Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (Bacillus licheniformis) isolated from gut contents of earthworm (Metaphire posthuma) in environmental remediation.

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L-1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 µg mL-1) at 5 mg mL-1 L-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L-1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.

Response of recalcitrant soil substances to reduced N deposition in a spruce forest soil: integrating laccase-encoding genes and lignin decomposition.

A long-term field experiment conducted in a Norway spruce forest at Solling, Central Germany, was used to verify and compare the response of lignin-decomposing fungal communities in soils receiving current and preindustrial atmospheric nitrogen (N) input for 14.5 years. Therefore, we investigated the decomposition of lignin compounds in relation to phenol oxidase activity and the diversity of basidiomycetes containing laccase genes in organic and mineral horizons. Lignin-derived CuO oxidation products and enzyme activity decreased with soil depth, while the degree of oxidative transformation of lignin increased. These patterns did not change with reduced atmospheric N input, likely reflecting a lasting saturation in available N. The laccase gene diversity decreased with soil depth in spring. In autumn, this pattern was only found in the control plot, receiving current N input. Principal component analysis confirmed the depth profile and distinguished a response of the fungal community to reduced N deposition for most organic layers in spring and a roof effect for the Oe layer in autumn. These responses of the fungal community did not translate into changes in enzyme activity and lignin content and decomposition, suggesting that transformation processes in soils are well buffered despite the rapid response of the microbial community to environmental factors.

Improving bioreactor cultivation conditions for sensitive cell lines by dynamic membrane aeration.

Although the importance of animal cell culture for the industrial (large scale) production of pharmaceutical products is continuously increasing, the sensibility of the cells towards their cultivation environment is still a challenging issue. In comparison to microbial cultures, cell cultures which are not protected by a cell wall are much more sensitive to shear stress and foam formation. Reactor design as well as the selection of 'robust' cell lines is particularly important for these circumstances. Nevertheless, even 'sensitive' cell lines are selected for certain pharmaceutical processes due to various reasons. These sensitive cell lines have even higher requirements regarding their cultivation environment. Important characteristics for the corresponding reactor design are a high (volumetric) gas mass transfer coefficient, low volumetric power input, low shear stress, low susceptibility to bio-fouling, the ability to cultivate sticky cells and sufficient mixing properties. Membrane aeration has been a long-known possibility to meet some of these requirements, but has not often been applied in recent years. The reasons lie mainly in low gas mass transfer rates, a limited installable volume-specific membrane surface area, restrictions in scalability and problems with membrane fouling. The dynamic membrane aeration bioreactor aeration is a simple concept for bubble-free oxygen supply of such sensitive cultures. It overcomes limitations and draw-backs of previous systems. Consisting of an oscillating, centrally arranged rotor (stirrer) that is wrapped with silicone membrane tubing, it enables doubling the gas mass transfer at the same shear stress in the investigated cultivation scales of 12, 20, 100, and 200 L. Continuous cultivation at these scales allows the same product output as fed-batch cultivation does at tremendously larger reactor volumes. Apart from introducing this novel technology, the presentation comprises selected cultivation results obtained for blood coagulation factor VIII in continuous mode and a therapeutic monoclonal antibody in fed-batch mode in comparison to reference trials.

Isotope and phase effects on the proton tautomerism in polycrystalline porphycene revealed by NMR.

Using high resolution solid state (15)N and (2)H spectroscopy and longitudinal relaxometry we have studied the tautomerism of porphycene in the solid state, corresponding to a double proton transfer in two cooperative hydrogen bonds. The tautomerism is degenerate above 225 K but the degeneracy is lifted below this temperature, indicating a phase transition. Thus, the high-temperature phase is characterized by a dynamic proton disorder and the low-temperature phase by a dynamic proton order. (15)N magnetization transfer experiments obtained under cross polarization (CP) and magic angle spinning (MAS) conditions reveal the presence of two nonequivalent molecules A and B in the unit cell of phase II, exhibiting slightly different equilibrium constants of the tautomerism. Rate constants of the tautomerism in phase I could be obtained by the analysis of the longitudinal (15)N and (2)H relaxation times. The former, obtained at 9.12 MHz, exhibit a T(1) minimum around 270 K and are consistent with proton transfer induced dipolar (1)H-(15)N relaxation mechanism. The latter, obtained at 46.03 MHz, exhibit a minimum around 330 K and arise from quadrupole relaxation. Within the margin of error, the rate constants of the HH and of the HD/DD tautomerism are the same, exhibiting a barrier of about 30 kJ mol(-1), as expected for an overbarrier reaction in a configuration with two compressed hydrogen bonds. By contrast, in the low-temperature phase a switch of the DD transfer kinetics into the nanosecond time scale is observed, exhibiting a non-Arrhenius temperature dependence which is typical for tunneling. This increase of the rate constants by lowering the temperature is discussed in terms of a switch from a concerted HH transfer in phase I to a stepwise transfer in phase II, where intermolecular interactions lower the energy of one of the cis-intermediates.

