Steady-state Continuous Cultures Research Articles

Metabolic response of Bacillus sphaericus 1593M for dual-substrate limitation in continuous and total-cell-retention cultures

A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source.

Adenine quantitation in yeast extracts and fermentation media and its relationship to protein expression and cell growth in adenine auxotrophs of Saccharomyces cerevisiae.

We have described a method to reliably measure the free adenine content of yeast extract powders or the adenine concentrations found in chemically-defined and complex fermentation samples. This method relies on the selective precolumn derivatization of adenine with chloroacetaldehyde to form the fluorescent adenine adduct 1,N6-ethenoadenine. The derivatized adenine can then be resolved from other components found in samples with reverse phase HPLC and selectively monitored with fluorescence. This method was then used to study the adenine nutritional requirements of adenine auxotrophs of recombinant Saccharomyces cerevisiae. The adenine content of individual yeast extract powders was examined in relation to the cell mass (dry cell weight, DCW) achieved in culture media formulated with these powders. A general increase in DCW was observed with increasing adenine concentration in the yeast extract. Conversely, we observed that as adenine concentration increased in complex media the expression levels of a heterologous protein decreased. This method also allowed us to examine the adenine/DCW ratio in both steady-state continuous culture and batch culture. In both cases, the total in vivo adenine content as measured by the amount of adenine utilized from the culture media was estimated to be ca. 25-40 mg/g DCW. However, data suggest that this value is in excess of what is strictly required for cell growth and represents the quantity of adenine required to saturate intracellular pools of adenine or adenine metabolites. A minimum requirement for cell growth is at least as low as 12.5 mg of adenine/g of cells.

Stabilization and increased production of insecticidal crystal proteins of Bacillus thuringiensis subsp. galleriae in steady- and transient-state continuous cultures

Bacillus thuringiensis subsp. galleriae, grown in continuous cultures, segregated to spontaneous asporogenic variants replacing the wild-type Spo+ Cry+ strains [Sachidanandham R, Jayaraman K (1993) Appl Microbiol Biotechnol 40:504–507]. Realizing that this was due to specific but unknown nutritional requirements, we undertook further continuous-culture studies to identify growth requirement(s) by pulsing various medium components and growth factors. While carbon, nitrogen and pulses of nutrients exhibited a neutral pulse response, a group of amino acids were shown to improve the stability and volumetric productivity of biomass. The formation of spores and insecticidal crystal proteins was found to be higher with amino acid supplementation. Comparison of carbon-limited steady-state continuous cultures under two different conditions of growth brought forth the stabilizing effects of the amino acid supplementation. Batch experiments carried out with these inputs demonstrated a better carbon utilization, resulting in a higher biomass as well as enhancement of bioinsecticidal activity.

Microbial consortia for multiple pollutant biodegradation

Abstract

Quantitative measurement of sulphur formation by steady-state and transient-state continuous cultures of autotrophic Thiobacillus species

Sulphur formation by the obligately chemolithoautotrophic Thiobacillus o and Thiobacillus neapolitanus was studied in aerobic, substrate-limited continuous cultures. The performance of transient-state and steady-state cultures was compared using different methods for measuring sulphur production. Below a dilution rate (D) of 0.3 h−1 (at 50% air saturation), sulphate-producing steady states were obtained, and cultures grown with sulphide or thiosulphate (at D = 0.06 h−1) showed similar characteristics (e.g. cell yields, oxidation capacities and CO2-fixation capacities). Elemental sulphur was a major product above D = 0.3 h−1, but steady states were difficult to achieve, because of adherence of sulphur to the fermentor surfaces and the accumulation of sulphide. These problems could be circumvented using transient-state experiments of 1 h. It was then found that elemental sulphur was formed under oxygen limitation or at high substrate load. The rates of sulphur formation obtained by sulphur analysis agreed with the values calculated from stoichiometric balances. Sulphide and thiosulphate proved to be equivalent substrates for both Thiobacillus species during elemental sulphur formation under the conditions tested. It is concluded that transient-state cultures of thiobacilli, pregrown as sulphate-producing steady-state cultures, provide experimental conditions for the quantitative assessment of sulphur formation from (labile) sulphide and from thiosulphate.

