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Microbiology and Biotechnology Letters

Research Article(보문)

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Fermentation Microbiology (FM)  |  Applied Microbiology

Microbiol. Biotechnol. Lett. 2024; 52(3): 314-324

https://doi.org/10.48022/mbl.2406.06009

Received: June 26, 2024; Revised: August 6, 2024; Accepted: August 12, 2024

Characterization of Pigment Production by Endophytic Rhodotorula mucilaginosa MGI from Tagetes erecta

Isswa Iqbal1, Warda Sarwar1, Qurban Ali1, and Safia Ahmed1,2*

1Department of Microbiology, Quaid-i-Azam University, Islamabad 45320, Pakistan
2Vice Chancellor office, Shaheed Benazir Bhutto Women University, Peshawar Khyber Pakhtunkhwa, Pakistan

Correspondence to :
Safia Ahmed,     safiamrl@yahoo.com

Abstract

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Due to the hazardous effects of synthetic pigments, natural pigments are gaining popularity. Among natural sources microorganisms have become a major source of numerous industrially essential items and their use for getting various natural products have expanded dramatically in recent years. In the present study, 9 endophytic fungal strains were isolated from Tagetes erecta. On screening, yeast strain MGI was selected for further study which was identified as Rhodotorula mucilaginosa MGI. The pigment was intracellular, and the color of the crude extract was orange. The extract was subjected to characterization by UV-visible spectrophotometer and was purified by column phase chromatography, after purification two pigmented fractions were obtained. These fractions on characterization by thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectrophotometer affirms that they belong to carotenoid group of pigments. Orange (F1) and yellow (F2) fractions were anticipated as astaxanthin and beta carotene respectively. Moreover, the bioactive potential of pigmented fractions was investigated which manifested that F1 exhibited a maximum antioxidant activity of about 77% and F2 showed the highest zone of inhibition of 38 mm against Staphylococcus aureus. Thus, this study reflects that an endophytic yeast strain has the potential for the production of bioactive microbial pigments.

Keywords: Tagetes erecta, endophytic yeast, Rhodotorula mucilaginosa, carotenoids, astaxanthin, beta carotene

Introduction

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Pigments are the colored compounds that possess bio-technological potential and offer promising avenues for various applications. Colors are the first variable that contribute to the sensory, aesthetic, nutritional value, safety, or freshness of industrial products [1, 2]. Pigments are of different types, synthetic or natural. Synthetic dyes are synthesized by chemical means while natural pigments are produced by natural sources like animals, plants, and microorganisms. Synthetic dyes are hazardous as they are cytotoxic, carcinogenic, or teratogenic [3, 4]. They cannot be degraded by living organisms and they lack safe waste disposal techniques [5, 6]. Due to these problems, they pose a threat to human beings and the environment. With the advent of the hazardous effects of chemically synthesized pigment demand for natural colors is increasing at a faster pace and new sources are being searched. Moreover, numerous industries also allocate resources towards research and development to discover new colors and sources, which in turn drives the creation of new market trends and fosters innovation [7].

Nature is replete with pigment-producing organisms: plants, animals, and microorganisms. Microbial pigments are cost-effective, they eliminate the need for large labor, they are easy to handle and can tolerate diverse conditions like pH, light, and heat effectively, and are more stable against physiological changes [2, 8]. They provide dynamic production and extraction means and they are readily available, and their availability is not dependent on seasons [9]. Microbial pigments being secondary metabolites possess several properties like antibacterial, antifungal, anti-cancerous, antioxidant, antimutagenic, antiproliferative, immunosuppressive, and anti-diabetic and they are biodegradable, nontoxic, and eco-friendly [10, 11]. Due to these properties, they have application in various industries like food, cosmetics, textile, leather, paper, and pharmaceutical industries [12, 13]. Thus, using microbial pigments in different industries provide sustainable developmental goals for mankind.

