Conidiation of Aspergillus niger in continuous culture

Summary Conidiation of Aspergillus niger was studied in carbon-limited and nitrogen-limited chemostat culture. Under citrate-limitation conidiation intensity varied inversely with dilution rate. Conidiophores were less complex than in aerial conidiation and at high dilution rates conidia occasionally developed from modified hyphal tips. Conidiation was difficult to achieve under glucose-limitation. At the low dilution rates that allowed limited conidiation steady state could not be maintained due to onset of autolysis. At higher dilution rates when steady state was readily obtained conidiation did not occur. The maximum yield constants under citrate-limitation and glucose-limitation were respectively 0.145 and 0.4 mg dry weight/mg substrate, while the relative specific maintenance values were 0.045 and 0.018 mg substrate/mg dry weight/h. Under ammonium-limitation with citrate as the carbon source there was no conidiation. When nitrate became the limiting nitrogen source conidiophore initiation occurred but biomass production was low and wash-out occurred at D=0.034 h^-1.     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr.]

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar ‘UA4805’ (UA) had achieved a leaf photosynthetic rate (P_n) of 23.2 μmol · m^?2 · s^?1, close to its steady‐state rate, while the slowest cultivar ‘Tachinagaha’ (Tc) had only reached 13.0 μmol · m^?2 · s^?1 and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of P_n to intercellular CO₂ concentration (C_i), it was found that the speed of apparent in vivo activation of ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.     

Kinetic behavior of mixed populations of activated sludge

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage isothermal continuous flow completely mixed aeration tank. A series of experiments were carried out at various dilution rates using glucose as the growth limiting substrate. The steady-state behavior of the system was observed at each dilution rate and the results were found to fit fairly well with the steady-state equation bayed on the Monod model with an endogenous respiration term included, i.e., μ = μ_mS/(K_s + S) ? K_d. The growth kinetics of cells harvested at steady state for each dilution rate were studied using batch experiments. The multiple response data of the system as functions of time were used to estimate the parameter values in the above kinetic model. It was found that values of the growth parameters changed significantly and systematically with cell population. For example, values of μ_m were high at high dilution rates and low at low dilution rates. It was also found that only those batch growth parameters from cells obtained at fairly high dilution rates are comparable with those estimated by the results of steady-state operations. The results of this investigation suggest that (1) different cell populations pre dominated at different steady-state dilution rates, with high dilution rates resulting in predominantly fast-growing organisms and low dilution rates resulting in predominantly slow-growing cells, and (2) risk exists in any randomly picked batch experiment to predict the steady-state behavior of the system when heterogeneous microbial populations must be used.     

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h^?1 with an initial cell mass of less than 0.6 OD₆₀₀. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD₆₀₀ or higher, or at low dilution rates (<0.05 h^?1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron‐supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron‐limited chemostat cultures was observed compared to iron‐supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955–964. © 2009 Wiley Periodicals, Inc.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Growth of Saccharomyces cerevisiae was investigated under aerobic conditions in a glucose limited chemostat. The steady state concentrations of cells, glucose and ethanol were measured in dependence of the dilution rate. The growth rate showed a biphasic dependence from the glucose concentration. A shift from respiratory to fermentative metabolism (Crabtree-effect) altering heavily the cell yield and the ethanol yield took place in the range of dilution rates between 0.3 h^-1 and 0.5 h^-1. Therefore the classical theory of continuous cultures is not applicable on aerobic growth of Saccharomyces cerevisiae under glucose limitation without introducing further premises. On the other hand the steady state cell concentration as a function of the dilution rate fits well the theoretically calculated curves, if cells are cultivated under conditions where only fermentation or respiration is possible.     

