World J Microbiol Biotechnol (2014) 30:2839–2850 DOI 10.1007/s11274-014-1710-4
ORIGINAL PAPER
Biodegradation of cefdinir by a novel yeast strain, Ustilago sp. SMN03 isolated from pharmaceutical wastewater A. Selvi • Jaseetha Abdul Salam • Nilanjana Das
Received: 28 April 2014 / Accepted: 24 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Cefdinir, a semi-synthetic third generation cephalosporin antibiotic being considered as an emerging pollutant, demands removal from aquatic ecosystems. A yeast strain isolated from pharmaceutical wastewater which was identified as Ustilago sp. SMN03 by molecular techniques and was found to be capable of utilizing cefdinir as a sole carbon source. The isolate was found to degrade 81 % of cefdinir within 6 days under optimized conditions viz. pH 6.0, temperature 30 °C, a shaking speed of 120 rpm, an inoculum dosage of 4 % (w/v) and an initial cefdinir concentration of 200 mg L-1. Kinetic studies revealed that cefdinir degradation followed the pseudo-first order model, a rate constant of 0.222 per day and a half-life period of 3.26 days. Using LC–MS analysis, six novel intermediates formed during the cefdinir degradation were identified and characterized. FT-IR analysis showed that the functional groups ranging from 1,766 to 1,519 cm-1, characteristic for lactam ring were completely removed during the cefdinir degradation. The opening of the blactam ring was one of the major steps in the cefdinir degradation process. Based on the results from the present study, a possible pathway of cefdinir degradation by Ustilago sp. SMN03 was proposed. To the best of our knowledge, this is the first report on microbial degradation of cefdinir by yeast. Keywords Biodegradation Cefdinir Kinetics LC–MS Pharmaceutical wastewater Ustilago sp.
A. Selvi J. A. Salam N. Das (&) Environmental Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India e-mail: [email protected]
Introduction Antibiotics have been considered as an emerging pollutants over the past few years (Homem and Santos 2011). Pharmaceutical industries involved in the production of antibiotics discharge their wastes openly which contain some quantity of active compounds which are toxic in nature. Globally, it is estimated that one million tons of antibiotics are consumed every year (Wise 2002) and are known to be present at a concentration ranging from a few micrograms to kilograms in water and soils posing a threat to the environment (Hamscher et al. 2002). Cefdinir is an advanced third generation semi-synthetic cephalosporin antibiotic, characterized by a vinyl group at C-3 and a (Z)-2-(2-amino-4 thiazolyl)-2-(hydroxyimino) acetyl moiety at C-7 and is used for the treatment of acute respiratory related disorders and mild skin infections. Figure 1 shows the chemical structure of cefdinir. The effluents released from cephalosporin production units were reported to release harmful compounds which are resistant to biodegradation, photo-transformation and natural degradation (Wang and Lin 2012). The presence of high concentration of cephalosporin in the environment leads to very high chemical oxygen demand, thus by increasing the toxic strength of the effluent (Duan 2009). Various physico-chemical methods have been used for the treatment of pharmaceutical wastewaters which are of limited applicability because of limitations such as inefficiency of remediating high strength wastewater, high operating cost, huge labour requirement, high equipment cost, intervention of toxic by-products etc. (Homem and Santos 2011). Thus, one way to solve these problems may be to use bioremediation technology involving microorganisms which are attracting increasing attention nowadays as a less expensive and more environmentally friendly
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the isolate was grown in YEPD broth containing 100 mg L-1 of cefdinir for 10 days at 28 °C in shaking conditions. Cefdinir degradation by yeast isolate
Fig. 1 Structure of cefdinir
alternative to the conventional treatment methods (Okoh 2006). There are reports on the use of bacteria viz. Pseudomonas putida and Pseudomonas fluorescens (Krishnan et al. 2012), Bacillus and Bacteriods (Wagner et al. 2011) for degradation of cephalosporin derivatives. However, no report is available on cefdinir degradation by yeast. Therefore, the present investigation is the first attempt to assess the potentiality of yeast Ustilago sp. SMN03 for the bioremediation of cefdinir and to propose the cefdinir degradation pathway.
Cefdinir degradation efficiency was tested by growing the isolated yeast in minimal broth (MB) containing ammonium sulphate (5 g L-1), potassium dihydrogen phosphate (1 g L-1), dipotassium hydrogen phosphate (2 g L-1), magnesium sulphate (0.5 g L-1), sodium chloride (0.1 g L-1), manganese chloride (0.01 g L-1), ferrous sulphate (0.01 g L-1), sodium molybdate (0.01 g L-1), at pH 7.2 ± 0.5. Cefdinir (100 mg L-1) was added into the MB after sterilization and thoroughly mixed. Yeast culture acclimatized in YEPD broth (OD600 = 0.1) was added into cefdinir containing MB and incubated at 28 ± 2 °C for 7 days on a rotary shaker. Samples were withdrawn at regular intervals for the measurement of cell growth. Optical density (600 nm) was measured and correlated to the biomass production. Gene sequencing and identification of the yeast isolate
Materials and methods Chemicals Cefdinir (99 % purity) was kindly donated by Orchid Pharmaceuticals, Chennai, India. Dimethyl sulphoxide (DMSO) procured from SRL Chemicals, India Ltd., was used to prepare a stock solution of cefdinir (10 g L-1). All other chemicals were of high quality and obtained from HiMedia India Ltd. and SRL Chemicals India Ltd. Collection and analysis of pharmaceutical wastewater Wastewater was collected from a pharmaceutical industry located in Ranipet, Vellore Dist., India. The effluent was collected aseptically in a sterile plastic container and sealed in a plastic bag. The collected sample was transported safely to the laboratory, stored at 4 °C and analyzed for various physico-chemical characteristics using standard methods (Kavitha et al. 2012). Isolation and acclimatization of yeast The pharmaceutical wastewater was serially diluted using sterile distilled water and dilutions were spread plated onto yeast extract peptone dextrose agar [YEPD; yeast extract (10 g L-1), peptone (20 g L-1), dextrose (20 g L-1), agar (20 g L-1)]. The plates were incubated at 28 ± 2 °C for 48 h. After incubation, the yeast colony was isolated and confirmed by microscopy. The isolated yeast was further streaked onto YEPD agar plates to obtain a pure culture. For acclimatization,
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Yeast cells were grown on YEPD broth for 48 h at 28 °C. Cells were harvested by centrifuging at 8,4009g for 10 min. High molecular weight DNA was obtained from the yeast cells by phenol/chloroform extraction protocol (Cheng and Jiang 2006). The 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:50 -TGTACACACCGCCCGTC-30 and reverse-UL28R:50 -ATCGCCAGTTCTGCTTAC-30 . 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 partial and complete sequences for the genes of 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA. The purified PCR products were characterized by partial and complete 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 available on the GenBank (Altschul et al. 1990). The phylogenetic analysis was performed using CLUSTAL W (DDBJ-DNA Data Bank of Japan). A phylogenetic tree was constructed by the neighbour-joining method using TREEVIEW software and bootstrap analysis was carried on with 1,000 random samples taken from multiple alignment (Felsenstein 1985). Nucleotide sequence accession number The assembled partial and complete 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA sequences were deposited in GenBank under accession number KF922222.1.
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Growth monitoring The growth of the yeast was determined by measuring the dry weight of biomass. Samples from the culture flasks were withdrawn at regular intervals and the cell suspension was centrifuged at 8,4009g for 10 min. The supernatant obtained was transferred into pre-weighed Petri dishes and dried at 105 °C for 20 min and the cell dry weight of yeast biomass was calculated.
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curve using cefdinir as standard. The percentage of cefdinir degradation was calculated as follows, Cefdinir degradation efficiency ð%Þ ¼
Ci Cf 100 Ci ð1Þ
where, Ci is the initial cefdinir concentration and Cf is the final cefdinir concentration. Kinetic studies on cefdinir degradation
Optimization of growth parameters The acclimatized yeast culture was added to Erlenmeyer flasks (100 ml) containing 25 mL of sterile MB and cefdinir (100 mg L-1) for optimization of growth parameters. The effect of various growth parameters viz. pH (4.0–9.0), incubation temperature (20–40 °C), shaking speed (80–140 rpm), inoculum dosage (1–5 %) and initial cefdinir concentration (50–300 mg L-1) on degradation efficiency of the yeast isolate were studied. During optimization of parameters, all the parameters were kept constant except for the optimizing parameter. While optimizing pH, parameters such as temperature 28 °C, shaking speed 120 rpm, inoculum dosage 2 % and initial cefdinir concentration 100 mg L-1 were kept constant, varying the pH values ranging from 4.0 to 9.0. A similar trend was followed for other parameters too. All the experiments were carried out in triplicates. An abiotic control was maintained for each experiment.
