Evaluation of Antimicrobial Activity and Identification of Yellow Pigmented Marine SpongeAssociated Fungi from Teluk Awur, Jepara, Central Java

Marine sponge associated fungi are known as potential source of metabolites with various biological activities. Natural pigment is one of metabolite which produced by microorganisms. Several researches reported the antimicrobial activity from natural pigment. Unfortunately there are lack of information about marine fungi natural pigment and its producer. The aims of this research were to identify yellow pigmented Indonesian marine sponge­associated fungi, to extract the pigment, and to study the antimicrobial activity of the pigment against clinical MDR bacteria and clinical pathogenic fungi. Sponge associated­fungus isolate MT23 was successfully identified as Trichoderma parareesei. The fungal pigment could be extracted only in methanol with yield 6.22±0.29%. The pigment could inhibited Salmonella typhi and Escherichia coli MDR strains. The biggest antibacterial activity was shown by concentration 1000μg/mL against S. typhi with inhibition zone was 4.03±0.06 mm.


Introduction
Pigments are chemical compounds which give colours because of its ability to absorb light in the wavelength range at visible region (DelgadoVargas et al., 2000).The utilization of pigments is increasing every year due to the increase of demands in food, beverages, pharmaceutical and cosmeceutical industries (Rymbai et al., 2011; Venil et al., 2013).Natural pigments are known to have biological activities as its advantages over the synthetic pigments (Rostami et al., 2016; Norman et al., 2016; Yolmeh et al., 2016).
Antimicrobial activity is one of biological activities showed by natural pigments.Salem et al. (2014) successfully extracted natural pigment from Carthamus tinctorius.The pigments displayed antimicrobial activity against MethicillinResistant Staphylococcus aureus (MRSA), Escherichia coli ATCC 25218 and Candida albicans.Another research reported that carotenoids pigment were able to inhibit Salmonella enteritidis (PTCC 1709) and E. coli (PTCC1260) (Rostami et al., 2016).In addition, crude extract of pigment from Streptomyces sp.D25 was reported to have antibacterial activity against biofilm forming bacteria such as Pseudomonas sp.P1., Bacillus sp.P13 and Alcaligens sp.M28 (Radhakrishnan et al., 2016).
Marine microorganisms are known as potential producer of natural pigment with great antibacterial activity.There are several microorganisms are known life associated with marine organisms especially sponges such as bacteria, actinomycetes and fungi (Idraningrat et al., 2016; Wu et al., 2016).Ibrahim et al. (2014) studied antimicrobial activity of prodigiosin (red pigment) from spongeassociated bacteria Serratia marcescens IBRL USM 84.This pigment greatly inhibited MRSA and had good activity inhibited Bacillus cereus, Bacillus licheniformis and Agrobacterium tumefaciens.In 2015, Suresh et al. (2015) observed antibacterial activity of red pigment produced by Halolactibacillus alkaliphilus MSRD1 which isolated from seaweed against S. aureus and Salmonella typhi.In addition, Srilekha et al. (2016) successfully isolated marine pigmented bacteria and extracted the pigment then studied its antimicrobial activity.Result showed the pigment had great antimicrobial activity against bacteria such as S. aureus, Proteus vulgaris, K. pneumoniae, E. coli, and fungi such as Fusarium sp., C. albicans, Mucor sp., and A. flavus with inhibition zone range 18-28 mm.These reports show great potential of natural pigments from marine bacteria as antibacterial agent.
However, there are still a few researches about the potential of natural pigments from marine fungi and its antibacterial activity against clinical multidrug resistant bacteria (MDR) and clinical pathogenic fungi.MDR bacteria are bacteria which already resistant to several antibiotics that should be effective to inhibit the growth of the bacteria (Magiorakos et al., 2012).This is a big issue for public health.The aims of this research were to identify Indonesian yellow pigmented spongeassociated fungi, to extract the pigment, and to study the antimicrobial activity of the pigment against clinical MDR bacteria and clinical pathogenic fungi.

