Soil Microcosms for Bioaugmentation With Fungal Isolates to Remediate Petroleum Hydrocarbon-contaminated Soil

The aim of this work was to isolate indigenous PAH degrading-fungi from petroleum contaminated soil and exogenous ligninolytic strains from decaying-wood, with the ability to secrete diverse enzyme activity. A total of ten ligninolytic fungal isolates and two native strains, has been successfully isolated, screened and identied. The phylogenetic analysis revealed that the indigenous fungi (KBR1 and KB8) belong to the genus Aspergillus niger and tubingensis. While the ligninolytic exogenous PAH-degrading strains namely KBR1-1, KB4, KB2 and LB3 were aliated to different genera like Syncephalastrum sp, Paecilomyces formosus, Fusarium chlamydosporum, and Coniochaeta sp., respectively. Basis on the taxonomic analysis, enzymatic activities and the hydrocarbons removal rates, single fungal culture employing the strain LB3, KB4, KBR1 and the mixed culture (LB3+KB4) were selected to be used in soil microcosms treatments. The Total petroleum hydrocarbons (TPH), fungal growth rates, BOD 5 /COD ratios and GC-MS analysis, were determined in all soil microcosmos treatments (SMT) and compared with those of the control (SMU). After 60 days of culture incubation, the highest rate of TPH degradation was recorded in SMT[KB4] by approximately 92±2.35% followed by SMT[KBR1] then SMT[LB3+KB4] with 86.66±1.83% and 85.14±2.21%, respectively.


Introduction
Over the last two decades, accelerated industrialization, and the massive use of aromatic compounds in explosives, dyestuffs, pesticides and pharmaceuticals has resulted in serious environmental contamination of soil, water, and air.
Oil spillage is a serious threat to all compartments of the ecosystem 1 . During extraction, transportation, storage and distribution operations, crude oil and its re ned products are frequently exposed to accidental spillage causing soil pollution 2 .
Soils contaminated with persistent organic pollutants (POPs) associated with petroleum such as PAHs, have high potential health risk because its ability to enter food chain and its a nity for accumulation in living organisms 3. Soil matrix properties and functions are closely related to the different activities occurring on land and xenobiotic structures like PAHs-associated with petroleum. Owing to the chemical stability of PAHs, their hydrophobicity and recalcitrance to microbial degradation, spilled oil may damage the biological and physico-chemical properties of the petroleum-polluted soil.
Petroleum hydrocarbons cause alteration of soil biological properties, affecting the microbial diversity and the enzymatic activities as well as the physico-chemical characteristics 4,5 . Certain essential soil function may be lost due to the high toxicity of such persistent aromatic hydrocarbon structures 6 . Indeed, spilled oil may develop anaerobic conditions and asphyxia in soil pore with consequent impacts on the microbial activities 7 . In this regard, Klamerus-Iwan et al. 8 demonstrated a signi cative decline of the Therefore, bioaugmentation approaches are necessary to enhance the performance of indigenous microbial population several fold through the introduction of microbes with speci c metabolic activities for an effective in-situ remediation of polluted areas 19 .
Typically, fungi are suited for bioremediation of crude oil in polluted sites owing of their diverse metabolic activities. They are able to secrete a board range of ligninolytic and non-ligninolytic enzymes to use petroleum hydrocarbons as a carbon and energy source and assimilate into fungal biomass 20 . Moreover, the e ciency of fungal culture to remove or degrade PAHs from petroleum contaminated soil is related to various factors, among them pollutant bioavailability, survival of microorganism and their metabolic diversity are essential for bioaugmentation 21 .
Previously, it was well demonstrated that Soil microcosms (SM) serve as test systems that may be adapted to various environmental conditions. Indeed, outcomes of microcosm studies are often used to develop remedial pilot process specifications 22 .
The current study highlights the application of newly fungal isolates (indigenous from the investigated soil and exogenous from decay wood) in the PAHs-contaminated soil remediation processes. Individual and mixed fungal cultures are selected based on their taxonomy and metabolic diversity, to enhance the bioremediation performance in soil microcosmos systems.
Soil Sampling, physico-chemical characteristics, and microbial population PAHs contaminated-soil samples were collected from spots around the oil well (7) located in Dammam city (Saudi Arabia). All samples of soils were taken at depth 5-10 cm from upper surface of topsoil. Samples were transferred to laboratory in nylon sterilized sac closed tightly and marked with relevant information (number, location speci c characteristics and date). Before utilization in the treatment study, all soil samples were mixed and sieved to remove particulars greater than 1.25 cm and saved under 4 °C.
The physicochemical analysis was performed by the Arabia Life Sciences Division-Environmental Saudi Arabia (ALS) which is diversi ed testing services organization. The pH, moisture content, biological oxygen demand (BOD), total petroleum hydrocarbon (TPH) semivolatile and volatile organic compounds-BTEX (benzene, toluene, ethylbenzene and xylene) of the petroleum-contaminated soil before and after fungal treatments were determined (Table 3).
For the estimation of the total Petroleum hydrocarbon (TPH) (semi-volatile), the USEPA 8015B method gas chromatography/ ame ionization detection (GC/FID) was conducted. Sample extracts were analyzed by Capillary GC/FID and quanti ed against alkane standards over the range C10 -C40.
TPH Volatiles / BTEX were determined using the method EPA 8260 Purge and Trap-gas chromatographymass spectrometry (GC/MS). Extracts are analyzed by Purge and Trap. Capillary GC/MS. Methanol Extraction of Soils for Purge and Trap was performed by the method (USEPA SW 846 -5030A) The soil pH was determined by digital pH meter in a soil water suspension (1:2.5) as described by Jackson 48 The moisture contents of soil were determined gravimetrically, based on weight loss over 12 hours drying period at 103-105 °C. This method is compliant with NEPM (2013).
Enumeration of bacteria from contaminated soil was performed by using serial dilution and plating technique. The microbial populations were counted in terms of Colony forming units (CFUs) 49 .

