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Induction of antimicrobial, antioxidant metabolites production by co-cultivation of two red-sea-sponge-associated Aspergillus sp. CO2 and Bacillus sp. COBZ21


The growing spread of infectious diseases has become a potential global health threat to human beings. According to WHO reports, in this study, we investigated the impact of co-cultivating the isolated endophytic fungus Aspergillus sp. CO2 and Bacillus sp. COBZ21 as a method to stimulate the production of natural bioactive substances. (GC/MS)-based metabolomics profiling of two sponge-associated microbes, Aspergillus sp. CO2 and Bacillus sp. COBZ21, revealed that the co-culture of these two isolates induced the accumulation of metabolites that were not traced in their axenic cultures. By detection of different activities of extracts of Bacillus sp. COBZ21 and Aspergillus sp. CO2 and coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2. It was noted that the coculture strategy was the reason for a notable increase in some different activities, such as the antimicrobial activity, which showed potent activity against Escherichia coli ATCC 25,922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC 10,231. The antibiofilm activity showed significant biofilm inhibitory activity toward Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10,145, and Staph aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922, while the antioxidant activity based on the DPPH assay showed maximum activity (75.25%). GC-MS investigations revealed the presence of variable chemical constituents belonging to different chemical categories, which reflected their chemical diversity. The main components are (+-) cis-Deethylburnamine (2.66%), Bis(3,6,9,12-tetraoxapentaethylene) crowno-N,N,N’,N’-tetra methylpphanediamine (2.48%), and 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[,8)0.0(3,7)] trideca-3,6-diene-10,12,13-trione (3.13%), respectively, for Bacillus sp. axenic culture, Aspergillus sp. CO2, Aspergillus sp. CO2, and Bacillus sp. COBZ21 coculture. By studying the ADME-related physicochemical properties of coculture extract, the compound showed log Po/w values above 5 (8.82). The solubility of the substance was moderate. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is important to note that the latter did not meet the criteria outlined in the rule of three (RO3). The toxicity prediction of the coculture extract was performed using the ProTox II web server, which showed that the selected compound has no pronounced toxicity.

Peer Review reports


Antimicrobial resistance is one of the significant challenges of the 21st century. The appearance of new infectious and multidrug-resistant microorganisms is emerging as a new threat to human health and global stability [1]. The imperative to identify novel medications with unique modes of action derived from natural sources is indisputable as a means to address the emergence of antimicrobial resistance and ensure their safety and efficacy [2]. Multi Drug Resistance (MDR) pathogens represent one of the most critical global health threats for both human and feed stocks due to their increased resistance to the known antibiotic categories, which makes the disease caused by one of the MDR groups inevitably lead to death.

In the ecological system, there are two distinct forms of microbial competition that might take place: interference competition and scramble competition. Interactions can occur either intra-specifically or inter-specifically. Interference competition occurs when a certain microbe actively restricts access to nutrients for another microorganism. The phenomenon of scramble competition occurs when a certain microbe depletes available nutrients prior to another microorganism. Indeed, the level of competition escalates in situations where there is limited availability of nutrients. Microbial co-culture exhibits considerable promise in facilitating the identification of continuous production of secondary metabolites, a feat that can be accomplished within controlled laboratory settings. Recent discoveries have highlighted the significance of mixed fermentation or microbial co-culture, as well as microbial transformation, genome mining, and unculturable bacteria, as crucial sources of new antibiotics [3].

Previous studies have extensively examined co-cultures, resulting in findings such as enhanced production of known metabolites or the discovery of new substances. The microbial co-culture technique, which involves replicating conditions seen in nature, has been shown to enhance the efficacy of antibiotics in crude extracts, augment the production of established secondary metabolites, generate analogs of known metabolites, and activate bioactive component pathways that were previously dormant. The fungal co-culture strategy has proven to be a successful method for activating static biosynthetic gene clusters in fungal strains, resulting in the production of hitherto undiscovered secondary metabolites. This strategy typically encompasses three distinct approaches, namely fungal-fungal, fungal-bacterial, and fungal-host co-cultures [4, 5].

