• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Rhamnolipids Induced by Glycerol Enhance Dibenzothiophene Biodegradation in Burkholderia sp. C3

    2020-09-12 03:11:18CamilaOrtegaRamirezAbrahamKwanQingLi
    Engineering 2020年5期

    Camila A. Ortega Ramirez, Abraham Kwan, Qing X. Li*

    Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA

    Keywords:Biodegradation Bioremediation Biosurfactant Biotransformation Glycerol Microbial metabolism Rhamnolipid

    A B S T R A C T In highly urbanized areas, pollution from anthropogenic activities has compromised the integrity of the land,decreasing soil availability for agricultural practices. Dibenzothiophene(DBT)is a heterocyclic aromatic hydrocarbon frequently found in urbanized areas, and is often used as a model chemical to study the microbial transformation of pollutants. The potential for human exposure and its health risk makes DBT a chemical of concern; thus, it needs to be environmentally managed. We utilized glycerol to stimulate Burkholderia sp. C3 in order to degrade DBT in respect to ①DBT biodegradation kinetics,② bacterial growth, ③ rhamnolipid (RL) biosynthesis, and ④ RL secretion. Under an optimum glycerol-to-DBT molar ratio,the DBT biodegradation rate constants increased up to 18-fold and enhanced DBT biodegradation by 25%-30% at day 1 relative to cultivation with DBT alone. This enhancement was correlated with an increase in bacterial growth and RL biosynthesis. Proteomics studies revealed the enzymes involved in the upper and main steps of RL biosynthesis. The RL congeners Rha-C10-C10,Rha-Rha-C10-C10, Rha-Rha-C10-C12, and Rha-Rha-C12-C12 were identified in the medium supplemented with glycerol and DBT, whereas only Rha-C12-C12 was identified in cultures without glycerol or with RL inhibitors. The studies indicated that glycerol enhances DBT biodegradation via increased RL synthesis and bacterial growth. The results warrant further studies of environmental biostimulation with glycerol to advance bioremediation technologies and increase soil availability for agricultural purposes.

    1. Introduction

    Soil and water are essential natural resources for agricultural practices. In highly urbanized areas, pollution from anthropogenic activities has compromised the integrity of agricultural lands and the water streams,leading to decreased soil functionality and food safety concerns. Regions such as North Africa and South Asia have used more than 90% of the available land [1]. In China, the use of polluted water for soil irrigation has resulted in soil pollution [2].Soil bioremediation can help to restore land for reutilization and crop production. Bioremediation uses microbial metabolisms for the removal of contaminants from the environment [3,4].Polycyclic aromatic hydrocarbons (PAHs) are a typical class of contaminants from anthropogenic activities[2].Dibenzothiophene(DBT) is a major sulfur-containing PAH [5] and is often used as a model chemical to assess PAH soil contamination [5]. DBT is a hydrophobic compound with a water solubility of 7.9 μmol·L-1and an octanol-water coefficient of 4.44 [6]. The lipophilic nature of DBT allows it to concentrate in the environment and bioaccumulate through the food chain, giving it both a food safety and ecotoxicological risk [7]. Dose-dependent exposure of DBT to zebrafish embryos was found to disrupt cardiac function, with higher doses being associated with morphological abnormalities and mortality [6]. One study showed that DBT and its metabolites act as estrogenic compounds in T47D human breast adenocarcinoma cells [8]. The high detection frequency of DBT in sediments and urbanized areas,the potential for human exposure,and the health threat make it a chemical of concern; thus, DBT needs to be environmentally managed [5,8].

    Bioremediation of hydrophobic pollutants such as DBT is frequently limited by the low abundance of microorganisms and by poor chemical bioavailability, resulting in low biodegradation kinetics [9]. In addition to catabolic enzymes [10-12], the intentional augmentation of contaminated soil with microorganisms adept at producing biosurfactants [13,14] is a method to improve chemical solubilization and bioavailability[15,16].The production of both biosurfactants and catabolic enzymes in bacteria [17-20]suggests an evolutionary adaptation by microorganisms to overcome low substrate availability.

    Burkholderia sp.C3 is a PAH-degrading bacterium isolated from a PAH-polluted site[21].It contains dioxygenase genes responsible for degrading PAHs such as phenanthrene [22,23]. This study was designed to investigate glycerol as a co-substrate to stimulate the strain C3 to degrade DBT. Our preliminary studies indicated that glycerol can enhance the biodegradation of DBT, unlike other substrates tested(e.g.,glucose).Apparent DBT solubilization,foam formation, and earlier DBT degradation by C3 were observed during the cultivation process when glycerol was supplemented.This was not observed in cultures supplemented with DBT alone.Such a difference suggested the secretion of a surfactant agent.Glycerol readily enters into the lipid metabolic pathway of β-oxidation and de novo fatty acid synthesis (FAS II), affecting the production of rhamnolipid (RL) biosurfactants and polyhydroxyalkanoic acids (PHAs) [24,25]. There have been few reports on RLs produced by Burkholderia sp.To our knowledge,this is the first report on a direct association between RL biosynthesis and DBT biodegradation induced by glycerol. Our studies indicated that biostimulation with glycerol can lead to the effective degradation of DBT in the environment by increasing the bioavailability of hydrophobic contaminants. The technology investigated in the present study may therefore be applicable to the bioremediation of hydrophobic contaminants such as pesticides.

    2. Materials and methods

    2.1. C3 cultivation and DBT biodegradation

    Test tubes were baked at 450 °C for 3 h. DBT dissolved in acetone was placed in a test tube, followed by complete evaporation of acetone under nitrogen(N2)gas.Next,5 mL of minimal medium(MM) [26] and an appropriate volume of 50% aqueous glycerol solution were added. The final concentration of DBT was 0.54 mmol·L-1(100 ppm (1 ppm = 10-6)), while the final concentration of glycerol was 0,0.05,0.5,5,50,200,or 500 mmol·L-1.C3 cells grown overnight in Luria-Bertani (LB) rich medium at 30 °C were washed three times with MM and adjusted to an optical density at 600 nm (OD600) of 0.5 in MM, of which 0.5 mL were inoculated to each of the tubes at a concentration of approximately 0.05 OD600. Cultures with glycerol but without DBT were also prepared. In the RL biosynthesis inhibition experiments, the final concentration of each 2-bromohexanoic acid (HEX) and 2-bromooctanoic acid (OC) was 2 mmol·L-1. Cultures were incubated in a rotary shaker at 30°C at 200 revolutions per minute.Autoclaved C3 cells were used as controls.

