Biodegradation of benzo[a]pyrene by Rhodotorula sp. NS01 strain isolated from contaminated soil sample

 

Sanjeeb Kumar Mandal, Nilanjana Das*

Bioremediation Laboratory, School of Bio Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India

 

ABSTRACT:

Microbial degradation provides a constructive approach to remove various toxic pollutants from the environment. There are reports on microorganisms as degraders of Benzo[a]pyrene (BaP), but very less report is available on yeast as potential degrader. Hence, the present work is focused on the isolation of yeast strain by a soil enrichment technique that possess the potentiality to degrade BaP. BaP is a high molecular weight polycyclic aromatic hydrocarbon (HMW PAHs) that has been considered as a harmful environmental persistent pollutant due to its high toxicity and carcinogenic nature. Molecular identification by 18S rRNA sequences revealed the isolate as, Rhodotorula sp. NS01. The course of the degradation was studied using Fourier transform infrared (FTIR) spectroscopy and high performance liquid chromatography (HPLC). The strain was found to utilize BaP as a sole carbon and energy source and efficiently degraded 52% of 10 mg/L of BaP within 7 days. The degradation data was tested with various kinetic models and the best fit was seen with first-order model with a calculated rate constant of 0.111 per day and a half-life period of 6.2 days. FTIR analysis revealed sharp peaks at 3388.93, 1724.36 and 1645.28 cm^-1 corresponding to hydroxyl, aldehydes, ketones along with the reduction of C-C and C-H stretch in ring which confirmed the effective BaP biodegradation by Rhodotorola sp. NS01.

 

 

 

 

INTRODUCTION:

Benzo[a]pyrene (BaP) is a highly recalcitrant, high molecular weight polycyclic aromatic hydrocarbon (HMW PAH) with high genotoxicity¹.  BaP has five fused benzene in its structure. It is produced by the partially combustion of organic substances such as fossil fuels, during industrial processes and from naturally occurring forest fires². BaP has been identified as a prime pollutant of concern by the US Environmental Protection Agency (EPA)³ and this compound is also known to be one of the most effective carcinogen amongst all known PAHs.

 

The toxicity of BaP in humans (smokers) as the causative agent for lung cancer has been reported³. It is metabolized in humans and animals to form a number of metabolites that elicit toxicity⁴. Therefore, the toxic effect of BaP in various living forms and environments necessitates its removal from the system.

 

The natural degradation of BaP is a very slow process and varies according to the soil type and environmental conditions. The physico-chemical methods for remediation of BaP include chemical oxidation, photolysis, bioaccumulation, volatilization and adsorption. These methods have some drawbacks such as, high operating cost, requires long term treatment and intervention of toxic by-products, etc.^5-6. Till date, microbial degradation is considered to be the major process which affects the persistence of PAHs in nature from chronically contaminated soils^7-9 by potentially breaking down into harmless end products such as water and carbon dioxide¹⁰. Bioremediation is considered as an inexpensive, environment friendly and highly efficient clean-up technology than other physico-chemical treatment methods available for BaP removal so far⁵.

 

There are reports on remediation of BaP using various microorganisms. Armillaria sp. F022, a white-rot fungus screened from a tropical rain forest utilised BaP as a source of sole carbon and energy ¹¹. T. viride, F. Solani and F. oxysporum strains isolated from benzo[a]pyrene contaminated soil possesses varying benzo[a]pyrene degrading ability was reported by Verdin et al. ¹². Similarly, batch experiments conducted by Rentz et al.¹³ to demonstrate the degradation of benzo[a]pyrene by Sphingomonas yanoikuyae JAR02. Xu et al. ¹⁴ studied the bacterial BaP biodegradation using Kocuria sp. P10 and reported 41.75% degradation efficiency. In another investigation by Ke et al. ¹⁵ BaP degradation with algae, Selenastrum capricornutum showed 8.6% degradation efficiency. Kotterman et al. ¹⁶ employed a fungal strain, Bjerkandera sp. BOS55 and reported 8.5% of BaP degradation efficiency.

