Evaluation of culture media and conditions of Amazonian filamentous fungi in an antimicrobial screening program

The rediscovery of bioactive compounds is a problem within natural product screening programs, because the chemical-genetic diversity of fungi is little explored and the standardization of cultivation conditions that allow obtaining new actives is critical in such programs. In this work, the impact of two solid mediums (rice and oats), a liquid medium (Czapeck broth) and different fermentation conditions were evaluated in order to explore new metabolic routes. Twelve filamentous fungi from Amazonian environments were used. UV-Vis spectrophotometry estimated the complexity of the extracts produced. The antimicrobial activity of the extracts was evaluated against an isolate of each Escherichia coli strain, Salmonella sp. and Staphylococcus aureus. Solid media proved to be more promising, as they allowed a wider range of active metabolites to be obtained. The oat medium provided a greater variety of metabolites, but due to the great complexity of the extracts obtained, the separation procedures were Research, Society and Development, v. 10, n. 14, e370101422065, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.22065 2 considerably more complex than for rice. Together, the rice culture medium and the use of 39 days of fermentation proved to be more promising conditions than the liquid medium normally used in screening programs in Brazil. The cultivation of Penicillium maximae (isolated for the second time in Brazilian territory) in solid medium provided the production of active fractions against E. coli in bioautography. In this study, it was observed that different fermentation conditions in solid culture are considerably promising in the search for bioactive natural products.


Introduction
Compounds isolated from biological sources represent the vast majority of drugs approved for clinical use, especially for cancer and infectious diseases (Dayanidhi et al., 2021;Atanasov et al., 2021). Approximately one quarter of the therapeutic compounds were isolated from fungi (Berdy, 2012). These microorganisms synthesize compounds that may be the key to solving emerging public health problems (Raghava Rao, Mani, Satyanarayana & Raghava, 2017). However, according to Palma Esposito et al. (2021) certain cultivation conditions do not always favor the expression of cryptic genes (genes silenced under a standard cultivation condition), and the rediscovery of bioactive molecules is a problem faced by natural product laboratories (Pan, Bai, Chen, Zhang & Wang, 2019).
Variation of cultivation parameters, an approach often known as OSMAC (One Strain Many Compounds) (Loffy et al., 2021), can have significant impacts on the quantity and diversity of secondary metabolites produced (Zutz et al., 2016).
However, in an antimicrobial screening program in which a large number of filamentous fungi are to be screened, the OSMAC approach is not always viable, so the pursuit of a culture medium and condition that will allow more efficient production of new Antimicrobials become a necessity (VanderMolen, Raja, El-Elimat & Oberlies, 2013).
Evaluating different fermentation parameters such as medium composition, aeration rate, temperature and pH change can also be a laborious process, so in this study we chose to analyze the impact of modifications on two critical parameters in the process. fermentatives: the culture media and the fermentation time. Furthermore, the use of conditions that impose stress on the microorganism may favor the detection and isolation of a more diverse range of bioactive compounds, with solid fermentation being more associated with metabolic stress of cultivated fungi than fermentation in broth (VanderMolen et al., 2013).
Thus, this study aimed to optimize the culture media and time in order to induce alternative metabolic pathways to obtain new compounds within an antimicrobial screening program.

Methodology
The study was carried out with twelve filamentous fungi, six isolateds from different types of Amazonian soil (unpublished data) and six air isolates (anemophiles) in a previous study (Cruz, Souza de & Abegg, 2019). These fungi were pre-selected for preliminary antimicrobial activity results (data not shown). Soil fungi were obtained by the use of two selective media with the intention of isolating low abundance microorganisms: HSUC and starch milk agar, according to Du et al. (2012).

Identification of Filamentous Fungi
The twelve isolates were identified by morphological techniques (macro and microscopic) . Of these twelve isolates, five isolates with marked antimicrobial potential were identified by sequencing the internal transcribed spacer in the ITS regions (ITS1-5.8S-ITS2 of the rDNA gene), according to the protocol of Tonial et al. (2015), the sequences being compared using the GenBank platform.

Pilot Scale Cultivation
The twelve isolates were grown on a pilot scale under two fermentation conditions, solid (rice and oats) and liquid (Czapeck broth). The media were prepared according to VanderMolen et al. (2013).