Fungal isolate
Fungus MT23 was culture collection of Marine Fungi Division of Tropical Marine Biotechnology Laboratory, Integrated Laboratory of Diponegoro University, Semarang, Central Java, Indonesia.This fungus was isolated from unidentified marine sponge from Teluk Awur Bay, Jepara, Central Java.

MDR bacteria and clinical pathogenic fungi isolates
MDR bacteria and clinical pathogenic fungi were obtained from Dr. Kariadi General Hospital Medical Center, Semarang, Central Java, Indonesia and Diponegoro National Hospital, Semarang, Central Java, Indonesia.Bacteria E. coli, S. typhi and fungi Malassezia furfur and Trichophyton rubrum were used in this study.

Fungal culture
Fungus was recultured according to Sibero et al. (2016) with particular modifications.Approximately 1 cm 2 fungus was taken from the stock culture then transferred to fresh Malt Extract Agar (MEA) and incubated in room temperature (23 o C) for 7 days with addition of chloramphenicol 5% (%w/w).After that, the morphology of the recultured fungus was compared to the culture stock.The same morphology indicate the fungus was not contaminated.Then fungus MT23 was reculture to new fresh MEA without chloramphenicol, pH 5.4, illumination using LED lamp (Philip LED 20 watt) and incubated in room temperature (23 o C) for 14 days.

MDR bacteria and clinical pathogenic fungi
Gramnegative bacteria E. coli and S. typhi were revified on MacConkey Agar while M. furfur and T. rubrum were revified on Potato Dextrose Agar (PDA).The bacteria were incubated for 24 h in 32 o C while the fungi were incubated in at 27 o C for 24 h.

Morphological approach
Fungus MT23 was recultured on MEA for 5 days with slide cultured method (Qiu et al. 2005).Sterilized MEA was cut with size 2 cm 2 then transferred and put onto object glass then covered by cover glass.After that, fungal mycelia were inoculated on each side of the MEA then incubated at room temperature (set at 23 o C).After 5 days of incubation, object and cover glasses were covered by fungal mycelia.MEA was separated from the glasses.Lactophenol cotton blue was dropped on the object and cover glasses then covered by its pairs.After that, the morphology of the fungi was observed under microscope.

Molecular approach
DNA of fungus MT23 was extracted using Chelex method from Turan et al. (2015) with particular modifications.Seven days old of mycelium were immersed in 100 µL ddH2O and 1000 µL of 0.5% saponin for 24 h in chilling temperature (4 o C).Then it was separated by centrifugator with 12000 rpm for 10 min.The supernatant was discarded while the template was added 100 µL of ddH 2 O followed by addition of 50 µL of 20% Chelex 100.Then it was heated in water bath at 80 o C for 5 min.After that, the mixture was homogenized using vortex for 10 s and reheated for 5 min.After reheated, the DNA in mixture were separated using centrifugation at 12000 rpm for 10 min.Then the supernatant were transferred to new microtube and stored at 20 o C for 24h.
DNA of fungus MT23 was amplified based on Internal Transcribed Spacer (ITS) region.The amplification using primer 1 µL of ITS 1 (5'TCC GTA GGT GAA CCT GCG G3') and 1 µL of ITS 4 (5'TCC TCC GCT TAT TGA TAT GC3').The primers were mixed with 12.5 µL of GoTaq Green Master Mix Promega, 0.5 µL of DNA extract and 10 µL of ddH 2 O.The PCR profile consisted of 3 min of preheat at 93 o C for 3 min, 30 cycles for denaturation at 95 o C (1 min), annealing at 56.1 o C (1 min) and extension at 72 o C (1 min).Final extension was done at 72 o C for 7 min.The quality of PCR product was checked using gel electrophoresis in agarose 1%.Then PCR product was sent to 1 st Base Laboratories Sdn Bhd, Malaysia for DNA sequencing.DNA sequence was analyzed to its homology using Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov).Phylogenetic tree was reconstructed using MEGA 7.0 software package (Tamura et al. 2011).Neighborjoining was applied as the statistical method with 1000 bootstrap replications.