In previous studies, the co-cultivation of two marine microorganisms, namely the fungus Emericella sp. and the actinomycete Salinispora arenicola, resulted in a significant enhancement of the expression of the emericellamide biosynthetic gene cluster 100-fold [6]. The co-cultivation of the marine-derived fungus Libertella sp. with the marine bacteria CNJ-328.8 resulted in the induction of the creation of novel cytotoxic diterpenes, namely libertellenones A-D. A further illustration entailed the synthesis of a novel antibiotic, pestalone, using the co-cultivation of the marine fungus Pestalotia sp. alongside an unidentified unicellular marine bacterium, strain CNJ-328 [7].

Therefore, the current study aimed to use a coculture strategy between fungal and bacterial isolates to increase the activity of different activities such as antimicrobial, antioxidant, and antibiofilm.

Materials and methods

Collection of sponge sample

The marine sponge Corella cyathophora was collected using SCUBA equipment from Hurghada at N 26°59’42.87”, E 33°54’4.02” at 10 m depth in the Hurghada region. Red Sea, Egypt. The collected sponge was brought into the laboratory, coded, photographed (Fig. 1), and kept in a cold place (5 °C) until the isolation of fungal and bacterium strains.

Fig. 1
figure 1

(a) Marine sponge morphology (b) Colony morphology of the fungus on PDA (c) Microscopic examination of the isolated fungus

Isolation of sponge-associated fungus and bacteria

The isolation of fungus and bacterium associated with the sponge commenced with the implementation of a surface sterilization procedure. This involved subjecting the sponge to a 1-minute treatment with 75% ethanol, followed by a 3-minute immersion in a solution of 0.5% sodium hypochlorite. Subsequently, the sponge was rinsed three times with sterilized, distilled H2O, and let to air dry under aseptic conditions. Following this, the desiccated segments of the sponge were crushed using a mortar. Subsequently, 1 gram of the powdery material was suspended in 9 milliliters of sterilized distilled water and vigorously mixed using a vortex mixer until a uniform mixture was achieved. Serial dilutions were performed up to the third order, and subsequently, 1 ml of the second and third dilutions were aseptically put onto Petri dishes containing rose Bengal agar. The Petri dishes were then incubated at a temperature of 30 °C for a duration of 5 days. Following the completion of the incubation period, the fungal colonies that had grown were then placed onto potato dextrose agar plates and subjected to incubation under the identical circumstances as before [8]. While, bacterial isolate was is incubated at a temperature of 37 °C for a duration of 3 days on nutrient agar plat.

Morphological identification

The most potent fungal isolate was selected based on antimicrobial screening (data not showed). The phenotypic characterization of the most potent fungal isolate was conducted by cultivating the fungus on potato dextrose agar plates for 10 days. The analysis encompassed the inspection of colony morphology, including shape, color, and medium pigmentation, as well as microscopic evaluation of the produced spores and mycelium.

Genetic identification of the isolated fungus and bacteria

The process of molecularly identifying fungal (CO2) and bacterial isolates involved the extraction of genomic DNA using the Qiagen DNeasy Mini Kit, following the guidelines provided by its designer [9]. The amplification reactions for fungal 18Sr RNA were performed using two primers: ITS1 (5TCCGTAGGTGAACCTGCG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC3), and for bacterial 16Sr RNA, two primers: 27 F (5AGAGTTTGATCCTGGCTCAG-3) and 1492R (5’GGTTACCTTGTTACGACTT’3). The PCR products were submitted to two commercial sequencing services, namely SolGent and Macrogen, located in South Korea. The sequences that were acquired were subjected to analysis using the BLASTN algorithm in order to examine their similarity and homology with the relevant target gene sequences present in the NCBI database. The construction of the phylogenetic tree was performed using the maximum-likelihood (ML) method, facilitated by the software MEGAX [10].


In the co-fermentation experiment, a 10 mL aliquot of a 3-day-old culture of Bacillus sp. COBZ21 was aseptically put into each of ten 2 L Erlenmeyer flasks. These flasks had previously been filled with 500 mL of ISP2 medium and inoculated with a 10 mL aliquot of a 5-day-old culture of Aspergillus sp. CO2.

Extraction of bioactive secondary metabolites

Following the fermentation process of axenic cultures and co-culture, a filtration step was carried out. Subsequently, the resulting supernatant was subjected to extraction using ethyl acetate (1.5 L), resulting in the formation of the ethyl acetate soluble fraction (800 mg) [11].