Cultivation in Solid Medium
The fungi were grown on 2% malt extract agar at 28 ºC for ten days. Ten plugs (7 mm) from the culture plates were transferred to 15 mL of YESD broth (20 g soy peptone, 20 g dextrose, 5 g yeast extract, 1 L H2O) and this broth was incubated for 5 days with shaking. orbit at 110 rpm and room temperature. The pre-fermentation broth containing the mycelial mass was poured onto the solid media (rice and oats), which were cultivated under static condition for 11 and 31 days. To obtain the extracts, 50 mL of methanol was added to each culture, the material was macerated and subjected to orbital agitation at 100 rpm for 24 hours. The cultures were filtered, the mycelia being discarded and the extracts concentrated in rotary evaporator (VanderMolen et al., 2013).

Liquid Culture
The fungi were grown on 2% malt extract agar at 28ºC for 28 days. Ten plugs (7 mm) were transferred to 250 ml Czapeck broth. The cultures were fermented under orbital agitation at 100 rpm for 14 and 21 days at room temperature. To obtain crude extracts, 250 mL of methanol was added to the liquid cultures. Extraction occurred under orbital agitation at 100 rpm for 24 hours and the extracts were concentrated by rotary evaporator.

Antimicrobial Activity Test
Dry methanolic extracts were resuspended (200 mg / mL) in sterile distilled water and tested for antimicrobial activity by the Spot technique (Fleming, Etchells, & Costilow,1975). Standard suspensions of the target bacteria were produced, OD600nm = 0.02 for E. coli and Salmonella sp. and 0.05 for Staphylococcus aureus. The suspensions were homogeneously distributed on the surface of 90 mm Mueller Hinton (AMH) agar plates, pending the drying of the agar surface in a biological safety cabinet. Subsequently, 10 µL of crude extracts were pipetted at three equidistant points per plate. The test was performed in triplicate and sterile distilled water was used as negative control. Results were interpreted based on the measurement of inhibition halos after 24 hours of incubation at 37ºC.

Breakdown of Active Extracts
The raw extracts produced in pilot scale were partitioned liquid-liquid, repeating the extraction process three times for each solvent. Solvents in increasing order of polarity (Hexane C6H14, Chloroform CHCl3 and Ethyl Acetate C4H8O2) were used in a 1: 1 (v / v) ratio (Etame et al., 2019). Four fractions were generated: Hexane Fraction (FH), Chloroform Fraction (FC), Ethyl Acetate Fraction (FA) and Remnant Fraction (FR). The obtained fractions were rotary evaporated and their masses determined. Fraction yield was calculated in grams and fractions were tested for antimicrobial activity by the agar diffusion method (Silveira, Olea, Mesquita, Cruz & Mendes, 2009).

Analysis of the Remaining Fraction Complexity
Scanning analyzes were performed on a UV-Vis Spectrophotometer (Gold Spectrum Lab. -UV-5200) (λ 250-600 nm) as a preliminary analysis of the complexity of the Remaining Fractions obtained on a pilot scale. Data were processed in Origin Software 2017 (Originlab).

Large Scale Solid Fermentation
Ten filamentous fungi were grown on 2% malt extract agar (2% MEA) for ten days. Twenty plugs (7 mm) from the cultures were transferred to 30 mL of YESD broth. Pre-fermentation broths were grown under orbital agitation at 100 rpm for five days at room temperature.
The pre-fermented material was transferred to 2 L flasks containing oat solid medium (400 g oats and 300 mL distilled water). This medium was chosen because of a seemingly larger range of metabolites generated at the pilot scale. Research, Society andDevelopment, v. 10, n. 14, e370101422065, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.22065 5 The solid medium was then homogenized by maceration and cultivation followed for 39 days in static condition and room temperature. For extraction, 1.5 L of methanol was added to each flask and extraction occurred for 12 hours under orbital agitation at 120 rpm. To confirm the antimicrobial activity of the large-scale crops the extracts were subjected to Spot activity testing (Fleming, Etchells, & Costilow,1975).

Partial Purification of Active Compounds
Two extracts (HSUC-6 and IS-9) produced from the cultivation of fungi Penicillium maximae and Fusarium oxysporum on a large scale were submitted to purification processes. These extracts were initially partitioned liquid-liquid three times using solvents in increasing order of polarity: Hexane (C6H14), Chloroform (CHCl3) and Ethyl Acetate (C4H8O2) in a 1:1 ratio (v/v) according Etame et al. (2019). The fractions with higher polarity obtained after partition were submitted to Column Chromatography (CC) by Merck®60 (0.063-0.200 mm).
The IS-9 ethyl acetate fraction and the remaining HSUC-6 fraction obtained after Column Chromatography, were treated with the chloroform: methanol and ethyl acetate: methanol systems, respectively, in 100 mL portions, with a 10% increment of methanol each eluted portion (Sanchez & Wang, 2012). Individually collected fractions from the column were analyzed by Thin Layer Chromatography (CCD) using standard silica-coated chromatographic plates (Merck®) (Azerang et al., 2019). The fractions were applied to the chromatoplates, eluted in chloroform: ethyl acetate (2: 8) and observed in UV light chamber (λ 250 nm and 350 nm). From the isolated bands Rf (Retention Factor) values were calculated and fractions with similar band patterns were pooled.

