Competition during submerged mixed culture of Geotrichum candidum and Penicillium camembertii on glucose and threonine

Geotrichum candidum and Penicillium camembertii were cultivated in pure and mixed cultures on glucose and threonine. In pure cultures, G. candidum used glucose as a carbon and an energy source and threonine only as a nitrogen source, even after glucose exhaustion. Contrarily, when growing in isolation, P. camembertii used simultaneously threonine and glucose as carbon sources. A diauxic growth was recorded during the mixed culture of both species, which competed for glucose, the sole carbon source available for G. candidum growth, leading to higher glucose consumption rates than those recorded during pure cultures, while after glucose exhaustion, low growth was recorded in a second step, showing a 'competition' for threonine, the sole remaining carbon and nitrogen sources, confirmed by the increase of 1.0+/-0.1 log of the G. candidum Colony Forming Units. 'Competition' between G. candidum and P. camembertii for the limiting substrate was found to have a positive effect on growth, since it did not lead to the annihilation of one species, as usually observed, but in their coexistence, leading to a rather similar final number of the CFUs for the two populations. 'Competition' resulted in the absence of assimilation of the second available carbon substrate (lactate) as previously observed, or its use only as a nitrogen source, as was the case for threonine in this work.     

The effect of lactate addition on the growth of Penicillium camembertii on glutamate

The effect of an additional carbon source, lactate, on Penicillium camembertii growth on glutamate as both carbon and nitrogen sources was examined. Glutamate (and lactate) was present in excess in both media. Throughout the whole culture, similar growth time-courses were recorded on both media, indicating the absence of a lactate effect on growth. During the first part of growth, corresponding to an increasing amount of viable biomass, the rate of glutamate consumption remained high, as well as the related ammonium production, indicating its use as a carbon source in addition to being nitrogen source. The low growth rates recorded during the last part of growth resulted in low glutamate consumption, while lactate consumption continued mainly by a maintenance mechanism for the energy supply. A clear differentiation appeared therefore between the carbon source and the energy source: glutamate was mainly used as C source (and N source) for biosynthesis, while lactate was mainly assimilated for energy supply. Carbon and nitrogen yield examinations confirmed this result. Indeed, the C/N ratio found for P. camembertii cellular material (8.14) was about twice that of glutamate (4.29). From this, about half of the available nitrogen was used for biomass formation during growth on glutamate-lactate based medium, as experimentally confirmed (constant yield nitrogen from biomass on nitrogen from glutamate was found (0.49), while the excess nitrogen was released as ammonium). The constant and close to unit (0.99) yield carbon from CO2 on carbon from lactate, also recorded during growth on glutamate-lactate based medium, confirmed that lactate was mainly used as an energy source.     

Carbon and nitrogen yields during batch cultures of Geotrichum candidum and Penicillium camembertii

Batch cultures of Geotrichum candidum and Penicillium camembertii were carried out on peptones as carbon and nitrogen source and in the presence of lactate as a second carbon source. Unless growth ceased, carbon and nitrogen yields remained constants, except yields involving lactate consumption by G. candidum, since this fungus preferentially metabolized peptones as a carbon source. For both fungi, nearly 40% of the available carbon was metabolized for cellular biosynthesis and the remainder (about 60%) as carbon dioxide, for the energy supply of both biosynthesis and viable cell maintenance. Moreover, in relation to their carbon content, amino acids contain excess nitrogen, which was released as ammonium. From all these, the yields of ammonium nitrogen on cellular nitrogen were in all cases higher than 1, and were especially high when the medium contained only peptones as a carbon source, 4.4 and 5.7 for G. candidum and P. camembertii respectively. Indeed, in this case, the excess nitrogen was especially pronounced.     

Reconstruction of the biomass history from carbon and nitrogen substrate consumption,ammonia release and proton transfer during solid cultures of <Emphasis Type="Italic">Geotrichum candidum</Emphasis> and <Emphasis Type="Italic">Penicillium camembertii</Emphasis>

Geotrichum candidumand 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.     

Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.     

