Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Molecular phylogenetic analysis of attached Ulvaceae species and free-floating Enteromorpha from Qingdao coasts in 2007

Based on the sequence data of the nuclear ribosomal DNA internal transcribed spacer (ITS) 1, 5.8 S, and ITS 2, the molecular phylogeny was analyzed on Ulvaceae species collected from Qingdao coasts in summer of 2007, including 15 attached Ulva and Enteromorpha samples from 10 locations and 10 free-floating Enteromorpha samples from seven locations. The result supported the monophyly of all free-floating Enteromorpha samples, implying the unialgal composition of the free-floating Enteromorpha, and the attached Ulvaceae species from Qingdao coasts were grouped into other five clades, suggesting that they were not the biogeographic origin of the free-floating Enteromorpha in that season.

Settling abalone veliger larvae in a free-swimming microalgal culture

In the current abalone hatchery in China, insufficient diatoms on vertically placed corrugated pvc plates at later stage often could not support the growth of postlarvae up to the stage that they can feed on live macroalgae. As a result, stripping the spats (35 mm) off by anaesthetization and switching the diet from live diatoms to artificial powdered diet in combination has to be performed in most of the abalone farms. This manipulation normally leads to more than 50% mortality. Here we report the direct use of the unicellular green alga Platymonas helgolandica Kylin var. tsingtaoensis as a potential alga to be used to settle the veliger larvae of the Pacific abalone Haliotis discus hannai and to feed the postlarvae. Settlement rate of 2-day-old veliger larvae in mono culture of P helgolandica could be as high as 92% ( +/- 4.2%) on day 10 in small scale trials, higher than that in the selected benthic diatom strain (53.6% +/- 12.7%) when settled in the water in which bacteria propagation was controlled by treatment of 2 ppm of benzylpenicillinum calcium and streptomycin sulfate. Postlarvae fed solely on P. helgolandica or the selected benthic diatom Navicula-2005-A grew at rates of 40.1 ( +/- 1.9) and 45.8 (+/- 13.4) mu m day(-1), respectively, when raised at 22 degrees C until day 50 postfertilization. P. helgolandica was shown to have distinct diurnal settling rhythm characterized with a peak of settled cells in the middle of the night for cell division and a peak of free-swimming cells in the middle of the day. High density of attached P. helgolandica cells on the inner surface of the culture facility in the night fits the nocturnal feeding behavior of the abalone spats. Judged by the promising larvae settling rate, growth and survival rates of the postlarvae fed with this alga, the free-swimming micro-green alga P. helgolandica constitutes a potential species for settling the veliger larvae and for supporting the growth of postlarvae as well. (c) 2006 Published by Elsevier B.V.

Factors affecting the estimation of gas hydrate and free gas saturation

Attenuations of different types of gas hydrate cementation in fluid-saturated porous solids are discussed. The factors affecting estimation of gas hydrate and free gas saturation are analyzed. It is suggested that porosity of sediment, the P wave velocity model and methods of calculating elastic modulus are key factors in the estimation of gas hydrate and free gas saturations. Attenuation of gas hydrate-bearing sediment is closely related with the cementation types of gas hydrate. Negative anomalies of quality factors indicate that gas hydrate deposits away from grain as part of fluid. Positive anomalies of the quality factors indicate that gas hydrate contacts with solid and changes the elastic modulus of matrix. Low frequency velocity and high frequency velocity models are used to estimate gas hydrate and free gas saturation in the Blake Ridge area according to the well log data of the hole 995 in ODP leg 164. The gas hydrate saturation obtained by low frequency velocity is 10% similar to 20% of the pore space and free gas saturation is 0.5% similar to 1% of the pore space. The gas hydrate saturation obtained by high frequency velocity is 5% similar to 10% of the pore space and free gas saturation is 1% similar to 2% of the pore space.

Extraction of Nitraria tangutorum seed oil by supercritical carbon dioxide and determination of free fatty acids by HPLC/APC/IMS with fluorescence detection

The seed oil from Nitraria tangutorum samples was obtained by supercritical carbon dioxide extraction methods. The extraction parameters for this methodology, including pressure, temperature, particle size and extraction time, were optimized. The free fatty acids in the seed oil were separated with a pre-column derivation method and 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as a labeling regent, followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion mode). HPLC analysis shows that the main compositions of the seed oil samples were free fatty acids (FFAs) in high to low concentrations as follows: linoleic acid, oleic acid, hexadecanoic acid and octadecanoic acid. The assay detection limits (at signal-to-noise of 3:1) were 3.378-6.572 nmol/L. Excellent linear responses were observed, with correlation coefficients greater than 0.999. The facile BDETS derivatization coupled with mass spectrometry detection allowed the development of a highly sensitive method for analyzing free fatty acids in seed oil by supercritical CO2 extraction. The established method is highly efficient for seed oil extraction and extremely sensitive for fatty acid profile determination. (C) 2007 Elsevier B.V. All rights reserved.

Determination of free fatty acids in bryophyte plants and soil by HPLC with fluorescence detection and identification by online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(ptoluenesulfonate)-ethyll-2-phenylimidazole-[4,5-f-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C-8 column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Determination of 30 free fatty acids in two famous Tibetan medicines by HPLC with fluorescence detection and mass spectrometric identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free Fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 degrees C with anhydrous K2CO3 as catalyst. A mixture Of C-1-C-30 fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C-8 column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were > 0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were < 3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H](+). The collision-induced dissociation of the molecular ion produced fragment ions at [MH - H2O](+), [MH - 2H(2)O](+), [MH - 3H(2)O](+). The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N - CO, O - CO, and C - OCC, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M + H]+ -> [MH - H2O](+), [MH - H2O](+), [MH - 2H(2)O](+), [MH-3H(2)O](+), 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was < 15% at broad linear dynamic ranges (0.0244-25nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.