Degradation study of lindane by novel strains Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W
© The Author(s) 2016
Received: 3 August 2016
Accepted: 14 December 2016
Published: 28 December 2016
This study was carried out to isolate and characterize the bacterial strains from lindane-contaminated soil and they were also assessed for their lindane-degrading potential.
In this study the enrichment culture method was used for isolation of lindane degrading bacterial isolates, in which the mineral salt medium (MSM) supplemented with different concentrations of lindane was used. Further, the screening for the potential lindane degrading isolates was done using the spray plate method and colorimetric dechlorinase enzyme assay. The selected isolates were also studied for their growth response under varying range of temperature, pH, and NaCl. The finally selected isolates DAB-1Y and DAB-1W showing best lindane degradation activity was further subjected to biochemical characterization, microscopy, degradation/kinetic study, and 16S rDNA sequencing. The strain identification were performed using the biochemical characterization, microscopy and the species identifies by 16S rDNA sequence of the two isolates using the standard 16S primers, the 16 S rRNA partial sequence was analyzed through BLAST analysis and phylogenetic tree was generated based on UGPMA clustering method using MEGA7 software. This shows the phylogenetic relationship with the related strains. The two isolates of this study were finally characterized as Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W, and their 16S rRNA sequence was submitted to GenBank database with accession numbers, KJ811539 and KX986577, respectively.
Out of the 20 isolates, the isolates DAB-1Y and DAB-1W exhibited best lindane-degrading activity of 94 and 98%, respectively, recorded after 8 days of incubation. The optimum growth was observed at temperature 30 °C, pH 7, and 5% NaCl observed for both isolates. Of the four isomers of hexachlorocyclohexane, isomer α and γ were the fastest degrading isomers, which were degraded up to 86 and 94% by isolates DAB-1Y and up to 93 and 98% by DAB-1W, respectively, reported after 8 days incubation. Isomer β was highly recalcitrant in which maximum 35 and 32% lindane degradation was observed even after 28 days incubation by isolates, DAB-1Y and DAB-1W, respectively. At lower lindane concentrations (1–10 mg/L), specific growth rate increased with increase in lindane concentration, maximum being 0.008 and 0.006/day for DAB-1Y and DAB-1W, respectively. The 16 S rRNA partial sequence of isolate DAB-1Y showed similarity with Kocuria sp. by BLAST analysis and was named as Kocuria sp. DAB-1Y and DAB-IW with Staphylococcus sp. DAB-1W. The 16S rDNA sequence of isolate DAB-1Y and DAB-1W was submitted to online at National Centre of Biotechnology Information (NCBI) with GenBank accession numbers, KJ811539 and KX986577, respectively.
This study has demonstrated that Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W were found efficient in bioremediation of gamma-HCH and can be utilized further for biodegradation of environmental contamination of lindane and can be utilized in bioremediation program.
KeywordsLindane (hexachlorocyclohexane, HCH) Degradation 16 S rRNA sequencing Spray plate assay
Lindane is an organochlorine compound; the ‘γ’ isomer of hexachlorocyclohexane (γ-HCH) primarily used as a fumigant and an insecticide against a wide range of insects. Out of the four isomers of hexachlorocyclohexane (α, β, γ, and δ), ‘γ’ is the only isomer having insecticidal property. Its agricultural use has been banned in most of the developed countries; however, some developing countries are continuing its use due to economic factors such as low cost (Johri et al. 1998). Due to its continuous use throughout the world, lindane-contaminated sites are prominent worldwide. Once HCH enters the environment, it can distribute globally (Simonich and Hitéis 1995) and can also persist in various environments (Abhilash 2009; Abhilash et al. 2008). The various sites of lindane contamination have been reported in different countries, viz., Europe (Concha-Grana, et al. 2006), America (Osterreicher-Cunha et al. 2003; Phillips et al. 2006), and Asia (Prakash et al. 2004; Zhu et al. 2005). The half-life period reported for lindane in soil and water are 708 and 2292 days, respectively (Beyer and Matthies 2001). Lindane causes various environmental impacts and persists in the soil for long periods. Therefore, toxicity and threats of environmental contamination are of great concern, and this problem can be solved through biodegradation-based approaches. The need of the hour is to develop procedures that could remove these toxic compounds by converting them to non-toxic form intermediate simple compounds. The various approaches of decontamination of HCH like chemical treatment, incineration, and land filling available, but they lack widespread application due to their cost factor and toxicity concerns to the living system. The bioremediation technology has been proposed as a promising tool for in situ detoxification of pesticide-contaminated sites. The various soil microorganisms capable of degrading and utilizing the organochlorine γ-hexachlorocyclohexane as a source of carbon have been reported over the last two decades at various places (Sahu et al. 1990; Adhya et al. 1996; Okeke et al. 2002; Nawab et al. 2003). Although the use of HCH has been banned in most of the countries, γ-HCH and its non-insecticidal isomers α, β, and δ still continue to pose environmental and health hazard (Pavilikova et al. 2012).
