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<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
Introduction<br />
Studies on biodegradation <strong>of</strong> pesticides contaminants soil by using<br />
Tremetes versicolor and Phanerochaete chrysosporium<br />
G. Umamaheswari * , V. Ramamurthy ** , S. Raveendran *** , K. Radhika *** and A. Kavitha Amirthanayagi ***<br />
* Post Graduate and Research Department <strong>of</strong> Biotechnology, Marudupandiyar College, Vallam,<br />
Thanjavur – 403. Tamil Nadu. ** Post Graduate and Research Department <strong>of</strong> Biochemistry,<br />
Marudupandiyar College, Vallam, Thanjavur - 403, Thanjavur District, Tamil Nadu.<br />
*** P.G & Research Department <strong>of</strong> Zoology, Khadir Mohideen College, Adirampattinam - 614 701,<br />
Thanjavur District, Tamil Nadu.<br />
Email : v.ramamoorthy07@gmail.com<br />
Corresponding Author<br />
V. Ramamurthy<br />
Post Graduate and Research<br />
Department <strong>of</strong> Biochemistry,<br />
Marudupandiyar College,<br />
Vallam, Thanjavur - 613 403,<br />
E-mail: v.ramamoorthy07@gmail.com<br />
Article History<br />
Received on 12 March, 2012;<br />
Revised in revised form 4 April,<br />
2012; Accepted 22 April, 2012<br />
Abstract<br />
Currently more number <strong>of</strong> possible mechanisms<br />
is available to clean up <strong>of</strong> pesticides in soil, viz<br />
chemical treatment, volatilization and incineration.<br />
Chemical treatment and volatilization are feasible and<br />
are problematic as large volumes <strong>of</strong> acids and alkalis<br />
are produced and subsequently must be disposed <strong>of</strong> by<br />
incineration, which is a very reliable method for<br />
destruction <strong>of</strong> these compounds, has met serious<br />
public opposition, because <strong>of</strong> its potentially toxic<br />
emissions, and its elevated economic costs (Kearney<br />
and Wauchope, 1998; Zhang and Chiao, 2002). Overall<br />
most <strong>of</strong> these physico-chemical cleaning technologies<br />
Among the various soil contaminants, pesticides are <strong>of</strong> primary<br />
importance due to their continuous entry into the soil environment. However, in<br />
un-sterilized and untreated soils, pesticide degradation rate was at the medium<br />
when compared to the sterilized soil indicating that the soil microorganisms play<br />
an important role in pesticide degradation. The pesticides were reduced in their<br />
level after the treatment <strong>of</strong> bacterial isolates. Totally three different pesticide such<br />
as simazine, trifluralin and dieldrin was recorded in soil extract through HPLC.<br />
Among the pesticide simazine was recorded maximum than trifluralin and<br />
dieldrin. The results obtained in this study provide valuable knowledge on the<br />
abilities <strong>of</strong> T.versicolor and P.chrysosporium might serve as a sound basis for the<br />
further exploitation <strong>of</strong> these species as fungal inoculants in biological remediation<br />
processes.<br />
Keywords: Pesticides, simazine, trifluralin, dieldrin, Tremetes versicolor and<br />
Phanerochaete chrysosporium<br />
are expensive and rather inefficient (Nerud et al., 2003<br />
and Schoefs et al., 2004) because the contaminated<br />
soil has to be excavated at a site and moved to a storage<br />
area where it can be processed. Due to environmental<br />
concerns associated with the accumulation <strong>of</strong> pesticides<br />
in food products and water supplies there is a great<br />
need to develop safe, convenient and economically<br />
feasible methods for pesticide remediation (Zhang and<br />
Chiao, 2002). For this reason several biological<br />
techniques involving biodegradation <strong>of</strong> organic<br />
compounds by micro- organisms have been developed<br />
(Schoefs et al., 2004).<br />
Page 35
<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
The use <strong>of</strong> microorganisms (fungi or bacteria),<br />
