Production of Coconut Aroma in Solid-State Cultivation: Screening and Identification of Trichoderma Strains for 6-Pentyl-Alpha-Pyrone and Conidia Production

The polyketide, 6-pentyl-alpha-pyrone (6-PP), has a characteristic coconut aroma and is produced by many Trichoderma species. In this study, we screened formerly isolated Trichoderma strains for 6-PP production grown using solid-state fermentation (SSF). Sugarcane bagasse supplemented with a nutrient solution was used as the support material for the culture. We detected out-standingly strong 6-PP production in the case of two Trichoderma strains in this experiment. The strains were identiﬁed based on internal transcribed spacer (ITS) sequence analysis as Trichoderma asperellum (G7 and G18). We also investigated the production of 6-PP in the case of the best Trichoderma strain selected ( T. asperellum G7) from two types of cultivation (SSF and liquid cultivation (LC)). The same liquid medium was used to impregnate sugarcane bagasse. Results led to a 6-PP production 80 times higher (85.11 μ g · g · DM − 1 ) in SSF than that found in LC (10.00 μ g · L − 1 ) after eight days of cultivation. For this, the SSF is the best option to produce 6-PP compared to liquid cultures.


Introduction
Solid-state fermentation (SSF) is an ancestral process, initially used for human alimentation [1].Numerous studies have shown advantages of SSF over its liquid homologues, advantages related to quantitative and qualitative aspects in the productions of enzymes and secondary metabolites by filamentous fungi [2,3].Moreover, one of the main driving forces in the gain of popularity of SSF over the last decades is the growing concern about sustainability in bioprocess development linked to the possibility of using agroindustrial wastes as cheap culture media to perform fermentations [4,5].Agroindustrial residues are generally considered the best substrates for solid-state fermentation processes, mainly due to their low cost [6,7].In SSF, the solid medium provides support and nutrients for the microorganism's development.A large number of microorganisms including bacteria, yeasts, and fungi have been used for cultivation on different solid substrates such as sugarcane bagasse, coffee husk, jatropha cake, sorghum pulp, olive pomace, and fruit seeds [8,9].However, filamentous fungi especially Hypocreacea are the best choice for secondary metabolites production [4].
e fungal genus Trichoderma, known since 1887 for its antagonistic properties, has been used as a biological control organism against several plant pathogens [10].During their development, Trichoderma produce biomass, primary metabolites (enzymes and organic acids), conidia, and secondary metabolites like 6-pentyl-alphapyrone (6-PP), a fungicidal compound [11].is lactone is also called "coconut lactone" because of its characteristic smell.Trichoderma has been well investigated because of its commercial importance used in food and pharmaceutical products [12].On the contrary, 6-PP has attracted considerable attention as a control agent due to its powerful antifungal activity.Hence, the ability of 6-PP to control the growth of Botrytis cinerea has been highlighted by Walter et al. [13].Several workers have also reported that 6-PP production is apparently related to the antagonism response of some Trichoderma species to disadvantageous culture conditions and that the solid-state culture can give higher yields of 6-PP than submerged fermentation [14][15][16].Furthermore, chemical synthesis of 6-PP requires high temperature (400 °C) and seven reaction steps [17].For this reason, production of this compound by a biotechnological way can be an interesting alternative to chemical synthesis.
A previous work has already been published for the production of 6-PP using SSF and Trichoderma strains [18].
is work aimed to screen Trichoderma strains derived from different natural sources on solid media for 6-PP production and to characterize the best strains able to produce a large quantity of 6-PP and conidia.Moreover, solid and submerged conditions were tested for the best Trichoderma strain selected to compare the production of 6-PP using the same composition of liquid culture medium.e fungal conidia were stored at 4 °C in a 5 ml bottle on potato dextrose agar (PDA) culture medium.

