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.     

Changes in intracellular ATP‐content of CHO cells as response to hyperosmolality

A variety of approaches has been published to enhance specific productivity (q_p) of recombinant Chinese hamster ovary (CHO) cells. Changes in culture conditions, e. g. temperature shifts, sodium butyrate treatment and hyperosmolality, were shown to improve q_p. To contribute to a better understanding of the correlation between hyperosmolality and enhanced q_p, we analyzed cellular kinetics and intracellular adenine nucleotide pools during osmotic shift periods. Known phenotypes like increased formation rates for lactate and product (anti‐IL‐8 antibody; q_lactate, q_p) as well as increased cell specific uptake rate for glucose (q_glucose) were found—besides inhibition of cell growth and G1‐arrest occurred during batch cultivations with osmotic shift. The analysis of intracellular AXP pools revealed enlarged ATP amounts for cells as response to hyperosmolality while energy charges remained unchanged. Enhanced ATP‐pools coincided with severely increased ATP formation rates (q_ATP) which outweighed by far the putative requirements attributed to regulatory volume increase. Therefore elevated q_ATP mirrored an increased cellular demand for energy while experiencing hyperosmotic shift. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1212–1216, 2015     

Molar growth yields,respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a chemostat

The effect of growth rate on the physiology of Beneckea natriegens was studied in chemostat culture. The molar growth yields (Y) from glucose and oxygen, the specific rates of oxygen (q _{O 2}) and glucose (q _glc) consumption and the specific rate of CO₂ production (q _{CO 2}) were linearly dependent on the growth rate over the dilution rate 0.17 h^-1 to 0.60 h^-1. Further increase in the dilution rate resulted in a decrease in growth yield and respiration rate and these changes were coincident with increases in the specific rate of glucose utilisation and of acetate production. The affinity of Beneckea natriegens for glucose was similar when measured either directly in chemostat culture or in a closed oxygen electrode system using harvested bacteria. The total content of cytochromes decreased with increasing growth rate. However, the quantity of CO-binding cytochromes remained independent of growth rate and correlated with the potential respiration rate.     

Regulation of photosynthetic electron-transport in Phaseolus vulgaris L., as determined by room-temperature chlorophyll a fluorescence

The regulation of photosystem II (PSII) by light-, CO₂-, and O₂-dependent changes in the capacity for carbon metabolism was studied. Estimates of the rate of electron transport through PSII were made from gas-exchange data and from measurements of chlorophyll fluorescence. At subsaturating photon-flux density (PFD), the rate of electron transport was independent of O₂ and CO₂. Feedback on electron transport was observed under two conditions. At saturating PFD and low partial pressure of CO₂, p(CO₂), the rate of electron transport increased with p(CO₂). However, at high p(CO₂), switching from normal to low p(O₂) did not affect the net rate of photosynthetic CO₂ assimilation but the rate of electron-transport decreased by an amount related to the change in the rate of photorespiration. We interpret these effects as 1) regulation of ribulose-1,5-bisphosphatecarboxylase (RuBPCase, EC 4.1.1.39) activity to match the rate of electron transport at limiting PFD, 2) regulation of electron-transport rate to match the rate of RuBPCase at low p(CO₂), and 3) regulation of the electron-transport rate to match the capacity for starch and sucrose synthesis at high p(CO₂) and PFD. These studies provide evidence that PSII is regulated so that the capacity for electron transport is matched to the capacity for other processes required by photosynthesis, such as ribulose-bisphosphate carboxylation and starch and sucrose synthesis. We show that at least two mechanisms contribute to the regulation of PSII activity and that the relative engagement of these mechanisms varies with time following a step change in the capacity for ribulose-bisphosphate carboxylation and starch and sucrose synthesis. Finally, we take advantage of the relatively slow activation of deactivated RuBPCase in vivo to show that the activation level of this enzyme can limit the rate of electron transport as evidenced by increased feedback on PSII following a step change in p(CO₂). As RuBPCase as activated, the feedback on PSII declined.Abbreviations and symbols J_C electron-transport rate calculated from CO₂-assimilation measurements - J_F electron-transport rate calculated from fluorescence parameters - PFD photon-flux density - q_E energy-dependent quenching - PSII photosystem II - q_Q Q-dependent quenching - QY quantum yield - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) C.I.W. publication No. 1015     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Dynamic modelling of limitations on improving leaf CO2 assimilation under fluctuating irradiance

