APPLICABILITY OF DISTILLATION-EXTRACTION TECHNIQUE FOR ISOLATION OF VOLATILE COMPOUNDS FROM DIFFERENT CULTURES OF MICROSCOPIC FUNGI

Elżbieta Gębarowska¹, Antoni Szumny², Stanisław J. Pietr^3
1 Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
² Department of Chemistry, Wrocław University of Environmental and Life Sciences, Poland
³ Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland

ABSTRACT

The aim of the study was to evaluate the effectiveness of this simple, solvent-free, one-step distillation-extraction method for the isolation of volatile metabolites from cultures of microscopic fungi applying a Deryng apparatus. For this study we tested liquid and solid-state cultures of filamentous fungi of Trichoderma viride, T. harzianum and Aspergillus niger and yeast fungi of Rhodotorula glutinis and Saccharomyces cerevisiae. The volatile compound, 6-pentyl-alpha-pyrone, was isolated via steam distillation, applying the Deryng apparatus as the only volatile metabolite from liquid and solid cultures of tested fungal cultures in the range from 2,1 to 109,6 μg in 1 mL or 1 g fungal cultures.

Key words: fungal volatile metabolites, lactones, Deryng apparatus, filamentous fungi, yeast.

INTRODUCTION

Numerous filamentous fungi from the genus Trichoderma, Aspergillus, Fusarium and Monilinia as well yeasts from the genus Rhodotorula are well known for producing several volatile compounds [1, 2, 15, 19]. They produce compounds, which belong to different structure classes such as mono-, sesqui-, diterpenes as well as corresponding terpenoids, alcohols, ketones, aldehydes, lactones, esters etc. [4, 10, 13, 16, 20]. They are products of secondary metabolism of microorganisms such as side- or end products from various intermediates. Microorganisms for this purpose can utilize various intermediates such as sugars, fatty acids and amino acids. These are available from agricultural and food industry by-products which are optically pure, extracellular, and suitable for commercial exploitation [3, 4, 13, 20].

There is a potential of volatile compounds for biotechnological applications within industry, medicine and agriculture. Some of them exhibit a phyto- and mycotoxic activities. In food and cosmetic industry, they can be used as flavoring and aroma agents, in medicine, as pharmaceuticals (antibiotic and immunosuppressant activity) [13, 16, 23]. Generally, these compounds have antimicrobial activity, but some of them often have obscure or unknown functions in the organism. Due to the antifungal, bactericidal and/or insecticidal activity, these compounds can be used as natural plant protection agents instead of synthetic pesticides in crop production. Moreover, fungal volatiles of soil-borne fungus may stimulate plant growth and natural defence responses in plants against pathogens [12, 13, 20, 25].

Natural origin lactone 6-pentyl-alpha-pyrone (CAS number 27593-23-3, syn. e.g. 6PAP, 6-n-pentyl-2H-pyran-2-one, 6-amyl-alpha-pyrone) is the most studied. It belongs to a group of unsaturated delta-lactones, and possesses an intensively coconut-like aroma. Usually it is biosynthesized by fungi from genus Trichoderma or Aspergillus [13, 16, 18, 20] and has fungicidal properties against a wide range of phytopathogens such as Botrytis cinerea, Chaetomium spp., Curvularia lunata, Aspergillus flavus [4, 9, 10, 13, 16, 26]. Production of 6PAP is also one of the key metabolite synthesized by Trichoderma species, which are commercialized as a biocontrol agent for plant production [13, 14, 20, 32].

For this purpose it’s a fast, simple and low-cost method of extraction of volatile metabolites as a potential for natural pesticides, which can be produced by different fungi utilizing divergent substrates as indicated.

There are various techniques for the extraction, e.g. solvent extraction methods, distillation methods (using Clevenger-type and Lickens-Nickerson apparatus), headspace techniques, sorptive techniques (Solid Phase MicroExtraction, Stirring Bar Sorptive Extraction, SBSE), liquid-surface immobilization system (Ext-LSI) [5, 17, 23, 27, 30].

Among the various techniques and methods of extraction of volatile compounds from fungal cultures, the most common used techniques are solvent extraction or less frequently, steam distillation mostly on Lickens-Nickerson apparatus. Both methods require large volumes of non-environmentally friendly organic solvent (e.g. dichloromethane, diethyl ether), vacuum rotary evaporators extractor etc. In the case of the distillation method, it requires a sophisticated apparatus [5, 15, 26, 29]. From our research data, each step of removing organic solvent (usually on rotary evaporator) could affect the composition of volatile compounds.

The aim of this work was to evaluate the effectiveness of the distillation-extraction for isolation of volatile compounds from different cultures of microscopic fungi applying a Deryng apparatus [8]. This apparatus is used for the isolation and determination of plant essential oils in the Central-European region and is modification of better known, Clevenger-type apparatus [11]. Moreover, the time of distillation using Deryng apparatus is much shorter then used Lickens-Nickerson.

