Effect of Lead and Copper on the Growth of Heavy Metal Resistance Fungi
Isolated from Second Industrial City in Riyadh, Saudi Arabia
Al-Kadeeb A. Siham
In this study, soil samples for isolation of heavy metal resistance
fungi were collected from different distance of Electric Meter manufactory
in Second Industrial City, Riyadh, Saudi Arabia. Soil samples were analyzed
chemically for heavy metal concentrations, total soluble salts and pH
and mechanically for composition of soils. Saturation percent were determined
also. Eighteen fungal isolates were grown in 10 mM concentration
of either lead or copper ions. Aspergillus was predominant and
represented by 6 species. Fusarium was represented by 4 species.
Mucor was represented by 3 species. Penicillium was represented
by 2 species. While Alternaria, Cephaliophora, Eurotium
were represented by one species each. The intraspecific variability in
growth response to Pb2+ and Cu2+ on agar and liquid
culture was studied among isolated fungi. The growth rate of some fungi
isolated on solid media was less sensitive to addition of lead or copper
than biomass production in liquid culture.
Heavy metals waste has increased rapidly since the industrial revolution.
Toxic metals species are mobilized from industrial activities and fossil
fuel consumption and eventually are accumulated through the food chain,
leading to both ecological and health problems. The influence of heavy metals
on the microbial biomass varied with the kind of heavy metal and with the
soil type. Lead tends to accumulate in soils due to its low solubility and
relative freedom from microbial degradation. It remains accessible to the
food chain far into the future (Alloway, 1990).
Since copper is a widely used material, there are many actual or potential
sources of copper pollution. Copper is essential to human life and health
but, like all heavy metals, is potentially toxic as well significant (Nuhoglu
et al., 2002). The addition of copper to soil was reported to significantly
decreased the amount of microbial biomass and to have a pronounced toxic
effect on the size of the biomass compared to certain metals such as Pb
and As (Aoyama and Nagumo, 1997).
Copper-resistance has been demonstrated in a number of microorganisms,
including Aspergillus niger, Penicillium chrysogenum and
Rhizopus stolonifer; Helicobacter pylori; Pseudomonas
pickettii; Candida guilliermondii and Pseudomonas putida
Strain S4 (Hashem, 1989; Ge and Taylor, 1996; Gilotra and Srivastava,
1997; Saxena and Srivastava, 1998, 2002).
Hashem (1995) found that radial growth of Aspergillus candidus
was decreased by high concentration of cadmium and low copper concentration
stimulates this growth. Abd-El Naby (1997) found that high concentration
of manganese, ferrous and copper reduced the growth of Aspergillus
Hashem (1997) demonstrated the effect of cobalt, copper, lead, molybdenum,
manganese and zinc on the growth of Alternaria alternata, Aspergillus
flavus, Cladosporium herbarum, Curvularia lunata and
Ulocladium chlamydosporum isolated from the industrial Al-jubail city,
Saudi Arabia, at concentration of 500, 1000 and 2000 μg mL-1.
Higher concentrations of Cu and Zn stimulated growth of tested fungi,
while higher concentrations of lead, manganese and molybdenum inhibited
growth of some fungi.
Richards et al. (2002) determined the sensitivity of 12 Frankia
strains to heavy metals by a growth inhibition assay. About the effect
of Pb2+ and Cu2+ on strains growth, they reported
that most of the strains were less sensitive to Pb2+ (6 to
8 mM). While most strains were sensitive to 0.1 mM Cu2+, four
strains were resistant to elevated levels of Cu2+ (2 to 5 mM
and concentrations as high as 20 mM).
Sharma et al. (2002) investigated the biosorption potential of
a fungal strain isolated from industrial wastewaters contaminated with
zinc and other heavy metals. Also the growth of organisms was monitored
in the presence of other metals such as Pb, Cu, Ni and Cr separately.
The organism was capable of growing in each case at 100 mg L-1
initial concentration. Tsekova and Todorova (2002) demonstrated the influence
of copper (II) ion on the growth of Aspergillus niger.