A dynamic model of leaf CO₂ assimilation was developed as an extension of the canonical steady‐state model, by adding the effects of energy‐dependent non‐photochemical quenching (qE), chloroplast movement, photoinhibition, regulation of enzyme activity in the Calvin cycle, metabolite concentrations, and dynamic CO₂ diffusion. The model was calibrated and tested successfully using published measurements of gas exchange and chlorophyll fluorescence on Arabidopsis thaliana ecotype Col‐0 and several photosynthetic mutants and transformants affecting the regulation of Rubisco activity (rca‐2 and rwt43), non‐photochemical quenching (npq4‐1 and npq1‐2), and sucrose synthesis (spsa1). The potential improvements on CO₂ assimilation under fluctuating irradiance that can be achieved by removing the kinetic limitations on the regulation of enzyme activities, electron transport, and stomatal conductance were calculated in silico for different scenarios. The model predicted that the rates of activation of enzymes in the Calvin cycle and stomatal opening were the most limiting (up to 17% improvement) and that effects varied with the frequency of fluctuations. On the other hand, relaxation of qE and chloroplast movement had a strong effect on average low‐irradiance CO₂ assimilation (up to 10% improvement). Strong synergies among processes were found, such that removing all kinetic limitations simultaneously resulted in improvements of up to 32%.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.     

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.     

A dynamic mathematical model of the chemostat

A number of experimental studies on the dynamic, behavior of the chemostat have shown that the specific growth rate does not, instantaneously adjust to changes in the concentration of limiting substrate in the chemostat following disturbances in the steady state input limiting substrate concentration or in the steady state dilution rate. Instead of an instantaneous response, as would be predicted by the Monod equation, experimental studies have shown that the specific growth rate experiences a dynamic lag in responding to the changes in the concentration of limiting substrate in the culture vessel. The observed dynamic lag has been recognized by researchers in such terms as an inertial phenomenon and as a hysteresis effect, but as yet a systems engineering approach has not been applied to the observed data. The present paper criticizes the use of the Monod equation as a dynamic relationship and offers as an alternative a dynamic equation relating specific growth rate to the limiting substrate concentration in the chemostat. Following the development of equations, experimental methods of evaluating parameters are discussed. Dynamic responses of analog simulations (incorporating the newly derived equations) are compared with the dynamic responses predicted by the Monod equation and with the dynamic responses of experimental chemostats.     

COMPARATIVE KINETIC STUDIES OF NITRATE-LIMITED GROWTH AND NITRATE UPTAKE IN PHYTOPLANKTON IN CONTINUOUS CULTURE1

A comparative study of nitrate-limited growth and nitrate uptake was carried out in chemostat cultures of Ankistrodesmus falcatus (Corda) Ralfs., Asterionella formosa Hass., and Fragilaria crotonensis Kit. In each species growth rate (μ) was related to total cell nitrogen or cell quota (q) by the empirical Droop growth function. Nitrate uptake was a function of both external N concentration and q. The apparent maximum uptake rate (V_m') at a given μ was inversely related to q – q₀, where q₀ is the minimum quota. The apparent half-saturation constant for uptake, (K_m') appears to show a slight inverse trend with μ, although statistical analysis shows that this trend is inconclusive. When q approaches q₀, V_m' is several orders of magnitude greater than μq, the calculated steady-state uptake rate. As q increases, however, the difference between these two variables decreases sharply until q approaches q_m, the cell quota for nitrogen-rich cells. At this point the difference between μq and V_m' disappears. This behavior is explained by the feedback regulation of N uptake. The inverse relationship between V_m' and q – q₀ can be described by an empirical three-parameter equation.     