Isolation of decay-wood decomposing fungi and indigenous soil fungi
Decay-wood samples were collected in sterilized and labeled plastic bag from different biotopes of the region of Barzah and Rahat from Khulais-Jeddah city, during March 2020. Malt Extract Agar (MEA) (30.0 g/L, pH 5.5.) supplemented with antibiotics (0.01% of ampicillin and streptomycin) was used as selective media for the isolation of fungal strains. The isolation of decay-wood decomposing fungi was carried out by direct plat method as suggested by Daâssi et al. 50 . The purity of the fungal strain was proofed by microscopic observation.
Both soil-plates and soil-suspensions methods were used for the isolation of soil fungi 23 . Plates were prepared by transferring 0.05 to 0.015 g of the contaminated-soil to be examined into a sterilized Petri dish. Cooled medium of MEA was added and the soil particles dispersed throughout the agar by gentle shaking of plates before the agar solidi es. Soil-suspension solution was prepared from 10 g of the dried contaminated soil dissolved in 100 mL of sterile physiological water (NaCl 9 g/L) and maintained for 20 min under agitation on a reciprocating shaker at 120 rpm. After shaken, serial dilutions and plating technique were performed according to Agrawal et al. 51 . All plates were incubated at 30 °C during 5 to 7 days. Fungal colony was sub-cultured on fresh MEA supplemented with 0.01% of ampicillin and streptomycin until getting a pure strain. Preliminary identi cation of the fungal isolates was performed through macroscopic and microscopic observation.
Selection of Hydrocarbon-degrading fungal isolates Preliminary screening of oil-degrading fungal isolates (both ligninolytic and native fungal isolates) was performed by agar well diffusion method. As sole source of carbon and energy, 5% of Diesel fuel (Saudi Aramco, de ned according to Daâssi et al. 52 has been spread on the surface of the MEA plates with glass rod. Fungal strains suspensions (in sterile water) was prepared and downloaded in MEA plate wells. The culture plates were incubated at 30 °C, and the appearance of substantial growth was daily monitored during 5 days. Also, the culture plates with and without addition of Diesel fuel were examined for growth.
Then the selected fungal strains on the agar well diffusion method, were checked for its ability to grow and mineralize the petroleum hydrocarbons in the contaminated soil. Cultures were carried out on Mineral (NH 4 ) 6 Mo 7 O 24 ⋅4H2O, 0.01 g. Before autoclaving, the pH of the solution was adjusted. 5 g of the soil sample was mixed with 20 mL of MM and inoculated with 1% of spore suspension of each isolate. Then the 250-mL Erlenmeyer asks were incubated in stationary-phase for 14 days at 30 °C.
Plate-agar tests for the investigation of the enzymatic activities Plates containing selective media (supplemented with the suit enzyme substrate) were used as a qualitative test to detect the enzymatic activities in the fungal collection. Under aseptic conditions, mycelial fraction was taking from each pure strain, and placing on the surface of the selective agar media. After incubation at 30 ºC, fungal strains were recorded as positive or negative based on the appearance of degraded halo surrounding mycelium growth.