Sani et al. (2003) studied the toxicity of Pb (II) to sulfate-reducing
bacteria (SRB) using Desulfovibrio desulfuricans G20 in a medium
specifically designed to assess metal toxicity. The effects of Pb (II)
toxicity were observed in terms of longer lag times. Lower specific growth
rates and in some cases no measurable growth. With an increase in medium
pH from 6 to 8, Pb (II) toxicity decreased. At all pH values, in the presence
of Pb (II) concentrations ranging from 3 to 15 μM, specific growth
rates decreased and lag times increased. The minimum inhibiting concentration
(MIC) of Pb (II) causing a complete inhibition in growth at pH 6 was 10
μM. The present study aimed to isolated heavy metal resistance fungi
from soils collected from different distance of Electric Meter manufactory
in Second Industrial City, Riyadh, Saudi Arabia with soil analysis for
this place. Also this study aimed to develop the effect of lead and copper
ions on mycelial growth of these fungi.
MATERIALS AND METHODS
Characteristics of soil: Method describe by Piper (1955) was used for
determination the soil type. Chemical analysis of the soil sample were
done by methods used by Chapman and Pratt (1961). Heavy metals (Al, Co,
Cu, Pb) in soil were measured by an atomic absorption spectrophotometer
after digestion with a mixture of HNO3-HCl (Soon and Abboud,
1993). Soil analysis was done with help of Soil Science Department, College
of Food and Agriculture Science, King Saud University.
Collection and isolation of fungi: Soil samples from different distance
0, 30, 60 and 100 m of Electric Meter manufactory in Second Industrial
City, Riyadh, Saudi Arabia were collected according to the method described
by Johnson et al. (1960) at a depth of 1-10 cm during the month
of February (2006) in which the temperature was 30°C and the percentage
humidity was 24% (five individual locations were selected within each
sampling site). The samples were stored in sterile plastic bags and transported
to the laboratory. Five collections of a total weight of 5000 g from the
same distance were mixed thoroughly. Approximately, half of the mixed
samples were used for soil analysis, the rest were sieved through screens
with a 0.5 and 0.1 mm diameter opening to remove stones and other debris
and used for isolation of fungal content. Soil plates method was used
for isolation fungal flora as described by Warcup (1957).
Each soil sample was culture on 3 replicates plates of peptone-dextrose
agar containing rose bengal and antibiotic (Martin, 1950) with 2 mmole
of either PbNO3 or CuSO4.5H2O. All plates
were incubated at 25±2°C for one week and examined daily. Further
inspections of the plates were made two weeks after plating to record
slow growing fungi. Fungal growing on these plates were regarded as having
at least low level of Pb2+ and Cu2+ tolerance and
were retreated on modified Dox agar (Naguib, 1967) plates for isolation
of single colonies.Screeing for high lead and copper tolerance fungi:
The isolated fungi from the previous experiment were, therefore, screened
for their abilities to tolerate the level of 10 mM concentration of lead
and copper ions. All isolates were separately inoculated onto modified
Dox agar plates with 10 mM concentration lead or copper ions. There were
3 replicates perisolate. All plates were incubated at 25±2°C
for one week. Isolates that did not grow were discarded whereas ones that
grew were regarded as being tolerant to a high level concentrations of
lead and copper ions and were used for further testing.
Identification of fungal isolate: Slides of hypha, conidiophores and
conidia were prepared by mounting with lacto-fuchsine and examined by
viewing at 1600 X magnification using a compound microscope. Size and
color of fungal colonies on media also were also recorded. All fungal
isolates were identified according to Samson et al. (1996) and
Ellis (1971, 1976).
Effect of lead and copper ions concentration on fungal growth rate and
mycelium dry weight: Inoculum (8 mm disk) from 7 day old culture of isolates
were inoculated on the center of modified Dox agar plates (9 cm) diameter
containing different concentrations i.e., 0, 2, 6, 12 mM of sulphate salt
of copper or nitrate salt of lead. There were three replicates for each
experiment. The media without metal served as control. After various periods
of incubation at 25±2°C the diameters of mycelia growth, in
four directions and the radial growth rates (in cm per day) were determined
(Babich and Stotzky, 1977). Mycelia growth was measured on days 3 after
incubation for Mucor circinelloides and Mucor racemosus,
on days 4 for Cephaliophora irrgularis and on 5 days for Alternaria
chlamydospora, Aspergillus niger, Aspergillus orchaceus,
Aspergillus oryzae, Aspergillus parasiticus, Aspergillus
tamarii, Aspergillus ustus, Eurotium herbarium, Fusarium
equiseti, Fusarium poae, Fusarium solani, Fusarium
sporotrichioids, Mucor plumbeus and Penicillium glabrum.