A variety of pesticides are being used in agriculture crops for the control of various insects. In spite of their agricultural benefits, pesticides are often considered a serious threat to the environment because of their persistence in environment for long period of time. Therefore the removal of pesticides from the environment source and ecological site is a topic of research interest for the scientists worldwide. In recent years, the use of degrading microorganisms or removing pesticides has been employed as the ecofriendly approach. This will enrich the in situ degradation and ex situ degradation as well. Therefore, the microbial bioresource has the great potential in solving current environmental problems. The bioremediation-based approaches possess high efficiency, sustainability, and their ecofriendly nature provides a solution to traditional physico-chemical remediation. Shrivastava et al. (2015) characterized a novel LinA type 3 δ-hexachlorocyclohexane dehydrochlorinase. The LinA gene involved the synthesis of first enzyme of the microbial degradation pathway of lindane, and leads to the dehydrochlorination of all four HCH isomers except beta-isomer. The two variants, LinA type 1 and LinA type 2, differ at 10 out of 156 amino acid residues. This study describes the characterization of a new variant of this enzyme, LinA type 3 gene was identified from a HCH-contaminated soil sample using metagenomic approach. Sun et al. (2015) conducted four pilot-scale test microcosms bioaugmentation study for the remediation of organochlorine pesticides (OCPs)-contaminated soil. They noted the effects on degradation of HCHs and dichlorodiphenyltrichloroethanes (DDTs) and found that nutrients/plant bioaugmentation enhanced the degradation of 81.18 and 85.4%, respectively). In order to develop the cleanup strategy of the polluted sites, recent study conducted by Laquitaine et al. (2016) has demonstrated the biodegradability of HCH in agricultural soils from Guadeloupe (French West Indies) and conducted studies which lead to the identification of the degrading genes among the characterized strains. The chlorinated pesticides viz. HCH, chlordecone and dieldrin, were used in agriculture until the start of 1990, this resulted in a contamination of the soil and water in the areas of banana production. They have conducted studies for lindane degradation in soil slurry microcosms. The 40% lindane degradation efficiency was reported in 30-day treatment experiment. During the course of this degradation study, the lindane concentration decreased from 6000 to 1330 and 800 to 340 ng/mL for the biotic and abiotic soils samples, respectively. Molecular studies for gene analysis indicating that HCH degradation was probably mediated by bacteria closely related to Sphingomonadaceae family bacteria.
Considering, the adverse environmental impacts of lindane and its toxicity, in the present study, we have attempted to isolate and characterize novel bacterial strains from lindane-contaminated soil collected from Lucknow, India. The enrichment culture method was used for the screening of the isolates using mineral salt medium supplemented with varying concentration of lindane as sole carbon source. Also, we have tried to study the kinetics of these two potent bacterial strains capable of degrading γ-HCH grown in the shake flask culture. The isolation and characterization of the strains from the ecological habits lead to find out the new genes and enzymes involved in the degradation of toxic compound by the activity of the microbial strains. Further new strains will enrich the gene pool for the biodegradation of lindane/or other toxic compounds present in the environment. These strains may be utilized as the microbial source for the degradative gene(s), and these genes can be transferred to non-degradative strains by recombinant DNA technology.
Chemicals, soil sampling, and isolation of bacterial strains
The technical grade HCH isomers (α, β, γ, and δ; 99.9% pure) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and other chemicals and reagents of technical/molecular grade were obtained from Qualichem, Ranbaxy etc. The media and molecular biology kits were obtained from Hi-Media, Mumbai India. Soil samples were collected in sterile polythene bags from a contaminated site of Lucknow, UP, India. The collected soil samples were brought to the laboratory, dried, and kept at 4 °C until further microbial isolation was performed. The pH value of the soil samples ranged between 6.5 and 7.8.
