Original Research Al-Razi Univ J Med Sci 2020; 4 (2)

Al-Razi University Journal of

Medical Sciences
RUJMS

Oil pollution in Lake Timsah, detection and bioremediation through rearing of

Mugil cephalus and Tilapia zillii
1
Nabil S. Shwtar, 1Hekmat M.M. Tantawy and 1Nour El-dein H. Saleh. 2Mohammed S. A. Al-Awar
1
Department of Zoology, Faculty of Science, Suez Canal University, Egypt. 2Department of Biology, Faculty
of Applied Science, Amran University, Yemen.

*Corresponding author: Nabil S. Shwtar, Department of Zoology, Faculty of Science, Suez Canal
University, Egypt: nabeelshs55@gmail.com

Abstract
This research assessed the capacity of bioremediation the complete pollution of petroleum produced by
burned motor oil in the lake Timsah, Egypt using four indigenous bacteria and four isolates of fungi,
separated from Lake and to record the effects on two species of fish, Mugil cephalus and Tilapia zillii, Fish
to assess the impacts of the water-solution (WSF) fraction of burning motor oil on the growth efficiency and
percentage of the fish's survival with regards to their clinical signs, lengths and weights alone and in
combination with 4 bacterial and 4 fungal strains, have been carried out on the toxicity and bioremediation
research. Burnt motor oil liquids was added to Aquarium water and four bacterial (Achromobacter sp.,
Bacillus sp., Clostridium sp. and Pseudomonas sp.), four fungal Isolates (Absidia corymbifera, Aspergillus
sydowii, Mucor circinelloides, and Penicillium sp.) were taken to treat the water in Lake aquarium with a
microbial application. The treatment was prolonged for 45 days. The results showed the great disparity
between both types of fish, mullet and tilapia, where the resistance of tilapia over mullet for each of the oil
and microorganisms has led the oil added to the aquaria is none treated to the rate of death of 40% of the
mullet fish, and 50% of the tilapia processing. A comparison between treatment and blank aquaria the
results has shown that the rate of death was more in blank which proves that the micro-organisms have been
used in the aquaria of oil additive treatment had improved and grown well. Microbiological analysis was
conducted to the muscles and liver of both types of fish and the results showed that the average of CFU of
bacterial colonies in fish treated with bacteria is much less compared with untreated fisf. This explain that
the growth rate of the tested fish keeping a similar as untreated groups. On the other hand, the GC / FID
showed a decrease in the concentration of (PAHs) and this promotes better fish in the treatment groups
compared with the control and also do not see any of the compounds of (PAHs) in the aquaria for the
treatment of tilapia fish with fungi and bacteria. The results proved that the microbial treatment using
combination of fungi and bacteria are more effective for the remediation of the burned motor oil
contaminated lake-water than fungi or bacteria alone.

Keywords: oil pollution, bioremediation, bacteria, fungi, Lake Timsah, Mugil cephalus and Tilapia
zillii..
………………………………………………………………………………………….

