Determination of Poly-β-Hydroxybutyrate and Poly-β-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography

A convenient gas-liquid chromatography procedure to quantify poly-β-hydroxybutyrate and poly-β-hydroxyvalerate in activated sludge was developed by combining lyophilization of the samples, purification of the chloroform phase by water reextraction, and the use of capillary columns. With a flame ionization detector the sensitivity was estimated at 10⁻⁵ g/liter.     

Determination of Pyrrolnitrin and Derivatives by Gas-Liquid Chromatography

A gas-liquid chromatographic technique was applied to the separation of pyrrolnitrin and its derivatives. The simultaneous use of a flame detector and an electron capture detector made possible the distinction between the nitro derivatives of pyrrolnitrin and the other metabolites. The metabolites could be readily quantitated with the electron capture detector, offering a much more sensitive assay than the flame detector.     

Determination of Chloropicrin in Fumigants by Gas-Liquid Chromatography

The present time, chloropicrin (CP) is commonly used as the fumigant for stored grain insects, nematodes, soil fungus and weed seeds. The quantitative determination of CP has been performed by total chlorine method¹⁾, colorimetric method²⁾, and polarography³⁾. However, these methods require a great deal of trouble. In the previous paper, the author reported on the operating conditions and retention datas for the determination of CP by gas-liquid chromatography (GLC)⁴⁾. The method reported here is an application of this work for analysis of liquid soil fumigants.     

Determination of Steam-Volatile Organic Acids in Fermentation Media by Gas-Liquid Chromatography

Five gas chromatographic liquid phases (25% Carbowax 20 M plus 4% H₃PO₄, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC-296 [poly (diethylene glycol adipate)] plus 2% H₃PO₄) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C₂ to C₅ was accomplished by the Carbowax 20 M plus H₃PO₄, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H₃PO₄ separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H₃PO₄ proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C₂ to C₅, except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C₂ to C₅) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H₂SO₄ and refrigeration at 4 C in stoppered tubes.     

Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography

A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds.     

Analysis of Poly-beta-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-beta-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Identification of Vibrio cholerae by Pyrolysis Gas-Liquid Chromatography

Cholera-like vibrios examined by pyrolysis gas-liquid chromatography could be distinguished from other common aerobic gram-negative bacilli, including oxidase-positive organisms, e.g., Aeromonas. Vibrios in Heiberg group I were subdivided into three types on the basis of differences in one complex in the chromatogram, and these closely corresponded with the identification as classical, El Tor, or "intermediate" biotypes of Vibrio cholerae by conventional methods.     

Identification of Abscisic Acid in Tulipa gesneriana L. by Gas-Liquid Chromatography with Electron Capture and Combined Gas-Liquid Chromatography and Mass Spectrometry

A major growth inhibitory substance of tulip bulbs (Tulipa gesneriana L. cv Paul Richter) has been unequivocally shown to be abscisic acid (ABA). The ABA methyl ester of the free ether-soluble acid fractions of tulip organs had the identical retention time on gas-liquid chromatography with electron capture detector as authentic ABA methyl ester. In addition, the mass spectra were the same. On a unit dry matter basis, the basalplate and floral shoot contained 3.6 and 2.6 times more ABA than the fleshy scales, respectively.     

Virulence Prediction of Yersinia enterocolitica by Pyrolysis Gas-Liquid Chromatography

[This corrects the article on p. 648 in vol. 40.].     

Quantitation of 1,4-Benzoxazin-3-ones in Maize by Gas-Liquid Chromatography

A gas-liquid chromatographic (GLC) procedure is reported for the quantitation of the trimethylsilyl (TMS) derivatives of substituted 2-hydroxy-2H-1,4-benzoxazin-3(4H)-ones (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one[HBOA]; 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[HMBOA];2,4- dihydroxy-2H-1,4-benzoxazin-3(4H)-one[DIBOA]; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[DIMBOA]; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one[DIM ₂BOA]) found in maize (Zea mays L.) extracts. Derivatized samples were chromatographed on columns with liquid phases of 2% DC-11 and 3% OV-17 and detected by flame ionization. Internal standards were methyl palmitate and methyl stearate on DC-11 and methyl behenate on OV-17. Detector response was linear to at least 5 nanomoles for TMS₂-HBOA and TMS₂-DIBOA and to 19 nanomoles for TMS₂-DIMBOA. Standard errors of 2% or less were obtained when four replicate samples were analyzed. For each of the 15 maize lines examined, the amount of DIMBOA determined by GLC was directly proportional to the amount of ferric chloride-reactive material determined colorimetrically.     

活性污泥合成聚羟基脂肪酸脂的研究进展

聚羟基脂肪酸( PHAs) 是许多原核微生物在不平衡生长条件下合成的细胞内能量和碳源储藏性物质,同时也是一种可完全生物降解的塑料,由于其良好的环境效应及机械性能而受到广泛关注.使用活性污泥合成PHA既能降低PHA的生产成本,又能充分利用活性污泥资源,减少对环境的污染.综述了活性污泥合成PHA的研究进展, 包括合成PHA的主要微生物、碳源及影响PHA积累的因素.     