    2.2. Extraction and analysis of DBT

    DBT was extracted and analyzed according to Ref.[27].To summarize, after the culture was acidified to approximately pH 2-3 with HCl, DBT was extracted with ethyl acetate three times. The DBT was then analyzed on an Agilent 1100 series highperformance liquid chromatograph(HPLC)equipped with an Aqua C18 column(150 mm×4.60 mm,5 μm particle size;Phenomenex,Inc., USA) and detected at 245 nm. The mobile phase was 60%aqueous acetonitrile (ACN).

    2.3. Data calculation

    Time course points were drawn with standard errors of the mean bars representing variations among three or six biological replicates.Degradation curves were fitted with a first-order kinetic equation, C =C0×e-kt, where k is the DBT biodegradation rate constant and C is the concentration measured at time t, and C0is the initial concentration (Table 1). The DBT half-life (t1/2) was calculated using t1/2=(ln 2)/k. Statistical tests were done with IBM SPSS Statistics 19. The tests performed were Tukey’s honestly significant difference (HSD), least significant difference (LSD), and Bonferroni.

    2.4. Protein extraction

    C3 cells were collected from cultures at day 2 and washed three times with filtered and sterilized distilled water followed by protein extraction, according to the reported method [28] with slight modifications. A lysis buffer was prepared by mixing 9 mL of a 9 mol·L-1urea solution with 1 mL of 10× protease inhibitor solution.Protease inhibitor solution was prepared from Sigma fast protease inhibitor tablets (Sigma-Aldrich, USA). After complete removal of the medium by centrifugation at 5850g(g=9.8 m·s-2),cell pellets were re-suspended in 700 μL of lysis buffer. This cell suspension was added to 300 μL of lysis buffer solution in a screw-cap vial that was 2:3 prefilled with 0.5 mm(diameter)glass beads(BioSpec Products,USA).Cell membranes were disrupted by six cycles of bead-beating at maximum speed for 60 s on a minibeadbeater (BioSpec Products, USA) and on ice for 60 s. After cell debris removal by centrifugation at 20 820g for 15 min,the supernatant was run through an Amicon Ultra-0.5 mL centrifugal filter(3 K cutoff; Millipore, USA) to concentrate proteins on the filter.The filter was then washed with 500 μL of Milli-Q (mQ) water.

    2.5. Protein sample preparation for liquid chromatography mass spectrometry analysis

    A protein amount of 36 μg was loaded onto a 12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie blue to visualize the protein bands. Each gel lane was fractionated (approximately 1 mm3)and washed with 25 mmol·L-1ammonium bicarbonate (NH4-HCO3)/50% ACN until the pieces became clear. Gel pieces were dehydrated with 100% ACN prior to dithiothreitol reduction at 56°C for 30 min and iodoacetamide alkylation at ambient temperature for 20 min. In-gel protein digestion was done with Trypsin/Lys-C Mix (Mass-Spec Grade, Promega, USA) at 37 °C for 16-18 h. Protein digests were desalted and concentrated with Pierce C18 tips (Thermo Scientific, USA), and then analyzed on a Bruker nanoLC-amaZon speed ion trap mass spectrometer (MS) system.The peptides were separated on a C18 analytical column (0.1 mm×150 mm,3 μm,200 ?,Bruker,USA)with a gradient elution from 5%to 65%ACN in 0.1%formic acid for 80 min after a 2 min running delay. After 90 min of elution, the mobile phase was changed to 95% ACN containing 0.1% formic acid and remained as such for 10 min,followed by column equilibration with 5%ACN containing 0.1% formic acid for 20 min for the next run. The flow rate was 800 nL·min-1. The MS parameters were set at a capillary voltage of 1600 and a capillary temperature of 149.5°C.A survey scan from mass-to-charge ratio (m/z) 400 to 3000 was followed by datadependent tandem mass spectrometry (MS/MS) of the 10 most abundant ions and 0.5 Da instrument error. Dynamic exclusion was set to repeat the same precursor ion twice, followed by excluding it for 0.8 min.

    2.6. Protein database and database search

    Raw files (file type BAF) were converted to mascot generic format (.mgf) with DataAnalysis software (Bruker, USA). The peakpicking algorithm was Apex. The absolute intensity threshold was set to 100.

    Table 1 Enhancement of biodegradation rate constant and half-life of DBT is dependent upon glycerol concentrations.

    The canonical and isoform protein sequence(492 entries)database was in FASTA format and was downloaded from the UniProt Knowledgebase(4 April 2016 at 9:35 a.m.).The database was constructed by using Burkholderia (UniProt taxonomy: 32008) as the fixed search term and the different protein names as the variable terms. The database search was performed with the MyriMatch search engine [29]. The configuration was as follows: instrument type as ion trap, precursor mass as auto, enzyme as trypsin/P(allowing identification of Trypsin/Lys-C Mix digests),average precursor tolerance 1.5 m/z, fragment tolerance at 0.5 m/z, and mono precursor tolerance at 10 ppm. The modifications were as follows: carbamidomethyl (fixed) and methionine oxidation(variable).

    2.7. Data normalization

    Spectral counts of the proteins identified in each treatment were obtained with IdPicker software.The treatments were as follows: (A) 0.54 mmol·L-1DBT; (B) 50 mmol·L-1glycerol; (C)50 mmol·L-1glycerol and 0.54 mmol·L-1DBT;and(D)50 mmol·L-1glycerol, 0.54 mmol·L-1DBT, and 2 mmol·L-1OC. The filters to match the peptide to its MS/MS spectra were as follows: a maximum false discovery rate(FDR)of 1%and a minimum of one spectrum per peptide and per match. In order to match peptides to proteins, a minimum of two distinct peptides and spectra and two minimum additional peptides were allowed [30,31]. Data were normalized according to the assumption in Ref.[32]—namely,that MS/MS intensities are equal to 1 and without considering the peptide length.A log(normalizedcount+1)was applied and the normalized data was compared with the raw data.An analysis of variance(ANOVA,p-value <0.05)was done to search for protein abundance changes with statistical significance using DEseq[33]. The p-value and log Fold change (FC) of the glycerol×DBT interaction term were calculated and are shown for each protein in Table 2. The log FC represents the effect size of the glycerol×DBT interaction term. When the log FC was upregulated, it indicated that the glycerol×DBT interaction had a positive effect on the protein abundance of the treatments that contained this interaction (i.e.,treatments (C) and (D)).