Introduction accidental oil spillage, leakage and break

pipelines1. Moreover, many studies reported
Crude oil production constitute a major sours that the oil contamination occurs due to the
of energy in the wide world and produce transportation of oil, surface runoff, illegal
serious environmental problems due to bilge water discharges and port activity2. Oil
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spills can cause deleterious impacts to aquatic of the environment conditions. All of these
and terrestrial environment and important bioremediation considers promise
human health3. However, its effects on techniques to be better alternatives, saver and
aquaculture could be serious on the grounds eco-friendly, than physicochemical methods.
that oil tends to bio-accumulation in the In co-culture degradation of PAHs, it has
tissues of mollusks, fish and mammals4. For been suggested that the procedure is started
the most part take-up of marine oil pollutants by the release of fungal extracellular proteins
by fish can happen through bioconcentration that separate particles that are too enormous
from water and additionally bioaccumulation to even think about passing through bacterial
from diet or potentially suspended sediments5. cell dividers, achieving a halfway oxidative
The U.S. Environmental Protection Agency corruption of the PAH. In co-culture
(EPA) lists 16 PAHs as priority pollutants6. degradation of PAHs by fungi, the process is
The environmental concern about PAH is due initiated by the release of fungal extracellular
to their potential to form highly carcinogenic enzymes that break down molecules that are
and mutagenic derivatives such as diols and very large to pass through bacterial cell walls,
epoxides7,8. The water shows limited solvency accomplishing a partial oxidative degradation
of PAHs so it has been shown that contact of of the PAH16. This initial ring oxidation
water with PAH-pollutants sediment that increases the potential for degradation and
could tend to toxicity of the water. Moreover, mineralisation by bacteria as the oxidised
Polluted sediment in surface waters represents metabolites have increased water solubility
a continuing resource of pollution in the and reactivity thereby eliminating this initial
aquatic food chain. Indeed, even minor ring oxidation as the rate-limiting step for
convergence of numerous PAHs could be bacteria17. This process not only addresses the
complemented through fish, representing a inability of the bacteria to transport the
possible danger to man, being at the head of molecule into the cell, it also prevents the
the food chain9,10. PAHs entering the water accumulation of the fungal metabolites.
framework can be first collected in fine- Previously fungal metabolites have been
grained sediments, suspended particles, then reported to accumulate and exhibit an
remobilized in the seawater, at that point inhibitory effect on degradation18. Before the
become bioavailable to local organisms Lastly development of the Suez Canal in the
accumulate in biota that has high rates of nineteenth century, the Bitter Lakes were
take-up or is unfit to effectively process the moderately little hyper-saline inland lakes
parent mixes (for example mussels and many encompassed by salt-encrusted sabkha. After
invertebrates)11. In the water biological the lakes were associated with both the
ecosystems, fungi assume a significant role Mediterranean and the Red Sea by the Suez
with their capacity in expelling risky Canal, they turned into a solitary marine
compounds from the water. Sediment body; their size expanded, and their saltiness
particles polluted with hydrocarbons from oil diminished, coming to somewhere in the
spills is one of the desired ecological range of 43 and 46 ppt19. The northern more
specialties to fungi which use them as carbon extensive finish of the water body is known as
source12. the Great Bitter Lake, while the southern
smaller part is known as the Little Bitter
The microorganisms involved bacteria, yeast Lake. The base is sandy and sparsely secured
and fungi represent the important groups in with vegetation. Agrarian land, traveler
the degradation of hydrocarbons3,13. There are advancement and intermittent zones of salt
two ways for bioremediation: bog outskirt the lakes on the western side,
bioaugmentation and biostimulation14,15. while the eastern side is generally sandy
Whereas, the bioaugmentation defined as the desert. Lake Timsah, one of the little lakes
addition of highly efficient oil-degrading that establish the Bitter Lakes and situated on
bacteria to improve and enhance the the north of the Suez Canal, is a land-
degradation, biostimulation means stimulate inundated embayment with an all out region
indigenous bacteria activity by modification of 15 km2. Lake Timsah is the most important
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asset in Ismailia city. It lies on Suez Canal at The lake is likewise the end-sink of aliphatic
the mid-path between Port Said and Suez and fragrant hydrocarbons that start from
urban communities; The Lake is the end- delivery exercises, ballasting water, upkeep
purpose of different outlets that release huge and sea works in the few harbors around. The
volumes of rural, civil and modern lake underpins fishing and the travel industry
wastewater. The lake is limited by Ismailia, businesses that utilize countless neighborhood
the primary city of the district that releases residents and give a critical segment of the
portions of its crude and somewhat rewarded locale incomes. A few investigations were led
metropolitan waste into the lake. The lake is to screen determined natural poisons in the
the primary wet dock of the city, a little port distinctive segment of the lake, including
that likewise harbors an assortment of marine diverse marine organism22. Polycyclic
works, including the upkeep work of the Suez aromatic hydrocarbons (PAHs) were the
Canal Authority and its partnered sea works compounds mapped out in these studies23.
and serves the significant wellspring of fish Generally take-up of marine oil pollutes by
which spread Ismailia city and its neighbored fish can happen through bioconcentration
region. As of late there are an incredible from water as well as bioaccumulation from
mindfulness that the lake water get a lot of diet as well as suspended sediments5. Also,
releases which include a huge scope of the Lake Timsah presented to some of extra
contaminations, for example, substantial oil contamination sources, from the boat
metals, harmful natural mixes and others. All building organizations which encompassing
sort of contaminations begin from cultivating the lake, for example, El-Timsah transport
and mechanical exercises in the region of the building organization, El-Timsah shipyard,
lake. These toxins are harmful to both sea- El-karakat workshop, and Arab Contractors
going biota and people. The disintegration of shipyard work shop20. The quality of life in
the lake has reached out to a genuine level the lake has been a major concern for the
where pressing activity is required promptly local authorities for the last few years. High
to reestablish the lake biological system 20. contamination level is the cause behind the
decline in the lake biodiversity and the
As the principal oil productive zone of the decline in fish quality harvested from the
world is the Middle East zone, the red Sea is lake23,24.
considered, still and will be for long time, the
primary transportation course for unrefined The present study aimed to Isolation and
petroleum. On account of this reality, the identification of some of the indigenous
hazard and capability of oil contamination are microorganisms (bacteria and fungi) capable
parallely increased in the Red Sea condition21 to biodegradation of polycyclic aromatic
what's more, thus Suez Canal, and Lake hydrocarbons (PAHs) and evaluate the ability
Timsah. A portion of the channels releasing of four bacterial and four fungal strains to
into the lake are heavy with an assortment of bioremediate the problems caused by motor
modern contaminations beginning from oil pollution through rearing M. cephalus and
mechanical zones in Cairo. T. zillii.

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MATERIALS AND METHODS Sampling: Surface water samples were
collected in wide neck glass bottles. Surface
Isolation and identification of Bacteria and sediment (1-5cm) samples were collected using
Fungi a hand shovel (trawel) and stored in sterile glass
The objective: Isolation and identification of jars. Five surface water and three surface
species of bacteria and fungi able to sediment samples were collected from different
biodegradation of oils. sites at Lake Timsah. (photo.1).

