Range Of Dilution Rates Research Articles

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.

Oscillations in continuous cultures of budding yeast: A segregated parameter analysis

Sustained oscillations have been observed in continuous cultures of Saccharomyces cerevisiae. These oscillations appear spontaneously under aerobic conditions and may constitute a severe limitation for process control. We have found that oscillations arise only in a well defined range of dilution rates and dissolved oxygen values. The period of the oscillations is related, but not equal, to the mass doubling time, and shows a relation ship with both the parent cells and daughter cells generation times. At high dilution rates two oscillatory regimens, with different periods, are observed. The analysis of the budding index shows a marked degree of synchronization of the culture, however significant differences, both in phase and in amplitude, are ob served if the budding index of parent cells and of daughter cells are considered separately. The complex changes of the cell population are clearly demonstrated by the continuous and periodic modification of both cell volume distributions and protein distributions. Ethanol is always accumulated before the drop of dissolved oxygen concentration and one of the peaks of budding index. We propose a model that explains the insurgence of these oscillation as a consequence of changes in cell cycle parameters due to alternate growth in glucose and in ethanol.

Effect of temperature, light intensity and dilution rate on the cellular composition of red algaPorphyridium cruentum in light-limited chemostat cultures

The fatty acid, polysaccharide and pigment contents ofPorphyridium cruentum at different growth temperatures, light intensities and dilution rates were studied in light-limited chemostat. The highest biomass yield was obtained at 25°C over the range of dilution rate studied (0.079 to 0.30 day−1). The production of fatty acids increased with increasing dilution rates. At low dilution rates, the contents of fatty acids at 20°C, 25°C and 30°C were comparable. Temperature did not affect the content of linolenic acid (18∶2) though fatty acids with less than two double bonds predominated at higher temperatures, those with more than two double bonds accumulated at lower temperatures. The yields of arachidonic acid (20∶4) increased under light of higher intensity. The maximum production of extracellular and cellular polysaccharides occured at low dilution rates under high light at 25°C. The amounts of chlorophylla, carotenoids and phycoerythrin all increased in the same proportions with dilution rate, and were highest at 25°C.

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.

Changes in intracellular sodium and potassium in chemostat‐cultivated Escherichia coli

AbstractEscherichia coli was continuously cultivated at constant dilution rate for a range of agitation rates, and at constant agitation rate over a range of dilution rates. Mean cell volume increased linearly both with increase in agitation rate (as we have previously found) and with increase in dilution rate. In each case, measurement of intracellular sodium and potassium content after rapid separation of cells from the medium by centrifugation through silicone oil showed these to increase as the mean cell volume increased. In absolute terms, the increase in sodium or potassium per unit cell volume was significantly greater for changes in agitation rate than for changes in dilution rate.

Double mutants of saccharomyces cerevisiae harbour stable plasmids: stable expression of a eukaryotic gene and the influence of host physiology during continuous culture

An investigation of the inheritance of a plasmid in a Saccharomyces cerevisiae ura3 furl double mutant has been performed in chemostat culture. The plasmid, bearing the gene for human α 1-antitrypsin and the yeast URA3 gene was observed to be stable over a range of dilution rates (0.1 h −1–0.3 h −1) corresponding to those growth rates most relevant to industrial bioprocesses using S. cerevisiae yeasts. The plasmid copy number remained constant in the respirative and in the oxidoreductive phases of growth. Stability of expression of the eukaryotic gene coding for α 1-antitrypsin was maintained at all dilution rates. However, the yield of α 1-antitrypsin was highest when the culture's carbon and energy source, glucose, was not completely utilized. The maximum respiratory capacity of the double mutant was observed to be typical for laboratory strains of S. cerevisiae. These data show that S. cerevisiae double mutants can be made to harbour stable plasmids which will stably express a eukaryotic gene. However, to achieve optimal recombinant product formation, careful attention must be paid to these yeasts' complex physiology.