    2.8. RL extraction and quantification

    RLs were extracted according to Ref. [34]. To summarize, cells were removed at day 2 by centrifugation at 5850g for 10 min and filtration with a 0.2 μm pore size. The medium was extracted with ethyl acetate three times. The extracts were combined and dried under a gentle stream of N2gas, followed by re-suspending the residues in 0.5 mL of methanol. The same procedure was applied to an RL standard (50 μg·mL-1) dissolved with 5 or 50 mmol·L-1glycerol in MM. RLs were quantified with an orcinol assay [35] (0.19% orcinol (w/v) in 53% sulfuric acid (v/v). A 250 μL aliquot of the sample was collected, dried, and resuspended in 250 μL of water.To 100 μL of RL extract,or to varying concentrations of a RL standard, a 900 μL aliquot of the orcinol solution was added. Mixtures were incubated for 30 min at 80 °C. Absorbance was read at 421 nm. An RL standard curve was prepared in a range from 0 to 500 μg·mL-1. The remaining 250 μL re-suspended in methanol was used for HPLC fractionation on an Agilent 1100 series HPLC (Agilent Technologies, USA)equipped with an Aqua C18 column (150 mm × 4.60 mm, 5 μm particle size; Phenomenex, Inc., USA) according to Ref. [34]. Two RL fractions were collected at 4-5 min (F1) and 5-6 min (F2) and dried to completion at 45 °C. Fractions were re-suspended in 10 μL of 10%ACN/water and desalted with Pierce C18 tips(Thermo Scientific, USA). The manufacturer protocol was modified as follows: Trifluoroacetic acid was not used. F1 and F2 samples were bound to the C18 tip ten times, washed five times with 5% ACN,and eluted with 10 μL of 70% ACN. Samples were fully dried at 45°C and re-suspended in 2 μL of 50%ACN containing 40 mg·mL-1of 2,5-dihydroxybenzoic acid (DHB) matrix. A total of 1 μL of matrix was spotted on the target plate and air dried, and then 1 μL of sample/matrix mixture was spotted.

    2.9. RL identification

    RL congeners were identified on a matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF) ultraflexIIImass spectrometer (Bruker, USA) operated in positive, reflectron mode following the published procedures[36].The instrument was calibrated with the degree of polymerization (DP) series: maltotriose hydrate (DP3, MW: 504.44 g·mol-1), maltotetraose (DP4, MW:666.57 g·mol-1), maltopentaose (DP5, MW: 828.71 g·mol-1), and maltohexaose(DP6,MW:990.85 g·mol-1)[37].A 500 μmol·L-1aliquot of each DP series was dissolved in mQ water.A total of 5 μL of each DP series was mixed with 20 μL of DHB matrix to a final concentration of 62.5 μmol·L-1. A 2 μL aliquot of this mixture was spotted on a MALDI target plate and allowed to air dry.The instrument m/z range was 300-1200 with 50 pulsations per laser shot and 38%-50% laser intensity. Ions were suppressed below 300 Da. Flexi Analysis and Compass Isotope Pattern software(Bruker,USA) were used for mass spectral analysis. RL peaks were assigned using an in silico database created according to the RLs reported [38], with a 0.5 Da error mass.

    An Agilent 6520A liquid chromatograph-quadrupole time-offlight mass spectrometer (LC-Q-TOF-MS) system (Agilent Technologies, Canada) was used for the MS/MS analysis of samples.The RL sample (20 μL) was separated on a Luna C18 column(100 mm × 2.0 mm, 3 μm particle size) (Phenomenex, Inc., USA)in gradient starting at 5% ACN in 5 mmol·L-1aqueous ammonium formate buffer (pH 3.3) to 95% ACN in 15 min, and then held for15 min.The mobile phase flow rate was 0.3 mL·min-1.The column was equilibrated for 10 min between runs.The electrospray ionization interface was set at the negative mode. The capillary voltage was 4000 V. The fragmentor and skimmer voltages were 180 and 80 V, respectively. When the system was in the MS/MS mode,the drying and nebulizing gas was nitrogen, while helium was the collision gas.The gas temperature was 350°C.The dry gas flow rate was 10 L·min-1. The nebulizer pressure was 25 psi (1 psi =6.895 kPa). Full-scan data were acquired via scanning from m/z 50-1700. The collision energy in the MS/MS mode was 20 eV.

    Table 2 Proteins identified mediating RL biosynthesis in Burkholderia sp. C3 at day 2 of incubation.

    3. Results and discussion

    3.1. Biostimulation with glycerol enhances DBT biodegradation and Burkholderia sp. C3 growth

    This study was designed to follow a biostimulation strategy to investigate the influence of glycerol on the DBT biodegradation ability of Burkholderia sp. C3. Co-substrate experiments with DBT and glycerol exhibited neither a carbon catabolite repression phenomenon nor an antagonistic effect, which has been reported for other co-substrate mixtures [16,39,40]. The results indicate that biostimulation with glycerol supports C3 cell growth while enhancing DBT biodegradation, as shown in Fig. 1. The experiments show that the enhancement of DBT biodegradation was dependent upon the molar ratios of glycerol to DBT used and on the incubation time. Significant differences were observed when the culture was stimulated with 0.5, 5, 50, and 200 mmol·L-1of glycerol(Tukey HSD;LSD;Bonferroni;p <0.001).At these concentrations, glycerol enhanced the DBT biodegradation by 25%-30%after one day of incubation.In cultures at a glycerol(50 mmol·L-1)-to-DBT molar ratio of 92.6:1, the strain C3 degraded 100% of 0.54 mmol·L-1(100 ppm)DBT after seven days of incubation.This optimal glycerol concentration was used for the proteomics and mass spectrometry experiments. At this concentration, the DBT half-life decreased to the lowest—from 27.5 to 1.5 days—and the DBT degradation rate constant showed an 18-fold increase(Table 1). Negligible degradation was observed in the control cultures with autoclaved bacteria, as shown by the negative fold change in Table 1. Similar to the DBT biodegradation kinetics at 50 mmol·L-1glycerol supplementation, C3 degraded 92% of 0.54 mmol·L-1DBT at day 10 in cultures containing 0.5 mmol·L-1glycerol. However, at this glycerol concentration, the C3 growth remained at 0.05 OD600, which indicated that the increase in biomass was not the only cause for the enhanced DBT biodegradation,and that additional molecular mechanisms were involved.Statistical analysis showed no significant difference in DBT biodegradation between days 7 and 10 (Tukey HSD; LSD; Bonferroni;p < 0.05). Therefore, seven days would be recommended for biodegradation under the conditions tested.

    DBT as a sole carbon source did not support strain C3 cell growth.The OD600remained at 0.05 after a 10-day incubation period,which was equal to the initial inoculum(Fig.1).PAH biodegradation in liquid cultures with a single carbon source depends on the bacterial strain, PAH structures, and PAH concentrations. For example, the strain C3 at day 7 degraded 94% of 40 ppm DBT[22]. Our results indicate that C3 degraded 11%-12% of 100 ppm DBT at day 7. At this concentration, DBT had no apparent benefit to the bacteria, and even a decrease in biodegradation efficiency was observed. This finding was supported by the fact that C3 cell growth in glycerol-DBT mixtures was inhibited in comparison with growth in glycerol alone. For example, mixtures of 0.54 mmol·L-1DBT with glycerol ranging from 50 to 500 mmol·L-1exhibited reduced growth, up to 0.3 OD600relative to glycerol alone, and started after one day of cultivation. DBT did not inhibit growth at lower concentrations of glycerol (0.05-5 mmol·L-1). Growth inhibition with 50 to 500 mmol·L-1of glycerol may be associated with the toxicity of DBT, DBT metabolites, or both. It was reported that PAHs’ hydrophobicity and carcinogenicity influence their toxicity[16,41,42].

