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Research Article, J Mar Biol Oceanogr Vol: 4 Issue: 2

Isolation and Investigation of Biodegradation Potential of Multiple Polycyclic Aromatic Hydrocarbons (PAHs) Degrading Marine Bacteria near Bhavnagar Coast, India

Rahul K Rajpara, Dushyant R Dudhagara, Jwalant K Bhatt, Chirag M Ghevariya, Tejal B Domadiya, Haren B Gosai, Anjana K Vala and Bharti P Dave*
Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Sardar Vallabhbhai Patel Campus, Bhavnagar - 364001, Gujarat, India
Corresponding author : Bharti P Dave
Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Sardar Vallabhbhai Patel Campus, Bhavnagar - 364001, Gujarat, India
Tel: 0278-2519824; Fax: 2521545
E-mail: [email protected]
Received: October 05, 2015 Accepted: December 03, 2015 Published: December 08, 2015
Citation: Rajpara RK, Dudhagara DR, Bhatt JK, Ghevariya CM, Domadiya TB, et al. (2015) Isolation and Investigation of Biodegradation Potential of Multiple Polycyclic Aromatic Hydrocarbons (PAHs) Degrading Marine Bacteria near Bhavnagar Coast, India. J Mar Biol Oceanogr 4:2. doi:10.4172/2324-8661.1000147

Abstract

Present work deals with modified isolation methods for indigenous microorganisms with a capability to use both low molecular weight (LMW) and high molecular weight (HMW)PAHs, which are pervasive recalcitrant pollutants. Methods such as biphasic enrichment, and specific isolation methods has resulted in the isolation of organisms such as Sphingomonas, Pseudomonas, Mycobacterium, Achromobacter, and Streptomyces species with efficacy to degrade majority of LMW PAHs up to 85% after four days of experiments, which is substantially rapid rate of degradation attributed by microorganisms. Moreover, the organisms had shown up to 30% degradation of HMW PAHs within the same time frame, making the isolation strategies more credible. The study thus, holds prime importance of conquering the difficulties in the isolation of multiple hydrocarbons degrading microorganisms, which can be further applied for the successful application for bioremediation of hydrocarbon impacted environments.

Keywords: Biodegradation; Multiple PAHs; Selective isolation; Marine microorganisms

Keywords

Biodegradation; Multiple PAHs; Selective isolation; Marine microorganisms

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are class of organic pollutants that are considered as most hazardous compounds due to their toxic, mutagenic, and carcinogenic properties. PAHs can be separated on the basis of their aromatic benzene rings; low-molecularweight (LMW) compounds consist of two or three rings, and highmolecular- weight (HMW) compounds, more than three rings. They are distributed naturally as coal and petroleum. Furthermore they are also instigated anthropogenically by burning coal, gas and vegetation, waste incineration, transportation and spillage of oil, forest fires, and volcanic eruptions [1-4]. They are listed as priority pollutants by United States Environmental Protection Agency (USEPA) due to their mutagenic and carcinogenic effects on biota.
Due to the wide variation in environmental conditions, microorganisms possess unique characteristics for adaptation to such hostile environments. Therefore, bacteria isolated from the marine environment have the capability to utilize PAHs and other recalcitrant pollutants. The main advantage of the use of these indigenous microbial communities is that it can be directly used in bioremediation study without any genetic alteration [5].
The indigenous diversity of marine bacteria has high potential to adapt to unfavorable conditions and its flux. Hence, they are considered to be important global players for the bioremediation of polluted habitats. In past years, many capable marine bacteria have been isolated to utilize LMW and HMW PAHs as carbon and energy source have been studied which includes Sphingomonas, Pseudomonas, Bacillus, Nocardia, Oceanospirillum, Alcanivorax, etc. [6].
Various microbial groups participate in the degradation of PAHs in the soil, but bacteria and actinomycetes are good candidates for soil bioremediation because they utilize a broad range of carbon sources including aromatic molecules. Moreover, sites of contamination contain multiple PAHs. It is meaningful to isolate strains that have stable degradation ability for mixture of PAHs reflecting the actual structural complexity of multicomponent PAHs in contaminated environment which is otherwise not observed in bacteria isolated from contaminated sediments having an individual PAH [2-7].
The focus of the present study has shifted to isolation of multiple PAHs degrading bacteria that can successfully mineralize both LMW and HMW PAHs from contaminated sediments, and can be exploited for the restoration of PAHs impacted sites.
Based on our previous studies, isolation methods for PAHs degrading marine bacteria require extensive modification of preexisting techniques for achieving an increase in bacterial PAHs degrading diversity with faster growth rates. Based on our experience and the difficulties encountered, we here in report various isolation and screening approaches that may overcome the pre-existing difficulties. The responses towards these modified strategies have resulted in the isolation of indigenous microorganisms capable of degrading high concentrations of multiple PAHs in polluted marine environments.