Fraction Antimicrobial Activity
The antimicrobial activity of semi-purified fractions was evaluated by bioautography technique (Dewanjee, Gangopadhyay, Bhattacharya, Khanra & Dua, 2015). The fractions were chromatographed on chromatographic plates (Merck®) with chloroform: ethyl acetate (2: 8; v / v) elution system. The plates were dried to full evaporation of the solvents and analyzed in a UV light chamber (λ 300 nm) to determine the Rf values. Suspensions of the target microorganisms (OD600nm 0.02 for E. coli and Salmonella sp. and 0.05 for S. aureus) were homogeneously distributed and dried on the surface of AMH plates. The eluted chromatoplates were applied upside down on the inoculated surface and kept in contact with the target bacteria for 3 hours. Then the chromatoplates were removed and the plates incubated for 24 hours at 37 ° C.
The results were interpreted based on the observation of inhibition halos in the previously observed bands.
The minimum inhibitory concentration (MIC) of the fractions of interest was determined by 96 well plate microdilution assay (Noumi et al., 2018). The E. coli test strain was grown at 37 ° C overnight and a suspension adjusted to OD600nm = 0.02. Semi-purified fractions were resuspended in 10% DMSO and tested at concentrations of 500 to 3.9 µg / mL. Inhibitory activity was evidenced with the aid of the rezasurine colorimetric indicator (0.01%).

Statistical Analysis
Analyzes were performed using Biostat (AnalystSoft) software using one -way ANOVA and Tukey 's test. Data were considered significantly different when p <0.05. Pearson's coefficient (ρ) was used to calculate the correlation between the evaluated data.

Identification of Filamentous Fungi
The twelve isolates were categorized into four genera: Aspergillus sp., Penicillium sp., Curvularia sp. and Fusarium sp. (Table 1). Marked active fungi in preliminary studies were identified at molecular level as representative of the species: Penicillium maximae, Penicillium rolfsii, Aspergillus ochraceus, Aspergillus melleus and Fusarium oxysporum (Table 1).
However, there are no reports of isolation of soil samples in Brazil.

Pilot Scale Fermentation
Considering the potential of filamentous fungi as antimicrobial suppliers (Adpressa & Loesgen, 2016), filamentous fungi isolated from the Amazonian environment were cultivated under different conditions in order to evaluate the most appropriate culture media and time for an antimicrobial screening program. The isolates were fermented in two distinct periods for antimicrobial potential determination and the choice of the evaluated nutritional conditions was based on the search for the expression of little explored metabolic pathways under normal fermentation conditions (Adpressa & Loesgen, 2016). This is because, substantial changes in these conditions can promote real effects on the final metabolic profile (Ochi, 2016 rotation scheme adopted in submerged crops and pre-inoculum of solid crops (pre-fermentation in YESD broth) was determined by the need for multiplication of the mycelial mass produced in each fermented group.
Regarding the yields obtained within each cultivation group (Fig. 1A), it was observed that the dry masses produced comparing the solid crops (rice and oats) did not present significant differences between the two evaluated periods (11 and 31 days). However, individually analyzed, some isolates increased the extract biomass in the 31-day oat substrate, when compared to the mass produced by the same fungus cultivated in rice. This increase in biomass occurred especially for TPP1.1, 8.4, 2.5 and 8.2 fungi grown on oat substrate during the 31 days of fermentation (data not shown).
Comparing the yield obtained in the solid cultivation rice (11 and 31 days) and Czapeck broth (14 and 21 days) (Fig.   1B), it was observed that there was no significant difference in relation to the masses produced (p = 0.3432).   There were no differences in yields comparing solid oat crops (11 days) and liquid crops (14 days) of fermentation (p = 0.1267). However, the 31-day cultivation on oat substrate produced a significantly higher mass than the 21-day broth cultivation (p = 0.0422) (Fig. 1C), which was also observed by VanderMolen et al. (2013) where crops grown in solid media (especially oat cultivation) produced extracts with masses one to two orders of magnitude larger than the same fungus grown in liquid media.