    3.2. The RL biosynthetic pathway is activated in Burkholderia sp. C3

    Glycerol is a good carbon source for RL production in Pseudomonas species [38]. It can be metabolically assimilated into the lipid pathways affecting PHA granule formation and RL production[24,38,43,44]. Table 2 lists the relative abundance of the enzymes detected in four treatments—namely (A) 0.54 mmol·L-1DBT; (B)50 mmol·L-1glycerol;(C)50 mmol·L-1glycerol and 0.54 mmol·L-1DBT; and (D) 50 mmol·L-1glycerol, 0.54 mmol·L-1DBT, and 2 mmol·L-1OC—along with the log FC and p-value.These enzymes are responsible for RL biosynthesis,as shown in Fig.2.Biosynthesis of RLs requires an R-3-hydroxydecanoyl-ACP or -CoA lipid precursor produced from the FAS II[45,46]and/or β-oxidation[24]pathways, respectively, and a dTDP-L-rhamnose sugar precursor [43].Once the lipid and sugar precursors are produced,RhlABC mediates the formation of mono- or di-RLs. The anabolic enzymes AlgC and RmlBCD involved in dTDP-L-rhamnose biosynthesis were detected in the four treatments (Fig. 2, Table 2). RmlD catalyzes the final step from dTDP-4-keto-6-deoxy-L-mannose to dTDP-rhamnose.The changes in the relative protein abundance of RmlD in the different treatments were significant (p <0.05), and the log FC was upregulated.The log FC represents the effect of the glycerol×DBT interaction term. Its upregulation indicates that this interaction had a positive effect on the treatments containing this term. The enzyme RmlC that catalyzes the previous step in the pathway was also upregulated. The relative abundance of both RmlD and RmlC suggests that the synthesis of RL sugar precursors is not inhibited by the RL biosynthesis inhibitor OC.

    Fig.1. DBT biodegradation kinetics by Burkholderia sp.C3 and its growth over a 10-day cultivation period with 0.54 mmol·L-1 DBT and different glycerol supplementation(0,0.05, 0.5, 5, 50, 200, and 500 mmol·L-1).

    Enzymes from the FAS II and/or β-oxidation pathways were detected in the four treatments (Fig. 2, Table 2). FabG is the enzyme catalyzing the final step in R-3-hydroxydecanoyl-ACP synthesis via the FAS II pathway [46].The relative abundance of FabG was >1 in all treatments,indicating that this protein was detected.However, the p-value was above 0.05, meaning that their relative abundances did not show significant differences(Table 2).The preceding step catalyzed by FabF was upregulated in treatment (C).The β-oxidation enzymes FadB and FadE were abundant in treatment (C) (Table 2). The results indicate that the later stages in the FAS II and β-oxidation pathways are active in C3 at day 2 of cultivation.

    RhlA produces 3-hydroxy alkanoic acid (HAA), which is then utilized by RhlB for mono-rhamnolipid biosynthesis [47]. RhlA was identified and the log FC showed upregulation. Rhamnosyltransferases RhlB and RhlC were also identified (Table 2). RhlB and RhlC are involved in the direct formation of monorhamnolipids [48,49] and di-RLs [50], respectively. The proteins identified in Table 2 suggest that glycerol induces lipid precursors for the biosynthesis of RL biosurfactants in C3 cells. The data suggest the involvement of both the FAS II and β-oxidation pathways in the synthesis of the lipid precursor. However, it is uncertain which pathway dominates, as these pathways have additional roles in the cells, such as cell proliferation.

    Fig.2. Identification of proteins involved on RL biosynthesis in Burkholderia sp.C3.Bolded proteins showed upregulation in the glycerol×DBT interaction term(log FC).FadB: enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; FadE: acyl-CoA dehydrogenase;FabF:β-oxoacyl-ACP synthase 2;ACP:acyl carrier protein;FabG:3-oxoacyl-ACP-reductase; RhlA: HAA synthase; HAA: 3-hydroxy alkanoic acid; RhlB:rhamnosyltransferase 1; RhlC: rhamnosyltransferase 2; AlgC: phosphoglucomutase/phosphomannomutase; RmlB: dTDP-glucose 4,6-dehydratase; RmlC: dTDP-4-dehydrorhamnose-3,5-epimerase; RmlD: dTDP-4-dehydrorhamnose reductase.

    3.3. Enhanced DBT biodegradation in Burkholderia sp. C3 is affected by RL biosynthesis and its inhibition

    DBT is hydrophobic [6] and its bioavailability is the first requirement for biodegradation [15]. DBT solubilization and foam formation were observed in the cultures when glycerol was supplemented.Such observations suggest the secretion of a surfactant agent. Our proteomics results indicate that the RL biosurfactant biosynthesis pathway is active in C3. Thus, the relevance of RLs to the enhanced DBT biodegradation induced by glycerol was investigated with an orcinol assay [51].

    Fig. 3. Association between (a) the amount of RL secreted at day 2 and (b) the amount of DBT degraded at day 1 by Burkholderia sp. C3 cultivated without inhibitor, with 2 mmol·L-1 of HEX or 2 mmol·L-1 of OC and different glycerol concentrations (0, 0.05, 0.5, 5, 50, and 200 mmol·L-1).

    Fig. 4. HPLC chromatograms of RLs secreted by Burkholderia sp. C3 cultivated with (a) 0.54 mmol·L-1 DBT and 50 mmol·L-1 glycerol, with (b) 2 mmol·L-1 of HEX, or(c)2 mmol·L-1 of OC,and cultivated with(d)0.54 mmol·L-1 DBT,with(e)2 mmol·L-1 of HEX,or(f)2 mmol·L-1 of OC.(g)MALDI-TOF-MS of 4 RL congeners(M1,M2,M3,and M4) identified from HPLC chromatogram a, fraction 2 (F2), and (h) LC-Q-TOF-MS2 of Rha-C10-C10 (M1).