Antimicrobial activity by plate assay method

The antimicrobial efficacy of the examined extracts was assessed against various microbial strains, including Gram-negative bacteria (Escherichia coli ATCC 25,922, Klebsiella pneumoniae, and Salmonella typhi), Gram-positive bacteria (Staphylococcus aureus NRRLB-767, Methicillin-resistant Staphylococcus aureus (MRSA), yeast (Candida albicans ATCC 10,231), and fungi (Aspergillus niger ATCC 10,231) [12, 13]. The experiment was conducted using 96-well flat polystyrene plates. A volume of 10 µl of test extracts, with a final concentration of 250 µg/ml, was mixed into 80 µl of lysogeny broth (LB broth). Subsequently, 10 µl of bacterial culture suspension in the logarithmic growth phase was added. The plates were then subjected to overnight incubation at a temperature of 37 °C. Following incubation, the observed antibacterial action of the examined extracts was manifested by clearance in the wells. Conversely, extracts that did not have any effect on the bacteria resulted in the growth media appearing opaque in the wells. The control group consisted of the pathogen without any kind of treatment. The measurement of absorbance was conducted around 20 h following the initiation of the experiment at wavelength 600 using a Spectrostar Nano Microplate Reader developed by BMG LABTECH GmbH, located in Allmendgrun, Germany.

Biofilm inhibitory activity

The biofilm inhibitory activity of the extracts obtained was assessed using a microtiter plate assay (MTP) conducted in 96 well-flat bottom polystyrene titer plates against four clinical microorganisms (Pseudomonas aeruginosa ATCC 10,145, Staphylococcus aureus NRRLB-767, Escherichia coli ATCC 25,922, and Bacillus subtilis ATCC 6633) [14]. In this experiment, each well was filled with 180 µL of LB broth, which consisted of tryptone (10 g), yeast extract (5 g), and NaCl (10 g/L). Subsequently, 10 µL of an overnight pathogenic bacterial culture was inoculated into each well. Following this, 10 µL of the desired samples were added to the experimental wells, while a blank control was used as a reference. The entire setup was then incubated at a temperature of 37 °C for a duration of 24 h. Following the incubation period, the contents within the wells were extracted and subsequently subjected to a washing process using 200 µL of phosphate buffer saline (PBS) at a pH of 7.2. This procedure was carried out in order to eliminate any bacteria that were not attached to the surface. The sessile bacteria were immobilized using a 2%sodium acetate solution and subsequently stained using a 0.1% crystal violet dye. The excessive discoloration was eliminated through a process of washing with deionized water and thereafter being lowed to dry. Additionally, the dried plates underwent a washing process with 95% ethanol. Subsequently, the optical density (OD) was measured at a wavelength of 595 nm using a microtitre plate reader (BMG LABTECH GmbH, located in Allmendgrün, Germany).

Assessment of antioxidant activity

The assessment of free radical scavenging activity (RSA) involved the measurement of decoloration in an ethanolic solution of DPPH radical, which was then examined using spectrophotometry at a wavelength of 517 nm [15]. The scavenging activity was calculated as follows:

$$\eqalign{&{\rm{Scavenging}}\,{\rm{ability }}\left( \%\right) \cr & = \left( {{{\rm{A}}_{{\rm{517}}\,{\rm{of}}\,{\rm{control}}}} - {{\rm{A}}_{{\rm{517}}\,{\rm{of}}\,{\rm{sample}}}}/{{\rm{A}}_{{\rm{517}}\,{\rm{of}}\,{\rm{control}}}}} \right) \times 100.}$$

GC-MS analysis

According to the reported procedures [16], GC-MS analysis was conducted utilizing a Thermo Scientific Trace GC Ultra/ISQ Single Quadrupole MS instrument, equipped with a TG-5MS fused silica capillary column of 30 m in length, 0.251 mm in diameter, and featuring a 0.1 mm film thickness. The experimental setup involved the utilization of an electron ionization system with an ionization energy of 70 electron volts (eV). Helium gas was employed as the carrier gas, maintaining a consistent flow rate of 1 ml/min for gas chromatography-mass spectrometry (GC-MS) detection. The temperature of the MS transfer line was adjusted to 280 °C, while the injector was also set at the same temperature. The oven was initially set to a temperature of 50 °C and maintained at this level for a few minutes. Subsequently, the temperature was increased at a rate of 7 °C per minute until it reached 150 °C. Following this, the temperature was further increased at a rate of 5 °C per minute until it reached 270 °C. The oven was then held at this temperature for 2 min. Finally, the temperature was raised to a final value of 310 °C at a rate of 3.5 °C per minute and maintained at this level for a few minutes. The investigation involved quantifying all the discovered components through the use of a percent relative peak area. A preliminary determination of the compounds was conducted by comparing the relative retention time and mass spectra with the NIST and WILLY library data of the GC-MS instrument.

