Degradation of oil and hydrocarbons derivatives by isolated yeast strains from Ras Tanura in Saudi Arabia

2.1 Sediment sampling

Manual sampling device was used to collect sediments from three points of Ras Tanura City (26°38′38″N and 50°9′33″E), eastern province, Kingdom of Saudi Arabia which located on the Arabian Gulf surrounded by seawater. Samples were kept into sterile bags and directly transferred to laboratory in iced containers in less than 24 h to carry out enrichment step.

2.2 Yeast isolation

To enrich isolate yeast, Nutrient Broth (NB) (Oxoid, RM175.00) was used. After incubation for three days at 30˚C and agitation 200 rpm, Nutrient Agar (NA) (Oxoid, CM0003B) media was applied for culturing 10^-3–10^{-5 sample} dilutions.

2.3 Preliminary test for degradation ability

To evaluate biodegradation capability for yeast isolates, Natural Sea Water agar (NSWA) plates containing petroleum oil 0.5 % (v/v) was applied. After incubation at 30 °C for one week, nominated yeast isolate were selected for further studies.

2.4 Molecular identification of yeasts

18S rRNA was applied as molecular marker to identify yeast isolates. Total genomic DNA was purified via Yeast DNA Extraction Kit (Thermo Fisher, USA, 78870) according to manufacturer protocol. General eukaryotic primers EukA (5-AACCTGGTTGATCCTGCCAGT-3) and EukB (5-TGATCCTTCTGCAGGTTCACCTAC-3) primers were employed to amplify 18S rRNA subunits (Petroni et al., 2002). Amplification was carried out through Peqstar gradient thermal cycler (VWR, Germany) with following conditions, 95 °C for 5 min; 94 °C for 1 min, 52 °C for 1 min and 72 °C for 2 min (35 cycles); and 72 °C for 10 min (Hassanshahian et al., 2012). To visualized data, 1.5 % agarose gel electrophoresis (Ausubel et al., 1999) was used via Digi-doc it gel documentation sys-tem (UVP, UK). Amplicons were purified through Gene JET PCR Purification Kit (Thermo Scientific). ABI PRISM 3100 Genetic Analyzer was applied for sequenced and performed by Macrogen In. Seal, Korea. Aligned sequences were analyzed on NCBI website (https://www.ncbi.nlm.nih.gov/webcite) using BLAST to identify yeast isolate. Multiple sequence alignment, and molecular phylogeny was performed using ClusteralW software analysis (https://www.Clus-teralW.com). Nucleotide sequence were submitted to NCBI database to get the accession numbers.

2.5 Monitoring yeast growth and degradation ability

Colorimetric method was employed to evaluate growth rate with biodegradation activity. NSWA media supplemented with0.5 % crude petroleum oil (M1) was employed for yeast culturing with shaking (150 rpm) at 30 °C for 72 h. after adjusting media pH was to 7.0, microbial growth (OD600) was measured as optical density (OD) at a wave length of λ600 nm. Chloroform extraction method was used to measure oil consumption gravimetrically in acidified medium (APHA, 1998).

2.6 Hydrocarbons and some derivatives biodegradation potentials

Aromatic derivatives hydrocarbons (naphthylamine, phenol, naphthaleneethyldiamine, phenanthrene, naphthalene 2-sulfonate and naphthalene) and aliphatic (nhexane, n-heptane and n-pentadecane) was applied as marker for biodegradation cabbalism for yeast isolates. 600 mg/l was used for each hydrocarbon or derivative in NSWA medium. Two original yeast cultures were applied for inoculating NSW media supplemented with separate different aromatic hydrocarbons after grown yeast cultures for three days at 30 °C with agitation at 200 rpm. Spectrophotomoteric method was used for growth tracking. GC (Shimadzu GC-17A) and HPLC (Beckman system Gold 126 Solvent Module) were used for evaluating residual non-degraded hydrocarbons. Varied concentrations of naphthalene and phenol (250 to 5000 mg/l) were applied to determine degradation efficiency of the isolate.

2.7 RAPD analysis

Random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting were studied via four random primers OPA-02 (5′-TGCCGCGCTG-3′), OPA-03 (5′-AGTCAGCCAC-3′), OPA-11(5′-CAATCGCCGT-3′) and OPA-15 (5′-TTCCGAACCC-3′) (Martorell et al., 2005). PCR master mix preparation, thermal cycler program and amplicons visualization were performed according to Martorell et al. (2005). Binary system was used to convert fragment profile to 0,1 matrix data with Digi-Doc it (gel documentation system, UVP, England) integrated with Totallab software analysis (Total.lab.v.1.1).

3.1 Biodegradation assays

As shown by Table 2, three yeast isolates showed varied potentials for oil consumption. A isolate reflected higest oil degradation (8.2 %) comparing with lowest degradation % (5.5 %) which remarked C isolate. Positive correlation was detected between Oil consumption % and growth rate. Isolate B revealed moderate biodegradation activity comparing with A and C isolates. Positive relation was detected between degradation ability and growth rate.

Many studies support our findings for employed yeast as oil biodegradation microorganism by Kazemzadeh et al., 2022; Oyewole et al., 2022).

More light was added by Hesham et al., (2006) for using enrichment technique to select potential yeast isolates. They cleared that enrichment technique consider a powerful tool for tracking promising oil biodegrading ability of yeast strains. Furthermore, different researches selected and identified hydrocarbon biodegrading yeasts from varied ecosystems (Manee et al., 1998; Zinjarde and Pant, 2002). In accordance with our findings for isolating yeast from water, hydrocarbon degrader yeast strains was identified and characterized as C. albicans and Candida maltose in lagoon water from Nigeria (Matthew et al. (2008). Our results are in agreements to those of Zinjarde et al. (1997); they identified Y. lipolytica from oil-polluted Indian sea water.

