15N Kinetic Analysis of N2O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of ¹⁵N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the “denitrifying enzyme” nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% ¹⁴N) and 0.4 mM nitrite (99% ¹⁵N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.     

Simultaneous Determination of Patulin and Penicillic Acid in Grains by Gas Chromatographic Method

Changes in aggregation and properties of alkali-treated soybean 7S and 11S globulins depend upon protein concentration during alkali-treatment. Such variables were investigated by viscosity, electrophoresis, circular dichroism, pulsed NMR, emulsion capacity and CaCl₂ precipitation measurements. In lower protein concentrations, the intrinsic viscosity decreased and the penetrative fractions into electrophoresis gel increased. The reduced contacts of proteins during neutralization resulted in smaller aggregates. Also specific fractions which were more sensitive to protein concentration on aggregation were observed for 11S globulin. The quantity of bound water depended only on pH at 7% concentration treatment. When the gel was formed, the bound water of protein increased, e.g., 0.085 g and 0.135 g/g protein at pH 10.6 and 13.2 treatment, respectively, whereas at 1% treatment, bound water showed almost no pH dependence (about 0.13 g/g protein). Furthermore, proteins prepared at higher protein concentrations were characterized by higher emulsion capacity and CaCI₂ precipitation ability. However, no protein concentration dependence was seen in the secondary structure of the aggregates.     

Determination of 15N Abundance in Nanogram Pools of NO3- and NO2- by Denitrification Bioassay and Mass Spectrometry

Suspensions of two strains of Pseudomonas aeruginosa (ON12 and ON12-1) were used to reduce NO₃^- and NO₂^-, respectively, to N₂O. The evolved N₂O was quantified by gas chromatography with electron capture detection, and the ¹⁵N abundance was determined by mass spectrometry with a special inlet system and triple-collector detection. Sample gas containing unknown N₂O pools as small as 0.5 ng of N was analyzed by use of a spike technique, in which a reference gas of N₂O of natural ¹⁵N abundance was added to obtain enough total N for the mass spectrometer. In NO₃^- or NO₂^- pools, the ¹⁵N abundance could be determined in samples as small as approximately 3.5 ng of N. No cross-contamination took place between the NO₃^- and NO₂^- pools. The excellent separation of NO₃^- and NO₂^- pools, small sample size required, and low contamination risk during N₂O analysis offer great advantages in isotope studies of inorganic N transformations by, e.g., nitrifying or denitrifying bacteria in the environment.     

Determination of Reductones by High-Pressure Liquid Chromatographic Method

Asaccharobacter celatus AHU1763 is a Gram-positive, obligate anaerobic, non-spore forming, rod-shaped bacteria that was successfully isolated from rat cecal content. Daizein was converted to equol via dihydrodaidzein by this bacterium. A crude enzyme that converted daidzein to dihydrodaidzein was detected mainly in the culture supernatant. The ability of this enzyme dropped after the culture supernatant was exposed to a normal atmospheric environment for even 5 min. Furthermore, the enzyme responsible for changing dihydrodaidzein to equol was detected mainly in the cell debris, which required anaerobic conditions for its activity.     

Gas Chromatographic Determination of Phytol in Plant Material

Phytol can be gas chromatographed without decomposition on an all-glass column with 2% Hyprose SP-80 on Chromosorb W; is stationary phase. Utilizing this a technique has been developed by which phytol of plant origin can be quantitatively determined. The practical limit of sensitivity is about 0.02 μg of phytol.     

Field evaluation of N2-fixation and N-utilization by phaseolus bean varieties determined by15N isotope dilution*

Summary Differences in N₂-fixation byPhaseolus vulgaris bean cultivars were successfully evaluated in the field using¹⁵N isotope dilution technique with a non-fixing test crop of a different species (wheat). The Phaseolus cultivars could have been similarly ranked for N₂-fixation capacity from either seed yield or total nitrogen yield, but the isotope method provided a direct measure of N₂-fixation and made it possible to estimate the proportion of fixed to total nitrogen in the crop and in plant parts. Amounts of nitrogen fixed varied between 24.59 kg N/ha for the 60-day cultivar Goiano precoce to 64.91 kg N/ha for the 90-day cultivar Carioca. The per cent of plant nitrogen due to fixation was 57–68% for the 90-day cultivars and 37% for Goiano precoce (60-day cultivar). Fertilizer utilization was 17–30% of a 20 kg N/ha fertilizer application. 100 kg N/ha fertilizer application decreased N₂-fixation without suppressing it totally. Differences in yield between the highest yielding (Carioca) and the lowest (Moruna) 90-day cultivars were also due apparently to varietal differences in efficiency of conversion of nitrogen to economic matteri.e. seed, as well as to differences in capacity of genotypes for N₂-fixation. The work described here was in part supported by IAEA Research Contract No. RC/2084 UNDP/IAEA Project BRA/78/006     

N Kinetic Analysis of N(2)O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the "denitrifying enzyme" nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% N) and 0.4 mM nitrite (99% N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.     

