Process Biochemistry 41 (2006) 1318–1324 www.elsevier.com/locate/procbio Impact of solid medium composition on the conidiation in Penicillium camemberti Isabelle Krasniewski a,*, Pascal Molimard b, Gilles Feron a, Catherine Vergoignan a, Alain Durand a, Jean-François Cavin a, Pascale Cotton c a INRA UMR 1082 de Microbiologie, INRA/ENSBANA/UB, 17 rue Sully BP86510, 21065 Dijon cedex, France b DEGUSSA Ferments d’Aromatisation, 16 rue de la Gare, 77260 La Ferté Sous Jouarre, France c Université Claude Bernard, Lyon I, Unité de Microbiologie et Génétique UMR 5122, Biologie Cellulaire Fongique, Bat Lwoff, 10 rue Dubois, 69622 Villeurbanne cedex, France Received 8 July 2005; received in revised form 7 December 2005; accepted 10 January 2006 Abstract Conidiation of an industrial strain of Penicillium (P.) camemberti, a ripening fungus, was examined on solid media. In order to evaluate the influence of nutritional factors on conidiation, we developed an inoculation and transfer procedure that allowed to obtain an homogenous mycelial biomass. Absence of conidiation was observed when ammonium sulphate and sodium nitrate were used as nitrogen sources. In contrast, conidiation increased significantly in the presence of ammonium phosphate or potassium nitrate with 4.8  106 and 12  106 spores ml 1, respectively. By using those optimal conditions, the influence of nutrient starvation or calcium supply on conidiation was studied. Under the conditions tested, nitrogen starvation and calcium supply were better inducers than carbon starvation. A high carbon-to-nitrogen (C/N) ratio provided the highest level of with 4.2  107 spores ml 1 and a sporulation index of 8.2 after 16 days of cultivation. # 2006 Elsevier Ltd. All rights reserved. Keywords: Penicillium camemberti; Conidiation; Solid media; Calcium; Nitrate; C/N ratio 1. Introduction Asexual sporulation is a widespread reproductive mode for filamentous fungi. It consists of a massive production of spores, called conidia in the case of Ascomycetes. Under favourable environmental conditions, each conidium is able to produce a young mycelium. This property contributed to the use of Penicillium camemberti conidia as cheese starter culture. Directly introduced in milk or seeding by surface pulverization, this white mold is used to ripen and flavor a variety of French soft cheeses. Suspensions of P. camemberti conidia that are used in the dairy industry are either marketed as concentrated, stabilized suspensions or as lyophilized preparations. Traditionally, P. camemberti conidia are produced by surface cultivation on agar media in Roux-flasks and harvested after 3 weeks [1]. However, * Corresponding author. Tel.: +33 3 80 69 36 60; fax: +33 3 80 69 32 29. E-mail address: krasni@free.fr (I. Krasniewski). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.01.005 this mode of production appears outdated, and consequently must be improved. Surface cultures present some drawbacks for the study of the conidiation. The medium is usually centrally inoculated, and the fungus grows as a circular heterogeneous colony, composed of zones differing in morphology and metabolic activities. Conidiation occurs mainly in the aged zone located at the centre of the culture. Besides growth of the vegetative mycelium, conidiophore development and conidiation often take place simultaneously in surface cultures adding difficulties to the distinction of changes associated with conidiation from those due to degeneration and aging of the vegetative hyphae [2]. To avoid these problems, different techniques have been developed to induce microcycle conidiation: temperature-shift experiments for P. digitatum [3] and P. cyclopium [4], and glutamate as sole nitrogen source for P. urticae [5]. A synchronised sporulation was developed in P. digitatum by using a vitamin mixture lacking p-amino-benzoic acid [6]. However, all these techniques have been developed in submerged cultures. Besides, conidiation of Penicillium species has been of interest for long [7–9], mainly in submerged culture that allow 1319 I. Krasniewski et al. / Process Biochemistry 41 (2006) 1318–1324 a