Commensalism during submerged mixed culture of Geotrichum candidum and Penicillium camembertii on glutamate and lactate

Geotrichum candidum and Penicillium camembertii were cultivated in pure and mixed cultures on glutamate- and lactate-based medium. In pure culture, P. camembertii assimilated simultaneously glutamate, as a nitrogen and carbon source for biosynthesis, and lactate as an energy source. On the contrary, G. candidum grew on glutamate alone. The mixed culture led to higher growth rates and then higher rates of substrate consumption and metabolite production than each pure culture; however, the behaviour recorded was similar to that observed during G. candidum pure culture, in particular the absence of lactate assimilation during growth, illustrating a commensalism between both species. The presence of G. candidum induced a form of “competition” and then a better assimilation by P. camembertii of the sole nitrogen source, glutamate, which was therefore used as an energy source in addition to be a carbon (and nitrogen) source. Lactate was only used for energy supply during stationary state, as also recorded during G. candidum pure culture.     

Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures

Saccharomyces uvarum, Geotrichum candidum, Endomycopsis burtonii and Hansemula canadensis have been isolated from deteriorated silages and/or spoiled lactic acid fermented foods. All yeasts could grow in relatively high (100 mmol/l) concentrations of acetate or lactate in Wickerham's assimilation broth but were inhibited by increasing concentrations of these acids. Propionate was much more inhibitory than acetate or lactate. Growth rate, but not final cell yield, was adversely affected by increasing concentrations of these acids. The three acids used in combination were synergistically inhibitory to H. canadensis and G. candidum. Saccharomyces uvarum was inhibited by synergistic mixtures of acetate/propionate. Endomycopsis burtonii and H. canadensis were inhibited by synergistic mixtures of acetate/lactate, acetate/propionate and lactate/propionate. Lactate and propionate had a synergistic inhibitory effect on G. candidum. The mechanism of inhibition of growth of S. uvarum by acetate and propionate was further investigated. Acetate and propionate each caused a reduction in cellular efficiency (Y, μg cells/μmole glucose consumed). Mixtures caused a drastic reduction in growth rate and Y. These results suggest that the cells may expend energy in reducing intracellular concentrations of acetate while propionate probably acts in a different manner.     

Modeling growth,substrate consumption and product formation of <Emphasis Type="Italic">Penicillium nalgiovense</Emphasis> grown on meat simulation medium in submerged batch culture

Penicillium nalgiovense is the most widely used starter mold for cured and fermented meat products. The development of a biomass film on the surface of these products prevents a large degree undesirable growth of various fungal contaminants and contributes to the ripening process with production of metabolites. This work presents an attempt to model the growth of P. nalgiovense and to relate it to substrate consumption and product release. Because of the extremely complex nature of the meat product fermentation, submerged culture was employed in a bioreactor system that enabled on-line monitoring, using a meat simulation medium, which contained peptones and lactate as carbon, nitrogen and energy sources. The unstructured model presented is based on a partial association of substrate assimilation and product formation with growth. Experimentally derived values for peptones and lactate were compared with model-derived values and their proportions corresponding to growth associated parts, used for biosynthesis, and non-growth associated parts, used for maintenance. The model was applied for the products ammonia, carbon dioxide and protons. Both peptones and lactate were used mainly for biosynthesis (85 and 80% of the total amounts provided, respectively). Assimilation of lactate and ammonia formation from amino acid metabolism resulted in a proton exchange, which was mainly growth associated. The contribution of the growth associated mechanism to the total proton exchange was estimated to be 75% while the contribution of the non-growth associated mechanism increased during the growth phase and reached a maximum of 25%. For carbon dioxide production, the contribution of a maintenance mechanism was evident at 40 h, while production was growth-associated and remained such even at the end of fermentation at 168 h when growth rate was very low. The partially growth associated model showed good agreement with the experimental data and allows accurate determination of the proportions of substrates or products related to biosynthesis and cell maintenance.     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of lignin peroxidase in the decolourization of black olive mill wastewaters by Geotrichum candidum

AIM: Decolourization of black olive mill wastewaters (OMW) by depolymerization of phenolic compounds by Geotrichum candidum. METHODS AND RESULTS: Our results show that G. candidum is able to grow on black OMW supplemented with carbon source and nitrogen. The Geotrichum growth decreased the pH and induced a 49% of colour removal when the black OMW was supplemented with glycerol and diammonium tartrate (20 mm ammonium). An improvement of 10% of colour removal was observed when the culture was supplemented with veratryl alcohol. The decolourization was inhibited with glutamate as nitrogen source. CONCLUSION: Our results suggest the potential use of G. candidum in black OMW decolourization and support the concept that lignin peroxidase (LiP) of G. candidum is involved in the depolymerization of phenolic compounds. Significance and Impact of the Study: This is the first report of LiP production by G. candidum on OMW.     

