Accelerated Death Kinetics of Aspergillus niger Spores under High-Pressure Carbonation

The death kinetics of Aspergillus niger spores under high-pressure carbonation were investigated with respect to the concentration of dissolved CO₂ (dCO₂) and treatment temperature. All of the inactivation followed first-order death kinetics. The D value (decimal reduction time, or the time required for a 1-log-cycle reduction in the microbial population) in the saline carbonated at 10 MPa was 0.16 min at 52°C. The log D values were linearly related to the treatment temperature and the concentration of dCO₂, but a significant interaction was observed between them.     

Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor

At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios. High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging.     

Strategies for improved dCO2 removal in large-scale fed-batch cultures

Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process. A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO(2) was measured using a fiber optic based probe. dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.). Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value. A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg). Titer at day 10 of culture improved by 1.5-fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.     

Effects of elevated dissolved CO2 levels on batch and continuous cultures of Aspergillus niger A60: an evaluation of experimental methods.

The effects of elevated levels of dissolved carbon dioxide (dCO2), produced by gassing with CO2-enriched gas mixtures, upon an industrial strain of Aspergillus niger (strain A60) producing citrate and gluconate were quantitatively assessed. Particular attention was paid to the reliability and accuracy of the steam-sterilizable dCO2 probe, especially in the presence of high concentrations of potentially interfering acidic species. The response of the organism to elevated dCO2 levels was assessed by using both batch and chemostat cultures, and the sensitivity of the organism in different growth phases (lag, exponential, and stationary) was examined. Chemostat cultures showed markedly less inhibition (in terms of biomass and organic acid synthesis) than did batch cultures. Studies in batch culture indicated that lag-phase cultures were especially sensitive to elevated dCO2 levels. Overall, the results of this study indicate that previous experimental methods used to examine dCO2 effects in submerged cultures (continuous CO2-enriched gassing of batch cultures from time zero) have been inappropriate and have led to systematic overestimation of the inhibitory effects of dCO2 on mycelial organisms.     

Flow cytometric detection of a two-step cell death induced by hyperthermia

A human leukaemic cell line (REH) was subjected to various temperatures approximately greater than 42 degrees C for various time intervals; the cells were stained with a mixture of ethidium bromide and acridine orange, and red and green fluorescence were analysed by flow cytometry. Nontreated cells appeared as one cluster (V) in the biparameter histograms, but with time of heat treatment, two further discrete clusters (D1,D2) of cells appeared successively. They were distinguished by both the degree of red and green fluorescence. The kinetics of transit from one cluster to the other was dependent on temperature, the time lag between both steps becoming shorter with higher temperatures. It was shown previously that the same effect occurred during incubation with various cytostatic agents, and that only the D2 stage correlated with the stage of cell death monitored by the usual trypan blue exclusion test. Therefore the ethidium bromide technique seems to monitor an earlier stage of cell death. The decrease in the number of dye-excluding (V) cells during heat exposure occurred in two phases. After an initial decrease a plateau of number of dye-excluding cells was reached; the duration and level of this plateau depended on the temperature. The plateau was followed by a second phase where the remaining cells ceased to exclude the dye.     

The influence of dissolved CO(2) concentration on the death kinetics of Saccharomyces cerevisiae

AIMS: The effects of temperature and concentration of dissolved CO(2) on the inactivation of Saccharomyces cerevisiae were investigated using a plug-flow system. METHODS AND RESULTS: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38 degrees C) were used. The D-values obtained were 0.14 min at 8 MPa and 38 degrees C, and 0.15 min at 10 MPa and 36 degrees C. The log D-values were related linearly to the treatment temperature and to the dissolved CO(2) concentration. The thermal resistance constant (zCO(2)(T)) was 9.5 degrees C in the media, including significant levels of CO(2), and the CO(2) resistance constant was z(temp.)(gamma)=7.2 gamma. CONCLUSION: This work has shown that inactivation followed first-order death kinetics, and the effects of temperature and CO(2) concentration were consistent through the critical temperature and pressure of CO(2). Therefore, it is feasible to estimate D-values at any temperature and any CO(2) concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.     

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA. Despite a significant decrease in the rate of O(2) evolution after this treatment, only small changes in the P680*+ reduction kinetics were observed. Loss of Ca(2+) did not affect P680*+ reduction associated with electron transfer from Y(Z). Since much larger changes in the P680*+ reduction kinetics of intact PS II occurred at comparable free-Ca(2+) concentrations in the presence of EGTA, we conclude that EGTA influenced the P680*+ reduction kinetics by directly interacting with PS II rather than by lowering the free Ca(2+) concentration of the surrounding media. Notwithstanding these effects, we show that useful information about Ca(2+) binding to PS II can be obtained when direct interaction of EGTA is taken into account.     

