Growth of Legionella pneumophila in continuous culture.

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

GROWTH OF COLPOMENIA PEREGRINA (PHAEOPHYCEAE) IN CULTURE: EFFECTS OF SALINITY,TEMPERATURE AND DAYLENGTH1

Increases in size of Colpomenia peregrina (Sauv.) Hamel microthalli were determined at six different combinations of salinity and temperature under laboratory conditions. Microthalli grown at low salinity (15%) did not have a growth rate significantly different from microthalli grown at high salinity (30%). However, more macrothalli were produced in higher salinity media. Fewer macrothalli were produced at lower temperatures. Growth of C. peregrina microthalli was also determined at six different combinations of daylength and temperature. Microthalli under 15:9 h (LD cycle) did not have a growth rate significantly different from microthalli under 9:15 h LD. However, more macrothalli were produced under long day conditions. Of the three different temperatures utilized (5, 13 and 20° C) only 5° C produced significantly different growth rates. Low temperature reduced growth to such an extent that macrothalli of a size that could be recognized in the field took approximately two months to produce in culture. These results may explain the seasonal presence/absence of the delophycean phase of C. peregrina in the field. Zoospore survivorship and macrothallus production of C. peregrina in culture indicated that both PES and an artificial medium were suitable for the laboratory cultivation of this plant.     

The kinetics and physiology of stipitatic acid and gluconate production by carbon sufficient cultures of Penicillium stipitatum growing in continuous culture

The yield from glucose of ammonia-grown carbon-limited continuous cultures of Penicillium stipitatum was ca. 20% higher than that of nitrate-grown cultures at all growth rates examined. However, the yield from oxygen was similar during growth on both nitrogen sources. Under phosphate limitation the specific rate of gluconic acid and stipitatic acid production increased with growth rate, but the former product accounted for virtually 100% of the excreted carbon. Stipitatic acid was not produced under nitrogen limitation, and glucose supplied to the culture in excess of that required for growth was virtually quantatively converted into gluconic acid. Productivities of 11.4 g gluconic acid/L/h were stably maintained in continuous culture. Under conditions of glucose excess the enzyme glucose oxidase was excreted into the culture. The specific activity of this extracellular enzyme increased when the input glucose concentration to the culture was progressively increased. The excretion of a protein under nitrogen limitation suggests that this enzyme plays an important role under these conditions. Indeed, it was demonstrated that nitrogen-limited cultures did not overmetabolize gluconate at either pH 6.5 or 3.5, although up to 29 g/L gluconate was present in the culture. The Y(gluconate) and YO(2) of C- and N-limited gluconate-grown cultures were similar indicating that the rapid conversion of glucose to gluconate probably affords a means of regulating carbon flow in this organism. Nitrogen-limited cultures of P. stipitatum overmetabolized glucose to a much greater extent than acetate, fructose, or gluconate.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system.

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.     

Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.     

A cell suspension ofLinum flavum (L.) in phosphate limited continuous culture

A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.     

Influence of produced acetic acid on growth of sulfate reducing bacteria

Summary A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under gás (H₂S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated acetic acid concentrations of approximately 54 mg/L.     

Citrate Fermentation by Lactococcus and Leuconostoc spp

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.     

Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures

It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.     

Production of brevicin 286 by Lactobacillus brevis VB286 and partial characterization

Brevicin 286 was produced by Lactobacillus brevis VB286 isolated from vacuum-packaged meat and was partially purified by ammonium sulphate precipitation, gel filtration and dialysis. The bacteriocin was susceptible to proteolytic enzymes, stable to heating at 100°C particularly under acidic pH conditions, and showed a relatively narrow spectrum of activity with notable activity against Listeria sp. Production of brevicin 286 was optimal during exponential growth at 20°C. Higher rates of cell growth occurred between 30 and 37°C but with little or no expression of brevicin 286. A food-grade formulation consisting of 4% yeast extract and 1% glucose was found to be adequate for optimal brevicin 286 production and the bacteriocin-containing culture supernate was successfully spray dried with full recovery of antibacterial activity in the resultant powder.     

Effect of growth rate and substrate limitation on the composition and structure of the cell wall of Saccharomyces cerevisiae

1. A study was made of the composition and structure of walls isolated from yeast grown in continuous culture at different rates, under three conditions of glucose limitation in which the concentrations of glucose and ammonium sulphate in the medium and the oxygen-transfer rate in the culture were varied, and one condition of NH(4) (+) limitation. 2. The contents of total glucan and total mannan in the walls were relatively little affected by the growth rate under any of the four sets of conditions. The phosphorus and protein contents of walls from yeast grown under each of the four conditions increased as the growth rate was decreased. Walls from yeast grown under NH(4) (+) limitation contained only half as much protein as walls from cells grown under glucose limitation. The proportion of lipid was greatest in walls from yeast grown under NH(4) (+) limitation. 3. A procedure was devised for fractionating isolated walls, based on the ease with which the glucan and mannan were extracted with water and with hot and cold 6% (w/v) potassium hydroxide solution. The percentage of glucan, mannan, protein and phosphorus in each of the fractions was affected by the rate of growth and by the nature of the substrate limitation. 4. The beta-fructofuranosidase activities of yeast grown under glucose limitation increased as the growth rate was lowered, but decreased at very low growth rates. The effects at low growth rates were probably due to repression of enzyme synthesis by residual glucose in the culture filtrate. The beta-fructofuranosidase activities of yeast grown under NH(4) (+) limitation were much lower than those from yeast grown under any of the conditions of glucose limitation. 5. Yeast cells grown at any of the rates under NH(4) (+) limitation were longer and thinner than those grown at the same rate under any of the conditions of glucose limitation. Mean cell volumes were dependent on growth rate but not on the nature of the substrate limitation. 6. Electron micrographs of thin sections of isolated walls showed that cells grown under NH(4) (+) limitation had a more porous structure than those from cells grown under any of the conditions of glucose limitation.     

