Introduction

In recent decades, environmental contaminations with petroleum compounds have gained much attention. Oil spillages into the environment followed by pollution of soils, rivers, and sea have become one of the major worldwide concerns [1]. These spills are threat to public health and destroy natural resources [2]. Therefore, remediation of petroleum-polluted soils is a fundamental need for the survival of living organisms. It can be carried out by different methods including physicochemical and biological approaches [3]. Bioremediation, based on the potentiality of microorganisms to detoxify environmental contaminants [4], is one of the most promising methods of reducing the environmental pollution caused by oil spills [5]. As compared to physicochemical methods, bioremediation might be a cost-effective, non-destructive, and environmentally friendly approach for the remediation of contaminated soils [6].

Nowadays, the combination of microbial species for the biodegradation of certain pollutants has attracted much attention [7]. The appropriate combination of bacteria and fungi demonstrated powerful potential in bioremediation processes [7–9]. A collaboration between different microbes under co-culture conditions such as co-metabolism or antagonism makes the system to perform better than a single microorganism [8, 10], though there have been few reports on the case of yeast species’ co-existing capacity to degrade toxic organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and produce biosurfactants [11, 12].

Since crude oil is a mixture of many different hydrocarbons such as PAHs, and individual microorganisms have the ability to metabolize only a limited range of compounds, bioremediation of petroleum hydrocarbons requires a mixture of microorganisms to degrade a wider range of hydrocarbons more effectively [9]. In 2012, Hesham et al. stated that over 80% of high molecular weight PAHs were degraded by the bioaugmentation system for the biologically treated oilfield effluent, using the co-culture of five exogenous yeast strains [13].

Efficient oil biodegradation depends on optimum conditions such as nutrient availability and physicochemical factors that support biological treatment. Microbial production or adding surface active agents to the contaminated site is one of the most effective factors for enhancing the degradation of hydrocarbon compounds [14]. The use of surfactants has been proposed as an approach to increase the bioavailability of hydrophobic organic compounds (HOCs) in soil [15]. Microbial originated surfactants, also called biosurfactants, are being considered. Biodegradability, low toxicity, ecological adequacy, and ability to be produced from renewable and less expensive substrates are some of their advantages over their chemically artificial equivalents [16]. Mainly, bacteria and yeasts are capable of synthesizing biosurfactants [11]. Several Candida species and Trichosporon asahii were reported to be yeast biosurfactant producers and grow well in oil-rich environments [13, 17].

Some microorganisms such as Mycobacterium, Rhodococcus, and Candida have been reported, to modify their cell surface hydrophobicity to improve uptake of hydrophobic substrates when exposed to these compounds. Increase in cell surface hydrophobicity and surface active agent production are two main approaches that facilitate the transportation of hydrophobic substrates into cells [18].

Previously, yeast strains that showed high efficiency in removing crude oil and producing biosurfactant were isolated from oil-contaminated soil samples (unpublished work). Sarocladium sp. and Cryptococcus sp. were shown to be the superior strains. Hence, little information exists on the feasibility of using yeast co-cultures; the present study focused on this aspect. A comparison of individual cultures and co-culture for oil degradation, biosurfactant production, emulsification activity, and cell surface hydrophobicity by these two yeast strains was also conducted. To the authors’ knowledge, no detailed scientific studies have so far been conducted on the relationship between these factors, and this study will promote understanding of the synergistic effects of yeast co-culture on oil degradation.

Materials and Methods

Isolate Selection and Culture Conditions

In previous studies, two yeast strains (Y17 and Y71 ) with crude oil-degrading potential were isolated from the soils of different regions of Iran (Kermanshah, Iran 34°18′46.0″N 46°11′21.4″E and Lorestan, Iran 33°25′44.7″N 48°10′35.4″E). Pure cultures were obtained by subculturing on fresh potato dextrose agar (PDA) plates. According to the crude oil-degrading ability, further studies were conducted on their crude oil removal, pyrene degradation, and surface activities. Plates were incubated at 28 °C for 2 days and then stored in a refrigerator at 4 °C. All the experiments were conducted at 28 °C on a rotary shaker at 120 rpm for broth cultures.

