Decrease In Specific Growth Rate Research Articles

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate.

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.

Ethanol-induced dimorphism and lipid composition changes in Mucor fragilis CCMI 142.

To study the effect of ethanol on morphology, lipid production and fatty acid profile of Mucor fragilis CCMI 142 cultures. Cell inhibition in shake flask cultures due to alcohol toxicity grew linearly from 0.418 mol x 1(-1) to 0.816 mol x 1(-1) ethanol corresponding to a decrease of specific growth rate. The growth inhibition constant took the value of 2.27 mol x 1(-1). The germination of fungal spores into hyphae is inhibited by concentrations from 0.418 mol x 1(-1) to 0.816 mol x 1(-1) ethanol. In this range, M. fragilis CCMI 142 spores form, exclusively, budding yeast-like cells instead of filaments. Below 0.418 mol 1-1 ethanol the formation of yeast-like cells was stimulated and there was a spore germination delay. The lipid content decreased as the concentration of ethanol increased, and was associated with an increase of unsaturated/saturated fatty acid ratio. The major conclusion of the study is the production of an enriched unsaturated fatty acids final product with particular emphasis to the presence of gamma-linolenic acid (18:3omega6) a biologically active compound with a useful impact in nutraceutical science.

Morphological characterization of Aspergillus nidulans: growth, septation and fragmentation.

The influence of the sepA gene on the growth of Aspergillus nidulans has been investigated by characterizing and comparing the parental strain A28 (pabaA6 biA1) with the sepA null mutant (sepA4DeltaBM:). The sepA gene is known to affect the septation process in A. nidulans, therefore the sepA4DeltaBM: strain does not produce any septa during the first hours of growth. During batch cultivations sepA4DeltaBM: shows an abrupt decrease in specific growth rate and more pronounced fragmentation (in response to elevated stirrer speed) than the parental strain. Higher specific fragmentation rates (q(frag)) were obtained for the sepA4DeltaBM: strain. The physiological reasons for the differences have been investigated by employing fluorescent stains. Computerized image analysis revealed that the more pronounced fragmentation in the mutant was due to the lower number and irregular spacing of septa (visualized by calcofluor white staining), which resulted in a weaker hyphal structure that is more vulnerable to shear stress and fragmentation than the parental strain. This led to a loss of active biomass (determined by Mag fura staining) from the hyphae of the mutant, which had failed to compartmentalize by formation of septa, in turn resulting in decreased specific growth rates for the culture.

Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma.

When subjected to hyperosmotic pressure resulting from NaCl addition, KR12H-2 transfectoma, like most hybridomas, displayed a decrease in specific growth rate (mu) and an increase in specific antibody productivity (q(Ab)). Elevation of medium osmolality from 285 to 425 mOsm/kg decreased mu by 20%, while it increased q(Ab) by 376%. Although cell mass also increased at higher osmolality, it was not the main factor in increasing q(Ab). Hyperosmotic pressure was found to enhance transcription levels of immunoglobulin (Ig) mRNAs preferentially, compared with non-IgG mRNA. The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs were enhanced as much as q(Ab). This result suggests that enhanced q(Ab) at higher osmolality was mainly due to enhanced transcription levels of Ig mRNA. However, these increased transcription levels of Ig mRNAs were not due to the enhanced stability of Ig mRNA. In fact, the stability of Ig mRNAs decreased at higher osmolality. Elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg decreased the half-lives of HC and LC mRNAs by 37% and 36%, respectively. A simple mathematical model revealed that transcription rates of Ig mRNAs increased by more than 476% at 425 mOsm/kg. These elevated transcription levels could, in turn, increase the translation rates of Ig polypeptides. However, the translation rates of Ig polypeptides were not enhanced as much as the transcription levels of Ig mRNAs and q(Ab). The elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg increased HC and LC mRNA specific translation rates by 172% and 240%, respectively. Taken together, the data suggest that (1) enhanced q(Ab) of KR12H-2 transfectoma at higher osmolality is due to elevated transcription rates of Ig mRNAs and expedited post-translational processing of Ig, and (2) antibody secretion by KR12H-2 transfectoma is most likely controlled at the level of Ig translation, particularly HC translation.