Laccase activity
To detect laccase-producing fungi, strains were grown on selective solid medium MEA supplemented with 150 µM copper sulfate (as Lac inducer) and 5 mM of 2,6-DMP/or 0.2 mM of ABTS (Lac substrates) then, incubated at 30 ºC. Fungal isolates which showed red-brown (with 2,6-DMP) and green halos with ABTS), were selected as Lac positive (Lac (+)) strains and transferred into 250 mL asks of MEB added with 150 µM CuSO4, as inducer, for further characterization.

Proteolytic activity
Sterile milk (250 mL L -1 (v/v)) was incorporated as fungal protease substrate in the nutrient agar medium (pH 5.5) containing 5 g/L of peptone and 3 g/L of yeast extract after sterilization and semi-cooling of the media. The presence of the degraded milk halo is evidence of the proteolytic activity 53 .

CMCase activity
Modi ed Mandels and Reese medium was used as screening medium to select CMCase positive (CMCase+) species 54 . Fungi were grown on the agar medium and incubated for 7 days at 30 °C. After fungal growth, CMC agar plates were stained with 1% (w/v) Congo-Red solution for 15 min and discolored with NaCl (1 M) for 15 min. CMCase activity is detected by the presence of a halo around the isolate 55 .

Lipase activity
For the detection of lipase activity, a selective medium of MEA amended by 1% olive oil and 0.001% rhodamine B was used. The fungal isolates were incubated at 30 °C during 7 days then revealed using 365 nm light. The positive strains showed uorescence under the UV-light 50 .

Amylase activity
For the α -amylase activity detection, starch agar plate method was performed. The fungal isolates were inoculated onto a starch plate and incubated at 30 °C until growth is seen. The petri dish is then ooded with an iodine solution to visualize the degraded halo. Amylase positive (Amyl+) species presents a clearing halo around the mycelial growth 56 .

Optical microscopy
Optical microscopy images of the suspended mycelium were taken after a seven-days old MEA-plate fungal cultures using an optical VHX-5000 digital microscope (Keyence).

Identi cation and phylogenetic tree of fungal isolates
The selected hydrocarbon-degrading fungal strains were cultivated in a 150 mL ask containing 50 mL of liquid Malt Extract Broth medium (MEB) for 5 days. Then mycelium was harvested by ltration and successive washings with sterile Milli-Q water. The genomic DNA was extracted from the fungal cells using a DNeasy Plant Mini Kit (QIAGEN). The purity and the quantity of DNA samples were estimated by the optical density ratio A260/A280. The molecular identi cation was carried out with the protocol suggested by Daâssi et al. 57 . The primers used for the ampli cation were ITS1 (5 -TCCGTAGGTGAACCTGCGG-3) and (3-TCCTCCGCTTATTGATATGC-5) 58 . Blastn analysis was used for the resulting sequences (www.ncbi.nlm.nih.gov/BlastN). The organisms were identi ed based on the subjected sequences in the databases showing the highest identity.
Multiple sequence alignment was achieved using ClustalW between the selected subjected sequences and the query ITS sequences of the isolated strains 59 .
Phylogenetic tree was inferred using the neighbor-joining method (NJ) 60 in the MEGA11 program with bootstrap values based on 1000 replicates 61 . Sequences have been deposited in GenBank.

Fungal inoculum preparation
Mycelial suspension from the selected hydrocarbon-degrading fungi exogenous ligninolytic (LB3; RB4) and indigenous (BKR1) was prepared as described by Potin et al. 62 . Seven-days old MEA-plate fungal cultures of the 3 selected isolates was washed with 5 mL of sterile physiological water to obtain the fungal suspension which further was ltrated through sterile glass wool to separate mycelia from spores.
The collected spore suspensions were estimated by Thoma cell counting chamber. 25 mL MEB amendment was added to each microcosm in order to induce spore germination in the microcosms. Spores were added to the medium in calculated volumes to give a nal total spore concentration of 10 4 spores g soil -1 .

