Biodegradation of mixed polycyclic aromatic hydrocarbons by Pseudomonas sp. isolated from estuarine sediment

Abstract Polycyclic aromatic hydrocarbons (PAHs) are released into the environment via several natural and anthropogenic sources leading to long-term consequences that severely affect the environment, ecosystem, human and animal health. In this study, the extent of PAH pollution was measured at two estuarine locations of the major rivers of Goa, Zuari and Mandovi. Out of the 16 PAHs marked as carcinogenic, 13 PAHs were detected from the sediment samples collected. Bacterial strains were isolated from these PAH-contaminated sediments using conventional plating method. Six of these selected bacterial isolates were checked for their efficacy to degrade both low (phenanthrene) and high molecular weight (fluoranthene and pyrene) hydrocarbons. Amongst them, two bacterial isolates exhibited over 80% degradation of phenanthrene. Isolate NIOSV7 (Pseudomonas pachastrellae) could degrade 83.33% phenanthrene, 40.22% fluoranthene and 45.28% pyrene, while isolate NIOSV8, identified as Pseudomonas oleovorans, showed 85.09% phenanthrene, 70.61% fluoranthene and 67.18% of pyrene degradation in 120 h, at 100 ppm initial concentration of phenanthrene and 75 ppm of fluoranthane and pyrene. Though many Pseudomonas sp. are documented for PAH degradation, this is the first report showing PAH-degradation by Pseudomonas oleovorans strain. This study also shows that the bacteria isolated from estuarine sediments contaminated with PAH have good potential to degrade low and high molecular weight PAHs. These toxic pollutants can be bio-transformed into nontoxic metabolites with the help of microorganisms isolated from the same habitat.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants generated from incomplete combustion of carbon-containing substances, including fossil fuel, biomass fuel, tobacco smoke, charred and smoked meat, forest fires and volcanic eruption (Behera et al. 2018). Their poor solubility in water and intrinsic chemical stability makes them persistent and pervasive in the environment (Paulik et al. 2016). PAHs tend to adsorb on particulate matter in water, air, sediment and soil; these characteristics make them less available for biological uptake and allow them to accumulate in the food chain. This bioaccumulation of PAH leads to a toxic, carcinogenic and mutagenic effect in aquatic and human life (Masiol et al. 2012;Abdel-Shafy and Mansour 2016;Alegbeleye, Opeolu, and Jackson 2017).
Due to anthropogenic activities, the marine ecosystem is prone to PAH contamination, including accidental oil spillage or oil seepage from ships, drilling, refining, transportation, and petroleum industries. The released PAH gets deposited at the bottom of the riverine and marine ecosystem; hence the sediments of the marine ecosystem act as the ultimate sink for PAH (Arulazhagan, Vasudevan, and Yeom 2010;Aziz et al. 2018). The priority pollutants listed by US EPA often found in marine and aquatic environments are phenanthrene (Phe), fluoranthene (Flt) and pyrene (Pyr), which contain three or four aromatic rings (Dudhagara et al. 2016;Ramzi et al. 2017;Veerasingam et al. 2015).
Over the past four decades, various treatment methods such as physical (incineration), chemical (photolysis, chemical precipitation, electrolysis and adsorption) and biological (bioremediation, phytoremediation, bio-augmentation) processes are demonstrated to treat hazardous contaminants. Even though various treatment methods are available to remove hydrocarbon contamination, bioremediation is a green alternative approach to treat these hazardous pollutants because of their nondeteriorating effect on the environment (Aziz et al. 2018). Bioremediation involves the degradation or transformation of hazardous pollutants into nontoxic products such as carbon dioxide and water. Bioremediation can be accomplished by biotic components, including bacteria, fungi, algae and plants (Pasumarthi, Chandrasekaran, and Mutnuri 2013;Alegbeleye, Opeolu, and Jackson 2017).
Bacterial degradation of PAHs is an efficient method to eliminate pollutants due to rapid growth, quick adaptability, cost-effectiveness and eco-friendly nature (Ghosal et al. 2016). As mentioned earlier, the marine environment is the ultimate sink of PAHs; the bacteria isolated from the marine origin have the intrinsic ability to degrade them at the fastest rate (Swaathy et al. 2014).
This study aims to isolate indigenous bacteria competent to degrade Phe, Flt, Pyr, and assess their capacity to clean up the PAH contamination. This study highlights biodegradation as a feasible tool to remove PAH contamination from the marine environment.

