Effective removal of naphthalene from contaminated soil using halotolerant bacterial strains and vermiremediation techniques

ABSTRACT The marine environment poses a serious threat due to accidental oil spill and industrial discharge specifically from refineries which has become a serious threat to the marine ecosystem. Most of the recalcitrant compounds belong to the group of polyaromatic hydrocarbons. Hence, the present study focuses on biodegradability of simple Polycyclic Aromatic Hydrocarbon (PAH) such as Naphthalene using halotolerant bacterial consortium isolated from oil-spilled environment. Two halotolerant bacterial consortiums were isolated based on the degradation of PAH which measured to be 69.38% by RSKVG1 and 30.61% by RSKVG2 after 96 hours. Naphthalene utilisation was up to 100 mg/L by RSKVG1 and 300 mg/L by RSKVG2. Further, the isolates were optimised for different parameters, optimum NaCl concentration was 30 g/L and 50 g/L by RSKVG1 and RSKVG2, respectively. The functional groups and secondary metabolites produced by the bacterial strains were characterised by FT-IR, HPLC, and GC-MS analysis. Biochemical, morphological and Molecular characterisation, and construction of phylogenetic tree was done which revealed the significant similarity of RSKVG1 and RSKVG2 to Bacillus thuringiensis and Bacillus pacificus respectively. The Naphthalene was also studied for the degradation using the vermiremediation technique.


Introduction
The marine environment poses a serious threat due to accidental oil spill and industrial discharge specifically from refineries. As a result of ever-increasing swift industrialisation, the saline and hypersaline environments are contaminated with recalcitrant pollutants. Petroleum, textile, leather, pesticide, herbicide, pharmaceutical, and dyestuff manufacturing industries discharge saline wastewater and these effluents in turn have an impact on water potability, aquatic life, and agriculture as they constitute recalcitrant compounds [1]. Fossil fuels, fossil oil serve as the main source of energy that drives the world's industrial activity. The reservoir water is produced in high quantities as a result of oil extraction and subsequently, the oil of analytical grade and obtained from Merck, India. The soil slurry reactor used for the experimental setup was purchased from a pot seller and the soil used for the bioremediation and vermiremediation process was collected from Stella Maris College garden.

Sample collection
The halotolerant naphthalene degrading bacterial strains were isolated from the soil samples collected from the oil-spilled contaminated site from Ennore Beach, Chennai, Tamil Nadu, India at latitude 13.2146ºN and longitude 80.3203ºE.

Mineral salts medium (MSM)
Mineral salts medium (MSM) includes Ammonium chloride-2.5 g, potassium dihydrogen phosphate-5.46 g, disodium hydrogen phosphate-4.76 g, magnesium sulphate-0.20 g, sodium chloride-30.0 g, and Distilled water-1 L at pH-7.4 ± 0.2 [6]. The final pH of the medium was adjusted to 7.4 with 0.1 N NaOH, and the medium was autoclaved (121°C for 15 min) before the addition of Naphthalene. The stock solution of Naphthalene (100 mg per litre) was freshly prepared in ethyl acetate each time the experiment was performed.

Enrichment and isolation of the naphthalene degrading bacterial strains
The soil sample was enriched in 100 ml of MSM broth with 30 g/L NaCl concentration and incubated using an orbital shaker at 150 rpm at 37°C for 48 hours. After enrichment, serial dilution was performed and plated on MSM agar plates with 100 parts per million (ppm) of Naphthalene and incubated at 37°C for 72 hours.

Biodegradation studies of naphthalene and protein estimation of the isolated bacterial strains
Biodegradation of Naphthalene concentration was studied using the two bacterial strains (RSKVG1 and RSKVG2) by inoculation in Mineral Salts Medium (MSM), with 100 ppm of Naphthalene at 30 g/L of NaCl concentration. Different degradation experiments were conducted for the growth and optimisation and were 1) Autoclaved MSM medium + 100 ppm of Naphthalene + Bacterial culture; 2) Autoclaved MSM medium + 100 ppm of Naphthalene (Control) and 3) Autoclaved MSM medium + Bacterial culture (Control).
All the combinations were prepared in duplicates and kept for incubation using an orbital shaker at 37°C at 150 rpm. The samples were analysed every 24 hours for 5 days and were centrifuged at 10,000 g for 10 minutes and the supernatant from the sample was analysed at OD 271 nm for the degradation efficiency of the bacterial strains. The biodegradation efficiency (%) was determined according to [7]: Biodegradation efficiency % ð Þ ¼ � 100 where C i = Initial concentration of Naphthalene, C f = Final concentration of Naphthalene.

