Introduction

Lindane (γ-HCH), an organochlorine compound is one of the most important pesticides used in agriculture during the last half century. It is synthesized by photochemical chlorination of benzene under UV light, along with the formation of four major stereo isomers (α, β, γ and δ HCH) (Phillips et al. 2005). Out of the four isomers, lindane is the only one with insecticidal property (Manickam et al. 2007). Lindane is listed as a priority pollutant by USEPA as it is seen as a possible carcinogen. The half-life period for lindane in soil and water was reported as 708 and 2,292 days respectively (Beyer and Matthies 2001). Although nowadays the use of lindane is restricted or altogether banned in most countries, residues of lindane are found all over the world in soil, water, air, plants, agricultural products, animals, food and humans (Pinero et al. 2007; Kidd et al. 2008; Herrero-Mercado et al. 2010; Fuentes et al. 2011; Alvarez et al. 2012).

Biodegradation is a more efficient and environmentally friendly method for detoxification of hexachlorocyclohexane (HCH) residues compared to physical and chemical methods (Zhang et al. 2010). A number of genera of bacteria (Chaudhary et al. 2006; Manickam et al. 2007; Zhang et al. 2010; De Paolis et al. 2013), filamentous fungi (Dritsa et al. 2009; Sagar and Singh 2011; Guillén-Jiméneza et al. 2012), actinomycetes (Fuentes et al. 2011; Alvarez et al. 2012) and microalgae (Zhang et al. 2012) have been reported with the ability to degrade lindane. But the reports are scanty on the potentiality of yeast as lindane degrader. In our previous work, we reported a yeast strain, identified as Rhodotorula sp. VITJzN03 with lindane degradation potentiality in liquid media (Salam et al. 2013). Removal of lindane depends on specific environmental conditions in the locality and on the biodegradation potential of the used microbial population. The knowledge of degradation kinetics enhances the understanding of lindane degradation and contributes towards the development of an appropriate in situ bioremediation strategy. It is further discovered that most studies lack information regarding the enzymatic mechanism of lindane degradation. A detailed study on enzymatic mechanisms and degradation pathway is thus an essential requirement for the successful implementation of microbes.

Therefore, the objectives of the present study were: (1) to isolate and identify novel yeast strain having potentiality to degrade high concentration of lindane (2) to study the kinetics of lindane degradation (3) to elucidate the involvement of key enzymes in the degradation and (4) to propose the possible metabolic pathway of lindane degradation.

Materials and methods

Chemicals

Lindane (γ-HCH, >99.9 % purity) and all enzyme substrates were purchased from Sigma-Aldrich (USA) while methanol, acetone and ethyl acetate were procured from SRL. India Ltd. A stock solution of lindane was prepared using 104 mg lindane dissolved in 100 mL methanol. The media components used for culture media preparation were purchased from Hi-media India Ltd. All the other chemicals used throughout the study were of high quality, obtained from standard manufacturers.

Soil Sampling

For isolation of lindane degrading yeast, rhizospheric soil was collected from sugarcane fields at Villianur (11° 55′ N, 79° 45′ E″), Pondicherry, India, which had a long history of lindane application for more than 15 years. Soil cores (0–20 cm) taken from selected spots were collected in sterile plastic bags. The physico-chemical property of the soil was analyzed from the National Agro Foundation, Chennai and listed in Table 1.

