Effect Of Specific Growth Rate Research Articles

Analysis of the effects of specific growth rate of Lactococcus lactis MG1363 on aerobic metabolism and its application to high-density culture

Lactic acid bacteria (LAB) are known to produce a large amount of lactate when cultured under non-aerated conditions, which inhibits their growth at high concentrations. Our previous studies have shown that LAB can be cultured without lactate production under aerated conditions at a low specific growth rate. In this study, we investigated the effects of specific growth rate on cell yield and the specific production rates of metabolites in aerated fed-batch cultures of Lactococcus lactis MG1363. The results showed that lactate and acetoin production could be suppressed at specific growth rates below 0.2 h-1, whereas acetate production was the highest at a specific growth rate of 0.2 h-1. When LAB was cultured at a specific growth rate of 0.25 h-1 with the addition of 5mg/L heme to assist ATP production by respiration, lactate and acetate production was suppressed, and cell concentration reached 19g-dry-cell/L (5.6× 10ˆ10cfu/mL) with a high cell yield of 0.42 ± 0.02g-dry-cell/g-glucose.

Genome-scale modeling for Bacillus coagulans to understand the metabolic characteristics.

Lactic acid is widely used in many industries, especially in the production of poly-lactic acid. Bacillus coagulans is a promising lactic acid producer in industrial fermentation due to its thermophilic property. In this study, we developed the first genome-scale metabolic model (GEM) of B. coagulans iBag597, together with an enzyme-constrained model ec-iBag597. We measured strain-specific biomass composition and integrated the data into a biomass equation. Then, we validated iBag597 against experimental data generated in this study, including amino acid requirements and carbon source utilization, showing that simulations were generally consistent with the experimental results. Subsequently, we carried out chemostats to investigate the effects of specific growth rate and culture pH on metabolism of B. coagulans. Meanwhile, we used iBag597 to estimate the intracellular metabolic fluxes for those conditions. The results showed that B. coagulans was capable of generating ATP via multiple pathways, and switched among them in response to various conditions. With ec-iBag597, we estimated the protein cost and protein efficiency for each ATP-producing pathway to investigate the switches. Our models pave the way for systems biology of B. coagulans, and our findings suggest that maintaining a proper growth rate and selecting an optimal pH are beneficial for lactate fermentation.

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity.

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μmax , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.

Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter

Carbon source and growth rate are two major parameters affecting recombinant protein production in Pichia pastoris. The effect of the most commonly used carbon sources (glycerol or glucose) and the specific growth rate (μ) has been studied on the production of a human antigen-binding fragment (Fab) in this cell factory under the constitutive GAP promoter in fed-batch cultures.Glycerol for batch phase and glucose for fed-batch phase was the most successful carbon source combination. During batch phase, by-products were detected when glucose was used, despite maintaining DO at values higher than 35%. Also, the presence of cell aggregates was detected affecting the reproducibility and operability of the bioprocess.Conversely, glucose was the best substrate for fed-batch phase. When working at C-limiting conditions, neither by-products nor aggregates were detected and Fab production levels were comparable to those obtained with glycerol. In addition, the lower heat yield (YQ/X) and oxygen to biomass yield (YO2/X) for glucose-supported cultures made this substrate the best alternative from an industrial operational point of view.In addition, the effect of specific growth rate on fed-batch Fab production was studied. Medium and high μ (0.10 and 0.15h−1) set-points showed similar Fab production yield. However, in terms of total and volumetric productivity, higher μ was the best process condition.

RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis.

BackgroundMean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different conditions of exponential growth.ResultsIt was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes). Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms.ConclusionOur results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and increased transcription of cellular maintenance genes at the expense of growth related genes. The strong link between P:N stoichiometry, RNA:protein ratio, ribosomal requirement for protein synthesis, and growth rate of microorganisms indicated by the study could be used to characterize the N and P economy of complex ecosystems such as soils and the oceans.

