Pseudomonas aeruginosa PR23 isolated from oil contaminated soil tolerate and degrades mixture of polyaromatic hydrocarbons and express novel proteins

doi:10.21203/rs.3.rs-3323379/v1

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Pseudomonas aeruginosa PR23 isolated from oil contaminated soil tolerate and degrades mixture of polyaromatic hydrocarbons and express novel proteins

https://doi.org/10.21203/rs.3.rs-3323379/v1

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published 07 Jul, 2024

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Pseudomonas aeruginosa PR23 isolated from the hydrocarbon contaminated soil tolerate and degrades mixture of polyaromatic hydrocarbons (PAHs) having initial concentration of 1300 ppm. The degradation and intermediates formed were assessed through GC-MS analysis. The isolated strain was able to degrade 59.2% mixture of PAHs in 3 days and 71.6% by day 15. Effect of PAHs on protein expression in Pseudomonas aeruginosa PR23 was studied through nano LC-MS/MS. Thirty-six proteins were observed that showed more than 2-fold increased expression in the presence of mixture of PAHs. Out of these proteins, 7 proteins are reported for their role in degradation of naphthalene, phenanthrene, and pyrene. Data also showed the presence of 16 proteins that are uniquely expressed in the presence of mixture of PAHs. A Twin-arginine translocation signal peptide (tat system) known for transportation of folded proteins across the cell membrane showed more than 8-fold increased expression in the presence of mixture of PAHs. Results indicate that isolated strain adopts the conditions in the presence of mixture of PAHs by modulating its metabolic and physiological processes. These findings suggest that Pseudomonas aeruginosa PR23 may be a suitable candidate to be used for the development of strategies for bioremediation of mixture of PAHs.

Bioremediation

degradation of PAHs

effect of PAHs on total proteome

mixture of PAHs

Pseudomonas aeruginosa PR23

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic and carcinogenic to living organisms (Kim et al. 2013; Lee et al. 2021), including humans (Alegbeleye et al. 2017). They can accumulate in the environment matrix (Chakravarty et al. 2022) and are harmful to human health and ecosystems (Priyadarshanee et al. 2022; Mallah et al. 2022). Therefore, it is crucial to develop effective and eco-friendly strategies to remediate PAH-contaminated environment. There are several reports of microbial bioremediation of various PAHs. Most of these studies include degradation of single PAHs by using one bacterium (Meena et al. 2016; Li et al. 2021; Abbasi et al. 2022; Imam et al. 2023). However, PAH-contamination in environment frequently occurs as combination of various PAHs making their bioremediation more difficult (Agrawal et al. 2019). Thus, it is important to search a bacterium that can degrade broad range of PAHs for the development of efficient bioremediation strategies. Earlier, an obligate marine bacteria Cycloclasticus sp. strain P1 was shown to utilize mixture of PAHs [(naphthalene (98%), phenanthrene (92%) and pyrene (57%)] having final concentration of 500 ppm within 5 days of incubation (Wang et al. 2018). In another study, Mycobacterium sp. strain PYR-1 could degrade 90% phenanthrene and 56% pyrene (500 ppm each) after 14 days of incubation (Kweon et al. 2011). PAHs degrading bacterial community isolated from PAHs polluted area degrades 52% LMW-PAHs and 35% HMW-PAHs within 16 days of incubation (Lu et al. 2019). Pseudomonas species is one of the most extensively studied bacterial genera (Sitaraman 2015) due to its ability to survive harsh environmental conditions and to metabolize a wide range of organic compounds for growth and energy (Koshlaf and Ball 2017). Pseudomonas species such as P. aeruginosa DQ8 (Zhang et al. 2011), P. alcaligenes PA-10 (Gordon and Dobson 2001), P. putida NCIB (Yang et al. 1994), P. fluorescens ACB (Moonen et al. 2008) are among the efficient PAHs degraders and have been reported to biodegrade naphthalene, phenanthrene, anthracene, pyrene, fluoranthene and chrysene (Wick et al. 2011; Cao et al. 2015; Yan and Wu 2017; Wu et al. 2019). Bacteria are known to secrete enzymes monooxygenases and ring-cleaving dioxygenases, which catalyze the initial steps of PAHs degradation (Kadri et al. 2017). Intracellular enzymes are responsible for the further catabolism of the initial degradation products. Therefore, the complete mineralization of PAHs requires the action of both extracellular and intracellular enzymes (Haritash and Kaushik 2009; Kadri et al. 2017). In this study, Pseudomonas aeruginosa PR23 was isolated from oil contaminated soil. Its potential to degrade the mixture of PAHs was evaluated by Gas chromatography-mass spectrometry (GC- MS) analysis. Effects of mixture of PAHs on extracellular enzymes and intracellular protein expression in isolated Pseudomonas aeruginosa PR23 was evaluated.