Pigment-producing microbes like bacteria, fungi, yeasts, and microalgae are present in tremendous resources. Some ecological niches inhabited by microbes are marine, terrestrial, and mangrove ecosystems. Thus, soil, rhizosphere of plants, freshwater, seawater, air, and even endophytes inhabiting tissue of healthy plants are home for number of pigments producing microbes [14]. Some common genera of microbes that produce various shades of color are Serratia, Bacillus, Rhodococcus, Pseudomonas, Dunaliella, Monascus, Paecilomyces, Penicillium, Rhodotorula, Cryptococcus, Sporobolomyces etc. [6, 15]. Endophytes are the store house of bioactive secondary metabolites and microbial pigments are one of them. An endophytic fungus Phyllosticta capitalensis isolated from dry deciduous forest produces a melanin pigment [14]. Another endophytic fungal strain SX01, obtained from the twigs of Ginkgo biloba produces red pigment [16]. Yeasts being unicellular in nature and high growth rate are effective in producing wide range of industrial products. As endophytic yeasts exist in phyto-biome and have industrial applications, so it is essential to further study endophytic yeasts [17].

Pigments are characterized into different types based on certain characteristics like functional groups, presence of specific chromophores and method of synthesis [18]. Thus, these microbial pigments can be organic, inorganic, polar, non-polar and intracellular or extracellular [19]. Some types of microbial pigments are carotenoids, melanin, prodigiosin, flavins, pyocyanin, violacein etc. [20, 21]. Carotenoids are the most abundant group of pigments which are sub-divided into lycopene, β-carotene, torulene, torularhodin, astaxanthin, zeaxanthin, canthaxanthin and lutein, etc. [22, 23]. Carotenoids as natural coloring products have vast range of applications. The global market of carotenoids is evolving and is expected to reach 2 billion dollars by 2027 with a compound annual growth rate (CAGR) of 2.6% [24]. Although the higher proportion shared at market level is of synthetic pigments but considering the side effects, global market of microbial pigments is progressing expeditiously. Moreover, use of natural pigments in numerous essential industrial products have received approval from various well-known organizations like World Health Organization (WHO), Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) [8].

Microbial pigments present promising alternatives to artificial dyes, prompting ongoing research aimed at transitioning these pigments from research laboratories to industrial markets. To facilitate this transition and to fulfil the demand of growing consumers, researchers are focusing on various aspects of microbial pigments like searching for new reservoirs of pigment producing microbes and searching for effective green and sustainable extraction or purification methods and evaluating the bioactive potential of microbial pigments. This study is based on the aspect of isolating pigment producing endophytic fungi from Tagetes erecta and characterizing pigment production of endophytic yeast by various means.

Materials and Methods

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Collection and processing of sample

T. erecta (Mari-gold flower) for studying microbial pigments was collected in sterile zipper bag from the botanical garden of Quaid-i-Azam University, Islamabad. To remove dirt or soil particles T. erecta flower was washed under the running tap water, different parts of flower i.e., petals, sepals and bud were separated. The sample surfaces were sterilized with series of solutions. Firstly surfaces were rinsed with autoclave distilled water for 1 min, then 70% ethanol was used for 1 min followed by 2% of Sodium hypochlorite for 2 to 3 min and at last final washing was done with 70% ethanol for 60 sec [25]. After sterilization segregated parts were cut horizontally and vertically into small pieces using a sterile blade and these tissues were then placed on potato dextrose agar (PDA) plates for isolation of endophytic fungi.

Isolation and screening of pigment producing microbes

Potato dextrose agar (PDA) plates for fungal growth were incubated at 28℃ for 7−14 days. The plates after incubation time were observed for growth and the isolates which showed some colors were sub-cultured by streak plate or point inoculation method on PDA plates. For screening these isolates were inoculated in broth media. After incubation time results were noted. For further study one isolate which showed good color production was selected.

