Toomas Paalme

The computer-controlled continuous culture of Escherichia coli with smooth change of dilution rate (A-stat)

Abstract A method of continuous cultivation of bacterial cells with computer-controlled smooth change of growth rate was developed. This method was used in parallel with batch and chemostat culture techniques for determination of the growth characteristics of Escherichia coli K12 on mineral salt medium with glucose. The maximum growth rate (0.5–0.55 h −1) for this strain determined in the batch culture was remarkably higher than the wash-out value ( μ wa = 0.38 h −1 ) observed in the conventional chemostat experiment. If in the continuous culture smooth increase of dilution rate (accelerostat = A-stat cultivation procedure) instead of stepwise change was applied, the culture conditions, corresponding to the steady state definition, were maintained up to a dilution rate of 0.47 h −1 . However, at higher dilution rates acetate started to accumulate and growth yield based on glucose consumption decreased. The maximum growth rate μ max observed in these experiments was 0.57 h −1 . The wash out of the culture at relatively low dilution rate ( D = 0.38 h −1 ) in the chemostat could be explained by the inhibitory effect of acetate, accumulating due to the shift up of glucose concentration occurring after the stepwise (too sharp) increase of dilution rate. The continuous cultivation with smooth and constant increase of dilution rate (A-stat) proved itself as a powerful experimental method for the study of the behaviour of growth characteristics.

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l−1) and yeast extract concentrations (4, 8 and 16 g l−1) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 106 ml−1) was obtained using a glucose concentration of 10 g l−1 and a yeast extract concentration of 8 g l−1 in the feeding medium. A yeast extract concentration of 16 g l−1 inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.

The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach

The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied. For all substrates studied the maximum value of specific respiration rate, QO2, remained between 14–18 mmol O2 h-1 g dwt-1 and the maximum growth rate varied from 0.22 h-1 on acetate to 0.77 h-1 on glucose + casamino acids. After the respiratory capacity of the cells was exhausted at growth rates µ < µcrit, the growth yield YXO2, increased slightly when the dilution rate increased. The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain. Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E. coli exceeds by two-fold that (theoretically) required to build up biomass. The experimental value of mATP < 4 mmol ATP h-1 g dwt-1 determined from A-stat cultivation data was low compared with the calculated ‘unproductive hydrolysis’ of ATP (64–103 mmole ATP g dwt-1).

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.

Evolution of Bacterial Consortia in Spontaneously Started Rye Sourdoughs during Two Months of Daily Propagation

The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1∶10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping.

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry

Aims:  The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid‐state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation.

A study on growth characteristics and nutrient consumption of Lactobacillus plantarum in A-stat culture

Lactobacillus plantarum was grown in complex media containing glucose and yeast extract. The maximum growth yield based on yeast extract consumption was 0.5 g dwt g-1. Growth yield YATP 15–17 g dwt mol ATP-1 was almost constant in the glucose limited A-stat experiment whereas in the yeast extract limited culture it increased with dilution rate. The maximum specific growth rate observed, 0.5 h-1, was similar for both A-stat and batch cultures. Specific oxygen consumption, QO2, reached the value of 1.8 mmol O2 h-1 g dwt-1. It was shown that Val, Ile, Leu, Tyr and Phe, were consumed mainly as free amino acids, while Asp, Pro, Lys and Arg were derived from peptides. Significantly more Asp, Ser, Glu, Val, Ile, Leu and Phe were consumed than needed to build up cell protein whereas some Pro, Gly, Ala and Lys was synthesized. A network of metabolic reactions in L. plantarum was proposed on the basis of the experimental data.

Structural changes of starch during baking and staling of rye bread.

Rye sourdough breads go stale more slowly than wheat breads. To understand the peculiarities of bread staling, rye sourdough bread, wheat bread, and a number of starches were studied using wide-angle X-ray diffraction, nuclear magnetic resonance ((13)C CP MAS NMR, (1)H NMR, (31)P NMR), polarized light microscopy, rheological methods, microcalorimetry, and measurement of water activity. The degree of crystallinity of starch in breads decreased with hydration and baking to 3% and increased during 11 days of storage to 21% in rye sourdough bread and to 26% in wheat bread. (13)C NMR spectra show that the chemical structures of rye and wheat amylopectin and amylose contents are very similar; differences were found in the starch phospholipid fraction characterized by (31)P NMR. The (13)C CP MAS NMR spectra demonstrate that starch in rye sourdough breads crystallize in different forms than in wheat bread. It is proposed that different proportions of water incorporation into the crystalline structure of starch during staling and changes in starch fine structure cause the different rates of staling of rye and wheat bread.

Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed Lactobacillus helveticus as the dominant species.

To assess the structure and stability of a dominant lactic acid bacteria (LAB) population during the propagation of rye sourdough in an industrial semi‐fluid production over a period of 7 months.

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants.

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.