Biochemical Profile by GC–MS of Fungal Biomass Produced from the Ascospores of Tirmania nivea as a Natural Renewable Resource

The edible fruiting bodies of desert truffles are seasonally collected and consumed in many regions of the world. Although they are very expensive, they are bought and sold as a result of considerable scientific reports confirming their health and nutritional benefits. This study aimed to conduct laboratory production of the fungal biomass of Tirmania nivea as a natural renewable resource of many active biological compounds using an artificial growth medium. The T. nivea collected from Hafar Al-Batin, which is north of Saudi Arabia, and their ascospores were harvested and used to produce fungal biomass in potato dextrose broth. The cultivation was conducted using a shaking incubator at 25 °C for two weeks at 200 rpm. The crud extracts of the fungal biomass and mycelium-free broth were prepared using ethyl acetate, methanol and hexane. Preliminary gas chromatography–mass spectrometry (GC–MS) analysis and their biological activity as antimicrobial agents were investigated. The results showed that the crude extracts have biological activity against mold, yeast and bacteria. The preliminary GC–MS analysis reported that the fungal biomass and extracellular metabolites in the growth medium are industrial renewable resources of several biological compounds that could be used as antifungal, antibacterial, antiviral, anticancer, antioxidant, anti-trypanosomal and anti-inflammatory agents.


Introduction
Tirmania nivea, which is locally called zubaidi, fagaa or kma'at, is an edible hypogenous fungal fruiting body. From a taxonomic point of view, the genus Tirmania belongs to the family Terfeziaceae and the order Pezizales. T. nivea is collected seasonally from several desert environments, including Saudi Arabia's desert. It is consumed because of its distinctive flavor and nutrient value, and it is relatively expensive due to its natural and seasonal growth associated with specific conditions and limited environments [1,2].
Generally, desert edible fungal fruiting bodies are ethnopharmacologically used to treat eye infections and fatigue, as well as promote fertility in men [3]. Chemical analysis showed that 100 g of a dry fruiting body of T. nivea consisted of fat, protein, carbohydrates and ash at 6.78, 28.8., 57.8 and 5/100 g, respectively; ascorbic acid, carotenoids and anthocyanins at 10.6, 1.1 and 29.1/100 g; and minerals such as potassium, calcium, magnesium, iron, sodium, phosphate, manganese and copper [4].
The major volatile organic compounds of the ascocarp of T. nivea include unsaturated fatty acids and hexa-decanoic acid [5]. Antioxidant chemicals extracted from the fruiting body of T. nivea vary according to its natural source. Generally, it contains anthocyanins, ascorbic acid, phenolics, flavonoids and carotenoids [6]. Aside from using truffles as food, many studies have proven that they are an important source of biological compounds, such as antioxidant, antibacterial, antifungal, antiviral, hepato-preservative, anticancer and anti-inflammatory agents [7]. A crude ethyl acetate extract obtained from T. pinoyi showed biological activity as an antimicrobial agent against Enterococcus sp., Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli [8]. Some alcoholic extracts produced from the ascocarp of T. nivea and Terfezia claveryi showed biological activity against the human liver cancer cell line HepG2 [9]. The aqueous extracts of some desert truffles could be used to produce nanoparticles, which could be introduced in some applications [10].
Ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is used to analyze fruiting bodies of T. nivea and T. claveryi. It was reported that T. nivea and T. claveryi fruiting bodies contain behenic acid, resveratrol, margaric acid, naringenin, oleic acid, and lauric acid and that the highest hit bio-compounds related to anti-inflammatory targets [11]. The fruiting bodies of these truffles have a distinctive aroma because they contain significant volatile bio-compounds such as esters, alcohols, aldehydes, ketones, and sulfurs. Their therapeutic features involve their biological activity as antioxidants, antiviral, antimicrobial, hepatoprotective, and inflammatory agents. The major bio-compounds responsible for curative properties include phytosterols, N-arachidonoylethanolamine (anandamide), phenolics, and steroidal glycosides (tuberoside) [12].
Although there have been successful attempts to cultivate some types of desert truffle, such as T. claveryi [13], there are many challenges related to climatic conditions and weather effects. Given the high yields of mycelia that can be produced in a laboratory, this work was designed to produce mycelia of T. nivea using artificial media in the laboratory and to perform a gas chromatography-mass spectrometry (GC-MS) analysis of several alcoholic extracts to determine the active biological compounds from the produced mycelia.