better homogeneity of the biomass and an easier upscaling and automation [10]. Such studies have shown that conidiation can be principally improved by: (i) calcium addition [8], (ii) type of nitrogen source [11], (iii) glucose starvation [2], (iv) levels of intermediates and derivatives of the Krebs cycle [12]. On the other hand, studies on the qualities of spores have been shown that aerial and submerged spores of P. oxalicum differ in hydrophobicity, viability and efficacy as biocontrol agent [13]. Moreover, differences in hydrophobicity have been shown to affect the viability of Trichoderma harzianum. Aerially produced spores were highly hydrophobic and showed longer viability after storage than submerged spores [14]. Furthermore, most filamentous fungi remain entirely vegetative in submerged culture, which is consistent with the findings that differentiated structures are characteristic of aerial mycelium [11]. The aim of the present work was to screen and optimize composition of the growth media that would allow a better conidiation of P. camemberti on solid media. 2. Materials and methods 2.1. Microorganism P. camemberti strain D1 was provided by Degussa Ferments d’Aromatisation, France. Stock culture of P. camemberti was stored on potato dextrose agar (PDA) slants at 20 8C. 2.2. Growth medium compositions The reference medium, based on Bockelmann’s medium [10], was separated into two parts, a glucose solution and a nitrogen solution, for its preparation in order to avoid Maillard reactions. It was prepared as follows: a glucose solution containing 10 g of D-glucose and 15 g of Agar–Agar were dissolved in 500 ml distilled water. A nitrogen solution containing 3.29 g (NH4)2HPO4, 4.4 g Na3-citrate, 20 ml trace elements solution and distilled water up to 500 ml were mixed. The pH of the two solutions was adjusted to 5.6 with 20% H2SO4 before autoclaving (121 8C, 30 min). After cooling, 500 ml of glucose solution were mixed to 500 ml of nitrogen solution and plated on 100 mm diameter Petri dishes, on the basis of 25 ml per dish. The trace element stock solution contained (per liter): 500 mg CuSO4
5H2O, 340 mg MnSO4
1H2O, 200 mg ZnSO4
7H2O, 15 g MgSO4
7H2O, 13 g KH2PO4, 5 g KCl and 5 g FeSO4
7H2O. The growth medium compositions with other nitrogen sources are shown in Table 1. All experiments were carried out in triplicate. 2.3. Inoculation procedure A Roux-flask, containing 150 ml of PDA medium, was inoculated with 2  105 spores ml 1, from a stored slant. After 10 days of cultivation at 23 8C, spores were harvested aseptically with 50 ml of 0.9% NaCl. The spore suspension was washed twice with 0.9% NaCl, centrifuged at 8000  g for 15 min and the pellet was suspended in 40 ml of distilled water. This suspension was diluted to obtain 3–4  107 spores ml 1. Sterile pre-weighed glass microfibre filters GF/C (Whatman), having a diameter of 90 mm, were soaked successively for 15 s in the spore suspension. The volume of inoculum, deposited on each filters, was 2.09  0.09 ml, corresponding to an average inoculation rate of 3  106 spores ml 1 of medium. Then the filters were immediately put on agar medium and the Petri dishes were incubated in darkness at 23 8C for up to 10 days. 2.4. Spores quantification Spores on surface culture were harvested with 0.9% NaCl with a sterile scraper and counted with a haemocytometer. Fungal biomass was determined by drying the whole invaded filters at 70 8C during 72 h. Sporulation index was defined as the number of spores (expressed in millions) divided by the dry weight (expressed in mg). 2.5. Analytical methods For pH, nitrogen and glucose determinations, the whole agar medium was equilibrated for 1 h by shaking in 100 ml of distilled water. The solution was then filtered through a 0.45 mm nylon filter and kept at 20 8C until analysis. Glucose concentration was measured by HPLC (Aminex HPX-87H column, Merck) [15]. The concentration of ammonium ions in the culture medium was determined by the Nessler reagent [16]. The culture medium was diluted 400-fold and 1/20 of Nessler reagent was added. The A395 was immediately measured, and the ammonium concentration was determined from a calibration curve of 25–200 mM ammonium sulphate. The concentration of nitrate ions in the culture medium was determined by Bioquant1 Nitrates KS compact (Calbiochem, Merck) according to the supplier’s instruction. 