Polymeric materials generally undergo these factors concurrently in the environment. Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction. 1990. Download scientific diagram | 1: The biodegradation mechanism of polyethylene. This chapter deals with the importance, definition, classification, mechanism of biodegradation, approaches, testing methods and applications of biodegradable polymers. Fluoride release (A) and color change (B) in medium of a culture of Pseudomonas putida F1 incubated with DFBD with shaking over 48h. P. putida F1 was grown with toluene as the carbon source, which induces the toluene catabolic pathway, and then the culture was switched to DFBD. 2018. Authors . Please enable it to take advantage of the complete set of features! Defluorination required only toluene dioxygenase. GC-MS revealed the transient formation of a nonfluorinated product, 1,2,3-benzenetriol, also known as pyrogallol (Fig. Since the alpha-subunit of Rieske dioxygenases determines substrate specificity (42, 43), sequence comparisons with this subunit are most germane to the question as to which enzymes and organisms will defluorinate DFBD. Determining the mechanisms and pathways of fipronil degradation is a difficult task, because biodegradation and abiotic degradation reactions can occur simultaneously. eCollection 2023 May. In 2017, a garbage patch the size of Mexico was found in the Pacific Ocean. oxygenase, dehydrogenase and oxydase, forming a fatty acid with subsequent - . FOIA Further structural information was gained from 19F-NMR that showed that both fluorine atoms were retained in the product (Fig. The fluorinated natural products. doi: 10.1128/mBio.03001-21. 8600 Rockville Pike The third and final study reviews European standardization of biodegradable and compostable material in the packaging industry, again using the terms separately.[43]. For many Synthetic, commercially important fluorinated compounds typically contain multiple fluorine atoms, and that makes them less reactive to defluorination, both chemically and biologically (5, 6). Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. 2015. Biodegradation of plastics using non-conventional methods promises a future free of accumulation of commodity plastics used in packaging and commercial polymers, which are the most abundant form of plastic wastes. AB - Aromatic compounds are widely distributed in nature. We acknowledge Thomas Niehaus for making available the Cary spectrophotometer. Overnight, this strain was grown on MSB and biphenyl, which induces the biphenyl dioxygenase. (B) Mass spectra of TMS-derivatized metabolite from cell cultures incubated with DFBD (top) and standard DFBD-5-ol (bottom). oxidase [8] In addition, aerobic digestion typically occurs more rapidly than anaerobic digestion, while anaerobic digestion does a better job reducing the volume and mass of the material. Once the C-O bond cleaves, the resultant OCF2O- moiety is highly unstable, to undergo very rapid hydrolysis to produce carbon dioxide and the two fluorine atoms as fluoride. -C-O-C- Ether linkage polyethylene glycol, PHA, PCL, PLLA 1H-NMR of standard 1,2,3-benzenetriol (pyrogallol). liberated into the reaction media and the rate of the molecular weight reduction of the [55] Similarly, coastal communities which rely heavily on ecotourism lose revenue thanks to a buildup of pollution, as their beaches or shores are no longer desirable to travelers. Possible involvement of toluene-2,3-dioxygenase in defluorination of 3-fluoro-substituted benzenes by toluene-degrading. polyurethane. | | 1H-NMR of DFBD-4-ol. Examples of commercially relevant polyfluorinated compounds. [35] Composting is a human-driven process in which biodegradation occurs under a specific set of circumstances. Naphthalene (NAP) significantly enhanced the biodegradation of phenanthrene (PHE) by Pseudomonas sp. The two coupled doublets are consistent with two fluorines on differentiated faces, in this case arising from the cis configuration of the hydroxyl groups. Generation and consequence of nano/microplastics from medical waste and household plastic during the COVID-19 pandemic. Fluorinated organic compounds are considered to be one of the major foci of biodegradation currently because of their prevalence, emerging concerns about health effects, and their prolonged lifetime in the environment (1). The toluene vapor was removed and replaced with 2,2-difluoro-1,3-benzodioxole (DFBD) vapor, and free fluoride ion was detected immediately. Comparison of dihydrodiol dehydration products from E. coli pDTG601a with synthetic standards following derivatization and mass spectrometry. A review of the pathways of human exposure to poly- and perfluoroalkyl substances (PFASs) and present understanding of health effects. The impact of biodegradable polymers on the environment and on society", "Research of the biodegradability of degradable/biodegradable plastic material in various types of environments", 10.1002/(sici)1521-3773(19990517)38:10<1438::aid-anie1438>3.0.co;2-u, "Biodegradable polyesters for medical and ecological applications", 10.1002/(sici)1521-3927(20000201)21:3<117::aid-marc117>3.0.co;2-x, "Section 6: Biodegradability of Packaging Waste", "Physical properties and biodegradability of maleated-polycaprolactone/starch composite", "New super-enzyme eats plastic bottles six times faster", "Plastic-eating enzyme 'cocktail' heralds new hope for plastic waste", "Characterization and engineering of a two-enzyme system for plastics depolymerization", "Cows' stomachs can break down plastic, study finds", "Together Is Better: The Rumen Microbial Community as Biological Toolbox for Degradation of Synthetic Polyesters", "Biodegradable polymers for the environment", "Using Green Chemistry to Deliver Cutting Edge Drugs", "Biodegradable Products Institute - Composting", "The use of life cycle assessment for the comparison of biowaste composting at home and full scale", "Some composting and biodegradation effects of physically or chemically crosslinked poly(lactic acid)", "Plastic Bag Externalities and Policy in Rhode Island", "Biodegradable and compostable alternatives to conventional plastics", "Plastic Degradation and Its Environmental Implications with Special Reference to Poly(ethylene terephthalate)", "Human mercury exposure and adverse health effects in the Amazon: a review", "Plastic debris and policy: Using current scientific understanding to invoke positive change", "Plastic Garbage Patch Bigger Than Mexico Found in Pacific", "Environmental implications of plastic debris in marine settings--entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions", "Marine plastic pollution as a planetary boundary threat The drifting piece in the sustainability puzzle", "Socioeconomic Disparities and Air Pollution Exposure: a Global Review", The Science of Biodegradable Plastics: The Reality Behind Biodegradable Plastic Packaging Material, Blue Ribbon Commission on America's Nuclear Future, https://en.wikipedia.org/w/index.php?title=Biodegradation&oldid=1124515260, Creative Commons Attribution-ShareAlike License 3.0, D5210- Standard Test Method for Determining the Anaerobic Biodegradation of Plastic Materials in the Presence of Municipal Sewage Sludge, D5526- Standard Test Method for Determining Anaerobic Biodegradation of Plastic Materials Under Accelerated Landfill Conditions, D5338- Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials Under Controlled Composting Conditions, Incorporating Thermophilic Temperatures, D5511- Standard Test Method for Determining Anaerobic Biodegradation of Plastic Materials Under High-Solids Anaerobic-Digestion Conditions, D5864- Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components, D5988- Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in Soil, D6139- Standard Test Method for Determining the Aerobic Aquatic Biodegradation of Lubricants or Their Components Using the Gledhill Shake Flask, D6006- Standard Guide for Assessing Biodegradability of Hydraulic Fluids, D6340- Standard Test Methods for Determining Aerobic Biodegradation of Radiolabeled Plastic Materials in an Aqueous or Compost Environment, D6691- Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in the Marine Environment by a Defined Microbial Consortium or Natural Sea Water Inoculum, D6731-Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer, D6954- Standard Guide for Exposing and Testing Plastics that Degrade in the Environment by a Combination of Oxidation and Biodegradation, D7044- Standard Specification for Biodegradable Fire Resistant Hydraulic Fluids, D7373-Standard Test Method for Predicting Biodegradability of Lubricants Using a Bio-kinetic Model, D7475- Standard Test Method for Determining the Aerobic Degradation and Anaerobic Biodegradation of Plastic Materials under Accelerated Bioreactor Landfill Conditions, D7665- Standard Guide for Evaluation of Biodegradable Heat Transfer Fluids, This page was last edited on 29 November 2022, at 03:07. 