The isolation of bacterial strains was carried out from soil samples using enrichment culture technique (Dams et al. 2007). Mineral salt medium (MSM) was prepared in 250 mL Erlenmeyer flask containing (per liter) potassium dihydrogen phosphate, 0.85 g; dipotassium hydrogen phosphate, 2.17 g; disodium hydrogen phosphate, 3.34 g; ammonium chloride, 0.1 g; magnesium sulfate, 0.5 g; calcium chloride, 0.5 g; ferrous sulfate, 0.01 g; sodium molybdate, 0.01 g; at a pH of 7.2 ± 0 5 (Sahu et al. 1990). Two grams of collected soil sample and 10 mg/L of lindane was weighed and added to 100 mL of sterile MSM broth. The stock solutions of the HCH were filter sterilized (0.22 micron Millipore syringe filter) and then aseptically transferred to autoclaved cooled MSM. After thorough mixing, the flask was incubated at 30 °C for 7 days in a rotary shaker at 120 rpm. Subsequently, 1 mL of the inoculum having a cell density of approximately 5 × 103 CFU/mL was taken from the flasks with a micropipette and was transferred to sterile medium (100 mL) containing the same amount of lindane concentration. The inoculum was transferred to fresh media each time with increasing lindane concentration from 10 to 100 mg/L in a stepwise manner. After acclimatization of the culture, the serial dilution up to 10−4 dilution was spread plated and used for isolation of bacterial colonies on mineral agar plates supplemented with 10 mg/L of lindane. The plates were incubated under aerobic conditions at 30 °C in incubator (Remi Instruments, India). After 24 h, colonies with unique morphology were sub-cultured on fresh agar plates in the form of single culture and preserved at 4 °C till further use.
Screening and selection of lindane-degrading bacteria (quantitative study)
The bacterial isolation was done using the enrichment culture technique and tested qualitatively for lindane-degrading activity by spray plate dehalogenase assay (Phillips et al. 2001; Manickam et al. 2008). Spray plates were prepared with MSM supplemented with 1.5% agar in petri dishes, and after solidification of the plates, the pure culture was streaked on the surface of these plates. The 0.5% of lindane solution was prepared by dissolving lindane powder in acetone and was sprayed on the surface of preset agar plates. These plates were further incubated for seven days at 28 ± 2 °C in incubator. This method was used for the isolation of lindane-degrading bacteria RP-1, RP-2 and RP-3 reported in our other study (Pannu and Kumar 2014).
Kinetic study for HCH degradation
μ g is a function of S.
This is because K d is neglected during exponential phase.
The kinetic study was performed for γ-HCH degradation for initial concentrations 1, 2, 5, 10, 15, 20, 30, 40, and 50 mg/L in different flasks and two control experiments (without biomass and without substrate) were carried out for each concentration. The samples at particular time interval were withdrawn from the flasks from shaker and analyzed for the bacterial growth by monitoring the optical density of the culture at 600 nm using UV–Vis spectrophotometer (Shimadzu, Japan).
Effect of physiological parameters on lindane biodegradation
Different physiological parameters like temperature (20–50 °C), pH (3–11), NaCl (1–5%), and incubation time (5–20 days) were optimized to study their effect on biodegradation by isolates DAB-1Y and DAB-1W. The Sphingobium japonicum (MTCC 6362) was used as reference control in all the experiments. Degradation study was carried out in mineral salt medium (MSM) containing lindane (10 mg/L) as sole carbon source and analyzed by varying one parameter at a time, keeping others constant. The culture showing growth, by measuring OD660 using UV–Vis spectrophotometer was taken as indicator of lindane utilization. The maximum value of OD660 values obtained was taken as optimized set of parameters for lindane degradation (data not shown).
