Ex- Situ Studies on Biodegradation of Artificially Enriched Kerosene and Diesel Soils by Fungal isolates

This article was downloaded by: [mansi banker]On: 23 July 2015, At: 03:59Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place,London, SW1P 1WG

Click for updates

Soil and Sediment Contamination: An InternationalJournalPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/bssc20

Ex- Situ Studies on Biodegradation of ArtificiallyEnriched Kerosene and Diesel Soils by Fungal isolatesShamiyan R. Khana, Nirmal Kumar J.I.a, Mansi Bankera & Rita N. Kumarb

a P.G. Department of Environmental Science and Technology, Institute of Science andTechnology for Advanced Studies and Research (ISTAR), VallabhVidya Nagar - 388 120,Gujarat, Indiab Department of Biological and Environmental Sciences, N.V. Patel College, VallabhVidyaNagar, Gujarat, India - 388 120Accepted author version posted online: 08 Jul 2015.

To cite this article: Shamiyan R. Khan, Nirmal Kumar J.I., Mansi Banker & Rita N. Kumar (2015): Ex- Situ Studies onBiodegradation of Artificially Enriched Kerosene and Diesel Soils by Fungal isolates, Soil and Sediment Contamination: AnInternational Journal, DOI: 10.1080/15320383.2015.1054555

To link to this article: http://dx.doi.org/10.1080/15320383.2015.1054555

Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a serviceto authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting,typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication ofthe Version of Record (VoR). During production and pre-press, errors may be discovered which could affect thecontent, and all legal disclaimers that apply to the journal relate to this version also.

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 1

Ex- Situ Studies on Biodegradation of Artificially Enriched Kerosene and Diesel Soils by

Fungal isolates.

Shamiyan R. Khan1, Nirmal Kumar J.I.

1*, Mansi Banker

1 and Rita N. Kumar

2

1*P.G. Department of Environmental Science and Technology, Institute of Science and

Technology for Advanced Studies and Research (ISTAR), VallabhVidya Nagar -388 120,

Gujarat, India. E mail: [email protected]

2Department of Biological and Environmental Sciences, N.V. Patel College, VallabhVidya

Nagar, Gujarat, India- 388 120

ABSTRACT

To demonstrate the potential of biodegradation of soils enriched with kerosene and diesel, an ex-

situ study with the objective of evaluating and comparing the effects of three different fungal

isolates P. janthenillum, P. decumbens and A. terreus was performed. The study dealt with the

biodegradation of artificially enriched kerosene and diesel soils by 5%, 10% and 15% (w/w). The

experiment was performed by ex-situ large scale tray method using 24 plastic trays 6” X 3” X 1”

in each containing 60 kg enriched soil. After 8 weeks of inoculation of the fungal isolates, P.

janthinellum found to be potential compared to the other two and displayed the highest kerosene

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 2

and diesel degradative capacity resulting 98.29%, 97%, 96% and 82%, 70% and 62%

degradation at 5, 10 and 15% kerosene and diesel enriched soils after 45 and 60 days

respectively. Moreover, the total fungal population was found to increase as a function of time. A

first-order kinetic model equation showed that the specific biodegradation rate constant ‘k’ value

were 0.1023 and 0.0285 day-1

for 5% kerosene and diesel enrichment by P. janthinellum

treatment strategy which was comparatively higher than the values for other two organisms

tested. Thus, the degree of effectiveness of these bioremediation strategies in the soils enriched

with kerosene and diesel is in the following order: P. janthenillum>P. decumbens>A. terreus.

Keywords: Ex-situ large scale tray method; first-order kinetic; fungal isolates; kerosene and

diesel enriched soils.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 3

INTRODUCTION

The worldwide high demand for petroleum and associated products as a source of energy

has resulted in increased oil exploration, production, and refining, and has consequently led to a

high level of environmental pollution. Oil spills due to blow outs, leakage from underground

storage tanks, tanker accidents, sabotage and accidental rupture of pipelines as well as dumping

of waste petroleum products introduce nonorganic, carcinogenic and growth-inhibiting chemicals

present in the crude oil and their toxicity to microorganism and man is well known (Okpokwasili

and Odokuma 1990). Also, it results in significant decline in the quality of soil and makes it unfit

for use (Shabir et al. 2008) as well as affects plants and animal (Plohl and Leskovsek, 2002).

Crude oil is an extremely complex mixture of aliphatic and aromatic hydrocarbons, including

volatile components of gasoline, petrol, kerosene, diesel, lubricant oil, and solid asphaltene

residues; however, the kerosene and diesel fractions pose the greatest pollution threats and

problems owing to their excessive use (Solano-Serena et al. 2000). Moreover Amellal et al.

(2001) had shown that PAHs which are one of the constituents of petroleum products can be

carcinogenic and/or mutagenic in some circumstances and have been classified as priority

pollutants.

Among several remediation technologies available for petroleum hydrocarbons removal

from the soil and groundwater, bioremediation technology is gaining prominence due to its

simplicity, environmental friendliness, higher efficiency, and cost-effectiveness (Mariano et al.