Chemicals
The standards of 16 PAHs were procured from Sigma Aldrich, USA (10 lg mL À1 ). Phe, Flt and Pyr were obtained from TCI, India. Organic solvents including n-Hexane, Acetone and Dichloromethane were purchased from Merck, India. The 10% stock solution of each Phe, Flt and Pyr was prepared by dissolving them in acetone. Zobell marine (ZM) agar and ZM broth were procured from Hi-media, India. The composition of minimal salt media (MSM) magnesium sulfate heptahydrate 0.2 g L À1 , calcium chloride 0.02 g L À1 , potassium dihydrogen phosphate 1.0 g L À1 , di-potassium hydrogen phosphate 1 g L À1 , ammonium sulfate 1 g L À1 , sodium chloride 10 g L À1 at pH 7. All the reagents and chemicals used for the study were of high purity and analytical grade.
Sample collection, isolation and screening of PAHdegrading bacteria The sediment samples were collected using a clean sediment grab from a fishing trawler at depths of 8 m for both sites in Mandovi, and 6 and 10 m in Zuari estuaries of Goa, India ( Figure  1). Immediately after collection, samples were stored in an amber-coloured bottle for PAH determination and sterile sampling bags for isolation of bacteria, and transported to the laboratory in an icebox. Sediment samples for PAH determination were kept at À20 C to avoid indigenous microbial activity. At the same time, the sediment samples for isolation of bacteria were immediately processed. Approximately 1 g sediment samples were serially diluted in seawater, and conventional spread plate technique was used to isolate bacterial strains on ZM agar plates. Pure bacterial isolates were obtained from these plates and maintained for further work. A single bacterial colony was inoculated in ZM broth and incubated overnight at room temperature on a shaker at 120 rpm. The overnight grown culture was inoculated to fresh ZM broth, and the culture broth was centrifuged to obtain a cell pellet. The cell pellet was washed and resuspended in MSM. The bacterial suspension was inoculated in MSM containing 50 ppm Phe, Flt and Pyr individually as a sole carbon source and incubated at 28 C, 120 rpm. Appropriate controls of MSM with PAHs without bacterial culture (to compensate for the abiotic loss due to volatilization) and MSM without PAHs with bacterial suspension were maintained at similar conditions. After 24 h incubation, the presence of phenolic intermediates in the culture broth during the degradation process was checked by Folin-Ciocalteu (FC) method (Bhatawadekar & Damare, unpublished work). Briefly, 100 lL of the mixture, each from the control and test, was transferred into a 96-well microtitre plate containing 100 lL of FC reagent, followed by 80 lL of 10% sodium bicarbonate to maintain an alkaline condition. This reaction mixture was incubated in the dark for 30 min and observed for the formation of a blue color complex to monitor the formation of intermediate metabolites. Resorcinol solution (0.5%) used as a positive control.

Identification of PAH-degrading bacteria
The pure bacterial isolates were grown in ZM broth for 24-48 h at 28 C, and the cell pellet was obtained from culture broth by centrifugation at 12,000 rpm for 2 min. Total genomic DNA was extracted using a DNA extraction kit (Nucleopore, Genetix Biotech, India) following the manufacturerspecified protocol. PCR amplification of 16S rDNA was carried out using universal primers 27F (5 0 -AGAGTTTGATCCTGGCTCAG-3 0 ) and 1492 R (5 0 -TACGGTTACCTTGTTACGACTT-3 0 ), followed by amplified product purification using PCR Clean-Up System (Promega Corporation, USA). Sequencing of nucleotide was performed using Genetic Analyzer 3730xl (ABI) based on the Big Dye terminator v 2.0 chain terminator chemistry. BLAST analysis was carried using the NCBI database. MEGA X (Kumar et al. 2018) was used for phylogenetic analysis of the sequences from the isolates along with other related type strain sequences obtained from NCBI.