transferred to 250ml Erlynmyer-flask,

containing 90ml sterile d. H2O and agitated for
10 min at 150 rpm, then 1ml was aseptically
removed and spread of on to half-strength
minimal medium plates (5 replica each). Plates
were incubated at 21oC for 3 days for
enumeration of fungal growth and overnight at
37°C for examination of bacterial growth. After
incubation, the developed colonies were
counted (as counts of total viable bacteria/
fungi (CFU).
Isolation of Bacteria and Filamentous Fungi
from Water
photo.1: Lake Timsah map showing sampling sites
(Google-earth) 10ml of each water samples was concentrated
ᴑ: sites of water collection (NO. from 1-5), by centrifugation at 7000 rpm for 10 min then
▲: sites of sediment collection (NO. from 1-3) precipitate was aseptically removed with a
sterile pipette and spreaded of onto half-
Preparing Media for Isolation of Bacteria Strength minimal medium plates (5 replica
and Fungi each). Plates were then incubated at 21oC for 3
The following medium was used for isolation days for enumeration of fungal growth and
and enumeration of bacteria and fungi (half- overnight for recognizing of bacterial growth.
Strength minimal medium). The composition of After incubation, the developed colonies were
the medium per liter was NaNO3 2.0g, K2HPO4 counted (as counts of total viable bacteria/fungi
1.0g, MgSO4.7H2O 0.5g, KCl 0.5g, FeSO4 (CFU).
0.01g, Agar 20g, 500 ml distilled water. For
Purification of Microorganisms: The
isolation of fungi, Chloramphenicol 250mg/l
procedure of purification of bacteria and fungi
and Rose-Bengal were added to prevent
was undertaken as described by26 with some
bacterial growth, autoclaved at 121oC for 20
modifications. Bacterial and fungal colonies
min. The components were allowed to cool to
were purified and subcultured on LB agar and
about 45oC under aseptic conditions, and 500ml
Czapeks-dox agar plates respectively and
sterile lake-water was added and mixed
incubated at 21oC for 2-3 days for fungi and at
thoroughly and then dispensed into sterile Petri-
37oC overnight for bacteria. The isolates were
dishes.
stored in refrigerator to be used later.
Isolation of bacteria and filamentous fungi Identification of Fungi: Growing fungi were
from sediment identified on agar plates and microscopic
The procedure of isolation was as described examinations were carried out according to the
by25, with some modifications.10g (wet weight) recommendations stated in compendium of soil
of each sediment sample was aseptically fungi27.
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Identification of Bacteria: Bacterial isolates aromatic fraction (F2) was eluted with 60ml of
were identified using various morphological hexane and dichloromethane (80:20; V/V).
and biochemical characteristics. For Both of F1 and F2 fractions were concentrated
identification of bacterial isolates to genus using a gentle stream of pure nitrogen to about
level, gram staining, motility tests28 were 0.2 ml, before being injected into GC/FID.
carried out. In addition to Morphological
parameters as colony color, size, shape and GC/FID analysis - according to31. The
margin were carried out according to29. extracted oil samples were analyzed using
Biochemical tests such as catalase, oxidase, Aglient model 6890 plus gas chromatography at
indole, methyl red, Vogus Presuker, glucose the National Research Center. The condition of
fermentation, citrate, urea and nitrate reduction analysis was as follows:
were performed using the taxonomic scheme of Chromosorb W-AWDCMS (100/120mesh)
Bergey’s Manual of Determinative coated with 3% OV-17 packed in a 1.8 m long
Bacteriology30. x 2 mm ID glass column with nitrogen carrier
gas at 40 ml/min flow rate. Column temperature
Biochemical tests – according to the Bergey’s held at 100oC for 4 min, then programed at
Manual of Systematic Bacteriology30. 8oC/min to final hold at 280oC. Detector: flame
Extraction of total hydrocarbons from Ionization Detector. Injector: splitless injection.
sediments Experiment for bioremediation of motor oils by
Soxhlet apparatus was used. 50-100g of wet bacteria and fungi through rearing fishs
weight sediment was extracted with 200ml of Objective: 1- Assessment of The efficiency of
n-hexane-dichloromethane mixture (1:1) v/v. bacteria and fungi and combination of both in
Mixture was then wormed to 40oC in water bath the process of bioremediation of motor oil
for 7 hours. The extract was then filtered through the period of experiment.
through watt-mar filter paper in the presence of
2- 6g of anhydrous sodium sulphate, and the 2-The impact of oil pollution on representative
extract was then concentrated to 2ml as samples of living organisms by
discussed previously. histopathological effects of motor oil in some
organ of the fishes such as livers and gills.
Extraction of total hydrocarbons from
water: Water samples (1L each) were extracted Sampling: Fish samples of fingers Mugil
with two volumes of 30ml n-hexane in a cephalus (av. Weight 0.68g& av. length
separating funnel with shaking thoroughly for 3.38cm) and Tilapia zillii (av. Weight 4.17&
15min at 150 rpm on a horizontal shaker. The av. length 5.29cm) were obtained from
extract was dried on anhydrous sodium sulphate fishermen at the shore of Lake Timsah over all
(2-6g) and then concentrated to 1ml as the periods of this study.
explained above. 1000 L of of Lake Timsah Water (pH: 7.93 -
8.56 and salinity: 30 – 35ppm)
Cleaning-up and fractionation process: Water collected from site 1.
Cleaning-up and fractionation was performed Dose of oil: 1ml motor oil, Dose of bacteria:
prior to gas chromatograph/flame ionization 0.01g (dry weight), Dose of fungi: 0.04g (dry
detector (GC/FID). The extracted volume was weight) and Temperature: 20 – 25 oC
passed through the silica column prepared by Fish stock
slurry packing with 10 g of silica, followed by
10 g of alumina and finally 1g of anhydrous Fishes were captured using fishing nets (1 mm
sodium sulphate. The aliphatic fraction (F1) mesh) from Lake Timsah. They were
was sequentially eluted from the column using transported and acclimated in two 100 L glass
25 ml of hexane. However, the unsaturated tanks filled with lake-water from the collecting
site (30-35ppt salinity, and 20-25ºC
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temperature). The tanks were equipped with 30 L lake-water at densities of 10
constant aeration and kept under natural mullet/aquarium.
photoperiod (13 L: 11 D). The fishes remained Experiment design : The experiment design
in the tanks for two- four weeks, according to32. was planned as the methods described by33 with
Before the fishes were distributed in the some modifications. 16 aquaria used in this
experimental aquaria, 10 individuals were experiment, 8 for Mugil cephalus and 8 for
randomly selected for the detection of the initial Tilapia zillii. All 8 aquaria distributed as a
weight and length. Then were transferred to
followin
eight glass aquaria (40 x 40 x 60 cm) filled with
Table 1 Distribution of M.cephalus and T. zillii in aquaria