Buoyancy regulation in phosphate-limited cultures ofMicrocystis aeruginosa

Buoyancy regulation was studied in P-limited continuous cultures of Microcystis aeruginosa grown on light-dark cycles of 8–16 h. Gas-vesicle content did not vary systematically over a range of dilution rates form 0.004 to 0.015 h−1. A reduction in irradiance did not cause a significant change in gas-vesicle content. The proportion of floating cells decreased during the photoperiod and increased during the dark period. At three dilution rates, parallel cultures were grown at growth-saturating irradiance and at a lower irradiance. The cultures at low irradiance had a higher proportion of floating cells and a smaller decrease in buoyancy during the light period. The buoyancy losses were not due to destruction of gas vesicles but, rather, to the accumulation of heavy substances. However, measured increases in polysaccharide ballast accounted for only 60% of the required ballast. The molecule(s) which comprised the remainder of the ballast are unknown. Upon relief of phosphate limitation, P-limited cultures increased their buoyancy when incubated in the dark or light. Buoyancy increases in the dark were correlated with a decrease in polysaccharide content, whereas there was an increase in gas vesicle content in the light.

Intracellular reaction rates, enzyme activities and biomass yields in Methylomonas L3: growth rate and substrate composition effects

The effects of specific growth rate and medium feed composition on the metabolic reactions of methanol incorporation and oxidation have been studied in carbon-limited, chemostatic cultures of Methylomonas L3. An in situ radioisotopic tracer technique was employed. The in vivo rates of substrate-carbon flow and the corresponding steady-state levels of several key RuMP-type methylotrophic enzymes were determined over a range of dilution rates from 0.19 to 0.41 h-1 on methanol and methanol/formaldehyde substrates. It was determined that an absolute correlation does not exist between the in vivo specific carbon flux and the in vitro specific activity of any of the key enzymes studied. Oxidation of substrate-carbon via 6-phosphogluconate dehydrogenase is not stringently regulated in this methylotroph and the extent of its operation may be dependent on kinetic factors which make immediate cellular detoxification of formaldehyde imperative. As such, the cyclic oxidation mechanism in this methylotroph does not appear to be coupled to efficient energy utilization, since it was observed that high levels of the cyclic oxidation flux are commensurate with depressed biomass yields.

PRODUCTION OF ACETONE AND BUTANOL BY CLOSTRIDIUM ACETOBUTYLICUM IN A PRODUCT LIMITED CHEMOSTAT

Abstract The solvents acetone and butanol are produced by Clostridium acetobutylicum during batch culture only after a considerable lag. The influence of limiting nutrients on the onset of solvent production has been investigated by studying the continuous fermentation under conditions of carbon, nitrogen and phosphate excess over a wide range of dilution rates. A maximum solvent concentration of 12 gl−1 was obtained at the lowest dilution rate investigated, namely 0.05 hr−1, and the average yield of solvents over the range of dilution rates approximated 0.3. These data confirm that a limitation of either of these nutrients is not essential for solvent production.

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.

Effects of dilution rate on biomass and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture.

Propionibacterium acnes, P. avidum and P. granulosum were grown in continuous culture at a range of dilution rates on a semi-synthetic medium. Dilution rates were chosen to allow the bacteria to grow at the same relative growth rates as compared to their respective mumax values. The steady-state levels and production rates of biomass and extracellular enzymes were determined. The lipase and hyaluronate lyase of P. granulosum and the proteolytic activity of P. acnes and P. avidum were growth linked enzymes (i.e. they were produced at constant amounts per unit of biomass). In contrast, the lipase, hyaluronate lyase and acid phosphatase of P. acnes and the lipase of P. avidum were shown to be non-growth linked enzymes.

Lactic acid productivity of a continuous culture of Lactobacillus delbreuckii

Maximum volumetric productivities of biomass (1.40 gl−1h−1) and lactic acid (8.93 gl−1h−1) for a continuous culture ofLactobacillus delbreuckii occurred between dilution rates 0.35h−1 and 0.40h−1. All major nutrients were in excess in these cultures. Glucose utilisation was complete at dilution rates of 0.1h−1 and lower. Product and biomass yields were constant in the dilution rate range studied (0.05h−1 to 0.50h−1).

