Marine Sponge-Derived Fungi: Fermentation and Cytotoxic Activity

Putu Oka Samirana1,2, Yosi Bayu Murti3, Riris Istighfari Jenie4, Erna Prawita Setyowati3* 1Doctoral Program in Pharmaceutical Science, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia 2Department of Pharmacy, Faculty of Mathematics and Natural Science, Udayana University, Bali 80361, Indonesia 3Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia 4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia


INTRODUCTION
The association between sponges and sponge symbionts has potential in drug discovery. Not only are secondary metabolites produced by sponges, but also sponge symbionts may synthesize secondary metabolites. Therefore, the microbial symbionts associated with sponges can be isolated and cultured to increase the production of certain bioactive compounds derived from sponges (Lee et al., 2001). Marine sponge-derived fungi are a source of secondary metabolites that are currently being studied intensively. Although research on fungi derived from marine sponges is still less than research on terrestrial fungi, several important findings derived from fungi associated with marine sponges have added to the value of these fungi in the discovery of natural products (Butler et al., 2014), such as various kinds of secondary metabolites which have antimalarial, antiviral, antibacterial, and anticancer or cytotoxic activity. It is a potential that should be explored to increase the number of medicines derived from marine life without damaging the marine biota itself (Debbab et al., 2011;Hikmawan et al., 2020;Huang et al., 2011).
The metabolites produced by fungi coming from marine sponges are the result of chemical communication between fungus and sponge which is mutually beneficial; moreover, in some cases, they can produce completely new metabolites (Pejin and Karaman, 2017). The symbiosis between fungi and sponges, among others, provides a source of nutrition, a place of defense and protection, and stabilization of the sponge structure, treats sponge waste, and produces bioactive compounds. Therefore, it is likely that the bioactive compounds are also produced by the associated fungi of these sponges (Proksch et al., 2002;Thomas et al., 2010). Data obtained from the National Cancer Institutes of the United States show that sponges are a potential source of compounds in producing cytotoxic effects (Brinkmann et al., 2017). Thus, marine sponge-derived fungi also have the potential to produce bioactive compounds as cytotoxic.
To produce bioactive compounds, the associated fungi derived from marine sponges must be fermented outside the sponge's body tissue using a fermentation media. The fermentation of microorganisms in this case is a fungus which will be influenced by physical and chemical factors. Physical factors affecting microorganisms comprise temperature, pH, and osmotic salinity, while chemical factors consist of sources of carbon, nitrogen, and nutrients in culture media (Pratiwi, 2008). The marine sponge-derived fungi depend on carbon, nitrogen, and salt (salinity) sources. Fungi associated with the marine environment based on several studies are strongly influenced by their growth in the presence of simple carbon sources, such as glucose and dextrose, and can even affect the production of secondary metabolites that affect their biological activity (Anuhya et al., 2017;Fuentes et al., 2015;Mahapatra et al., 2013;Miller et al., 1981;Ripa et al., 2009;Sguros and Simms, 1963). The increase in salt content (salinity) beyond seawater causes several species of associated fungi from the sea to decrease their growth rate, but the unavailability of salinity inhibits the growth of these associated fungi from the sea (Amon and Yei, 1982;Huang et al., 2011;Jones, 2000;Venkatachalam et al., 2019).
The objective of this review is to summarize the components extracted from the fermentation of marine spongederived fungi which have cytotoxic activity, identify the potential cytotoxic activity of these components based on the IC 50 value, and find out what influences the fermentation conditions from marine sponge-derived fungi to produce cytotoxic bioactive compounds. The advantage of this review is to determine the relationship between the cytotoxic activity of fungi derived from marine sponges and their fermentation medium in producing cytotoxic compounds which have the potential to be developed as future cancer drug candidates. In the future, this review can be used as a reference to produce potential cytotoxic compounds from fungi from marine sponges using optimal fermentation medium conditions.

METHOD
A systematic search was conducted to find all publications related to the topic up to June 2020 on PubMed and Google Scholar. The keywords used to browse the articles were "fungi, sponge-derived, cytotoxic" or "fungi, sponge-associated, cancer". The data included in this review were primary articles in English regarding cytotoxic studies of components produced from fungi derived from marine sponges and the conditions of fermentation, as shown in Table 1. Articles were excluded from primary articles if they were review articles, conference articles, and thesis, and no data were available for retrieval. All synthetic derivatives of natural metabolites that occur in sponges are not mentioned in this review. The variables assessed in this review include sponge species/genera of sponges, fungi-associated species/genera, fermentation medium, extracted specimens from fermentation products, components of the extracted product, type of cancer, type of cell line, and the cytotoxic effect of these components.