Fed-batch xylitol production with two recombinant Saccharomyces cerevisiae strains expressing XYL1 at different levels, using glucose as a cosubstrate: A comparison of production parameters and strain stability

Xylitol production with two recombinant Sacharomyces cerevisiae strains expressing the XYL1 gene, coding for xylose reductase (XR), at different levels, the 'low XR strain' at 0.51 U/mg and the 'high XR strain' at 10.8 U/mg, was compared in batch and fed-batch culture. Xylose was not consumed in the presence of high glucose concentrations, because both sugars are transported by the glucose transport system, which has a higher affinity for glucose than for xylose. When glucose was fed gradually to the culture, high concentrations were avoided, and xylose was converted to xylitol with a specific productivity of 0.10 g g(-1) h(-1) attained with the low XR strain and 0.19 g g(-1) h(-1) with the high XR strain, indicating that factors other than the XR-activity control the rate of xylose conversion.The overproduction of XR put a substantial protein burden on the high XR strain, contributing to a 50% decrease in specific growth rate and reduced biomass yield compared with the low XR strain. Despite the use of selective medium, the stability of the high XR strain was poor in long fed-batch and chemostat cultures, whereas the low XR strain was stable. The high XR strain lost its XR activity almost completely in some fed-batch cultures and in chemostat culture. In chemostat cultivation, part of the population lost the plasmid harboring the XR gene. This was due to the fact that leucine was released into the broth from plasmid containing cells, which enabled some cells to grow without the plasmid containing the LEU2 auxotrophic complementation selection marker. Furthermore, isolation and analysis of plasmids from a population that had lost its XR activity, showed that in addition to the original plasmid, a rearranged form of the plasmid, retaining the selection marker but not the expression of active XR, was present. However, these observations could only partly explain the decrease in XR activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 391-399, 1997.

Effect of nonylphenol on growth of Neurospora crassa and Candida albicans.

The effects of the nonionic surfactant nonylphenol on the growth and morphologies of the filamentous fungus Neurospora crassa and the diploid yeast Candida albicans have been examined. Nonylphenol inhibited respiration and growth of N. crassa, effecting a 10-fold decrease in organism yield at 25 microM. Severe morphological defects were also induced: cell shape was abnormal and apical dominance was lost. Nonylphenol monoethoxylate (the parent compound of nonylphenol) was a less potent growth inhibitor and morphogen. The growth of the yeast form of C. albicans was sensitive to nonylphenol (inducing an order of magnitude decrease in specific growth rate with a 10-fold increase in dose concentration) but not nonylphenol monoethoxylate. Similarly, C. albicans ATP content was reduced and glucose-induced extracellular acidification was inhibited only by nonylphenol. Although estrogens may induce the dimorphic transition of C. albicans, nonylphenol (as an environmental estrogen mimic) failed to trigger germ tube formation under nonpermissive conditions and inhibited it under permissive conditions. The effects of nonylphenol are most readily explained as the result of uncoupling of respiration, which produces multiple physiological effects.

Enhancement of eicosapentaenoic acid (EPA) and γ‐linolenic acid (GLA) production by manipulating algal density of outdoor cultures of Monodus subterraneus (Eustigmatophyta) and Spirulina platensis (Cyanobacteria)

The effect of algal density on cell growth and composition with special reference to the production of eicosapentaenoic acid (EPA) and γ‐linolenic acid (GLA) was investigated in semi-continuous outdoor cultures of Monodus subterraneus and Spirulina platensis. In both species, an exponential decrease in specific growth rate and a positive skewed pattern in biomass productivity were associated with an increase in density. In M. subterraneus, the highest EPA cell content (3·8% of dry weight) occurred at the optimal density of c. 4gl−1 i.e. the density that yields the highest output rate of biomass per culture volume or area. In S. platensis, high density was associated with a decrease in total fatty acid content, but the relative abundance of GLA increased. GLA content was therefore fairly stable at c. 1% of dry weight throughout the range of algal density tested. Fatty acid desaturation seemed to be associated with an increase in culture density of S. platensis, resulting in an increase in the proportion of the fatty acids 16:1 and GLA and a decrease in 16:0 and 18:2. Using a flat plate reactor with a narrow light‐path and intensive stirring which facilitated high cell concentration, we obtained maximal EPA productivity of 58·9mg1−1 day−1 in M. subterraneus and maximal GLA productivity of 26·4 mg1−1 day−1 in S. platensis. The former is the highest EPA production rate reported to date for any microalga, and the latter is the first report on GLA productivity by microalgae. The data presented herein suggest that maintaining very high, yet optimal culture densities (i.e. 4−10gl−1) in enclosed reactors represents an efficient operating mode for enhancing productivity of desired polyunsaturated fatty acids.