Materials and Methods

Sample collection
Polluted surface sediment samples were collected from crude oil contaminated sites near Bhavnagar coast (latitude 21.46 °N, longitude 72.11 °E, latitude 21.12 °N, longitude 72.3 °E and latitude 21.74 °N and longitude 72.12 °E) Gujarat, India using sterilized amber glass bottles and stored at 4 °C for further use.
Media and chemicals
All media were purchased from Hi-Media Laboratories, India. Solvents, reagents and chemicals used were of HPLC grade and purchased from Fishers Scientific, India. Naphthalene (Nap), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flt), pyrene (Pyr), and chrysene (Chry) were purchased from Sigma-Aldrich, Germany. Glasswares used were amber to avoid photooxidation of volatile PAHs.
Enrichment using selective media
Nagel and Andreesen’s (NA) medium [8] and Bushnell-Hass (BH) [9] medium were used for enrichment of PAHs degrading bacteria from crude oil polluted marine sediment samples.1g sediments were added to 50 mL of above medium, amended with 50 ppm of each of the hydrocarbon (ƩPAHs = 300 ppm). The flasks were kept on environmental shaker (New Brunswick, USA) at 150 rpm at 30 °C. After seven days of enrichment, 100 μL samples from each flask were spreaded on different selective media such as Pseudomonas isolation agar, R2A agar, Humic acid-Vitamin (HV) agar, Lowenstein-Jensen (LJ) agar, actinomycetes isolation agar (AIA) and glucose asparagine agar (GAA) (Table 1).
Table 1: Various selective methods for isolation of multiple PAHs degraders.
Identification of marine bacteria
Identification of marine isolates had been done based on the utilization of 95 different carbon sources coated on BiologTM Microtitre plate. The results of the reactions were captured in the corresponding database of BiologTMMicroLog software version 4.2.
Extraction and GC-MS analyses of residual PAHs
For extraction of PAHs, equal volume of dichloromethane was added to the BH broth and sonicated for 5 min thrice with one minute of rest. Solvent phase was collected and the same procedure was repeated twice. Aqueous phase was removed by Na2SO4 and collected solvent phase pooled and evaporated with gentle stream of N2 gas. After evaporation residual fluoranthene was dissolved in dichloromethane and injected into GC-MS [10].
Residual PAHs were estimated by GC-MS (Shimadzu QP2010+, Japan). The conditions kept were as suggested by Mohajeri et al. [11] in which Rtx-5 capillary column (Restek, USA) (0.25 mm internal diameter, 0.25 μm film thickness, 30 m long) had been used. The MS was operated in the selected ion monitoring mode with a temperature program of 60°C for 1 min hold, then increased by 10°C min-1 up to 160 °C then 10 min hold followed by further increase up to 280°C at a rate of 5°C min-1 hold for 10 min. Pure helium (99.999%) was used as a carrier gas at flow rate of 1.5 mL min-1. 1 μL sample was injected into the injector with temperature of 280°C using splitless injection mode and the interface temperature was maintained as above [11,12]. Prior to GC–MS analysis, standard PAHs mixture was injected, for internal calibration and their quantification.