In silico predictions of absorption, distribution, metabolism, and excretion (ADME)-related physicochemical properties and toxicity

The physicochemical features of the molecule were anticipated using the SwissADME web tools, which are specifically designed to assess ADME-related characteristics, Additionally, the toxicity of the selected compound has been predicted via ProTox II web server [17, 18].

Statistical analysis

The data were presented as mean ± SE. Data obtained were analyzed by ANOVA one-way, t-test (n = 3 replicates) was used in comparisons.

Results and discussion

Microbial isolation

The fungus CO2 was isolated from the marine sponge Corella cyathophora (Fig. 1.a). The main morphological features were examined using a light microscope. The isolates exhibited notable differentiation when considering their morphological properties, including colony structure, texture, and medium pigmentation.

Identification of the most potent fungal isolate

The morphological and microscopic inspection of the mycelium and spores cultivated on PDA plates provided confirmation that the isolate is classified as Aspergillus sp. based on the taxonomy established by [19] (Fig. 1.b and 1.c).

Genetic identification of the associated fungus and bacteria

The genetic identification of the isolated fungus and bacteria was conducted by employing sequencing techniques targeting the 18 S rRNA and 16 S rRNA genes, respectively. The DNA was subjected to extraction, amplification, sequencing, and alignment with known sequences stored in the GeneBank database using the Basic Local Alignment Search Tool (BLAST). The results obtained demonstrated a high degree of similarity between the acquired sequence and the fungal isolate CO2, with a homology of 98.84% corresponding to Aspergillus sp. Similarly, the bacterial isolate COBZ21 exhibited a similarity of 99.68% with Bacillus sp. The identification of the CO2 isolate as Aspergillus sp. and the bacterial isolate as Bacillus sp. COBZ21 was determined through the examination of the DNA sequence and physical characteristics. The fungal and bacterial isolates have been archived in GenBank under the accession numbers ON859093.1 and ON859024.1, respectively. The evolutionary history was deduced by employing the neighbor-joining method, as proposed by Saitou and Nei [20]. The tree that demonstrates optimality is depicted. The bootstrap test was used to determine the percentage of duplicate trees in which the associated taxa grouped together [21]. These percentages are displayed adjacent to the branches. The illustrated tree has been accurately represented in terms of scale, where the lengths of the branches correlate to the evolutionary distances utilized in the inference of the phylogenetic tree (Fig. 2a and b). The Maximum Composite Likelihood technique was employed for determining the evolutionary distances and are in units of the number of base substitutions per site [10]. The current investigation included a total of 13 nucleotide sequences. This study included an analysis of the codon positions, which included the first, second, third, and noncoding locations. The paired deletion option was employed to remove all occurrences of ambiguous locations in each pair of sequences. The ultimate dataset comprised a total of 1755 sites. The software MEGA X was utilized to conduct evolutionary analysis [22].

