Hans-Dietrich Lüdemann

N-15 and C-13 CPMAS and solution NMR studies of N-15 enriched plant material during 600 days of microbial degradation

Abstract Nitrogen-15 enriched plants ( Lolium perenne (rye grass) and Triticum sativum (wheat)) have been composted under controlled conditions up to 630 days. The composts were characterized by C-13 and N-15 CPMAS spectra. For some composts N-15 HR NMR solution spectra of the NaOH extracts were studied. The composts were characterized by weight loss and elementary analysis. Most of the nitrogen detectable is assigned to amide/peptide structures (80–90%), the remaining intensities being found in the chemical shift region of amino- and ammonium groups. Signals for nitrogen-containing heterocycles were not observed. Less than 5% of the intensity of the low field side of the main peptide/amide signal might be ascribed to indole/imidazole/uric acid derivatives. Combined analysis of C-13 CPMAS spectra, the elemental analysis and mass balance, yield the result that all chemical structures visible in the C-13 spectra are altered in the composting process, although at different rates.

13C- and 15N-NMR spectroscopic examination of the transformation of organic nitrogen in plant biomass during thermal treatment

Abstract Structural changes in lignocellulosic biomass heated under conditions comparable to those encountered in several types of natural or planned burnings have been studied by solid-state 13C- and 15N-CPMAS NMR spectroscopy of 15N-enriched ryegrass (Lolium rigidum) after being subjected to progressive thermal treatment. The solid-state 15N-NMR spectra of biomass subjected to severe heating revealed amide-N in forms which are resistant to the thermal treatment. Progressive burning was found to occur in two well-defined stages: In the early stage the free amino acid and some NH2 groups were removed, but no substantial disruption of the peptide structure was observed. In the final stage of burning the amide-N was converted to heterocyclic structures such as pyrroles, imidazoles and indoles. Some evidence for the presence of pyridines and phenazines was also found at this stage. These findings suggest that a major portion of the N is in forms that may survive most natural fires and that their stability towards further microbial degradation is increased by the heating. The solid-state 13C-NMR spectra revealed that the carbohydrate fraction is converted into condensed dehydrated material producing intense signals in the aromatic region.

The Chemical Nature of Nitrogen in Native Soil Organic Matter

H. Knicker, R. Frtind and H.-D. Ltidemann Institut for Biophysik und physikalische Biochemie der Universit~it, W-8400 Regensburg, FRG Fossil fuels and soil organic matter (SOM) together contain approximately five times more carbon than the biota and the atmosphere. Of this, soil organic matter accounts for about 30 % of the carbon present. In addition, SOM has an average carbon/nitrogen ratio of 10/1 and contains a huge fraction of the total ni- trogen available for plant growth [1]. Taking into account that the abundance of nitrogen in the earths crust is much lower than that of carbon, this is a signif- icant fraction of the total nitrogen acces- sible to the biosphere. Under natural soil conditions, without the addition of miner- al fertilizers, SOM provides the majority of the nitrogen necessary for plant growth. It is also thought to be respon- sible for the interaction between agricul- tural biocides and the soil [5-7]. The chemical structure of this ubiquitous material, SOM, and especially the chem- ical nature of the nitrogen are thus of great and general importance. The mo- lecular structure of the nitrogen- containing fraction is, however, still a matter of controversy [2-4]. Structural models based on partial chem- ical analysis claim that a significant part of the nitrogen is present in the form of heteroaromatic structures, while NMR- spectroscopic studies on lSN-enriched composts and recent humic material found approximately 85 % of the signal intensity in the amide/peptide region of chemical shift and no signals in the range typical for heteroaromatic nitrogen. A major fraction of the native soil organic matter has been in the soil for several hundred to several thousand years [8, 9]. Compared to these time spans, laboratory-produced material has been fermented for at most 1 year, and it could be argued that heteroaromatic structures are only produced after much longer fermentation periods. This criti- cism may be overcome by the study of lsN-CPMAS spectra of soil organic mat- ter with natural lSN levels. This has not been achieved hitherto, because the low natural abundance (0.4 %) and the small gyromagnetic ratio of the 15N nucleus and therefore its low sensitivity in NMR experiments appeared to make this experiment an impossible one. The most abundant 14N-isotope (99.6 %) cannot be studied by high-resolution NMR because its large nuclear quadrupole moment leads to very broad and unresolved signals, especially in solid-state NMR [101. In previous systematic studies on 15N- enriched composts and organic soil extracts [11] our group optimized all spectral parameters for the ~sN-CPMAS experiment. A crude estimate showed that it should be possible to obtain 15N spectra with a tolerable signal-to-noise ratio after the accumulation of approximately one million transients. In the present paper we report on the first successful results of such experiments. Six German soils were studied as detailed in Table 1. In Figs. 1 and 2 some of the spectra obtained are shown. They fully corrobo- rate the conclusions drawn from the stud- ies of short-term composting exper-

