INTRODUCTION
Many fungi from various environments on earth have been identified to produce an extensive variety of chemical compounds which possess bioactivity. The formation of these chemical compounds can be through primary and secondary metabolism. Chemical compounds generated by fungi cannot be thoroughly demonstrated by their function in the metabolism of these fungi. Generally, these chemical compounds can function as a detoxification mechanism for fungi and have the ability to communicate between fungi and the surrounding environment (Cometto-Muñiz et al., 2005; Humphris et al., 2002). One kind of metabolites discovered in fungi is a volatile compound. Terpenes, alcohols, hydrocarbons, ketones, esters, carboxylic acids, and some volatile sulfur-containing compounds are among the diverse chemical classes represented in the secondary metabolites of fungi. Fungi from oceanic regions are also able to produce these volatile chemical compounds, in addition to those produced by fungi from lands. Researchers are currently interested in studying marine fungi because they produce a variety of chemical compounds that are distinct from those produced by their terrestrial counterparts. An example of a fungus revealed in marine environments is one connected to marine sponges. The marine sponge is a marine biota whose body is almost 60% in symbiosis with other microorganisms; the one incorporating them is a fungus. Marine sponge-associated fungi contribute significantly to the metabolic chain in the sponge body (Brinkmann et al., 2017; Taylor et al., 2007; Zhang et al., 2005). There are several factors causing fungi originating from marine areas, particularly marine sponge-associated fungi, to own different chemical compounds from their relatives that grow in land areas, one of which is extreme environmental conditions or experiencing biological stress such as limited carbon supply and insufficient sunlight, and the osmotic pressure is relatively high. It causes marine-derived fungi to develop their own metabolic pathways to generate new secondary metabolites with various biological activities (Debbab et al., 2011; Huang et al., 2011; Samirana et al., 2021a).
One of the marine sponges with the species Stylissa flabelliformis has been understood to possess several quite diverse marine sponge-associated functions. Sponge S. flabelliformis was obtained from the waters of Menjangan Island, West Bali National Park (Indonesia). The marine sponge-derived fungi that have been isolated from the S. flabelliformis sponge incorporate with Aspergillus flavus UPMZ02 (SAL 1), Aspergillus fumigatus CD1621 (SAL 2), Trichoderma reesei JCM 2267 (SAL 3), Aspergillus nomius KUB105 (SAL 4), Aspergillus sp. TLWK-09 (SAL 5), A. flavus MC-10-L (SAL 6), Penicillium sp. RMA-2 (SAL 7), Aspergillus sp. TLWK-09 (SAL 9), A. fumigatus (SAL 9), and T. reesei TV221 (SAL 10). The marine sponge-associated fungus S. flabelliformis with codes SAL 5, SAL 6, SAL 7, SAL 9, and SAL 10 owned antimicrobial activity on Staphylococcus aureus American type culture collection (ATCC) 29213, Escherichia coli (EC) ATCC 25922, and methicillin-resistant S. aureus (MRSA) which were assessed from their zone of inhibition. In accordance with the further research, among the five marine sponge-associated fungi of S. flabelliformis that possess antimicrobial bioactivity, T. reesei TV221 owns the most antimicrobial potential (Samirana et al., 2021b; Setyowati et al., 2018, 2017). In accordance with the results of this study, the fungus T. reesei TV221 associated with S. flabelliformis sponges is tremendously potential to be advanced as an antimicrobial agent in the future and can be detected to have bioactive compounds that act as antimicrobial agents.
The search for antimicrobial bioactive compounds from the fungus T. reesei TV221 can be performed by utilizing a metabolomics approach. The metabolomics approach is a process of discovering bioactive compounds by employing the chemical profile of natural products. The combination of chemical profiles with bioactivity data produces big data that will lead to bioactive compounds from these natural products (Xu et al., 2006; Yuliana et al., 2011). As previously identified, most of the secondary metabolites of fungi are volatile chemical compounds. A reliable chemical profile which is able to represent the active components and their chemical characteristics are acquired through appropriate instrumentation techniques. Precise instrumentation enhances separation capability, measurement precision, and selectivity and reduces instrument interference. The most appropriate instrumentation for the separation of volatile compounds is by employing the gas chromatography-mass spectroscopy (GC-MS) technique. The GC-MS technique in research associated with metabolomics requires the separated metabolites to be volatile and thermostable (Han et al., 2009; Samirana et al., 2022).
