Modelling of aerobic growth of Saccharomyces cerevisiae in a pH-auxostat

A model for growth and overflow metabolism of Saccharomyces cerevisiae was applied to simulate continuous cultivations in a pH-auxostat. The concentrations of glucose, biomass and ethanol are controlled by the flow ratio r between fresh medium and titrant solution, both of which are pH-regulated. A critical value of r could be derived, below which the culture becomes substrate depleted, resulting in a stop-flow condition with retained biomass but without growth. At r-values slightly above the critical value the pH-auxostat is substrate limited and unstable. Further increase of the r value results in a stable continuous culture growing at w_max. Thus, the pH-auxostat complements the chemostat in the growth range at or close to w_max. Even at w_max conditions, the ethanol concentration can be controlled at a low level.     

On the coexistence of competing microbial species in a chemostat under cycling

If a microorganism has a growth coupled production or consumption of acid or alkali, it is possible to use the pH-auxostat as a means of control in continuous fermentation. In using the pH-auxostat, it is possible to separate the inlet substrate flow in two different streams. These will both be pH controlled, with one main flow, consisting of nutrients and a second minor but concentrated flow, of acid or alkali. Hereby, it is possible to vary the difference in pH between the fermentor and the inlet medium. This pH difference is proportional to the steady-state cell mass concentration.(1,2) It is shown that by separating the inlet flow in two different streams and cultivating without any substrate limitation, the maximum growth rate may be obtained while the cell mass concentration will be controlled. This will also give the possibility to reach high cell mass concentrations at mu(max) without the risk of wash-out. A modified expression, based on hydrogen, of the steady-state bio-mass concentration, X, is developed as \documentclass{article}\pagestyle{empty}\begin{document}$$ X = Y_{X/H} \cdot [F_{{\rm Hin}} /(F_{{\rm Hin}} + F_{{\rm Min}} )] \cdot (C_{{\rm Hin}} - C_{{\rm HFERM}} ) $$\end{document} where Y(X/H) is the yield coefficient of cell mass per acid produced. The indexes Hin and Min refer to the inflows of alkali and medium, respectively; C(Hin) is the inlet concentration of hydrogen ions. The boundary condition for the cell mass shows that S(in) > X/Y(X/S), where S(in) is the medium substrate concentration and Y(X/S) is the yield of biomass per consumed substrate. It is shown that when the cell mass concentration exceeds this value, the flow stops. The applicability of the pH-auxostat method is then verified from different experiments. It is hereby used to detect a deviation from the maximal growth rate showing effects on the microbial physiology. With Escherichia coli used as the model organism, the effect on the growth rate of temperature and high concentration of ammonia were investigated.     

Studies on the stability of solvent production by Clostridium acetobutylicum in continuous culture

Various methods of continuous flow culture of Clostridium acetobutylicum NCIB 8052 were investigated, with the aim of obtaining prolonged production of acetone and butanol. In ammonia-limited chemostat culture, maximal concentrations of solvents were obtained at pH 5–5 at a relatively high biomass concentration of 1.3–2.0 g/1 dry weight maintained at a dilution rate of 0.06/h. Similar dependence of solvent production on the sustenance of a relatively high cell density was observed in magnesium- or phosphate-limited chemostat cultures. Solvent production was always transient, however, with a shift to production of only acetic and butyric acids being observed after 4–16 volume changes. Longer term solvent production was obtainable under conditions of glucose limitation but the solvent yield was low. Cultivation in a pH-auxostat permitted solvent production in reasonably high yield over at least 70 volume changes with no signs of culture degeneration. Although none of the continuous flow cultures achieved a true steady state, we conclude that turbidostat or pH-auxostat culture are the methods of choice for continuous solvent production by Cl. acetobutylicum NCIB 8052.     

