Characterization and Dynamic Analysis of Dissolved Proteins in Lake Biwa Using Two-Dimensional Electrophoresis.

Concentrated protein-like fluorophores of dissolved organic matter (DOM) in Lake Biwa were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). As a result, it was found that the protein-like fluorophores in Lake Biwa are likely to be dissolved proteins released from phytoplankton. SDS-PAGE analysis revealed that protein-like fluorophores from Lake Biwa usually consist of proteins with molecular weights of 33 - 35, 42 - 45, 56 - 59 and 62 - 66 kDa. The molecular weights (MWs) and isoelectric points (pIs) of dissolved proteins in Lake Biwa were firstly clarified using 2-DE. The 2-DE patterns of proteins in Lake Biwa were compared with those for algal DOM released from four kinds of lake phytoplankton, Microcystis aeruginosa, Staurastrum dorsidentiferum, Cryptomonas ovata, and Fragilaria capucina. Some of the protein spots in 2-DE of Lake Biwa were similar to those of algal DOM released during cultivation. The relations between monthly changes in the electrophoretic gel images of lake water samples and those in the community structure of phytoplankton in Lake Biwa were observed. These results suggest that some of the dissolved proteins in Lake Biwa are likely derived from phytoplankton.

Biodegradation of dissolved organic matter (DOM) released from phytoplankton in Lake Biwa.

The biodegradation study of algal dissolved organic matter (DOM) released from Microcystis aeruginosa, Staurastrum dorsidentiferum and Cryptomonas ovata was carried out. The algal DOM released from Microcystis aeruginosa and Staurastrum dorsidentiferum is relatively stable, while a part of the algal DOM released from Cryptomonas ovata may be easily decomposed. Before biodegradation, two fulvic-like fluorescence peaks (A and B) and a protein-like fluorescence peak (C) and another peak with E(x)/E(m) values of 320 - 330/390 nm (peak D) were observed in the algal DOM released from three kinds of phytoplankton. The fulvic-like fluorophores may be refractory regardless of the kinds of phytoplankton, while protein-like fluorophores released from Microcystis aeruginosa and Staurastrum dorsidentiferum may be relatively refractory and those from Cryptomonas ovata may be unstable. Peak D in the surface water of Lake Biwa may be attributed to low-molecular-weight substances produced during cultivation and/or biodegradation of several kinds of phytoplankton. The ratios of the fluorescence intensities (RFI/DOC) of peak A to peak B in algal DOM (< 1.0) were lower than those in soil Dando FA (1.8). On the other hand, no relationships between peak A and peak C were observed for three kinds of phytoplankton.

Characterization of protein-like fluorophores released from lake phytoplankton on the basis of fractionation and electrophoresis.

Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.

Characterization of fluorophores released from three kinds of lake phytoplankton using gel chromatography and fluorescence spectrophotometry.

Three kinds of phytoplankton were cultivated, and the contribution of dissolved organic matter (DOM) released from the phytoplankton was examined to clarify the cause of organic pollution of Lake Biwa. Microcystis aeruginosa, Staurastrum dorcidentiferum, and Cryptomonas ovata were evaluated with regard to cultivation. A significant peak (M(w): <3000 Da) was mainly detected in the algal DOM released from plankton during cultivation by gel chromatography with a fluorescence detector (E(x) = 340 nm, E(m) = 435 nm). Since this peak corresponds to a peak with lower molecular weight in three peaks detected in the surface water of Lake Biwa, it can be concluded that the algal DOM released from the plankton during cultivation makes a considerable contribution to the refractory organic matter in Lake Biwa. Three fluorescence maxima were observed in the cultivation of three kinds of phytoplankton: two fulvic-like fluorescence peaks (A and B) and a protein-like fluorescence peak (C). These peaks became larger as their cell counts of plankton increased. As for the fractionations of algal DOM using DAX-8, the ratio of hydrophilic DOM is fairly high in DOM produced by three kinds of phytoplankton. The order of the amount of algal DOM per cell volume during cultivation was Cryptomonas ovata > Microcystis aeruginosa > Staurastrum dorcidentiferum. These results suggest that the increase of the refractory organic matter in Lake Biwa may be attributed to a change of the predominant phytoplankton.

Characterization of dissolved organic matter released from Microcystis aeruginosa.