Isolation and identification of bacterium degrading mixture of PAHs (Naphthalene, phenanthrene, pyrene)

Sample collection, media preparation and enrichment of microbial consortia. Soil samples were collected from a petroleum-contaminated site (28^°35ꞌ33.3"N 77^°1ꞌ23.7"E) at a depth of 5 cm and stored in a sterile container at -20˚C. One gram of sample soil was added to 50 mL of Luria Bertani (LB) broth media and incubated at 37°C and 150 rpm for 48 h. The 48 h culture was then serially diluted to 10^− 2 to avoid soil particulate matter. Mineral Salt Medium (MSM) was used having working concentrations as follow: KH₂PO₄ (1.0 g/L), Na₂HPO₄.2H₂O (1.25 g/L), MgSO₄.7H₂O (0.5 g/L), CaCl₂.2H₂O (0.05 g/L), FeSO₄.2H₂O (0.005 g/L) and (NH₄)₂SO₄ (1.0 g/L) (Lakshmi et al. 2011) and as used previously in our laboratory (Jindal and Aggarwal 2023). Aliquots (1% v/v) from 10^− 2 serial diluted suspension were transferred to MSM containing glucose (1 g/L) and incubated at 30˚C, 150 rpm till O.D_600nm reached to 0.6. Stock solutions of the PAHs (naphthalene, phenanthrene, and pyrene) were prepared in acetone and added to 50 mL MSM at desired working concentrations (naphthalene 0.05 g/L; phenanthrene 0.5 g/L, pyrene 0.1 g/L). The medium was incubated overnight in sterile conditions to evaporate acetone from the media. Inoculum aliquots (1% v/v) of MSM containing glucose were transferred to flasks containing MSM only and MSM containing a mixture of PAHs (0.05 g/L, 0.5 g/L, 0.1 g/L). Uninoculated MSM and MSM supplemented with only mixture of PAHs was used as blanks. The growth kinetics of soil microbial consortia were studied for 15 days at 30°C in the dark.

Isolation and identification of mixture of PAH-degrading bacterium. After 15 days of growth in MSM containing mixture of PAHs, culture was serially diluted from 10^− 1 to 10^− 5. Each diluted suspension culture was surface plated on MSM agar containing mixture of PAHs and incubated at 30℃ for 24 h. Bacterial colonies were purified by repetitive streaking on nutrient agar. The isolated bacteria were identified using 16S rRNA gene sequencing.

Effect of mixture of PAHs on Pseudomonas aeruginosa PR23

On growth. The isolated strain was cultured in MSM flask containing glucose (1.0 g/L) for 24 h at 30°C and 150 rpm till the O.D_600nm reached to 0.6–0.8. The inoculum from the culture was incubated in MSM containing mixture of PAHs (naphthalene 0.1 g/L; phenanthrene 1.0 g/L, pyrene 0.2 g/L), MSM containing glucose, MSM containing glucose and mixture of PAHs and in MSM only for 15 days at 30℃ in dark condition and growth was observed at every 3rd day by measuring absorbance at 600 nm. Uninoculated MSM and MSM containing mixture of PAHs served as blanks.

On protein expression. Culture as above were centrifuged at 8000 rpm for 10 min, 4℃ at every 3rd day till 15th day. The culture liquid was collected and protein was estimated using Bradford’s assay (Bradford 1976). Intracellular protein was extracted using the slightly modified acetone-triton X-100 protocol (Bhaduri and Demchick 1983).

On enzymes. Effect of mixture of PAHs on alkaline phosphatase, acid phosphatase, protease, β-glucosidase activities was assessed in the extracellular liquid following protocols as mentioned (see Supplemental Table S1) and as previously done in the laboratory (Jindal and Aggarwal 2023).