The experimental data on the degradation kinetics of cefdinir were fitted with various kinetic models like zero order, first order, second order and pseudo-first order respectively. The equations used for the kinetic models and their half-life are as follows: Zero order (Petrucci et al. 2007), Ct C0 ¼ k0 t
ð2Þ
t1=2 ¼ C0 =2k0
ð3Þ
First order (Dykaar and Kitanidis 1996), ln Ct ¼ k1 t þ lnC0
ð4Þ
t1=2 ¼ ln2=k1
ð5Þ
Second order (Capellos and Bielski 1972), 1=Ct ¼ 1=C0 þ k2 t
ð6Þ
t1=2 ¼ 1=k2 C0
ð7Þ
Pseudo-first order (Capellos and Bielski 1972), 0
Degradation studies All the experiments on degradation of cefdinir were carried out in triplicates. The required quantity of the cefdinir stock solution was dispensed into Erlenmeyer flasks (100 mL capacity) containing sterile MB (25 mL) and was inoculated with the yeast culture. The flasks used for inoculum development were incubated on a rotary shaker (120 rpm) at 30 °C. The flasks were removed at regular intervals for analysis of residual cefdinir concentration. Uninoculated flasks were maintained as an abiotic control. Estimation of residual cefdinir concentration Degradation of cefdinir by yeast isolate was demonstrated by collecting 2 ml of cell free supernatant from each of the triplicates which were removed after 0, 1, 2, 3, 4, 5, 6, and 7 days for the estimation of residual cefdinir. The analysis was done using UV–visible spectrophotometer (ShimadzuUV-2450) following the method of Cabri et al. (2006) with minor modifications. The absorbance was measured at 285 nm. The concentration was computed from a calibration
Ct ¼ C0 ek t
ð8Þ
t1=2 ¼ ln0:5= k0
ð9Þ
where C0 is the initial concentration of cefdinir in the medium, Ct is the concentration of cefdinir at time ‘t’ K is the degradation rate constant, t1/2 is the biodegradation half-life period of cefdinir. Extraction of metabolites and LC–MS analysis The degradation products were analyzed using liquid chromatography mass spectrometer (Mashelkar and Renapurkar 2010). Bruker, HCT plus model, LC–ESI–MS analysis was performed with an HPLC system (reversed phase C18 column of 75 mm 9 4.6 mm inner diameter and 5 lm beads size, Zorbax) coupled with a mass spectrometer. For liquid chromatography, the mobile phase consisted of 90:10 (acetonitrile: water) with 0.1 % formic acid. The mass spectrometry was carried out in electrospray ionization (ESI) positive ion mode employing conditions such as heated capillary temperature at 300 °C, nebulizer gas (Nitrogen) at 35 psi and spray voltage of
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Table 1 Physicochemical characteristics of pharmaceutical wastewater Parameters
Pharmaceutical wastewater collected from Ranipet, Vellore Dist., India
Colour
Pale yellow
pH
5.0–6.0
Temperature
20–25 °C
Biological oxygen demand (mg L-1)
6,000–7,000
Chemical oxygen demand (mg L-1)
24,000–27,000
Cephalosporin (Cefdinir) (mg L-1)
125–175
Total suspended solids (mg L-1)
300–500
Total dissolved solids (mg L-1)
18,000–19,500
Total Kjeldhal nitrogen (mg L-1)
2,000–2,180
Ammonical nitrogen (mg L-1)
1,800–1,900
Chlorides (mg L-1)
90–105
Sulphates (mg L-1)
5,000–6,600
4,000 V. The flow rate of the solvent was 0.5 mL/min. The injection volume of the sample was 10 lL and a total run time of 15 min was maintained for each analysis. The detection wavelength was 285 nm. Fourier transform-infrared spectroscopy (FTIR) Infrared spectra were obtained using an IR affinity-1 FT-IR spectrophotometer (Shimadzu) using KBr pellets. The sample was prepared by collecting the cell free supernatant by centrifugation at 10,000 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 scanning range was kept from 4,000 to 500 cm-1 and the spectral resolution was 4 cm-1 (Aleem et al. 2008).
Results Analysis of pharmaceutical wastewater The physico-chemical characteristics of the pharmaceutical wastewater collected from Ranipet, Vellore Dist., India were analyzed in National Agro Foundation, Chennai and Tamilnadu Pollution control board (TPCB), Katpadi and the data is presented in Table 1. The concentration of cefdinir analyzed was in the range of 125–175 mg L-1.
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Isolation of yeast A yeast isolate, designated as SMN03 was obtained from pharmaceutical wastewater and selected on the basis of its colony characteristics and appearance under 1009 objective of the microscope. Pure cultures were made by successive sub-culturing onto YEPD plates. Stock cultures were stored on agar slants containing cefdinir (100 mg L-1).
Identification of the yeast isolate and phylogenetic analysis Colonies of strain SMN03 (Fig. 2a) on YEPD agar after 48 h of incubation were pink in colour, and about 0.7–1.0 mm in diameter, circular, rough, wrinkled, flat, opaque and convex with an undulate margin. Staining revealed ovoid, elongated budding cells of the yeast (Fig. 2b). Partial 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA gene regions were amplified and sequenced using designed primers for polymerase chain reaction (PCR). The size of the sequence was 1946 nucleotides long. The analysis of the assembled pairwise sequence alignment showed 100 % sequence similarity and 53 % query coverage with Ustilago sparsa KVU892 (accession no: JN367335.1) in similarity search using BLAST program. The phylogenetic analysis was performed and a tree was constructed with closely related organisms and presented in Fig. 3. Based on the BLAST results obtained, our strain was found to be closely related to Ustilago sparsa KVU892 (accession no. JN367335.1) and hence the isolated yeast strain was named as Ustilago sp. SMN03. Growth of Ustilago sp. SMN03 and cefdinir degradation The growth of Ustilago sp. SMN03 was studied with respect to cefdinir degradation. The yeast was found to utilize cefdinir as sole carbon and energy source. This was evident with its growth pattern in MB containing cefdinir of 100 mg L-1. There was a lag phase of 1 day before the growth and substrate degradation started. The growth was scanty at the end of day 1, producing a dry cell weight of only 0.22 g L-1. Later the growth was initiated well from day 2 and continued up to day 5 after which stationary phase was attained. Cefdinir degradation was found to be significant from the day 2. Figure 4 showed a positive correlation with an increase in yeast biomass to the degradation percentage of cefdinir. No significant difference in the cefdinir concentrations was noted in case of abiotic controls. To study the effect of DMSO (used to dissolve cefdinir) on the growth of yeast isolate, control plates were maintained using cefdinir with and without DMSO. The
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Fig. 2 a Morphology of Ustilago sp. SMN03 on YEPD after 48 h, b morphology of Ustilago sp. SMN03 under 1009 objective of bright field microscope
Fig. 3 Phylogenetic analysis of strain SMN03 and related species by neighbour-joining method. Phylogenetic tree of partial and complete gene sequences of 18S rRNA, ITS-1, 5.8S rRNA and 28S rRNA. The tree was tested by bootstrap taking 1,000 samples. The boot strap values are given at each branch and Genbank/EMBL accession numbers are included in parentheses
addition of DMSO did not show any effect on yeast growth in liquid and solid medium.
and initial cefdinir concentration of 200 mg L-1 (Fig. 5e). The isolate was able to degrade 81 % of 200 mg L-1 cefdinir after 6 days under optimized conditions.