Improved performance of intensive semicontinuous cultures of Scenedesmus by biomass recirculation

The microscopic green alga, Scenedesmus obliquus, was used in a semicontinuous culture system for the tertiary treatment of urban wastewater, with the simultaneous production of usable biomass. Partial biomass recycling was used to increase the productivity of the system by overcoming the limits imposed by the low maximal growth rate of the alga. The biomass to be recycled was collected by simple gravity settling of the removed culture.The culture system was operated at different dilution rates and its productivity measured at each rate. An evaluation of the crude nutrient composition of the algae produced at each dilution rate was also carried out.The system was found to operate stably at dilution rates of up to 0.8 day(-1) which represents a 20% net increase over the maximum dilution rate allowed under the same conditions in a system without recirculation. The composition of the biomass produced varied little over a range of dilution rates, which may be of relevance to its projected end-use.The study indicated that such a system can exploit available light to the full and should be of particular value for the treatment of low-strength wastes such as we employed.     

Effect of antecedent growth conditions on sensitivity of Escherichia coli to chlorine dioxide.

Bacterial resistance to inactivation by antibacterial agents that is induced by the growth environment was studied. Escherichia coli was grown in batch culture and in a chemostat, and the following parameters were varied: type of substrate, growth rate, temperature, and cell density during growth. Low doses (0.75 mg/liter) of chlorine dioxide were used to inactivate the cultures. The results demonstrated that populations grown under conditions that more closely approximated natural aquatic environments were more resistant than those grown under commonly employed batch culture conditions. In particular, bacteria grown at submaximal rates were more resistant than their counterparts grown at mumax. The most resistant populations encountered in this study were those grown at D values of 0.02 h-1 and 0.06 h-1 at 25 degrees C. Growth at 15 degrees C led to greater resistance than did growth at 37 degrees C. The conditions that produced relatively resistant phenotypes were much closer to those found in most natural environments than are the typical conditions of batch culture methods. The importance of major physiological changes that can be induced by the antecedent growth environment is discussed in light of the possible modes of action of several disinfectants.     

Factors affecting the production of an antimicrobial agent, plantaricin F, by Lactobacillus plantarum BF001

Lactobacillus plantarum BF001 produced plantaricin F in MRS broth but it was detected only after ca a 50-fold concentration. Growth on MRS broth and appearance of plantaricin F were similar under aerobic and anaerobic conditions. No growth occurred at pH 3 or at 4°C. Plantaricin F appeared first at early stationary growth phase (24 h) and was stable thereafter (pH 2). Amounts found in liquid cultures were ca 2–3 times higher than those from solidified MRS medium, and specific activities were ca 6 times higher in liquid culture (48 h). Maximal amounts of plantaricin F were found (48 h) when medium had an initial pH of 4 and growth was at 30°C. Under these conditions, cell growth and fermentation were partially uncoupled. Plantaricin F was not produced endogenously, organic nutrients were necessary. A molecular weight range of 500–3500 Da was indicated. Plantaricin F appears to be a secondary metabolite.     

Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters

The present work reports on the daily cyclic variation of oxygen generation rates, carbon consumption rates, photosynthetic activities, growth rates and biochemical composition of the biomass in a pilot plant continuous outdoor culture of the microalgae Porphyridium cruentum. A linear relationship between the external irradiance and the average irradiance inside the culture was found. In addition, the oxygen generation and carbon consumption rates were found to be a function of the average irradiance inside the culture. A reduction in photosynthetic activity of the cells at noon and recovery in the afternoon was also observed. Therefore, the cells showed a short-term response of parameters such as oxygen generation rate as well as carbon consumption rate with external and average irradiance; a model of photosynthesis rate considering photoinhibition is proposed. This model is a useful tool for the operation and scaleup of tubular photobioreactors, and can be used for determining CO₂ requirements of the system. The higher the photosynthesis rates, the lower the carbon losses, ranging from 25% at noon to 100% during the night. The growth rate showed a linear relationship with the daily mean average irradiance inside the culture with a long-term response. Likewise, a linear relationship among the oxygen generation rate and the growth rate was obtained. With respect to the biochemical composition of the biomass, the cells showed a long-term response of metabolic routes to mean daily culture conditions. During the illuminated period, energy was stored as carbohydrates and synthesis of proteins was low. During the night, the stored carbohydrates were consumed. The fatty acid dry weight (DW) content decreased during the daylight period, whereas the fatty acid profile, as total fatty acids, was a function of growth rate. A short-term variation of exopolysaccharides synthesis with solar irradiance was also observed, i.e. the higher the external irradiance the higher the excretion of exopolysaccharides as a protection against adverse culture conditions.     

Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu). Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2). In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2). The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.