Introduction

Now a days, crude oil remains a major source of energy and chemical raw material. Large-scale production, transport, use and disposal of petroleum globally have made it a major contaminant in both prevalence and quantity in the environment¹.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous oil contaminants in the environment with potential mutagenicity and carcinogenicity. They are generated from natural combustion processes as well as from human activities². Industrialization & accidental spillage have increased the pollution of Hydrocarbon compounds in the soil as well as water ³. Among the released compounds followings are of major concern: biphenyl, polychlorinated biphenyls (PCBs), petroleum chemicals including (PAHs) and BTEX (benzene, toluene, ethylbenzene and xylenes), pesticides, dioxins, furans, and flame retardants⁴.

Naphthalene, a hydrocarbon with two fused aromatic rings, the first member of PAH group and one of the 16 PAHs is classified as priority pollutants by Environmental Protection Agency (EPA) of United States, is a frequent pollutant established in nature ⁵. Naphthalene is an abundant component of coal tar. Also petroleum commonly contains naphthalene. Naphthalene is a raw chemical for industrial syntheses, in particular of phthalic anhydride. Human exposure to naphthalene was formerly not uncommon due to its (now obsolete) use as a moth repellent. Naphthalene at high concentration may cause hemolytic anemia and some other conditions, whereas the tumorigenic potential is presently considered low ⁶. Naphthalene has long been used as a model compound in PAH bioremediation studies.

PAH bioremediation is considered as an effective and environmentally benign cleanup technology as it involves the partial or complete bioconversion of these pollutants to microbial biomass, carbon dioxide and water⁷. Also, bioremediation has low cost and limited impact on the environment². The extent and rate of biodegradation depends on many factors including bioavailability of the carbon substrate, pH, temperature, oxygen, microbial load and strain, degree of acclimation, accessibility of nutrients, chemical structure of the compound, cellular transport properties, and chemical partitioning in growth medium [5,8]. A variety of bacteria have shown the potential of organic pollutants biodegradation, such as Arthrobacter spp., Bacillus spp., Pesaudomonas spp. and Mycobacterium spp. ⁹.

Microbial consortia have been widely used in cleanup of a number of pollutants in laboratory and field bioremediation studies. It is generally thought that microbial consortia are more effective than pure cultures in biodegradation of PAHs. This is possibly because of the broader enzymatic capacity achieved and the formation of toxic intermediate metabolites is counteracted by the selection of these dead-end products formed mainly by co-metabolism processes ⁵.

The aim of this study was to isolate Bacillus sp. from soil samples, test their ability as aconsortium to degrade naphthalene in liquid medium and compare it to pure cultures.

Materials and Methods

Sample Collection

Soil samples were collected from ten sites in Tehran andaround Tehran. The samples were collected in pre-sterilized sample bottles and the samples duly labeled and stored at 4 ^o C for further analysis.

Chemicals

Naphthalene, culture medium powders and mineral salts were purchased from Merck^® chemical company.

Isolation and characterization of crude oil– degrading Bacillus sp

The soil samples were passed through a sieve to remove large debris pieces and vegetation.1 g of each soil sample was dissolved in 9 mL of distilled water and heated at 80°C for 20 minutes. The samples were cultivated in 50 ml MSM medium with naphthalene concentration of 50 ppm for two weeks. At the end of each week, the colonies were isolated by pour plate technique on nutrient agar and MSM agar and incubated at 37 ^oC for 48 h. [10].The developed colonies were counted in plates and the average number of colonies was determinedper plates. The number of total bacteria (CFU) was determinedper gram dry weight of soil. The composition of the MSM medium used in this study was as follows (g/L):

NaNO₃ (2.0 g/L), NaCl(0.8 g/L), KCl (0.8 g/ L),CaCl₂.2H₂O(0.1 g/L), KH₂PO₄(2.0 g/L), Na₂HPO₄.12 H₂O(2.0 g/L), MgSO₄(0.2 g/L), FeSO₄.7H₂O (0.001 g/L); 2 mL trace element stock solution composed of (g/L): FeCl₃.6H₂O(0.08 g/L), ZnSO₄.H₂O(0.75 g/L),COCl₂.6H₂O(0.08g/L),CuSO₄.5H₂O(0.075 g/L), MgSO₄. H₂O (0.75 g/L), H₃BO₃ (0.15 g/L), Na₂MoO₄. 2H₂O (0.05 g/L). The initial pH was adjusted at 6.8¹¹.