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l(-1). However, high initial concentrations of oleic acid up to 50 g l(-1) were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml(-1) after 48 h with 5 g l(-1) of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l(-1) and the extracellular lipase activity was 6.3 U ml(-1) at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h(-1)) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l(-1) when the set point of specific growth rate (mu(set)) was 0.02 h(-1). High specific growth rate (0.04 and 0.08 h(-1)) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml(-1) when mu(set) was 0.02 h(-1), while the highest lipase productivity was 0.31 U ml(-1) h(-1) at mu(set) of 0.08 h(-1).

The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei

Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0.022-0.033 h(-1), the highest specific rate of total protein production being 4.1 mg g(-1) h(-1) at the specific growth rate 0.031 h(-1). At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6-8 % at high specific growth rates, 0.045-0.066 h(-1). To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0.031 h(-1). However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.

Degradation of EDTA by a chemostat culture of bacterial strain DSM 9103

Characteristics of growth and EDTA degradation by chemostat culture of bacterial strain DSM 9103 were investigated. The effect of specific growth rate ( μ) on mass cell yield ( Y X/S), specific rate of EDTA uptake ( q S) and the rate of Mg–EDTA degradation by a cell suspension ( q S rest) was studied. Based on the experimental data, kinetic constants of dependencies q S( S) and μ( S) were determined. The rates of substrate and energy expenditures for cell maintenance, m S and m e, were 0.02 and 0.014 h −1, respectively. High efficiency of DSM 9103 growth on EDTA in a chemostat was established; the maximum mass cell yield ( Y X/S m) and the maximum energy cell yield ( η X/S m) were as high as 35.3 and 50.5%, respectively. Results indicated that oxidation of side chains in the EDTA molecule by monooxygenase is coupled with energy utilization by the cells. Cells harvested from a chemostat (resting cells) retain the capability of EDTA degradation, although at a rate about four to five times lower than growing cells.

Effect of specific growth rate on the production of a recombinant nuclease by Escherichia coli

The effect of specific growth rate μ on the production of Vibrio vulnificus nuclease was investigated in fed-batch cultures of a high-yield recombinant Escherichia coli. A pseudo-exponential feeding method for controlling μ was employed to obtain the desired data. It was found that the nuclease content of cells γ P varied with μ, with a maximum occurred at μ=0.05 h −1; however, the specific nuclease production rate q p increased with increasing μ. The synthesis of nuclease was dependent on μ, irrespective of the feeding methods. When concerning the compromise between the nuclease yield and its production rate, the linear-gradient feeding method, being simple and adaptable, was shown to be adequate for the nuclease production.

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae.

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers' yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h(-1) under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h(-1) and 0.40 h(-1). Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.

Effect of specific growth rate on fermentative capacity of baker's yeast.

The specific growth rate is a key control parameter in the industrial production of baker's yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h-1, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol . g of biomass-1 . h-1 at D = 0.40 h-1. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h-1). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.025 h-1 to 20.5 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.28 h-1. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.40 h-1. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.

Effect of Growth Rate on the Production of β-Galactosidase from Escherichia Coli in Bacillus Subtilis Using Glucose-Limited Exponentially Fedbatch Cultures

The effect of specific growth rate (μ) on the production of β-galactosidase from Escherichia coli in Bacillus subtilis was assessed using glucose-limited exponentially fedbatch cultures (EFBC). A B subtilis strain carrying a chromosomal copy of the lacZ gene under the control of the subtilisin (aprE) regulatory region was used in this study. This strain also carries the hpr2 and degU32(Hy) mutations which are known to overexpress the subtilisin (aprE) gene. Constant μ in the range of 0.058–0.35 h −1 were maintained using mineral media and computer-controlled predetermined glucose feeding profiles. At all growth rates tested, a pseudo-steady state in μ was obtained; however, a sudden decrease in the slope of log of biomass concentration against time and accumulation of glucose and acetic acid indicated that control on μ was lost at all growth rates tested. Cultures maintained at a μ value close to the maximum obtained in batch cultures had a specific enzymatic activity relatively constant during the fedbatch phase. In contrast, cultures maintained at a μ value lower than 0.2 h −1 had specific and volumetric β-galactosidase activities that decreased during the fedbatch phase, reaching a value close to zero before glucose feeding was stopped. The maximum β-galactosidase activity after the EFBC phase was obtained at the maximum μ tested whereas it strongly decreased at lower μ values. This suggests that glucose limitation and μ reduction during exponential growth phase represses β-galactosidase production during the exponential and stationary phases.