 

Table 1: Reported works on degradation of benzo[a]pyrene by various microorganisms

 

Table 1 summarizes the reported works on the biodegradation of BaP by various microorganisms. But, reports are scanty on the use of yeast as a BaP degrader. Hence, in this study, we report a potential yeast strain identified as Rhodotorula sp. NS01 showing potentially to degrade. Therefore, the objective of the present study is: (a) to isolate and identify a novel yeast strain having the potentiality to degrade high concentration of BaP, (b) to study the kinetics of BaP degradation, and (c) to analyse the BaP degradation by HPLC and FTIR analysis.

 

MATERIALS AND METHODS:        

Chemicals:

Benzo[a]pyrene (≥ 96 % purity by HPLC) were procured from Sigma-Aldrich (St. Louis, USA). A stock solution of BaP prepared at a concentration of 1 mg/mL in chloroform. All other chemicals were of high quality and acquired from Hi-Media Laboratories (Mumbai, India) and SRL Pvt. Ltd. (Mumbai, India).

 

Soil Sampling

For isolation of PAHs degrading yeast, soil sample was collected from Katpadi [12.968 °N, 79.149 °E], Vellore, Tamil Nadu, India. Soil cores (5-20 cm) were collected in sterile plastic bags and brought to the laboratory, stored at 4 °C until microbial isolation. The soil was air dried at room temperature and sieved to a particle size of <2mm.

Enrichment culture for isolation and screening of BaP degrading yeast strain:

Yeast isolation was carried out from the soil sample by standard enrichment technique. The experiments were carried out in sterilized 250 mL Erlenmeyer flask to which the required quantity of working solution of BaP (0.1 mg/L) was added and allowed to evaporate overnight or till dry before the inoculation ²¹. 100 mL of mineral medium (MM) containing per litre of potassium dihydrogen phosphate 1 g, dipotassium hydrogen phosphate 1 g, sodium chloride 5 g, ammonium sulphate 0.3 g, magnesium sulphate 0.3 g and calcium chloride 0.02 g, at pH 6.8 ± 0.5 ²² was added to the same flask containing BaP. 1g of the soil sample was added to the medium and incubated for 5 days at 28 ± 2 ºC in rotary shaker. 10 mL of the enrichment culture was shifted every 5 days to fresh sterile medium, incubated under the above conditions and the concentration of BaP in the enrichment culture was increased from 0.1 to 1.0 mg/L in a stepwise manner. After 2 weeks of the evaluation period, the spread plate and subculture method were used to obtain pure isolates. The isolate thus obtained was named as NS01 and maintained on yeast extract peptone dextrose (YEPD) agar slants containing yeast extract; 10 g/L, peptone; 20 g/L, dextrose; 20 g/L and agar; 20 g/L with BaP (1.0 mg/L) and stored at 4ºC.

 

Gene sequencing and identification of the yeast isolate:

Yeast cells were grown on YEPD broth for 48 h at 28°C. Cells were harvested by centrifuging at 8400×g for 10 min. High molecular weight DNA was acquired from the yeast cells by phenol/chloroform extraction protocol²³. The DNA samples were dissolved in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and were used for PCR amplification. The primers used were as follows: forward- UL18F:5'-TGTACACACCGCCCGTC-3' and reverse- UL28R:5'-ATCGCCAGTTCTGCTTAC-3'. PCR amplification was carried out for 35 cycles at following conditions: 30 s at 95°C, 40 s at 60 °C, 40 s at 72 °C. The amplicon comprised of complete and partial sequences for the genes of 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28SrRNA. The purified PCR products were characterized by complete and partial sequence analysis. DNA sequencing was done using the same primers as mentioned above. A BLAST (Basic Local Alignment Search Tool) program was implied for similarity search from the database obtainable on the GenBank²⁴. The phylogenetic analysis was performed using CLUSTAL W (DDBJ- DNA Databank of Japan). A phylogenetic tree was constructed by the neighbor-joining method using TREEVIEW software for displaying phylogeny²⁵. The assembled partial and complete 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA sequences of strain Rhodotorula sp. NS01 were deposited in the GenBank database under accession number KP300039.