Mug. , Tilap. Meaning Content of the aquarium

A1, A1/ Control Lake-water+ fish
A2, A2/ Control+Oil Lake-water + fish +oil
B1, B1/ blank Lake-water + fish +mix bacteria
B2, B2/ blank Lake-water + fish +mix fungi
B3, B3/ blank Lake-water + fish +mix fungi and bacteria
C1, C1/ Treatment Lake-water + fish + mix bacteria +oil
C2, C2/ Treatment Lake-water + fish +mix fungi+ oil
C3, C3/ Treatment Lake-water + fish +mix fungi and bacteria+ oil

One as a normal control (sea water and Fish), and acclimiation, 15 days for oil burned
one as a poisonous control (sea water, Fish and treatments and 15 days for washing process.
oil burned of tractor 1ml), three as blank (sea The feeding regime was applied at 5% body
water, Fish and mix of bacteria or fungi or mix weight per day throughout the experiment, the
of bacteria & fungi) and final three aquaria used frequency of feeding was maintained as twice a
as treatment (sea water, Fish, mix of bacteria or day for six days a week. The artificial diet was
fungi or a consortium of bacteria & fungi and analyzed for moisture, crude protein, ether
(motor oil 1ml) photo2. extract and ash according to standard AOAC
methods33
The experimental period was extended for 45
days; it was divided into 15 days for adaption

photo 2. Photographs (a, b, c) shows experimental frame work and the used glass aquaria filled with
untreated lake-water (control) (A, A/); the bacteria in contact with M. cephalus, T. zillii blank (B1, B1/)
respectively; the oily contaminated lake-water under treatments (C1,C2, C3) with M. cephalus and (C1/,
C2/, C3/) with T. zillii. The feeding regime

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Sampling processes and microbial Analyses
examination processes in the oily
contaminated lake-water Gain in weight (g/fish) = Av. final weight (g) –
Av. Initial weight (g)
Water samples for microbiological analyses Daily weight gain = Gain in weight (mg/fish) /
were taken regularly from the rearing fish Days
aquaria after zero, 3, 5, 10, 15, 21 and 30 days
of the treatment. 1ml of these lake-water Daily length gain (mm/fish/day) = Gain in
samples was used aseptically to inoculate 9ml length (mm/fish) / Days
of sterile culture medium (nutrient broth) and Specific growth rate (SGR)(% day-1) = [(ln
then incubated for 24hr at 37ºC. Then, the dry final weight(g) – ln initial weight (g))/days of
weight of the bacterial growth was estimated in the expr.] *100
mg/100 ml according to34.
Instant Daily Growth: IDG= 100 * (Ln final
Examination processes of fish in aquarium weight- Ln initial weight) / days
1. Examination of skin: The skin of three Statistical analyses
treated fish per aquarium were swapped and The statistical analyses of the data were carried
resuspended in 5 ml sterile phosphate-buffered out in triplicates using ANOVA test and the
saline (PBS), which is composed of (g/l): 8.0 of least significant difference L.S.D.
NaCl; 0.3 of KCl; 0.73; of NaH2PO4 and 0.2 of
K2HPO4. Complete to 1 liter with dis H2O, RESULTS AND DISCUSSION
adjust pH to 7.4. All samples were 10-fold RESULTS
diluted, squeezed by hand for few minutes and
then spreaded onto nutrient agar plates which Isolation of Bacteria and Fungi from several
composed of (g/l): 3; beef extract; 5 peptone contaminated sites
and 20 agar using a glass spreader (5 cm). The
total bacterial count ‚ [colony forming unit In this study, it was attempted to demonstrate
(cfu/ml)] was estimated in each sample the potential degradability of motor oil and a
according to35. Sterile gloves, bags, swabs, and range of hydrocarbon substrates by
glass beakers were used for sampling. microorganisms isolated from contaminated
sites of surface sediments and water around
2. Examination of muscultre and liver: 1 g of Lake Timsah. Total count of bacteria isolates
the muscle part (or all the liver) of each from contaminated sites in Lake Timsah
examined fish was removed under sterile presented in Table 2 ranged from 63.66±2.51 to
condition, using sterile forceps, and transferred 195±66.64 of water and samples ranged from
to sterile tubes each, contains 1 ml PBS. All 37.33±36.35 to 110.33±8.73 of sediments
samples were 10-fold diluted, squeezed by hand samples.
for a few minutes and spread onto nutrient agar
plates as mentioned above. cfu/ml was
estimated in each sample according to35.

Determination of PAHs through rearing

Fish: Water samples for PAHs analyses were
taken regularly from the rearing fish aquaria
after 1, 2, and 4 weeks, 1litr of these lake-water
samples used for determination of
hydrocarbons by the method described above.

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Table 2 Enumeration of Bacterial colonies in Water and Sediment samples.

