Enhanced Biodegradation of Fluoranthene and Pyrene in Saline Condition Using Microbial Consortia

Enhanced Biodegradation of Fluoranthene and Pyrene in Saline Condition Using Microbial Consortia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced Biodegradation of Fluoranthene and Pyrene in Saline Condition Using Microbial Consortia Aziz Ahmed, Mohib Kakar, Zafar Ullah Jattak, Imran Iqbal, Farid Shokry Ataya, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5400344/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that exert acute toxic effects and/or possess carcinogenic, tumorigenic, and genotoxic properties. This study focused on the potential of a single bacterium and an enriched consortium to degrade high-molecular-weight (HMW) PAHs such as Pyrene (Pyr) and Fluoranthene (Flu) under saline conditions. The potential PAH degraders were isolated from mangrove sediments and identified as Ochrobactrum anthropi, Stenotrophomonas acidaminiphila , and Aeromonas salmonicida ss salmonicida. The findings revealed that the single culture degraders degraded Flu by 60%, 53%, and 47%, respectively, as well as Pyr by 58%, 51%, and 42%, respectively, from an initial concentration of 20 mg/L in seawater (28 ppm of NaCl) after 8 days. Meanwhile, the consortium degraded 85% of Flu and 81% of Pyr from an initial concentration of 50 mg/L after 8 days. The consortium also degraded a mixture of Flu and Pyr by about 60%. Biodegradation ability by the consortium for Flu and Pyr at different temperatures decreased in the order 30°C > 25°C > 35°C. The results revealed that, the consortium showed enhanced degradative capacity of more than 80% as compared to single isolates for degradation of Flu and Pyr and additionally the mixture of both PAHs showed 60% rate of degradation in saline environments. Therefore it can be concluded that the pooled microbial consortium has higher potential of degradation of PAHs especially for Flu and Pyr and this could be used as a method of removing PAHs pollution from the contaminated environment. Bacterial consortium Biodegradation Mangrove PAHs Seawater Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Over the past few decades, the Malaysian coasts have experienced significant environmental challenges arising from rapid industrialization, urbanization, and anthropogenic activities. These activities have culminated in the release of substantial quantities of environmental contaminants, particularly along the vital Klang Strait. This strait holds immense strategic importance due to its role in fishing, transportation, navigation, and tourism, making it one of the busiest shipping routes globally 1 . However, the relentless pace of industrial and commercial development, coupled with urbanization and motorization, has posed continuous threats to the Klang Strait's marine environment, leading to contamination and degradation. Among the numerous pollutants affecting marine ecosystems, polycyclic aromatic hydrocarbons (PAHs) represent a significant concern. PAHs, characterized by their aromatic structure comprising two or more fused rings, are pervasive neurotoxic pollutants with well-documented genotoxic, tumorigenic, mutagenic, and carcinogenic properties 2 . Their lipophilic nature enables them to accumulate in fatty tissues, posing ecological and public health risks through bioconcentration and biomagnification along food chains. Fluoranthene (Flu) and Pyrene (Pyr), both four-ring PAHs, are prominent among the pollutants found in the region. Originating from petroleum sludge and incomplete combustion of hydrocarbons, these compounds exhibit recalcitrant and hydrophobic characteristics 3 ’ 4 , along with cytotoxic, mutagenic, and potentially carcinogenic effects 5 . Microbial degradation of Flu and Pyr is complicated given their chemical structure and hydrophobic nature, which restrict their cellular uptake by microbes 6 ’ 7 . Despite their environmental persistence, Flu and Pyr are among the priority pollutants identified by environmental protection agencies worldwide, necessitating their quantification and monitoring in aquatic environments. Malaysia possessed the second largest tidal halophytic mangrove forests that cover 11.7% by area in Southeast Asia 8 . Mangrove swamps are coastal wetlands found in tropical and subtropical inter-tidal zones and participate in providing food and suitable environmental conditions for marine and estuarine microorganisms. The mangroves of the Klang Strait are particularly vulnerable to contamination from both petrogenic and pyrogenic sources of PAHs, endangering their ecological integrity 9 . Previous studies have highlighted the presence of PAHs in mangrove sediments ranged at 20–112 ng/g on a dry-weight along the Strait of Malacca, emphasizing the urgent need for remediation efforts. Mangrove ecosystems harbor a rich diverse microbial community that can degrade PAHs, making them an ideal environment for the microbial breakdown of these compounds 10 . Therefore, PAH-degrading bacterial strains were enriched for isolation and degradation of Pyr and Flu from mangrove sediments in this study. Subsequently, high potential isolates were selected for further analysis under varying conditions, including temperature, co-culture, and mixture of PAHs. Microbial degradation emerges as a promising approach for mitigating PAH contamination due to its efficiency, convenience, and environmental acceptability 11 . Studies reported that several bacterial genera have the potential to degrade Pyr and Flu in environment 10 ’ 12 . However, the successful application of biodegradation relies on various factors, including temperature, salinity, oxygen levels, pH, nutrient availability, hydrophobicity, degree of acclimation, contaminant concentration, and microbial populations 13 ’ 14 . Among them, temperature is of vital importance during PAH biodegradation, where the elevation or depression of temperature alters the physiology and structure of PAH-degrading bacteria and PAH properties, such as solubility, distribution, and bioavailability 15 . In saline environments, the efficacy of microbial degradation is often hampered by the limited metabolic activity of microorganisms and osmotic pressure imbalances 16 . Hence, application of halotolerant or halophilic PAH degraders in saline environments is crucial to assure the success of bioremediation of HMW PAH contamination. As for as we know, there has been no research conducted on the degradation of Flu and Pyr and their mixture under saline environments by S. acidaminiphila and A. salmonicida ss salmonicida , either individually or in co-culture. To address these challenges, this study focuses on isolating native PAH-degrading bacteria from aerobic mangrove sediments and assessing their degradation capabilities, particularly towards Flu and Pyr and their mixture. Additionally, the study aims to explore the synergistic effects of bacterial consortia in degrading these pollutants and determine the optimal temperature conditions for their degradation. METHODS AND MATERIALS 2.1 Culture Media and Chemicals Pyr and Flu used in this study were of high purity, purchased from Sigma–Aldrich (Selangor, Malaysia). The Carbon Free Mineral Medium (CFMM) consisted of the following: 2.8 g of NH 4 NO 3 , 2.0 g of KH 2 PO 4 , 0.8 g of Na 2 HPO 4 ·12H 2 O, 0.1 g of MgSO 4 ·7H 2 O, 50 mg of FeCl 3 ·6H 2 O, and 50 mg of CaCl 2 ·2H 2 O in 1 L distilled water was prepared according to 17 . Prior to the addition of PAHs, the medium was adjusted to pH 7.0–7.2 with 1 N H 2 SO 4 /NaOH and autoclaved at 121°C for 15 min. Stock solutions of each PAH (300 mg/L) were prepared in ethyl acetate, kept in a brown bottle at 4°C, and wrapped to avoid any exposure to light prior to dilution. Solid media plates were prepared by adding 20 g of agar into 1 L of CFMM medium. 2.2 Isolation and Characterization of PAH-degrading Bacterial Consortium Fresh mangrove sediments were collected from Sementa mangrove forest, Klang, Malaysia. The sediment sample was transformed to the laboratory on the ice and homogenized thoroughly to form a composite sample. 5 g of homogenized sediment was added into sterile flasks that contain 45 mL of CFMM and placed in a laboratory shaker overnight. The medium was allowed to settle for 2 hrs, and 5 mL of subculture supernatant was transferred into fresh CFMM that contain 50 mg/L of Pyr and Flu as the carbon and energy sources. Enriched medium was incubated in shaker at 30°C and 200 rpm for 8 days. This method was repeated for five consecutive times to obtain a relatively stable PAH-degrading bacterial consortium. Once the enrichment appeared turbid, 0.1 mL from diluted culture broth of 10 3 and 10 5 was spread onto CFMM agar plate. The plate sprayed with a layer of Pyr and Flu (0.5 mg/mL). Plates were incubated at 30°C and regularly checked for clear zones, which were scored as positive 18 . Positive bacterial colonies were transferred to nutrient agar plates until pure cultures were isolated. Identification of the pure cultures was performed using Biolog Automated System 2009 (Biolog Microstation System- MicroStation™). For identification, the isolated bacterial cells were regrown on NA plates at 30 o C for (24–28) hours so as to keep away metabolic vigor and loss of viability whilst is typical of most organism at the static phase. The inoculation fluid (IF) were applied as inoculum for focused cells using protocols-A (IF-A catalogue No. 72401) and B (IF-B catalogue No. 72402) at particularized turbidity range 95–98% T. This was accomplished using a specific cotton tipped inoculators swab (Catalogue No. 3353) to select almost 3 mm diameter are of the cell development from the very surface of the agar plate, and ultimately dipping it into the required IF. Then to ensure consistent suspension, some cell clumps were cautiously smashed against tube wall. The ensuring cell suspensions were poured into the multi-channel pipette reservoir. A particular 8-channels automated pipette was applied to distribute 100-µl of suspension into every cell in MicroPlates (Catalogue no.1030). The well consisted of 71 carbon-source utilization assays (Columns 1–8) and 22 chemical sensitivity assays (Columns 10–11), thus they can be recognized at species level that is based on the “Phenotypic Fingerprint” of the microorganisms arranged by a specific panel. MicroPlates were placed on Omnilog reader wherein they were carefully read by the use of Biolog’s Microbial Identification Systems software, and the recognized microbes were correctly recorded. 