 

Growth monitoring:

The growth of the yeast was determined by measuring the dry weight of biomass. Yeast culture acclimatized in YEPD broth (OD₆₀₀ = 0.1) was added to the series of flasks containing YEPD with and without BaP of conc (10 mg/L). The flasks were incubated at 28 ± 2 ºC for 8 days on a rotary shaker at 120 rpm. Samples were removed at regular intervals and the cell suspension was centrifuged at 10,000×g for 10 min. Then it was transferred into pre-weighed Petri dishes and dried at 105 ºC for 45 min and the dry weight of biomass was calculated.

 

Biodegradation of BaP:

All the experiments on degradation of BaP were carried out in triplicates. Degradation of BaP was performed in 100 mL of MM broth with added 10 mg/L of BaP as sole carbon and energy source and inoculated with culture broth of NS01 strain as required. The flasks used for inoculum development as well as the experimental flasks were incubated on a rotary shaker (120 rpm) at 30 ºC for 7 days. The flasks were removed at required intervals for analysis of residual substrate BaP. Uninoculated flasks were maintained as a control. The residual BaP in the culture medium was calculated using the formula:

Residual BaP (%) =                                  (1)

where, C_i is the initial concentration BaP in the medium and C_f is the final concentration of BaP.

 

Kinetics studies on BaP biodegradation:

 The experimental data of the degradation kinetics of BaP were fitted with various kinetics models like zero order²⁶, first order²⁷ and second order²⁸ respectively. Kinetic models were used to define the BaP degradation in mineral medium.

 

Extraction and characterization of degraded products by HPLC:

The degradation products were analyzed using Waters 1525 binary HPLC system equipped with a C18 column (150 mm × 4.5 µm) with dual λ absorbance detector (Waters 2487) and Rheodyne manual injector. The mobile phase consists of 40:60 (acetonitrile: water) moving at a constant flow rate of 0.8 mL/min in an isocratic mode at room temperature. Retention time for each signal was recorded at a wavelength of 254 nm with an injection volume of 20 µL and the data was processed with Empower software (developed by Waters Corporation). The total run time of 15 min was maintained for each analysis.

 

Fourier transform-infrared spectroscopy (FTIR) Analysis:

Infrared spectra were obtained using an IR affinity-1 FTIR spectrophotometer (Shimadzu) using KBr. The sample was produced by collecting the cell free supernatant by centrifugation at 10000 rpm for 10 min. The supernatant was kept for drying in vacuum drier. The dried sample was finely ground to powder and was mixed thoroughly with KBr. The range of scanning was kept from 4000 to 500 cm^-1 and the spectral resolution was 4 cm^-1.

 

RESULTS AND DISCUSSION:

Isolation, acclimation and screening of the yeast isolates:

A yeast isolate, designated as NS01 was obtained by soil enrichment culture is selected on the basis of its colony characteristics and appearance under 100X objective of the microscope after 48 h. Figure 1a, b shows the colony characteristics of the yeast isolate grown in mineral medium (MM) with added BaP (10 mg/L). Microscopic image of the yeast cells grown in medium without BaP is shown in figure 1c. The staining images (figure. 1b and 1c) showed no physiological changes in the yeast cells with response to BaP presence in the medium. This proved the capability of the yeast isolate in utilizing BaP as sole carbon source. To study the effect of chloroform (used to dissolve BaP) on the growth of the yeast isolate, control plates were maintained using BaP with and without chloroform. Addition of chloroform in BaP did not show any effect on the yeast growth in liquid as well as in the solid medium.

 

 

Figure 1: Morphological characteristics of Rhodotorula sp. NS01. (a) Rhodotorula sp. NS01 grown on MM agar after 48hr with BaP (10 mg/L), and (b) Rhodotorula sp. NS01 grown in MM agar with BaP under bright microscope (×100), and (c) Rhodotorula sp. NS01 grown in medium without BaP under bright microscope (×100).

