Introduction

Pigments produced by microorganisms are the important characteristic feature of some microorganisms like bacteria which might also be beneficial in characterization and identification. Microbial pigments have a variety of functions due to their higher environment-friendly property. Bacterial pigments have numerous useful properties like immunosuppressive, anticancer, biodegradability, antiproliferative, antibiotic, and so forth (Raju & Radha, 2015). Different pigment producing microorganisms like fungi, bacteria, mould and yeast are used for the industrial manufacturing and have wide range of application generally in material industries, pharmaceutical industries and food industries (Kumar, Vishwakarma, Singh, Dwivedi, & Kumar, 2015). Some other advantages of microbial pigments: acts as antioxidant agent, anticancer, antimicrobial, protects from Ultra Violet rays, extreme cold and hot, acquisition of macro or micro nutrients such as carbon, iron and nitrogen (Pankaj & Kumar, 2016). Pigments can be classified based on their origin as organic and inorganic. Synthetic and natural pigments are organic compounds; Inorganic pigments are observed in nature or produced through synthesis. Naturally occurring pigments are produced by using living organisms such as animal, plant and micro-organisms. The benefits of pigment manufacturing from microorganisms contain effortless and quick growth in the inexpensive culture medium, independence from climate conditions and colorings of one-of-a-kind shades (Sinha, Choubey, Kumar, & Bhosale, 2017).

Pigments are coloring compounds that exchange the colour of transmitted or reflected light as the end result of wavelength selective absorption. Colors have been used to add or enhance the aesthetic value of compound/products that we use (Samyuktha & Mahajan, 2016). Colors play an essential role in human lifestyles and in human culture. The color produced by microorganisms is known as bio colors (Bhatt, Khan, & Amin, 2013). Microbial communities are various and every place differs and has different microbial community which leads to various colors producing organisms. They have aesthetic traits like formation of different pigments, degradation of complex chemicals, by various microorganisms and can be isolated (Rokade & Pethe, 2016).

Bacterial pigment is an alternative source of natural pigments and have an extremely great potential for food utilization due to their natural coloration with safety to use, medicinal resources, nutrient like vitamins, manufacturing being independent of season and graphical climate with controllable and expected yield (Joshi, Attri, Bala, & Bhushan, 2003; Sharma, 2014).

MATERIAL AND METHODOLOGY

Water sample from Mula-mutha River in Pune and Soil samples from Bhosari dumping ground, Pune were collected. Sample were transported immediately to the laboratory and processed for further analysis.

Isolation of pigment producing bacteria

Soil & water samples were used serially diluted up to 10⁶. 10^-4 - 10^-6 dilutions were spreaded on sterile Nutrient agar (NA) plates by spread plate technique and incubate for at 37℃ for 24hrs to 48hrs.

The Nutrient agar plates had been found for growth after 48hrs of incubation time. The pigment producing microorganisms were used for in addition studies.

The selected pigmented colonies were selected and streaked on Nutrient agar slants and Nutrient agar plates to obtain a pure culture of the colony (Siddharthan, Sandhiya, & Hemalatha, 2020).

Characterization and Identification of isolated pigmented micro-organisms

The selected pigment producing bacterial isolates were identified on the basis of their morphological and biochemical characteristics. Colony characterization of pigment producing bacteria was done based on its size, shape, color, margin, opacity, consistency, elevation, Gram staining and motility. Later, the organism was identified using Bergey’s Manual of Determinative Bacteriology (Goswami & Bhowal, 2014; Holt, Krieg, Sneath, & Staley, 1994).

Enrichment of pigment producing bacteria

Identified bacterial isolates were inoculated in hundred ml sterile (NB) nutrient broth. Glycerol (2%) was added to the NB for enhancing the pigment production and one was inoculated without addition of glycerol to see the effect of glycerol in pigment production (Joshi, Yewatkar, Ghumare, & Trupti, 2019). This was kept on shaker incubator at 100 rpm for 3-7 days and was observed for pigment production till it was darkly pigmented and nearly opaque (Waghela & Khan, 2018).

Extraction of pigment

The yellow pigmented colony after incubation on rotatory shaker for 3 days was subjected to centrifugation at 6,000rpm for 15 minutes. The supernatant was discarded; the cells were washed with sterile distilled water and again centrifuged for 10 minutes at 4,000rpm. 4ml of ethyl acetate was added to the pellet and suspended it. The water bath was set at 60℃ and the obtained suspension was incubated in it for 15 minutes till all the visible pigments were extracted (Joshi et al., 2019). This was again subjected to centrifugation at 4,000rpm for 15 minutes. The extracts were analyzed by scanning the absorbance in the wavelength region of 200nm to 800nm using the spectrophotometer (Chandran, Duraipandi, Yuvaraj, Vivek, & Parthasarathy, 2014; Sinha et al., 2017).

Optimization of pH, Temperature and NaCl concentration

pH

To determine the optimum pH for the selected pigmented colony, equal amount of the microbial isolate was inoculated in 50 ml sterile nutrient broth in 100ml flask with different pH viz, 5.0, 6.0, 7.0 and 8.0 and incubated at 37°C for 48 hrs, on rotatory shaker for 2 to 3 days. Then the O.D was taken at 540nm to assay for maximum production of pigment (Goswami, Chaudhuri, & Dutta, 2010; Joshi, Attri, & Rana, 2011; Ratnakaran, Bhoir, & Gupta, 2020).

Temperature

To determine the optimum temperature for the selected pigmented colony, isolates were streaked on sterile nutrient agar (NA) plates and incubated at different temperature viz 4℃, 25℃, 30℃ and 37°C for 48hrs after that it was observed for pigmented colony production (Ratnakaran et al., 2020).