Optimization of growth parameters Kinetics of cefdinir degradation by Ustilago sp. SMN03 Figure 5a–e shows the effect of various parameters on growth and degradation of cefdinir by the yeast strain Ustilago sp. SMN03. The yeast showed maximum cell biomass and degradation efficiency at pH 6.0 (Fig. 5a), temperature 30 °C (Fig. 5b), shaking speed 120 rpm (Fig. 5c), inoculum dosage 4 % g dry weight L-1 (Fig. 5d)
The experimental data for the degradation kinetics of cefdinir was fitted with various kinetic reaction models (Fig. 6a–d) and the best fit was found to be with pseudofirst order reaction model. The degradation rate constant (k), half-life periods (t1/2) and regression values (R2) were
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FTIR analysis
Fig. 4 Growth of Ustilago sp. SMN03 in MB and cefdinir degradation (%), (blue square) cell dry weight, (red triangle) Residual cefdinir concentration and (green circle) Abiotic control. (Color figure online)
calculated with respect to the optimal concentration (200 mg L-1) and were presented in Table 2. The half–life period of cefdinir was recorded as 3.26 days. The regression coefficient was the highest (0.986) in case of pseudofirst order which implied that the removal of cefdinir was concentration dependent process. Metabolite characterization by LC–MS The LC–MS spectra of cefdinir and its metabolites formed during the degradation by Ustilago sp. SMN03 were recorded at different time intervals viz, 0,2,4,6 and 7th day are shown in Fig. 7a–e. In Fig. 7a, the day 0 sample showed a high intense peak of retention time (RT) of 1.6 min, corresponding to the m/z value of 396.1 in the positive ion mode which confirmed the presence of the parent compound, cefdinir. On day 2 (Fig. 7b), the onset of biodegradation of cefdinir by Ustilago sp. SMN03 was observed by the presence of a new peak at the RT of 4.1 min, which corresponded to the first metabolite M1 of m/z value 364.1 in the MS spectra. On day 4 (Fig. 7c), the occurrence of parent peak with reduced intensity and two other new peaks of RT 1.9 min and 2.3 min were observed. These peaks corresponded to two new metabolites M2 (m/z = 326.1) and M3 (m/z = 338.4) respectively. The disappearance of M1 was observed on the same day too. The metabolite M2 structure showed the opened b-lactam ring of cefdinir. On day 6, the reduction in the peak intensities of M2 and M3 was observed along with the peak of the parent compound with no new compounds formed (Fig. 7d). But on day 7 (Fig. 7e), two other metabolites M4 (RT 1.2 min) and M5 (RT 0.8 min) with m/z value of 170.2 and 99.4 were observed along with the parent compound cefdinir which was found to degrade 81 % as calculated from the liquid chromatogram. The mass spectra of cefdinir and its biodegradation metabolites are given in Table 3.
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Figure 8 illustrates FTIR spectra of cefdinir before and after degradation by Ustilago sp. SMN03. The FTIR spectrum of pure cefdinir (Fig. 8a) showed the characteristic absorption peaks at 3,556.74–3,115.04 cm-1 (O–H stretch of hydroxyl group COOH), 3,028.24–2,877.79 cm-1 (C–H stretch of the cyclic ring), 1,766.80–1,681.93 cm-1 (C=O stretching of COOH group), 1,622.13 cm-1 (C=C stretching of aromatic ring), 1,598.99 cm-1 (N–H bends), 1,301.95–1,240.23 cm-1 (C–N stretching) and 746.45–623.01 cm-1 (C–S stretching). The IR spectrum of degraded products (Fig. 8b) showed complete absence of characteristic peaks of cefdinir. The absorption peak at 3,201.83 cm-1 showed the presence of O–H stretch of hydroxyl group COOH in degraded products. The absence of sharp absorption peaks between 1,700 and 1,400 cm-1 conformed the cleavage of b-lactam ring. Sharp absorption peaks at 1,402.25, 1,111.00 and 617.22 cm-1 indicated the presence of aromatic, aliphatic amines and thiol ester groups in degraded products. Possible pathway for cefdinir degradation From the LCMS results, a putative pathway of aerobic cefdinir degradation by Ustilago sp. SMN03 was initiated and shown in Fig. 9. The degradation of cefdinir initiated with removal of a hydroxyl group and lateral methylene group resulting in (7-[22(2-amino-thiazol-4yl)-2-imino-acetylamino]-3-methyl-8-oxo -5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (M1). This was further degraded, giving rise to two possible metabolites 4-{2-[2-(2-amino-thiazol-4-yl)-2-hydroxyimino-acetylamino]-vinylsulfanyl}-but-2-enoic acid (M2) in which 4-membered b-lactam ring structure was opened and ((6R,7R)7-((Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) acetamido)8-Oxo-5-Thai-1-azabicyclo [4.2.0] Oct-2-en-3-l) methylium (M3) in which carboxylic acid group was removed. The degradation pathway proceeded further while M3 gave rise to a transient metabolite, (6R,7R)-&-amino-8-oxo-3-vinyl-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (not detected in the LC–MS) and two other metabolites, 5-Vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (M4) and Thiazol-2amine (M5) which are structurally simpler compared to the parent compound inferring the potentiality of degradation by our yeast isolate.
Discussion Environmental pollution caused by the release of wide range of cephalosporin compounds into the pharmaceutical wastewater has caused serious problems of today’s life (Krishnan et al. 2012). Poor biodegradability of such compounds having blactam ring have been reported (Alexy et al. 2004). Our studies
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Fig. 5 The effect of culture conditions on growth and degradation by Ustilago sp. SMN03 which was grown in mineral medium with concentration of cefdinir (100 mg L-1) for a period of 6 days. The effect of pH, temperature, shaking speed, inoculum dosage and initial cefdinir concentration were evaluated. a Effect of pH (4.0) (blue square), 5.0 (red triangle), 6.0 (green triangle), 7.0 (lavender triangle), 8.0 (blue triangle), 9.0 (orange circle), residual cefdinir conc (%) at pH 6.0 (blue triangle). b Effect of Temperature (20 °C) (blue diamond), 25 °C (red square), 30 °C (green triangle), 35 °C (lavender triangle), 40 °C (blue circle), residual cefdinir conc (%) at 30 °C (blue square). c Effect of shaking speed (80 rpm) (lavender
diamond), 100 rpm (red triangle), 120 rpm (green triangle), 140 rpm (lavender triangle), residual cefdinir conc (%) at 120 rpm (blue square). d Effect of inoculum dosage (w/v): 1 % (lavender triangle), 2 % (red square), 3 % (green triangle), 4 % (lavender triangle), 5 % (blue circle), residual cefdinir conc (%) at 4 % inoculum dosage (blue square). e Effect of initial cefdinir concentration (mg L-1): 50 (blue diamond), 100 (red square), 150 (green triangle), 200 (lavender triangle), 250 (blue circle), 300 (orange circle) residual cefdinir conc (%) at 200 mg L-1 (blue triangle), (red diamond) abiotic control. Error bars on the curves represent the standard deviation of triplicate samples (P B 0.001). (Color figure online)
gain importance by reporting the degradation of cefdinir, a blactam antibiotic, using yeast isolated from pharmaceutical effluent. Molecular characterization suggests that the yeast belongs to the genus Ustilago and was named as Ustilago sp. SMN03. Although there are reports on biodegradation of cephalosporin derivatives using bacteria (Krishnan et al. 2012; Wagner et al. 2011; Jiang et al. 2010), no report is available for degradation of cefdinir using microbes. Therefore, our report is the first attempt for biodegradation of cefdinir by yeast.