Biological Uptake of Phosphorus by Activated Sludge

The ability of activated sludge to remove phosphates was studied by adding carrier-free (32)P to raw sewage and measuring incorporation of the radioactivity into the cells over a period of time. Radioisotope determinations indicated that 48% of the (32)P radioactivity was removed by 12 hr. However, chemical methods indicated that only 30% of the orthophosphate apparently disappeared from the sewage during this period. Experiments with sludge prelabeled with (32)P indicated that considerable phosphate turnover occurred. The cells released large amounts of radioactivity as they were incorporating fresh phosphates. Starvation in isotonic saline for 18 hr caused the sludge to dump phosphate. When introduced into fresh sewage containing (32)P, the starved sludge removed about 60% of the radioactivity in 6 hr with little phosphate turnover. The ability of sludge to remove (32)P was inhibited approximately 83% by 10(-3)m 2,4-dinitrophenol. This inhibition was at the expense of the cell fraction that contained ribonucleic acid and deoxyribonucleic acid. The sludge cells released orthophosphate when exposed to the chemical agent. Experiments using (45)Ca indicated that calcium phosphate precipitation plays a minor role in phosphate removal under our experimental conditions.     

Lipid Patterns of Selected Microorganisms as Determined by Gas-Liquid Chromatography

The cellular lipid patterns of seven strains of microorganisms were examined by gas-liquid chromatography in this preliminary study. The chloroform methanol-soluble lipids were extracted by the Soxhlet method from dried cultures which had been grown at 25 +/- 2 C for 18 hr with mechanical shaking. The cellular extract was methylated by use of a low temperature sulfuric acid method, and the resulting methyl esters were chromatographed. Considerable differences in the lipid patterns among the seven microorganisms tested indicated that this method might be useful for the identification of closely related microbial genera, and possibly for species differentiation.     

Degradation of Mono-Fluorophenols by an Acclimated Activated Sludge

Acclimated activated sludge was examined for its ability to degrade mono-fluorophenols as the sole carbon source in aerobic batch cultures. The acclimated activated sludge degraded fluorophenol efficiently. It degraded 100 mg/l 3-fluoropheno and 4-fluorophenol in 16 h with, respectively, 99.85% and 99.91% fluoride anion release and it degraded 50 mg/l 2-fluorophenol in 15 h with 99.26% fluoride anion release. The aerobic biodegradability of the mono-fluorophenols decreased in the order: 4-fluorophenol > 3-fluorophenol > 2-fluorophenol, resulting mainly from a different octanol/water partition coefficient and different steric parameter of the fluorophenols. The mechanism study revealed that the initial step in the aerobic biodegradation of mono-fluorophenols by the activated sludge was their transformation to fluorocatechol. Following transformation of the fluorophenol to fluorocatechol, ring cleavage by catechol 1, 2-dioxygenases proceeded via an ortho-cleavage pathway, then defluorination occurred.     

Analysis of Streptococcal Cell Wall Fractions by Curie-Point Pyrolysis Gas-Liquid Chromatography

A streptococcal strain, classified as Z(3)III was differentiated from its mutant strain, Z(3), lacking the type III polysaccharide antigen, by Curie-point pyrolysis gas-liquid chromatography. Differences observed in pyrograms of whole cells or cell envelopes of both strains could be directly related to the pyrolysis pattern of the purified type III antigen. The same results were obtained when streptococcus F III and its mutant were analyzed. Whereas the pyrolysis patterns of the type III antigen extracted from Z(3)III and F III bacteria were identical, marked differences were found in pyrograms of the serologically identical type III antigen isolated from the culture medium. Type III antigen was also easily differentiated from the purified type I, II and IV antigens. From the above findings it was concluded that pyrolysis gas-liquid chromatography can be used as a tool for the quality control and identification of streptococcal cell wall components.     

Viscous Product from Activated Sludge by Methanol Fermentation

Aeration of activated sludge with 3 to 4% added methanol for 5 to 7 days yields an odorless, highly viscous (5,000 to 10,000 centipoise), black, pudding-like product containing glycan(s) linked other than α-1-4 or β-1-3. Backseeding gives maximum thickening in 3 to 4 days. Incomplete acid hydrolysis of the black product gives a 0.27% solution of reducing sugars (75% glucose) which is an 11.4% yield from the added methanol. Backseeding into either centrifuge supernatant or 0.1% yeast extract in tap water gives a light-colored polymer. Viscosity decreases during extended sterile cold storage. A 5% salt addition lowers viscosity one-half. From 6 to 12 colony types appear on plating backseeded media, but none of these isolates is a reliable polymer former.     

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.     

活性污泥产酸发酵研究进展

有机物的厌氧生物处理一般经过三个阶段:水解阶段、产酸发酵阶段和产甲烷阶段;研究证明,产酸相不同发酵类型的形成对产甲烷相乃至整个工艺的稳定运行具有至关重要的作用,此外,污泥厌氧消化过程所产生的大量的挥发性脂肪酸(VFAs),如乙酸、丙酸、丁酸及戊酸等,还可作为化工原料用于发酵工业生产各种高附加值产品.近年来,产酸发酵受到越来越多的关注,该文主要对污泥产酸阶段的产酸发酵类型、产酸发酵细菌的生态学、产酸过程的影响因素和生态因子以及产酸发酵的液相末端产物VFAs的测定方法进行了论述.     

Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H₂N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.