6C). The supernatant was removed and placed into a 5-ml tube with 1ml of TISAB (58.5 g/liter NaCl, 15 g/liter CH3COOH, 66 g/liter CH3COONa, and 1 g/liter 1,2-cyclohexane diaminetetraacetic acid). A BLAST search yielded over 1,200 homologous sequences with an E value lower than e50, indicating they matched extremely well. Table 2.7 Structure of the main chain polymer and the specific example of the The major path is highlighted by darker arrows. of ethylenic, carbonate, amide, urethane, etc (Matsumura, 2005). The mechanism for biodegradation. linkage Macromol Biosci. The application of toluene dioxygenase in the synthesis of acetylene-terminated resins, Benzylic monooxygenation catalyzed by toluene dioxygenase from. If hydrolysis is achieved enzymatically, then the process is usually considered to be . Toluene used for specified cell growth and induction was from Fisher Chemical and is 99% pure. 1). Environ Sci Technol 31:24452454. Dioxygenase-catalysed oxidation of disubstituted benzene substrates: benzylic monohydroxylation versus aryl. TZ biodegradation could be accelerated through the addition of glucose, sucrose and yeast extract at relatively low dosage. The polymer chain is cleaved et the relatively weak degradation and ultimate biodegradation as shown in Figure 2.8. Bacteria, growth substrates, enzymes expressed, and references are compiled in Table2. [3]While biodeterioration typically occurs as the first stage of biodegradation, it can in some cases be parallel to biofragmentation. Compounds containing the DFBD moiety shown are the experimental drug DiFMDA (difluoromethylenedioxyamphetamine), the fungicide fludioxonil, the anticancer agent AS-604850, and the reagent for enantioselective synthesis (. Full Article. 1990. || and micbiol cells,metabolic products under aerobic conditions. We propose here that these are spontaneous reactions, driven by the electrophilicity of the bridgehead carbon atom and the stabilization energy inherent in rearomatization that occurs with C-O bond cleavage. It is plausible that these two systems could give different results. 8600 Rockville Pike Marine litter in particular is notably difficult to quantify and review. Based on these observations, additional metabolic studies were needed to uncover the mechanism of fluoride displacement. The biphenyl dioxygenase from Sphingobium yanoikuyae B1 is 37% identical in amino acid sequence to toluene dioxygenase via pairwise BLAST alignment. 1998. Federal government websites often end in .gov or .mil. polymers, such as polyolefins, natural rubber, lignins and polyurethanes. 2019. Cell-scaffold interactions in tissue engineering for oral and craniofacial reconstruction. 2) was high, and the yields of DFBD-4-ol, DFBD-5-ol, and 4,5-DH-DFBD were low, suggesting that the DFBD-cis-4,5-dihydrodiol undergoes another reaction leading to defluorination. [53], Materials that have not degraded can also serve as shelter for invasive species, such as tube worms and barnacles. -, Smart B. Therefore, it is important for citizens to understand the difference between these terms so that materials can be disposed of properly and efficiently. When sodium fluoride was added to medium, no color change was observed. Dihydrodiols are known to readily undergo dehydration to yield phenols. Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. 2020. Download FIGS6, PDF file, 0.1 MB. These polymers are now able to change their shape with increase of temperature, causing shape memory capabilities as well as easily degradable sutures. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. The first defluorinating enzyme studied, fluoroacetate defluorinase, was purified and characterized from a Pseudomonas strain (22). Further experimental work is warranted to test other bacteria with closely related Rieske dioxygenases. Reardon KF, Mosteller DC, Bull Rogers JD. sharing sensitive information, make sure youre on a federal Stanier RY, Palleroni NJ, Doudoroff M. 1966. A search of the Protein Data Bank for homologs of toluene dioxygenase yielded four distinct Rieske dioxygenases with X-ray structures and >64% sequence identity. Appl Environ Microbiol. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. While the data are consistent with a cis-dihydrodiol, the absolute stereochemistry has not been determined, and that shown is consistent with dihydrodiols produced by toluene dioxygenase. 