3.2 Hydrocarbons and some derivatives degradation potentials of C.tropicalis strain A

Three strains capability for using varied hydrocarbons and some derivatives at 600 mg/L level was assayed after incubation for 72 h in NSW medium supplemented with selected compounds as a sole carbon source.

As shown by Table 3 and Fig. 2, biodegradation ability for hydrocarbons derivatives was varied significantly. Yeast strain C was superior for utilizing naphthylamine, phenol, naphthyl ethyl diamine and phenanthrene. Furthermore, negative correlation was detected between growth rate and length of aliphatic chain. Generally, remarked n-pentadecane with expanded chain length caused decreasing growth rate for three isolates.

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Fig. 2

Consumption % of hydrocarbons derivatives for three yeast isolates.

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Interestingly, isolate A did not show any biodegradation activity against n-pentadecane. Lowest biodegrading rate was characterized n-pentadecane with 0.55 and 0.74 of consumption % for A and B respectively. By contrary, three isolates (A, B and C) reflected highly phenol consumption rates (53.60, 66.31 and 71.25 %) respectively. Based on previous data, n-pentadecane and phenol degradation abilities for isolate C were estimated via supplemented NSW medium with different concentrations ranging from 200 to 4000 mg/L. As shown by Table 3, negative correlation was detected between isolate C growth rate and naphthalene concentrations. Highly naphthalene removing was detected at 200 mg/l. on the other hand, increasing naphthalene concentration at 4000 mg/l due to dramatic decrease of naphthalene removal (25 %). Highly phenol removal (52 %) was recorded at moderate concentration (1200 mg/L).

Our obtaining results for varied biodegradation abilities of yeast against hydrocarbons derivatives was in agreements with Kottb et al. (2019). They explained decreasing biodegrading naphthylamine and naphthalene 2- sulphonate cause of their lethal effect. More support was added to our results for increasing yeast biodegrading ability after incubation by (Lopes et al., 2019). They cleared that transferred oxygen mass was increased which due to an improvement on cellular growth and reducing sugars consumption. Also, increasing special receptor sites for binding hydrocarbons which uptake hydrocarbon uptake and may be play a key role for transfer hydrocarbons into the cell. Furthermore, catabolic pathway was accelerated via energy which generate via enzymes that introduce oxygen into the carbon.

To evaluate genetic similarity among three yeast biodegrading strains (A, B and C). Random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting with five primers was applied. As shown by Fig. 3 and table, all yeast strains showed different amplified fragments and polymorphism %. Generally, isolate A was superior for total amplified, polymorphic fragments and decreasing of genetic stability % comparing with isolates B and C. A yeast isolate reflected 75 fragments as total fragments with 64.1 % as polymorphism %. 73 amplicons with 50.3 % as polymorphism % were characterized B isolate. Lowest amplicons (71) and polymorphism % (36.1 %) were recorded for C isolate. A yeast isolate showed lowest genetic stability % (35.9 %) comparing with B and C isolates which reflected moderate and high polymorphism % (49.7 and 63.9) respectively. Distinguishable decreasing of genetic stability for isolate A may be considering the key role for biodegrading superiority. Based on RAPD data phyllogenetic tree was constructed. As a result for remarked similarity, isolates B and C were located in the same cluster with 55 % of genetic similarity. A isolate was represented as separate cluster due to lacking of genetic stability %. In accordance with our results for applied Random amplified polymorphic DNA-PCR as fingerprinting technique to understand variation of biodegradation activity among three yeast isolates, Hesham et al., (2018) used RAPD and proved correlation between random amplified polymorphic DNA profile and the geographic origin of seven toluene-degrading red yeasts (A5, A6, A12, A20, A26, A29, and A38). More support was added to our RAPD results by Meroth et al., (2003) and EL-Fikyet al., (2012). They indicated that RAPD-PCR technique consider a powerful useful tool for yeast identification and genetic diversity evaluation.

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Fig. 3

Random Amplified Polymorphic DNA (RAPD) fingerprinting results for five random primers for A, B and C yeast isolates.

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3.3 Protein profile study

Investigating protein profile for three yeast isolates consider a powerful tool to clear molecular base for genetic diversity among yeast isolates with varied biodegrading ability. Different polymorphism % was detected for three isolates. As shown by Fig. 4 and Table 5, isolate A reflected highest polymorphism % (69.2 %). On the other hand, lowest protein polymorphism % was recorded for isolates C (20 %). Isolate B showed moderate polymorphism % (37.5) based on protein profile data. Designed phyllogenetic tree based on protein profile indicated superior diversity for isolate A which located separately at 8 % of genetic similarity. Unique protein profile for isolate A could be led to its distinguishable activity for oil biodegradation.

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Fig. 4

Protein fingerprinting profile (a), band detection (b) and molecular weight calculation (c) and phyllogentic tree (d) for A, B and C yeast isolates.

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Our findings for using SDS-PAGE to explain biodegradation activity based on protein profile diversity was supported by Hofling et al. (2018). They used gel electrophoresis to investigate yeast soluble proteins of detect biodegradable plastic-degrading enzyme (PaE) from Pseudozyma antarctica JCM 10317. Corresponding patterns showed unique 36 polypeptides ranged from 14.4 to 200 kDa. Different nutritional compositions could explain protein patterns diversity as a result to express varied proteins derived from alternatives metabolic pathways. More light was added to our findings by Yu et al., (2015). They applied SDS-PAGE and N-terminal sequencing to understand Polyacrylamide (PAM) degrading by Pseudomonas putida which indicated conversion PAM amide groups into carboxyl groups by a PAM-induced extracellular enzyme from the aliphatic amidase family.