Diffusion of lactate and ammonium in relation to growth of Geotrichum candidum at the surface of solid media

Geotrichum candidum was cultivated at the surface of solid model media containing peptone to simulate the composition of Camembert cheese. The surface growth of G. candidum induced the diffusion of substrates from the core to the rind and the diffusion of produced metabolites from the rind to the core. In the range of pH measured during G. candidum growth, constant diffusion coefficients were found for lactate and ammonium, 0.4 and 0.8 cm(2) day(-1), respectively, determined in sterile culture medium. Growth kinetics are described using the Verlhust model and both lactate consumption and ammonium production are considered as partially linked to growth. The experimental diffusion gradients of lactate and ammonium recorded during G. candidum growth have been fitted. The diffusion/reaction model was found to match with experimental data until the end of growth, except with regard to ammonium concentration gradients in the presence of lactate in the medium. Indeed, G. candidum preferentially assimilated peptone over lactate as a carbon source, resulting in an almost cessation of ammonium release before the end of growth. On peptone, it was found that the proton transfer did not account for the ammonium concentration gradients. Indeed, amino acids, being positively charged, are involved in the proton transfer at the beginning of growth. This effect can be neglected in the presence of lactate within the medium, and the sum of both lactate consumption and ammonium release gradients corresponded well to the proton transfer gradients, confirming that both components are responsible for the pH increase observed during the ripening of soft Camembert cheese.     

Amino acid degradation by the mesophilic sulfate-reducing bacterium Desulfobacterium vacuolatum

Desulfobacterium vacuolatum strain IbRM was able to grow using casamino acids as a source of carbon, energy and nitrogen. Growth was accompanied by utilization of several amino acids and sulfide production. Proline and glutamate were used preferentially and to the greatest extent. Glycine, serine and alanine were used more slowly and only after proline and glutamate were used. Isoleucine, valine, leucine and aspartate decrease was slowest and occurred in a linear fashion throughout the growth phase. Amino acids used from casamino acids, excluding aspartate, were also used as single carbon, energy and nitrogen sources. As a single amino acid, aspartate could only be used as a nitrogen source. Aspartate was not used as an electron acceptor. No growth occurred on any amino acid in the absence of sulfate. As single substrates, isoleucine, proline and glutamate were oxidized without formation of acetate and with molar yields of 13.1, 9.4 and 7.7 g mol^–1, respectively. Received: 24 June 1997 / Accepted: 10 September 1997     

Nitrogen utilization in bacterial isolates from the equine cecum.

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Nitrogen utilization in bacterial isolates from the equine cecum

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.     

Nitrogen metabolism in the phototrophic bacteria Rhodocyclus purpureus and Rhodospirillum tenue.

Studies of the nitrogen nutrition and pathways of ammonia assimilation in Rhodocyclus purpureus and Rhodospirillum tenue have shown that these two seemingly related bacteria differ considerably in aspects of their nitrogen metabolism. When grown photoheterotrophically with malate as carbon source, R. purpureus utilized only NH4+ or glutamine as sole nitrogen sources and was unable to fix N2. By contrast, R. tenue was found to utilize a variety of amino acids as nitrogen sources and was a good N2 fixer. No nitrogenase activity was detected in cells of R. purpureus grown on limiting ammonia, whereas cells of R. tenue grown under identical conditions reduced acetylene to ethylene at high rates. Regardless of the nitrogen source supporting growth, extracts of cells of R. purpureus contained high levels of glutamate dehydrogenase, whereas R. tenue contained only trace levels of this enzyme. Alanine dehydrogenase activity was absent from both species. We conclude that R. purpureus is incapable of fixing molecular nitrogen and employs the glutamate dehydrogenase pathway as the primary means of assimilating NH4+ under all growth conditions. R. tenue, on the other hand, employs the glutamine synthetase/glutamate synthase pathway for the incorporation of NH4+ supplied exogenously or as the product of N2 fixation.