2007). This bioremediation technology relies on the natural ability of microorganisms to carryout

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 4

the partial degradation or mineralization of petroleum hydrocarbons (or organic chemicals) to

carbon dioxide and water (Atlas 1981; Duarte da Cunha and Leite 2000; Margesin and Schinner

2001). Crude oil as well as other commercial hydrocarbons could be sparsely biodegradable in

soils (Shabir et al. 2008); however, differences in the extent of biodegradation depending on soil

and hydrocarbon source type, concentration of total hydrocarbons, and oxygen and nutrient

availability have been reported by Bento et al. (2003). Studies have been conducted on petroleum

hydrocarbons degradation by microbial activities (Morgan and Watkinson 1989; Bossert and

Compeau 1995; Shamiyan et al., 2015b(In Press)), but much work has not been emphasized on

the biodegradation of some commercial petroleum products such as kerosene and diesel.

Shamiyan et al. (2013, 2014) carried out soil characterization and isolated certain petroleum

degrading fungal strains from aged petroleum affected natural soils. However, there is little or no

information available in the literature on the bioremediation of artificially enriched kerosene and

diesel soils Ex-situ by large scale tray methods. Therefore in the present investigation an attempt

has been made to carry out Ex- situ studies on biodegradation of artificially enriched kerosene

and diesel soils with an application of a first-order kinetic model equation in order to determine

the most efficiently degrading fungal isolate.

MATERIALS AND METHODOLOGY

Fungal Isolates

Fungal strains Penicillium decumbens PDX7, Penicillium janthinellum SDX7 and

Aspergillus terreus PKX4 were isolated and screened for potential kerosene and diesel

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 5

degradation from aged petroleum hydrocarbon affected soil sites of Anand, Gujarat, India. The

total petroleum hydrocarbon (TPH) concentrations encountered were 17,510 mg/kg and 17,949

mg/kg for kerosene and diesel soils respectively. Fungal isolates were identified based on

morphological, molecular (18S rRNA) methods and the sequences were submitted to NCBI gene

bank (Shamiyan et al. 2013, 2014) (Table 1).

Preparation of carrier based inoculums.

Hundred milliliters of axenic and exponentially growing dense cultures (Axenic spore

suspensions of the fungal isolates of about 105spores/mL) were prepared in sterile Mineral Salt

Media (NaNO3(2.0 g/L), NaCl(0.8 g/L), KCl(0.8 g/L), CaCl2.2H2O(0.1 g/L), KH2PO4(2.0 g/L),

Na2HPO4.12 H2O(2.0 g/L), MgSO4(0.2 g/L), FeSO4.7H2O (0.001 g/L), (pH- 6.6) and inoculated

on 1.0 kg of broken autoclaved rice grains in autoclavable plastic bags under aseptic conditions.

The fungal isolates were allowed separately to sporulate on autoclaved broken rice grains in dark

at 30±2o C for ten days. These carrier based inoculums were used for the Ex-situ studies on

degradation of kerosene and diesel enriched soils (Facundo et al. 2001)

Ex-situ Experimental set up

Soil sample was randomly collected with a Dutch auger at a depth of 15 cm from an

uncontaminated agricultural field in Anand, Gujarat, India, brought to the laboratory and were

homogenized, dried, sieved, passed through a 2 mm mesh screen, and stored in a polythene bag

at room temperature. Soil samples were analyzed three times i.e. initial execution of the work,

after enrichment with petroleum products and at the end of the experiment for particle size by the

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 6

international pipette method (Gee and Bauder 1986), bulk density by metal core sampler method

(Blake and Harte 1986), porosity and moisture content according to Maiti (2003). The pH and

electric conductivity of the soil samples were determined in soil/water (1:1, v/v) suspension by

pH meter and conductivity meter (Sension 5, HACH, USA). Total organic carbon (TOC)

concentration was determined with the titration method (Walkley and Black 1934). Total

nitrogen was measured by Kjeldahl digestion (Gerhardt, Turbotherm, Germany) and steam

distillation method (Black 1965).

The soil of 1600 kg was taken and divided into 8 heaps of 200 Kg each and were

artificially enriched with 5, 10 and 15% of kerosene and diesel separately and remaining 2 heaps

were used s control. The soils were thrice enriched with the petroleum products at an interval of

3 days up to 9 days. They were then divided into 24 plastic trays each containing 60 Kg of

treated soils (Figure 1). One Kilogram of carrier based fungal inoculum was added in each tray

and six control trays were maintained simultaneously without the carrier based inoculums. The

study was carried out during the winter season from January to March, 2014 when the

temperature ranged between 18 to 25 oC. The water (moisture) content of soil in each tray was

adjusted every week by addition of sterile distilled water to a moisture holding capacity of 50%.

In order to avoid anaerobic conditions, contents of the trays were aerated by tailings every 3

days. Samples were excavated every fifteen days and analyzed for residual kerosene, diesel and

fungal population. The whole experiments were carried out in triplicates (Embar et al. 2006).

Biodegradation activity and Total Petroleum Hydrocarbons (TPH)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 7

The determination of the biodegradation activity of the three fungal isolates was carried

out by collecting 2 g of soil samples at four places in each tray and combined to form a

composite sample for further analysis. The residual petroleum hydrocarbon was recovered by 1:1

of Chloroform: Dichloromethane (Chaillan et al. 2004). Analysis of the biodegradation activity

was made using a computerized capillary gas chromatography with flame ionized detector (GC-

FID, Perkin Elmer-Auto System) equipped with HP 3390A Integrator, split injector (split ratio

20/1) and flame ionization detector set at 300 oC. The carrier gas was nitrogen at flow rate of 1.5

mL/min. The column used was polydimethylsiloxane (length 30 m, internal diameter 0.32 mm,

film thickness 0.25 μm) and the temperature was programmed to increase from 60 to 320 oC at 4

oC min

-1. The total petroleum hydrocarbon (TPH) degradation by the fungal isolates was

calculated according to the following equation.

