Measurement of biological parameters during fermentation processes

Efficient fermentation control requires the measurement of biological parameters. Three techniques were tested for monitoring. In the first, the NADH-fluorescence of micro-organisms was measured in batch and in continuous cultures under aerobic and anaerobic conditions, providing information on the metabolic status of the cells. The effects of cell concentration and of different substrates (glucose, ethanol and oxygen) were studied. The second technique is the calorimetric determination of various substrates, such as penicillin or enzymes, by an enzyme/thermistor device. With immobilized penicillin acylase (E.C. 3.5.1.11) or penicillinase (E.C. 3.5.2.6), penicillin was determined selectively in a fermentation broth. The thermistor was also used to measure penicillin acylase activity. The third technique is laser flow cytometry. A commercial double-beam flow cytometry system was used to determine cell size, light scattering and the protein, DNA and RNA contents of single cells. Flow cytometry allows rapid and sensitive control of fermentation processes with genetically modified E. coli 5K (pHM12) cells. The results of monitoring the cell size, light scattering, and protein and DNA contents of different micro-organisms during fermentation are outlined.     

You cannot fight the pressure: Structural rearrangements of active pharmaceutical ingredients under magic angle spinning

Although solid-state nuclear magnetic resonance (NMR) is a versatile analytical tool to study polymorphs and phase transitions of pharmaceutical molecules and products, this work summarizes examples of spontaneous and unexpected (and unwanted) structural rearrangements and phase transitions (amorphous-to-crystalline and crystalline-to-crystalline) under magic angle spinning (MAS) conditions, some of them clearly being due to the pressure experienced by the samples. It is widely known that such changes can often be detected by X-ray powder diffraction (XRPD); here, the capability of solid-state NMR experiments with a special focus on ¹H-¹³C frequency-switched Lee–Goldburg heteronuclear correlation (FSLG HETCOR)/MAS NMR experiments to detect even subtle changes on a molecular level not observable by conventional 1D NMR experiments or XRPD is presented. Furthermore, it is shown that a polymorphic impurity combined with MAS can induce a crystalline-to-crystalline phase transition. This showcases that solid-state NMR is not always noninvasive and such changes upon MAS should be considered in particular when compounds are studied over longer time spans.     

The use of an automatic on-line system for monitoring penicillin cultivation in a bubble-colum loop reactor

Penicillium chrysogenum is cultivated by a fed-batch mode to produce penicillin V. During fermentation, the concentrations of the medium components must be held at predetermined levels, which will change during fermentation, e.g., in the growth phase the concentration of the carbon sources and the nitrogen sources (urea and ammonium) must be high enough to maximize biomass production, whereas in the production stage these sources should be limited. To achieve optimal substrate concentrations, continuous measurement of various components in the fermentation broth is necessary. This is done by using a sterilizable ultrafiltation sampling probe and an air-segmented automatic flow analysis system to determine reducing sugars, dissolved organic carbon, ammonium, urea, sulfate, phosphate and penicillin V concentrations; spectrophotometric and gas-sensing electrodes are used in order to guarantee dependable results throughout the 290-h fermentation process, the analysis system is automatically cleaned and calibrated, and blanks are determined. The results are stored and evaluated by computer.     

Aggregation of donor base stabilized 2-thienyllithium in a single crystal and in solution: distances from X-ray diffraction and the nuclear Overhauser effect

Various 2-thienyllithium derivatives were investigated in the solid state by X-ray diffraction and in solution by 2D NMR experiments. The determined structures of [(Et(2)O)Li(C(4)H(3)S)](4) (1), [(THF)(2)Li(C(4)H(3)S)](2) (2), [(DME)Li(C(4)H(3)S)](2) (3), [(TMEDA)Li(C(4)H(3)S)](2) (4), and [(PMDETA)Li(C(4)H(3)S)] (5) (DME = 1,2-dimethoxyethane, TMEDA = N,N,N',N'-tetramethylethylene-1,2-diamine, and PMDETA = N,N,N',N",N"-pentamethyldiethylenetriamine) were solved in nondonating toluene and provide firm ground for diffusion-ordered NMR spectroscopy as well as heteronuclear Overhauser enhancement NMR spectroscopy. The distance relation of nuclear Overhauser effects with a factor of r(-6) is employed to gain further insight into the aggregation degree of 1-5 in solution. Comparison of the slope provided by the linear region of the buildup curves and of the ∑r(-6) calculated distances from the crystal structures offers a handle to judge the structure retention versus conversion in solution. The structures of 3-5 are maintained in toluene solution. The data of 2, however, indicate a partial dissociation or a rapid exchange between the vertices of a tetrameric core and free THF molecules. Auxiliary exchange spectroscopy investigations showed that the signals of the nitrogen donor base containing compounds 4 and 5 exchange with the signals of nonlithiated thiophene. This is explained by exchange of the deuterium by a hydrogen atom via lithiation of toluene molecules.     

