Appl Biochem Biotechnol DOI 10.1007/s12010-014-0921-3
Studies on Production and Biological Potential of Prodigiosin by Serratia marcescens Rahul K. Suryawanshi & Chandrashekhar D. Patil & Hemant P. Borase & Bipinchandra K. Salunke & Satish V. Patil
Received: 26 September 2013 / Accepted: 14 April 2014 # Springer Science+Business Media New York 2014
Abstract Efficacy of Serratia marcescens for pigment production and biological activity was investigated. Natural substrates like sweet potato, mahua flower extract (Madhuca latifolia L.), and sesam at different concentrations were taken. As a carbon source microorganism favored potato powder was followed by sesam and mannitol, and as nitrogen source casein hydrolysate was followed by yeast and malt extract. The effect of inorganic salts on pigment production was also studied. At final optimized composition of suitable carbon, nitrogen source, and trace materials and at suitable physiological conditions, prodigiosin production was 4.8 g L−1. The isolated pigment showed antimicrobial activity against different pathogenic bacteria and fungi. Extracted pigment was characterized by spectroscopy, Fourier transform infrared (FTIR), and thin layer chromatography (TLC) which confirm production of biological compound prodigiosin. This study suggests that use of sweet potato powder and casein can be a potential alternative bioresource for commercial production of pigment prodigiosin. Keywords Serratia . Prodigiosin . Production . Optimization
Introduction Red-pigmented prodigiosin compound was first isolated from Serratia marcescens and identified as secondary metabolite so named from Bacillus prodigiosus, bacterium which was later renamed as S. marcescens, historically well known for “bleeding bread” report. Prodiginines share a common pyrrolyldipyrromethene core structure which can also be isolated from, Vibrio psychroerythrous, Alteromonas rubra, Pseudomonas magneslorubra, S. rubidaea, Actinomycetes, such as Streptoverticillium rubrireticuli and Streptomyces longisporus ruber [1]. Prodigiosin have a wide variety of biological properties, including antibacterial, antifungal, R. K. Suryawanshi : C. D. Patil : H. P. Borase : B. K. Salunke : S. V. Patil (*) School of Life Sciences, North Maharashtra University, Jalgaon 425001 Maharashtra, India e-mail: [email protected] S. V. Patil North Maharashtra Microbial Culture Collection, North Maharashtra University, Jalgaon, Maharashtra, India
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antimalarial, antibiotic, immunosuppressive, and anticancer [2–7]. These properties make this pigment as most powerful research candidate. Considering industrial applications of prodigiosin it is all time need to develop cost effective process for its production using alternate bioresource. Serratia like other enterobacteriaceae grow well on different synthetic and complex media using single carbon and nitrogen source, at the same time pigment production is highly sensitive to temperature and does not produce pigmentation at or above 37 °C. This study describes efficiency of local strain to produce biologically active pigment using some nonconventional carbon sources such as powder of Madhuca latifolia, sweet potato, sesam and nitrogen sources like casein, beef extract, urea etc. In addition to this, biological activity of pigment has also been investigated against Gram-positive and Gram-negative pathogenic bacteria, fungi and has been compared with standard antibiotics.
Materials and Methods Microbial Samples The microbial cultures were taken from culture collection of, North Maharashtra University, Jalgaon (NMCC). The bacterial cultures used were Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi, whereas fungal isolates were Aspergillus flavus, Fusarium oxysporium, and Penicillium notatum. Pigment Extraction and Isolation Cell mass was collected by centrifugation (7,000×g for 10 min) of pigmented cultures. Pigment was extracted using acidified methanol. The crude extract was evaporated by heating in water bath at 70 °C. The dried pigment was redissolved in methanol. Purification and Identification of the Pigment The crude extract of bacterial pigment was purified by thin layer chromatography (TLC) in (Hi-250 F) silica gel plates. Solvent system used was butanol: hexane (2:1). Separated spot of pigment was recovered by dissolving in methanol and dried by evaporation. The pigment concentration was determined as gram % on dry weight basis (w/v). The dried product was further dissolved in DMSO and was subjected to UV–visible spectrophotometry in the range of 700–350 nm. The Pigment was further characterized using Fourier transform infrared (FTIR) spectrophotometer (Testscan Shimadzu FTIR 8400, Japan). Dried pigment was mixed with KBr powder and pressed into pellet for FTIR spectroscopy with frequency range of 4,000–400 cm−1. Cell Weight Determination Cell weight was determined as per gravimetric method briefly; bacterial cells were grown for the period of 24 to 72 h at 28 °C and in various physicochemical conditions. Culture was centrifuged at 7,000×g for 10 min. Cell pellet was washed thrice with 0.85 % physiological saline and air dried on filter paper followed by drying at 105 °C for 3 h in an oven.
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Optimization of Prodigiosin Production Effect of Agitation on Pigment Production Various agitation speeds were used to study the influence of aeration on pigment, and biomass production. The 24 h grown culture of bacteria was inoculated in pre modified nutrient broth (carbon source as 1 % mannitol). Flasks were incubated on different shaking speeds of 100 to 200 rpm at 28 °C. Effect of pH on Pigment Production Active culture of S. marcescens was inoculated in nutrient broth with pH range of 3 to 10. The flasks were incubated at 28 °C for 24 h. The prodigiosin production was estimated after incubation. The pH at which microorganism gave maximum pigment production was kept constant for further studies. Effect of Temperature on Pigment Production Effect of temperature on biomass and prodigiosin production was investigated by keeping the cultivation temperature 25, 30, 35, and 40 °C in shaker incubator at the interval of 24 to 72 h. Effect of Inorganic Salts on Pigment Production S. marcescens was grown with nutrient broth containing different concentrations of inorganic salts. The concentrations used for calcium carbonate and ferrous sulfate ranged between 10 and 100 mg l−1. For sodium and potassium chloride, it was 0.2 to 2.4 g l−1, and for dipotassium phosphate and magnesium sulfate, the concentrations ranged between 20 and 220 mg l−1. After incubation time, the pigment production was determined. Carbon Sources and Nitrogen Sources Throughout the experiment, synthetic as well as natural polymeric carbon sources were used, i.e., glucose, maltose, lactose, sucrose, starch, sweet potato powder, M. latifolia extract, and sesam extract. While sodium nitrate, ammonium sulfate, casein, yeast extract, and peptone were compared as nitrogen source. Active bacterial culture was inoculated in nutrient broth containing (0.1 % w/v) carbon and nitrogen source. After incubation, pigment production was determined. The carbon and nitrogen source responsible for higher pigment production was further tested at different concentrations (0.2 to 2 g l−1) for determination of exact concentration responsible for high yield. Antimicrobial Assay The antimicrobial activity of the pigment was evaluated by antimicrobial susceptibility test. The bacterial and fungal cultures were grown in nutrient and Czapek dox broth at 37 and 28 °C, respectively. From 24 h grown culture of each microorganism, 100 μl at a concentration of 106 cells ml−1 was spread on the surface of nutrient agar for bacteria and on Czapek dox agar for fungi in Petri plates. Wells of 8 mm diameter were made with a metal borer. Extracts of 100 μl each was added in the wells at various concentrations of respective solvents. The plates were kept at 4 °C for 2 h to allow prediffusion then incubated at 37 °C for 24 h for
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bacteria and 28 °C for 48 h for fungi. Zones of inhibition were measured in millimeter from the circumference of the wells to the circumference of the inhibition zone. The assays were repeated three times with solvent serving as controls. Results were compared with standard antibiotic discs (HiMedia, Mumbai, India).