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (∼1%–2% of total acid production) of branched and straight-chain C3−C5 volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.

Control of the syntheses of bacteriochlorophyll, c- and b-type cytochromes, and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.

Metabolism of the rumen bacterium Selenomonas ruminantium grown in continuous culture

Selenomonas ruminantium 0078A was grown in a glucose-limited chemostat over a dilution rate range of 0.049-0-137/h. Fermentation products were acetate, propionate, succinate, lactate and C02; traces of ethanol were also detected. Succinate accounted for up to 52% of the substrate glucose carbon. When dilution rate was increased without a concomitant increase in glucose supply per unit time there were changes in the fermentation pattern which were not apparent when both dilution rate and glucose supply were simultaneously increased; the molar proportion of acetate increased at the expense of propionate.

Changes in metabolism of the rumen bacterium Streptococcus bovis H13/1 resulting from alteration in dilution rate and glucose supply per unit time.

Streptococcus bovis H13/1 was grown in a glucose-limited chemostat. A concomitant increase in dilution rate and glucose supply per unit time caused both an increase in lactate production per mole of glucose fermented and a linear increase in growth yield over the dilution rate range 0.052 to 0.141/h. When the dilution rate was increased with no change in glucose supply per unit time there was a reduction in lactate production and an increase in that of acetate and ethanol coinciding with a non-linear increase in growth yield. YMaxglu = 38.6 and a maintenance coefficient, ms = 0.290 mmol/l glucose/g cells/h were calculated. The results also suggested an interaction between the formate and CO2 pools.

Effect of Nitrogen and Phosphate on the Levels of Intermediates in Bakers' Yeast Grown in Continuous Culture

Bakers’ yeast (Saccharomyces cerevisiae) has been grown in continuous culture using a control medium and media which contained low levels of ammonium and phosphate. The effects of medium composition and growth rate on the levels of intermediates of the glycolytic pathways, the tricarboxylic acid cycle and the glyoxylate cycle were investigated. The energy charge varied only between 0·7 and 0·9 over the range of dilution rates studied; however, the level of ATP decreased by 50% at higher aerobic growth rates. Intermediates of the Embden-Meyerhof-Parnas pathway were higher at the low aerobic growth rates and decreased as the dilution rate was increased. However, higher levels of these intermediates were also observed at even higher dilution rates at which ethanol formation and fermentative metabolism occurred. Significant differences in levels of intermediates were observed between control experiments and fermentations using the low nitrogen and phosphate media. The greatest differences were observed in the levels of glucose 6-phosphate, 6-phosphogluconate, pyruvate, citrate and glyoxylate. Twenty-one different steady states were investigated and each was found to have a unique composition.

Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).

Respiratory activity, molar growth yields, and ATP content in an Arthrobacter sp. ammonium-limited chemostat culture

Arthrobacter fluorescens was grown in chemostat culture under ammonium-limited conditions and respiratory activity; molar growth yields and ATP content were determined over a wide range of dilution rates. Within a range of dilution rates between 0.10 h−1 and 0.20 h−1, morphological transition occurred, the ratio of cocci to rods appearing inversely related to growth rate. Molar growth yields for both glucose and ammonium decreased with increasing dilution rates because of the higher intracellular polysaccharide content at the lower growth rates. Different metabolic activities were shown in cocci, in rods, and during morphogenesis. A sharp decrease in in situ and potential oxygen quotient (Q(O2)) and in ATP content was observed in the range of dilution rates in which morphological changes occurred.

A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture.

The oleaginous yeast, Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates. Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogen-limited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D = 0.05/hr) in a chemostat. Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D = 0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized. The relative proportions of the major fatty acids (16:0, 18:0, 18:1, 18:2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions. However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed. Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18:0) 15% and the lowest linoleic acid (18:2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18:1) 51% and decreased palmitic acid (16:0) 25%.