either naturally occurring or introduced, to degrade<br />
pollutants is called bioremediation (Pointing, 2001).<br />
Microbial metabolism is probably the most important<br />
pesticide degradation process in soils (Kearney and<br />
Wauchope, 1998) and is the basis for bioremediation,<br />
as the degrading microorganisms obtain carbon,<br />
nitrogen or energy from the pesticide molecules (Gan<br />
and Koskinen, 1998). The goal <strong>of</strong> bioremediation is to<br />
at least reduce pollutant levels to undetectable, nontoxic<br />
or acceptable levels, i.e. within limits set by regulatory<br />
agencies (Pointing, 2001) or ideally completely mineralize<br />
organ is pollutants to carbon dioxide. From an<br />
environmental point <strong>of</strong> view this total mineralization is<br />
desirable as it represents complete detoxification (Gan<br />
and Koskinen, 1998). The use <strong>of</strong> bioremediation to<br />
remove pollutants is typically less expensive than the<br />
equivalent physico-chemical methods. This technology<br />
<strong>of</strong>fers the potential to treat contaminated soil and<br />
groundwater on site without the need for excavation<br />
(Balba et al., 1998), it requires little energy input and<br />
preserves the soil structure (Hohener et al., 1998).<br />
Perhaps the most attractive feature <strong>of</strong> bioremediation is<br />
the reduced impact on the natural ecosystems, which<br />
should be more acceptable to the public.<br />
Application <strong>of</strong> fungal technology for the<br />
cleanup <strong>of</strong> contaminants has shown promise since 1985<br />
when the white rot species Phanerochaete chrysosporium<br />
was found to be able to metabolize a number <strong>of</strong><br />
important environmental pollutants. This ability is<br />
generally attributed to the lignin degrading enzymatic<br />
system <strong>of</strong> the fungus and a similar degrading capacity<br />
was later described for other white rot fungal species<br />
(Sasek, 2003). An environmental factor that may well<br />
have a crucial effect on bioremediation is soil water<br />
availability, as it varies naturally throughout the<br />
year. Nevertheless, very few studies look at the effects<br />
<strong>of</strong> water availability on bioremediation. This study was<br />
aimed to isolate the fungi from pesticide-contaminated<br />
soil and to determine pesticide degradation rates in soil<br />
extract liquid broth after the incubation with fungal<br />
inoculation.<br />
Materials and Methods<br />
Collection <strong>of</strong> soil samples<br />
For this present study the soil samples were<br />
collected from agricultural field <strong>of</strong> Kurichi village,<br />
Pattukkottai taluk, Thanjavur District, Tamilnadu,<br />
India. The soil samples were collected from the study<br />
site at a depth within 15 cm from the surface <strong>of</strong> the soil<br />
at random during the study period. The collected soil<br />
samples were brought to the laboratory in sterilized<br />
polythene bags, handpicked, air dried and stored in<br />
containers for future use.<br />
Isolation <strong>of</strong> fungi<br />
One gram <strong>of</strong> the soil sample was suspended in<br />
100 ml <strong>of</strong> sterilized Minimal media, without sample<br />
considered as control. Both were incubated at 37 0 C for<br />
24 to 48 hrs. 0.1 ml <strong>of</strong> inoculum was transferred to<br />
petriplate having PDA medium by spread plate<br />
technique. Population <strong>of</strong> fungi was isolated from the<br />
soil samples by serial dilution technique. Fungi were<br />
identified by using standard manuals, viz Mannual <strong>of</strong><br />
Soil Fungi (Gillman, 1947), Dematiaceous Hyphomycetes<br />
(Ellis, 1971).<br />
Physico-chemical properties <strong>of</strong> the soil<br />
Shallow, duplicate soil cores were collected<br />
from the study site with a piston corer (54.3 cm 3 , 12 cm<br />
long x 1.2 cm radius) and stored in watertight plastic<br />