Results Optimization of Prodigiosin Production Effect of Aeration, Temperature, and pH on Pigment Production Increase in prodigiosin and biomass production was observed with increased aeration (maximum at 180 rpm for 48 h). Prodigiosin production was highest (0.23 g% w/v) at 120 rpm, and it decreased considerably after 200 rpm. The biomass and prodigiosin production was increased with increase in temperature up to 30 °C and decreased after 35 °C. The highest production (0.24 g%) was obtained when bacteria were cultivated at 30 °C for 48 h. The pH of the culture medium has been reported to play a key role in pigment synthesis. Serratia was cultivated at different initial pH values (3 to 10) in shake flask cultures. Organism showed growth and pigmentation in wide range of pH with the maximum (0.23 g%) being at the pH 7. Effect of Inorganic Salts on Pigment Production Prodigiosin production was almost constant with 10–100 mg l−1 concentration of calcium carbonate (Fig. 1b). Variation in the concentration of sodium and potassium chloride did not affect pigment and cell mass production and their concentration beyond 2 g l−1 w/v showed inhibitory effect on pigment production. Phosphate (KH2PO4) at concentration of 80 mg l−1 showed maximum prodigiosin production (0.17 g%), and a further increase in phosphate reduced pigment production (Fig. 1a). In case of ferrous sulfate (FeSO4), prodigiosin production was almost constant (0.19 g%) at the 30–50 mg l−1 and considerably decreased beyond 50 mg l−1 (Fig. 1c), while magnesium sulfate (MgSO4) at 10–200 mg l−1 concentration showed increase in pigment (0.310 g%) production, and above 200 mg l−1 concentration, prodigiosin production was decreased (Fig. 1d). Effect of Carbon Sources on Pigment Production Sweet potato extract gave maximum recovery of prodigiosin (0.42 g%), followed by sesame (0.361 g%) and maltose (0.359 g%), as compared to other carbon sources after 48 h (Fig. 2a). The effective concentration of potato powder was 1 g l−1. Pigment yield was comparatively less, below 0.6 g l−1 and above 1.2 g l−1 concentrations of potato powder (Fig. 2b). Yield of pigment was comparatively less with glucose, lactose, mannitol, sucrose, and M. latifolia floral extract as a carbon source. Effect of Nitrogen Sources on Pigment Production Casein at 0.6 g l−1 gave maximum amount pigment (0.48 g%) after 24 h (Fig. 3b), while yeast extract at 0.8 g l−1 showed comparable pigment production (0.5 g%) after 36 h. (Fig. 3c). Other nitrogen sources used were inferior for pigment production (Fig. 3a).
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Fig. 1 Effect of inorganic salts (a inorganic phosphate, b CaCO3, c FeSO4, and d MgSO4) on prodigiosin production
Identification Thin layer chromatography (TLC) was performed for isolated pigment on standard silica gel 60 F254 plates with butanol : hexane (2 : 1) as a solvent system. The Rf value of the sample and standard was found to be 0.86 (Fig. 4a). Spectrophotometric analysis of pigment showed single peak at 536 nm (Fig. 4b). In FTIR (Fig. 5), red pigment from Serratia showed strong and broad absorption at 3,378 cm−1 (O–H stretch), 2,951 cm−1 (C–H and C=O stretch) and 1,556 cm−1 (aromatic C=C, NO2 stretch) represents respective bonds, prodigiosin exhibits
Fig. 2 Effect of carbon source on pigment production a using different carbon sources and b with different concentrations of potato powder
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Fig. 3 Effect of nitrogen source on pigment production using a different nitrogen sources, b different concentrations of casein, and c different concentrations of yeast extract
similar absorptions in CHCl3 at 1,630 and 1,602 cm−1 [8]. N–H group and phenyl rings were evident at fingerprint region, which was characterized by weak intensity at 1,646 cm−1 (aromatic C=0). Absorption at 1,191 and 1,197 cm−1 showed C–N bend (amines) and C–O
Fig. 4 a TLC image of standard prodigiosin (S) and pigment extracted and purified from Serratia marcescens (T). b Spectroscopic analysis of pigment from S. marcescens
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Fig. 5 FTIR analysis of pigment from Serratia marcescens
(carboxylic) stretch, whereas 863 and 706 cm−1 indicate C–H phenyl ring bend, and weak absorption at 1,445 cm−1 also shows bending of C–H. Biological Effect Antibacterial Effect of Prodigiosin Prodigiosin showed inhibitory activity against range of Gram-positive and Gram-negative bacteria with zone of inhibition in the range of 14 to 21 mm in diameter at 100 μg ml−1 concentration (Fig. 6a). Minimum inhibitory concentration (MIC) for bacteria was in the range
Fig. 6 Antibacterial activity of pigment. a Zone of inhibition against different bacteria. b Minimum inhibitory concentration against different bacteria
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of 5 to 23 μg ml−1 (Fig. 6b). Higher concentrations of prodigiosin (100 and 50 μg ml−1) showed considerable growth inhibition of Gram-negative bacteria. Antifungal Effect of Prodigiosin The zone of inhibition for fungi was in the range of 17 to 18 mm in diameter at 100 μg ml−1 concentration (Fig. 7a). MIC of prodigiosin for the tested fungal isolates was in the range of 8 to 21 μg ml−1 (Fig. 7b).