Lasers for materials processing: specifications and trends

An overview is given of the types of lasers dominating the field of laser materials processing. The most prominent lasers in this field are the CO₂ and the Nd: YAG laser. The domain of CO₂ lasers is applications which demand high laser powers (up to 30 kW are available at present), whereas the domain of Nd:YAG lasers is micro-machining applications. In the kilowatt range of laser output power, the two types of lasers are in competition. New diffusion-cooled CO₂ laser systems are capable of output laser powers of several kilowatts, with good beam qualities, while still being quite compact. The output power and beam quality of Nd:YAG lasers has been improved in recent years, so that Nd:YAG lasers are now an alternative to CO₂ lasers even in the kilowatt range. This is especially true for applications that demand optical fibre transmission of the laser beam, which is possible with Nd:YAG laser light but not with the longerwavelength light emitted by CO₂ lasers. The main problem in solid-state lasers such as Nd:YAG is the thermal lensing effect and damage due to thermal stresses. In order to reduce thermal loading, cooling has to be enhanced. Several alternative geometries have been proposed to reduce thermal loading and, by this, thermal lensing effects. There are now slab and tube geometries which allow much higher output powers than the conventionally used laser rods. A very new scheme proposes a thin slab whose cooled side is also used as one of the laser mirrors, so that thermal gradients occur mainly in the direction of the beam propagation and not perpendicular to it, as is the case in the other geometries. As well as CO₂ and Nd:YAG lasers, semiconductor laser diodes are very promising for direct use of the emitted light or as pump sources for Nd:YAG and other solid-state lasers. When packaging together thousands of single laser diodes, output powers of several kilowatts can be realized. Major problems are collimation of the highly divergent laser beams and cooling of the laser diode bars.     

The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data

A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NMR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NMR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis.     

Pair distribution function and 71Ga NMR study of aqueous Ga3+ complexes

The atomic structures, and thereby the coordination chemistry, of metal ions in aqueous solution represent a cornerstone of chemistry, since they provide first steps in rationalizing generally observed chemical information. However, accurate structural information about metal ion solution species is often surprisingly scarce. Here, the atomic structures of Ga³⁺ ion complexes were determined directly in aqueous solutions across a wide range of pH, counter anions and concentrations by X-ray pair distribution function analysis and ⁷¹Ga NMR. At low pH (<2) octahedrally coordinated gallium dominates as either monomers with a high degree of solvent ordering or as Ga-dimers. At slightly higher pH (pH ≈ 2–3) a polyoxogallate structure is identified as either Ga₃₀ or Ga₃₂ in contradiction with the previously proposed Ga₁₃ Keggin structures. At neutral and slightly higher pH nanosized GaOOH particles form, whereas for pH > 12 tetrahedrally coordinated gallium ions surrounded by ordered solvent are observed. The effects of varying either the concentration or counter anion were minimal. The present study provides the first comprehensive structural exploration of the aqueous chemistry of Ga³⁺ ions with atomic resolution, which is relevant for both semiconductor fabrication and medical applications.

With changing pH four different structural regions in Ga³⁺ aqueous solutions are observed. In contrast the effects of different anions and concentrations are minimal.     

7Li residual quadrupolar couplings as a powerful tool to identify the degree of organolithium aggregation

Lithium in the gel: The NMR spectroscopic properties of common organolithium and lithium amide reagents are investigated in the anisotropic environment of a stretched polystyrene (PS) gel. PS is stable towards reactive organometallic compounds and can be used at low temperatures. The residual quadrupolar couplings (RQCs) from a single (7)Li?NMR spectrum can distinguish between high (hexamer, tetramer) and low (dimer, monomer) aggregation states (see scheme).