Crude Oil Removal Assay

For crude oil removal assay, pre-cultures were prepared in Erlenmeyer flasks of 50 ml, containing 10 ml of the Bushnell-Haas Broth supplemented with 1% glucose [19] as the carbon source for 1 day. Ten percent (v/v) inoculum was applied in the same medium as pre-culture without addition of glucose supplemented with 1% crude oil as the sole source of carbon. One milliliter of each individual culture broth at optical density of OD600 = 1 was harvested by centrifugation, mixed, and resuspended in a final volume of 1 ml sterile normal saline as inoculum [20]. Afterward, the flasks were incubated on a rotary shaker (120 rpm at 28 °C) for 24 days. Samples were collected at 6, 12, 18, and 24 days for further assay. The crude oil removal assay was carried out by dissolving residual crude oil of the medium with the same volume (10 ml) of toluene as culture broth, and the optical density of the oil extract was measured against a blank at 420 nm [19]. The test was done in triplicate for each sample. Biomass formation was measured by wet cell weight so that the biomass in 1 ml culture was immediately weighted after centrifugation and culture broth disposal.

Molecular Identification of Isolates

PCR-based molecular identification was performed. The region of Internal Transcribed Spacers (ITS) of ribosomal DNA (rDNA) was amplified using the primers ITS1 (5′TCC GTA GGT GAA CCT GCG G 3′), which hybridizes at the end of the 18S rDNA, and ITS4 (5′TCC TCC GCT TAT TGA TAT GC 3′), which hybridizes at the beginning of the 28S rDNA, and the D1/D2 region of the 28S rDNA, large subunit (LSU) rDNA, was amplified by NL1 and NL4 (NL1: 5′-GCA TAT CAA TAA GCG GAG GAA AAG and NL4: 5′-GGT CCG TGT TTC AAGACG G) synthesized by Bioneer Corporation, South Korea. Amplification was performed with a SensoQuest™ thermal cycler. The PCR mixture consisted of 12.5 μl of Taq DNA Polymerase Master Mix Red (2×) Amplicon™, 0.1 mM of each primer, and approximately 200 ng of extracted DNA as a template in a total volume of 25 μL. The PCR program was as follows: 95 °C for 5 min, followed by 30 cycles at 96 °C for 30 s, 55 °C for 90 s, and 72 °C for 30 s. Finally, there was an extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis on 1% (w/v) agarose gel, and purification was done for sequencing [21]. Analysis of the sequences was achieved for the taxonomic characterization of the isolated strains [22]. Sequence similarities were attained using Centraalbureau voor Schimmelcultures (CBS) and National Center for Biotechnology Information (NCBI) databases.

Biosurfactant Production and Measurement of Surface Tension Reduction

For biosurfactant production, a pre-culture was prepared in GPY medium without hydrophobic substrate to reach OD600 = 1; then, 10% v/v was used to initiate growth in 50 ml medium in 250-ml Erlenmeyer flasks containing 0.3% NaNO3, 0.025% KH2PO4, 0.025% MgSO4·7H2O, 0.1% yeast extract [23], and 300 mg l−1 pyrene as a hydrophobic carbon source. Flasks were incubated at 28 °C on a rotary shaker at 120 rpm. Measurements of surface tension were carried out on the cell-free broth obtained by the centrifugation of cultures at 4000×g. Surface tension reduction was measured using the Do Nouy ring method with a Kruss K7 tensiometer (Kruss Optronic, Germany). To assay surface tension, the sample temperature was adjusted to 25 °C and 20 ml of each sample was poured into the sample container of the tensiometer. Distilled water and un-inoculated medium were used as controls [24].

Emulsification Activity

The emulsification activity was carried out by adding 2 ml motor oil (diesel) and 2 ml acetate buffer to the 2 ml cell-free broth in a test tube. The resulting uniform emulsion was allowed to stand for 10 min, and its absorbance was measured at 540 nm. The blanks were prepared the same way as the samples but with 2 ml un-cultured broth. One unit of emulsification activity is defined as the amount of biosurfactant that affects an emulsion with an absorbance of 1.0 at 540 nm [25]. The emulsification activity was also compared with Triton X-100 (5%) and sodium dodecyl sulfate (SDS) as a chemical surfactant.