Effect of mixture of PAHs on total microbial proteome of Pseudomonas aeruginosa PR23

Effect of mixture of PAHs on total microbial proteome was studied in the presence and absence of mixture of PAHs was done by nano LC-MS/MS. Microbial culture grown in MSM containing inoculum and mixture of PAHs and MSM containing only inoculum (control) were taken at day 15 and centrifuged at 8000 rpm, 4℃. Pellets obtained were collected and total protein was extracted from each sample following acetone-triton X-100 protocol as mentioned above (Bhaduri and Demchick 1983). Total protein extracted were analysed by LC-MS/MS by service provider Vproteomics.

Degradation of mixture of PAHs by Pseudomonas aeruginosa PR23

Catechol-1,2-dioxygenase and Catechol-2,3-dioxygenase enzyme assay. C-1,2-O and C-2,3-O enzyme activity was assayed using catechol as substrate. MSM containing mixture of PAHs was inoculated (10%) and incubated for 15 days. On every 3rd day the culture liquid was collected and intracellular and extracellular activity was assessed. A reaction mixture containing crude enzyme, 3 mM catechol, and 50 mM phosphate buffer (pH 7.5) was incubated at RT. Optical density was measured at 260 nm to assess the C-1,2-O activity and 375 nm to assess the C-2,3-O activity. The formation of the product cis-cis muconic acid (ccMA) and 2-hydroxy semialdehyde muconic acid (2-HMS) respectively was determined (Kim et al. 1992).

Estimation of cis-cis muconic acid and 2- hydroxy muconic acid in culture liquid. The intermediate products formed during PAHs degradation (ccMA and 2-HMS) were estimated in culture liquid. The characteristic peak for ccMA was read at 260 nm and concentration of ccMA was estimated by using the molar extinction coefficient (ε) of 16 M^− 1 cm^− 1 for ccMA at 260 nm. The characteristic peak for 2-HMS was read at 375 nm and concentration of 2-HMS was estimated by using ε = 33000 M^− 1 cm^− 1 for 2-HMS at 375 nm (Xiao et al. 2007; Takeo et al. 2007).

GC-MS analysis

Intermediate metabolites produced during degradation of mixture of PAHs was analyzed using GC-MS. Isolated PAHs degrading strain was grown in MSM with mixture of PAHs for 15 days. Intermediate products of PAHs degradation were extracted with ethyl acetate on day 3 and day15. Uninoculated MSM with mixture of PAHs was used as blank. Metabolites were extracted from both inoculated and uninoculated media with ethyl acetate three times. The ethyl acetate fractions were air dried at room temperature. The dried fractions were resuspended in methanol and applied to GC-MS analysis by service provider (AIRF, JNU) using a Rxi® − 5Sil MS column with film thickness of 0.25µm. An inert carrier gas at a flow rate of 1.21 mL/min was used. Temperature was kept at 100℃ for 2 min and then set to 280℃ at the rate of 10℃/min for 20 min. Samples were injected in the split mode (1:10) at a linear velocity of 40.9 cm/s, and the scan range was m/z 40–650. The detected metabolites were searched in National Institute of Standards and Technology (NIST) library as described by Heitkamp et al. (1998).

The isolated strains were designated as PR22, PR23, PR24 and the sequences were submitted to the NCBI GenBank with accession number OR287182, OR287183, OR287184 respectively.

The pseudomonas aeruginosa PR23 is not publicly available due to ongoing research but may be available from the corresponding author on reasonable request.

Isolation and identification of mixture of PAHs degrading bacterium

Microbial consortia was grown in MSM containing mixture of PAHs and growth was studied for 15 days at 30℃ by measuring absorbance at 600 nm at every 24 h (Fig. 1). The absorbance increased till 2nd day (short lag phase and long log phase) and remain constant till 4th day (stationary phase) and then declined on 5th day (death phase). After 5th day consortia showed an increase in absorbance till 15 days (Fig. 1). After 15 days of incubation, microbial culture was plated on MSM agar containing mixture of PAHs. Three morphologically different colonies showing growth were isolated. The isolated colonies were identified through 16S rRNA sequencing and found to belongs to genus pseudomonas and aeruginosa species. The isolated strains were designated as PR22, PR23, PR24 and the sequences were submitted to the NCBI GenBank with accession number OR287182, OR287183, OR287184 respectively.