Production and extraction of pigment by selected isolate

Yeast isolate was inoculated in 150 ml Erlenmeyer flask of Potato dextrose broth (PDB) and in mineral media, these flasks were then incubated in rotary shaker at 150 rpm for 4−7 days at 27℃. The mineral media is composed of 1% w/v glucose, 0.1% w/v yeast extract, 0.1% w/v (NH4)2SO4, 0.2% w/v NaCl, 0.2 M, KH2PO4, and 0.0075% w/v CaCl2·H2O [26].

An accurate and effective method for extraction was evaluated. After the incubation time, for extraction of metabolites, the broth was centrifuged at 7800 rpm at 30℃, for 15 min. The centrifugation confirmed the nature of pigment i.e., either intracellular or extracellular [27]. The supernatant and pellet were observed. If the color appeared in pellet, pigment was intracellular and if the color appeared in supernatant the pigment was extracellular.

After the centrifugation, the supernatant was discarded, and the extraction was done through cell pellet. To determine the accurate solvent for extraction of pigment the solubility of the pellet was checked in different polar and non-polar solvents i.e., water, acetic acid, ethanol, methanol, acetone, ethyl acetate, n-hexane, tetrahy-drofuran, chloroform, butanol. The pellet was transferred in different Eppendorf’s, and solvent was added in them in equal ratio and extraction efficiency of each solvent was noted by visual and spectrophotometric analysis. The solvent which proved effective was used for further process. The selected solvent was added to the pellet along with the glass beads. The cells in the solvent were then subjected to sonication. After sonicating the sample for 20 min the sample was centrifuged again under the same conditions. After centrifugation colored supernatant was stored and the pellet was used again for extraction and this process was repeated until the color of the cells were faded [27].

Identification of selected isolate

For identification of yeast, morphological, microscopical and biochemical tests were performed. For morphological characteristics colour, shape and texture were noted. Microscopical analysis was performed by Lactophenol cotton blue staining. In addition to that some bio-chemical tests like urease, lipase, fermentation test (glucose, maltose, and sucrose), catalase, and oxidase tests were also performed [28].

For molecular identification of yeast strain, DNA was extracted by phenol chloroform method followed by PCR amplification using ITS1 and ITS4 primers. The sequence of universal primers of ITS1 and ITS4 are 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′ respectively [29]. After purification of PCR product, unidirectional Sanger sequencing was performed. The obtained sequence was analysed in BLASTn database and FASTA sequences of close neighbourings were downloaded and were aligned using Clustal W. Finally, the phylogenetic tree was constructed by bootstrap method in MEGA-11 software using the neighbor-joining algorithm with 1000 replicates. The sequence was submitted to the GenBank database.

Characterization of pigment by spectrophotometric analysis

For determining the wavelength maxima of the extracted pigment, scanning was done ranging from 400 to 600 nm on spectrophotometer. Thus, this absorption maxima gave an idea about the class of pigment.

Quantification of biomass and pigment yield

The strain was inoculated in 2.5 litre medium for large scale cultivation. The biomass was dried in hot air oven at 105℃ [30]. which was then weighed on an analytical balance and the difference between weights of the falcon tubes with the pellet and without pellet was considered as the dry weight of yeast cells [26]. The total carotenoid content in mg/l was calculated using the following formula with slight modifications [26, 31].

Total Carotenoid =Amax×V×104E×W(g)

Where A max is absorbance of total extract at 480 nm, V is the volume of the extract in ml, E is extinction coefficient of total carotenoid (0.16), W is dry weight of yeast cells.

Purification of pigment by column chromatography

For purification, pigment extract was subjected to column phase chromatography. Different fractions were obtained by using Silica Gel as stationary phase and different non-polar and polar solvents were used as mobile phases in different concentrations. Following mobile phases were used: n-hexane (100%), n-hexane: ethyl acetate (75:25), n-hexane: ethyl acetate (50:50), n-hexane: ethyl acetate (25:75), ethyl acetate (100%), ethyl acetate: methanol (75:25), ethyl acetate: methanol (50:50), ethyl acetate: methanol (25:75), methanol (100%).

