Dynamics and modeling of temperature-regulated gene product expression in recombinant yeast fermentation

The dynamic response of temperature-regulated gene expression in the recombinant yeast Saccharomyces cerevisiae, strain XK1-C2 carrying plasmid pSXR125, to temperature changes during fed-batch and continuous (chemostat) cultures was studied. The production of the gene product, beta-galactosidase, in the yeast cell is sensitive to the growth temperature. Gene expression of this product was fully turned on or off by temperature shifts between 24 and 30 degrees C. However, the response for gene turn-on and turn-off in this recombinant yeast was slow, requiring from several hours to over 10 h to fully appear. The continuous reactor took 30-60 h after the temperature shift to reach a new steady state. A dynamic process model was developed to simulate the reactor and cell responses to temperature shift. A first-order model was used to account for the effect of dilution rate on the change of protein concentration in the chemostat. It was found that cell response in gene expression to temperature shift followed first-order plus dead-time dynamics. Also, the response time for gene expression to temperature shift varied with specific growth rate or dilution rate of the continuous reactor. In general, the response was slower at a higher dilution rate and for gene turn-on than for gene turn-off. (c) 1996 John Wiley & Sons, Inc.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Effect of operating parameters on specific production rate of a cloned-gene product and performance of recombinant fermentation process

A two-stage continuous system in combination with a temperature-sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high-expression recombinant. In order to optimize the two-stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 DeltaH1 Deltatrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38 degrees C, a significant decrease in the colony-forming-units (CFU) of the plasmid-harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno-type from colony-forming to non-colony-forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40 degrees C, the varying dilution rate D in the range of 0.35-0.90 h(-1) did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h(-1), this factor remained practically constant up to 41 degrees C but increased rapidly beyond 41 degrees C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (mu(2) (app)) of the second stage constant at or near mu(2) (app) = 0.26 h(-1). Under a constant dilution rate, D(2) = 0.35 h(-1), the maximum specific production rate obtained was about q(p, max) = 38 units TrpA/mg cell/h at 41 degrees C. At a constant temperature, T(2) = 40 degrees C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D(2) = 0.35-0.90 h(-1). Based on the results of our study, the optimal operating conditions found were dilution rate D(2) = 0.35 h(-1) and operating temperature T(2) = 41 degrees C at the apparent specific growth rate of 0.26 h(-1). Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.     

A method for quantitative determination of ice nucleating agents in insect hemolymph

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 10³, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph.This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.     

Managing high-density commercial scale wine fermentations

AIMS: To investigate the effects of grape juice dilution and different temperature/nitrogen addition regimes on commercial-scale, high-density Shiraz and Chardonnay fermentations. METHODS AND RESULTS: Duplicated fermentations (30 hl) were conducted at two temperatures for Shiraz and for Chardonnay. Two additional tanks of Chardonnay and Shiraz were diluted. Nitrogen was added once at inoculation or in aliquots over several days. Yeast concentration and viability was determined by flow cytometry. Fermentation chemistry was monitored by Fourier transform infrared spectroscopy. Fermentations arrested in both of the undiluted, higher temperature duplicate tanks of Shiraz. Different fermentation temperature resulted in sensorially different Shiraz, but not Chardonnay, wines made from undiluted musts. The converse was observed for wines made from diluted musts. CONCLUSIONS: High-density musts can be fermented completely using reduced fermentation temperature coupled with incremental nitrogen addition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in duplicated, commercial-scale, high-density grape juice fermentations to address temperature, nitrogen addition, and juice dilution effects on stuck fermentation potential and wine sensory properties.     

Continuous cultivation of the yeastSaccharomyces cerevisiae at different dilution rates and glucose concentrations in nutrient media

The influence of glucose concentration in nutrient media on the specific growth rate and biomass yield in the course of continuous fermentation ofSaccharomyces cerevisiae was investigated. An increase of glucose content in media decreased the specific growth rate and the biomass yield. Glucose concentration had significant effects on protein and phosphate contents of cells. However, an increased glucose concentration increased the fermentative power ofS. cerevisiae (SJA-method). An increase of the dilution rate decreased the cell concentration in the fermentor. Specific growth rate approached the values of the dilution rate. The best agreement has been obtained at a dilution rate of 0.20/h. This dilution rate proved to be most convenient for the investigated microorganism and cultivation conditions (media composition, pH, aeration intensity and temperature). Biomass yield proved to be decreased by an increase of the dilution rate.     