 

Identification of the yeast and phylogenetic analysis:

Colonies of strain NS01 on YEPD agar after 48 h of incubation were pink, pigmented and about 1–3 mm in diameter, ovoid, mucoid, glistening, opaque and convex with an entire margin (figure not shown). Using designed primers for PCR, 18S rRNA, ITS1, 5.8SrRNA, ITS2 and 28S rRNA regions were amplified and sequenced. The size of the sequence was 1739 nucleotides long.

 

The sequence analysis of the 18S rRNA gene showed 99% sequence coverage and homology with Rhodotorula sp. GM5 (accession no: KF543865.1) in similarity search using BLAST program. Phylogenetic analysis (figure 2) also revealed that the strain NS01 was closely related to Rhodotorula sp. GM5 (accession no: KF543865.1). Therefore, the isolate NS01 is identified as Rhodotorula species and designated as Rhodotorula sp. NS01. The sequence results were submitted to the GenBank, NCBI database and the accession no. KP300039 was obtained.

 

Figure 2: Phylogenetic relationship of Rhodotorula sp. NS01 based on the 18S rRNA gene nucleotide sequences and related species by the neighbour-joining approach. The Genbank accession numbers are included in the parentheses.

 

Growth of Rhodotorula sp. NS01 on BaP:

The growth pattern of Rhodotorula sp. NS01 in the presence and absence of BaP as a function of time is presented in Figure 3. The metabolism of BaP by Rhodotorula sp. NS01 was assessed by the increase in the cell dry weight. Initially, the growth was found to be low in the presence of BaP but after acclimatization to BaP, the culture was capable of growing rapidly, exhibiting high growth rate. Figure 3 showed that the amount of cell dry weight produced in the medium containing BaP was much higher than the growth in the absence of BaP. This indicated the temporal course of substrate which degradation correlated well with the cell growth. After day 7, there was a decrease in the cell dry weight indicating the approach of stationary phase. No significant difference in the BaP concentration was noted in case of abiotic control. Hence, it was found that the isolated yeast strain NS01 could utilize BaP as a sole carbon and energy source showing 52% of BaP degradation at the end of 7 days of incubation.

 

Though, there are reports on BaP degradation using different microorganisms as discussed earlier, our present study gain importance by reporting the potentiality of yeast strain NS01 to degrade highest concentration of BaP (10 mg/L) in less time in comparison with the studies reported so far.

Kinetics of BaP degradation by Rhodotorula sp. NS01:

 The experimental data for the degradation kinetics of BaP (10 mg/L) was fitted with three mathematical kinetic reaction models as shown in figure 4 a-c. The results showed that, the degradation kinetics of BaP by Rhodotorula sp. NS01 can be described well with first order reaction model. The degradation rate constants (k), half-life periods (t_1/2), regression values (R²) and the regression equations under specified conditions for each reaction model are presented in Table 2. The regression coefficient, R² is highest (0.9822) in first order kinetics model, indicating that the degradation kinetics of BaP by Rhodotorula sp. NS01 followed first order reactions well. This implied that the removal of BaP by Rhodotorula sp. NS01 is a time dependant process. The calculated degradation rate constant k is 0.111 per day and the theoretical half-life of BaP is 6.26 days.

 

Similar results on BaP degradation following first order kinetics using an integrated chemical-biological treatment has been previously reported by Zeng et al. ²⁹. The calculated half-life of BaP in the present study is shortened than the earlier findings which proved the astonishing potentiality of Rhodotorula sp. NS01 on BaP degradation.

 

 

Figure 3: Growth performance of Rhodotorula sp. NS01 in the presence and absence of benzo[a]pyrene having concentration of 10 mg/L.

 

Figure 4: Kinetic plot of (a) zero order, (b) first order and (c) second order reaction model for benzo[a]pyrene degradation by Rhodotorula sp. NS01.