Selection of an efficient strain

The efficiency of isolated strains to degrade mixture of PAHs was investigated by growing them in the presence of PAHs (Naphthalene 0.1g/L + Phenanthrene 1.0g/L + Pyrene 0.2g/L) for 30 days at 30℃ and growth was recorded (Fig. 2) by measuring the absorbance at 600 nm. The PAHs degradation and utilization efficiency of pseudomonas aeruginosa PR22, PR23, and PR24 was calculated based on the increase in biomass as suggested by You et al. (2018). Strain PR23 showed better growth in comparison with other strains in the presence of mixture of PAHs and therefore was selected to study the degradation of mixture PAHs.

Pseudomonas aeruginosa PR23 tolerate, degrade and utilize mixture of PAHs for growth

Pseudomonas aeruginosa PR23 was grown in the presence of mixture of PAHs for 15 days. Control was setup by growing the strain PR23 in only MSM. The maximum growth was observed on 3rd day in the presence of MSM containing mixture of PAHs indicating that P. aeruginosa PR23 was able to tolerate and utilize PAHs as carbon source as no growth was observed in inoculum grown in MSM only. The absorbance remains same till 12th day in the presence of PAHs. The growth in the presence of glucose and PAHs with glucose declined on 6th day (Fig. 3).

Pseudomonas aeruginosa PR23 showed increased protein content in culture liquid in the presence of mixture of PAHs

The effect of mixture of PAHs on P. aeruginosa protein expression was determined by growing strain PR23 in the presence of mixture of PAHs, glucose, and mixture of PAHs with glucose for 15 days (Fig. 4a). The culture liquid showed higher amount of protein in the presence of mixture of PAHs as compared to only MSM. The protein content showed increasing concentration in the presence of mixture of PAHs from day 3 to day 15 whereas in the presence of MSM only, maximum protein content reached at day 9 and thereafter it decreased.

Effect of mixture of PAHs on microbial enzyme activities of Pseudomonas aeruginosa PR23

Effect of mixture of PAHs on release of enzymes alkaline phosphatase, acid phosphatase, protease and beta-glucosidase in the culture liquid was assessed. Alkaline phosphatase (ALP) showed increase in activity from day 3 to day 15 of growth in the presence of mixture of PAHs. No significant change in the ALP activity was observed in the medium containing only MSM (without any carbon source) whereas in the presence of glucose, it showed increase in activity from day 3 to day 15 with peak at day 12 (Fig. 4b). Acid phosphatase (ACP) activity detected in the culture liquid in the presence of mixture of PAHs, MSM and PAHs with glucose was low as compared to the activity in the presence of glucose only (Fig. 4c). The strain PR23 showed increasing trend in protease activity in the culture liquid in all the cases from day 3 to day 15. Higher protease activity was observed in culture liquid that was incubated with mixture of PAHs at day 15 (Fig. 4d). No beta-glucosidase activity was observed in the presence of mixture of PAHs.

Effect of mixture of PAHs on microbial proteome of Pseudomonas aeruginosa PR23

LC-MS/MS analysis of microbial proteome showed 1378 proteins in control and 1166 proteins were detected in the presence of mixture of PAHs. Data showed that among 1166 proteins, 36 proteins showed higher expression than control. Interestingly, expression of 16 new proteins were observed in the presence of mixture of PAHs that were absent in control (Table 1). Among these higher expressed 36 proteins, 7 proteins (Table 2) are reported in degradation pathways of naphthalene (Peng et al. 2008), phenanthrene (Ghosal et al. 2016), and pyrene (Liang et al. 2006). These proteins were searched in KEGG database and used to deduce the metabolic pathways followed by isolated strain to degrade mixture of PAHs (Fig. 5). Other highly expressed proteins were known to be involved in various biological processes such as purine and pyrimidine metabolism, RNA metabolism, nucleic acid binding activity, fatty acid synthesis, pentose phosphate pathway, glutamate metabolism, protein translation, glycolysis, nitrogen metabolism and fixation (see Supplemental Table 2). Interestingly, one protein showed more than 8.0-fold high expression in the presence of mixture of PAHs stress is ‘Twin-arginine translocation pathway signal protein” (Tat system) (Palmer and Berks 2012).

