2 3 Characterization and Enhanced Production of Prodigiosin from the Spoiled Coconut

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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering andBiotechnology ISSN 0273-2289Volume 166Number 1 Appl Biochem Biotechnol (2012)166:187-196DOI 10.1007/s12010-011-9415-8

Characterization and EnhancedProduction of Prodigiosin from the SpoiledCoconut

R. Siva, K. Subha, D. Bhakta,A. R. Ghosh & S. Babu

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Characterization and Enhanced Productionof Prodigiosin from the Spoiled Coconut

R. Siva & K. Subha & D. Bhakta & A. R. Ghosh & S. Babu

Received: 27 May 2011 /Accepted: 18 October 2011 /Published online: 10 November 2011# Springer Science+Business Media, LLC 2011

Abstract Many bacterial secondary products are bioactive substances that play animportant role in biotechnology and pharmacology (e.g., as antibiotics or antitumoragents). Over the past few years interest in prodigiosin has been increased due to itspromising anti-cancer activity. Prodigiosin is also of potential clinical interest because it isreported to have anti-fungal, anti-bacterial, anti-protozoal/anti-malarial, and immunosup-pressive activity. Thus there is a need to develop a high-throughput and cost-effectivebioprocess for the production of prodigiosin. In the present study, Serratia rubidaea wasisolated from colored portion of a spoiled coconut and further it was authenticated byMTCC, India. The various parameters like temperature, pH, salt concentration, andprecursors were optimized for the production of prodigiosin. We now report that thepigment production was higher in our isolated strain than S. marcescens. It was observedthat prodigiosin binds with plastic, paper, and fibers and thus in near future, it can also beused as a natural dye.

Keywords Serratia rubidaea . Serratia marcescens . Prodigiosin . Natural dye

Introduction

The red pigment, prodigiosin, is a member of the prodiginines produced by Serratiamarcescens, some actinomycetes, and a few other bacteria [1]. Prodigiosin is a lineartripyrrole and a typical secondary metabolite, appearing only in the later stages of bacterialgrowth [2]. Although prodigiosin have no defined role in the physiology of producingstrains, but have been reported to have anti-fungal, anti-bacterial, and anti-protozoal/anti-malarial activities. Prodigiosin is of great interest due to its immunosuppressive andantiproliferative properties [3] as well as its ability to induce apoptosis in certain cancercells [4]. It seems to have potential for applications in medical industries. Montaner et al.[5] showed that prodigiosin extracted from S. marcescens could induce apoptosis in

Appl Biochem Biotechnol (2012) 166:187–196DOI 10.1007/s12010-011-9415-8

R. Siva (*) : K. Subha :D. Bhakta :A. R. Ghosh : S. BabuSchool of Bio Sciences and Technology, VIT University, Vellore 632 014 Tamil Nadu, Indiae-mail: [email protected]

Author's personal copy

hematopoietic cancer cell lines including acute T-cell leukemia, myeloma, and Burkitt’slymphoma, with little effect on non-malignant cell lines. It has also been shown to induceapoptosis in human primary cancer cells in the case of B and T cells in B-cell chroniclymphocytic leukemia samples [6].

The prodigiosin groups of natural products are a family of tripyrrole red pigments thatcontain a common 4-methoxy, 2–2 bipyrrole ring system. The biosynthesis of the pigmentis a bifurcated process in which mono- and bipyrrole precursors are synthesized separatelyand then assembled to form prodigiosin [7]. However, pigmentation is only present in asmall percentage of isolated cultures. Pigment production is highly variable among speciesand is dependent on many factors such as species, incubation time, pH, temperature,nutritional factors etc. [8, 9].

In the light of their potential commercial values, there is a demand to develop a high-throughput and cost-effective bioprocess for the production of these prodigiosin andprodigiosin-like pigments and also to explore more prodigiosin-producing microbes. Thepigment prodigiosin has a number of medicinal properties but the application of thepigment as a natural dye is yet to be explored. In this context, we have isolated a bacteriumfrom pink-colored portion of a spoiled coconut (Fig. 1a) and the microbiological,biochemical, physiological, and cultural tests were performed and the organism wasidentified as Serratia rubidaea with prodigiosin production (Fig. 1b). This paper deals withthe identification of the pigment by chemical characterization, optimization of parametersfor the pigment production, and probable application of the pigment as a natural dye.

