Crude oil and pyrene degradation by halotolerant fungi Embellisia sp. KJ59 and Alternaria sp. KJ66 isolated from saline soils

ABSTRACT In recent years, investigating halophilic and halotolerant fungi enzymes has confirmed their significant potential in the biodegradation of crude oil. The aim of the current study was to evaluate the biodegradation of crude oil by halotolerant fungal Embellisia sp. KJ59 and Alternaria sp. KJ66. The fungal strains were isolated on Czapek Dox Agar (CDA), potato dextrose agar (PDA), and malt extract agar (MEA) containing 10% NaCl. Seven strains that could grow in salinity, ranging from 0 to 17% were selected. Out of the seven isolates, isolates KJ59 and KJ66 were selected as superior strains because they were more efficient in the biodegradation of crude oil on Minimal Salt Medium containing 1% crude oil and different salinity. The isolates were identified as Embellisia sp. and Alternaria sp. based on its nucleotide sequence of internal transcribed spacer gene. They could biodegrade petroleum, ranging from 81.3 to 95.1% in NaCl concentrations of 0, 2.5, and 5% on PDB over a period of 14 days. Pyrene removal by these strains was over 88.3% in saline and non-saline MSM containing 100 mg L−1 pyrene. Finally, Embellisia sp. KJ59 and Alternaria KJ66 were introduced fungal strains for crude oil biodegradation as the sole or supplementary carbon source in the presence of different salt concentrations. Furthermore, they are potential fungal strains for bioremediation of crude oil in non-saline and contaminated saline environments.


Introduction
9Nowadays, crude oil has an essential role in our daily life due to the fact that it is the primary source of energy and it is used as a primary material in some important industries.Therefore, petroleum and its derivatives are likely to cause pollution during production, transportation, and consumption [1].Every year, billions of gallons, including a significant amount of oily wastewater, are produced during production, transportation, and refining, which are crucial factors of the environment and soil pollution [2].Most petroleum hydrocarbons are carcinogenic and cause liver failure in humans, and they are also toxic to animals [3].Pyrene is one of the toxic low molecular weight compounds of crude oil, which belongs to polycyclic aromatic hydrocarbons (PAHs).The studies show that pyrene is the most carcinogenic substance of PAHs, and long-term exposure to pyrene results in cancer [4].Some fungi such as Sarocladium sp. and Cryptococcus sp. were suggested that are able to degrade pyrene in non-saline environments [5].
Different ranges of salinity in polluted areas with oil hydrocarbons contribute to enzyme denaturation and plasma membrane fragmentation because of high osmosis pressure.Consequently, it is difficult to use different methods of bioremediation in saline and hypersaline regions [6,7].Moreover, increasing salinity diminishes the quantity and variety of substrates that are degraded by microorganisms [8].
Halophilic and halotolerant bacteria and fungi are the most effective microorganisms for the biodegradation of spilled oil in saline environments [9].Some fungi are more successful than bacteria in the bioremediation of crude oil [1,10].The presence and activity of fungal strains in natural saline and hypersaline regions were demonstrated more than 80 years later from its primary research on the presence of bacteria in these environments [11].Halophilic fungi are defined as fungi that are frequently isolated from universal hypersaline regions at the salinity of over 1.7 M and can grow in vitro at the salinity of 3 M; whereas, halotolerant fungi can occasionally be isolated from hypersaline environments and can grow in vitro at 3 M NaCl concentration [11].
The previous study illustrates that halophilic and halotolerant fungi can biodegrade crude oil hydrocarbons [9,12].For instance, Phanerochaete chrysosporiumis known as a fungus which able to remove spilled oil in salinity between 0 and 4% [9].Furthermore, Fusarium lateritium, Drechslera sp.andPapulaspora sp. are reported as fungi that are able to degrade crude oil in saline media [8].Despite the reports on fungal and bacterial strains for PHA biodegradation in saline conditions such as Aspergillus sydowii, Mucor sp.F2, Rhodotorula mucilaginosa, Mycobacterium sp.B2, Pseudomonas sp.LZ-Q, and Hortae sp.B15, introducing new species of pyrene degrading microorganisms seems to be necessary [13][14][15][16].Since some crude oil degrading microbial strains may be able to remove only the aliphatic components, evaluating the capacity of aromatic portion removal in the isolated and identified strains is an important issue.The current study was aimed to evaluate the potential of isolated fungal strains, Embellisia sp.KJ59 and Alternaria sp.KJ66, in crude oil and pyrene removal as a sole or supplementary source of carbon in different NaCl concentrations.