Table 1

Nano LC-MS/MS based detection of novel proteins in the presence of mixture of PAHs
Accession ID	Protein description
P27017	Aliphatic amidase expression-regulating protein
Q9HZG8	DUF883 domain-containing protein
Q59653	Aspartate carbamoyltransferase
Q9I0I6	Methyl-accepting chemotaxis protein
Q9I0H0	Uncharacterized protein
Q9HUG0	Probable carbamoyl transferase
Q9HUZ7	Lipase LipC
Q9HYG8	GTP cyclohydrolase 1 1
Q9I1F7	(S)-ureidoglycine aminohydrolase cupin domain-containing protein
Q9I120	Filamentous haemagglutinin FhaB/tRNA nuclease CdiA-like TPS domain-containing protein
Q9I791	Probable hemagglutinin
Q9HTW9	Probable FAD-dependent monooxygenase
Q9HVT4	DUF2135 domain-containing protein
Q9I0F3	Type VI secretion system spike protein VgrG1c
Q9I4M1	Lysozyme inhibitor LprI N-terminal domain-containing protein
Q9I0A9	Virulence protein

Table 2 Nano LC-MS/MS based detection of proteins involved in degradation of naphthalene, phenanthrene, and pyrene

Accession ID	Protein description	Fold change	Log 2-fold change	Biological process	References
Q9I2C4	NAD⁺ dependent aldehyde dehydrogenase	14.29420815	3.837358796	Polycyclic aromatic hydrocarbon degradation (Naphthalene, phenanthrene, and pyrene degradation)	Keum et al. 2008; Haritash and Kaushik 2009; Mohapatra and Phale 2021; Abbasi et al. 2022
Q9HX05	Probable aldehyde dehydrogenase	13.0801058	3.709302305
Q9I6D0	Probable oxidoreductase	3.866243579	1.950932532
Q9HZ51	Probable ATP-binding component of ABC transporter	2.345324112	1.22978731
Q9I0X3	Muconate cycloisomerase I	1.447170236	0.533234641
Q9I2H1	Probable hydroxylase large subunit	1.276411827	0.352093882
Q9HTW9	Probable FAD-dependent monooxygenase*
*Only expressed in PAH treated sample

Degradation of mixture of PAHs by Pseudomonas aeruginosa PR23 and detection of intermediates using GC-MS analysis

The potential of P. aeruginosa PR23 to degrade mixture of PAHs (1300 ppm) was evaluated using GC-MS analysis on day 3 and day 15 of growth. GC-MS analysis detected intermediates as 4,5-dihydropyrene, 1,9-dihydropyrene, hexadecenoic acid, naphtho[2,1-b] thiophene, 6,7-benzothiophene, 6,7-benzoquinoline, 2,9-dimethylphenanthroline, eicosanoic acid, octadecanoic acid, and glycidyl palmitate (Fig. 6). The PAHs degradation efficiency of P. aeruginosa PR23 was found to be 59.2% in 3 days that increased to 71.6% on day 15 (Fig. 7).

Pseudomonas aeruginosa PR23 follow ortho- and meta- pathways to degrade mixture of PAHs

Enzymes involved in PAHs degradation i.e., catechol-1,2-dioxygenase (C-1,2-O) and catechol-2,3-dioxygenase (C-2,3-O) were assessed in intracellular medium and in the culture liquid. The intracellular C-1,2-O activity was higher than the C-2,3-O activity in general. Both enzymes showed maximum activity at day 9 of growth (Fig. 8a). Whereas the catechol-1,2-dioxygenase activity in the culture liquid was maximum at day 6 after that it showed decline in the activity. The catechol-2,3-dioxygenase activity in culture liquid remained the same until day 12 and then increased on day 15. In culture liquid also C-1,2-O showed higher activity than C-2,3-O (Fig. 8b) The culture liquid was analysed for the production of ccMA and 2-HMS which are produced after the degradation of catechol as an intermediate in the degradation pathways of PAHs. Presence of both ccMA and 2-HMS were detected in the culture liquid whereas concentration of ccMA was more than 2-HMS (Fig. 8c).

Polycyclic aromatic hydrocarbons (PAHs) contamination of environment is associated with serious harmful effects on environment and almost all the living organisms. PAHs contamination of environment is increasing with increase in population and industrialization. Therefore, it is important to develop cost-effective strategies for bioremediation of PAHs from the environment. Microbial bioremediation of PAHs is one of such approaches. Reports on microbial potential to degrade PAHs are available (Raquel et al. 2013; Gaur et al. 2018; Sangkharak et al. 2020; Mohapatra and Phale 2021; Sharma et al. 2022). However, in environment PAHs are rarely present in isolation but are present as mixture of PAHs. Thus, studying the response of a bacterium against mixture of PAHs would be desirable. (Wu et al. 2018; Medić et al. 2020; Zhang et al. 2021) for understanding the capabilities of a strain to tolerate and degrade pollutants.