Site Sample Isolates Dilution 102(CFU)

1 water Bacteria 10-2 97.7±9.29
2 water Bacteria 10-2 92.66±2.51
3 water Bacteria 10-2 63.33±25.16
4 water Bacteria 10-2 143±68.46
5 water Bacteria 10-2 195±66.64
1 sediment Bacteria 10-2 102.33±22.50
2 sediment Bacteria 10-2 110.33±8.73
3 sediment Bacteria 10-2 37.33±36.35

Total count of Fungi isolates from contaminated sites (Table 3) ranged from 2.33±0.57 to
56±57.23 of water and samples ranged from 6±3 to 57±55.83.
Table 3 Enumeration of fungal colonies in Water and Sediment samples.

Site Sample Isolates Dilution 102(CFU)

1 water Fungi 10-2 9.67±6.11
2 water Fungi 10-2 56±57.23
3 water Fungi 10-2 2.33±0.57
4 water Fungi 10-2 7.33±4.04
5 water Fungi 10-2 16.67±7.24
1 sediment Fungi 10-2 57±55.83
2 sediment Fungi 10-2 6±3
3 sediment Fungi 10-2 8.33±3.21

Identification of Bacterial Strains (Gram Stain and Biochemical Tests)

Seven bacterial isolates Pseudomonas sp., Achromobacter sp., Acinetobacter sp., Pseudomonas
aerogenosa, Bacillus sp., Clostridium sp., and Aeromonas sp. were isolated from five contaminated
sites around Lake Timsah from water and sediment. Morphological and biochemical characteristics of
all bacterial isolates were determined by Gram’s staining and biochemical tests according to the
Bergey’s Manual of Systematic Bacteriology. All the isolates were tested for selective biochemical
tests which are presented in Table 4.

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 Table 4 Biochemical tests of selected bacteria Species used in the Treatment
Sourc Locatio
Species g.s s.f Shape Cat. O In. Nit. G.F V.P m.r Cit. U M
e n
a&b 1,4,5 Clostridium sp + + Rods - + - + + - + + - -
a&b 3,5 Bacillus sp + + Rods + + - + + + + + + +
Pseudomonas Short
B 3 - - + + - Zn+ - - - - + +
aerogenosa bacilli
A 1,4 Achromobacter - - Rods + + - + - + - + - +
B 3 Acinetobacter sp - Bacilli + - - - + - + - + +
- Mono,bi.po
B 1,3 Pseudomonas sp _ + + - Zn+ - - - - - -
ly,bacilli
Short,mono
A 5 Aeromonas sp - _ + + + - - - + - - +
,bi,bacilli
Foot notes: a = water, b = sediments, g.s = gram stain, s.f = Spore forming, cat. = catalase, O = oxidase, In =
Indole, Nit. = nitrate, V.P = Voges-ProskauerG.F = glucose fermentation, m.r = Methyl red ,Cit. = citrate,
U = urea , M = motility .

Whereas, 10 fungal isolates Pencillium sp., stolonifer, were identified on agar plates and
Mucor hiemalis, M. mucedo, M. circinelloides, microscopic examinations were carried out
Aspergillus flavus, Alternaria sp., Absidia according to the recommendations stated in
corymbifera, Fusarium sp., A. sydawii, Rizopus compendium of soil fung
Extraction and detection of PAHs from sediments and water in Lake Timsah
Resuts of extraction and detection of PAHs from sediments and water in Lake Timsah are presented
in Figures (1 and 2) and Table 6.

Fig.1 GC/FID chromatogram of PAHs content of a sediment sample from site 2 in Lake Timsah.

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Fig.2 GC/FID chromatogram of PAHs content of a water sample from site 2 in Lake
Timsah.
Table.6 PAHs content in sediment and water samples from site 2 in Lake Timsah.

Standard PAHs Sediment (µg/L) Water(µg/L)

Naphthlaene N.D N.D

1-methylnaphalene N.D N.D
2-methylnaphalene N.D N.D
Acenaphthalene 29.31 N.D
Acenaphthylene 42.26 13.17
Fluorene 65.94 N.D
Anthracene 48.60 4.17
Phenanthrene 62.05 N.D
Fluoranthene 14.22 N.D
Pyrene N.D N.D
Benzo(a)anthracene N.D N.D
Chrysene N.D N.D
Benzo(b)anthracene N.D N.D
Benzo(k)fluoranthene N.D N.D
Indopyrene N.D N.D
TOTAL 261.93 17.34

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 The effect of the microbial motor oil treatments
Experiment for bioremediation of motor oils on the growth performance, survival percentage
by bacterial and fungal strains through and feed utilization parameters of M. cephalus
raring fishes and T. zillii, after 0, 45 days of rearing are
4.5. Estimation of growth performance and presented in (Table 7., 8) respectively.
the survival percentage of M. cephalus
and T. zillii

Table: (7) The effect of the microbial motor oil treatments on the growth performance, survival percentage
and feed utilization parameters of M. cephalus after 0, 45 days of rearing.

Foot notes F=Fish, O=Oil, B=Bacteria, Fun=Fungi

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Table 8 The effect of the microbial motor oil treatments on the growth performance, survival
percentage and feed utilization parameters of T. zillii after 0, 45 days of rearing

Foot notes F=Fish, O=Oil, B=Bacteria, Fun=Fung

The data presented in Fig. 3 (A, B) showed that The data presented in Fig. 4 (A, B) showed that
with 1ml oil in these tested aquaria the with 1 ml oil in these tested aquaria the
maximum fungal dry weight in M. cephalus and maximum bacterial dry weight in M. cephalus
T. zillii was increased to 0.08g/L and it was was increased to 0.05g/L and it was obtained
obtained mainly after the 4th day of treatment. mainly after the 7th and 13th day of treatment.