The pink-colored portion of the spoiled coconut (Fig. 1a) was used as source of thebacterium. This organism is further used for the production of prodigiosin. The organismwas repeatedly subcultured onto nutrient agar (NA; Himedia, India) to obtain pure cultureand to study the morphological characters of the colony such as size of the colony, theperiphery, surface texture, elevation, and pigmentation. Primarily, samples were subculturedonto NA, Mueller Hinton agar (MHA, Himedia, India), and potato dextrose agar (PDA,Himedia, India). But there was no growth found on PDA, less growth in MHA, and profusegrowth with red coloration onto NA. Hence NA was selected for further studies.

A reference strain of S. marcescens obtained from our culture collection, was used forcomparative analysis. The microscopic examination of the bacterium was done by differenttypes of staining such as simple staining, grams staining, and spore staining and motilitytest by hanging drop method [10].

Fig. 1 a Spoiled coconut; b isolation of S. rubidaea

188 Appl Biochem Biotechnol (2012) 166:187–196


The isolated strain was subjected to a number of biochemical tests to characterize itsbiochemical behavior. Production of certain enzymes, utilization of some substrates, the endproducts formed etc. are characteristic feature of certain group of organisms. These serve asbiochemical markers for the identification of the organisms. The biochemical tests such asIndole test, methyl red test, Voges-Proskauer test, citrate utilization test, triple sugar irontest, catalase test, oxidase test, carbohydrate utilization tests, amino acid utilization tests likelysine decarboxylase, arginine dihydrolase, ornithin decarboxylase, and phenyl alaninedeaminase test, other biochemical tests like, decarboxylase test, nitrate reduction test,phosphatase test, urease test, hydrolysis tests for casein, esculin, gelatin, starch, Tween 20,Tween 40, Tween 80, and o-nitrophenyl-β-D-galactopyranoside test were performed toidentify the organism. The procedures followed are based on the standard biochemical tests[10]. Besides, antibiotic sensitivity tests, study on the growth of the organism both ontosolid (nutrient agar, HiMedia, India) and liquid media (using peptone water, nutrient broth,and Luria Bertani broth—HiMedia, India) were carried out.

Three common broths, available in a microbiology laboratory, were used for this study:peptone water (1%), nutrient broth, and Luria Bertani broth. For qualitative assessment,primary inoculum of 3×106 colony forming units was inoculated into respective broth andwas incubated at 25 and 37 °C, respectively, for 24, 48, and 72 h in still and in stirred(150 rpm) condition.

Quantitative analysis of growth versus pigment production was carried out using peptonewater (PW). Besides, fresh coconut kernel was contaminated with pure culture of the strain andincubated at 25 and 37 °C, respectively, for 24, 48, and 72 h for growth and color production.

The chemical characterization of the dye was done after extracting the pigment. Thebacterium was grown in PW and cells were harvested in phosphate-buffered saline (pH 7.2).

Fig. 2 Growth of S. rubidaea and prodigiosin production in peptone water

Table 1 Growth and prodigiosin production in different media

Media Growth (24 h) Prodigiosin synthesis

28 °C 37 °C 43 °C 28 °C 37 °C 43 °C

Peptone water + + +/− + − −Nutrient broth + + − +/− − −Luria Bertani broth + + − − − −

Appl Biochem Biotechnol (2012) 166:187–196 189


It was mixed well in a vortex mixer and centrifuged at 10,000 rpm for 10 min. Thesupernatant and pellet were collected separately. Both the solvent and acid extractions wereperformed. Solvent extraction of the supernatant was performed by using dichloromethaneas solvent and acid extraction using 10% of 3 N HCl in acetone. The supernatant,dichloromethane extracted, and acetone extracted solutions were examined in the UV–Visible spectrophotometer (Amersham biosciences ultraspec1100 pro) for maximumabsorbance.

The LC-MS analysis was done for both acetone-extracted and dichloromethane-extracted compounds. The LC-MS was carried out in positive mode ionization inPhenomenex LUNA C18 column with two mobile phases. Mobile phase A consisting of10% methanol/90% water/0.1% TFA and mobile phase B consisting of 90% methanol/10%water and 0.1% TFA. The flow rate used for the analysis was 5 ml/min.

The different parameters for production of prodigiosin such as media, pH of the media,incubation temperature, time, precursors, and salt concentration were studied.

The application of the dye for coloring different materials such as plastic, cotton, andfabric was examined.

