Solid-state culture of Geotrichum candidum and Penicillium camembertii on a glutamate and lactate based medium

Geotrichum candidum and Penicillium camembertii have been cultivated at the surface of a glutamate, lactate-based medium. Glutamate has been chosen since it is a convenient carbon source for both fungi, in addition to a nitrogen source.The surface growth of both fungi induced the diffusion of substrates from the core to the rind. However, significant substrate concentrations (glutamate and lactate) always remained at the top of the gel, showing the absence of diffusional limitation of growth, in addition to the absence of substrate limitation. An absence of diffusional limitation, when the medium contained peptones instead of glutamate, was also indirectly deduced from the comparison of both media. Indeed, peptones are too complex for a possible identification of diffusional limitation from gradients analysis.An inhibitory effect of pH limited growth: at the end of the linear growth (oxygen limitation), inhibitory pH values were observed at the surface of the medium, even if it was not the case for the mean pH values. Since growth was limited by the alkaline pH at the surface of the gel, to account for this inhibition, an additional term has been introduced in the expression for the partial linking between consumption or production and growth. The diffusion coefficients for glutamate, lactate and ammonium have been also considered. Then, the concentration gradients for glutamate, lactate and ammonium have been calculated (second Fick law) and found to match with the experimental gradients.     

Diauxic growth of Penicillium camembertii on glucose and arginine

The growth of Penicillium camembertii during batch culture in a synthetic medium containing glucose and arginine was examined. The diauxic growth observed can be well characterized. Indeed, in a first phase, glucose and arginine were, respectively, assimilated as carbon and nitrogen sources, with an acidification of the medium (until 3.5), since arginine was taken up in exchange for protons. During this phase of growth, arginine, in addition to glucose, was also assimilated as an energy source, resulting in the release of the arginine carbon content as CO₂. Then, in a second phase, characterized by reduced growth rates after glucose depletion, arginine was assimilated as a carbon and nitrogen source, as well as an energy source, resulting in ammonium release which raised the pH (final pH 6.3), despite the amino acid/H⁺ exchange, since amino acids contain excess nitrogen in relation to their carbon content for fungi.     

Organic or mineral nitrogen source during Penicillium camembertii growth on a glucose limited medium

Penicillium camembertii was cultivated on a carbon-limited medium (glucose). Two nitrogen sources were compared, a mineral, ammonium, and an organic nitrogen source, lysine. Among the amino acids convenient nitrogen sources for P. camembertii, lysine was chosen since it cannot be assimilated as a carbon source for cell biosynthesis. During culture on glucose and ammonium, a decline phase immediately followed growth after glucose depletion, since no energy source remained in the medium. On the contrary, on glucose and lysine, a stationary state was recorded after glucose depletion, since lysine was used as the energy supply for cell maintenance, leading to the release of the corresponding carbon as CO₂, while nitrogen from lysine was released as ammonium.     

Assimilation of peptides and amino acids and dissimilation of lactate during submerged pure cultures of Penicillium camembertii and Geotrichum candidum

The behavior of Penicillium camembertii and Geotrichum candidum growing in submerged pure cultures on simple (glutamate) or complex (peptones) substrates as nitrogen and carbon sources and an lactate as a second carbon source was examined. Similar to the behavior previously recorded on a simple substrate (glutamate), a clear differentiation between the carbon source and the energy source was also shown on peptones and lactate during P. camembertii growth, since throughout growth, lactate was only dissimilated, viz., used for energy supply by oxidation into CO2, whereas peptides and amino acids from peptones were used for carbon (and nitrogen) assimilation. Because of its deaminating activity, G. candidum preferred peptides and amino acids to lactate as energy sources, in addition to being assimilated as carbon and nitrogen sources. From this, on peptones and lactate, G. candidum grew faster than P. camembertii (0.19 and 0.08 g/l/h, respectively) by assimilating the most readily utilizable peptides and amino acids; however, owing to its lower proteolytic activity, the maximum biomass was lower than that of P. camembertii (3.7 and 5.5 g/l, respectively), for which continuous proteolysis and assimilation of peptides were shown.     

Two Penicillium camembertii mutants affected in the production of cyclopiazonic acid

Penicillium camembertii was mutated and screened for cyclopiazonic acid-negative mutants. With a simple and rapid mini-extraction method for detection of cyclopiazonic acid production, we were able to isolate two strains which were affected in the production of this metabolite. One strain had completely lost the ability to synthesize detectable amounts of this secondary metabolite, whereas the other mutant produced 50 to 100 times less cyclopiazonic acid than the wild type. Also, the former strain had a changed morphology compared with the wild type. This morphological alteration appears to be coupled to the inability to produce cyclopiazonic acid because morphological revertants were able to synthesize cyclopiazonic acid to a level comparable to the wild type. The second mutant accumulated a new metabolite which was detectable by two-dimensional thin-layer chromatography. This new metabolite, however, appears not to be a direct precursor of cyclopiazonic acid.     

Two Penicillium camembertii mutants affected in the production of cyclopiazonic acid.

Penicillium camembertii was mutated and screened for cyclopiazonic acid-negative mutants. With a simple and rapid mini-extraction method for detection of cyclopiazonic acid production, we were able to isolate two strains which were affected in the production of this metabolite. One strain had completely lost the ability to synthesize detectable amounts of this secondary metabolite, whereas the other mutant produced 50 to 100 times less cyclopiazonic acid than the wild type. Also, the former strain had a changed morphology compared with the wild type. This morphological alteration appears to be coupled to the inability to produce cyclopiazonic acid because morphological revertants were able to synthesize cyclopiazonic acid to a level comparable to the wild type. The second mutant accumulated a new metabolite which was detectable by two-dimensional thin-layer chromatography. This new metabolite, however, appears not to be a direct precursor of cyclopiazonic acid.     