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 a

0.09

0.08
b
0.07

0.06 control
oil + fish
0.05
blank 1
0.04 blank 2
0.03 oil+fish+fungi

0.02 oil+fish+f+b

0.01

0
2 days 4 days 8 days 14 days 27 days

Fig.3 The fungal dry weight (g/L) in the surrounding media of the contaminated aquaria (O&F,
O&Fun&F, O&F&Fun&B, F&Fun, F,B,Fun and the control) during 30 days of oil treatment of (a) M.
cephalus and (b) T. zillii.

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 0.06

a
0.05

0.04

0.03

control
0.02

0.01

0
2 days 4 days 8 days 13 days 27 days

0.06

b
0.05

0.04 control
oil + fish

0.03 blank 1
blank 2
0.02 oil+fish+fungi
oil+fish+f+b
0.01

0
2 days 4 days 8 days 13 days 27 days

Fig.4 The bacterial dry weight (g/L) in the surrounding media of the contaminated aquaria (O&F,
O&Fun&F, O&F&Fun&B, F&Fun, F,B,Fun and the control) during 30 days of oil treatment of (a) M.
cephalusand (b) T. zillii.

The microbiological examinations of M. cephalus and T. zillii after 45 days of rearing shwed in
Table 9 and Table 10 respectively.

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Table 9. The microbiological examinations of M. cephalus after 45 days of rearing.

Bacterial count CFU × 102/100 ml

treatments
Skin Musculature Liver Mean*

Control 27.00 28.00 17.00 24a

F&B 0.13 0.64 0.83 0.53b
F&B&Fun 0.57 1.19 0.63 0.80b
O&F&B 1.08 0.10 6.53 2.57b
O&F&B&Fun 0.15 0.03 4.74 1.64b
Mean* 5.79c 5.99c 5.95c

*Mean values in the same column or in the same row which have the same letter are insignificantly
different at P < 0.05 while the mean values with different letters are significantly different at P < 0.05.
F=Fish, B=Bacteria, Fun=Fungi, O=Oil.

Table 10 The microbiological examinations of T. zillii after 45 days of rearing.

Bacterial count CFU × 102/100 ml
treatments
Skin Musculature Liver Mean*

Control 25.00 24.00 20.88 23.29a

F&B 0.14 2.40 9.80 4.11b
F&B&Fun 0.11 0.07 0.09 0.09b
O&F&B 0.03 2.79 3.71 2.18b
O&F&B&Fun 27.24 28.20 26.00 27.15a
Mean* 10.50c 11.49c 12.10c
*Mean values in the same column or in the same row which have the same letter are insignificantly
different at P < 0.05 while the mean values with different letters are significantly different at P < 0.05.

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Bioremediation of some fractions of PAH in treated aquaria
Table 11: Bioremediation of motor oil using selected isolates of bacteria and fungi (Oil
& fungi & Bacteria & T. zillii)

After 1 week After 2 weeks After 4 weeks

of treatment of treatment of treatment

Sample Additive( B+Fun+O+F( Remediation B+Fun+O+F

µg/L) µg/L) rate
methylnaphalene 96.907 28.541 70.55% N.D
Acenaphthalene 71.478 0.293 99.59% N.D
Fluoranthene 42.094 9.419 77.26% N.D

TOTAL 335.063 38.253 88.58% N.D

B= bacteria, Fun= fungi, O= oil, F= fish

Table 12: Bioremediation of motor oil using selected isolates of fungi (Oil & fungi & T.
zillii).

After 1 week of After 2 weeks After 4 weeks

treatment of treatment of treatment

Sample Additive( µg/L) Fun+F+O( Remediation Fun+F+O

µg/L) rate
Acenaphthylene 13.841 12.551 9.36% N.D
Fluorene 25.102 1.831 92.71% N.D
Phenanthrene 27.195 14.380 47.12% N.D
Pyrene 11.042 4.602 58.33% N.D

TOTAL 77.18 33.364 56.77% N.D

The Fig .5 Showed results of GC/FID chromatogram of PAHs content of a water samples
standard sample, additive sample, and treated sample by B & Fun after two week, and treated
sample by Bacteria & Fungi after four week from aquaria of M cephalus

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Table 13: Bioremediation of motor oil using selected isolates of bacteria (Oil & bacteria
& M. cephalus).

After 1 week of After 2 weeks After 4 weeks

treatment of treatment of treatment

Sample Additive( µg/L) B+F+O( µg/L) Remediation B+F+O

rate
Acenaphthalene 71.478 50.327 29.59% 25.311
Acenaphthylene 3.841 7.535 45.56% 4.226
Fluorene 25.102 10.128 59.64% 6.394
Anthracene 40.845 7.882 80.71% 4.663

TOTAL 151.266 75.872 49.84% 40.594

a
b

c
d

Fig. 5 GC/FID chromatogram of PAHs content of a water samples (a) standard sample,
(b) additive sample, (c) treated sample by B & Fun after two week, (d) treated sample by
Bactera & Fungi after four week from the aquaria.

Fig. 5 GC/FID chromatogram of PAHs content of a water samples (a) standard sample,
(b) additive sample, (c) treated sample by B & Fun after two week, (d) treated sample by
Bacteria & Fungi after four week from the aquaria.

