Pigment production from a mangrove Penicillium

A mangrove Penicillium producing red pigment was cultured in an optimized medium that was designed by the authors previously and used in this study. The purpose of this study was to identify the pigment and also to study the effect of bio elements on pigment production. Pigment from the medium was efficiently extracted using chloroform, ethyl acetate and n-butanol. Most of the red pigment was extracted into ethyl acetate and further purified by preparative thin layer chromatography. From H and C NMR data supported by electronic imaging mass spectrometry, structure of the compound was elucidated as 2-(4-acetyl phenyl) acetic acid. The yield of pigment produced was studied with respect to various salts and bio elements. Salts at high concentrations (sodium chloride, ammonium sulfate, and sodium nitrite) had a drastic effect on pigment yield because most of the pigment remained adhered to the mycelium instead of diffusing into the medium. Also, when bio elements were supplemented to the medium; calcium, iron, and zinc enhanced pigment yield whereas; potassium, magnesium, copper and manganese did not have significant impact on pigment production. Lead had a drastic negative effect on the pigment yield. Therefore, this study proves that salts and bio elements play a major role in the production of various metabolites from mangrove fungi.

in foodstuff, cosmetics, and pharmaceutical manufacturing processes (Francis, 1987;Kim et al., 1995).Microbial pigments can become highly significant when the production yield is high and the pigments are highly stable (Cho et al., 2002).
The ocean environment is a rich source for novel metabolite producing microbes, but only during last two decades the studies on marine metabolites have increased.Some previous studies showed that the marine Penicillium produced pigments (PP-V and PP-R) and these are similar in structure to the pigments produced from Monascus (monascorubrine and monascuscorubramine) (Ogihara and Oishi, 2002); pigments from Monascus are being used in the food industry since long time.Mohan and Vijay-Raj (2009) also described pigment production and radical scavenging activity from a Penicillium sp NIOM-2 isolated from marine sediment in India.Three other halophilic fungal strains Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum isolated from the salterns in the eastern coast of the Adriatic Sea, produced melanin pigments at saturated concentrations of sodium chloride (Tina et al., 2006).Details of several other studies on marine fungi producing significant metabolites are seen but literature on industrial application of potential marine fungal pigments is very minimal and this drives our interest to study the pigment potential of a mangrove Penicillium.
The purpose of this research was to isolate fungal pigments from mangrove sediments because mangroves exist under conditions of high salinity, extreme tides, high temperature, and various other stress conditions (Kathiresan and Bingham, 2001).Therefore microbes growing under these conditions will have the potential to produce different significant metabolites to cope with these stresses.
Research on mangrove pigments is very less; it has been recorded that microorganisms from mangroves open up new areas for potential biotechnological exploitation (Gopal and Chauhan, 2006).During this study period, we isolated nearly 100 mangrove fungi from Godavari mangroves of India.Most of the fungi were pigment producers and a red pigment-producing Penicillium was selected to study the pigment, its optimization conditions and the effect of metals and salts on pigment yield.In this paper, pigment extraction method, pigment structure and the impact of different bio elements and salts on pigment yield were explained.The main objective of this study was to identify which bio elements have a positive effect on pigment production and also to study if salts effect pigment production even when the fungus was from a marine origin.

Microorganism and inoculum preparation
The mangrove fungus DLR-7 isolated from Godavari Delta, Andhra Pradesh, India was identified as Penicillium sp. according to Alexopolus and Mims (1979).Stock cultures of (Karuna et al., 2009) this Penicillium were maintained on potato dextrose agar slants prepared with 50% aged seawater; the cultures were revived every month and stored at 4C until used in the experiment.Inoculum for these studies was prepared by growing the fungus initially at 25C on potato dextrose agar (PDA) plates for seven days.Plates having uniform growth and sporulation were selected and a 0.7 cm 2 plug from the outer zone of the colony was punched with a sterile cutter.The plugs were transferred to 100 ml of culture medium in 250 ml flasks and incubated under static conditions at 25C until maximum pigment was produced (Gunasekaran and Poorniamaal, 2008).

Culture conditions
An optimized basal culture medium was designed for red pigment production using potato extract prepared in the laboratory.Two hundred grams of potatoes were cleaned, sliced and cooked for 30 min with 500 ml of distilled water; the cooked potato slices were mashed and the liquid was filtered through a muslin cloth (Aneja, 2003).Then, 500 ml of seawater was added to the medium and autoclaved.Optimization of culture medium was carried out using various carbon and nitrogen sources and altering different physical parameters (Lathadevi et al., 2014).Results from the optimization experiments concluded that xylose (2% w/v) and glycine (1% w/v) when supplemented to potato extract and pH adjusted to 3.0, produced a high yield of pigment.Therefore, this medium was further used to extract and identify the pigment.The medium was also supplemented with different salts and bio elements to study if they have any effect on the concentration of the pigment produced.