Table 1 Selected physico-chemical properties of the soil used for isolation of yeast, VITJzN04
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Enrichment culture for isolation and screening of lindane degrading yeast strain

Yeast isolation was carried out from the rhizospheric soil samples by standard enrichment technique. The experiments were conducted in 250 mL Erlenmeyer flasks with 100 mL Mineral medium (MM; contained per litre-ammonium sulfate; 5 g, potassium dihydrogen phosphate; 1 g, dipotassium hydrogen phosphate; 2 g, magnesium sulfate; 0.5 g, sodium chloride; 0.1 g, manganese chloride; 0.01 g, ferrous sulfate; 0.01 g, sodium molybdate; 0.01 g, at pH 7.2 ± 0.5) and 10 mg L−1 lindane. 5 g of the soil sample was added and incubated for 5 days at 28 ± 2 °C in rotary shaker. 10 mL of the enrichment culture was transferred every 5 days to fresh sterile medium, incubated under above conditions and the concentration of lindane in the enrichment culture was increased from 10 to 100 mg L−1 in a step wise manner. After 2 weeks evaluation period, the spread plate and subculture method were used to obtain pure isolates. The isolate thus obtained was named VITJzN04 and maintained on yeast extract peptone dextrose (YEPD) agar slants with lindane (100 mg L−1) at 4 °C.

Identification of the yeast strain

The lindane degrading yeast was morphologically and physiologically characterized, classified and further identified in a polyphasic approach. Primary morphological characterization was done using bright field microscopy and atomic force microscopy. The isolate which resembled like yeast were further subjected to biochemical characterization using Vitek-2, compact yeast card reader with software version V2C 1.01 (CFRD, Kerala, India).

Taxonomic characterization of the yeast strain

The yeast strain was characterized and identified based on microscopic, cultural characteristics and rDNA (18S, ITS regions and D1/D2 domains) gene analysis. The genomic DNA of the isolated yeasts was amplified by using the primers, UL18F:5′-TGTACACACCGCCCGTC-3′ and UL28R:5′-ATCGCCAGTTCTGCTTAC-3′ (universal primers; both designed by Acme progen biotech India pvt ltd). The purified PCR products were characterized by partial and complete sequence analysis of the genes. The resulting consensus rDNA sequences were then compared to the non-redundant NCBI database using BLAST similarity searches (Altschul et al. 1990). A collection of closely related sequences from this database was used to perform multiple alignment analysis using the CLUSTAL X program. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 (Tamura et al. 2011). The partial and complete rDNA sequences of the lindane degrading yeasts were submitted to the GenBank database under the accession number JX454449.

Batch cultures of yeast strain in mineral medium supplemented with lindane

Yeast inoculums were prepared in YEPD broth. For inoculum preparation, 48 h cultures were pelleted out, washed with phosphate buffer and resuspended into MM supplemented with lindane (100 mg L−1) as sole carbon and energy source. The cultures were incubated for 10 days. After incubation, the cell suspension was centrifuged at 10,000×g for 10 min. The pellet was washed with methanol to remove residues of lindane. Then it was transferred into pre weighed Petri dishes and dried to constant weight at 105 °C for 45 min and the dry weight of biomass was calculated.

To study the effect of environmental factors on the growth and lindane gradation by the yeast, the flasks with 25 mL MM containing 100 mg L−1 lindane was inoculated with 1 mL of YEPD broth culture of VITJzN04 (OD600 = 0.1) and incubated at 28 ± 2 °C on a rotary shaker for 10 days. The effect of the pH of the medium (3–8), incubation temperature (20–40 °C), inoculum dosage (0.02–0.1 g L−1 dry weight) and initial lindane concentration (200–1,000 mg L−1) were studied. The tests were conducted in triplicates. Abiotic and biotic controls were maintained for each experiment.

Kinetics studies on lindane biodegradation by VITJzN04

Biodegradation kinetic studies for VITJzN04 were performed in 100 mL flasks in triplicates. Each flask contained 25 mL MM spiked with lindane (600 mg L−1) and optimized dosage yeast inoculum. The broth cultures were incubated in an orbital shaker (120 rpm) at 30 °C for 8 days. The flasks were removed at required intervals for analyses of inorganic chloride, residual substrate lindane, and intermediary metabolites. The residual lindane in the culture medium was calculated using the formula:

$$ {\text{Residual}} \, {\text{lindane}}\,( \%) = \left( {\frac{{C_{t} }}{{C_{0} }}} \right) \times 100 $$
(1)

where, C0 is the initial concentration of lindane in the medium and Ct is the concentration at time t.