Continuous microalgae cultivation in a photobioreactor

New biomass sources for alternative fuels has become a subject of increasing importance as the nation strives to resolve the economic and strategic impacts of limited fossil fuel resources on our national security, environment, and global climate. Algae are among the most promising non‐food‐crop‐based biomass feedstocks. However, there are currently no commercially viable microalgae‐based production systems for biofuel production that have been developed, as limitations include less‐than optimal oil content, growth rates, and cultivation techniques. While batch studies are critical for determining basic growth phases and characteristics of the algal species, steady‐state studies are necessary to better understand and measure the specific growth parameters. This study evaluated the effects of dilution rate on microalgal biomass productivity, lipid content, and fatty acid profile under steady‐state conditions with continuous illumination and carbon dioxide supplemention for two types of algae. Continuous cultures were conducted for more that 3 months. Our results show that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) when the dilution rate varied from 0.08 to 0.64 day^?1. The biomass productivity of C. minutissima reached a maximum value (137 mg/L/day) at a dilution rate of 0.33 day^?1, while the productivity of Dunaliella tertiolecta varied from 46 to 91 mg/L/day at a dilution rate of 0.17 to 0.74 day^?1. The biomass productivity of D. tertiolecta reached a maximum value of 91 mg/L/day at a dilution rate of 0.42 day^?1. Moreover, the lipid content had no significant change with various dilution rates. Biotechnol. Bioeng. 2012; 109: 2468–2474. © 2012 Wiley Periodicals, Inc.     

Dynamics and stability analysis of the growth and astaxanthin production system of Haematococcus pluvialis

This paper investigated high cell density cultivation of Haematococcus pluvialis for astaxanthin production in 3.7-L bioreactors. A biomass concentration of 2.74 g L⁻¹and an astaxanthin yield of 64.4 mg L⁻¹ were obtained. Based on the experimental results, a new and simple dynamic model is proposed, differing from Monod kinetics, to describe cell growth, product formation and substrate consumption. Good agreement was found between the model predictions and experimental data. The model revealed that there was cell growth inhibition on product formation and product feedback compensation for substrate consumption, but no substrate inhibition or product inhibition of cell growth. Stability analysis demonstrated that no multiplicity of steady states was observed; the unique positive steady state was locally asymptotically stable; and the effect of dilution rate on steady states was greater than that of the initial substrate concentration. Received 23 February 1999/ Accepted in revised form 08 June 1999     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Glucose limited growth of a respiratory deficient mutant of Saccharomyces cerevisiae was studied in continuous culture under steady state conditions. The maximal growth rate, the Michaelis constant, the cell yield, the maintenance coefficient and the ethanol yield of the growing cell population were determined. The steady state concentrations of cells, glucose and ethanol were measured as functions of the dilution rate and compared with theoretical predictions. A far-reaching agreement between theory and experiment was observed. The decrease of the cell yield in the range of low dilution rates is well explained by introducing the concept of maintenance energy in the general theory of continuous cultures. A deviation of the cell yield from the predicted values, which has been found in the range of high dilution rates, is discussed.     

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.     

Nonlinear control of bioreactors with input multiplicities—an experimental work

The phrase input multiplicities means that an input variable with more than one value produces the same output value as if there were a single input–single output process. With input multiplicities, the value of the process gain changes as the manipulated variable changes, and beyond a certain input value, the sign of the gain also changes. A conventional PI controller for processes with input multiplicities may give unstable, less economical, or oscillatory responses. In the present work, control problems of a continuous bioreactor exhibiting two input multiplicities in the dilution rate on productivity were experimentally analyzed. A regulatory problem for the evaluation of controllers was taken up, i.e. a step change was made in the feed substrate concentration from 20 to 25 g/l at steady state conduction at lower (0.09386 h⁻¹) and higher (0.2278 h⁻¹) dilution rates for the same productivity of 2.9 g/l h. The nonlinear PI controller gave a more stable and fast response at both input dilution rates. The linear PI controller designed for a lower input dilution rate was stable, with some oscillations at the lower dilution rate, but the response was unstable at a higher dilution rate due to the input multiplicity behaviour of the process. Thus, nonlinear PI controller performance was found to be superior to that of the linear controller, and earlier reported theoretical results have been validated by the present experimental work.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Genuscandida berkhout