Microbial Adhesion to Hydrocarbon Test

Cell surface hydrophobicity was determined by the microbial adhesion to hydrocarbon (MATH) method, according to Rosenberg et al. [26]. Selected isolates were inoculated in 50 ml mineral salt medium containing 1 g KH2PO4, 0.5 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 0.5 g yeast extract, 1000 ml distilled water, and pH 6 supplemented with 100 mg l−1 pyrene as the hydrophobic carbon source and 100 mg l−1 glucose as the hydrophilic carbon source. At the end of each 7-day cycle, subcultures (5% v/v) were transferred to the same medium to obtain the fourth generation of subcultures. According to Deng et al., Saccharomyces cerevisiae was used as negative control [18]. In the third generation, after reaching the late exponential phase (0.5 < OD600 < 1), the yeast cells were harvested from 4 ml of the culture by centrifugation at 4000×g for 10 min and washed twice with phosphate urea magnesium (PUM) buffer, 22.2 g K2HPO4·3H2O, 7.26 g KH2PO4, 1.8 g urea, 0.2 g MgSO4·7H2O, and 1000 ml distilled water, at pH of 7.1. The cells were resuspended in 4 ml PUM buffer to fit an optical density of ca.1.0 (A0). Toluene (4 ml) was added to 4 ml of the microbial suspension and vortexed for 2 min. Optical density was measured at 600 nm with a UV-visible spectrophotometer (UV-160A Shimadzu, Japan). The optical density of the aqueous phase was measured (A1) after 10 min, and the degree of hydrophobicity was calculated as [1 − (A0 − A1) / A0 × 100%] [18].

High-Pressure Liquid Chromatography for Pyrene Degradation Measurement

Pyrene degradation analysis was conducted using the high-pressure liquid chromatography (HPLC) method. For this purpose, the pre-culture of each strain and their co-culture prepared in the Bushnell-Haas medium were incubated to reach OD600 = 1. Ten percent (v/v) of pre-cultures was inoculated in 50-ml Erlenmeyer flasks containing 10 ml of the Bushnell-Haas medium with 100 mg l−1 pyrene as a sole carbon source. Flasks were incubated for 15 days at 28 °C, and sample analysis was done every 3 days. The blank medium was prepared without inoculation [27]. The remaining pyrene in the flasks was extracted using an equal volume of n-hexane. After solvent addition, the mixtures were placed on a rotary shaker for 15 min and then centrifuged for 15 min at 4000×g for better separation of the two phases. The organic phase of the samples was then cautiously removed and collected in glass vials, after solvent evaporation. The dilution of samples was performed using 1 ml acetonitrile, 100 μl of which was injected into the HPLC (Agilent Technologies, USA), and chromatographic separations were performed on an ACE LiChrosorb C18 column (250 × 4.6 mm ID, particle size 5 μm, ACE-121-2546) and thermostated at 28 °C. The mobile phase consisted of acetonitrile and distilled water in the ratio 80:20, 1 ml min−1 flow rate, and detection at 254 nm using a UV detector. A standard curve was obtained with 25, 50, 75, and 100 mg l−1 of pyrene [28].

Results and Discussion

Yeast Identification

ITS and LSU rDNA genes are commonly used for fungal identification. The sequence alignment of Y17 and Y71 in the CBS and NCBI databases showed 100% similarity, which is closely related to the genera Sarocladium and Cryptococcus, respectively. Considering these similarities, these two yeast strains were classified into the genera Sarocladium and Cryptococcus and the sequences were deposited in GenBank under accession numbers KU752201 and KX268614, respectively. Previous studies identified these genera among hydrocarbon-degrading microorganisms [29–31]. Ma et al. showed that the co-culture of Acremonium (very closely related genus to Sarocladium) with Bacillus subtilis enhanced dehydrogenase activity and crude oil degradation [29]. Also, Chandran and Das isolated the Cryptococcus yeast strain from hydrocarbon-contaminated soil [31]. This study’s primary screening results indicated that these two strains have high potential in oil degradation and biosurfactant production (data not shown). Co-culture strategy and evaluation of biosurfactant production were assessed according to these primary results.