In this study Pseudomonas aeruginosa PR23 has been isolated from consortia of bacteria grown in the presence of mixture of PAHs. Microbial consortium was able to utilized mixture of PAHs (0.05 g/L naphthalene + 0.5 g/L phenanthrene + 0.1 g/L pyrene) as sole carbon and energy source for its maintenance under aerobic conditions within 15 days of incubation. There are reports of degradation of mixture of PAHs by microbial consortia isolated from ore waste (Blanco-Enríquez et al. 2018). Microbial consortia could remove 90% of naphthalene, phenanthrene, and pyrene having initial concentration of 100 mg/L in 14 days. Microbial consortia consisting of genera Sphingobium and Pseudomonas were shown to utilize 600 mg/L phenanthrene and 100 mg/L dibenzothiophene in 5 days and 9 days respectively (Zhang et al. 2021). In this study also, microbial consortia from oil contaminated soil showed good growth in the presence of mixture of PAHs probably by utilizing them as carbon source. Zhang et al. (2021) has concluded degradation of phenanthrene and naphthalene by bacterial consortia based on bacterial growth. Another study showed good bacterial growth associated with biodegradation of crude oil (Kiamarsi et al. 2019). In our study also microbial consortia showed good growth from day 3 to day 15 with maximum absorbance of 0.18 at day 15.

Pseudomonas aeruginosa PR23 was selected from this microbial consortium for studying the degradation of mixture of PAHs based on its growth in the presence of mixture of PAHs. The efficiency of P. aeruginosa PR23 to utilize mixture of PAHs as carbon source on the basis of growth was 63.8%. In the presence of glucose as carbon source, P. aeruginosa PR23 showed 70% growth efficiency and in the presence of mixture of PAHs with glucose together exhibited 81% growth efficiency. These results indicated that P. aeruginosa PR23 utilize mixture of PAHs as carbon source with significant efficiency when compared with glucose as the carbon source. From Fig. 4a it seems mixture of PAHs affects expression of different proteins at different growth time. Isolated strain PR23 showed increased expression of proteins in the presence of mixture of PAHs than MSM containing inoculum only. It is possible that the strain PR23 shifted their metabolic processes to focus on the degradation of mixture of PAHs, rather than the production of proteins required for the growth. This shift in metabolism could have led to an increase in the overall protein concentration in the cells. Additionally, the higher concentration of PAHs in the media may be causing stress to the bacteria, leading to the increase in protein production that helped the bacteria to overcome the stress. Increase in the protein content can be correlated with the adaptation and degradation of PAHs that released more energy which may have been used for growth by the strain PR23. Thus, strain PR23 adapted to the PAHs by producing proteins that are involved in degradation of PAHs as evident from nano LC-MS/MS data also.

