Isolation and Characterization of Three New Crude Oil Degrading Yeast Strains, Candida parapsilosis SK1, Rhodotorula mucilaginosa SK2 and SK3

Abstract

Bioremediation using yeasts is an alternative way to minimize the effects of oil spillage on soil. This paper aims to establish a bioremediation protocol involving the optimization of physicochemical parameters. In this regard, three new yeast strains, SK1, SK2 and SK3, were isolated from hydrocarbon-contaminated samples from the Fez-Meknes region, Morocco. These isolates were identified as new species of Candida parapsilosis (SK1) and Rhodotorula mucilaginosa (SK2 and SK3), respectively, based on the similarity of their ITS region. The kinetic analysis of the process of degradation of petroleum oils are highlighted. These analyses were based on the degradation kinetics, and biomass formation using gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. The strains were able to degrade 68% of the total petroleum hydrocarbon in 21 days, as the sole carbon source. The addition of glucose increased the rate at which crude oil was consumed by the isolates. Our results suggest that inoculants based on Candida parapsilosis (SK1) and Rhodotorula mucilaginosa (SK2 and SK3) cells have potential application in the biodegradation of crude oil and possibly in the degradation of other related aromatic compounds.

1. Introduction

Soil and water pollution by oil and its derivatives is now a serious and widespread ecological hazard. The world’s use of petroleum and its derivatives, and their transportation, has made them major contaminants in prevalence and quantity in the environment. The impact of an oil spill can be easily understood by the fact that one barrel of crude oil can make 1 million barrels of water undrinkable [1].

Soil and water contamination by hydrocarbons causes a significant damage to life in the environment, as the accumulation of pollutants in animal and plant tissues can lead to death or mutations [2].

Agricultural soils in the Fez-Meknes region of Morocco are subject to contamination by various organic pollutants, including hydrocarbon discharges from storage tank spills. This pollution has an impact on all health, economic and environmental aspects.

Biodegradation is an alternative that has been widely used to reduce hydrocarbon pollutants in the environment by spreading microorganisms hosting specialized degradative machinery. It is also accepted worldwide as an in situ, self-propelled, and eco-friendly treatment that leads to complete mineralization at a low cost. [3]

The main microorganisms degrading hydrocarbons in polluted areas are bacteria, yeasts and fungi [4,5]. Walker et al., reported that yeasts are better degraders of crude oil than bacteria [6]. In addition, according to Obuekwe et al., Ashraf and Ali, yeasts can utilize n-alkanes as a unique source of carbon and energy [7,8]. Several yeast species, such as Candida lipolytica and Geotrichum sp., Trichosporon mucoides and Yorrowia lipolytica, isolated from contaminated water, were reported to degrade petroleum compounds [9,10].

Recently, Benmessaoud et al. conducted a study on the biotypology and diversity of soil yeasts in this region, which is a good indicator to assess the extent of ecosystem disturbance by oil pollution. [11]. However, this needs to be explored more in the process of biodegradation of different hydrocarbons. In this sense, and in order to enrich and deepen the knowledge of biodegradation of soil yeasts, especially those contaminated by organic pollutants, a study was initiated by the laboratory of Biotechnology, Conservation and Valorization of Natural Resources in Fez, Morocco.

The objectives of this work were to: (i) isolate a number of cultivable aerobic hydrocarbon degrading yeasts from hydrocarbon contaminated sites in the Fez-Meknes region of Morocco; (ii) identify the isolates at the species level using molecular techniques; (iii) evaluate the biodegradation capacity of the selected yeast strains and assess the influence of any physico-chemical parameters on biodegradation to obtain a better understanding of bioremediation problems.

2. Materials and Methods

2.1. Sampling and Isolation of Yeast Candidates

From an environmental point of view, the yeasts used in this study were directly isolated from soils polluted by hydrocarbons. These soils contain an abnormal concentration of hydrocarbon substances that are potentially dangerous for human life as well as for fauna and flora.

Soil samples were taken from different locations in the Fez-Meknes region between the periods of December 2015 and April 2016. Twelve stations were selected; their locations and coordinates are shown in

Table 1. Locations and coordinates of different studied stations.