Fatty acid patterns of genetically modified Cry3Bb1 expressing Bt-maize MON88017 and its near-isogenic line.

Fatty acid (FA) profiles of the Bt-maize line MON88017 expressing the Cry3Bb1 protein and its near-isogenic line DKC5143 were examined. Plant compartments under study included leaves taken from different internodes and roots. Sample preparation involved pressurized liquid extraction (PLE) of the biomass, transmethylation of the extracted lipids to give fatty acid methyl esters (FAMEs), and finally GC-MS analysis. The essential quality parameters for the FA profiles included total FA and sum of saturated FA, as well as double-bond index (DBI). FA profiles of the roots--characterized by high concentrations of homomorphic FA including palmitic and stearic acid, along with low concentrations of polyunsaturated surrogates--revealed high similarity between the genetically modified and the near-isogenic line. In contrast, FA profiles of the leaves showed significant differences: higher total FA concentrations and higher DBI were observed for the near-isogenic line. This was overwhelmingly associated with lower concentrations of alpha-linolenic acid (18:3omega3,6,9ccc) in the genetically modified leaf samples. These differences were particularly pronounced for leaves taken from the fourth elongated, above-ground internode. Given the large reported variability in the population of maize lines, MON88017 and its near-isogenic line can be regarded as equivalent with regard to their fatty acid profiles, despite the differences observed for the leaves. Further experiments are needed to assess whether the genetic modification of Bt-maize plants might induce unintended effects with regard to FA profiles.

Molecular level lignin patterns of genetically modified Bt-maize MON88017 and three conventional varieties using tetramethylammonium hydroxide (TMAH)-induced thermochemolysis.

Bt-maize MON88017, its near-isogenic line DKC5143, and the two conventional varieties DK315 and Benicia were subjected to tetramethylammonium hydroxide (TMAH)-induced thermochemolysis to reveal molecular level lignin patterns. MON88017 is genetically modified to express the Cry3Bb1 protein aimed at the Western corn rootworm Diabrotica virgifera virgifera, a serious threat for European maize production. The results indicated that roots of the Bt-maize were characterized by a slightly enhanced total lignin content (by approximately 7%) compared to the near-isogenic line, whereas the molecular-based patterns, expressed by the relative fractions of p-hydroxyphenyl, guaiacyl, and syringyl breakdown products (P-, G-, and S-units, respectively) were virtually identical for both lines. No effects regarding either total lignin or molecular-based lignin patterns could be observed for leaves, indicating that biogenesis of lignin was not pleiotropically affected by the genetic modification. Significant differences for both total lignin and different lignin proxies existed between the conventional maize lines. Molecular level lignin analysis by means of TMAH-induced thermochemolysis is able to distinguish conventional maize varieties. Further work is necessary to evaluate lignin-related pleiotropic effects in genetically modified maize plants. The validation and application of a commonly accepted method for lignin analysis, capable of characterizing lignin at the molecular level, is a prerequisite.

NMR studies of ultrafast intramolecular proton tautomerism in crystalline and amorphous n,n'-diphenyl-6-aminofulvene-1-aldimine: solid-state, kinetic isotope, and tunneling effects.