Biotransformations by Clostridium beijerinckii NCIMB 8052 in pH-auxostat culture

Solvent-producing cultures of Clostridium beijerinckii NCIMB 8052 can reduce a variety of aldehydes and ketones to the corresponding alcohols, but the enzymes that catalyse these biotransformations have not been identified. The possibility that butanol dehydrogenases were involved was tested by comparing the ability of solvent- and acid-producing pH-auxostat cultures to reduce representative biotransformation substrates. The ability of the cultures to produce solvents was manipulated by controlling the biomass concentration, and this was achieved by varying the glucose concentration in the inflowing medium. The solvent-producing culture could reduce cyclohexanone and benzaldehyde. In contrast, very little reduction of these substrates occured in the acid-producing culture. This suggested that one or more butanol dehydrogenases did indeed catalyse these biotransformations.     

Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat

Summary The fermentation of glycerol to 1,3-propanediol, acetate, and butyrate by Clostridium butyricum was studied with respect to growth inhibition by the accumulating products. The clostridia were grown in a pH-auxostat culture at low cell density and product concentration and near maximum growth rate. The products were then added individually to the medium in increasing concentrations and the resulting depression of growth rate was used as a quantitative estimate of product inhibition. Under these conditions growth was totally inhibited at concentrations of 60 g/l for 1,3-propanediol, 27 g/l for acetic acid and 19 g/l for butyric acid at pH 6.5. Appreciable inhibition by glycerol was found only above a concentration of 80 g/l. In a pH-auxostat without added products but with high cell density as well as in batch cultures the product proportions were different. The 1,3-propanediol concentration may approach the value of complete inhibition while the concentrations of acetic and butyric acids remained below these values by at least one order of magnitude. It was therefore concluded that 1,3-propanediol is the first range inhibitor in this fermentation.     

Bistat--a novel method of continuous cultivation

Bistat is the method of continuous cultivation developed on the basis of pH-auxostat. It provides the stability of cultivation in each point of mu(S) dependence. This article describes the principle of bistat operation, the theory of bistat dynamics, its instrumentation, and some experimental results.     

Der substratlimitierte pH-Auxostat – Eine neue Methode zur kontinuierlichen Kultur

A method for continuous cultivation of microorganisms is demonstrated, the substrate limited pH-auxostat. The limiting substrate only is added with constant velocity. In this culture the cells grow with high utilization of the limiting substrate and with the highest specific growth rate possible at the given conditions. Yield coefficients and dilution rates of stable K⁺-limited steady states in yeast cultures with different pH-values and biomass concentrations were measured.     

Modification of A-stat for the characterization of microorganisms

Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics. The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter. Physiological studies of Saccharomyces sp. and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch. The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates. Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.     

The effect of automatic ph maintenance on steady state cultivation regimes and their stability. A theoretical study of the case of a perfect ph controller and perfect stirring

The dynamics of pH-controlled chemostat and pH-auxostat is considered. The medium flow governed by pH-controller is calculated using the ionic (mass-charge) balance equations. The generalized equations of pH-controlled continuous fermentation are developed for the case of two variables: cell biomass and residual substrate concentrations. The study of steady state solutions has shown a substantial dependence of the variable patterns on a concentration of alkali (or acid) in titrant medium flow. The generalized analytical expressions for eigenvalues (reciprocals of transient characteristic times) of the culture dynamics have been obtained. The rates of transients during the steady state establishment are considered using these eigenvalues.     

Effect of conditions od Trichosporon pullulans culturing on the synthesis of immunomodulating glycoprotein]

An extracellular glycoprotein (GP) exhibiting immunomodulating activity produced by the yeast Trichosporon pullulans grown in a defined ethanol-containing medium differed substantially in its composition from that of the yeast cell walls: therefore, it cannot be considered a structural component of the cell walls. In batch culture, the greatest GP production (40 mg/l) occurred in the exponential phase of the yeast growth. Under continuous cultivation, in both chemostat and pH-auxostat regimes, the specific rate of GP synthesis (qGP) increased with the increasing specific growth rate (mu) and reached 1.55 mg/(g h) at mumax. Under limitation of the yeast growth by zinc qGP was three times lower than under nitrogen or iron limitation. The rate of GP production depended inversely on the oxygen concentration.     