Results and Discussion

Enrichment using selective media
Numbers of bacterial colonies were obtained on various selective media (Figure 1). R2A agar had been used for the isolation of Sphingomonads. Sphingomonas sp. is versatile in nature regarding their metabolic activity and is commonly isolated from PAHs polluted environment. It has been extensively reported to utilize recalcitrant compound like PAHs, dye, polychlorinated biphenyl (PCB) [13]. Sphingomonads have the ability to utilize a broad range of PAHs. They have also shown the ability to co-metabolize HMW PAHs [14]. The present study also demonstrates the diversity of Sphingomonas species. A total of nine colonies were picked on the basis of their morphological characteristics from R2A agar plate and subsequently assayed for screening on solid agar media coated with various PAHs. Pseudomonas as the most versatile genus and the metabolic pathways with the majority of the PAHs are well documented as described by [15]. In the present study, six different colonies based on colony morphology were selected for subsequent screening.
Figure 1: Total Number of colonies found on different selective media.
Selective isolation of Mycobacteria
Humic acid–Vitamin agar olive oil emulsification: From the HV-agar plate total of nine isolates have been selected based on variation in morphological features, for further study. The organisms were able to utilize humic acid as a sole carbon and nitrogen source. The HV agar plate has the advantage for the selective isolation of actinommycetes as they decompose and utilize humic acid, which otherwise resist biological decomposition. However, reports suggest that not all Actinomycetes can utilize Humic acid. Another mechanism is the probable activation of actinomycetes spores upon germination when plated on HV agar hence, the number of colonies on HV plate can be increased [16].
SDS–Cetrimide selection: By using SDS-Cetrimide selection method, five isolates have been selected on the basis of morphological features. Method for the selective isolation of Mycobacteria from crude oil polluted soil samples as proposed by Yamamura [17]. They have the ability to utilize both LMW and HMW PAHs and can efficiently degrade soil sorbet PAHs particles and thereby play a vital role in cleaning of environment pollutants directly or cometabolically [18,19]. Demulsification of olive oil- SDS emulsion (DOSE) is a of fast growing mycobacteria. On combining it with HV agar plates can result in the selective isolation from the environmental samples. It is one of the decontamination protocols where organisms other than mycobacterial species can be differentially removed. SDS in combination with NaCl and cetrimide could result in efficient recovery of mycobacteria from environment samples.
Selective isolation of actinomycetes
CaCO3 and chitin treatment: As a result, a total eight colonies were selected on the basis of morphological characteristics for further screening. CaCO3 and Chitin were the most efficient as it resulted in higher number of Actinomycetes colonies with very low relative number of bacteria and fungi [20].
Filtration method: Filtration method allows only filamentous cells with polymorphic cell shape and excludes the majority of bacteria which are abundant in the sample and also allows cultivation of some uncultured diversity [21]. In this method, a total seven colonies were selected from AIA medium for further screening study.
Biphasic technique: In the biphasic aqueous-organic system, conversion of the substrate takes place in the interfacial area. Silicone oil is widely used in biphasic technique due to its hydrophobic properties and its higher thermal stability [22]. Microorganisms grow at the interfacial area between aqueous and non-aqueous phase. In the present approach, a total six colonies were selected for the screening of multiple PAHs degrading bacteria. It has been reported that the accelerated degradation of PAHs can be obtained when biphasic water- silicone oil system is used compared to monophasic aqueous system. The difference between these two systems can be explained by the partitioning effect of the substrate in the silicone oil phase. This phase may facilitate a shift in reaction equilibrium by supplying the substrate between the phases. Moreover, silicone oil reduces the evaporation of volatile PAHs. Additionally, the high microbial activity obtained in the biphasic water-silicone oil system could have been the result of the optimization of the microenvironment.