In this study, to obtain an adequate variety of chemical profiles, variations were conducted on the fermentation medium of the fungus T. reesei TV221 in the form of variations in the Dextrose content and salinity of the fermentation medium. Furthermore, fermented products such as supernatant and biomass were extracted with ethyl acetate to acquire different types of extracts. It is expected that with numerous factors of variation, the chemical profile and antimicrobial activity produced can also vary which will later be able to lead to bioactive compounds that function as antimicrobials.
MATERIAL AND METHOD
Materials
Marine sponge-derived fungus T. reesei TV221 from the marine sponge S. flabelliformis was obtained from the waters of Menjangan Island, West Bali National Park (Indonesia). The test microbes employed in the antimicrobial study were S. aureus (SA) ATCC 25923 and EC ATCC 25922, Sabouraud Dextrose Agar (SDA) (Oxoid, UK), Dextrose (Oxoid, UK), Tryptone (Oxoid, UK), Peptone P (Oxoid, UK), nutrient agar (Oxoid, UK), nutrient broth (Oxoid, UK), ethyl acetate (Merck, German), methanol (Merck, German).
Fermentation and extraction of secondary metabolites from fungi
The T. reesei TV221 fungal isolate associated with the S. flabelliformis sponge was used in a previous study and had been stored at the Indonesian Center for Biodiversity and Biotechnology Bogor (ICBB) for recultivation (Setyowati et al., 2018). Identification and authentication have also been carried out at ICBB. The fungus T. reesei TV211 associated with sponge S. flabelliformis was recultured on SDA with 30 parts per trillion (ppt) salinity seawater as a solvent to enhance the number of fungi. Fungal cultures were incubated for 7 days at room temperature. Fermentation was administered with medium variations in Dextrose and salinity levels in accordance with Table 1 for 12 days at 25°C and placed on a rotary shaker (120 rpm). After 12 days of fermentation, the supernatant and biomass were separated. The supernatant and biomass extraction processes followed the procedures performed in previous studies (Samirana et al., 2021b).
Antimicrobial activity
Antimicrobial testing in this study utilized the determination of the zone of inhibition. The bacteria employed in the antimicrobial test were EC ATCC 25922 and S. aureus ATCC 25923. The antimicrobial testing procedure in this study followed the procedures administered in previous studies with minor adjustments (Samirana et al., 2021b; Syukri et al., 2021).
GC-MS chemical profiling
The supernatant and biomass extract samples were dissolved in methanol at a rate of 1 mg/ml. GC-MS preparation for chemical profile determination was administered in accordance with the procedure in a previous study (Setyowati et al., 2017). The profile data obtained are compared with the WILEY7.LIB library.
Statistical analysis
After obtaining the GC-MS chromatogram profile data, the mass spectrum data were correlated with the WILEY7.LIB library. The matching factor or Similarity Index (SI) is implemented at least 80%. The metabolomics approach in this study was examined by chemometric methods such as principal component analysis (PCA) and cluster analysis with a hierarchical approach employed to assess the differences between the chromatogram profiles.
RESULTS AND DISCUSSION
Antimicrobial activity
Trichoderma reesei TV221 marine sponge-associated fungi from S. flabelliformis were obtained from the waters of Menjangan Island, West Bali National Park, Indonesia. The fungi T. reesei have been understood to embody the highest antimicrobial activity under fermentation by employing Sabouraud Dextrose Broth medium (1l SDB containing 2% b/v Dextrose, 0.5% b/v Peptone P, and 0.5% b/v Tryptone), salinity of 30 ppt, and fermentation time of 12 days. It was also affected by the growth curve of the fungus T. reesei TV221 which acquired its peak growth on day 12 (Samirana et al., 2021b; Sibero et al., 2018). The production of secondary metabolites that possess bioactivity as antimicrobial is affected by nutrition during the fermentation process. Carbon sources and medium salinity influence the production of secondary metabolites from marine sponge-associated fungi that own bioactivity (Samirana et al., 2021a; Stanbury et al., 1995; Ukhty et al., 2017).