3. Results 3.1. Production of homogeneous mycelia In order to study the effect of different parameters on the conidiation, a cultivation technique was developed on solid medium in order to obtain a homogeneous mycelium. For this purpose, a glass microfibre filter was soaked in a calibrated spores suspension of P. camemberti. Contrary to cellophane discs usually used for transfert experiments, glass microfibre material allows a uniform repartition of the inoculum on the surface of the medium. Moreover, the cellulases constitutively secreted by Penicillium [17,18] could degrade the cellulose present in the cellophane filters and consequently interfere with the measurement of glucose consumption during the growth of the fungus. Once inoculation and transfer conditions were defined, we studied the effects of parameters frequently quoted in the Table 1 Growth media with different nitrogen sources Medium Glucose (g l 1) C (g l 1) Nitrogen source C (g l 1) N (g l 1) Na3-citrate (g l 1) (NH4)2SO4 (NH4)2HPO4 KNO3 NaNO3 10 10 10 10 4 4 4 4 3.77 3.29 5.77 4.85 0.8 0.8 0.8 0.8 4.4 4.4 4.4 4.4 In addition to nutrients cited in the table, all media contain 20 ml l 1 of trace element solution and 15 g l 1 of agar–agar. 1320 I. Krasniewski et al. / Process Biochemistry 41 (2006) 1318–1324 literature for their capacity to promote P. camemberti conidiation in liquid cultures [19–21]. 3.2. Effect of the nature of nitrogen source on conidiation in P. camemberti The influence of four nitrogenous sources [(NH4)2SO4, (NH4)2HPO4, NaNO3 and KNO3] on the initiation of conidiation was analysed for this purpose, each nutrient source added at a final concentration of 0.8 g l 1 of nitrogen (Table 1). The four nitrogen sources tested did not similarly affect the sporulation process. Moreover, sporulation was not observed in all conditions tested. During growth in the presence of (NH4)2HPO4 and KNO3, 4.8  106 and 12  106 spores ml 1 1 were, respectively, collected (Fig. 1A). Microscopic observations revealed that conidiophores appeared after 3 and 4 days of growth in the presence of these sources, respectively, while the mycelium remains in a vegetative form in the presence of (NH4)2SO4 and NaNO3. Analysis of the nitrogen source influence on the total biomass revealed that ammonium salts promoted a better growth than nitrate salts, with a two times higher production on day 4 (Fig. 2A). As a consequence, the sporulation index was Fig. 2. Influence of the nitrogen sources on biomass production (A), glucose consumption (B) and pH (C). P. camemberti was grown for 10 days, without transfer, on reference medium with one of these four nitrogenous sources: (NH4)2HPO4, (NH4)2SO4, KNO3 and NaNO3. Arrows indicated the onset of the conidiation on KNO3 or on (NH4)2HPO4. Data are average of three determinations and standard deviations. Fig. 1. Influence of the nitrogen source nature on spore number (A) and sporulation index (106 conidia mg 1 dry weight) (B). P. camemberti was grown for 10 days, without transfer, on reference medium with one of these four nitrogenous sources: (NH4)2HPO4, (NH4)2SO4, KNO3 and NaNO3. Data are average of three determinations and standard deviations. close to four times higher on KNO3 than that on (NH4)2HPO4, if the maximum were considered (Fig. 1B). If glucose consumption was uncorrelated to the onset of conidiation, it perfectly matched kinetics of total biomass production and occured more rapidly in the presence of ammonium salts than nitrate ones. Thus, depletion of glucose occured after 3 days of growth in the presence of ammonium instead of 8–10 days, in relation to media containing nitrates (Fig. 2B). Changes in pH during the growth period of P. camemberti were measured daily during 10 days (Fig. 2C). The initial pH I. Krasniewski et al. / Process Biochemistry 41 (2006) 1318–1324 Fig. 3. Profiles of nitrogen concentration in media