bags until analysis <strong>of</strong> pH, nutrients and organic content.<br />
The soil sample was air dried and powdered to pass<br />
through a sieve (2 mm). They were collected in a plastic<br />
container, sealed and stored at 4°C for further use.<br />
Page 36
<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
Determination <strong>of</strong> pH was done by a digital<br />
pH meter, electrical conductivity by a conductivity<br />
meter (Elico). The moisture content was determined<br />
after drying at 105°C for 24 hrs. Organic matter and<br />
organic carbon was determined titrimetrically (Walkley<br />
and Black, 1934) and expressed as percentage (%). The<br />
phosphorus content was estimated by Olsen et al.,<br />
(1954). The nitrogen and potassium was estimated<br />
according to the method <strong>of</strong> Sankaram (1966).<br />
Micronutrients viz Zinc, Copper, Manganese and Iron<br />
were estimated by Lindsay and Norwell (1978).<br />
Inoculation with fungi, sampling and dry weight<br />
determination<br />
A soil extract liquid broth was used in this<br />
study. 100 ml <strong>of</strong> soil extract was taken in Erlenmeyer<br />
flasks (250 ml) to which four plugs <strong>of</strong> actively growing<br />
mycelium <strong>of</strong> Tremetes versicolor and Phanerochaete<br />
chrysosporium were inoculated in each flask. It was<br />
maintained at 27±1 o C, for 25 days with constant<br />
agitation at 150 rpm. After the incubation period the<br />
mycelium was filtered through whatman No. 1 filter<br />
paper and biomass determined by drying the mycelium<br />
for 48 hours at 80 o C. The fresh filtrate was frozen at<br />
20 o C and used later for pesticide quantification<br />
determination.<br />
Pesticide analysis<br />
The HPLC was used to determine the amount<br />
<strong>of</strong> simazine, trifluralin and dieldrin (Eliassy, 1997).<br />
Results<br />
The present study was undertaken to assess the<br />
distribution and occurrence <strong>of</strong> fungi from pesticides<br />
polluted soil, which were collected from agricultural<br />
field <strong>of</strong> Kurichi village, Pattukkottai, Thanjavur<br />
District, Tamilnadu, India. Totally 8 species <strong>of</strong> fungi<br />
belongs to 5 genus were isolated from the soil samples.<br />
Among them the genus Aspergillus was recorded with<br />
3 species viz., A. niger, A. terreus, and A. flavus followed<br />
by Tremetes with 2 species viz., T. socotrana and<br />
T.versicolor. The genus Phenerocheate, Cladosporium and<br />
Fusarium were recorded with single species each<br />
(Table - 1).<br />
The physico-chemical characteristics <strong>of</strong> soil<br />
samples <strong>of</strong> the study sites were given in the Table 2.<br />
The texture and colour <strong>of</strong> the soil was sandy and brown<br />
respectively in the soil samples. The pH and electrical<br />
conductivity values were reported viz., 7.8 and 0.32<br />
respectively in the soil samples. The percentage <strong>of</strong><br />
organic matter was 0.56%. The macronutrients content<br />
in the study site were given in the Table - 2.<br />
The impact <strong>of</strong> the two fungal inoculants such<br />
as T. versicolor and P. chrysosporium on degradation<br />
<strong>of</strong> pesticides were observed and recorded for soil<br />
sample. Initially 3 different types <strong>of</strong> pesticides such<br />
as Simazine (5.1ppm), Trifluralin (3.34 ppm) and<br />
Dieldrin (1.76 ppm) were observed through HPLC.<br />
These pesticides were reduced in their level after the<br />
treatment <strong>of</strong> fungal isolates. Among the pesticides<br />
content the Simazine was reduced the maximum levels<br />
when compared to Trifluralin and Dieldrin. The fungi<br />
P. chrysosporium removed maximum amount <strong>of</strong> all<br />
pesticides than the T. versicolor in the present soil<br />
sample analysis (Table - 3).<br />
Discussion<br />
Successful bioremediation is dependent on an<br />
interdisciplinary approach involving such disciplines as<br />