The zero order (Petrucci et al. 2007), first order (Dykaar and Kitanidis 1996), second order (Capellos and Bielski 1972) and pseudo first order (Capellos and Bielski 1972) kinetic models were used to define the lindane degradation in mineral medium.

Effect of H2O2 on lindane degradation

Since H2O2 has been commonly used to facilitate the degradation of chemical pollutants, especially some of the persistent organic pollutants, the effect of H2O2 in the enhancement of lindane degradation by VITJzN04 was studied. In this experiment, the H2O2 levels were set as 0, 1, 5, 10, 12 m mol L−1 and added to MM with the yeasts, incubated under the optimized condition on a rotary shaker. Cultures were withdrawn on the 3rd day for estimation of lindane degradation. Controls were run simultaneously with lindane and H2O2 to assess the effect of lindane removal by H2O2 alone.

Enzyme assays

The activities of lindane degrading enzymes were checked in the yeast culture at different time intervals. The yeast cells were grown in the MM with 100 mg L−1 lindane as carbon and energy source. For cell-free extracts, cells were harvested and washed twice in 100 mM Tris–HCl (pH-7.4) and resuspended in the same buffer. The re-suspended cells were incubated in ice, followed by sonication in three pulses of each 10 s. This cell free extract was used for all enzyme assays. The enzyme activity in culture supernatant was also estimated simultaneously. The crude extracts from yeast cells grown in MM without lindane was used as controls.

The enzymes viz. lindane dechlorinase and lindane dehalogenase were assayed following the method described by Zhang et al. (2009) with minor modifications using lindane and 1-chlorobutane (1-CB) as substrates respectively. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of chloride ion per min under the experimental conditions. The release of chloride ion was monitored according to the procedure of Bergmann and Sanik (1957). 2,5-dichlorohydroquinone (2,5-DCHQ) reductive dechlorinase activity was assayed using 2,5-DCHQ as substrate (Miyauchi et al. 1998). Laccase (Lac) activity was determined by the oxidation of ABTS method (More et al. 2011). Lignin peroxidase (Lip) and Manganese peroidase (MnP) activities were determined as described by Hussaini et al. (2011), using veratryl alcohol and MnSO4 as substrates.

Analytical methods

Colorimetric assay for lindane dechlorination

Mineralization of lindane by yeast isolate was confirmed by estimating the release of free chloride ion by mercuric thiocyanate method (Bergmann and Sanik 1957). 2 mL of cell free supernatant from each of the triplicates were removed after regular intervals for the estimation of free. The color developed was measured at 460 nm by a spectrophotometer (UV-1800-Shimadzu). The amount of chloride was computed from calibration curve using NaCl as standard.

Extraction and characterization of degraded products by GC–MS and FTIR

Residual lindane and the degraded products in the culture broths were determined by GC–MS and FTIR analysis. Flasks were withdrawn at regular intervals for analysis of degraded products. The residual lindane as well as the degradation products were extracted and analysed by GC–MS as described by Salam et al. (2013). The spectra were compared with respective spectra of authentic compounds and also with the mass profile of the same compound available in the National Institute of Standard Technology (NIST) library, USA.

The FTIR spectra of lindane and degraded products were used to determine the vibrational frequency changes in functional groups. The extracted degraded products dissolved in ethyl acetate, were mixed with KBr and made in the form of pellets (13 mm in diameter and 1 mm thickness). IR spectroscopy was investigated with an IR affinity-1 FT-IR spectrophotometer (Shimadzu). The scanning wavenumber ranged from 4,000 to 400 cm−1 and the spectral resolution was 4 cm−1.

Statistical analysis

Mean of three replications was considered as the final reading for all the analysis. Data were analyzed using analysis of variance (ANOVA) using Prism6 software (GraphPad Inc.). The significant difference (P ≤ 0.001) was calculated.