Schedule dependent variation in human lymphocyte sensitivity to bleomycin and repair of chromosomal aberrations in G2.

Bleomycin (BLM) induced chromosomal damage in G2 phase and its repair kinetics in normal human lymphocytes were studied following different treatment schedules. As a first step, a dose-response curve was obtained (concentrations of 5-50 micrograms/ml). For repair kinetics studies, blood samples were treated with BLM at a concentration of 20 micrograms/ml. Continuous treatment produced equal numbers of breaks per cell (br/c) when the cells were treated 3, 4 or 5 h before fixation. If the treatment time was extended to 6 h, the level of br/c was increased 2-fold (p < 0.001) as a result of an increased number of cells with more than 3 br/c. The curves obtained after pulse treatment showed maximal chromosome damage at time 3 (45 min BLM treatment, followed by 2 h repair in drug free medium). When the time after treatment was extended to 4 h (treatment time 5), a 50% reduction in chromosome damage was measured. It was found out that at treatment points 3, 4 and 5 the differences in breaks per cell at the different schedules applied were statistically highly significant. If caffeine (CAF) was added, the continuous treatment, BLM+CAF, induced a statistically significant increase in the frequency of br/c at every treatment point, but the shape of the curve illustrating the kinetics of chromosomal damage remained unchanged. Moreover, the addition of CAF at continuous BLM treatment brings the level of br/c close to that measured at the pulse BLM treatment except for treatment time 3. When applied in a combination with BLM, CAF considerably modified the kinetics of chromosome damage for a pulse (BLM alone) treatment. The possible reasons for the changes in the level of br/c as well as a tentative scheme for assessment of chromosome damage repair capacity after BLM treatment are discussed.     

A vitalistic model to describe the thermal inactivation ofListeria monocytogenes

Summary Thermal inactivation of microorganisms has traditionally been described as log-linear in nature, that is the reduction in log numbers of survivors decreases in a linear manner with time. This is despite a plethora of data that shows consistent deviations from such kinetics for a wide range of organisms and conditions and that cannot be accounted for by experimental artifacts. Existing thermal death models fail to take such deviations into account and also fail to quantify the effects of heating menstruum on heat sensitivity. The thermal inactivation ofListeria monocytogenes ATCC 19115 has been investigated using a factorially-designed experiment comparing 45 conditions of salt concentration, pH value and temperature. Heating was carried out using a Submerged Coil heating apparatus that minimized experimental artifacts. Low pH values increased, whilst high salt concentrations decreased heat sensitivity. Results showed a significant and consistent deviation from log-linear kinetics, particularly at low temperatures. A number of distributions were tested for suitability to describe the variability of heat sensitivity within the population of heated cells (vitalistic approach). The use of the logistic function and log dose (log time) allowed the development of an accurate unifying predictive model across the whole range of heating conditions. It is proposed that this approach should be tested as a generalized modeling technique for death kinetics of vegetative bacteria.     

Death and injury of Staphytococcus aureus during thermal treatment of milk

Staphylococcus aureus isolated from milk and grown in milk was heated in milk. The phenomena of death as well as injury was investigated in the range of 50 to 75 degrees C. The D60 value (decimal reduction time on salt-free medium) was 0.87 min, the D'60 value (decimal reduction time in salt-containing medium) was 0.62 min. Cultures were injured as soon as heating started. This initial thermal shock increased with increasing temperature. At 50-60 degrees C injury was more rapid than death. At greater than 60 degrees C death became faster than injury and the two processes coincided at 70 degrees C. The Z value was 9.46 degrees C and the Z' value was 9.93 degrees C.     

Growth and cometabolic reduction kinetics of a uranium- and sulfate-reducing Desulfovibrio/Clostridia mixed culture: Temperature effects

Bioremediation of contaminated soils and aquifers is subject to spatial and temporal temperature changes that can alter the kinetics of key microbial processes. This study quantifies temperature effects on the kinetics of an ethanol-fed sulfate-reducing mixed culture derived from a uranium-contaminated aquifer subject to seasonal temperature fluctuations. The mixed culture contains Desulfovibrio sp. and a Clostridia-like organism. Rates of growth, ethanol utilization, decay, and uranium reduction decreased with decreasing temperature. No significant uranium reduction was observed at 10 degrees C. While both Monod saturation kinetics and pseudo second-order kinetics adequately described the rates of growth and utilization of electron donor (ethanol), model parameters for the pseudo second-order expression had smaller uncertainties. Uranium reduction kinetics were best described by pseudo second-order kinetics modified to include a term for inactivation/death of cells.     