Continuous cultivation of bakers' yeast: Change in cell composition at different dilution rates and effect of heat stress on trehalose level

The cell composition of bakers' yeast in a continuous culture was determined for different dilution rates. Also, the cellular response to heat stress in terms of trehalose, RNA, glycogen and protein was determined at a specified dilution rate of 0.1/h. The amount of storage saccharides, trehalose and glycogen, was found to decrease whereas the amount of RNA and protein increased with increasing dilution rates. As the dilution rate was increased from 0.1 to 0.4/h at 0.05 intervals the steady-state trehalose content decreased from 33 to 8.6 mg/g biomass, and glycogen content from 150 to 93 mg/g biomass. On the other hand, the protein content increased from 420 to 530 mg/g biomass and the RNA content from 93 to 113 mg/g biomass. Heat stress was applied by increasing the medium temperature from 30 to 36, 38 or 40°C at constant dilution rates. The highest amount of trehalose accumulation, 108 mg/g biomass, was achieved when heat stress at 38°C was applied. The protein content, on the other hand, decreased from 350 to 325 mg/g biomass at the end of the experiment.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

Regulation of ribosome synthesis in Escherichia coli: effects of temperature and dilution rate changes

The effect of temperature on the synthesis of ribosome in Escherichia coli K-12 was investigated. In continuous fermentation, the total and functioning ribosome contents decreased with increasing temperature, while the non-functioning ribosome content remained unchanged. Cells contained higher amounts of functioning ribosome at lower temperatures to compensate for the decrease in translational activity. A transient study was performed to investigate the dynamic response of the cell to changes in the dilution rate. In response to the dilution rate shift-up, the cell mass decreased until the cells produced a sufficient amount of ribosomes to support the new higher growth rate. However, the response to the dilution rate shift-down resulted in an immediate increase in cell mass. This may be due to the fact that the cell already contains enough ribosomes to support a lower growth rate corresponding to the new low dilution rate. Based on the experimental results, a mathematical model was developed to describe the cell growth at transient as well as steady states. The total ribosome content was included as a variable because it affects the growth rate of the cell. (c) 1996 John Wiley & Sons, Inc.     

Growth Rates of Sulfolobus acidocaldarius in Nature

Turnover times for water passing through several Sulfolobus acidocaldarius-containing springs were determined by measuring the dilution rates of small amounts of sodium chloride that were added to the springs. Chloride was diluted out exponentially, while concentrations of the bacteria remained constant. Additionally, temperature, pH, and chemical composition of the springs also remained constant during the time that the chloride was being diluted. The springs are thus steady-state systems, and since the rates of bacterial growth must be at least equal to the chloride dilution rates, minimal doubling times for the bacterial populations can be calculated. Half-times for chloride dilution, equivalent to bacterial doubling times, were on the order of 10 to 20 h for springs ranging in volume from about 20 to 2,000 liters, but approximately 30 days for two larger springs of about 1 million liters. Formaldehyde-fixed cells of a serologically distinguishable strain of S. acidocaldarius were also added as markers to four of the smaller springs, and the dilution rates of these bacteria were compared with the chloride dilution rates. The rates agreed reasonably well, thus verifying the growth rates obtained from the chloride dilution rates. In three springs, exponential growth was studied by draining the springs and allowing them to refill with bacteria-free water. Exponential doubling times were on the order of a few hours, much more rapid than steady-state doubling times. The methods used in this work may have wider utility in aquatic environments.     

Calcium transport in diluted or cooled ram semen

Ram semen was subjected to various dilution rates and temperatures of dilution, and was also subjected to slow cooling and rewarming. Calcium ion movements across sperm membranes were measured using the radioisotope 45Ca2+. It was shown that even 2- to 4-fold dilution caused an increase in intracellular calcium content. An increase in intracellular calcium also occurred in proportion to a decreased temperature of dilution. After an initial increase in intracellular calcium content, spermatozoa appeared able to restore a low intracellular calcium level over a period of 2 h at 22 degrees C or above. This ability was lost at 16 degrees C or below. However, if undiluted semen was slowly cooled (0.125 degrees C/min) even to 5 degrees C and rewarmed to 22 degrees C before dilution, the spermatozoa regained this ability. In contrast, spermatozoa rapidly cooled to 5 degrees C and rewarmed to 22 degrees C before dilution were not able to restore the low intracellular calcium level.     