Kinetic analysis of morphological differentiation and protease production in Streptomyces albidoflavus SMF301.

The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of Streptomyces albidoflavus SMF301 were determined in chemostat cultures. Production of three types of proteases: chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP) were analysed in relation to mycelium growth and spore formation. Production of CTP was closely linked to mycelium growth, whereas spore formation, TLP synthesis and MTP synthesis were inversely related to growth. Evaluation of various kinetic parameters [specific production rates of spores (q spo), TLP (q TLP), MTP (q MTP) and CTP (q CTP)] showed that mycelium growth rate and CTP production were optimal at 0.1 h-1, but submerged spore formation, TLP production and MTP production were optimal at 0.025 h-1. Changes in specific nutrient uptake rates [glucose (q glu), ammonium ion (q amn) and phosphate (q pho)] affected sporulation and protease production; limitation of carbon, nitrogen and phosphate stimulated spore, TLP and MTP production.

A study of the effect of specific growth rate and acetate on recombinant protein production of Escherichia coli JM107

The growth of Escherichia coli in fed-batch and continuous culture is examined and results show that α-amylase production is strongly dependent on specific growth rate (dilution rate) of the culture and production is greatest at an intermediate rate. Using continuous culture, it has also been found that the presence of acetate above a certain concentration reduces both μmax, and the production of recombinant protein.

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.

Iron chelator, exopolysaccharide and protease production in Staphylococcus epidermidis: a comparative study of the effects of specific growth rate in biofilm and planktonic culture.

The growth rate of Staphylococcus epidermidis was controlled for populations growing as a biofilm and perfused with supplemented, simple-salts medium. Production of iron chelators, extracellular protease and exopolysaccharide (EPS) by these populations was assessed as a function of specific growth rate and compared to that by planktonic populations grown in the same medium within a chemostat. Perfused biofilms increased their iron chelator and protease production with increasing growth rate. Chemostat populations decreased their production of iron chelators with increasing growth rate, whilst showing much enhanced production of proteases at intermediate growth rates (mu 0.15-0.25 h-1). Production of iron chelator and protease was generally 2-50 times higher by biofilms than by planktonic populations. EPS production was low and relatively unaffected by growth rate for the chemostat cultures (about 0.2 micrograms per unit cell mass) but high for the attached biofilms, particularly at slow growth rates (about 4 micrograms per unit cell mass). EPS production within the biofilms decreased markedly with increasing growth rate. At growth rates of 0.35 h-1 and above, the levels of EPS for biofilms and planktonic populations were equivalent. The results of this study clearly indicate that growth as a biofilm markedly influences extracellular virulence factor production by S. epidermidis.

Metabolic characterization of a L‐lysine‐producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.

The effect of specific growth rate and death rate on monoclonal antibody production in hybridoma chemostat cultures

AbstractExperimental data collected from bench‐scale chemostat cultures of mouse‐mouse hybridoma cells have been used for the development of a kinetic model of monoclonal antibody production. The specific antibody productivity was found to be proportional to the specific death rate, indicating a stimulating effect of stress on the ability of the cells to produce antibody. The death rate was found to be an exponential function of the average cell age in the culture. Furthermore, a Generalized Maintenance Energy (GME) model is proposed, to take account of the effects of culture stress on the nutrient uptake rates. The proposed relationships agreed very well with other mouse‐mouse hybridoma chemostat culture data in the literature.

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.