15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid,model compounds,and lignocellulose

The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.     

N-15 NMR study of the immobilization of 2,4- and 2,6-dinitrotoluene in aerobic compost

Large-scale aerobic windrow composting has been used to bioremediate washout lagoon soils contaminated with the explosives TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) at several sites within the United States. We previously used 15N NMR to investigate the reduction and binding of T15NT in aerobic bench-scale reactors simulating the conditions of windrow composting. These studies have been extended to 2,4-dinitrotoluene (2,4DNT) and 2,6-dinitrotoluene (2,6DNT), which, as impurities in TNT, are usually presentwherever soils have been contaminated with TNT. Liquid-state 15N NMR analyses of laboratory reactions between 4-methyl-3-nitroaniline-15N, the major monoamine reduction product of 2,4DNT, and the Elliot soil humic acid, both in the presence and absence of horseradish peroxidase, indicated that the amine underwent covalent binding with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and non-heterocyclic condensation products. Liquid-state 15N NMR analyses of the methanol extracts of 20 day aerobic bench-scale composts of 2,4-di-15N-nitrotoluene and 2,6-di-15N-nitrotoluene revealed the presence of nitrite and monoamine, but not diamine, reduction products, indicating the occurrence of both dioxygenase enzyme and reductive degradation pathways. Solid-state CP/MAS 15N NMR analyses of the whole composts, however, suggested that reduction to monoamines followed by covalent binding of the amines to organic matter was the predominant pathway.     

Solid-state 2H MAS NMR studies of TNT absorption in soil and clays

The binding of TNT to a soil and soil components is studied with 2H MAS NMR. This NMR technique clearly shows different interactions for deuterated TNT with soil, quartz, and clays. Basically, this NMR method relies on the well-defined 2H MAS NMR characteristics of a deuterated methyl group attached to an aromatic ring. Stationary rings and rings executing 2- or 3-fold jump motions all yield different NMR spectra. The technique has good sensitivity; these studies were conducted at a TNT loading of 22 mmol/kg. For the soil sample studied, TNT showed binding similar to a TNT/K10-montmorillonite sample.     

Simplified identification of aerobic spore-formers in the investigation of foods

Bacillus spp. are among the main spoilage organisms in food due to their versatile metabolism and heat-resistant spores. Except for B. cereus, no specific identification method is available, although information about Bacillus spp. would be useful in monitoring good manufacturing practice. A simplified identification scheme for aerobic spore-formers for routine laboratory use was elaborated by reducing the number of tests to be performed; 20 Bacillus spp. common in foods were considered. Besides cell morphology six biochemical characters were selected that proved most efficient in identification. In addition, methods were developed and modified to determine these characters by only 3 tests. By applying the simplified identification scheme, 766 strains of bacilli isolated from various foods were tested and species identity of 714 strains (93%) could be determined. Most aerobic spore-formers from foods were B. subtilis, B. pumilus and B. licheniformis. The spectrum of mesophilic bacilli isolated from natural sources was broad and comprised some 20 different species. Only one-third of the thermophilic isolates proved to be identical with, or similar to, B. stearothermophilus, while the rest were identified as five other species. The simplified method merits main application in ecological studies of foods.     

Characterization of bacteriocin N15 produced by Enterococcus faecium N15 and cloning of the related genes

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.     

Alkaline hydrolysis/polymerization of 2,4,6-trinitrotoluene: characterization of products by 13C and 15N NMR

Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and 15N-labeled TNT were analyzed by elemental analysis and 13C and 15N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp3- and 86% sp2-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp3- and 67% sp2-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The 15N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to microbial or chemical degradation of the polymeric materials remain unknown.     

Formation and characterization of aerobic granules in a sequencing batch reactor treating soybean-processing wastewater

Aerobic granules were cultivated in a sequencing batch reactor (SBR) fed with soybean-processing wastewater at 25+/-1 degrees C and pH 7.0+/-0.1. The granulation process was described via measuring the increase of sludge size. The formation of granules was found to be a four-phase process, that is, acclimating, shaping, developing, and maturated. A modified Logistic model could well fit with the granule growth by diameter and could be employed to estimate the maximum diameter, lag time, and specific diameter growth rate effectively. Both normal and log-normal distributions proved to be applicable to model the diameter distribution of the granules. The granule-containing liquor was shear thinning, and their rheological characteristics could be described by using the Herschel-Buckley equation. The suspended solids concentration, pH, temperature, diameter, settling velocity, specific gravity, and sludge volume index all had an effect on the apparent viscosity of the mixed liquor of granules. The matured granules had fractal nature with a fractal dimension of 1.87+/-0.34. Moreover, 83% of matured granules were permeable with fluid collection efficiencies over 0.034. As compared to activated sludge flocs, the aerobic granules grown on the soybean-processing wastewater had better settling ability, mass transfer efficiency, and bioactivity.     

NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on soil components

Phosphorus-31 nuclear magnetic resonance (31P NMR) was used to follow the decomposition of chlorpyrifos (an organothiophosphate pesticide) adsorbed on soil, humic acid, partially hydrated kaolin clay, and partially hydrated montmorillonite clay at high concentration (typically 2-10 wt %). Solid-state 31P NMR (using magic-angle spinning and cross polarization or direct polarization) and liquid-solution 31P NMR of DMSO and acetone extracts indicate that chlorpyrifos is initially physisorbed, appearing by solid-state 31P NMR to exhibit significant motion on the molecular level, which results in almost liquidlike solid-state spectra. Over periods ranging from hours to years, the signals due to unreacted chlorpyrifos sorbed on the clays diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, mineralization, and oxidation reactions. The 31P NMR signal characteristics indicate that these decomposition products are much more tightly bound to the clay than is chlorpyrifos. Solid-state 13C and 27AI NMR spectra were less useful for following the decomposition of chlorpyrifos than those obtained by 31P NMR. Solid-state 31P NMR results indicate that a chlorpyrifos loading level of 10% by weight, used in some of the samples to facilitate 31P NMR detection of less-than-dominant decomposition products, exceeds the adsorption capacity of the soil, humic acid, and kaolinite tested, but not Ca2+ -exchanged montmorillonite. This pattern is consistent with intercalation into the montmorillonite, but only surface adsorption on kaolinite.     

NMR investigation of the behavior of an organothiophosphate pesticide, methyl parathion, sorbed on clays

The decomposition of methyl parathion (an organothiophosphate pesticide) sorbed on partially hydrated kaolin and montmorillonite clays (in Ca2+, Cu2+, Zn2+, and Al3+ forms) at high concentration (typically 1-10 wt %) has been examined by nuclear magnetic resonance (NMR), using solid-state 31P NMR (based on magic-angle spinning and cross polarization or direct polarization) and liquid-state 31P NMR of DMSO and acetone extracts. The results indicate that methyl parathion is initially physisorbed, appearing by solid-state 31P NMR to exhibit substantial molecular-level motion. The signals due to unreacted methyl parathion diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, and oxidation reactions over periods ranging from hours to years. 31P NMR characteristics indicate that these decomposition products are much more tightly bound to the clay than is methyl parathion. Methyl parathion decomposition is most effectively catalyzed by partially hydrated Cu(II)- and Al-montmorillonites (but with different product distributions); Ca-montmorillonite and kaolin were least effective.     

NMR investigation of the behavior of an organothiophosphate pesticide, chlorpyrifos, sorbed on montmorillonite clays

Phosphorus-31 nuclear magnetic resonance (31P NMR) was used to explore the decomposition of chlorpyrifos (an organothiophosphate pesticide) sorbed at high concentration (typically 2-10 wt %) on partially hydrated montmorillonite clays in four different cation-exchanged forms (Ca2+, Cu2+, Zn2+, and Al3+). Solid-state 31P NMR (using magic-angle spinning and cross polarization or direct polarization) and liquid-solution 31P NMR of DMSO and acetone extracts indicate that chlorpyrifos is initially physisorbed, appearing by solid-state 31P NMR to exhibit significant motion on the molecular level, which results in almost liquidlike solid-state spectra. Over periods ranging from hours to years, the signals due to unreacted chlorpyrifos diminish and are replaced by new 31P NMR peaks resulting from hydrolysis, isomerization, mineralization, and oxidation reactions. The 31P NMR signal characteristics indicate that these decomposition products are much more tightly bound to the clay than is chlorpyrifos. Partially hydrated Cu(II)- and Al-montmorillonites most effectively catalyzed chlorpyrifos decomposition (but with different product distributions); Ca-montmorillonites (and, as previously shown, kaolin) were least effective. Solid-state 13C and 27Al NMR spectra were less useful for following the decomposition of chlorpyrifos than those obtained by 31P NMR. Pesticide loading levels (1-10% w/w) that are very much higher than those typically found in the environment were used to facilitate 31P NMR detection of less-than-dominant decomposition species.     

Experimental investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

The enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and the enzymatic ATP regeneration were performed simultaneously in an ultrafiltration hollow-fiber reactor using both the purified enzymes and the crude cell extract of Bacillus stearothermophilus. The process consisted of the three-step synthetic reactions catalyzed by glucokinase, phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. The experimental results of the yield and the recycle number agreed well with the theoretical predictions calculated using a computer program developed in our preceding study. This proved the validity of this computer program for predicting or analyzing the reactor performance.     