Individual cultures were preliminarily identified by morphological and biochemical techniques using the taxonomic scheme of Bergey’s Manual of Determinative Bacteriology. Then, isolated bacteria were identified by molecular method as well.

16SrRNA sequencing method was used for identification. Universal primers were used for PCR (Polymerase Chain Reaction):

27F: 5′-AGTTTGATCCTGGCTCAG-3′

1495R: 5′-CTACGGCTACCTTGTT-3′

The reaction mixture (25 µL) contained: each primer at a concentration of 0.5 µL, 12.5 µL of master mix, 1 µL of DNA and 10.5 µL of double distilled water. The following thermal profile was used for the PCR (My Cycler, Bio-Rad Laboratories, and United States): 94 ^oC, 2min; 35 cycles of 94^oC, 1 min; 55 ^oC, 1min; 72 ^oC, 2 min; 1 cycle of 72 ^oC, 10 min¹². The samples were sent to Zist Takapou Company for sequencing. Then we used BLAST (Basic Local Alignment Search Tool) algorithm to compare the sequences.

Surface Tension Measurement

The isolated Bacillus cultivated on Blood Agar and incubated at 30°C for 72h. The bacteria with β haemolytic activity were selected for measuring the surface tension. The surface tension was measured using the Du Neouy ring method by tensiometer (KRUSS GmbH, Germany). All the measurements have done three times ¹³.

Emulsification test

This test was conducted and measured by using Cooper and Goldenberg method: 4 mL of Mineral Salt Medium after 48h incubation, and 6 mL hydrocarbon (oil) was added to each test tube. The mixture was vortexed at high speed for 2min, and then the test tubes were leaved to stand for 24h. The E24 index is given as percentage of height of emulsified layer (cm) divided by total height of the liquid column (cm) Cooper and Goldenberg ¹¹.

Determination of Growth Curve

The strains were cultivated in nutrient brothmedium and placed on shaker (K5501, IKA^®-Works,Germany) with 150 rpm shaking speed. O.D_600nm of the medium was taken by using spectrophotometer (T90 UV-Vis,PG instruments Ltd, England) after every 12 hour for 8 days and the growth curve was depicted.

The Ability of Growth in Different Naphthalene Concentrations

The naphthalene concentration in culture medium was gradually increased up to 1000 ppm. O.D_600nm of the medium was measured every day for a period of nine days and the naphthalene tolerance chart was drawn.

The Optimization of Growth Conditions

To maximize naphthalene degradation, parameter optimization was done using the Taguchi experimental design. The experimented parameters were PH (2, 4, 6, 8), shaking speed (100,120,150,200), nitrogen source (ammonium sulfate, sodium nitrate, yeast extract, peptone) and naphthalene concentration of the medium (50, 100, 150, 200 ppm). After this experiment surface tension of consortium was measured again.

G.C. Analysis

For GC (Gas Chromatography) analysis, the culture medium was first transferred to a 250 ml pre-ash conical flask by addition of 20 μL m-terphenyl as internal standard (prepared in acetone with concentration of 1000 mg.L⁻¹) and 25 ml ethyl acetate and shacked for 15 min. After separation from the water layer, the organic solvent layer was transferred to a clean conical flask before the second extraction by another aliquot of 25 ml ethyl acetate. Finally, two extracts were combined together, dried by anhydrous Na₂SO₄ and the volume was adjusted to 50 ml. About 1 ml of the extract sample was transferred into a 1.5 ml brown vial and analyzed by GC-FID. Three vials were prepared, including control, fourth day and eighth day samples. .The GC-FID was equipped with a HP-5MS fused silica capillary column (60 m × 0.25 mm ID × 0.25 lm thicknesses, Agilent Technologies, USA) with the injector and detector temperature of 280°C and 300°C, respectively. Helium was used as the carrier gas. The oven temperature program was set as follows: The oven temperature program was from 80°C (for 2 min) to 120°C at a rate of 10°C/min and from 120°C to 300°C at a rate of 4°C/minute and held at 300°C for 15 min. The identification and quantification of chemicals were conducted based on matching their retention times of standards¹⁴.

Identification of catabolic genes

To reveal the proportion of colonies hybridizing for each probe, dot blots were prepared with 3 µg of genomic DNA from each isolate applied to a positively charged nylon membrane. Southern blot consisting of 10 Wl of PCR products amplified from DNA extracted directly from each soil sample and resolved on an agarose gel, were also blotted to a positively charged nylon membrane for analysis by hybridization with the corresponding ³²P-labelled probes.

