Introduction

Color is an integral part of human life that enhance the aesthetic values and it is probable one of the first characteristics perceived by the senses [5, 10]. Natural colors are generally extracted from fruits, vegetables, roots and microorganisms and are often called as “biocolor” because of their biological origin[8].The biocolors have been classified into three types 1.)Natural color : chlorophylls, carotenoids etc. 2.) Browning color : color produce during baking, sugar caramelization etc. 3.) Additives: anthocyanins, vitamins etc.[10]. Natural pigments have been used widely in the field of pharmaceutical industry, dairy industry, fish industry, printing industry and textile industry[13].

Natural pigments can be obtained from two major sources : plants and microorganisms. Microbial pigments are of industrial interest because they are often more stable, highly producible and easily available. The various types of microbial pigments are carotenoids, melanin, flavin, monascins, violacein and indigo[4]. Among which carotenoids are majorly present intracellular as yellow pigment in pigmented microorganisms.

Carotenoids are nothing but lipid soluble classes of molecule associated with the lipid fractions which are sensitive to oxygen, heat and light[2].These pigments have an important function to act as protective agents against oxidative damage[12].Inhibition of various types of cancers and enhancement of immune response[7].Due to anti-oxidant activity and pro vitamin A function, they protect us from “life style related” diseases such as cardiovascular disease and age related muscular degradation[14].In food industry carotenoids are used as colorant in food to pigment salmon, trout and poultry and also in the identification of egg yolk color[6].

Various subtypes of carotenoids like astaxanthin, xanthomonadins, flexirubin, zeaxanthin, lutein etc. are produced by various yellow pigmented microorganisms includes Micrococcus luteus, Pseudomonas cynzatha, Xanthomonas, Bacillus subtillis, Cytophaga sp. , Flavobacterium sp ,Erwinia sp, etc. They are widely spread in every ecosystem including soil, water and air. Some are also capable to produce amylase enzyme.

The present study was aimed at isolation and identification of bacterial isolates producing yellow pigment mainly carotenoid from different soil sources and determines its ability to produce amylase enzyme

Materials And Methods

Sampling

Different types of soil samples like wet soil, dusty soil, agricultural soil, garden soil etc. are collected from different areas of Surat district, Gujarat. Samples were collected by scrapping of the soil surface with sterile spatula and about 10 gram of soil were obtained from a depth of 2-5 cm[5] in sterile unused zip locked polythene bags.(Table-1)

Table 1 : Samples from different sites of Surat city,Gujarat,India.

Serial number Sample code Sites
1 CS Soil.from.Sarvajanik education.society campus
2 CS1 Grassy soil
3 I Ugat botanical Garden soil
4 I1 Garden soil near pond of Ugat botanical garden
5 A Agricultural soil of wheat farm.
6 LS Soil near humic lake present at Bhimpor
7 MS Soil from Golden beach, Dumas
8 SM Salt marsh soil, Dumas
9 DS Dusty soil of open ground
10 CUS Soil where building construction is going on at Athwagate

Isolation of yellow pigmented microorganisms

From the collected soil samples, soil suspensions were prepared using sterile distilled water[10].Samples were serially diluted up to 10-6 and dilution sample were plated on nutrient agar(NA) medium[3] and the plates were incubated at 37°C for 48 hours. Only the yellow pigmented bacterial agar plates for further studies. Pure cultures are maintained on nutrient agar slants at 10°C every 10 days[9].

Morphological and biochemical identification

The isolates were characterised by colony morphology on Nutrient agar plates and gram reaction. The biochemical characteristics (Indole production, Voges-Proskauer’s, Citrate utilisation, Urease production, Gelatin hydrolysis, Oxidase production, Catalase production, H2S production, Nitrate reduction and Casein hydrolysis) tests were also carried out using standard reference Bergey’s Manual of systematic bacteriology to confirm the identity of the cultures[5].

Determination of efficiency of amylase production

Isolates were transferred on 1% starch agar plates by spot inoculation and incubated at 37° C for 24 hours. The amylase production is determined qualitatively by flooding plates by Lugol’s Iodine solution. Zone of clearance is measured.