(Eq. 1)

Where B is biodegradation, TPHC is Total petroleum hydrocarbon of Control and TPHS is Total

petroleum hydrocarbon of Sample.

Statistical analysis

Student’s t-test was used to study the significant differences between treatments for aromatic and

aliphatic hydrocarbon degradation using KY plot (2.0 beta).

Kinetics of Degradation

The rate of petroleum hydrocarbon biodegradation was measured by the application of

first-order kinetic model equation (Equation 2) to the biodegradation data, which has generally

been used for biodegradation of petroleum hydrocarbons in soil (Abassi and Shquirat 2008;

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 8

Adesodun and Mbagwu 2008). Applying the biodegradation data obtained for each soil treatment

to first-order kinetic model using the linear regression routine of MATLAB 7.0 software package

made possible further evaluation and comparison of the applicability of the various

biodegradation treatment strategies.

S = So –Kt

(Eq. 2)

Taking the natural logarithm of Equation 1

ln(S/So) = −kt (Eq. 3)

Where S, So, k, and t are the initial petroleum hydrocarbon concentration, final petroleum

hydrocarbon concentration, specific degradation rate constant and time, respectively.

Estimation and identification of total fungal population

Both the control and enriched soils were assessed for total fungal population using the

colony forming unit (CFU) method (Lily et al. 2009). For that, 1 g of each soil sample was

suspended into 10 mL of sterile distilled water and was aseptically serially diluted further up to

10-7

dilution. An aliquot of 0.1 mL from each diluted soil suspension was poured onto Potato

Dextrose Agar plates using the spread plate technique. Plates were incubated for 3 to 5 days at 30

°C and the results were recorded as Log CFU. The fungal colonies appearing on the plates were

identified morphologically classified according to taxonomical keys in many literatures (Nelson-

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 9

Smith, 1973; Malloch, 1997) as well as molecular 18S rRNA based gene sequence identification

according to Shamiyan et al. (2015a).

RESULTS AND DISCUSSION

Soil Physico-chemical characterization.

The soil physico-chemical characterization of uncontaminated, post enriched and post

degraded kerosene and diesel displayed low porosity and high bulk density. Diesel enriched soils

exhibited maximum bulk density 0.93 g mL–1

due to the presence of high concentration of

hydrocarbons which cause an increase in soil hydrophobicity and therefore leading to decrease in

the moisture holding capacity of soil (Balks et al. 2002). Our results showed highest value 7.78%

of total nitrogen in diesel enriched soil and lowest 2.45% in uncontaminated soils which

corroborated with the findings of Ujowundu et al. (2011), who studied the biochemical and

physical characterization of diesel contaminated soil in south-eastern Nigeria. The increase could

be derived from the nitrogen content of the refined petroleum fuels (Slavica et al. 2003). High

amount of organic carbon 4.23% and 5.19% in kerosene and diesel enriched soil samples could

be due to the high carbon content in the petroleum products. The artificial enrichment resulted in

the acidic pH values which were found to restore to alkaline pH during the passage of the

degradation process. The low pH may have caused a reduction in the fungal population growth in

the enriched soils. A study by Verstrate et al. (1975) was conducted on optimal activity for

microbial degradation at a pH of 7.4 and considerable inhibition at pH 4.5 and above 8.5. A

reduction of nutrient content of the enriched soils might have caused lowest conductivity value

217 µS cm–1

followed by 235 µS cm–1

in diesel and kerosene, respectively where the values

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 10

heaved up as the degradation progressed as a function of time (Table 2). The change in the

physicochemical properties of soils could be due to the enrichment of kerosene and diesel

(Hawrot and Nowak 2006).

Kerosene Degradation

The degradation profile of kerosene as a function of time for each bioremediation

treatment by the three fungal isolates revealed that kerosene degradation initiated during the first

fifteen days of the experiment in all the treatments which slowly continued up to 45 days. The

percentage degradation of kerosene was achieved 99%, 98%, 97.5% in 5%, 10% and 15% (w/w)

by P janthenellum SDX7, whereas P. decumbens PDX7 displayed comparatively lower levels of

degradation corresponding to 97%, 94% and 89% followed by A. terreus PKX4 exhibiting 93%,

85% and 79.5% of kerosene enrichments (Figure 2). The percent Aromatic/ Aliphatic

hydrocarbons >0.8 indicate the efficiency of these isolates in eliminating the aromatic

hydrocarbons which are more complex to breakdown however, P. janthenellum SDX7 degraded

a maximum of 97% and 95% of aliphatic and aromatic fraction of 5% diesel with no statistical

difference (t-test, P≥0.05) (Table 3). Similar results were also reported by Okerentugba and

Ezeronye (2003) who showed utilization of petroleum hydrocarbons by single and mixed

hydrocarbon degrading fungal-bacterial consortia. The biodegradation of kerosene was

confirmed by the reduction in the area under the aliphatic and aromatic hydrocarbon peaks of the

chromatograms when compared to that of the abiotic control (without organism) suggesting the

removal of kerosene hydrocarbon (Figure 3). In the chromatographic images, it has been

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 11

recorded that the sharp and highest peaks stand for the n-alkanes and the peaks between them

comprise the naphthenes and aromatics corresponding to carbon atoms (C8 to C18). The

chromatograms showed that n-alkane fractions are easily degraded as the days progress by the

tested fungal isolate however, the rate is lower for branched alkanes followed by n-alkyl

aromatics, cyclic alkanes and polynuclear aromatics. These results are in agreement with the

findings obtained by Wang et al. (1998) who studied the comparison of crude oil composition

changes due to biodegradation and physical weathering using various fungal and bacterial

isolates.

