Yves Prigent

Growth and lactic acid production coupling for Lactobacillus helveticus cultivated on supplemented whey: influence of peptidic nitrogen deficiency

For Lactobacillus helveticus growing on supplemented whey permeate, some simple criteria have been proposed for assessing carbon and nitrogen source limitations: n• nirrespective of supplementation, carbon limitation coincided with the end of lactic acid production. n n• ncarbon limitation in absence of nitrogen deficiency led to an abrupt cessation of growth. n n• nnitrogen deficiency in the absence of carbon limitation resulted in an increase of the overall product on biomass yield YP/X during the second half of the cultivation. n n n n nAt pH near to the optimum (pH=6), even at the end of reaction, the final concentration of free lactic acid was below the inhibitory threshold (0.45–0.60 g l−1). In these conditions the shift from total to partial growth-associated production was tightly controlled by the concentration of available peptidic nitrogen.

Growth of Geotrichum candidum and Penicillium camembertii in liquid media in relation with the consumption of carbon and nitrogen sources and the release of ammonia and carbon dioxide

The growth of the filamentous fungi Geotrichum candidum and Penicillium camembertii on peptones as a sole carbon and nitrogen source, and in the presence of a second carbon source, lactic acid, have been compared. On both media, G. candidum exhibited similar kinetics until the end of growth, since it preferentially metabolized peptones as a carbon source, and lactic acid only for cellular maintenance during stationary phase. Growth of G. candidum was then nitrogen limited (peptones). On the contrary, fewer amino acids were convenient carbon sources for P. camembertii, resulting in a simultaneous consumption of peptones and lactic acid, and a cessation of growth due to the complete consumption of lactic acid. Morever, a lower amount of ammonia was produced since this metabolite resulted from the deamination of only carbon and nitrogen source amino acids. The production of ammonia induced an alkalinization of the broth (from 4.5 to 7.1). Morever, it was demonstrated that lactic acid consumption contributed also to media alkalinization (final pH 8.4). In absence of lactic acid, the medium contained a lower amount of available carbon, resulting in the absence of stationary state, deceleration growth phase was immediately followed by the death phase.

A novel concept of bioreactor: Specialized function two-stage continuous reactor, and its application to lactose conversion into lactic acid

Abstract From reactors performing a continuous production of an inhibitory metabolite like lactic acid, high product concentrations are obtained only at very low dilution rates. For such systems, volumetric productivity may be improved with the help of a novel two-stage reactor equipped with a separate feeding line in the second stage. By pumping a medium optimized for growth in the first stage and not controlling the pH of this reactor, an active seed culture (containing a small concentration of lactic acid) was prepared in continuous mode; this inoculum and a medium optimized for lactic acid production were fed in the second stage. Since 97% of the total amount of lactic acid was produced in the second stage, the system has been called ‘specialized function two-stage reactor’; the volumetric productivity obtained from this novel configuration was close to batch one in similar conditions.

Analysis of growth and production coupling for batch cultures of Lactobacillus helveticus with the help of an unstructured model

Abstract An unstructured model has been developed by assuming a sigmoidal variation with time of specific growth rate and introducing an additional term in Luedeking and Piret production kinetics. The model fitted growth and production kinetic data. The model was helpful for examining the influence of initial yeast extract concentration on growth and production coupling. In richly supplemented media, the growth associated production mechanism prevailed, while in the case of poor nitrogen supplementation, most lactic acid production was non-growth-associated. Both contributions might be calculated from model parameters; this was especially interesting when the forms of growth and production kinetics did not allow a clear conclusion. The additional term in the production rate expression was a convenient way to characterize ‘usable nitrogen’ concentration in the medium. From the model a clear criterion has been derived for optimizing the nitrogen supplementation from an economic point of view.

Carbon and nitrogen substrates consumption, ammonia release and proton transfer in relation with growth of Geotrichum candidum and Penicillium camemberti on a solid medium

It is difficult to determine fungal biomass growing on a solid medium directly. Therefore, indirect ways of measuring this growth have to be used instead. For this purpose, the kinetics of growth, of substrate consumption and of release of metabolites must be correlated. This has been carried out for Geotrichum candidum and Penicillium camemberti growing on a gelified lactic acid+peptone medium. Peptone was shown to be preferred by G. candidum as a carbon and energy source, in contrast to the behaviour observed for P. camemberti. At the end of growth, G. candidum released about three times more ammonia than P. camemberti. In both cases, the total number of protons consumed during growth was close to the sum of the number of moles of metabolized lactic acid and formed ammonia. During the main part of growth, constant biomass on substrate yields (peptone, lactic acid) was observed for both microorganisms. Therefore, in pure solid cultures, the biomass concentration of both species could be deduced either from DeltapH or from the reduction of substrates. From the above, an estimate of G. candidum biomass in mixed culture could be derived from the determination of ammonia release.