Extraction of pigment
Extraction of pigment from the liquid culture media was carried out by different solvents viz.non polar to polar (Padmavathi and Prabhudessai, 2013).Chloroform, ethyl acetate, and n-butanol were used for the extraction process; all the solvents used were obtained from Qualigens Fine Chemicals Pvt. Ltd., (Mumbai, India).Five liters of culture medium were prepared and about 500 mL were dispersed in ten 1 L conical flasks.The media were autoclaved and about 0.7 cm 2 plugs from the outer zone of the Penicillium culture plates were transferred to the media.The inoculated flasks were incubated under static conditions at 25C (Gunasekaran and Poorniamaal, 2008) for 12 days and the cultures were then harvested.The culture medium was then passed through filter paper (No. 1; Whatman, India Liasion Office, Mumbai, India).A three stage multi-contact/counter current extraction method was used to extract the pigment.Three 500 ml separating flasks were used, 200 ml of the filtered broth was added to the flasks, and 100 ml of distilled solvent was added to the first flask, shaken well and allowed to stand until the aqueous and organic layers separated.Organic layer was transferred to the second flask treated as in the previous step and repeated with the third flask as shown in Figure 1.
Finally, solvent with the pigment was transferred to a clean conical flask and extraction was repeated until no more pigment diffused into the solvent.The entire culture broth was extracted in the same way with chloroform, ethyl acetate, and n-butanol.Finally, the solvents were stripped off with the help of a rotary vacuum evaporator and the amount of pigment was weighed and purified by chromatography.Absorbance of the extracts was measured using a UV-visible spectrophotometer (Model 117, Systronics, India).

Chromatographic analysis
Thin layer chromatography (TLC, Sigma-Aldrich, Hyderabad, India), ).These trace metal concentrations were used at concentrations typically found in sea water.Stock solutions ranging from 10 5 to 10 6 ppb were prepared for calcium, magnesium and potassium.From the stock, different working concentrations (10 5 ppb to 15 × 10 5 ppb) were prepared and added to the culture media.For metals such as iron, copper, manganese, lead and zinc, a pre-stock solution was prepared and the required volumes were taken to obtain the respective concentrations of 0.05 ppb to 1 ppb.50 ml of media was prepared with pH 3.0, sterilized and inoculated with the test organism as described above and incubated at 25C for 12 days.All experiment treatments were performed in triplicates and compared to control media (media without trace elements).

Statistical analysis
Data analysis for treatments with salts and trace elements was performed to study their effect on pigment production.Multiple comparative analyses were performed using R version 2.15.3 for Statistical Computing (http://www.r-project.org/).Linear regression function "lm ()" in R was used to determine significant differences between the treatments using {lm (formula = y ~ x)}.Treatments statistically significant (p < 0.05) are considered to be positively affecting pigment production.Regression coefficients and p values of significant treatment comparisons were depicted in the table.

RESULTS AND DISCUSSION
The absorption maxima (λmax) and weight of the pigment fractions derived by solvent extraction are presented in Table 1.The chloroform fraction was colorless whereas, the ethyl acetate and n-butanol fractions were deep red.Chloroform and ethyl acetate extracts had fewer components and were fairly pure, but the butanol fraction had a large number of compounds.As the primary interest of this research was to investigate the red pigment, the ethyl acetate fraction was selected and further purified to elucidate the structure of the red pigment.Preparative TLC of the ethyl acetate fraction yielded a crystalline compound with a melting point of 156°C.It had a strong blue fluorescence on the TLC plate under UV; however, it appeared as a green arrow headed spot after spraying with 5% sulfuric acid in methanol.
From 1 H, 13 C nuclear magnetic resonance (NMR) data supported by electronic impact mass spectroscopy (EIMS) , the structure of the compound was elucidated as [2-(4-acetyl phenyl) acetic acid] as presented in Figure 2.However, during the chromatographic separation, the compound turned out to be colorless.the trace elements studied, calcium produced maximum pigment (1,490 mg/L) while the control yielded 1,065 mg/L of pigment.Bio elements such as zinc and calcium effect fungal growth and gene expression (Cho et al., 2002).
Limited literature is available on pigments from mangrove fungi.This is the first study providing the investigations on the effect of trace elements on mangrove fungal pigments.Observations recorded will significantly contribute for further work in this area.Pigments from terrestrial fungi like Monascus are being industrially used in the food industry, but there are no studies on the industrial use of pigments from mangrove fungi.The pigment component 2-(4-acetylphenyl) acetic acid isolated during this study is similar to Ganbajunin B, which is a brownish red pigment isolated from a mushroom Thelephora vialis.This compound has strong antioxidant properties and this mushroom is a favorite food in China due to its strong flavor and taste.
The pigment component isolated from mangrove fungus in this study has significant characters such as the compound isolated from the mushroom (T.vialis) and further detailed studies such as toxicity tests are required.These studies will be helpful to explore the industrial uses of mangrove fungal pigments.
Figure 4. Effect different bio elem

Table 1 .
Characteristics of the pigment fractions after solvent extraction.