with four nitrogenous sources: (NH4)2HPO4, (NH4)2SO4, KNO3 and NaNO3. Data are average of three determinations and standard deviations. value of the media containing ammonium was 5.6 and then decreased to 4.2 and 4.9 on day 2, followed by an increase to 7.0 and 7.5 on day 3, whereas in media containing nitrate, the pH, initially 5.6, progressively increased to 7.9 by day 3. Conidiation occurred beyond this time, when external pH remained alkaline and relatively constant. However, alkalinization probably played a great part in the onset of the conidiation, it was not absolutely necessary since P. camemberti remained in a vegetative form in two cases. Nevertheless, pH kinetic shape could be related to the biomass production. In the presence of ammonium, the acidification was certainly due to the consumption of this cation during the exponential growth. In all cases, the alkalinization started a short time after growth and continued after cessation of growth by a maintenance mechanism. It has been reported that P. camemberti was able to deaminate some amino acids, releasing ammonia probably involved in the alkalinization of the media [22]. Patterns of nitrogen consumption were similar on the three media which sporulated poorly or not at all, that is in the presence of (NH4)2SO4, (NH4)2HPO4 and NaNO3 (Fig. 3). Nitrogen decreased linearly by 75% from the first hour of growth up to the end of the experiment. During cultivation in the presence of KNO3, nitrogen consumption started 3 days after inoculation with a lower final decrease of 28%. Finally, KNO3-induced conidiation was completed before limitation of any major nutrient occurred within the medium. 1321 From the data above, it is apparent that (NH4)2HPO4 allowed a suboptimal conidiation; consequently, this nitrogen source was used in this experiment in order to detect the slight effects that the tested parameters might have on conidiation. After 3 days of cultivation on the reference medium, as glucose was completely metabolised, the filters invaded by mycelium were transferred on reference medium eventually modified as follow: lacking glucose or nitrogen source, supplemented with 100 mM malic acid, with 35 mM glutamic acid or with 15 mM CaCl2
2H2O. Quantification of spores revealed that sporulation required glucose as the spore number was 60–70 times lower on reference medium without glucose. The presence of malic acid or glutamic acid caused similar effects than a glucose deficiency. On the contrary, after 2 days of growth, sporulation was precociously induced by the presence of calcium (Fig. 4). The spore number was eight times higher on day 2 after growth on a calcium-supplemented medium than on the reference medium or reference medium without nitrogen source. While sporulation started more rapidly in presence of calcium, after 3 days of growth, spore numbers and sporulation indices were equivalent on the three media. Thereafter, the presence of calcium or the lacking of nitrogen source improved sporulation. 3.3. Effect of others nutrients on conidiation in P. camemberti For a better understanding of the sporulation process in P. camemberti, we have checked the influence of intermediates and derivatives of the Krebs cycle (malic and glutamic acids) [12] and calcium ions which are frequently considered for their ability to enhance the conidiation of this fungus in liquid culture [19–21]. Carbon and nitrogen starvation that are considered as inducers of the conidiation of P. chrysogenum and Aspergillus (A.) nidulans, was also tested [23,24]. Fig. 4. Influence of nitrogen starvation or calcium supply on spore production (A) and sporulation index (106 conidia mg 1 dry weight) (B). P. camemberti was grown for 3 days on reference medium, and then transferred to reference medium, reference medium without nitrogen and reference medium complemented with 15 mM CaCl2