^15N标记蛋白GAL4（62）的2D NMR实验

利用自编的脉冲程序,采用预饱和和自旋锁定对水峰进行双重抑制的方法,得到了＾１５Ｎ标记蛋白ＧＡＬ４（６２）的２Ｄ＾１Ｈ－＾１５ＮＨＳＱＣ、ＨＳＱＣ－ＮＯＥＳＹ、ＨＳＱＣ－ＴＯＣＳＹ谱,并对这几个谱在蛋白质＾１Ｈ谱的归属中所到的作用作了讨论。     

Gas Chromatographic Analysis of Volatile Esters in Wines

We isolated a new aerobic gram negative collagenolytic bacterium, strain CRZV₁. This strain is yellow pigmented, non motile; it poorly degrades the sugars and its proteolytic activity corresponds to one enzyme, a collagenase which is produced in media with collagen, or collagen like substrates. The strain CRZV_l, presents many similarities with Flavobacteria. However, the GC content of the DNA, which is 65%, excludes our collagenolytic strain from the genus Flavobacterium, where all the species have GC content between 30 and 42%. All the strains, already described as Flavobacteria, the GC contents of which are situated between 55 and 70%, are actually unclassified. They could be included in the genus Empedobacter. This genus is not in the approved list of bacterial names, so we only consider this new collagenolytic bacteria as a gram negative, aerobic, yellow pigmented bacteria.     

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25°C), stoppered bottles incubated and sampled at 37 to 85°C showed more methane upon analysis than bottles incubated at 25°C: values at 50, 63, and 85°C were 109, 126, and 125%, respectively, of the 25°C value. All variation between 4 and 50°C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63°C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.     

Gas Chromatographic Separation and Determination of Optical Isomers of Chrysanthemic Acid

Separation and determination of the optical isomers of chrysanthemic acid has been carried out by gas chromatography. The diastereoisomeric esters of chrysanthemic acid with l-menthol were separated on a column of 2% QF-1 coated on Chromosorb W. d-trans, l-trans and dl-cis Chrysanthemic acids were resolved but d-cis and l-cis acids were not separatable from one another on any column tested. These isomers of chrysanthemic acid were not isomerized during esterification and gas chromatographic operation, and their ratios were determined from their peak area ratios. The l-bornyl esters of the isomers of chrysanthemic acicf were not so easily separatable as the l-menthyl esters.     

Rapid Gas Chromatographic Determination of Micro-quantities of N-Methyl Carbamates as their 2, 4-Dinitrophenyl Derivatives

A rapid and sensitive method is presented for the determination of micro-quantities (1 to 100 μg) of N-methyl carbamates as dinitrophenyl methylamine (DNP-MA) by electron capture gas liquid chromatography (GLC). The method described is characterized by rapidity and simplicity of procedure due to an improvement made in the present investigation, i.e., hydrolysis of an N-methyl carbamate and dinitrophenylation of the resulting methylamine with 2, 4-dinitro-1-fluorobenzene (DNFB) were accomplished simultaneously in a single reaction mixture. A novel approach was also made to eliminate unreacted DNFB by conversion to dinitrophenyl glycine (DNP-glycine).     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

Detection of Australian Field Isolates of Citrus Tristeza Virus by Double-Stranded RNA Analysis

Double-stranded RNA analysis indicated that widespread latent infection by citrus tristeza virus (CTV) occurs naturally in a citrus variety collection at Merbein, Australia. Detection was based on the presence of the putative replicative form (RF) of the virus (ca 19. 5 kilobase pairs) and the presence or absence of three previously known putative subgenomic dsRNAs with molecular sizes of ca 3. 1,1.7 and 0.8 kilobase pairs. With the exception of some citrus relatives normally used as rootstocks, the dsRNAs were readily detectable in all the trees tested. Variability was noticed among the dsRNAs profiles of individual seedling trees of Microcitrus australasica var. sanguinea. By contrast, dsRNA patterns of trees of open-pollinated Ellendale tangor were indistinguishable. A number of dsRNA bands found in West Indian lime infected with CTV were not detected when this isolate was graft-inoculated to healthy sweet orange seedlings. A simple method for enhancing the sensitivity of detection of the CTV dsRNA bands by silver staining is described which involves incubating the gels in RNase A prior to staining; this allowed detection of the putative CTV RF in only 40 mg of green hark material.     