2.3 Biodegradation of Pyr and Flu by Enriched Bacterium and Consortium The degradation of Pyr and Flu using single bacterium and enriched consortium was evaluated. To demonstrate degradation experiments, 5 mL of inoculum with OD 600 of 0.1 to 0.2 was inoculated into 49 mL of seawater and supplemented with Flu and Pyr. The concentration of Flu and Pyr used in this study were 20 mg/L for single bacterium and/or 50 mg/L for enriched consortium. Seawater containing equal proportion of Flu and Pyr with no inoculum was set as a control to monitor abiotic losses during the experimental process. Pyr and Flu dissolved in ethyl acetate were then evaporated at room temperature by gently shaking the flask before inoculation. The pre-sterilized conical flasks were prepared in triplicates, covered with aluminium foil and incubated in a dark rotary shaker at 150 rpm and 30°C for 8 days. Residual concentrations of Flu and Pyr were quantified at the beginning and at the end of the incubation period. Samples were stored at − 20°C before analysis by gas chromatography equipped with a flame ionization detector (GC-FID). To evaluate the PAH degradation ( ER ), the following equation was applied described earlier 19 : where ER (%) is the percentage of removal efficiency; C i is the initial PAH concentration; and C f is the final PAH concentration. 2.4 The effect of Temperature on Flu and Pyr Degradation Pyr and Flu degradation was evaluated at different temperatures (25, 30, and 35°C), to examine the effect of temperature on degradation efficiency. The medium was augmented with each 50 mg/L of Pyr and Flu and incubated at different temperatures over a shaker for 8 days in dark. Residual Pyr and Flu were sampled at Day 0 and Day 8 to record Pyr and Flu concentration by using the same procedure. 2.5 Degradation Kinetic Model Flu and Pyr biodegradation by the single bacterium and the enriched consortium was fitted into a first-order kinetic model and expressed as in Eq. ( 2 ): $$\:\:{C}_{t}=\:{{C}_{o}e}^{-kt}$$ 2 where C t is the concentration (mg/L) of residual PAH at time t; and C o is the initial PAH concentration (mg/L) in the medium. Biodegradation rate constant ( k ) day − 1 was determined from the straight line slope of plot ln [C t /C o ] versus time. Biodegradation half-life ( t 1/2 ) = ln 2/ k , was also determined. 2.6 Analytical Procedure The residual extraction of Pyr and Flu from liquid medium was conducted by liquid-liquid technique. Biomass was separated from the culture via filtration, and the culture was introduced into a separating funnel with an equal volume (v/v) of ethyl acetate. These funnels were vigorously shaken for 10 mins to extract entire residual PAHs and allowed to rest for separation. This procedure was repeated twice and the combined organic solvent was filtered through a glass wool containing anhydrous sodium sulphate. The collected solutions were concentrated using a rotary evaporator until the volume reduced to approximately 1 mL which then was transferred into a 1.5 mL dark vial for GC–FID determination. PAH profile and concentration was determined using Agilent Technologies-7890A GC-FID. HP-5MS fused silica capillary column was used for analyses (30 m × 0.25 mm ID × 0:25 µm film thickness) under temperature-programmed settings of 60°C for 2 mins, 60°C to 120°C at a rate of 10°C/min, 120°C to 300°C at a rate of 3°C/min, and 300°C for 10 min. Detector and injector temperatures were set at 300 and 280°C, respectively. The split less sample was injected onto capillary column (HP-5MS) and nitrogen served as the carrier gas. PAH biodegradation was determined by calculating the remaining PAH concentration in the medium. 2.7 Statistical analysis Flu and Pyr degradation experiments were performed in triplicates and average readings were taken. The Statistical analysis using one-way ANOVA with Turkey’s test was done with IBM SPSS version 22. RESULTS AND DISCUSSION 3.1 Isolation and Characterization of Enriched PAH-degrading Bacterial Consortium Based on the clearing zones on CFMM solid agar plates sprayed with Flu and Pyr, three distinct types of PAH-degrading bacteria were isolated after incubation for 16–18 days. The PAH-degrading bacteria were O. anthropi, S. acidaminiphila , and A. salmonicida ss salmonicida. O. anthropi and S. acidaminiphila grew as light green and yellow pigmented colonies in Pyr-enriched media, respectively, whereas A. salmonicida ss salmonicida grew as a white pigmented colony in Flu-enriched media. O. anthropi is an aerobic, Gram-negative, motile, non-lactose-fermenting bacillus. O. anthropi isolated from saline soil contaminated by polycyclic aromatic hydrocarbons (PAHs) was previously reported to have the potential to degrade PAHs 20 . S. acidaminiphilia is a mesophile and gram-negative bacteria. Previous studies indicate that this organism has the potential to degrade polycyclic aromatic hydrocarbons 21 . Study by 19 also indicated that A. salmonicida ss salmonicida has the potential to degrade PAH especially benzene (a) pyrene in their study. This indicates that, the microbes’ isolates has the potential to be further explored for degradation of PAH, focusing on Flu and Pyr under saline condition. 3.2 Degradation of Pyr and Flu by Single Bacterium Degradation of Flu and Pyr by a single bacterium namely by S. acidaminiphila, O. anthropi, and A. salmonicida ss salmonicida in seawater (28 ppm of NaCl) was recorded at its initial concentration of 20 mg/L and incubation period of 8 days. Abiotic loss of PAH in the flask was ˂8% due to volatilization and/or adsorption to the glass, or possibly from the accumulation of PAH substrate in the cell matrix. The results of Flu degradation by S. acidaminiphila , O. anthropi , and A. salmonicida ss salmonicida reached up to 53%, 60%, and 47%, respectively (Fig. 1 ), whereas Pyr degradation was 51%, 58%, and 42%, respectively (Fig. 2 ). Flu degradation was better than Pyr degradation even though both are isomers. Pyr is the most persistent PAH within the context of this study. The low degradation rate recorded for Pyr was expected because its structure is a peri-fused PAH. The present study also revealed that O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida were able to degrade Flu and Pyr under saline conditions. This observation might be a result of enzyme specificity from these isolates. Among the three bacterial isolates, O . anthropi showed a higher capability of PAH degradation than S . acidaminiphila and A . salmonicida ss salmonicida . The biodegradation rate of Flu and Pyr by a single organism was comparable with other halotolerant Flu and Pyr degraders, but their culture condition and behaviour are different in some circumstances. Ochrobactrum anthropi strain VA1 can degrade 84% of Pyr under saline conditions similar to that in O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida . Studies from 22 suggested that Acinetobacter pittii and Enterobacter cloacae was able degrade 194 and 242 mg/L of Pyr. 23 reported that, Thalassospira sp. strain TSL5-1 can remove Flu and Pyr by 41.4% and 42.3%, respectively, in 5% (w/v) salt medium. Thalassospira sp. strain TSL5-2 was capable of degrading Pyr by 53.5% and Flu by 60% under 5% (w/v) saline condition. 24 asserted that Rhodococcus ruber strain L9 efficiently removed 76% of Pyr. 25 isolated Cycloclasticus strains, NY93E, PY97M, and PY97N from Yellow Sea sediment, which potentially degraded 52–63% of Pyr at 3% salt concentration. 26 found that LOP-9 Staphyloccous aureus and GWP-2 Mycobacterium vaanbaalenii were able to degrade Pyr 86% and 82.1% respectively. In a similar study, 27 postulated that Kocuria flava and Rhodococcus pyridinivorans effectively removed 3 ring PAHs from petroleum-contaminated soil. Numerous reports revealed the metabolic potential of Ochrobactrum sp. in Flu and Pyr degradation, suggesting the involvement of this bacteria in the degradation of several PAHs. Among these reports, Ochrobactrum anthropi BPyF3 isolated from hydrocarbon-contaminated soil has the ability to degrade 49.85% and 50.39% of Flu and Pyr, respectively, from an initial concentration of 50 mg/L after 7 days of incubation. The higher degradation of Flu and Pyr by O. anthropi can be also associated with the distribution of the organism widely in the in environmental sources. This is also supported by findings by 28 , indicates that O. anthropi isolated from contaminated soil showed effective degradation of various polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and as well other compounds including hexane, heptane, hexadecane and pesticides. 29 also isolated O. intermedium from crude oil-contaminated site, which degraded Flu 100% and Pyr 72.4%. In addition, Ochrobactrum anthropi had the ability to degrade and grow in Pyr, benzo(k)fluoranthene, and benzo(e)pyrene under saline conditions by utilizing carbon as energy source. Another report showed that Ochrobactrum sp. BL01 degraded 21% of benzo[a]pyrene during 14 days of incubation. Furthermore, Ochrobactrum sp. has been identified as a PAH-degrader of 2,4,6-tribromophenol through reductive dehalogenation and production of biosurfactants. The Stenotrophomonas genus is metabolically diverse, reportedly degrades various types of organic pollutants, and has an inherent resistance to many heavy metals. S. maltophilia VUN 10,003 was reported in HMW PAH degradation, but the potential of S. acidaminiphila was not completely explored for Flu and Pyr degradation. The degradation potential of S. acidaminiphila towards Pyr in this present study was higher than those from the previous studies by 30 who reported 68% Pyr degradation by Pseudomonas sp. and by 31 and 32 who also reported 19% and 34% Pyr degradation by Ochrobactrum sp. and Rhodococcus sp. P14 within 30 days, respectively, from an initial Pyr concentration of 50 mg/L. In comparison with the results from previous research, S. acidaminiphila has a significant biodegradative potential for Flu. In fact, 33 reported 13.7% of Flu degradation by Herbaspirillum chlorophenolicum strain FA1 isolated from activated sludge. Pasteurella sp. strain B-2 isolated from an activated sludge was able to degrade 40% of 20 mg/L Flu after 30 days of incubation. According to 12 , Stenotrophomonas sp. and Advenella sp. were able to remove Flu. 34 reported 30% degradation of 50 mg/L Flu, after incubation for 12 days by Bacillus thuringiensis strain NA2 isolated from a petroleum oil-contaminated site. Very few studies have reported the involvement of Aeromonas strains in the biodegradation of PAHs, such as anthracene, phenanthrene, and Pyr, but reports that specifically mentioned the use of A. salmonicida ss salmonicida strains for Flu and Pyr degradation under saline condition are non-existent. This study revealed that, A. salmonicida ss salmonicida had the ability to effectively degrade Pyr and Flu in saline condition. Statistical analysis showed that, S . acidaminiphila , O . anthropi , and A . salmonicida ss salmonicida were able to significantly degrade elevated concentrations of Pyr and Flu compared with abiotic loss ( p < 0.05). 