Nitrogen demand of different yeast strains during alcoholic fermentation. Importance of the stationary phase

The nitrogen demand of industrial yeast strains were compared. Substantial differences were found between strains. These did not change regardless of the initial medium composition and added nitrogen source. To separately study growth and stationary phases, we ran fermentations with different nitrogen feeding profiles: a) exponentially fed fermentations with a long growth phase, and b) constant rate fermentations with nitrogen addition during the stationary phase. Differences between stains mostly appeared during the second phase. Measuring nitrogen requirements under such conditions would thus be an interesting complementary test when selecting new strains especially for enological purposes since most fermentation kinetics are nitrogen limited.     

Evaluation of bacterial contamination in a fed-batch alcoholic fermentation process

Alcoholic fermentation by a commercial baker's yeast in a fed-batch process with cell recycling and high-test molasses as substrate was strongly inhibited by Lactobacillus fermentum CCT 1407 after a few recycles. When total acidity (mainly lactic acid) exceeded 4.8 g/l broth it seriously interfered with yeast bud formation and viability and above 6.0 g/l it decreased alcoholic efficiency.     

Miscibility studies of HPMC/PVA blends in water by viscosity, density, refractive index and ultrasonic velocity method

Hydroxy propyl methyl cellulose (HPMC)/polyvinyl alcohol (PVA) blends are edible polymer films used for food packing and directly in foodstuffs. However they are water-soluble in ordinary temperature and have good mechanical properties. The miscibility of HPMC/PVA blend in water was studied by viscosity, ultrasonic velocity, density and refractive index techniques at 30 and 50 °C. Using viscosity data, the interaction parameters μ and α were calculated. These values revealed that HPMC/PVA blend is miscible when the HPMC content is more than 60% in the blend at 30 and 50 °C. And also the result revealed that the change in temperature has no significant effect on the miscibility of HPMC/PVA polymer blend.     

In situ monitoring by quantitative Raman spectroscopy of alcoholic fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under high pressure

We monitored alcoholic fermentation in Saccharomyces cerevisiae as a function of high hydrostatic pressure. Ethanol production from 0.15 M glucose was measured by Raman spectroscopy in situ in a diamond-anvil cell. At 10 MPa, fermentation proceeds three times faster than at ambient pressure and the fermentation yield is enhanced by 5% after 24 h. Above 20 MPa, the reaction kinetics slows down with increasing pressure. The pressure above which no more ethanol is produced is calculated to be 87 ± 7 MPa. These results indicate that the activity of one or several enzymes of the glycolytic pathway is enhanced at low pressure up to 10 MPa. At higher pressures, they become progressively repressed, and they are completely inhibited above 87 MPa. Although fermentation was predicted to stop at ca. 50 MPa, due to the loss of activity of phosphofructokinase, the present study demonstrates that there is still an activity of ca. 30% of that measured at ambient pressure at 65 MPa. This study also validates the use of Raman spectroscopy for monitoring the metabolism of living microorganisms.     

On-line monitoring of lipid storage in yeasts using impedance spectroscopy

Bioremediation technologies and many environmentally sound biosyntheses rely on the catalytic potential of whole cells. For analyzing and controlling such processes robust real-time indicators for the concentration of intact cells such as impedance are required. The conventional method measures the capacitances of cell suspensions at one or two frequencies and correlates them with biomass concentrations. However, cell inclusions such as lipid droplets or overproduced enzymes may block intracellular ion paths, thereby possibly modifying the dielectric properties of the cells. To test the hypothesis that the total impedance spectrum into the analysis may provide useful information about cell inclusions, the impedance spectrum of a technical culture of the oleaginous yeast Arxula adeninivorans was measured and evaluated every 15 s. This yeast is a good test object since it stores the excess of assimilated carbon in experimentally controllable lipid droplets. Upon correction for possible impedance signal interferences, we derived different empirical methods suitable to indicate incipient lipid formation. The methods were designed to act on-line and are thus principally suited for real-time monitoring of cell inclusions. In search for optimised bioprocess monitoring we tested a heuristic spectrum analysis using integrative statistics (RDA). With this approach we were able to accurately detect the formation of cell inclusions, which is potentially valuable for future bioprocess control strategies.     