Russian Article pp. 255–266

The growth of the yeast Candida utilis VKM-Y-2332 was investigated during the cultivation on a mineral medium with addition of ethanol in the regime of the chemostat and the pH-auxostat. The composition of the macroelements of the biomass produced by Candida utilis VKM-Y-2332 was studied. Stoichiometric equations of the growth process of the examined yeast strain on ethanol were presented. The growth of Candida utilis on ethanol and glucose was also characterized by the way of comparision.     

Development of a multivariable auxostat

Summary Auxostat operation was extended to two set points in an aerobic yeast fermentation of Saccharomyces cerevisiae. The concentration of NH₄ ⁺ was controlled directly with an on-line NH₃ probe and control of pH indirectly fixed the glucose concentration, thus demonstrating an NH₄ ⁺:pH-auxostat. Multivariable control in the auxostat was superior to a chemostat for studying nutrient interaction and exploring multisubstrate limitations on metabolism.     

The effect of specific growth rates on the recovery of amino acids

Three specific growth rates, 0.23, 0.45 and 0.51 h^–1, were used to cultivate Corynebacterium glutamicum in a pH-auxostat. The specific formation rates of most amino acids increased by raising the specific growth rates. The highest specific growth rate, 0.51 h^–1, favors the production of LEU; whereas the highest production yield for ALA and GLU were at = 0.23 h^–1. A correlation among specific growth rates, glucose consumption rate, and production yields of amino acids was obtained.     

High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system

Thraustochytrids, in particular Schizochytrium spp., are used for the production of the valuable polyunsaturated fatty acid, docosahexaenoic acid (DHA; 22:6 n-3). Growth of Schizochytrium sp. G13/2S in a defined medium was initially made in shake-flask cultures to determine the optimum concentrations of glucose (100-200 g l(-1)) and ammonia ( approximately 300 mg l(-1)) that could be used by this microorganism. In subsequent fermenter cultures, a pH-auxostat method was used to maintain NH(3) from 200-300 mg l(-1). During the first 49 h of fermentation, 150 g glucose l(-1) produced 63 g cell dry wt l(-1). Although growth was not limited by the supply of nitrogen, total fatty acids were at 25% cell dry wt which is more than half the final lipid content of commercially-grown Schizochytrium biomass which uses N-limited medium in the final stages for maximum lipid accumulation. This strategy is therefore useful for the cultivation of Schizochytrium to a high cell density up to the point when lipid accumulation can be triggered by N exhaustion.     

Substrate controlled development of anaerobic acidifying aggregates at different shear rates in a gas lift reactor.

The influence of liquid shear rates on the development of acidifying mixed-culture aggregates was studied in a gas-lift reactor. The glucose concentration was kept at a constant and relatively high level by operating the reactor in pH-auxostat mode. Size, strength, and wet density of aggregates cultivated at different superficial gas velocities (Ug) were investigated. Image analysis showed that the Sauter mean diameter (Ds) decreased with increasing Ug. A stirred tank was used to characterize the surface detachment rate (Rd) under non-growth conditions. An exponential decrease was observed in Rd with the applied Ug during cultivation, i.e., aggregates became stronger. The increased strength coincided with an increase in aggregate wet density. Size classified aggregates showed an increase in Rd with the square of the aggregate diameter (Dp), however, this contribution was much smaller than the effect of adaptation. Experiments in a similar gas-lift reactor under dynamic conditions without adaptation, showed that Rd increased exponentially with increasing Ug. So, two important contributions to Rd can be distinguished: adaptation, which induces stronger aggregates, and aggregate size, which makes them less susceptible to hydrodynamic shear. A general expression for Rd was derived, which depends on Dp and Ug. Combining this equation with the surface biomass growth rate (Rg) allowed for the estimation of the maximal diameter (Dmax) aggregates can reach at any Ug, and it was found that the estimated and measured Dmax were in good agreement.     