Two strains ofCandida albicans, one of low, the other of high virulency, were cultivated in a semi-synthetic medium with glucose concentrations 3 and 0.4%. The validity of the mathematical theory of single stage continuous cultivation was verified. The standard hyperbolic course of the curves expressing mutual relations between the substrate and microorganisms concentrations (S and X respectively), and dilution rate D gets lost at the concentration of 3%. For the 0.4% glucose concentration, reasonable agreement between theoretical and experimental values was obtained at lower dilution rates, so far as the yield coefficient remained constant. At greater dilution rates D, the yield coefficient decreased and the experimental values deviated from the theoretical ones. Specific rate of oxygen consumption\(Q_{O_2 } \), specific rate of CO₂ formation\(Q_{CO_2 } \), the specific rate of the substrate consumption q, alcohol formation P and total acids formation have been followed. By calculation, the smallest dilution rate “a”, was determined for which steady state conditions are still valid. This magnitude has been experimentally verified for the strain “19”. The steady state conditions are possible at 0.4% glucose concentration for the following range of dilution rates D: Strain “19” from D=0.0039 to 0.42 h^?1 strain “109” from D=0.003 to 0.41 h^?1. All calculated values were evaluated by statistical t-test. Significant differences between strains have been found for the yield coefficient at the concentration of 0.4%. This difference is probably caused by a small difference in the initial concentration of glucose S₀.     

Influence of specific growth limitation and dilution rate on the phosphorylation efficiency and cytochrome content of mitochondria of Candida utilis NCYC 321

With Candida utilis cells that had been removed directly from a 61 chemostat culture, in steady state, well-coupled mitochondria generally could be isolated. This required a modified snail-gut enzyme procedure that allowed the total processing time to be decreased to 3 h, or less. Examination of these mitochondria in an oxygraph showed the presence of 3 sites of energy conservation when the cells were grown at various dilution rates between 0.1 and 0.45 h^-1 in environments that were, successively, glucose-, ammonia-, magnesium-, phosphate- and sulphate-limited. Potassium-limited cells also apparently possessed 3 sites of oxidative phosphorylation when growing at dilution rates greater than 0.2 h^-1, but only 2 sites when growing at lower dilution rates. Analysis of cytochrome spectra obtained with these intact mitochondria revealed large quantitative (but not qualitative) differences, depending on the environmental conditions under which the yeast had been cultured. In particular, comparison of the ratio of cytochrome b to cytochrome a showed a pattern of change with dilution rate in mitochondria from potassium-limited cells that was distinctly different from those evident in mitochondria from cells that had been limited in their growth by the availability of other nutrients.     

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

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 (Q_p), polyphosphate (Q_pp) and chlorophyll a increased with increasing dilution rate, and the growth rate of the alga was described by Q_p as well as Q_pp in the Droop model. The ratio Q_pp/Q_p 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 Q_pp/Q_p 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 (μ_m) for the vegetative cells was 0.21 h^?1.     

Continuous fermentation to produce xanthan biopolymer: Laboratory investigation

Xanthan biopolymer has been produced by single-stage continuous fermentation with Xanthomonas campestris NRRL B-1459 in a medium of glucose, minerals, distillers' solubles, and urea for as long as 20 days. At the highest dilution rate studied (D = 0.0285 hr^?1), the steady state rate of xanthan production was 0.36 g/kg/hr and the steady state yield, basis glucose consumed, was 68%. Observations indicate that xanthan production rate is a function of pH and D.     

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y _glucose ^max and values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h^–1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K⁺ or Mg²⁺)-limited cultures expressed the highest metabolic rates and with the K⁺ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K⁺-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K⁺ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.     

Effect of Dissolved Oxygen on Continuous Production of Methionine

Methionine production by a mutant strain of Corynebacterium lilium was studied at different dilution rates and different dissolved oxygen concentrations, based on a statistical central composite design. The effect of dissolved oxygen levels between 2 % and 60 % and dilution rate levels between 0.04 to 0.29 on continuous production of methionine was studied. The process is described by the Leudeking‐Piret model. The experimentally obtained maxima for methionine and biomass productivities, observed at the same dilution rate of 0.17 h^–1, also corresponds to the theoretical prediction based on this model. This experimental observation is further supported by the surface response prediction of a dilution rate of 0.16 h^–1 for maximum methionine productivity and 0.15 h^–1 for cell mass productivity. Also, beyond the critical value of 30 % dissolved oxygen, the apparent benefits of oxygen transfer become less significant.     

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

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