Sample Collection
Edible desert truffles (T. nivea) were collected from Hafar Al-Bati, which is north of Saudi Arabia (coordinates: 28 • 26 3 N 45 • 57 49 E), between 1 March and 30 April 2021. The samples were immediately transferred using iceboxes to the Microbiology Laboratory, Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia. The morphological features of the samples were recorded, and the samples were washed three times using water. The external surface of the samples was sterilized three times using an ethanol solution (70% ethanol), and then the samples were washed three times using sterile water. The samples were put in sterile plastic boxes and preserved at −20 • C until further analysis.

Macroscopic and Microscopic Study
Morphological characteristics of sporocarp (fruiting body) and ascospore were recorded. A thin layer of the internal part of the samples was examined using digital light microscopy (Motic plus 2 ML, Xiamen, China) without using any stains and with lactophenol cotton blue stain (Sigma-Aldrich, St. Louis, MO, USA). The characteristics of the asci and ascospores were recorded. Colony characteristics grown from the ascospores were studied on potato dextrose agar (PDA) (Oxoid Ltd., Basingstoke, UK) after incubation at 25 • C for 48 h. then the fungal hyphae were examined using light microscopy.

Mycelia Production
The spore suspension was formulated from the internal part of the sample prepared, as mentioned above. The internal part of the sample was vigorously crushed, and centrifugation (Universal 320, Hettich, Zentrifugen, Germany) was conducted at 3000× g for 10 min. The supernatant was collected, and a direct microscopic count using Petroff-Hausser counting chambers and a standard plate count using the pour plate method were used to count and determine the viability of the ascospores. The mycelia of T. nivea were produced from the ascospore suspension using potato dextrose broth (Oxoid Ltd., Basingstoke, UK) (0.4 g of potato extract produced from 20 g of infused potato and 2 g of dextrose per 100 mL of distilled water) at 25 • C for two weeks at 200 rpm (IKA ® KS 4000 i control, Staufen, Germany). Mycelia were collected from the broth using centrifugation at 2000× g for 10 min. The collected mycelia were washed three times using sterile normal saline solution (0.89% sodium chloride). The wet biomass was collected, and the yield (g of wet biomass per 100 mL of the medium used for cultivating the mycelium of T. nivea) was calculated.

Alcoholic Extracts
The production of crude alcoholic extracts was performed using methanol (Sigma-Aldrich, USA), ethyl acetate and hexane (Avonchem, Macclesfield, UK), and n-hexane (Sigma-Aldrich, St. Louis, MO, USA). Exactly 100 mL of the mycelium-free broth or 10 g of wet mycelial biomass was extracted by 100 mL of solvent through the maceration method at room temperature using a shaking incubator at 200 rpm (IKA ® KS 4000 i control, Staufen, Germany). The extraction was repeated three times, and a fresh solvent was used each time. The crude extracts were filtered, and the solvent was removed using a rotary evaporator (IKA ® Rv 10, Staufen, Germany).

GC-MS Analysis
Agilent GC 7890A jointed with a triple-axis detector 5975 C single quadrupole mass spectrometer were used for GC-MS analysis. The column of chromatographic was an Agilent HP 5MS column (30 m × 0.25 mm × 0.25 µm film thickness), with gas carrier (highpurity helium, at a flow rate of 1 mL/min). The temperature of the injector was 280 • C and it was equipped with a splitless injector at 20:1. The temperatures were set at 230 • C and 150 • C for the source temperature of MS and the Quad temperature, respectively. The initial oven temperature was 40 • C for 1 min, then it was increased to 150 • C at 10 • C min −1 for 1 min, increasing further to 300 • C at 10 • C min −1 for 1 min. The scanning range was set at 40 to 600 mass ranges at 70 eV electron energy with solvent holdback of 3 min. Eventually, unknown compounds were identified by comparing the spectra with those of the NIST 2008 (National Institute of Standard and Technology library). The single sample analysis was 29 min as required total time for analysis.

Biological Activity of Crude Alcoholic Extracts
The preliminary biological activity of the crude extracts against bacteria, mold and yeast was tested using a disk diffusion assay. A 6-mm sterile disk was loaded with 4 mg of the crude extract, and the test protocol was followed according to the studies of Desbois and Smith [14] and Khadka et al. [15] using E. coli ATCC 25922, S. aureus ATCC 29213, Candida albicans ATCC 60193 and Aspergillus niger Wild strain.

