Carbon and nitrogen sources differently influence Penicillium sp . HC 1 conidiation in solid and liquid culture

1 Grupo de Biotecnología Ambiental e Industrial – GBAI, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, BogotáD.C., Colombia. 2 Instituto de Biotecnología, Universidad Nacional de Colombia, Bogotá D.C., Colombia. 3 Facultad de Ingeniería, Universidad Nacional de Colombia, Ciudad Universitaria, BogotáD.C., Colombia. 4 Unidad de Investigaciones Agropecuarias – UNIDIA, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., Colombia.

formation (asexual propagules) is very important for reproduction and rapid dissemination and can lead to producing mycelia rapidly in favourable environmental conditions.Such property, and the fact that they are structures having greater tolerance to different types of stress than vegetative cells, has led to conidia suspensions being widely used in biotechnological-based industry for producing seed cultures or obtaining a formulated final product (Feofilova et al., 2011).
Solid state fermentation (SSF) or submerged fermentation (SmF) is used for large-scale fungi culture.SSF has been used since ancestral times, offering several advantages over SmF, the most important being that it can reproduce the natural process of fungal growth, thereby leading to higher yields of metabolites, growth or asexual propagule formation.Nevertheless, SSF has numerous disadvantages concerning SmF; these would include low mixture efficiency, difficulty in scaling-up, difficulty in controlling different culture parameters, such as pH, temperature, aeration, oxygen transfer, and the great impurity of the products so obtained, thereby increasing recovery costs (Couto and Sanromán, 2006).The foregoing means that SmF continues to be used more in large-scale industrial processes.However, the greatest problem regarding SmF is the culture system per se (Grimm et al., 2005), because fungi may have different structural forms throughout their lifecycles influencing the culture's rheological properties and fungal metabolism and thus metabolite production (Grimm et al., 2005;Znidarsic and Pavko, 2001;Papagianni, 2004).In addition, SmF conditions are not ideal for conidia formation (Znidarsic and Pavko, 2001;Hadley and Harrold, 1958;Morton, 1961;Thomas et al., 1987;Boualem et al., 2008) and conidiogenesis is not easily achieved in SmF, due to the relatively good availability of nutrients.Mechanisms controlling asexual propagule formation differ between species (Roncal and Ugalde, 2003;Znidarsic and Pavko, 2001) and most still remain unknown.
Specifically, concerning the genus Penicillium, inducing conidiogenesis in SmF has been studied for many years, given the commercial interest shown regarding some species from this genus.Foster et al. (1945) showed that Penicillium notatum conidia could be produced in SmF, having morphology and activity similar to that obtained in surface cultures.However, conidia formation only occurred if the culture medium contained a high calcium concentration (0.5 to 5%) (Foster et al., 1945).Such finding has been proven for several Penicillium species, such as Penicillium cyclopium, Penicillium griseofulvum, Penicillium paxilli, Penicillium bilaii and Penicillium oxalicum (Roncal and Ugalde, 2003).Nutrient limitation is another factor determining the induction of conidiogenesis.Contrary to submerged hyphae, aerial hyphae grow outside basal medium separated from the nutrients, leading to aerial hyphal detecting nutrient limitation which could thereby induce the start of conidiogenesis (Roncal and Ugalde, 2003).Hadley and Harrold (1958) found that conidiogenesis in P. notatum was connected to nitrogen metabolism, since reduced nitrate levels in the medium increased the ability to produce conidia and reduced calcium requirement (Hadley and Harrold, 1958).Nitrogen limitation provokes conidiogenesis in most Penicillium sp.(Roncal and Ugalde, 2003).However, conidiogenesis induction due to carbon limitation has also been reported.For example, low glucose concentration in P. chrysogenum restricts vegetative growth, thereby inducing conidia formation (Righelato et al., 1968).Other nutritional conditions could induce conidiogenesis; regarding P. griseofulvum, neither conidiophores nor conidia are formed in submerged culture in culture medium containing glucose and nitrate, even with nitrogen limitation, but may be induced in the presence of very high glucose concentrations or by adding defined concentrations of calcium or copper (Morton, 1961).
It has been reported that culture conditions, such as pH, oxygen, and exposure to visible light during mycelial growth affect conidia formation in terms of their amount and their morphological and physiological characteristics, as tolerance to thermal and oxidative stress by ultraviolet (UV) radiation.This pattern has been studied in entomopathogenic fungi, such as Beauveria bassiana (Chong-Rodríguez et al., 2011), Metarhizium anisopliae (Hallsworth and Magan, 1994), Metarhizium robertsii (Rangel et al., 2011), Paecilomyces farinosus (Hallsworth and Magan, 1994) and Paecilomyces fumosoroseus (De la Torre and Cárdenas-Cota, 1996;Vidal et al., 1998), and phytopathogens, such as Colletotrichum acutatum (de Menezes et al., 2015) and Colletotrichum truncatum (Jackson and Schisler, 1992).Few reports regarding the genus Penicillium have dealt with the relationship between culture conditions and the characteristics of the conidia so obtained.Pascual et al. (2000), found that P. oxalicum conidia viability, hydrophobicity, and efficiency (in terms of biocontrol) differed when produced in liquid culture or in solid culture, those produced in solid medium being more efficient (Pascual et al., 2000).
The present work studies how carbon and nitrogen sources and culture condition (solid or liquid) affect conidiophore and conidia formation and also their tolerance to different types of stress in Penicillium sp.HC1, a fungus of industrial interest given its ability to degrade lignocellulose residues.