Theoretical investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

The performance of an ultrafiltration hollow-fiber reactor, in which enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and enzymatic ATP regeneration are performed simultaneously, was analyzed theoretically. The reaction system consists of three-step synthetic reactions catalyzed by glucokinase (GK), phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. Based on a simple analytical model developed previously in which the liquid flowing in a tube was assumed to be plug flow and the radial concentration gradients in the tubes and shell side space were both neglected, a computer program was developed to calculate the concentration profiles of all the components along the flow direction in the tubes and shell side space of the reactor. From the FDP concentrations at the reactor outlet calculated under various operational conditions, reactor performances such as the FDP yield and the ATP recycle number were determined. The calculation showed the interesting phenomenon that under some conditions the FDP yield was higher when GK concentration was lower.     

Variation in the natural enrichment of 15N in apple trees and soils

Leaf, wood and soil materials taken from an established apple orchard contained ¹⁵N in excess of atmospheric levels. Enrichments were highest (0.380 atom%) in surface soil and lowest in leaves (0.376 atom%). Enrichments varied over the course of a single season, being generally higher in late summer (August) and autumn (November) than in June and July. The patterns of variation in enrichment suggested that assessments of tree to tree variation for leaves should be made; but time variation for surface soil would be more important.     

The influence of small‐ and large‐scale composting on the dissipation of pesticide residues in a biopurification matrix

BACKGROUND: Pesticides are efficiently retained and degraded in the organic matrix of a biopurification system. However, as this matrix mineralizes slowly over time, nutrients will start to become depleted and thus a decay in biomass will probably occur. At that moment, the efficiency of the system decreases and the matrix should be replaced. The spent matrix might still contain residues of pesticides. Hence treatment of this matrix is essential. In this study we opted to use composting or incubation as an effective and environmentally friendly treatment strategy. RESULTS: Small‐ and large‐scale composting/incubation trials were set up to treat the presence of linuron, bentazone, metalaxyl and bifenthrin in a contaminated matrix. Large‐scale composting, performed in an industrial composting facility, resulted in decreased concentrations of metalaxyl, linuron and bentazone. Degradation of bifenthrin was very limited. In the small‐scale incubation process, a decrease in concentration was noted for bifenthrin, metalaxyl and bentazone. A reduction in extractable pesticide concentration does not, however, always indicate degradation but could be attributed to the formation of non‐extractable residues. CONCLUSION: Industrial and small‐scale composting/incubation reduced the concentration of some pesticides during the timeframe studied, although little reduction was obtained for the persistent pesticide bifenthrin in the industrial composting process and for linuron in barrel incubation. Copyright © 2010 Society of Chemical Industry     

Start-up, microbial community analysis and formation of aerobic granules in a tert-butyl alcohol degrading sequencing batch reactor

We demonstrate that compact well-settling aerobic granules can be developed in a sequencing batch reactor (SBR) for the biological removal of tert-butyl alcohol (TBA) using a strategy involving step increases in TBA loading rate achieved through increasing TBA concentrations in the influent. A moderate selection pressure that included a cycle time of 24 h and a start-of-cycle TBA concentration of 100 mg/L was initially introduced to encourage the growth and retention of biomass and avoid biomass loss from hydraulic washout. Start-of-cycle TBA concentrations were increased to 150, 300, 450, and 600 mg/L on days 90, 100, 121, and 199, respectively. These increases were only introduced after complete TBA removal was accompanied by visible improvements in biomass concentration and biomass settling ability. This acclimation strategy produced incrementally higher biomass concentrations and better settling biomass with higher specific TBA biodegradation rates. Effluent TBA concentrations were consistently below the detection limit of 25 microg/L. Aerobic granules were first observed about 180 days after reactor start-up. The granules had a clearly defined shape and appearance, settled significantly faster than the suspended sludge in the reactor, and eventually became the dominant form of biomass in the reactor. The adapted granules were capable of complete TBA removal and contained a stable microbial population with a low diversity of sequences of community 16S rRNA gene fragments. This study indicates that it is possible to use aerobic granules for TBA remediation and will contribute to a better understanding of how microbial acclimation can be exploited in the SBR to biologically remove recalcitrant xenobiotics.     

A comparison of the labeled magnitude (LAM) scale,an 11-point category scale and the traditional 9-point hedonic scale

Schutz and Cardello [Schutz, H. G. & Cardello, A. V. (2001). A labeled affective magnitude (LAM) scale for assessing food liking/disliking. Journal of Sensory Studies, 16, 117–159] proposed the labeled magnitude (LAM) scale for measuring food acceptance. The LAM is a line scale anchored at its end points with the phrases “greatest imaginable like” and “greatest imaginable dislike” and uses as intermediate anchors the nine phrases of the traditional hedonic scale. In this study, three hedonic scales were compared, including the widely-used 9-point hedonic scale, the LAM scale, and an 11-point category scale using the LAM’s verbal anchors as category labels. Three groups of consumers (N = about 100 each) used one of the three scales to evaluate the acceptability of highly liked foods (orange juices, potato chips, cookies, and ice cream, with four samples of each). Scales were evaluated primarily on their ability to show differences in acceptability, the correspondence of acceptance ratings to preference ranking and the correspondence of stated product usage (e.g., purchase of pulp vs. non-pulp orange juice) to the product scoring highest. All three scales performed equally well, with no one scale showing a consistent superiority over another. All three scales were able to differentiate acceptability of the orange juices, chips and cookies. No scale differentiated among the ice creams, which had equal and high acceptability. All scales showed a strong correspondence between liking and preference rankings and also between the product rated highest and the type of product usually consumed, within each of the product categories.     

