Increasing Dilution Rate Research Articles

Performance assessment of two patent strains ofZymomonas mobilis in batch and continuous fermentations

The performance ofZymomonas mobilis strains ATCC 31821 and ATCC 31823 was assessed in batch and continuous culture. In batch culture using a medium containing 250 g/l glucose, identical maximum specific growth rates of 0.16/h were found, though final biomass concentration and growth yield were significantly lower for ATCC 31 823 than for ATCC 31 821. Final ethanol concentrations in this medium were about 110 g/l vor both organisms. In continuous culture at increasing dilution rates using a medium containing 100 g/l glucose, no significant differences were seen between the two strains with respect to the fermentation parameters studied. For ATCC 31 821, maximum rates of glucose uptake (Qs) and ethanol produktion (Qp) of 8.7 g glu/g/h and 4.4 g eth/g/h, respectively, were found. Both strains showed a similar performance at a fixed dilution rate of 0.1/h, where maximum ethanol concentrations of about 68 g/l were reached at a feed glucose concentration of about 139 g/l. At this dilution rate the maximum values of Qs and Qp were about 5.8 g glu/g/h and 2.8 g eth/g/h, respectively. Test tube experiments showed that growth, measured as optical density, decreased with increasing concentrations of exogenous ethanol with complete inhibition of growth at ethanol concentrations >8% (v/v). As evidenced by the results presented here, we have been unable to practice the invention as described in U.S. Patent 4,403,034 (Rogers and Tribe 1983).

Hydrogen gas production from continuous fermentation of glucose in a minimal medium with Clostridium butyricum LMG 1213t1

Summary We investigated the formation of hydrogen gas by Clostridium butyricum strain LMG 1213t1 in a chemostat in glucose limitation. At pH 5.1 the fermentation did not reach a steady-state. Within the pH range 5.5 to 7.1 in the culture vessel, and at constant dilution rate D no marked change in product spectrum was found. The produced gas consisted of approximately 60% H 2 and 40% CO 2 . About 1.75 mol H 2 was produced per mol glucose consumed. Hydrogen gas production rate increased proportionally with dilution rate D. At the highest dilution rate investigated 21.7 mmol H 2 was produced per hour and per liter reactor. Slightly higher efficiencies of hydrogen gas formation were obtained with acetate or butyrate (sodium salts) in the inflow medium. Acetate and butyrate were produced in a ratio of approximately 1.8 to 3 in all conditions tested. With increasing dilution rate D and at constant pH, slightly less fatty acids and slightly more cell material were formed. Y glucose max and Y ATP max values were 28.56 and 12.30 g dry weight/mol glucose or ATP, respectively. No change in fermentation pattern was observed with increasing concentrations of acetate or butyrate (up to 102.7 mM and 77.2 mM respectively) in the inflow medium.

Continuous cultivation of recombinant Escherichia coli: Existence of an optimum dilution rate for maximum plasmid and gene product concentration

Growth yield factors, plasmid stability, cellular plasmid content, and cloned gene product activity for Escherichia coli HB101 containing plasmid pDM246 were measured at several dilution rates in continuous culture. Cell mass yield per mass of glucose consumed declined with increasing dilution rate. There was no evidence of plasmid segregational instability in any experiments, none of which employed selective medium. Plasmid content per cell varied with population-specific growth rate as observed in earlier batch experiments with the same strain. Plasmid content declined with increasing specific growth rate following indication of a maximum number of plasmids per cell at specific growth rates of ca. 0.3 h(-1). Cloned gene product (beta-lactamase) activity exhibited a sharp maximum with respect to dilution rate in continuous culture. Qualitatively different results were observed in previous experiments in batch cultivation in which specific growth rate changes were effected by altering medium composition.

Protein degradation in anaerobic digestion: influence of volatile fatty acids and carbohydrates on hydrolysis and acidogenic fermentation of gelatin

The hydrolysis and fermentation of gelatin in the presence of a carbohydrate by gelatin-adapted mixed anaerobic bacterial populations in putatively carbon-limited chemostat cultures is investigated. It was shown that the degradation of the protein is progressively retarded with increasing dilution rates, as well as with increased concentrations of carbohydrates present in the feed as a second substrate. That this is not due to high concentrations of fermentation products in the reactor was established. Moreover, the carbohydrate is totally fermented at all dilution rates. It is concluded that for optimal performance of an anaerobic digestion system purifying waste waters containing carbohydrate/protein mixtures, fermentation of carbohydrates should be spatially separated from hydrolysis and fermentation of the protein.