Discussion Optimization of Prodigiosin Production Effect of Agitation, Temperature, and pH on Pigment Production Increase in pigment and biomass production was observed with increased agitation suggesting a key role of shaking speed in pigment production, few references show comparable effect of aeration with carotenoids production by yeast [9–13]. Aerobic conditions influence the metabolism of cells which increase the requirement of amino acids and hence cause more production of pigment [14]. Results for effect of temperature on biomass and pigment production are in agreement with the observation of Chang et al.[15] for S. marcescens strain 389. Qadri and Robert [16] reported no prodigiosin production at 38 °C; however, with same culture, pigment production was observed when temperature was shifted to 27 °C. A complete inhibition in prodigiosin was observed in most of the basically used media tested at 37 °C, which was similar to the result observed by Pryce and Terry [17]. The effect of temperature may be due to sensitivity of monopyrrole and bipyrrole to higher temperatures [18]. Synthesis of amino acids is also affected by the change in temperature [19]. The pH of the culture medium has been reported to play a key role in pigment synthesis. It is essential to set a definite pH as sugar uptake depends on external pH, and it may affect function of different proteins involved in the metabolism of essential amino acids required for synthesis of prodigiosin [20]; therefore, adequate control of pH value is essential in both batch and
Fig. 7 Antifungal activity of pigment. a Zone of inhibition against different fungal strains. b Minimum inhibitory concentration of pigment against different fungal strains
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continuous production of pigment. pH 7 was found suitable for maximum pigment formation. Effect of Inorganic Salts on Pigment Production Inorganic salt, such as calcium carbonate, magnesium sulfate, and potassium phosphate, has a role in physiology and growth of organism. However, no substantial increase or inhibition in prodigiosin production was found with different concentrations of sodium and potassium chloride, and with increased concentration of these salts, pigment production was inhibited. This observation is in accordance with San et al. [21]. Calcium carbonate has decreased pigment formation (Fig. 1). It is been reported that calcium carbonate may be added to maintain the pH; at specific concentration, it stimulates sugar uptake by decreasing Km of sugar transport, whereas high sugar concentration inhibits this reaction [22]. Removal of sodium chloride and inorganic phosphate has found to enhance prodigiosin production [23]. In contrast to this, Ryazantseva et al. [24] reported stimulative action of sodium chloride on prodigiosin production. Although it has been said that inorganic phosphate decreases pigment production by S. marcescens [25], we have observed that up to 100 mg l−1, it stimulates pigment production but further concentration of phosphate inhibitory to pigment formation (Fig. 1). The inhibitory effect of inorganic phosphate might be due to inhibition of enzymatic activity like that for alkaline phosphatase [26], provided that the activity of enzyme is found to increase just before synthesis of secondary metabolites [27, 28]. Synthesis of two immediate precursors is found to be inhibited by inorganic phosphate [26]. Similarly, ferrous sulfate increases pigment production (Fig. 1). Effect of Carbon Sources on Pigment Production The type of carbon source used may play crucial role in pigment formation. Serratia was cultivated in the basal medium substituting different carbon sources. From a total of nine carbon sources, sweet potato extract (0.42 g%) gave maximum yield. Pigment production with maltose (0.359 g%) is in accordance with Sundaramoorthy et al. [29] and Shahitha and Poornima [30], who found maltose as a better carbon source which gave 3 g l−1 of prodigiosin. Pigment production was highest at 1 g l−1 potato powder. A higher production of prodigiosin with starch is comparable with the results obtained by Wei et al. [31], which gave 2.3 g l−1, and after optimizing the carbon : nitrogen ratio, it was further enhanced to 6.7 g l−1. Due to polymeric nature, starch might be utilized at slower rate, and hence the catabolic repression is avoided. On the other hand, pigment yields were less than 60 % with glucose, lactose, mannitol, and sucrose. Pigment production by these sources was comparable with the production by M. latifolia floral extract (Fig. 2a). The M. latifolia L. flower extract mainly contains sucrose as a principal component of the sugars, and interestingly, sucrose as a carbon source was least preferred by Serratia showing low pigment synthesis. Comparatively lower production of prodigiosin in presence of glucose and lactose was observed (Fig. 2a), which is similar to the previous report of Harned [32] who obtained a yield of 5 mg l−1 using a medium with glucose as a carbon source. Report says that pentose sugars inhibit prodigiosin production [25] and oligosaccharide is better than glucose for pigment production by Serratia [33]. By many reasons, glucose is an inferior source for prodigiosin production. It is responsible for the production of glucose-6-phosphate dehydrogenase which inhibits pigment production and causes the lowering of pH [34]. The addition of glucose and maltose also causes catabolic
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repression [35]. Serratia sp. is not lactose-fermenting, therefore pigment production with lactose was low [36]. Effect of Nitrogen Sources on Pigment Production Casein and yeast extract were found to be the most effective nitrogen sources as compared to beef extract, sodium nitrate, ammonium sulfate, and urea for production of pigment and biomass (Fig. 3). The highest amount (0.48 g% w/v) of pigment was produced in the medium containing 1 g l−1 potato extract and 0.6 g l−1 casein with 120 rpm after 24 h while with the use of casein, fermentation time was also reduced by few hours. Yeast extract showed 0.5 g% prodigiosin after 36 h. These results are similar with Song et al. [8] who used casein as nitrogen source for prodigiosin production and obtained 0.43 g%. Results for casein are in accordance with Robert [37] who observed induction of biosynthesis of prodigiosin after the addition of casein hydrolysate to suspension of washed, nonpigmented, nonproliferating cells. Casein hydrolysate contains aspartic acid, glutamic acid, proline, and alanine, which cause pigment production when added individually. Structurally methyl group on C6 position of prodigiosin comes from methionine [38]. Methionine and cysteine increase prodigiosin production [20]. At high concentration of casein and yeast extract, both pigment and cell mass decreased possibly because Serratia spp. is an osmotically sensitive strain showing impaired growth and partial lysis with leakage of culture fluid. Peptone and yeast extract are not commonly added to media of Gram-negative bacteria as they usually cause pleomorphism and make cell wall fragile [39, 40]. Glycine is a principal constituent of peptone, casein, and yeast extract, which causes interference with peptiodoglycan synthesis, results in the accumulation of UDP-glycopeptide precursor in cytoplasm, and makes osmotically fragile cells. Similar effects were demonstrated in case of peptone supplemented in fermentation medium of Azotobacter vinelandii for PHB synthesis [41]. Ammonium sulfate, nitrite, urea, meat extract, malt extract, and sodium nitrite found to give low pigment production. Low prodigiosin production with ammonium sulfate was also observed by Rockem and Weitzman [42]. Ammonium is poor nitrogen donar in Schiff base conversion of orange to red pigment [43]. Identification The Rf (0.86) value of separated spots was exactly matched with reference material (standard). The FTIR transmission pattern of the pigment is similar for the functional groups present in prodigiosin. The FTIR analysis, TLC, and spectroscopic analysis showed prodigiosin as a principal molecule in the extract (Fig. 4 and 5). The results are in accordance with Song et al. [8]. In our previous report, the result for high-performance TLC (HPTLC) of prodigiosin is available showing a single peak at 535 nm [44]. Biological Effect Antibacterial Effect of Prodigiosin Antibacterial effect against P. aeruginosa, E. coli, and S. aureus is in comparison with previous reports [3, 45]. The least MIC was for Gram-positive bacteria (Fig. 6b), but at higher concentrations of prodigiosin (100 and 50 μg ml−1), considerable growth inhibition of Gram-negative bacteria was observed. Prodigiosin affect intracellular pH gradient of the organism [46].