Total protein content of the halotolerant bacterial strains
In a sterilised 100 ml flask, 50 ml of autoclaved MSM medium was added which was supplemented with 100 ppm of naphthalene and inoculated with the two bacterial strains (RSKVG1 and RSKVG2) in separate flasks. Protein estimation from the 0th day to the 4th day was examined at 660 nm to check for the growth of the organisms [8].

Intermediates production through FT -IR, HPLC, and GC -MS
To examine the naphthalene degradation, and the metabolites produced by the bacterial strains were identified through FT-IR, HPLC, and GC-MS. The extraction was extracted using separating funnel using ethyl acetate as a solvent phase. The extracted sample was purified using Whatman Filter paper No. 1 to remove the debris. The filtrate was fed into FT-IR and the spectra were recorded using KBr pellets [9]. Shimadzu Prominence Binary Gradient HPLC System with a C 18 column was used with isocratic elution as a methanol gradient. The condensed sample was filtered using a 0.22 µm syringe filter, injected into the HPLC system maintaining the flow rate of 1.0 ml min −1 . Metabolites were detected by a Knauer UV detector at 254 nm and identified by comparing their retention times with those compounds analysed under the same conditions [10]. Hewlett-Packard 6890 gas chromatograph equipped with 5973 mass spectrometers with HP-5 MS was used. The column temperature can hold 100°C for 1 min, 15°C/min to 160°C and 5°C/min to 300°C hold for 7 min. The injector was maintained at 280°C with a splitless period of 3 min. The carrier phase was maintained by helium at a flow rate of 1 ml/min by using electronic pressure control. The intermediates produced were confirmed using the standards and controls [11].

Morphological, biochemical and molecular characterisation of naphthalene degrading bacterial strains
The bacterial strains RSKVG1 and RSKVG2 were morphologically characterised by Gram's staining and Scanning Electron Microscopy (SEM) according to the method of Prior and Perkins [12]. Molecular identification of the isolates was done by 16S rRNA sequencing. region was amplified using universal primers by PCR from the isolated DNA. The purified PCR amplicon was sequenced using the Universal forward (5ʹ-TTTGATCCTGGCTCAG-3ʹ) and reverse (5ʹ-AAGGAGG TGATCCAGCCGCA-3ʹ) primers. Sequencing was carried out using the BDT v3.1 Cycle sequencing kit on ABI 3500 Genetic Analyser. The evolutionary relationship between the bacterial strains was determined by constructing a phylogenetic tree using FASTA format, the tree was constructed using MEGA 7.0 software.

Biodegradation of naphthalene using vermiremediation and bioremediation
The experiment was performed as duplicates setup in mud pots, each filled with 1 kg of 2 mm sieved soil. Five sets of treatments, set A (Abiotic control), set B (Naphthalene + Earthworms), and set C (Earthworms) are set up to remediate the soil. Three sets of treatments, set A (Abiotic control), set D (Co-culture) are set up to the remediate soil using encapsulated cells of two microbial strains. For bioremediation, the experiment was carried out in pots (Control, Cocultures of the bacterial strains RSKVG1 and RSKVG2 in duplicates). About 100 ml of Nutrient broth was prepared and sterilised at 121 o C at 15 psi for 15 minutes. The broth when cooled was added with 0.50 g of sterilised biochar and mixed thoroughly. The co-cultures were then introduced into the soil in the Pots D and D1. For vermiremediation, the experiment set up consisted of 5 pots (Control, Pot B and B1-Naphthalene + earthworms and Pot C and C1-Earthworms) containing 1 kg of soil each sieved through 2 mm sieve and autoclaved ( Figure 1). The adult earthworms with well-developed clitellum were chosen for this study. Ten earthworms were introduced into each pot [13]. The worms were supplemented with Naphthalene in pots B and B1 and were without Naphthalene in pots C and C1. Due to the addition of secondary feed materials (cow dung and kitchen waste) in the contaminated soil, significant dilution of Naphthalene was expected [14]. The soil samples were collected at regular intervals for various physicochemical analyses.