The contribution of dissolved organic matter (DOM) released from phytoplankton (Microcystis aeruginosa) during cultivation and biodegradation was examined to clarify the causes of the organic pollution of Lake Biwa. Two peaks, peak 2 (retention time (RT) = 32 min) and peak 3 (RT = 35 min), were detected in the algal DOM released from Microcystis aeruginosa during cultivation and biodegradation by gel chromatography with a fluorescence detector (Ex = 340 nm, Em = 435 nm). As these peaks correspond with the peaks detected in the surface water of Lake Biwa, one can conclude that the algal DOM released from Microcystis aeruginosa during cultivation and biodegradation makes a considerable contribution to the refractory organic matter in Lake Biwa. Three fluorescence maxima were observed in the cultivation of Microcystis aeruginosa: a fulvic-like fluorescence peak (peak A) with Ex/Em values of 320/430 nm, a protein-like fluorescence peak (peak C) with Ex/Em values of 280/360 nm, and another peak with Ex/Em values of 240/370 nm. The fluorescence material of peak C has a larger MW than that of peak A. The algal-derived DOM from Microcystis aeruginosa has similar fluorescence to fulvic acid of soil origin but exhibits mainly hydrophilic characteristics. In the biodegradation of Microcystis aeruginosa, a fulvic-like fluorescence peak (peak B) with Ex/Em values of 250/440 nm and a peak with Ex/Em values of 320/380 nm were observed.

Development of a portable instrument for the continuous analysis of volatile organic compounds (VOCs) and its application to environmental monitoring.

A small, time efficient and sensitive instrument for the continuous analysis of very volatile organic compounds (VOCs) with a boiling point lower than 100 degrees C in addition to the analysis of VOCs with a boiling point in the range of 100-150 degrees C was developed and applied to the measurement of VOCs in the course of university research and environmental monitoring. VOCs, such as n-hexane, acetone, ethyl acetate, alcohols, benzene, toluene and xylene, were continuously measured once every 30 min. The detection limits of hexane, ethyl acetate, benzene and toluene at a preconcentration time of 10 min were 0.41 microg/m(3) (0.12 ppb), 0.67 microg/m(3) (0.19 ppb), 0.22 microg/m(3) (0.07 ppb) and 0.22 microg/m(3) (0.06 ppb), respectively. The relative standard deviations of VOCs were less than 5%. The sensitivities of the present method VOCs were higher than those of the conventional method. The temporal changes in VOC concentrations in several laboratories and at a plant for the disposal of organic liquid wastes were measured, and the behavior of VOCs was analyzed. All the VOC concentrations, except that of ethyl acetate, determined using the portable instrument were slightly lower than those determined using a passive sampler. The portable instrument developed in the course of this study can be used for the risk assessment and management of chemicals.

Size distribution and characteristics of chemical components in ambient particulate matter.

Ambient particulate matter and gas in Kyoto were investigated by gravimetric analysis, X-ray fluorescence spectrometry, and ion chromatography in order to clarify their behavior and origin. The size distribution and characteristics of the chemical components in ambient particulates collected on PTFE membrane filters using an Andersen air sampler were examined from August 2001 to April 2004. A four-stage filter pack method was used to sample the atmosphere for the determination of gas (SO2, HNO3, HCl, NH3) and particulate matter (SO42, NO3, Cl-, Na+, K+, Ca2+, Mg2+, NH4+) concentrations from October 2002 to April 2004. The concentration of SPM mass was in the range of 6.7 - 80.2 microg/m3. The size distributions of SPM mass were bimodal, peaking at around 0.65 - 1.1 and 3.3 - 4.7 microm, and 40 - 85% of SPM mass was fine particles (< 2.1 microm). Na, Mg, Al, Si, Ca, Cl, and Fe were mainly present in coarse particles (2.1 to 11.0 microm), while S was present in fine particles. The concentrations of Al, Si, Ca, Mg, and Fe in fine particles increased from March to April in 2002, and those in coarse particulates increased in November 2002 and from March to April in 2004. This may be the effect of the continental yellow sand "Kosa." The differences in the size distributions of Al, Si, Ca, Mg, and Fe in particles may depend on differences in their place of occurrence and course of transport from China to Japan. The concentration of HCl gas was higher than that of particulate chloride ion in summer. Nitric acid gas existed at higher concentrations in summer, but fine particulate nitrate ion was observed in winter. The gaseous-to-fine aerosol nitrate fraction became higher at warmer temperatures. Coarse sulfate was below 10%, and SO2 gas and fine particulate sulfate were above 90%.