MATERIALS AND METHODS

Tested fungi
Five strains of filamentous fungi (Trichoderma viride strain F74 and Tv1, T. harzianum strain 263 and Th1, Aspergillus niger strains F12) and two strains of yeast fungi (Saccharomyces cerevisiae strain Sc1, Rhodotorula glutinis strain Rg1) were used in this experiment. Tested strains came from the collection of the Department of Agricultural Microbiology of the Wrocław University of Environmental and Life Sciences. All strains were maintained on potato dextrose agar (PDA) medium, incubated at 26°C and stored in 4°C until use.

Liquid and solid state of fungal cultures
The possibility of using apparatus Deryng for the distillation-extraction of volatile metabolites from liquid and solid culture of fungi was investigated in the present study. The cultivation of fungi cultures were conducted under different growth conditions.

Filamentous fungi were cultivated on liquid Czapek Dox Broth medium (Emapol, Poland) and yeast was cultivated on Bacto Yeast Extract medium (Difco Laboratories Inc., USA). One hundred millilitres of media were inoculated with 10 mL of aqueous suspension (1 × 105 mL^-1) of spores and mycelium of fungi or cells yeast. Trichoderma strains were incubated for 14 days, A. niger F12 for 10 days and yeast for 7 days. The cultures of these fungi were incubated on a rotary shaker at 120 rpm at 27°C in the dark. After incubation, the entire cultures of fungi (mycelium with supernatant) were directly transferred to round-bottom flasks (500 mL volume) and the volatile metabolites were isolated by distillation-extraction applying the Deryng apparatus. As a control for this procedure was a culture of S. cerevisiae, which did not produce 6PAP.

The ability to produce volatile metabolites was also studied on solid media. For this purpose solid Potato Dextrose Agar (Difco Laboratories Inc., USA) and the sugar beet bagasse were tested. Petri dishes with 20 mL of PDA each were inoculated with 5 mm diameter plugs cut from five-day-old cultures T. viride F74 and T. harzianum 263, and were incubated at 27°C in the dark for 7 days. After incubation, the medium with mycelium and spores from 5 plates (100 mL) was transferred to a round-bottom flask and 200 mL distilled water was added before distillation-extraction. One hundred grams of sterile sugar beet bagasse in a conical flask was inoculated with 20 mL of an aqueous suspension T. harzianum 263 with spores and mycelium fragments. After mixing the medium with fungal inoculum the flask was stationary incubated at 27°C in the dark for 14 days. The whole culture was transferred to a round-bottom flask, and 200 mL distilled water was added before distillation-extraction. Our previous experiment data showed, that commercially available anti-foaming agents (like Antifoam 204, A, B Sigma-Aldrich) after distillation gave  traces of impurities on GC-MS chromatograms. Because of this fact, we decided to use, for prevention of foam forming, the cut part (about 5 g) of a clean steel wire ball placed into Deryng apparatus pipe.

Extraction and analysis of volatile metabolites
The whole one-step process, distillation-extraction using the Deryng apparatus was carried out for 1 hour in the presence of 1 mL of cyclohexane as an extracting agent with 200 μg of delta-decanolactone as the internal standard. The vapours were condensed by means of a cold refrigerant. After distilling, whole cyclohexane, containing the volatile metabolites, was collected and kept in a tightly closed vial at -15°C until GC–MS analyses were performed. As a control for this procedure (recovery percent), we distilled 100 mL of all used in experiment media (Czapek or PDA medium), containing 1 mL of pure delta-decanolactone (Sigma-Aldrich Co., USA) according to the procedure described above. After distillation-extraction exactly 1 mL of the delta-decanolactone was collected in the Deryng apparatus.

The chemical composition of the volatile compounds were identified using a gas chromatograph (GC) coupled to a mass spectrometer (MS), a Saturn 2000 MS Varian Chrompack with ZB-1 (Phenomenex) column (30 m × 0.25 mm film × 0.25 mm ID). The MS was equipped with an ion-trap analyzer set at 1508 for all analyses with an electron multiplier voltage of 1350 V. Scanning (1 scan/s) was performed in the range of 39–400 m/z using electron impact ionization at 70 eV. The analyses were carried out using helium as a carrier gas at a flow rate of 1.0 mL min^-1 in a split ratio of 1:20 and the following program: 60°C at the beginning and hold 3 min; 3°C min^-1 to 120°C; 15°C min^-1 to 300°C. The injector and detector were held at 200 and 300°C, respectively. The compounds were identified by using 3-analytical methods: Kovats indices (KI), GC/MS retention times of authentic chemicals-standards (S) and mass spectra of compounds and NIST08 spectral library collection (MS). The retention index standards used in this study consisted of a mixture of aliphatic hydrocarbons ranging from C5 through C20 dissolved in methanol.

