Influence of immobilization parameters on growth and lactic acid production by Streptococcus thermophilus and Lactobacillus bulgaricus co-immobilized in calcium alginate gel beads.

Streptococcus thermophilus and Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1-1.5 M) and alginate (1-2%, w/v) concentrations. Highest lactic acid production was 35 g l(-1) when both bacteria were in high viscosity beads (1%, w/v alginate) hardened in 0.1 M CaCl2 . The gel bead composition affected size and distribution of entrapped lactic acid bacteria.

A relationship between RP4 plasmid acquisition and phenotypic changes in Pseudomonas fluorescens R2fN.

The physiological behaviour of Pseudomonas fluorescens strain R2fN was compared to that of transconjugants [R2fN(RP4)], and two aggregation phenotypes were identified (Agr- and Agr+). Agr+ phenotype is characterized by the appearance of macroscopic aggregates when cells are growing in liquid media. Transconjugants exhibited Agr+ phenotype whereas wild type strain represented Agr-. Evidence is presented to support correlation between Agr+ phenotype acquisition and the presence of the broad-host range plasmid RP4 in strain R2fN. In addition, according to bacterial adherence to hydrocarbon test the transconjugant cells appeared to be very hydrophilic whereas wild type R2fN cells were hydrophobic.

alpha-Amylase production by free and immobilized Bacillus subtilis.

The effect of glucose on the alpha-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific alpha-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the alpha-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific alpha-amylase production with the free-cell system was produced by the immobilized-cell batch culture.

Relevance and isotopic assessment of hexose-6-phosphate recycling in micro-organisms.

Some pathways of hexose-6-phosphate recycling--those involving a breakdown of the hexose skeleton--through carbohydrate metabolism of micro-organisms were analyzed for both metabolic and isotopic effects. Two modes of recycling were proposed based on the degree of alteration of the hexose molecule through the catabolic part of the cycle. Simulated operation of most of these pathways resulted in increased synthesis of hexose-6-phosphate and NADPH, and reduced the NADH and moreover the ATP synthesis within the carbohydrate metabolism. A basic model for the quantitative assessment by means of isotopic studies of the processes of hexose-6-phosphate recycling is presented. The model was initially designed for the study of micro-organisms producing polysaccharides, but it can be extended to other situations.

Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti.

The pathways of polysaccharide biosynthesis were investigated in cells of Sinorhizobium meliloti (strain Su47) using a stable isotope approach. The isotopic labeling of the periplasmic beta-1,2-glucans synthesized from glucose labeled at various positions evidenced the involvement of catabolic pathways, namely the pentose-phosphate and Entner-Doudoroff pathways, into the early steps of polysaccharide synthesis. The exopolysaccharides produced at the same time had a labeling pattern similar to that of the beta-glucans, indicating similar early steps for both polysaccharides. The results emphasized a cyclic organization of the carbohydrate metabolism in S. meliloti, in which the carbons of the initial hexose were allowed to re-enter the catabolic pathways many times. The metabolic incidences of such metabolic topology are discussed.

Conjugative plasmid transfer between Pseudomonas strains within alginate bead microcosms: effect of the internal gel structure.

Because microorganisms frequently live in an immobilized state in natural habitats, a cell-confined system was used to study bacterial conjugation. Two Pseudomonas putida strains were introduced together within calcium alginate gels. Different alginate beads were designed by varying the polysaccharide and the gelation solution concentrations. Microscopic examinations showed that 2% gels were quite homogeneous, but that 1.5% and 1% gels were rather heterogeneous. In these two last cases, shaft-shaped macrostructures were present. They were colonized during the culture by great densities of highly motile bacteria. Gene transfers due to conjugation were investigated in such alginate gel bead microcosms, in batch and continuous cultures. High-initial transfer frequencies were detected whatever the gel, but no conjugation events seemed to occur with further growth in the beads. Transfer frequency values were roughly similar in the different tested systems. Alginate gels used as artificial microcosms may be valuable to study the effect of cell microenvironment on genetic transfers in complex systems.

1H-NMR on line monitoring of water activity during lipase catalysed esterification.