Effect of growth conditions on heat resistance of Arizona bacteria grown in a chemostat.

The effects of various growth conditions on the heat resistance of Arizona bacteria grown in a continuous-culture device (chemostat) were studied. Using either glucose, NH4Cl, NaH2PO4, or MgCl2 as the rate-limiting nutrient, it was found that the heat resistance, in all cases depended on the dilution rate and, hence, growth rate of the culture. Cells grown at high dilution rates were less heat resistant than those grown at low dilution rates. If, however, the dilution rate was maintained at a constant rate, the higher the growth temperature, the more heat resistant were the cells. Also at any given dilution rate, the cells were most heat resistant when grown at a near neutral pH. Most survival curves were biphasic in shape, indicating the presence in the population of two fractions of cells, one fraction being more resistant than the other. The size of the more heat-resistant fraction varied from almost 100% in very slow-growing cultures to practically 0% in cultures grown at a dilution rate of 0.67 h-1.     

The effects of limiting nutrients, dilution rate, culture pH, and temperature on the yield constant and anthocyanin accumulation of carrot cells grown in semicontinuous chemostat cultures

With carrot cells grown in semicontinuous culture with phosphate as limiting nutrient. Dougall and Weyrauch (1980) found that the steady-state culture density was different at different dilution rates. They suggested that the yield constant for biomass was different at different dilution rates. Here the yield constant for biomass for PO(4) (3-), NH(4) (+), Mg(2+), and glucose-limited semicontinuous cultures has been measured directly at two dilution rates. The yield constants for PO(4) (3-), NH(4) (+), and Mg(2+) but not for glucose are different at the two dilution rates. The effects of pH and temperature on the biomass yield constant was measured to extend the number of system parameters examined. Biomass yield constant was changed little with change from 25 to 28 degrees C or from pH 4.2 to pH 5.5. The steady-state levels of anthocyanin were also measured. The responses of anthocyanin levels to the system parameters are different to the biomass responses. The data suggest that at different values of each of the system parameters, the composition and metabolic activities of the cells at steady state in semicontinuous cultures are different.     

酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法的建立

目的:建立一种酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法,提高基因芯片检测的灵敏度。方法:待测靶基因与固定在玻片上的探针杂交,依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺及链霉亲和素标记的量子点,37℃孵育,然后加入银增强试剂显色,最后用可视化生物芯片扫描仪扫描并记录结果;以牛布鲁菌210105株为检测对象,以酪胺信号放大-荧光素Cy3(TSA-Cy3)检测法为对照方法,测定酪胺信号放大-量子点标记银染增强(TSA-QDS)检测法的灵敏度。结果:确定了基因芯片量子点标记银染增强可视化检测方法的检测流程,优化了检测条件,并考察了检测灵敏度。优化的检测条件为:酪胺-生物素稀释比例为1∶4000,链酶亲和素标记的量子点稀释比例为1∶50,37℃孵育时间为25~30 min,银染增强时间为6~7 min。检测牛布鲁菌的灵敏度为103CFU/mL。结论:该方法实现了基因芯片高灵敏度可视化检测,其灵敏度与荧光法相当,并且有可视化的优势。     

Continuous butanol production from whey permeate with immobilized Clostridium beyerinckii LMD 27.6

Summary The aim of this study was to find the conditions necessary for the continuous butanol production from whey permeate with Clostridium beyerinckii LMD 27.6, immobilized in calcium alginate beads. The influence of three parameters on the butanol production was investigated: the fermentation temperature, the dilution rate (during start-up and at steady state) and the concentration of calcium ions in the fermentation broth. It was found that both a fermentation temperature of 30° C and a dilution rate of 0.1 h^-1 or less during the start-up phase are required to achieve continuous butanol production from whey permeate. Butanol can be produced continuously from whey permeate in reactor productivities sixteen times higher than those found in batch cultures with free C. beyerinckii cells on whey media.     