3.3 Biodegradation kinetics of Pyr and Flu under single isolates Various kinetics models have previously been applied for biodegradation process 35 ’ 36 . However, the first-order rate equation widely used model and showed best fitted with the equation PAH. Table 1 presents the first-order kinetic model and half-lives used to determine Flu and Pyr degradation by single bacterium. In comparison with the Flu control that had a rate constant ( k ) of 0.010 day − 1 and t 1/2 of 69.3 days, O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida had k values of 0.118, 0.095, and 0.078 day − 1 , respectively, and t 1/2 of 5.8, 7.2, and 8.8 days, respectively. In comparison with the Pyr control that had k of 0.011 day − 1 and t 1/2 of 63 days, O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida had k values of 0.108, 0.089, and 0.067 day − 1 , respectively, and t 1/2 of 6.4, 7.7, and 10.3 days, respectively. O . anthropi showed highest rate constant for degradation of Flu and Pyr in comparison S . acidaminiphila , and A . salmonicida ss salmonicida . The half-lives of Flu was shorter than that of Pyr. In soil, persistency is indicated by a t 1/2 ≥ 180 days 37 , whereas, t 1/2 < 10 days signifies rapid degradation. Consequently, after an acclimatization process, O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida can be effective species for removal of 4-ring PAHs. 38 asserted that compounds with short t 1/2 indicated modest accumulation. Interestedly, in this study half-lives value obtained were lower than reported in literature. Table 1 kinetic parameters for Pyr and Flu degradation by isolates after 8 days PAH Single isolates Degradation percentage % Rate of Pyr and Flu degradation ( k ) day − 1 Half-life (ln2/ k ) (days) F-value ( p < 0.05 ) Flu Ochrobactrum anthropi 60 0.118 5.8 93.432 Flu Stenotrophomonas acidaminiphila 53 0.095 7.2 Flu Aeromonas salmonicida ss salmonicida 47 0.078 8.8 Flu Control 8 0.010 69.3 Pyr Ochrobactrum anthropi 58 0.108 6.4 16.416 Pyr Stenotrophomonas acidaminiphila 51 0.089 7.7 Pyr Aeromonas salmonicida ss salmonicida 42 0.067 10.3 Pyr Control 9 0.011 63 3.4 Biodegradation of Flu and Pyr by Enriched Bacterial Consortium An enriched bacterial consortium using the three isolates S. acidaminiphila , O. anthropic , and A. salmonicida ss salmonicida was also investigated for the degradation of Flu and Pyr with an initial concentration of 50 mg/L in seawater (28 ppm of NaCl) for 8 days. The recorded abiotic loss of PAHs was below 10%. Figure 3 shows that, the degradation rate of Flu and Pyr by the enriched bacterial consortium were 85% and 81%, respectively. These results indicated that, an enriched bacterial consortium enhanced the degradation of Flu and Pyr degradation compared with single bacterial isolates, reflecting the synergistic action among bacterial isolates. Statistical analysis showed that, the consortium was capable of degrading Pyr and Flu at higher concentrations compared with abiotic loss ( p < 0.05). The degradation rates of the enriched consortium were 5.21 and 5.06 mg/L − day for Flu and Pyr, respectively. Hence these rates were higher than those of the bacterial consortium reported by Lafortune et al. 2009 39 which recorded 4.7 mg/L − day for Pyr. Isaac et al. 2015 40 asserted that, the mixed culture C15 removed 1.05 mg/L − day of Pyr. These results were also higher than the single isolates of Rhodococcus sp. P14 at 0.57 mg/L − day for Pyr as previously reported by 32 Klebsiella pneumoniae PL1 at 1.25 mg/L − day Pyr, and Ochrobactrum anthropi BPyF3 at 3.6 and 3.56 mg/L − day for Pyr and Flu degradation, respectively. Burkholderia cepacia also showed 0.88 and 0.85 mg/L − day degradation for Pyr and Flu, respectively, whereas Porphyrobacter sp. B51 degraded 4.0 mg/L − day of Pyr. However, variations in pollutant reduction among the microbial consortia recorded by the aforementioned authors and the present study may be a result of culture conditions, differences in initial concentrations, and the microbial strain used. A previous study by 41 reported that, Burkholderia fungorum and Mycobacterium gilvum degraded 100 mg/L of Pyr and Flu within 20–24 days by 98.6% ± 1.9% and 99.6% ± 0.7%, respectively. 25 asserted that, both Flu and Pyr biodegradation by a bacterial consortium of Cycloclasticus sp. PY97M and Marinobacter nanhaiticus D15-8WT was faster than a single bacterial isolate and observed synergic effects. 19 recently reported that, HMW PAH biodegradation by bacterial consortium was higher than that by a single bacterium under saline conditions. 42 also reported the synergistic impact of co-culture in Flu and Pyr degradation, where a single bacterial isolate of Sphingomonas sp. or Mycobacterium sp. was able to degrade Pyr and Flu within 14 days. A co-culture of Sphingomonas and Mycobacterium strains enhanced the degradation and/or completely removed the two PAHs in 7 days. The activity amid the bacterial isolates was credited to the synergistic effect in the bacterial consortium by complementary degradative abilities among the microbes of consortia and elimination of toxic effects during degradation. First-order kinetic model fitted with biodegradation data was used to determine the Flu and Pyr biodegradation rates of the enriched consortium. Table 2 illustrates that, bacterial consortium degradation rate constants were 0.208 and 0.247 day − 1 and t 1/2 of 3.3 and 2.8 days for Flu and Pyr, respectively, compared with the abiotic and autoclaved control at k values of 0.010 and 0.012 day − 1 and t 1/2 of 69.3 and 57.7 days for Pyr and Flu, respectively. Table 2 kinetic parameters for Pyr and Flu degradation by enriched consortium after 8 days PAH Isolates Degradation percentage% Rate of Pyr and Flu degradation ( k ) day − 1 Half-life (ln2/ k ) (days) F-value ( p < 0.05 ) Pyr A + B + C 81 0.208 3.3 265.296 Pyr Control 8 0.010 69.3 Flu A + B + C 85 0.247 2.8 Flu Control 9 0.012 57.7 Mixture of PAHs (Flu) A + B + C 69 0.145 4.7 150.15 Pyr A + B + C 64 0.129 5.3 Flu Control 7 0.009 77 Pyr Control 9 0.011 63 A = Ochrobactrum anthropi ; B = Stenotrophomonas acidaminiphila ; C = Aeromonas salmonicida ss salmonicida 3.5 Degradation of Mixture of PAHs by bacterial Consortium Biodegradation by the cultivated consortium using seawater containing a mixture of Flu and Pyr at 50 mg/L each was investigated. The consortium was able to degrade 69% of Flu and 64% of Pyr after 8 days of incubation as shown in Fig. 4 . Statistical analysis showed that, the consortium was capable of significantly degrading the PAH mixture compared with abiotic loss at ( p < 0.05). This results indicated that, the degradation by the bacterial consortium was higher than those reported by Guo et al. which asserted that the enriched consortia degraded less than 40% of mixture of PAHs (Phe + Fla + Pyr). 43 also reported that Stenotrophomonas sp. and Pseudomonas sp. potentially degraded 14% and 7% of Pyr, in a mixture of five PAHs, respectively. 44 reported that Kocuria flava and Rhodococcus pyridinivorans degraded 55.6%, 59.5%, and 59.1% of 10 mg L − 1 of mixed PAHs (phenanthrene, anthracene, fluorene, and pyrene) respectively, within 15 days. The combined metabolism by bacterial consortium may increase the degradation of mixed HMW PAHs, because the intermediary biotransformation of products from individual strains may serve as primary substrate for catabolism and growth by others. 45 observed that, degradation of a PAH mixture may be a result of collaborative processes involved in the consortium of isolates with complementary capacities. The main source of complementary degradation in the bacterial consortium might also result from the synergy between versatile genetic formation and degradative enzymatic specificity. A first-order kinetic model was applied to determine the biodegradation rate of Flu and Pyr mixture by the enriched consortium. Table 2 indicates that, degradation by bacterial consortium (A + B + C) had k values of 0.145 and 0.129 day − 1 for the Flu and Pyr mixture, respectively, and t 1/2 of 4.7 and 5.3 days. The consortium was more promising than the control and autoclaved set-ups, which had k values of 0.009 and 0.011 day − 1 and t 1/2 of 77 and 63 days, respectively. 3.6 Effect of Temperature on Pyr and Flu Degradation The effect of temperature was on Pyr and Flu degradation was also investigated in this study. The temperature effect was tested was in range of 25 to 35°C at pH 7.8 and agitation speed of 150 rpm in seawater (28 ppm NaCl). The temperature range might alter the biodegradation process because some authors considered this range as narrow. In the present study, great differences from temperature variations were observed with an optimum at 30°C for bacterial consortium applied to PAHs, which was also near the natural conditions where the samples were initially collected. Pyr degradation was prominent at 30°C as compared to 25°C and 35°C. However, the Pyr degradation at 35°C was lower by 13.2% compared with that at 30°C (Fig. 5 ). The Flu degradation rate at 30°C was higher than those at both 25 and 35°C, whereas the rate at 25°C increased by 11.0% compared with that at 35°C (Fig. 6 ). The degradative abilities of bacterial consortium towards Flu and Pyr were lower at 25 and 35°C at every stage during the study period. Statistical analysis showed that, the consortium significantly degraded Pyr and Flu at 30°C compared with the control ( p < 0.05). However, the difference in degradation efficiency observed at different temperatures by the bacterial consortium was not significant. These data indicated that, the bacterial consortium had great potential in degrading Pyr and Flu at 30°C. In a similar research, 46 asserted that, NJ2 strain degraded 60% of Pyr at 37°C. Al-Thukair and Malik, 2016 47 found that, B. fungorum removed 56% and 59% of Pyr at 37°C and 25°C, respectively, whereas, Caulobacter sp removed 35–36% of Pyr at 37 and 25°C. This study also agreed with 48 who reported that, Klebsiella pneumonia PL1 degraded Pyr faster at 30°C than at 20 and 40°C. Similar results were recorded when Acinetobacter strain USTB-X biodegraded 63% of Pyr at 30°C. Optimum Pyr degradation occurs above room temperatures. 33 isolated Herbaspirillum chlorophenolicum strain FA1 from activated sludge, which degraded 13.7% Flu at 30°C after 30 days of incubation. Thus, temperature is one of the crucial environmental factors in PAH degradation because it influences the metabolic pathways of the bacteria. Table 3 presents the results of the kinetic Pyr and Flu degradation by bacterial consortium (A + B + C) under different temperatures. The effect of temperature on the degradation rate constant shows k values of 0.177–0.179 day − 1 at 25°C, 0.199–0.201 day − 1 at 30°C, and 0.138–0.146 day − 1 at 35°C, for both Pyr and Flu respectively, and t 1/2 of 3.7–3.8, 3.2–3.4, and 4.7–5.0 days for Pyr and Flu, respectively. The abiotic and autoclaved rate had k values of 0.012–0.014, 0.015–0.016, and 0.013–0.016 day − 1 of Pyr and Flu, respectively, with t 1/2 = 49.5–53.3, 43.3–46.2, and 43.5–53.4 days, at 25°C, 30°C, and 35°C, respectively. Table 3 kinetic parameters for Pyr and Flu degradation by enriched consortium at different temperature after 8 days Temp o C PAH Isolates Degradation percentage% Rate of Pyr and Flu degradation ( k ) day − 1 Half-life (ln2/ k ) (days) F-value ( p < 0.05 ) 25 Pyr A + B + C 76 0.179 3.7 90.330 30 Pyr A + B + C 80 0.201 3.4 35 Pyr A + B + C 67 0.138 5 25 Pyr Control 11 0.014 49.5 30 Pyr Control 12 0.015 46.2 35 Pyr Control 10 0.013 53.4 25 Flu A + B + C 77 0.177 3.8 229.158 30 Flu A + B + C 81 0.199 3.2 35 Flu A + B + C 69 0.146 4.7 25 Flu Control 10 0.012 53.3 30 Flu Control 12 0.016 43.3 35 Flu Control 12 0.016 43.5 A = Ochrobactrum anthropi ; B = Stenotrophomonas acidaminiphila ; C = Aeromonas salmonicida ss salmonicida CONCLUSION Three high molecular weight PAH-degrading bacteria have been isolated from mangrove sediments and characterized as O . anthropi , S . acidaminiphila , and A . salmonicida ss salmonicida for their capacity to breakdown Pyr and Flu. This study revealed that, a single bacterium and an enriched consortium exhibited excellent degradation capability to degrade Flu and Pyr and their mixture under saline conditions. Though, single bacterium had lower degradation potential, while the enriched consortium had more effective and a wider range of substrate degradation. The individual isolates and enriched consortium were able to efficiently removed Pyr and Flu at optimal at 30°C compared with those at 25°C or 35°C. These isolates have an excellent biodegradation potential in saline conditions, and therefore, suggesting the application at the field scale as well as contaminated mangrove ecosystem and coastal area. In the future, it would be provident to further study the molecular verification of microbial community and complete degradation pathway of Pyr and Flu by individual isolates. Declarations Acknowledgements Funding The authors extend their appreciation to Researchers Supporting Project number (RSPD2024R693), King Saud University, Riyadh, Saudi Arabia. Data Availability The data supporting the conclusions of this study are available within the article. Conflict of Interest : The authors declare that they have no conflict of interest. Contributions Aziz Ahmed work for final draft and supervision, Mohib Ullah Kakar and Zafar Ullah Jattak performed the experiments, Imran Iqbal perform experiments validation, Farid Shokry Ataya, Dalia Fouad performed the analysis of results and interpretation. References Zulkifli, N., Ismail Raja Ibrahim, R., Azlan Abdul Rahman, A. & Fawwaz Mohd Yasid, A. Maritime Cooperation in the Straits of Malacca (2016-2020): challenges and recommend for a new framework. Asian J. Res. Educ. Soc. Sci. 2 , 10–32 (2020). Zhou, H. et al. 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Phylogeny and polycyclic aromatic hydrocarbons degradation potential of bacteria isolated from crude oil-contaminated site. J. Environ. Sci. Heal. 52 , 897–904 (2017). Ae, O. S. O. et al. Pyrene-degradation Potentials of Pseudomonas Species Isolated from Polluted Soils. World J. Microbiol. Biotechnol. 24 , 2639–2646 (2008). Yirui, W., Tengteng, H., Zhong, M., … Y. Z.-J. of & 2009, U. Isolation of marine benzo pyrene-degrading Ochrobactrum sp. BAP5 and proteins characterization. J. Environ. Sci. 21 , 1446–1451 (2009). Song, X. et al. Isolation, characterization of Rhodococcus sp. P14 capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons and aliphatic hydrocarbons. Mar. Pollut. Bull. 62 , 2122–2128 (2011). Xu, H. X. et al. Degradation of fluoranthene by a newly isolated strain of Herbaspirillum chlorophenolicum from activated sludge. Biodegradation 22 , 335–345 (2011). Maiti, A., Das, S., Sci, N. B.-J. & 2012, U. Bioremediation of high molecular weight polycyclic aromatic hydrocarbons by Bacillus thuringiensis strain NA2. J. Sci. 72 , (2012). Goveas, L., Selvaraj, R., Kumar, P., Chemosphere, R. V.- & 2022, U. Biodegradation kinetics and metabolism of Benzo (a) fluorene by Pseudomonas strains isolated from refinery effluent. Chemosphere 307 , 136041 (2022). Rabodonirina, S., Rasolomampianina, R., … F. K.-J. of environmental & 2019, U. Degradation of fluorene and phenanthrene in PAHs-contaminated soil using Pseudomonas and Bacillus strains isolated from oil spill sites. J. Environ. Manage. 232 , 1–7 (2019). Klecka, G., Boethling, R., Franklin, J., Grady, L. & Graham, D. Evaluation of Persistance and Long-range Transport of Organic Chemicals in the Environment . (2000). Wang, C. et al. PAHs biodegradation potential of indigenous consortia from agricultural soil and contaminated soil in two-liquid-phase bioreactor (TLPB). J. Hazard. Mater. 176 , 41–47 (2010). Lafortune, I. et al. Bacterial diversity of a consortium degrading high-molecular-weight polycyclic aromatic hydrocarbons in a two-liquid phase biosystem. Microb. Ecol. 57 , 455–468 (2009). Isaac, P., Martínez, F. L., Bourguignon, N., Anchez, L. A. S. & Ferrero, M. A. Improved PAHs removal performance by a defined bacterial consortium of indigenous Pseudomonas and actinobacteria from Patagonia, Argentina. Int. Biodeterior. Biodegradation 101 , 23–31 (2015). Darmawan, R., Nakata, H., … H. O.-… of B. & & 2015, U. Isolation and evaluation of PAH degrading bacteria. J. Bioremediation Biodegredation 6 , 1 (2015). Guo, C., Dang, Z., Wong, Y., & N. T.-I. B. & 2010, U. Biodegradation ability and dioxgenase genes of PAH-degrading Sphingomonas and Mycobacterium strains isolated from mangrove sediments. Int. Biodeterior. Biodegradation 64 , 419–426 (2010). Zhu, X. et al. Biodegradation of mixed PAHs by PAH-degrading endophytic bacteria. Int. J. Environ. Res. Public Health 13 , 805 (2016). Sakshi, Singh, S. K. & Haritash, A. K. Bacterial degradation of mixed-PAHs and expression of PAH-catabolic genes. World J. Microbiol. Biotechnol. 39 , (2023). Bouchez, M., Blanchet, D. & Vandecasteele, J. P. Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism. Appl. Microbiol. Biotechnol. 43 , 156–164 (1995). Singh, S., Kumari, B., Upadhyay, S., … S. M.-B. & 2013, U. Bacterial degradation of pyrene in minimal salt medium mediated by catechol dioxygenases: enzyme purification and molecular size determination. Bioresour. Technol. 133 , 293–300 (2013). Al-Thukair, A., Biodegradation, K. M.-I. B. & & 2016, U. Pyrene metabolism by the novel bacterial strains Burkholderia fungorum (T3A13001) and Caulobacter sp (T2A12002) isolated from an oil-polluted site in the Arabian. Int. Biodeterior. Biodegradation 110 , 32–37 (2016). Ping, L. et al. Isolation and characterization of pyrene and benzo[a]pyrene-degrading Klebsiella pneumonia PL1 and its potential use in bioremediation. Appl. Microbiol. Biotechnol. 98 , 3819–3828 (2014). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5400344","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":375070543,"identity":"a7dd48f4-901a-429a-954b-3492135d32d3","order_by":0,"name":"Aziz 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University","correspondingAuthor":false,"prefix":"","firstName":"Farid","middleName":"Shokry","lastName":"Ataya","suffix":""},{"id":375070548,"identity":"fdd8605f-0e69-46eb-bb59-2d2ee8a58e73","order_by":5,"name":"Dalia Fouad","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"","lastName":"Fouad","suffix":""}],"badges":[],"createdAt":"2024-11-06 07:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5400344/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5400344/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69655360,"identity":"1e64f876-5a8f-40f9-b7ad-d01523e49f8b","added_by":"auto","created_at":"2024-11-22 17:17:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of Flu by different bacterial isolates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/2c47c4ba3fa2a724ee91fdf0.png"},{"id":69655359,"identity":"22471b5d-a267-4095-a5c9-78f73aa54569","added_by":"auto","created_at":"2024-11-22 17:17:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of Pyr by different bacterial isolates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/8b34350d6b92112c69092dbd.png"},{"id":69655356,"identity":"8e060648-e732-4561-9443-e43579687e3e","added_by":"auto","created_at":"2024-11-22 17:17:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of Flu and Pyr by bacterial consortium\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/66b64a009db0bb88289bba87.png"},{"id":69655361,"identity":"f38fc2fd-f6d3-4906-a4a7-1a8e5b858e47","added_by":"auto","created_at":"2024-11-22 17:17:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of mixture of Flu and Pyr by bacterial consortium\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/ab4dd07cf1055fad43982232.png"},{"id":69655787,"identity":"fa21c983-9d22-4676-8df0-626f30a8baeb","added_by":"auto","created_at":"2024-11-22 17:25:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of Pyr by bacterial consortium at different temperature\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/4e6b242405ca403f54ed65c8.png"},{"id":69655357,"identity":"30d3ce53-4871-4492-8833-e3c01995d257","added_by":"auto","created_at":"2024-11-22 17:17:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of Flu by bacterial consortium at different temperature\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/ca917382796a6939b2850eb0.png"},{"id":87416215,"identity":"396f7cd5-26e5-40e5-b590-afec4f946b55","added_by":"auto","created_at":"2025-07-23 14:39:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1504981,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5400344/v1/d51bcc5f-0ef2-48c7-a38f-338f0b4bcc5b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Biodegradation of Fluoranthene and Pyrene in Saline Condition Using Microbial Consortia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOver the past few decades, the Malaysian coasts have experienced significant environmental challenges arising from rapid industrialization, urbanization, and anthropogenic activities. These activities have culminated in the release of substantial quantities of environmental contaminants, particularly along the vital Klang Strait. This strait holds immense strategic importance due to its role in fishing, transportation, navigation, and tourism, making it one of the busiest shipping routes globally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the relentless pace of industrial and commercial development, coupled with urbanization and motorization, has posed continuous threats to the Klang Strait's marine environment, leading to contamination and degradation.\u003c/p\u003e \u003cp\u003eAmong the numerous pollutants affecting marine ecosystems, polycyclic aromatic hydrocarbons (PAHs) represent a significant concern. PAHs, characterized by their aromatic structure comprising two or more fused rings, are pervasive neurotoxic pollutants with well-documented genotoxic, tumorigenic, mutagenic, and carcinogenic properties\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Their lipophilic nature enables them to accumulate in fatty tissues, posing ecological and public health risks through bioconcentration and biomagnification along food chains.\u003c/p\u003e \u003cp\u003eFluoranthene (Flu) and Pyrene (Pyr), both four-ring PAHs, are prominent among the pollutants found in the region. Originating from petroleum sludge and incomplete combustion of hydrocarbons, these compounds exhibit recalcitrant and hydrophobic characteristics\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, along with cytotoxic, mutagenic, and potentially carcinogenic effects\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Microbial degradation of Flu and Pyr is complicated given their chemical structure and hydrophobic nature, which restrict their cellular uptake by microbes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite their environmental persistence, Flu and Pyr are among the priority pollutants identified by environmental protection agencies worldwide, necessitating their quantification and monitoring in aquatic environments.