Fig. 2
figure 2

Phylogenetic trees of (a) Aspergillus sp. CO2 and (b) Bacillus sp. COBZ21

Metabolomic profiles of the fungal and bacterial axenic and co-culture extracts

Gas chromatography-mass spectrometry (GC-MS) analysis was employed to analyze the chemical profiles of the fungi Aspergillus sp. CO2 and Bacillus sp. COBZ21 (Figs. 1 and 2S) following their fermentations, both under axenic and co-fermentation conditions. The metabolomic profile of the co-culture extract exhibited the stimulation of various metabolites from distinct chemical classes in contrast to those observed in the two separate cultures. The total peak areas of the discovered constituents in the axenic culture of Bacillus sp. amount to 40.08%. The chemical structures of these identified compounds are documented in Table 1. The main detected compounds are (+-) cis-Deethylburnamine (2.66%) and 2,7,12,17-Tetraethyl-3,5:8,10:13,15:18,20-tetrakis (2,2-di methylpropano) porphyrin (2.60%). Moreover, the analysis of Aspergillus sp. CO2 extract using gas chromatography-mass spectrometry (GC-MS) resulted in the detection and identification of a total of 28 components (Table 2). The total peak areas of the identified ingredients constitute 49.56%, the prospects of the chemical structures of the identified compounds are recorded in Table 2. The main detected compounds are Bis(3,6,9,12-tetraoxapentaethylene)crowno-N,N,N’,N’-tetra methylpphanediamine (2.48%), Lipo-3-episapelin A (2.52%), 2,3-Diacetoxy-6,7,10,11-tetrapentyloxytriphenylene (2.27%), and 3-(2-Dimethylaminoethyl)-5-hydroxy-4-(3-hydroxypropyl)indole (2.23%). The study of Aspergillus sp. CO2 and Bacillus sp. COBZ21 coculturing extracts by GC-MS resulted in the discovery of 28 compounds, as shown in Table 3. The cumulative peak areas of the detected components account for 55.98% of the total. The chemical structures of these identified compounds are documented in Table 3. The main detected compounds are 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[,8)0.0(3,7)]trideca-3,6-diene-10,12,13-trione (3.13%), 2-[3’,5’-di(t-Butyl)-4’-hydroxyphenyl)-3-methyl-1,4-naphthoquinone (3.04%), 21-(1-methoxycarbonylethyl)-à,á,.delta.,ç-tetramethyl porphyrine (2.68%). The mixed fermentation of Aspergillus sp. CO2 and Bacillus sp. COBZ21 resulted in the formation of various chemicals, as indicated by the chemical structure composition analysis (refer to Table 3). The observed induction may arise from either environmental competition or chemical defense systems. Following that, the samples obtained from two pure cultures and a mixed culture were analyzed for their antibacterial, antibiofilm, and antioxidant properties.

Fig. 3
figure 3

Biofilm inhibitory activity of bacterial, fungal and mixing culture

Bac: extract of Bacillus sp. COBZ21, Asp: extract of Aspergillus sp. CO2 & Ba-Asp: extract of coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2

B. subtilis: BacillussubtilisATCC 6633, P. aeruginosa: Pseudomonas aeruginosa ATCC 10,145, S. aureus: Staphylococcus aureus NRRLB-767, E. coli: Escherichia coli ATCC 25,922

Table 1 Chemical compositions of Bacillus sp. COBZ21
Table 2 Chemical compositions of Aspergillus sp. CO2
Table 3 Chemical compositions of Aspergillus sp. CO2 and Bacillus sp. COBZ21 coculturing

Biological evaluation

Antimicrobial activity

Antimicrobial resistance (AMR) arises as a consequence of the evolutionary adaptation of bacteria, viruses, fungi, and parasites, resulting in the loss of their capacity to effectively react to antibiotics. This phenomenon renders infections more challenging to manage, amplifying the likelihood of disease transmission, severe morbidity, and mortality. The efficacy of antibiotics and other antimicrobial treatments is compromised due to the emergence of drug resistance, rendering the treatment of illnesses increasingly challenging or even unattainable [12]. The antimicrobial activity of the three extracts was evaluated against pathogenic bacteria and fungi using 96-well flat polystyrene plates. The obtained results revealed that the bacterial extract displayed moderate antibacterial activity against S. aureus NRRLB-767 and E. coli ATCC 25,922. Furthermore, the fungal extract (ASP) showed low antibacterial activity against Klebsiella, Salmonella, and S. aureus NRRLB-767, while it displayed pronounced activity against E. coli ATCC 25,922. Additionally, the co-culturing extract (Ba-Asp) exhibited potent antibacterial activity against E. coli ATCC 25,922 and S. aureus NRRLB-767, while it showed low antibacterial activity against Salmonella and Klebsiella and antifungal activity against C. albicans ATCC 10,231 (Table 4). The MIC of three extracts was measured toward positive strains and presented in Table 5. The study conducted by Moubasher et al., [23] showed that the co-culturing of fungi and bacteria holds promise as a viable approach for generating secondary metabolites that possess antibacterial properties. The findings from the co-culture experiment revealed that bacteria subjected to chemical stress exhibited diverse reactions, leading to the production of fungal secondary metabolites that possess antibacterial properties. During the co-culture of eight Streptomyces strains and nine fungi, Nicault et al., [24] examined a total of 72 distinct interaction zones involving Streptomyces-fungus interactions (SFIZs). Two of the samples were chosen due to their demonstration of an increase in antibacterial activity when compared to the individual cultures. The examination of these SFIZs revealed that co-cultivation exerted a significant influence on the metabolic expression of each participant, hence facilitating the production of distinct chemicals. The findings indicated that investigating the metabolic capabilities of Streptomyces and fungi through the replication of biotic interactions observed in this particular ecological niche holds considerable potential as a research direction.