Pigment extraction
Yellow pigment from fungus MT23 was extracted using solidliquid extraction (Manikkam et al. 2015).The mycelia were separated from media, then the media were cut into smaller size and dried using silica gel in desiccator for 48 h.After that, solvent optimization for pigment extraction was done by immersing the dried agar in methanol, ethyl acetate and nhexane with agitation using a shaker (118 rpm) for 24 h then filtered using filter paper (MachereyNagel 640d•Ø 1125 mm).The filtrate was evaporated using rotary evaporator (Eyela SB1100) set at 38 o C. The pigment yield percentage was calculated using following equation:

Antibacterial activity
Antibacterial activity of the pigment was evaluated using agardisc diffusion method from Balouiri et al. (2016) with several modifications.Pigment extract was dissolved in methanol and diluted to five concentrations (50, 125, 250, 500 and 100 µg/mL).S. typhi and E. coli strain MDR were grown on MacConkey Agar for 24 h (32 o C) then diluted in physiological saline solution into 0.5 Sibero et al. I.J.Biotech.
McFarland.Then the bacterial solution was inoculated to MHA using sterile cotton swab.Each extract (10 µl) was injected onto sterilized paper disc then placed on the MHA and incubated at 32 o C for 24 h.Amoxicillin + Clavulanic acid (30 µg/disc) was used as positive control.The presence of clear zone indicated the antibacterial activity of the pigment extract.

Antifungal activity
This assay was done according to Bhalodia and Shukla (2011) and Balouiri et al. (2016) with modifications.Pigment extract was dissolved in methanol and diluted to five concentrations (50, 125, 250, 500 and 100 µg/mL).M. furfur and T. rubrum were grown on PDA for 24 h at 27 o C. Then the fungi were inoculated using pour plate method.Each extract (10 µL) was injected onto sterilized paper disc then placed on the PDA and incubated at 27 o C for 24 h.Nystatin was used as positive control in this study.The presence of clear zone on PDA indicated the antifungal activity of the pigment extract.

Data analysis
The impact of solvents to pigment yields was calculated with One Way Analysis of Variance (ANOVA) with P<0.05 while antibacterial and antifungal activities were calculated using Two Way ANOVA with P<0.05.The data was analyzed using SPSS 5.0.