13C and 15N NMR analysis of some fungal melanins in comparison with soil organic matter

A variety of fungal melanins with natural 15N abundance are characterized by solid-state 13C and 15N NMR spectroscopy and are compared to solid-state 13C and 15N NMR spectra of organic matter from representative soils. In all solid-state 15N NMR spectra the peptide/amide region (−220 to −285 ppm) dominates with more than 70% of the total intensity. The region between −285 and −375 ppm, assigned to amino and ammonium groups, always contains more than half of the remaining intensity. The area in the region from −30 to −220 ppm, where aromatic heterocycles would show signals, makes up less than 10% of the total intensity. These findings call into question common structural models for melanins. The solid-state 13C NMR spectra, on the other hand, reveal large differences when the melanins are compared to each other, and to composts and soils. The concentration of the aromatic carbon varies from 5 to 40% in the melanin series. The ratio CaroNtot and CaliNtot were calculated, and confirm that nitrogen in these samples is bound in Ca-groups rather than in aromatic heterocyclic structures.

Transformation of lignin-related compounds with laccase in organic solvents

Abstract The extracellular laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) of Trametes versicolor was isolated from culture medium and immobilized by entrapment of the enzyme in a solvent-resistant hydrophilic matrix like Sepharose-CL-6B. The gel-enzyme association has been shown to be stable in water containing organic solvents. The efficiency of the immobilized laccase in different organic solvents was comparable with the activity shown in a buffered aqueous system. The immobilized laccase in organic solvents showed a good stability and a high tolerance to elevated temperatures. Water-insoluble organosolv lignin (OL), dissolved in dioxane/water, was readily converted by immobilized laccase from Trametes versicolor . The transformed lignin showed an increase in phenolic groups, changes in the quantity of conjugated elements, and a significant modification of both the aliphatic and aromatic carbon moieties of the lignin molecule. The changes in the lignin molecule were analyzed by UV-, IR-spectroscopy, and 13 C-NMR solid-state spectroscopy. High-performance size exclusion chromatography (HPSEC) of the lignin transformed with the laccase—Sepharose complex revealed a pronounced increase in weight-average molecular weight. Polymerization of the lignin in the organic solvent proved to be 4 times more effective than polymerization of the same compound in an aqueous system. Water-insoluble organosolv lignin as well as a variety of lignin-related aromatics, solubilized in dioxane-H 2 O (7 : 3), was readily converted by laccase preparation either in batch or in continuous flow column. Reaction of laccase with the solubilized lignin generates in the reaction media reduced oxygen species able to reduce the cytochrome c. For the first time it is now possible to perform enzymatically catalyzed reactions with lignin in an organic solvent. This is a first step towards an enzymatically derivatization of lignin, the formation of polymer blends on the basis of lignin by an enzymatically catalyzed reaction.

The density dependence of self-diffusion in some simple amines

Self-diffusion coefficients for monomethylamine and trimethylamine are given, taken in the temperature range between the melting pressure curve and 423 K at pressures up to 200 MPa. In the same T,p-range intradiffusion coefficients for an equimolar mixture of trimethylamine and ammonia were measured. For the evaluation of the diffusion data the densities of trimethylamine were determined between 273 and 373 K up to 200 MPa. These data permit the description of diffusion in this fluid by the rough hard sphere (RHS) model. The result of the data analysis of all systems studied under inclusion of the previously investigated ammonia is that hydrogen bonding has no influence upon the dynamics in any of these systems.

