Simultaneous biodegradation of phenolics and petroleum hydrocarbons from semi-coking wastewater: Construction of bacterial consortium and their metabolic division of labor.

Phenols and petroleum hydrocarbons were the main contributors to COD in semi-coking wastewater, and their removal was urgent and worthwhile. The microbial strains were selected to construct microbial community for the wastewater treatment. The concentration of phenols was decreased from 2450 ± 1.2 mg/L to 200 ± 0.9 mg/L, and the removal rate of petroleum hydrocarbons was up to 97.08 ± 0.09 % by microorganisms. After phenolic compounds with high toxicity were removed by bioaugmentation, the treated semi-coking wastewater was more biodegradable, and its water quality has been significantly improved. Through GC-MS and high-through sequencing technology, the metabolic division of labor in degradation of phenols, ring-cleavage of aromatic compounds, mineralization of metabolites was further revealed. The microbial community consisting of Pseudomonas stutzeri N2 and Rhodococcus qingshengii FF could effectively and simultaneously remove phenols and petroleum hydrocarbons, and these two strains possess great potential of being applied in aerobic biological treatment process of large-scale semi-coking wastewater.

Toxicity evaluation of the metabolites derived from the degradation of phenanthrene by one of a soil ubiquitous PAHs-degrading strain Rhodococcus qingshengii FF.

Rhodococcus qingshengii strain FF is a soil ubiquitous strain that has a high polycyclic aromatic hydrocarbons (PAHs) biodegradation capability. In this work, phenanthrene was used as a PAH model compound. The accumulated pattern of the metabolites of phenanthrene by strain FF was investigated, and their toxicity to Vibrio fischeri, effect on microbiota diversity of farmland soil and influence on seed of wheat were evaluated. Total of 29 main intermediates were observed for the phenanthrene degradation process. Pyrogallol was the predominant accumulated metabolite, and 59% of the accumulated metabolites were oxygen-containing PAHs that have only one benzene ring. The acute toxicity assessment showed the accumulated metabolites in later phase were more toxic to Vibrio fischeri. Microbe and wheat seed response to the different stages of phenanthrene metabolites indicated pollution significantly decreased microbial richness and evenness of farmland soil and lower germinal length, root length or root number of wheat seed. These results indicated that not only the elimination of PAHs, but also the easily accumulated metabolites produced during the PAHs degradation process should be paid enough attention. The comprehensive evaluation of toxicity during the degradation process would provide useful information for the use of microbe-orientated strategies in PAHs bioremediation.

A novel Ca2+ indicator for long-term tracking of intracellular calcium flux.

The major drawback of using Fluo-4 AM is that it requires an organic anion transporter inhibitor, such as probenecid, to prevent leakage. This can hinder the studies that require extended monitoring time and longer cellular retention. To address the issue, a novel Ca2+ indicator, Calbryte 520 AM, was developed. We compared the performance of Fluo-4 AM and Calbryte 520 AM following prolonged incubation periods after cell loading. Cells loaded with Calbryte 520 AM retained the dye for up to 24 h while exhibiting significant fluorescence brightness and superior Fmax/F0 ratios (Fmax: fluorescence intensity upon stimulation; F0: intensity before stimulation). It demonstrated that the longer retention of Calbryte 520 AM can be exploited to accommodate for the extended time required when monitoring calcium dynamics.

Optimization and characterization of rhamnolipid production by Pseudomonas aeruginosa NY3 using waste frying oil as the sole carbon.