The finally selected isolates, DAB-1Y and DAB-1W, were characterized by morphological and biochemical characterization; 16S rRNA sequencing and one isolate (DAB-1W) in addition to 16S rRNA were also characterized by GC-FAME technique. The former technique is based on the sequencing using universal primers to the 16S rDNA of our isolates and later is based on the analysis of the fatty acid profile of the bacteria. As the fatty acid profile of the bacterial species is the unique signature to be helpful in detection on the bacterial species from the varied samples. The fatty acid is also produced by the bacteria under specific media supplementation and studied by Ehrhardt et al. (2010), but in our study, the identification is done based on the fatty acid which was produced in the customized medium and experiment was conducted in duplicate along with control. Therefore, the fatty acid uniquely produced during our study was characterized and detected through FAME, and the identification was done using Sherlock identification system which identifies fatty acids from our sample from the screening of fatty acid library. The fatty acid profile identifies the microbial species. A variety of microorganisms degrade the HCH isomers and their degradation studied by many researchers (reviewed by Alvarez et al. 2012).
The identification of the bacteria involved in degradation was proceeded mainly by 16S rRNA sequencing for strain identifications, but one isolate was also characterized by GC-FAME analysis as well. The 16S rRNA is approx 1500 bp region of genomic region of bacteria and considered as genetic signature for the identification of the bacteria. A number of studies were conducted based on this technique, and it was considered that 16S region is conserved during the course of evolution among bacterial linage. Therefore, it helps microbiologists and pathologists in the characterization of new strain isolated from various sources. Similarly, GC-FAME-based identification was also considered rapid and reliable technique for the identification of microorganisms based on the pattern of cellular fatty acid in bacteria, which is unique signature and aids in their identification and classification. The only commercial available database for the identification by fatty acid methyl ester (FAME) analysis is Sherlock microbial identification system (MIS) developed by Microbial ID, Inc (MIDI). This was developed by Sasser et al. (2001) for the identification of aerobic bacteria. Therefore, it represents rapid, accurate, less expensive approach for the identification of more than 1500 microbial species (www.midi.com/pages).
Morphological and biochemical characterization
The different morphological studies of the selected lindane-degrading isolates, DAB-1Y and DAB-1Y, were carried out to determine their cell shape, cell size, color, cell motility, capsule formation, colony morphology, pigmentation etc., by growing the culture on nutrient agar plates followed by preliminary identification using Gram’s staining. The isolates were further identified up to genus level by biochemical tests, 16S rRNA sequencing, and GC-FAME analysis.
The isolates, DAB-1Y and DAB-1W, were identified biochemically according to the method given by Karn et al. (2011) in which Kocuria sp. CL2 showed similar biochemical results with respect to colony shape, cell grouping starch and casein hydrolysis, gelatin liquefaction, methyl red and vogues–proskauer test, catalase test, adonitol, sorbitol, citrate, ornithine, lysine and urea utilization, nitrate reduction, phenylalanine deamination, triple sugar iron test, H2S production, and various carbohydrate fermentation tests like mannitol, galactose, xylose, rhamnose, sucrose, glucose, arabinose and lactose) with the standard protocols given in Bergey’s Manual of Determinative Microbiology (Holt et al. 1994). The KB10 kit (Hi-Media, Mumbai, India) was used for biochemical characterization and other biochemical tests were carried out using standard methods (Cappuccino and Sherman 2010). The gelatin liquefaction test was performed by method given by Clarke (1953).
Molecular characterization of isolates
Genomic DNA extraction
Total DNA from the bacterial isolates was isolated using the Alkaline lysis method as described by Kate Wilson (1987). In each case, 5 mL of an overnight culture was centrifuged at 5590×g for 5 min and the resulting pellet was resuspended in 574 μL of TE (10 mM Tris, 1 mM EDTA, pH 8) buffer. Into this, 300 μL of cell lysis buffer containing lysozyme, SDS, CTAB, and proteinase-K was added and the mixture was incubated for 1 h at 37 °C. Subsequently, DNA was separated from the cell lysate using phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). By the adding of isopropyl alcohol, the DNA was precipitated and the pellet was resuspended in 50 μL of TE buffer. Then RNase was added to remove RNA before storage of the samples at −20 °C. The quality of the extracted DNA was analyzed by electrophoresis in 1% agarose gel supplemented with ethidium bromide (10 mg/mL). The DNA bands were visualized in UV-gel documentation system (BioRad, USA).
