE 1: Concentrations of lead and Zinc and some
other parameters of the various soils samples analyzed
Soil sample distance from pit |
Pb
mg/g |
Zn
mg/g |
pH
|
Temp.
0C |
Organic matter
mg/g |
Soil moisture
% |
Pit soil |
360.52 |
217.47 |
5.6 |
30.2 |
2.57 |
37.00 |
5m |
305.46 |
176.32 |
6.2 |
2.9.6 |
3.67 |
35.00 |
10m |
216.24 |
106.81 |
6.8 |
29.7 |
5.14 |
34.00 |
100m |
105.31 |
81.77 |
7.2 |
29.6 |
7.12 |
32.00 |
Control |
36.16 |
40.67 |
7.5 |
29.5 |
7.41 |
30.00 |
*Values are the mean values of three times sampling.
TABLE 2: Prevalence of bacterial species isolated from the various
samples
Organisms |
Pit |
5m |
10m |
100m |
Control |
Pseudomonas
species |
30.00 |
30.00 |
40.00 |
60.00 |
30.00 |
Micrococcus
species |
20.00 |
30.00 |
40.00 |
60.00 |
60.00 |
Xanthomonas
speccies |
- |
- |
20.00 |
30.00 |
20.00 |
Azotobacter
species |
20.00 |
20.00 |
40.00 |
60.00 |
60.00 |
E. coli |
- |
20.00 |
60.00 |
80.00 |
90.00 |
Salmonella
species |
- |
- |
- |
20.00 |
10.00 |
Chromobacterspecies |
20.00 |
30.00 |
40.00 |
60.00 |
40.00 |
Bacillus
species |
30.00 |
40.00 |
80.00 |
100.00 |
100.00 |
Staphylococcus species |
20.00 |
20.00 |
30.00 |
60.00 |
70.00 |
Lactobacillus species |
- |
- |
20.00 |
40.00 |
50.00 |
*Values are mean percentages of isolatesobtained from ten times
sampling; - Not observed
TABLE 3:Bioloads of various groups of bacterial species from the various
soil samples
Bacterial group |
Pit |
5m |
10m |
100m |
Control |
THBC |
2.3x104 |
6.1x104 |
4.3x105 |
5.7x106 |
6.4x106 |
NBC |
1.2x101 |
2.9x102 |
4.1x103 |
2.8x104 |
3.6x104 |
CBC |
3.1x102 |
4.1x103 |
2.9x104 |
2.1x104 |
3.2x106 |
PRBC |
1.6x102 |
1.8x102 |
2.7x103 |
2.7x103 |
2.1x103 |
ZRBC |
2.1x102 |
2.5x102 |
3.1x103 |
2.3x103 |
2.7x103 |
*Bioload values are mean values of five
times estimation; THBC: Total Heterotrophic Bacterial Count; NBC: Nitrifying
Bacterial Count; CBC: Coliform Bacterial Count; PRBC: Lead (Pb) Resistant
Bacteria Count; ZRBC: Zinc (Zn) Resistant Bacteria Count
TABLE 4: Soil enzymatic activities
in the various soil samples examined
Enzymes
|
Pit
|
5m
|
10m
|
100m
|
Control
|
Dehydrogenase
mg g-1 6h-1
|
0.72
|
0.99
|
1.31
|
4.11
|
4.42
|
Urease
mg g-1 24h-1
|
0.61
|
0.92
|
2.2
|
3.10
|
3.32
|
Polyphenol oxidase
mg g-1 3h-1
|
0.91
|
1.51
|
1.74
|
2.1
|
2.61
|
Hydrogen oxidase
mL g-1 1h-1
|
0.93
|
1.32
|
1.82
|
2.4
|
2.52
|
Acid phosphatase
(µmol-p-nitrophenol)
|
0.54
|
0.72
|
1.01
|
1.53
|
1.62
|
Alkaline phosphoatase
(µmol-p-nitrophenol)
|
0.97
|
1.12
|
12a
|
1.4b
|
1.61
|
Soil enzymatic activities were significantly lower in the soil
from the pit compared to other soil sampling points. However, the activities
increased with distance away from the excavation pit correlating negatively
with the heavy metal concentrations in soil. The most sensitive enzyme activity
was the dehydrogenase, which had 6.13 times activities less in the pit compared
to the control and 4.3 times less in the 100m away. Urease also showed high
sensitivity to Pb and Zn poisoning with 5.44 times less than the value obtained
in the control (3.32). The phenol oxidase was 2.86 times less in pit than
control. Acid phosphatase and hydrogen peroxidase, also showed similar gradient
in activities (Table 4). The least sensitive i.e more resistant enzyme was the
alkaline phosphatase which had only 1.66 time less activities in the most heavy
metal contaminated soil pit.