Yield and cost are two major factors limiting the widespread use of rhamnolipids (RLs). In the present study, waste frying oil (WFO) was used as the sole carbon source to produce environmentally friendly RLs by Pseudomonas aeruginosa NY3. The Plackett-Burman design (PBD) and Box-Behnken design (BBD) methods were used to maximize the production yield of RL. The PBD results showed that the concentrations of NaNO3 , Na2 HPO4 , and trace elements were the key factors affecting the yield of RL. Furthermore, the BBD results showed that at NaNO3 , Na2 HPO4 , and trace elements concentrations were 4.95, 0.66, and 0.64 mL/L, respectively, the average RL yield reached 9.15 ± 0.52 g/L, 1.58-fold higher than that observed before optimization. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-ion trap-time of flight mass spectrometry (LCMS-IT-TOF) were used to elucidate the diversity of RL congeners. The results showed that, after optimization, the RL congener diversity increased, and the major RL constituent was converted from di-RLs (64.04%) to mono-RLs (60.44%). These results suggested that the concentrations of the components contained in the culture medium of P. aeruginosa NY3 influenced not only the yield of RL, but also its congener distribution.

Immobilization of Rhodococcus qingshengii strain FF on the surface of polyethylene and its adsorption and biodegradation of mimic produced water.

Low pH and high salinity characteristic of produced water (PW) posed a big challenge for the direct biological treatment. The immobilization of R. qingshengii strain FF, which degraded petroleum effectively under low pH, and application of immobilized R. qingshengii strain FF in treating mimic PW was studied in this work. The immobilization of R. qingshengii strain FF on the surface of polyethylene foam (PEF), one type of waste packaging materials, was optimized using the response surface methodology. Under optimum conditions, cell density of R. qingshengii strain FF immobilized on the surface of PEF reached 388 mg (cells)/g(PEF). In addition, a few factors, including hydraulic retention time (HRT), pH and salinity, were studied for treating mimic PW using immobilized R. qingshengii strain FF. The result of this study demonstrated that TPH degradation efficiency of PW by immobilized R. qingshengii strain FF reached above 90% when HRT was longer than 8 h. Weak acid and high salinity conditions only moderately decreased TPH. Asphalt, alkanes and aromatic hydrocarbon contained in petroleum can be degraded to some extent. These results indicated that immobilized R. qingshengii strain FF can be used as a highly efficient strain which could be used in biological treatment of real PW.

Homogeneously catalytic oxidation of phenanthrene by the reaction of extracellular secretions of pyocyanin and Nicotinamide Adenine Dinucleotide.

Application of biological methods on polycyclic aromatic hydrocarbons (PAHs) treatment is always limited by its low degradation efficiency. In this work, a catalytic oxidation pathway of phenanthrene resulted by extracellular secretions of P. aeruginosa NY3 was proposed. Results of the in vitro experiments showed that, the extracellular secretions of Pyocyanin (Pyo) and Nicotinamide Adenine Dinucleotide (NADH) acted as homogeneous catalysts because which produced H2O2, hydroxyl free radical and superoxide anion radical continuously under aerobic conditions. These produced reactive oxygen species oxidized the phenanthrene in aqueous solution, leading to the cleavage of the phenanthrene ring and the formation of phthalates products and low molecular weight metabolites (such as alkanoic acids). The ratio of BOD5/COD of phenanthrene-containing wastewaters was greatly improved after treating with Pyo and NADH. Results of the in vivo experiments showed that, pre-degradation of phenanthrene by extracellular fluid simultaneously containing Pyo and NADH, promoted cell growth of P. aeruginosa NY3, which confirmed the improvement of bioavalability of phenanthrene-containing wastewaters by the catalytic oxidation of Pyo and NADH. Further details of the free radical detection indicated that, the increase in secretion of Pyo by a bacterium was favorable to the production of H2O2 in the extracellular fluid.

A highly selective fluorescent probe for the intracellular measurement of magnesium ion.

Magnesium ion (Mg+2) plays an important role in various biological processes. All the commercial indicators available share a common drawback, i.e., they have a higher affinity towards calcium ions (Ca+2) than Mg+2. In this study, we reported a new robust green fluorescent indicator, Mag-520, for detection of Mg+2 in live cells. Our results showed that Mag-520 has 10 fold higher affinity towards Mg+2 than Ca+2, while mag-fluo-4 has less than 0.5 fold affinity to Mg+2 than Ca+2 under the same conditions using flow cytometry and fluorescence microscopy. The results demonstrated that Mag-520 provides a better tool to measure Mg+2 with less interference from Ca+2.