Results

Isolation and identification of VITJzN04

Microorganisms associated with rhizospheric soil are considered efficient agents to remediate pesticide (Elcey and Kunhi 2010). In this study, highly efficient indigenous yeast strain was obtained from sugarcane fields by enrichment culture, which was capable of utilizing lindane as a sole carbon and energy source in mineral medium. The yeast strain VITJzN04 was identified using a polyphasic approach, combining morphological, physiological and genetic identity and characters. The yeast strain when cultured on YEPD agar plates for 48 h (Fig. 1a), formed colonies approximately 5 mm diameter, which appeared circular, convex, creamy white colour with an entire margin. The microscopic images of VITJzN04 under bright field and atomic force microscopes (AFM) are given in the Fig. 1b, c respectively.

Fig. 1
figure 1

Morphological characteristics of strain VITJzN04 a on YEPD agar after 48 h, b under bright field microscope (×100) and c under atomic force microscope (×25 μm)

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The biochemical characterization was used to identify the isolate to genus level, which was performed by rapid identification using a Vitek-2 compact yeast card reader. The results of rapid biochemical test (Supplement Table 1) revealed that the lindane degrading yeast showed maximum identity with genus Candida with a confidence level of 99 %.

The sequence and phylogenetic analysis of the strain VITJzN04 were performed by neighbour-joining algorithm as shown in Fig. 2. The partial rDNA sequence (1,415 bp) was matched with those published in the NCBI database, and close relatedness was seen with the genus Candida. Therefore, the isolated yeast strain was named as Candida sp. VITJzN04 and accessible in the international culture maintenance MCC in NCCS, Pune, India.

Fig. 2
figure 2

Phylogenetic analysis of VITJzN04 and related species by the neighbor-joining approach. The Genbank accession numbers are included in the parentheses. Boot strap values are given at branch node. The scale bars represent 0.05 substitutions per site

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Degradation characteristics of lindane by VITJzN04

Growth kinetics

In the growth experiments, strain VITJzN04 grew well, utilizing lindane as the sole carbon and energy source (Fig. 3a). When grown in an initial concentration of 100 mg L−1, the complete utilization of lindane was noted within 3rd of incubation and the culture approached stationary phase on the 4th day. A maximum biomass of 1.3 g L−1 was marked in this case. The simultaneous chloride release into the medium was examined and significant correlation between chloride release, lindane degradation and biomass yield was noted.

Fig. 3
figure 3

a Growth of VITJzN04 in MM with lindane (100 mg L−1) corresponding to lindane degradation and chloride ion release; Optimization of growth parameters b pH, c temperature, d inoculum dosage and e lindane concentration. The bar graphs represent the cell dry weight and line graphs represent the lindane degradation. Error bars on the curves represent standard deviation of triplicate samples. *P ≤ 0.0001

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Optimization of the lindane degrading conditions by strain VITJzN04

The optimum range of the five significant factors: pH 3–8 (Fig. 3b); temperature 20–40 °C (Fig. 3c); inoculum dosage 0.02–0.1 g L−1 (Fig. 3d) and lindane concentration 200–1,000 mg L−1 (Fig. 3e) affecting degradation were determined in separate experiments. The growth and utilization of lindane by VITJzN04 was found greatly influenced by the tested factors. Optimum pH for lindane degradation by VITJzN04 was found as 7. At pH below 5 and above 7, growth of yeast strain was found to be low. At an incubation temperature of 30 °C, initial lindane concentration was reduced by 92 %, therefore this temperature was considered as optimum. The optimum dosage of inoculum of VITJzN04 for the degradation of 100 mg L−1 was found as 0.06 g L−1. At this dosage, the degradation process reached equillibrium within 2 days. In the present study, VITJzN04 showed great efficacy of degrading lindane until an initial concentration 600 mg L−1 in 6 days, after which the degradation efficiency decreased significantly with the increase in concentration. Therefore, initial concentration of 600 mg L−1 was the optimized concentration of lindane.

