Carbon Dioxide Emission in Relation to the Growth of Geotrichum candidum in Solid Cultures

Geotrichum candidum plays an important role in the ripening of Camembert‐type cheeses. However, the direct measurement of the biomass concentration is rather tedious and, therefore, the development of alternative methods for monitoring the growth on solid media would be very useful. For this purpose, a non‐structured model was previously developed to describe the CO₂ emission during the growth of G. candidum in liquid cultures. The CO₂ production was assumed to be partially associated with growth: a part resulted from growth and the remaining from cellular maintenance. This model has also been validated in solid cultures on peptone‐lactate based medium. The coefficients for growth‐associated and non‐growth‐associated production were found to be 0.301 and 0.123 per day, respectively. Therefore, the CO₂ production may be a non‐destructive and useful tool to monitor fungal growth in solid cultures. In the case of mixed cultures of both fungi (Geotrichum candidum and Penicillium camembertii) involved in the ripening of Camembert cheeses, CO₂ emission can be related to the total viable biomass, while ammonia and volatile sulphur compounds can be linked to G. candidum biomass. Indeed, it was previously shown that Penicillium camembertii released only very low amounts of both compounds.     

Sporulation and the Macromolecular Composition of the Mycelium and Arthrospores of Geotrichum candidum

In submerged culture the mycelium of Geotrichum candidum breaks into fragments (arthrospores) which are either cylindrical or ellipsoidal in shape; the proportion of each spore type depends upon the glucose concentration in the medium. Above 0.2% glucose the ellipsoidal type prevails, whereas the cylindrical type is more abundant at lower glucose concentrations. The cylindrical spores and the mycelium have a very similar macromolecular composition, but the ellipsoidal spores have less RNA and protein and more carbohydrate than the mycelium.     

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.     

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.     

Changes of Exponential Growth Rates in Relation to Differentiation of Geotrichum candidum in Submerged Culture

Growth and differentiation of the imperfect fungus Geotrichum candidum were followed in submerged cultures containing a simple synthetic glucose salt medium. Uptake of glucose, ammonium and oxygen from the medium were measured during the entire growth perod. In 0.1% glucose the fungus grows with one exponential growth phase until all the glucose has been consumed. The arthrospores are formed in the stationary phase. In 0.5% glucose the growth curve has two exponential growth phases, one with a doubling time of 1.8 h and a second one with a doubling time of 4.9 h. The second exponential growth phase, which starts when less than 15% of the glucose and less than 30% of the ammonium have been consumed, is shown to be the sporulation phase. During this growth phase the oxygen saturation in the culture remained constant at about 50%.     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.

On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.     

Scale down and optimization of olive mill wastewaters decolorization by Geotrichum candidum

Microbiol flora acclimated in continuous pilot scale bubble column fed with OMW was analysed. The most efficient isolated fungus was identified to white-rot fungus Geotrichum candidum. Decolorization of OMW by Geotrichum candidum was investigated by using Hadamard's matrix for screening the important parameters and optimize them in order to control the biological decolorization. Agitation favours the conversion of COD removed into Geotrichum candidum biomass especially with high arthoconodia and few mycelium. Dilution of OMW and aeration enhanced the mycelium growth and rammification which that allowed polyphenols hydrolysis and then a decolorization. The initial pH of OMW is suitable for its decolorization by Geotrichum candidum growth. Ammonium sulfate concentrations tested with different OMW dilutions showed that the COD:N:S ratio of 100:5:2 is suitable for higher black colour removal. With optimized conditions Geotrichum candidum growth on OMW in laboratory scale bubble column, the OD removal reached 70% and all fractions of polyphenolic compounds of OMW were oxidized.     

Inhibition ofGeotrichum candidum by bicarbonate

Summary In a Sabouraud liquid medium system with NaHCO₃ or KHCO₃ at as low as 0.1% (w/v), the mycelial growth ofGeotrichum candidum was inhibited by greater than 90%. Bicarbonate may have a potential use as a microbial inhibitor in a variety of food systems.

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Inhibition de Geotrichum candidum par le bicarbonate

Résumé La croissance mycélienne deGeotrichum candidum a été inhibée à plus de 90% dans le milieu liquide de Sabouraud contenant le NaHCO₃ ou le KHCO₃ à des concentrations aussi faibles que 0.1%. Le bicarbonate peut ainsi avoir un usage potentiel comme inhibiteur microbien dans divers systèmes nutritifs.

     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Utility of ionic liquid for Geotrichum candidum-catalyzed synthesis of optically active alcohols

Ionic liquids have recognized as a solvent for Geotrichum candidum-catalyzed optical resolution and/or deracemization of racemic secondary alcohols, giving optically active alcohols. The immobilized Geotrichum candidum proceeded the enantioselective oxidation of alcohols, producing chiral alcohols in an ionic liquid. Further, deracemization of racemic alcohols was proceeded to give the corresponding chiral alcohols in high yield with excellent stereoselectivity by the Geotrichum candidum–NaBH₄ system in the mixture of MES buffer solution and ionic liquid.     

Toxic action of citric acid onGeotrichum candidum

Citric acid had a toxic effect on the mycelial growth ofGeotrichum candidum. At equal pH, citric acid was more toxic than HCl but its effect was minimized by adding CaCl₂. The results indicate that the toxic effect of citric acid is due to the undissociated component and its ability to sequester the essential cations present in the growth medium.