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Fig. 8 a Antibacterial and b antifungal effects of standard antibiotics against target microorganisms
Antifungal Effect of Prodigiosin The tripyrrole pigment prodigiosin not only inhibits bacteria but also fungi (Fig. 7). In previous reports prodigiosin have shown inhibition against Candida parapsilosis, Cryptococcus sp. [3], Candida albicans [1], and Didymella applanata [47]. Action of S. marcescens in vitro may be associated with specific properties of the cell wall of fungus [47]. Prodigiosin can affect mitochondrial dysfunction by the uncoupling of electronic chain transport of protons to mitochondrial ATP synthase and causes reduction in adenosine triphosphate (ATP) production without decreasing oxygen consumption. It also acts as proton pump inhibitor [2, 48]. Comparison with Standard Antibiotics Comparative analysis of inhibition studies showed that prodigiosin has more potential for inhibition of S. aureus and S. subtilis compared to antibiotics, such as cefatoxime and vancomycin; similarly, inhibitory potential of prodigiosin against P. aeruginosa and S. typhi was greater than vancomycin and cefatoxime, respectively. Inhibition of S. aureus by prodigiosin is comparable with inhibition showed by penicillin and ampicillin, whereas inhibition of S. subtilis, P. aeruginosa, E. coli, and S. typhi was less when compared to these antibiotics (Figs. 6 and 8). In case of antifungal effect, nystatin showed greater inhibition than prodigiosin (Figs. 7 and 8).
Conclusion After optimization of culture conditions for S. marcescens, a profound increase was observed in prodigiosin production. The highest pigment formation was observed in the presence of potato starch powder (1 g l−1) as a carbon source, while casein (0.6 g l−1) and yeast extract 1 g l−1) as a nitrogen source. The effect of inorganic salts has also been studied, which indicates that the presence of phosphate (80 mg l−1), FeSO4 (50 mg l−1), and MgSO4 (200 mg l−1) enhances pigment production, whereas CaCO3 inhibits pigment formation. Similarly, biological activity of the prodigiosin and its comparison with standard antibiotics provides useful information. Acknowledgments The author RKS is thankful to UGC BSR RSSMS scheme (Ref.: F.7-137/2007), New Delhi, India for the financial assistance. We are also thankful to North Maharashtra culture collection (NMCC) for providing the microbial cultures.
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Appl Biochem Biotechnol 25. Lawanson, A. O., & Sholeye, F. O. (1975). Inhibition of prodigiosin formation in Serratia marcescens by adenosine triphosphate. Experientia, 32, 439–440. 26. Frank, R. W., Mark, L. F., & Weinberg, E. D. (1977). Phosphate inhibition of secondary metabolism in Serratia marcescens. Applied and Environmental Microbiology, 33, 1042–1046. 27. Mertz, F. P., & Doolin, L. E. (1973). The effect of inorganic phosphate on the biosynthesis of vancomycin. Canadian journal of microbiology, 11, 765–777. 28. Weinberg, E. D. (1974). Secondary metabolism: control by temperature and inorganic phosphate. Developments in Industrial Microbiology, 15, 70–81. 29. Sundaramoorthy, N., Yogesh, P., & Dhandapani, R. (2009). Production of prodigiosin from Serratia marcescens isolated from soil. Indian Journal of Science and Technology, 10, 32–34. 30. Shahitha, S., & Poornima, K. (2012). Enhanced prodigiosin production in Serratia Marcescens. Journal of Applied Pharmaceutical Science, 2, 138–140. 31. Wei, C. C., Wan, J. Y., Chia, C. C., Jo, S. C., Shih, H. H., Chih, H. C., et al. (2013). Enhancing production of prodigiosin from Serratia marcescens C3 by statistical experimental design and porous carrier addition strategy. Biochemical Engineering Journal, 78, 93–100. 32. Harned, R. L. (1954). The Production of prodigiosin by submerged growth of Serratia marcescens. Journal of Applied Microbiology, 2, 365–368. 33. Martin, J. F., & Demain, A. L. (1980). Control of antibiotic biosynthesis. Microbiological Reviews, 44, 230– 251. 34. Gargallo, D., Lorén, J. G., Guinea, J., & Vinas, M. (1987). Glucose-6-phosphate dehydrogenase alloenzymes and their relationship to pigmentation in Serratia marcescens. Applied and Environmental Microbiology, 53, 1983–1986. 35. Giri, A. V., Anandkumar, N., Muthukumaran, G., & Pennathur, G. (2004). A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil. BMC Microbiology, 4, 1–10. 36. Bhathini, V. P., Francis, S. P., Muthusamy, P., & Jayaraman, A. (2013). Optimization and production of prodigiosin from Serratia marcescens mbb05 using various natural substrates. Asian Journal of Pharmaceutical and Clinical Research, 6, 34–41. 37. Robert, P. W. (1973). Biosynthesis of prodigiosin, a secondary metabolite of Serratia marcescens. Applied Microbiology, 25, 396–402. 38. Hussain, Q. S. M., & Robert, P. W. (1973). Role of methionine in biosynthesis of prodigiosin by Serratia marcescens. Journal of Bacteriology, 116, 1191–1198. 39. Tchan, Y. T. (1984). Family II. Azotobacteraceae, In: Bergey’s manual of systematic bacteriology, 1, 219– 234. 40. Vela, G. R., & Rosenthal, R. S. (1972). Effect of peptone on Azotobacter morphology. Journal of Bacteriology, 111, 260–266. 