Physicochemical analysis of soil samples from bioremediation and vermiremediation studies
The experiment was performed for 15 days and the sample was taken once in 3 days for analysis of the following parameters. The pH, temperature, total organic carbon (TOC) [15], and Total Kjeldahl Nitrogen (TKN) by micro Kjeldahl method were analysed (APHA).

Isolation of naphthalene degrading halotolerant bacterial strains
The halotolerant bacterial strains were enriched with the soil samples collected from Ennore Creek, a backwater along the Bay of Bengal's coromandel coast. The Ennore creek obtains wastewater from various sources, including untreated wastewater and treated effluents from industrial sources. For the isolation of halotolerant bacterial strains, the sampling area was explicitly selected as the sampling environment had both the contaminants from anthropogenic sources and a salinity. The site served as the best source to isolate the acclimatised halotolerant bacterial strains. The enrichment cultures supplemented with the naphthalene showed many isolates out of which two of the bacterial strains showed the best degradation ability. The halotolerant bacterial strains were isolated from the enrichment culture supplemented with 100 ppm of naphthalene in which only two bacterial colonies (RSKVG1 and RSKVG2) of different morphologies appeared on Naphthalene containing MSM agar plates ( Figure S1 in Supplementary data). Bulbul Gupta et al. [5] had reported the isolation of naphthalene degrading bacteria from the soil samples collected from the crude oil soil near fuel filling stations in Chandigarh.

Screening of naphthalene degradation using isolated halotolerant bacterial strains
The biodegradation of naphthalene and their growth by both the bacterial strains. The bacterial strain RSKVG1 utilised naphthalene which was provided to it as the carbon source (100 ppm) and was able to degrade up to 69.38% 3rd day. Whereas the percentage of degradation by the bacterial strain RSKVG2 was a maximum of 30.61% on 3rd day. Bulbul Gupta et al. [5] in their work had supplemented the medium with Naphthalene as the sole carbon source.

Optimisation of various parameters on naphthalene degradation
RSKVG1 was able to degrade a maximum of 63.15% on 3rd day with 100 ppm of naphthalene ( Figure S2 (a) in the Supplementary data). RSKVG2 was able to degrade NAPH at 300 ppm with a degradation percentage of 42.85% on 3rd day. At pH 7, on 3rd day there was a maximum degradation of 82.45% and 66.31% by RSKVG1 and RSKVG2, respectively. As the pH increased, there was a decrease in the degradation efficiency approximately in the range of 30% to 52%, which in turn showed a decline in the growth of the organism, it revealed that the organism can degrade only at neutral pH. RSKVG1 showed a degradation percentage of 42.39% on 3rd day at 37 o C and RSKVG2 showed a maximum percentage of degradation of Naphthalene of 46.55% on 3 rd day at 25 o C. RSKVG1 showed a maximum degradation percentage of 60% with 3% NaCl on 3rd day and RSKVG2 showed an efficiency of degradation at 5% NaCl concentration with 56% degradation on the 3rd day ( Figure S2 (b) in the Supplementary data).
Arulazhagan et al. [6] explained the optimisation of NaCl concentration used for the degradation of PAH. The concentrations of NaCl used were 30 g/L, 60 g/L, and 90 g/L. They reported that degradation occurred nearly 80% to 99% in 7 days of incubation with 30 g/L of NaCl, 65% in 10 days with 60 g/L of NaCl, and with 90 g/L of NaCl, there was a gradual reduction in the degradation in 6 days ( Figure S5 (a,b) in the Supplementary data). The concentration of PAH used as about 500 ppm. The pH and temperature maintained were 7.4 and 37 o C ( Figure S3, S4 (a,b) in the Supplementary data) respectively.

Metabolites formed after degradation of naphthalene
The FT-IR spectra of the control naphthalene (100 ppm) showed peaks at 3170.97 cm −1 and 1666.50 cm −1 which corresponds to the -C ≡ C-H: C-H stretching of alkynes and alkenes in the non-biodegraded naphthalene (Figure 2). The metabolites formed after degradation of naphthalene by the halotolerant bacterial strains RSKVG1 and RSKVG2 showed various peaks and the results of RSKVG1 (Table 2) and RSKVG2 (Table 3) were correlated using standard ( Table 1). The stretching frequency of O-H bond, C-C bond, S=O bond, C-Br bond were common to both the bacterial strains RSKVG1 and RSKVG2.
Revathy et al. [16] explained the FT-IR spectra of naphthalene before and after degradation. The peaks at 3170.97 cm −1 and 1666.50 cm −1 which corresponds to -C≡C-H: C-H stretch of alkynes and alkenes represent the naphthalene before degradation. The absence of peaks at 3170.97 cm −1 , 1666.50 cm −1 , 989.48 cm −1 , 937.40 cm −1 , 1109.07 cm −1 and 867.97 cm −1 indicates the degradation of naphthalene by the bacterial strain.    Abo-State et al. [17] reported that HPLC analysis for degraded naphthalene compound after 21 days using methanol/water ratio as the mobile phase. About 93% of naphthalene was been degraded after 21 days of incubation in the HPLC analysis.