Cytotoxic activity of marine sponge-derived fungi
The number of articles that has been searched to June 2020 was 86 primary articles (Table 1). We identified that the 30 genera of sponge and 30 genera of fungi derived from the sponge investigated were related to their cytotoxic activity. The genera of sponges that are most frequently studied are Callyspongia, Halichondria, Phakellia, and Petrosia. The most frequently studied sponge-derived fungal genera are Aspergillus, Penicillium, Trichoderma, and Gymnascella. Figure 1 shows the number of studies that have been conducted for the cytotoxic activity of marine sponge-derived fungi, of which the number of publications is increasing from year to year. The highest number of publications published in 2019 was 14 articles, followed by 2018 and 2017 with 12 and 11 articles, respectively. The number of publications from 1997 to 2016 continued to increase, but the number of articles per year has not exceeded 2017-2019. It indicates that the focus of research in 1997-2016 was still on marine sponges explored for their bioactive components. Excessive exploration causes damage to the ecosystem of the sponge, which made the species decrease, and is not balanced with sponge growth. It creates a new trend, in which many scientists are interested in researching endophytic samples from sponges, including endophytic fungi from sponges, and it absolutely reduces the damage to sponge the habitat, which is increasingly rare in nature (Carroll et al., 2019;Thomas et al., 2010). Secondary metabolites from sea sponges have also been studied and have the potential in the medical world, including antiviral, antitumor, antimicrobial, antimalarial, and cytotoxicity (Guo et al., 2019;Hikmawan et al., 2020;Setyowati et al., 2009;Wang, 2006). Several studies have reported that the bioactive compounds obtained from sea sponges are most likely secondary metabolite compounds produced by the associated microbes in the bodies of marine sponges. It is caused by 40%-50% of the body tissue of marine sponges, which consists of microbes (Proksch et al., 2002;Thakur and Müller, 2004). The marine sponge association microbes can be fungi (Thomas et al., 2010).
Marine sponge-derived fungi produce bioactive compounds depending on the surrounding environment. The original habitat of these fungi is symbiotic in sponge tissues; thus, the production of compounds depends on the results of symbiosis with the host. When this fungus is outside its host, the active compound produced depends on the medium growth for the fungus. To be able to produce bioactive compounds similar to those produced in symbiosis with a sponge, the growth medium is made as closely as possible to the situation in its host. In addition to obtaining active compounds, it is also to reduce the occurrence of mutations that occur in fungi (Debbab et al., 2011;Huang et al., 2011;Kjer et al., 2010;Lee et al., 2001). Research on cytotoxic agents derived from marine sponges and their symbiotic microbes is still the concern of natural product researchers. More than 10% of cytotoxic activity comes from marine sponges that have been identified to date. The symbiosis of microorganisms from sponsors is proven to have an important role in bioactive compounds as cytotoxic agents. A review from 1955 to 2016 of marine sponges acting as cytotoxic agents reported that 107 new cytotoxic agents originated from marine sponges and were thought to have originated in symbiosis with the microbes present in these sponges .

Classification of cytotoxic activity of compound from marine sponge-derived fungi
In this review, we provide an overview of the bioactive metabolites extracted and isolated from marine sponge-derived fungi exhibiting in vitro cytotoxic activity in cell line cancer. By comparing the IC 50 values, the units in nM, M, and ng/ml are converted into μg/ml unit by adjusting the molecular weight of the compound. All components were classified based on the IC 50 value following the definition of Weerapreeyakul et al. (2012), which classifies the activity of cytotoxic components into "very strong cytotoxic": IC 50 is < 10 μg/ml; "strong cytotoxic": IC 50 is 10-100 μg/ml; and "moderate cytotoxic": IC 50 is 100-500 μg/ ml. What needs to be noted is that the test was administered with different cell line cancers, so there is a possibility that the inactive component in one cell line cancer could have a different IC 50 value in another type of cell line cancer. It would be wise to reevaluate the activity of the inactive components obtained from marine sponge-derived fungi using other cancer cell lines (Badisa et al., 2009;Sutejo et al., 2016;Weerapreeyakul et al., 2012).