Inoculum (8 mm disk) of isolated fungi were added to 100 mL aliquots
of liquid modified Dox media containing the same concentration of copper
sulphate or Nitrate salt of lead in order to determine the dry weight
of isolated fungi. Each salts treatment had three replicates. This assay
had positive control (no salts but with fungal inoculum). All flasks were
incubated at 25±2°C and the mycelium was harvest at 5 day intervals.
The mycelium was transferred to pre weight filter papers thoroughly washed
with distilled water and drained by suction. The mycelial pellets were
then placed in a hot air dry in oven at 28°C for 24 h. The mycelia
were left to dry till constant weight.
RESULTS AND DISCUSSION
Characteristics of soil: Table 1 observed that the
pH value of the soil samples tested was neutral alkaline (range 7.58-7.76).
The concentration of Ca2+ are the highest compared with another
cations ranged between 53-80 meq-1, followed by Mg2+ ranged
between 31.2-66.6 meq-1, while Na+ and K+
concentrations are low ranged between 6.0-8.3 and 5.16-10.32 meq-1,
respectively. However the concentration Ca2+, Mg2+ and
K+ were less than that reported before for Saudi Arabia soils
(Hashem, 1993; Hashem and Al-Johany, 1994), while Al-Kadeeb (2006) reported
highest concentration for Ca2+, Mg2+ and Na+
ions, but lowest in Mg2+ions concentration when
compared with this study. Also Table 1 show that the
concentration of Cl- was the highest compared with another
anions ranged between 190-350 meq-1, but it was less than that
reported before for Saudi Arabia soil (Al-Kadeeb, 2006).
Table 2 show that the concentrations of Al3+
in the tested sample soil was less than reported before for Saudi Arabia
soils (Hashem, 1990, 1993), but was close agreement with that reported
by Al-Kadeeb (2006).
In the present study also Co concentration of tested sample soil was
less than reported before for samples collected from industrial Yanbu
city, from Al-Madeinah and from industrial Areas (Hashem, 1993; Hashem
and Al-Johany, 1994; Abed and Al-Wakel, 2002). Lead concentration in the
tested soil sample was to be similar to those studies reported before
by Hashem (1993) and Al-Kadeeb (2006), but was highest than that study
reported by Hashem and Al-Johany (1994). The concentration of copper in
this study was less than other studies reported before in some Saudi Arabia
soils (Hashem, 1990, 1993).
Effect of copper and lead concentration on fungal growth rate and mycelium
dry weight: Results (Table 3) indicated that the mycelium
dry weight of Aspergillus niger, Aspergillus oryzae, Aspergillus
tamarii, Aspergillus ustus, Cephaliophora irregularis,
Eurotium herbariorum, Fusarium equiseti, Fusarium poae,
Fusarium solani, Fusarium sporotrichioides, Mucor circinelloides,
Mucor plumbeus, Mucor racemosu, Penicillium roqueforti
decrease consistently with increasing level of lead ions concentration
in the growth medium, while mycelium dry weight of Alternaria chlamydospora
and Penicillium glabrum was stimulated in 2 mM concentration of
lead ions, by about 23 and 9.6%, respectively, while it decrease in concentration
6 and 12 mM of lead ions by about 17 and 29%, respectively for Alternaria
chlamydospora and by about 39 and 44%, respectively for Penicillium
glabrum, when compared with that of control, also mycelium dry weight
of Aspergillus ochraceus and Aspergillus parasiticus was
increase in 2 and 6 mM concentration of lead ions by about 43 and 4%,
respectively for Aspergillus ochraceus and 6 and 4%, respectively
for Aspergillus parasiticus and it decrease in concentration 12
mM by about 4% for Aspergillus ochraceus and 15% for Aspergillus
parasiticus, when compared with that of control.