Determination of pigment production rate

All the isolates were recultured in nutrient agar plates at 37°C.After 24,36 and 48 hours the development of yellow color was observed[3].All the isolates were also recultured on luria burtini(LB) agar and incubated at 37°C for 24 hours.

Extraction of carotenoid form yellow pigmented bacterial isolates

The yellow pigmented bacterial isolates were grown in LB broth in a rotary shaker at 180 rpm in 37°C for three days. After three days, cells were harvested by centrifugation at 8,000 rpm for 15 minutes. Then the pellet was washed with sterile distilled water and spin for 15 minutes at 4,000rpm. Five ml of methanol was added to the pellet and suspended it. Then it was incubated in a water bath at 60°C for 15 minutes until all visible pigments are extracted and centrifuged at 4,000 rpm for 15 minutes. The colored supernatant was separated then it was filtered through Whatman no. 1 filter paper [11]. The carotenoid extracts were analysed by scanning the absorbance in the wavelength region of 450 nm using the spectrophotometer. The total carotenoid content in the methanol extract was estimated by measuring the absorbance at λmax(450 nm)[5].Isolate having highest carotenoid content was selected for further study.

Effect of pH, salt and temperature on growth and pigment production

The effect of pH and NaCl concentration on the carotenoid production by screened bacterial isolate was determined by inoculating the pure cultures in sterile LB broth. For effect of pH, sterile LB broth with pH 2, 4, 6, 7, 8 and 10 was used whereas for effect of NaCl, sterile nutrient broth with 0.5%, 1%, 2%, 4%, 6% and 8% NaCl concentration was used and incubated at 37 ºC for about 24-48hr[1]. After incubation, cell mass density and carotenoid production by taking optical density at 600 nm and 450 nm are determined.

Results

Isolation and Identification

In this study, 24 isolates of yellow pigmented bacteria were isolated from 10 different types of soil samples from different areas of Surat city.(fig. 1,2)Total number of isolates from each sample are listed in table 2.

Table 2 : Total number of isolates from each sample

Sample Number of isolates
CS 3
CS1 2
I 2
I1 3
A 4
LS 3
MS 2
SM 1
DS 3
CUS 1

Bacterial colonies on nutrient agar(NA) plates were studied under a light microscope at a magnification of 10X and 15X for size, pigmentation, elevation and margin .Gram reaction and motility were also studied.(Table 3)(fig 1)For identification of Staphylococcus aureus, isolates were cultivated on Mannitol salt agar(MSA) plates.Isolate CSc showed yellow colonies with turn the medium yellow which confirms it as S.aureus.(fig 2)

image 1

Table 3 : Morphological characterization of pigment producing microbes

Isolates Colony Characteristics Grams reaction Motility
CSa Yellow pigmented irregular, Opaque, Rough, Slightly raised Gram positive.Cocci in bunch Non-motile
CSb Yellow pigmented, Circular, Opaque, Rough, Slightly raised Gram positive cocci in tetrads Non-motile
CSc Pale yellow pigmented, Circular, Opaque, Smooth, Convex Gram positive cocci in bunch Non-motile
CS1a Lemon yellow pigmented, Circular, Opaque, Rough, Convex Gram positive cocci inbunch Non-motile
CS1b Pale yellow pigmented Circular, Translucent, Smooth, Slightly raised Gram positive rods Non-motile
Ia Pale yellow pigmented, Circular, Opaque, Smooth, Convex Gram negative rods Motile
Ib Yellow pigmented, Circular, Translucent, Smooth, Convex Gram positive short rods Motile
I1a Yellow pigmented, Circular, Opaque, Smooth, Convex Gram negative coccobacilli Motile
I1b Bright yellow pigmented, Circular, Opaque, Rough, Raised Gram positive rods in chain Motile
I1c Pale yellow pigmented, Circular, Opaque, Smooth, Flat Gram positive cocci in bunch Non-Motile
A1 Yellow pigmented, Elliptical, Opaque, Smooth, Convex Gram positive cocci in bunch Non-motile
A2 Pale yellow pigmented, Circular, Opaque, Smooth, Convex Gram positive short rods Motile
A3 Light yellow pigmented, Circular, Translucent, Smooth, Slightly raised Gram positive cocci in bunch Non-motile
A4 Golden yellow pigmented, Congregated, Opaque, Smooth, Raised Gram positive cocci in bunch Non-motile
LS3 Yellow pigmented, Circular, Opaque, Smooth, Convex Gram.positive.cocci.in.tetrads Non-motile
LS4 Lemon yellow pigmented, Circular, Opaque, Smooth, Convex Gram positive cocci in bunch Non-Motile
LS5 Light yellow pigmented, Irregular, Translucent, Smooth, Flat Gram negative rods Motile
MS1 Lemon yellow pigmented, Circular, Opaque, Smooth, Convex Gram positive cocci in tetrads Non-motile
MS2 Yellow pigmented, Circular, Opaque, Rough, Convex Gram positive cocci in tetrads Non-motile
SM2 Lemon yellow pigmented, Circular, Opaque, Rough, Convex Gram positive cocci in tetrads Non-motile
DS1 Bright yellow pigmented, Circular, Opaque, Smooth, Raised Gram positive cocci in tetrads Non-motile
DS2 Yellow pigmented, Circular, Translucent, Smooth, Convex Gram positive cocci in tetrads Non-motile
DS3 Light yellow pigmented, Circular, Translucent, Smooth, Slightly raised Gram positive cocci in tetrads Non-motile
CUS1 Pale yellow pigmented, Circular, Opaque, Smooth, Convex Gram positive cocci in tetrads Non-motile