The fungus T. reesei TV221 in previous studies has been identified to possess antimicrobial activity on several bacteria which are S. aureus ATCC 29213, EC ATCC 25922, and MRSA. The fermented component that owns the highest antimicrobial activity is the supernatant portion compared to the biomass portion (Samirana et al., 2021b; Setyowati et al., 2018). In this study, antimicrobial tests were administered on S. aureus ATCC 29213 and EC ATCC 25922 by varying the components of the fermentation medium. Variations in the components of the fermentation medium are hoped to produce diverse amounts of secondary metabolites so that various antimicrobial activities are obtained for the objective of tracing antimicrobial bioactive compounds with a metabolomic approach by administering the GC-MS technique. The antimicrobial activity test results of the supernatant ethyl acetate extract and fermented biomass with various medium components are demonstrated in Figure 1.
The most potent antimicrobial activity of supernatant extracts and biomass was displayed in the culture of S. aureus ATCC 25923. Staphylococcus aureus ATCC 25923 bacteria were employed in this study to represent Gram-positive bacteria. The highest antimicrobial activity in S. aureus ATCC 25923 was revealed in the ethyl acetate supernatant extract with variations in the numbers 8 and 9 media, that is, with 2% Dextrose concentration and 30 and 15 ppt salinities. It is in accordance with the previous study, in which the ethyl acetate supernatant extract from the fermentation of the fungus T. reesei TV221 for 12 days with Sabouraud Dextrose Broth medium (2% Dextrose) and 30 ppt salinity presented the most potent antimicrobial activity against S. aureus bacteria (Samirana et al., 2021b; Setyowati et al., 2017). The antimicrobial activity of the supernatant extract and fermented biomass with various medium variations (Dextrose and salinity) on EC ATCC 25922 presented lower results than the antimicrobial activity of S. aureus ATCC 25923 bacteria. This result is in accordance with previous studies conducted by previous researchers (Samirana et al., 2021b; Setyowati et al., 2017). The ethyl acetate extract of biomass fermented with various medium variations did not present antimicrobial activity on EC ATCC 25922. EC ATCC 25922 bacteria were administered in this study to represent Gram-negative bacteria. It can be displayed here that the antimicrobial activity of the ethyl acetate extract, both supernatant, and biomass is robustly affected by the variation of the medium. The concentration of Dextrose is revealed to possess an effect on antimicrobial activity, particularly when it was perceived against the bacteria S. aureus ATCC 25923, in which the higher the concentration of Dextrose is, and the more potent antimicrobial activity is. Dextrose is one of the carbon sources required for fungi in their growth cycle. Fungi require large amounts of carbon sources as these carbon sources will later be administered in the formation of fungal cell walls. Simple carbon sources such as Dextrose are more easily processed by fungi in their metabolism; thus, their levels in the fermentation medium possess an effect on fungi in generating bioactive compounds, particularly here as antimicrobials (Kjer et al., 2010; Moore-Landecker, 1996; Muthukumar and Venkatesh, 2013; Samirana et al., 2021a). The level of salinity in the fermenting medium seems to possess little effect on the metabolism of the fungi T. reesei TV221. It can be perceived in the antimicrobial activity of S. aureus ATCC 25923 and EC ATCC 25922 which did not own a significant effect. Although the fungi T. reesei TV221 originated from marine sponges, it is uncovered that the salinity of the medium did not significantly influence the production of secondary metabolites that function as antimicrobials. Several Trichoderma genera originating from the marine environment possess good growth with salinity levels ranging from 0.5% to 2% (5–20 ppt). Salinity levels exceeding 3% (30 ppt) are able to decrease the growth of fungal colonies of the genus Trichoderma (Mishra et al., 2016; Sánchez-Montesinos et al., 2019).