Determination of PAH content in the sediment
Extraction of PAH from sediment samples was carried out using the modified US EPA 3540C method (US EPA 1996). Briefly, the freeze-dried and homogenized sediment sample was weighed (10 g) and mixed with anhydrous sodium sulfate to remove moisture and subjected to soxhlet extraction for 72 h using hexane and dichloromethane (1:1). Extracts were concentrated using the rotary evaporator and purified using a silica gel column. The purified extract was resuspended in 1 mL of acetonitrile for GC-MS analysis (Shimadzu, QP2010 plus). Rtx-5MS capillary column (0.32 mm internal diameter, 0.25 mm film thickness, 30 m long) was used. The MS was operated in the full scan mode with a temperature programme of 50 C for 5 min hold, then increased by 4 C min À1 up to 310 C hold for 10 min. Pure helium (99.99%) was used as a carrier gas at a flow rate of 2.0 mL min À1 . Sample (1 mL) was injected using split-less injection mode. Before GC-MS analysis, calibration and quantification were performed using a standard PAH mixture (2000 to 10,000 ng g À1 ). Total PAH concentration was reported in ng g À1 .

Bio-degradation of PAH by bacterial isolates
Initially, the bacterial inoculum was grown in ZM broth (20 mL in 100 mL conical flask) for 24 h, centrifuged to get cell pellet, washed with MSM and resuspended in MSM. This bacterial suspension was inoculated in MSM supplemented with 100 ppm Phe, 75 ppm Flt and 75 ppm Pyr as a sole carbon source and incubated at 28 C, 150 rpm for 5 days. Appropriate controls of MSM with the same concentrations of Phe, Flt, and Pyr were maintained at similar conditions to ascertain the abiotic loss due to volatilization.

Quantitative estimation of biodegradation of PAH
The residual Phe, Flt and Pyr were extracted with hexane. Briefly, an equal volume of hexane was added to the culture medium and kept in shaking condition for 1 h at 160 rpm, followed by vigorous shaking in the separating funnel and allowed to stand until two phases were separated. The extraction process was repeated thrice. First, the organic phase was pooled together and dried over anhydrous sodium sulfate. Then solvent in the organic extract was concentrated using the rotary evaporator, resuspended to a final volume of 1 mL, and analyzed using flame ionization detector on Shimadzu GC 2010 plus chromatogram equipped with fused silica capillary column of 30 m length, 0.32 mm internal diameter, and 0.25 mm film thickness. The injector and detector temperature were maintained at 300 C, and oven temperature programme was initiated with 50 C hold for 5 min and ramped with 4 C per minute to 280 C and final hold time for 10 min. Highpurity nitrogen (99.99%, bone dry) was used as a carrier gas at a flow rate of 3 mL min À1 . The degradation percentage was calculated as follows: Phe, Flt, Pyr per cent degradation where Ci ¼ initial concentration of PAH; Cf ¼ final concentration of PAH (residual PAH concentration).

PAH concentrations in the sediments
The total concentration of extractable PAHs in the sediment samples ranged from 1647 ng g À1 to 24,712 ng g À1 dry weight (Figure 2). The total PAH contamination present in the sediment was higher at Zuari than at Mandovi sites. The order of PAH concentrations found in Zuari sediment samples (ghi)perylene, and in Mandovi sediment samples was Fluoranthene > Pyrene > Naphthalene > Phenanthrene > Acenaphthylene > Fluorene respectively (Table 1). The total concentration of high molecular weight PAHs found in Mandovi and Zuari was 1240 and 3094 ng g À1 , respectively. The low molecular weight PAHs were high in Zuari (21,618 ng g À1 ) than Mandovi (407 ng g À1 ). The common PAHs found at both sites were Nap, Flu, Phe, Flt and Pyr with a major contribution of high molecular weight PAHs, Flt and Pyr.

Isolation and screening of PAH-degrading bacteria
A total of 80 bacterial strains were isolated from sediment samples spread plated on ZM agar. All the bacterial strains were screened for their ability to degrade Phe, Flt and Pyr (50 ppm each) using FC assay. The 12 bacterial strains showing PAH degradation were further analyzed for quantifying the degradation using GC. Six bacterial strains (NIOSV3, NIOSV7, NIOSV8, NIOSV20, NIOSV33 and NIOSV89) exhibiting more than 30% phenanthrene degradation were selected for further detailed studies. Isolates NIOSV3, NIOSV33 and NIOSV89 were obtained from Mandovi estuary, while NIOSV7, NIOSV8, and NIOSV20 were from Zuari estuary. The results revealed that isolates from the Zuari estuary are comparatively more efficient for degradation than the isolates from the Mandovi estuary.