Organisms and inoculation
Penicillium sp.HC1 was selected from a screening study of cellulolytic microorganisms isolated from rhizosphere soils of rice crops located at Tolima and Meta, Colombia (Gutiérrez-Rojas et al., 2012).This isolate has been deposited in the Centraalbureau voor Schimmelcultures Fungal Biodiversity Center (CBS-KNAW) as CBS 136205.The inoculum for all experiments consisted of a suspension having 10 8 conidia.ml - which was prepared from a
Cultures were incubated at 28°C.Liquid medium was prepared in 100 ml Erlenmeyer flasks with 20 ml working volume and inoculated with 2 ml of the conidia suspension, incubated at 28°C and shaken at 100 rpm on an orbital shaker.After 4 days' incubation, a sample was taken from both solid and liquid media for morphological characterisation by image analysis on an optical microscope (Leica DM1000) with a digital camera (Leica, ICC50 HD).Conidia suspensions were obtained after 8 days' incubation in which the amount of conidia was determined by haemocytometer as well as their viability and tolerance to stress.All experiments were performed in triplicate.

Germination percentage
Germination percentage was evaluated for determining conidia viability.Three 5 µl aliquots of a 10 8 conidia.ml - suspension were inoculated on Petri dishes containing water agar and incubated at 28°C for 18 to 20 h.Conidia germination percentage was calculated by counting under a microscope, a minimum of 100 conidia (germinated and non-germinated) per each 5 µl aliquot.All evaluations were carried on in triplicate.

Tolerance to thermal stress
Aliquots containing 1 ml of 10 7 conidia.ml - suspension were heated at 45 and 50°C for 1 h; control was kept at room temperature (RT) (García-Rico et al., 2011).Germination percentage was calculated after treatment time had elapsed.All experiments were done in triplicate.

Tolerance to oxidative stress
A suspension of 10 8 conidia.ml - was mixed with hydrogen peroxide to reach a final concentration of 0, 100, 110, 120, 130, 140, and 150 mM, and then incubated for 30 min at RT (García-Rico et al., 2011).After this incubation time, germination percentage was calculated.All experiments were done in triplicate.

Tolerance to UV radiation
A 10 ml of 10 8 conidia.ml - suspension was submitted to UV radiation in a laminar flow chamber (Streamline laboratory products EN 1822.1) at 20 cm constant distance from UV lamp.A 1 ml sample was taken at different intervals of time: 0, 0.5, 1, 2, 3, 5, and 10 min according to Rangel et al. (2011), with some modifications.Germination percentage was calculated.All experiments were done in triplicate.

