Introduction
Production of aromatic compounds play great importance in industrial world. It is estimated that the world market for aromatic compounds reaches about 18.6 million USD in 2008, and tend to increase higher today (1). Thus, it means that production of aromatic compounds seems to be promising.

Aromatic compounds can be found in perfumes, essential oils and food as flavors (2).Aromatic compounds can be produced by chemical synthesis or extraction from natural material (3). However, production from natural is more preferred since it is more environmental friendly and safe for organisms. The problem is, extraction from natural is sometimes limited by their low concentration. To overcome this, production of aromatic compounds based on microbial synthesis or bioconversion is commonly used.

Around 100 aromatic compounds have been produced by microbial fermentation. Solid-state fermentation (SSF) has been known as fermentation method that allow the utilization of agro-industrial wastes as substrates to produce aromatic compounds. In another view, it gives added-value in solving the pollution problem. Some researchers have reported the use of cassava bagasse, sugarcane bagasse, apple pomace, giant palm bran, andcoffee husk as substrates for microbial aromatic production by SSF(4).Some studies reported the successful ofTrichoderma viridae in producing aromatic compounds using sugarcane bagasse as substrates (5,6), while another researches alsoreported the use of Trichoderma harzianumin sugarcane bagasse which both of the strain produced 6-pentyl-a-pyrone (6-PP), an unsaturated D-lactone with strong coconut aroma-like (7,8).

In this research we tried to explore the potential production of aromatic compounds by SSF of Trichoderma viridae in Pandanus tectorius fruits as substrates. Pandanus tectorius fruit is commonly found as wastes in south coastal of Yogyakarta Province, Indonesia since none is known about the advantages of this fruit. The fruit textures is really fibrous and causing skin itches, therefore it is inedible for human or animal consumptions (Fig.1)

Figure:1 Morphology of Pandanus tectoriusfruit fiber. Courtessy of (9) Click here to view figure

So far, research about production of aromatic compounds by SSF of Trichoderma viridae in Pandanus tectorius fruitsas substrates has not been reported yet. We investigate and report this work with the hope that it will give contribution for the next research about production of aromatic compounds using SSF.

Material and Methods

Microorganism and inoculum preparation : Fungal strain of Trichoderma viridaewas gained from the culture collection of Food and Nutritional Centre Studies UGM, Yogyakarta. The fungal was grown and transferred periodically in Potato Dextrose Agar (PDA) slants medium then kept at 4°C for storage. Spore suspensions was prepared from 7-days old culture by adding 0,2 mLTween 80 dissolved in sterile distilled water. The spore were then counted by Neubauer Chamber until it reached concentration about108 spore / mL.

Substrate preparation : Pandanus tectoriusfruits used in this research were obtained from Watu Kodok Beach, Gunung Kidul, Yogyakarta, Indonesia. Samples were taken in month of June (dry season) at 12.00 noon. Basal mesocarp which was the soft and fibrous part of the fruit were used in this research. It was cutted, juiced, then the fiber pulps were dried and grinded until reached fine particle size. Nutritional composition of the substrates were then analyzed e.g. protein (Kjedahl method), lipid (Soxhlet extractions), total sugars (Phenol sulphate) and moisture (Gravimetry method).

Solid- State Fermentation (SSF): Solid-state fermentation were doneby using 3 g of dry substrates in 250 mL flasks. The flasks were covered with cotton, sterilized in 121°C for 15 minutes, and inoculated with 108spores/mL. Fermentations were carried outinpH 6.0, 76,6% moisture, at 30°C temperature for 10 days.

Extraction of aromatic compounds: Aromatic compounds was extracted by adding fermented product with 30 mL distilled water and further extracted with 30 mL dichlormethane. The mixture were then dried over sodium sulphate anhydrous before continued to GC/MS analysis.

GC/MS analysis: The GC/MS analysis was done using GC/MS Agilent HP 5 MS UI apparatus, 30 m DB methyl silicone with 0,25 mm ID and 0.25 μm film thickness with helium as carrier gas. The gas velocity was 25,9 cm/s. GC oven was programmed in 70°C for 5 min for initial temperature until it reached 280°C for 34 min. The ion temperature was 250°C, EI ionization was 70 eV with range scan was m/z 25-600. GC/MS data is then further processed with the available software, using NIST 12 LIB, NIST 62 LIB and WILEY 229 LIB library.

Results

The potential of material to be used as fermentation substrates is determined by their nutritional compositions. From the proximate analysis of substrates, we found that dried Pandanus tectorius fruits contained 13,015% total sugars, 3% proteins, 1,39% lipids and 76,6% moisture (Table 1).

Table 2 : Aromatic compounds produced by SSF ofTrichoderma viridae inPandanus tectorius Fruits

Aromatic compounds identified from SSF of Trichoderma viridae were mostly consisted of alkanes(tetradecane, tertracosane, tetracosahexaene, pentadecane, hexacosane, heptadecane, dan octadecane). While other compounds such as alcohol (phenol), amide (9-octacenamide) and monoterpene aldehid (9-octadecenal) only found in little compositions.

Discussion

The isolate of Trichodermaviridae exhibited slow growth in Pandanus tectorius solid substrates. This was probably because of low total sugar content in Pandanus tectorius which was important to provide energy sources for microbial growth and aromatic compounds production (6). According to Soares et al.(10),glucose has directional effects on microbial metabolism pathways and volatile compounds production. To overcome fungal slow growth, soluble sugars may be added to the materials in order to support microbial growth (11).