Nitrification and Denitrification in Lake and Estuarine Sediments Measured by the 15N Dilution Technique and Isotope Pairing

The transformation of nitrogen compounds in lake and estuarine sediments incubated in the dark was analyzed in a continuous-flowthrough system. The inflowing water contained ¹⁵NO₃^-, and by determination of the isotopic composition of the N₂, NO₃^-, and NH₄⁺ pools in the outflowing water, it was possible to quantify the following reactions: total NO₃^- uptake, denitrification based on NO₃^- from the overlying water, nitrification, coupled nitrification-denitrification, and N mineralization. In sediment cores from both lake and estuarine environments, benthic microphytes assimilated NO₃^- and NH₄⁺ for a period of 25 to 60 h after darkening. Under steady-state conditions in the dark, denitrification of NO₃^- originating from the overlying water accounted for 91 to 171 μmol m^-2 h^-1 in the lake sediments and for 131 to 182 μmol m^-2 h^-1 in the estuarine sediments, corresponding to approximately 100% of the total NO₃^- uptake for both sediments. It seems that high NO₃^- uptake by benthic microphytes in the initial dark period may have been misinterpreted in earlier investigations as dissimilatory reduction to ammonium. The rates of coupled nitrification-denitrification within the sediments contributed to 10% of the total denitrification at steady state in the dark, and total nitrification was only twice as high as the coupled process.     

Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance

Nitrogen (N) is often the most limiting nutrient in organic cropping systems. N₂ fixing crops present an important option to improve N supply and to maintain soil fertility. In a field experiment, we investigated whether the lower N fertilization level and higher soil microbial activity in organic than conventional systems affected symbiotic N₂ fixation by soybean (Glycine max, var. Maple Arrow) growing in 2004 in plots that were since 1978 under the following systems: bio-dynamic (DYN); bio-organic (ORG); conventional with organic and mineral fertilizers (CON); CON with exclusively mineral fertilizers (MIN); non-fertilized control (NON). We estimated the percentage of legume N derived from the atmosphere (%Ndfa) by the natural abundance (NA) method. For ORG and MIN we additionally applied the enriched ¹⁵N isotope dilution method (ID) based on residual mineral and organic ¹⁵N labeled fertilizers that were applied in 2003 in microplots installed in ORG and MIN plots. These different enrichment treatments resulted in equal %Ndfa values. The %Ndfa obtained by NA for ORG and MIN was confirmed by the ID method, with similar variation. However, as plant growth was restricted by the microplot frames the NA technique provided more accurate estimates of the quantities of symbiotically fixed N₂ (Nfix). At maturity of soybean the %Ndfa ranged from 24 to 54%. It decreased in the order ORG > CON > DYN > NON > MIN, with significantly lowest value for MIN. Corresponding Nfix in above ground plant material ranged from 15 to 26 g N m^-2, with a decreasing trend in the order DYN = ORG > CON > MIN > NON. For all treatments, the N withdrawal by harvested grains was greater than Nfix. This shows that at the low to medium %Ndfa, soybeans did not improve the N supply to any system but removed significant amounts of soil N. High-soil N mineralization and/or low-soil P availability may have limited symbiotic N₂ fixation.     

Gas Chromatographic Analysis of Furfural and Hydroxymethyl-furfural in Wine

The thermal denaturation of 7S globulin has been examined as a function of ionic strength from 0 to 4.0 by using immunochemical methods, disc electrophoresis and turbidity. Changes in immunochemical properties were a measure of the extent of denaturation and provided information on the structural changes in 7S globulin at the molecular level. The 7S globulin did not lose its antigenicity even after heating, and both dissociates and aggregates maintained partial cross reactivity against anti-7S. The nature of thermal denaturation was divided into two main types at an ionic strength of approximately 2.0. At an ionic strength near zero, stable dissociation products were actually formed, and at ionic strengths from 0.1 to 2.0, the presence of salts increased in aggregate formation. The stabilizing effect of salt on thermal denaturation appeared at 2.0 ~ 2.2 μ. In the range of 3.5 ~ 4.0 μ, 7S globulin was heated to 100°C for 5min without changes in both antigenic specificity and electrophoresis pattern.