Environmental factors such as pH, incubation temperature, shaking speed, inoculum dosage and the initial concentration of the substrate influence the growth of microbes and their degrading abilities (Elcey and Kunhi 2010). It was reported that rates of hydrolysis are a function of environmental conditions involving temperature and pH as the most important parameters during hydrolysis of three b-lactam antibiotics (Mitchell et al. 2014). Higher susceptibility to basic hydrolysis of cefdinir in aqueous solution was reported
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Fig. 6 Kinetic plots of cefdinir degradation by Ustilago sp. SMN03. a Zero order, b first order, c second order and d pseudo-first order reaction model Table 2 The kinetic parameters for degradation of cefdinir at 200 mg L-1 by Ustilago sp. SMN03 Kinetic models Zero order
First order
Parameters Regression equation
Ct = -12.22t ? 100.1
k (d-1)
12.22
t1/2
4.095
R2
0.971
Regression equation -1
Second order
Pseudo-first order
ln Ct = -0.222t ? 4.714
k (d )
0.222
t1/2
3.622
R2
0.980
Regression equation
1/Ct = -0.004t ? 0.006
k (d-1)
0.004
t1/2
125
R2
0.922
Regression equation -1
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Ustilago sparsa SMN03
Ct = 0.109 e-0.222t
k (d ) t1/2
0.222 3.26
R2
0.986
(Okamoto et al. 1996a, b). Hydrolysis of Cephalosporin C were reported by Konecny et al. (1973). Ceftiofur, a third generation cephalosporin antibiotic was reported to be broken down in both acidic and alkaline environments (Wagner et al. 2011). Variation in pH did not have any significant effect on the biodegradation of antibiotics (Nnenna et al. 2011). But in the present study, pH was found to play an important role during the growth of the yeast isolate as well as degradation of cefdinir at an optimal pH of 6.0. The concentration of target pollutant is an important factor which affects the microbial growth as well as the rate of degradation. In this study, the initial concentration of 200 mg L-1 was found to be optimum and the concentrations above optimal did not support the growth of the yeast isolate which was evident from the poor cell dry weight. Alexy et al. (2004) assessed the biodegradation of antibiotics in the closed bottle system using mixed bacterial population and the biodegradation rate of cefuroxime and ceftriaxone were found to be 77 and 30 % after 28 days of incubation. Sundararaman and Saravanane (2010) reported
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Fig. 7 Metabolite characterization by LC–MS. a Chromatogram on day 0 (inset MS spectra and parent compound structure), b chromatogram on day 2 (inset MS spectra, metabolite M1 structure), c Chromatogram on day 4 (inset MS spectra, metabolite M2 and M3 structure), d chromatogram on day 6, e chromatogram on day 7 (inset MS spectra, metabolite M4 and M5 structure)
81 % of antibiotic removal efficiency at a maximum concentration of 175 mg L-1 using anaerobic cephalosporin degrading enrichment culture, after 90 days of operation. Biodegradation of cephalosporin drugs viz. cefoxitin sodium, ceftiofur sodium and ceftriaxone sodium by P. putida and P. fluorescens was reported which showed maximum degradation efficiency of 64, 58 and 52 % respectively (Krishnan et al. 2012). In another study, Hamrapurkar et al. (2011) reported cefdinir degradation of 48.83 % by base hydrolysis method. Therefore, it was noteworthy that the isolated yeast strain Ustilago sp. SMN03 of the present study could tolerate 200 mg L-1 of cefdinir and degrade 81 % at the end of 6 days, which was found to be quite comparable to other biological and physico-chemical methods of degradation of cephalosporin derivatives reported so far. This observation is extremely
important from the perspective of environmental pollution control. The kinetic analysis of cefdinir degradation by the yeast Ustilago sp. SMN03 was done by comparing with zero order, first order, pseudo first order and second order kinetic equations. It was found that the degradation of cefdinir was dependent on the substrate concentration which was well explained by pseudo first order equation. Brites et al. (2013) reported the degradation kinetics of cephamycin C, a class of cephalosporin antibiotic through pseudo-first order model with the rate constant 1.5 9 10-3 and half life 459 h (19.13 days). The calculated half life of cefdinir, in the present study is shortened (3.26 days) compared to the earlier findings of cephamycin C indicating the astonishing potentiality of the yeast strain Ustilago sp. SMN03 in cefdinir degradation.
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Table 3 LCMS retention times and mass spectra of cefdinir and various metabolites produced by Ustilago sp. SMN03 Days
LCMS-RT (min)
Mass spectrum (m/z)
Suggested metabolites
Control
1.6
396.1
Cefdinir (parent compound)
2 days
4.1
364.1
(7-[2-2(2-amino-thiazol-4yl)-2-imino-acetylamino]-3-methyl8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (M1)
4 days
2.3
326.1
1.9
338.4
4-{2-[2-(2-amino-thiazol-4-yl)-2-hydroxyimino-acetylamino]vinylsulfanyl}-but-2-enoic acid (M2) ((6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-en-3-l) methylium (M3)
6 days 7 days
2.3
326.1
(M2)
1.9
338.4
(M3)
1.2
170.2
5-Vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (M4)
0.8
100.14
Thiazol-2-amine (M5)
Fig. 8 FTIR analysis of cefdinir degradation by Ustilago sp. SMN03. a before degradation and b after degradation
LC–MS based metabolite analyses have profound implications for prediction of the structure of degraded products of cefdinir based on their mass values (Mashelkar and Renapurkar 2010). The intermediate obtained in the present study were entirely different from other cefdinir degradation intermediates reported by other workers (Mashelkar and Renapurkar 2010; Okamoto et al. 1996a, b). Based on the characterization of metabolites, a hypothetical biodegradative pathway of cefdinir degradation of yeast has been proposed. The complex parent compound cefdinir was broken into simpler compounds
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by yeast isolate Ustilago sp. SMN03 within 6 days. The opening of the b-lactam ring was reported as one of the major steps in the degradation of cefdinir by various researchers (Okamoto et al. 1996a, b; Cabri et al. 2006; Mashelkar and Renapurkar 2010) which was also noticed in our study. Mitchell et al. (2014) observed the degradation of cefoxitin through side chain cleavage, b-lactam ring lysis and carbamate group. Common hydrolysis reaction sites on antibiotics include labile carbonyl moieties such as esters, lactones, and lactams (Waterman et al. 2002). In the present study, most of the major
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Fig. 9 Proposed pathway of cefdinir biodegradation by Ustilago sp. SMN03
functional groups such as carboxyl group, hydroxyl group, lactam group and carbonyl group were removed showing that the parent compound was biodegraded to simpler intermediates for utilization as carbon and energy source by the yeast isolate. It can be concluded that the novel yeast strain Ustilago sp. SMN03 isolated from pharmaceutical wastewater may be considered as a potential microbe for bioremediation of cefdinir-contaminated environments based on its ability to take up and degrade high concentrations of cefdinir from aqueous medium. Further research to elucidate the enzymatic mechanism of cefdinir degradation by yeast is in progress. Acknowledgments The authors express their gratitude to Mr. BMN. Moorthy, Effluent Manager, Orchid pharmaceuticals, Chennai, India for providing the pure cefdinir sample. We also extend sincere thanks to Dr. Thirumalai, Assistant Professor, Dept. of Chemistry, Thiruvalluvar University, Vellore for helping us during this study. We also take this opportunity to thank BMD labs, Chennai for LC–MS analysis, Acme Progen Biotech, Salem for identification of yeast isolate and VIT University, Vellore for providing financial assistance and laboratory facilities.
References Aleem O, Kuchekara B, Porea Y, Lateb B (2008) Effect of cyclodextrin and hydroxypropyl-cyclodextrin complexation on
physicochemical properties and antimicrobial activity of cefdinir. J Pharm Biomed Anal 47:535–540 Alexy R, Kumpel T, Ku¨mmerer K (2004) Assessment of degradation of 18 antibiotics in the closed bottle test. Chemosphere 57:505–512 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 5:403–410 Brites LM, Oliveira LM, Barboza M (2013) Kinetic study on Cephamycin C degradation. Appl Biochem Biotechnol 171:2121–2128 Cabri W, Ghetti P, Alpegiani M, Pozzi G, Justo-Erbez A, PerezMartınez JI, Villalo´n-Rubio R, Carmen Monedero-Perales M, Munoz-Ruiz A (2006) Cefdinir: a comparative study of anhydrous vs. monohydrate form microstructure and tabletting behaviour. Eur J Pharm Biopharm 64:212–221 Capellos C, Bielski BH (1972) Kinetic systems: mathematical description of chemical kinetics in solution. Wiley-Inter science, New York Cheng HR, Jiang N (2006) Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 28:55–59 Duan H (2009) Study on the treatment process of wastewater from cephalosporin production. J Sustain Dev 2:133–136 Dykaar BB, Kitanidis PK (1996) Macrotransport of a biologically reacting solute through porous media. Water Res 32:307–320 Elcey CD, Kunhi AAM (2010) Substantially enhanced degradation of hexachlorocyclohexane isomers by a microbial consortium on acclimation. J Agric Food Chem 58:1046–1054 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 Hamrapurkar P, Patil MG, Mitesh P, Sandeep P (2011) A developed and validated stability-indicating reverse phase high performance liquid chromatographic method for determination of cefdinir in the presence of its degradation products as per