Moreover, Nocentini et al. (2000) while studying the bioremediation of soil contaminated

by hydrocarbon mixture displayed various bacterial species to be potential kerosene degraders.

The present results indicate that the three isolates employed in this study are extremely efficient

to degrade kerosene. However, P. janthenellum SDX7 showed the highest potential to eliminate

kerosene from soil could be due to adaption of these fungi to hydrocarbon composition, as well

as the high oxidative enzyme activities (Macera-Lopez et al. 2008). A meager reduction in

kerosene concentration was observed in control setup due to combined effects of abiotic

degradation, evaporation losses of kerosene and soil indigenous microorganisms (Shabir et al.

2008).

The first-order kinetic model using the linear regression shown the highest ‘k’ value

(0.1023 day−1

) for the biodegradation of kerosene in soil by P. janthenellum SDX7 proves

exceedingly suitable for remediation of kerosene contaminated soils. P. decumbens PDX7 and A.

terreus PKX4 comparatively displayed lower ‘k’ values 0.0490 and 0.0352 day−1

respectively

(Table 4). Our present results very well corroborated with the findings of Agarry et al. (2010)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 12

while studying the bioremediation strategies for kerosene degradation also reported similar

findings. Hence, the degree of effectiveness was in the order P. janthenellum SDX7> P.

decumbens PDX7> A. terreus PKX4 for remediation of kerosene enriched soils. Similar

observation reported by Adekunle and Adebambo, (2007) in which the isolated Rhizopus species

from the seed of Detarium Senegalense (J. F Gmelin) showed the highest ability to degradation

of kerosene amongst Aspergillus flavus, Aspergillus niger, Mucor and Talaromyces. Shamiyan et

al. (2015b) also reported upto 96% of kerosene degradation while evaluation the enzymatic

degradation by fungal isolate P. janthenellum In vitro.

Diesel Degradation

The soils enriched by 5%, 10% and 15% diesel contained hydrocarbons ranging from

11,305 mg/kg, 19,950 mg/kg and 45,058 mg/kg respectively. The highest diesel degradative

capacity of P. janthinellum SDX7 exhibiting 82% for 5%, followed by 70% and 62% for 10 and

15% diesel enriched soils respectively after 60 days. Similarly about 75%, 61% and 54%

degradation of hydrocarbons by P. decumbens PDX7 was encountered at 5, 10 and 15% diesel

enriched soils. However, A. terreus PKX4 degraded diesel hydrocarbons by 68%, 57% and 49%

for the three concentrations (Figure 2). The percent of Aromatic/ Aliphatic hydrocarbons >0.8

indicate the efficiency in removing the aromatic hydrocarbons of diesel by P. janthinellum SDX7

is more as compared to the other two fungal isolates (Table 5). The chromatographic images

depict the smaller peaks of naphthenes and aromatics corresponding to carbon atoms (C10 to

C22) between the sharp and tallest peaks of the n-alkanes. P. janthenellum SDX7 degraded a

maximum of 69% and 65% of aliphatic and aromatic fraction of 5% diesel (t –test, P≥0.05)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 13

where the results are very well substantiated with the findings of Manee et al. (1998) who proved

the higher efficiency of the fungal isolates to degrade the aliphatic fractions compared to the

aromatic fractions during crude oil degradation. The biodegradation of diesel was confirmed by

the reduction in the peak area under the aliphatic and aromatic hydrocarbon of the

chromatograms when compared to that of the abiotic control which reveals the removal of these

diesel hydrocarbons (Figure 4). The chromatograms showed n-alkane fractions are easily

degraded as a function of time and completely eliminating C10 to C15 which is corroborated

with the findings of Dussan and Numpaque (2012) who stated the degradation of diesel fuel by

certain bacterial species. However, the rate of degradation was lower for branched alkanes

followed by n-alkyl aromatics, cyclic alkanes and polynuclear aromatics by the tested fungal

isolates.

The results indicate that the ‘k’ value was maximum 0.0285 day−1

for P janthenallum

SDX7, while the lowest ‘k’ value (0.0112 day−1

) was registered for A. terreus PKX4 during

diesel degradation (Table 4). Therefore, value of the kinetic parameter showed that the degree of

effectiveness of these bioremediation strategies in the cleanup of soil contaminated with diesel

using fungal isolates is in the following order: P. janthenellum SDX7> P. decumbens PDX7> A.

terreus PKX, Similar results were reported by Dadrasnia and Agamuthu (2013) who studied

the first order kinetics to compare 5% diesel degradation by using different organic wastes,

where use of soy cakes were proved to be the best degradation strategy. The study revealed

P. janthinellum SDX7 was found to be more efficient than the other two species. The

hydrocarbon degradation abilities of Aspergillus sp. and Penicillum sp. are similar to the findings

of April et al. (2000) who showed that these two organisms were among the sixty-four species of

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 14

hydrocarbon-degrading filamentous fungi isolated from flare pit soils in northern and western

Canada. Macera-Lopez et al. (2008) also reported very efficient hydrocarbon degradation ability

of the two fungal species. Earlier findings of Oudot et al. (1993) and April et al. (2000)

supported the role of Syncephalastrum sp., Trichoderma sp., Neurospora sitophila, Rhizopus

arrhizus and Mucor sp. isolated from petroleum contaminated sites in active degradation of

petroleum hydrocarbons.