Kinetic studies of lindane degradation by VITJzN04

The degradation of lindane (600 mg L−1) was fitted with the four mathematical kinetic models as shown in Fig. 4a–d. The results show that the degradation kinetics of lindane by VITJzN04 can be described well by both first-order and pseudo-first-order reaction kinetics with respect to the aqueous concentration of lindane. The apparent kinetic rate constants (K), half-lives (T 1/2) and the regression equations under given conditions for each reaction model are presented in Table 2. The regression coefficient, R2 is highest (0.954) in both the first-order and pseudo-first order kinetic model, indicating that the degradation kinetics of lindane by VITJzN04 follows first-order and pseudo-first-order reactions well. This implies that the removal of lindane by VITJzN04 is a time dependant process. The calculated degradation rate constant K is 0.588 day−1 and the theoretical half-life of lindane is 1.17 days.

Fig. 4
figure 4

Kinetic Plot of a zero order; b first-order; c second-order and d pseudo-first order reaction model for lindane degradation by VITJZN04. e Effect of H2O2 on degradation of 600 mg L−1 lindane by VITJzN03 at 30 °C for 3 days (*P ≤ 0.0001)

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Table 2 The kinetic parameters for the degradation of lindane at 600 mg L−1 by VITJzN04
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Effect of H2O2 in the enhancement of lindane degradation

The effect of H2O2 at various concentrations on enhancement of lindane degradation by VITJzN04 was studied. As seen in Fig. 4e, within the range of 0–10 mmol L−1, H2O2 exhibited significant promotion of lindane degradation by the yeast strain. About 3–12 % lindane was oxidized when the concentration of H2O2 was increased from 1 to 12 mmol L−1 in the medium without yeast culture. The addition of H2O2 in the culture medium of VITJzN04 enhanced the removal of lindane from 60 to 92 % on the third day of incubation.

Elucidation of lindane degradation pathway

Intermediates of lindane identified by GC–MS measurement

As shown in Fig. 5a, GC–MS analysis of lindane and its products after complete degradation identified six possible metabolites using an NIST library identification program. Lindane, which eluted with a retention time of 15.7 min, was gradually degraded into infinitesimal quantities. Sample withdrawn on the day 2, indicated the presence of 2 possible new intermediate peak that eluted at 21.3 and 24.06 min was captured and identified as γ-pentachlorocyclohexane (γ-PCCH) and trichloro-2,5-cyclohexadiene-1-ol (TCCH-1-ol), both of which was detected on day 4 as well, indicating that they were formed during the initial phase of degradation and was then transformed into other compounds for further degradation. Another three apparent metabolites that were eluted at 18.59, 14.02 and 9.12 min were seen on 4th day of incubation and was confirmed to be 2,5-Dichlorohydroquinone (2,5-DCHQ), 2-Chlorohydroquinone (2-CHQ), Hydroxymuconicsemialdehyde (HMSA), in the order, according to the NIST library. On the 6th day, a new product eluted at 8.06 min, identified as maleyl acetate (MA) appeared in chromatogram, simultaneously with the absence of parent lindane peak, indicating complete transformation of lindane into metabolites by VITJzN04. Eventually, no persistent accumulative product was detected by GC–MS analysis and base peak was detected on the final day of assay. The mass spectra of the lindane and degraded products are given in Table 3.