\u003c/p\u003e \u003cp\u003eMalaysia possessed the second largest tidal halophytic mangrove forests that cover 11.7% by area in Southeast Asia\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Mangrove swamps are coastal wetlands found in tropical and subtropical inter-tidal zones and participate in providing food and suitable environmental conditions for marine and estuarine microorganisms. The mangroves of the Klang Strait are particularly vulnerable to contamination from both petrogenic and pyrogenic sources of PAHs, endangering their ecological integrity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Previous studies have highlighted the presence of PAHs in mangrove sediments ranged at 20\u0026ndash;112 ng/g on a dry-weight along the Strait of Malacca, emphasizing the urgent need for remediation efforts. Mangrove ecosystems harbor a rich diverse microbial community that can degrade PAHs, making them an ideal environment for the microbial breakdown of these compounds\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, PAH-degrading bacterial strains were enriched for isolation and degradation of Pyr and Flu from mangrove sediments in this study. Subsequently, high potential isolates were selected for further analysis under varying conditions, including temperature, co-culture, and mixture of PAHs.\u003c/p\u003e \u003cp\u003eMicrobial degradation emerges as a promising approach for mitigating PAH contamination due to its efficiency, convenience, and environmental acceptability\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Studies reported that several bacterial genera have the potential to degrade Pyr and Flu in environment \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the successful application of biodegradation relies on various factors, including temperature, salinity, oxygen levels, pH, nutrient availability, hydrophobicity, degree of acclimation, contaminant concentration, and microbial populations\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Among them, temperature is of vital importance during PAH biodegradation, where the elevation or depression of temperature alters the physiology and structure of PAH-degrading bacteria and PAH properties, such as solubility, distribution, and bioavailability\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In saline environments, the efficacy of microbial degradation is often hampered by the limited metabolic activity of microorganisms and osmotic pressure imbalances\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Hence, application of halotolerant or halophilic PAH degraders in saline environments is crucial to assure the success of bioremediation of HMW PAH contamination. As for as we know, there has been no research conducted on the degradation of Flu and Pyr and their mixture under saline environments by \u003cem\u003eS. acidaminiphila\u003c/em\u003e and \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e, either individually or in co-culture.\u003c/p\u003e \u003cp\u003eTo address these challenges, this study focuses on isolating native PAH-degrading bacteria from aerobic mangrove sediments and assessing their degradation capabilities, particularly towards Flu and Pyr and their mixture. Additionally, the study aims to explore the synergistic effects of bacterial consortia in degrading these pollutants and determine the optimal temperature conditions for their degradation.\u003c/p\u003e"},{"header":"METHODS AND MATERIALS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Culture Media and Chemicals\u003c/h2\u003e\n \u003cp\u003ePyr and Flu used in this study were of high purity, purchased from Sigma\u0026ndash;Aldrich (Selangor, Malaysia). The Carbon Free Mineral Medium (CFMM) consisted of the following: 2.8 g of NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e, 2.0 g of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.8 g of Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO, 0.1 g of MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 50 mg of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, and 50 mg of CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO in 1 L distilled water was prepared according to\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Prior to the addition of PAHs, the medium was adjusted to pH 7.0\u0026ndash;7.2 with 1 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e/NaOH and autoclaved at 121\u0026deg;C for 15 min. Stock solutions of each PAH (300 mg/L) were prepared in ethyl acetate, kept in a brown bottle at 4\u0026deg;C, and wrapped to avoid any exposure to light prior to dilution. Solid media plates were prepared by adding 20 g of agar into 1 L of CFMM medium.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Isolation and Characterization of PAH-degrading Bacterial Consortium\u003c/h2\u003e\n \u003cp\u003eFresh mangrove sediments were collected from Sementa mangrove forest, Klang, Malaysia. The sediment sample was transformed to the laboratory on the ice and homogenized thoroughly to form a composite sample. 5 g of homogenized sediment was added into sterile flasks that contain 45 mL of CFMM and placed in a laboratory shaker overnight. The medium was allowed to settle for 2 hrs, and 5 mL of subculture supernatant was transferred into fresh CFMM that contain 50 mg/L of Pyr and Flu as the carbon and energy sources. Enriched medium was incubated in shaker at 30\u0026deg;C and 200 rpm for 8 days. This method was repeated for five consecutive times to obtain a relatively stable PAH-degrading bacterial consortium. Once the enrichment appeared turbid, 0.1 mL from diluted culture broth of 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e5\u003c/sup\u003e was spread onto CFMM agar plate. The plate sprayed with a layer of Pyr and Flu (0.5 mg/mL). Plates were incubated at 30\u0026deg;C and regularly checked for clear zones, which were scored as positive\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Positive bacterial colonies were transferred to nutrient agar plates until pure cultures were isolated. Identification of the pure cultures was performed using Biolog Automated System 2009 (Biolog Microstation System- MicroStation\u0026trade;). For identification, the isolated bacterial cells were regrown on NA plates at 30 \u003csup\u003eo\u003c/sup\u003eC for (24\u0026ndash;28) hours so as to keep away metabolic vigor and loss of viability whilst is typical of most organism at the static phase. The inoculation fluid (IF) were applied as inoculum for focused cells using protocols-A (IF-A catalogue No. 72401) and B (IF-B catalogue No. 72402) at particularized turbidity range 95\u0026ndash;98% T. This was accomplished using a specific cotton tipped inoculators swab (Catalogue No. 3353) to select almost 3 mm diameter are of the cell development from the very surface of the agar plate, and ultimately dipping it into the required IF. Then to ensure consistent suspension, some cell clumps were cautiously smashed against tube wall. The ensuring cell suspensions were poured into the multi-channel pipette reservoir.\u003c/p\u003e\n \u003cp\u003eA particular 8-channels automated pipette was applied to distribute 100-\u0026micro;l of suspension into every cell in MicroPlates (Catalogue no.1030). The well consisted of 71 carbon-source utilization assays (Columns 1\u0026ndash;8) and 22 chemical sensitivity assays (Columns 10\u0026ndash;11), thus they can be recognized at species level that is based on the \u0026ldquo;Phenotypic Fingerprint\u0026rdquo; of the microorganisms arranged by a specific panel. MicroPlates were placed on Omnilog reader wherein they were carefully read by the use of Biolog\u0026rsquo;s Microbial Identification Systems software, and the recognized microbes were correctly recorded.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Biodegradation of Pyr and Flu by Enriched Bacterium and Consortium\u003c/h2\u003e\n \u003cp\u003eThe degradation of Pyr and Flu using single bacterium and enriched consortium was evaluated. To demonstrate degradation experiments, 5 mL of inoculum with OD\u003csub\u003e600\u003c/sub\u003e of 0.1 to 0.2 was inoculated into 49 mL of seawater and supplemented with Flu and Pyr. The concentration of Flu and Pyr used in this study were 20 mg/L for single bacterium and/or 50 mg/L for enriched consortium. Seawater containing equal proportion of Flu and Pyr with no inoculum was set as a control to monitor abiotic losses during the experimental process. Pyr and Flu dissolved in ethyl acetate were then evaporated at room temperature by gently shaking the flask before inoculation. The pre-sterilized conical flasks were prepared in triplicates, covered with aluminium foil and incubated in a dark rotary shaker at 150 rpm and 30\u0026deg;C for 8 days. Residual concentrations of Flu and Pyr were quantified at the beginning and at the end of the incubation period. Samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C before analysis by gas chromatography equipped with a flame ionization detector (GC-FID).\u003c/p\u003e\n \u003cp\u003eTo evaluate the PAH degradation (\u003cem\u003eER\u003c/em\u003e), the following equation was applied described earlier\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1732295359.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cem\u003eER\u003c/em\u003e (%) is the percentage of removal efficiency; C\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the initial PAH concentration; and C\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e is the final PAH concentration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 The effect of Temperature on Flu and Pyr Degradation\u003c/h2\u003e\n \u003cp\u003ePyr and Flu degradation was evaluated at different temperatures (25, 30, and 35\u0026deg;C), to examine the effect of temperature on degradation efficiency. The medium was augmented with each 50 mg/L of Pyr and Flu and incubated at different temperatures over a shaker for 8 days in dark. Residual Pyr and Flu were sampled at Day 0 and Day 8 to record Pyr and Flu concentration by using the same procedure.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Degradation Kinetic Model\u003c/h2\u003e\n \u003cp\u003eFlu and Pyr biodegradation by the single bacterium and the enriched consortium was fitted into a first-order kinetic model and expressed as in Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e):\u003c/p\u003e\n \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$\\:\\:{C}_{t}=\\:{{C}_{o}e}^{-kt}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere C\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e is the concentration (mg/L) of residual PAH at time t; and C\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e is the initial PAH concentration (mg/L) in the medium. Biodegradation rate constant (\u003cem\u003ek\u003c/em\u003e) day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was determined from the straight line slope of plot ln [C\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e/C\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e] versus time. Biodegradation half-life (\u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;ln 2/\u003cem\u003ek\u003c/em\u003e, was also determined.