Fungal identification
Fungus MT23 is a marine sponge associated fungi which is isolated from sponge from Teluk Awur, Jepara, Central Java.Macroscopic characteristics of fungus MT23 were white colour mycelium with green radial growth ring, hyphae concentrated and produced yellow colour in the media.This fungus grown well in room temperature (23 o C) and changed the colour of MEA from slightly orange to yellow colour.Colour change was started at fourth day of cultivation and successfully changed the colour of whole media at day 14 (Figure 1).Physical conditions of environment including pH and temperature were reported to give impact to the fungal pigment production.A fungus Penicillium purpurogenum GH2 produced highest yield of red pigment after 240 h (10 days) of cultivation with pH 5 at 24 o C (Mendéz et al. 2011).On the other hand, Velmurungan et al. (2010) induced fungal pigment production using colour of lights.They figured out that the darker lights like red (780-622 nm) and blue (492-455) gave higher pigment productions compared to green (577-492 nm), yellow (597-577 nm) and white lights in 5 different fungi.This phenomenon could be explained by the postulating of the existance of photoreceptor in fungi.However, there was no clear explanation about the mechanisms of the light to induce pigment productions in fungi.Furthermore, Blumenstein et al. (2005) suggested that a phytochrome type of system may be operative in particular fungi, in his case was A. nidulans.The phytochrome suggested able to sense the red and farred light through photointerconversion between the two stable conformations.
Figure 2 shows the morphology of fungus MT23 under microscope.This fungus had hyaline and thin mycelium with septum.The conidiophore were branching with lageniform phialide with green conidia, smooth, eguttulate, and ellipsoidal to cylindrical form.According to its characteristics, fungus MT23 was judged as member of genus Trichoderma (Atanasova et al., 2010; Rahman et al., 2011; Qin and Zhuang, 2016).To confirm the accuracy of morphological identification, we did identification through molecular approach.
Fungal DNA was extracted using Chelex 100 method (Turan et al., 2015).This method is commonly used to extract microorganisms' DNA (Liu et al., 2015; Susilowati et al., 2015).Chelex 100 able to prevent DNA damage by inhibiting DNase activity (Walsh et al., 1997).Fungal mycelium was immersed in saponins and ddH2O for overnight.Saponins is detergent agent to lyse the cells (Dong et al., 1997).To amplify fungal DNA, ITS1 and ITS4 were used as primer.These primers will amplify DNA from end of 18S rRNA gene region, internal transcribed spacer 1, 5.8S rRNA gene I.J.Biotech.region, internal transcribed spacer 2 region and the beginning of 28S rRNA gene region (White et al., 1990; Larena et al., 1999; Anderson et al., 2003; Bellemain et al., 2010; Schoch et al., 2012).Figure 3 shows the location which amplified by ITS primer.
Fungal DNA amplification was done by using polymerase chain reaction (PCR) the quality of PCR product was checked using gel electrophoresis.The result of visualization of fungal DNA quality is shown by Figure 4.
Figure 4 shows that the fungal DNA was successfully amplified in region between 500 to 1000 bp.This result is supported by the statement of Nilsson et al. (2009) who stated that the fungal DNA is normally obtainable in a single round of Sanger DNA sequencing in 650 bp region.PCR product was sequenced to get the sequence of fungal DNA, and then the sequence was compared to that of the database in gene bank using BLAS homology.According to fungal DNA comparison, fungus MT23 was 99% similar to Trichoderma parareesei ATCC MYA4777 (Houseknecht et al., 2011).Phylogenetic tree of fungus MT23 is shown by Figure 5.
Result of morphology observation was supposed fungus MT23 was member of genus Trichoderma and supported by the molecular identification which successfully identified fungus MT23 as T. parareesei.Genus Trichoderma is well known as terrestrial fungus and give advantages for agricultural sector (Rinu et al., 2013; Saravanakumar et al., 2013; El Komy et al., 2015; Hamed et al., 2015).The finding of T. parareesei as marine sponge associated fungi showed that this fungus was a marine facultative (marine derived) fungi.Several species from Trichoderma such as T. longibrachiatum, T. harzianum, and T. atroviride were reported to tolerate to the increasing of salinity (GalHemed et al. 2011).Genus of Trichoderma was also known to produced pigment and bioactive compounds (Rubeena et al., 2013; Vacondio et al., 2015; Benkada et al., 2016).