1H-NMR spectroscopy is used to determine simultaneously the water activity (aw) and the time course of an esterification reaction catalysed by a lipase. Chemical shifts signals of hydroxylic hydrogens in fast exchange (i.e the average hydroxylic signal of acid, alcohol and water) varies with water activity and ester content. Calibration curves have been established from model media composed of the substrates and various ester contents, at different water activities, in order to mimic a reaction medium. One relationship is established between water activity, hydroxylic hydrogen signal chemical shift and ester content. In order to estimate the water activity evolution as a function of time, this last relationship is applied to the hydroxylic hydrogen chemical shift measured in a reaction medium where the Rhizomucor miehei lipase in a powder form is suspended in the liquid substrates. This alternative way of determining the water activity based on hydroxylic hydrogen chemical shift presents some advantages over more classical means, i.e. time saved and inaccuracies avoided by monitoring without handling the sample.

Response surface analysis of chlortetracycline and tetracycline production with K-carrageenan immobilized Streptomyces aureofaciens.

A full-factorial experimental design at three levels with two independent variables, carrageenan concentration (1.0, 1.5, and 2.0%) and potassium chloride concentration (0.3, 0.7, and 1.1 M) was studied in order to analyze the effect of both factors on the antibiotic production of K-carrageenan-immobilized mycelia of Streptomyces aureofaciens. The response surfaces obtained have indicated that both carrageenan and potassium chloride concentrations have a pronounced effect on the yield of chlortetracycline (CTC) and tetracycline (TC) produced by S. aureofaciens. By exclusively varying the immobilization conditions, the tetracycline production can be enhanced more than eight times (12.3 mg g-1 biomass for immobilized cells vs. 1.5 mg g-1 biomass for free cells) in comparison with free-cell mycelial cultures.

Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR.

The dehydrogenation of [1-(13)C]- and [2-(13)C]glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains. The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a gamma-lactone being detected in addition to the expected delta-lactone. These lactones--as well as the gluconate--could be further metabolized by the cells. The delta-lactone was utilized faster than the gamma-lactone. The presence--in significant amounts--and the relative stability of the lactones raise the question of their possible physiological significance.

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti.

The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated. During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected. A glucuronan lyase was identified, this new bacterial lyase produces d.p. 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity.

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads.

Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 µg·g-1 FWB) within beads and excreted it into the culture medium (up to 8 µg·g-1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 µg·g-1 FWB), no scopolin has been found into the culture medium.

NMR on-line monitoring of esterification catalyzed by cutinase.

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.

Stability of plasmid pHV1431 in free and immobilized cell cultures. Effect of temperature.

The segregational and structural stability of pHV1431 has been examined in Bacillus subtilis grown at 30 and 37 degrees C in continuous cultures without selection pressure. Immediately after appearance of plasmid-free cells in the reactor, a competition was observed between bacteria that favored plasmid-free cells because of the faster growth. A stronger instability was found at 30 degrees C compared to that at 37 degrees C. At 30 degrees C after 50 hours of culture, 2% of the cells carried the plasmid, whereas at 37 degrees C this percentage was reached after 130 hours. In both cases, no structural instability was observed. To improve the stability, the recombinant Bacillus subtilis (pHV1431) was immobilized in kappa-carrageenan gel beads. In comparison to free cell systems, a higher cell concentration was obtained. Moreover, the plasmid was maintained stable for longer periods; after 150 hours of culture 40% of cells in the reactor still carried the plasmid at both temperatures.

Water activity by 1H nuclear magnetic resonance spectroscopy: application to the study of water exchange in biphasic media.

The water activity (alpha w) of the liquid phase is investigated by means of 1H NMR for both monophasic and biphasic systems. The chemical shift or the area of the signal of the hydroxylic hydrogens is compared to calibration curves obtained from mixtures equilibrated at different water activities, thus allowing determination of the alpha w of the system. The chemical shift varies linearly as a function of the alpha w of the system. Through simple calculation the hydroxylic hydrogen concentration obtained from the area gives the water concentration in the medium; the sorption curve is thus obtained through NMR. The application of this method to a biphasic system composed of a liquid phase (an equimolar mixture of acid-alcohol) and a solid phase (a lipase), equilibrated separately at two different water activities, gives information on the water exchange between both phases during the lag phase of the esterification reaction. Light and slow water exchanges are observed from the solid phase at high alpha w toward the liquid phase at low alpha w. The lag phase of the esterification reaction is too short for the water activity equilibrium to be reached before the reaction starts.

