Scale-up of stirring as foam disruption (SAFD) to industrial scale

Foam disruption by agitation—the stirring as foam disruption (SAFD) technique—was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD—foam entrainment—was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised. Electronic Publication     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.     

Effects of ozone and mild drought stress on gas exchange, antioxidants and chloroplast pigments in current-year needles of young Norway spruce [Picea abies (L.) Karst.]

 To investigate the effects of ozone exposure and soil drought, singly and in combination, on gas exchange, antioxidant contents and pigments in current-year needles of Norway spruce [Picea abies (L.) Karst.] 4-year-old seedlings were fumigated in growth chambers with either charcoal-filtered air or with 100 nl l^–1 ozone for 106 days. After 3 weeks a 20% reduction in gas exchange was observed in ozone-treated seedlings. However, no further decrease occurred in spite of continued ozone exposure. Whole needle ascorbate and apoplastic ascorbate increased until the end of the experiment and contents were 62% and 82%, respectively, higher than in ozone-free controls. This increase in ascorbate might have protected net photosynthesis from further decline. Ozone pre-treated plants and ozone-free controls were subjected to soil drought for 38 days which caused stomatal narrowing. Thereby ozone uptake was reduced when compared to well watered seedlings. At the end of the experiment drought alone, and even more in combination with ozone, had also caused an increase in ascorbate. Glutathione increased only in drought-stressed seedlings. The redox states of the ascorbate and the glutathione pools were not affected by any treatment. Superoxide dismutase activity declined under both stresses but was most reduced by ozone alone. While chlorophyll and neoxanthin contents remained unchanged, carotenes were significantly decreased upon drought. The combination of O₃ and drought induced increased lutein contents, an increased pool size of the xanthophyll cycle as well as an increased epoxidation status of the xanthophyll cycle. These results suggest that spruce needles seem to be able to acclimate to ozone stress but also to drought stress by increasing their ascorbate pools and protecting pigments. Received: 15 September 1997 / Accepted: 24 March 1998     

Factors determining the midday depression of photosynthesis in trees under monsoon climate

 Field studies of gas exchange of Populus deltoides, Prosopis juliflora and Acacia auriculiformis showed large diurnal changes in net photosynthesis (A) and stomatal conductance (g_s) during autumn. P. deltoides and P. juliflora undergo pronounced midday depression in A and g_s while A. auriculiformis showed a one-peak response. Several factors indicative of photosynthetic performance were found to be reversibly affected during afternoon decline. These include (i) decrease in initial slope of the CO₂ response curve (carboxylation efficiency), (ii) substantial increase in CO₂ compensation point and (iii) decrease in overall quantum yield of photosystem II. The phenomenon can be duplicated in potted plants by simulating a typical daily pattern of PPFD and VPD. It is found that high VPD induces significant decline in A and g_s at moderate temperature and saturating PPFD (800 μmol m^–2 s^–1) whereas these parameters are only marginally affected at high PPFD and low VPD. Fluorescence data show that the tree species under study have a high capacity for safe dissipation of excessive excitation energy. The activation of photorespiration, as evident from an increase in CO₂ compensation point, maintains constant internal CO₂ concentration (C_i) which may aid in minimizing photoinhibition during stomatal closure at midday. In case of P. deltoides and P. juliflora the stomata seem to be quite sensitive to the changes in humidity whereas this does not appear to be essential in case of A. auriculiformis because of its phyllode structure that endows it with mechanisms for conserving water without undergoing large-scale stomatal changes. Received: 16 October 1997 / Accepted: 5 March 1998     

华法令诱导大鼠体内主动脉的钙化作用

目的:研究华法令对大鼠体内主动脉的钙化作用。方法:采用华法令(3 mg/g饲料)的饲料喂养诱导大鼠体内动脉钙化。实验分3组(n=6),对照组、6 W钙化组、12 W钙化组。Von Kossa染色观察钙结节形成,邻甲酚酞络合酮比色法定量测定钙沉积含量。采用TUNEL染色法检测大鼠主动脉组织内血管平滑肌细胞(Vascular Smooth Muscle Cell,VSMC)凋亡。Western blot法检测大鼠主动脉组织中生长抑制特异蛋白6(Growth Arrest Specific Gene 6 Protein,Gas 6)蛋白表达水平。结果:钙化组钙结节、钙沉积含量及VSMC凋亡均高于对照组(P0.01)有显著差异。钙化形成与凋亡表达呈显著正相关(R2=0.8853,P0.0001);钙化组Gas 6蛋白表达量较对照组下调(P0.01)有显著差异。结论:华法令通过下调Gas 6蛋白诱导大鼠体内主动脉钙化形成。     

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.     