Stations		X (m)	Y (m)	Long (°)	Latitude (°)	Altitude (m)
Fez	St1	536,812.29	385,356.78	−4.99950384	34.06655874	417.80
Ain Taoujdat	St2	516,521.40	372,299.99	5.21961543	33.94932340	466.62
Meknes	St3	486,955.04	362,381.44	5.53941429	33.85992761	558.21
El-Hajeb	St4	500,657.74	343,619.64	−5.39130449	33.69079909	872.69
Sefrou	St5	551,896.96	358,566.53	−4.83761684	33.82433591	819.27
Azzaba	St6	564,183.82	358,165.06	4.70472032	33.82004776	83.80
Bir tam tam	St7	564,346.54	369,849.05	−4.70227923	33.92540590	580.83
Ifrane	St8	524,585.96	330,314.77	−5.13350818	33.57051778	1648.16
Guigou	St9	550,800.08	309,567.03	−4.85224822	33.38246581	1489.58
Boulman	St10	569,872.57	301,040.56	−4.64786418	33.30449076	1710.10
Outat el haje	St11	658,570.94	305,612.54	−3.69442105	33.33630542	796.13
Tahla	St12	592,005.51	384,107.56	−4.40159734	34.05195780	571.20

.

Around 100 g of soil was sampled, put into a sterile plastic bag and transported to the laboratory and kept cold (4 ± 0.1 °C). A total of 1 g of sample soil transferred into 10 mL of sterile distilled water and stirred for 10 min. A total of 1 mL of suspension was used to inoculate malt–yeast–glucose–peptone agar (yeast extract (3 g); malt extract (3 g), peptone (5 g), glucose (10 g); agar (15 g), H₂O distillate 1 L), supplemented with chloramphenicol (200 mg/L), and incubated at 30 ± 0.1 °C for up to 72 h. The yeast-like colonies were isolated for additional purification [12].

The yeast strains were cultivated in 150 mL Erlenmeyer flasks containing 50 mL of mineral basal medium (MM), containing, per liter, 3.4 g K₂HPO₄, 4.3 g KH₂PO₄, 0.3 g MgCl₂⋅2H₂O and 1 g (NH₄)₂SO₄, and supplemented with 1 mL of a solution of trace elements contained in 1 L (without complexing agents): HCl, 25%, 10 mL; FeCl₂ 4H₂O, 1.5 g; CoCl₂⋅6HaO, 190 mg; MnCl₂ 4H₂O, 100 mg; ZnCl₂, 70 mg; H₃BO₃, 62 mg; Na₂MoO₄ 2H₂O, 36 mg; NiCl₂⋅6H₂O, 24 mg; CuCl₂ 2H₂O, 17 mg [13].

Two types of degradation mechanism were tested: gratuitous biodegradation using crude oil as the only carbon source, and cometabolism, using a second carbon source (glucose), easily assimilated by the three yeast strains.

A concentration of 3.74% (w/v) of the crude oil “Arabia Length (AL)” was added to the MM medium as a carbon source. The flasks were inoculated with 100 µL cultures pre-grown in MM medium plus 1 g/L of glucose. After this, they were incubated 30 ± 0.1 °C in a dry shaker at 100 rpm for one week.

The experiment was repeated three times for each isolat in order to estimate the error values.

2.2. Biodegradation Ability and Biomass Formation

The biodegradation assay for Petroleum compounds and derivatives as carbon sources was carried out in Erlenmeyer flasks containing 100 mL of the Bushnell Has Medium (KH₂PO₄ (1 g), K₂HPO₄ (1 g), NH₄NO₃ (1 g), MgSO₄ 7H₂O (0.2 g), FeCl₃ (0.05 g), CaCl₂ 2H₂O (0.02 g), H₂O distillate 1 L) [14]. Experiments were carried out in triplicate, under shaking (100 rpm), and at 28 ± 2 °C, for 21 days. A total of 100 μL of the preculture was used for each selected yeast strain and 3.74% (w/v) hydrocarbons were used as the sole carbon source. Several hydrocarbon substrates were used to evaluate the biodegradation potential of the selected strains (crude oil, Undecane, Hexadecane, diesel, Gasoline, Naphthalene, Toluene and Pyruvic acid). Two control flasks were also maintained for each series.

Biomass concentration was measured using a spectrophotometer at λ_600nm (JENWAY 6300/UV-visible), based on the predetermined correlation between optical density and dry weight of biomass. These measurements were carried out 0, 4, 15 and 21 days, respectively.