Culture fluorescence studies on aerobic continuous cultures ofSaccharomyces cerevisiae

Fluorometric measurements were performed in continuous aerobic cultures ofSaccharomyces cerevisiae in order to study the effect of substrate concentration and residence time on the intracellular NADH-level. A modified Beyelermicrofluorometer probe (Beyeler et al. 1981) was used for the experiments. It was possible to use this sensor continuously up to five weeks without problems. The relative NADH-values obtained by the on-line monitoring of the NADH-dependent culture fluorescence were compared to the enzymatically determined NADH-content. Biomass estimation from fluorescence data was performed. During oxidative-reductive catabolism the deviation between calculated and measured data were below 5%. The differences between oxidative and oxidative-reductive catabolism were studied regarding glucose addition, dilution rate increase and aerobic-anaerobic transition. For synchronized continuous cultures, changes in dilution rate resulted in changes of the oscillating behaviour. Flow cytometric studies in comparison with fluorometric studies showed changes in budding behaviour during the oscillations.

Transient responses of continuously growing yeast cultures to dilution rate shifts: A sensitive means to analyze biology and the performance of equipment

Transient responses of continuously growing yeast cultures to step changes in dilution rate were influenced by (1) the preshift conditions of the culture, (2) the magnitude of the dilution rate increase, (3) the organism involved, and (4) the type of bioreactor used. The sensitivity of transient responses to seemingly minor differences in the design of experiments indicated that this type of investigation may be a means to analyze kinetics of microbial cultures as well as the effectiveness of bioreactors.

Continuous culture with complete cell recycle to obtain high cell densities in product inhibited cultures; cultivation ofStreptococcus lactis for production of superoxide dismutase

A cultivation system is described for cultivatingStreptococcus lactis in continuous culture with complete cell recycle. The aim was to obtain high cell densities for the production of su-peroxide dismutase (SOD) while avoiding the growth inhibiting effects of the lactic acid produced. This type of cultivation was performed both at constant and at increasing dilution rates. Comparisons made include those between cell mass productivity and SOD productivity in recycling cultivations and batch cultivations. In the recycling cultivation at increasing dilution rates a cell mass of 19 g/1 was obtained after 22 h of cultivation and the SOD productivity was 43.103 U/1.h which is four times higher than for batch cultivations. The effect of recyclingS. lactis was also considered and no damage of the microorganisms was observed.

INTERACTING EFFECTS OF LIGHT AND NUTRIENT LIMITATION ON THE GROWTH RATE OFSYNECHOCOCCUS LINEARIS(CYANOPHYCEAE)1

ABSTRACTThe blue‐green algaSynechococcus linearis(Naeg.) Kom. was grown in P‐ and N‐limited chemostats over a range of potentially limiting irradiances in order to determine the combined effects of light and nutrient limitation on some aspects of the composition and metabolism of this alga. Over a narrow range of low irradiances, simultaneous limitation of growth rate by light and either N or P was shown. This simultaneous limitation of growth rate by a nutrient and a physical factor can be explained by the ability of an increased supply of one to compensate in part for a decreased supply of the other. At all irradiances, the internal concentration of the limiting nutrient increased with increasing dilution rate, and the results could be fitted to the Droop relationship. With decreasing irradiance, the internal concentration of the limiting nutrient increased. There appeared to be little or no effect of light on the minimum internal concentration of P but that of N increased with decreasing light. Both chlorophyllaand biliprotein per unit particulate C increased with increasing dilution rate and decreasing irradiance. The critical N/P ratio increased with decreasing light as the N requirement of N‐limited cells increased faster than did the P requirement of P‐limited cells. The composition of exponentially growing cells in complete medium varied much less with light. Neither dilution rate nor irradiance during growth had a great effect on saturated rates of P or N uptake or alkaline phosphatase activity. Calculated assimilation ratios increased with light and dilution rate. The role of the flexibility of nutrient composition in adaptation to adverse conditions and the implications of the results for the use of physiological indicators of nutrient status are discussed.

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.