Determination of ethyl glucuronide in hair samples of Chinese people by protein precipitation (PPT) and large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS)

Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R2=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse.     

Accurate analysis of urea in milk and milk powder by isotope dilution gas chromatography–mass spectrometry

A high order method for measuring urea concentrations in milk and milk powder was developed. The method can be applied to certify the concentration of urea in some new milk and milk powder CRMs. This high accurate method for analysis of milk is valuable given the inherent challenges associated with the complexity of the sample matrix. A measurement procedure based on gas chromatography/isotope dilution mass spectrometry (GC/IDMS) was developed. Samples were pre-treated with acetonitrile to remove proteins and the method was applied to determine urea concentrations in milk and milk powder. Excellent precision was obtained, with within- and between-set coefficients of variation of 0.15–0.46 and 0.18–0.65%, respectively. The measurement uncertainty is evaluated. The method can trace to mass.     

Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS

In the present study we describe a method, which is based on solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) and which can be used for the profiling of microbial volatile organic compounds (MVOCs) in the headspace (HS) of cultures of filamentous fungi. The method comprises the following successive steps: 1. growth of the fungus on a solid culture medium directly in headspace vials, 2. measurement of volatiles by HS-SPME-GC-MS, 3. deconvolution of mass spectra, 4. identification of volatiles by comparison of measured, deconvoluted mass spectra and linear temperature programmed retention indices (LTPRI) on two stationary GC phases with database entries and LTPRI published in the literature, and 5. profiling of the identified MVOCs.The developed method was successfully applied to cultures of the biocontrol fungus Trichoderma atroviride. An in-house library consisting of mass spectra and LTPRI values of fungal VOCs was established and used to study the profiles of MVOCs of this fungus. In total, 25 different MVOCs were identified by applying strict criteria (spectral match factor at least 90% and a maximum relative deviation of LTPRI of ± 2% from literature values). The MVOCs were assigned to the compound classes of alcohols, ketones, alkanes, furanes, pyrones (mainly the bioactive 6-pentyl-alpha-pyrone), mono- and sesquiterpenes, 13 of which have never been reported to be produced by Trichoderma spp. before. Eleven of these volatiles have been additionally confirmed using authentic standards. Finally, time course experiments and cultivation of T. atroviride in the presence of the mycotoxin fusaric acid demonstrated the potential of the method to study the dynamics of MVOC profiles as well as the effect of different environmental/biological conditions on the expression of MVOCs of filamentous fungi.     

Microextraction and Gas Chromatography/Mass Spectrometry for improved analysis of geosmin and other fungal “off” volatiles in grape juice

Geosmin is a volatile fungal metabolite with an earthy aroma produced in grape products from rotten grapes. The accumulation of geosmin in grapes is caused by the interaction of Botrytis cinerea and Penicillium expansum. Solid Phase Microextraction (SPME) has great utility for collecting volatile compounds in wine. However, contamination with earthy odours may have occurred previously in the must and novel methods are required for this commodity. In the present report, several parameters of the SPME were evaluated to optimize geosmin extraction. The method permitted quantification of geosmin and other fungal volatiles by Gas Chromatography-Mass Spectrometer (GC-MS) at very low concentrations. Limits of detection and quantification (L_D and L_Q) for geosmin were 4.7 ng L⁻¹ and 15.6 ng L⁻¹ respectively. The RSD was 4.1% and the recovery rates ranged from 115% to 134%. Uniquely, haloanisoles were analyzed by using only one internal standard (2,3,6-trichloroanisole) thus avoiding the synthesis of deuterated anisole analogues that are used as internal standard in other methods. The method was used for the analysis of grape juice samples inoculated with B. cinerea and P. expansum. Geosmin and methylisoborneol were the compounds that appeared to contribute most to earthy odours, although other fungal compounds which are claimed to cause earthy or mouldy off-odours were detected (e.g. 1-octen-3-ol and fenchol).