A New Model for the Reconstruction of Biomass History from Carbon Dioxide Emission during Batch Cultivation of Geotrichum candidum.

A non-structured model has been developed to describe the CO2 emission during growth of Geotrichum candidum on a lactate + peptone-based liquid medium. From the nitrogen and carbon mass balances, it was shown that about 50% of the total CO2 released was from the metabolism of the energy supply for biosynthesis, and the remaining from that for maintenance; thus, CO2 production was considered to be partially associated with growth. The model fitted the experimental data as long as a net growth was observed (0-50 h). The coefficients for growth- and non-growth-associated CO2 production were A = 0.646 (dimensionless) and B = 0.017 h(-1), respectively. From the coefficients of the model and the CO2 history data, the biomass kinetics has been reconstructed, and the calculated biomass concentrations agree fairly well with the experimental data. From this, measurement of the CO2 evolved may be used as an indirect and non-intrusive method of monitoring fungal growth during the first 50 h of cultivation.

Reconstruction of the biomass history from carbon and nitrogen substrate consumption, ammonia release and proton transfer during solid cultures of Geotrichum candidum and Penicillium camembertii

Abstract.Geotrichum candidum and Penicillium camembertii were cultivated on the surface of a gelified medium, simulating the composition of the aqueous phase of a Camembert cheese. The relation of their growth with substrate consumption (carbon or nitrogen), metabolite production (ammonia), or proton transfer (deduced from pH by means of the buffer capacity of the medium) was examined. The coefficients associated with cellular biosynthesis and resulting from cellular maintenance were determined. From these coefficients and the considered substrate utilization or metabolite production kinetics, the growth kinetics were reconstructed until the end of growth. The model allowed analysis of biosynthesis and cellular maintenance contributions to the considered kinetics. At the end of growth, almost all the peptone was used for G. candidum biosynthesis, while most of the lactic acid (62%) was used for cellular maintenance. P. camembertii metabolized fewer amino acids as carbon sources, resulting in use of peptone for maintenance (12%), and lactic acid (80%) for cell biosynthesis. For both microorganisms, ammonia production was growth-associated, since this production resulted from the deamination of carbon- and nitrogen-source amino acids, in close relation with peptone consumption.

Proton transfer in relation to growth of Geotrichum candidum and Penicillium camemberti in synthetic liquid media

Since continuous monitoring of mycelial growth in liquid media is still difficult, indirect ways of calculating on-line the total biomass concentration are of great interest. Growth of fungi like G. candidum and P. camemberti (widely used in cheese ripening) in liquid media may induce large pH changes. When nitrogen is supplied by glutamic acid (also able to supply carbon), an alkalinization of the medium is recorded, irrespective of the presence of an extra carbon source; when nitrogen is provided by lysine (unable to supply carbon) supplemented by glucose, an acidification is observed, Depending on the amino acid used, the pH variation is linearly correlated with growth (glutamic acid) or not (lysine). A simple model predicts that the concentration of proton transferred vary in proportion to that of the total biomass: the former variable may be easily derived from the pH history and the buffer capacity of the medium. For all combinations of nitrogen and carbon sources tested, the model has been experimentally validated for both microorganisms during the main growth period: following a preliminary calibration, fungal growth may be monitored on-line by pH measurements. 1999 Elsevier Science Inc. All rights reserved.

Substrate and metabolite diffusion within model medium for soft cheese in relation to growth of Penicillium camembertii

Penicillium camembertii was cultivated on a jellified peptone—lactate based medium to simulate the composition of Camembert cheese. Diffusional limitations due to substrate consumption were not involved in the linear growth recorded during culture, while nitrogen (peptone) limitation accounted for growth cessation. Examination of gradients confirmed that medium neutralization was the consequence of lactate consumption and ammonium production. The diffusion of the lactate assimilated from the core to the rind and that of the ammonium produced from the rind to the core was described by means of a diffusion/reaction model involving a partial linking of consumption or production to growth. The model matched experimental data throughout growth.

Identification and experimental validation of a criterion allowing prediction of cellular activity for preculture of lactic acid bacteria

Cellular activity of seed cultures of lactic acid bacteria is usually quantified in an indirect way by means of growth and production rates observed after transfer into a fresh culture medium. However, direct evaluation of the activity seems more suitable. Since inhibition of growth by undissociated lactic acid accumulated in the broth is now undoubtedly proven, the best performance can be expected from cells able to grow at a fast rate in the presence of high undissociated lactic acid concentrations. From this, the following criterion was proposed in order to evaluate cellular activity: the target function to be maximized is the product of the specific growth rate and the undissociated acid concentration. Such a criterion was used to optimize the medium composition, seed culture duration, and pH of precultures. In all cases, it was experimentally verified that the best volumetric productivity was obtained, after transfer to fresh medium, from cells corresponding to the highest value for the target function.