These bacteria were then identified and characterized with the help of morphological and biochemical characteristics. Their identification at genus level were done with the help of Bergey’s mannual of systematic bacteriology.(Table 4)

Table 4 : Biochmical characterization of isolates

Isolates Biochemical tests Probable
Indole Methyl Red Voges-Prosceur Citrate Urease Gelatin.Hydrolysis Catalase oxidase H2S production Nitate reduction Caseinhydrolysis IdentityToGenuslevel
CSa + + + + + Staphylococcus.sp.
CSb + + + Micrococcus sp.
CSc + + + S.aureus
CS1a + + + Staphylococcus sp.
CS1b + + Microbacterium sp.
Ia + + + Xanthomonas sp.
Ib + + + + Xanthobacter sp.
I1a + + + Xanthomonas sp.
I1b + + + + Bacillus sp.
I1c + + + + + Exiguobacterium sp.
A1 + + + + Micrococcus sp.
A2 + + Erwinia sp.
A3 + + + Staphylococcus sp.
A4 + + + Staphylococcus sp.
LS3 + + + + Micrococcus sp.
LS4 + + + Staphylococcus sp.
LS5 + + Erwinia sp.
MS1 + + + + + Micrococcus sp.
MS2 + + + + Micrococcus sp.
SM2 + + + + + + Micrococcus sp.
DS1 + + + + + Micrococcus sp.
DS2 + + + + + Micrococcus sp.
DS3 + + + + + + Micrococcus sp.
CUS1 + + + + Micrococcus sp.

Efficiency of Amylase production

After 24 hours of incubation at 30°C when starch agar plates were flooded with Lugol’s iodine solution, the violet color is observed due to the presence of starch. Isolates capable of producing amylase showed the zone of clearance to its colony surrounding.(fig 3)From 24 isolates 12 isolates able to produce amylase. The diameter of zone of clearance are measured by rular which is listed in table 5.By measuring the diameter, it was came to know that isolate A1(Micrococcus sp.) gave highest zone of clearance which means it can produce more amylase comparative to others.so it can be utilise further for microbial amylase production.

image 2

Table 5 : Diameters of zone of clearance of amylase producing isolates

Isolates Probable identity Diameter of zone of clearance on starch agar plate(mm)
CS1b Micrococcus sp. 5
Ia Xanthomonas sp. 15
Ib Xanthobacter sp. 3
I1a Xanthomonas sp. 6
I1b Bacillus sp. 5
A1 Micrococcus so. 18
A2 Erwinia sp. 2
A3 Staphylococcus sp. 3
LS4 Staphylococcus sp.. 7
LS5 Erwinia sp. 2
DS1 Micrococcus sp. 5
DS3 Micrococcus sp. 4