Fungal Population Study

The fungal communities observed during the initial sampling in the soil prior enrichment with

petroleum products included species Aspergillus versicolor, A. niger, Fusarium oxysporum,

Penicillium janthinellum, Rhizopus oryzae and Sympodiomycopsis paphiopedili however at the

time of enrichment the three inoculated strains P. janthenellum, P. decumbens and A. terreus

flourished in the petroleum product enriched soils, Hence they were responsible for the

petroleum hydrocarbon degradation initially along with the hydrocarbon resistant indigenous

species Hawrot and Nowak (2006). Moreover as the degradation progressed, the fungal

population also increased owing to the reduction in the petroleum contamination thereby making

the soil more suitable for the survival of the indigenous fungal species (Obire, 1988; Facundo et

al., 2001). Okoh (2006) demonstrated the presence of C5 – C10 in the petroleum fraction shown

to be inhibitory to the majority the microbial community which tend to disrupt phospholipids and

lipoprotein membrane structures of microorganisms. The lower microbial population and

depletion of indigenous fungal species in the enriched soils could be due to the effect of the

petroleum hydrocarbons which lead to impairment of gaseous exchange and retention of soil

carbon dioxide (Ujowundu et al. 2011). The fungal growth rate expressed in colony forming

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 15

units reviled that there was a sharp depletion in the fungal population at the initial time of the

enrichment of the soil with kerosene and diesel. It is been further encountered the 1.6X 105

CFU/g in 15 % kerosene enriched soil and 1.1X 105

CFU/g in 15 % diesel enriched soil.

However, the Log CFU/g values were found to increase along with the degradation of kerosene

and diesel displaying highest values upto 6.6X 106 CFU/g for both the treatments after 15 days

(Figure 5). These results were in agreement with the findings of Mulkins-Philip and Stewart

(1974) and Obire (1988) who emphasized an increase in the microbial population due to the

decline in the xenobiotic compounds and attenuation of the indigenous species.

CONCLUSION

In present study revealed the rate of biodegradation of kerosene and diesel in artificially enriched

soils could be enhanced by the addition of carrier based inoculum of fungal isolates by Ex-situ

tray methods. The three fungal isolates used in the study exhibited high degree of degrading

potential in the order P. janthenellum SDX7>P. decumbens PDX7>A. terreus PKX4. Thus, the

use of fungal isolate P. janthenellum SDX7 for the degradation of petroleum hydrocarbons in

kerosene and diesel enriched soils may be recommended for Ex Situ bioremediation strategies for

environmental cleanup.

Acknowledgements

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 16

The authors are highly thankful to University Grants Commission (UGC) for financial support

and also thankful to Sophisticated Instrumentation Centre for Advanced Research and Testing

(SICART) for analysis of samples on GC.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 17

References

Abassi, B. E. and Shquirat, W. D. 2008. Kinetics of indigenous isolated bacteria used for ex-

situ bioremediation of petroleum contaminated soil, Water Air Soil Pollut. 192, 221–226.

Adekunle, A. A. and Adebambo, O. A. 2007. Petroleum Hydrocarbon utilization by fungi

isolated from Detarium Senegalense (J. F Gmelin) Seeds. J. Am. Sc. 3(1), 69-76.

Adesodun, J. K. and Mbagwu, J. S. C. 2008. Biodegradation of waste lubricating petroleum

oil in a tropical alfisol as mediated by animal droppings, Bioresour. Technol. 99, 5659–5665.

Agarry, S. E., Solomon, B. O. and Audu, T. O. K. 2010. Substrate utilization and inhibition

kinetics: Batch degradation of phenol by indigenous monoculture of Pseudomonas

aeruginosa, Inter. J. Biotech. Mol. Biol. Res. 1(2), 22 – 30.

Amellal, N., Portal, J. M. and Berthelin, J. 2001. Effect of soil structure on bioavailability of

polycyclic aromatic hydrocarbons within aggregates of a contaminated soil, Appl. Geochem.

16, 1611-19.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 18

April, T. M., Foght, J. M. and Currah, R. S. 2000. Hydrocarbon-degrading filamentous fungi

isolated from flare pit soils in Northern and Western Canada. Can. J. Microbiol. 46(1), 38-

49.

Atlas, R. M. 1981. Microbial degradation of petroleum hydrocarbons: An environmental

prospective, Microbiol. Rev. 45, 180–209.

Balks, M. R., Paetzold, R. P., Kimble, J. M., Aislabie, J. and Campbell, I. B. 2002. Effects of

hydrocarbon spills on the temperature and moisture regimes of Cryosols in the Ross Sea

region, Antarc. Sci. 14 (4), 319–326.

Bento, F. M., Camargo, F. A., Okeke, B. and Frankenberger, T. W. 2003. Bioremediation of

soil contaminated by diesel oil. Braz. J. Microbiol. 34, 65–68.