Insect cell growth evaluation during serum-free adaptation in stirred suspension cultures

Sf-9 insect cells were adapted to three different serum-free media (SF900II, EXCELL 401 and IPL/41 supplemented) in 125 ml stirred vessels by gradually reducing serum concentration from 10 to 0% (v/v). TC100 medium sup-plemented with 10% fetal bovine serum was used as control. With this procedure it was possible to obtain cells fully adapted to SF900II and EXCELL 401 in 5 weeks. The adapted cells could be frozen in serum-free medium and thawed without any decrease in specific growth rate or maximum cell concentration. Even after 4 months of culture in stirred vessels at 170 rpm the specific growth rate and maximum cell concentration (0.031 h and 4.8 × 10 cells/ml, respectively) remained constant.

Expression parameters for target gene cloned in Escherichia coli in response to phosphate supply

Recombinant strain of E.coli 1727 overexpressed target gene at enhanced level when supplied with excess of inorganic phosphate. Rate of target gene expression, yield coefficient for target gene product and plasmid copy number increased significantly: 50%, 100% and 40% respectively at 125 mM excess phosphate. This was, however, accompanied by 26% decrease in specific growth rate and 30% in cellular growth yield coefficient.

Correlation of Aspergillus niger broth rheological properties with biomass concentration and the shape of mycelial aggregates

Aspergillus niger was grown in a 7-L chemostat at biomass levels of 7 to 9 gL(-1); dilution rates of 0.03, 0.05, 0.075, and 0.009 h(-1); and dissolved oxygen tensions of 7%, 12%, and 40% of air saturation. Broth rheological measurements were made on-line, while off-line image analysis was used to measure mycelial morphology, including characterization of mycelial aggregates (clumps). Under all conditions, more than 87% of the hyphase were in clumps, the shape of which determined the rheological characteristics of the broth. In particular, the power law consistency index could be correlated with the biomass concentration and the roughness factor of the clumps, which describes their hairiness. A decrease in specific growth rate decreased roughness, possibly due to changes in the amount of clump breakup. However, decreases of roughness with increasing dissolved oxygen tension might rather imply some effect on hyphal-hyphal interactions within the clumps.

The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts

Summary: The response of 31 ascomycetous yeasts to a reduction in water activity (aW) adjusted with D-glucose or NaCl was investigated. The growth of most yeasts was more tolerant to glucose than to NaCl at equivalent aW. Zygosaccharomyces rouxii was the most osmotolerant yeast examined. Natural abundance 13C-NMR spectroscopy and HPLC analyses of eight yeasts indicated that glycerol and arabitol or mannitol were accumulated intracellularly in response to aW. reduction. Pichia sorbitophila, Candida cacaoi, Candida magnoliae and Zygosaccharomyces bisporus responded to reduced aW by a decrease in specific growth rate and cell volume, and an accumulation of glycerol. The other polyol accumulated did not increase in concentration with aW reduction to the same degree as glycerol. A polyol concentration ratio (intra/extracellular) as high as 800-fold was attained across the membrane. Greater amounts of polyols were produced at equivalent aW values when adjusted with glucose than with NaCl. The ability to accumulate high concentrations of polyols appears to be the most important criterion in determining osmotolerance.

Ammonium Transport in the Alkalophilic Diazotrophic Cyanobacterium Nostoc calcicola: Influence of Phosphate Limitation and Metabolic Inhibitors

Ammonium transport in the diazotrophic cyanobacterium Nostoc calcicola was investigated in phosphate-sufficient and deficient conditions. Cells grown under phosphate-deficient conditions were characterized by a decrease in specific growth rate, phycocyanin content (50 %), oxygen evolution (50 %), reduced ATP pool and derepressed alkaline phosphatase activity. Under these conditions ammonium transport exhibited no change in K m (10.0 µM) in comparison to phosphate-sufficient cells, but a change in V max (41.0 nmol mg -1 protein min -1 ) was observed. The reduced rate of ammonium transport was attributed to unavailability of sufficient energy. Conditions that lowered or abolished the ATP pool (dark incubation or treatment with metabolic inhibitors DCMU, DCCD or CCCP) also reduced ammonium transport via two transport systems. The results indicated that unavailability of phosphate in the immediate environment may limit ammonium transport by a diazotrophic cyanobacterium.