Fig. 5
figure 5

a GC–MS analysis of lindane and its degradation products at 2, 4, 6 and 8 days; b FTIR analysis of lindane before (pure lindane) and after degradation by strain VITJzN04 for 3 days

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Table 3 GC retention times and mass spectra of lindane and the various metabolites produced by VITJzN04
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FTIR spectra

FTIR spectra of control lindane (Fig. 5b) showed the specific peaks in CH stretch (above 3,000 cm−1), C–C skeletal vibrations (1,350–1,000 cm−1), CH–Cl stretch (800–700 cm−1) and benzene rings corresponding to the peak at 1,650–1,600 cm−1. The second spectra illustrated, degraded products of lindane on day 3 showed peak at 3,600 cm−1 for hydroxyl group for aromatic primary alcohols representing hydroxyl derivatives of chlorocyclohehaxadienes; peak at 1,680 cm−1 for quinone stretch supporting the formation hydroquinones; peak at wave number 1,300–1,400 cm−1 indicated aldehyde stretches and benzene rings. Peak around 800–600 cm−1 indicated the presence of C–Cl stretch.

Proposed lindane degradation pathway by strain VITJzN04

From the number of aromatics identified by the detection methods discussed above, a possible degradation pathway is proposed (Fig. 6). In other words, the parent lindane was first dechlorinated to form, penta-chloro and subsequently the tetra-chloro derivative. Further by multi step reduction and dechlorination, 2,5-DCHQ is formed which is reduced to 2-CHQ. The closed ring is then opened and hydrolysed to produce HMSA. HMSA is oxidized to form maleyl acetate which would be incorporated directly into cellular tricarboxylic acid cycle for generation of energy. Some undetected intermediates such as dichlorocyclohexa-2,5-diene-1,4-diol (DCCH-2,5-diene-1,4-diol) and hydroquinone (HQ) are shown within doted-lines. It is supposed that these compounds may have accumulated to trace amounts and was spontaneously transformed. The existence of these transient metabolites can be inferred from subsequent compounds detected. Similar degradation route has been observed in a bacterial strain Sphingomonas japonica UT26 (Nagata et al. 2007).

Fig. 6
figure 6

The proposed lindane degradation pathway by strain VITJzN04. The complete arrows represent products identified by GC–MS. The doted arrows represent transient metabolites, which were not detected in GC–MS

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Enzymatic activities during lindane degradation

Table 4 represents the enzyme activities in the culture supernatant and cell lysate on the 2nd, 4th and 6th day of lindane degradation by VITJzN04. On the initial phase of lindane breakdown, lindane dechlorinase had profound activity both in the supernatant and the cell lysate whereas the reverse trend was observed for other degradative enzymes studied. Lindane dechlorinase and dehalogenase activities reduced with time, as the chlorinated compounds seemed to reduce with time. The DCHQ reductive dechlorinase activity was negligible on the day 2, but was found to be increased on the 4th day and further decreased over incubation time. Similar activity was seen in case of other enzymes such as Mn peroxidase and lignin peroxidase. The activity of lindane dechlorinase, dehalogenase and peroxidases was more in cell supernatant than in the cell lysate which implicated the extracellular nature of these enzymes.

Table 4 Enzyme activities (U/mL) in the culture supernatant and cell free extracts of VITJzN04
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Discussion

Lindane, a broad spectrum synthetic organochlorine pesticide, has been heavily used throughout the world for pest control both in agriculture and medicine during the past few decades, resulting in widespread contamination. Bioremediation employs the use of living microorganisms to degrade and detoxify pollutants, and is generally implied to be most significant route determining the fate and behaviour of xenobiotics in the environment (Singh 2009). Several microorganisms capable of degrading lindane have been isolated from diverse geographic locations. Yeast possesses the biochemical and ecological capacity to degrade environmental organic chemicals, either by chemical modification or by influencing chemical bioavailability (Singh 2009). But the potential use for yeast in bioremediation of lindane and other pesticides has not received the attention it deserves. This report is a complete elucidation of the mechanism of lindane degradation by the yeast species.

In the present study, isolation of lindane-degrading yeast by enrichment culture from sugarcane fields, allowed us to obtain a very potent and fast growing yeast strain, VITJzN04, which was identified was Candida sp. based on morphological and 18S rDNA gene analysis. Previous research has shown that the potential lindane-degrading microorganism were mostly from genera Pseudomonas, Sphingomonas, Streptomyces, and Fusarium (Salam et al. 2013), while Candida sp. VITJzN04 appeared to be a new species that was found greatly effective in degrading lindane and its metabolites. Members of this genus are ubiquitous in the environment and some isolates have antagonistic effect on pathogen due to their production of bioactive secondary metabolites (Bello et al. 2008). However, very little knowledge is available regarding the use of these yeast to degrade and detoxify pollutants. Therefore, the current findings expand their use.