On-line sensitive lightness measurement of cell mass in Saccharomyces cerevisiae culture

An industrial spectrophotometer was used as a very accurate on-line biomass sensor to investigate fast dynamic changes in yeast culture in the range of 0.5–5 g/l. High sensitive variation in biomass concentration of 0.015 g/l was detected. A fast dynamic response is induced in a steady state continuous culture of Saccharomyces cerevisiae by an acetate pulse and biomass concentration profile clearly determined by this sensor.

Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline, glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg−1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na+ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h−1 and 0.4 h−1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.

A combined cell‐cycle and metabolic model for the growth of hybridoma cells in steady‐state continuous culture

The Model presented in this work demonstrates the combination of cell-cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady-state continuous culture. The cell-cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling-and apoptotic-cell population, which together form the viable-cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic-cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod-type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance-energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady-state values of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable-cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature.

The microaerophily and photosensitivity of Propionibacterium acnes.

The effect of oxygen on the in vitro propagation of Propionibacterium acnes was investigated under defined culture conditions. This micro-organism is the predominant bacterial resident within the pilosebaceous follicles of sebum-rich areas of human skin. The organism was grown in continuous culture in defined synthetic medium with glucose as the main carbon-energy source at various air saturation concentrations and in the presence and absence of light. Steady state continuous cultures were achieved at very low oxygen tensions in the presence of light, and at higher levels of oxygen when non-illuminated. Culture biomass yields were higher than those of anaerobic cultures. Bacterial cells were inactivated in the presence of light at high oxygen concentrations because of photosensitization reactions involving excess oxygen and microbial porphyrin species.

Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool.

Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I. Vasconcelos, L. Girbal, and P. Soucaille, J. Bacteriol. 176:1443-1450, 1994). We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio. A high substitution of glucose by pyruvate [P/(G+P) value of 0.67 g/g] provided a carbon-limited culture with butanol and butyrate as the major end products. In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K. A. Walter, R. V. Nair, R. V. Carry, G. N. Bennett, and E. T. Papoutsakis, Gene 134:107-111, 1993).

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

We examined phosphate (Pi) uptake by two well-characterized microorganisms: a green alga (Selenastrum capricornutum) and a heterotrophic yeast (Rhodotorula rubra). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of Pi. We found that, under these conditions, both organisms had very high transport rates for Pi. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store Pi more efficiently than the alga under dilute Pi continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

We examined phosphate (Pi) uptake by two well-characterized microorganisms: a green alga (Selenastrum capricornutum) and a heterotrophic yeast (Rhodotorula rubra). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of Pi. We found that, under these conditions, both organisms had very high transport rates for Pi. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store Pi more efficiently than the alga under dilute Pi continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.

In vivo manipulation of the xanthophyll cycle and the role of zeaxanthin in the protection against photodamage in the green alga Chlorella pyrenoidosa.

Chlorella pyrenoidosa was grown in steady-state continuous cultures in either high or low light. Samples of these cultures were incubated in darkness (violaxanthin state) or in saturating light (zeaxanthin state). These samples were kept in the respective preadapted states throughout the entire photodamage treatment. Photodamage involved exposure to single-turnover flashes fired at a low (non-actinic) frequency. The damage caused by the light stress thus applied was monitored by changes in photosynthetic properties and pigment composition. Cells preadapted in the light resisted photodamage better than those kept in darkness. The low light grown cells were more vulnerable to photodamage than the high light grown cells. Our experimental approach permitted the equilibria between the components that participate in the xanthophyll cycle to be set without addition of inhibitors. Regardless of the total amount of violaxanthin being present, its conversion to anthera- and zeaxanthin is a prerequisite for protection. The protection is most effective for photosystem II. It appeared that antheraxanthin accumulates as a result of photodamaging flashes provided that these are fired in the presence of background light, i.e. with zeaxanthin present. From this, it is newly derived that the xanthophyll cycle operates in full in the light, including epoxidation of zeaxanthin. The latter conversion was also demonstrated in vitro, via nonenzymatic oxygen-dependent turnover of zeaxanthin into violaxanthin.