Assessment of Growth Rate and Crude Oil Degradation Between Individual and Co-Culture of Sarocladium sp. and Cryptococcus sp.

In the current study, the crude oil biodegradative potential of individual yeast cultures and co-culture was evaluated. The results clearly showed that the remaining oil in the individual cultures were 64 and 63% in comparison with 50% in the co-culture (Fig. 1). Also, Fig. 1 shows that crude oil degradation is completely related to biomass production. These results revealed the ability of isolates to consume hydrocarbons as sole sources of carbon and energy which was apparent from biomass formation. However, biomass formation initiated approximately in the first 6 days was in relation to crude oil removal that presented its major reduction after day 6 (Fig. 1).

Fig. 1
figure 1

Oil removal profiles (a) and growth (b) of individual and co-culture of Sarocladium sp. and Cryptococcus sp. in Bushnell-Haas broth with 1% crude oil as sole carbon source

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The results of the current study show that oil removal and biomass production were about 28 and 35% higher in the co-culture than individual cultures of Sarocladium sp. and Cryptococcus sp., respectively (Fig. 1). Other studies have described the ability of co-cultures to degrade 30–50% diesel oil [10], up to 95% of aliphatic hydrocarbons in oily sludge [7], and approximately 70% PAHs [8]. In the current study, 50% crude oil removal by co-culture indicated that this yeast consortium has potential to be used in the bioremediation of petrochemical polluted environments.

Evaluation of Surface Tension and Emulsification Activity in Individual and Co-Culture of Sarocladium sp. and Cryptococcus sp.

Reducing surface tension by producing biosurfactant is a strategy for growth on hydrocarbon and oil substrate [18]. Table 1 presents the results of surface tension measurements for the two yeast isolates and their co-culture. According to Deng and colleagues (2010), a good biosurfactant producer microorganism can reduce the surface tension of the growth media to 40 mN m−1 or lower [18]. In the present study, the surface tensions of all the culture media were below 40 mN m1 (Table 1). The most efficient biosurfactant production was achieved in the case of a co-culture which was 31 mN m−1 as compared to Sarocladium sp. and Cryptococcus sp. with surface tensions of about 34 and 33 mN m−1, respectively (Table 1). A comparison between the produced biosurfactant and chemical surfactants such as SDS and Tween 80 showed that an individual culture of each yeast strain could reduce the surface tension the same way as SDS and about 10% better than Tween 80 (Table 2). These results are in agreement with the findings of Chandran, using biosurfactant produced by a single culture of T. asahii [17]. The results of this study showed that the co-culture fermentation broth could further reduce the surface tension more, by 10 and 20%, than SDS and Tween 80, respectively (Table 1).

Table 1 Comparison of surface tension and emulsifying properties in various samples
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Table 2 Cell surface hydrophobicity of yeast strains after growth on pyrene
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The oil spreading assay results indicated that the maximum production of surface active agent by individual culture and co-culture was achieved on the third day (Fig. 2). But in co-culture fermentation, surfactant production was 28% more than one detected in individual cultures. According to the results, it was concluded that the biosurfactant produced by this two yeast strains and their consortium was unstable and reduced after maximum production on the third day (Fig. 2).

Fig. 2
figure 2

Biosurfactant production by individual and co-culture of Sarocladium sp. and Cryptococcus sp.

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Some yeast strains are emulsifier producers in the presence of hydrophobic compounds [18]. Both strains were found to produce biosurfactant by their ability to emulsify the motor oil with emulsification activity (EA) 0.329 and 0.412 for Sarocladium sp. and Cryptococcus sp., respectively (Table 1). With the aim of a better connection between the microorganism and hydrophobic substrate, emulsion formation is a good strategy for enhancing the contact surface, thereby increasing the biodegradation rate. The EA of the biosurfactant revealed that it could be used as an emulsion-forming agent for hydrocarbons and oils, so as to improve the biodegradation process [33]. Both individual cultures of Sarocladium sp. and Cryptococcus sp. showed good emulsification activity (0.329 and 0.412, respectively), but that of their co-culture was more (33 to 67%) as compared to separate cultures (Table 1). However, the results showed that the emulsification activity of the individual culture for Sarocladium sp. (0.329), Cryptococcus sp. (0.412) and co-culture (0.549) were lower than the one detected in Candida lipolytica (2.19) [16] and lower than that of other synthetic commercial surfactants such as Tween 80 (2.19) and Triton X-100 (2.4) [16].