Halotolerance test
Salinity tolerance for isolated fungi strains was tested on PDA with 0, 10, 17 and 20% w/v of NaCl.pH was adjusted to 7. Plates were incubated for 7 days at 28°C.Colony formation and diameter were evaluated for each strain [21].

Crude oil biodegradation assay in the presence of NaCl
Biodegradation of petroleum hydrocarbons as a sole source of carbon was tested for a screening of degrading fungi strains in various NaCl concentrations.Minimal salt medium [22] with 0, 2.5 and 5% w/v NaCl was provided.Moreover, 1% v/v crude oil was added.A volume of 10 ml of the culture media was put in 50 ml Erlenmeyer flasks, and each flask was inoculated by 5 × 10 6 of the isolate spores except blank samples.pH was adjusted to 7. Furthermore, blank samples without inoculation were provided.The test was carried out in triplicate for each salinity.The inoculated flasks were incubated in shaker flasks for 14 days at 28°C and 170 rpm [9].Crude oil removal was assayed by measurement of total petroleum hydrocarbons [23] with the spectrophotometer.Toluene was used as a solvent, and dilution of 10 −1 was provided from residual petroleum and toluene.It was assayed with the spectrophotometer at a wavelength of 420 nm [5].

Morphological and molecular identification
According to salinity tolerance and crude oil degradation, the superior strains were identified morphologically according to form and colour of colonies on PDA with NaCl concentration of 5%, form, and arrangement of spores [24].Microscopic characteristics were studied by slide culture technique.Spores and mycelia were stained with lactophenol cotton blue and examined with an optical microscope [25].Furthermore, superior strains were identified with molecular approaches.Fungal cell lysis was carried out by adding liquid nitrogen and pounding frozen cells.DNA extraction was performed by the phenol-chloroform approach.Genomic DNA extraction was followed by PCR amplification with ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) primers [26].After an initial denaturation of DNA at 96°C for 5 min, 30 cycles of amplification were performed.Each cycle consisted of denaturation at 92°C for 20 s, annealing at 58°C for 45 s, extension at 72°C for 1 min and a final extension at 72°C for 5 min.PCR products were sent to Macrogen Corporation in South Korea for sequencing.Sequences similarities were obtained from NCBI database.Mega6 was used for drawing phylogenetic trees with bootstrap replications of 1000 in a neighbour-joining algorithm.The isolates were deposited in the University of Tehran Microorganisms Collection, and their sequences were registered in the GenBank.

Assessment of petroleum biodegradation by isolated fungi strains in different salt concentrations
The test was performed on Potato Dextrose Broth (PDB) containing 0, 2.5 and 5% w/v NaCl and 1% v/v crude oil.A volume of 10 ml of the culture medium was added to 50 ml Erlenmeyer flasks.A volume of 1 cm 2 segments of fresh Embellisia sp.KJ59 and Alternaria sp.KJ66 on plates containing PDA with a salinity of 5% was added to each flask.Fungal mycelia pellets were separated from the culture medium by centrifuge at 4000 g, and they were washed with sterile distilled water.After that, they were homogenised, and 5% v/v homogenised suspension of 5 × 10 6 spore ml −1 were added to each flask.The test was done in triplicate.Flasks were incubated in a shaker flask at 28°C and 170 rpm for 14 days.For assessment of biomass production, fungal mycelia pellets were separated from the culture medium by centrifuging at 4000 g.Then, they were washed with distilled water and dried in an oven at 50°C for 1 day.Weights of dried biomass were measured.Flasks were analysed on Day 2, 5, 7, 10, 12, and 14, and the graphs of crude oil biodegradation and biomass production were provided [8].