    Gutierrez et al. [25] showed that inhibition of RhlA by bromoalkanoic acids suppresses the production of RLs and PHA in Pseudomonas species, and that this inhibition is dependent on the bromoalkanoic acid used. Inhibition of RLs and PHA has also been described in the literature[24].2-Bromoalkanoic acids(HEX or OC)were therefore used to probe the roles of RLs in DBT biodegradation by the strain C3. If RLs play a role in DBT biodegradation,HEX and OC should decrease DBT biodegradation via RL biosynthesis inhibition.An increase in RL secretion was quantified when the strain C3 was cultivated in the glycerol/DBT mixture relative to cultivation in DBT alone. RL biosynthesis and secretion, induced by varying concentrations of glycerol (Fig. 3(a)), were strongly associated with the amount of DBT degraded (Fig. 3(b)), as was RL biosynthesis inhibition by HEX and OC.The results agreed with the proteomics findings; RL biosynthesis occurs in C3 and is strongly associated with its biodegradation ability.

    3.4. RL congeners are secreted by Burkholderia sp. C3

    An RL standard containing a mixture of Rha-Rha-C10-C10 and Rha-C10-C10 congeners was used to identify RLs in experimental samples. RL congeners were eluted between 4 and 6 min (Fig. 4).The HPLC chromatograms of the experimental samples matched well with those of the RL standards (data not shown). Two fractions named F1 and F2 were collected from the experimental samples with 0.5 mmol·L-1DBT and either 50 or 0 mmol·L-1glycerol.Fractions from the cultures treated with 2 mmol·L-1of HEX or OC inhibitors were also collected. The peak intensity on the HPLC chromatogram of the extract from the 50 mmol·L-1glycerolsupplemented culture was approximately 10-fold higher than that on the 0 mmol·L-1glycerol HPLC chromatogram.Glycerol induced an increase in RL production. It is noteworthy that multiple peaks occurred at the OC chromatograms (Figs. 4(c) and (f)).

    The most intense peaks were assigned to RL congeners. In the 50 mmol·L-1glycerol samples, C3 secreted the congeners Rha-C10-C10 (M1 + Na+), Rha-Rha-C10-C10 (M2 + Na+), Rha-Rha-C10-C12—which were identified in sodium (M3 + Na+) or potassium(M3 + K+) adduct ion forms—and Rha-Rha-C12-C12 (M4 + Na+).The mono-rhamnolipid congener Rha-C12-C12 was identified in the 0 mmol·L-1glycerol samples and in the samples with HEX or OC inhibitors (either in 50 or 0 mmol·L-1glycerol) (data not shown). The peaks M1 and M2 were confirmed with MS/MS. The mass spectrum of Rha-C10-C10 is shown in Fig. 4(h). Loss of C10 acyl chain and rhamnose sugar fragments (Rha) were observed. It is concluded that glycerol supports C3 cell growth and enhances the biodegradation of DBT via an RL-mediated mechanism.

    Our findings suggest that the utilization of glycerol could lead to the effective biodegradation of persistent organic pollutants—including organochlorine pesticides and PAHs—in contaminated soil and could thus be used in the restoration of land functionality,making it available for agricultural purposes. This biostimulation strategy could be applied in combination with the bioaugmentation of a suitable microbial consortium. Our findings suggest that glycerol may trigger a superior response in a bacterial population with a mixture of PAH degraders and RL producers.An initial analysis of the bacterial population and cellular respiration,along with PAH biodegradation monitoring over time,would be required for a proper assessment of the bioremediation site.

    4. Conclusions

    The amphipathic properties of RLs suggest multiple functions for RL producers and PAH degraders. Some of the functions are related to hydrophobic chemical solubilization, chemical uptake,and biological assimilation. Our studies indicated a 30% increase in DBT biodegradation with glycerol biostimulation after one day of incubation. This enhancement was associated with increased di-RL biosynthesis by Burkholderia sp. C3 and bacterial growth. In addition,we report the use of 2-bromoalkanoic acid for RL biosynthesis inhibition in a Burkholderia species.To our knowledge,this is the first report correlating glycerol supplementation to DBT biodegradation and RL biosynthesis.The results suggest a potential practicality of the biostimulation strategy described here to improve bioremediation efficiencies and facilitate such processes.

    Acknowledgements

    This work was supported in part by Grant N00014-12-1-0496 from the Office of Naval Research and a subcontract with the Western Center for Agricultural Health and Safety (NIOSH grant 2U54OH007550). The authors thank Dr. Margaret R. Baker for her assistance with MALDI/TOF.

    Compliance with ethics guidelines

    Camila A. Ortega Ramirez, Abraham Kwan, and Qing X. Li declare that they have no conflict of interest or financial conflicts to disclose.