Macroscopic and Microscopic Characteristics
T. nivea has a white edible fruiting body with a size that can sometimes reach more than 10 cm. The white color of the fruiting body changes to dark yellow over time as a result of exposure to air. The amyloid ascus of T. nivea has an ellipsoid shape, with eight smooth ascospores ( Figure 1). Figure 2 shows the morphological culture and microscopic characteristics of colony produced from T. nivea ascospores on PDA. Hairy white colonies (2.5 cm in size) after incubation for 48 h, with a light black center and light yellow pigment, can be shown in the reverse view of the colony. The diameter of the septate-hyphae is approximately 4 µm.

Mycelial Biomass Production
The ascospores of T. nivea can be cultivated on potato dextrose broth to produce a spherical mass of mycelium ( Figure 3A). The morphological characteristics of the biomass produced from laboratory cultivation are shown in Figure 3B. The yield (g of biomass/100 mL of broth) was 6 ± 1 (N = 3).

Mycelial Biomass Production
The ascospores of T. nivea can be cultivated on potato dextrose broth to produce a spherical mass of mycelium ( Figure 3A). The morphological characteristics of the biomass produced from laboratory cultivation are shown in Figure 3B. The yield (g of biomass/100 mL of broth) was 6 ± 1 (N = 3).

GC-MS Analysis of Crud Extracts
The GC-MS analysis of crude methanol, ethyl acetate and hexane extracts obtained from the wet mycelial biomass was performed to screen the active biological compounds that could be produced from the mycelium. The results showed that the crude ethyl acetate, methanol and hexane extracts could be renewable resources of many biological compounds (Tables 1-6). The results showed that the crude extracts are sources of many important compounds such as hexanal, dodecane, 4-hydroxyphenylacetic acid, heptadecane, 1-eicosene, hexa-decanoic acid-methyl ester, 7-pentadecyne, phenol, 2,4-bis(1,1-dimethylethyl), methyl palmitate, methyl linolelaidate, methyl oleate, methyl stearate, 9,12-Octadecadienoic acid (Z,Z)-, heptadecane, 1-octadecene, 5-hydroxymaltol, 11-dodecen-1ol trifluoroacetate, and other compounds of medicinal and industrial importance listed in the tables.     Table 5. The GC-MS analysis of the crude hexane extract obtained from wet mycelial biomass produced using ascospores of T. nivea.

Biological Activity of Crude Alcoholic Extracts
The data from the disk diffusion test showed that all the extracts had biological activity against all the tested microorganisms. The inhibition zones resulting from the activity of the extracts ranged from 8 mm to about 13 mm. The findings showed that crude methanol hexane extracts of the mycelia-free broth had the highest biological activity against tested microbes among all extracts investigated in this work (Table 7).