GC-MS analysis showed that the p. aeruginosa strain PR23 could degrade 59.2% mixture of PAHs in 3 days and 71.6% by day 15. Pseudomonas aeruginosa has been shown to degrade 58% of petroleum hydrocarbons in 14 days (You et al. 2018) and was able to tolerate phenanthrene up to 0.9 g/L concentration. (Kafilzadeh et al. 2016). Isolated strain PR23 was found to tolerate initial concentration of naphthalene (0.1 g/L), phenanthrene (1.0 g/L), and pyrene (0.2 g/L) and degrade 99% naphthalene, 27% phenanthrene, and 78% pyrene in 15 days of growth. There are reports on Pseudomonas aeruginosa that can degrade multiple PAHs. For example, strain L10 was able to degrade 79.3% of n-alkanes, 79.7% of naphthalene, 71.8% of phenanthrene, and 34.7% of pyrene with final concentration of 200 mg/L in 10 days of incubation (Wu et al. 2018). Strain DN1 was able to degrade 84.47% of 50 µg/ml fluoranthene in 9 days (He et al. 2018). Strain san ai was able to degrade n-hexadecane (80%), n-nonadecane (98%), fluorene (96%), phenanthrene (50%), and pyrene (41%) having initial concentration 20 mg/L (Medić et al. 2020). Another study on bioremediation of mixed PAHs revealed that the klebsiella pneumoniae could degrade only 35.20% of mixed PAHs (Premnath et al. 2021). Our results indicate that isolated strain PR23 can tolerate and degrade higher concentrations of PAHs and may serve as better candidate for PAHs bioremediation. GC-MS analysis showed the presence of 4,5-dihydropyrene and 1,9-dihydropyrene as intermediate metabolites of PAHs degradation. Mycobacterium austroafricanum also yield 1,9-dihydropyrene as an intermediate in the degradation of pyrene (Paterek et al. 2001). Intermediates detected in our study through GC-MS such as hexadecenoic acid, naphtho[2,1-b] thiophene, 6,7-benzothiophene, 6,7-benzoquinoline, 2,9-dimethylphenanthroline, eicosanoic acid, octadecanoic acid, and glycidyl palmitate have also been reported in other studies (Keum et al. 2008; Zhang et al. 2021). P. aeruginosa PR23 degrades 59.2% PAHs (naphthalene, phenanthrene, and pyrene) having initial concentration of 1300 ppm in 3 days and 71.6% by day 15.

Dioxygenase and monooxygenases are key enzymes for the degradation of PAHs (Peng et al. 2008). Detection of C-1,2-O and C-2,3-O activities in intracellular medium and in culture liquid also supports the potential of strain PR23 to degrade PAHs. Presence of both C-1,2-O and C-2,3-O activities in intracellular medium suggests that the strain PR23 mediate the degradation of PAHs mainly via o- and m- cleavage of catechol yielding ccMA and 2-HMS. This is supported by detection of ccMA and 2-HMS in culture liquid. Pseudomonas putida strain P8 showed the simultaneous expression of both o- and m- cleavage pathway in the benzoate biodegradation (Cao et al. 2008). Whereas, Rhodococcus sp. strain DK17 and DK180 showed that high concentration of benzene induced the ortho-cleavage pathway (Kim et al. 2002). Pseudomonas putida F1 degrades aromatic compounds via meta- cleavage pathway (Zylstra et al. 1997). Selection of degradation pathway is strain specific and depends on availability of growth substrate (Cao et al. 2008). Isolated strain PR23 showed both the C-1,2-O and C-2,3-O activity in the presence of mixture of PAHs indicates it follows both the cleavage pathways. Bacteria may overcome stress caused by PAHs by expressing proteins that are involved in the degradation of PAHs (Seo et al. 2009). Nano LC-MS/MS based analysis of proteins expressed by strain PR23 in the presence of mixture of PAHs showed 36 proteins with higher expression as compared to control (with no carbon source). KEGG database-based search identified proteins that were directly involved in the degradation of naphthalene, phenanthrene, and pyrene. LC-MS/MS data (Table 2) showed 13-fold increased expression of aldehyde dehydrogenase in the presence of PAHs known to mediate the conversion of 1-hydroxy-2-naphthaldehyde into 1-hydroxy-2-naphthoate. Muconate cycloisomerase showed 1.4-fold increased expression that mediate the conversion of cis-cis muconic acid to muconolactone in phenanthrene degradation pathway. Oxidoreductase showed more than 3.8-fold increased expression which mediate the conversion of Trans-4,5-Dihydroxy-4,5-dihydropyrene to 4,5-Dihydroxypyrene in pyrene pathway, Cis-3,4-dihydroxy-3,4-dihydrophenanthrene to 3,4-dihydroxy-phenanthrene in phenanthrene pathway and 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene in naphthalene pathway (Kanehisa 2000). Data also showed that FAD-dependent monooxygenase (Table 1) induced only in the presence of mixture of PAHs, responsible for breakdown of polyaromatic hydrocarbons in the initial steps of degradation. Higher expression of proteins that are involved in a variety of biological process such as purine and pyrimidine metabolism, RNA metabolism, nucleic acid binding activity, fatty acid synthesis, pentose phosphate pathway, glutamate metabolism, protein translation, glycolysis, nitrogen metabolism and fixation suggest that the strain PR23 has adapted to mixture of PAHs by modulating its metabolic pathways (Li et al. 2019). Eight-fold higher expression of Twin-arginine translocation pathway signal protein in the presence of PAHs is observed. The Tat system is a protein transport system that is used by bacteria to transport proteins across the membrane. It is a two-step process; in the first step a signal peptide is added to the proteins and in the second step TatD (Tat-dependent translocase) uses its energy to transport the protein across the membrane (Biswas et al. 2009; Palmer and Berks 2012). The higher expression of Tat system signal peptide in the presence of mixture of PAHs suggests that P. aeruginosa PR23 may be exporting proteins, enzymes or the intermediates in the environment that are involved in the degradation of PAHs. Although, the role of the Tat system in the presence of PAHs is not fully understood. However, the Tat system can be a promising target for the development of new PAHs bioremediation strategies with increased efficiency of PAHs degradation.