2.3. Extraction of Residual Crude Oil

To quantify the degradation of n-alkanes present in crude oil by the selected yeasts, the total petroleum hydrocarbon (TPH) was determined by the gravimetric weight loss technique.

Cell-free supernatants of the culture media containing petroleum oil or derivatives were extracted three times using 10 mL of dichloromethane and dried with anhydrous sodium sulphate [15].

The dichloromethane was evaporated by simple distillation at 60 °C, the amount of residual TPH was determined using a standard method for oil gravimetric. The controls were treated under the same conditions.

Biodegradation degree was calculated as:

$$\frac{\left(X_0-X_t\right)}{X_0}\ast 100(\%)$$

where X₀ is the initial amount of hydrocarbon and X_t is the remaining hydrocarbons after biodegradation [16,17]. The prepared samples then extracted and prepared for GC/MS analyses.

2.4. Hydrocarbon Analysis

The quantitative analysis of the hydrocarbons biodegradation by yeasts was based on GC/MS according to the method described by Gargouri et al. [17,18,19]. A total of 1 µL of the alkane fraction was analyzed by gas chromatography analysis GC/MS (trace GC 1300 TSQ 8000 evo) using aHP- 5 MS chromatographic column (5% phenyl Methyl Siloxane) of size 30 m × 0.25 mm × 0.25 µm. The temperature was programmed to vary linearly from 70 °C to 230 °C at the rate of 20 °C min⁻¹, then from 230 °C to 300 °C at 40 °C min⁻¹ and 10 min at 300 °C. The helium was the carrier gas.

2.5. Identification and Phylogenetic Affiliation of the Yeast Strains

The selected yeasts were identified by their macroscopic, microscopic and physiological characteristics [12,20,21]. Potential isolates SK1, SK2 and SK3 were identified using molecular techniques by extracting genomic DNA and sequencing of ITS region as per Masneuf-Pomar‘ede et al. [22] and White et al. [23]. The Internal Transcribed Spacer (ITS) primers (forward ITS 1-5′ TCC GTA GGT GAA CCT GCG G 3′ and reverse ITS 4- 5′ TCC TCC GCT TAT TGA TAT GC 3′), which amplify a fragment of approximately 580 bp containing ITS1, 5.8 S and ITS 4 region, were used for this purpose. The amplification reaction was performed using a DNA thermal cycler™ Veriti™ in a 50 µL reaction mixture containing 5x GoTaq reaction buffer (Promega), 0.25 mM each dNTP, 0.2 µM each primer, 50 ng DNA template, and 2.5 U GoTaq DNA polymerase.

The Polymerase Chain Reaction program was executed with the following instructions: After an initial denaturation at 95 °C for 5 min, amplification was carried out through 30 cycles, each consisting of denaturation at 94 °C for 1 minute, annealing at 56 °C for 45 s, an extension step at 72 °C for 1 min and a final extension at 72 °C for 10 min.

Preliminary identifications were made on the assembly of basis sequence and by searching the National Center for Biotechnology Information (NCBI) database. Strains were assigned to a particular genus when sequence similarity to a strain type was at least 98%, and to a given species when sequence similarity was at least at least 99.5%.

The related sequences were aligned using Clustal W under MEGA X software. The aligned ITS- DNA gene sequences were used to construct a phylogenetic tree using the Neighbour-Joining (NJ) Method, and yeast species were identified based on the phylogenetic analysis results [24,25].

2.6. Statistical Analysis

Statistical analysis was carried out by ANOVA combination of the Shapiro–Wilk test and Levene test using software R package version 3.3.1 [26]. The purpose of the Tukey test is to refine the analysis, and specify which groups are statistically different.

3. Results

3.1. Identification of Yeasts

Based on the principal characteristics of potential isolates, the strains of SK1, SK2 and SK3 belong to genera Candida and Rhodotorula (

Table 2. Biochemical and physiological characteristics of the yeast strains.