Pigment extraction
Fungus MT23 changed the colour of MEA to yellow because it produced extracellular pigment.Extracellular pigments production is indicated by colour changing of the environment where the fungi live.Previous research successfully identified black extracellular pigment which produced by endophytic fungi as melanin having photoprotector activity (Sibero et al., 2016).Fungus MT23 produced yellow pigment started at the fourth day of cultivation and   The fungus had highest pigment absorbance (AU 500nm) at temperature 28-30 o C, pH 5.5, glucose as carbon source, peptone as nitrogen source and cultivated for 8 days.Yellow pigment from fungus MT23 were extracted using organic solvents.Environmental conditions have important role in fungi growth.Pradeep and Pradeep (2013) studied the optimum cultivation condition for growth of fungus Fusarium moniliforme.The fungus had highest biomass (g/L) at temperature 28 o C, pH 5.5, glucose as carbon source, peptone as nitrogen source, KH 2 PO 4 as source of salt and addition of methionine as additional amino acid.Stationary phase of this fungus was on eighth day of cultivation.In this study, we used Malt Extract Agar (MEA) from HiMedia M173 which contained malt extract and mycological peptone.Peptide and amino acid in peptone are easily metabolized by fungi which induced the production of fungal metabolites, including pigment (Calestino et al., 2014). Da Costa Souza et al. (2016) used several media including malt extract to induced pigment production in filamentous fungi.As a result, pigment production in malt extract was higher than other medium.In addition, nutrient content in media can regulate the expression of genes to activate the metabolic path way for pigments production (Pradeep et al., 2013).
Organic solvents such as nhexane, ethyl acetate, chloroform, methanol and dichloromethane were usually used to extract natural pigments from microorganisms (Saravanan andRadhakrishnan, 2016; Weber et al., 2016).In this research, nhexane, ethyl acetate and methanol were used to extract fungal pigment.The purpose of this step was to know the best solvent to obtain highest fungal pigment yield.The result of pigment extraction is shown by Figure 6 and Table 1.
Table 1 shows that only methanol was able to extract fungal extracellular pigment from fungus MT23.Statistic analysis showed that solvents gave significant difference to the fungal pigment yield (P<0.05).The polarity of pigment will influence the suitability of  optimum solvent to extract the pigment.Several pigments such as astaxanthin, chlorophyll a, chlorophyll b and xanthophyll were able to be extracted with methanol (Sasidharan et al., 2013, Sumanta et al., 2014; Bhat and Marar 2015).

Antibacterial and antifungal activities
Fungal pigments are known to produce antimicrobial activity.Geweely (2011) successfully investigated antimicrobial of several fungal pigments.The result showed that fungus Aspergillus nidulans, Fusarium moniliforme, Penicillium purpurogenum, and Phoma herbarum had weak to moderate antimicrobial activity against dermatophyte fungi, non dermatophyte fungi and several bacteria.Another research showed antibacterial activity of astaxanthin pigment from marine yeast (Ushakumari and Ramanujan, 2013).Fungal pigment from fungus MT23 was tested against several clinical pathogenic microorganisms.The result of antibacterial and antifungal is shown in Table 2.
According to Table 2, fungal pigment from MT23 showed weak antibacterial activity against clinical MDR bacteria S. typhi and E. coli.The biggest antibacterial activity showed at the concentration of 1000 µg/mL against S. typhi.The pigment didn't show any antifungal activity against clinical pathogenic fungi.The weak antimicrobial activity means that this fungal pigment is not potential as a source of antibiotic against clinical pathogenic and MDR microorganisms.Carotenoid is one of natural pigment which produced by fungi with yellow, orange to reddish colors.The production of this pigment is related to stress tolerance or with the synthesis of physiologically active by product (Avalos and Limόn, 2015).Carotenoid pigment is mostly reported has antimicrobial and antioxidant potential (Ernawita et al., 2016; Yoo et al., 2016).The antimicrobial mechanisms of this pigment could lead to the accumulation of lysozyme enzyme that digest bacterial cell walls (Abu Ghannam et al., 2013).

Conclusions
Sponge associatedfungus isolate MT23 was successfully identified using morphological and molecular approaches as member of Trichoderma and had 99% similarity to Trichoderma parareesei.The fungal pigment could be extracted only in methanol with yield 6,22±0,29%.The pigment showed weak antibacterial activity against S. typhi and E. coli strain MDR, furthermore the pigment did not show any activity against clinical pathogenic fungi.

Figure 1 .
Figure 1.a) 4 days old of mycelium, b) 4 days old of reverse side, c) 14 days old of reverse side.

Figure 3 .
Figure 3. Diagram of the rDNA region of fungi and ITS primer (Source: Bellemain et al., 2010).

Figure 4 .
Figure 4. Visualization of DNA band of fungus MT23.

Figure 6 .
Figure 6.The result of maceration of agar media in organic solvents: a) in ethyl acetate, b) in nhexane, c) in methanol.

Table 1 .
Fungal pigment yield with different solvents.

Table 2 .
Antibacterial and antifungal activities of fungal pigment MT23.