PCR amplification was carried out in 20 mM TrisHCl (pH 8.4); 50 mM KCl; 1.25 mM MgCl2; dNTPs at 200 WM each, 2.5 units PLATINUM Taq DNA polymerase; 0.2 µM forward and reverse primers; and template DNA at 0.1 µg per 50 µl reaction. The cycling conditions (Techne Cyclogene Thermal Cycler) were 5 min at 94³C, followed by 25 cycles (40 cycles when amplifying from DNA isolated from soil) of 94 ^oC for 2 min, 55 ^oC (nah primers) for 1 min, 72³C for 1 min, maximal ramp rates throughout, with the final 72 ^oC segment of the cycle extended to 10 min beforecooling to 4 ^oC. PCR products amplified from genomic DNA of PAH-degrading isolates were visualized by agarose gel electrophoresis¹⁵.

Statistical Analysis

We used one way ANOVA test for statistical analysis, also Taguchi experimental design was analyzed by factorial ANOVA,at a level of significance of p< 0.05.

Results

Isolation and identification

Four morphologically different Bacillus colonies were isolated from the samples. The maximum numbers of bacteria 4.1* 10⁴ and the minimum numbers of PAHs degrading bacteria 1.8*10⁴ of view.

The isolates were gram positive, rode shaped and had endospores. Figure 1 shows gram and malachite green stains’ results. The results of biochemical tests have shown in table1.

Figure 1: a- Gram stain, b-Malachite green stain of isolates   Click here to View figure

The strains a, b, c and d were identified as Bacillus cereus, Paenibacillus lactis, Bacillus fusiformis and Bacillus subtilis respectively by 16SrRNA sequencing method. A 100bp marker was used. Figure 2 shows the band positions that indicated in the gel

Figure 2: The band positions indicated in the gel. a: Bacillus cereus. b: Paenibacillus lactis. c: Bacillus fosiformis. d: Bacillus subtilis   Click here to View figure

Surface Tension Experiments

All fourisolates have shown colonies on Blood agarmedium, therefore they were selected for surface tension experiment.

The initial surface tension was 70 (mN/m), which was reduced to 38,33,35,37 and 29 (mN/m) by Bacillus cereus, Paenibacillus lactis, Bacillus fosiformis and Bacillus subtilis and consortium of four isolates respectively.

Emulsification test

The ability of emulsifying for the consortium was 85% and for Bacillus cereus, Paenibacillus lactis, Bacillus fosiformis and Bacillus subtilis were 76%,73%,77% and 79% respectively.

Growth Curve Study

Figure 3 shows the growth curve of the isolates in nutrient broth medium. Generation time for the isolates was 24 hours.

Naphthalene Toleration by Consortium

According to results, the consortium had ability to biodegrade 1000 ppm of naphthalene concentration in MSM liquid medium (Figure4). Growth on MSM agar medium after nine days confirmed this result.

Table1: Biochemical Tests’ Resultsfor Bacillus sp Isolated from Soil of Tehran and around Tehran

Optimization Experiment

The results of optimization experiment showed that the optimumpH for consortiumgrowth was in MSM medium; also the best shaking speed for the growth was 150rpm. The best additional nitrogen source was yeastextract and naphthaleneconcentration at 200ppm had the best effect on growth. (Figure 5) And surface tension amountwas 26 (mN/m)in this culture.

Figure 3: Growth curve of isolates   Click here to View figure

Figure 4: Naphthalene tolerance capacity of consortium   Click here to View figure

Figure 5: a. The comparison of consortium growth rate in different pH, b, and the comparison of consortium growth rate indifferent shaking speeds, c; the comparison of consortium growth rate in different extranitrogen sources. A.S: Ammonium sulfate. S. Nitrite: Sodium nitrite. S. Nitrate: Sodium nitrate. Y.E: Yeast extract and P: Peptone, d. the comparison of consortium growth rate in different concentrations of naphthalene   Click here to View figure

G.C. Analysis

In order to determine naphthalene degradation and intermediate metabolites formation we used GC analysis. The chromatograms were shown in Figure 6.

Identifications of Catabolic genes

All of naphthalene degrading isolates possessed nah genes.

Statistical Analysis

There was a significant difference 0.05 between the average of the bacteria in medium with and without PAHs. Also data was significantfor surface tension and optimization experiment.