Some observations on protease production in continuous suspension cultures of Bacillus firmus

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study.

Linear growth and poly(beta-hydroxybutyrate) synthesis in response to pulse-wise addition of the growth-limiting substrate to steady-state heterotrophic continuous cultures of Aquaspirillum autotrophicum.

Heterotrophic pyruvate-limited steady-state continuous cultures of the bacterium Aquaspirillum autotrophicum were perturbed with a pulse injection of a small volume of concentrated pyruvate solution. These cultures exhibited an instantaneous change in the growth dynamics, turning from steady state to apparently linear growth. These transient growth-responses had no lag phase and were clearly distinct from unlimited exponential growth according to the initial rates of increase of biomass and substrate disappearance kinetics. A linear accumulation with time of poly(beta-hydroxybutyrate) was observed within the cells. Slopes of these linear responses were negatively correlated with the dilution rate. Physiological bases of linear growth are discussed in the light of the models of H. E. Kubitschek. Poly(beta-hydroxybutyrate) synthesis in the absence of exogenous limitation may serve to protect the cells against a transient metabolic overflow.

Cybernetic modeling of iron‐limited growth and siderophore production

The cybernetic modeling framework developed by Ramkrishna and co-workers has been applied to a case of bacterial metabolite production, namely the production of siderophores (iron-chelating agents) associated with iron-limiting fermentation conditions. Experimental growth data showed that, even though final biomass levels were controlled by exhaustion of the carbon source, iron-limiting conditions also affected the biomass yield. A structured model which includes the process of an iron-limiting energy resource production was able to quantitatively account for this apparent dual-substrate limitation over a wide range of batch and continuous operating conditions. The experiments data also showed quite large difference in iron uptake over the wide range of operating condition and iron levels investigated. The inclusion in the model of the processes of low and high (siderophore-mediated) affinity iron transport, and siderophore production led to simulation results that were in good quantitative agreement with the siderophore, medium and cell iron levels, in both batch and steady-state continuous culture operating conditions.

The biooxidation of methanol, ethanol and isopropanol by a defined co-culture at elevated temperatures

The introduction of more imaginative enrichment and isolation procedures has permitted the isolation of pure cultures of thermotolerant methylotrophic bacteria, a group that was previously unknown. One potential application for such bacteria is in the aerobic biotreatment of petrochemical industry wastewaters at elevated temperatures. Here, the growth and biooxidation characteristics of one such bacterium, Bacillus sp. NCIB 12522, in co-culture with a Gram-negative thermophilic non-methylotrophic solvent utilizing bacterium, NA 17, on a mixture of methanol, ethanol and isopropanol, under both steady and transient state continuous flow culture conditions are reported. The results indicate that at dilution rates <0.2 h−1 effective biooxidation can be achieved at temperatures between 50° and 57 °C. As a result of step increases in bioreactor feed concentrations, the fraction of the methylotroph present in the co-culture changed according to whether the methanol or the ethanol concentrations were increased, but when isopropanol was increased, no change in the methylotroph fraction occurred between the initial and final steady states.

Continuous-Culture Responses of Candida shehatae to Shifts in Temperature and Aeration: Implications for Ethanol Inhibition

Temperature and aeration shifts were used to perturb steady-state continuous cultures to determine the effects of ethanol on xylose metabolism by Candida shehatae. The accumulation of ethanol exerted a delayed inhibitory effect on the specific rate of substrate utilization. A second effect was also observed in which the specific rate of xylitol production increased at the expense of the specific rate of ethanol production. Both effects were enhanced at higher temperature. Inhibitory effects also occurred in glucose metabolism.

Studies on beta-galactosidase production in transient operation cultures

Beta-galactosidase production in transient operation culture was studied considering cycles of 90 and 180 min. The results obtained after 14 cycles periods proved that significant increases in the especific enzyme activity are attained in transient operation namely 3.83 to 4.81 umg−1 compared with those obtained in steady state continuous culture which ranged between 1.71 to 2.2 Umg−1.