Table 4 Antimicrobial activity of different extracts
Table 5 Minimum inhibitory concentration different extracts

Antibiofilm assay

Bacterial biofilms are intricate assemblages of bacteria that adhere to surfaces and are held together by matrices composed primarily of polysaccharides, proteins, and extracellular DNA [25]. Bacterial biofilms have been identified as significant contributors to bacterial persistence and are a source of nosocomial infection that is engaged in a variety of infectious illnesses [26]. The antibiofilm efficacy of the extracts obtained from both axenic and coculture conditions was evaluated through the utilization of the MTT test against four distinct pathogenic bacterial strains, including P. aeruginosa ATCC 10,145, S. aureus NRRLB-767, E. coli ATCC 25,922, and B. subtilis ATCC 6633. The obtained results showed that the bacterial extract (BAC) has low to moderate antibiofilm activity toward S. aureus NRRLB-767 and E. coli ATCC 25,922 only, with biofilm inhibition ratios of up to 45.38 and 25.02%, respectively, while the fungal extract (Asp) displayed very low biofilm inhibition activity against B. subtilis ATCC 6633, P. aeruginosa ATCC 10,145, and S. aureus NRRLB-767, with biofilm inhibitory activity reaching 17.02, 10.25, and 20.73%, while no activity was measured against E. coli ATCC 25,922. On the other hand, mixed culture (Bac-Asp) showed significant biofilm inhibitory activity toward B. subtilis ATCC 6633, P. aeruginosa ATCC 10,145, and S. aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922 (Fig. 3).

Antioxidant activity

The disparity between the protective capacity of antioxidants and the generation of reactive species is a significant concern, as it contributes to the development of various diseases by inducing harm to the genome and other macromolecules. There is a significant amount of literature indicating that increased intake of antioxidants through dietary supplementation is associated with a reduced likelihood of developing a range of diseases. Furthermore, the inclusion of nutraceuticals containing natural chemicals, particularly antioxidants, is vital within the realm of food products [27]. So, in this study, the antioxidant activity of bacterial, fungal, and mixed extracts based on the DPPH assay (200 µg/ml) showed the most antioxidant agent, showing maximum DPPH scavenging activity (75.25%), followed by fungal extract alone (60.26%), then bacterial extract (41.22%) (Table 6). Study on the antioxidant activity of a coculture extract derived from the mycelia of Ganoderma lucidum and Flammulina velutipes in a submerged fermentation process. The assessment of antioxidant activity was conducted using the quantification of ABTS radical scavenging activity. The IC50 values for the extracts of G. lucidum and F. velutipes, as well as their monocultures and cocultures, were determined to be 1.54, 1.99, and 1.64 mg/mL, respectively. The aforementioned findings substantiated that the monoculture of G. lucidum exhibited superior antioxidant activity in comparison to the monoculture of F. velutipes. Furthermore, the coculture showed antioxidant activity that was equal to that of G. lucidum monoculture [28]. Sutthiphatkul, [29] attempts to determine the most favorable co-culture ratio of Acetobacter pasteurianus AJ605 and Zygosaccharomyces bailii YN403 for the fermentation of kombucha. The findings of the study indicate that the maximum antioxidant activity was observed when a co-culture ratio of 8:2 (v/v) of A. pasteurianus AJ605 and Z. bailii YN403 was used over a 10-day fermentation period. The resulting DPPH IC50 value was measured at 25.76 µl/mL, while the ABTS IC50 value was determined to be 8.84 µl/mL. The antioxidant activity of the co-culture ratio of 8:2 was found to be statistically indistinguishable (p > 0.05) from that of the symbiotic culture of bacteria and yeasts (SCOBY) alone.