Assessment of pyrene biodegradation in different salt concentrations
For evaluation of pyrene biodegradation, 10 ml of MSM was added to 50 ml flasks in addition to 100 mg L −1 pyrene as a sole carbon source and 0, 2.5, and 5% w/v of NaCl.For the preparation of inoculum, 1 cm 2 of fresh cultured Embellisia sp.KJ59 and Alternaria sp.KJ66 on Petri dish was added to each flask.Erlenmeyer flasks were incubated in a shaker incubator at 28°C and 170 rpm.Fungal mycelia pellets were separated from the culture medium by centrifuge at 4000 g and were washed with sterile distilled water.After that, they were homogenised, and 5% v/v homogenised suspension of 5 × 10 6 spore ml −1 was added to each flask.They have incubated in a shaker flask for 14 days at 28°C and 170 rpm.The test was conducted in triplicate.For pyrene extraction, 4 ml of hexane was added to each flask.Hexane was evaporated for one day.After that, 2 ml acetonitrile was added to the residue of pyrene.The amount of residual pyrene was analysed by HPLC (Knauer, Germany) at 265 nm.Column C18 was utilised to detect the pyrene.The HPLC elution program consisted of a 20 min linear gradient of acetonitrile/water (80:20, v v −1 ).The flow rate was 1 ml min −1 .The amount of pyrene degradation was analysed in comparison with a standard graph with different pyrene concentrations and blank samples [5].

Quality assurance and quality control
In order to provide reliable and accurate data, all analytical experiments were conducted in triplicate.If necessary, additional runs were also performed for more reassurance.All data were expressed as the mean ± standard deviation.The flasks and containers were rinsed twice with dH 2 O and oven dried.Crude oil or pyrene containing culture media without fungi was considered as the negative control during the analytical measurements which were not subjected to any change in the initial concentration throughout the experiments.

Statistical analysis
Treatment differences in crude oil and pyrene removal were analysed using one-way ANOVA followed by the post hoc Tukey's test.

Collection of soil samples and isolation
In this study, 20 samples were collected from saline soils of different areas in Iran for the isolation of fungi that are adapted to high salinity.pH range of soil samples was from 6.4 to 8, and EC range was from 8.4 to 194.7 mS cm −1 .73 fungal isolates were isolated on PDA, CDA, and MEA culture media containing 10% w/v NaCl.
Margesin and Schinner [27] reported that lots of regions which are polluted with crude oil have an extreme condition like high or low temperature, acidic or alkaline pH, high salt concentrations and high pressure.So, indigenous and extremophilic petroleumdegrading microorganisms are suitable for these environments due to the fact that they are adapted to these conditions.The Persian Gulf is one of the most polluted areas with oil hydrocarbons due to the fact that they produce nearly 60% of crude oil all over the World [28].Iran is located in the desert zone.Therefore, soils in places where there is crude oil pollution, have a wide range of salinity [29], so the microorganisms that can grow in saline soils and marine environments are suitable for these environments.

Halotolerance test
Table 1 indicates the halotolerance test data for the isolated fungi in salinity of 0, 10, 17, and 20% w/v on PDA over the period of seven days.The results showed that all studied isolates could grow in salinity of 0, 10, and 17% with different diameters of colonies but only two strains, KJ59 and KJ66 could grow in 20% salinity.However, the diameters of colonies were less than 1 cm in this condition.All isolates grow in non-saline media.High salinity restricts petroleum biodegradation because salinity inhibits the activities of microorganisms and the fungi capable to grow in vitro at 17% salinity are classified as halophilic and halotolerant strains [11].The media enriched with 50% glucose or 17% NaCl have a water activity (aw) less than 0.85.Therefore, all of the isolated seven fungal strains belonged to halophiles and halotolerant groups and could grow in media with low water activity.Ion homoeostasis, compatible solute accumulation and changing the plasma membrane fluidity are reported as the main fungal strategies to tolerate salinity [11].

Assessment of petroleum biodegradation by isolated fungi strains in different salt concentrations
Results of crude oil removal by seven isolated strains, at various salinities, were summarised in Table 2.The isolates KJ43, KJ59, KJ65, and KJ66 showed considerable percentage of crude oil degradation in non-saline conditions.However, biodegradation potential of KJ43 and KJ65 was less than the other studied fungi.The isolate KJ59 removed a percentage of 62.1% crude oil in non-saline MSMduring 14 days.Furthermore, in salinity of 2.5 and 5%, it could degrade 44.7 and 37.3% of crude oil, respectively.Isolate KJ66 could biodegrade 59.1, 58.9, and 17.1% in NaCl concentrations of 0, 2.5, and 5% w/v, respectively.Consequently, the isolates KJ59 and KJ66 were selected as the superior strains for further studies for evaluation of pyrene and crude oil removal in saline conditions.