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Analytical Procedure\u003c/h2\u003e\u003cp\u003eThe residual extraction of Pyr and Flu from liquid medium was conducted by liquid-liquid technique. Biomass was separated from the culture via filtration, and the culture was introduced into a separating funnel with an equal volume (v/v) of ethyl acetate. These funnels were vigorously shaken for 10 mins to extract entire residual PAHs and allowed to rest for separation. This procedure was repeated twice and the combined organic solvent was filtered through a glass wool containing anhydrous sodium sulphate. The collected solutions were concentrated using a rotary evaporator until the volume reduced to approximately 1 mL which then was transferred into a 1.5 mL dark vial for GC\u0026ndash;FID determination.\u003c/p\u003e\u003cp\u003ePAH profile and concentration was determined using Agilent Technologies-7890A GC-FID. HP-5MS fused silica capillary column was used for analyses (30 m \u0026times; 0.25 mm ID \u0026times; 0:25 \u0026micro;m film thickness) under temperature-programmed settings of 60\u0026deg;C for 2 mins, 60\u0026deg;C to 120\u0026deg;C at a rate of 10\u0026deg;C/min, 120\u0026deg;C to 300\u0026deg;C at a rate of 3\u0026deg;C/min, and 300\u0026deg;C for 10 min. Detector and injector temperatures were set at 300 and 280\u0026deg;C, respectively. The split less sample was injected onto capillary column (HP-5MS) and nitrogen served as the carrier gas. PAH biodegradation was determined by calculating the remaining PAH concentration in the medium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003eFlu and Pyr degradation experiments were performed in triplicates and average readings were taken. The Statistical analysis using one-way ANOVA with Turkey\u0026rsquo;s test was done with IBM SPSS version 22.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and Characterization of Enriched PAH-degrading Bacterial Consortium\u003c/h2\u003e \u003cp\u003eBased on the clearing zones on CFMM solid agar plates sprayed with Flu and Pyr, three distinct types of PAH-degrading bacteria were isolated after incubation for 16\u0026ndash;18 days. The PAH-degrading bacteria were \u003cem\u003eO. anthropi, S. acidaminiphila\u003c/em\u003e, and \u003cem\u003eA. salmonicida ss salmonicida. O. anthropi\u003c/em\u003e and \u003cem\u003eS. acidaminiphila\u003c/em\u003e grew as light green and yellow pigmented colonies in Pyr-enriched media, respectively, whereas \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e grew as a white pigmented colony in Flu-enriched media. O. \u003cem\u003eanthropi\u003c/em\u003e is an aerobic, Gram-negative, motile, non-lactose-fermenting bacillus. O. \u003cem\u003eanthropi\u003c/em\u003e isolated from saline soil contaminated by polycyclic aromatic hydrocarbons (PAHs) was previously reported to have the potential to degrade PAHs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. S. \u003cem\u003eacidaminiphilia\u003c/em\u003e is a mesophile and gram-negative bacteria. Previous studies indicate that this organism has the potential to degrade polycyclic aromatic hydrocarbons \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Study by\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e also indicated that \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e has the potential to degrade PAH especially benzene (a) pyrene in their study. This indicates that, the microbes\u0026rsquo; isolates has the potential to be further explored for degradation of PAH, focusing on Flu and Pyr under saline condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Degradation of Pyr and Flu by Single Bacterium\u003c/h2\u003e \u003cp\u003eDegradation of Flu and Pyr by a single bacterium namely by \u003cem\u003eS. acidaminiphila, O. anthropi, and A. salmonicida ss salmonicida\u003c/em\u003e in seawater (28 ppm of NaCl) was recorded at its initial concentration of 20 mg/L and incubation period of 8 days. Abiotic loss of PAH in the flask was ˂8% due to volatilization and/or adsorption to the glass, or possibly from the accumulation of PAH substrate in the cell matrix. The results of Flu degradation by \u003cem\u003eS. acidaminiphila\u003c/em\u003e, \u003cem\u003eO. anthropi\u003c/em\u003e, and \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e reached up to 53%, 60%, and 47%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), whereas Pyr degradation was 51%, 58%, and 42%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFlu degradation was better than Pyr degradation even though both are isomers. Pyr is the most persistent PAH within the context of this study. The low degradation rate recorded for Pyr was expected because its structure is a peri-fused PAH. The present study also revealed that \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e were able to degrade Flu and Pyr under saline conditions. This observation might be a result of enzyme specificity from these isolates. Among the three bacterial isolates, \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e showed a higher capability of PAH degradation than \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe biodegradation rate of Flu and Pyr by a single organism was comparable with other halotolerant Flu and Pyr degraders, but their culture condition and behaviour are different in some circumstances. \u003cem\u003eOchrobactrum anthropi\u003c/em\u003e strain VA1 can degrade 84% of Pyr under saline conditions similar to that in \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e. Studies from\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e suggested that \u003cem\u003eAcinetobacter pittii\u003c/em\u003e and \u003cem\u003eEnterobacter cloacae\u003c/em\u003e was able degrade 194 and 242 mg/L of Pyr. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e reported that, \u003cem\u003eThalassospira\u003c/em\u003e sp. strain TSL5-1 can remove Flu and Pyr by 41.4% and 42.3%, respectively, in 5% (w/v) salt medium. \u003cem\u003eThalassospira\u003c/em\u003e sp. strain TSL5-2 was capable of degrading Pyr by 53.5% and Flu by 60% under 5% (w/v) saline condition. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e asserted that \u003cem\u003eRhodococcus ruber\u003c/em\u003e strain L9 efficiently removed 76% of Pyr. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003eisolated \u003cem\u003eCycloclasticus\u003c/em\u003e strains, NY93E, PY97M, and PY97N from Yellow Sea sediment, which potentially degraded 52\u0026ndash;63% of Pyr at 3% salt concentration. \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003efound that LOP-9 \u003cem\u003eStaphyloccous\u003c/em\u003e aureus and GWP-2 \u003cem\u003eMycobacterium vaanbaalenii\u003c/em\u003e were able to degrade Pyr 86% and 82.1% respectively. In a similar study, \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003epostulated that \u003cem\u003eKocuria flava\u003c/em\u003e and \u003cem\u003eRhodococcus pyridinivorans\u003c/em\u003e effectively removed 3 ring PAHs from petroleum-contaminated soil.\u003c/p\u003e \u003cp\u003eNumerous reports revealed the metabolic potential of \u003cem\u003eOchrobactrum\u003c/em\u003e sp. in Flu and Pyr degradation, suggesting the involvement of this bacteria in the degradation of several PAHs. Among these reports, \u003cem\u003eOchrobactrum anthropi BPyF3\u003c/em\u003e isolated from hydrocarbon-contaminated soil has the ability to degrade 49.85% and 50.39% of Flu and Pyr, respectively, from an initial concentration of 50 mg/L after 7 days of incubation. The higher degradation of Flu and Pyr by \u003cem\u003eO. anthropi\u003c/em\u003e can be also associated with the distribution of the organism widely in the in environmental sources. This is also supported by findings by\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, indicates that \u003cem\u003eO. anthropi\u003c/em\u003e isolated from contaminated soil showed effective degradation of various polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and as well other compounds including hexane, heptane, hexadecane and pesticides.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e \u003c/sup\u003ealso isolated \u003cem\u003eO. intermedium\u003c/em\u003e from crude oil-contaminated site, which degraded Flu 100% and Pyr 72.4%. In addition, \u003cem\u003eOchrobactrum anthropi\u003c/em\u003e had the ability to degrade and grow in Pyr, benzo(k)fluoranthene, and benzo(e)pyrene under saline conditions by utilizing carbon as energy source. Another report showed that \u003cem\u003eOchrobactrum\u003c/em\u003e sp. \u003cem\u003eBL01\u003c/em\u003e degraded 21% of benzo[a]pyrene during 14 days of incubation. Furthermore, \u003cem\u003eOchrobactrum\u003c/em\u003e sp. has been identified as a PAH-degrader of 2,4,6-tribromophenol through reductive dehalogenation and production of biosurfactants.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eStenotrophomonas\u003c/em\u003e genus is metabolically diverse, reportedly degrades various types of organic pollutants, and has an inherent resistance to many heavy metals. \u003cem\u003eS. maltophilia\u003c/em\u003e VUN 10,003 was reported in HMW PAH degradation, but the potential of \u003cem\u003eS. acidaminiphila\u003c/em\u003e was not completely explored for Flu and Pyr degradation. The degradation potential of \u003cem\u003eS. acidaminiphila\u003c/em\u003e towards Pyr in this present study was higher than those from the previous studies by\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e who reported 68% Pyr degradation by \u003cem\u003ePseudomonas\u003c/em\u003e sp. and by\u003csup\u003e31\u003c/sup\u003e and \u003csup\u003e32\u003c/sup\u003ewho also reported 19% and 34% Pyr degradation by \u003cem\u003eOchrobactrum\u003c/em\u003e sp. and \u003cem\u003eRhodococcus\u003c/em\u003e sp. P14 within 30 days, respectively, from an initial Pyr concentration of 50 mg/L. In comparison with the results from previous research, \u003cem\u003eS. acidaminiphila\u003c/em\u003e has a significant biodegradative potential for Flu. In fact, \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003ereported 13.7% of Flu degradation by \u003cem\u003eHerbaspirillum chlorophenolicum\u003c/em\u003e strain FA1 isolated from activated sludge. \u003cem\u003ePasteurella\u003c/em\u003e sp. strain B-2 isolated from an activated sludge was able to degrade 40% of 20 mg/L Flu after 30 days of incubation. According to\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e sp. and \u003cem\u003eAdvenella\u003c/em\u003e sp. were able to remove Flu. \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003ereported 30% degradation of 50 mg/L Flu, after incubation for 12 days by \u003cem\u003eBacillus thuringiensis\u003c/em\u003e strain NA2 isolated from a petroleum oil-contaminated site.