41. Page, W. J., & Cornish, A. (1993). Growth of Azotobacter vinelandii UWD in fish peptone medium and simplified extraction of poly-beta-hydroxybutyrate. Applied and Environmental Microbiology, 59, 4236– 4244. 42. Rokem, J. S., & Weitzman, P. (1987). Prodigiosin formation by Serratia marcescens in a chemostat. Enzyme and Microbial Technology, 9, 153–155. 43. Hardjito, L., Huq, A., & Colwell, R. R. (2002). The influence of environmental conditions on the production of pigment by Serratia marcescens. Biotechnology and Bioprocess Engineering, 7, 100–104. 44. Rahul, S., Chandrashekhar, P., Hemant, B., Chandrakant, N., Laxmikant, S., & Satish P. Nematicidal activity of microbial pigment from Serratia marcescens. In press (ID: 904310 doi:10.1080/14786419.2014.904310). 45. Antony, V. S., Chandana, K. S., & Narendra, K. G. (2011). Optimization of prodigiosin production by Serratia marcescens SU-10 and evaluation of its bioactivity. International Research Journal of Biotechnology, 2, 128–133. 46. Francisco, R., Pérez-Tomás, R., Gimènez-Bonafé, P., Soto-Cerrato, V., Giménez-Xavier, P., & Ambrosio, S. (2007). Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines. European Journal of Pharmacology, 572, 111–119. 47. Duzhak, B., Panfilova, Z. I., Duzhak, T. G., Vasyuninal, E. A., & Shternshis, M. V. (2012). Role of prodigiosin and chitinases in antagonistic activity of the bacterium Serratia marcescens against the fungus Didymella applanata. Biochemistry (Moscow), 77, 910–916. 48. Sato, T., Konno, H., Tanaka, Y., Kataoka, T., Nagai, K., Wasserman, H. H., et al. (1998). Prodigiosins as a new group of H+/Cl− symporters that uncouple proton translocators. The Journal of Biological Chemistry, 273, 21455–21462.
Studies on Production and Biological Potential of Prodigiosin by Serratia marcescens Rahul K. Suryawanshi & Chandrashekhar D. Patil & Hemant P. Borase & Bipinchandra K. Salunke & Satish V. Patil
Received: 26 September 2013 / Accepted: 14 April 2014 # Springer Science+Business Media New York 2014
Abstract Efficacy of Serratia marcescens for pigment production and biological activity was investigated. Natural substrates like sweet potato, mahua flower extract (Madhuca latifolia L.), and sesam at different concentrations were taken. As a carbon source microorganism favored potato powder was followed by sesam and mannitol, and as nitrogen source casein hydrolysate was followed by yeast and malt extract. The effect of inorganic salts on pigment production was also studied. At final optimized composition of suitable carbon, nitrogen source, and trace materials and at suitable physiological conditions, prodigiosin production was 4.8 g L−1. The isolated pigment showed antimicrobial activity against different pathogenic bacteria and fungi. Extracted pigment was characterized by spectroscopy, Fourier transform infrared (FTIR), and thin layer chromatography (TLC) which confirm production of biological compound prodigiosin. This study suggests that use of sweet potato powder and casein can be a potential alternative bioresource for commercial production of pigment prodigiosin. Keywords Serratia . Prodigiosin . Production . Optimization
Introduction Red-pigmented prodigiosin compound was first isolated from Serratia marcescens and identified as secondary metabolite so named from Bacillus prodigiosus, bacterium which was later renamed as S. marcescens, historically well known for “bleeding bread” report. Prodiginines share a common pyrrolyldipyrromethene core structure which can also be isolated from, Vibrio psychroerythrous, Alteromonas rubra, Pseudomonas magneslorubra, S. rubidaea, Actinomycetes, such as Streptoverticillium rubrireticuli and Streptomyces longisporus ruber [1]. Prodigiosin have a wide variety of biological properties, including antibacterial, antifungal, R. K. Suryawanshi : C. D. Patil : H. P. Borase : B. K. Salunke : S. V. Patil (*) School of Life Sciences, North Maharashtra University, Jalgaon 425001 Maharashtra, India e-mail: [email protected] S. V. Patil North Maharashtra Microbial Culture Collection, North Maharashtra University, Jalgaon, Maharashtra, India
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antimalarial, antibiotic, immunosuppressive, and anticancer [2–7]. These properties make this pigment as most powerful research candidate. Considering industrial applications of prodigiosin it is all time need to develop cost effective process for its production using alternate bioresource. Serratia like other enterobacteriaceae grow well on different synthetic and complex media using single carbon and nitrogen source, at the same time pigment production is highly sensitive to temperature and does not produce pigmentation at or above 37 °C. This study describes efficiency of local strain to produce biologically active pigment using some nonconventional carbon sources such as powder of Madhuca latifolia, sweet potato, sesam and nitrogen sources like casein, beef extract, urea etc. In addition to this, biological activity of pigment has also been investigated against Gram-positive and Gram-negative pathogenic bacteria, fungi and has been compared with standard antibiotics.