PCR amplification of 16S rDNA
The polymerase chain reaction (PCR) amplification for targeting the 16S rDNA from the genomic region of isolate DAB-1Y was carried out using 8F (5′-AGAGTTTGATCMTGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′) forward and reverse primers, respectively. Similarly, the isolate DAB-1W DNA was amplified through PCR using 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primer(s), respectively. Two µL of purified genomic DNA was used as template. The 16S rRNA gene was amplified using GeneAmp 2700 PCR systems (BioRad, USA). The reaction mixture for the PCR contained 4 µL of 10× PCR buffer with MgCl2; 10 mM of dNTPs; 0.5 µL of each primer; 10 mM and 0.5 µL of 5 U/µL Taq DNA polymerase (Sigma, USA) in a final volume of 25 µL. The PCR was performed with an initial denaturation at 92 °C for 2 min followed by 35 cycles of 92 °C for 1 min, 48 °C for 30 s, and 72 °C for 2 min, and a final extension of 72 °C for 6 min was given. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized under gel documentation system after staining with ethidium bromide.
Sequencing and phylogenetic analysis
The amplified PCR products were sent for sequencing to Yaazh Xenomics, Chennai, India. The 16S rDNA sequence obtained checked for any chimeric sequence using online search-based DECIPHER bioinformatics tool (Wright et al. 2012). The comparison of the sequences obtained was done with the GenBank database using Basic Local Alignment Search Tool (BLAST)N program using National Centre of Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and similar sequences were downloaded and aligned with Muscle; a phylogenetic tree was constructed using UGPMA clustering method using MEGA7 software by Neighbor-joining method (Kumar et al. 2016).
16S rRNA accession numbers
The two PCR product amplified by the 16S rRNA universal primers in this work was deposited to NCBI GenBank with accession numbers KJ811539 and KX986577, respectively.
Identification of strain by GC-FAME
Gas chromatography-fatty acid methyl ester (GC-FAME) is an alternative tool to classical microbiological identification techniques. The genomic expressions (i.e., DNA/RNA homology, lipid composition, protein pattern etc.) are conserved among a group of microbial species and they seem to be the reliable indicators for the identification. This analysis makes use of short chain fatty acids to characterize genera and species of bacteria. As compared to FAME, 16S rRNA-based species characterization is more reliable technique because it is molecular biology-based technique. The 40 mg of pre grown bacterial cells was harvested and saponified. After this, the fatty acids were extracted and methylated to form fatty acid methyl esters (FAME). The samples were sent to Royal Life Science, Hyderabad, India; for GC-FAME analysis. These fatty acids from the sample were analyzed using gas chromatography with the help of MIDI Sherlock software for FAME pattern analysis. The aerobic library (RTBSA 6.21) was referred for analysis of the DAB-1W strain and was performed as per the method given by Sasser (2001).
Results and discussion
The isolates DAB-1Y and DAB-1W were found potential HCH degrading strains and seem to have great potential for the gamma-HCH degradation program. The results and discussion based on their characterization are presented below based on biochemical, microscopy, degradation analysis, and molecular identification of their species based on molecular (16S rRNA sequencing and also identification by the GC-FAME.
Enrichment, isolation, and screening of lindane-degrading bacteria
The selected bacterial isolates were tested qualitatively for lindane-degrading activity using spray plate method. The formation of lindane clearance zone surrounding bacterial colonies indicated the utilization of lindane. The chloride ions released during the degradation study were analyzed based on the titration-based assay, and the change of color from yellow to pink is observed (Fig. 1c) Similar lindane degradation zones were observed for bacterium Pseudomonas paucimobilis (Senoo and Wada 1989), fungus Conidiobolus 03-1-56 by (Nagpal et al. 2008), and for yeast Rhodotorula sp. VITJzNo3 (Salam et al. 2013). The clear halo zones also appeared on agar plates containing precipitated γ-HCH around colonies of lindane-degrading isolates reported by Thomas et al. (1996). The production of halo zones around culture growth observed after incubation leads to the conclusion that the enzymes involved in γ-HCH dechlorination are extracellularly produced and the secretion of these enzymes from the bacterial culture leads to the production of clear haloes around colony.
Degradation study of lindane (quantitative analysis of chloride ion release)
Comparison of lindane degradation by the bacterial strains reported in different studies
Bacterial strain reported
Kocuria sp. DAB-IY and Staphylococcus sp. DAB-1W
Streptomyces sp. M7
Sineli et al. (2016)
Sphingomonas baderi sp.