Candida sp. VITJzN04 utilized lindane as sole carbon and energy source for growth in MM. During the incubation the yeast in MM with lindane, the organism attained exponential phase on the day 1 itself, furthermore complete removal of lindane was noted at the end of stationary phase. This was complemented with the 100 % release of chloride ions from the lindane, which is clear indication of complete and rapid mineralization. This observation is extremely important from the perspective of environmental pollution control.

In our previous study with yeast Rhodotorula VITJzN03, the optimum pH for degradation of lindane was found to be 6.0 (Salam et al. 2013). Other workers reported that a neutral range of pH 6–8 was most favourable for lindane degradation (Elcey and Kunhi 2010). In the case of incubation temperature, the behaviour of Candida sp. VITJzN04 was similar to the results obtained in other studies (Zhang et al. 2010, 2012; Salam et al. 2013) all of which documented 30 °C as optimum for lindane degradation. However, the fungal degradation of lindane is also reported at lower temperatures (Rigas et al. 2007; Dritsa et al. 2009). Guillen-Jimenez et al. (2012) described that the use of larger inoculum size increased the degradation efficiency and decreased the time of degradation of highly toxic compounds. The elevation of microbial load will increase the microbial activity and thus the number metabolic pathways involved. Increasing the inoculum size from 0.02 to 0.06 mg L−1 promoted the degradation efficiency from 40 to 100 % for the same incubation time. When the initial inoculums dosage was increased further, there was no significant difference in the degradation rate. Similar effect on lindane degradation upon increased inoculum size was noted in other studies as well (Chaudhary et al. 2006). The concentration of the target pollutant is an important factor which affects the microbial growth as well as the rate of degradation. Low concentration of pollutant might not be able to induce the enzymes of degradative activity while high levels may be toxic to the microorganisms (Awasthi et al. 2000). It was noteworthy that this particular strain could tolerate till a high concentration of 1,000 mg L−1 degrading about 42 % of lindane. However, the specific degradation rate decreased with increase in initial lindane concentration. These observations indicated that increased lindane concentration had a profound effect on degradation performance of strain VITJzN04, but did not lead to complete inhibition. The other yeast strain, Rhodotorula sp. VITJzN03, already reported as the best known lindane degrader completed degradation of 600 mg L−1lindane within 8 days. Therefore, the presently isolated yeast Candida sp. VITJzN04 proved higher potentiality on lindane degradation making it the most efficient agent of all the lindane degraders so far studied.

Hydrogen peroxide is known to facilitate the degradation of chemical pollutants especially persistent organic pollutants (Svrcek et al. 2010). In the present study, addition of H2O2 into the MM facilitated the lindane dechlorination by Candida sp. VITJzN04. It might be possibly due to increased availability of electron donor for the removal of Cl− and oxidation of lindane. Similar response on the use of H2O2 to facilitate the degradation of other organic pollutants was reported by other workers (Svrcek et al. 2010; Ye et al. 2013).

The kinetic analysis of lindane degradation by the yeast Candida sp. VITJzN04 was done by comparing, with zero-order, first-order, pseudo-first order and second order kinetic equations. It was found that the degradation of lindane was dependant on the substrate concentration, which was well explained by first order equation. Previously, the degradation kinetic of lindane was also studied in other organisms, where the calculated half-life of lindane were described as follows—1.66 days for Rhodotorula sp. VITJzN03 (Salam et al. 2013); 4.78 days for Anabeana azotica (Zhang et al. 2012). Clearly, the calculated half-life of lindane, in the present study is shortened (1.17 days) than in the earlier findings indicating the astonishing potency of the strain in lindane degradation.