Soil microcosms assays for bioaugmentation studies
Soil microcosms were used in this study for the mycoremediation of the PHC-contaminated soil. Each microcosm contained 50 g of 6 mm sieved soil mixed with 125 mL of MEB inside 400 mL glass bottles sealed with rubber caps and aluminum seals and incubated at room temperature for 60 days.
Control soil Microcosms (zero day/untreated/ without fungal inoculation) and test soil microcosm (treated) for each fungal culture (mono or co-culture) were set up during 60 days of treatment in order to evaluate the biotic vs abiotic degradation of the PHC contaminated soil. All the microcosm experiments were conducted in triplicate.
Soil microcosm treatments (SM) were designed: (SMU-sterile): control microcosms conducted by air-dried untreated contaminated soil (sterile soil) to assess abiotic losses of hydrocarbons.
(SMU-Not sterile): control microcosms formed by untreated not sterile soil to assess biotic degradation. All the microcosm treatments were inoculated with an initial concentration of 10 4 spores per gram of soil.
Soil samples from each microcosm were collected manually with clean and sterilized (ethanol 70%) stainless steel spatulas on days 15, 30 and 60 for the analytic analysis to assess hydrocarbons degradation in the soil microcosms.
Assessment of Petroleum-contaminated soil degradation by fungal isolates

Biomass estimation
The variation in the rate/biomass of fungal cultures (mono or co-cultures) was determined gravimetrically. The weight of ask was taken before and after incubation period.

Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD)
The rate of degradation and the e ciency of the fungal isolates in the treatment of the PAHscontaminated soil, were evaluated by the BOD and COD analysis performed by standard methods 63 (APHA, 2001 and IS-3025)

Total Petroleum Hydrocarbon (% TPH) and Gas chromatography-Mass Spectrometry (GC-MS) analysis
The extraction of the residual petroleum hydrocarbons from the contaminated soil was carried out by mechanical shaking as described by Siddique et al. 64 with some modi cations.
For each culture incubation period (15, 30 and 60 days), the remaining petroleum hydrocarbons (PHCs) was extracted from each soil microcosms (untreated/treated) using of 30 mL of Dichloromethane (DCM). 10g of soil sample were put in glass bottle added with anhydrous sodium sulphate (Na 2 SO 4 ) to remove moisture. The mixture was acidi ed with of concentrated HCl (12 N) to avoid further degradation and shaken on a reciprocating shaker at 120 rpm for 3 hours. Then, the he reactional mixture was separated by centrifugation (10 min, 8000, at 4 °C). The supernatant was transferred in separating funnel (250 mL) to remove the aqueous layer and sequentially extracted twice with 2 volumes of dichloromethane, respectively. Finally using a rotator evaporator, the dichloromethane was evaporated in 55 °C and the extracts were concentrated to near dryness then re-dissolved in 1 mL dichloromethane solution. The samples were kept at -20 C until being analyzed. The residual PHC concentration of the untreated and the treated by the mono or the co-fungal cultures was determined.
The percentage of Total Petroleum Hydrocarbon (TPH) by gravimetrically and results were expressed as percentages of respective controls 51 .
The treated/extracted petroleum hydrocarbons were analyzed by gravimetric analysis and gas chromatography (GC) by Agilent GC-MSD (6890N-5973) with pole temperature kept at 80 °C for 4 min, then increased at a rate of 5 °C·min -1 to 250 °C and maintained at 250 °C for 20 min.

Results And Discussion
Isolation of decay-wood decomposing fungi and soil native fungi Pieces of decaying wood samples used in this study were collected from different biotopes of western Saudi Arabia (Khulais-Jeddah). Barzah and Rahat are two natural habitats from the Khulais, selected for the decaying wood sampling during March 2020.
Primary identi cation of the ligninolytic fungal isolates was based on plate morphology while the purity of the isolates was proofed using microscopic parameters ( Fig. 1a-f).
From the decay wood samples, a total of ten pure wood-decomposing fungal isolates were obtained and proofed by microscopic observation.
Ten morphologically different fungal strains were isolated successfully, two abundant strains from the polluted soil and ten from the decay-wood, then were maintained as pure cultures in MEA.
Both soil-plate and soil-suspension methods were used for the isolation of soil native fungi 23 . Two fungal strains presented the highest abundance and growth ability in the soil sample were examined by the soil plate and soil suspension isolation method.
These native fungi designed by KBR8 and KBR1 were chosen and identi ed by morphological characters and taxonomical keys. Based on the morphological aspects and microscopic observation, the strains KBR1 and KBR8 demonstrated the general characteristics of the genus Aspergillus.