Interactive effect of warming,nitrogen and phosphorus limitation on phytoplankton cell size

Cell size is one of the ecologically most important traits of phytoplankton. The cell size variation is frequently related to temperature and nutrient limitation. In order to disentangle the role of both factors, an experiment was conducted to determine the possible interactions of these factors. Baltic Sea water containing the natural plankton community was used. We performed a factorial combined experiment of temperature, type of nutrient limitation (N vs. P), and strength of nutrient limitation. The type of nutrient limitation was manipulated by altering the N:P ratio of the medium (balanced, N and P limitation) and strength by the dilution rate (0% and 50%) of the semicontinuous cultures. The negative effect of temperature on cell size was strongest under N limitation, intermediate under P limitation, and weakest when N and P were supplied at balanced ratios. However, temperature also influenced the intensity of nutrient imitation, because at higher temperature there was a tendency for dissolved nutrient concentrations to be lower, while the C:N or C:P ratio being higher…higher at identical dilution rates and medium composition. Analyzing the response of cell size to C:N ratios (as index of N limitation) and C:P ratios (as index of P limitation) indicated a clear dominance of the nutrient effect over the direct temperature effect, although the temperature effect was also significant.     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

Effects of dilution on dissolved oxygen depletion and microbial populations in the biochemical oxygen demand determination

The biochemical oxygen demand (BOD) value is still a key parameter that can determine the level of organics, particularly the content of biodegradable organics in water. In this work, the effects of sample dilution, which should be done inevitably to get appropriate dissolved oxygen (DO) depletion, on the measurement of 5-day BOD (BOD₅), was investigated with and without seeding using natural and synthetic water. The dilution effects were also evaluated for water samples taken in different seasons such as summer and winter because water temperature can cause a change in the types of microbial species, thus leading to different oxygen depletion profiles during BOD testing. The predation phenomenon between microbial cells was found to be dependent on the inorganic nutrients and carbon sources, showing a change in cell populations according to cell size after 5-day incubation. The dilution of water samples for BOD determination was linked to changes in the environment for microbial growth such as nutrition. The predation phenomenon between microbial cells was more important with less dilution. BOD₅ increased with the specific amount of inorganic nutrient per microbial mass when the natural water was diluted. When seeding was done for synthetic water samples, the seed volume also affected BOD due to the rate of organic uptake by microbes. BOD₅ increased with the specific bacterial population per organic source supplied at the beginning of BOD measurement. For more accurate BOD measurements, specific guidelines on dilution should be established.     

Continuous ethanol production from pineapple cannery waste using immobilized yeast cells

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).     

Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA.

The heat of dissociation of the second proton of 1,2-dimyristoylphosphatidic acid (DMPA) was studied as a function of temperature using titration calorimetry. The dissociation of the second proton of DMPA was induced by addition of NaOH. From the calorimetric titration experiment, the intrinsic pK0 for the dissociation reaction could be determined by applying the Gouy-Chapman theory. pK0 decreases with temperature from ca. 6.2 at 11 degrees C to 5.4 at 54 degrees C. From the total heat of reaction, the dissociation enthalpy, delta Hdiss, was determined by subtracting the heat of neutralization of water and the heat of dilution of NaOH. In the temperature range between 2 and 23 degrees C, delta Hdiss is endothermic with an average value of ca. 2.5 kcal.mol-1 and shows no clear-cut temperature dependence. In the temperature range between 23 and 52 degrees C, delta Hdiss calculated after subtraction of the heat of neutralization and dilution is not the true dissociation enthalpy but includes contributions from the phase transition enthalpy, delta Htrans, as the pH jump induces a transition from the gel to the liquid-crystalline phase. The delta Cp for the reaction enthalpy observed in this temperature range is positive. Above 53 degrees C, the pH jump induces again only the dissociation of the second proton, and the bilayers stay in the liquid-crystalline phase. In this temperature range, delta Hdiss seems to decrease with temperature. The thermodynamic data from titration calorimetry and differential scanning calorimetry as a function of pH can be combined to construct a complete enthalpy-temperature diagram of DMPA in its two ionization states.