 | Table 1. Variation of fermentation medium (Dextrose and salinity levels). [Click here to view] |
In this antimicrobial study, measurements were made of the diameter of the inhibition zone against the bacteria S. aureus ATCC 25923 and EC ATCC 25922. The diameter of the inhibition zone was able to illustrate the strength of the sample in inhibiting microbial growth. The category of antimicrobial power strength, an inhibition zone diameter of 5 mm or less, was categorized as weak; an inhibition zone of 5–10 mm was classified as moderate; an inhibition zone of 10–20 mm was classified as strong; an inhibition zone of 20 mm or more was categorized as tremendously strong (Davis and Stout, 1971; Samirana et al., 2021b). Figure 2 depicts the inhibition zone in broad strokes. The ethyl acetate supernatant extract with a medium variation of eighth (14.33 mm) and the ethyl acetate supernatant extract with a variation of the ninth medium (13.67 mm) both discovered the diameter of the inhibition zone in the antimicrobial study of the bacteria EC ATCC 25922, while the medium variations 3 and 4 displayed the narrowest zone of inhibition (6.33 mm). While this was progressing on, the ethyl acetate extract of biomass with different medium variations did not demonstrate that bacteria possessed an inhibition zone. Based on the inhibition zone strength category, the supernatant extract with medium variations 8 and 9 owned robust antimicrobial activity, while the supernatant ethyl acetate extract in medium variations 3–7 possessed moderate antimicrobial activity. It implies that the supernatant ethyl acetate extract with variations 8 and 9 owns the potential as an antimicrobial agent in EC ATCC 25922 representing Gram-negative bacteria. In the antimicrobial study of S. aureus ATCC 25923, the diameter of the widest inhibition zone was implied by the ethyl acetate supernatant extract with a medium variation of 8 (17.33 mm) followed by ethyl acetate supernatant extract with a medium variation of 9 (16.67 mm); then for the lowest inhibition zone narrow was unveiled by the ethyl acetate extract of biomass with a medium variation of 3 (6.33 mm). The biomass ethyl acetate extract with medium variations 1 and 2 did not present any inhibition zones. In accordance with the strength category of the zone of inhibition, the ethyl acetate supernatant extract with a medium variation of 4–9 possessed a category of robust antimicrobial activity, while the ethyl acetate supernatant extract with a medium variation of 1–3 and biomass ethyl acetate extract with a medium variation of 3–9 owned moderate antimicrobial activity. It implies that the supernatant ethyl acetate extract with variations 8 and 9 possesses the potential as an antimicrobial agent in S. aureus ATCC 25923 which represents Gram-positive bacteria. The results displayed that the antimicrobial activity was more active in S. aureus ATCC 25923 compared to EC ATCC 25922; it could be due to the fact that EC ATCC 25922, which are Gram-negative bacteria, possess a more complex cell wall structure that owns more lipid composition and is thicker than the bacteria in S. aureus ATCC 25923 which are categorized as Gram-positive bacteria (Pelczar and Chan, 2008a, 2008b). In this study, the negative control (CO−) administered was the solvent implemented to dissolve the extract, that is, methanol. The utilization of negative control aims to ensure that the solvent utilized is inert and does not possess antimicrobial activity. The positive control (CO+) used in this study was chloramphenicol. Chloramphenicol is a broad-spectrum antibiotic that is tremendously effective against Gram-negative and Gram-positive bacteria (Tjay and Rahardja, 2015).