Determination of pigment production rate

When all bacterial isolates were re-cultivated on nutrient agar plates, 8 isolates were clearly developed yellow pigment colony after 24 hours, 1 isolate need 36 hours and other 14 isolates need 48 hours to develop pigment at 37°C incubation. Here Xanthobacter sp. and Bacillus sp. developed pigment in 48 hours while Exiguobacterium sp. took 24 hours. Micrococcus sp. , Staphylococcus sp. Xanthomonas sp. and Erwinia sp. Can develop yellow pigment in 24 to 48 hours depending upon their species.(Fig 4)

image 3

Isolates produce yellow pigment more efficiently and rapidly (in 24 hours) on LB agar plate then on nutrient agar plate. (fig 5)

image 4

Extraction of carotenoid form yellow pigmented bacterial isolates

Carotenoid extraction for the yellow pigmented isolates was carried out using methanol as a solvent. Out of 24 isolates only 4 isolates showed high amounts of carotenoid content viz., DS2>LS5>CS1a>LS4>A2>DS3 (Figure 6). From this results, isolate DS2 produced significantly higher amount of carotenoids when compared to other isolates. The absorption spectrum of methanol extract of the cell pellet from the bacterial isolates showed maximum absorption at 450 nm which was identical to the absorption spectrum of β-carotene reference sample.DS2 isolate is further used for lab-scale carotenoid production.h

image 5

Effect of pH, salt and temperature on growth and pigment production

Maximum growth was observed at pH 6, at 30°C and 0.5% NaCl concentration.The intensity in pigmentation is highest at pH 7, at 25°C and 2 % and 6% salt concentration.(Fig 7,8,9)

image 6

image 7

image 8

Discussion

As Indra A. et al.(2005) suggested, the study revealed that yellow pigmented bacteria are widely present in soil and based on the isolates found they can effectively produce amylase enzyme and carotenoid pigment which has a potential for application as food supplement and as antioxidant. The results of our study show that the isolate DS2 screened from dusty soil produced a considerable amount of carotenoid. Samyuktha S. and Sayali N.(2016) stated that yellow pigment producing organisms shows sensitivity towards pH and salt concentration.Same result found in our study,too. Among all isolates Micrococcus sp. were diversely present in almost all types of soil,50% isolates can produce amylase efficiently as Sailu and Bolanle K. said(2009).. Thus, due to higher carotenoid production,as Samyukhtha S. and Sayali N. suggested(2016) it can be used as a potential source for pharmaceutical and other cosmetic industries. Recent developments in the molecular biology of carotenoid biosynthesis from organisms that accumulated different carotenoid product have provided a variety of genes that can be employed as tools for a new strategy of heterologous expression in different host organisms. Engineering of microbial pathway enzyme can produce high amount of carotenoids in industrial process from this Micrococcus sp.

Conclusion

The study highlights the isolation, identification ,pigment production rate, amylase producing efficiency of yellow pigmented bacteria and effect of pH, temperature and NaCl concentration on carotenoid production by chromogens found in various types of soil. A total 24 isolates were obtained. Isolates were belongs to Micrococcus sp. ,Staphylococcus sp. , Xanthobacter sp. Xanthomonas sp. ,Bacillus sp. , Erwinia sp. ,Microbacterium sp. and Exiguobacterium sp. accouring to Bergey’s manual systematic bacteriology based on their morphological and biochemical characteristics. Among all isolates Micrococcus sp. were diversely present in almost all types of soil.50% isolates can produce amylase efficiently.Pigment production in nutrient agar medium occurs in 24-48 hours at 37°C but when organisms were provided yeast extract as a sole nitrogen source,as in luria burtini agar medium; they could produce yellow pigment more effectively in 24 hours at 37°C.For the extraction of carotenoid from chromogenic organism ,methanol is an excellent solvent.Micrococcus sp. proves to produce highest carotenoid, which is further used for production of carotenoid in optimised media by pH, temperature and salt concentration.It shows organisms grow well and also produce pigment in moderate temperature (25°C and 30°C).lower salt concentration is required for cell growth but they can produce pigment in salt concentration upto 6%.Organisms grow well in neutral and alkaline pH while pigment production seems highest at neutral pH. Thus, by providing such optimised condition, carotenoid production by using yellow pigmented organism can be carried out.