Thermal resistance of Bacillus subtilis var. niger in a closed system.

The heat resistance of Bacillus subtilis var. niger has been measured from 85 to 125 degrees C using moisture levels of percent relative humidity (%RH) less than or equal to 0.001 to 100 in a closed system. Five curves have been presented to characterize the thermal destruction, using thermal death times defined as F values at a given combination of three moisture and temperature conditions. Reductions of 99.99% (4-log10 cycles) of the initial population were estimated for the three moisture conditions. At 110 degrees C, the expected time for a 4-log10 reduction was 1.1 h at %RH = 100, 3.1 h at %RH less than or equal to 0.1 and 54 h at %RH = 10.7. Goodness-of-fit tests to examine the adequacy of three polynomial models failed to indicate a trend. The linear model (from which estimates of D are obtained) was satisfactory for estimating the thermal death times (%RH less than or equal to 0.1) in the plate count range. The estimates based on observed thermal death times and D values for the %RH = 100 diverged so that D values generally gave a more conservative estimate over the temperature range 90 to 125 degrees C. Estimates of ZF and ZL ranged from 32.1 to 58.3 degrees C for the %RH less than or equal to 0.1 and 100. A ZD value of 30.0 was obtained for data observed at %RH less than or equal to 0.1. The ZF results were obtained from plotting observed log times to achieve a 99.99% reduction in the initial population versus temperature. Estimates of ZL and ZD were obtained by using linear estimates of L100 approximately equal to 4D and D values in a similar plot.     

Thermal resistance of Bacillus subtilis var. niger in a closed system.

The heat resistance of Bacillus subtilis var. niger has been measured from 85 to 125 degrees C using moisture levels of percent relative humidity (%RH) less than or equal to 0.001 to 100 in a closed system. Five curves have been presented to characterize the thermal destruction, using thermal death times defined as F values at a given combination of three moisture and temperature conditions. Reductions of 99.99% (4-log10 cycles) of the initial population were estimated for the three moisture conditions. At 110 degrees C, the expected time for a 4-log10 reduction was 1.1 h at %RH = 100, 3.1 h at %RH less than or equal to 0.1 and 54 h at %RH = 10.7. Goodness-of-fit tests to examine the adequacy of three polynomial models failed to indicate a trend. The linear model (from which estimates of D are obtained) was satisfactory for estimating the thermal death times (%RH less than or equal to 0.1) in the plate count range. The estimates based on observed thermal death times and D values for the %RH = 100 diverged so that D values generally gave a more conservative estimate over the temperature range 90 to 125 degrees C. Estimates of ZF and ZL ranged from 32.1 to 58.3 degrees C for the %RH less than or equal to 0.1 and 100. A ZD value of 30.0 was obtained for data observed at %RH less than or equal to 0.1. The ZF results were obtained from plotting observed log times to achieve a 99.99% reduction in the initial population versus temperature. Estimates of ZL and ZD were obtained by using linear estimates of L100 approximately equal to 4D and D values in a similar plot.     

Inhibition by 1,25 dihydroxyvitamin D3 of c-myc down-regulation and DNA fragmentation in cytosine arabinoside-induced erythroid differentiation of K562 cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on DNA fragmentation, altered expression of the heat shock protein (hsp) 70 gene, and protooncogenes c-myc and c-myb was studied during chemical induction of erythroid differentiation in K562 cells. Preincubation of K562 cells with 1,25(OH)2D3 did not alter the concentration of hemoglobin in cells which did differentiate, but led to a reduction in the accumulation of low molecular weight DNA generated by Ara-C administration. The extent of this reduction was similar to the degree of inhibition of hemoglobin formation in the culture as the whole. Preincubation with 1,25(OH)2D3 had no effect on the increase of hsp 70 gene expression induced by a 48-hr treatment with Ara-C, but prevented the Ara-C-induced down-regulation of the protooncogene c-myc. The protooncogene c-myb was down-regulated after 15 min of treatment with Ara-C, and exposure to 1,25(OH)2D3 prior to Ara-C caused a further down-regulation of its expression. The data suggest that the events associated with erythroid differentiation may be separable into at least two groups; one of these may have an influence on the kinetics of the cell cycle traverse, and the other may be related to the expression of the erythroid phenotype.     