Statistical analysis
Differences between conidiophore and conidia morphology as well as the amount of conidia produced under all conditions (in solid and liquid) and tolerance to stress were evaluated by analysing one-way variance (ANOVA) and Tukey test having 0.05 significance level.Two-way analysis of variance (ANOVA) was also used for determining the influence of the variables separately and the interaction between them on the amount of conidia produced.
Pearson correlation was used for measuring the correlation between conidiophore morphology and the tolerance of the conidia produced.SPSS version 21 and Design Expert version 7 statistical software was used for all the analysis.

Effect of the type of carbon and nitrogen source on conidia formation
Figures 1 and 2 show the results of microscopic characterisation.Differences were observed between the conidiophores obtained using simple carbon sources (SS) and complex sources (CS) in solid medium.The conidiophores were wider in the latter (CS: 14.73 ± 4.48 μm; SS: 9.41 ± 3.58 μm), having more phialides (CS: 9 ± 2; SS: 5 ± 2) and the phialides, in turn, were much bigger (CS: 8.67 ± 1.76 μm; SS: 8.21 ± 1.57 μm) and wider (CS: 2.18 ± 0.4 μm; SS: 1.92 ± 0.33 μm).Reproductive structures were not observed in all liquid media, for that reason it is not possible to stablish differences between types of sources.However, the data did show that conidiophores obtained in submerged condition were shorter (12.98 ± 2.62 μm), narrower (7.37 ± 1.92 μm) and had less phialides (2 ± 1) than those obtained in solid media with any of the sources evaluated here.Differences were only found regarding the conidia size obtained in solid medium with complex carbon sources, being larger (2.50 ± 0.49 μm × 2.16 ± 0.46 μm) than those obtained in solid medium with simple sources (2.04 ± 0.41 μm × 1.77 ± 0.39 μm) or in liquid medium (1.87 ± 0.42 μm × 1.67 ± 0.39 μm).

Effect of the type of carbon and nitrogen source on conidia tolerance
Figure 4 shows the tolerance to thermal stress (50°C) of  conidia obtained in all solid and liquid media.The relative germination percentage of conidia obtained from solid media was 16% on average, with 56.7% coefficient of variation, whilst this was 37.02%, with 99.1% coefficient of variation, from liquid media.The incidence of complex or simple carbon and organic or inorganic nitrogen sources in solid media was not evident.Conidia obtained from liquid media had a clear tendency towards high tolerance (close to 100%) in media having complex carbon sources (cassava starch, wheat bran, and rice flour) combined with inorganic nitrogen sources ((NH 4 ) 2 HPO 4 and KNO 3 ).The conidia obtained from liquid media 11 (cassava starch + (NH 4 ) 2 HPO 4 ) and 19 (rice flour + (NH 4 ) 2 HPO 4 ) showed tolerance at 45°C, oxidative stress and UV radiation.Conidia obtained from PDA (8 days culture) were used as the standard for comparison (Figure 5).These results showed that the conidia obtained from in these two media  were not only more tolerant to temperature (Figure 5A) but also to oxidative and stress caused by UV radiation (Figure 5B and C).

Correlation between conidiophores' morphological characters and conidia's tolerance to thermal stress
Pearson correlation between the conidiophores' morphological characteristics and the results of the 50°C tolerance test, regarding conidia produced in different culture conditions, was analysed.Positive and statistically significant correlations were obtained in all cases (Table 3).The results suggested that the conidia obtained from larger structures tended to be more tolerant; this was evident regarding solid media where the most tolerant conidia were obtained from cassava medium starch as carbon source.The structures having most phialides were also obtained with such media.However, lower tolerance values were not obtained in all liquid media; in fact, the most tolerant conidia were obtained in liquid media with complex carbon sources and inorganic nitrogen sources (media 11, 15, and 19).This result suggests that this ratio is not always direct and does not just depend on one condition, such as solid or liquid medium or simple or complex carbon source, but rather on the interaction of many factors.