Microbial Surface Hydrophobicity

Cell surface hydrophobicity was linked to biosurfactant secretion, helping in the adhesion of microorganisms to the hydrocarbon substrate [17, 26]. The cell surface hydrophobicity of these two yeast strains in pyrene was used as a measure of potential cell affinity for hydrophobic substrates. As presented in Table 2, a comparison of the first and fourth generation clearly showed enhancement of cell surface hydrophobicity. Yeast cell treatment with pyrene as a hydrophobic substrate in four generations increased the hydrophobicity values of Sarocladium sp. and Cryptococcus sp. (38 and 85%, respectively) (Table 2); S. cerevisiae was used as a negative control [18]. Increase in cell hydrophobicity guarantees strong adhesion of cells to a hydrophobic substrate so as to form an efficient uptake system intended for biodegradation [18]. Coimbra et al. showed that Candida strains increased their produced surfactants and surface hydrophobicity when grown on hydrophobic compounds, while neither cell-associated nor extracellular emulsification activity was observed in the glucose medium [34]. Cell hydrophobicity is associated with the conformation of the mannoprotein fibrils of the yeast cell wall. Thus, in response to environmental changes, microbes can modify their surface properties by changing surface proteins [18]. Likewise, Table 2 shows that both strains used in this study originally possessed relatively high hydrophobicity. Comparing the values for the first and fourth generations showed that the change in hydrophobicity, especially for Cryptococcus sp., required induction time (Table 2).

Relation Between Oil Degradation and Biosurfactant Production in Co-culture of Sarocladium sp. and Cryptococcus sp.

As shown in Fig. 1, there was an obvious correlation between cell growth and crude oil degradation. On the other hand, as stated above, there was also a relationship between biosurfactant production, cell surface hydrophobicity, and biodegradation. As shown in Fig. 2, biosurfactant production was initiated at the first 24 h and reached its maximum value at day 3. Moreover, crude oil degradation was accelerated from the third day to the fourth day indicating the role of biosurfactant in the emulsification and solubilization of crude oil, therefore increasing its bioavailability for hydrocarbon-degrading strains. Hence, the biosurfactant-producing strains can participate in the biodegradation process, enhancing oil and hydrocarbon digestion. Increase in degradation rate by means of biosurfactant was also displayed in pyrene degradation (Fig. 3). The results of the current study clearly indicated the maximum rate of pyrene degradation in the first 3 days together with biosurfactant production (Figs. 2 and 3).

Fig. 3
figure 3

Pyrene biodegradation in MSM medium containing 100 mg l−1 pyrene as a sole carbon source

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Assessment of Pyrene Degradation in Individual and Co-culture of Sarocladium sp. and Cryptococcus sp.

The degradation rates of pyrene by yeast strains and their co-culture were determined and presented in Fig. 3. Approximately, 50% pyrene removal was achieved by individual cultures, whereas about 70% removal was achieved by their co-culture. These results are in accordance with the findings of Kim and Lee in which their co-culture performed more efficiently in the removal of four PAHs: anthracene, fluoranthene, phenanthrene, and pyrene [8]. The time of pyrene degradation showed that Sarocladium sp. and Cryptococcus sp. metabolized the hydrocarbon at the same rate and the removal of co-culture is more efficient than the individual strains (Fig. 3).

Conclusion

This study investigated the ability of yeast co-culture in biosurfactant production accompanied by pyrene biodegradation. Crude oil degradation, biosurfactant production, and pyrene degradation assays revealed the great potential of the co-culture as compared to individual cultures of Sarocladium sp. and Cryptococcus sp. Hence, the co-culture of Sarocladium sp. and Cryptococcus sp. was found to be an optimal combination for the removal of hydrocarbons and PAHs. Thus, this co-culture could be suitable for application in the bioremediation of hydrocarbon-contaminated sites.