Copper effect on the growth kinetics of Methylococcus capsulatus (bath)

Summary Bistat and pH-auxostat fermentations have been used to study the effect of Cu⁺⁺ on Methylococcus capsulates (Bath) growing on natural gas. An optimum for growth rate and yield was found to be within the range of 3–6 mole Cu/g dry cells. The cells absorb quantitatively Cu⁺⁺ from surrounding broth until 6 mole Cu/g dry cells has been reached. Also the effect of copper on sMMO activity has been investigated. A complete suppressing of sMMO is obtained with 1.5 M Cu/g dry cells.     

Distribution of ATP and reducing equivalents in Corynebacterium glutamicum during amino acid resolution

The distributions of ATP and reducing equivalents were estimated with the aid of a pre-constructed bionetwork describing the growth characteristics of Corynebacterium glutamicum cultivated in a pH-auxostat. Results showed that the specific growth rate (μ) imposed different degrees of influences on both ATP and reducing equivalents. The ATP generated from the TCA cycle increased about 33% as μ was raised from 0.23 to 0.45 h⁻¹, while the ATP obtained from the EMP pathway decreased from 32.90 to 2.12% over the same growth span. In contrast, the reducing equivalents contributed from both the TCA cycle and the EMP pathway varied slightly. The ATP obtained through glucose utilization was insufficient to balance the microbial demands. As a result, the transformation among amino acids to compensate the ATP requirement was noticed. To enhance product resolution from an amino acid mix, metabolic nodes at AKG, PYR, PEP, and RU5P have been identified and could be considered concurrently as the potential nodes for manipulating metabolic flows in C. glutamicum.     

Bacterial strain characterizing by EDTA requirement

A novel strain of bacteria (LPM-4) was isolated that is characterized by a unique EDTA requirement for cell growth. Suspensions of washed cells of strain LPM-4 degrated EDTA complexes with Ba2+, Mg 2+, Ca2+, and Mn2+ at constant rates (0.310-0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 +/- 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrating bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095 h(-1)) and lower mass cell yield (0.219 g cells/g EDTA) that is promising for its practical applications for EDTA removal in wastewater treatment plants.     

Bacterial Strain Characterizing by EDTA Requirement

A novel strain of bacteria (LPM-4) characterized by a unique EDTA requirement for cell growth was isolated. Suspensions of washed cells of strain LPM-4 degraded EDTA complexes with Ba²⁺, Mg²⁺, Ca²⁺, and Mn²⁺ at constant rates ( 0.310 ± 0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 ± 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrading bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095^{? 1}) and lower mass cell yield (0.219 g cells/g EDTA), which is promising for its practical applications for EDTA removal in wastewater treatment plants.     

Inhibition of Candida valida growth by copper ions]

The high toxicity of copper ions for Candida valida growth was established at pH-auxostat regime. The value of mu max decreased even at the residual Cu2+ concentration 1.0 mg/l. The inhibition constant (Ki) that characterized a copper ion concentration at which yeast specific growth rate was halved was equal to 7.7 mg/l. A linear dependence of 1/mu max on a residual concentration of copper ions indicates that yeast growth inhibition is due to inhibition of one enzymic reaction which is the most sensitive to copper. Yeast growth inhibition by copper was accompanied by accumulation of Cu2+ ions in biomass, a decrease in nucleic acid and true protein contents, and changes in amino acid composition of protein. The amounts of cystine and cysteine in protein increased and tryptophane content decreased with inhibition of yeast growth. Yeast growth inhibition by copper did not affect the lipid content but significantly reduced the degree of unsaturation due to a decrease in the amounts of polyunsaturated linoleic and alpha-linolenic acids.