 | Figure 1. Antimicrobial activity of supernatant extract (sample code A–I) and biomass extract (sample code J–R); CO− = negative control; CO+ = positive control. (a) EC ATCC 25922; (b) S. aureus ATCC 25923. [Click here to view] |
In an antimicrobial assay employing the disc method, the type of inhibition zone produced signifies the sensitivity of bacteria to antimicrobial or antibiotic agents from the sample. The radical and irradical zone are two types of inhibition zones of antibacterial activities. The radical zone is the uncontaminated area that is free of bacterial growth and has a distinct boundary from the bacterial colony. The inhibition zone is referred to as the irradical zone if it appears faint and there is no discernible boundary between the bacterial colony and the inhibition zone (Davis and Stout, 1971; Pelczar and Chan, 2008b; Samirana et al., 2021b). Table 2 presents the types of inhibition zones from antimicrobial studies on bacteria S. aureus ATCC 25923 and EC ATCC 25922 from ethyl acetate supernatant extracts and fermented biomass with various medium variations (Dextrose and salinity). The type of inhibition zone in the antimicrobial test of S. aureus bacteria ATCC 25923 possesses a radical zone type for ethyl acetate supernatant extract and an irradical zone type for biomass ethyl acetate extract. It implies that the supernatant ethyl acetate extract was more effective in killing bacterial colonies of S. aureus ATCC 25923 than the biomass ethyl acetate extract which was merely able to inhibit the growth of bacterial colonies. For the type of inhibition zone in the study of antimicrobial bacteria, EC ATCC 25922 owned a radical zone type for the supernatant ethyl acetate extract, while the biomass ethyl acetate extract did not possess antimicrobial activity on EC ATCC 25922. It implied that the ethyl acetate supernatant extract was effective in eradicating colonies of bacteria EC ATCC 25922 although it owns a diameter that is not extensive enough. Due to the type of inhibition zone from antimicrobial studies on EC ATCC 25922 and S. aureus ATCC 25923, it can be indicated that the supernatant ethyl acetate extract possesses the potential to inhibit bacterial colonies. In the future, to ensure its antibacterial strength, it is necessary to conduct minimum inhibitory concentration and minimum bactericidal concentration tests.
 | Figure 2. Value of antibacterial inhibition zone diameter at (a) EC ATCC 25922 and (b) S. aureus ATCC 25923. [Click here to view] |
GC-MS chemical profile
The working principle of GC-MS is separation by employing the gas chromatography (GC) technique, which employs differences in the vapor points of volatile compounds. This GC is correlated with a Quadrupole Mass Analyzer and an ionizing electron (EI) source. EI is one of the ionization techniques functioning by striking electron ions into molecules resulting from GC elution so as to produce pieces of ions from the molecular mass. The mass spectrum of EI was acquired under standard conditions (electron energy of 70 eV and ion source temperature of 200°C–250°C); thus, compounds, particularly volatile compounds, could be identified easily and could compare the obtained mass spectra with the reference mass spectra of pure compounds collected through specialized libraries (Schauer et al., 2005).
In this study, there were 18 samples, the GC-MS chemical profile of which was identified. In Figure 3, there are five GC-MS chemical profiles from the sample, which are the ethyl acetate supernatant extract with medium variations of 3 (C), 6 (F), 7 (G), 8 (H), and 9 (I). All GC-MS chemical profiles of all extracts from both the supernatant and biomass were documented on the area under the curve and retention time (Rt) data, which were then normalized to the Rt data. In Figure 3, it is displayed that there are similarities in the patterns formulated in the three GC-MS chemical profiles; only there are differences in the area of the produced peaks. This difference in peak area is because of variations in the fermentation medium causing different amounts of secondary metabolites to be generated. It is what causes differences in antimicrobial activity resulting from each of these extracts. This difference in area and activity can later be scrutinized with a metabolomic approach to be able to tell which peaks possess an essential role in antimicrobial activity (Yuliana et al., 2011; Yulianto et al., 2016).
 | Table 2. Inhibition type of antimicrobial activity on EC ATCC 25922 and S. aureus ATCC 25923. [Click here to view] |
Figure 3 displays that there are several peaks that possess increase in height and breadth. The changes in peak area and height were associated with the number of secondary metabolites of the fungus T. reesei TV221 generated during the fermentation process. In addition to the ability of the bioactivity of the extract, as previously mentioned, it is affected by the nutrients and conditions of the fungal fermentation medium. In the variation of the fermentation media 7 (G), 8 (H), and 9 (I), in which there is only variation in the salinity of the medium, there are variations in the spectrum pattern that occurs. It can be performed for instance at the peak with Rt 27,423 minutes, in which the peak looks wider and higher as the salini