Kaur et al. (2013)
Arthrobacter florescens and Arthrobacter giacomelloi
De Polis et al. (2013)
Actinobacteria sp. and Streptomyces sp.
1.66 mg/L optimum for degradation
Kocuria rhizophila, Microbacterium resistens, Staphylococcus equorum, Staphylococcus cohnii subsp.ureolyticus
Abhilash et al. (2011)
300 μg/mL after 108 h
Elcey and Kunhi (2010)
Azotobacter chroococcum JL102
Anupama and Paul (2010)
Sphingomonas sp. NM05
Manickam et al. (2008)
Sphingomonas sp. NM05
Manickam et al. (2008)
Lodha et al. (2007)
Pseudomonas aerogenosa ITRC5
5 mg/mL soil
Kumar et al. (2006)
Okeke et al. (2002)
Bacillus brevis and Bacillus circulans
Gupta et al. (2000)
Arthobacter citreus BI-100
Datta et al. (2000)
Effect of different physiological parameters on lindane degradation
The incubation temperature of 30 °C has been found as optimum temperature for lindane degradation (Zhang et al. 2010, 2012; Salam et al. 2013). However, rapid degradation of lindane by Sphingobium strains has been reported by Zheng et al. (2011) even at low temperature (4 °C) indicating that lindane degradation could be achieved even in colder contaminated regions. We have also tested the degradation of the inoculated strains in the liquid culture at 4 °C and checking the dechlorinase activity of the culture filtrate received after centrifugation. No activity was observed in DAB-1W and DAB-1Y strains. The percentage lindane degradation was studied for different incubation times ranging from 2 to 8 days. It was observed that lindane degradation increased with increase in incubation time from 2 to 28 days (Fig. 2). It was 13, 15, and 20% reported after 2 days for DAB-1Y, DAB-1W, and MTCC 6362, respectively. The degradation increased to 24, 27, and 30% after 4 days and increased to 38, 47, and 60% after 6 days for DAB-1Y, DAB-1W, and MTCC 6362, respectively. The maximum lindane degradation was observed 98% for MTCC 6362 after 8 days incubation. Our isolates DAB-1Y and DAB-1W demonstrated comparable results to standard strains and achieved 94 and 98% degradation, respectively, after 8 days of incubation under standard conditions.
Morphological and biochemical characterization of isolates DAB-1Y and DAB-1W
Rods in chains
Colony morphology on NA plates
Growth at 4 °C
Growth at 8 °C
Growth at 25 °C
Growth at 45 °C
Growth with 1% NaCl
Growth with 5% NaCl
Growth with 7% NaCl
Growth with 10% NaCl
Cell motility (by hanging drop method)
Growth with 12% NaCl
Growth at pH 2
Growth at pH 4
Growth at pH 5
Growth at pH 7
Growth at pH 8
Growth at pH 9
Growth at pH 11
Kinetics of HCH degradation
The bacterial cell morphology viz. cell shape, size, color of colony, motility, capsule formation, colony margins etc. was used for the grouping of our two isolates, and biochemical tests were performed to know the metabolic characteristic. The results obtained in this study are presented below.
Morphological and biochemical characterization
Degradation behavior of lindane isomers by isolates, DAB-1Y and DAB-1W
Incubation period (days)
Degradation by isolate DAB-1Y (5 µg/mL) in %
Degradation by isolate. DAB-1W
(5 µg/mL) in %
The isolate DAB-1W was found to be Gram negative, white cocci, no pigmentation observed, negative for citrate utilization, urease, adonitol, and lactose, and showed positive tests for lysine, ornithine, nitrate reduction, TDA, H2S production, glucose, arabinose, and sorbitol. Grows at 25, 45 °C, but not at 4 and 8 °C. Its growth was observed at 1, 5, and 7% NaCl, but not at 10 and 12% NaCl concentration. Grows at pH 5, 7, 8, 9, but not at pH 2, 4, and 11. No sporulation was observed; it hydrolysed starch; and no gelatin hydrolysis was observed (Table 3). Similar results were reported by Abhilash et al. (2011) for Staphylococcus equorum and Staphylococcus cohnii. The S. equorum was Gram positive, motile; showed growth from 10 to 42 °C temperature, pH 5.2–9, and NaCl concentration 2 to 10%, hydrolyzed citrate; showed positive methyl red, nitrate reduction, and urea tests; and utilized arabinose, glucose, mannose, xylose, sucrose, and fructose. S. cohnii was gram positive, non-motile; showed growth from 25 to 42 °C, pH 5.2–9, and NaCl 2 to 10%; showed positive for nitrate reduction, urea tests; and utilized mannose and fructose.
