Pedogenetic Activities of Soil Microbes as Influenced by Trivalent Cationic Chromium
We investigated the effects of different levels of chromium
(111) (0, 10, 50, 100, 250, 500 and 1000 mg Cr kg-1 d.w.) on
selected soil microbial parameters, such as total and active fungal mycelia,
total microbial biomass carbon (Cmic) and soil potential activity
(respiration) on an Isohyperthermic Arenic Kandiudult of Otamiri River
floodplain, southeastern Nigeria. Field sampling was conducted in 2006
and collected soil samples were put in plastic containers before application
of 7 treatments of Cr (111) as indicated. Soil samples were kept under
laboratory conditions (water holding capacity = 30%; temperature = 25Â°C).
Treatments were arranged using Completely Randomized Design (CRD) with
5 replicates. Routine and special analyses were conducted on investigated
parameters. Results indicated significant (p<0.001) reduction in active
and total fungal mycelia caused by additions of Cr (111) to the soil.
A significant decrease (p<0.01) in Cmic was detected only
in soils with 1000 mg Cr kg-1 d.w. Soil microbial activity
declined to 17.07% in contaminated soils when compared with soils for
control experiments. There were good relationships between soil microbial
respiration and active fungal mycelium, suggesting their use in soil quality
Soil is a product of interactions of complex pedogenic processes
(Birkeland, 1999), including the plant-rock-soil association (Darmody et
al., 2001, 2004). Part of pedogenesis is the contribution of the microbial
populations which play fundamental role in ecosystem functioning, especially
in organic matter decomposition and nutrient cycling. Activity of soil microbes
becomes a very sensitive and reliable indicator of changes in the pedosphere.
But soil microbial responses are often altered by anthropogenic activities,
most of which are deleterious (Aiyesanmi, 2006). Ekpo and Nwankpa (2006) reported
that high levels of crude oil pollution caused significant depression in the
growth of fungi possibly due to high heavy metals content. Apart from crude
oil spillage and other exploration activities, heavy metals enter the soil system
through the use of organic and inorganic fertilizers.
One of such biotoxic heavy metals is chromium, which among
other forms can appear as a trivalent cation Cr (III) and stable in the soilsphere
(Stepniewska et al., 2004). Chromium is both a mutagen and a carcinogen
even at sub-ppm levels (Stewart et al., 2003). Chromium is highly soluble
in an aquatic environment (Babel and Opiso, 2007) and readily adsorbed by living
organisms. In humans, accumulation of Cr beyond permissible limits can cause
severe health problems (Kurniawan, 2002), such as lung cancer, liver damage,
kidney and reproductive problems.
Chromium has no less important effects on microbial growth
and activity. Negative correlations were reported between Cr and microbial performance
(Kizilkaya et al., 2004) while no effect was also recorded among some
authors (Majer et al., 2002). However contrasting effects among environmental
factors make it difficult to pin down a single factor in a cause-effect relationship
with another biological variable. It is in the light of the above fact that
we studied the effects of different rates of Cr (III) on microbial activities
related to soil formation and this is consistent with earlier suggestions (Dick,
1997; Gilier et al., 1998; Gigliotti and Farini, 2002). With increasing
population and conflictive land uses in southeastern Nigeria coupled with the
spate of land degradation, it becomes necessary to determine levels of Cr (III)
content of soils considered biotoxic to the microbial community. Based on the
above, the major objective of our study was to determine the minimum Cr (III)
concentration in studied soils at which a significant decline in the activity
of soil microbes was observed.
MATERIALS AND METHODS
Study Area: Soils of Otamiri River floodplain Southeastern Nigeria are
located between latitudes 4Â°15100.22011 and 5Â°30110.31011N
and longitudes 6Â°47121.31011 and 7Â°01105.41011
E. Soils of the area were classified as lsohyperthermic Arenic Kandiudult
(Onweremadu et al., 2006). Soils are derived from Coastal Plain Sands
(Benin formation) of the Oligocene-Miocene geological era. The study site belongs
to the lowland areas of southeastern Nigeria. It is humid tropical, having an
average annual rainfall of 2400 mm and annual temperatures ranging from 20-29Â°C.
The study area has a rainforest vegetation and farming is a major socioeconomic
Soils Sampling: Surface soil samples were collected before rains in
2006 at the midslope of Otamiri River Slope. The site was considered part of
the river valley with low chromium concentration (5.2 mg kg-1 d.w.)
and uncultivated river vegetation Soil samples were air-dried and sieved using
2 mm sieve.