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2850 international conference on harmonization guidelines. Pharm Methods 2:15–20 Hamscher G, Sczesny S, Hoper H, Nau H (2002) Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal Chem 74:1509–1518 Homem V, Santos L (2011) Degradation and removal methods of antibiotics from aqueous matrices. J Environ Manage 92:2304–2347 Jiang M, Wang L, Ji R (2010) Biotic and abiotic degradation of four cephalosporin antibiotics in a lake surface water and sediment. Chemosphere 80:1399–1405 Kavitha RV, Makam KMR, Asith KA (2012) Physico-chemical analysis of effluents from pharmaceutical industry and its efficiency study. Int J Eng Res Appl 2:103–110 Konecny J, Felber E, Gruner J (1973) Kinetics of hydrolysis of cephalosporin C. J Antibiot 26:135–141 Krishnan S, Roach B, Kasinathan K, Annamalai P, Nooruddin T, Gunasekaran M (2012) Studies on the biodegradation of cephalosporin drugs in pharmaceutical effluent using Pseudomonas putida and Pseudomonas fluorescence. Botany. July 7–11 Mashelkar UC, Renapurkar SD (2010) A LCMS compatible stabilityindicating HPLC assay method for cefdinir. Int J ChemTech Res 2:114–121 Mitchell SM, Ullman JL, Teel AL, Watts RJ (2014) pH and temperature effects on the hydrolysis of three b-lactam antibiotics: ampicillin, cefalotin and cefoxitin. Sci Total Environ 466–467:547–555 Nnenna FP, Lekiah P, Obemeata O (2011) Degradation of antibiotics by bacteria and fungi from the aquatic environment. J Toxicol Environ Health Sci 3:275–285 Okamoto Y, Kiriyama K, Namiki Y, Matsushita J, Fujioka M, Yasuda T (1996a) Degradation kinetics and isomerization of cefdinir: a
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World J Microbiol Biotechnol (2014) 30:2839–2850 new oral cephalosporin in aqueous solution. J Pharm Sci 85:976–983 Okamoto Y, Kiriyama K, Namiki Y, Matsushita J, Fujioka M, Yasuda T (1996b) Degradation kinetics and isomerization of cefdinir, a new oral Cephalosporin, in aqueous solution. 2. Hydrolytic degradation pathway and mechanism for b-lactam ring opened lactones. J Pharm Sci 85:984–989 Okoh AI (2006) Biodegradation alternative in the cleanup of petroleum hydrocarbon pollutants. Biotechnol Mol Biol Rev 2:38–50 Petrucci RH, Harwood WS, Herring G, Madura JD (2007) General chemistry: principles and modern applications, 9th edn. Pearson Education, Upper Saddle River Sundararaman S, Saravanane R (2010) Effect of loading rate and HRT on the removal of cephalosporin and their intermediates during the operation of a membrane bioreactor treating pharmaceutical wastewater. Water Sci Technol 61:1907–1914 Wagner RD, Johnson SJ, Cerniglia CE, Erickson BD (2011) Bovine intestinal bacteria inactivate and degrade Ceftiofur and ceftriaxone with multiple b-lactamases. Antimicrobiol Agents chemother 11:4990–4998 Wang X-H, Lin AY (2012) Phototransformation of Cephalosporin antibiotics in an aqueous environment results in higher toxicity. Environ Sci Technol 46:12417–12426 Waterman KC, Adami RC, Alsante KM, Antipas AS, Arenson DR, Carrier R (2002) Hydrolysis in pharmaceutical formulations. Pharm Dev Technol 7:113–46 Wise R (2002) Antimicrobial resistance: priorities of action. J Antimicrob Chem 49:585–586
ORIGINAL PAPER
Biodegradation of cefdinir by a novel yeast strain, Ustilago sp. SMN03 isolated from pharmaceutical wastewater A. Selvi • Jaseetha Abdul Salam • Nilanjana Das
Received: 28 April 2014 / Accepted: 24 July 2014 / Published online: 3 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Cefdinir, a semi-synthetic third generation cephalosporin antibiotic being considered as an emerging pollutant, demands removal from aquatic ecosystems. A yeast strain isolated from pharmaceutical wastewater which was identified as Ustilago sp. SMN03 by molecular techniques and was found to be capable of utilizing cefdinir as a sole carbon source. The isolate was found to degrade 81 % of cefdinir within 6 days under optimized conditions viz. pH 6.0, temperature 30 °C, a shaking speed of 120 rpm, an inoculum dosage of 4 % (w/v) and an initial cefdinir concentration of 200 mg L-1. Kinetic studies revealed that cefdinir degradation followed the pseudo-first order model, a rate constant of 0.222 per day and a half-life period of 3.26 days. Using LC–MS analysis, six novel intermediates formed during the cefdinir degradation were identified and characterized. FT-IR analysis showed that the functional groups ranging from 1,766 to 1,519 cm-1, characteristic for lactam ring were completely removed during the cefdinir degradation. The opening of the blactam ring was one of the major steps in the cefdinir degradation process. Based on the results from the present study, a possible pathway of cefdinir degradation by Ustilago sp. SMN03 was proposed. To the best of our knowledge, this is the first report on microbial degradation of cefdinir by yeast. Keywords Biodegradation Cefdinir Kinetics LC–MS Pharmaceutical wastewater Ustilago sp.
A. Selvi J. A. Salam N. Das (&) Environmental Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India e-mail: [email protected]
Introduction Antibiotics have been considered as an emerging pollutants over the past few years (Homem and Santos 2011). Pharmaceutical industries involved in the production of antibiotics discharge their wastes openly which contain some quantity of active compounds which are toxic in nature. Globally, it is estimated that one million tons of antibiotics are consumed every year (Wise 2002) and are known to be present at a concentration ranging from a few micrograms to kilograms in water and soils posing a threat to the environment (Hamscher et al. 2002). Cefdinir is an advanced third generation semi-synthetic cephalosporin antibiotic, characterized by a vinyl group at C-3 and a (Z)-2-(2-amino-4 thiazolyl)-2-(hydroxyimino) acetyl moiety at C-7 and is used for the treatment of acute respiratory related disorders and mild skin infections. Figure 1 shows the chemical structure of cefdinir. The effluents released from cephalosporin production units were reported to release harmful compounds which are resistant to biodegradation, photo-transformation and natural degradation (Wang and Lin 2012). The presence of high concentration of cephalosporin in the environment leads to very high chemical oxygen demand, thus by increasing the toxic strength of the effluent (Duan 2009). Various physico-chemical methods have been used for the treatment of pharmaceutical wastewaters which are of limited applicability because of limitations such as inefficiency of remediating high strength wastewater, high operating cost, huge labour requirement, high equipment cost, intervention of toxic by-products etc. (Homem and Santos 2011). Thus, one way to solve these problems may be to use bioremediation technology involving microorganisms which are attracting increasing attention nowadays as a less expensive and more environmentally friendly
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the isolate was grown in YEPD broth containing 100 mg L-1 of cefdinir for 10 days at 28 °C in shaking conditions. Cefdinir degradation by yeast isolate
Fig. 1 Structure of cefdinir
alternative to the conventional treatment methods (Okoh 2006). There are reports on the use of bacteria viz. Pseudomonas putida and Pseudomonas fluorescens (Krishnan et al. 2012), Bacillus and Bacteriods (Wagner et al. 2011) for degradation of cephalosporin derivatives. However, no report is available on cefdinir degradation by yeast. Therefore, the present investigation is the first attempt to assess the potentiality of yeast Ustilago sp. SMN03 for the bioremediation of cefdinir and to propose the cefdinir degradation pathway.
Cefdinir degradation efficiency was tested by growing the isolated yeast in minimal broth (MB) containing ammonium sulphate (5 g L-1), potassium dihydrogen phosphate (1 g L-1), dipotassium hydrogen phosphate (2 g L-1), magnesium sulphate (0.5 g L-1), sodium chloride (0.1 g L-1), manganese chloride (0.01 g L-1), ferrous sulphate (0.01 g L-1), sodium molybdate (0.01 g L-1), at pH 7.2 ± 0.5. Cefdinir (100 mg L-1) was added into the MB after sterilization and thoroughly mixed. Yeast culture acclimatized in YEPD broth (OD600 = 0.1) was added into cefdinir containing MB and incubated at 28 ± 2 °C for 7 days on a rotary shaker. Samples were withdrawn at regular intervals for the measurement of cell growth. Optical density (600 nm) was measured and correlated to the biomass production. Gene sequencing and identification of the yeast isolate
Materials and methods Chemicals Cefdinir (99 % purity) was kindly donated by Orchid Pharmaceuticals, Chennai, India. Dimethyl sulphoxide (DMSO) procured from SRL Chemicals, India Ltd., was used to prepare a stock solution of cefdinir (10 g L-1). All other chemicals were of high quality and obtained from HiMedia India Ltd. and SRL Chemicals India Ltd. Collection and analysis of pharmaceutical wastewater Wastewater was collected from a pharmaceutical industry located in Ranipet, Vellore Dist., India. The effluent was collected aseptically in a sterile plastic container and sealed in a plastic bag. The collected sample was transported safely to the laboratory, stored at 4 °C and analyzed for various physico-chemical characteristics using standard methods (Kavitha et al. 2012). Isolation and acclimatization of yeast The pharmaceutical wastewater was serially diluted using sterile distilled water and dilutions were spread plated onto yeast extract peptone dextrose agar [YEPD; yeast extract (10 g L-1), peptone (20 g L-1), dextrose (20 g L-1), agar (20 g L-1)]. The plates were incubated at 28 ± 2 °C for 48 h. After incubation, the yeast colony was isolated and confirmed by microscopy. The isolated yeast was further streaked onto YEPD agar plates to obtain a pure culture. For acclimatization,
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Yeast cells were grown on YEPD broth for 48 h at 28 °C. Cells were harvested by centrifuging at 8,4009g for 10 min. High molecular weight DNA was obtained from the yeast cells by phenol/chloroform extraction protocol (Cheng and Jiang 2006). The 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:50 -TGTACACACCGCCCGTC-30 and reverse-UL28R:50 -ATCGCCAGTTCTGCTTAC-30 . 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 partial and complete sequences for the genes of 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA. The purified PCR products were characterized by partial and complete 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 available on the GenBank (Altschul et al. 1990). The phylogenetic analysis was performed using CLUSTAL W (DDBJ-DNA Data Bank of Japan). A phylogenetic tree was constructed by the neighbour-joining method using TREEVIEW software and bootstrap analysis was carried on with 1,000 random samples taken from multiple alignment (Felsenstein 1985). Nucleotide sequence accession number The assembled partial and complete 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA sequences were deposited in GenBank under accession number KF922222.1.