Effect of Growth Rate on the Synthesis of Penicillin by Penicillium chrysogenum in Batch and Chemostat Cultures

The kinetics of penicillin production by Penicillium chrysogenum Wis 54-1255 in a glucose-limited chemostat and in batch cultures are reported. The specific production rate of penicillin, q_pen (units per milligram of dry weight per hour) was independent of specific growth rate over the range 0.014 to 0.086 hr^-1. Growth was stopped by restricting the glucose supply to the “maintenance ration,” that is, the glucose requirement of the organism at zero growth rate with all other nutrients in excess. Under such conditions, the organism dry weight remained constant, but the q_pen fell approximately linearly to zero at a rate inversely related to the previous growth rate. Glucose supplied in excess of the maintenance ration inhibited the decay of q_pen. At a critical growth rate between 0.009 and 0.014 hr^-1, the decay was completely inhibited. Quantitative expressions for the q_pen of growing and nongrowing cultures were derived and used to predict the steady-state concentrations of penicillin accumulating in one- and two-stage continuous processes. A rational explanation of the kinetics of penicillin accumulation in batch cultures is given, relating the rate of penicillin synthesis to growth rate. It is concluded that an important role of corn steep liquor (CSL), a heterogeneous carbon and nitrogen source commonly used in penicillin production media, is the provision of substrates which allow a high concentration of mold to be reached before the growth rate falls below the critical value. CSL had no significant effect on q_pen.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution. The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4‐dichlorophenoxyacetate (2,4‐D) were cultivated in a chemostat on 2,4‐D in the presence of (R)‐2‐(2,4‐dichlorophenoxy)propionate. Long‐term cultivation led to enhanced 2,4‐D degradation, as demonstrated by improved values of the Michaelis–Menten constant K_m for 2,4‐D and the catalytic efficiency k_cat/K_m of the initial degradative key enzyme (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2‐DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4‐dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

NEUTRAL LIPID AND CARBOHYDRATE PRODUCTIVITIES AS A RESPONSE TO NITROGEN STATUS IN ISOCHRYSIS SP. (T‐ISO; HAPTOPHYCEAE): STARVATION VERSUS LIMITATION1

Partitioning of the carbon (C) fixed during photosynthesis between neutral lipids (NL) and carbohydrates was investigated in Isochrysis sp. (Haptophyceae) in relation to its nitrogen (N) status. Using batch and nitrate‐limited continuous cultures, we studied the response of these energy reserve pools to both conditions of N starvation and limitation. During N starvation, NL and carbohydrate quotas increased but their specific growth rates (specific rates of variation, μ_CAR and μ_NL) decreased. When cells were successively deprived and then resupplied with NO₃, both carbohydrates and neutral lipids were inversely related to the N quota (N:C). These negative relationships were not identical during N impoverishment and replenishment, indicating a hysteresis phenomenon between N and C reserve mobilizations. Cells acclimated to increasing degrees of N limitation in steady‐state chemostat cultures showed decreasing NL quota and increasing carbohydrate quota. N starvation led to a visible but only transient increase of NL productivity. In continuous cultures, the highest NL productivity was obtained for the highest experimented dilution rate (D = 1.0 d^?1; i.e., for non N‐limited growth conditions), whereas the highest carbohydrate productivity was obtained at D = 0.67 d^?1. We used these results to discuss the nitrogen conditions that optimize NL productivities in the context of biofuel production.     

Kinetics and energetics of reduced sulfur oxidation by chemostat cultures of Thiobacillus ferrooxidans

Thiobacillus ferrooxidans was grown in chemostat cultures with thiosulfate and tetrathionate as the limiting substrates. The yields at steady state on both substrates at different dilution rates were calculated. In a few experiments the air supply was supplemented with 2% CO₂ (v/v). This resulted in a slightly increased yield.Cells from the chemostat cultures were used to study the kinetics of thiosulfate, tetrathionate, sulfite and sulfide oxidation. With all substrates mentioned the Ks values were in the micromolar range. The values for thiosulfate and tetrathionate were 2 orders of magnitude lower that those published previously.     

Dynamic analysis of a microbial process: A systems engineering approach

A general mathematical model of the chemostat system is developed in order to define an experimental program of dynamic testing. A glucose-limited culture ofSaccharomyces cerevisiae was grown in a chemostat using chemically defined medium. The chemostat was perturbed from an initial steady state by changes in input glucose concentration, dilution rate, pH, and temperature. Dynamic responses of cell mass, glucose, cell number, RNA, and protein concentrations were measured. A number of simulation techniques were used in developing a dynamic mathematical model and in comparing the developed model with experimental data as well as the Monod model. The resulting model was found to be quantitatively accurate and superior to the Monod model. The developed model was interpreted in the light of cell physiology. Adjustment of intracellular RNA fraction was found to be rate limiting in acceleration of cell specific growth rate.