Thus, by incorporating physically and chemically inert organic phase - silicone oil, adaptation of xenobiotic compound degrading microorganisms can be accelerated and in turn elevated degradation can also be achieved. Hence, the biphasic system could be highly significant for both – the selection of multiple PAHs degrading microorganisms and degradation of poorly water soluble PAHs [22].
Screening of multiple PAHs degrading marine bacteria
From the above selective media, collectively a total of fifty isolates (designated as LS 1 to LS 50) had been obtained based on various morphological criteria as depicted in Fig. 1. These isolates were further examined for the utilization of PAHs, based on the zone of clearance on plates coated with individual PAHs is mentioned in Table S1. . Based on the zone of clearances fourteen isolates that exhibited utilization of four or more LMW and HMW PAHs were selected for further studies. Amongst them, eleven were obtained from liquid culture experiments whereas the remaining three were obtained from biphasic technique.
Identification of isolates
Multiple PAHs degrading fourteen isolates as above had been identified by BiologTM Microtitre plates, by using Micro Log software version 4.2 (Table 2).
Table 2: Identification of isolates using BIOLOG system.
Utilization of multiple PAHs in liquid culture
A total eleven isolates obtained from liquid culture were selected for the utilization of both LMW and HMW PAHs by liquid culture experiment (Figure 2). Streptomyces sp. showed considerable 11.23% and 8.39% degradation of Nap and Phe, respectively; followed by Pyr (6.32%) and Ant (4.22%) on the 4th day of incubation. The results of Balachandran [23] who had also worked on Streptomycetes sp. isolated from oil contaminated soil to degrade Nap, Phe, and diesel oil. Similar work has also been carried out by Ferradji et al. [24], who have isolated Streptomycessp. AB1, AH4, and AM2 from the North of Algeria which degraded Nap 82.36%, 85.23%, and 81.03% respectively after 12 days of incubation.
Figure 2: Degradation of multiple PAHs in liquid culture experiment by different isolates on 4th day.
Nocardia carnca showed 23.06% and 14.43% degradation of Nap and Phe, respectively while no apparent difference has been observed in the degradation of Ant (8.69%) and Pyr (8.32%) on 4th day of incubation. Nocardia Otitidiscaviarum strain isolated from petroindustrial area, able to degrade Nap as demonstrated by Zeinali et al. [25].
Three Sphingomonas species identified as Sphingobium yanoikuyae, S.paucimobilis, and Sphingomonas sp. were able to utilize LMW and HMW PAHs. Sphingobium yanoikuyae exhibited 99.66% degradation of Nap, followed by 66.32% and 44.79% degradation of Phe and Ant, respectively (Figure 2). The isolate has showed inadequate 26.66% and 15.35% degradation of Pyr and Flt on the 4thday of incubation. The results are in harmony with Zhong et al. [14] who has also worked on Sphingomonas sp. which showed the ability to co-metabolize Ant and Flt when growing in phenanthrenecontaining mineral salts medium. S. paucimobilis showed 95.09% and 94.52% degradation of Nap and Phe, respectively on 4thday, followed by 45.33% (Pyr), 25.37% (Ant), and 16.23% (Flt) on 4th day of incubation. The results are in accordance to the degradation of HMW PAHs (Pyr and BaP) by S. paucimobilis after four days of incubation [26]. Sphingomonas sp. showed a striking 79.56% degradation of Nap and 68.23% degradation of Phe, followed by 45.13% and 12.45% degradation of Ant and Pyr, respectively on the 4thday. Sphingomonads have been found to Nap maximally, followed by Phe; whereas, Flt was the least degraded hydrocarbon.
In the present study, three Pseudomonas species have been identified as Pseudomonas aeruginosa, P. putida and Pseudomonas sp. (Table 2). Pseudomonas aeruginosa has exhibited 79.38% degradation of Nap, followed by 50.32%, 22.30%, and 15.39% degradation of Phe, Ant, and Pyr degradation, respectively on the 4th day of incubation. Pseudomonas sp., and P. putida both showed extensive 90.66% and 56.50% degradation of Nap, 94.52% and 35.30% degradation of Phe, and 8.89% and 15.56% degradation of Ant, respectively on 4th day of incubation (Figure 2). Nap and Phe were the most degraded hydrocarbons amongst all LMW PAHs. Similar results have been extrapolated by Jacques et al. [27] in which three species of Pseudomonas from long term petrochemical polluted sludge have been isolated. Among these, P. aeruginosa have multiple PAH degrading capability and showed 71% degradation of anthracene on 2nd day of incubation, whereas, P. citronellolis and P. aeruginosa showed 51% and 24.4% degradation of Ant, respectively.