	SK1	SK2	SK3
Asexual reproduction
Filamentous growth	+	-	-
Acetic acid production	-	-	-
Urease	-	+	+
Diazonium Blue B reaction	-	+	+
Glucose	1	1	1
Inulin	0	1	1
Sucrose	1	1	1
Raffinose	0	1	1
Melibiose	1	1	1
Galactose	1	1	1
Lactose	1	1	1
Trehalose	1	1	1
Maltose	0	1	1
Melezitose	1	1	1
Methyl-α-d-glucoside	1	1	0
Soluble starch	1	1	1
Cellobiose	0	0	0
Salicin	0	0	0
l-Sorbose	1	1	1
l-Rhamnose	1	1	1
d-Xylose	1	1	0
l-Arabinose	1	1	1
d-Arabinose	0	1	1
d-Ribose	0	0	0
Methanol	0	0	0
Ethanol	1	1	0
Glycerol	1	1	1
Erythritol	0	0	0
Ribitol	1	1	1
Galactitol	0	1	0
d-Mannitol	0	0	0
d-Glucitol	1	1	0
myo-Inositol	1	0	0
dl-Lactate	0	1	0
Succinate	1	1	0
Citrate	1	0	0
d-Gluconate	1	1	1
d-Glucosamine	0	0	0
n-Acetyl-d-glucosamine	0	0	0
Hexadecane	0	0	0
Nitrate	1	1	1
Vitamin-free	1	1	1
Nitrite	0	1	1
d-Glucuronate	0	0	0
Xylitol	1	1	1
l-Tartaric acid	1	1	1
Saccharic acid	0	0	0
p-Hydroxybenzoic acid	0	1	1
m-Hydroxybenzoic acid	0	1	1
Gallic acid	0	0	0
Gentisic acid	0	1	1
Vanillic acid	0	1	1
Ferulic acid	0	1	1
Veratric acid	0	1	1
Cycloheximide 0.01%	0	1	0
Cycloheximide 0.1%	0	1	0
Growth at 25 °C	1	1	1
Growth at 30 °C	1	1	1
Growth at 35 °C	1	1	1
Growth at 37 °C	1	1	1
Starch formation	0	0	0

).

Molecular identification of those isolates was based on ITS sequencing. According to sequence similarities and multiple alignments, the following isolates were identified: SK1 Candida parapsilosis (99%), SK2 Rhodotorula sp. Isolate (99%) also shared similarities with Rhodotorula mucilaginosa (100%), and SK3 Rhodotorula dairenensis (82.42%) shared similarities with Rhodotorula mucilaginosa (82.42%). The phylogenetic relationships between the sequence of the ITS-region of the yeast strains are shown in (Figure 1).

The phylogenetic relationship between strains was inferred using the Maximum Likelihood method and Tamura-Nei model [25,27]. The selected tree represents a consensus topology between the different reconstructions. The 16 S rRNA gene sequences of strains used in this study were deposited in the NCBI GenBank database and are available under the following accession numbers: MZ148416-MZ148418, for Candida parapsilosis strain SK1, Rhodotorula mucilaginosa strain SK2, and Rhodotorula mucilaginosa strain SK3, respectively.

3.2. Screening of Yeast Isolates for Oil Degradation

The aim of this test is to select the most efficient strains regarding their capacity to degrade crude oil. Among the 36 tested strains, only three yeasts, namely, SK1, SK2 and SK3, were selected as candidates in this study, which showed an important turbidity in the cultures between interactions as a sign of growth (Figure 2). Subsequently, these three strains were grown separately and mixed in equal proportions for further analysis, which consisted of quantifying their biodegradation rates.

To characterize the biodegradation abilities of the yeast against the crude oil, the amount of biodegraded substrate was measured using the gravimetric weight-loss technique. This technique requires very careful precision. The use of abiotic flasks must not be overlooked to quantify the losses related to substrate leakage; particularly, to ensure that the yeast uses the carbon source that is put into culture. This will make it possible to validate the results obtained for the calculation of the degradation rate and the mineralization yield by considering the values of the residual hydrocarbon concentrations.

The results show that, after each incubation period of 4, 15 and 21 days at 30 °C in Bushnell Has medium containing 3.74% crude oil, and under a shaking of 100 rpm, the three tested strains have a crude oil biodegradation potential that increases with incubation time and exceeds 60%. In addition, the degradation of crude oil is estimated at 68 ± 0.9% for SK1, 68 ± 1.23% for SK2, SK3 68 ± 1.18% (Figure 3). Moreover, the best degradation rates for 4 and 15 days are observed in strain SK2 with a rate of 15.87 ± 0.1% and 56.04 ± 0.07%, respectively. The addition of glucose as the second carbon source allowed for the removal of about 78.6%, 89.6% and 78.7% of total petroleum hydrocarbon for SK1, SK2 and SK3, respectively (Figure 4).