    Nomenclature

    ACN acetonitrile

    CoA coenzyme A

    FabZ 3-hydroxy-ACP-dehydratase

    FDR false discovery rate

    HEX 2-bromohexanoic acid

    HPLC high-performance liquid chromatograph

    HSD honestly significant difference

    LSD least significant difference

    DHB dihydroxybenzoic acid

    DP degree of polymerization

    OC 2-bromooctanoic acid

    DBT dibenzothiophene

    FAS II de novo fatty acid synthesis

    FadB enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase

    FadE acyl-CoA dehydrogenase

    FabF β-oxoacyl-ACP synthase 2

    ACP acyl carrier protein

    FabG 3-oxoacyl-ACP-reductase

    RhlA HAA synthase

    HAA 3-hydroxy alkanoic acid

    RhlB rhamnosyltransferase 1

    RhlC rhamnosyltransferase 2

    AlgC phosphoglucomutase/phosphomannomutase

    RmlB dTDP-glucose 4,6-dehydratase

    RmlC dTDP-4-dehydrorhamnose-3,5-epimerase

    RmlD dTDP-4-dehydrorhamnose reductase

    MALDI/TOF matrix-assisted laser desorption ionization time-offlight

    LC-Q-TOF-MS liquid chromatograph-quadrupole time-of-flight mass spectrometer

    MM minimal medium

    MS mass spectrometry

    PAH polycyclic aromatic hydrocarbon

    PHA polyhydroxyalkanoic acid

    RL rhamnolipid

    成人午夜高清在线视频| 叶爱在线成人免费视频播放| 岛国在线免费视频观看| 中文亚洲av片在线观看爽| 国产亚洲av高清不卡| 美女大奶头视频| 舔av片在线| 后天国语完整版免费观看| 一本精品99久久精品77| 日韩中文字幕欧美一区二区| 深夜精品福利| 免费看十八禁软件| 伦理电影免费视频| 亚洲成人免费电影在线观看| 国产成年人精品一区二区| 欧美成人一区二区免费高清观看 | 亚洲美女视频黄频| 国产精品久久久久久人妻精品电影| 国产视频内射| 色综合婷婷激情| 老熟妇仑乱视频hdxx| 免费电影在线观看免费观看| 最近最新免费中文字幕在线| 丰满人妻熟妇乱又伦精品不卡| 国产免费男女视频| 国产成人系列免费观看| 久久久久久久精品吃奶| 国产精品 欧美亚洲| 岛国在线观看网站| 国产伦精品一区二区三区视频9 | 麻豆成人午夜福利视频| 啦啦啦观看免费观看视频高清| 一二三四在线观看免费中文在| 国产又色又爽无遮挡免费看| 嫩草影院入口| 国产精品永久免费网站| 成年版毛片免费区| 国产真人三级小视频在线观看| 日本免费a在线| 老司机午夜十八禁免费视频| 脱女人内裤的视频| 国产爱豆传媒在线观看| 亚洲av电影在线进入| 国产麻豆成人av免费视频| 成人欧美大片| 日本五十路高清| 淫秽高清视频在线观看| 最新在线观看一区二区三区| 亚洲国产精品合色在线| 国产蜜桃级精品一区二区三区| 91久久精品国产一区二区成人 | 国内精品久久久久精免费| 精品国产超薄肉色丝袜足j| 国产欧美日韩一区二区三| 久久精品91无色码中文字幕| 午夜久久久久精精品| 在线a可以看的网站| 九九久久精品国产亚洲av麻豆 | 9191精品国产免费久久| www日本黄色视频网| 91在线精品国自产拍蜜月 | 不卡av一区二区三区| 人人妻人人澡欧美一区二区| 成在线人永久免费视频| 成人三级做爰电影| 黑人巨大精品欧美一区二区mp4| 婷婷六月久久综合丁香| 禁无遮挡网站| 在线国产一区二区在线| 亚洲成人精品中文字幕电影| 啪啪无遮挡十八禁网站| 国产人伦9x9x在线观看| 午夜视频精品福利| 久久久久久久午夜电影| 首页视频小说图片口味搜索| 国产伦一二天堂av在线观看| 久久精品夜夜夜夜夜久久蜜豆| 老鸭窝网址在线观看| 日韩欧美在线二视频| 国产精品一区二区精品视频观看| 热99在线观看视频| 免费看十八禁软件| 国产高清三级在线| 亚洲中文字幕一区二区三区有码在线看 | 久久精品91无色码中文字幕| 看免费av毛片| 黄色 视频免费看| 亚洲精品国产精品久久久不卡| 精品熟女少妇八av免费久了| 看片在线看免费视频| 男人舔奶头视频| 九九热线精品视视频播放| 成年人黄色毛片网站| 国产成人av激情在线播放| 身体一侧抽搐| 19禁男女啪啪无遮挡网站| 美女 人体艺术 gogo| 黑人操中国人逼视频| 亚洲成人精品中文字幕电影| cao死你这个sao货| 久久久久久久久免费视频了| 午夜福利高清视频| 亚洲精品中文字幕一二三四区| 久久精品国产99精品国产亚洲性色| 免费在线观看视频国产中文字幕亚洲| 亚洲激情在线av| 国产亚洲av嫩草精品影院| 国内精品久久久久精免费| 18禁观看日本| 综合色av麻豆| 欧美一级毛片孕妇| 亚洲国产高清在线一区二区三| 国产欧美日韩精品亚洲av| 99久久国产精品久久久| 欧美不卡视频在线免费观看| 亚洲av免费在线观看| 久久久国产成人免费| 人人妻人人看人人澡| 村上凉子中文字幕在线| 亚洲国产精品成人综合色| 国产av一区在线观看免费| 国产99白浆流出| 国产精品日韩av在线免费观看| 18禁黄网站禁片午夜丰满| 9191精品国产免费久久| 一进一出好大好爽视频| 一级作爱视频免费观看| 欧美精品啪啪一区二区三区| 免费av毛片视频| 国产欧美日韩一区二区精品| 免费看光身美女| 国产精品久久久人人做人人爽| 久久久色成人| 欧美日韩国产亚洲二区| 97超视频在线观看视频| 免费在线观看亚洲国产| 国产成人精品久久二区二区91| 国内久久婷婷六月综合欲色啪| 一区二区三区激情视频| 国产精品av久久久久免费| 