Characterization of pigment by thin layer chromatography (TLC) and by Fourier Transform Infrared-Spectroscopy (FTIR)

The extract of pigment obtained by column phase chromatography was characterized by Thin Layer Chromatography (TLC). The stationary phase used was aluminum plates coated with 0.25 mm thick silica gel and different mobile phases like Toluene: ethyl acetate (3:1) and Toluene: acetone (3:1) etc. were used in different concentrations. The spot of extracted pigment was applied on the labelled line on TLC plate and was dipped in the mobile phase. After the solvent front reached the marked line, TLC plate was visualized under UV transilluminator. If the bands appeared under UV-light, these bands were marked by the lead pencil and the Rf value of the spot was calculated by using formula [32].

Rf=Distance travelled by the pigmentDistance travelled by the solvent

The bands that appeared on the thin layer chromatography plates were then subjected to Fourier transform infrared spectrophotometer (FTIR) at a wavelength range of 4000−500 cm-1 for detection of functional groups.

Antioxidant activity of purified fractions

Free radical scavenging activity by DPPH (2,2′-diphenyl-1-picrylhydrazyl radical) assay was performed with slight modifications [33]. The sample having concentration of 200 µg/ml was added to 1 ml of 3 mM freshly prepared DPPH solution and was incubated in dark at 37℃for 30−45 min. The positive control used in assay was Ascorbic acid (200 µg/ml) and negative control was DMSO. After incubation absorbance was recorded at 517 nm and % of scavenging activity was calculated by using the formula given below:

% scavenging =Absorbance of control-Absorbance of sampleAbsorbance of control×100%

Antimicrobial activity of purified fractions

The antimicrobial activity of fractions were analyzed against gram positive Staphylococcus aureus (ATCC 25923), gram negative Escherichia coli (ATCC: 25922) and Candida albicans (ATCC 10231) strains by agar well diffusion assay [34]. The 100 µl of sample having a concentration of 1 mg/ml were added in wells aseptically, N-hexane (100 µl) was used as negative control and levofloxacin (10 µg) and nystatin (50 µg) were used for bacterial strains and fungal strains respectively. The zone of inhibition was reported in milli-meters (mm).

Statistical analysis

The results of antioxidant and antimicrobial activity were computed as mean ± standard deviation. After representing in form of mean and standard deviation the statistical significance of results was checked by one-way ANOVA followed by Tukey HSD test using Statistix 10 software. The values were considered significant when p < 0.05.

Results

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Isolation and screening of pigment producing microbes

For isolation of pigment producing fungi marigold flower (T. erecta) was collected (Fig. 1A). A total of 9 fungal isolates were obtained from petals, sepal, and bud of flower. Description of sources of isolates is given in Table 1. Among these endophytic fungal isolates 5 showed colored colonies on Potato dextrose agar (PDA) plates. These isolates were named as MGA, MGB, MGC, MGD, MGI. Screening of these 5 isolates for pigment production was done by observing the color on both solid and broth media and results are represented in Table 2. Among all fungal isolates, yeast isolate MGI obtained from the bud of marigold flower showed good color production in both solid and submerged state media. So, it was used for further study.

Table 1 . Fungal isolates from Tagetes erecta.

Parts of marigold flowerFungal isolates
Petal2
Sepal4
Bud3
Total9

Table 2 . Observation of colors on agar media and broth media of Endophytic fungal isolates.

IsolatesColor observed on plate (front view)Color observed on plate (back view)Color observed in broth
MGY-IPinkPinkPink
MGY-AYellow center green edgesYellowNo change
MGY-BYellow center white edgesBrownBrown
MGY-COff-white to green center white edgesYellowYellow
MGY-DWhiteYellowNo change

Figure 1.Isolation and extraction of pigment from endophytic yeast isolate MGI. (A) Sample of Marigold flower (Tagetes erecta), (B) Plate containing endophytic yeast isolate MGI, (C) Pigment production in Potato dextrose broth (PDB) media, (D) Color of crude extract.