Environmental conditions affect the microbe growth pattern and their ability to release enzymes in extracellular medium to degrade organic compounds (Peng et al. 2008). The PAHs in the soil is responsible for disturbing the biological parameters of soil and analysing the diversity of microorganisms and enzymatic activity helps in defining the intensity of organic and mineral transformation in the soil environment (Lipińska et al. 2014). In the present study, alkaline and acid phosphatase activity, protease activity and beta glucosidase activity were assessed. Alkaline phosphatase and protease activities showed increase trend suggesting that availability of carbon increased the enzymatic activities. However, acid phosphatase activity in the culture liquid decreased from day 3 to day 15 of the experiments. Also, no significant activity of beta-glucosidase was observed in the presence of mixture of PAHs but was observed in the presence of glucose only. Similar observations were reported by Serrano et al. (2009). They showed that urease, phosphatase, beta-glucosidase, aryl sulphatase activity decreased significantly in the first 50 days of experiment but then increased afterwards. These increased enzymes activities have been correlated with the availability of utilizable substrates (Blagodatsky et al. 2000; Tejada and Gonzalez 2007). In the present study the increasing trends of alkaline phosphatase after day 3 and protease activity after day 6 to day 12 suggest that the strain PR23 degrades mixture of PAHs and utilize them as source of carbon and energy till 15 days of experiment. Similar observations were reported by (Pilipović et al. 2015) which showed 50% decrease in acid phosphatase activity in oil contaminated soil and Ohiri et al. (2013) reported the strong inhibition of acid and alkaline phosphatase activity in the presence of crude oil. Whereas the rate of phosphatase measured was increased with the degree of soil contamination with PAHs (Lipińska et al. 2014).

In conclusion Pseudomonas aeruginosa PR23 have the potential to tolerate and degrade mixture of pyrene, phenanthrene and naphthalene at high concentration at 30℃. As in the environment PAHs contamination often occurs in combination of various PAHs. The ability of isolated strain PR23 to tolerate and degrade makes it a suitable strain to be used for bioremediation strategies. However, further detailed research is needed at genomics and proteomics level to examine the optimal conditions for use in bioremediation applications.

Acknowledgement

Authors acknowledge GGS Indraprastha University for providing research facilities and academic environment to do this kind of work. KKA acknowledges Faculty Research Grant Scheme to conduct this research. SJ acknowledges Indraprastha Research Fellowship and Short Term Research Fellowship of Guru Gobind Singh Indraprastha University.

Author contribution All the authors contributed to this study. KKA conceived and designed research. SJ conducted experiments. KKA and SJ analyzed data. KKA and SJ wrote the manuscript. All authors read and approved the manuscript.

Funding This work was supported by research grant to KKA under Faculty Research Grant Scheme of Guru Gobind Singh Indraprastha University, vide no. (GGSIPU/DRC/FRGS/2020/1988/22 and GGSIPU/DRC/FRGS/2021/594/19).

Data availability statement

All data generated and analysed during this study are included in this article and its supplementary information files. The pseudomonas aeruginosa PR23 is not publicly available due to ongoing research but may be available from the corresponding author on reasonable request.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest The authors declare no competing interests.

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Pseudomonas aeruginosa PR23 isolated from oil contaminated soil tolerate and degrades mixture of polyaromatic hydrocarbons and express novel proteins

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Abstract

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Introduction

Materials and Methods

Isolation and identification of bacterium degrading mixture of PAHs (Naphthalene, phenanthrene, pyrene)

GC-MS analysis

Results

Isolation and identification of mixture of PAHs degrading bacterium

Selection of an efficient strain

Discussion

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References

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Supplementary Files

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