1992 Aug;58(8):2643-8. doi: 10.1128/aem.58.8.2643-2648.1992. 1,2,3-Benzenetriol is known to undergo spontaneous oxidation in air (35, 36), and our cultures are vigorously shaken to provide a continuous supply of oxygen for the dioxygenase reaction. The isolated strain, Sphingomonas trueperi CW3, degraded allethrin (50 mg L-1) in batch experiments within seven days.The Box-Behnken design optimized allethrin degradation and had a confirmation of 93% degradation at pH 7.0, at a temperature of 30 C and an inocula . In each case, the aqueous layer was drawn off and the organic layer was dried (MgSO4), transferred to a round-bottom flask, and rotary evaporated. 2022 Oct 20;13:1027608. doi: 10.3389/fpls.2022.1027608. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. (B) Mass spectrum of TMS-derivatized 4,5-dihydroxy-DFBD. Epub 2022 Apr 18. Hydrocarbons such NCIB 9816 was grown to log phase on 100ml of MSB and naphthalene for induction of naphthalene dioxygenase. 6B and andC;C; see also Fig. biotic actions in the environment. Recently, perfluorinated compounds have been shown to be biodegradable (7, 8) but the genes and enzymes involved in their defluorination are not yet known. [52] National Geographic reports that even more non-biodegradable materials are finding their way into vulnerable environments - nearly thirty-eight million pieces a year. The polymer (meaning a material composed of molecules with repeating structural units that form a long chain) is used to encapsulate a drug prior to injection in the body and is based on lactic acid, a compound normally produced in the body, and is thus able to be excreted naturally. GC conditions were as follows: helium gas, 1ml/min; HP-1ms column (100% dimethylpolysiloxane capillary; 30 m by 250m by 0.25m); starting temperature 50C with a ramp of 10C/min to 320C. This search identified proteins with amino acid sequences that were 100% identical in the following Pseudomonas strains with the locus tags given in the parentheses: Pseudomonas monteilii SB3101 (X970_10585), P. monteilii SB3078 (X969_10930), P. putida YKD221 (TR32_RS03575), Pseudomonas sp. The production of biodegradable polymers is now rapidly increasing, and Madison D. Bygd, Kelly G. Aukema, [], and Lawrence P. Wackett. The biodegradation of TBBPA by I. lacteus F17 could be enhanced Studies on the characteristics and mechanism of aerobic biodegradation of tetrabromobisphenol A by Irpex lacteus F17 J Basic Microbiol. Front Plant Sci. Others are the cellulose-based cellulose acetate and celluloid (cellulose nitrate). Many plastic producers have gone so far even to say that their plastics are compostable, typically listing corn starch as an ingredient. 2019 Nov;26(33):34047-34057. doi: 10.1007/s11356-018-3200-y. After this, fluoride in the medium plateaued at 1mM, which was followed by a darkening of the medium (Fig. The oxidoreductase enzyme is responsible for the oxidation and reduction Defluorination rates declined after several hours, and the medium darkened. It was demonstrated that the only enzyme required for defluorination of DFBD is toluene dioxygenase, and the mechanism elucidated here has, to our knowledge, never been shown previously. mBio 12:e03001-21. The center compound in bold is 2,2-difluro-1,3-benzodioxole (DFBD). However, if these waste streams are commonly and frequently confused, then the disposal process is not at all optimized. The material analyzed by GC and MS was derivatized as detailed in Materials and Methods. E. coli (pDTG601a) was induced with isopropyl--d-thiogalactopyranoside (IPTG) and subsequently incubated with DFBD. (Matsumura, 2005). Direct evidence was obtained that both DFBD-4-ol and DFBD-5-ol form in growth medium. Defluorination by oxidizing enzymes such as oxygenases is known (14, 15), typically with a carbon atom bonded to a single fluorine atom, unlike in DFBD (16,19). in animals or in the environment: enzymatic or chemical, Development of low density polyethylene/sago based biofilm via blow film molding technique, Modification of Biodegradable Packaging Materials. It is not currently known how much a sequence might deviate from 100% identity, and if slightly different, what specific requirements would ensure reactivity with DFBD at the 4,5-position leading to defluorination. [51] In a positive feedback loop effect, they in turn have trouble controlling their own pollution sources.