2H2O. Data are average of three determinations and standard deviations. 1322 I. Krasniewski et al. / Process Biochemistry 41 (2006) 1318–1324 During growth of the fungus, except on media without glucose, the biomass doubled within 24 h of transfer to reach between 12 and 13.7 g l 1 (Fig. 5A). Due to autolysis, values decreased after 24 h. As shown in Fig. 5B, glucose was completely metabolised from 1 to 2 days after transfer, corresponding to a rapid biomass production. Due to the glucose depletion, nitrogen consumption was stopped after 1 day. The presence of malic acid used as C-source in the growth medium allowed nitrogen consumption to continue (Fig. 5C). Glutamic acid was completely consumed within 24 h (data not shown). The carbon and nitrogen supply, brought by malic or glutamic acid, could explain the low conidiation in presence of these two nutrients. 3.4. Influence of the carbon-to-nitrogen ratio on the conidiation in P. camemberti Fig. 5. Influence of factors on biomass production (A), glucose consumption (B) and (NH4)2HPO4 consumption (C). P. camemberti was grown for 3 days on reference medium, and then transferred to reference medium, reference medium without glucose, reference medium without nitrogen, reference medium complemented with 15 mM CaCl2
2H2O, reference medium complemented with 100 mM malic acid or reference medium complemented with 35 mM glutamic acid. Data are average of three determinations and standard deviations. Results above showed that conidiation required the presence of glucose but was improved by a nitrogen starvation. Therefore, to improve the spore production, we had the carbon-to-nitrogen ratio (C/N) vary (Table 2) and used the nitrogen source the most favourable to the conidiation, the KNO3. As expected, fungal biomass production was positively correlated to the glucose concentration in the culture medium, while the KNO3 concentration did not affect the vegetative growth (Table 2). For the same C/N ratio, sporulation given by spore count was about six times lower for C/N: 5 compared to C/N: 5bis, for which the glucose concentration was twice higher (Table 2). Sporulation was also reduced in media with a C/N ratio of 0.5. The absence of conidiation was mainly due to the low vegetative growth, consecutive to glucose limitation. A drastic glucose depletion inhibited conidiation when nitrogen was in a high (C/N: 0.5) or low ratio (C/N: 5). For the same nitrogen concentration, spore numbers and even sporulation indices were significantly higher when glucose concentration was higher: 1 > 0.5, 20 > 5 and 10 > 5bis. Regardless of the glucose concentration, spore yields were enhanced when nitrogen concentration decreased. While vegetative growth on media with a C/N ratio of 1 and 5bis is equivalent in 16 days, the spore number and the sporulation index in C/N: 5bis were significantly higher than those in C/N: 1 culture. In the same way, the spore number and the sporulation Table 2 Effect of carbon-to-nitrogen ratio on growth and sporulation of P. camemberti after 16 days of cultivation (A) C/N ratio Glucose and C (g l 1) KNO3 and N (g l 1) 0.5 5 (2) 28.8 (4) 5 5 (2) 2.9 (0.4) 1 10 (4) 28.8 (4) 5bis 10 (4) 5.8 (0.8) 10 20 (8) 5.8 (0.8) 20 20 (8) 2.9 (0.4) (B) Biomass (g l 1) Spores count (106 ml 1) Sporulation index 2.63 (0.02) 2.00 (0.15) 0.76 1.50 (0.12) 1.56 (0.21) 1.04 3.10 (0.15) 3.56 (0.15) 1.15 2.69 (0.04) 9.85 (0.83) 3.66 5.23 (0.17) 27.43 (3.41) 5.25 5.05 (0.14) 41.54 (4.08) 8.23 A, C/N ratio values and initial concentrations in glucose, carbon (in brackets), KNO3 and nitrogen (in brackets); B, indicators of development in P. camemberti. Data are average of three determinations and standard deviations (in brackets). I. Krasniewski et al. / Process Biochemistry 41 (2006) 1318–1324 index were around 1.6 times higher in C/N: 20 culture than those in C/N: 10 culture. 