Selection of hydrocarbon-degrading fungal isolates
The indigenous fungal isolates and the ligninolytic fungi were tested for their ability to degrade petroleum hydrocarbons. Primary screening was conducted on culture plates basis on the agar well diffusion method (Fig. 2).
Out of the twelve isolated strains investigated in the culture plate experiment, six were recorded as petroleum hydrocarbon-degrading via a degraded halo surrounding mycelium growth. The diameter of the halo demonstrates the ability of fungus to utilize petroleum hydrocarbons.
In all petri dishes the highest growth diameter was around the mycelia of KBR1, KBR8 (native soil isolates) and KB4, LB3 (ligninolytic isolates) indicating good ability to degrade diesel hydrocarbons among the other isolated strains. However, KBR1-1 and KB2 showed a tight halo during fungal growth.
In addition, the mix culture consisting of LB3+KB4 showed the highest growth diameter.
Lot nasabasl et al. 24 in related study on fungal isolated strains from soil hydrocarbon-polluted samples indicated that isolated fungi can be used in hydrocarbon bioremediation processes however, their e ciency varied within the species and the metabolic diversity according to the fungi.
Later, a con rmatory assay for hydrocarbon degradation, potentials of the isolated fungi was conducted on Erlenmeyer asks with the investigated soil. The gravimetric determination of the residual hydrocarbon after biodegradation was performed by weighing the quantity of the petroleum hydrocarbons against the control.
The estimated crude oil degradation e ciency after 14 days demonstrated that the ligninolytic isolate KB4 showed maximum ability to utilize crude oil, giving the highest percent degradation of 29.32%, followed by the native isolates KBR1 and KBR8 indicating 22.68 and 20.34% degradation, respectively (Fig. 3).
These results are in line with those recorded by Lot nasabasl et al. 24 who reported a highest remediation rate belongs to Aspergillus niger (20.55%) and the lowest rate belongs to Penicillium sp (16.453%).
Additionally, the mycelial growth and the biomass gain pro les indicated that all the selected strains previously tested on the culture plate were able to grow and use the petroleum hydrocarbons as carbon source. Figure 3 shows an increase in rates of fungal growth in the media containing petroleum contaminated soil compared with inoculated media (MM) with not polluted soil. This result demonstrates the ability of the fungal strains to assimilate petroleum hydrocarbons molecules using diverse extra cellular enzymes for their growth.
In this regard, Oboh et al. 25 reported the ability of Aspergillus sp., Penicillum, Rhizopus and Rhodotorula species to grow on crude petroleum as the sole source of carbon and energy.
Fungal culture on petroleum polluted soil proofed the potent of the selected fungi in the degradation of TPH from the contaminated soil and were thus selected for further study.
The degradation ability of different genera from different habitats makes their catabolic potential even more versatile to transform persistent organic compounds into inert and non-toxic molecules 26 .
Among the Twelve isolated fungi, the most interesting fungal strains in term of crude oil degradation (LB3, KBR1, KBR8, KB2, KBR1-1) were cultivated and run for molecular identi cation based on the analysis of the ampli ed nucleotide sequences of the nuclear ribosomal ITS1-5.8-ITS4 region.