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Growth monitoring The growth of the yeast was determined by measuring the dry weight of biomass. Samples from the culture flasks were withdrawn at regular intervals and the cell suspension was centrifuged at 8,4009g for 10 min. The supernatant obtained was transferred into pre-weighed Petri dishes and dried at 105 °C for 20 min and the cell dry weight of yeast biomass was calculated.
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curve using cefdinir as standard. The percentage of cefdinir degradation was calculated as follows, Cefdinir degradation efficiency ð%Þ ¼
Ci Cf 100 Ci ð1Þ
where, Ci is the initial cefdinir concentration and Cf is the final cefdinir concentration. Kinetic studies on cefdinir degradation
Optimization of growth parameters The acclimatized yeast culture was added to Erlenmeyer flasks (100 ml) containing 25 mL of sterile MB and cefdinir (100 mg L-1) for optimization of growth parameters. The effect of various growth parameters viz. pH (4.0–9.0), incubation temperature (20–40 °C), shaking speed (80–140 rpm), inoculum dosage (1–5 %) and initial cefdinir concentration (50–300 mg L-1) on degradation efficiency of the yeast isolate were studied. During optimization of parameters, all the parameters were kept constant except for the optimizing parameter. While optimizing pH, parameters such as temperature 28 °C, shaking speed 120 rpm, inoculum dosage 2 % and initial cefdinir concentration 100 mg L-1 were kept constant, varying the pH values ranging from 4.0 to 9.0. A similar trend was followed for other parameters too. All the experiments were carried out in triplicates. An abiotic control was maintained for each experiment.
The experimental data on the degradation kinetics of cefdinir were fitted with various kinetic models like zero order, first order, second order and pseudo-first order respectively. The equations used for the kinetic models and their half-life are as follows: Zero order (Petrucci et al. 2007), Ct C0 ¼ k0 t
ð2Þ
t1=2 ¼ C0 =2k0
ð3Þ
First order (Dykaar and Kitanidis 1996), ln Ct ¼ k1 t þ lnC0
ð4Þ
t1=2 ¼ ln2=k1
ð5Þ
Second order (Capellos and Bielski 1972), 1=Ct ¼ 1=C0 þ k2 t
ð6Þ
t1=2 ¼ 1=k2 C0
ð7Þ
Pseudo-first order (Capellos and Bielski 1972), 0
Degradation studies All the experiments on degradation of cefdinir were carried out in triplicates. The required quantity of the cefdinir stock solution was dispensed into Erlenmeyer flasks (100 mL capacity) containing sterile MB (25 mL) and was inoculated with the yeast culture. The flasks used for inoculum development were incubated on a rotary shaker (120 rpm) at 30 °C. The flasks were removed at regular intervals for analysis of residual cefdinir concentration. Uninoculated flasks were maintained as an abiotic control. Estimation of residual cefdinir concentration Degradation of cefdinir by yeast isolate was demonstrated by collecting 2 ml of cell free supernatant from each of the triplicates which were removed after 0, 1, 2, 3, 4, 5, 6, and 7 days for the estimation of residual cefdinir. The analysis was done using UV–visible spectrophotometer (ShimadzuUV-2450) following the method of Cabri et al. (2006) with minor modifications. The absorbance was measured at 285 nm. The concentration was computed from a calibration
Ct ¼ C0 ek t
ð8Þ
t1=2 ¼ ln0:5= k0
ð9Þ
where C0 is the initial concentration of cefdinir in the medium, Ct is the concentration of cefdinir at time ‘t’ K is the degradation rate constant, t1/2 is the biodegradation half-life period of cefdinir. Extraction of metabolites and LC–MS analysis The degradation products were analyzed using liquid chromatography mass spectrometer (Mashelkar and Renapurkar 2010). Bruker, HCT plus model, LC–ESI–MS analysis was performed with an HPLC system (reversed phase C18 column of 75 mm 9 4.6 mm inner diameter and 5 lm beads size, Zorbax) coupled with a mass spectrometer. For liquid chromatography, the mobile phase consisted of 90:10 (acetonitrile: water) with 0.1 % formic acid. The mass spectrometry was carried out in electrospray ionization (ESI) positive ion mode employing conditions such as heated capillary temperature at 300 °C, nebulizer gas (Nitrogen) at 35 psi and spray voltage of
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Table 1 Physicochemical characteristics of pharmaceutical wastewater Parameters
Pharmaceutical wastewater collected from Ranipet, Vellore Dist., India
Colour
Pale yellow
pH
5.0–6.0
Temperature
20–25 °C
Biological oxygen demand (mg L-1)
6,000–7,000
Chemical oxygen demand (mg L-1)
24,000–27,000
Cephalosporin (Cefdinir) (mg L-1)
125–175
Total suspended solids (mg L-1)
300–500
Total dissolved solids (mg L-1)
18,000–19,500
Total Kjeldhal nitrogen (mg L-1)
2,000–2,180
Ammonical nitrogen (mg L-1)
1,800–1,900
Chlorides (mg L-1)
90–105
Sulphates (mg L-1)
5,000–6,600
4,000 V. The flow rate of the solvent was 0.5 mL/min. The injection volume of the sample was 10 lL and a total run time of 15 min was maintained for each analysis. The detection wavelength was 285 nm. Fourier transform-infrared spectroscopy (FTIR) Infrared spectra were obtained using an IR affinity-1 FT-IR spectrophotometer (Shimadzu) using KBr pellets. The sample was prepared by collecting the cell free supernatant by centrifugation at 10,000 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 scanning range was kept from 4,000 to 500 cm-1 and the spectral resolution was 4 cm-1 (Aleem et al. 2008).
Results Analysis of pharmaceutical wastewater The physico-chemical characteristics of the pharmaceutical wastewater collected from Ranipet, Vellore Dist., India were analyzed in National Agro Foundation, Chennai and Tamilnadu Pollution control board (TPCB), Katpadi and the data is presented in Table 1. The concentration of cefdinir analyzed was in the range of 125–175 mg L-1.
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Isolation of yeast A yeast isolate, designated as SMN03 was obtained from pharmaceutical wastewater and selected on the basis of its colony characteristics and appearance under 1009 objective of the microscope. Pure cultures were made by successive sub-culturing onto YEPD plates. Stock cultures were stored on agar slants containing cefdinir (100 mg L-1).