Establishment and application of a novel fluorescence-based analytical method for the rapid detection of viable bacteria in different samples.

A rapid method for readily detecting the total numbers of viable bacterial cells in numerous samples (including surface water, solid inoculants, and soil samples) is reported using a newly developed hand-held fluorometer and a fluorescent dye Calcein UltraGreen™ AM. Compared to the traditional plate counting method that requires 48 hours of cultivation, the newly established method does not require any incubation time, making the detection method faster and more convenient. The portable rapid detection fluorometer has a wide dynamic range of relative fluorescence intensity from 45 to 30 133. It can detect bacterial concentration ranging from 105 to 1010 cells per mL. This newly established method has good applicability for accurately and quickly detecting the cell number of viable bacteria in various samples. The results of the fluorescence-based method were compared with those of the traditional plate counting method, and it was found that the relative standard deviation was less than 6%. This new rapid measurement system provides a robust method for the rapid on-site detection of viable bacteria.

Biological treatment of high salinity and low pH produced water in oilfield with immobilized cells of P. aeruginosa NY3 in a pilot-scale.

Produced water (PW) in oilfield, as the largest waste streams in the oil and gas production, has posed a huge threat to the ecosystem. In this work, an environmentally friendly and recyclable biofilms have been developed for treating PW. We discovered that the cells of P. aeruginosa NY3 could be easily immobilized on the surface of polyurethane foam (PUF). Removal efficiency of oil and suspended solids (SS) by immobilized P. aeruginosa NY3 was keeping above 80% and 76% both in a laboratory scale and a pilot scale under suitable pH. Low pH and high value of SS had negative effect on the degradation of oil and SS by P. aeruginosa NY3. Recovery test showed that, the activity of biofilms P. aeruginosa NY3 after running in a pilot scale could be recovered in 5 days. Removal ability of oil in the real PW by the recovered biofilms of P. aeruginosa NY3 was even higher than that of the freshly prepared biofilms. These results indicated that, with a simple pH adjustment, immobilized P. aeruginosa NY3 could be recycled for removing oil and SS in the raw PW resulted from oil production.

Evidences of extracellular abiotic degradation of hexadecane through free radical mechanism induced by the secreted phenazine compounds of P. aeruginosa NY3.

The aim of this work was to investigate the effects of secreted extracellular phenazine compounds (PHCs) on the degradation efficiency of alkanes by P. aeruginosa NY3. Under aerobic conditions, the PHCs secreted by P. aeruginosa NY3 initiate the oxidation of alkanes outside cells, in coupling with some reducing agents, such as ß-Nicotinamide adenine dinucleotide, reduced disodium salt (NADH) or reduced glutathione (GSH). This reaction might be via free radical reactions similar to Fenton Oxidation Reaction (FOR). P. aeruginosa NY3 secretes pyocyanin (Pyo), 1-hydroxyphenazine (HPE), phenazine-1-carboxylic acid (PCA), and phenazine-1-amide (PCN) simultaneously. The cell-free extracellular fluid containing these four PHCs degrades hexadecane effectively. The observation of Electron Spin Resonance (EPR) signals of superoxide anion radical (O2-), hydroxyl radical (OH) and/or carbon free radicals (R) both in vivo and in vitro suggested the degradation of hexadecane could be via a free radical pathway. Secretion of PHCs has been found to be characteristic of Pseudomonas which is often involved in or related to the degradation of organic pollutants. Our work suggested that certain organic contaminants may be oxidized through ubiquitously extracellular abiotic degradation by the free radicals produced during bio-remediation and bio-treatment.

Fluorescent real-time quantitative measurements of intracellular peroxynitrite generation and inhibition.