免费看日本二区| 网址你懂的国产日韩在线| 日韩欧美一区二区三区在线观看| 亚洲人成电影免费在线| 国产精品国产高清国产av| 久久这里只有精品中国| 69av精品久久久久久| 老司机福利观看| 一个人看的www免费观看视频| 国产精品久久久久久精品电影| 18美女黄网站色大片免费观看| 国产成人啪精品午夜网站| 亚洲成人免费电影在线观看| 久久久久久国产a免费观看| 国产69精品久久久久777片 | 操出白浆在线播放| 在线十欧美十亚洲十日本专区| 他把我摸到了高潮在线观看| 麻豆国产av国片精品| 9191精品国产免费久久| 免费电影在线观看免费观看| 久久精品国产99精品国产亚洲性色| 国产精品98久久久久久宅男小说| 国产一区二区三区在线臀色熟女| 最近最新中文字幕大全电影3| 日韩人妻高清精品专区| 中亚洲国语对白在线视频| 国内精品久久久久精免费| 最好的美女福利视频网| 18禁裸乳无遮挡免费网站照片| 亚洲av中文字字幕乱码综合| 国产欧美日韩一区二区三| 日韩有码中文字幕| 亚洲精品一卡2卡三卡4卡5卡| 88av欧美| 精品久久久久久成人av| 此物有八面人人有两片| 欧美成人性av电影在线观看| xxx96com| 欧美在线黄色| 三级毛片av免费| 久久久久国产精品人妻aⅴ院| 黄片小视频在线播放| 国产精品美女特级片免费视频播放器 | 国产主播在线观看一区二区| 午夜福利在线观看吧| 亚洲精品乱码久久久v下载方式 | 99在线人妻在线中文字幕| 国产毛片a区久久久久| 亚洲av成人不卡在线观看播放网| 亚洲精品色激情综合| 18美女黄网站色大片免费观看| 狂野欧美激情性xxxx| 国产亚洲精品av在线| 日本黄色视频三级网站网址| 精品久久久久久,| 床上黄色一级片| 久久九九热精品免费| 国产精品亚洲av一区麻豆| xxx96com| 又紧又爽又黄一区二区| 国产精品av视频在线免费观看| 18禁美女被吸乳视频| 久久久久性生活片| 99国产精品99久久久久| av在线蜜桃| 亚洲熟妇中文字幕五十中出| 久久午夜亚洲精品久久| xxx96com| 国产午夜福利久久久久久| 在线免费观看的www视频| 桃红色精品国产亚洲av| 国产又黄又爽又无遮挡在线| 亚洲成人免费电影在线观看| 91av网一区二区| 18禁裸乳无遮挡免费网站照片| 亚洲国产欧美网| 午夜免费观看网址| 色综合欧美亚洲国产小说| 两个人的视频大全免费| 这个男人来自地球电影免费观看| 欧美日韩瑟瑟在线播放| 成在线人永久免费视频| 99久久精品一区二区三区| 一区福利在线观看| 性欧美人与动物交配| 国内精品美女久久久久久| 岛国在线免费视频观看| 国产亚洲精品一区二区www| 亚洲真实伦在线观看| 美女黄网站色视频| 亚洲第一欧美日韩一区二区三区| 成人国产一区最新在线观看| 又大又爽又粗| 欧美不卡视频在线免费观看| 日韩欧美免费精品| 麻豆一二三区av精品| 久久久成人免费电影| 老司机午夜十八禁免费视频| 国产高清视频在线播放一区| 欧美黄色片欧美黄色片| 99精品欧美一区二区三区四区| 欧美乱色亚洲激情| 久久精品91蜜桃| 亚洲av成人不卡在线观看播放网| 亚洲国产欧洲综合997久久,| 国模一区二区三区四区视频 | 成人av一区二区三区在线看| 国产av不卡久久| 麻豆成人午夜福利视频| 国产1区2区3区精品| 男人舔奶头视频| 日韩欧美免费精品| 成人亚洲精品av一区二区| 亚洲av五月六月丁香网| 国产精品av视频在线免费观看| 又大又爽又粗| 精品国内亚洲2022精品成人| 此物有八面人人有两片| 亚洲欧美一区二区三区黑人| 美女扒开内裤让男人捅视频| 亚洲自偷自拍图片 自拍| 亚洲中文字幕一区二区三区有码在线看 | 波多野结衣高清无吗| 久久久国产欧美日韩av| 亚洲国产欧美人成| 成人特级av手机在线观看| 亚洲精品美女久久久久99蜜臀| 国产精品女同一区二区软件 | 白带黄色成豆腐渣| 在线观看免费视频日本深夜| 亚洲电影在线观看av| 伊人久久大香线蕉亚洲五| 99re在线观看精品视频| 精品国产乱码久久久久久男人| 又大又爽又粗| 一本精品99久久精品77| 三级男女做爰猛烈吃奶摸视频| 五月玫瑰六月丁香| 男人的好看免费观看在线视频| 淫妇啪啪啪对白视频| 国产爱豆传媒在线观看| 少妇裸体淫交视频免费看高清| 法律面前人人平等表现在哪些方面| 日本成人三级电影网站| 日本精品一区二区三区蜜桃| 国产美女午夜福利| 亚洲无线观看免费| www日本黄色视频网| 这个男人来自地球电影免费观看| 脱女人内裤的视频| 亚洲成人精品中文字幕电影| 中亚洲国语对白在线视频| 91老司机精品| 香蕉av资源在线| 久久草成人影院| 精品电影一区二区在线| 麻豆成人午夜福利视频| 人妻夜夜爽99麻豆av| 18美女黄网站色大片免费观看| 免费在线观看视频国产中文字幕亚洲| 国产精品av久久久久免费| 美女被艹到高潮喷水动态| 精品乱码久久久久久99久播| 伦理电影免费视频| 在线永久观看黄色视频| 欧美国产日韩亚洲一区| 亚洲片人在线观看| 法律面前人人平等表现在哪些方面| 精品久久久久久,| 精品一区二区三区四区五区乱码| www.自偷自拍.com| 18禁黄网站禁片午夜丰满| 身体一侧抽搐| 一本一本综合久久| 亚洲中文字幕一区二区三区有码在线看 | 成人精品一区二区免费| 国产精品九九99| 天堂网av新在线| 久久天堂一区二区三区四区| 99久久成人亚洲精品观看| 美女免费视频网站| 婷婷丁香在线五月| 国产不卡一卡二| 欧美黄色淫秽网站| 少妇人妻一区二区三区视频| 精品久久蜜臀av无| 美女午夜性视频免费| 又爽又黄无遮挡网站| 嫩草影视91久久| 亚洲人成电影免费在线| 亚洲专区中文字幕在线| 精品熟女少妇八av免费久了| 国产乱人视频| 国产成人av教育| 悠悠久久av| 久久性视频一级片| 麻豆久久精品国产亚洲av| 国产亚洲精品久久久com| 在线观看66精品国产| 91久久精品国产一区二区成人 | 久久香蕉精品热| 最近最新免费中文字幕在线| 精品不卡国产一区二区三区| 高清毛片免费观看视频网站| 在线观看66精品国产| 男女视频在线观看网站免费| 免费av不卡在线播放| 亚洲欧美日韩卡通动漫| 手机成人av网站| av视频在线观看入口| 超碰成人久久| 免费看美女性在线毛片视频| 两性午夜刺激爽爽歪歪视频在线观看| 性色av乱码一区二区三区2| 国产亚洲精品av在线| 久久久精品欧美日韩精品| 人人妻人人看人人澡| 国产成人福利小说| 一级毛片高清免费大全| 国产黄a三级三级三级人| 久久草成人影院| 国产高清视频在线播放一区| 无人区码免费观看不卡| 亚洲国产高清在线一区二区三| 久久中文字幕人妻熟女| 国产成人精品久久二区二区免费| 亚洲九九香蕉| 天天一区二区日本电影三级| 夜夜爽天天搞| 亚洲中文字幕日韩| 国产欧美日韩一区二区精品| 久久精品人妻少妇| 99久久精品国产亚洲精品| 99国产极品粉嫩在线观看| xxxwww97欧美| 一级a爱片免费观看的视频| www国产在线视频色| 成人三级黄色视频| 国产成人啪精品午夜网站| 草草在线视频免费看| www.