Molecular and phylogenetic analysis of PAHdegrading bacteria
The bacterial strains able to degrade PAH were identified by sequencing the 16S rDNA gene. Molecular analysis shows that the potential PAHdegraders belong to the phylum Proteobacteria and Firmicutes (Figure 3). The bacterial isolates NIOSV3, NIOSV7, NIOSV8, NIOSV20, NIOSV33 and NIOSV89 were similar to Bacillus megaterium, Pseudomonas pachastrellae, Pseudomonas oleovorans, Pseudoalteromonas sp., Bacillus megaterium and Psychrobacter marincola, respectively. The phylogenetic tree was constructed using the Neighbour-Joining method at a bootstrap value of 1000 ( Figure 3). The 16S rDNA gene sequences were submitted to NCBI, and accession numbers are MZ5313893-MZ5313898. The phylogenetic tree shows that the two major PAH-degrading bacterial isolates, NIOSV7 and NIOSV8, are related to Pseudomonas pachastrellae and Pseudomonas oleovorans, respectively, which belong to class Gammaproteobacteria.

Degradation of phenanthrene, fluoranthene and pyrene
The degradation potential of select bacterial isolates was assessed in a 100 mL flask containing MSM with 100 ppm Phe, 75 ppm Flt and 75 ppm Pyr as a sole carbon source. Changes in the concentration of PAHs were assessed using gas chromatography. Six bacterial isolates showed Phe degradation in the range of 35%-85%, Flt degradation in 11%-71%, and Pyr 6%-67% degradation from each PAHs' concentration within 120 h  incubation at 28 C (  Figure 1). The degradation efficiencies shown by Pseudomonas oleovorans NIOSV8 and Pseudomonas pachastrellae NIOSV7 were much higher as compared to other four strains studied (Table 2).