Plastic The efficiency of the pigment to be used as a dye was tested by coloring plasticcontainers such as beakers, Eppendorfs, and Falcon tubes. Both the crude extract and the

Fig. 3 Growth of S. rubidaea in coconut incubated at room temperature

Fig. 4 Growth curve against prodigiosin production



extracted compound (acid extraction and organic solvent extraction) were used for coloring.The light sensitivity was also examined by exposing it to sunlight repeatedly and thefastness by repeated washing.

Cotton and Fabrics The cotton to be colored was dipped in it and placed in boiling waterbath for 20 min. After removal of the cotton from the solution, it was washed with coldwater and then allowed to dry in air. After drying it was again soaked in water for 20 min tocheck the fastness of the color. The cotton was air dried. The light sensitivity of the dye waschecked by exposing it to light for prolonged period.

The MS data revealing two peaks obtained at retention times 1.891 minute and 2.154 minute

The fragmentation pattern of the peak obtained at retenion time 1.81 minute

The fragmentation pattern of the peak obtained at retention time 2.154 minute

Fig. 5 LC-MS confirmation of the pigment prodigiosin



Isolation and Identification

The bacterium that was grown in the nutrient agar was examined for colony morphology. Itwas found to be entire, dome shaped, elevated, circular, smooth and shiny, opaque withpink-colored pigmentation. In brief, the strain isolated with red pigment was a gramnegative, motile, rod shaped, highly mucoid colonies, oxidase negative, reduced nitrate,indole negative, Vogues-Proskauer positive, and Simmon’s citrate positive. It grew wellbetween pH 4.5 and 9.0, between 15 and 42 °C, and in 2–7% of NaCl salt concentration.Thus, the strain isolated is identified as S. rubidaea (further it was authenticated by MTCC,Microbial Type Culture Collection and Gene Bank, India).

The indication of sugar utilization by color change from red (phenyle red) to yellow (dueto acid production as an end product) for 18 and 24 h was examined. The strain, S.rubidaea (MHA120308) utilized most of the sugars used with the exception of rhamnose,sorbitol, cellobiose, and starch. Cellobiose was utilized after 48 h of incubation whilefructose and mannitol were used within 18 h. The strain was found to be resistant tomultiple antibiotics such as ampicillin, bacitracin, oxacillin, penicillin G, streptomycin, andvancomycin.

Inoculation in the broth showed color production and growth in peptone water (Fig. 2).In case of nutrient broth and LB broth, growth was present but color production was notobserved (Table 1).

The growth and color production in peptone water with and without carbohydrates(fructose) showed that carbohydrate source is not essential for the growth as well as forcolor production. The presence of peptone is sufficient for both growth and colorproduction. The absence of color production in LB broth may be due to the absence of thepeptic digest of the animal tissue or due to other components present in the media. Innutrient broth with Hiveg (digested peptone from vegetable origin, Himedia, India) extract,the color formation was less in comparison to the nutrient broth with peptic digest ofanimal. The peptic digest of animal might have some nutrient that can enhance theproduction of the pigment. The growth of the pigment in broth is dependent on mediacomponents, temperature of incubation, and the rpm at which it is kept for shaking. Staticcultures show no color production and the broth incubated at higher temperatures (43 °C)shows no color production. So aeration, temperature, and media components are importantfactors that determine the color production in liquid media.

The re-inoculation in the coconut shows that the coconut when kept at room temperatureshows growth and color production whereas both are comparatively limited when incubated

Fig. 6 Effect of temperature on prodigiosin production



at 37 °C. Figure 3 shows the growth of S. rubidaea in coconut incubated at roomtemperature and at 37 °C.

The growth pattern of S. rubidaea was observed at different time intervals. The organismshowed a short lag phase and a distinct log phase with stationary phase starts from 16 h ofgrowth and extended up to 44 h. The pigment production was observed after 8 h ofincubation and till 44 h of incubation. The maximum absorbed peak (A535) for pigmentproduction was seen at 26th hour (Fig. 4). From the figure, it may be examined that thestationary phase begins from 20 h. So the optimum pigment production is observed duringthe stationary phase.

The spectroscopic analysis shows that the mixture has maximum absorbance at threedifferent wavelengths. Maximum absorbance was observed at 535 nm. Various researchershave reported that the primary pigment responsible for color production in Serratia speciesis prodigiosin and that has the maximum absorbance at 535 nm [1, 9]. The UV–visiblespectrum analysis (Fig. 4) and the mass analysis with LC-MS show that the pigment isprodigiosin (Fig. 5).