Distribution of 15N in amino acids during 15N-leucine infusion: impact on the estimation of endogenous flows in dairy cows

The distribution of ¹⁵N in AA during [¹⁵N]Leu infusion and its impact on the estimation of endogenous nitrogen (EN) flows in dairy cows was evaluated in 4 lactating cows equipped with ruminal, duodenal (n = 4), and ileal (n = 2) cannulae fed a silage-based diet during a 35-d experimental period. To label EN, starting on d 27, an infusion of L-[¹⁵N]Leu (0.45 mmol/h) was performed for 200 h. Samples of feed, duodenal and ileal digesta, feces, blood, urine, and mucosa of the rumen and duodenum were taken at 0900, 1100, 1300, and 1500 h on d 34 and at 0800, 1000, 1200, and 1400 h on d 35. The enrichment and fluxes of total N and individual AA were determined and used to calculate the EN flows at the duodenum, ileum, and in the feces. Based on the concept that EN comprises desquamation and secretions, EN flows were estimated, using as representative of the enrichment of EN only the enrichment of the gut mucosa (upper limit) or the average of the mucosa and the export protein enrichment (assumed to have a similar enrichment to casein; lower limit). Estimations of duodenal and fecal EN flows using the isotope dilution of ¹⁵N-total and ¹⁵N-Leu were not different and EN was an important fraction of duodenal and fecal flows, representing 14 to 30% of the duodenal flow and 18 to 31% of the fecal flow, depending on the dilution method used. The total EN flow at the duodenum is present in approximately equal proportions as either free EN or EN incorporated into bacterial protein. Ileal EN flow was 18% greater than the fecal EN flow. Using the combination of the gut and export protein, the duodenal and fecal EN flows estimated with the isotopic dilution of Leu vs. other labeled AA were less different than when estimated using the enrichment of gut mucosa alone. The current approaches have highlighted that present prediction schemes probably underestimate EN flows at the duodenum and, in consequence, overestimate net protein and AA supply. Refinement of the procedures may allow direct and accurate estimation of metabolic fecal protein, an important component of the so-called maintenance requirement of dairy cows.     

Microbial communities in the garbage composting with rice hull as an amendment revealed by culture-dependent and -independent approaches

The diversity and succession of microbial communities during the garbage composting with rice hull as an amendment were studied by denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal primers. Based on temperature changes, the composting process could be divided into thermophilic, cooling-down, and maturing stages. The DGGE profiles and clone library analysis revealed that the microbial community drastically changed during the composting process from the thermophilic to the maturing stages. The dominant bacterial group changed from the phylum Firmicutes in the thermophilic stage to the phylum Bacteroidetes in the maturing stage. This change in microbial communities may be significant for the composting process. The diversity of cultivated bacteria isolated from samples taken at various stages of the composting process was low. A total of 87 isolates were classified as belonging to only four different groups. These groups were also detected in the DGGE profiles and by the clone library analysis. Our study indicated that a combination of culture-dependent and -independent approaches could be very useful for monitoring both bacterial diversity and the succession of communities during the composting process. This study would be beneficial for assessing the ecological consequences of disposal of organic waste.     

烤烟对饼肥和秸秆肥中15N的吸收与利用

应用15N同位素示踪技术,研究了饼肥和秸秆肥与无机肥配比及不同施肥方法条件下,烤烟对饼肥和秸秆肥中氮素的吸收与利用规律。结果表明:①烤烟对饼肥和秸秆肥中15N素的吸收基本符合“S”型曲线,以25%饼肥 75%硝酸铵和25%秸秆肥 75%硝酸铵全部作基肥的处理,烟株对肥料中氮素的吸收利用率较高,分别达到94.31%和59.58%;②烤烟根系从饼肥和秸秆肥中吸收的氮素首先供应营养器官中生长最旺盛的部位———顶,然后是上部叶和下部叶,茎中的氮素供应较少,根中最少;③烟株从饼肥和秸秆肥中吸收的氮素在烟株各器官中的分布为:顶>上部叶>中部叶>下部叶>茎>根,且以25%饼肥 75%硝酸铵和25%秸秆肥 75%硝酸铵全部作基肥处理的烟叶氮含量较适宜。