Joseph Boudrant

The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus

Production of lactic acid from date juice by fermentation has been studied using Lactobacillus casei subsp. rhamnosus as the producer organism. The optimum substrate concentration, expressed in its glucose content, was 60 g l(-1). Various nitrogen sources were compared with yeast extract in terms of their efficiency for lactic acid production. None of these nitrogen sources gave lactic acid concentrations as high as that obtained with yeast extract. As yeast extract supplementation was not economically attractive, different proportions of (NH4)2SO4 and yeast extract were used. When the elemental nitrogen ratio of(NH4)2SO4 to yeast extract was 4:1, the substrate use and efficiency of lactic acid production were the same as in date juice supplemented with 20 g l(-1) yeast extract (0:5).

Microbial processes for ascorbic acid biosynthesis: a review

L-Ascorbic acid is an important product currently made using the Reichstein process, which is mainly chemical. Recently, bacteria have been identified that are able to transform in a very efficient way glucose to 2,5-keto-D-gluconic acid and this product to 2-keto-L-idonic acid, precursor of L-ascorbic acid. When the corresponding strains are used together, it is possible to get 2-keto-L-idonic acid directly from glucose. Moreover, new strains have been constructed by introducing a gene from a strain responsible for the second step into a strain responsible for the first step. By using one of the new strains, the transformation can be performed in a single step with only one strain. However, the classical process still remains the most competitive.

Characterization of a Highly Thermostable Alkaline Phosphatase from the Euryarchaeon Pyrococcus abyssi

ABSTRACT This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of theE. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far,P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition,P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

Use of waste date products in the fermentative formation of baker's yeast biomass by Saccharomyces cerevisiae

Abstract This work was an approach to waste date products valorization through biomass production with the yeast Saccharomyces cerevisiae. The carbon and nitrogen sources of a semi-synthetic fermentation medium were substituted by date-coat (fleshy part) sugar extract, date-seed hydrolysate, and ammonium nitrate. This modified medium was enriched with date-seed ash and date-seed lipid. Date-coat sugar extract as a carbon source was found to be satisfactory at a concentration of 25 g/l (expressed as its glucose concentration) and date-seed hydrolysate as a nitrogen source was equally suitable at 25 g/l. The addition to the medium of 1·0 g/l ammonium nitrate increased the efficiency of yeast biomass formation, as did phosphorus, which produced a maximum when the medium was supplemented with about 6·0 g/l KH 2 PO 4 . The presence of 1 g/l date-seed lipid in the medium also increased the efficiency of biomass formation. Finally, the addition of date-seed ash (0·6 g/l), as a mineral source, to the fermentation medium could substitute for MgSO 4 and CaCl 2 of the semi-synthetic medium.

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain of Escherichia coli.

SummaryThe influence of complex compounds on the growth of a recombinant strain ofEscherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.

Use of date products in production of the thermophilic dairy starter strain Streptococcus thermophilus

Date-coat sugar extract and date-seed hydrolysate were utilized as the main constituents of a medium for the production of a thermophilic dairy starter strain. Date-coat sugar extract was used as the carbon source, while date-seed hydrolysate was used as the nitrogen source. A suitable concentration of date-coat sugar was in the range of 50 mg sugar/ml. Addition of various amounts of date-seed hydrolysate as the sole nitrogen source in the medium showed that, in spite of a nitrogen insufficiency found in the hydrolysate, the production of the starter strain increased with date-seed hydrolysate (nitrogen) concentration, but the maximum production of biomass was less than that observed with other nitrogen sources. Therefore, various amounts of urea were added and a mixture of urea (6 mg/ml) and of date-seed hydrolysate (4.0 mg/ml) allowed an increase in the concentration of the biomass. The addition of date-seed ash as a mineral source, at a concentration of 1.0 mg/ml in the medium containing date-coat sugar extract, date-seed hydrolysate, and urea could substitute for MgSO4, and MnSO4 of the usual medium. This medium gave the maximum production of the thermophilic lactic acid bacteria (0.57 mg/ml) and lactic acid (2 mg/ml), very close to what was obtained with the Elliker broth medium.

Improved Performances and Control of Beer Fermentation Using Encapsulated α‐Acetolactate Decarboxylase and Modeling

The use of the enzyme α‐acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate‐limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated α‐acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on α‐acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated α‐acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free α‐acetolactate decarboxylase. The advantage of immobilized α‐acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.

Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture ofEscherichia coli in a non-selective medium

SummaryExperimental results were obtained withEscherichia coli C600 galK (GAPDH), a genetically engineered strain that synthetizes a large quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (80 % of the soluble proteins). Data concerning the stability of plasmid-containing cells and gene expression as a function of dilution rate have been obtained in continuous cultures. Contrary to other studies, our results show a clear indication that the rate of the recombinant activity was dependent on dilution rate. The results support the finding that the apparent stability of the plasmid decreases with dilution rate.

Flocculation dispersion in Kluyveromyces lactis

Yeast flocculation is a complex phenomenon. The present work concerns the effect of different factors on the flocculation dispersion of Kluyveromyces lactis. This study employs mother and hybrid strains, the latter being obtained by mating a flocculent and a non-flocculent mother strain. The optimum pH of flocculation was around 4·5. Flocculation dispersion depends upon the nature of carbohydrate present, galactose being the most effective. Protease action depends upon both strains and enzymes. Data analysis of the influence of temperature shows identical values for activation energies of the flocculation dispersion reactions. These results lead to a biochemical flocculation mechanism which involves the binding of a surface lectin to carbohydrate receptors.

Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E. coli gapA gene.

At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.