Black, C. A. 1965. Methods of Soil Analyses. America Society of Agronomy and Soil

Science Society of America, Madison, Wisconsin, USA

Blake, G. R. and Harte, K. H. 1986. Methods of soil Analysis. America Society of Agronomy

and Soil Science Society of America, pp. 363-375. Madison, Wisconsin, USA.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 19

Bossert, I. D. and Compeau, G. C. 1995. Clean up of petroleum hydrocarbon contamination

in soil. In: Microbial Transformation and Biodegradation of Toxic Organic Chemicals, pp.

77–125. (Young, Y. L. and Cerniglia, C. E. Eds) Wiley-Liss, New York

Chaillan, F., Fleche, A. L., Bury, E., Phantavong, Y. H., Grimont, P., Saliot, A. and Oudot, J.

2004. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading

microorganisms. Res. Microbiol. 155, 587-595.

Dadrasnia, A. and Agamuthu, P. 2013. Dynamics of diesel fuel degradation in

contaminated soil using organic wastes. Int. J. Env. Sci. Tech. 10(4), 769-778

Duarte da Cunha, C. and Leite, S. G. F. 2000. Gasoline biodegradation in different soil

microcosms. Braz. J. Microbiol. 31, 45–49.

Dussán, J. and Numpaque, M. 2012. Degradation of diesel, a component of the explosive

ANFO, by bacteria selected from an open cast coal mine in La Guajira, Colombia. J.

Bioproces. Biotechniq. 2(4), 1-5.

Embar, K., Forgacs, C. and Sivan, A. 2006. The role of indigenous bacterial and fungal soil

populations in the biodegradation of crude oil in a desert soil. Biodegrad. J. 17, 369-377.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 20

Facundo, J., Márquez, R., Hernández, R. V. and Teresa, L. A. 2001. Biodegradation of diesel

oil in soil by a microbial consortium. Water, Air, and Soil Poll. 128, 313–320.

Gee, G. W. and Bauder, J. W. 1986. Methods of soil analysis. America Society of Agronomy

and Soil Science Society of America, Madison, Wisconsin, USA

Hawrot, M. and Nowak, A. 2006. Effects of Different Soil Treatments on Diesel Fuel

Biodegradation. Polish J. Environ. Stud. 15(4), 643-646.

Lily, M. K., Bahuguna, A., Dangwal, K. and Garg, V. 2009. Degradation of Benzo[a] Pyrene

by a novel strain Bacillus subtilisBMT4i(MTCC 9447). Braz. J. Microbiol. 40(4), 884–892.

Maiti, S. K. 2003. Handbook of methods in Environmental studies, Vol:2 Air, Noise, Soil

and overburden analysis, 1st ed. Oxford book Company, Jaipur, Rajasthan, India.

Macera-López, M. E., Esparza-García, F., Chávez-Gómez, B., Rodríguez-Vázquez, R.,

Saucedo-Castañeda, G. and Barrera-Cortés, J. 2008. Bioremediation of an aged hydrocarbon-

contaminated soil by a combined system of biostimulation-bioaugmentation with filamentous

fungi. Int. Biodeter. Biodegr. 61, 151-160.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 21

Manee, P., Prayad, P., Edward, S. U. and Ladda, T. 1998. Biodegradation of crude oil by soil

microorganisms in the tropics. Biodeg. J. 9, 83-90.

Margesin, R. and Schinner, F. 2001. Bioremediation (natural attenuation and biostimulation)

of diesel oil contaminated soil in an Alpine Glacier Skiing area. Appl. Environ. Microbiol.

67, 3127– 3133.

Mariano, A. P., Kataoka, A. G., Angelis, D. F. and Bonotto, D. M. 2007. Laboratory study on

the bioremediation of diesel oil contaminated soil from a petrol station. Braz. J. Microbiol.

38, 346– 353.

Malloch, D., 1997. Moulds Isolation, Cultivation and Identification. Department of Botany

University of Toronto. Available from:

http://www.botany.utoronto.ca/Researchlabs/MallochLab/Malloch/Cultivation.html

Morgan, P. and Watkinson, R. J. 1989. Hydrocarbon degradation in soils and methods for

soil biotreatment. CRC Crit. Rev. Biotechnol. 8, 305–328.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 22

Mulkins-Phillips, G. J. and Stewart, J. E. 1974. Effects of environmental parameters on

bacterial degradation of bunker crude oils and hydrocarbons. Appl. Microbiol. 28 (6), 915-

922.

Nelson-Smith, A., 1973. Oil Pollution and Marine Ecology. Plenum Press, New York.

Nocentini, M., Nocentini, D. and Pinelli, F. 2000. Bioremediation of a soil contaminated by

hydrocarbon mixtures: the residual concentration problem. Chemosphere. 41, 1115-1123.

Obire, O. 1988. Studies on the biodegradation potentials of some microorganisms isolated

from water systems of two petroleum producing areas in Nigeria. Niger. J. Bot. 1, 81-90.

Okerentugba, P. O. and Ezeronye, O. U. 2003. Petroleum degrading potentials of single and

mixed microbial cultures isolated from rivers and refinery effluent in Nigeria. Af. J.

Biotechnol. 2 (9), 288-292.

Okoh, A. I. 2006. Biodegradation alternative in the cleanup of petroleum hydrocarbon

pollutants. Biotechnol. Mol. Biol. Rev. 1(2), 38-50.