Production and extraction of pigments by yeast isolate MGI

The yeast isolate MGI showed pink colored colonies (Fig. 1B) and after inoculating in broth medium it produces pink color in potato dextrose broth (PDB) and in mineral media as shown in Fig. 1C. After centrifugation, the pink color was observed in the pellet, and the supernatant was colorless, so the pigment was intracellular in nature.

For effective extraction from pellet different solvents were tested. The solvents which proved effective were acetone, ethanol, and methanol as these solvents acquire the color of pigment. Some solvents which showed slight color change were water or chloroform (trichloromethane). On the other hand, solvents like ethyl acetate, n-hexane, and tetrahydrofuran do not attain any color and were proved least effective for extraction. Thus, for extraction of pigment from pellet, acetone was added in pellet which was then sonicated and was further centrifuged. The color was observed in the supernatant, so it was collected, and the pellet was reused. The process was repeated thrice until the color of cells faded away. Initially, the color of supernatant in the first extraction was dark orange which after each extraction becomes light. The color of the extracted pigment was orange as represented in Fig. 1D.

Morphological and biochemical characteristics of yeast isolate MGI

The colonies observed on plate were circular, mucoid, pink, smooth, raised, and entire. In broth media they showed growth in form of sediments (Table 3). Lactophenol cotton blue staining of yeast isolate MGI under microscope showed oval shaped cells and multiple buds on some cells were observed while ascospores and pseudohyphae were absent as depicted in Fig. 2. Yeast isolate MGI showed positive results for urease, lipase, and catalase and showed negative results for fermentation of sugars (glucose, lactose, and maltose), for production of acid and for oxidase test as shown in Table 4.

Table 3 . Morphological characteristics of Rhodotorula mucilaginosa MGI.

CharacteristicsObservations
ColorPink
ShapeCircular
MarginEntire
ElevationRaised
TextureMucoid
SurfaceSmooth
Growth in broth mediaGrowth in the form of sediments

Table 4 . Biochemical tests of Rhodotorula mucilaginosa MGI.

Biochemical TestResults
Urease+
Lipase+
Glucose fermentation–
Lactose fermentation–
Maltose fermentation–
Acid production–
Catalase+
Oxidase–

+ indicates positive and – indicates negative results.
Figure 2.Microscopical examination of endophytic Rhodotorula mucilaginosa isolate MGI.

Molecular Identification of yeast isolate MGI

The sequence obtained from sequencing of the ITS and 5.8S regions of the ribosomal genes of the yeast was submitted to GenBank with accession number OQ155212. The phylogenetic tree as represented in Fig. 3 was constructed and according to the results yeast isolate MGI showed 99% homology with R. mucilaginosa isolate J36. Hence on basis of these analysis it was concluded that yeast isolate MGI is R. mucilaginosa.

Figure 3.Phylogenetic tree constructed using the neighborjoining method from yeast isolate MGI (OQ155212). Numbers at nodes represent bootstrap values (expressed as percentages of 1000 replicates).

Characterization and Quantification of pigment using spectrophotometer

High absorbance of crude lies at a range from 440 nm to 500 nm. The absorption maxima of crude pigment were recorded at 480 nm and at 460 nm. The carotenoid yield was quantified as 950 mg/l.

Purification of pigment by column phase chromatography

After column phase chromatography three different types of fractions were obtained in terms of color i.e., orange, yellow and transparent. Orange fraction was obtained in non-polar mobile phases, and yellow fraction was eluted in other different combination of solvents in different concentrations as shown in Table 5.

Table 5 . Profile of column phase and thin layer chromatography.

FractionsSolventsRf value
OrangeN-hexane (100%),
N-hexane and ethyl acetate (75:25)		0.58
YellowEthyl acetate and Methanol
(75:25 and 25:25)		0.92


Characterization of pigment by thin layer chromatography (TLC) and FTIR analysis

Orange fraction gave orange colored band on silica gel plate (TLC), the pigment was separated with mobile phase i.e., Toluene: ethyl acetate (3:1). The Rf value of the band was 0.58. For yellow fraction similar mobile phase was used and Rf value calculated was 0.92 (Table 5).