Identification of the yeast isolate and phylogenetic analysis Colonies of strain SMN03 (Fig. 2a) on YEPD agar after 48 h of incubation were pink in colour, and about 0.7–1.0 mm in diameter, circular, rough, wrinkled, flat, opaque and convex with an undulate margin. Staining revealed ovoid, elongated budding cells of the yeast (Fig. 2b). Partial 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA gene regions were amplified and sequenced using designed primers for polymerase chain reaction (PCR). The size of the sequence was 1946 nucleotides long. The analysis of the assembled pairwise sequence alignment showed 100 % sequence similarity and 53 % query coverage with Ustilago sparsa KVU892 (accession no: JN367335.1) in similarity search using BLAST program. The phylogenetic analysis was performed and a tree was constructed with closely related organisms and presented in Fig. 3. Based on the BLAST results obtained, our strain was found to be closely related to Ustilago sparsa KVU892 (accession no. JN367335.1) and hence the isolated yeast strain was named as Ustilago sp. SMN03. Growth of Ustilago sp. SMN03 and cefdinir degradation The growth of Ustilago sp. SMN03 was studied with respect to cefdinir degradation. The yeast was found to utilize cefdinir as sole carbon and energy source. This was evident with its growth pattern in MB containing cefdinir of 100 mg L-1. There was a lag phase of 1 day before the growth and substrate degradation started. The growth was scanty at the end of day 1, producing a dry cell weight of only 0.22 g L-1. Later the growth was initiated well from day 2 and continued up to day 5 after which stationary phase was attained. Cefdinir degradation was found to be significant from the day 2. Figure 4 showed a positive correlation with an increase in yeast biomass to the degradation percentage of cefdinir. No significant difference in the cefdinir concentrations was noted in case of abiotic controls. To study the effect of DMSO (used to dissolve cefdinir) on the growth of yeast isolate, control plates were maintained using cefdinir with and without DMSO. The
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Fig. 2 a Morphology of Ustilago sp. SMN03 on YEPD after 48 h, b morphology of Ustilago sp. SMN03 under 1009 objective of bright field microscope
Fig. 3 Phylogenetic analysis of strain SMN03 and related species by neighbour-joining method. Phylogenetic tree of partial and complete gene sequences of 18S rRNA, ITS-1, 5.8S rRNA and 28S rRNA. The tree was tested by bootstrap taking 1,000 samples. The boot strap values are given at each branch and Genbank/EMBL accession numbers are included in parentheses
addition of DMSO did not show any effect on yeast growth in liquid and solid medium.
and initial cefdinir concentration of 200 mg L-1 (Fig. 5e). The isolate was able to degrade 81 % of 200 mg L-1 cefdinir after 6 days under optimized conditions.
Optimization of growth parameters Kinetics of cefdinir degradation by Ustilago sp. SMN03 Figure 5a–e shows the effect of various parameters on growth and degradation of cefdinir by the yeast strain Ustilago sp. SMN03. The yeast showed maximum cell biomass and degradation efficiency at pH 6.0 (Fig. 5a), temperature 30 °C (Fig. 5b), shaking speed 120 rpm (Fig. 5c), inoculum dosage 4 % g dry weight L-1 (Fig. 5d)
The experimental data for the degradation kinetics of cefdinir was fitted with various kinetic reaction models (Fig. 6a–d) and the best fit was found to be with pseudofirst order reaction model. The degradation rate constant (k), half-life periods (t1/2) and regression values (R2) were
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FTIR analysis
Fig. 4 Growth of Ustilago sp. SMN03 in MB and cefdinir degradation (%), (blue square) cell dry weight, (red triangle) Residual cefdinir concentration and (green circle) Abiotic control. (Color figure online)
calculated with respect to the optimal concentration (200 mg L-1) and were presented in Table 2. The half–life period of cefdinir was recorded as 3.26 days. The regression coefficient was the highest (0.986) in case of pseudofirst order which implied that the removal of cefdinir was concentration dependent process. Metabolite characterization by LC–MS The LC–MS spectra of cefdinir and its metabolites formed during the degradation by Ustilago sp. SMN03 were recorded at different time intervals viz, 0,2,4,6 and 7th day are shown in Fig. 7a–e. In Fig. 7a, the day 0 sample showed a high intense peak of retention time (RT) of 1.6 min, corresponding to the m/z value of 396.1 in the positive ion mode which confirmed the presence of the parent compound, cefdinir. On day 2 (Fig. 7b), the onset of biodegradation of cefdinir by Ustilago sp. SMN03 was observed by the presence of a new peak at the RT of 4.1 min, which corresponded to the first metabolite M1 of m/z value 364.1 in the MS spectra. On day 4 (Fig. 7c), the occurrence of parent peak with reduced intensity and two other new peaks of RT 1.9 min and 2.3 min were observed. These peaks corresponded to two new metabolites M2 (m/z = 326.1) and M3 (m/z = 338.4) respectively. The disappearance of M1 was observed on the same day too. The metabolite M2 structure showed the opened b-lactam ring of cefdinir. On day 6, the reduction in the peak intensities of M2 and M3 was observed along with the peak of the parent compound with no new compounds formed (Fig. 7d). But on day 7 (Fig. 7e), two other metabolites M4 (RT 1.2 min) and M5 (RT 0.8 min) with m/z value of 170.2 and 99.4 were observed along with the parent compound cefdinir which was found to degrade 81 % as calculated from the liquid chromatogram. The mass spectra of cefdinir and its biodegradation metabolites are given in Table 3.
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Figure 8 illustrates FTIR spectra of cefdinir before and after degradation by Ustilago sp. SMN03. The FTIR spectrum of pure cefdinir (Fig. 8a) showed the characteristic absorption peaks at 3,556.74–3,115.04 cm-1 (O–H stretch of hydroxyl group COOH), 3,028.24–2,877.79 cm-1 (C–H stretch of the cyclic ring), 1,766.80–1,681.93 cm-1 (C=O stretching of COOH group), 1,622.13 cm-1 (C=C stretching of aromatic ring), 1,598.99 cm-1 (N–H bends), 1,301.95–1,240.23 cm-1 (C–N stretching) and 746.45–623.01 cm-1 (C–S stretching). The IR spectrum of degraded products (Fig. 8b) showed complete absence of characteristic peaks of cefdinir. The absorption peak at 3,201.83 cm-1 showed the presence of O–H stretch of hydroxyl group COOH in degraded products. The absence of sharp absorption peaks between 1,700 and 1,400 cm-1 conformed the cleavage of b-lactam ring. Sharp absorption peaks at 1,402.25, 1,111.00 and 617.22 cm-1 indicated the presence of aromatic, aliphatic amines and thiol ester groups in degraded products. Possible pathway for cefdinir degradation From the LCMS results, a putative pathway of aerobic cefdinir degradation by Ustilago sp. SMN03 was initiated and shown in Fig. 9. The degradation of cefdinir initiated with removal of a hydroxyl group and lateral methylene group resulting in (7-[22(2-amino-thiazol-4yl)-2-imino-acetylamino]-3-methyl-8-oxo -5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (M1). This was further degraded, giving rise to two possible metabolites 4-{2-[2-(2-amino-thiazol-4-yl)-2-hydroxyimino-acetylamino]-vinylsulfanyl}-but-2-enoic acid (M2) in which 4-membered b-lactam ring structure was opened and ((6R,7R)7-((Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) acetamido)8-Oxo-5-Thai-1-azabicyclo [4.2.0] Oct-2-en-3-l) methylium (M3) in which carboxylic acid group was removed. The degradation pathway proceeded further while M3 gave rise to a transient metabolite, (6R,7R)-&-amino-8-oxo-3-vinyl-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (not detected in the LC–MS) and two other metabolites, 5-Vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (M4) and Thiazol-2amine (M5) which are structurally simpler compared to the parent compound inferring the potentiality of degradation by our yeast isolate.
Discussion Environmental pollution caused by the release of wide range of cephalosporin compounds into the pharmaceutical wastewater has caused serious problems of today’s life (Krishnan et al. 2012). Poor biodegradability of such compounds having blactam ring have been reported (Alexy et al. 2004). Our studies
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Fig. 5 The effect of culture conditions on growth and degradation by Ustilago sp. SMN03 which was grown in mineral medium with concentration of cefdinir (100 mg L-1) for a period of 6 days. The effect of pH, temperature, shaking speed, inoculum dosage and initial cefdinir concentration were evaluated. a Effect of pH (4.0) (blue square), 5.0 (red triangle), 6.0 (green triangle), 7.0 (lavender triangle), 8.0 (blue triangle), 9.0 (orange circle), residual cefdinir conc (%) at pH 6.0 (blue triangle). b Effect of Temperature (20 °C) (blue diamond), 25 °C (red square), 30 °C (green triangle), 35 °C (lavender triangle), 40 °C (blue circle), residual cefdinir conc (%) at 30 °C (blue square). c Effect of shaking speed (80 rpm) (lavender
diamond), 100 rpm (red triangle), 120 rpm (green triangle), 140 rpm (lavender triangle), residual cefdinir conc (%) at 120 rpm (blue square). d Effect of inoculum dosage (w/v): 1 % (lavender triangle), 2 % (red square), 3 % (green triangle), 4 % (lavender triangle), 5 % (blue circle), residual cefdinir conc (%) at 4 % inoculum dosage (blue square). e Effect of initial cefdinir concentration (mg L-1): 50 (blue diamond), 100 (red square), 150 (green triangle), 200 (lavender triangle), 250 (blue circle), 300 (orange circle) residual cefdinir conc (%) at 200 mg L-1 (blue triangle), (red diamond) abiotic control. Error bars on the curves represent the standard deviation of triplicate samples (P B 0.001). (Color figure online)
gain importance by reporting the degradation of cefdinir, a blactam antibiotic, using yeast isolated from pharmaceutical effluent. Molecular characterization suggests that the yeast belongs to the genus Ustilago and was named as Ustilago sp. SMN03. Although there are reports on biodegradation of cephalosporin derivatives using bacteria (Krishnan et al. 2012; Wagner et al. 2011; Jiang et al. 2010), no report is available for degradation of cefdinir using microbes. Therefore, our report is the first attempt for biodegradation of cefdinir by yeast.