Effect of the carbon:nitrogen ratio on conidia tolerance
Conidia were obtained in two conditions (solid and liquid), in culture medium with simple carbon source (glucose) and inorganic nitrogen source (KNO 3 ), and then submitted to thermal stress (50°C) for establishing the effect of C:N ratio on conidia tolerance.It was seen that nitrogen limitation for both conditions increased conidia tolerance (Figure 6).In a non limiting nitrogen ratio (5:1), conidia obtained in solid medium had 1.41 ± 0.87 germination percentage and those in liquid medium 25.66 ± 0.87; whilst a limiting nitrogen ratio (40:1) increased such percentage to 5.34 ± 0.33 in solid media and to 53.66 ± 2.70 in liquid media.This effect was more noticeable in submerged (p<0.000,F = 55.322)than in solid condition (p=0.003,F=10.805).Two-way analysis of variance (ANOVA) showed that this culture condition had greater influence on response (p<0.0001,F=80.13) than C:N ratio (p<0.0001,F=11.72).

DISCUSSION
Studying culture conditions' influence on conidia production has been limited to entomopathogenic fungi of industrial interest (Jackson and Schisler, 1992;Hallsworth and Magan, 1994;De la Torre and Cárdenas-Cota, 1996;Vidal et al., 1998;Chong-Rodríguez et al., 2011;Rangel et al., 2011;de Menezes et al., 2015), but has been little studied in the genus Penicillium.The present work aimed at evaluating the influence of carbon source, nitrogen source, and their interaction on solid and  submerged culture, regarding conidiophore and conidia morphology, the amount of conidia produced and their tolerance to conditions of stress in the cellulolytic fungi Penicillium sp.HC1.Krasniewski et al. (2006) studied the effect of culture medium composition in solid culture on conidia production in Penicillium camemberti and found that not just concentration, but also the type of nitrogen source influenced conidiogenesis in this fungi; KNO 3 stimulated conidia production whilst (NH 4 ) 2 SO 4 was inhibitory, using glucose as carbon source.Whether such clear tendency regarding the type of nitrogen source favouring conidiation (or not) could not be established in our work, probably due to the effect not just being caused by the nitrogen source, but also interaction with the carbon source.
The conidiation pattern was different in liquid media where a clear tendency for obtaining greater conidia production with complex carbon sources emerged.Given the nature of complex sources, some other component could have been exercising an influence on conidiogenesis, which could only be observed in the submerged condition.Mycelium air contact was the dominant stimulus for Penicillium sp.conidiophore formation in solid medium (Morton, 1961;Roncal and Ugalde, 2003); such situation did not occur when Penicillium sp.HC1 grew submerged, meaning that other inducing factors would have been playing a dominant role.Such factors might have been ions; it has been shown that calcium is fundamental for conidiophore formation in submerged culture in differing Penicillium sp.(Roncal and Ugalde, 2003).Roncal et al. (2002) identified a diterpenoide in P. cyclopium, they named it conidiogenone, which could act as a hormone at very low concentrations (10 -7 to 10 -8 mol.L -1 ), thereby inducing conidiogenesis at some calcium concentration.According to their results, conidiogenone and conidiogenol (conidiogenone precursor) were produced from very early growth phases onwards and were continuously released to culture medium, where they became accumulated until reaching a concentration which induced conidiogenesis.It seems that calcium reduces the threshold concentration required in liquid medium for such induction in a yet-to-be-understood way, but which is probably related to this cation's binding to the hyphae external surface (Roncal et al., 2002).The inducing role of other ions, such as Mg, K, Cu, and PO 4 in liquid media has also been reported in P. griseofulvum, P. chrysogenum (Morton, 1961), and P. camemberti (Bockelmann et al., 1999).