Materials and Methods Microbial Samples The microbial cultures were taken from culture collection of, North Maharashtra University, Jalgaon (NMCC). The bacterial cultures used were Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhi, whereas fungal isolates were Aspergillus flavus, Fusarium oxysporium, and Penicillium notatum. Pigment Extraction and Isolation Cell mass was collected by centrifugation (7,000×g for 10 min) of pigmented cultures. Pigment was extracted using acidified methanol. The crude extract was evaporated by heating in water bath at 70 °C. The dried pigment was redissolved in methanol. Purification and Identification of the Pigment The crude extract of bacterial pigment was purified by thin layer chromatography (TLC) in (Hi-250 F) silica gel plates. Solvent system used was butanol: hexane (2:1). Separated spot of pigment was recovered by dissolving in methanol and dried by evaporation. The pigment concentration was determined as gram % on dry weight basis (w/v). The dried product was further dissolved in DMSO and was subjected to UV–visible spectrophotometry in the range of 700–350 nm. The Pigment was further characterized using Fourier transform infrared (FTIR) spectrophotometer (Testscan Shimadzu FTIR 8400, Japan). Dried pigment was mixed with KBr powder and pressed into pellet for FTIR spectroscopy with frequency range of 4,000–400 cm−1. Cell Weight Determination Cell weight was determined as per gravimetric method briefly; bacterial cells were grown for the period of 24 to 72 h at 28 °C and in various physicochemical conditions. Culture was centrifuged at 7,000×g for 10 min. Cell pellet was washed thrice with 0.85 % physiological saline and air dried on filter paper followed by drying at 105 °C for 3 h in an oven.
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Optimization of Prodigiosin Production Effect of Agitation on Pigment Production Various agitation speeds were used to study the influence of aeration on pigment, and biomass production. The 24 h grown culture of bacteria was inoculated in pre modified nutrient broth (carbon source as 1 % mannitol). Flasks were incubated on different shaking speeds of 100 to 200 rpm at 28 °C. Effect of pH on Pigment Production Active culture of S. marcescens was inoculated in nutrient broth with pH range of 3 to 10. The flasks were incubated at 28 °C for 24 h. The prodigiosin production was estimated after incubation. The pH at which microorganism gave maximum pigment production was kept constant for further studies. Effect of Temperature on Pigment Production Effect of temperature on biomass and prodigiosin production was investigated by keeping the cultivation temperature 25, 30, 35, and 40 °C in shaker incubator at the interval of 24 to 72 h. Effect of Inorganic Salts on Pigment Production S. marcescens was grown with nutrient broth containing different concentrations of inorganic salts. The concentrations used for calcium carbonate and ferrous sulfate ranged between 10 and 100 mg l−1. For sodium and potassium chloride, it was 0.2 to 2.4 g l−1, and for dipotassium phosphate and magnesium sulfate, the concentrations ranged between 20 and 220 mg l−1. After incubation time, the pigment production was determined. Carbon Sources and Nitrogen Sources Throughout the experiment, synthetic as well as natural polymeric carbon sources were used, i.e., glucose, maltose, lactose, sucrose, starch, sweet potato powder, M. latifolia extract, and sesam extract. While sodium nitrate, ammonium sulfate, casein, yeast extract, and peptone were compared as nitrogen source. Active bacterial culture was inoculated in nutrient broth containing (0.1 % w/v) carbon and nitrogen source. After incubation, pigment production was determined. The carbon and nitrogen source responsible for higher pigment production was further tested at different concentrations (0.2 to 2 g l−1) for determination of exact concentration responsible for high yield. Antimicrobial Assay The antimicrobial activity of the pigment was evaluated by antimicrobial susceptibility test. The bacterial and fungal cultures were grown in nutrient and Czapek dox broth at 37 and 28 °C, respectively. From 24 h grown culture of each microorganism, 100 μl at a concentration of 106 cells ml−1 was spread on the surface of nutrient agar for bacteria and on Czapek dox agar for fungi in Petri plates. Wells of 8 mm diameter were made with a metal borer. Extracts of 100 μl each was added in the wells at various concentrations of respective solvents. The plates were kept at 4 °C for 2 h to allow prediffusion then incubated at 37 °C for 24 h for
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bacteria and 28 °C for 48 h for fungi. Zones of inhibition were measured in millimeter from the circumference of the wells to the circumference of the inhibition zone. The assays were repeated three times with solvent serving as controls. Results were compared with standard antibiotic discs (HiMedia, Mumbai, India).
Results Optimization of Prodigiosin Production Effect of Aeration, Temperature, and pH on Pigment Production Increase in prodigiosin and biomass production was observed with increased aeration (maximum at 180 rpm for 48 h). Prodigiosin production was highest (0.23 g% w/v) at 120 rpm, and it decreased considerably after 200 rpm. The biomass and prodigiosin production was increased with increase in temperature up to 30 °C and decreased after 35 °C. The highest production (0.24 g%) was obtained when bacteria were cultivated at 30 °C for 48 h. The pH of the culture medium has been reported to play a key role in pigment synthesis. Serratia was cultivated at different initial pH values (3 to 10) in shake flask cultures. Organism showed growth and pigmentation in wide range of pH with the maximum (0.23 g%) being at the pH 7. Effect of Inorganic Salts on Pigment Production Prodigiosin production was almost constant with 10–100 mg l−1 concentration of calcium carbonate (Fig. 1b). Variation in the concentration of sodium and potassium chloride did not affect pigment and cell mass production and their concentration beyond 2 g l−1 w/v showed inhibitory effect on pigment production. Phosphate (KH2PO4) at concentration of 80 mg l−1 showed maximum prodigiosin production (0.17 g%), and a further increase in phosphate reduced pigment production (Fig. 1a). In case of ferrous sulfate (FeSO4), prodigiosin production was almost constant (0.19 g%) at the 30–50 mg l−1 and considerably decreased beyond 50 mg l−1 (Fig. 1c), while magnesium sulfate (MgSO4) at 10–200 mg l−1 concentration showed increase in pigment (0.310 g%) production, and above 200 mg l−1 concentration, prodigiosin production was decreased (Fig. 1d). Effect of Carbon Sources on Pigment Production Sweet potato extract gave maximum recovery of prodigiosin (0.42 g%), followed by sesame (0.361 g%) and maltose (0.359 g%), as compared to other carbon sources after 48 h (Fig. 2a). The effective concentration of potato powder was 1 g l−1. Pigment yield was comparatively less, below 0.6 g l−1 and above 1.2 g l−1 concentrations of potato powder (Fig. 2b). Yield of pigment was comparatively less with glucose, lactose, mannitol, sucrose, and M. latifolia floral extract as a carbon source. Effect of Nitrogen Sources on Pigment Production Casein at 0.6 g l−1 gave maximum amount pigment (0.48 g%) after 24 h (Fig. 3b), while yeast extract at 0.8 g l−1 showed comparable pigment production (0.5 g%) after 36 h. (Fig. 3c). Other nitrogen sources used were inferior for pigment production (Fig. 3a).