Molecular characterization and phylogenetic analysis
Potential lindane-degrading microorganisms reported in other research were mostly from genera Pseudomonas, Sphingobium, Streptomyces, Fusarium (Salam et al. 2013), and K. rhizophila, Microbacterium resistens, Staphylococcus equorium, Staphylococcous cohnii subsp. urelyticus (Abhilash et al. 2011) and Bacillus brevis and Bacillus circulans isolated from HCH-contaminated soil (Gupta et al. 2000). Salam et al. (2013) isolated a novel yeast strain from sugarcane field, and this has found the ability to use lindane as sole carbon source in the mineral salt medium. The yeast strain was identified and named as Candida sp. VITJzN04 based on polyphasic approach which was based on morphological, biochemical, and 18 rDNA sequencing, D1/D2, and ITS sequence analysis. The strain efficiently degraded 600 mg/L of lindane, 6 days of incubation in MSM under optimal conditions (pH 7, temperature 30 °C, and inoculum 0.06 g/L) with half life of 1.17 days and 0.588/day degradation constant. The lindane degradation also tested through kinetic studies as well. Wang et al. (2015) isolated Arthrobacter nicotianae DH19 from Ginseng rhizospheric soil using morphological, biochemical tests, and 16S rRNA gene sequencing. This strain utilizes pentachloronitrobenzene (PCNB) as a sole carbon course for growth when it was inoculated in mineral salt medium. It grows at pH 6.85, 30 °C, and inoculum concentration 1.45 g/L and degrade efficiently 90 5 PCNB in 7 days. This strain degrades dichlorodiphenyl trichloroethane, hexachlorocyclohexane, cypermethrin, and cyhalothrin. The metabolites from PCNB degradation were identified using GC–MS. This was first report of PCNB-degrading strain DH19 isolated from rhizospheric soil and therefore, DH19 can be employed in bioremediation of PCNB in the environmental sites cleanup. In the similar study Karn et al. (2011) isolated Kocuria sp. CL2 from secondary sludge of pulp and paper mill and this strain was found degradation acitivity against pentachlorophenol (PCP).
Lindane is a chlorinated cyclic saturated hydrocarbon used extensively for the control of agricultural pests and mosquitoes. Due to its continuous use in the past decade throughout world, the lindane-contaminated sites are prominent, and thus there is an urgent need to develop its cleanup strategies. Bioremediation is one such technology that could be employed for decontamination of pesticides contaminated soil/sites. Microbial biotechnology possesses ample scope to work in this direction; the new techniques of molecular identification based on high throughput sequencing techniques lead to fine identification of the resistance genes among the strains in less time and cost. In the current work, two novel bacterial strains were isolated which successfully degraded lindane exhibiting 94 and 98% of degrading activity. The results of the present investigation indicate that the strains reported in this study viz Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W may be used as an important biological mean for bioremediation of lindane-contaminated sites.
- SI value:
similarity index value
mineral salt medium
gas chromatography-fatty acid methyl ester analysis
polymerase chain reaction
American Type Culture Collection, USA
DK and AK conducted the research, DK also supervised the research, and JS helped in writing the manuscript. All authors read and approved the final manuscript.
The financial support received from University Grants Commission (UGC), New Delhi India, is thankfully acknowledged to carry out this research work in the form of major research project (F.No.42-449/2013 (SR) awarded to DK. The AK/JS also wish to thank UGC for providing financial assistance in the form of research fellowship under this project. The authors also thank DCR University of Science and Technology Murthal, Sonepat Haryana, India for providing necessary facilities during this study. The authors also thank Editor and reviewers for their constructive suggestions that have lead to the improvement in the manuscript.
The authors declare that they have no competing interests.
This work was supported by UGC, New Delhi, India in the form of major research project with Grant Number F.No.42-449/2013 (SR).
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