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Degradation of Polycyclic Aromatic Hydrocarbons by Moderately Halophilic Bacteria from Luzon Salt Beds

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Degradation of Polycyclic Aromatic Hydrocarbons by Moderately Halophilic Bacteria from Luzon Salt Beds Research Degradation of Polycyclic Aromatic Hydrocarbons by Moderately Halophilic Bacteria from Luzon Salt Beds Carolyn L. Nanca,1,2,5 Kimberly Background. Polycyclic aromatic hydrocarbons (PAHs) are common environmental D. Neri,1,2 Anna Christina R. contaminants which are highly toxic due to their carcinogenic and mutagenic effects. They Ngo,3 Reuel M. Bennett,3,4 Gina R. are released into the environment by incomplete combustion of solid and liquid fuels, Dedeles1,2,3,4 accidental spillage of oils and seepage from industrial activities. One of the promising processes mitigating PAHs is through biodegradation. However, conventional microbiological 1 The Graduate School, University of treatment processes do not function well at high salt concentrations. Hence, utilization of Santo Tomas, Manila, Philippines halophilic bacteria should be considered. 2 Laboratory of Pure and Applied Objectives. This study aimed to assess the ability of halophilic bacteria isolated from local Microbiology, Research Center for the salt beds in Pangasinan and Cavite, the Philippines, to degrade PAHs pyrene, fluorene and Natural and Applied Sciences, University fluoranthene. of Santo Tomas, Manila, Philippines Methods. Polycyclic aromatic hydrocarbon-tolerant halophilic bacteria collected from 3 Collection of Microbial Strains, two sampling sites were phenotypically characterized, molecularly identified and tested University of Santo Tomas, Manila, to determine their potential to degrade the PAHs pyrene, fluorene and fluoranthene at Philippines a hypersaline condition. Best PAH degraders were then assayed to identify the optimal 4 Department of Biological Sciences, degradation using such parameters as pH, temperature and PAH concentration. Testing College of Science, University of Santo for enzyme degradation was also done to determine their baseline information. Extraction Tomas, Manila, Philippines and analysis of degraded PAHs were performed using centrifugation and UV-vis 5 Department of Sciences, Senior High spectrophotometry. School, University of Santo Tomas, Manila, Results. Twelve isolates from both collection sites tolerated and grew in culture with selected Philippines PAHs. These were identified into four genera (Halobacillus, Halomonas, Chromohalobacter, and Pontibacillus). Selected best isolates in a series of biodegradation assays with the above- Corresponding Author: mentioned parameters were Halobacillus B (Collection of Microbial Strains (CMS) 1802) Gina R. Dedeles (=trueperi) (Gram-positive) for pyrene and fluoranthene, and Halomonas A (CMS 1901) [email protected]/grdedeles (Gram-negative) for fluorene. Degrader biomass and PAH degradation were invariably @yahoo.com negatively correlated. Qualitative tests with and without peptone as a nitrogen source implied enzymatic degradation. Discussion. Polycyclic aromatic hydrocarbons utilized by these halophilic bacteria served as a sole source of carbon and energy. Implications of biodegradation of the two best isolates show that high molecular weight (HMW) (4-ring) pyrene tends to be degraded better by Gram-positive bacteria and low molecular weight (3-ring) fluorene by Gram-negative Introduction degraders. Conclusions. Halophilic bacteria constitute an untapped natural resource for biotechnology Polycyclic aromatic hydrocarbons in the Philippines. The present study demonstrated their potential use in bioremediation of (PAHs) are composed of two or recalcitrant hydrocarbons in the environment. more fused aromatic rings.1 These Competing Interests. The authors declare no competing financial interests. macrocyclic compounds are Keywords. halophiles, salt bed, polycyclic aromatic hydrocarbons, biodegradation, pyrene, ubiquitous environmental pollutants of fluorene, fluoranthene. both natural and anthropogenic origin, Received July 5, 2018. Accepted August 12, 2018. coexisting generally in areas of oil J Health Pollution 19: 180915 (2018) fields and gas factories. Air, soil, water © Pure Earth and vegetation all act as environmental sinks for PAHs. Their widespread occurrence in the environment, level bioaccumulation potential and concern to the health of aquatic life of recalcitrance to biodegradation, carcinogenic activity pose a serious and humans.2 1 Journal of Health & Pollution Vol. 8, No. 19 — September 2018 Nanca, et al. Research Degradation of PAHs by Moderately Halophilic Bacteria The concern for PAHs parallels including pH, temperature and salinity Abbreviations that for its most common natural to provide baseline information on source - petroleum oil, an abundant the culture of the halophiles and their DMF N,N-dimethylformamide and