Three different strains of Mycobacteria have been isolated namely Mycobacterium aurum, M. vanbaalenii and Mycobacterium sp. All three isolates exhibited the ability to utilize multiple PAHs as carbon and energy sources (Figure 2). Mycobacterium aurum had exhibited 86.65% Nap followed by 63.15% Phe on the 4th day of incubation. Whereas, Ant (15.56%) and Flt (10.53%) has been degraded to a lesser extent by the same isolate. Strains of Mycobacterium which were isolated from creosote-contaminated soil and their ability to utilize Pyr and Flt as sole source of carbon and energy has also been demonstrated by [28]. M. vanbaalenii had been found to degrade five of the hydrocarbons, out of which Nap (56.49%) was found to be the most degraded hydrocarbon followed by Phe (41.72%), and Ant (30.45%), while Pyr (23.56%), and Flt (16.08%) were least degraded hydrocarbons. Similar study has also been conducted by Moody et al. [29], in which Mycobacterium strain PYR-1 had also shown degradation of 92% Ant and 90% Phe after 14 days of incubation. Mycobacterium sp. had been able to degrade 41.64% Nap and 36.12% Phe whereas, 13.56% Ant and 9.03% Pyr had been degraded on the 4th day of incubation.
Utilization of multiple PAHs in Biphasic technique
This technique was very constructive for hydrocarbon degradation as they have very low solubility and very high hydrophobicity in an aqueous system. Three isolates viz. Achromobacter xylosoxidans, Sphingomonas sp., Pseudomonas stutzeri have been screened. A. xylosoxidans was found to be the most efficient isolate amongst all three having the ability to degrade six different hydrocarbons. The isolate degraded 57.56% Nap followed by 45.46%, 43.12% and 38.31% of Phe, Pyr, and Ant respectively on the 7thday. The isolate also degraded Chry (29.73%) and Flt (23.56%) on the 7th day of incubation (Figure 3).
Figure 3: Degradation of multiple PAHs using biphasic technique with different isolates on 7th day.
Sphingomonas sp. showed 68.45% Nap removal on the 7th day, followed by Phe (59.23%), and Ant (24.26%). Pyr (31.32%) and Chry (8.40%) had degraded in less extent. A novel strain of Sphingomonas sp. with the ability to degrade 50% Chry in biphasic technique on the 5thday of incubation reported by Willison et al. [30-32].
P. stutzeri showed 86.76% Nap degradation, followed by 57.23% Phe, 39.35% Pyr degradation on 7thday, whereas, Chry (23.43%) and Ant (20.13%) had been degrading less after 7th day of incubation.
It has been observed that Nap and Phe had been degraded maximally followed by Ant amongst LMW PAHs, amongst HMW PAHs, degradation of Pyr was found to be higher as compared to Flt and Chry. It has been observed that LMW PAHs have been preferentially degraded over HMW PAHs amongst the mixtures of PAHs. It clearly indicates the difficulty in the degradation of HMW PAHs by the isolates. Moreover, fourteen isolates did not show the ability to assimilate various hydrocarbons as their carbon and energy source indicating the level of difficulty to exploit microorganisms for hydrocarbon bioremediation.
The study thus holds the importance so as to bridge the gap between the pre-established methods and newer efficient techniques of isolation of multiple PAHs utilizing microorganisms.

Conclusion

The study was subjected to isolate potent multiple PAHs degraders using various isolation strategies. The major findings of the study are the organisms belonging to the groups such as Mycobacterium, Rhodococcus, Nocardia, and Proteobacteria were found to be predominant. Fourteen potent PAHs degrading isolates had been obtained having tremendous capability to degrade multiple LMW and HMW PAHs within considerably less time i.e., four days. The methods thus can be employed so as to isolate indigenous microorganisms having inherent properties to degrade multiple hydrocarbons in hostile environment, making them suitable agents for bioremediation of polluted environment.

Acknowledgments

Authors are thankful to Gujarat State Biotechnology Mission (GSBTM) Gandhinagar, Gujarat and Earth System Sciences Organization (ESSO), Ministry of Earth Science, Government of India, New Delhi.

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