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Fig. 1 Effect of inorganic salts (a inorganic phosphate, b CaCO3, c FeSO4, and d MgSO4) on prodigiosin production
Identification Thin layer chromatography (TLC) was performed for isolated pigment on standard silica gel 60 F254 plates with butanol : hexane (2 : 1) as a solvent system. The Rf value of the sample and standard was found to be 0.86 (Fig. 4a). Spectrophotometric analysis of pigment showed single peak at 536 nm (Fig. 4b). In FTIR (Fig. 5), red pigment from Serratia showed strong and broad absorption at 3,378 cm−1 (O–H stretch), 2,951 cm−1 (C–H and C=O stretch) and 1,556 cm−1 (aromatic C=C, NO2 stretch) represents respective bonds, prodigiosin exhibits
Fig. 2 Effect of carbon source on pigment production a using different carbon sources and b with different concentrations of potato powder
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Fig. 3 Effect of nitrogen source on pigment production using a different nitrogen sources, b different concentrations of casein, and c different concentrations of yeast extract
similar absorptions in CHCl3 at 1,630 and 1,602 cm−1 [8]. N–H group and phenyl rings were evident at fingerprint region, which was characterized by weak intensity at 1,646 cm−1 (aromatic C=0). Absorption at 1,191 and 1,197 cm−1 showed C–N bend (amines) and C–O
Fig. 4 a TLC image of standard prodigiosin (S) and pigment extracted and purified from Serratia marcescens (T). b Spectroscopic analysis of pigment from S. marcescens
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Fig. 5 FTIR analysis of pigment from Serratia marcescens
(carboxylic) stretch, whereas 863 and 706 cm−1 indicate C–H phenyl ring bend, and weak absorption at 1,445 cm−1 also shows bending of C–H. Biological Effect Antibacterial Effect of Prodigiosin Prodigiosin showed inhibitory activity against range of Gram-positive and Gram-negative bacteria with zone of inhibition in the range of 14 to 21 mm in diameter at 100 μg ml−1 concentration (Fig. 6a). Minimum inhibitory concentration (MIC) for bacteria was in the range
Fig. 6 Antibacterial activity of pigment. a Zone of inhibition against different bacteria. b Minimum inhibitory concentration against different bacteria
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of 5 to 23 μg ml−1 (Fig. 6b). Higher concentrations of prodigiosin (100 and 50 μg ml−1) showed considerable growth inhibition of Gram-negative bacteria. Antifungal Effect of Prodigiosin The zone of inhibition for fungi was in the range of 17 to 18 mm in diameter at 100 μg ml−1 concentration (Fig. 7a). MIC of prodigiosin for the tested fungal isolates was in the range of 8 to 21 μg ml−1 (Fig. 7b).
Discussion Optimization of Prodigiosin Production Effect of Agitation, Temperature, and pH on Pigment Production Increase in pigment and biomass production was observed with increased agitation suggesting a key role of shaking speed in pigment production, few references show comparable effect of aeration with carotenoids production by yeast [9–13]. Aerobic conditions influence the metabolism of cells which increase the requirement of amino acids and hence cause more production of pigment [14]. Results for effect of temperature on biomass and pigment production are in agreement with the observation of Chang et al.[15] for S. marcescens strain 389. Qadri and Robert [16] reported no prodigiosin production at 38 °C; however, with same culture, pigment production was observed when temperature was shifted to 27 °C. A complete inhibition in prodigiosin was observed in most of the basically used media tested at 37 °C, which was similar to the result observed by Pryce and Terry [17]. The effect of temperature may be due to sensitivity of monopyrrole and bipyrrole to higher temperatures [18]. Synthesis of amino acids is also affected by the change in temperature [19]. The pH of the culture medium has been reported to play a key role in pigment synthesis. It is essential to set a definite pH as sugar uptake depends on external pH, and it may affect function of different proteins involved in the metabolism of essential amino acids required for synthesis of prodigiosin [20]; therefore, adequate control of pH value is essential in both batch and
Fig. 7 Antifungal activity of pigment. a Zone of inhibition against different fungal strains. b Minimum inhibitory concentration of pigment against different fungal strains
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continuous production of pigment. pH 7 was found suitable for maximum pigment formation. Effect of Inorganic Salts on Pigment Production Inorganic salt, such as calcium carbonate, magnesium sulfate, and potassium phosphate, has a role in physiology and growth of organism. However, no substantial increase or inhibition in prodigiosin production was found with different concentrations of sodium and potassium chloride, and with increased concentration of these salts, pigment production was inhibited. This observation is in accordance with San et al. [21]. Calcium carbonate has decreased pigment formation (Fig. 1). It is been reported that calcium carbonate may be added to maintain the pH; at specific concentration, it stimulates sugar uptake by decreasing Km of sugar transport, whereas high sugar concentration inhibits this reaction [22]. Removal of sodium chloride and inorganic phosphate has found to enhance prodigiosin production [23]. In contrast to this, Ryazantseva et al. [24] reported stimulative action of sodium chloride on prodigiosin production. Although it has been said that inorganic phosphate decreases pigment production by S. marcescens [25], we have observed that up to 100 mg l−1, it stimulates pigment production but further concentration of phosphate inhibitory to pigment formation (Fig. 1). The inhibitory effect of inorganic phosphate might be due to inhibition of enzymatic activity like that for alkaline phosphatase [26], provided that the activity of enzyme is found to increase just before synthesis of secondary metabolites [27, 28]. Synthesis of two immediate precursors is found to be inhibited by inorganic phosphate [26]. Similarly, ferrous sulfate increases pigment production (Fig. 1). Effect of Carbon Sources on Pigment Production The type of carbon source used may play crucial role in pigment formation. Serratia was cultivated in the basal medium substituting different carbon sources. From a total of nine carbon sources, sweet potato extract (0.42 g%) gave maximum yield. Pigment production with maltose (0.359 g%) is in accordance with Sundaramoorthy et al. [29] and Shahitha and Poornima [30], who found maltose as a better carbon source which gave 3 g l−1 of prodigiosin. Pigment production was highest at 1 g l−1 potato powder. A higher production of prodigiosin with starch is comparable with the results obtained by Wei et al. [31], which gave 2.3 g l−1, and after optimizing the carbon : nitrogen ratio, it was further enhanced to 6.7 g l−1. Due to polymeric nature, starch