Assessment of Diesel Degrading Potential of Fungal Isolates from Sludge Contaminated Soil of Petroleum Refinery, Haryana
Application of micro organisms for effective removal of hydrocarbon contamination
from soil has been considered by several workers since decontamination of polluted
soil by other methods leads to production of toxic compounds and these techniques
are non-economic also. In the present study, soil samples from five different
petroleum sludge contaminated sites were studied for assessment of their diesel
degrading potential. The average heterogeneous fungal count in different soil
samples ranged from 35.67±5.69 to 51.33±7.64 and the average count
of diesel utilizing fungi ranged from 3.33±1.15 to 26.00±4.00.
Total heterogeneous fungal count and diesel utilizing fungal count varied significantly
in sludge production and disposal site, as compared to control soil. Thirteen
native fungi species of six fungal genera were isolated from different soil
samples. The identified fungal genera included Aspergillus, Fusarium, Cladosporium,
Penicillium, Rhizopus and Alternaria. Biodegradation ability of all
isolates was confirmed by shake flask culture and vapour phase transfer method.
The results showed that indigenous fungal isolates Aspergillus sp.,
Alternaria sp., Penicillium sp. and Fusarium sp.
displayed highest capability for biodegradation of diesel. Hence, these
fungal species can be used for bioremediation of sludge and diesel contaminated
to cite this article:
Smita Chaudhry, Jyoti Luhach, Vandana Sharma and Chetan Sharma, 2012. Assessment of Diesel Degrading Potential of Fungal Isolates from Sludge Contaminated Soil of Petroleum Refinery, Haryana. Research Journal of Microbiology, 7: 182-190.
Received: March 06, 2012;
Accepted: April 18, 2012;
Published: June 21, 2012
Petroleum products continue to be used as the principle source of energy; however,
despite its important usage, petroleum hydrocarbons also act as a globally environmental
pollutant. Since, the petroleum hydrocarbons are used widely, oil spills are
inevitable even in virtually inhabited areas like Antarctica. Hydrocarbons are
biopersistent, bioaccumulative and can cause deleterious effects to aquatic
fauna and flora as well as to humans (Benson et al.,
2007). However, not all hydrocarbon contamination is anthropogenic. Approximately
five million tonnes of crude oil and refined oil enter the environment each
year as a result of anthropogenic sources such as oil spill (Hinchee
and Kitte, 1995). Extensive changes in marine, as well as terrestrial ecosystem
resulting from the grounding of Exxon Valdez, 1989; Erica spill, 1999 and Prestige
spill, 2002 have recently increased the attention of environmentalist, chemists,
biotechnologists and engineers.
Polycyclic Aromatic Hydrocarbons (PAHs) are organic molecules with two or more
benzene rings in which the number and arrangement of the rings result in diverse
physical and chemical properties (Leahy and Colwell, 1990).
Petroleum industries are responsible for the generation of large amounts of
organic residues causing pollution of soil, sea and rivers. In the petrochemical
sector, as on 01-10-2009, India has 20 refineries (17 in Public sector and 3
in Private sector) throughout the country with a total refining capacity of
177.956 MMTPA at present and by expansion of existing refineries there will
be an addition of 17.146 MMTPA to the refining capacity. At present, total sludge
generated from all refineries is 28,200 tons per annum. This amount will increase
further by proposal of expansion and new refineries (Bhattacharya
and Shekdar, 2003).
Petroleum hydrocarbon compounds bind to different soil components and these
are difficult to remove or degrade (Erdogan and Karaca,
2011). Bioremediation process is an attractive approach for better management
of this huge amount of sludge and cleaning up of hydrocarbons from environment
because it is simple technique, easy to maintain, applicable over large areas,
cost-effective and leads to the complete destruction of the contaminant (Bento
et al., 2005; Achal et al., 2011).
Main reason for this concept is that the majority of the molecules in the crude
oil and refined product are biodegradable. Biodegradation of petroleum and other
hydrocarbons in the environment is a complex process whos quantitative
and qualitative aspects depend on the nature and amount of oil or hydrocarbon
present, the environmental conditions and the composition of the autochthonous
microbial community (Ekpenyong et al., 2007).