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Résumé L'acide citrique a un effet toxique sur la croissance mycélienne deGeotrichum candidum. A même pH, l'acide citrique est plus toxique que l'HCl mais son effet est minimisé par l'ajout de CaCl₂. Les résultats indiquent quel'effect de toxicité de l'acide citrique est dû à sa forme non dissociée et à sa capacité de séquestrer les cations essentiels presents dans le milieu de croissance.

     

Kinetics of growth and medium de-acidification for Geotrichum candidum and Penicillium camemberti cultivated on complex liquid media

Growth kinetics of Geotrichum candidum and Penicillium camemberti in submerged cultures under conditions of low aeration rate and uncontrolled pH were continuously recorded turbidimetrically. In these conditions the exponential growth phase was short, and ceased at a total biomass concentration of about 0.5 gl^–1 for G. candidum and 0.8 gl^–1 for P. candidum. The succeeding linear growth phase was also short, and its end corresponded to a biomass of 1.5 and 2.5 gl^–1 for G. candidum and P. camemberti respectively. A fair growth was recorded for P. camemberti on peptone-lactate medium, but G. candidum required addition of trace elements. For neither of these species growth was stimulated by growth factors of yeast extract. In peptone-lactate medium, the final pH did not depend on supplementation: 8–8.4 was recorded for G. candidum and 8.7–8.8 for P. camemberti.     

Rapid conversion of cyclohexenone,cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum candidum CCT 1205

?-Caprolactone (?-CL) was obtained with excellent conversion and short reaction times from the substrates cyclohexenone, cyclohexanone and cyclohexanol using whole cells of Brazilian Geotrichum candidum (CCT 1205). The reactions were monitored over time by gas chromatography, and the intermediates of the one-pot cascade biotransformation involving reductions of C=C and C=O bonds as well as the Baeyer–Villiger oxidation were identified and quantified. The Baeyer–Villiger monooxygenase (BVMO) enzyme was predominant, and all three substrates were completely converted into ?-CL. Furthermore, the whole cells of Geotrichum candidum were recycled and reutilized in the biotransformation of cyclohexanone, producing ?-CL at least six consecutive times without a significant loss of activity, reaction yields or product purity.     

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.     

Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

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.     

Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers

Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5′-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.     

Improvement of the kinetics of anaerobic digestion of molasses by the removal of phenolic compounds

Summary Removal of most of the phenolic compounds (88.5%) present in molasses by an aerobic pretreatment with Geotrichum candidum considerably increased the kinetics of anaerobic digestion of this wastewater. Inhibition phenomena were not observed in the range of COD studied (2.3–12.9 g/l). The yield coefficient of methane was 51 % higher than for untreated molasses.     

Use of cheese whey to enhance <Emphasis Type="Italic">Geotrichum candidum</Emphasis> biomass production in olive mill wastewater

Geotrichum candidum is a yeast-like filamentous fungus that has attracted industrial interest. The present work investigated G. candidum biomass production in agro-industrial wastewaters (olive mill wastewater (OMW) and cheese whey (CW)) as the only substrate. Different solid media (Sabouraud dextrose agar (SDA), CW, OMW, and OMW/CW mixtures in different proportions) were tested. OMW/CW mixtures proved to be suitable for optimal mycelia growth of G. candidum with a very high hyphae density. The highest fungal and expansion rate growth of 83 ± 1 mm and 12.4 day⁻¹, respectively, were obtained on a 20:80 mixture of OMW/CW, which was incubated for 7 days. This optimal mixture was used to study the biomass production and the OMW decolorization ability of G. candidum in the presence of CW in liquid medium. Liquid cultures were also conducted in OMW and CW separately. After 5 days of incubation, fungal biomass reached 9.26 g l⁻¹ in the OMW/CW mixture and only 2.83 g l⁻¹ in CW, while no biomass production was observed in OMW alone. OMW decolorization and dephenolization by G. candidum also improved in the presence of CW with a decolorization efficiency of 54.5% and a total phenolic reduction of 55.3%, compared with the control which yielded values of about 10% and 15%, respectively. These results suggested that OMW/CW—as the only substrate—could be used as a cost-effective medium to produce G. candidum biomass, without the need for water dilution or supplementation with other nutriments.     

Aerobic degradation of 2,4,6-trinitrotoluene by the yeast strain <Emphasis Type="Italic">Geotrichum candidum</Emphasis> AN-Z4

The yeast strain Geotrichum candidum AN-Z4 isolated from an anthropogenically polluted site was able to transform 2,4,6-trinitrotoluene (TNT) via the formation of unstable intermediate hydride Meisenheimer complexes with their subsequent destruction and accumulation of nitrite and nitrate ions as the end mineral forms of nitrogen. Aeration of the medium promoted more profound destruction of this xenobiotic by the strain G. candidum AN-Z4 than static conditions. The yeast strain was shown to produce citrate, succinate, and isocitrate, which sharply acidified the medium and influenced the TNT destruction. Two possible pathways of TNT biodegradation were confirmed experimentally: (1) via the destruction of the TNT-monohydride complex (3-H⁻-TNT) and (2) via the destruction of one protonated TNT-dihydride complex (3,5-2H⁻-TNT · H⁺). The strain G. candidum AN-Z4, due to its ability for TNT degradation, may be promising for bioremediation of TNT-contaminated soil and water.