The bioactive components studied were 132 components extracted and isolated from marine sponge-derived fungi. These 132 components consist of 16 extracts, 5 fractions, and 111 isolates. As shown in Figure 2, among the observed bioactive components, there are 56 components with very strong cytotoxic  activity, 31 components with strong cytotoxic activity, and 15 components with moderate cytotoxic activity against various cell line cancers. There are 16 components that cannot be classified because they have IC 50 values > 500 μg/ml and have less accurate and clear IC 50 values reported in the article. Furthermore, they cannot be included in that classification. There are 14 components reported to have no cytotoxic activity. The component is inactive only in some cell line cancers, and testing on other types of cell line cancers has not been conducted to see its cytotoxic activity (Badisa et al., 2009;Sutejo et al., 2016;Weerapreeyakul et al., 2012).
In this review, 27 types of cancer were found used in the study to determine the cytotoxic activity of marine sponge-derived fungi. The most common types of cancer are human leukemia, human colon cancer, human lung cancer, human cervix cancer, and human breast cancer. It is in line with the report by Bray et al. (2018) in which this type of cancer is reported to have a high incidence rate worldwide in humans, and even this type of cancer is included in the top 10 cancers causing death in humans. It has triggered many researchers to focus on these five types of cancer by exploring new compounds coming from the sea, especially marine sponge-derived fungi (Thomas et al., 2010). Potential compounds from marine sponge-derived fungi are expected to be used as new drugs in cancer treatment. To conduct a brief test of anticancer activity, researchers used in vitro cell line cancer to facilitate the screening of the anticancer activity of components obtained from marine sponge-derived fungi. In this review, we identified 317 types of cell line cancers used in determining the cytotoxic activity of components obtained from marine sponge-derived fungi. The most common types of cell line cancer are human cervix cancer (HeLa), human lung cancer (A-549), human leukemia (HL-60), mouse leukemia (P338), human liver cancer (HepG-2), human colon cancer (HCT-116), and human breast cancer (MCF-7). The use of this type of cell line cancer is based on the cancer incidence rates mentioned previously. Furthermore, there are factors which considered the use of this cell line cancer. These factors include easiness to handle and manipulate, high homogeneity, high degree of similarity with the initial tumor, large number and variety of cancer cell lines available, immediate accessibility to the scientific community, unlimited autoreplicative source, continuous cell lines, easy substitution of contaminated cultures for the respective frozen cell lines, and reproducibility of results in the correct conditions (Ferreira et al., 2013). Moreover, cell lines which are normal cells are also used. The use of normal cell lines aims to determine the cytotoxic strength of a sample that only damages cancer cells and does not damage normal cells of living things. The comparison of the IC 50 value between normal cell line and cancer cell line produces a value called selectivity index (SI). Compounds or extracts having a SI > 3 have high selectivity in certain cancer cells (Badisa et al., 2009;Sutejo et al., 2016). The normal cell lines that are often used in cytotoxic-related research in this review are Vero (monkey epithelial kidney) and HL7702 (human normal liver).
The components isolated from marine sponge-derived fungi classified as very strong cytotoxic are shown in Table 2. Xanthone derivatives are metabolites generally distributed in higher plants and several types of fungi. This metabolite has several biological activities, such as antimicrobial, antiviral,   antitubercular, and anticancer. Anomalin A (1) is one of the xanthones derived from the fungus Arthrinium sp. which is associated with the sponge Geodia cydonium (see Fig. 3) (Abdel-Lateff et al., 2003;Ebada et al., 2011;Morel et al., 2000;Peres et al., 2000). Bioactive polyketide derivatives include bromophilone A (2), epoxyphomalin A (6), heterocornol A (9), heterocornol O (10), and oxalicumone A (14) (Fig. 3). Bromophilone A (2) is a polyketide azaphilone group with a bicyclic core and conjugated chromophore which has a bromide atom as a substituent. This metabolite is the combined result of the fungal fermentation media of Penicillium canescens with NaBr. Epoxyphomalin A (6) derived from the fungus Phoma sp. associated with the sponge Ectyplasia perox is a very active component in many cancer cell lines and this component has the potential to be developed for future cancer therapy. Heterocornol A (9) and heterocornol O (10) are polyketide derivatives derived from the fungi of the genera Pestalotiopsis associated with the sponge Phakellia fusca. Both these components