Successful application of bioremediation methodology to contaminated systems
is affected by several physical and biological parameters (Al-Turki,
2009) like characteristics of the site and the parameters that affect the
microbial biodegradation of pollutants (Jain et al.,
2011; Sabate et al., 2004). Many physical,
chemical and environmental factors like temperature, nutrients, oxygen, biodegradability,
photo-oxidation, bio-availability, soil moisture, soil acidity and alkalinity
etc. affects the process of biodegradation of hydrocarbons (Rahman
et al., 2003; Venosa and Zhu, 2003; Delille
et al., 2004; Pelletier et al., 2004;
Maki et al., 2001; Trindade
et al., 2005).
Whenever, PAHs are unavailable to biological systems, then toxicity is reduced
but process of biodegradation is inhibited because they are partially unavailable
for microbial degradation (Al-Turki, 2009). It is known
that greater degradation of oil pollutants is carried out in situ by a consortium
of microorganisms and more than 200 species of bacteria, fungi and even algae
can biodegrade hydrocarbons (Onifade and Abubakar, 2007).
Many native strains including ligninolytic fungi have great potential for remediation
of pentachlorophenol (PCP) and polycyclic aromatic hydrocarbon from diesel-contaminated
soils in oil refinery sites (Low et al., 2008).
The advantages associated with fungal bioremediation lay primarily in the versatility
of the technology and its cost efficiency compared to other remediation technologies.
The use of fungi is expected to be relatively economical because they can be
grown on inexpensive agricultural or forest wastes such as corncobs and sawdust.
In one such study, Davies and Westlake (1979) examined
60 fungal isolates for their ability to grow on n-tetradecane, toluene, naphthalene
and seven crude oils of various compositions. Forty cultures, including 28 soil
isolates, could grow on the crude oils. In another study, Fusarium sp.
F092 was found degradation capacity for aliphatic fraction in crude oil under
saline conditions (Hidayat and Tachibana, 2012).
Keeping better degradation ability of native strains of fungi in mind and to
find an appropriate cost effective solution to the above mentioned problems
by bioremediation, the objectives of this study were therefore, to isolate and
identify indigenous fungal flora from contaminated soils of petroleum refinery
and to evaluate the diesel degrading potential of the potent isolates.
MATERIALS AND METHODS
Sampling site: The soil samples for present study were collected from
different locations of the refinery of Indian Oil Corporation Limited, Panipat.
Panipat Refinery set up in 1998, is the seventh refinery of the Indian Oil.
It is located in the state of Haryana and is about 23 km away from Panipat city.
Its refining capacity is 15 MMTPA (Million Metric Tonnes Per Annum). Soil samples
were taken from nearby of sludge processing and disposal areas contaminated
with sludge and oil waste.
Soil sampling: Soil samples from surface soil (0-5 cm depth) were collected
from different sites (sample 1-uncontaminated soil from Effluent Treatment Plant
(ETP) (control soil), sample 2- sludge contaminated soil from sludge production
site in ETP, sample 3-contaminated soil from sludge drying pits in ETP, sample
4-contaminated soil from final disposal of sludge at nearby site and sample
5-composite sample of first four samples. Soil samples from four different sites
were taken consecutively after tilling with a sterile scoop and transferred
into sterile polythene bags for microbiological determination. Then, small parts
of soil from all collected samples from the four sites were mixed to form a
composite sample or sample 5 for biodegradation experiments. The sample was
transported to the laboratory and kept in a refrigerator (in order to keep the
organisms viable and free from any contaminant) before analysis.
Media and chemicals: The media used for isolation of fungi was Potato
Dextrose Agar (PDA) containing potato (200 g), glucose (30 g), distilled water
(1 L) and agar (20 g). For isolation of diesel utilizing fungi, oil agar media
was used. The media was prepared by adding 1% diesel (v/v) sterilized with 0.22
μm pore size Millipore filter paper (Moslein France) to the mineral salt
medium (MSM) that was prepared according to modified Mills
et al. (1978). The composition of the medium was NaCl (10.0 g), MgSO4.7H2O
(0.42 g), KCl (0.29 g), KH2PO4 (0.83 g), Na2HPO4
(1.25 g), NaNO3 (0.42 g), agar (20 g), distilled water (1 L) and
pH of 7.2. For testing degradation capability of indigenous fungal isolates,
Bacto Bushnell-Haas broth containing MgSO4 (0.2 g L-1),
CaCl2 (0.02 g L-1), KH2PO4 (1 g
L-1), K2HPO4 (1 g L-1), FeCl2
(0.05 g L-1) and NH4NO3 (1 g L-1)
was used. Tween 80 (0.1%), redox reagent (2%) and diesel (1%) were all incorporated
into the broth.
