Release of aminopeptidase from Lactobacillus casei sp. casei by cell disruption in a Microfluidizer

Cell disruption in a Microfluidizer was a function of both operating pressure and number of passes. The operating pressure had greater effect on the disruption than did the number of passes as indicated by the magnitude of the constants from the cell disruption equation. Protein release correlated with aminopeptidase release by Lactobacillus casei sp. casei. The optimum operating pressure for enzyme extraction was 76 MPa with loss of enzyme activity about 15 to 20%.     

Disruption of native and recombinant Escherichia coli in a high-pressure homogenizer

The disruption of native and recombinant strains of Escherichia coli was studied using a high-pressure homogenizer (Microfluidizer). The cells were grown in both batch and continuous fermentations. Cell suspensions ranging from 4 to 175 g dry wt/L were investigated at disruption pressures ranging from 30-95 MPa and at up to five passes. For both types of cells, the fraction of cells disrupted was dependent on the growth rate and concentration of the cells, the disruption pressure, and the number of passes through the disrupter. A model is presented that correlates the fractional disruption with these operating variables. The recombinant strain disrupted more readily than the native strain; 95 to 98% disruption of the former was achieved in two to three passes at a pressure of 95 MPa.     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Simulation of particle size distribution changes occurring during high-pressure disruption of bakers' yeast

Measurements of size distributions are provided for the breakage of commercial packed bakers' yeast cells as a function of operating pressure and number of passes through a Manton Gaulin high-pressure homogenizer. A two parameter model was developed, based upon the use of a Boltzmann function, to simulate the changes in size distribution that accompany the cell breakage process. The effects of operating pressure and number of passes are incorporated in the model and the result is used to simulate the particle size distribution of the cell homogenate. The results show that there is little breakage below a threshold pressure of 115 bar and above which breakage is critically dependent upon the pressure and number of passes through the homogenizer. The analysis provides a means of studying the efficiency of centrifugation that may follow cell disruption and provides the basis for further studies of size distribution changes accompanying cell disruption. (c) 1996 John Wiley & Sons, Inc.     

Disruption of Candida utilis cells in high pressure flow devices*

The disruption of Candida utilis cells in suspensions subjected to different types of stress was investigated. Stresses caused by impingement of a high velocity jet of suspended cells against a stationary surface were found to be significantly more effective for disruption than either shear or normal stresses. The fraction of cells disrupted by impingement is a first order function of the number of passes through the disruptor and, over a prescribed range of operating pressures, is a power function of pressure. These results indicate that impingement is the predominant mechanism causing cells disruption in high pressure flow devices such as Manton–Gaulin homogenizers. The impingement results suggest that cells grown in cyclic batch culture are easier to disrupt than cells grown at a lower specific growth rate in continuous culture. In addition to determining the fraction of cells disrupted, the release of invertase activity was determined for the impingement experiments. The fraction of total invertase activity released was found to be somewhat greater than the fraction of cells disrupted.     

Effect of process variables on supercritical fluid disruption of Ralstonia eutropha cells for poly(R-hydroxybutyrate) recovery

This research focuses on the disruption of the gram-negative bacterium Ralstonia eutropha cells by supercritical CO2 for poly(R-hydroxybutyrate) (PHB) recovery. The variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical CO2 pressure, have been studied. Effect of this disruption technique on PHB molecular mass was also investigated. PHB recovery was examined using a combination of this method and chemical pretreatments. For salt pretreatment, the cells were exposed to 140 mM NaCl and heat (60 degrees C, 1 h). The cells were also exposed to 0.2-0.8% (w/w) NaOH to examine the effect of alkaline pretreatment. Bacterial cells treated in growth phase exhibited less resistance to disruption than nutrient-limited cells in the stationary phase. It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells. Pretreatment with a minimum of 0.4% (w/w) NaOH was necessary to enable complete disruption with two times pressure release. Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock. The proposed method is economic and comparable with other recovery methods in terms of the percentage of PHB recovery and energy consumption, while it is environmentally more benign.     

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.     

Enhanced disruption of Candida utilis using enzymatic pretreatment and high-pressure homogenization

The enhancement of the overall disruption of a native strain of Candida utilis (ATCC 9226) was studied using a combination of two methods, namely, pretreatment in the form of partial enzymatic lysis by Zymolyase followed by mechanical disruption in a Microfluidizer high-pressure homogenizer. The cells were grown in both batch and continuous cultures to examine the effect of specific growth rate on disruption. Cell suspensions ranging in concentration from 7 to 120 g DW/L were disrupted with and without enzymatic pretreatment. For yeast grown in batch culture, final total disruption obtained using the combined protocol approached 95% with four passes at a pressure of 95 MPa, as compared with only 65% disruption using only mechanical homogenization. A modified model was developed to predict the fraction disrupted by the enzymatic pretreatment-mechanical homogenization two-stage process. Predicted disruptions agreed favorably with experimental observations (maximum deviation of 20%) over a wide range of operating conditions. (c) 1994 John Wiley & Sons, Inc.     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

Growth in cadmium-containing medium induces resistance to heat in E. coli

We have shown that 10 microM Cd2+ in the growth medium can induce resistance to subsequent heat treatment in E. coli B/r. Resistance was shown by cells during an extended lag phase and, especially, during log phase. The results contrast with the effect of Cd2+ exposure on radiation lethality, for which sensitization was previously reported in cells from lag and stationary phase cultures.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Regulation of mitochondrial biogenesis. Occurrence of non-functioning components of the mitochondrial respiratory chain in Saccharomyces cerevisiae grown in the presence of proteinase inhibitors: evidence for proteolytic control over assembly of the respiratory chain.

