Uptake and accumulation of B-group vitamers in Saccharomyces cerevisiae in ethanol-stat fed-batch culture.

The uptake and accumulation of the B-group vitamins thiamine, riboflavin, nicotinamide, pantothenic acid and pyridoxine in Saccharomyces cerevisiae was studied by gradually increasing the specific dosage of vitamins in an ethanol-stat fed-batch culture. Thiamine, nicotinamide, pantothenic acid, and pyridoxine were almost completely taken up at low vitamin dosages. Thiamine was determined to be the major accumulating form of vitamin B1 while most of the assimilated nicotinamide and pantothenic acid accumulated in cofactor forms. Despite the obvious uptake of pyridoxine, accumulation of B6 vitamers was not observed. In contrast with the other vitamins studied, riboflavin began accumulating in the culture medium immediately after vitamin addition was initiated. By the end of the experiment, the apparent uptake of all vitamins exceeded their accumulation in the cells. Variations in the growth rate of yeast at different vitamin dosages demonstrate the importance of balancing the vitamins in the media during cultivation.

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

AIMS: 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. METHODS AND RESULTS: The sourdough was started from a 6-year-old freeze-dried sourdough originating from the same bakery. A unique microbial consortium consisting mainly of bacteria belonging to species Lactobacillus helveticus, Lactobacillus panis and Lactobacillus pontis was identified based on culture-dependent (Rep-PCR) and culture-independent [denaturing gradient gel electrophoresis (DGGE)] methods. Three of the isolated Lact. helveticus strains showed remarkable adaptation to the sourdough conditions. They differed from the type strain by the ability to ferment compounds specific to plant material, like salicin, cellobiose and sucrose, but did not ferment lactose. CONCLUSION: We showed remarkable stability of a LAB consortium in rye sourdough started from lyophilized sourdough and propagated in a large bakery for 7 months. Lactobacillus helveticus was detected as the dominant species in the consortium and was shown to be metabolically adapted to the sourdough environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an established and adapted microbial consortium as a starter is a good alternative to commercial starter strains.

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. METHODS AND RESULTS: Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power-time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD-time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long-run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O(2) protective effect of rye sourdough. CONCLUSIONS: Measurements of the power-time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.

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.

13C-NMR study of acetate assimilation in Thermoproteus neutrophilus.

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2-Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.

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 mu > mu crit, 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).

Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.