Culturing, isolation and enumeration of heterotrophic indigenous fungi in
soil samples: Collected sample was homogeneously mixed and carefully sorted
to remove stones and other unwanted soil debris using 2.0 mm sieve. Isolation
and enumeration of heterotrophic fungi was done by serial dilution agar plating
method. This method is based on the principle that when material containing
microorganism is cultured, each viable microorganism will develop into a colony,
hence colonies appearing on the plates represent the living organisms present
in the sample (Aneja, 2005). Potato Dextrose Agar (PDA)
culture media was used to isolate the fungal species that were present in all
soil samples. Sterile saline, i.e., 0.85% (w/v) sodium chloride was used as
diluent for inoculum preparation. 1.0 g of homogenized, soil sample was aseptically
transferred into a sterile test tube containing 9.0 mL of the diluent. This
gave 10-1 dilution. Subsequently 10-3 serial solutions
were prepared from the 10-1 dilution. Then, 0.1 mL aliquot of 10-3
dilution of each soil sample was aseptically removed with a sterile pipette
and separately spread plated with flame-sterilized glass spreader on PDA plates
in triplicates. The cultured plates were incubated at 27°C for 5-7 days.
After incubation, the colonies appeared on PDA plates were recorded as counts
of total viable heterotrophic fungi for all five soil samples.
Isolation and enumeration of diesel utilizing indigenous fungi in soil samples:
For isolation and preliminary identification of diesel utilizing capability
of fungi, oil agar media was used. Oil agar plates were inoculated in triplicate
with 0.1 mL aliquots of 10-3 dilutions of each soil sample and incubated
at 27°C for 7 days. Colonies appeared on oil agar plates were counted after
a week and recorded as substantial growth of diesel utilizing molds for different
soil samples. The colonies counted were expressed as Colony Forming Unit (CFU)
per gram soil. The counts of diesel degraders was further calculated and expressed
as a percentage of the total heterotrophic diesel degraders
population of fungi.
Identification of indigenous fungal isolates: For the purification of
fungal isolates, the grown cultures of heterotrophic fungi and diesel utilizing
fungi were further carefully and aseptically sub-cultured on same culture media
(PDA), which were stored on potato dextrose agar slants for subsequent characterization
and identification tests. The inoculated plates were identified on the basis
of cultural (colour and colonial appearance of fungal colony) and morphological
characteristics in lacto-phenol cotton blue wet mount by compound microscope
and the software Honstech-TVR and VT Size-5 were used to identify the different
fungal species. Observed characteristics were recorded and compared with the
established identification key (Nelson-Smith, 1973; Malloch,
1997; Aneja, 2005).
Primary step for confirming biodegradation potentials of fungal isolates:
For confirming biodegradation ability of indigenous fungal isolates, Bacto Bushnell-Haas
broth was used, which is a modified method used by Desai
et al. (1993). Two agar plugs (1 cm2 each) of a pure growth
of each isolate were inoculated into Bacto Bushnell-Haas broth (50 mL/250 Erlenmeyer
flask) incorporated with sterile diesel (1% v/v). During this set up the control
flask had no organism. Incubation was done at room temperature (27°C) with
constant shaking at 180 revolution/min for 7 days. The aliquots in the flasks
were monitored daily for colour change from deep blue to pink (initially) and
then colourless (finally). On a daily basis, 5 mL of the aliquots were collected
from each flask and the absorbance was noted on spectrophotometer at 600 nm
Final confirmation for biodegradation potential of fungal isolates:
Fungal isolates showing better performance (fastest colour change) in primary
step were further tested by vapour phase transfer diesel utilization test (Thijsse
and van der Linden, 1961). This test was carried out for the confirmatory
identification of actual diesel-utilizing moulds. The composition and preparation
of the diesel utilization test medium was the same as that of oil agar medium
except that diesel was made available via vapour phase transfer. Putative diesel-utilizing
fungal isolates in first step of confirmation were streaked on plates of agar
medium (one isolate per plate). Inner side of Petri dish was covered with a
sterile filter paper (Whatman No. 1) saturated with filter-sterilized diesel.