have an IC 50 ranging from 2 to 10 µg/ml in some cancer cells. Oxalicumone  A (14) is a chromone-type bioactive polyketide derivative to be precise, dihydrothiophene-condensed chromone. This bioactive component comes from Aspergillus sp. LS34 associated with the sponge Haliclona sp. possessing very strong cytotoxic activity in the cancer cell lines CCRF-CEM and K562 (Frank et al., 2019;Gao et al., 2013;Lei et al., 2017aLei et al., , 2019Li et al., 2019;Mohamed et al., 2009;Sun et al., 2013;. Cytochalasins are a group of metabolites that are often found in fungi in several genera, such as Phomopsis, Chalara, Hyposylon, Xylaria, Daldinia, Pseudeurotium, and Phoma exigua. Cytochalasin K (3) is a metabolite of the marine spongederived fungi Arthrinium arundinis ZSDS1-F3 (Fig. 3). This class of metabolites is unique in its structure with a macrocyclic ring with antitumor, antibacterial, and HIV-1 protease inhibition activity. The most unusual activity of this metabolite is the ability to make the cell secrete its nucleus resulting in the formation of a cell without a nucleus Wang et al., 2015). Diorcinols L (4) is a phenol derivative metabolite (Fig. 3). This metabolite comes from the fungus Didymellaceae sp. SCSIO F46 in association with Callyspongia sp. sponge. These phenol derivatives have strong cytotoxic activity in a number of cancer cell lines, including A-549, DU145, H1975, HeLa, HL60, Huh-7, andMCF-7 (Tian et al., 2018a). Acremonium sp. associated with the Teichaxinella sp. sponge produces several types of bioactive metabolite groups from the polyketides, hydroquinones, ketideterpenes, alkaloids, and terpene glycosides. Efrapeptin G (5) is a new bioactive metabolite from the peptaibiotic class derived from the fungus (Fig. 3). This bioactive metabolite has a very strong cytotoxic activity in several cancer cell lines, such as HCT-116, HeLa, HT-29, and SK-BR3 (Boot et al., 2006).

Influences of the fermentation conditions from marine spongederived fungi to produce cytotoxic metabolite
Metabolites produced by microbes are divided into two, primary metabolites and secondary metabolites. The production of primary metabolites is considered important, for instance, ethanol, citric acid, polysaccharides, acetone, butanol, and vitamins. Secondary metabolites produced by microbes include antibiotics, growth promoters, enzyme inhibitors, and others (Stanbury et al., 1995). Marine sponge-derived fungi produce a large number of new bioactive secondary metabolites, some of which exhibit new molecular structures that have never been previously found in nature. To be able to produce bioactive metabolites, the fungi associated with the sponge must first be isolated from the host and then fermented with a liquid medium of which composition is as close as possible to the state when it is in the host (Kjer et al., 2010).
In this review, we identified 11 types of carbon sources used in the fermentation media for marine sponge-derived fungi, including rice (38 media), glucose (33 media), malt extract (30 media), dextrose (9 media), and potato (8 media). Fungi require a greater amount of carbon than other essential elements because half of the dry weight of the fungal cell is estimated to consist of carbon which is important in the formation of the fungal cell wall (Moore-Landecker, 1996). The source of complex carbon in the medium is converted by the fungus into a simpler form that can be metabolized. Currently, rice and malt extract is widely used by researchers as a source of carbon in the medium. These two complex carbon sources, after being sterilized by heating, split into simpler carbon which could be used by fungi in their metabolism, with the result that these two carbon sources are widely used in the protocol for fermentation of marine sponge-derived fungi to produce new bioactive compounds, especially those useful for cancer (Kjer et al., 2010;Muthukumar et al., 2013). Some of the fungal isolates associated with the marine environment include Culcitalna achraspora Meyers and Moore, Humicola alopallonella Meyers and Moore, Orbomyces spectabilis Linder, Halosphaeria mediosetigera Cribb and Cribb, Penicillium decumbens, Penicillium chrysogenum, Acremonium strictum, Fusarium fujikuroi, and Fusarium sporotrichioides, which have a dry weight of mycelia developing with increasing levels of carbon sources in the fermentation media (Fuentes et al., 2015;Sguros and Simms, 1963). Trichoderma lignorum has increased conidia and hyphae growth when the carbon source is increased, but its growth decreases when the concentration of the carbon source exceeds 10 times of the frequent use (Seto and Tazaki, 1975). The effect of various carbon sources on the growth of Trichoderma viride species shows that the maximum production of secondary metabolites resulting from the highest to lowest production is influenced by the carbon sources of sucrose, glucose, cellulose, maltose, and carboxymethyl cellulose with an optimum level of 1%-1.5% (Gautam et al., 2010).