Molecular identi cation of the selected petroleum hydrocarbon-degrading fungi
The molecular identi cation of the strains was performed by BLAST alignment tool of the National Center for Biotechnology Information (NCBI) database. Closely related sequences were obtained from the GenBank database with similarity greater than 95% (Table 1).
The ITS regions of the fungal strains were sequenced at Macrogen (Republic of Korea) and submitted to GenBank database with accession numbers; MZ817958, MW699896, MW699897, MZ817959, MW699895 and MW699893.
Based on the percentage of similarity (Table 1)  Based on the multiple alignment of the ITS sequences provided by Clustal Omega program, the phylogenic analysis was run to nd the evolutionary relationships of the newly fungal isolated to previously characterized species (Fig. 4).
A phylogenetic tree was created by Neighbor Joining Method (NJ) based on alignment of the ITS sequences of exogenous fungal strains (KBR1-1, KB4, KB2 and LB3) and indigenous strains (KBR1 and KB8) with their homologue sequences obtained from NCBI database.
Analysis of 18S rRNA genes of the genus Coniochaeta revealed that the taxon appears as a monophyletic group related to teleomorphs of the genus Lecythophora.
According to Lopez et al. 28 Lecythophora (Coniochaeta) is a lamentous ascomycetous which belongs to the Coniochaetaceae family and Sordariales order.
The strain KB4 (Accession no. MW699897) showed 98.39% ITS identi es with Paecilomyces formosus, Thermoascaceae sp. and Penicillium sp. and closed to the genus Byssochlamys spectabilis (98.12%). The morphological traits of the fungus were determined to a liate the isolate to the genus Paecilomyces formosus (yellow septate hyphae, conidia unicellular).
The genus Paecilomyces was rst described by Bainier 29 as a genus closely related to Penicillium and comprising only one species, P. variotii Bainier.
Accordingly, previous study of Moreno-Gavíra et al. 30 reported that the genus Paecilomyces has yellowish septate hyphae, with irregularly branched conidiophores and smooth walls. The conidia are unicellular; in chains; and the youngest conidium is at the basal end.
The indigenous isolates (KBR8 and KBR1) were a liated to the genus Aspergillus based on BLAST analysis of the ITS sequences. In addition, the phylogenetic analysis showed that the two indigenous isolates clustered in a clade comprising exclusively Aspergillus species, with high bootstrap values for each branch. ITS sequences of the fungal isolates were deposited at GenBank under accession numbers MW699895 and MW699896 for KBR8 and KBR1 respectively.
It can be inferred from the phylogenetic tree that the strain closest to isolate KBR1-1 is the species Syncephalastrum racemosum. The related sequence, corresponding to strain KBR1-1 was deposited under the accession no. MZ817958.
In the present work, Aspergillus niger (KBR1), Lecythophora (Coniochaeta) (LB3), Paecilomyces formosus (KB4), Syncephalastrum racemosum (KBR1-1), Aspergillus tubingensis (KBR8), and Fusarium chlamydosporum (KB2) were the perfect fungal isolates demonstrated e ciency to biodegrade petroleum hydrocarbons. Our results agree with results of Gesinde et al. 31 who reported that Aspergillus niger have very active degradation capability of Nigerian and Arabian Crude Oils. Furthermore, in the same study, the genera, Aspergillus, Penicillium and Fusarium species were demonstrated as the most e cient metabolizers of hydrocarbons.in comparison with other isolates.

Screening of the enzyme activity for the newly isolates
The capacity of the fungal isolates to produce several enzymatic activities such as lipases, proteases, amylases, cellulases, and laccases enzymes was investigated on selective solid media.
Out of the twelve tested isolated strains, seven strains were recorded to secrete laccases, 10 strains were cellulases positive, 4 strains were amylases positive, 21 strains were found to produce proteases and 4 strains were able to produce lipases ( Table 2).
Hence, according to Lopez et al. 28 the ascomycete Coniochaeta ligniaria NRRL was able to produce lignocellulose-degrading enzymes including cellulase, xylanase and two lignin peroxidases (manganese peroxidase, MnP and lignin peroxidase, LiP), but no laccase activity was recorded.
Due to the complexity of the lignin-cellulosic materials, ligninolytic fungi are involved in the cycling of nutrients versatile metabolic activities such as (hydrolases, oxidoreductases and esterases) for the degradation of the complex organic molecules into simplest 32,33 .
Recent researches reported the role of lipase activity in the petroleum hydrocarbons degradation.
Similarly, Ramdass and Rampersad 34 demonstrated the presence of lipase activity in ve newly isolates from crude oil polluted soil.
Our results of the enzyme activity screening demonstrate a high metabolic diversity of the isolated fungal strains makes their catabolic potential in the PAHs remediation processes. Basis on the taxonomic analysis and the metabolic diversity, the isolates KBR1, LB3, KB4 and the mixed culture of LB3+LB4 were selected to be used for the PAH-contaminated soil remediation in microcosm systems.