4. Discussion For the strain tested in this work, glucose starvation could not trigger a significant conidiation. This was not in agreement with previous studies reporting that glucose starvation was a contributory factor implicated in the onset of conidiation [2,23– 25]. Our results showed that conidiation occurred before total glucose consumption. Furthermore, the cultures sporulated at a much reduced level when the initial glucose concentration was lower than 10 g l 1. It could be argued that conidiation requires a minimal concentration of glucose, which may be related to the enhance of the biosynthetic demand during conidiation [12]. Our study on the influence of the C/N ratio on conidiation showed that a carbon concentration higher than nitrogen concentration was required for conidiation. The highest sporulation index detected in our experiments was for a C/N ratio of 20. Even though nitrogen concentration was important, the type of nitrogen source also played an essential part in conidiation. KNO3 stimulated conidiation, while (NH4)2SO4 was inhibitory. This fact was already established in Aspergillus spp. [24,26] and Monascus spp [27]. In Penicillium cyclopium, addition of NH4+ in concentrations higher than 6 mM to cultures with glutamate or glutamine evoked vegetative growth [4]. Besides, a shift from (NH4)2HPO4-containing to (NH4)2HPO4-free media produced an abundant conidiation; while a shift from (NH4)2HPO4 to (NH4)2HPO4-glutamate media produced a low spore number. Marzluf [28] has reported that some nitrogenous compounds, like ammonia, glutamine and glutamate, are preferentially used by fungi. However, when these primary nitrogen sources are not available or are present in concentrations low enough to limit growth, nitrogen derepression occurs and various other nitrogen sources, like nitrate, can be used [28]. Moreover, in P. roqueforti, the existence of a nitrogen regulatory circuit was highlighted [29]. As regulator areA in Aspergillus, expression of the regulator nmc was subjected to nitrogen control, enhanced under nitrogen derepression conditions (nitrate) and reduced under nitrogen repression (ammonium) [29]. Results in this work suggested that conidiation of P. camemberti was regulated by nitrogen repression. On the other hand, (NH4)2HPO4 favoured conidiation more than NaNO3. The type of salts associated with ammonium or nitrate sources should be taken also into account. K+ and PO43 must be required for conidiation, as it was already shown for P. cyclopium [30], Fusarium solani [31] and A. oryzae [32]. Among external minerals, calcium was the most effective to trigger conidiation. Three days after transfer, the spore number and the sporulation index were higher when mycelia were cultivated in presence of calcium. Calcium has been reported to induce sporulation of several members of the genus Penicillium in submerged liquid culture: P. notatum [8], P. griseofulvum [11], P. notatum [9], P. cyclopium [20] and P. oxalicum [21]. It has been suggested that Ca2+ affects conidiation by binding the extracellular hyphal surface [19], and thereby influencing 1323 metabolism. In P. cyclopium, externally bound calciuminduced conidiation, causing changes in plasma membrane function which disrupted the pH gradient observed during apical growth [20]. In P. notatum, calcium-induced cultures swung to a primarily Entner-Doudoroff and pentose-phosphate based catabolism, and further diminished oxidative Krebs cycle capacity [33]. In the chytridiomycete Blastocladiella emersonii, induction of sporulation was closely dependent on extracellular calcium. A calcium influx was likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein had been suggested to mediate calcium events in sporulation [34]. In P. cyclopium, recent findings have shown that calcium does not really act as an inducer, but enhances the action of the autoinducer, the conidiogenone. The mechanisms of induction are not yet understood but probably related to the binding of the cation to the extracellular hyphal surface [35,36]. Thus, factors which promote conidiation seem to act by perturbating the normal progress of cellular metabolism. It would then be interesting to study the impact of these factors on the spore qualities and on the expression of genes, such as brlA, abaA or wetA, implicated in the sporulation events of A. nidulans [37] and some Penicillium species [38,39]. According to results obtained in this work, the presence of potassium nitrate, a glucose concentration of at least 10 g l 1 and the addition of calcium were the best conditions to favour conidiation on P. camemberti. 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