microbiology, engineering, ecology, geology and<br />
chemistry (Boopathy, 2000). To evaluate the outcome<br />
<strong>of</strong> bioremediation it is critical to assess some<br />
microbiological and biochemical parameters all giving<br />
information on soil quality. It is difficult to choose<br />
which parameters are more reliable, as the relationship<br />
Page 37
<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
between an individual biochemical property and the<br />
totalmicrobial activity is not always clear, in complex<br />
systems like soils where the microorganisms and<br />
processes involved in the degradation <strong>of</strong> organic<br />
compounds are highly diverse (Nannipieri et al., 1990).<br />
White rot fungi possess a number <strong>of</strong> advantages<br />
that can be exploited in bioremediation systems. Because<br />
key components <strong>of</strong> their lignin-degrading system are<br />
extracellular, these fungi can degrade insoluble<br />
chemicals such as lignin or an extremely diverse range<br />
<strong>of</strong> very persistent or toxic environmental pollutants<br />
(Barr and Aust, 1994). The mycelial growth habit is<br />
also advantageous as it allows rapid colonization <strong>of</strong><br />
substrates, and hyphal extension enables penetration <strong>of</strong><br />
soil reaching pollutants in ways that other organisms<br />
cannot do (Reddy and Mathew, 2001). This can<br />
maximise physical, mechanical and enzymatic contact<br />
with the surrounding environment (Maloney, 2001). In<br />
addition, these fungi use inexpensive and abundant<br />
lignocellulosic materials as a nutrient source. They can<br />
tolerate a wide range <strong>of</strong> environmental conditions, such<br />
as temperature, pH and moisture and do not require pre-<br />
conditioning to a particular pollutant, because their<br />
degradative system is induced by nutrient deprivation<br />
(Barr and Aust, 1994).<br />
In a more recent study by biological<br />
degradation <strong>of</strong> benzene and toluene by T.versicolor was<br />
analyzed and the biomass were determined by Demir<br />
(2004). Within an incubation period <strong>of</strong> 48 hrs, it was<br />
observed that, removal was completed after 4 hours<br />
when initial toluene concentration was 50 mg/l and was<br />
completed in 36 hrs when this was 300 mg/l.<br />
Biodegradation was completed by the end <strong>of</strong> the 4th hr<br />
at benzene concentrations <strong>of</strong> 50mgl -1 while it continued<br />
for 42 hrs at 300 mgl -1 . With the addition <strong>of</strong> veratryl<br />
alcohol, laccase inducers, to the basic feed medium, the<br />
operation <strong>of</strong> the enzyme system was enhanced and<br />
biodegradation completed in a shorter time period.<br />
Table - 1. Fungal flora in the soil sample<br />
Sl. No<br />
Name <strong>of</strong> the organism<br />
1 Aspergillus niger<br />
2 A. terreus<br />
3 A. flavus<br />
4 Phenerochete chrysosporium<br />
5 Tremetes socotrana<br />
6 T.versicolor<br />
7 Fusarium sp<br />
8 Cladosporium sp<br />
Table - 2. Physico-chemical characteristics <strong>of</strong> soil<br />
sample<br />
Sl.No Parameters Values<br />
1. Soils texture Sandy<br />
2. Colour Brown<br />
3. pH 7.8<br />
4. Ecdcm -1 0.32<br />
5. Organic carbon % 0. 56%<br />
6. Nitrogen (mg/g) 91.2<br />
7. Potassium (mg/g) 78.1<br />
8. Phosphorus (mg/g) 9.5<br />
9. Zinc (ppm) 0.93<br />
10. Copper (ppm) 0.36<br />
11. Iron (ppm) 3.23<br />
12. Manganese (ppm) 8.83<br />
The microbial population <strong>of</strong> a site<br />
contaminated with pesticides may be eliminated,<br />
significantly reduced or altered; but alternatively,<br />
microbes may adapt to the presence <strong>of</strong> toxic<br />
compounds and can survive by degrading them as some<br />
microorganisms can utilize pesticides as a nutrient<br />
source. As soil microorganisms are not equal resistant<br />
to xenobiotics, some <strong>of</strong> them are very sensitive and do<br />