Experiment: Four kilograms of sieved fresh soil was incubated in plastic
containers and added with chromium (III) sulphate solution at increasing concentrations
of 0, 10, 50, 100, 250, 500 and 1000 mg Cr kg-1 dry weight). The
soil condition was controlled by having a constant gravimetric moisture content
(Water holding capacity = 30%) and temperature (Temperature = 25Â°C) and
kept in the dark for 60 days. Soil water was recharged every two days to make
up for evaporated moisture loss. The control soil samples were given the conditions
as those treated with chromium sulphate solution but there was no chromium application
on it. Five replicates were administered for each Cr concentration, giving a
total of 35 samples for the study and arranged in a Completely Randomized Design
Laboratory Analyses: Particle size analysis was determined by hydrometer
method (Gee and Or, 2002). Soil pH was measured potentiometrically in I:1 soil/solution
and a clear supernatant was estimated using a microprocessor ionanalyzer/901
(Orion Research, Beverly, M.A.), involving a combination of glass and Calomel
electrode (Beckman Tullerton C.A.) Total Organic Carbon (TOC) and Total Nitrogen
(TN) in air-dried soil samples (<180 Î¼m) were analyzed in a Vario Max-ELEMENTAR
CN-analyzer (D-63452 Hanau, Germany). Cation exchange capacity was estimated
by summation of Mehlich -3 extracted cations (Darmody et al., 2000) while
exchangeable cations were got through inductively coupled plasma spectroscopy
(Mehlich, 1984). Silt-Clay Ratio (SCR) and Carbon-Nitrogen ratio (C/N) were
calculated by dividing silt content by clay and carbon content by nitrogen,
During the first week after incubation, biological analyses
were carried out on fresh soils stored at 4Â°C. Total and active fungal mycelia
were measured by filter technique (Sundman and Sivela, 1978). One gram of fresh
soil was mixed with 100 mL of K-phosphate buffer 60 mM at pH 7.5 and the suspensions
were oscillated at 600 rpm for 2 min and 0.5 mL was transferred to a polypropylene
prefilter, having 0.6 Î¼m mesh size. A prefilter was treated with aniline
blue in 80% lactic acid, to determine total fungal mycelium while another prefilter
was treated with fluorescein diacetate to estimate active fungal mycelium as
described by Soderstron (1977). Thereafter, prefilters were observed at a magnification
of 400 x (Zeiss Azioskop MC 100 Spot Microscope, equipped with a mercury lamp).
Length of fungal mycelium was determined by the intersection method and converted
to fungal mass on the basis of average values of cross-section, density and
dry mass of hyphae (Berg and Soderstrom, 1979). Total microbial biomass carbon
was estimated by fumigation extraction method using 5 g oven-dry soil, calculating
microbial biomass carbon as 2.64 Ec values (Vance et al., 1987), where
Ec is the difference between extractable C from fumigated and non-fumigated
samples. The potential activity of soils was measured as soil respiration determined
by gas chromatograph as CO2 evolved from fresh soil samples incubated
for 60 min in standard conditions of 30% water holding capacity and 25Â°C
temperature and kept in the dark. Values were expressed as mg CO2
g-1 d.w. h-1.
Total Cr on the soil was determined using a modification of
EPA method 3052. The soil was digested in a CEM microwave model MDS-8ID, with
hydrofluoric and nitric acid. Boric acid was added before sample analyses to
facilitate the removal of hydrofluoric acid from solution though the formation
of fluoroboric acid.
Statistical Analyses: Descriptive statistics, namely means and standard
deviations were calculated on the 5 replicates for each treatment. Variability
in soil incubated with different Cr (III) concentrations were assayed by one-way
analysis of variance (ANOVA), followed by the SNK Student-Newman-Keuls Test
(p<0.05, N = 5). Relationship between Cr (III) concentrations and biological
parameters were analyzed by correlation coefficient using PC SAS version 8.2
RESULTS AND DISCUSSION
Soil Properties: Soils were sandy and highly weathered (SCR = 0.3) and
strongly acidic which is attributable to parent material, climate, fluvial depositions
and land use. Low CEC, is possibly due to low organic carbon and clay contents
of the soil. Values of these soil properties are shown in Table
1. However, Oti (2007) reported that land degradation by soil erosion has
altered soil properties negatively, resulting to soil productivity decline.
Carbon-nitrogen ratio of soils before treatment, often used as an indicator
of state of soil microbial biomass (Moore et al., 2000), was greater
than 6 (C/N = 12), indicating higher soil fungi population compared with bacteria
(Moore et al., 2000).
||Selected soil properties in the study site
Cr = 5.2 mg kg-1
d.w., SCR = Silt-Clay Ratio, SOC = Soil Organic Carbon, TN = Total Nitrogen,
CEC = Cation Exchange Capacity, d.w. = Dry weight
||Distribution of total and active fungal mycelium
(meanÂ±SD) in soil samples at different rates of Cr (III) concentrations
|SD = Standard Deviation, Cr = Chromium, d.w.