3.3. Biomasses

The three best-performing isolated yeast strains, SK1, SK2 and SK3, were tested individually against some short-chain alkanes such as undecane and gasoline, long-chain n-alkanes such as hexadodecane, and aromatic hydrocarbons and other hydrocarbon substrates (toluene, naphthalene, diesel, pyruvic acid and crude oil).

In the presence of these different hydrocarbons, the optical density changed as a function of time for each isolate (Figure 5). The turbidity of the culture was estimated by measuring the growth in the strains at regular intervals (0, 4, 15 and 21 days) using a spectrophotometer (UV-visible type JENWAY 6300) at 600 nm. This reflects the degradation potential of the selected hydrocarbons by the selected yeasts.

The follow-up of the growth kinetics, in the presence of aromatic hydrocarbons for the strains, showed a weak growth throughout the incubation time.

The growth in these yeasts in the other types of hydrocarbons was linear between time 0 and day 15 of incubation, which means that these strains use xenobiotics as a source of energy for their growth (oxidation). After 15 days of incubation, the growth was limited, because, in this period, the chemical compound was not used for yeast growth but was degraded due to metabolic activity (co-metabolism).

The results show that SK1, SK2 and SK3 have a greater capacity to degrade crude oil and hexadecane.

In addition to their ability to grow on crude oil and hexadecane as the only carbon source, the strains can also grow on some refinery by-products, such as undecane, diesel fuel, gasoline and naphthalene (Figure 5). Hexadecane and crude oil were the best substrates to support the growth in the yeast strains.

The growth intensity is variable depending on the strain; the SK2 strain shows a strong cloudiness in the culture medium compared to the other strains.

However, the growth in strain SK3 in the presence of crude oil showed an extended degradation time.

3.4. Gas Chromatographic Analysis

The degradation of the crude oil by Candida and Rhodotorula species was analyzed by gas chromatography coupled to mass spectrometry (GC/MS). The analysis revealed that the crude oil exhibited a distribution of n-alkanes between C7 and C28 (Figure 6). The proportion of hydrocarbons in the mixture was highly variable and ranged from 97% in the lighter oils to 50% in the heavier oils.

The crude oil degradation capacity of the isolates was evaluated by comparing the peaks in the chromatogram of the control to that of the samples. Candida parapsilosis and Rhodotorula mucilaginosa represented the efficient utilization of the n-alkane fraction compared to the untreated control.

GC/MS analysis revealed that suspension cells were capable of degrading aliphatic fractions, showing that all detectable hydrocarbon peaks were fully or partially utilized within 21 days by the yeast isolates. All three strains could degrade C7 to C28 components. They were able to reduce the relative abundance of toluene to 0.4%. The strains were completely used in the C8–C11 range. Rhodotorula mucilaginosa showed the complete degradation of the components phenol, hexadecane and ar-tumerone. In contrast, SK3 showed a nominal phenol degradation at 18%. However, Candida parapsilosis completely degraded components C16–C28 (

Table 3. Gas chromatographic analysis data showing percentage of crude oil degradation by selected isolates.

Oil Fractions %	Control	SK1	SK2	SK3
C7	2.2	0.4	0.4	0.4
C9	5	0	0	0
C10	2.6	0.6	0.2	1.2
C11	10	4	2	8
Phenol	45	20	0	18
Ar-tumerone	3	0	0	0.8
C16	21.2	0	0	0
C28	10	0	0.2	1

).

4. Discussion

The increased diversity of yeast can degrade several types of organic pollutants that exist at ground level (biodegradation). There is interest in the degradation of hydrocarbons and their intermediates as new sources of carbon in the soil. Yeast feed on hydrocarbons and transform them into water and CO₂. The yeasts are isolated from environments polluted with petroleum hydrocarbons because they are the best at degrading these pollutants [28,29].

In the present study, some crude-oil-degrading yeasts were isolated from Morocco. Among these isolates, three strains were identified as Candida parapsilosis (SK1), Rhodotorula mucilaginosa (SK2) and Rhodotorula mucilaginosa (SK3). The property of these natural isolates in a petroleum-oil-contaminated area, motivated an investigation into the potentiality of those isolates to be used for petroleum biodegradation. Many investigators have reported the involvement of bacteria and yeast in crude oil biodegradation [30,31]. On the contrary, there is scant information that yeasts are better hydrocarbon-degraders than bacteria [17,32].