Yeast was grown in glucose- or galactose-containing media without or with proteinase inhibitors, phenylmethanesulphonyl fluoride and pepstatin. Culture growth was practically not affected by these compounds. Yeast growth on glucose in the presence of either phenylmethanesulphonyl fluoride or pepstatin entails accumulation of cytochromes c, c1, b and aa3 to a 25--30% excess above the control by the stationary phase, while cell respiration is unaffected. During growth on galactose the maximal cytochrome content (per unit weight of biomass) is reached in the mid-exponential phase and then decreases by 30--40% towards the stationary phase, while cell respiration remains constant. Addition of phenylmethanesulphonyl fluoride or pepstatin in the mid-exponential phase blocks the decrease in cytochrome levels and has no effect on cell respiration. Mitochondrial populations isolated from stationary-phase control and phenylmethanesulphonyl fluoride-grown cells glucose cultures display identical succinate oxidase and partial-respiratory-chain activities, despite the differences in cytochrome contents. However, the activities of individual respiratory complexes measured after maximal activation are nearly proportional to the amounts of corresponding components. The same situation holds true for mitochondrial populations from mid-exponential-phase, stationary-phase control and stationary-phase inhibitor-grown cells of galactose cultures. The findings suggest that the 'surplus' respiratory-chain components do not participate in electron flow because of the lack of interaction with adjacent carriers.     

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.     

Why do rotifer populations present a typical sigmoid growth curve?

To determine the underlying processes to population growth in the rotifer Brachionus plicatilis, we conducted an experiment using 1.5 ml cultures for 70 days. All individuals were transferred daily to culture media containing algae, and the number of individuals, clutch sizes and number of deaths were counted. The population dynamics showed a typical sigmoid curve. The population density increased exponentially from 10 to 682 individuals during the first 7 days (exponential growth phase), and gradually up to about 1500 individuals during the next 30 days (post-exponential growth phase). The population density then remained at a constant level with small fluctuations during the rest of the experimental period (stationary phase). Mortalities appeared from the post-exponential growth phase and were almost constant at about 2% throughout the experimental period. The clutch size decreased from 5 to 1 during the first 5 days, and afterwards females laid only one egg each. The proportion of non-reproductive females increased from 30% (exponential growth phase) to 80% (post-exponential growth phase) to 90% (stationary phase). These results suggest that the exponential growth phase resulted from the imbalance between a high birth rate and a low death rate, while the stationary phase was maintained by the compensation between low birth and death rates.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

The release of virus-like particles from recombinant Saccharomyces cerevisiae: effect of freezing and thawing on homogenization and bead milling

Recombinant cells of Saccharomyces cerevisiae, expressing virus-like particles (Ty-VLPs), can be readily disrupted in a high pressure homogenizer and show identical disruption kinetics to the untransformed host strain. When the cells are freeze/thawed before disruption, they become about four times more resistant to homogenization. This effect increases with the number of freeze/thaw cycles, but is independent of the time the cells remain frozen. The freeze/thaw effect is observed with cells harvested during both the logarithmic and stationary phase of growth, and occurs with the untransformed host strain as well as the transformed one. Freeze/thawed cells are twice as resistant to disruption in the bead mill as fresh cells. (c) 1994 John Wiley & Sons, Inc.     

Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.     

Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis

Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space. Received: 27 February 1998 / Received revision: 21 August 1998 / Accepted: 3 September 1998     

Cell arrangement,mass and dimension ofStreptococcus faecalis during growth

During exponential growth ofStreptococcus faecalis, the distribution of cell arrangements remains constant, but depends on the growth rate. The predominant cell arrangements are diplococci (about 60–80% of total cells) the amount of which varies only little with the growth rate. A clear correlation exists for cells growing as chains; the amount decreases from about 20% at μ=2.0 to about 6% at μ=0.45. After cessation of growth in the stationary phase, the number of diplococci and chains decreases and the number of monococci increases; after 10 h in the stationary phase, more than 50% of the cells have become monococci. The dry weight of 2.5×10⁻¹⁰ mg/cell remains constant at different growth rates, while cell size shows small differences on different growth media. Treatment of exponentially growing cultures with crystal violet or nitrofurantoin results in faster sedimentation on sucrose gradients of treated cultures compared to untreated cultures. While crystal violet effects an increased chain formation, treatment with nitrofurantoin results in an increase of the size of the individual cell.     

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria

Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.