Discussion
PAHs are the primary concern for the environmental pollution that cause toxic effects on marine animals, plants and humans. Oil spillage caused due to barge and ship traffic in Mandovi and Zuari estuaries is a significant source of hydrocarbon pollution (Pasumarthi, Chandrasekaran, and Mutnuri 2013;Kessarkar et al. 2015;Suneel et al. 2019). The total concentrations of the 16 PAHs (US EPA Priority listed pollutants) in the collected samples ranged up to 24,712 ng g À1 , indicating moderate to a very high level of PAH concentration (Dudhagara et al. 2016) at the sampled sites of Mandovi and Zuari estuary. Phe, Flt, and Pyr were the PAHs reported from 3 of the 4 sampled sites. Flt and Pyr were selected for degradation studies as representative HMW PAHs. Phe was chosen as an LMW PAH having both bay and k regions and designated as prototypic PAH for studying PAH contamination (Ghosal et al. 2016).
Biodegradation of these toxic PAHs using microbes to reduce pollution in sediment is very important. As sediments act as a sink for the constant deposition of hydrocarbon pollutants, the microbial communities of the polluted environment have evolved and developed the ability to utilize these hydrocarbons as carbon sources for growth and energy (Tam et al. 2002). The number of different hydrocarbon-degrading bacterial strains was significant in the indigenous community and could degrade complex hydrocarbons (Bibi et al. 2018; Al-Thukair, Malik, and Nzila  Psychrobacter marincola 73.39 ± 4.40 11.28 ± 1.53 11.3 ± 3.75 Control (without cultureto ascertain abiotic loss) 1.74 ± 0.04 1.56 ± 0.04 1.1 ± 0.04 2020). The bacterial population in the anthropogenic polluted environment develop a mechanism for detoxifying and metabolizing these pollutants via acclimatization and adaptation (Tam et al. 2002). The primary source of Phe is urban pollution. Phe is a potent inhibitor of gap junctional intercellular communication, photosensitiser of skin and mild allergen, and it can also impair immune function (Alegbeleye, Opeolu, and Jackson 2017). Phe biodegradation has been widely studied using various bacterial genera, including Mycobacterium, Brevibacterium, Sphingomonas, Rhodotorula, Aeromonas, Arthrobacter, etc. (Alegbeleye, Opeolu, and Jackson 2017). However, only a few studies have focused on biodegradation of a mixture of Phe, Flt and Pyr (Xu et al. 2016). Previous reports of Phe (100 ppm) degradation using Bacillus sp. showed <64% degradation within 7 days (Sukhdhane et al. 2019). The other study reported Pseudomonas citronellolis to degrade 94% of 100 ppm Phe in 15 days (Oyehan and Al-Thukair 2017). Xu et al. (2016) reported Phe degradation using strains of Sphingomonas sp. and Klebsiella sp. Sphingomonas sp. showed 74.32% degradation of 100 ppm Phe, while Klebsiella sp. showed 70.29% degradation of 50 ppm Phe, within 15 days. Compared to the previous studies, we report here 85 and 83.33% degradation of 100 ppm Phe by Pseudomonas oleovorans (Isolate NIOSV8) and Pseudomonas pachastrellae (Isolate NIOSV7), respectively, within 5 days.
Flt and Pyr are the HMW PAHs mainly composed of 4 aromatic rings. Flt is a non-alternant PAH that is ubiquitously present in the environment (Reddy et al. 2018;Ghosal et al. 2016), while pyrene is alternant and peri-condensed PAH and possesses symmetrical structure. They are genotoxic, mutagenic and carcinogenic (Gupta, Kumar, and Pal 2019 (Nwinyi, Ajayi, and Amund 2016;Gupta, Kumar, and Pal 2019).
Pseudomonas plecoglossicida and Pseudomonas sp. isolated from soil of former industrial sites have shown 5% and 7% degradation of 97 ppm Flt in 21 days (Nwinyi, Ajayi, and Amund 2016). Other strains, Sphingomonas sp. and a consortium of Mycobacterium sp. and Sphingomonas sp. showed 58.18% degradation of 50 ppm and 71% degradation of 10 ppm Flt within 30 and 7 days, respectively (Xu et al. 2016;Zhong et al. 2011). Compared to this, Pseudomonas oleovorans (Isolate NIO SV8) reported in this study showed higher (70.61% of 75 ppm) degradation of Flt within 5 days. Reddy et al. (2018) have reported Flt degradation higher than the present study, with 96% degradation of 240 ppm Flt using Paenibacillus sp. within 2 days.
Many Pseudomonas species have been reported to degrade PAHs (Nwinyi, Ajayi, and Amund 2016;Gupta, Kumar, and Pal 2019;Oyehan and Al-Thukair 2017). Pseudomonas pachastrellae was reported to degrade petroleum hydrocarbon, along with other bacterial strains like Rhodococcus sp., Acinetobacter sp., etc. (Kostka et al. 2011;Perdigão et al. 2020). Pseudomonas oleovorans has been reported for polyhydroxyalkanoate (PHA) production using n-octane as a carbon source (Santhanam and Sasidharan 2010) and for biodegradation of Tetrahydro-furan and BTEX (Zhou et al. 2011;Chen et al. 2013). However, Pseudomonas pachastrellae and Pseudomonas oleovorans are not reported for degradation of Phe, Flt and Pyr. The results of this study reveal that Pseudomonas oleovorans and Pseudomonas pachastrellae could be efficiently used for bioremediation of sites contaminated with Phe, Flt, and Pyr.

Conclusion
Six different indigenous bacteria with varied PAH-degradation capacity were isolated from Mandovi and Zuari estuaries in this study. All the isolates belonging to the Phylum Proteobacteria showed comparatively more degradation ability than Firmicutes. Pseudomonas oleovorans and Pseudomonas pachastrellae were highly potent, phylogenetically related to known PAH-degraders, degraded more than 80% Phe in 120 h. Pseudomonas oleovorans showed 70 and 67% degradation for Flt and Pyr (HMW PAH), respectively. The other strain, Pseudomonas pachastrellae, showed more than 40% degradation of both Flt and Pyr within 120 h. The other strain Psychrobacter marincola exhibited good Phe degradation ability. These results indicate that the bacteria isolated from contaminated sites of the estuary can serve as prominent PAH-degraders. Our future study aims at conducting a microcosm experiment for PAH-contaminated sediment using these potential PAH-degrading bacterial strains.