Factors influencing the affinity and capacity of T3-binding sites in hepatic nuclei of rainbow trout,Oncorhynchus mykiss

The effects of several variables were tested on the in vitro binding affinity (Ka) and maximum binding capacity (MBC) of saturable 3,5,3′-triiodo-L-thyronine (T3)-binding sites (putative thyroid hormone receptors) extracted from hepatic nuclei of immature rainbow trout held on a 12L:12D photocycle at 12°C. Neither Ka nor MBC varied during the daily photocycle, nor were they influenced by the growth rate, food ration, or a 13-fold chronic elevation in plasma T3. However, MBC was inversely correlated with fish size and was depressed significantly by two injections of 17β-estradiol (E2) or starvation for 3 or 12 days. The Ka was uninfluenced in either case. Since both E2 and starvation also depress T3 levels in the blood, the decrease in MBC could be secondary to this change. The drug ipodate, which depresses plasma T3, also lowered the MBC. However, MBC remained low in ipodate-treated trout given exogenous T3, thus suggesting that the effect of ipodate was not due to T3 deprivation. It remains to be shown, therefore, that plasma T3 depression itself can modify MBC. It is concluded that increasing body size and catabolic states, both of which are associated with a decrease in specific growth rate, tend to decrease the abundance of hepatic T3 receptors.

Effects of Some Environmental Factors on Growth Characteristics of Candida utilis on Peat Hydrolysates

Samples of peat from Pine Island and Brookston bogs in Minnesota were hydrolyzed with various concentrations of HCl or H(2)SO(4) solutions, before or after debituminization (an extraction process used to remove waxy materials, bitumens, from peat), to produce peak hydrolysates as growth substrates for Candida utilis. Hydrolysates were neutralized with concentrated NaOH solution to pH 3.5, 4.5, 5.0, 5.5, 6.0, and 7.0. The precipitated humates were removed by filtration. The resulting peat hydrolysates were amended with reagent-grade K(2)HPO(4), K(2)SO(4), and MgSO(4), 200, 100, and 50 mg per liter of peat hydrolysate, respectively. The debituminized peat produced more total nitrogen (TN) and reducing substances (RS) than the nondebituminized peat. Peat hydrolysates produced by HCl solutions contained slightly higher RS and TN than those produced by H(2)SO(4) solutions; however, the latter were better growth substrates than the former. The yield coefficients in both H(2)SO(4) and HCl hydrolysates initially decreased at 12 to 24 h and then increased gradually over the remaining incubation period (24 to 96 h). As TN and RS were increased, an increase in cell density, biomass, and productivity was observed. In contrast, a decrease in specific growth rate occurred as the RS and TN were increased. The generation time of C. utilis was affected by the concentrations of RS and TN. A peak substrate yield coefficient was found at pH 5.0 in HCl hydrolysates and at pH 6.0 to 6.5 in H(2)SO(4) hydrolysates. Good linear correlation coefficients were found between RS and biomass of C. utilis. The coefficients of correlation increased as the TN level in hydrolysates was increased.

Regulation of synthesis of serine hydroxymethyltransferase in chemostat cultures of Escherichia coli.

Serine hydroxymethyltransferase was synthesized as a constant fraction of total protein of Escherichia coli over a wide range of specific growth rates. This was observed in all strains when grown in glucose-limited chemostat cultures; in thymine-requiring mutants during thymidine-limited growth; and in met A and met B auxotrophs, defective in homocysteine biosynthesis, during methionine-limited growth. This behavior has been referred to by others as "metabolic control." In addition, the synthesis of serine hydroxy-methyltransferase was subject to specific active control mechanisms, which responded to the needs of the cell for purine biosynthesis, methylation reactions, as well as to serine limitation. Under purine limitation, the rate of enzyme synthesis increased with decreasing growth rate, that is with increasing purine limitation. During methionine-limited growth of met E and met F auxotrophs (mutants unable to methylate homocysteine) the rate of enzyme synthesis increased with a decrease in specific growth rate from 0.65 to 0.30 h-1 but declined with further decrease in growth rate. Under serine limitation the rate of enzyme synthesis remained proportional to the growth rate, but at a rate twice that observed in unrestricted or glucose-limited growth. When purines were added to unrestricted or glucose-limited cultures, the rate of enzyme synthesis decreased by 40%, but remained proportional to growth rate. Addition of methionine or serine alone had no effect.