Simple analysis of volatile organic compounds (VOCs) in the atmosphere using passive samplers.

A simple analysis of volatile organic compounds (VOCs), such as benzene, toluene, m,p-xylene, and o-xylene, at low levels in the atmosphere was conducted using passive samplers. The methods were applied to analyzing the behavior and origin of VOCs in Kyoto City. The passive samplers were exposed for 7 - 14 days at sampling sites in Kyoto City and for 30 days in the mountains (Mt. Hiei and Mt. Daimonji). Shibata gas-tube samplers packed with activated carbon were used for the determination of VOCs. The absorbed VOCs were extracted into carbon disulfide (CS2) and measured by FID-GC. The determination limits and relative standard deviations for VOCs were 0.3 microg/m3 and 3%, respectively. The samplers were set up at 5 sites in March, 2001 and at 13 stations on Mt. Hiei in November, 2002. The average concentrations of ambient benzene, which were higher than the environmental criterion (3.0 microg/m3), except for those on Mt. Daimonji from March, 2001, to February, 2002, decreased to below 3.0 microg/m3 from March, 2002, to February, 2003. The decrease in ambient benzene may have been due to a decrease in the benzene content in gasoline by the end of 1999, and also by implementation of the Pollutant Release and Transfer Register (PRTR) Act in 2001.

Determinations of humic substances and other dissolved organic matter and their effects on the increase of COD in Lake Biwa.

Humic substances and other dissolved organic matter (DOM) in Lake Biwa and the surrounding rivers were investigated to elucidate their origins and behavior. An annual increase in chemical oxygen demand (COD) has been observed in the northern basin of Lake Biwa since 1985. The concentrations of dissolved organic carbon (DOC) in the northern and southern basins of Lake Biwa were 1.7-2.4 mgC/l and 1.9-2.6 mgC/l, respectively. The DOC concentrations tended to be high in summer and low in winter, and the seasonal changes in the concentrations of humic substances were small. The humic substances content of DOM was considered to be comparatively small because the ratio of the concentration of humic substances to DOC was in the range of 0.14-0.32. From the results of the fractionation of DOM in lake waters, it was estimated that hydrophobic acids, such as humic substances and hydrophilic acids, were about 25% and 45%, respectively. The main origin of hydrophobic acids in Lake Biwa may be humic substances from soils around the rivers that flow into Lake Biwa, while hydrophilic acids may be due to the inner production by phytoplankton. Therefore, the increase of COD in the northern basin of Lake Biwa may be attributed to the contributions of not only humic substances but also hydrophilic acids.

Preconcentration of trace aluminum from environmental water samples with 5'-chloro-5-dodecyl-2,4,2'-trihydroxyazobenzene impregnated macroporous resin.

5'-Chloro-5-dodecyl-2,4,2'-trihydroxyazobenzene (CDTA) impregnated XAD-4 resin (CDTA resin) was prepared, and applied to the preconcentration of trace aluminum from environmental water samples with satisfactory results. Aluminum was measured by flow-injection analysis (FIA) with fluorescence detection by using lumogallion after the preconcentration of aluminum with CDTA resin. Fe3+ and other coexisting substances had no interference on the determination of aluminum by the present method. Chemical speciation of trace aluminum in environmental water was carried out by using the CDTA resin after the separation of aluminum species with ionic exchangers without changing the pH. About 50% of water-soluble aluminum in Lake Biwa was found to be aluminum organic species (anion species).

Speciation of aluminum in soil extracts using cation and anion exchangers followed by a flow-injection system with fluorescence detection using lumogallion.

Flow-injection analysis (FIA) with fluorescence detection of aluminum using lumogallion was applied to the chemical speciation of aluminum in soil extracts after the separation of aluminum species with ionic exchangers. Aluminum complexes with organic substances (anion species) can be specified from other species by using a strongly acidic cation exchanger in the pH range of 3 to 5. Furthermore, aluminum can be separated into three categories, namely, (i) the Al3+ and Al-OH complex, (ii) aluminum organic complexes (cation species), and (iii) its anion species by using a strongly acidic and a weakly acidic cation exchanger at around pH 5. A considerable percentage of water-soluble aluminum in soils was found to be complexes with humic substances.