DISCUSSION
Results obtained in this work showed that soil pH and organic
matter content were adversely affected by high Pb and Zn concentrations
observed nearest the pit. The values obtained at the pit and 5m away were above
the acceptable levels26 hence affected the pH and organic matter
content. Oliveira and Pampulha1, Babich and Stotzky27 and
Christensen28 agree that low pH (acidic) reduces solubility and
speciation of metals in soil and soil solution which directly rubs off in soil
organic matter. This was the case in this study where pH and organic matter in
the pit soil were lower than others. This could have caused the high adverse
effect observed in the soil at the pit and 5m away. This assertion tally well
with Nwaugo et al29 and Chinyere30 that pollutants
have highest concentrations at the discharge point or sources.
Though ten bacterial species were observed in this work, only five
were seen in the immediate vicinity of the pollution pit. The bacteria
increased in prevalence and diversity away from the pit indicating a negative
correlation with Pb & Zn concentrations. Fagade and Adetutu10,
Nwuba31 and Abdou et al13 stated that heavy metal
suppressed microbial growth but allows resistance ones to grow slowly which was
observed in this work. However, away from the high Pb and Zn concentration,
microbial prevalence and diversity increased. This observation was further
buttressed by results obtained in the bioload analysis as bioload of most
bacterial groups were higher distances away from the pit. Lee et al2,
Kuperman and Carreiro32, and Fagade and Adetutu10
reported that most heavy metals are toxic to soil micro-organisms at high
concentrations and even inhibit the enzymes resulting in low microbial
occurrence in such polluted soils.
All the various groups of bacteria examined were not affected at
the same rate. This work agrees with Nwaugo et al33, Martensson34
and Oliveira and Pampulha1 that nitrifying bacteria which is a
complex group of phylogenetically and physiologically diverse bacteria are very
sensitive to pollution. This gives a very positive indication that the nitrifying
bacteria can be used as good indicators of anthropogenic pollution of the soil.
There was less effect on THB, which could be understood as the group is the sum
total of the heterotrophic (all variable and culturable) bacteria present in the
soil at that point. The suppression of even the Pb and Zn resistant bacteria
groups of the pit and 5m away show that no group of bacteria is spared by
adverse environmental conditions, though the extent varies.
The significance of soil enzymatic activities assessment is
enormous as even the activities of the unculturable bacterial types are equally
assessed with the culturable ones. This is because the enzymatic activities
correlate well with their parent organisms1,2,35-37. Analysis of
this work show that the oxidoreductases used in the work (dehydrogenase, urease, phenol
oxidase, perioxidase and the phosphatases-(alkaline and acid) were sensitive to
Pb and Zn contamination. However dehydrogenase,
urease, phenol oxidase and acid phosphatase were more sensitive to the heavy
metals pollution. The work agrees with Quilchairo and Maronon38,
Leiros et al39, Konapka et al3 and a host
of others that soil enzymatic activities could easily be applied in assessing
soil quality, especially the intracellular ones which are directly attached to
the organisms.
The soil fertility could also be assessed by these enzymatic
activities along with the nitrifying bacterial bioload. These determine the
level of Nitrogen in the soil in question which agrees with Mantellin and Touraine40.
Dehydrogenase activity, urease and acid phosphatase were very sensitive to the
heavy metal contamination. However, the work disagrees with Wyszkowska and
Kuchariski41 who stated that acid phosphatase was less sensitive in
the soil. This difference could be due to the type of soil contaminant examined
as Wyszkowska and Kuchariski41 had worked on petrol-contaminated
soil.
Lee et al2, Wyszkowska and Kuchurski41,
and Mantellin and Touraine40 reported that plants assimilate and
accumulate these heavy metals in their tissues which could be dangerous to man
if such plants are consumed. In conclusion, this work therefore suggests that
the excavation for Pb and Zn with the consequent inappropriate disposal of the
resultant wastes, cause high concentration of these elements in the soil. The
concentration of the contaminants gradually decreased with distance away from
the source of pollution, which agrees
with Nwaugo et al42. The Pb and Zn
contamination adversely affected the soil microbial quality in both prevalence
and diversity and calls for remediation if the soil must be used for
agricultural purposes.
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© 2008 Nigerian Society for
Experimental Biology
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