Antimicrobial Activity of Raw Extracts
The antimicrobial potential of extracts produced in different crops was evaluated. In each growing condition, moderate activity was observed against one or more strains tested. In particular, solid media extracts were active against Gramnegative E. coli bacteria, with inhibition halos up to 15 mm in diameter (Table 2).
In this study, eight strains of the genus Aspergillus sp. (IS-6, TPP1.1, TPP1.2, IS-13, 7.4, 7.3, 2.5 and IS-7) produced extracts with antimicrobial activity against E. coli and S. aureus in at least one of the evaluated solid media. In another study, it was observed that the cultivation of fungi of the genus Aspergillus sp. in solid rice medium, for example, it generates extracts with antimicrobial potential against E. coli, S. aureus, Bacillus subtilis, Proteus mirabilis and Malassezia furfur (Chagas et al., 2017). Indeed, solid fermentation allows the production of a range of bioactive compounds (Li et al., 2016) such as fusarialin produced by Fusarium sp., with inhibitory activity against S. aureus in rice, in the study by Tchoukoua et al. (2018).
Currently, solid crops have been used in the literature as an alternative for the exploitation of fungal metabolic chemiodiversity. This is because these nutritional sources may favor the production of compounds that are sometimes not biosynthesized in standard liquid medium cultures (Li et al., 2019). Compared to liquid cultures, solid cultures produce greater diversity of compounds with antimicrobial and antifungal potential (Özkaya et al., 2018;Yue et al., 2015;Hemphill et al., 2017;Wang et al., 2015).
The antimicrobial potential of extracts produced in solid crops compared to extracts produced in liquid crops is relatively attractive. In particular, the extract produced in oat cultivation by the fungus P. maximae (HSUC-6) was active against all strains tested, with inhibition halos ranging from 7-15 mm in diameter. In contrast, the same broth fungus did not produce potentially active extracts in the shortest fermentation periods (14 days). At 21 days of fermentation, the extracts were active against E. coli and Salmonella sp. with inhibition halos up to 15 mm.

Antimicrobial Activity of Fractions
Methanolic crude extracts that showed antimicrobial activity were partitioned and fractional yields were analyzed indicating how much of the crude extract solubilized in the different solvents. Also, the degree of polarity of the extracts with antimicrobial potential was evidenced, according to table 3.
Among the four fractions evaluated for antimicrobial activity in rice cultivation, only the remaining fractions were active, in this case against E. coli and S. aureus. When tested the same fractions against Salmonella sp., the remaining fraction showed the highest antimicrobial potential.
Oat cultivation fractions, when tested against E. coli, showed that most of the antimicrobial activity occurred in the Remnant fraction (p = 0.001175), with inhibition halos between 7 -11 mm in diameter. Among the fractions obtained from oat cultivation tested against Salmonella sp., there was a prevalence of antimicrobial activity of the remaining fraction in relation to the other fractions tested (p = 0.0004619). In addition, the remaining fractions obtained from oat cultivation were also active against S. aureus (Table 3).  The fractions obtained from broth cultures (14 and 21 days) were active in all fractions tested, with no prevalence of antimicrobial activity for a particular fraction. Moreover, when evaluating the yield of the fractions obtained from the broth cultivation, for the lower polarity fractions the yield was lower than for the higher polarity fractions when compared to the results with solid cultivation, suggesting that in the broth fermentation there was a lower production of nonpolar compounds.
In contrast, in oat cultivation, larger amounts of nonpolar compounds were produced when compared to broth cultivars, whereas the hexane fraction, although not showing antimicrobial activity, had significantly higher yield (p = 0.0132) when compared to the others. fractions (data not shown). This was also observed in the study by VanderMolen et al. (2013), where the authors mention that, after degreasing solid extracts from oat cultivation, there was a reduction in total dry mass, since hexane washing eliminated some common and unwanted metabolites (eg fatty acids). Thus, although the yield obtained in oat cultivation is relatively higher than that produced in broth cultivation, part of this mass is represented by (undesirable) nonpolar compounds, suggesting the need for refinement in the purification processes of the compounds of interest (VanderMolen et al., 2013).