The self-diffusion and hydrogen bond interaction in neat liquid alkanols: a molecular dynamic simulation study

Self-diffusion of methanol, ethanol, 1-propanol and 2-propanol has been studied by molecular dynamics simulation in the temperature range between the melting pressure curve and 478 K at pressures up to 300 MPa. The simulation results on self-diffusion of methanol, ethanol and 2-propanol (for 2-propanol, at high temperatures) agree well with experiment, which suggests that the simulation method is a powerful tool to obtain self-diffusion coefficients over wide range of temperature and pressure, under which it is rather difficult for experiments. The local structures of methanol, ethanol and 2-propanol are investigated by calculating the radial distribution functions, H-bond numbers, coordination numbers and the ratios of H-bond number divided by coordination number. The correlation between self-diffusion and structural properties, and the influence of temperature and pressure on them are discussed. The degree of forming H-bond space network in methanol, ethanol and water is higher than that in 2-propanol, and they are all higher than those in ammonia and methylamine. The simulation results demonstrate that the effect of hydrogen bonding on the translational dynamics in methanol and ethanol is more pronounced than that in 2-propanol.

T,p Dependence of intradiffusion in binary fluid mixtures with carbon dioxide as one component

Intradiffusion coefficients Dii were measured by the pulsed field gradient NMR technique for five binary systems with carbon dioxide as one component, the second component being benzene, hydrogen, palmitic acid methyl ester, acetic acid or methanol. The studies are limited for most systems to low temperatures because of the onset of crystallisation. They are extended to a maximum pressure of 200 MPa. The concentration dependence of the hydrogen-containing mixtures shows, at the carbon dioxide rich side of the Dii s. x diagram, a fairly steep increase in diffusivity with hydrogen concentration. The analysis of the methanol/carbon dioxide system is complemented by studies of the hydroxyl proton chemical shift. The x, T, p dependence of the chemical shift is described by a linear association model. These data show clearly that the carbon dioxide molecule does not form any hydrogen bonds with the methanol hydroxyl group.

T,p-Dependence of the self-diffusion and spin–lattice relaxation in fluid hydrogen and deuterium

Self-diffusion coefficients D were studied for fluid hydrogen and fluid deuterium at pressures up to 200 MPa and in the temperature range 171–372 K by the spin echo method. Spin–lattice relaxation times of both nuclei were measured in the same p,T range. The diffusion coefficients D are described by the rough hard sphere (RHS) model invoking the rotation translation coupling parameter A = 1. Activation parameters are also derived. The dynamic isotope effect of D given by Dr = D(H2)/D(D2) is a function of p and T and varies from Dr≈1.1 at the lowest pressures and highest temperatures to Dr≈1.3 for the highest pressures and lowest temperatures reached. The spin–lattice relaxation is for both compounds dominated by the spin–rotation interaction. Only for T1(H2) at T = 171 K can a minor contribution from dipole–dipole relaxation be derived from the data.

Intradiffusion in binary fluids. The influence of dipoles and hydrogen bonding

Abstract Intradiffusion coefficients D ii of three binary fluid systems as function of temperature and pressure have been obtained by the pulsed field gradient spin echo technique. The nonpolar component for all three systems is tetramethylsilane (TMS). To this compound pivalonitrile (PN) was added in order to study the influence of large dipole moments upon translational mobility. The other two mixtures contain pivalic acid (PA) and 2,2-dimethyl-1-propanol (DMP). With these compounds the influence of hydrogen bonding was studied. The D ii of these systems were studied at pressures up to 200 MPa and between the onset of crystallization in the mixtures at the low temperature side and 423 K. For DMP/TMS analysis of the chemical shifts of the hydroxyl protons as function of c,T , and p yields information about the position of the hydrogen bond equilibria.