Table 2:  Kinetic parameters for degradation of benzo[a]pyrene at 10 mg/L by Rhodotorula sp. NS01

Kinetics Model	Parameters	Rhodotorula sp. NS01
Zero order	Regression equation	Ct =  -0.7517t + 9.8733
Ct-Co=Kt	K (day-1)	-0.7517
T1/2= Co/2Ko	T1/2	6.56
	R2	0.9653
First order	Regression equation	lnCt= -0.1106t + 2.3276
lnCt = K1t +  lnCo	K (day-1)	-0.1106
T1/2=ln2/K1	T1/2	6.26
	R2	0.9822
Second order	Regression equation	1/Ct = 0.017t + 0.0901
1/Ct=1/Co + K2t	K (day-1)	0.017
T1/2=Co/2K2	T1/2	652.8693
	R2	0.9784

 

Analytical methods for bioremediation of BaP:

HPLC was used to monitor the degradation of BaP¹³. The extraction procedures are efficient in extracting the BaP residues from the MM medium for HPLC analysis. The HPLC analysis revealed the efficient BaP degradation by NS01 strain grown in MM medium utilized BaP, thus confirming the potentiatily of the yeast strain. The degraded product peaks of BaP obtained at the end of 7 days are compared with the HPLC peaks for BaP standard (Figure not shown). Day 0 sample showed a single high intense peak of retention time (RT) 1.641 min confirming the presence of the parent compound, BaP. At the end of day 7, the biodegradation of BaP by Rhodotorula sp. NS01 was observed by the presence of two other new peaks at RT of 1.007 and 1.464 min. These peaks suggested that the BaP degraded products by Rhodotorula sp. NS01. Previous findings of BaP degradation by microorganisms reported the ring fission products (30), reduction of C-H ring structure forming BaP enantiomers and oxidised products ^{16, 31-33} as most possible metabolites.

 

The FTIR spectral bands, before and after degradation validated a positive indication of BaP degradation by the isolated yeast strain, Rhodotorula sp. NS01. These results suggest that the parental compound structure has undergone significant change after degradation by the isolate. The FTIR spectrum (figure 5a) of pure BaP (day 0) revealed characteristic absorption peaks at 1467.83 cm^-1 and 1176.58-1346.31 cm^-1 corresponding to C-C stretch in aromatic ring.  The peaks at 3028.24 cm^-1 and 686.66-871.82 cm^-1 corresponds to C-H stretch. The spectra of the degraded products of BaP at the end of day 7 (figure 5b) showed a sharp shift at 3388.93 cm^-1 corresponding to O-H stretch of alcohol. Other characteristic peaks such as 2916.37 cm^-1 corresponding to C-H stretch, 1724.36 cm^-1 and 1645.28 cm^-1 corresponding to C=O stretch of aldehydes, ketones. The presence of peaks at 1463.97 cm^-1 and 800.46-617.22 cm^-1 indicate reduction of C-C and C-H stretch in ring confirming the occurrence of biodegradation. These results suggest that the isolated yeast strain has affinity for C-H aromatic stretches and hence degrades the parent compound³³.

 

Figure 5: (a) FTIR spectrum of benzo[a]pyrene as control. (b) FTIR spectrum of benzo[a]pyrene after degradation by Rhodotorula sp. NS01.

 

CONCLUSION:

A yeast isolate, Rhodotorula sp. NS01 obtained by enrichment method from the soil environment was found to be capable of degrading BaP at a concentration of 10 mg/L in aqueous medium utilizing BaP as sole carbon and energy source. The effectiveness of degradation was confirmed by analytic methods like HPLC and FTIR studies. Thus, the results of the present study confirmed the potentiality of benzo[a]pyrene degrading yeast strain which could serve as bioresource for the efficient degradation of benzo[a]pyrene polluted environment. Further research to elucidate the enzymatic mechanism of BaP degradation by yeast is in progress.

 

CONFLICT OF INTERESTS:

The authors declare that there is no conflict of interest regarding the publication of this paper.

 

 

ACKNOWLEDGMENTS:

We thank Acme Progen Biotech Pvt. Ltd., Salem for yeast identification. Financial assistance and laboratory facilities provided by VIT University, Vellore, India are acknowledged.

 

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Accepted on 31.05.2017        © RJPT All right reserved