= Dry weight
Distribution of total
carbon and soil potential activity (meanÂ±SD)
at different rate of Cr (III) concentrations
|d.w. = Dry weight, Cr = Chromium
Chromium Concentrations and Microbial Performance: There were significant
differences (p<0.001) in the distribution of these mycelial types given different
rates of Cr (III) application (Table 2). There were significant
(p<0.001) reductions in mycelia forms as Cr (III) concentrations were increased
and these results are consistent with the findings of Ekpo and Nwankpa (2006)
in crude oil polluted soils of Nigeria. Similar findings were reported by scholars
in agroecologies other than the study site (Hiroki, 1992; Mainville et al.,
2006), where marked effects of heavy metals pollution on fungal mycelium were
Soil microbial biomass carbon (Cmic) was less affected
by increasing doses of Cr (III) additions when compared with the effect of this
heavy metal on total and active fungal mycelia (Table 3). Significant
reductions (40.90%) for the total microbial biomass as shown in Table
3 was only found in soils with 100 mg Cr kg-1, suggesting that
higher concentrations of Cr (III) in soil is inhibitory to accumulation of Cmic.
However, tillage practices like higher Cr (III) concentrations, could reduce
soil microbial biomass C significantly (Acosta-Martinez et al., 2004),
noting that Cmic in perennial pasture was greater than value of Cmic
from continuously cultivated cotton soils. In other studies (Franzluebbers et
al., 1995), Cmic was increased with conservation while the same
tillage type modified soil microbial community (Pankhurst et al., 2002).
Total soil activity represented by rate of respiration is shown in Table
3, indicating a significant reduction (p<0.001) in soils treated with
1000 mg kg-1. There was 17.07% reduction in respiration in soils
treated with trivalent cationic chromium when compared with control soils. Lower
values of respiration (soil activity) could be a result of inhibition of intracellular
enzymatic activities in fungi. Similar findings Klose and Tabatabai (2002) and
Renella et al. (2002) reported that fumigation with chemicalsubstances
inhibited proteases and consequently reduced intracellular activity (Acosta-Martinez
et al., 2004) and microbial biomass while microbial biomass was positively
affected by enzyme B-Î²-glucosidases activity at 0-7.5 cm depth (Ndiaye
et al., 2002). The implication of reduced microbial performance following
increasing applications of chromium is the inability of these soil organisms
to optimally play their role in sustaining environmental quality and agricultural productivity.
Entry of chromium and other heavy metals alters soil system adversely, leading
to either sub-optimal or total extinction of soil life (Brady and Weil,1999).
It becomes necessary to assess microbial biomass in soils especially in soils
vulnerable to contamination by heavy metals (Brookes et al., 1986).
||Relationships between Cr (III) concentration
and soil biological parameters
|Cr = Chromium, vs = Versus
Chromium and Biological Parameters: There was a significant positive
correlation (R = 0.91; p<0.0001) with minimal coefficient of alienation between
Cr (III) content in soils and potential respiration. Results also indicated
that Cr (III) had high relationship with soil microbial respiration than microbial
biomass, suggesting that the former has more predictive capacity than the latter.
Also, there was a negative correlation between Cr (III) and active fungal mycelium,
indicating that higher concentrations of Cr (III) certainly inhibit activity
of fungal mycelium (Table 4).
Soil potential activity (respiration) in chromium-treated soils
was related to some biological parameters (Table 4). There
was a positive relationship between soil potential activity and active fungal
mycelium (R = 0.62; p<0.01) when compared with total fungal mycelium (R =
0.43; p<=0.05), while microbial biomass carbon (R = 0.36; p<=0.05) had a very
poor relationship with respiration. This result is consistent with the findings
of Brookes (1995) that soil microbiological properties, such as respiration
and microbial biomass carbon (Cmic) respond quickly to environmental
conditions than inherent soil organic matter content.
This study revealed that Cr (III) concentrations had varying
quantitative influences on both total and active fungal mycelia. Very low values
of Cr (III) had significant (p<0.001) reductions in fungal performance while
microbial biomass carbon showed least sensitivity to Cr (III) treatment. There
were good relationship between soil potential respiration and active fungal
mycelium when soils were treated with Cr (III). These results show that soil
microbial respiration and activity of fungal mycelium can be very useful in
modelling soil environmental quality and other soil toxicological tests.
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