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Discussion hydrocarbons (PAHs) were investigated in
The effect of oil contamination and its water, sediment and fish of Timsah Lake
removal from the environment considered (Suez Canal, Egypt) using high
one of strategies for environmental performance liquid chromatography
restoration of oil polluted sites. The oil (HPLC) have reported that the
involves hydrocarbons that naturally concentration ranged from 52.46–
occurring organic compounds and the 3393μg/L, 585.9-8592.8μg/L for water and
ability to utilize hydrocarbons are widely sediment, respectively. 43 are also reported
distributed among diverse microorganisms. the total petroleum hydrocarbon (TPH)
several researchers documented lists content in water of Lake Timsah samples
containing bacteria and fungi that can reaching 103mg/l, in sediment samples
remove a wide range of contaminants36,37. reaching 635mg/kg. In This study amounts
moreover, several authors illustrated that of pollutants are somewhat alarmingly
the fungi play an significant role in the high; the total PAHs content in sediment
bioremediation products of oil and the most sample reaching 261.93 µg/L (Fig.1 and
of these fungi attributes to the following Table.6) Whereas total PAHs content in
genera: Candida, Cephalosporium, water samples reaching 17.34 µg/L Fig.2
Cladosporium, Alternaria, Aspergillus, and Table.6 PAHs are contaminants of
Fusarium, Geotrichum, Gliocladium, marine coastal sediments because of their
Paecilomyces, Penicillium, Pleurotus, hydrophobic character (water solubility
Polyporus, Rhizopus, Torulopsis, Mucor, between 10-10 and 10-13 mol/l) they are
Rhodotolura, Saccharomyces, and easily sorbed onto suspended
38-42 44,45
Talaromyces . This work has shown the particulate . In this form, they are more
occurrence of pure strains of various persistent to biodegradation in comparison
bacteria and fungi Tables (4, 5) from to dissolved PAHs45,46. This explains why
contaminated sediment and lake water. their concentration in sediments could be
Surface water and sediment samples were higher than that in the overlaying water
collected from contaminated sites allover samples. 47 reported that the P. aeruginosa
Lake Timsah (Ismailia, Egypt). The (in respect to M. cephalus), (F&B) let to
contaminating hydrocarbons were 6.7and 10.7 %, decrease percentage in the
extracted from each sample to be analyzed daily length gain and the daily weight gain
qualitatively and quantitatively using respectively, compared to the control.
standard methods. Results obtained Furthermore, the biological treatment using
indicate that generally all sites are microbes which carried out in the aquaria
contaminated with PAHs which were (O&F&B) also led to a reduction in the
affected by weathering to different degrees daily weight gain and a reduction in the
as detected by gas chromatographic daily length gain compared to the control.
(GC/FID)23. who reported that the levels of In this study, the survival percentage and
PAHs residues detected in the sediment growth performance of M. cephalus and T.
samples from Lake Timsah a variety of 15 zilliiwere estimated in the examined oily
PAHs were detected in all sampling sites, contaminated lake-water after 30 and 45
with total concentrations ranging from 54.6 days of the rearing process are presented in
mg kg-1 to 27,784 mg kg-1. In the study (Tables 7, 8). The obtained data in
of20, for the nature, origin and distribution (Table.7) showed in general that the
of the listed reference US Environmental bacterial treatment led to improve on the
Protection Agency (EPA) priority survival percentage and the growth
pollutants; 16 polycyclic aromatic performance of the M. cephalus compared
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to the untreated fish (control), while the 0.08g/L and it was obtained mainly after
fungal treatment showed slight impacts on the 4th day of treatment.
both the survival percentage and the
growth performance of M. cephalus The data presented in Fig.4 (a, b) showed
compared to the control. Moreover, the mix that with 1 ml oil in these tested aquaria the
of bacterial fungal treatment showed slight maximum bacterial dry weight in M.
impacts on survival percentage and cephalus was increased to 0.05g/L and it
improve on the growth performance of the was obtained mainly after the 7th and 13th
M. cephalus compared to the untreated fish day of treatment. The impact of the
(control). microbial treatments on the total bacterial
count of the muscle, skin and the internal
Moreover, it was observed that after 45 organs of the tested M. cephalusand and T.
days of the rearing period the addition of zilliiwere presented in Table 6.8 & 6.9
1ml motor oil in the tested aquaria led to respectively. And the impact of the
moderate effects on the growth rates of M. microbial treatments on the total bacterial
cephalus on fungal treatment. But the count of the muscle, skin and the internal
bacterial and fungal treatment have organs of the tested M. cephalus was
improved the daily weight gain and the observed that the most affected fish part
daily length day compared to the control. 47 was the skin followed by the internal
who recorded that the microbial treatment organs.
using P. aeruginosa is more effective for Table 9 Showed Mugil was observed that
the remediation of the crude oil the most affected fish part was the muscle
contaminated seawater and also for followed by the liver. The lowest bacterial
keeping the growth performance of the count was estimated in the fish skin. The
tested fish as similar as the untreated fries. microbial treatment led to decrease the fish
The obtained data in (Table8.) showed that susceptibility towards the bacterial
the bacterial and combination of bacteria accumulation compared to the control. It
and fungi treatments led to slight improve showed a high significant difference at P <
on the growth performance and good 0.05 in accumulating the bacterial counts in
growth in survival percentage of T. zillii these examined fish the mean bacterial
compared to the untreated fish (control). count was 2.57 × 102 cfu/100 ml and 1.64
But the fungal treatments led to mild × 102 cfu/100 ml in bacterial and bacterial
impact on the growth rate and no effect in &fungal treatment respectively compared
survival percentage of T. zillii compared to to that of the control (20 × 102 cfu/100 ml).
the untreated fish (control). Moreover, it
was observed that after 45 days of the Table 10 Showed T. zilliiwas observed that
rearing period the addition of 1ml motor oil the most affected fish part was the liver
in the tested aquaria, O&F&B led to slight followed by the muscle. The lowest
effects on the survival of T. zillii but it not bacterial count was estimated in the fish
affected on the growth rates. In contrast, skin. The microbial treatment led to
the addition of 1ml motor oil in the decrease the fish susceptibility towards the
untreated fish showed mild to serius effects bacterial accumulation compared to the
in both M. cephalus and T. zillii. The data control. It showed a high significant
presented in Fig.3 (a, b) showed that with difference at P < 0.05 in accumulating the
1ml oil in these tested aquaria the bacterial counts in these examined fish the
maximum fungal dry weight in M. mean bacterial count was 2.18 × 102
cephalus and T. zillii was increased to cfu/100 ml in bacterial treatment compared