Modelling Biolog profiles' evolution for yeast growth monitoring in alcoholic fermentation

Aims: A research was undertaken to explore the possibility to express with suitable mathematical models Biolog metabolic curves obtained for oenological yeasts and to use such models for monitoring yeast growth in alcoholic fermentation. Methods and Results: Experimental curves of metabolic activity in Biolog YT microplates, obtained in a previous work for various oenological yeast strains in pure cultures and mixed populations, at various cell concentrations, have been modelled with Gompertz’s, Gompertz’s modified and Lindstrom’s mathematical equations. Lindstrom’s model proved to be the most suitable to fit the curves of the oenological yeasts under study, providing the highest correlation coefficients between experimental and calculated data. The model made it possible to recognize, in mixed yeast populations, the presence of active dry yeasts used for guided fermentations. Model’s constant parameters were used for a numerical characterization of yeast curves. Conclusions: The application of the model to the experimental data resulted to be suitable for an early prediction of the successive evolution of yeast growth. Significance and Impact of the Study: The results obtained indicate the possibility to develop protocols for monitoring yeast presence during alcoholic fermentation, with an early assessment of the correct evolution of their growth, especially when active dry yeasts are employed.     

超声波在生物发酵工程中的应用

一定强度的超声波作用于发酵过程中缩短发酵时间,改善生物反应条件,提高生物产品的质量和产量,微弱超声可用于在线检测某些发酵过程参数,本简要介绍了超声波作用于生物发酵过程的基本原理,并详细讨论了超声波在遗传育种 ,改善发酵工艺有发酵产物的提取与分离,发酵产物浓度的在线检测等方面的主要应用。     

Continuous monitoring of nitrogenase activity in Azotobacter vinelandii fermentation using off-gas mass spectrometry

This study evaluated the feasibility of monitoring nitro-genase activity in situ through measurement of N(2) uptake rate (NUR) using off-gas mass spectrometry. Four 50-L cultures of Azotobacter vinelandii were grown aer-obically in nitrogen-free medium to cell densities of 1.0-1.3gL(-1) magnetic-sector mass spectrometer was used to monitor NUR along with other gas exchange rates. The small specific uptake rate (1.2 mmol g(-1) h(-1)) and low cell density were found to lead to a NUR below the measurement accuracy limits under normal conditions. An operating strategy and feed gas mixture (40% O(2), 45% N(2) 15% Ar) were designed to improve the signal-to-noise ratio while maintaining dissolved O(2) and N(2) levels in desired ranges. The fraction of N(2) removed from the air stream was increased approximately 5-fold from 0.2% to 1.0% and the measurement noise was reduced 25-fold from a baseline of +/-5to +/-0.2 mmol L(-1) h(-1). The NUR measurements were compared against in vivo and in vitro acetylene reduction assays as well as on-line cell growth rate measurements. While electron transfer requirements predict an NUR-to-acetylene reduction rate ratio of 0.33, measured ratios for the in vivo and in vitro assays were 0.8 and 0.44, respectively. This suggests that other rate-limiting steps were present in the case of the in vivo assay. In accordance with reports in the literature, no concomitant hydrogen evolution was detected. This is the first reported continuous and direct measurement of NUR in fermentation and demonstrates a novel approach for improving measurement accuracy through rational adjustment of operating conditions. The technique has potential to provide useful insight for development and control of microbial nitrogen fixation processes.(c) John Wiley & Sons, Inc.     

On-line monitoring of lipolytic activity by sequential injection analysis

An automated sequential injection analysis using stop-flow technique for the on-line determination of lipolytic activity has been developed. It is based on a colorimetric method using a chromogenic substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester. The system permits a linear range analysis between 5–100 lipolytic activity units ml^–1, without external dilution of the sample, a sampling frequency of 5 samples per hour and a relative standard deviation (RSD) of 5%. The analyser has been used for the on-line monitoring of Candida rugosa fed-batch fermentation with excellent performance, regarding its reliability and reproducibility.     