RESULTS AND DISCUSSION

The objective of this study was to develop a simple, fast and reliable method for the investigation of volatile metabolites excreted from liquid or solid cultures of filamentous fungi and yeast without typical organic solvent. For this purpose we applied distillation-extraction using Deryng apparatus.

Results of the isolation of volatile compounds from different tested cultures of fungi applying a Deryng apparatus are presented Table 1. We identified 6-pentyl-alpha-pyrone (6PAP) as the only one volatile metabolite from liquid and solid cultures of tested fungi (e.g. Fig. 1, Fig 2). The amount of 6PAP determined in 1 mL or 1 g cultures ranged from 2,1 to 109,6 μg. The production of 6PAP proved to be dependent on both fungal species and growth conditions (liquid or solid, aerated or stationary cultures, incubation time). The greatest amount of 6PAP was produced in liquid and aerated cultures of T. viride strain F74. In cultures of T. viride Tv1, T. harzianum Th1 and S. cerevisiae Sc1 this lactone was not determined.

In the literature, production of 6PAP by fungi, especially from genus Trichoderma, is well documented [9, 13, 18, 20, 24]. The fact is that, some fungi can produce only one antibiotic compound, while others produce a number of them. In addition, the production of antifungal substances is varied with isolate, even within the same species.. Also, a particular Trichoderma strain may produce different metabolites at different stages of growth or under varying culture conditions [6, 7, 13]. Moreover, production of 6PAP was related to carbon and nitrogen sources [24, 28]. For example Sarhy-Bagnon et al. [28] from liquid culture of T. harzianum isolated ≈55 μg 6PAP mL^-1. When Trichoderma spp. growth on solid state cultures (sugarcane bagasse) production of 6PAP was higher (3 mg 6PAP g^-1).

Surprisingly, contrary to other authors [15, 29] in the obtained extracts, 6PAP was a pure compound (>98% according to CG) that we analyzed on gas chromatograph as well as on TLC. However, when we used sugar beet bagasse as a medium, we observed a decreased purity of ≈18% by volatile metabolites.

This method of isolation of volatile metabolites from fungi has many advantages. It does not require using larger amounts of chemical reagents (e.g. organic solvents as the extractive phase, drying agents) and specialized equipment (e.g. vacuum evaporator, SPE cartridges, chromatographic column) as was described in the literature [20, 26]. This approach for the isolation of volatile metabolites is simple, facile, and possible to perform in every laboratory. The described procedure is also inexpensive and environmentally friendly. This method of extraction may be used as a fast and simple evaluation of the efficacy of production of volatile metabolites by different microbes utilizing divergent substrates. In particular, it might be employed in a case where microorganisms comprised of multicomponent mixtures of metabolites or when raw materials are used for  production. In contrast to classic methods of isolation, the described procedure allows for the easy separation the non-volatile from the volatile metabolites. It is particularly important for the GC or GC-MS analysis, where the presence of non-volatile compounds in extracts, like lipids, could cause column as well as ion-source damage.

The other advantage of our method is a lack of influence of impurities presented in solvent used in extraction methods. These results have not been analyzed in known published literature. For example Siddique et al. [29], found in cultures of Trichoderma spp. metabolites (obtained via extraction by different organic solvents like metanol, ethyl acetate and n-hexan) compounds like toluene, heptane, octane, ethylbenzene, indane as original metabolites. Other researchers, Sunesson et al. [31] also found in extracts from cultures of Aspergillus versicolor and other filamentous fungi compounds like heptane, xylen, octane, acetone. Also Larsen and Frisvad [21] found artifacts as a metabolites of Trichoderma spp., like 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT), octane and hexen after distillation on Lickens-Nickerson apparatus. The absence of such contaminants in our tests could be related to the lack of organic solvent extraction steps, and then compacting of the sample.

This method of extraction may be used as a fast and easy evaluation of the efficacy of different microbes and substrates for the production of volatile metabolites, particularly in cases when microorganisms comprising multicomponent mixtures of metabolites or raw material are used for the production. Opposite to classical methods of isolation, the described procedure allows for an easy way to separate non-volatile metabolites from volatiles. It is particularly important for GC or GC-MS analysis, where presence of non-volatile compounds like lipids in extracts could cause damage of column.

CONCLUSIONS

1. A new approach for the isolation of volatile compounds from  fungi cultures has been developed. This procedure did not require solvents and high-cost equipment, such as evaporators.
2. This is a easy method of evaluation of fungi for the production of volatile metabolites. The strains which produce a large quantity of volatile metabolites are potential candidates for use in biological plant protection within the cosmetic and food industry or for the production of fragrances.
3. Applying the developed procedure 6-pentyl-alpha-pyrone in the range from 2.1 to 109.6 μg in 1 mL or 1 g from tested fungal cultures was isolated.

Accepted for print: 20.09.2014

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