Inoculum Preparation.
e preculture medium had the following composition: glucose: 30.0 g•L −1 ; KH 2 PO 4 : e pH was adjusted to 5.6 before sterilization.Bottles (100 ml) containing 50 ml of this medium were inoculated with 5-day-old strains (1.0 cm 2 from PDA medium) and incubated under agitation (150 rpm, 5 cm diameter stroke) at 25 °C for 4 days.e mycelium was homogenized in an Ultra-Turrax homogenizer, and this mycelial homogenate was used as the inoculum.

Culture Media and Conditions for SSC.
e solid substrate comprising 4.0 g sugarcane bagasse was placed in 250 ml bottles supplemented with 10 ml of the same nutriment solution used for liquid culture.e humidity was set to 66% before sterilization.Sugarcane bagasse and nutriment solutions were individually autoclaved at 120 °C during 1 hour.Each bottle was then inoculated with 3.0 mL of mycelial cell suspension.e incubation temperature was 27 °C.Culture conditions such as incubation temperature, moisture level, inoculum concentration, and pH are fixed according to previous studies [18].
2.5.Production Kinetics.Kinetic profiles of 6-PP, conidia (for SSF), biomass, pH, and glucose were studied at the second, fourth, sixth, eighth, and tenth day of culture.

Conidia and 6-PP Analysis.
All the results being expressed in gram of dry matter, the water content of each sample was measured as follows: 1.0 g of fermented material was introduced into a lab oven at 105 °C to analyze the relative humidity of the sample.
Conidia suspension was prepared by mixing 1.0 g of the fermented material and 100 mL of distilled water containing tween 80 (0.01% v : v).
e conidia were counted using a traditional hemocytometer as described by Roussos et al. [19].
Sugar concentration was determined after each enzyme reaction according to the amount of resulting reducing sugar using 3,5-dinitro salicylic acid (DNS) following the assay method described by Miller [20].
e extraction of aroma produced by fungi in SSF was performed by the Soxhlet system from 10 g of wet solid fermented material using pure heptane (99.7%, Sigma-Aldrich, USA).For the extraction of 6-PP produced in the liquid medium, a 10 ml aliquot of the filtered fermented broth was extracted with 100 ml of heptane.
Samples were then stirred for 20 minutes and concentrated under a rotatory evaporator at 40 °C. 1 ml of the obtained heptanoic extract was then filtered with a 0.2 μm Millipore filter into vials and then analyzed with a gas chromatograph 7890A (GC) (Agilent Technology, USA) equipped with a split/splitless injector (T � 200 °C) and a flame ionization detector (T � 260 °C).6-PP was separated using a Supelcowax capillary column (internal diameter: 0.25 mm; length: 60 m; film thickness: 0.25 μm).e carrier gas was dihydrogen (column flow 1 mL•min −1 ), and the split ratio was 2 : 1. e oven temperature was set as follows: 30 min at 180 °C, from 180 to 230 °C at 10 °C•min −1 , and 10 min at 230 °C.Quantitative analysis of 6-PP was carried out using the internal calibration method, using c -undecanolactone (99%, Sigma-Aldrich) as an internal standard.e amplified ITS fragments were sequenced (GATC Biotech).Phylogenetic analyses were performed using MEGA7 [21].