An on-line sampling system for fermentation monitoring using membrane inlet mass spectrometry (MIMS): application to phenoxyacetic acid monitoring in penicillin fermentation

A sampling system for on-line monitoring of organic compounds of low volatility in complex fermentation media uses membrane inlet mass spectrometry (MIMS). A Syringe pump draws a continuous flow of microfiltered broth from the reactor and circulates it after acidification through a membrane inlet, in which a membrane is the only interface between the sample and the high vacuum of a mass spectrometer. All operations run automatically, i.e., sampling, acidification measurement, and calibration. The on-stream acidification enables MIMS monitoring of carboxylic acids, as they must be undissociated in order to pass the hydrophobic membrane. The performance of the monitoring system was tested by measurements of standard solutions of phenoxyacetic acid (POAA, the sie chain precursor of penicillin-V) as well as on POAA during 200 h penicillin-V fermentation. During the entire fermentation POAA was monitored n low millimolar concentrations with high accuracy and fast response to step changes in POAA concentration. Tandem mass spectrometry (MS/MS) allowed direct identification of peaks in the mass spectrum of the broth that were not accounted for by POAA. These peaks were identified as SO(2) and SCO. (c) 1994 John Wiley & Sons, Inc.     

Possible use of Biolog methodology for monitoring yeast presence in alcoholic fermentation for wine‐making

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

聚唾液酸的发酵动力学研究

对以大肠杆菌K235发酵法生产聚唾液酸的动力学特性进行了研究,提出了细胞生长动力学,基质消耗动力学,聚唾液酸生成动力学模型,通过对实验数据的计算,模型的模拟结果较好地和实验值吻合,正确地分析 聚唾液酸的发酵过程及其动力学机制。     

Discovery of alcohol dehydrogenase from mushrooms and application to alcoholic beverages

Saccharomyces cerevisiae is the main microorganism used in alcoholic beverage brewing, because this microbe has alcohol dehydrogenase (ADH) activity. We have recently discovered that some genera of mushrooms produce alcohol dehydrogenase, and made wine, beer and sake using mushrooms in place of S. cerevisiae. The highest alcohol concentrations in the wine, beer and sake were achieved with Pleurotus ostreatus (2648 mM, 12.2%), Tricholoma matsutake (1069 mM, 4.6%) and Agaricus blazei (1736 mM, 8.0%). In the case of wine made using A. blazei, the same alcohol concentration (1736 mM, 8.0%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β- -glucan, which is known to have preventive effects against cancer. The wine made using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, alcoholic beverages made using mushrooms seem to be a functional food source which can be expected to have preventive effects against cancer and thrombosis.     

产弹性蛋白酶细菌的发酵动力学研究

基于14L的发酵罐分批发酵实验数据,建立了发酵过程菌体生长、产物生成及基质消耗随时间变化的数学模型。Logistic方程、Luedeking—Piret方程能够很好地分别描述产弹性蛋白酶菌体生长;发酵产酶过程和基质消耗过程。并将3个动力学模型的预测值和实验值进行了比较,所建立的分批发酵动力学模型能较好地反映弹性蛋白酶分批发酵过程。     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Development of a kinetic model for the alcoholic fermentation of must

We Propose a kinetic expression which accounts for the temperature dependence of ethanol yield losses in batch alcoholic fermentation. Moreover, the characteristic parameters of the microbial growth equation have been calculated for Saccharomyces cerevisiae under typical wine industry conditions. A substrate consumption equation is established which minimizes possible model deviations in the latter process stages. Experimental data were obtained in the laboratory and the proposed equations were then applied at an industrial level (2.5 x 10(4) L) where they described the data well.     

Real-time monitoring of shake flask fermentation and off gas using triple disposable noninvasive optical sensors

Bioprocess development is a data-driven process requiring a large number of experiments to be conducted under varying conditions. Small-scale upstream bioprocess development is often performed in shake flasks because they are inexpensive and can be operated in parallel. However, shake flasks are often not equipped to accurately monitor critical process parameters such as pH, dissolved oxygen, and CO2 concentrations. Therefore, there is no definitive information on oxygen supply of growing cells, CO2 formation, and pH changes. Here we describe several shake flask fermentations where all three parameters are monitored by disposable noninvasive optical sensors. The sensitive element of these sensors is a thin, luminescent patch affixed inside the flask. Small electronic devices for excitation and fluorescence detection are positioned outside the shake flask for noninvasive monitoring. By measuring the process parameters throughout the course of the E. coli fermentations, we obtain information that is not routinely available in shake flask fermentations. For example, for cultures with only a few millimeters liquid depth, oxygen limitation can occur at relatively low agitation speeds. Under certain conditions oscillations in dissolved oxygen can occur. An increase in shaker speed and a decrease in culture volume can increase the oxygen availability and reduce the duration of oxygen limitation.     

Reaction kinetics of -glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of -glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of -glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.