It is important to remember that the three selected strains showed strong biodegradation because they are already adapted to this type of product. In this present work, the SK1, SK2 and SK3 strains were able to degrade 68% of the total petroleum hydrocarbon in 21 days as the only source of carbon.

The hydrocarbons are transformed according to a chain oxidation reaction, which is carried out by the successive degradation of a more complex molecule until simple by-products are obtained, which are represented by H₂O and CO₂, with renewal of the biomass [33].

Several investigations have used the co-metabolism as an effective biodegradation mechanism in media that contain a substrate, which can induce the metabolic pathway.

Moreover, the presence of an easily assimilable substrate (co-metabolisme) such as glycose may allow for an organism to degrade a recalcitrant compound by providing the energy or reducing the necessary enzyme activity [34,35]. Due to the variability in the hydrocarbon composition of crude oil, microorganisms develop several adaptive mechanisms to utilize a wide range of hydrocarbons as substrates. It has been shown that they are able to produce enzymes that are adapted to degrade hydrocarbons.

For example, a rapidly metabolizable carbon source such as glutamate increases pentochlorophenol’s biodegradation rate by Flavobacterium sp, and the glucose increases the phenol’s biodegradation rate by Aureobasidium pullulans [36,37,38]. In this case, the involved substances are considered primary growth sources, since they are directly involved in yeast growth.

In our studies, for three yeast strains, we noticed that the maximal increase in biodegradation is correlated by adding glucose as an easily assimilable substrate and as the second carbon source. This allowed Candida parapsilosis and Rhodotorula mucilaginosa to efficiently remove petroleum hydrocarbon by about 78.6%, 89.6% and 78.7% respectively.

Statistical tests show that the biodegradation of crude oil supplemented with glucose by the SK2 strain was statistically significant and different from the other two strains. According to Kumar (2022), yeasts did not metabolize xenobiotic compounds as the only source of carbon and energy; they required a second source of carbon for the degradation of pollutants. The growth properties of the tested yeast strains were correlated with the carbon source that was used [39].

The investigated yeasts showed an impressive ability to degrade most of the tested compounds but showed a distinctive preference for hexadecane. Besides their capacity to grow on hexadecane, the three yeasts showed adequate potential to grow on some refinery subproducts, such as crude oil, diesel fuel and Undecane. Measurement of these isolates’ biomass using hexadecane, undecane, diesel oil, and crude oil as substrates showed that the yeasts were able of utilizing a wide range of intermediate carbon chain length n-alkanes, probably because of the less toxic nature of the long-chain n-alkanes [40].

The analysis of gas chromatography coupled with mass spectrometry (GC/MS) shows that the strains assimilate the alkane fraction compared to the untreated control. The complete or partial use of hydrocarbon peaks reveals that these three yeast isolates could degrade the aliphatic fractions in 21 days. The n-alkanes differ in the attack by the isolates and could be ranked based on the availability of these hydrocarbons to these yeasts for mineralization. R. mucilaginosa also showed a considerable utilization of components from an aromatic fraction as a sole source of carbon and energy. While C. parapsilosis could not completely degrade aromatic hydrocarbons as the sole carbon source. This is due to the presence of a high enzymatic capacity, which allows these strains to degrade complex hydrocarbons.

According to Omar and Rehm, Candida parapsilosis effectively degraded n-alkanes and the highest degree of degradation of the alkane mixture (C12–C18) was achieved by cells immobilized on granular clay [41]. This result is in agreement with other research, where hydrocarbon-degrading microbes showed the preferential degradation of n-alkanes > branched alkanes > low-molecular-weight aromatics > cyclic alkanes > high-molecular-weight aromatics > asphaltene fractions.

After all, the results obtained in the laboratory must be adapted to the natural environment. Therefore, we should examine the behaviour of these strains in mesocosm experiments to clarify this way of acting in the natural environment for the bioremediation of soils and wastewater that are polluted by hydrocarbons.

5. Conclusions

In our study, we isolated and characterizated three new crude-oil-degrading yeast strains, Candida parapsilosis SK1, Rhodotorula mucilaginosa SK2 and SK3, which developed coping mechanisms to survive in the hostile environments of these soils. These yeasts will be used to establish a bioremediation protocol, involving the optimization of physico-chemical parameters, for the bioremediation of environments contaminated by hydrocarbons.