\u003c/p\u003e \u003cp\u003eVery few studies have reported the involvement of \u003cem\u003eAeromonas\u003c/em\u003e strains in the biodegradation of PAHs, such as anthracene, phenanthrene, and Pyr, but reports that specifically mentioned the use of \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e strains for Flu and Pyr degradation under saline condition are non-existent. This study revealed that, \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e had the ability to effectively degrade Pyr and Flu in saline condition. Statistical analysis showed that, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e were able to significantly degrade elevated concentrations of Pyr and Flu compared with abiotic loss (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Biodegradation kinetics of Pyr and Flu under single isolates\u003c/h2\u003e \u003cp\u003eVarious kinetics models have previously been applied for biodegradation process\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u0026rsquo;\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, the first-order rate equation widely used model and showed best fitted with the equation PAH. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the first-order kinetic model and half-lives used to determine Flu and Pyr degradation by single bacterium. In comparison with the Flu control that had a rate constant (\u003cem\u003ek\u003c/em\u003e) of 0.010 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 69.3 days, \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e had \u003cem\u003ek\u003c/em\u003e values of 0.118, 0.095, and 0.078 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 5.8, 7.2, and 8.8 days, respectively. In comparison with the Pyr control that had \u003cem\u003ek\u003c/em\u003e of 0.011 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 63 days, \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e had \u003cem\u003ek\u003c/em\u003e values of 0.108, 0.089, and 0.067 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 6.4, 7.7, and 10.3 days, respectively. \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e showed highest rate constant for degradation of Flu and Pyr in comparison \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e. The half-lives of Flu was shorter than that of Pyr. In soil, persistency is indicated by a \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e \u0026ge; 180 days \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, whereas, \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e \u0026lt; 10 days signifies rapid degradation. Consequently, after an acclimatization process, \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e can be effective species for removal of 4-ring PAHs. \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003easserted that compounds with short \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e indicated modest accumulation. Interestedly, in this study half-lives value obtained were lower than reported in literature.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003ekinetic parameters for Pyr and Flu degradation by isolates after 8 days\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSingle isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDegradation percentage %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRate of Pyr and Flu degradation (\u003cem\u003ek\u003c/em\u003e) day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHalf-life (ln2/\u003cem\u003ek\u003c/em\u003e) (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF-value (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eOchrobactrum anthropi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAeromonas salmonicida ss salmonicida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eOchrobactrum anthropi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16.416\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAeromonas salmonicida ss salmonicida\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Biodegradation of Flu and Pyr by Enriched Bacterial Consortium\u003c/h2\u003e \u003cp\u003eAn enriched bacterial consortium using the three isolates \u003cem\u003eS. acidaminiphila\u003c/em\u003e, \u003cem\u003eO. anthropic\u003c/em\u003e, and \u003cem\u003eA. salmonicida ss salmonicida\u003c/em\u003e was also investigated for the degradation of Flu and Pyr with an initial concentration of 50 mg/L in seawater (28 ppm of NaCl) for 8 days. The recorded abiotic loss of PAHs was below 10%. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that, the degradation rate of Flu and Pyr by the enriched bacterial consortium were 85% and 81%, respectively.\u003c/p\u003e \u003cp\u003eThese results indicated that, an enriched bacterial consortium enhanced the degradation of Flu and Pyr degradation compared with single bacterial isolates, reflecting the synergistic action among bacterial isolates. Statistical analysis showed that, the consortium was capable of degrading Pyr and Flu at higher concentrations compared with abiotic loss (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The degradation rates of the enriched consortium were 5.21 and 5.06 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e for Flu and Pyr, respectively. Hence these rates were higher than those of the bacterial consortium reported by Lafortune et al. 2009 \u003csup\u003e39\u003c/sup\u003e which recorded 4.7 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e for Pyr. Isaac et al. 2015 \u003csup\u003e40\u003c/sup\u003e asserted that, the mixed culture C15 removed 1.05 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e of Pyr. These results were also higher than the single isolates of \u003cem\u003eRhodococcus\u003c/em\u003e sp. P14 at 0.57 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e for Pyr as previously reported by\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e PL1 at 1.25 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e Pyr, and \u003cem\u003eOchrobactrum anthropi\u003c/em\u003e BPyF3 at 3.6 and 3.56 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e for Pyr and Flu degradation, respectively. \u003cem\u003eBurkholderia cepacia\u003c/em\u003e also showed 0.88 and 0.85 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e degradation for Pyr and Flu, respectively, whereas \u003cem\u003ePorphyrobacter\u003c/em\u003e sp. B51 degraded 4.0 mg/L\u003csup\u003e\u0026minus;\u0026thinsp;day\u003c/sup\u003e of Pyr. However, variations in pollutant reduction among the microbial consortia recorded by the aforementioned authors and the present study may be a result of culture conditions, differences in initial concentrations, and the microbial strain used.\u003c/p\u003e \u003cp\u003eA previous study by\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e reported that, \u003cem\u003eBurkholderia fungorum\u003c/em\u003e and \u003cem\u003eMycobacterium gilvum\u003c/em\u003e degraded 100 mg/L of Pyr and Flu within 20\u0026ndash;24 days by 98.6% \u0026plusmn; 1.9% and 99.6% \u0026plusmn; 0.7%, respectively. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003easserted that, both Flu and Pyr biodegradation by a bacterial consortium of \u003cem\u003eCycloclasticus\u003c/em\u003e sp. PY97M and \u003cem\u003eMarinobacter nanhaiticus\u003c/em\u003e D15-8WT was faster than a single bacterial isolate and observed synergic effects. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003erecently reported that, HMW PAH biodegradation by bacterial consortium was higher than that by a single bacterium under saline conditions. \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003ealso reported the synergistic impact of co-culture in Flu and Pyr degradation, where a single bacterial isolate of \u003cem\u003eSphingomonas\u003c/em\u003e sp. or \u003cem\u003eMycobacterium\u003c/em\u003e sp. was able to degrade Pyr and Flu within 14 days. A co-culture of \u003cem\u003eSphingomonas\u003c/em\u003e and \u003cem\u003eMycobacterium\u003c/em\u003e strains enhanced the degradation and/or completely removed the two PAHs in 7 days. The activity amid the bacterial isolates was credited to the synergistic effect in the bacterial consortium by complementary degradative abilities among the microbes of consortia and elimination of toxic effects during degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst-order kinetic model fitted with biodegradation data was used to determine the Flu and Pyr biodegradation rates of the enriched consortium. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates that, bacterial consortium degradation rate constants were 0.208 and 0.247 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 3.3 and 2.8 days for Flu and Pyr, respectively, compared with the abiotic and autoclaved control at \u003cem\u003ek\u003c/em\u003e values of 0.010 and 0.012 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 69.3 and 57.7 days for Pyr and Flu, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003ekinetic parameters for Pyr and Flu degradation by enriched consortium after 8 days\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDegradation percentage%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRate of Pyr and Flu degradation (\u003cem\u003ek\u003c/em\u003e) day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHalf-life (ln2/\u003cem\u003ek\u003c/em\u003e) (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF-value (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e265.296\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixture of PAHs (Flu)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e150.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eA\u0026thinsp;=\u0026thinsp;\u003cem\u003eOchrobactrum anthropi\u003c/em\u003e; B\u0026thinsp;=\u0026thinsp;\u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e; C\u0026thinsp;=\u0026thinsp;\u003cem\u003eAeromonas salmonicida ss salmonicida\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Degradation of Mixture of PAHs by bacterial Consortium\u003c/h2\u003e \u003cp\u003eBiodegradation by the cultivated consortium using seawater containing a mixture of Flu and Pyr at 50 mg/L each was investigated. The consortium was able to degrade 69% of Flu and 64% of Pyr after 8 days of incubation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Statistical analysis showed that, the consortium was capable of significantly degrading the PAH mixture compared with abiotic loss at (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This results indicated that, the degradation by the bacterial consortium was higher than those reported by Guo et al. which asserted that the enriched consortia degraded less than 40% of mixture of PAHs (Phe\u0026thinsp;+\u0026thinsp;Fla\u0026thinsp;+\u0026thinsp;Pyr). \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003ealso reported that \u003cem\u003eStenotrophomonas\u003c/em\u003e sp. and \u003cem\u003ePseudomonas\u003c/em\u003e sp. potentially degraded 14% and 7% of Pyr, in a mixture of five PAHs, respectively. \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003ereported that \u003cem\u003eKocuria flava\u003c/em\u003e and \u003cem\u003eRhodococcus pyridinivorans\u003c/em\u003e degraded 55.6%, 59.5%, and 59.1% of 10 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of mixed PAHs (phenanthrene, anthracene, fluorene, and pyrene) respectively, within 15 days. The combined metabolism by bacterial consortium may increase the degradation of mixed HMW PAHs, because the intermediary biotransformation of products from individual strains may serve as primary substrate for catabolism and growth by others. \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003eobserved that, degradation of a PAH mixture may be a result of collaborative processes involved in the consortium of isolates with complementary capacities. The main source of complementary degradation in the bacterial consortium might also result from the synergy between versatile genetic formation and degradative enzymatic specificity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA first-order kinetic model was applied to determine the biodegradation rate of Flu and Pyr mixture by the enriched consortium. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicates that, degradation by bacterial consortium (A\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C) had \u003cem\u003ek\u003c/em\u003e values of 0.145 and 0.129 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the Flu and Pyr mixture, respectively, and \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e1/2\u003c/em\u003e\u003c/sub\u003e of 4.7 and 5.3 days. The consortium was more promising than the control and autoclaved set-ups, which had \u003cem\u003ek\u003c/em\u003e values of 0.009 and 0.011 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e1/2\u003c/em\u003e\u003c/sub\u003e of 77 and 63 days, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of Temperature on Pyr and Flu Degradation\u003c/h2\u003e \u003cp\u003eThe effect of temperature was on Pyr and Flu degradation was also investigated in this study. The temperature effect was tested was in range of 25 to 35\u0026deg;C at pH 7.8 and agitation speed of 150 rpm in seawater (28 ppm NaCl). The temperature range might alter the biodegradation process because some authors considered this range as narrow. In the present study, great differences from temperature variations were observed with an optimum at 30\u0026deg;C for bacterial consortium applied to PAHs, which was also near the natural conditions where the samples were initially collected. Pyr degradation was prominent at 30\u0026deg;C as compared to 25\u0026deg;C and 35\u0026deg;C. However, the Pyr degradation at 35\u0026deg;C was lower by 13.2% compared with that at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Flu degradation rate at 30\u0026deg;C was higher than those at both 25 and 35\u0026deg;C, whereas the rate at 25\u0026deg;C increased by 11.0% compared with that at 35\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The degradative abilities of bacterial consortium towards Flu and Pyr were lower at 25 and 35\u0026deg;C at every stage during the study period. Statistical analysis showed that, the consortium significantly degraded Pyr and Flu at 30\u0026deg;C compared with the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the difference in degradation efficiency observed at different temperatures by the bacterial consortium was not significant. These data indicated that, the bacterial consortium had great potential in degrading Pyr and Flu at 30\u0026deg;C. In a similar research, \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003easserted that, NJ2 strain degraded 60% of Pyr at 37\u0026deg;C. Al-Thukair and Malik, 2016 \u003csup\u003e47\u003c/sup\u003e found that, \u003cem\u003eB. fungorum\u003c/em\u003e removed 56% and 59% of Pyr at 37\u0026deg;C and 25\u0026deg;C, respectively, whereas, \u003cem\u003eCaulobacter sp\u003c/em\u003e removed 35\u0026ndash;36% of Pyr at 37 and 25\u0026deg;C. This study also agreed with\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e who reported that, \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e PL1 degraded Pyr faster at 30\u0026deg;C than at 20 and 40\u0026deg;C. Similar results were recorded when \u003cem\u003eAcinetobacter\u003c/em\u003e strain USTB-X biodegraded 63% of Pyr at 30\u0026deg;C. Optimum Pyr degradation occurs above room temperatures. \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003eisolated \u003cem\u003eHerbaspirillum chlorophenolicum\u003c/em\u003e strain FA1 from activated sludge, which degraded 13.7% Flu at 30\u0026deg;C after 30 days of incubation. Thus, temperature is one of the crucial environmental factors in PAH degradation because it influences the metabolic pathways of the bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the results of the kinetic Pyr and Flu degradation by bacterial consortium (A\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C) under different temperatures. The effect of temperature on the degradation rate constant shows \u003cem\u003ek\u003c/em\u003e values of 0.177\u0026ndash;0.179 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 25\u0026deg;C, 0.199\u0026ndash;0.201 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 30\u0026deg;C, and 0.138\u0026ndash;0.146 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 35\u0026deg;C, for both Pyr and Flu respectively, and \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e of 3.7\u0026ndash;3.8, 3.2\u0026ndash;3.4, and 4.7\u0026ndash;5.0 days for Pyr and Flu, respectively. The abiotic and autoclaved rate had \u003cem\u003ek\u003c/em\u003e values of 0.012\u0026ndash;0.014, 0.015\u0026ndash;0.016, and 0.013\u0026ndash;0.016 day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Pyr and Flu, respectively, with \u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e = 49.5\u0026ndash;53.3, 43.3\u0026ndash;46.2, and 43.5\u0026ndash;53.4 days, at 25\u0026deg;C, 30\u0026deg;C, and 35\u0026deg;C, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ekinetic parameters for Pyr and Flu degradation by enriched consortium at different temperature after 8 days\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemp\u003c/p\u003e \u003cp\u003e\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDegradation percentage%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRate of Pyr and Flu degradation (\u003cem\u003ek\u003c/em\u003e) day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHalf-life (ln2/\u003cem\u003ek\u003c/em\u003e) (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF-value (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.330\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e229.158\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eA\u0026thinsp;=\u0026thinsp;\u003cem\u003eOchrobactrum anthropi\u003c/em\u003e; B\u0026thinsp;=\u0026thinsp;\u003cem\u003eStenotrophomonas acidaminiphila\u003c/em\u003e; C\u0026thinsp;=\u0026thinsp;\u003cem\u003eAeromonas salmonicida ss salmonicida\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThree high molecular weight PAH-degrading bacteria have been isolated from mangrove sediments and characterized as \u003cem\u003eO\u003c/em\u003e. \u003cem\u003eanthropi\u003c/em\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eacidaminiphila\u003c/em\u003e, and \u003cem\u003eA\u003c/em\u003e. \u003cem\u003esalmonicida ss salmonicida\u003c/em\u003e for their capacity to breakdown Pyr and Flu. This study revealed that, a single bacterium and an enriched consortium exhibited excellent degradation capability to degrade Flu and Pyr and their mixture under saline conditions. Though, single bacterium had lower degradation potential, while the enriched consortium had more effective and a wider range of substrate degradation. The individual isolates and enriched consortium were able to efficiently removed Pyr and Flu at optimal at 30\u0026deg;C compared with those at 25\u0026deg;C or 35\u0026deg;C. These isolates have an excellent biodegradation potential in saline conditions, and therefore, suggesting the application at the field scale as well as contaminated mangrove ecosystem and coastal area. In the future, it would be provident to further study the molecular verification of microbial community and complete degradation pathway of Pyr and Flu by individual isolates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their appreciation to Researchers Supporting Project number (RSPD2024R693), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The data supporting the conclusions of this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e:\u0026nbsp;The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAziz Ahmed work\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e for final draft and supervision, Mohib Ullah Kakar and Zafar Ullah Jattak performed the experiments, Imran Iqbal perform experiments validation, Farid Shokry Ataya, Dalia Fouad\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eperformed the analysis of results and interpretation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZulkifli, N., Ismail Raja Ibrahim, R., Azlan Abdul Rahman, A. \u0026amp; Fawwaz Mohd Yasid, A. 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Biotechnol.\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 3819\u0026ndash;3828 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacterial consortium, Biodegradation, Mangrove, PAHs, Seawater","lastPublishedDoi":"10.21203/rs.3.rs-5400344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5400344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants that exert acute toxic effects and/or possess carcinogenic, tumorigenic, and genotoxic properties. This study focused on the potential of a single bacterium and an enriched consortium to degrade high-molecular-weight (HMW) PAHs such as Pyrene (Pyr) and Fluoranthene (Flu) under saline conditions. The potential PAH degraders were isolated from mangrove sediments and identified as \u003cem\u003eOchrobactrum anthropi, Stenotrophomonas acidaminiphila\u003c/em\u003e, and \u003cem\u003eAeromonas salmonicida ss salmonicida.\u003c/em\u003e The findings revealed that the single culture degraders degraded Flu by 60%, 53%, and 47%, respectively, as well as Pyr by 58%, 51%, and 42%, respectively, from an initial concentration of 20 mg/L in seawater (28 ppm of NaCl) after 8 days. Meanwhile, the consortium degraded 85% of Flu and 81% of Pyr from an initial concentration of 50 mg/L after 8 days. The consortium also degraded a mixture of Flu and Pyr by about 60%. Biodegradation ability by the consortium for Flu and Pyr at different temperatures decreased in the order 30\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;25\u0026deg;C\u0026thinsp;\u0026gt;\u0026thinsp;35\u0026deg;C. The results revealed that, the consortium showed enhanced degradative capacity of more than 80% as compared to single isolates for degradation of Flu and Pyr and additionally the mixture of both PAHs showed 60% rate of degradation in saline environments. Therefore it can be concluded that the pooled microbial consortium has higher potential of degradation of PAHs especially for Flu and Pyr and this could be used as a method of removing PAHs pollution from the contaminated environment.\u003c/p\u003e","manuscriptTitle":"Enhanced Biodegradation of Fluoranthene and Pyrene in Saline Condition Using Microbial Consortia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-22 17:17:01","doi":"10.21203/rs.3.rs-5400344/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1d18fc79-a9ae-4832-b66a-0c86aedc9cfd","owner":[],"postedDate":"November 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-23T14:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-22 17:17:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5400344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5400344","identity":"rs-5400344","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}