Peroxynitrite (ONOO-), a strong oxidant species, is produced by the reaction of nitric oxide (NO) and superoxide (O2.-) radicals. It plays an important role as a biological regulator in numbers of physiological and pathological processes. In this study, we developed fluorescence-based real-time quantitative measurements to detect intracellular ONOO-. The probe DAX-J2 PON Green showed high selectivity toward ONOO- over other competing species, and has been successfully applied in microplate reader and flow cytometer to quantitatively measure endogenous ONOO- production. Moreover, the results demonstrated the inhibitory effects of curcumin on intracellular ONOO- generation.

New violet-excitable reagents for multicolor flow applications.

We have recently added three new fluorophores-BD Horizon™ V450, BD Horizon V500, and BD Horizon V550 (V450, V500, and V550; BD Biosciences, San Jose, CA) to our existing AmCyan product, forming a group of four violet-excitable dyes from which we have produced functional antibody conjugates. These conjugates, with emission maxima that range from 450 to 535 nm, are compatible with multilaser flow cytometry (FCM) and can be used for polychromatic FCM in three-color or two-color combinations; in fact, V500 fills a spectral opening that has thus far not been exploited by other manufacturers of FCM reagents. We here report that conjugates based on BD Horizon dyes performed well within a useful sensitivity range, established by testing a representative group of violet-excitable FCM reagents currently available, and that V500 has better compatibility with FITC in multicolor applications than does AmCyan.

3-Carboxy-6-chloro-7-hydroxycoumarin: a highly fluorescent, water-soluble violet-excitable dye for cell analysis.

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (approximately 0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450-antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450-antibody conjugates are functional and stable.

A novel fluorogenic coumarin substrate for monitoring acid phosphatase activity at low pH environment.

This article described the synthesis and application of 6-chloro-8-fluoro-4-methylumbelliferone phosphate (CF-MUP) in analyzing acid phosphatase activity. Compared to the existing MUP, the new coumarin phosphate, CF-MUP, demonstrateed much higher sensitivity and was more robust for detecting the activity of acid phosphatase than the classic substrate 4-methylumbelliferone phosphate (MUP). The product of enzyme reaction, 6-chloro-8-fluoro-4-methylumbelliferone (CF-MU) possesses strong fluorescence at approximately 450 nm with low pKa (4.7), high fluorescence quantum yield and pH independence in the physiological pH range. This new fluorescence dye, CF-MU, is a convenient tool for assays with buffer pH between 4.5 and 8.

Real-time and high throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye.

Adenosine-3',5'-cyclic monophosphate (cAMP) conveys the signals from G-protein coupled receptors (GPCRs) and regulates a variety of downstream cellular events. However, there are few robust assays available for measuring cAMP in live cells. Most of the existing cAMP assays require cell lysis and/or have relatively low throughput. We report a live cell-based cAMP assay that has been developed to record the real-time changes in intracellular cAMP. By employing a mutated cyclic-nucleotide-gated ion channel (CNGC) as the cAMP biosensor, the change of cAMP level is coupled to the change of transmembrane potential that is measured through a new fluorescent membrane potential (MP)-sensitive dye, HLB 021-152. We have successfully used HLB 021-152 for homogeneously monitoring cAMP stimulations in live cells under both serum-containing and serum-free environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-152 increases rapidly. It has much greater assay dynamic range than DiSBAC2(3), the existing "gold standard" dye for measuring cellular MP. This new MP dye can be readily formulated for high throughput screening of GPCR modulators either with serum or without serum.

N-DEVD-N'-morpholinecarbonyl-rhodamine 110: novel caspase-3 fluorogenic substrates for cell-based apoptosis assay.

A novel caspase-3 substrate N-Ac-DEVD-N'-MC-R110, which is a fluorogenic substrate cleavable in a single step, has been prepared. It has a significantly higher enzyme turnover rate and sensitivity for detecting caspase-3 activity both in solution and living cells than existing fluorogenic substrates.

Fluorescent sodium ion indicators based on the 1,7-diaza-15-crown-5 system.

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels.

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.