Continuous growth kinetics of Candida utilis in pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent as the sole carbon and energy source in a chemostat at dilution rates between 0.10 and 0.62 h(-1) to determine the growth kinetics. The principal sugars in the effluent were sucrose, glucose, and fructose. The cell yield coefficient on carbohydrate varied with dilution rate and a maximum value of 0.63 was observed at a dilution rate of 0.33 h(-1). The steady-state concentrations of carbohydrate, reducing sugar, and chemical oxygen demand (COD) appeared to follow Monod saturation kinetics with increasing dilution rate, although none of the measured parameters represented a pure substrate. The maximum specific growth rate and reducing sugar saturation constant were 0.64 h(-1) and 0.060 g/L, respectively. A maximum cell mass productivity of 2.3 g/L h was observed at a dilution rate of 0.51 h(-1). At this dilution rate, only 68% of the COD was removed. A 95% COD removal was attained at a dilution rate of 0.10 h(-1). Optimal yeast productivity and COD reduction occurred at a dilution rate of 0.33 h(-1).

Continuous bacterial coal desulfurization employing Thiobacillus ferrooxidans

The leaching of pyrite sulfur from coal employing Thiobacillus Ferrooxidans was studied in a continuous stirred tank reactor at a variety of dilution rates (0.02-0.11 h(-1)) and coal surface areas (0.25-1.0 m(2)/mL). The bacterial leaching rate was found to increase with increasing coal surface area concentration and increasing dilution rate. The bacterial concentration on the coal surface was related to the bacterial concentration in solution by a irreversible second-order (of the second kind) kinetic equation. The concentration of bacteria on the coal in all experiments was the concentration at saturation. Step changes in the coal concentration were observed to result in dramatic declines in bacterial concentration in solution. A bacterial mass balance model was employed to calculate the specific growth rate on the solid which was observed to increase with increasing dilution rate.

Mixed culture kinetics of stringent and relaxedEscherichia coli cells in glucose-limited chemostat

The mixed culture kinetics of stringent and relaxed Escherichia coli cells were investigated in a glucose-limited chemostat at different dilution rates. Independent of the dilution rate the stringent cells competed out the relaxed cells. But the number of generations necessary for displaying the relaxed cells by the stringent ones increased with increasing dilution rate. The results are discussed as a consequence of the regulatory role of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) which is known to be present at different concentrations in stringent and relaxed cells under conditions of nutrient limitation. In addition, it is postulated that the coefficient of the maintenance metabolism according to PIRT (1965) is slower in stringent cells than in relaxed cells of E. coli.

Effect of pH and temperature on morphology of batch and chemostat cultures of Penicillium chrysogenum

Batch cultures of Penicillium chrysogenum were grown on solid and liquid media. The mean hyphal extension rate of mycelia on solid medium was a function of the length of the organism's hyphal growth unit and its specific rate. The optimum temperature for colony expansion was 20°C, although the fastest specific growth rate was observed at 30∘. The reduction in colony radial growth rate at 30° was associated with a decrease in hyphal growth unit length. When P. chrysogenum was grown in glucose-limited chemostat cultures at stirrer speeds of 1000 or 1200 rev./min, hyphal growth unit length decreased with increase in dilution rate. However, at a stirrer speed of 500 rev./min, hyphal growth unit length increased with increase in dilution rate. When the mould was grown at a constant dilution rate but at various temperatures or pH values, maximum hyphal growth unit lengths were observed at 30∘ and pH 6.0. A direct relationship was observed between dilution rate and the nitrogen content of glucose-limited chemostat cultures. Mycelia grown in ammonium-limited chemostat cultures contained only about half the nitrogen content of mycelia grown at the same dilution rate in glucose-limited cultures.

Biochemical activities during lipid accumulation in Candida curvata.

Intracellular and extracellular concentrations of citrate and the specific activities of ten different enzymes in Candida curvata D were examined in relation to lipid biosynthesis in batch and continuous culture. Citrate was found to accumulate prior to lipid production and declined markedly as lipid accumulated in batch culture. The cells excreted citrate as the culture became nitrogen-limiting after 30 hr of growth, but little more was expelled after 40 hr when lipid accumulation was more marked. In continuous culture, only low levels of citrate were detected at the lower dilution rates and citrate was completely absent from both the cells and medium above a dilution rate of 0.1/hr. The activity of malic enzyme, malate dehydrogenase and ATP:citrate lyase increased in batch culture on lipid accumulated and, in continuous culture, both malic enzyme and ATP:citrate lyase varied in parallel with the specific rate of lipid synthesis which increased with increasing dilution rate. Activity of malate dehydrogenase, citrate synthase and glucose-6-phosphate dehydrogenase decreased with increasing dilution rate. The regulatory significance of these enzymes in lipid accumulation by C. curvata is discussed.