Morphological and molecular identification
ITS gene sequence similarity in NCBI database showed that KJ59 belonged to Embellisia sp. with 100% similarity.Isolate KJ66 has 100% similarity with Alternaria sp.Some species of Alternaria and Embellisia are known as the locoweed endophytes [30].Up to date, many secondary metabolites like indolizidine alkaloids, O-Heterocyclic embeurekols, embellicines and α-Pyrone derivatives have been extracted and identified from Embellisia spp [31].Also, an strain of Embellisia has been isolated from Antarctica and reported as the protease producer [32].Several researches have focussed on environmental applications of Alternariadifferent species.A. alternata CMERI F6 has been shown to decolourise Congo red as an azo dye [33].Another strain of A. alternata has been reported to degrade natural rubber [34].In addition, A. tenuissimawas capable to degrade polymeric materials such as pyridine-based polyether polyurethanes [35].The sequences of Embellisia sp.KJ59 and Alternaria sp.KJ66 were registered in the GenBank under the accession numbers KU293683 and MG008684, respectively.Table 3 shows morphological and molecular characteristics of Embellisia sp.KJ59 and Alternaria sp.KJ66.Furthermore, Figure 1 shows microscopic images of them.KJ66. Figure 2 is phylogenetic trees of isolates KJ59 and KJ66 with bootstrap replications of 1000 in the neighbor-joining algorithm.
The current study is the first report of pyrene biodegradation by a strain of genus Embellisia; however, some species of Alternaria such as Alternaria alternatawere have reported previously to remove crude oil and PAHs [36].

Assessment of petroleum degradation in different salt concentrations
According to our results, Embellisia sp.KJ59 could remove 95.1% of crude on PDB without NaCl over a period of 14 days.In salinity of 2.5 and 5% w/v, it could degrade 92.4 and 87.3% of crude oil, respectively.Degradation of crude oil by Alternaria sp.KJ66 was 89.1, 85, and 81.3% on PDB in NaCl concentrations of 0, 2.5 and 5% w/v, respectively.Dried weight biomass of Embellisia sp.KJ59 was 61.2 g L −1 on non-saline PDB, and in salinity of 2.5%, and 5% w/v, biomass production was 56.9 and 43.2 g L −1 , respectively.Biomass production of Alternaria sp.KJ66 was 54.5, 53.3, and 40.2 g L −1 in salinity of 0, 2.5, and 5%, respectively.Figure 3 illustrates the relationship between crude oil removal and biomass production by Embellisia sp.KJ59 and Alternaria sp.KJ66 on PDB with 1% v/v crude oil in NaCl concentrations of 0%, 2.5% and 5% w/v.Results of our studies show that biodegradation of petroleum by Embellisia sp.KJ59 and Alternaria sp.KJ66 in salinity of 5% was less than in 0 and 2.5% salinity on MSM medium.Therefore, salinity may have a significant effect on oil removal by these strains.Maximum crude oil removal was observed in the non-saline medium.Furthermore, Embellisia sp.KJ59 has better performance in salinity of 0 and 5% than Alternaria sp.KJ66.However, in NaCl concentration of 2.5%, Alternaria sp.KJ66 was able to remove more crude oil in comparison with Embellisia sp.KJ59.
The graph illustrates crude oil removal dried biomass weight increased sharply in the first seven days.Then, there is a slight growth during the next 14 days, and the most of the crude oil is removed over the period of these days.It shows the fungal isolates may produce more enzymes for the degradation of crude oils during the first 7 days.Moreover, there was an apparent correlation between crude oil biodegradation and biomass production.With regard to the blank sample, there is no significant change in the amount of crude oil.
Behnood et al. [9] reported biodegradation of crude oil as a sole source of carbon, by Phanerochaete chrysosporium in salinity of 0, 1, 2, 3, and 4%.The best performance of the strain was 58, 1% in salinity of 1% for 12 days.The maximum amount of salinity was 4%, such that oil removal was sharply reduced to 21.8%.Nevertheless, Embellisia sp.KJ59 could remove 37.3% of crude oil in salinity of 5% on MSM culture without supplementary carbon and 44.7% in 2.5% salinity for 14 days.
In this study, the amount of biomass production of Embellisia sp.KJ59 and Alternaria sp.KJ66 on PDB containing 1% crude oil decreases, when the amount of salinity increases.As a result, NaCl may have an adverse effect on growth.This negative effect can be attributed to the cell proteins denaturation and cell membrane disruption due to the changes of osmotic pressure.Also, PDB was used as a medium, which has a supplementary source of carbon.It caused growth in pace and amount of crude oil biodegradation.Therefore, supplementary source of carbon can raise the speed and amount of crude oil removal and growth of strains (Figure 2).
Obuekwe et al. [8] studied the growth and crude oil removal by the strain Papulaspora sp. on PDB with the salinity of 0 and 5%.The crude oil was introduced as a supplementary source of carbon and energy.The strain could remove 21.7 and 93.8% in salinity of 0 and 5%, respectively.Biomass production was 4.7 and 7 g L −1 in salinity of 0% and 5%, respectively.In the current study, the growth of Embellisiasp.KJ59 and Alternaria sp.KJ66 was more on PDB in NaCl concentrations of 0 and 5% for 14 days when compared with the previous study.Studies which are mentioned above are similar due to the fact that they were examined fungi that are able to remove crude oil in saline media.Table 4   Alternaria sp.KJ66 are appropriate strains for bioremediation of crude oil as a sole or supplementary source of carbon in oil-polluted environments.