Discussion

Isolation of Bacillus sp

In this study, ten soil samples were collected from Tehran and around Tehran. Five samples were from contaminated sites and five samples were from uncontaminated sites. Three of the contaminated samples contained Bacillus naphthalene degraders and one of the samples from uncontaminated sites contained Bacillus degraders, because the ability of native microbial population in biodegradation of contaminants were shown in former studies¹¹.

According to the previous studies Bacillus cereus and Bacillus subtilis were isolated from soil samples which could degrade naphthalene and other PAHs, but to the best of our knowledge, Paenibacillus lactis with degradation abilities hasn’t isolated from soilsbefore^11,16,17.

Surface Tension Measurement and Emulsification Test

The surface tension amount for consortium was the lowest; therefore it had the ability to produce more amount of biosurafctant than pure cultures and biodegrade naphthalene more effectively.

For the first time Banat in 1993 performed both haemolytic and surface tension experiment to determine biosurfactant production. Also in other studies hemolytic and surface tension experiment were used on Bacillus subtilis^10,18. We selected the isolates with surface tension amount lower than 40 (mN/m), which contained all of strains. The consortium showed the lowest surface tension. That means it produced the maximum amount of biosurfactant ¹⁹. Akhavan etal in 2008 used emulsification test for Bacillus spp.¹¹

Figure 6: a. Chromatogram of solvent (ethyl acetate), b. Chromatogram of control sample, c. Chromatogram of sample collected from fourth day after growth, d. Chromatogram of sample collected from eight days after growth.   Click here to View figure

Growth Curve

The isolates had almost steady growth for the first day (Lag phase). After that, the growth rate was exponentially increased (Log phase)for the next six days. At the end, the growth rate was remained steady (Stationary phase). Growth curves of four isolates were approximately similar.

In 2012 a research conducted by kafilzadeh and growth curve of Pseudomonas sp. and Corynebacterium sp. on pyrene, phenantherene   and naphthalene was studied and it involves all stages we saw in this study ²⁰.

Growth in High Concentrations of Naphthalene

Figure 4 suggests an effective degradation of consortium and ability to adapt with naphthalene concentrations up to 1000ppm.

The Optimization study for consortium

The results of a previous study showed that the best nitrogen source for Bacillus growth is yeast extract ²¹. Also pH=7 was shown to be the best amount of pH for Bacillus fusiformis growth in the medium²².

G.C. Analysis

Figure 6 showed that naphthalene concentration in MSM liquid medium was reduced in eight days and intermediate metabolites from its degradation (like 1,2-dihydroxy 1,2-dihydro naphthalene) were increased.

This method was used to determine alkanes’ degradation by bacteria in India. The results showed that Bacillus subtilis is the best degrader²³. In another study, itwas used on phenantherene and fluorene degradation by a consortium enriched from mangrove sediments¹⁴.

Identifications of Catabolic genes

Lloyd-Jones etal investigated catabolic genes involved in naphthalene and phenantherenein soils of New Zealand. nah genes were identified in Pseudomonas spp.¹⁵.

The Comparison of Pure Cultures and Consortium in Naphthalene Biodegradation

According to the results of this study the consortium of four isolates could biodegrade naphthalene more effectively than each culture. In a former study, a consortium of Bacillus spp. reduced surface tension of medium from 72 (mN/m) to 28 (mN/m), therefore it was more effective than pure cultures²⁴. In another study, the consortium showed more ability for biodegradation of 2,4-dichlorophenol ¹⁶.Results of another study showed that a consortium of Psedumonas, Bacillus and Staphylococcus has more capability for biodegradation than pure cultures ²⁵. Abdel et al in 2008 got the same results in their study on a Bacillus consortium.²⁶

Conclusion

In this study we isloated Bacillus sp from soil samples and measured naphthalene biodegradation ability on pure cultures and their consortium. Results of phenotypic and genotipic identification showed that these isolates wereBacillus cereus, Paenibacillus lactis, Bacillus fusiformis and Bacillus subtilis with 99% of similarity index.The results of surface tension experiment showed that the consortium of four isolates produce more amount of biosurfactant and in emulsification test we understand the consortium was a better emulsifier. Therefore it was more effective in naphthalene biodegradation in comparison to pure cultures. The findings of this study suggest a potential bioremediation role of this Bacillus consortium in the removal of naphthalene from contaminated soils.

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