Environmental factors such as pH, incubation temperature, shaking speed, inoculum dosage and the initial concentration of the substrate influence the growth of microbes and their degrading abilities (Elcey and Kunhi 2010). It was reported that rates of hydrolysis are a function of environmental conditions involving temperature and pH as the most important parameters during hydrolysis of three b-lactam antibiotics (Mitchell et al. 2014). Higher susceptibility to basic hydrolysis of cefdinir in aqueous solution was reported
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Fig. 6 Kinetic plots of cefdinir degradation by Ustilago sp. SMN03. a Zero order, b first order, c second order and d pseudo-first order reaction model Table 2 The kinetic parameters for degradation of cefdinir at 200 mg L-1 by Ustilago sp. SMN03 Kinetic models Zero order
First order
Parameters Regression equation
Ct = -12.22t ? 100.1
k (d-1)
12.22
t1/2
4.095
R2
0.971
Regression equation -1
Second order
Pseudo-first order
ln Ct = -0.222t ? 4.714
k (d )
0.222
t1/2
3.622
R2
0.980
Regression equation
1/Ct = -0.004t ? 0.006
k (d-1)
0.004
t1/2
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R2
0.922
Regression equation -1
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Ustilago sparsa SMN03
Ct = 0.109 e-0.222t
k (d ) t1/2
0.222 3.26
R2
0.986
(Okamoto et al. 1996a, b). Hydrolysis of Cephalosporin C were reported by Konecny et al. (1973). Ceftiofur, a third generation cephalosporin antibiotic was reported to be broken down in both acidic and alkaline environments (Wagner et al. 2011). Variation in pH did not have any significant effect on the biodegradation of antibiotics (Nnenna et al. 2011). But in the present study, pH was found to play an important role during the growth of the yeast isolate as well as degradation of cefdinir at an optimal pH of 6.0. The concentration of target pollutant is an important factor which affects the microbial growth as well as the rate of degradation. In this study, the initial concentration of 200 mg L-1 was found to be optimum and the concentrations above optimal did not support the growth of the yeast isolate which was evident from the poor cell dry weight. Alexy et al. (2004) assessed the biodegradation of antibiotics in the closed bottle system using mixed bacterial population and the biodegradation rate of cefuroxime and ceftriaxone were found to be 77 and 30 % after 28 days of incubation. Sundararaman and Saravanane (2010) reported
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Fig. 7 Metabolite characterization by LC–MS. a Chromatogram on day 0 (inset MS spectra and parent compound structure), b chromatogram on day 2 (inset MS spectra, metabolite M1 structure), c Chromatogram on day 4 (inset MS spectra, metabolite M2 and M3 structure), d chromatogram on day 6, e chromatogram on day 7 (inset MS spectra, metabolite M4 and M5 structure)
81 % of antibiotic removal efficiency at a maximum concentration of 175 mg L-1 using anaerobic cephalosporin degrading enrichment culture, after 90 days of operation. Biodegradation of cephalosporin drugs viz. cefoxitin sodium, ceftiofur sodium and ceftriaxone sodium by P. putida and P. fluorescens was reported which showed maximum degradation efficiency of 64, 58 and 52 % respectively (Krishnan et al. 2012). In another study, Hamrapurkar et al. (2011) reported cefdinir degradation of 48.83 % by base hydrolysis method. Therefore, it was noteworthy that the isolated yeast strain Ustilago sp. SMN03 of the present study could tolerate 200 mg L-1 of cefdinir and degrade 81 % at the end of 6 days, which was found to be quite comparable to other biological and physico-chemical methods of degradation of cephalosporin derivatives reported so far. This observation is extremely
important from the perspective of environmental pollution control. The kinetic analysis of cefdinir degradation by the yeast Ustilago sp. SMN03 was done by comparing with zero order, first order, pseudo first order and second order kinetic equations. It was found that the degradation of cefdinir was dependent on the substrate concentration which was well explained by pseudo first order equation. Brites et al. (2013) reported the degradation kinetics of cephamycin C, a class of cephalosporin antibiotic through pseudo-first order model with the rate constant 1.5 9 10-3 and half life 459 h (19.13 days). The calculated half life of cefdinir, in the present study is shortened (3.26 days) compared to the earlier findings of cephamycin C indicating the astonishing potentiality of the yeast strain Ustilago sp. SMN03 in cefdinir degradation.
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Table 3 LCMS retention times and mass spectra of cefdinir and various metabolites produced by Ustilago sp. SMN03 Days
LCMS-RT (min)
Mass spectrum (m/z)
Suggested metabolites
Control
1.6
396.1
Cefdinir (parent compound)
2 days
4.1
364.1
(7-[2-2(2-amino-thiazol-4yl)-2-imino-acetylamino]-3-methyl8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (M1)
4 days
2.3
326.1
1.9
338.4
4-{2-[2-(2-amino-thiazol-4-yl)-2-hydroxyimino-acetylamino]vinylsulfanyl}-but-2-enoic acid (M2) ((6R,7R)-7-((Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino) acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-en-3-l) methylium (M3)
6 days 7 days
2.3
326.1
(M2)
1.9
338.4
(M3)
1.2
170.2
5-Vinyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid (M4)
0.8
100.14
Thiazol-2-amine (M5)
Fig. 8 FTIR analysis of cefdinir degradation by Ustilago sp. SMN03. a before degradation and b after degradation
LC–MS based metabolite analyses have profound implications for prediction of the structure of degraded products of cefdinir based on their mass values (Mashelkar and Renapurkar 2010). The intermediate obtained in the present study were entirely different from other cefdinir degradation intermediates reported by other workers (Mashelkar and Renapurkar 2010; Okamoto et al. 1996a, b). Based on the characterization of metabolites, a hypothetical biodegradative pathway of cefdinir degradation of yeast has been proposed. The complex parent compound cefdinir was broken into simpler compounds
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by yeast isolate Ustilago sp. SMN03 within 6 days. The opening of the b-lactam ring was reported as one of the major steps in the degradation of cefdinir by various researchers (Okamoto et al. 1996a, b; Cabri et al. 2006; Mashelkar and Renapurkar 2010) which was also noticed in our study. Mitchell et al. (2014) observed the degradation of cefoxitin through side chain cleavage, b-lactam ring lysis and carbamate group. Common hydrolysis reaction sites on antibiotics include labile carbonyl moieties such as esters, lactones, and lactams (Waterman et al. 2002). In the present study, most of the major
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Fig. 9 Proposed pathway of cefdinir biodegradation by Ustilago sp. SMN03
functional groups such as carboxyl group, hydroxyl group, lactam group and carbonyl group were removed showing that the parent compound was biodegraded to simpler intermediates for utilization as carbon and energy source by the yeast isolate. It can be concluded that the novel yeast strain Ustilago sp. SMN03 isolated from pharmaceutical wastewater may be considered as a potential microbe for bioremediation of cefdinir-contaminated environments based on its ability to take up and degrade high concentrations of cefdinir from aqueous medium. Further research to elucidate the enzymatic mechanism of cefdinir degradation by yeast is in progress. Acknowledgments The authors express their gratitude to Mr. BMN. Moorthy, Effluent Manager, Orchid pharmaceuticals, Chennai, India for providing the pure cefdinir sample. We also extend sincere thanks to Dr. Thirumalai, Assistant Professor, Dept. of Chemistry, Thiruvalluvar University, Vellore for helping us during this study. We also take this opportunity to thank BMD labs, Chennai for LC–MS analysis, Acme Progen Biotech, Salem for identification of yeast isolate and VIT University, Vellore for providing financial assistance and laboratory facilities.
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