Complexity of Pilot Scale Fractions
The remaining fractions obtained from the pilot crop (rice, oats and Czapeck broth) were analyzed by UV -Vis absorbance spectrophotometer and showed differences regarding the complexity of the extracts produced between different crops ( Figure 2). The UV spectra of HSUC-6 (Penicillium maximae) extracts indicated that different substances may have been produced when the fungus was grown on solid media (oats and rice), as shown in Figure 2, because the UV spectra of solid crops show peaks. not observed in the UV spectra of the liquid cultures, presuming a greater complexity of the mixture. The study by Gao et al. (2012) reinforces this idea by evaluating HPLC chromatograms of extracts produced by Penicillium commune QDS-17 fungus, where it was observed that the fungus produced nine new compounds only when grown on solid substrate, and this production of a wider range of compounds. was assumed from the higher number of peaks when compared to liquid cultures.
This was also observed in the comparative study of the metabolic profile of the fungus Aspergillus carneus associated with sea sponge (Agelas oroides). The fungus was cultivated in solid medium rice with sea salt, rice without sea salt and in modified Czapeck broth. Of the three new isolated compounds, isopropylchaetominin was observed in the three culture media used. Isoterrelumamide A was produced only when the fungus was grown in modified Czapeck and 5'-epi-averufanin was isolated only when the fungi were grown in solid rice with or without sea salt (Özkaya et al., 2018), adding to the idea that the fermentation condition may alter the metabolic pathway of a given microorganism in a culture environment, favoring the production of new bioactive compounds (Romano, Jackson, Patry & Dobson, 2018).

Large Scale Fermentation
Large-scale oat yields obtained after 39 days of fermentation were compared with oat-crop yields at 11 and 31 days of smaller-scale fermentation (Figure 3) by dividing the yield obtained (in grams) by the amount of substrate used in the fermentation (in grams). The mass produced in the 39 days of cultivation was considerably larger than the one produced in smaller scale (p = 0,01171). However, this condition seems to depend directly on the metabolism of each fungus grown on this substrate. Source: The Authors.

Antimicrobial Activity of Partially Purified Fractions
The HSUC-6 and IS-9 extracts obtained from large-scale solid cultivation were partially purified generating fractions, such as which were tested for antimicrobial activity (Table 4).

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The three partially purified fractions of the HSUC-6 extract (produced by Penicillium maximae) were active against at least two of the three microorganisms evaluated (E.coli, Salmonella sp. and S. aureus). It is worth noting that the fungus P.
maximae is of great importance as it is little explored due to its recent isolation. Table 4. Antimicrobial potential of partially purified fractions.
Of the partially purified fractions of the IS-9 extract (produced by Fusarium oxysporum), three fractions showed antimicrobial activity, especially against E.coli and Salmonella sp.. In the study by Tchoukoua et al. (2018) Furthermore, as active fractions produced by large-scale cultivation were tested for antimicrobial activity using the Bioautography technique, as shown in Figure 4.  Figure 4A: Antimicrobial activity test of HSUC-6 fractions after disk diffusion partition. Figure 4B: Thin Layer Chromatography of partially purified fractions. Figure 4C: Bioautography of HSUC-6 fractions against inhibition-spotted E. coli (Rf = 0.80). Figure 4D: Minimum inhibitory concentration test of HSUC-6 PAP Fil3 fraction of P. maximae against E. coli at concentrations; 500, 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 µg / mL. Source: The Authors.
The active fractions ( Figure 4A) of the IS-9 (Fusarium oxysporum) and HSUC-6 (Penicillium maximae) were tested for antimicrobial activity by bioautography. Activity was observed from one of the bands of the HSUC-6 PAP Fil3 fraction of P. maximae against E. coli ( Figure 4C). However, in the MIC test, the fraction was not active against E. coli at any of the concentrations tested ( Figure 4D). Presumably the bioactive compounds were in insufficient concentration in the fraction tested to evidence the antimicrobial activity in this test.

Conclusion
In this study, although preliminary, it was observed that solid media appear to provide a more diverse range of antimicrobial active secondary metabolites when compared to liquid cultures. In addition, solid crops can guarantee sufficient extract yield for the isolation of the bioactive compounds of interest. However, in liquid cultures extracts with smaller amounts of nonpolar compounds are produced, facilitating later antimicrobial isolation steps.
In the antimicrobial screening program of this working group, considering the problem of rediscovery in this area and based on the results observed here, solid cultivation in rice medium and static fermentation for 39 days at room temperature will be employed in subsequent works.
For future work, the cultivation of filamentous fungi under static fermentation conditions for 39 days is suggested, which may be promising for obtaining bioactive compounds with antibacterial potential. In addition, it is necessary to search for strategies that optimize the conditions for purification and isolation of bioactive compounds, in order to facilitate the subsequent stages of analysis.