There are five types of nitrogen sources that we can identify in this review including peptone (30 media), yeast extract (16 media), glutamate (6 media), NaNO 3 (2 media), and tryptophan (2 media). The nitrogen source in the marine sponge-derived fungi fermentation media does not appear to be present in the medium given its use which is not as much as the carbon source. Nitrogen sources are one of the important elements in the growth of endophytic fungi; however, nitrogen sources are not very influential in the growth of fungi and in the formation of secondary metabolites, except for metabolites containing nitrogen in their molecules. The use of peptone as a nitrogen source in the medium gives a high increase in mycelia dry weight compared to the use of inorganic nitrogen sources such as NaNO 3 and NH 4 Cl (Hussain et al., 2003;Khattabi et al., 2004;Muthukumar et al., 2013).
Halophilic microorganisms can grow at high levels of salinity, for instance, in the sea with 3% NaCl. The salinity of a microorganism growth environment causes differences in osmotic pressure. Increasing levels of salinity exceeding the salinity of seawater resulted in several species of associated fungi from the sea decreasing their growth rates, but the unavailability of salinity inhibits the growth of these associated fungi from the sea. Several genera of associated fungi including Penicillium (32 strains), Aspergillus (10 strains), Mycelia sterilia (3 strains), Fusarium (1 strain), and Paecilomyces (1 strain) were isolated from several samples, such as seaweed, underwater sediments, and mangrove roots which have optimal growth and have the widest colony diameter found in fungi grown on medium with 3%-6% NaCl. The antimicrobial activity of these associated fungi in C. albicans showed the highest activity when treated with 6%-9% NaCl (Huang et al., 2011). The use of seawater or sea salt in the marine sponge-derived fungi fermentation media is very important because when living in its host, the surrounding environment of the fungus is a sea with salinity levels adjusting to the surrounding sea conditions. In this review, we identified 70 fermentation media using conditions such as in the sea, using natural seawater and sea salt, artificial seawater, and sea salt, while 26 media do not use conditions such as the origin of the fungus. The identification results show that the medium using seawater components (natural or artificial) produces bioactive components with very strong cytotoxic activity. The addition of components such as KH 2 PO 4 , MgSO 4 , MnCl 2 , and KCl in a medium which do not use seawater components also makes the fungi produce active metabolites as cytotoxic. Several genera of Trichoderma associated with the marine environment have optimal growth and dry weight mycelial at salinity levels of 1%-2%, but salinity levels that exceed 3% reduce the growth of fungal colonies (Bheemaraya et al., 2013;Mishra et al., 2016;Sánchez-Montesinos et al., 2019).
In this review, we classify the extracted specimens to obtain bioactive components from marine sponge-derived fungi into medium part, part mycelia, and both. The extraction process using both parts of mycelia and medium is the decision that most researchers do to extract bioactive components from fermentation. It is possible because the bioactive components are not yet known whether they are in the fungal cell or excreted out of the cell; therefore, the use of the extraction process for these two parts results in an optimal extraction. Furthermore, the extraction results using these two parts on average produce bioactive components containing a very strong cytotoxic activity (Kjer et al., 2010).

CONCLUSION
The data presented in the review show the potential of marine sponge-derived fungi as producing metabolites with cytotoxic activity and can reduce exploitation of rare sponges to produce bioactive components in cancer therapy. The components have been summarized and the most promising components are polyketide derivatives, lipopeptides, cyclodepsipeptides, decalin derivatives, xanthone derivatives, phenol derivatives, cytochalasins, peptaibiotics, phthalides, anthraquinone, terpenes, decalin derivatives, and lactones. In producing bioactive metabolites for cytotoxicity, the fermentation media is essential. Carbon sources, nitrogen, salinity, and extracted specimens are factors in the production of bioactive metabolites for cytotoxic fungi from marine sponges. A comprehensive approach is needed to evaluate the specific mechanism of action of the bioactive component as an anticancer. For further large-scale development in evaluating the production of bioactive metabolites from marine sponge-derived fungi, it may be necessary to develop components of the fermentation media which are more specific to certain fungi.