Previous reports have proved the ability of microbial consortium to supersede the ability of individual microorganisms in biodegradation and remediation of toxic waste from contaminated soil as well as industrial effluents (Pant and Adholeya 2010). In the present study, the yeast strain VITJzN04 was found to attain complete degradation of high concentration of lindane within a period of 6 days, marking its outstanding potentiality in lindane remediation. The performance of the organism may be enhanced by providing a microbial consortium for lindane degradation in aqueous and soil environments.

GC–MS-based metabolite analysis has profound implications for discovering the mode of action of pesticides and helps to unravel the effect of altered gene expression on metabolism and organism performance in biotechnological applications (Du et al. 2013). This technique is irreplaceable because of its unique ability to detect compounds with high volatilities, low boiling temperatures and low molecular weights with high sensitivity. In our study, the metabolites formed during the degradation of lindane by Candida sp. VITJzN04 was identified by GC–MS. In our earlier report on lindane degradation by yeast Rhodotorula sp. VITJzN03, chlorobenzene derivatives were detected during the lindane degradation. In the present study, the presence of metabolites such as derivates of hydroquinone and HMSA signifies that different enzymatic mechanism for lindane degradation exist and can lead to the differential accumulation of compounds. The major metabolites reported for fungi in the existing papers during lindane degradation are γ-PCCH and benzoic acid derivatives (Guillén-Jiméneza et al. 2012).

Although, several fungi capable of degrading lindane have been isolated and studied, yet no proper report on the enzymatic mechanism of degradation is available. A possible reason attributed to this might be the involvement of complex and nonspecific enzyme systems, due to high degree of chlorination in lindane molecule. Oxidative enzymes and cytochrome p450 enzymes have been reported to be responsible for pesticide degradation in eukaryotes (Van Eerd et al. 2003). Recent studies have focused mostly on the activities of the enzymes viz. laccase, Mn peroxidase and lignin peroxidase to understand the role of fungi in lindane degradation (Nagpal et al. 2008; Guillén-Jiméneza et al. 2012). On the other hand, lin-operon, a set of enzymes holds responsible for bacterial degradation of lindane (Lal et al. 2010). In the present study, activities of both fungal and bacterial enzymes were tested in lindane containing cultures. Significant enzyme activities viz. Mn-peroxidase and lignin peroxidase along with lindane dechlorinase, lindane dehalogenase and reductive dechlorinase was noted over the incubation period. In contrast, laccase activities were not shown in the cultures. This is the first report on the involvement of the enzymes viz. lindane dechlorinase and lindane dehalogenase during lindane degradation in yeast species.

Lignolytic enzymes namely lignin peroxidase, laccase and manganese peroidases were found to play important role in organic pollutant remediation (Pant and Adholeya 2009a, b). Our research findings can enhance the understanding of the role of yeasts towards enzyme mediated remediation of lindane. Solid state fermentation of the yeast Candida sp. VITJzN04 can improve the production of enzymes which can be used for enzyme technologies and remediation purposes (Pant and Adholeya 2009b).

Conclusions

In the present study, Candida VITJzN04 obtained from sugarcane field exhibited greater potential for complete degradation of 600 mg L−1 lindane and its hydrolytic metabolites within 6 days in aqueous medium. The kinetic studies revealed a very good compliance with the first and pseudo first order models. Enzyme analysis indicated the involvement of lindane dechlorinase, lindane dehalogenase, DCHQ reductive dechlorinase, Mn peroxidase and lignin peroxidase enzymes during lindane degradation by the yeast strain. Based on our results, possible hypothetical degradation route is proposed. To our knowledge, this is the first report on the detailed biodegradation mechanism of lindane in yeast. It can be concluded that the yeast strain Candida sp. VITJzN04 may serve as a potential bioresource for efficient decontamination of lindane polluted environment.