Microcosms for petroleum-contaminated soil remediation
The petroleum-contaminated soil samples were collected from the oil well (7) located in Dammam city (Saudi Arabia), and maintained in plastic containers.
The soil samples were collected from the surface layer (5-10 cm) at different spots around petroleum pipe line spillage. The soil samples were mixed together forming a composite sample which will used to represent areas of contamination in this study. The composite sample was sieved with a 6 mm grid before soil microcosm treatments and 2 mm grid for soil characterization.
The primary physical properties of the soil were the dark color, the pH of 6.8, and the average moisture content of 1.6 ± 0.2%. (Table 3).
Similar high content of TPH was shown in the study of Torres et al. 35  Soil microcosms batch were conducted in several treatment systems (Table 4), to assess the potential of single or mixed fungal strains in the remediation of petroleum-polluted soil. The investigated soil was not sterilized to preserve its microbial indigenous ora as well as its physico-chemical properties 37 . Indeed, the indigenous soil ora constitute an important heterogenous microbial population for the enhancement of the biodegradation process Soil microcosms were treated with selected fungal strain shown ability to degrade petroleum hydrocarbons and with broad enzymatic capacities.

Pro le of the TPH in soil microcosms
To study the petroleum hydrocarbon removal ability of the fungal strains, the TPH in each SM system was followed at different periods during the treatment (Fig. 5). The kinetic of TPH during the soil microcosms treatments (Fig.5) demonstrated a rapid decrease of the hydrocarbons in the SMT[LB3+KB4] compared with others SM inoculated by single strain. This result highlights the essential role of the co-occurrence of different microbial species to enhance the biodegradation yields 40,41 . While, previous ndings obtained by Okerentugba and Ezeronye 42 , demonstrated that single fungal culture found to be better than mixed cultures.
Allover results highlight the improvement of the biodegradation yields by bio-augmenting soil microcosms by indigenous or exogenous fungi.

BOD5 and COD in soil microcosms
For the evaluation and the monitoring of the degradation process, BOD and COD were estimated at different period of the soil microcosm treatments. The results of organic removal are shown in Fig. 7.
BOD 5 and COD of the control microcosm (SMU) were 57 and 145 mg/L, respectively as seen in Table 3.
The average BOD 5 and COD percent removal e ciency in this study was approximately 86.5% and 57.8%, respectively.
The BOD 5 /COD ratio can give indication on the biodegradability of the petroleum contaminated soil.
Polluted samples may be biodegradable when the BOD 5 /COD ratio value was between 0.4 and 0.8 43 .
The BOD 5 /COD ratio within the same time interval was found as 0.28.
The ratio of BOD 5 (Figure 7), the samples can still not be considered as highly biodegradable (BOD 5 /COD > 0.4).

GC-MS analysis for soil microcosms
The remained petroleum hydrocarbons (PHCs) were extracted and characterized by GC-MS for each soil microcosm (SM) treatment system after 60 days of cultures incubation. Fig. 8 illustrated the superposed pro les of PHCs in the degrading soil microcosm by GC-MS analysis.
PHCs remained in the soil microcosms, showed decrease in the area of major peaks compared to control SMU, suggesting degradation of the main compounds; while the appearance of new peaks in these samples indicated the breakdown of products or presumed metabolites. As seen in the Fig. 9, chromatograms revealed a signi cant reduction in the intensity of PHC peaks after SM treatment by fungal bioaugmentation (Fig. 9b-d) compared with the control (Fig. 9a).
GC-MS analysis performed after biodegradation showed that the biodegradation patterns of petroleum hydrocarbon fractions in SM treated by single strain and SM treated by mixed species, were markedly different throughout time, compared to the control microcosm.
GC-Ms pro les demonstrated the e ciency of the newly isolates fungal strains to remediate petroleum contaminated soil in the microcosm system.
Further quantitative and qualitative identi cation of the main compounds in the extracted PHCs were conducted at different SM treatment systems ( Table 5). The control system (SMU) (Not sterile soil) represented the biotic effect of the native microbial comities in the contaminated soil, was incubated in same experimental conditions as the treated SM.

Conclusion
The Present study revealed that, the fungal ligninolytic isolates and the indigenous fungi collected from petroleum contaminated soil samples holds promise for the effective PAHs-bioremediation. Further, to enhance the biodegradation e ciency, bio-stimulation, the properties of biosurfactant and enzymes and mechanism of degradation is necessary.     Phylogenetic tree for the hydrocarbon-degrading isolated fungi (exo and indigenous isolates) and related sequences based on the BLAST alignment of ITS sequences. The ClustalW program was used to generate the phylogenetic trees using the NJ method with bootstrap replicates.
Page 34/38  Degradation e ciency (%) in soil microcosm for each treatment against control microcosm.  Supplementary Files