The main aim was to supply diesel (source of hydrocarbons) as sole source of
carbon and energy for the growth of the microorganisms on the mineral salts
agar medium surface through vapour phase transfer. All the plates were inverted
and incubated at 27°C for 7-14 days (Okpokwasili and
Amanchukwu, 1988). Colonial growth of different fungi which appeared on
the mineral salts agar medium was noted as confirmed diesel-utilizers.
According to results of the enumeration of indigenous fungi, the average counts
of total heterogeneous fungi on PDA plates and the average counts of diesel
utilizing fungi in the oil agar media were expressed as (x103 CFU
|| Identification of native fungal isolates from all soil samples
|| Average count (x103 CFU/g soil) of total heterogeneous
fungi and diesel utilizing fungi in soil samples
The counts of total heterogeneous fungi ranged 38 to 45 with an average count
of 41.33±3.51 for sample 1; 31 to 42 with an average count of 35.67±5.69
for sample 2; 31 to 46 with an average count of 39.67±7.77 for sample
3; 43 to 53 with an average count of 51.33±7.64 for sample 4 and 46 to
56 with an average count of 50.67±5.03 for sample 5. While the counts
of diesel-utilizing fungi in the soil samples ranged from 2 to 4 with an average
of 3.33±1.15 for sample 1; 13 to 15 with an average count of 13.67±1.15
for sample 2; 8 to 12 with an average count of 10.33±2.08 for sample
3; 20 to 25 with an average count of 22±2.65 for sample 4 and from 22
to 30 with an average count of 26±4.00 CFU g-1, respectively
(Fig. 1). When diesel-utilizing fungal counts were expressed
as percentage (%) of the corresponding total fungal counts in the soil samples,
then for sample 1,2, 3, 4 and 5 the percentage varied as 8, 38, 26, 42 and 51%,
During present investigation, thirteen heterotrophic fungal species belonging
to a total of six genera were isolated from all five soil samples. These include
Aspergillus (A. flavus, A. niger, A. fumigatus); Fusarium sp.;
Alternaria sp.; Cladosporium sp.; Penicillium sp. and Rhizopus
sp. (Table 1). Of these total six genera, five genera
viz.; Aspergillus sp.; Fusarium sp.; Penicillium sp.; Alternaria
sp. and Rhizopus sp. were found diesel-utilizers.
During primary step for confirming biodegradation potentials of fungal isolates,
these isolates produced a colour change in the Bacto Bushnell-Haas broth medium.
The absorbance of broth medium changed according to degradation extent in each
flask. Total colour change (blue to colourless) was also observed in some flasks
while in other flasks colour changes up to some extent. Among the better performing
nine isolates that produced total colour change, Aspergillus flavus, Alternaria
spp., Penicillium spp. and Fusarium spp. displayed
the fastest onset colour change (decrease in absorbance of broth medium) and
hence, highest capability of biodegradation (Fig. 2). This
figure shows the decrease in the absorbance of the Bacto Bushnell Haas broth
medium after fungal treatment.
|| Variation in the absorbance of BH broth media by different
Control here refers the media without inoculation. Almost all fungal isolates
showed change in color from blue to pink, indicate that these native cultures
have ability to grow and to degrade diesel contaminants. More the change in
color of broth more will be the degradation ability of fungal isolates.
For confirmatory identification of actual diesel-utilizing moulds, all fungal
isolates which were taken in primary step were further tested by vapour phase
transfer diesel utilization test. After 10 days of incubation, the six fungal
isolates i.e., Aspergillus flavus, Aspergillus niger, Fusarium sp,
Alternaria sp., Penicillium sp. and Rhizopus sp. showed
better growth during diesel treatment by vapour phase transfer method. This
further confirms diesel biodegradation potentials of these fungal isolates.