Okpokwasili, G. C. and Odokuma, L. O. 1990. Effect of salinity on biodegradation of oil

spill dispersants. Waste Manage. 10, 141-146.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 23

Oudot, J., Duport, J., Haloui, S. and Roquebert, M. F. 1993. Biodegradation potential of

hydrocarbonassimilating tropiocal fungi. Soil Biol. Biochem. 25, 1167-1173.

Plohl, K. H. and Leskovsek, B. M. 2002. Biological degradation of motor oil in water. Acta.

Chim. Slov. 49, 279–289.

Shabir, G. M., Afzal, F., Anwar, R. And Tahseen, K. Z. M. 2008. Biodegradation of kerosene

in soil by a mixed bacterial culture under different nutrient conditions. Int. J. Biodeter.

Biodegrad. 61, 161– 166.

Shamiyan, R. K., Nirmal, K. J. I., Rita, N. K. and Jignasha, G. P. 2013. An assessment of

physicochemical properties, heavy metal enrichment and fungal characterization of refined

kerosene impacted soil in Anand, Gujarat, India. Inter. J. Environ. 2(1), 165-175.

Shamiyan, R. K., Nirmal, K. J. I., Rita, N. K. and Jignasha, G. P. 2014. Baseline study for

bioremediation of diesel Contaminated soil site of Anand, Gujarat, India. Inter. Res. J. Chem.

4: 37- 53.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 24

Shamiyan, R. K., Nirmal, K. J. I., Rita, N. K. and Jignasha, G. P. 2015a. In vitro study on

assessment of petrol, kerosene and diesel degrading potential of indigenous fungal isolates

from different petroleum product effected soils. Inter. J. Rec. Res. Rev. VIII(1): 8-15.

Shamiyan, R. K., Nirmal, K. J. I., Rita, N. K. and Jignasha, G. P. 2015b. Enzymatic

evaluation during biodegradation of kerosene and diesel, by locally isolated fungi from

petroleum contaminated soils of Western India. Soil Sediment Contam. 24(5), (In Press).

Slavica, S. D., Slavica, B. and Brantner, B. A. 2003. Comparison of ultrasonic extraction and

soxhlet extraction of polycyclic aromatic hydrocarbons from soil. Umweltanalytsches labor,

Sachenplatz13, A-1200 Vienna, Austria.

Solano-Serena, F. R., Marchal, S., Casaregola, C., Vasnier, J. M., Lebeault, Vandecasteele, J.

P. 2000. A mycobacterium strain with extended capacities for degradation of gasoline

hydrocarbons. Appl. Environ. Microbiol. 66, 2392–2399.

Ujowundu, C. O., Kalu, F. N., Nwaoguikpe, R. N., Kalu, O. I., Ihejirka, C. E., Nwosunjoku,

E. C. and Okechukwu, R. I. 2011. Biochemical and physical characterization of diesel oil

contaminated soil in southeastern Nigeria. Res. J. Chem. Sci. 1(8), 57-62.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 25

Verstrate, W., Vancooke, R., Berger, R. and Verlinde, A. 1975. Modelling of the breakdown

and the motilization of hydrocarbon and the soil layers. In: Proceedings of the 3rd

International Biodegradation Symposium (Sharpley, J. N. and Kaplan, A. M., Eds), Applied

Science Publisher, Ltd, London

Walkley, A. and Black, I.A. 1934. An examination of the Degtjareff method for determining

organic carbon in soils: Effect of variations in digestion conditions and of inorganic soil

constituents. Soil Science. 63, 251-263.

Wang, Z., Fingas, M., Blenkinsopp, S., Sergy, G., Landriault, M., Sigouin, L., Foght, J.,

Semple, K. and Westlake, D. W. S. 1998. Comparsion of oil composition changes due to

biodegradation and physical weathering in different oils. J. Chromatogr. A. 809, 89–107.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 26

Table1: Fungal isolates identified based on 18S rRNA identification methods.

Sequence

ID

TOP BLAST Similarity NCBI GenBank

accessions numbers.

PKX4 Aspergillus terreus 100 KC545862 KC545863

PDX7 Penicillium decumbens 99 KC545840 KC545841

SDX7 Penicillium janthinellum 100 KC545842 KC545843

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 27

Table 2: Physicochemical properties of uncontaminated, Post enrichment and post degradation of

kerosene and diesel soil by the fungal isolates.

Parameter Uncontaminated

soil

Post Enrichment Post Degradation

Kerosene Diesel Kerosene Diesel

Sand (%) 42.4±1.6 40.4±1 40.4±1.8 41.8±1.2 41.8±1.1

Silt (%) 25.2±0.8 26.2±1.8 26.2±1.2 25.2±1.3 25.2±0.9

Clay (%) 32.4±1.2 33.4±1.6 33.4±1.3 33±1.5 33±1.1

Texture class Clay loam Clay loam Clay loam

Bulk density (g mL–1) 0.68±0.2 0.93±0.4 0.99±0.1 0.73±0.7 0.75±0.2

Porosity (%) 69.95±2 64.3±2 62.2±2.8 68.9±1.1 67.89±2.9

pH 7.2±0.4 5.4±0.6 5.4±1 7.1±0.4 7.0±0.5

Electrical

conductivity (µS cm–1

) 428±4 235±5 217±4.5 415±5 405±6

Salinity (%) 1.4±0.2 1.9±0.4 1.9±0.1 1.54±0.3 1.5±0.2

Total Nitrogen (%) 2.45±0.5 6.08±1.2 7.78±1.7 2.50±0.5 3.43±0.7

Available phosphorus

(%) 6.18±1.2 6.12±0.9 6.17±1.3 4.23±1.3 4.56±0.8

Total Organic Carbon

(%) 0.86±0.4 4.23±1.2 5.19±1.5 0.98±0.2 1.02±0.4

Sulphate (mg kg–1) 879±6 1100±20 1350±30 890±11 920±14

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 28

Table 3: Kerosene hydrocarbon degradation by the three fungal isolates after 45 days