Discussion
This study is the first of its kind to use the ascospores of T. nivea to produce mycelial biomass as a renewable resource of biological compounds. The yield of wet biomass and the compounds detected in fungal-cell free broth are encouraging for the consideration of industrial applications that might depend on this source. The GC-MS analysis of the mycelial biomass and by-products biosynthesized as extracellular compounds in a growth medium (potato dextrose broth) reported that mycelial biomass and its by-products are natural renewable resources of several important biological compounds that have many applications in the medical and industrial fields. The production of biological compounds from the fruiting body of T. nivea requires a long period of time and is affected by climate change, unlike the current method, in which all production conditions are controlled inside a laboratory.
The preliminary screening of chemical compounds of the crude ethyl acetate of wet mycelia biomass showed that the extract contained the following important biological compounds: hexanal (hexanaldehyde), which is applied in the flavor industry [16]; anthracene, which is used to produce dyes [17]; 8-methyloctahydrocoumarin, which can be used as a coumarin derivative [18]; dodecane, which is used as a solvent and a scintillator [19]; phenol, 2, 4-bis (1,1-dimethylethyl), which has many applications in medication and food [20]; 4-hydroxyphenylacetic acid, ethyl ester and tert-butyl-dimethyl-silyl, which is a 4-hydroxyphenylacetic acid derivative that plays an important role in metabolic reactions in plant, fungi, animals and humans [21]; heptadecane, which is used in the production of essential oils [22]; oleic acid, which has many applications in human and animal food as emulsifying agent and in cosmetics [23]; 1-eicosene, which is used as a monomer to produce several chemical copolymers [24]; hexa-decanoic acid methyl ester, which is an anticancer compound [25]; tetra-decanoic acid (i.e., myristic acid used in cosmetics) [26]; 9,12-octadecadienoic acid and methyl ester (linoleic acid), which is used as a hypocholesterolemic, anticancer and anti-inflammatory agent, among others [27]; and 7-pentadecyne, which is an anticancer compound [25].
Similar biological compounds were extracted using ethyl acetate from both wet mycelia biomass and mycelia-free broth, such as phenol, 2, 4-bis (1,1-dimethylethyl), heptadecane, hexa-decanoic acid methyl ester (which was also found in the crude methanol extract and crude hexane extract of wet mycelia biomass) and 1-eicosene. All the biological compounds listed in Table 2 were detected only in the crude ethyl acetate of wet mycelia biomass, except for the compounds mentioned above and cis-9-octadecenamide (i.e., oleamide, a drug for anxiety disorders) [28], which was also found in the crude hexane extract of wet mycelial biomass. The compounds presented in Table 2 include those that could be used in industrial applications, such as octadecanamide (stearamide) [29,30], stigmasta derivatives [31], 1-octadecene (which has an important role in nanoelectronics production) [32] and methyl decanoate [33]. Table 3 presents the following compounds, also found in other extracts: dodecane (found in crude ethyl acetate), 7-pentadecyne (found in crude ethyl acetate), hexa-decanoic acid methyl ester (found in the crude extract of ethyl acetate and crude hexane extract of wet mycelial biomass), 9,12-octadecadienoic acid (Z,Z)-methyl ester (found in crude ethyl acetate and hexane extracts of wet mycelial biomass), 9-octadecenoic acid (Z)-methyl ester (found in crude ethyl acetate and hexane extracts of wet mycelial biomass), methyl 16-methyl-heptadecanoate (found in the crude hexane extract of mycelium-free medium) and n-hexa-decanoic acid (found in the crude methanol extract of wet mycelial biomass and considered an anti-inflammatory agent [34] and a potential anticancer agent [35].
In industrial mycology, there are two major two forms of applied production, the first from fungal biomass (fungal cells) and the second from metabolic products which are frequently extracted from the cell-fee broth. In the present work, the findings reported significant variety between the compounds detected in mycelia-free broths and fungal biomass for all extracts used. For example, octa-decanamide (which has several industrial applications [30] was extracted using ethyl acetate from fungal cell-free broth but not from fungal cell biomass. The same applies to carbamic acid (whose esters are commonly used as insecticides [53] and its derivates. With respect to crude methanol extracts, for example, the dodemorph (fungicide agent [54] can be produced from the fungal cell-free broth using methanol in the primary extraction stage. Among all extracts obtained in this works, the crude methanol extract obtained from the mycelia-free broth was the only resource of several compounds such as dodemorph, 1-piperidineethanol, and maltol. The same result was repeated with regard to crude hexane extracts, where it was confirmed that fungal cell-free broth is considered a resource of many compounds that are absent from the fungal biomass. For example, caryophyllene, heptacosane, Tetratriacontane, dibutyl phthalate, and docosene. Thomas et al., [55] reported that there are many industrial applications of Truffles including T. nivea. The applications include food, medicinal, and nanoparticles. In general, truffles (for example, T. nivea) are considered as one the most important sources of antioxidant agents such as phenolics, β-carotene, and ascorbic acid [56]. There are several antimicrobial agents detected in this work such as 1-Piperidineethanol and its derivatives (in the crude methanol extract of mycelia-free medium), Benzimidazole (in crude hexane extract of mycelial biomass and its derivatives), and dodemorph (in crude methanol extracts of mycelia-free medium). Some studies have reported that the fruiting body of T. nivea contains many antimicrobial agents [7,11] but there no work investigated the biomass produced from ascospores T. nivea as a source of potential antimicrobial agents.

Conclusions
This study investigated the preliminary chemical screening of the major volatile organic compounds of mycelia biomass produced from the ascospores T. nivea and metabolites secreted as extracellular products. The yield of wet mycelium biomass reached about 6 gm per 100 mL of medium; furthermore the fungal cell-free broth was the source of many important compounds. The findings show that the crude extracts of ethyl acetate, methanol and hexane obtained in this study could be natural renewable resources in several biological activity compounds that play the role of antibacterial, antifungal (for example, piperidine-ethanol, benzimidazole, and dodemorph), antiviral, anticancer, antioxidant, anti-trypanosomal and anti-inflammatory agents. The crude hexane and methanol of fungal cell-free broth extracts could be the best source of antimicrobial agents among all tested extracts. In addition, many compounds were found in all the crude extracts that could be used in industrial applications. We suggest confirming the benefits of this novel and renewable resource through small-scale production of its products and further investigation of its biological activity.