Regarding tolerance to temperature (50°C), a clear tendency for obtaining conidia having high tolerance (close to 100%) was obtained in liquid culture with complex carbon sources and inorganic nitrogen sources.Such result was confirmed for conidia obtained in liquid media 11 (cassava starch and (NH 4 ) 2 HPO 4 ) and 19 (rice flour and (NH 4 ) 2 HPO 4 ) where high tolerance to oxidative stress and stress caused by UV radiation was also obtained.The influence of culture medium composition on conidia tolerance has been reported for other genera.Hallsworth and Magan (1994) found differences in polyhydroxy alcohol and trehalose content in conidia from three entomopathogenic fungi (Beauveria bassiana, Metarhizium anisopliae, and Paecylomices farinosus) when they were cultured in different carbon sources and concentrations (Hallsworth and Magan, 1994).The accumulation of polyols, such as mannitol, and trehalose is a mechanism, which cells use for protecting themselves from stress.Trehalose, for example, can replace water at low water activity and stabilise proteins during desiccation, thereby preserving membrane integrity (Hallsworth and Magan, 1996;Rangel et al., 2008).Cells accumulate these compounds in response to thermal shock, freezing, dehydration, osmotic stress, and carbon limitation and also to stress caused by other agents like UV radiation (Rangel et al., 2008).The interaction of the three factors (submerged culture condition, complex carbon source, and inorganic nitrogen source) may have caused a stressful environment for Penicillium sp.HC1, leading it to accumulating compounds, such as those reported in other species, therefore conidia produced in these conditions increased tolerance to the stressing conditions evaluated here.
However, not just the type of carbon and nitrogen source affect conidia tolerance to stress; the C:N ratio also influences such characteristic.Different C:N ratios were evaluated using a simple carbon source; this led to low tolerance (compared to that obtained with complex sources) and these conidia's tolerance we observed to be increased by increasing nitrogen restriction, even though to the detriment of the amount of conidia produced in very limiting ratios (data not shown).The influence of C:N ratio on fungal conidia activity and characteristics has been studied in fungi, such as Talaromyces flavus (Engelkes et al., 1997), B. bassiana, andPochonia chlamydosporia (Gao andLiu, 2010a), Paecilomyces lilacinus, and M. anisopliae (Gao and Liu, 2010b), Lecanicillium lecanii and Trichoderma viride (Gao and Liu, 2009).The relationship between carbon concentration and C:N ratio with conidia production and quality has been clear in all cases; however, this relationship was different for each species studied and depended on factors such as type of carbon source, type of nitrogen source and culture system.The latter was evident in our results as fungal response to nitrogen limitation in solid medium was very different to that obtained in the liquid media where the effect was much clearer.
It was clear that culture medium composition and culture system (solid or liquid) were the critical factors determining the amount and tolerance of conidia, therefore, these factors must be carefully defined for guaranteeing conidia survival in field conditions.However, further studies are needed for establishing which mechanisms are involved regarding the differences in tolerance observed in the culture conditions evaluated here.This work is the first report in which all these parameters (carbon source, nitrogen source, C:N ratio, culture system, amount, and tolerance of conidia) have been evaluated for Penicillium sp.

Figure 2 .
Figure 2. Morphological characteristics of Penicillium sp.HC1 conidiophores and conidia obtained from different solid and liquid culture media.The same letters indicate no significant difference according to Tukey test (95% significance).

Figure 3 .Figure 4 .
Figure 3. Production of conidia in different culture media.(A) Solid media -simple carbon sources.(B) Solid mediacomplex carbon sources.(C) Liquid media -simple carbon sources.(D) Liquid media -complex carbon sources.Black bars represent the higher responses, white bars represent the lower responses and squared bars represent the intermediate responses, according to Tukey test (p<0.05).

Figure 5 .
Figure 5. Tolerance to thermal stress (A), oxidative stress (B) and UV radiation (C) of Penicillium sp.HC1 conidia in liquid (11 and 19) and PDA media.

Table 1 .
Combinations of carbon and nitrogen sources to evaluate their influence on growth and conidia production in Penicillium sp.HC1, on solid and liquid media.

Table 2 .
Effect of carbon and nitrogen sources on conidia production, on solid and liquid media.

Table 3 .
Pearson correlation coefficients of survival percentage at 50°C of conidia obtained from different culture conditions and morphological characteristics of conidiophores