S- Significant (P≤0.05); N.S- Non Significant (P≥0.05)

Fungal

isolate

Aliphatic

hydrocarbons (%)

Aromatic

hydrocarbons (%)

Aromatic/Aliphatic

hydrocarbons (%)

Student

‘t’ test

t(cal) 5% 10% 15% 5% 10% 15% 5% 10% 15%

P.

janthinellum

SDX7

97±2 96±1 95±2 95±4 92±1 91±2 0.97 0.95 0.95 2.5

(S)

P.

decumbens

PDX7

92±3 90±1.5 86±2 88±5 82±4 74±4 0.95 0.86 0.86 1.809

(N.S)

A. terreus

PKX4 88±4 82±2 75±4 79±4 71±4 62±5 0.89 0.86 0.82

1.779

(N.S)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 29

Table 4: Specific Degradation Rate Constant (k) and Correlation Coefficient (R2) during

Kerosene and Diesel Degradation.

(SDX7-5, SDX7-10 and SDX7-15% - P. janthenellum treated with 5, 10 and 15% kerosene

and diesel; PDX7-5, PDX7-10 and PDX7-15% - P. decumbens treated with 5, 10 and 15%

kerosene and diesel and PKX4-5, PKX4-10 and PKX4-15% - A. terreus treated with 5, 10

and 15% kerosene and diesel)

Soil

treatments

Kerosene Degradation Diesel Degradation

k (day−1

) R2 k (day

−1) R

2

SDX7-5% 0.1023 0.8121 0.0285 0.9184

SDX7-10% 0.0869 0.7954 0.0200 0.907

SDX7-15% 0.0819 0.778 0.0161 0.8875

PDX7-5% 0.0779 0.8083 0.0231 0.8833

PDX7-10% 0.0625 0.7722 0.0156 0.894

PDX7-15% 0.0490 0.7499 0.0129 0.8961

PKX4-5% 0.0590 0.8426 0.0189 0.8778

PKX4-10% 0.0421 0.8327 0.0140 0.8848

PKX4-15% 0.0352 0.8185 0.0112 0.8979

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 30

Table 5: Diesel hydrocarbon degradation after 60 days by the three fungal isolates.

S- Significant (P≤0.05); N.S- Non Significant (P≥0.05)

Fungal

isolate

Aliphatic

hydrocarbons (%)

Aromatic

hydrocarbons (%)

Aromatic/Aliphatic

hydrocarbons (%)

Student

‘t’ test

t(cal) 5% 10% 15% 5% 10% 15% 5% 10% 15%

P.

janthinellum

SDX7

68±2 60±1 52±2 65±4 55±1 49±2 0.86 0.81 0.8 2.479

(S)

P.

decumbens

PDX7

65±3 52±1.5 40±2 52±5 38±4 28±4 0.8 0.73 0.7 1.296

(N.S)

A. terreus

PKX4 55±4 45±2 32±4 40±4 32±4 22±5 0.72 0.71 0.68

1.498

(N.S)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 31

Figure 1: Schematic diagram showing Ex-situ experimental setup of 5, 10 and 15%

kerosene and diesel artificially enriched soils treated with three fungal isolates (SDX7- P.

janthenellum, PDX7- P. decumbens, PKX4- A. terreus) and Control.

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 32

Figure 2: Kerosene degradation by the three fungal isolates as a function of time (SDX7-5, SDX7-

10 and SDX7-15% - P. janthenellum treated with 5, 10 and 15% kerosene and diesel; PDX7-5,

PDX7-10 and PDX7-15% - P. decumbens treated with 5, 10 and 15% kerosene and diesel and

PKX4-5, PKX4-10 and PKX4-15% - A. terreus treated with 5, 10 and 15% kerosene and diesel)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 33

Figure 3: GC-FID chromatograms of Kerosene degradation by most potential fungal

isolate P. janthenellum SDX7 after 45 days (a- Control, b- 5% kerosene enrichment, c- 10%

kerosene enrichment and d- 15% kerosene enrichment).

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 34

Figure 4: GC-FID chromatograms of Diesel degradation by most potential fungal isolate P.

janthenellum SDX7 after 60 days (a- Control, b- 5% diesel enrichment, c- 10% diesel

enrichment and d- 15% diesel enrichment).

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT 35

Figure 5: Effect of kerosene and diesel degradation on fungal population. (SDX7-5, SDX7-

10 and SDX7-15% - P. janthenellum treated with 5, 10 and 15% kerosene and diesel;

PDX7-5, PDX7-10 and PDX7-15% - P. decumbens treated with 5, 10 and 15% kerosene

and diesel and PKX4-5, PKX4-10 and PKX4-15% - A. terreus treated with 5, 10 and 15%

kerosene and diesel)

Dow

nloa

ded

by [

man

si b

anke

r] a

t 03:

59 2

3 Ju

ly 2

015