Using solid-state NMR spectroscopy, we have detected and characterized ultrafast intramolecular proton tautomerism in the N-H-N hydrogen bonds of solid N, N'-diphenyl-6-aminofulvene-1-aldimine ( I) on the microsecond-to-picosecond time scale. (15)N cross-polarization magic-angle-spinning NMR experiments using (1)H decoupling performed on polycrystalline I- (15)N 2 and the related compound N-phenyl- N'-(1,3,4-triazole)-6-aminofulvene-1-aldimine ( II) provided information about the thermodynamics of the tautomeric processes. We found that II forms only a single tautomer but that the gas-phase degeneracy of the two tautomers of I is lifted by solid-state interactions. Rate constants, including H/D kinetic isotope effects (KIEs), on the microsecond-to-picosecond time scale were obtained by measuring and analyzing the longitudinal (15)N and (2)H relaxation times of I- (15)N 2, I- (15)N 2- d 10, and I- (15)N 2- d 1 over a wide temperature range. In addition to the microcrystalline modification, a novel amorphous modification of I was found and studied. In this modification, proton transfer is much faster than in the crystalline form. For both modifications, we observed large H/D KIEs that were temperature-dependent at high temperatures and temperature-independent at low temperatures. These findings are interpreted in terms of a simple quasiclassical tunneling model proposed by Bell and modified by Limbach. We obtained evidence that a reorganization energy is necessary in order to compress the N-H-N hydrogen bond and achieve a molecular configuration in which the barrier for H transfer is reduced and tunneling or an over-barrier reaction can occur.

Patchiness and spatial distribution of laccase genes of ectomycorrhizal, saprotrophic, and unknown basidiomycetes in the upper horizons of a mixed forest cambisol.

Decomposition of plant litter by the soil microbial community is an important process of controlling nutrient cycling and soil humus formation. Fungal laccases are key players in litter-associated polyphenol degradation, but little is known about the diversity and spatial distribution of fungal species with laccase genes in soils. Diversity of basidiomycete laccase genes was assessed in a cambisolic forest soil, and the spatial distribution of the sequences was mapped in a 100-m(2) plot by using polymerase chain reaction (PCR) on soil DNA extracts. Diversity of laccase sequences was higher in the organic horizon and decreased with the depth. A total of 167 different sequences sharing 44-96% oligonucleotide similarity was found in 13 soil cores harvested in the 100-m(2) plot. Dissimilarity in laccase sequence content was 67% between adjacent cores; 45.5%, 35.5% and 19% of laccase sequences were attributed to ectomycorrhizal, unknown and saprotrophic basidiomycetes, respectively. Most dominant sequences were attributed to the extramatrical hyphae of known ectomycorrhizal taxa (e.g., Russulaceae) and restricted to small patches (<0.77 m(2)) in a specific soil horizon. Soil fungi with laccase genes occupied different niches and showed strikingly variable distribution patterns. The distribution of laccase sequences, and corresponding fungi, likely reflected a part of the oxidative potential in soils.

Molecular composition of leaves and stems of genetically modified Bt and near-isogenic non-Bt maize--characterization of lignin patterns.

Transformation of crops, including maize (Zea mays L.), with the cry1Ab gene from Bacillus thuringiensis to combat lepidopteran pests results in pleiotropic effects regarding lignin biosynthesis. Lignin patterns in stems and leaves of two genetically modified Bt-maize varieties (Novelis T and Valmont T) were studied along with their non-Bt near-isolines (Nobilis and Prelude, respectively). Molecular-level based thermochemolysis using tetramethylammonium hydroxide (TMAH) in combination with gas chromatography-mass spectrometry (GC-MS) was used to quantitate the total lignin contents and to identify monomeric lignin subunits including p-hydroxyphenyl (P), guaiacyl (G), and syringyl (S) moieties. The results were supplemented and confirmed by cupric oxide oxidation. The stems of the transgenic lines had higher concentrations of total lignin than the respective isogenic lines: Valmont T/Prelude by 18% and Novelis T/Nobilis by 28%. In contrast, differences in the total lignin concentration of leaves between the transgenic and the respective near-isogenic lines were marginal. There were significant modifications in the ratio of p-hydroxyphenyl/guaiacyl/syringyl molecular marker units of stem lignin between transgenic and isogenic lines. The guaiacyl units (in particular the G18 marker) accounted chiefly for the higher total lignin contents in the transgenic lines. The leaf lignin patterns did not show significant differences in molecular markers between isogenic and transgenic lines. TMAH-induced thermochemolysis--conducted in both the on-line and off-line modes--provided detailed information on the molecular composition of lignin, thus proving superior to the established "wet chemistry" methods of lignin determination.

6-Aminofulvene-1-aldimine: A Model Molecule for the Study of Intramolecular Hydrogen Bonds.

An unusually substantial coupling is observed across the hydrogen bond of fully 15 N-labeled compound 1 when it is studied by 1 H and 15 N NMR spectroscopy. The structure was determined by X-ray diffraction and shown to correspond to tautomer 1 a (both in the solid state and in solution). These results open up a new field of hydrogen-bond research by NMR spectroscopic methods.