Growth of Bacteroides fragilis in Continuous Culture and in Batch Cultures at Controlled pH

Bacteroides fragilis NCTC 9343 has been grown in continuous cultures with glucose as growth-limiting factor. At pH 7.0 and at a dilution rate of 0.07 per h, glucose limited growth in concentrations up to 0.6%. Maximal cell yield and productivity were obtained with 0.87% glucose in the inflowing medium. A pH of 7.0 was optimal for growth. With 0.6% glucose in the fresh medium and at pH 7.0, cell yield and productivity were highest at a dilution rate of 0.07 per h and 0.11 per h, respectively. At dilution rates higher than 0.07 per h, glucose was no longer growth limiting, and at dilution rates above 0.11 per h, another compound seemed to have replaced glucose also as energy source. When grown in batch cultures at pH 7.0, the best yields of B. fragilis was achieved with 0.6% glucose in the fresh medium. The highest specific growth rate (mum) determined from viable counts was 0.45, corresponding to a mean generation time of 92 min.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

The effects of limiting nutrients, dilution rate, culture pH, and temperature on the yield constant and anthocyanin accumulation of carrot cells grown in semicontinuous chemostat cultures

With carrot cells grown in semicontinuous culture with phosphate as limiting nutrient. Dougall and Weyrauch (1980) found that the steady-state culture density was different at different dilution rates. They suggested that the yield constant for biomass was different at different dilution rates. Here the yield constant for biomass for PO(4) (3-), NH(4) (+), Mg(2+), and glucose-limited semicontinuous cultures has been measured directly at two dilution rates. The yield constants for PO(4) (3-), NH(4) (+), and Mg(2+) but not for glucose are different at the two dilution rates. The effects of pH and temperature on the biomass yield constant was measured to extend the number of system parameters examined. Biomass yield constant was changed little with change from 25 to 28 degrees C or from pH 4.2 to pH 5.5. The steady-state levels of anthocyanin were also measured. The responses of anthocyanin levels to the system parameters are different to the biomass responses. The data suggest that at different values of each of the system parameters, the composition and metabolic activities of the cells at steady state in semicontinuous cultures are different.     

Effect of growth conditions on heat resistance of Arizona bacteria grown in a chemostat.

The effects of various growth conditions on the heat resistance of Arizona bacteria grown in a continuous-culture device (chemostat) were studied. Using either glucose, NH4Cl, NaH2PO4, or MgCl2 as the rate-limiting nutrient, it was found that the heat resistance, in all cases depended on the dilution rate and, hence, growth rate of the culture. Cells grown at high dilution rates were less heat resistant than those grown at low dilution rates. If, however, the dilution rate was maintained at a constant rate, the higher the growth temperature, the more heat resistant were the cells. Also at any given dilution rate, the cells were most heat resistant when grown at a near neutral pH. Most survival curves were biphasic in shape, indicating the presence in the population of two fractions of cells, one fraction being more resistant than the other. The size of the more heat-resistant fraction varied from almost 100% in very slow-growing cultures to practically 0% in cultures grown at a dilution rate of 0.67 h-1.     

Growth and anthocyanin accumulation rates of carrot suspension cultures grown with excess nutrients after semicontinuous culture with different limiting nutrients at several dilution rates, pHs, and temperatures

When carrot cell cultures, after growth in semicontinuous culture, were transferred to media containing excess nutrients, they grew at different rates. The growth rates were generally higher after semicontinuous culture at higher dilution rates. There appears to be a limit on dilution rate above which growth rate does not increase. These changes were also displayed by clones from the parental culture. The possibility that these changes in growth rate reflect a need for the cultures to adapt to their new conditions is discussed. The growth rates of the cultures is markedly increased at 25 degrees C compared with 22 degrees C with little further increase at 28 degrees C. Growth rate is altered by less than 20% when pH is changed from 4.5 to either 5.5 or 4.2. The rates of anthocyanin accumulation by the cultures were similar under all conditions tested except at 22 degrees C. They were larger than the rates of dry weight accumulation. In contrast, the amounts of anthocyanin accumulated in the clones and in the parental cultures grown at pH 5.5 instead of pH 4.5 were very different. The observations were interpreted as showing that the clones differ in the rate of metabolism but not in the rate of synthesis of anthocyanins and that at pH 5.5 the rate of metabolism of anthocyanins but not the rate of synthesis is higher than it is at pH 4.5. The use of semicontinuous cultures as a source of inoculum for batch cultures rather than as a source of biomass for extraction of chemicals is discussed.     

Selection of Protease-Positive and Protease-Negative Variants of Streptococcus cremoris

Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E₈, HP, and Wg₂ at pH values higher than 6.0 in milk. For S. cremoris E₈ this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not found in all media tested. The poor growth of the protease-negative variant at low pH was not due to lower internal pH values. By growing S. cremoris E₈ and Wg₂ in acidified milk (pH 5.9) the proteolytic activity of the cultures could be stabilized. In continuous cultures under amino acid limitation the wild type S. cremoris E₈ and HP strains had a selective advantage over the protease-negative variants at low dilution rates (D < 0.2) at all pH values of the medium. This was apparently due to a lower affinity-constant (K_s) of the protease-positive variants for amino acids. Finally, a high fraction of protease-positive variants could be maintained in continuous cultures by using a growth medium with low concentrations of casein as a nitrogen source. At high dilution rates nearly all cells were protease positive.     

Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Chemostat production of pediocin SM‐1 by Pediococcus pentosaceus Mees 1934

Production of the bacteriocin pediocin SM‐1 by Pediococcus pentosaceus Mees 1934 was investigated in pH‐controlled batch and chemostat cultures using a complex medium containing glucose, sucrose or fructose. In chemostat cultures operated at 150 rpm, 30°C, 60% dissolved oxygen tension, pH 6.5, and D = 0.148 h^?1, the pediocin titer reached 185 AU/mL representing an increase of 32% compared with batch cultures in which glucose was used as the carbon source. Pediocin biosynthesis was markedly affected by the growth rate of the producer microorganism. For all carbon sources tested, pediocin production appeared to take place only at dilution rates lower than μ_max. However, only glucose supported production at the very low dilution rate of 0.05 h^?1 indicating a direct regulation of pediocin biosynthesis by the carbon source. Glucose supported higher biomass productivity and higher pediocin titers and yields compared with the other sugars used. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1481–1486, 2015     

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Methanogenesis from Ethanol by Defined Mixed Continuous Cultures

Methanogenesis from ethanol by defined mixed continuous cultures was studied. Under sulfate-free conditions, a Desulfovibrio strain was used as the ethanol-degrading species producing acetic acid and hydrogen. In a two-membered mutualistic coculture, the hydrogen was converted to methane by a Methanobacterium sp. and pH was maintained at neutrality by the addition of alkali. Introduction of a third species, the acetate-utilizing Methanosarcina mazei, obviated the need for external pH control. Methanogenesis by the co-and triculture was studied at various dilution rates in the steady state. The mutualistic coculture performed like a composite single species, as predicted from the theory of mutualistic interactions. Coupling between the mutualistic coculture and the acetate-utilizing methanogen was less tight. Increasing the dilution rate destabilized the triculture; at low dilution rates, instability was soon recovered, but at higher dilution rates imbalance between the rates of production and removal of acetic acid led to a drop in pH. Flocs formed in the triculture. An annulus of the Methanobacterium sp. and Desulfovibrio sp. was retained around the Methanosarcina sp. by strands of material probably derived from the Methanosarcina sp.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Growth and cell wall composition of glucose-limited bean cells (Phaseolus vulgaris L.)

Glucose-limited bean cells (Phaseolus vulgaris L.) were grown in a modified bacterial fermentor at a constant pH of 4.8. The cultures were kept in steady state at different specific growth rates varying from 0.00216 h^–1 to 0.0106 h^–1. Culture conditions are described that are needed to start a continuous culture. First, it was essential to use log-phase cells as starting material. Second, it was important to increase the dilution rate gradually, otherwise cells in the culture aggregated. Cells grown at the highest dilution rate employed contained twice as much protein per gram dry weight as cells grown at the lowest dilution rate. The composition of the cell walls also varied with the dilution rate in contrast to their relatively constant composition when grown in batch culture.     

Co-culture of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron in arabinogalactan-limited chemostats: effects of dilution rate and pH

The effects of dilution rate (D = 0.04-0.38/h) and pH (5.0-6.5) on co-cultures of Bifidobacterium adolescentis and Bacteroides thetaiotaomicron were studied in arabinogalactan-limited chemostats. B. thetaiotaomicron outcompeted B. adolescentis at all dilution rates at culture pH values between 5.0 and 6.0, although the bifidobacterium was always detected in the fermenters. At pH 6.5, however, B. adolescentis predominated in co-cultures at dilution rates above 0.24/h. Arabinogalactan degrading enzymes (beta-galactosidase, alpha-arabinofuranosidase) were strongly catabolite repressed in bacteroides at high dilution rates, but were constitutive and growth rate-associated in B. adolescentis. The increased competitiveness of B. adolescentis at high specific growth rates was not related to its ability to synthesise increased levels of depolymerising enzymes. Measurements of residual carbohydrate in pure and mixed culture chemostats showed that the bacteroides extensively digested the galactose backbone of the polymer, and to a lesser degree, the arabinose sidechains. Nevertheless, arabinose monomers and oligosaccharides (d.p. < 10) accumulated in these cultures under all growth conditions. In contrast, the bifidobacterium utilized considerably less arabinogalactan than the bacteroides, and this was reflected in the mixed culture studies. These experiments demonstrate that B. thetaiotaomicron was able to compete most successfully for this plant cell wall polysaccharide under nutritional, physiological and environmental conditions broadly similar to those encountered in the human colon, and indicate the existence of synergistic interactions between the two organisms that were growth rate dependent.     

Mass balance of heavy metal uptake by encapsulated cultures ofKlebsiella aerogenes

Dialysis was employed as a method of speciating heavy metals in cultures of an extracellular polymer forming strain ofKlebsiella aerogenes. A noncapsulated strain of the same bacterium was used as a control, and a mass balance of copper, cadmium, cobalt, nickel, and manganese in batch culture at pH 4.5 and pH 6.8 and in continuous culture at pH 6.8 was constructed. Copper and cadmium were accumulated by the cell during rapid proliferation whereas all 5 metals were bound nonspecifically by extracellular polymer produced during stationary phase and at low dilution rates. The presence of extracellular polymer appeared to inhibit cellular uptake of nickel. At the lower pH, metal uptake was considerably reduced. The results are discussed in the context of metal removal in the activated sludge process of waste water treatment.