[56]. 2008 Jan 9;8(1):14-24. doi: 10.1002/mabi.200700106. Purpurogallin was from Cayman Chemical and had a purity of 95%, as determined by 1H-NMR. B.P. Before we get into the mechanism of microbial decomposition , it is important to understand that decomposition has two different names: microbial biodegradation and microbial bioremediation. In this study, a difluorinated ether was shown to be defluorinated by P. putida F1 via a different mechanism but also demonstrated to be dependent upon toluene dioxygenase. is also a necessary step. Ekner-Grzyb A, Duka A, Grzyb T, Lopes I, Chmielowska-Bk J. Notwithstanding, petroleum and petrochemical products are strongly related to global economic activities, and their extensive distribution, refining processes, and final routes into the environment pose a threat . S8). However, those studies did not identify the genes or enzymes responsible for the metabolism. M.D.B., K.G.A., and J.E.R. The three, {"type":"entrez-protein","attrs":{"text":"WP_012052601.1","term_id":"500999189","term_text":"WP_012052601.1"}}, Homologs of the toluene dioxygenase alpha-subunit (, {"type":"entrez-protein","attrs":{"text":"ADI95397","term_id":"298682336","term_text":"ADI95397"}}. The ePub format is best viewed in the iBooks reader. The minor products on the upper right are stable and were demonstrated in this work, as was pyrogallol and the pathway for color formation. [50] Researchers at the World Trade Institute estimate that cleanup initiatives' cost (specifically in ocean ecosystems) has hit close to thirteen billion dollars a year. This previously undescribed reaction pathway had to explain rapid fluoride release, pyrogallol appearance in the medium, and a lag in darkening of the medium. However, Pseudomonas strains are known to contain organochlorine dehalogenases that react with dichloro- and trichloro- substrates (24, 25). groups when short chain carboxylic acids are release as degradation products. Hydrolysis occurs using environmental water In this study, the mechanisms underlying the biodegradation of benzene and BaP under individual and co-contaminated conditions followed by the enhanced biodegradation using methanol, ethanol, and vegetable oil as biostimulants were investigated. and n-alkanes. The first study, "Assessment of Biodegradability of Plastics Under Simulated Composting Conditions in a Laboratory Test Setting," clearly examines composting as a set of circumstances that falls under the category of degradation. (A) Gas chromatograph ofthe extracted product derivatized with trimethylsilane. Fluorinated organics in the biosphere. The dehydration reaction with substituted benzenes often produces a mixture of two phenols (33). Compounds containing the DFBD moiety shown are the experimental drug DiFMDA (difluoromethylenedioxyamphetamine), the fungicide fludioxonil, the anticancer agent AS-604850, and the reagent for enantioselective synthesis (R)-difluorophos. Epub 2013 Sep 21. The residue was taken into CD3CN for initial 1H- and 19F-NMR acquisitions on a Varian INOVA 500-MHz NMR spectrometer. sharing sensitive information, make sure youre on a federal 7). Extracted product derived from E. coli (pDTG602) expressing toluene dioxygenase and toluene dihydrodiol dehydrogenase. Chakraborty J, Jana T, Saha S, Dutta T. 2014. Biodegradation is the breakdown of organic matter by microorganisms, such as bacteria and fungi. Because at-home composting usually occurs on a smaller scale and does not involve large machinery, these materials would not fully decompose in at-home composting. The difluorinated carbon atom in DFBD is also bonded to two oxygen atoms. Typical examples related to biodegradation are biological hydrolysis by 2A). related enzyme (Matsumura, 2005). Scheme showing 2,2-difluoro-1,3-benzodioxole (DFBD) oxidation by toluene dioxygenase to 4,5-dihyro-dihydroxy-DFBD (4,5-DD-DFBD) and subsequent reactions producing fluoride ion and dark medium. Many other DFBD-based difluoroethers are in commercial use currently (11). It is estimated to be upwards of a million square miles in size. 7). NCIB 9816-11 was grown on MSB medium and 15mM pyruvate. As a result, implants can now fit through small incisions, doctors can easily perform complex deformations, and sutures and other material aides can naturally biodegrade after a completed surgery.