||Chemical analysis of soil samples
|* Saturation percent, ** Electric conductivity, ***
Sodium saturation ratio
||Mechanical analysis as well as heavy metals content
of soil sample
||Change in mycelia dry weight during growth of the isolated
fungi at 2, 6 and 12 mM concentrations of lead and copper ions on
modified Dox media over 5 day incubation at 25±2°C
Table 3 also demonstrated that mycelium dry weight
of Aspergillus ochraceus was stimulated in 2 mM concentration
of copper ions by about 34%, when compared with that of control, while
it decrease in 6, 12 mM concentration of copper ions, also mycelium dry
weight of Cephaliophora irregularis, Eurotium herbariorum,
Fusarium equiseti and Penicillium glabrum was increase in 2
and 6 mM concentration of copper ions by about 30 and 18%, respectively
for Cephaliophora irregularis, by about 16 and 15%, respectively
for Eurotium herbariorum, by about 42 and 9%, respectively for
Fusarium equiseti and by about 37 and 29%, respectively for Penicillium
glabrum, when compared with that of control and it decrease in concentration
12 mM. Hashem (1989) reported stimulation of fungal growth in some species
by low levels of copper. Also he demonstrated a decrease in mycelium dry
weight for the other fungi with increasing of copper ions concentration.
Aspergillus ustus dry weight stimulate at concentration 2 mM of copper
ions by about 6%, while 6, 12 mM concentration of copper ions inhibit
fungal growth by about 15 and 24%, respectively as compared to the control.
Richards et al. (2002) reported that most of 12 Frankia strains
were less sensitive to Pb2+ (6 to 8 mM). While most strains
were sensitive to 0.1 mM Cu2+, four strains were resistant
to elevated levels of Cu2+ (2 to 5 mM and concentrations as
high as 20 mM), when they determined the sensitivity of 12 Frankia
strains to heavy metals by a growth inhibition assay.
Results (Table 4) shows adecrease in colony diameter
of Aspergillus oryzae, Aspergillus tamarii, Eurotium
herbariorum, Fusarium solani and Penicillium roqueforti
was observed with increased of lead or copper ions concentrations. The
growth of Aspergillus niger was no affected at lower concentrations
(2 mM) of lead or copper ions. Also high concentration (6 and 12) of these
metals caused little inhibition in fungal growth by about 2 and 6%, respectively,
when compared with that of control. While Abd-El Naby (1997) found that
high concentration of copper reduced the growth of Aspergillus niger.
Aspergillus ochraceus, Aspergillus parasiticus Fusarium equiseti
and Mucor circinelloides growth was no affected at lower concentrations
(2 mM) of lead or copper ions, but high concentrations of these metals
caused inhibition in fungal growth when compared with that of control.
Growth of Aspergillus ustus and Fusarium poae inhibited
with increasing lead ions concentration, also 6 and 12 mM concentration
of copper ions inhibited their growth, while 2 mM concentration of copper
ions stimulated their growth by about 11 and 7.6%, respectively, when
compared with that of control. A decrease in colony diameter of
Cephaliophora irregularis, Fusarium sporotrichioides and Mucor
racemosu was observed with increase of copper ions concentration and
with 6 and 12 mM lead ions concentration when compared with that of control,
while 2 mM lead ions concentration did not affect on fungal growth.
||Growth of isolated fungi at 2, 6 and 12 mM concentrations
of lead and copper ions on modified Dox agar plates over 3, 4 or 5
day incubation at 25±2°C
Stimulation of Penicillium glabrum growth was observed by about
11% at 2 mM copper ions concentration, while 2 mM lead ions concentration
did not affect on fungal growth. A decreased in fungal colony diameter
with 6 and 12 mM concentrations of both lead and copper ions by about
6 and 39%, respectively for lead and 3 and 17% for respectively for copper,
when compared with that of control.
Mucor plumbeus growth was highly tolerant of both lead and copper
ions concentrations and the tolerance values were all around 100%.
These results demonstrated that, the growth rate of some fungi isolated
on solid media like Mucor plumbeus, Aspergillus niger and
Aspergillus oryzae was less sensitive to addition of lead or copper
than biomass production in liquid culture. Also these results cleared
that fungi isolated from Second Industrial City in Riyadh, Saudi Arabia
resistance heavy metals and we can used to remove these metals from contaminated
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