Page 38
<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
Table - 3. Analysis Pesticide level in soil extract by HPLC<br />
not grow when toxic compounds are present in high<br />
concentrations or constitute a low carbon and energy<br />
source, while other are able to adapt and grow well<br />
(Guirard et al., 2003). Han et al. (2004) studied the<br />
degradation <strong>of</strong> phenanthrene by T.versicolor and its<br />
laccase was purified. After 36 hrs incubation, about<br />
46 and 65% <strong>of</strong> 100mgl -1 <strong>of</strong> phenanthrene added in<br />
shaken and static fungal cultures were removed,<br />
respectively. Although the removal percentage was<br />
highest (76.7%) at 10mgl -1 <strong>of</strong> phenanthrene, the trans-<br />
formation rate was maximal (0.82mgh -1 ) at 100mgl -1 <strong>of</strong><br />
phenanthrene in the fungal culture. When the purified<br />
laccase <strong>of</strong> T. versicolor reacted with phenanthrene, the<br />
compound was not transformed. Another interesting<br />
example <strong>of</strong> contaminant degradation and enzyme activity<br />
was in the study described by Barr and Aust (1994).<br />
They described cyanide to be quite toxic to spores <strong>of</strong><br />
P.chrysosporium (50% inhibition <strong>of</strong> glucose metabolism<br />
at 2.6mgl -1 ).<br />
Min et al. (2001) reported that increasing<br />
concentrations <strong>of</strong> butachlor in soil enhanced the activity<br />
<strong>of</strong> dehydrogenase with the highest activity on the<br />
16 th day after application <strong>of</strong> 22mg/kg soil <strong>of</strong> but achlor.<br />
Baran et al. (2004) reported high dehydrogenase<br />
activity in soil contaminated with PAH. Felsot and<br />
Dzantor (1995) described the effect <strong>of</strong> alachlor and<br />
organic amendment on soil dehydrogenase activity<br />
and on pesticide degradation rates. Amendment <strong>of</strong> soil<br />
Control<br />
(ppm)<br />
with corn meal caused fasterdegradation <strong>of</strong> alachlor.<br />
At very high concentrations <strong>of</strong> alachlor (750mg/kg soil)<br />
dehydrogenase activities in amended soils surpassed<br />
levels in corresponding nopesticide controls after<br />
21days with coincident alachlor degradation >50%<br />
during the same period. They suggested that stimulation<br />
<strong>of</strong> microbial activity by addition <strong>of</strong> organic amendments<br />
might enhance co-metabolism <strong>of</strong> high concentrations <strong>of</strong><br />
pesticides in soil. Totally three different pesticide such<br />
as simazine, trifluralin and dieldrin was recorded in soil<br />
extract through HPLC. Among the pesticide simazine<br />
was recorded maximum than trifluralin and dieldrin.<br />
The results obtained in this study provide valuable<br />
knowledge on the abilities <strong>of</strong> T.versicolor and<br />
P. chrysosporium might serve as a sound basis for the<br />
further exploitation <strong>of</strong> these species as fungal<br />
inoculants in biological remediation processes.<br />
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1. Simazine 5.1 5.1 2.48 2.96<br />
2. Trifluralin 3.34 3.31 2.63 2.77<br />
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<strong>International</strong> <strong>Journal</strong> <strong>of</strong> <strong>Advanced</strong> <strong>Life</strong> <strong>Sciences</strong> (IJALS)<br />
Int. j. Adv. Lif. Sci., Available online on at www.<br />
Int. J. Adv. Lif. Sci., Available online on at www. ijals.com<br />
ISSN<br />
2277 – 758X<br />
Umamaheswari et al., IJALS, Vol.3. May - 2012. RESEARCH ARTICLE<br />
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Corresponding Author : V. Ramamurthy, Post Graduate and Research Department <strong>of</strong> Biochemistry,<br />
Marudupandiyar College, Vallam, Thanjavur - 613 403, Thanjavur District, Tamil Nadu, India, E-mail :<br />
v.ramamoorthy07@gmail.com.<br />
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