Screening and Identification of 6-PP Producing Fungi.
e 6-PP production by a total of 17 strains of Trichoderma strains isolated from different natural sources was examined.
ese experiments were performed on solid media with sugarcane bagasse impregnated with a nutritive solution.All strains were cultivated at 27 °C and harvested after 8 days of total incubation.Only seven produced 6-PP (Table 1).
Characterization by ITS sequences analyses (Table 1) and morphological observations (Table 2) of the selected strains were conducted on the strains that produced the highest amount of 6-PP.Although they did not produce 6-PP, the strains G13 and G17 were also identified in order to compare the relationship between species and 6-PP and conidia production.Morphological characterization was performed on pure cultures grown for 3-6 days on PDA and was based on the ten characters (Table 2).Most discriminating ones were conidial shape, mycelial form, colony color, and culture smell.
e molecular identification of our fungal strains was performed by sequencing the ITS 1 regions of rDNA and compared NCBI GenBank nucleotide database.e BLAST analyses showed that the ITS sequences of all sequenced strains were at least 99% similar to the corresponding GenBank sequences.All belonged to Trichoderma, with only two species identified: five strains were affiliated to the species Trichoderma asperellum (G7, G18, G3, G11, and G9) while three proved to be Trichoderma longibrachiatum (G13, G14, and G17).One of the T. asperellum strains (G7) showed the best 6-PP production with an average of 86.69 μg•g•DM −1 of 6-PP (Table 1).Two other strains, T. asperellum G18 and T. asperellum G3, produced 80.79 μg•g•DM −1 and 39.83 μg•g•DM −1 , respectively.e highest value of conidia production was shown by T. asperellum G9 (5.78 × 10 8 conidia•g•DM −1 ) followed by T. asperellum G18 and T. longibrachiatum G17 with 3.95 and 2.81 × 10 8 conidia•g•DM −1 , respectively.Comparing the production of conidia and 6-PP, the maximum conidia production was showed by T. asperellum G9; however, this strain also showed the most limited amount of 6-PP (16.30μg•g•DM −1 ).Currently, Trichoderma strains are able to produce a large quantity of conidia and a green-colored fermented product characteristic due to conidia color.In this experiment, T. asperellum G7, presenting a limited conidia production and white to transparent color, is defined the best strain to produce 6-PP under the conditions tested.Based on these results, in order to evaluate the effect of conidia on 6-PP production and due to the interesting 6-PP properties, we selected T. asperellum G7 strain for further 6-PP production survey.
Glucose concentration, biomass, and 6-PP production in culture medium (Figure 1) were determined for 12 days in a stirred liquid culture of T. asperellum G7.As can be seen from Figure 1, the biomass increased sharply to a level of 5.84 g•L −1 at the end of 4 days, and thereafter, the increase was gradual, reaching a final value of 14.57 g•L −1 .
e residual sugar decreased sharply to a level of 5.66 g•L −1 at the end of fermentation, indicating rapid utilization of sugar.6-PP production was started after 6 days of incubation, the maximum value of 6-PP production (10.00 μg•L −1 ) was observed after the exponential phase, which is a common characteristic of secondary metabolites, and then decreased rapidly to attain values of 1 μg•L −1 after 10 days.ere was a wide pH variation in the liquid culture, and the pH decreased from 6.0 to 4.7, after 8 days.

Conidia Production by T. asperellum G7 Cultivated in SSF.
In order to compare the production of 6-PP by T. asperellum G7 in liquid and in SSF, the same liquid medium tested in both cultures was used to impregnate sugarcane bagasse.6-PP and conidia production was determined during 10 days of culture (Figure 2).Under SSF conditions, conidia production was initiated after 1 day of incubation.e conidia production increased sharply to a level of 3.7 × 10 7 conidia•g•DM −1 at the end of the second day, and thereafter, the increase was gradual, reaching a final value of 5.1 × 10 7 conidia•g•DM −1 .After 2 days, 6-PP production started.e maximum value of 6-PP production was observed after 8 days (85.11 μg•g•DM −1 ).After that, the 6-PP production decreased progressively to attain the value of 58.14 μg g•DM −1 at 10 days.As seen in Figure 2, conidiation and 6-PP production follow a similar trend at the beginning of cultivation, and the production of 6-PP was started after one day of conidiation.