CHARACTERISTICS OF PHOSPHORUS LIMITATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE) AND ITS PALMELLOIDS1

ABSTRACT Chlamydomonas reinhardtii Dang, was grown in a chemostat culture under phosphate limitation. The steady state concentration of phosphate was below the detection limit (< 1 μg P/L) in all runs. The cellular content of phosphorus (Qp), polyphosphate (Qpp) and chlorophyll a increased with increasing dilution rate, and the growth rate of the alga was described by Qp as well as Qpp in the Droop model. The ratio Qpp/Qp and the activity of alkaline phosphatase were maximal at high and low growth rates, respectively. Palmelloids of Chlamydomonas were found at high dilution rates (D > 0.12 h−1) and became attached to the wall of the culture vessel. They differed from the vegetative stage in both chemical composition and growth rate. Their contents of phosphorus and chlorophyll a were low, as in the vegetative cells, which grew at a low growth rate, whereas the ration Qpp/Qp and the activity of alkaline phosphatase were comparable with those of fast growing vegetative cells. The growth rate of the palmelloids was 0.03 h−1 whereas maximum growth rate (μm) for the vegetative cells was 0.21 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.

Modelling of Sustained Oscillations Observed in Continuous Culture of Saccharomyces Cerevisiae

Oscillations in continuous cultures of S.cerevisiae can be observed at conditions when only small amounts of ethanol are produced. Decreasing the dilution rate and the oxygen supply to the culture increases the oscillation period. This means that the period is not only a function of the mean generation time of the culture. Two variable models with Monod growth kinetics and variable yield (Y = a + bS) lead to oscillations. Contrary to the experiments this type of model yields increasing oscillation periods with increasing dilution rate. With a/b proportional to the dissolved oxygen concentration, this model approximately describes the influence of oxygen on the existence and the period of oscillations. The direction in the phase plane plot is opposite to that observed in the experiments. Models with only biomass and dissolved oxygen concentration and a variable yield function also lead to oscillations, which only are practically observable at very low kLa values. A more complex model based on biochemical pathways gives a better description of the experiments. This model considers storage of carbohydrate reserve material (glycogen and trehalose) and feedback control of their respective hydrolytic enzymes through concentration levels. The model qualitatively describes the existence of oscillations, their frequenpy and also gives the right direction in the phase plane plot.

Lipid from yeast fermentation: Effects of cultural conditions on lipid production and its characteristics of rhodotorula glutinis

AbstractTo produce lipids from microbial origins, Rhodotorula glutinis (syn. Rhodotorula gracilis) NRRL Y‐1091 was cultured in batch and continuous systems under nitrogen‐ and carbon‐limited conditions. The lipid production patterns are shown to be different from each other depending on growing conditions. In continuous cultures under nitrogen‐limited conditions, the maximum lipid accumulation was observed at the lowest dilution rate examined, giving the efficiency of substrate conversion of 16.4 g lipid per 100 g glucose consumed. As the dilution rate increased, cell biomass, lipid content, lipid productivity and lipid yield decreased. In carbon‐limited continuous cultures, cell biomass decreased with increasing dilution rate, but lipid content remained almost constant. Neutral lipid portions in nitrogen‐limited cultured yeast cells decreased as the dilution rate increased, and glyco‐ and phospholipid portions showed the reverse trend. Major components in the neutral lipid portions in yeast cells are triglyceride, free fatty acid, steryl ester and sterol. Phosphatidylserine was the predominant phospholipid in yeast cells. The dilution rate also affected the fatty acid composition of all lipid portions; polyunsaturated fatty acids increased and saturated and monounsaturated fatty acids decreased with increasing dilution rates. The degrees of unsaturation of each lipid class and total lipids were also increased by increasing the dilution rate.

Calorimetric analysis of Escherichia coli in continuous culture

The heat evolution rate was measured in continuous culture of Escherichia coli k-12 on glucose-limiting medium. The specific heat production rate based on the dry cell mass increased in proportion to increase of dilution rate. The specific heat production rate based on a viable cell increased nonlinearly because the cell size varied in each dilution rate. Several thermochemical data were calculated for each dilution rate from the stoichiometric equation which was derived from elementary analysis of the cells. Growth yields based on energy measured directly was found to be less influenced by dilution rate than the growth yield based on consumed glucose. This suggested that the energy efficiency of utilization of catabolic energy for anabolism was almost constant.

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 and a maximum prodigiosin concentration of 80 ng of prodigiosin . mg of cell protein were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h), and maximum prodigiosin concentrations were higher (1,500 ng . mg of protein). 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 KH(2)PO(4) 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, 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) was observed in chemostat cultures at a dilution rate of 0.057 h. 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, 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.