Effects of salts on production and on O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain.

Acetylation determined by 1H-nuclear magnetic resonance spectroscopy and production of the glucuronan excreted by the Rhizobium meliloti M5N1CS strain during cultivation in RCS medium with and without added magnesium salts have been studied. These salts induce an increase in the degree of substitution and the molar ratio of 2,3-di-O-acetyl residues. A decrease in production is observed after 75 h of fermentation as the magnesium salt concentration increases. The presence of manganese and sodium salts in the culture induces inhibition of exopolysaccharide (EPS) production. However, the structure of the EPS is similar to that of the EPS produced by standard fermentation, without modification in the degree of substitution.

Cyclic (1-->2)-beta-D-glucans excreted by the glucuronan-producing strain Rhizobium meliloti M5N1CS (NCIMB 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1.

During fermentation, the mutant strain Rhizobium meliloti M5N1CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1-->4)-beta-D-glucuronan. In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1-->2)-beta-D-glucans with degrees of polymerization from 17 to 30. Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.

Physico-chemical properties of the encapsulation matrix and germination of carrot somatic embryos.

Carrot somatic embryos were encapsulated in alginate gel beads. To improve the quality of a "synthetic seed" coating, the rheology and dehydration properties of different matrices were tested. By increasing alginate and CaCl(2) concentrations, additional mineral elements were shown to increase resistance to rupture, and to depress the germination of somatic embryos. A polysaccharide addition was found to slow the alginate matrix dehydration; alginate-gellan gum and alginate-kaolin matrices could preserve the viability of somatic embryos at low relative humidities (30% to 35% germinations at 50% relative humidity) to a greater extent than other matrices.

Study of parameters affecting poly(3-hydroxybutyrate) quantification by gas chromatography.

Poly(3-hydroxybutyrate) (PHB) quantification has been developed mostly using acidic methanolysis followed by GC analysis of the 3-hydroxybutyrate methyl ester. However, under our experimental conditions, only 62% of the ester was detected by GC analysis. Following the study of the different steps involved in this method (i.e., hydrolysis, esterification, and recovery of the ester), the recovery was shown to be limiting. Addition of water to the organic phase, required for its purification before injection, led to the partition of the ester between the organic and the aqueous phase. The influence of the length of acidic methanolysis time on the amount of ester detected was also investigated. NMR analysis was used to show that secondary products were absent in both phases, regardless of heating time. Moreover, increasing acid concentration and the use of lyophilized cells were shown to lead to the decrease of the treatment time. Concerning internal standard choice, methyl benzoate was found to meet all the requirements to correct injection volume errors or to follow organic phase volume changes as a function of acid and water concentrations. The validity of the method was checked on Rhizobium meliloti M5N1 cells, which are shown to produce about 60% PHB (w/w) when cultivated with fructose as the carbon source.

Physicochemical properties of extracellular (1-->4)-beta-D-glucuronan produced by the Rhizobium meliloti M5N1CS strain during fermentation: evidence of degradation by an exoenzyme activated by Mg2+.

The mutant Rhizobium meliloti M5N1CS (NCIMB 40472) produced a partially acetylated (1-->4)-beta-D-glucuronan during fermentation. The polysaccharide (EPS) extracted from broth during fermentation by microfiltration and ultrafiltration (using a 100 kDa cut-off membrane) was characterized by size exclusion chromatography. The weight-average molecular weight (Mw) of the EPS decreased from 7.5 x 10(5) to 5 x 10(5) between 27 and 75 h of fermentation. When MgSO4.7H2O (0.8 gl-1 per day) was present in the medium during the same period, the Mw of the EPS decreased to 1.5 x 10(5). Since EPS degradation was detected after microfiltration of the fermentation medium supplemented with magnesium, the Mw decrease of the EPS extracted during fermentation may be explained by enzymic degradation of the polymer activated by Mg2+ ions.