Characterisation of naphthalene degrading halotolerant bacterial strains
The isolated bacterial strains were studied for the morphological and biochemical characterisation. The biochemical characterisation was concluded as a positive/negative using control and the results are tabulated in Table 5. Scanning Electron Microscope (SEM) of RSKVG1 and RSKVG 2 was analysed under 12,000 X and 30,000 X magnifications (Figures 8

Physicochemical analysis of bioremediated and vermiremediated soil
The control sample (A) showed a slow increase in pH as the days increased and were in the range of 6.3 to 7.7, whereas the co-cultures in Pot D showed a rapid increase in pH from Day 0 to 15 in the range of 6.4 to 8.9. The pH of the vermiremediated samples in Pot B and C showed a range from 6.1 to 9.2 and 6.2 to 9, respectively. The fluctuation in the pH is attributed in any biological degradation process and the degradation is active in the neutral range [19]. The temperature in the control ranged between 29°C and 32°C whereas the cocultures temperature was reported to be between 29°C and 33°C whereas for Pot B it was up to 32°C and for Pot C it was up to 33°C this was also in line with Mizwar et al. [20]. A reduction in the TOC content was observed in both co-cultures and in soil remediated by earthworms was observed. Which showed the utilisation of naphthalene as the sole carbon source by both the bacterial strains and the vermiremediated samples.
In this study, the control showed a range between 6.9% and 8.2%, and the TOC was highly reduced in bioremediated soil about 2.5% to 7.3% than in vermiremediated soil about 3.7% to 7.3% ( Figure 12). The control (Pot A) showed 432.24 mg/kg of TKN content in the soil on Day 0. The TKN content on Day 3 for Pot B and D was reported as 416.28 mg/ kg and 540.24 mg/kg, whereas, the TKN content for Pot B and D on Day 15 was determined to be 420.28 mg/kg and 424.84 mg/kg, respectively. Therefore, the TKN content was quantitatively high in the co-cultured soil remediated sample when compared to that of the vermiremediated soil as shown in Figure 13. This showed the mineralisation of the organic content was more with the added cocultures in bioremediation than the earthworms used in the vermiremediation process. The reason could be the direct addition of bacterial strains aided to increase the bioremediation process than the earthworm microflora. Karthika Arumugam et al. [21] explained the physicochemical analysis of the contaminated soil with PAH for both bioremediated and vermiremediated soil. pH varied from 6.14 to 7.56 for bioremediation and 6.18 to 8.01 for vermiremediated soil. TOC was reported to be 37.47% for bioremediation and was lesser in vermiremediation about 26.52%. The TKN content was in the range of 1.75% and 1.39% for bioremediated and vermiremediated soil, respectively. Figure S6 pH analysis for the naphthalene remediated soil using co-cultures and earthworms.

Conclusions
The present study focused on different optimisation parameters of the bacterial strains to degrade the low molecular weight aromatic compound -Naphthalene and the biodegradation of this compound which was enhanced with the halotolerant bacterial strains. The bioremediation of Naphthalene with the encapsulated form of the isolated halotolerant bacterial strains with biochar was implemented to remove the Naphthalene from the contaminated soil. Apart from this vermiremediation also plays a major role in remediating the soil and degrading the Naphthalene present in the soil. Earthworms harbouring the microbial consortium also tend to reduce the Naphthalene contamination in the soil by degrading it effectively. Hence, these halotolerant bacterial strains can be used as co-cultures or can be immobilised on a carrier material which could increase the effectiveness of Naphthalene degradation. In the further study addition of cocultures along with vermiremediation of naphthalene will prove to be an effective integrated biological approach for the remediation of polyaromatic hydrocarbons in the contaminated environment.