Discussion
e production of secondary metabolites is an actual topic that is investigated following several ways, including bioreactors design, medium composition, and fungal strains [22,23].Concerning medium, substrates for SSF are usually homogeneous agroindustrial by-products which offer an economic advantage in this fermentation process [24].Even so, to be fully both economically competitive and ecofriendly, the SSF must, to a larger extent, be carried out on local by-products.
It is well known that sugarcane bagasse is particularly suitable for SSF because of its porosity allowing good water absorption, indispensable to carry out the microbial metabolism [25].Moreover, in terms of volume, it is the major solid agroindustrial by-product generated [26].
In this study, the use of sugarcane bagasse as support for fungal growth for conidia and secondary metabolites production using the solid-state culture was evaluated.Our main objective was to screen and identify Trichoderma strains producing an considerable amount of 6-PP.Among the 17 strains tested, 7 Trichoderma strains demonstrated the ability to produce 6-PP.e characterization of these strains by morphological and ITS gene analysis indicated that they were related to T. asperellum and T. longibranchiatum.
Interestingly, while 6-PP is detected in traces in all the cultures of the tested T. longibrachiatum strains, the species asperellum is much capable of producing 6-PP under these conditions.
Among the strains that produce 6-PP, our results indicated that T. asperellum G7 was the best strain with an average 6-PP production of 85.11 μg•g•DM −1 .With a maximum value of 86.69 μg•g•DM −1 , our value was significantly higher than those reported in literature studies for other species (T.harzianum IM206040, T. atroviride IM206040, and T. harzianum 4040) cultivated under the same conditions; [12,15,[27][28][29].When comparing the two types of cultures (solid and liquid culture), a significant difference on 6-PP biosynthesis was observed.e maximum 6-PP concentration in SSF was 80 times higher than the one obtained in LC. ese results confirm those previously obtained by Sarhy-Bagnon et al. [15] and Galindo et al. [30]; these authors explain that the inhibitory effect of 6-PP and the acidification observed in LC appeared to be responsible for the low concentrations obtained in the liquid medium.
ese results underline the differences between SSF and LC already observed in the literature.Indeed, for many secondary metabolites, SSF exhibits increased production when compared to its liquid counterpart [31].
On the contrary, a very marked pattern was observed on the relationship between conidiogenesis and 6-PP production: growth on the solid medium showed that conidia and 6-PP production followed a similar trend at the beginning of cultivation for T. asperellum G7. is does mean that the production of conidia and secondary metabolites from the genus Trichoderma is more related to the culture conditions which cause an increase in the concentration of 6-PP and conidia. is effect has a direct impact on the technical feasibility of conidia production because under these culture conditions, it is possible to use reactors such as trays, bags, or bioreactors to scale up the fermentation process for 6-PP and conidia production.
T. asperellum G7 selected by this screening is a promising strain, thanks to its ability to produce considerable quantities of 6-PP.
e behavior of this strain should be investigated against phytopathogenic fungi such as, for example, Fusarium oxysporum, Botrytis cinerea, Crinipellis perniciosa, and Rhizoctonia solani [12,13,32,33].For this reason, it is important to produce 6-PP, conidia, or biomass in large amounts to meet field requirements.In addition, the opportunity for local agroindustrial by-products of low commercial value to be used in a biotechnological process makes this process an important valorization approach.

Conclusion
Trichoderma strains isolated from different natural sources and derived from different laboratories (Mexico, Tunisia, Greece, and France) were screened for 6-PP production.6-PP production could be detected on solid media in the case of 7 Trichoderma strains.Among those, five belonged to the species T. asperellum (G7, G18, G3, G11, and G9).However, strains belonging to longibrachiatum species were unable to significantly produce 6-PP.e present study shows for the first time a new strain "T.asperellum G7" able to produce large amount of coconut aroma.Comparison between SSF and LC revealed that, for this strain, 6-PP production was continuous during 10 days of incubation on the solid medium, whereas in LC, 6-PP production decrease was observed in addition to being 80 times inferior compared to SSF. is indicates a clear advantage of SSF over LC for this process.

Figure 1 :
Figure 1: Evolution of 6-PP, glucose, and biomass in the liquid medium with Trichoderma asperellum G7.

Figure 2 :
Figure 2: Evolution of conidia and 6-PP production in the solid medium, during the culture of T. asperellum G7 on solid medium.
Identification.Total DNA was extracted from 50-200 mg of mycelium (fresh weight) using the

Table 1 :
Comparison of 17 strains of Trichoderma for their ability to produce 6-PP and conidia after 8 days of cultivation and molecular identification by ITS1 rDNA regions sequencing.

Table 2 :
Morphological characters of Trichoderma selected strains grown on PDA Petri dish and incubated at 27 °C for 4-5 days.