Minimal Requirements for Exponential Growth of Lactococcus lactis

A minimal growth medium containing glucose, acetate, vitamins, and eight amino acids allowed for growth of Lactococcus lactis subsp. lactis, with a specific growth rate in batch culture of μ = 0.3 h^-1. With 19 amino acids added, the growth rate increased to μ = 0.7 h^-1 and the exponential growth phase proceeded until high cell concentrations were reached. We show that morpholinepropanesulfonic acid (MOPS) is a suitable buffer for L. lactis and may be applied in high concentrations.     

Amino acid consumption by Chloroflexus aurantiacus in batch and continuous cultures

Amino acid consumption was studied with batch and continuous chemostat cultures of Chloroflexus aurantiacus grown phototrophically in complex medium with casamino acids (Pierson and Castenholz 1974). Amino acids like Arg, Asx, Thr, Ala, Tyr, which were utilized during the early exponential phase by cells grown in batch cultures were consumed in chemostat cultures essentially at any of the dilution rates employed (0.018–0.104 h^-1). Those amino acids which were taken up during subsequent phases of growth were consumed in chemostat cultures preferentially at low dilution rates. For example, the consumption of Glx was enhanced during the late exponential phase and at low dilution rates. At high dilution rates Glx was not consumed at all. Since Glx utilization largely paralleled bacteriochlorophyll formation, it is discussed that formation of the photopigment depends on the intracellular availability of Glu as the exclusive precursor for tetrapyrrole synthesis.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (P_I versus P_III type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing α_s1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the P_I-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a P_III-type proteinase.     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.     

A continuous culture study of the regulation of extracellular protease production inVibrio SA1

During growth ofVibrio SA1 in a lactate-limited chemostat in the presence of 2mm phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h^-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h^-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h^-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h^-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5mm cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5mm) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis inVibrio SA1.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l^–1 h^–1, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml^–1, by constant addition of sucrose at a feeding rate of 7 g l^–1 h^–1; an increase in 58% over that of the batch culture (2,658 IU ml^–1). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Quantitative physiology of Lactococcus lactis at extreme low‐growth rates

This paper describes the metabolic adaptation of Lactococcus lactis during the transition from a growing to a non‐growing state using retentostat cultivation. Under retentostat cultivation, the specific growth rate decreased from 0.025 h^?1 to 0.0001 h^?1 in 42 days, while doubling time increased to more than 260 days. Viability of the overall culture was maintained above 90% but included approximately 20% damaged cells, which had lost their colony forming capacity on solid media. Although culture biomass and viability had reached a steady‐state after 14 days of retentostat cultivation, the morphology of the cells changed from coccus‐to‐rod shape at later stages of retentostat cultivation, by which the cell's surface to volume ratio was estimated to increase 2.4‐fold. Furthermore, the metabolic patterns switched between homolactic and mixed‐acid fermentation during the retentostat cultivation. Retentostat cultivation enabled the calculation of accurate substrate‐ and energy‐related maintenance coefficients and biomass yields under non‐growing conditions, which were in good agreement with those calculated by extrapolation from chemostat cultivations at high dilution rates. In this study, we illustrate how retentostat cultivation allows decoupling of growth and non‐growth associated processes in L. lactis, enabling the analysis of quantitative physiological responses of this bacterium to near zero‐specific growth rates.     

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.     

Regulation of Proteolytic Activity in the Hyperthermophile Pyrococcus furiosus

Pyrococcus furiosus was shown to grow on casein or peptides as the sole carbon, energy, and nitrogen sources, while maltose could be used as a carbon and energy source only if peptides were present in the medium. A mixture of all 20 single amino acids could not replace the peptide requirement. Specific intracellular proteolytic activity was induced under low casein or tryptone levels and was decreased by the addition of maltose to both peptide-limiting and peptide-rich media in batch and continuous cultures. In a peptide-limited chemostat, activity towards azocasein and MeO-Suc-Arg-Pro-Tyr-p-nitroanilide reached a maximum at a dilution rate of 0.28 h, while activity toward l-lysine-p-nitroanilide reached a maximum at 0.50 h. Under peptide-limiting conditions, levels of the 66-kDa protease (S66) were enhanced relative to those of other cell proteins. Preliminary evidence suggests that this protease is immunologically related to the eukaryotic multicatalytic proteinase complex (proteosome).     

The effect of levofloxacin concentration on the development and maintenance of antibiotic-resistant clones of Escherichia coli in chemostat culture

A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg l⁻¹) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h⁻¹). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h⁻¹). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h⁻¹). At levofloxacin concentrations less than 0.03 mg l⁻¹, the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin. Journal of Industrial Microbiology & Biotechnology (2002) 29, 155–162 doi:10.1038/sj.jim.7000295 Received 26 September 2001/ Accepted in revised form 13 June 2002     

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation

A mutant of fast milk-coagulating (Fmc⁺) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc⁻) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc⁺ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.     

Simultaneous and successive induction of synthesis of β-Galactosidase and tryptophanase inEscherichia coli K 12 in the chemostat

β-Galactosidase and tryptophanase were induced either simultaneously or successively during continuous cultivation of the inducible strainEscherichia coli K 12 in the chemostat. Growth was limited by glycerol and the dilution rate was 0.1 h⁻¹. During both the simultaneous and successive induction specific rates of synthesis, as well as maximum enzyme levels, were identical with those obtained after independent induction of individual enzymes. As compared with batch cultivation, β-galactosidase reached the same specific rate of synthesis in the chemostat, whereas the specific rate of synthesis of tryptophanase in the chemostat was up to five times higher.