Lactose continuous fermentation with cells recycled by ultrafiltration and lactate separation by electrodialysis: model identification

Summary An off-line parameter estimation method has been developed to predict the dynamic behaviour of a continuous lactose fermentation system. The model used is an unstructured model taking into account cell growth, substrate consumption, and metabolite production (lactic acid). This method, based on the Hooke-Jeeves non-linear-programming technique, results in a good estimation of the biological parameters of the model, and so gives a better understanding of the different phenomena involved in lactose fermentation.Nomenclature Cp, Cs, Cz, Dp, Ds, Dz coefficients in system (A) - Fe bioreactor influent flow rate (1/h) - I current in the ED unit (A) - J lactate flux in the ED unit (g/h) - Kd mortality constant (h^-1) - Kp product inhibition constant (g/l) - Ks strbstrate saturation constant (g/l) - P ₀ product concentration in the bioreactor (g/l) - P ₁ product concentration in the D tank (g/l) - P _0r estimation of P ₀ (g/l) - Q ₀ retentate flow rate (UF influent) (1/h) - Q ₁ permeate flow rate (1/h) - Q ₂₂ cell bleed flow rate (1/h) - Q ₃ recycling flow rate in the ED (influent) (1/h) - Se substrate concentration in the influent (g/l) - S ₀ supstrate concentration in the bioreactor (g/l) - S ₁ substrate concentration in tank D (g/l) - S _0r estimation of S ₀ (g/l) - t time (h) - V ₀ fermentation broth volume (1) - V ₁ tank D volume (1) - X ₀ biomass concentration in the bioreactor (g/l) - Y _P/S (=1/Y _S/P) lactic acid yield coefficient (g lactic acid/g lactose consumed) - Y _X/S (=1/Y _S/X) cell yield coefficient (g cells produced/g lactose consumed) - Y _X/Z (=1/Y _Z/X) second cell yield coefficient (g cells produced/g nitrogen consumed) - Y _x, Y _m input mathematical parameters of the linear system (M ₂) - Ze nitrogen concentration in the influent (g/l) - Z ₀ nitrogen concentration in the bioreactor (g/l) - Z ₁ nitrogen concentration in tank D (g/l) - Z _0r estimation of Z ₀ (g/l) - , constants of the Luedeking and Piret's model - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1)     

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.     

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens

A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase. Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and can be carried out on whole broth.     

Interactions between pyruvate and lactate metabolism in Propionibacterium freudenreichii subsp. shermanii: in vivo (13)C nuclear magnetic resonance studies

In vivo (13)C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichii subsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 micromol. min(-1). g(-1) (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 micromol. min(-1). g(-1) when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO(2), succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.     

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS

Background

FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp.

Results

Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L^-1 of GDH activity (57 mg L^-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source.

Conclusions

The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.     

Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

Concentrated starter productions in a two stage cell-recycle bioreactor plant

Summary Continuous production of concentratedLactobacillus acidophilus andBifidobacterium bifidum cells is described in two successive cell-recycle bioreactors. This process allows the study of the relationships between two microorganisms without mixed cultures.