NaCl Concentration

To determine the optimum NaCl concentration for the selected pigmented colony, equal amount of the bacterial isolate was inoculated in 50 ml sterile nutrient broth in 100ml flask with different NaClviz, 0.5%, 1%, 2%, 4% and after that incubated at 37°C on rotatory shaker for 2 to 3 days. The O.D was taken at 540nm to assay for maximum pigment production (Ratnakaran et al., 2020).

Antioxidant activity test

This approach is totally based on the principle of enlarge in the absorbance of the response mixtures. The absorbance will increase with the antioxidant activity will increases. The complete activity of antioxidant can be measured by way of the ferric reducing antioxidant power assay (FRAP). The phenolic and flavonoids acids both are presnt in the medicinal plant show off strong antioxidant activity and which is relying on their potential to form the complicated with metallic atoms, specially copper and iron (Rajurkar & Hande, 2011). The antioxidant compound existing in the samples varieties a colour complex with potassium ferricyanide, Trichloroacetic acid and ferric chloride, which is measured at seven hundred nm by way of Spectrophotometer (Fernandes et al., 2016; Vijayalakshmi & Ruckmani, 2016).

Antibacterial assay

The antibacterial assay was performed by Kirby-Bauer Disk Diffusion method (Hudzicki, 2009). The sample micro-organisms were collected and pigment was tested for its antibacterial activity. The Nutrient agar plate was once seeded with 24hrs old microbial culture and wells had been bored in the NA plates. The wells have been filled by suitable quantity 20μlof pigment, it was once stored in fridge for half an hour after that it used to be incubated at 37⁰C for 24hrs and the end result used to be found by using measuring zone of inhibition in diameter (Singh et al., 2012; Waghela et al., 2018).

Results and Discussion

The water & soil samples collected from different parts of Pune city have been used for isolation of pigment producing bacteria. Two pigment producing micro-organism have been identified and characterized which had been yellow and orange in coloration (Figure 1).

Characterization of micro-organism was done by morphologically and various biochemical tests. This test gave us confirmatory results for identification of Microorganism. Thus given biochemical test followed by referring Bergey’s manual (Holt et al., 1994) gave confirmation of species as Micrococcus and Arthrobacter (Table 2; Table 1).

Various type methods had been used for the extraction of pigment producing micro-organism like filtration and centrifugation with ethanol addition so that cell gets lysed and intracellular pigment can be easily extracted. The extracted pigment had been yellow and orange in coloration (Figure 2). The optical-density of the pigment had been measured and in addition processed for antibacterial and antioxidant activity.

The impact of pH concentration on the production of pigment through each and every bacterial isolate was once determined through inoculating the pure cultures in sterile Nutrient broth. For impact of pH, sterile nutrient broth with pH 5, 6, 7, and eight was once used and incubated at 37⁰C for about 24-48 hrs. Investigation on this study, both isolates show maximum production recorded on pH 7. Hence pH 7 was maintained optimization studies. This suggests significance of pH in the media considering that its altered value can both extend and limit the quantity of pigment (Figure 3). Isolates increase used to be most at impartial pH and pigmentation was once found most when stored under refrigeration.

By observing the absorbance values obtained at different NaCl concentrations for yellow and orange pigmented colony. It can be concluded that at 0.5% NaCl concentration for yellow pigment and at 2% NaCl concentration for orange pigment production is the highest and can be considered as the optimum NaCl concentration for the production of pigment in large amount (Figure 4).

The yellow and orange pigment colony streaked on NA plates and incubated at different temperatures was observed for growth. It was observed that the yellow pigmented colony grows at 30℃ and 37℃ and orange pigmented colony grows at 25℃ and 30℃. Therefore, it can be concluded that the yellow colony can grow between 30℃-37℃and orange colony can grow between 25℃-30℃ (Figure 5).

Total antioxidant was determined by using FRAP assay. The Absorbance used to be measured at seven-hundred nm used UV-VIS Spectrometer. The optical density at 700 nm for orange extract was 1.16, for yellow pigment it was 0.93 and Ascorbic acid as standard it was 0.203. The orange pigment has more antioxidant activity compared to yellow pigment.

Antibacterial activity was measured against 2 organisms like Escherichia coli and Pseudomonas. The antibacterial activity of orange pigment showed against the bacteria Pseudomonas and yellow pigment has no effect against Pseudomonas. Both yellow and orange pigment showed antibacterial activity against the E.coli. The Inhibition zone used to be measured to consider activity of antimicrobial (Figure 6).

From the results obtained, we observed that soil and water samples contain diverse microorganisms that have the ability to produce pigment as their secondary metabolite. Addition of glycerol tends to increase the pigment production. The pigment producing bacteria grows at neutral pH.

Conclusion

The results of present study confirm that extracted pigments can be used in various industries like pharmaceutical, paper printing industries etc. antibacterial and antioxidant activity of pigment can be of great benefit compared to synthetic dyes which are toxic and harmful to nature. They can be used as an achievablesource in textile, paper printing & food companies. From optimization results we can conclude that these organisms’ best grow at 30℃ temperature, neutral pH and 0.5% - 2% NaCl concentration. Soil and water are diverse in nature and can be screened for more pigment producing organism’s and use their pigment as an alternative for synthetic dyes. More efforts are to be made for the use of cheaper substrates for the pigment producing organisms.

Acknowledgement

We wish to express our sincere thanks and gratitude to Head of the department of Microbiology Mr. Vasudevan Patturajan, Dr. D. Y. Patil ACS College, Pimpri, Pune, for providing the necessary facilities to carry out this research.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that there is no conflict of interest for this study.