High density production of sorbose from sorbitol by fed-batch culture with DO-stat.

Gluconobacter suboxydans was cultivated in a fed-batch culture with a DO-stat under the condition that none of the components in the basal medium limited the growth of the microorganism and the production of sorbose. Final concentration of sorbose reached 460 g/l after 16 hrs with CSL medium and 628 g/l after 14 hrs with YE medium, respectively. Conversion ratio of sorbitol to sorbose was almost maintained at about 0.9 for CSL medium and about 1.0 for YE medium during the cultivation. The effect of sorbose concentration on growth was investigated in test tube culture. Decrease in specific growth rate and a lag phase were observed with increase in sorbose concentration in culture medium. The growth was inhibited considerably at 300 g/l of sorbose concentration and was not observed at sorbose concentrations over 510 g/l. The effect of osmotic pressure and water activity on growth was examined. Growth rate and sorbose production rate decreased as osmotic pressure increased or water activity decreased due to the accumulation of sorbose.

The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate.

The uptake of thirteen essential amino acids by mouse LS cells in suspension culture was determined by bacteriological assay methods. Chemostat continuous-flow cultures were used to determine the effect of different cell growth rates on the quantitative amino acid requirements for growth. The growth yields of the cells ( Y = g cell dry weight produced/g amino acid utilized) were calculated for each of the essential amino acids. A mixture of the non-essential amino acids, serine, alanine and glycine increased the cell yield from the essential amino acids. The growth yields from nearly all the essential amino acids in batch culture were increased when glutamic acid was substituted for the glutamine in the medium. The growth yields from the amino acids in batch culture were much less at the beginning than at the end of the culture. The highest efficiencies of conversion of amino acids to cell material were obtained by chemostat culture. When glutamic acid largely replaced the glutamine in the medium the conversion of amino acid nitrogen to cell nitrogen was 100 % efficient (that is, the theoretical yield was obtained) at the optimum growth rate (cell doubling time, 43 h). The maximum population density a given amino acid mixture will support can be calculated from the data. It is concluded that in several routinely used tissue culture media the cell growth is limited by the amino acid supply. In batch culture glutamine was wasted by (1) its spontaneous decomposition to pyrrolidone carboxylic acid and ammonia, and (2) its enzymic breakdown to glutamic acid and ammonia, but also glutamine was used less efficiently than glutamic acid. Study of the influence of cell growth rate on amino acid uptake rates per unit mass of cells indicated that a marked change in amino acid metabolism occurred at a specific growth rate of 0.4 day -1 (cell doubling time, 43 h). With decrease in specific growth rate below 0.4 day -1 there was a marked stimulation of amino acid uptake rate per cell and essential amino acids were consumed increasingly for functions other than synthesis of cell material.

Morphology of Liberian Negro deciduous teeth I. Odontometry.

AbstractAn odontometric study of the deciduous teeth of 21 Liberian Negro children is reported. These data were obtained from dental casts alone. Statistical comparisons were made of the mesiodistal diameter alone with two American White populations and the three Japanese populations. Maxillary teeth, with the exception of the cuspids were larger in the Negro sample, while only the Negro mandibular molars were significantly wider. This situation resulted in an alteration of Negro maxillary tooth size order. Comparisons were made with other populations reported in the literature which led us to stress the fact that, as yet, no satisfactory sample of deciduous tooth measurements are available for any population. A discussion of tooth form differences stresses the point that teeth differ in both attributes of form, i.e., in shape as well as in size between the several major racial groups. The processes by which these shape differences are brought about were discussed and it was suggested that differing relative growth rates may well be one cause. An allometric analysis of fetal molar tooth growth showed, again, that a sharp interphase occurs in all teeth, with a subsequent decrease in specific growth rate. This interphase corresponds to the initiation of cuspal calcification.