The FTIR analysis of the orange fraction showed major absorbance peaks at following wave numbers 2921, 2852, 1709, 1410, 1279 and 721 cm-1 as shown in Fig. 4A. 2921 cm-1 and 2852 cm-1 indicates aliphatic C-H stretch, 1709 cm-1 showed stretch of ketone C=O, 1279 cm-1 represents aromatic ester C-O, 1410 cm-1 peak indicates O-H stretch and 721 cm-1 shows presence of C=C bond. The FTIR analysis of yellow fraction showed major absorbance peaks at following wave numbers 2924, 1715, 1363 cm-1 as represented in Fig. 4B. 2924 cm-1 indicates aliphatic C-H stretch, 1715 cm-1 shows an aromatic C-H, and 1363 cm-1 shows CH3 bend.

Figure 4.Fourier transform Infrared (FTIR) spectrum of pigmented fractions produced by Rhodotorula mucilaginosa MGI. (A) FTIR spectrum of orange fraction, (B) FTIR spectrum of yellow fraction.

Antioxidant and Antimicrobial activity of purified fractions

Both purified fractions showed scavenging free radical activity but the highest antioxidant activity of about 77% was exhibited by orange fraction F1. The percentages of scavenging free radical activity of fractions, positive control and negative are portrayed in Fig. 5A.

Figure 5.(A) Antioxidant potential of purified fractions. (B) Antimicrobial potential of purified fractions. Error bars represent the standard deviation. Data with capital letters indicate the significant differences (p < 0.05) in antioxidant and antimicrobial activity respectively.

The pigmented fractions F1 and F2 possessed antibacterial and antifungal activities against test strains. In case of S. aureus highest activity was showed by F2 with zone of inhibition of about 38 mm which was even greater than zone of inhibition of positive control. Like-wise, F2 also showed maximum zone of inhibition against E. coli and manifested highest antifungal activity against C. albicans. The results are shown in Fig. 5B.

Discussion

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The present study was done to isolate pigment producing microbes from the T. erecta (marigold flower) and characterization of pigments produced from the selected yeast isolate MGI. In studies conducted by Mani et al. and Sarang et al., endophytic fungal isolates were isolated from different samples having the ability to produce color pigments [35, 36]. Similarly, Otero et al. and Karanjgaokar and Tarfe isolated pigmented yeast by processing ornamental flowering plant and flower samples [37, 38].

Most of the morphological traits of pink yeast MGI resemble with Rhodotorula genus as they showed correspondence with similar studies conducted by Elsanhoty et al., Lau et al. and Chae et al. [39−41]. Pale pink to pink colored colonies of some species of Cryptococcus genus were also reported [42]. Biochemical tests performed for identification showed that the isolate did not ferment sugars and lacked the ability to produce acid while it has a potential to hydrolyze urease and produce lipase enzyme. According to some previous studies, similar biochemical results were reported for Rhodotorula genus [28, 43]. By 18S rRNA sequencing, the strain was identified as R. mucilaginosa MGI.

R. mucilaginosa MGI produced intracellular pigment as color was confined to the cells pellet. Several species of yeasts were reported to produce intracellular pigments [44]. In study of Poddar et al., pigment produced was intracellular in nature, and it was also extracted by similar steps with slight modifications [27].

On characterizing with UV spectrophotometer, obtained wavelength range predicts that this pigment belongs to the class of carotenoid as they absorb wavelength in visible region from 400 to 500 nm due to the presence of conjugated double bounded structures [45, 46]. Similarly, as in some studies 480 nm wavelength was reported for astaxanthin and 465 nm for beta carotene so astaxanthin and beta carotene class of carotenoid may exist in crude pigment [38, 47].