熟女人妻精品国产| 宅男免费午夜| 成人一区二区视频在线观看| 色综合站精品国产| 欧美日韩国产亚洲二区| 性色av乱码一区二区三区2| 日本熟妇午夜| 国产精品久久久久久人妻精品电影| 亚洲色图av天堂| 欧美黑人巨大hd| 免费av不卡在线播放| 成人无遮挡网站| 国产亚洲av高清不卡| 日本一二三区视频观看| 日韩欧美 国产精品| 久99久视频精品免费| 国产三级中文精品| 少妇的丰满在线观看| www日本黄色视频网| 亚洲男人的天堂狠狠| 久久精品国产亚洲av香蕉五月| 国产精品永久免费网站| 88av欧美| 中文字幕高清在线视频| 男女之事视频高清在线观看| 婷婷亚洲欧美| 丝袜人妻中文字幕| 成人av一区二区三区在线看| xxxwww97欧美| 国产精品香港三级国产av潘金莲| 国产综合懂色| 19禁男女啪啪无遮挡网站| 看黄色毛片网站| av在线天堂中文字幕| 久久精品亚洲精品国产色婷小说| 免费人成视频x8x8入口观看| 日韩中文字幕欧美一区二区| 亚洲成人久久爱视频| av天堂在线播放| 免费看日本二区| av欧美777| 亚洲欧洲精品一区二区精品久久久| 丰满人妻一区二区三区视频av | 在线观看舔阴道视频| 免费大片18禁| 国产精品98久久久久久宅男小说| 欧美成狂野欧美在线观看| 悠悠久久av| 国内精品久久久久久久电影| 中文字幕人成人乱码亚洲影| 国产aⅴ精品一区二区三区波| 五月玫瑰六月丁香| 国产亚洲精品综合一区在线观看| 狠狠狠狠99中文字幕| 白带黄色成豆腐渣| 成年人黄色毛片网站| 日韩人妻高清精品专区| 亚洲精品456在线播放app | 看免费av毛片| 久久中文字幕人妻熟女| 一个人看的www免费观看视频| 黄片大片在线免费观看| 欧美+亚洲+日韩+国产| 国产精品一及| 男女那种视频在线观看| 国产精品日韩av在线免费观看| 亚洲无线观看免费| 亚洲精华国产精华精| 久久精品91蜜桃| 国产成人aa在线观看| 亚洲真实伦在线观看| 午夜福利成人在线免费观看| 热99在线观看视频| 一级毛片女人18水好多| 国产精品自产拍在线观看55亚洲| 白带黄色成豆腐渣| 亚洲色图 男人天堂 中文字幕| 99视频精品全部免费 在线 | 麻豆久久精品国产亚洲av| 操出白浆在线播放| 国产男靠女视频免费网站| 夜夜夜夜夜久久久久| 18禁黄网站禁片免费观看直播| 首页视频小说图片口味搜索| 男人舔女人下体高潮全视频| 日韩欧美国产在线观看| 天堂动漫精品| 真人做人爱边吃奶动态| av在线天堂中文字幕| 一个人看视频在线观看www免费 | tocl精华| 欧美+亚洲+日韩+国产| 两人在一起打扑克的视频| 久久国产乱子伦精品免费另类| 亚洲欧美精品综合久久99| av欧美777| 黑人欧美特级aaaaaa片| 亚洲精品国产精品久久久不卡| 久久久久久久久中文| 国产精品女同一区二区软件 | 国产av在哪里看| 色综合欧美亚洲国产小说| 欧美午夜高清在线| 中文字幕久久专区| 中文字幕熟女人妻在线| 亚洲va日本ⅴa欧美va伊人久久| 免费人成视频x8x8入口观看| 成人特级黄色片久久久久久久| 在线观看免费午夜福利视频| 国产野战对白在线观看| 欧美日韩亚洲国产一区二区在线观看| 一进一出好大好爽视频| 脱女人内裤的视频| 亚洲狠狠婷婷综合久久图片| 在线十欧美十亚洲十日本专区| 最近最新免费中文字幕在线| 国产高清视频在线播放一区| 欧美成人免费av一区二区三区| 欧美黄色片欧美黄色片| 久久久久免费精品人妻一区二区| 国产精品久久视频播放| 好男人电影高清在线观看| 亚洲av电影不卡..在线观看| 哪里可以看免费的av片| www日本在线高清视频| 国产亚洲精品久久久com| 亚洲真实伦在线观看| 高清在线国产一区| 午夜福利免费观看在线| 黄色日韩在线| 熟女人妻精品中文字幕| 日韩 欧美 亚洲 中文字幕| 精品国产乱子伦一区二区三区| 在线观看美女被高潮喷水网站 | 中文字幕久久专区| 日本a在线网址| 亚洲精品在线美女| 国产亚洲欧美98| 精品国产美女av久久久久小说| 波多野结衣巨乳人妻| 亚洲欧美日韩卡通动漫| 午夜视频精品福利| 午夜福利成人在线免费观看| 19禁男女啪啪无遮挡网站| 久久久久国产精品人妻aⅴ院| 精品欧美国产一区二区三| 亚洲成人精品中文字幕电影| 亚洲人成网站高清观看| 黄频高清免费视频| 国产极品精品免费视频能看的| 成人鲁丝片一二三区免费| 美女黄网站色视频| 又黄又粗又硬又大视频| 桃红色精品国产亚洲av| 此物有八面人人有两片| av黄色大香蕉| 听说在线观看完整版免费高清| 久久国产精品影院| 亚洲欧美激情综合另类| 国产精品免费一区二区三区在线| 国产亚洲精品av在线| a在线观看视频网站| 国产三级在线视频| 国产高清三级在线| 18禁美女被吸乳视频| 小说图片视频综合网站| 欧美成狂野欧美在线观看| 国产精品综合久久久久久久免费| 麻豆成人午夜福利视频| 老汉色∧v一级毛片| 在线观看午夜福利视频| 精品一区二区三区视频在线观看免费| 99精品久久久久人妻精品| 91字幕亚洲| 国产97色在线日韩免费| 日韩成人在线观看一区二区三区| 法律面前人人平等表现在哪些方面| 1024香蕉在线观看| 国内精品久久久久精免费| 中文字幕熟女人妻在线| 在线十欧美十亚洲十日本专区| а√天堂www在线а√下载| 亚洲国产日韩欧美精品在线观看 | 欧洲精品卡2卡3卡4卡5卡区| 一个人免费在线观看的高清视频| 日本成人三级电影网站| 国产97色在线日韩免费| 亚洲精品美女久久久久99蜜臀| 又黄又爽又免费观看的视频| 国产精品美女特级片免费视频播放器 | 一本一本综合久久| 少妇丰满av| 国产成人精品久久二区二区免费| 高清毛片免费观看视频网站| 91av网一区二区| 美女被艹到高潮喷水动态| 精品久久久久久久末码| 国产野战对白在线观看| 久久久久久久久免费视频了| 欧美激情在线99| 好男人电影高清在线观看| 亚洲天堂国产精品一区在线| 国产黄色小视频在线观看| 两个人的视频大全免费| 成人18禁在线播放| 香蕉丝袜av| 国产高清视频在线播放一区| 高清在线国产一区| 观看美女的网站| 国产高潮美女av| 99久久成人亚洲精品观看| 少妇裸体淫交视频免费看高清| 国产高潮美女av| 国产高清videossex| 男女之事视频高清在线观看| 国内少妇人妻偷人精品xxx网站 | or卡值多少钱| 欧美激情久久久久久爽电影| 午夜激情欧美在线| 欧美黄色片欧美黄色片| 激情在线观看视频在线高清| 精品熟女少妇八av免费久了| 久久99热这里只有精品18| 国产成+人综合+亚洲专区| 成人性生交大片免费视频hd| 亚洲激情在线av| 欧美精品啪啪一区二区三区| 精品一区二区三区四区五区乱码| 日本精品一区二区三区蜜桃| 欧美日韩精品网址| 欧美丝袜亚洲另类 | 亚洲一区二区三区不卡视频| 男人和女人高潮做爰伦理| 草草在线视频免费看|