might be utilized at slower rate, and hence the catabolic repression is avoided. On the other hand, pigment yields were less than 60 % with glucose, lactose, mannitol, and sucrose. Pigment production by these sources was comparable with the production by M. latifolia floral extract (Fig. 2a). The M. latifolia L. flower extract mainly contains sucrose as a principal component of the sugars, and interestingly, sucrose as a carbon source was least preferred by Serratia showing low pigment synthesis. Comparatively lower production of prodigiosin in presence of glucose and lactose was observed (Fig. 2a), which is similar to the previous report of Harned [32] who obtained a yield of 5 mg l−1 using a medium with glucose as a carbon source. Report says that pentose sugars inhibit prodigiosin production [25] and oligosaccharide is better than glucose for pigment production by Serratia [33]. By many reasons, glucose is an inferior source for prodigiosin production. It is responsible for the production of glucose-6-phosphate dehydrogenase which inhibits pigment production and causes the lowering of pH [34]. The addition of glucose and maltose also causes catabolic
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repression [35]. Serratia sp. is not lactose-fermenting, therefore pigment production with lactose was low [36]. Effect of Nitrogen Sources on Pigment Production Casein and yeast extract were found to be the most effective nitrogen sources as compared to beef extract, sodium nitrate, ammonium sulfate, and urea for production of pigment and biomass (Fig. 3). The highest amount (0.48 g% w/v) of pigment was produced in the medium containing 1 g l−1 potato extract and 0.6 g l−1 casein with 120 rpm after 24 h while with the use of casein, fermentation time was also reduced by few hours. Yeast extract showed 0.5 g% prodigiosin after 36 h. These results are similar with Song et al. [8] who used casein as nitrogen source for prodigiosin production and obtained 0.43 g%. Results for casein are in accordance with Robert [37] who observed induction of biosynthesis of prodigiosin after the addition of casein hydrolysate to suspension of washed, nonpigmented, nonproliferating cells. Casein hydrolysate contains aspartic acid, glutamic acid, proline, and alanine, which cause pigment production when added individually. Structurally methyl group on C6 position of prodigiosin comes from methionine [38]. Methionine and cysteine increase prodigiosin production [20]. At high concentration of casein and yeast extract, both pigment and cell mass decreased possibly because Serratia spp. is an osmotically sensitive strain showing impaired growth and partial lysis with leakage of culture fluid. Peptone and yeast extract are not commonly added to media of Gram-negative bacteria as they usually cause pleomorphism and make cell wall fragile [39, 40]. Glycine is a principal constituent of peptone, casein, and yeast extract, which causes interference with peptiodoglycan synthesis, results in the accumulation of UDP-glycopeptide precursor in cytoplasm, and makes osmotically fragile cells. Similar effects were demonstrated in case of peptone supplemented in fermentation medium of Azotobacter vinelandii for PHB synthesis [41]. Ammonium sulfate, nitrite, urea, meat extract, malt extract, and sodium nitrite found to give low pigment production. Low prodigiosin production with ammonium sulfate was also observed by Rockem and Weitzman [42]. Ammonium is poor nitrogen donar in Schiff base conversion of orange to red pigment [43]. Identification The Rf (0.86) value of separated spots was exactly matched with reference material (standard). The FTIR transmission pattern of the pigment is similar for the functional groups present in prodigiosin. The FTIR analysis, TLC, and spectroscopic analysis showed prodigiosin as a principal molecule in the extract (Fig. 4 and 5). The results are in accordance with Song et al. [8]. In our previous report, the result for high-performance TLC (HPTLC) of prodigiosin is available showing a single peak at 535 nm [44]. Biological Effect Antibacterial Effect of Prodigiosin Antibacterial effect against P. aeruginosa, E. coli, and S. aureus is in comparison with previous reports [3, 45]. The least MIC was for Gram-positive bacteria (Fig. 6b), but at higher concentrations of prodigiosin (100 and 50 μg ml−1), considerable growth inhibition of Gram-negative bacteria was observed. Prodigiosin affect intracellular pH gradient of the organism [46].
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Fig. 8 a Antibacterial and b antifungal effects of standard antibiotics against target microorganisms
Antifungal Effect of Prodigiosin The tripyrrole pigment prodigiosin not only inhibits bacteria but also fungi (Fig. 7). In previous reports prodigiosin have shown inhibition against Candida parapsilosis, Cryptococcus sp. [3], Candida albicans [1], and Didymella applanata [47]. Action of S. marcescens in vitro may be associated with specific properties of the cell wall of fungus [47]. Prodigiosin can affect mitochondrial dysfunction by the uncoupling of electronic chain transport of protons to mitochondrial ATP synthase and causes reduction in adenosine triphosphate (ATP) production without decreasing oxygen consumption. It also acts as proton pump inhibitor [2, 48]. Comparison with Standard Antibiotics Comparative analysis of inhibition studies showed that prodigiosin has more potential for inhibition of S. aureus and S. subtilis compared to antibiotics, such as cefatoxime and vancomycin; similarly, inhibitory potential of prodigiosin against P. aeruginosa and S. typhi was greater than vancomycin and cefatoxime, respectively. Inhibition of S. aureus by prodigiosin is comparable with inhibition showed by penicillin and ampicillin, whereas inhibition of S. subtilis, P. aeruginosa, E. coli, and S. typhi was less when compared to these antibiotics (Figs. 6 and 8). In case of antifungal effect, nystatin showed greater inhibition than prodigiosin (Figs. 7 and 8).
Conclusion After optimization of culture conditions for S. marcescens, a profound increase was observed in prodigiosin production. The highest pigment formation was observed in the presence of potato starch powder (1 g l−1) as a carbon source, while casein (0.6 g l−1) and yeast extract 1 g l−1) as a nitrogen source. The effect of inorganic salts has also been studied, which indicates that the presence of phosphate (80 mg l−1), FeSO4 (50 mg l−1), and MgSO4 (200 mg l−1) enhances pigment production, whereas CaCO3 inhibits pigment formation. Similarly, biological activity of the prodigiosin and its comparison with standard antibiotics provides useful information. Acknowledgments The author RKS is thankful to UGC BSR RSSMS scheme (Ref.: F.7-137/2007), New Delhi, India for the financial assistance. We are also thankful to North Maharashtra culture collection (NMCC) for providing the microbial cultures.
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