Microbiological Aspects of Ethylene Oxide Sterilization: III. Effects of Humidity and Water Activity on the Sporicidal Activity of Ethylene Oxide

An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 +/- 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed.     

Modeling of permeabilization process in Pseudomonas putida G7 for enhanced limonin bioconversion

A facile process of enhanced whole cell biotransformation to debitter the triterpenoid limonin in citrus juices was optimized in this work. To maximize bioconversion, permeabilization conditions were modeled using response surface methodology. A central composite rotatable design with four significant variables (concentration, temperature, pH, and treatment time) was employed. The second order polynomial equations with R2 values above 0.9 showed good correspondence between experimental and predicted values. The concentration, temperature, pH, and treatment time as well as their interactions had significant effects (p?相似文献   

Characterization and influence factors of Fe(III) reduction of Shewanella cinica D14T]

This is the first time to described the dissimilatory Fe(III) reducing characteristics of Shewanella cinica D14T. The effects of O2, light, temperature and pH on dissimilatory Fe(III) reduction were examined. The results suggested that the rate of Fe(III) reduction decreased with increasing Fe(III) concentration. Fe(III) reduction was partially inhibited by the presence of either O2 or light. The optimum temperature for Fe(III) reduction is 37 degrees C. At pH 6.0-10.0, strain D14T can reduce Fe(III). The soluble Fe(III) is more easy to be reduced than the insoluble one. Results of protein denaturants SDS and OGP suggest that the Fe(III) reduction activity of S. cinica is mostly localized to the soluble outer membrane fraction. The azo dye decolorization and Fe(III) reduction in strain D14T were enhanced in the presence of Fe(III) and dye.     

Kinetics of biocide kill

The efficient use of biocides to control microbial contamination is dependent upon selecting the most potent agent at the anticipated end-use concentration. This is based upon an accurate determination of two basic parameters:

• 1.(1) The time taken by the biocide to achieve a total kill (death rate or decimal reduction time).
• 2.(2) The effect of biocide concentration on the death rate or decimal reduction time.

The time taken to achieve a total kill can be calculated from the death rate. In the simplest case a plot of the natural logarithm of survivors declines linearly when plotted against time and the slope of that line is the death rate. However, the plot of the line of survivors against time is frequently non-linear. Concave curves may result from attempts to control a mixed population with different degrees of tolerance to the biocide; the shape of the curve being a combination of two or more different linear declines. Convex curves, or curves with a shoulder, may be due to one of three phenomena. The target organisms may adhere together in clumps of two or more; the nature of the reaction of biocide with the target organism is one where the organism first changes from a resistant to a susceptible state; or the nature of the biocide molecule is such that uptake is relatively slow and death only commences when a critical concentration has accumulated within the cell. With all such convex curves the decline eventually becomes linear and again the slope is the death rate.The relationship between death rate and biocide concentration is rarely proportional and usually exponential. Thus halving the concentration may cause a disproportionate increase in the decimal reduction time. This relationship is controlled by the concentration coefficient or concentration exponent. When the logarithm of concentration is plotted against the log of death rate or log of decimal reduction time, a linear relationship is obtained whose slope is the concentration coefficient. Thus if the death rates at two or more concentrations are known, the concentration coefficient can be determined and the effect of dilution predicted.The mathematical interpretation of these phenomena and their influence on biocide treatment are discussed.     

Evidence of enzymatic catalysis of oxygen reduction on stainless steels under marine biofilm

Cathodic current trends on stainless steel samples with different surface percentages covered by biofilm and potentiostatically polarized in natural seawater were studied under oxygen concentration changes, temperature increases, and additions of enzymic inhibitors to the solution. The results showed that on each surface fraction covered by biofilm the oxygen reduction kinetics resembled a reaction catalyzed by an immobilised enzyme with high oxygen affinity (apparent Michaelis-Menten dissociation constant close to K(O(2))(M) ≈?10 μM) and low activation energy (W ≈ 20 KJ mole(-1)). The proposed enzyme rapidly degraded when the temperature was increased above the ambient (half-life time of ～1 day at 25°C, and of a few minutes at 50°C). Furthermore, when reversible enzymic inhibitors (eg sodium azide and cyanide) were added, the cathodic current induced by biofilm growth was inhibited.