Temperature and pH are the common environmental factors that play a crucial role incell growth and prodigiosin production of Serratia strains [11]. For instance, it has beenreported that the production of prodigiosin was inhibited when the temperature was lowerthan 20 °C or higher than 37 °C. In the present study, the effect of temperature onprodigiosin production was observed and shown in Fig. 6. It shows that there is no colorproduction at temperatures above 37 °C and there is no growth above 40 °C. The colorformation is more at lower temperatures and is high at 28 °C as reported by the previous

Fig. 7 Effect of pH on prodigiosin production

Fig. 8 Effect of salt concentration on prodigiosin production



researchers. This is seen in case of both the reference strain (S. marcescens) and the isolatedstrain (S. rubidaea). The optimum temperature for production of the pigment was found tobe 28 °C.

The optimum pH for prodigiosin production was also determined by observingprodigiosin production at culture pHs of 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. The resultsobtained for the effect of pH was observed and shown in Fig. 7. From the graph it can beseen that the production of prodigiosin increase is more at pH 4.5 and it progressivelydecreases with increase in pH. This shows that the color production is more efficient atacidic pH than at alkaline pH.

The effect of salt concentration on pigment production was studied and the resultsare shown in Fig. 8. The effect of salt concentration on prodigiosin production showsthat the pigment production decreases as the salt concentration increases in the isolatedstrain. The optimum salt concentration for the pigment production was found to be 1%NaCl in the media. In case of the reference strain the optimum concentration for thepigment production was 3% and 4%. In the reference strain the pigment productionincreases with increase in salt concentration and then it decreases above 4%. It was also

Fig. 9 Effect of precursor on prodigiosin production

Fig. 10 The application prospective of prodigiosin in successfully staining the plastics (a and b) as well aswool (c) and fiber (d)



observed that the growth and pigment production was present in the isolated strain up to9% salt where as in the reference strain no growth is observed at salt concentration of 7%and above. Hence the isolated strain is a salt-tolerant bacterium. This result is supportedby the previous work [12].

There is evidence that the precursor may play a crucial role in synthesis of prodigiosinby S. marcescens species [12–14]. Some amino acids, such as tryptophan, proline, andhistidine, contain pyrrole structures; therefore, they may stimulate prodigiosin production.This motivated us to investigate the effect of amino acid supplementation on prodigiosinproduction by the isolated strain. The results obtained for the effect of precursors is shownin the Fig. 9. Among the combinations of amino acids used, the methinone with prolineshowed highest production of the pigment and the other combinations such as methioninewith leucine and leucine with alanine showed increase in color production than the control(without induction). The other precursor acetate also showed good inducing property. Theeffect of precursor shows that the proline when used individually or in combination withmethionine act as a good precursor.

The plastic in Eppendorf, beakers, and Falcon tubes were efficiently colored by thispigment. The color was retained even after repeated washing and was resistant to light. Thecotton and fabric were also efficiently colored by the pigment. Since the pigment can bindto plastics, cotton as well as to fabric it can be used as a dye (Fig. 10a–d).

From literature survey it is observed that a large number of microbes are capable ofproducing pigments and the Serratia species are producers of a red-colored pigment,prodigiosin. Most of the work done in the pigment is from the source Serratia marcescens.This is the first report on the optimization of prodigiosin production from S. rubidaea. Thevarious parameters like temperature, pH, salt concentration, and effect of precursor werestudied for the optimization of prodigiosin production. Again, the pigment prodigiosin has anumber of medicinal properties but the application of the pigment as a natural dye is yet tobe explored.

There is interest in the dyeing of textile fibers with natural dyes, on account of their highcompatibility with the environment, and because of their lower toxicity and allergicreactions. As a result of the use of natural products with low toxicity, a decrease in theoverall reduction of exposure to harmful chemicals is expected for both textile workers andwearers of the cloths. It is traditionally believed that many of these natural dyestuffs areeffective against insect attack and have some medicinal value. It is speculated that theprodigiosin will make good market in the near future as a natural dye in textile industry.

Acknowledgments The authors are thankful to VIT Management for their support and encouragements.

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2 3 Characterization and Enhanced Production of Prodigiosin from the Spoiled Coconut

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