By purifying the pigment with column phase chromatography two colored fractions orange and yellow implies that they are carotenoids as carotenoids are red, yellow and orange in color [23]. On TLC plate, Rf value of orange and yellow fraction were recorded as 0.58 and 0.92. In similar research study, Rf value of 0.6 was reported for astaxanthin monoester and Rf value of about 0.94 was reported for beta carotene [48−50]. Hence, this anticipates that these orange and yellow fractions may belong to astaxanthin and beta carotene group respectively. Rf values also predicts about the polarity of compounds. Compound that travels a greater distance from origin is said to be non-polar in nature while one which covers a shorter distance is said to be polar [27, 51]. As Rf value of orange fraction was 0.58 so it is slightly polar while Rf value of yellow fraction was 0.92 so it is non-polar in nature.

FTIR spectrophotometric results of orange fraction implies that this purified pigment is astaxanthin as some of the functional groups like hydroxyl (OH), methyl (C-H), ketone (C=O), ester (C-O) was also reported in study for astaxanthin [52]. Similarly, presence of carbon and hydrogen indicates the presence of hydrocarbon functional group which predicts that this yellow purified pigment is a carotene [23]. As some of the peaks showed close correspondence with study of FTIR analysis of beta carotene so the obtained pigment might be a beta carotene [53].

Pigmented fraction of R. mucilaginosa MGI manifested antioxidant potential and can neutralize the effect of reactive oxygen species, ROS (a special group of free radicals) and can aid in treating various life-threatening diseases [54]. According to similar studies antioxidant potency of carotenoid compounds have been reported. It was studied that astaxanthin poses more antioxidant potential than any other class of carotenoid [55]. Like-wise in the following study F1 (orange fraction) showed greater scavenging activity as compared to F2 (yellow fraction). The pigmented fractions manifested antimicrobial activity which showed that they have a potency to be used as an antibacterial or antifungal product. The extracted carotenoid pigment from R. mucilaginosa MGI showed greater susceptibility towards gram positive bacteria, S. aureus while showed lesser susceptibility towards gram negative bacteria, E. coli. The zone of inhibition in case of our study against S. aureus and E.coli were greater than zones recorded for Rhodococcus kroppenstedtii which were 21.0 ± 0.2 mm and 10.0 ± 0.42 mm respectively [56]. These differences occur because anti-microbial activity is dependent upon the cell wall composition of test strains and structure and composition of the produced pigment [57].

In summary, 9 endophytic fungal strains were isolated from Tagetes erecta. Pink endophytic yeast isolate MGI on screening was selected for further study and it belongs to Rhodotorula genus and molecular analysis identify it as R. mucilaginosa. It produced an intracellular pigment, and the color of crude pigment was orange. On characterization by spectrophotometer, absorption maxima were recorded at 480 nm and 460 nm. At these wavelengths pigment was quantified. The pigment was purified by column phase chromatography and two pigmented fractions were obtained. These fractions were subjected to further characterization by thin layer chromatography (TLC) and Fourier transformer infrared radiations (FTIR). Results confirmed that these pigments are carotenoids and their Rf values, polarity and functional groups showed correspondence with astaxanthin and beta carotene respectively. The pigmented fractions exhibited antibacterial, antifungal and antioxidant potentials. Therefore, they can be used effectively in the food and pharmaceutical industry.

Abbreviations

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PDA: Potato dextrose agar

PDB: Potato dextrose broth

ITS: Internal transcribed spacer

PCR: Polymerase chain reaction

Blast-n: Nucleotide Basic Local Alignment Search Tool

MEGA 11: Molecular Evolutionary Genetics Analysis Version 11 Rpm: Revolution per minute

AU: Absorbance units

TLC: Thin layer chromatography

FTIR: Fourier-transform infrared spectroscopy

Rf: Retention factor

UV: Ultraviolent radiations

ATCC: American Type Culture Collection

Acknowledgments

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The authors acknowledge the department of Microbiology of Quaid-i-Azam university, Islamabad to support the research.

Author Contributions

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Isswa Iqbal conceptualized the work, performed experiments, analyzed results under the supervision of Safia Ahmed and prepared the draft. Warda Sarwar and Qurban Ali provide help while performing experiments and analyzing results. Safia Ahmed supervised and approved the version to be published.

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