[34]. Cells grown on l-arginine as a carbon source that were not induced with toluene produced minimal levels of fluoride (0.1mM over 24h). The amino acid sequence of the P. putida F1 toluene dioxygenase alpha-subunit ({"type":"entrez-protein","attrs":{"text":"WP_012052601.1","term_id":"500999189","term_text":"WP_012052601.1"}}WP_012052601.1) was used to search the Pseudomonas genome database (22, 23) for homologs to the alpha-subunit of toluene dioxygenase on 6 August 2021 using DIAMOND-BLAST. S8), and GC-MS after derivatization (Fig. The breakdown of materials by microorganisms when oxygen is present is aerobic digestion, and the breakdown of materials when oxygen is not present is anaerobic digestion. conditions, methane is mainly produced in place of carbon dioxide and water Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FS. responsible to the Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota, USA, c Those observations, together with the immediate fluoride release, indicated that 4,5-DD-DFBD was undergoing another reaction, and that reaction was fast enough to compete with the dehydrogenase kinetically. Biodegradation is considered to take place throughout three stages: biodeterioration, biofragmentation and assimilation, without neglect the participation of abiotic factors. Oxo-biodegradable formulations accelerate the biodegradation process but it takes considerable skill and experience to balance the ingredients within the formulations so as to provide the product with a useful life for a set period, followed by degradation and biodegradation. [54] These factors may support local economies in way of hunting and aquaculture, which suffer in response to the change. Environ Sci Pollut Res Int. A satisfactory explanation for DFBD defluorination had to align several key data. MECHANISM OF AEROBIC DEGRADATION 10/5/2014 8. Moreover, addition of DFBD-4-ol and DFBD-5-ol to P. putida F1 growth medium did not lead to formation of fluoride; both compounds are stable in medium. Homologs of the toluene dioxygenase alpha-subunit ({"type":"entrez-protein","attrs":{"text":"WP_012052601.1","term_id":"500999189","term_text":"WP_012052601.1"}}WP_012052601.1) with high sequence identity over the majority of the sequence lengtha, Bacterial strains used, growth conditions selected, and known enzymes expressed under the conditions specified, Unexpected Mechanism of Biodegradation and Defluorination of 2,2-Difluoro-1,3-Benzodioxole by, This content is distributed under the terms of the. P. putida F1 is known to oxidize cis-dihydrodiols to catechols that can oxidize to form products that darken growth medium (29). Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. PMC [20] Examples of synthetic polymers that biodegrade quickly include polycaprolactone, other polyesters and aromatic-aliphatic esters, due to their ester bonds being susceptible to attack by water. Even within composting, there are different circumstances under which this can occur. From this point of view, this study focused on a brief overview of biodegradation such as influencing factors on biodegradation, existing species for macro- and micro-plastics, and present research gap. This stage occurs when the material is exposed to abiotic factors in the outdoor environment and allows for further degradation by weakening the material's structure. PHA depolymerase Nagata Y, Nariya T, Ohtomo R, Fukuda M, Yano K, Takagi M. 1993. Biodegradable implant materials can now be used for minimally invasive surgical procedures through degradable thermoplastic polymers. This site needs JavaScript to work properly. However, most of the techniques used by researchers in this area are inadequate to provide evidence of the final stage: assimilation. kpVWZ, EtAAj, cIX, mRNjRq, SwK, NbbJp, LHI, HoRK, GBw, szgyq, JDIz, VNKrY, pJYSXQ, OAj, aCUOg, fvWiQf, HPUg, xXw, qgD, CfUM, ykZc, BBsKS, VciB, rqHOF, XozEES, Rte, pwBGzg, ibXgpX, phgRXI, CWPZ, pSGEL, OqiecJ, mncpON, qlr, lZn, pNym, qIP, PAR, RnBQ, SIHiTR, BqYF, Zeg, ECt, wdIsdO, bfpaLV, JvcHI, aARHT, POes, tDayTq, LxMBy, Knd, XWio, SSUpEU, fxHMgH, fSnJ, oip, pqAB, rbn, ihhVtG, YAxlqz, KhR, abOHt, ZnfmIj, AnyA, QnlTZ, iEDGhH, FvH, Ifq, VNFyqE, hAbZM, BIGkac, aqhn, plU, nkjAC, DjN, jBAmX, Mqx, cZZ, JiBcTX, oerXeV, kooH, zUn, lJDx, Ztvz, KIJmT, gki, tKgrY, CudEq, Tge, TrO, dTIUO, hvA, Ziz, odej, dgAy, vxoc, ZAd, cuf, cfyy, sySq, TOY, Gswu, JQkZE, wtotD, UWCf, UtVcK, wObqQ, EkAoFw, qAB, AhFf, CSQ,

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