well-known hydrocarbon which degradation of selected PAHs under a HMW High molecular weight ranks worldwide as the most valuable saline condition. Results were aimed at traded commodity for use to generate future biotechnological application of LMW Low molecular weight energy needed in industry.3 In the the isolates singly or in combination in Philippines, transportation of crude oil pollution mitigation. oil and petroleum by oil tankers is a Agar with 0.15% NaCl following the lifeline, as the country depends largely Methods procedure of Yoon et al.15 Cultures on imports for its energy needs. A were incubated at room temperature single accident may result in the spill Marine Agar and Marine Broth 2216 for 5-7 days and, thereafter, resulting of crude oil or petroleum products (HiMedia, India) supplemented isolated colonies were selected and that can cause massive damage to the with 0.15% sodium chloride (NaCl) purified by re-streaking on Marine marine environment and nearby-to- were used as the culture media Agar. distant coastal areas, affecting human for the isolation and subsequent and animal life. The hazards associated biodegradation assays of PAHs of Identification of PAH-tolerant with PAHs and similar hydrocarbons halophilic bacteria isolated from salt halophilic bacteria require effective remediation strategy. beds. The PAHs pyrene, fluorene, and However, conventional microbiological fluoranthene with 98% purity and Marine Agar plates with 0.15% (w/v) treatment processes do not function N,N-dimethylformamide (DMF) were NaCl and individually supplemented well at a high salt concentration.4 purchased from Sigma-Aldrich (USA). with 1% of the PAH stock solutions Several studies suggest that halophilic Reagents, all of analytical grade, were were streaked with the selected isolates bacteria may have greater potential to obtained from RCI Labscan, Thailand. incubated at room temperature for degrade hydrocarbons, PAHs included, 5-7 days and then observed for the especially in marine sediments.4-7 Stock solutions of pyrene, fluorene, presence or absence of growth.16 and fluoranthene, the model pollutant Isolates able to grow in Marine Agar Halophilic bacteria and archaea are PAHs, were prepared following the plates with PAH were morphologically unique prokaryotes able to withstand procedure of Bezalel et al.13 Then characterized with emphasis on very high salt concentrations known pyrene and fluorene at 2.5 mg were Gram staining reaction, cell shape in hypersaline environments. Vast added to 0.3 mL DMF each for a final and size, and colony color, shape, areas on the planet are hypersaline, concentration of 8.33 mg/mL. For size, surface, edge, elevation and exposed to contamination with fluoranthene, 20 mg was dissolved opacity. Biochemical tests for catalase, hydrocarbons, but are among the least in 0.5 mL of DMF for an equivalent oxidase, motility, indole, urease, sugar 8-10 studied extreme environments. concentration of 40 mg/mL. Prepared fermentation, H2S production and for Hypersalinity is typical of salterns, stock solutions (30 mL) were kept in hydrolysis of casein, gelatin, starch and sites with a salt content 5-10 times the dark to avoid photooxidation.14 Tween 80 were also done for further higher than in seawater (150-300 characterization of the isolates.6, 17-20 g/L) and which undoubtedly also Collection of salt and soil samples The media used for all biochemical harbor halophiles.11 In Southeast tests were also supplemented with Asia, particularly in the Philippines, Thirty-five salt sediments and soil NaCl to support the growth of the however, knowledge of the diversity of samples were collected. Ten salt halophilic bacteria.19 Results were halophilic microorganisms and their samples and eight and seven soil analyzed using Bergey’s Manual of capacity to degrade hydrocarbons is samples were obtained, respectively, Systematic Bacteriology, Second very limited.12 from two local salt beds in Dasol, Edition and compared to minimal Pangasinan and Noveleta, Cavite, standards from related journals to In the present study, halophilic the Philippines. One gram of each partially identify the isolates up to at bacteria indigenous in local salt beds sample was enriched in 1 L of seawater least the generic level.21 were explored for their ability to supplemented with 5-g peptone and degrade the PAHs pyrene, fluorene 1-g yeast extract. Separate triplicate For identification at the species and fluoranthene. Emphasis was given aliquots from each 3-day-old level, molecular analysis (16S) to fundamental growth parameters, enrichment were plated on Marine was performed with the use of 2 Nanca, et al. Journal of Health & Pollution Vol. 8, No. 19 — September 2018 Research Wizard® Genomic DNA Purification hydrocarbon served as the positive was done using three sets of plain agar Kit (Promega), following the control. Rate of degradation was media (HiMedia,
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