Despite low total sugar contents in substrates, protein and moisture contents of Pandanus tectorius fruits were nearly ideal for SSF growth (50-70%) (12). So it is possible that Pandanus tectorius fruits may provide suitable conditions for aromatic compounds production by SSF. The optimization of fermentation conditions are needed further, in order to create better yields in microbial biomass growth and higher aromatic compounds concentration.

Aromatic compounds produced by SSF of Trichoderma viridae had almost similarities with Trichoderma atroviridae detected by HS-SPME. The microbial aromatic compounds detected in the culture samples of Trichoderma atroviridae consisted of the compound classes of alkanes, alcohols, ketones, pyrones, furanes, monoterpene and sesquiterpenes (13), however aromatic compounds from SSF of Trichoderma viridae constituted lower and different members in classes than Trichoderma atroviridae.

Aromatic compounds were also expected to be indigenous resulted by SSF of Trichoderma viridae in Pandanus tectorius fruits since it showed difference aromatic compounds compositions obtained by SSF of Trichoderma viridae and Trichoderma harzianumgrown in sugarcane bagasse (5,6,7,8). Therefore we concluded that aromatic compounds were potentially produced by SSF of Trichoderma viridaein Pandanus tectorius fruits.

Acknowledgments

We thank to Indonesian Ministry of Research, Technology and Higher Education for State University Operational Assistance Fund 2015- Research Grants Scheme for Faculty of Biology UGM; Laboratory of Chromatography, Faculty of Pharmacy Universitas Gadjah Mada for GC/MS analysis.

References

1. Rossi ,S.C., L.P.S. Vandenberghe  B.M.P. Pereira, F.D. Gago , J.A. Rizzolo, C.R. Soccol , A.B.P. Medeiros. Improving fruity aroma production by fungi in SSF using citric pulp. Food Res. Int. 2009; 42: 484–486
   CrossRef
2. Solange I. Mussatto, Lina F. Ballesteros, S. Martins and José A. Teixeira. In : Use of agro-industrial wastes in solid-state fermentation processes, industrial waste (Prof. Kuan-Yeow Show, Ed). ISBN: 978-95351-0253-3, InTech, Available from: http://www.intechopen.com/books/industrial-waste/use-of-agro-industrialwastes-in-solid-state-fermentation-processes, 2012; pp 121-140.
3. Longo, M.A. and M.A. Sanroman.Production of food aroma compounds. Food Technol. Biotechnol.2006; 44 (3): 335–353
4. Medeiros, A.B.P., Ashok Pandey, Luciana P. S. V G M. Pastore and C R. Socco. Production and Recovery of Aroma Compounds Producedby Solid-State Fermentation Using Different Adsorbents.Food Technol. Biotechnol. 2006; 44 (1): 47–51
5. De Aráujo,A.A.D.. G.M. Pastore, and R. G. Berger. Production of coconut aroma by fungi cultivation in solid-state fermentation. App. Biochem.Biotech. 2002;98-100
   CrossRef
6. Fadel, H.H. M G Mahmoudb, M M S Askerb, S N Lotfy. Characterization and evaluation of coconut aroma produced by Trichoderma viride EMCC-107 in solid state fermentation on sugarcane bagasse. Electron. J. Biotechnol. 2015; 18 (1)
   CrossRef
7. Carreón, L.S., K. Balderas-Ruíz, E. Galindo, M. Rito-Palomares. Production and biotransformation of 6-pentyl-a-pyrone by Trichoderma harzianum in two-phase culture systems.Appl Microbiol.Biotechnol. 2002; 58:170–174
   CrossRef
8. De Penha, M.P., M.H.M. da Rocha Leao, and S.G.F. Leite. Sugarcane bagasse as substrate for the production of coconut aroma for solid state fermentation. Biores. 2012; 7 (2) : 2366-2375
   CrossRef
9. Thomson, L.A.J., L. Englberger, L. Guarino, R.R. Thaman, and C. Elevitch : Pandanus tectorius (pandanus), ver. 1.1. In:Species Profiles for Pacific Island Agroforestry (C.R. Elevitch, ed.). Hōlualoa: Permanent Agriculture Resources (PAR). http://www.traditionaltree.org, 2006; pp 1-29
   CrossRef
10. Soares M, Christen P, Pandey A& Soccol CR. Fruity flavor production by Ceratocystis fimbriata grown on coffee husk in solid-state fermentation.Proc. Biochem. 2000; 35 : 857-861.
    CrossRef
11. Manpreet, S., Sawraj, S., Sachin, D., Pankaj, S. and Banerjee, U.C. Influence of Process Parameters on the Production of Metabolites in Solid-State Fermentation. Mal. J. Microbiol. 2005; 1(2) : 1-9
    CrossRef
12. Pandey A., P. Selvakumar, C. R. Soccol, Poonam Nigam. Solid state fermentation for the production of industrial enzymes.Curr. Sci. 2001; 77 (1) : 149-162
13. Stoppacher, N., B. Kluger, S. Zeilinger, R. Krska and R. Schuhmacher. Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS.J. Microbiol. Methods. 2010; 81 : 187–193
    CrossRef