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to that of the control (23.29 × 102 cfu/100 that the use of both bacteria and fungi in
ml). However, it showed insignificant the bioremediation of oil was more
difference at P < 0.05 in accumulating the efficient compared to using bacteria in the
bacterial counts in these examined fish the treatment aquaria (Fig.5). Fungi also
mean bacterial count was 27.15 × 102 showed more efficient for bioremediation
cfu/100 ml in bacterial treatment compared of PAHs than bacteria.
to that of the control (23.29 × 102 cfu/100 Table 11 infers that when selected isolates
ml). The lowest bacterial count was of bacteria and fungi were used for
estimated in the fish muscle. bioremediation of motor oil through
rearing T. zillii, 71% of 1-
Biodegradation is one of the major means methylnaphalene, 100% of Acenaphthalene
by which hydrocarbon pollutants can be and 77% of Fluoranthene fractions of PAH
removed from the environment48. A wide were removed after 2 weeks of the
range of organisms are involved in this beginning of the experiment while no
process, often acting as consortia. The residues were detected by GC/FID after 4
polycyclic aromatic hydrocarbons, weeks (Fig.5). This result also presented in
especially the high molecular weight Table 12 showed that the fungi removed
PAHs, are regulated contaminants at sites 56.77% of total PAHs after 2 weeks of the
polluted with crude oil49. It is often beginning of the experiment while no
difficult to find organisms that will residues were detected after 4 weeks. For
individually degrade all the fractions of M. cephalus results from the polycyclic
crude oil (aliphatics, alicyclic, and aromatic hydrocarbon analyses are
aromatic). The four organisms isolated of summarized in Table 13 which showed
bacteria (pseudomonas sp., bacillus sp., Lower bioremediation rate 49.84% after 2
Clostricium sp and Achromobacter sp) and weeks of the beginning of the experiment
isolated of fungi (Aspergillus sydawii, but 40.594 µg/L residues were detected
Mucor sp., Pencillium sp., Fusarium sp., after 4 weeks when bacteria were worked
and Absidia corymbifera) in this study together in the aquaria of M. cephalus. In
which were identified as species of bacteria another study, strains were isolated from
and fungi possess the ability of grow on petroleum polluted soil and identified as
aromatic fraction of petroleum. These are Pseudomonas pseudoalcaligenes, Bacillus
of great interest because previous findings firmus, Bacillus alvei, Penicillium
have demonstrated broad substrate spectra funiculosum, Aspergillus sydowii and
of the genus not only on hydrocarbons but Rhizopus sp., and they removed 79%, 80%,
also on diverse range of xenobiotic 68%, 86%, 81% and 67% of TPH
compounds50,51. 52 who recorded that respectively54.
Aspergillus and Penicillium species were
55
the most efficient metabolizers of reported that the Genus
hydrocarbons. In addition to degrading Stenotrophomonas, Bacillus, Brevibacillus,
hydrocarbons directly, fungal mycelia can Nocardiodes and Pseudomonas were used
penetrate oil, thereby increasing the surface in combination and give a degradation rate
area available for biodegradation. 53 of 67% after only 12 days of treatment. On
reported that fungi can grow under the other hand the bioremediation rates
environmentally stressed conditions such were relatively lower when selected
as low pH and poor nutrient status, where isolates of bacteria were used alone.
bacteria growth might be limited. In the Histopathological changes in liver and gills
present study. GC/FID analysis showed were reported in published paper56.

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CONCLUSION 6. Tao, S., et al. (2004). Sci. Total
Environ. 320:11.
The use in the treatment of water polluted 7. Stegeman, J. J. and J. J. Lech (1991).
with oil fractions of the bacterial or fungal "Cyrochrome P-450 onooxygenase
strains isolated from Lake Timsah has led system in aquatic species: Carcinogen
to the bioremediation of the polycyclic metabolism and biomarkers for
aromatic hydrocarbons (PAHs) as carcinogen and pollutant exposure."
important fraction of motor oil and Environmental Health Perspectives 90:
Improvement of the condition of the fish 101-109.
tissues with decrease of microbial 8. Baird, C. (1995). "Environmental
accumulation. In addition, bacteria and Chemistry." W.H. Freeman and
fungi were more effective to be used Company New York: 276-278 pp.
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therefore, that the use of such an embedded aromatic hydrocarbons environmental
microbial scheme (fungi & bacteria) for the pollution and bioremediation." Trends
bioremediation of crude oil in marine in Biotechnology 20: 243-248.
contaminated fields could be helpful. 10. Ali, H., et al. (2006). "Assessment of
Polycyclic Aromatic Hydrocarbons
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