Assessment of pyrene degradation by halotolerant fungi strains
Results of pyrene removal, as a model heavy Polycyclic Aromatic Hydrocarbons (PAHs), were analysed and illustrated in comparison with the standard graph of pyrene.Pyrene biodegradation by Embellisia sp.KJ59 was 98.6, 96.6 and 94.8% in NaCl concentrations of 0, 2.5, and 5% w/v, respectively.Moreover, Alternaria sp.KJ66 could remove 95.6, 95.2, and 88.3% of pyrene in 0, 2.5 and 5% salinity, respectively.Pyrene degradation chromatograms are provided (Supplementary material).Pyrene degradation by Embellisia sp.KJ59 and Alternaria sp.KJ66 in this study represented that salinity has no remarkable effect on degradation rate.Romero et al. [38] studied the amount of pyrene degradation by Fusarium solaniand Rhodotorula glutinison Bushnell Haas minimal medium.F. solanicould degrade 32% of pyrene for 17 days.R. glutinis could also remove approximately 37% of pyrene for this period.In this study, Embellisia sp.KJ59 and Alternaria sp.KJ66 could remove pyrene, as a toxic heavy polycyclic aromatic hydrocarbon (PAH) and a sole source of carbon, more efficiently in nonsaline and saline MSM in a 14-day period.Consequently, They are capable strains for the biodegradation of pyrene in non-saline and saline environments.Table 5 indicates pyrene biodegradation by different strains in different conditions.Alternaria spp.belonging to the white rot fungi group and their potential in producing peroxidase enzyme is reported in several studies.These enzymes play a significant role in the degradation of different environmental contaminants by microorganisms.

Conclusion
Embellisia sp.KJ59 and Alternaria sp.KJ66, which are isolated from saline soils are potential strains for bioremediation of crude oil in saline and hypersaline regions due to the fact that they are halotolerant and can remove crude oil as a sole or supplementary source of carbon and pyrene.The current study is the first report of petroleum hydrocarbons biodegradation by strains of genus Embellisia.Nonetheless, crude oil degradation by some species of genus Alternaria was reported.

Figure 3 .
Figure 3. Crude oil removal and biomass production by A) Embellisia sp.KJ59 and B) Alternaria sp.KJ66 in PDB containing 1% v/v crude oil in different salinity during 14 days.

Table 1 .
Growth of selected fungal isolates (based on diameters of colonies) on PDA in different concentrations of NaCl in seven days.

Table 2 .
Percentage of crude oil removal by selected isolates on MSM in different salinity during 14 days.
represents the biodegradation of 1% crude oil by different strains in various conditions.The results indicate Embellisiasp.KJ59 and

Table 4 .
Biodegradation of 1% crude oil by different strains in different condition.

Table 5 .
Pyrene removal by different strains in different condition.