The results of heterogeneous fungi and diesel utilizing fungi in all five soil
samples suggest that the diesel utilizing fungi were adapted to the quantity
of hydrocarbons in the environment; hence the increase in the counts of petroleum-utilizing
fungi in heavy sludge polluted areas. Obire and Nwaubeta
(2001, 2002) in a related study on bacteria have
reported similar findings. In all five samples, the reduction in heterogeneous
fungi population in sample of sludge production and drying site was because
of toxicity of hydrocarbons. But in sample of sludge disposal site, the fungal
count increased because in disposal area toxic effects of sludge got diluted.
The toxicity of crude oil or petroleum products varies widely and the scale
of pollution depends on the quantity of oil and the damage done to the environment
(Colwell et al., 1977). Almost same trend was
observed in case of diesel utilizing fungi, where highest average numbers of
diesel utilizing fungal isolates were observed in composite soil sample followed
by soil samples of sludge disposal site, indicating the adaptability of native
fungal isolates in contaminated environment. The percentage of diesel (hydrocarbon)
utilizers in a particular environment appears to be an index of the presence
of hydrocarbons in that environment and environmental exposure to petroleum
hydrocarbons. These results agree with the reports of Mulkins-Phillips
and Stewart (1974).
The occurrence of a variety of fungal genera (i.e., fungal diversity) of both
heterotrophic fungi and diesel degrading fungi in the sludge disposal site and
composite soil samples was found to be higher. The addition of sludge to the
soils resulted in selective increases and decreases in the numbers of fungal
populations and enrichment of various fungal genera. Some of these organisms
have earlier been reported as hydrocarbon bio-degraders by April
et al. (2000).
During primary step for confirming biodegradation potentials of fungal isolates,
the ability of these isolates to produce a colour change in the Bacto Bushnell-Haas
broth medium is presumably due to the reduction of the indicator by the oxidized
products of hydrocarbon degradation. The total colour change (blue to colourless)
supports the fact that the isolates are potential hydrocarbon oxidizers. Better
performing nine isolates which produced total colour change, Aspergillus
flavus, Alternaria spp., Penicillium spp. and Fusarium
spp. displayed the fastest onset colour change and hence, highest
capability of biodegradation. The high rate of diesel (hydrocarbon) degradation
by the four fungi could emanate from their massive growth and enzyme production
responses during their growth phases. This could be supported by the findings
of Bogan and Lamar (1996), which showed that extracellular
ligninolytic enzymes of white rot fungi are produced in response to their growth
phases. The utilization of 0.1% of Tween 80 during the assay and the implication
of these three organisms in hydrocarbon degradation from our results is similar
to the findings of April et al. (2000).
In vapour phase transfer diesel utilization test after 10 days of incubation
for confirmatory identification of actual diesel-utilizing moulds, the six fungal
isolates i.e., Aspergillus flavus, Aspergillus niger, Fusarium sp,
Alternaria sp., Penicillium sp. and Rhizopus sp. showed
better growth, further confirming diesel biodegradation potentials of these
In this study, we observed that higher biodegradation efficiency was exhibited
by Aspergillus flavus, Alternaria sp., Penicillium sp. and
Fusarium sp, providing these fungi to be better hydrocarbon degraders.
Thus, they can be effectively utilized for the degradation of sludge and for
bioremediation of oil polluted farm lands especially those located within the
vicinity of the petroleum processing and disposal sites. Since microorganisms
play an essential role in biogeochemical cycling, interference with microbial
metabolic activities by pollutants in the environment can have far reaching
ecological consequences. During field applications for bioremediation of sludge
contaminated sites, after large scale production of the potent fungal organisms,
adequate carriers or extenders like tween 80 can be used for enhancement of
degradation process. Inert stickers or adhesives like molasses, corn syrup,
skim milk, casein and latexes may be incorporated into the formulation in order
to prevent run offs. A good sticker combined with charcoal can serve as a protectant
for reducing the effects of ultra-violet light, desiccation and other detrimental
environmental factors. During application, the sludge and oil contaminated sites
should first be tilled to loosen the soil. Thereafter, the loosened soil should
be enriched with adequate nutrients necessary for the growth of the organism
before applying the microbial inoculants which must be properly mixed with the
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