Table 6 DPPH scavenging activity (%) of different extracts

The ADME-related physicochemical properties

Based on the GC-mass analysis of the mixed culture of Aspergillus sp. CO2 and Bacillus sp. COBZ21, Physicochemical properties of the first compound 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[,8)0.0(3,7)]trideca-3,6-diene- 0,12,13-trione was detected using the SwissADME online server [18]. The estimations are based on drug-likeness guidelines. Accordingly, the compound did not pass the Lipinski du due to two violations (MW > 500, MLOGP > 4.15), Ghose rules, due to four violations (MW > 480, WLOGP > 5.6, MR > 130, #atoms > 70). The compound exhibited 0.17% oral bioavailability, which reflects its poor properties as an oral medication (Table 7). Furthermore, the expeditious assessment of drug-likeness was conducted by the graphical representation of bioavailability. A radar map was generated in order to assess and compare the two compounds using six physicochemical properties, namely size, polarity, lipophilicity, solubility, flexibility, and saturation. The depicted region in the diagram corresponds to the optimal range of values for each parameter. Based on the diagram provided, the compound exhibited an optimal range (shown by the pink area) for all criteria, with the exception of size, flexibility, insolubility, and lipophilicity (Fig. 4). Lipophilicity, an additional physicochemical property of significance, serves as an indicator of the compound’s capacity to permeate the cell membrane [30, 31]. The compound showed Log Po/w values above 5 (8.82), suggesting bad permeability and absorption across the cell membrane. Moreover, the solubility of a molecule is a critical factor that significantly impacts the absorption of the compound throughout the formulation process [18]. According to the ESOL topological model, it may be inferred that the chemical exhibits a modest level of solubility. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is worth noting that the molecule in question did not meet the criteria outlined by the rule of three (RO3). Table 8 shows the compound pharmacokinetic parameters measured using the vector machine algorithm (SVM) model [18].

Table 7 ADME-related physicochemical parameters of Ba-Asp
Table 8 Pharmacokinetics parameters of Ba-Asp
Fig. 4
figure 4

Bioavailability Radar plot of Ba-Asp The pink area shows the optimal range for each property (Lipophilicity: XLOGP3 between − 0.7 and + 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds)

Toxicity prediction via ProTox II

Using the ProTox II website, the compound’s toxicity prediction was carried out [32]. Results in Table 9 showed that, the selected compound has no pronounced toxicity, as predicted by ProTox II. The primary purpose of the toxicity radar chart is to provide a concise visual representation of the level of certainty associated with positive toxicity outcomes in relation to the average value within each respective category (Fig. 5).

Table 9 In silico toxicity prediction of Ba-Asp compound
Fig. 5
figure 5

Toxicity radar chart


The fungal species Aspergillus sp. CO2 and the bacterial species Bacillus sp. COBZ21 have been obtained by isolation procedures from the sea sponge Corella cyathophora. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the chemical profiles of the crude extracts derived from Aspergillus sp. CO2, Bacillus sp. COBZ21, and their coculture. The employment of a coculture approach including Bacillus sp. COBZ21 and Aspergillus sp. CO2 resulted in a notable augmentation of many activities, including antibacterial, antibiofilm, and antioxidant properties. Subsequently, utilizing gas chromatography-mass spectrometry examination of the heterogeneous culture the ADME-related physicochemical parameters of the first drug were evaluated utilizing the SwissADME web server. The toxicity assessment of the coculture extract was conducted using the ProTox II web site, which indicated that the chosen chemical does not exhibit significant toxicity.

Data availability

The datasets generated and/or analyzed during the current study are available in the GenBank repository, [Accession numbers ON859093.1 and ON859024.1].


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A.A.H., M.A.G., M.A. and M.E.E: Conceptualization, methodology, software, formal analysis, investigation, writing—original draft preparation.A.K.K., M.A., H.A.K. and N.R.S.: Methodology, investigation, writing—original draft preparation.All Authors verify that all experiments were performed in accordance with relevant guidelines and regulations.

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Correspondence to Ahmed A. Hamed or Mosad A. Ghareeb.

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Hamed, A.A., Ghareeb, M.A., Kelany, A.K. et al. Induction of antimicrobial, antioxidant metabolites production by co-cultivation of two red-sea-sponge-associated Aspergillus sp. CO2 and Bacillus sp. COBZ21. BMC Biotechnol 24, 3 (2024).

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