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.     

Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii.

A new lipase from Penicillium camembertii U-150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin-A-Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalin-A-Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.     

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.     

Purification and characterization of mono-and diacylglycerol lipase isolated from Penicillium camembertii U-150

Summary A novel enzyme hydrolysing mono- and diacylglycerol was found in the culture filtrate of an isolated fungus, Penicillium camembertii. The enzyme was separated into two forms (A- and B-enzyme) with almost the same molecular weight (37,000–39,000), amino acid composition and identical N-terminal amino acid sequence. B-Enzyme, a major component, was purified approximately 210-fold with an activity yield of 2.6%. The B-enzyme was specific to mono- and diacylglycerols and hydrolysed long-chain monoacylglycerols most efficiently. Triacylglycerols were completely inert as substrates for the enzyme. The B-enzyme preferred to attack -position to -position of monoacylglycerol, but showed no stereospecificity on mono- and diacylglycerol. Both Fe³⁺ and Hg²⁺ inhibited B-enzyme activity significantly.Offprint requests to: S. Yamaguchi     

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.     

Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3

The culture medium including nitrogen source, carbon source and metal ions, for lipase from Penicillium camembertii Thom PG-3 was optimized and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28 °C for 96 h. The lipase activity produced was enhanced 3.9-fold and reached 500 U/ml. The lipase was purified 19.8-fold by pH precipitation, ethanol precipitation and ammonium sulphate precipitation as well as DEAE-cellulose chromatography. The purified lipase showed one polypeptide band in SDS-polyacrylamide gel electrophoreses (SDS-PAGE) with molecular weight 28.18 kDa. The optimal pH and temperature for activity of lipase were 6.4 and 48 °C, respectively, which are higher than those lipases from other penicillium sources. The P. camembertii Thom lipase is 1,3-positional specificity for hydrolysis of triglyceride and hydrolyses plant oil preferentially to animal oil. The lipase can be used in short chain ester synthesis with an esterification degree of 95%.     

Enzymatic synthesis of monoglycerides by esterification reaction using Penicillium camembertii lipase immobilized on epoxy SiO2-PVA composite

Glycerol-fatty acid esterification has been conducted with lipase from Penicillium camembertii lipase immobilized on epoxy SiO₂-PVA in solvent-free media, with the major product being 1-monoglyceride, a useful food emulsifier. For a given set of initial conditions, the influence of reaction was measured in terms of product formation and selectivity using different fatty acids as acyl donors. Results were found to be relatively dependent of the chain length of the fatty acids, showing high specificity for both myristic and palmytic acids attaining final mixture that fulfills the requirements established by the World Health Organization to be used as food emulsifiers.     

Modelling the inhibitory effect of copper sulfate on the growth of Penicillium expansum and Botrytis cinerea

Aims: This study aimed to investigate the effect of copper sulfate (from 0 to 8 mmol kg^?1) on radial growth rate and lag time of two moulds responsible for vine grapes spoilage: Penicillium expansum strain 25·03 and Botrytis cinerea, strains BC1 and BC2. Methods and results: A new model was developed to describe tailing and shoulders in the inhibition curves. Because of tailing, the minimum inhibitory concentration (MIC), was not defined as the concentration at which no growth was observed, but as the concentration at which the lag time was infinite. The concentrations at which μ = μ_opt/2, (Cu₅₀), were in the range of 2·2–2·6 mmol kg^?1. Radial growth rate of P. expansum and the reciprocal of the lag time were linearly correlated (r = 0·84). In contrast, in the range 0–4 mmol kg^?1, an inhibition of growth of B. cinerea was observed whereas germination remained unaffected (i.e. the lag time was constant). In the range 4–8 mmol kg^?1, the radial growth rate of B. cinerea was almost constant (c. 1 mm day^?1), but germination was inhibited (i.e. the lag time was increased). Conclusions: The MIC values were 4·7 mmol kg^?1 for P. expansum, 8·2 and 7·3 mmol kg^?1 for B. cinerea strain BC1 and BC2, respectively, demonstrating that some isolates of these moulds are resistant to copper. Significance and Impact of the Study: Copper concentrations at 4 mmol kg^?1 would be sufficient to control the development of these isolates, but the toxicity of copper should be extended to other isolates and evaluated in vineyards.     

Intra- and extracellular concentrations of glutamate, lactate and acetate during growth of Corynebacterium glutamicum on different media

Corynebacterium glutamicum ATCC 17965 was cultivated in a 4-L batch aerated fermentor with glucose, fructose and mixtures of these two sugars in various proportions as carbon sources and with different concentrations of minerals and vitamins. A multilayer centrifugation technique was devised to obtain cell extracts in order to assess intracellular production of glutamate and partitioning between intracellular and extracellular spaces for lactate and acetate, the main by-products produced during the growth phase. Glutamate production increased with the proportion of glucose in the carbon source. The average value for the intracellular concentration of glutamate obtained with basic glucose medium was increased three-fold when initial concentrations of vitamins and minerals were increased four-fold. In this case, overall production of glutamate (16.3 mM) reached the highest value obtained. Production of acetate was weak on all media types (< 1.6 mm). it was the same for lactate synthesis in media where glucose remained the major carbon source (< 2.3 mm). production of lactate was significantly higher on media where fructose was the main carbon source (> 10 mM to 60 mM). The increase in lactate production and the decrease in glutamate production were correlated to a modification of carbon flux distribution between the metabolic pathways as the fructose proportion was increased. An increase in the concentration of minerals favoured production of glutamate during growth. This was correlated with an increase in the NADPH,H⁺ production rate. Received 16 January 1996/ Accepted in revised form 14 January 1997