Prodigiosin from marine bacterium: production, characterization and application as dye in textile industry.
Prodigiosin is a tripyrrole first characterized from Serratia marcescens, which forms beautiful pillar box red colonies and S. marcescens are the major producers of prodigiosin . Prodigiosin has several biological activities such as immunomodulatory, antibacterial, antimycotic and antimalarial activities and so on [2,3]. Serratia rubidaea N-1, isolated from the Ariake Sea, a bay located in the Kyusyu region, Japan, produce red pigment prodigiosin .
The production of prodigiosin in S. marcescens is susceptible to temperature and is substantially inhibited at temperatures higher than 37[degrees]C . Conventional media used for the biosynthesis of prodigiosin by S. marcescens strains are complex media that are rich in a variety of nutrients [1, 5, 6]. Certain nutrients, such as thiamine  and ferric acid , are particularly crucial for prodigiosin production, whereas phosphate , adenosine triphosphate, and ribose  have inhibitory effects on prodigiosin yield. It was observed that novel peanut seed broth gave rise to a significant enhancement of prodigiosin production . Moreover, it was reported that the addition of silica-gel carriers to a liquid culture of S. marcescens led to marked increase in cell growth and the production of prodigiosin . In addition, since prodigiosin is often located on the cell envelope, the addition of surfactants, such as sodium dodecyl sulphate (SDS), could also enhance the recovery efficiency for prodigiosins . Identification of optimized medium composition to achieve more efficient production of a prodigiosin-like pigment (PLP) from S. marcescens SM[DELTA]R, which is a SpnR-defective isogenic mutant of S. marcescens SS-1 was reported . LB broth was shown to be an effective growth medium for S. marcescens SM[DELTA]R, leading to the production of a biosurfactant and also a prodigiosin-like-pigment [13, 14]. The components of LB broth (tryptone, NaCl, and yeast extract) were examined individually for their effects on prodigiosin production. The effect of vegetable oil supplementation on prodigiosin production was also reported .
In the light of its potential commercial values, there is a demand to develop high-throughput and cost effective bioprocesses for prodigiosin production. Recently medium optimization by statistical methods has proved to be a powerful and useful tool for designing and development of cost effective bioprocesses for the large scale production of any desired product of commercial value. Plackett-Burman design offers good and fast screening procedure and mathematically computes the significance of large number of factors in one experiment, which is time saving and maintain convincing information on each component. Only the most effective factors with positive significance would be selected for further optimization studies, while those showing high negative effect on the bioprocess may be dropped in all further experiments . This indicates the effectiveness of Plackett-Burman design as a tool for elucidating the most important variables affecting the response. Applying Box-Behnken design is an efficient method to optimize the selected factors for maximal production that tests the effect of factors interaction. Besides, it converts the bioprocess factor correlations into a mathematical model that predicts where the optimum is likely to be located. It is worthwhile to advise the microbial industry sponsors to apply these experimental designs to maintain high efficiency and profit bioprocesses . To the best of our knowledge there is no report available on the statistical optimization of pigment production of prodigiosin pigment by any bacteria.
It has been reported that prodigiosin could induce apoptosis in various kinds of cancer cells, such as haematopoietic, colorectal and gastric cancer cells [17, 18]. Whereas, its application in textile industry is not well studied. The textile industry is one among the rapidly growing industries worldwide. In India it accounts for 14 % of the total industrial production, and contributes to nearly 30 % of the total exports. Textile industry utilizes enormous amounts of synthetic dyes and consequently the textile effluent, which is often very difficult to treat and dispose, poses serious threat to the environment and has become a very grave problem in environment conservation. Further they also pollute the ground water resources of drinking water and agriculture practices. Several attempts are made to evolve ideal processes for safe and effective disposal of dye effluent from industries. Environment protection becoming so imperative, development of environment friendly technologies has become the need of the hour. Hence natural pigments have drawn the attention of industry as safe alternative. Among the natural sources of colourants, microorganisms offer great scope and hope.
In this communication we report the isolation and identification of a prodigiosin like pigment produced by a marine Serratia sp. BTWJ8 and optimization of the process variables including the composition of the medium for the maximum production of prodigiosin, that have potential for application as a dye in textile industry, employing statistical models Plackett-Burman design and Response Surface Methodology.
Materials and Methods
Source of Microorganism
Serratia sp. BTWJ8, isolated from seawater, near Cochin, west coast of India, and identified as a potential strain that produced bright red prodigiosin pigment was used. This strain was maintained on Zobells marine agar medium (HiMedia, India). The organism was subcultured at regular interval of 1 month and stored at 4[degrees]C.
Selection of suitable solvent for extracting the bacterial pigment
Initially the solvent that could support maximal yield of pigment on extraction of culture broth was standardized, using different solvents viz; ethanol, acetone, methanol, petroleum ether, ethyl acetate, chloroform, hexane, diethyl ether and distilled water.
Selection of media for pigment production
A preculture was prepared by transferring a loop full of the agar slope culture onto 5 ml of autoclaved Mineral Salts Tryptone Sucrose (MSTS) medium taken in a boiling tube and incubated for 18 h at room temperature (28 [+ or -] 2[degrees]C), at 150 rpm in an orbital shaker. Using this preculture as inoculum, 50 ml of MSTS medium taken in an Erlenmeyer flask was inoculated [1 % (v/v) level] and incubated at room temperature (28 [+ or -] 2[degrees]C) in an orbital shaker for 18 h. Later, one ml of this bacterial culture (Optical density 1.00) was used as inoculum for all the experiments unless otherwise mentioned.
After conventional "one-variable-at-a-time" approach the optimized MSTS medium conditions used in the study for maximal pigment included the following: Incubation time-30 h; incubation temperature: 25[degrees]C; initial pH of the media-6.0; agitation-150 rpm; dextrose-30 mM; yeast extract-1% (w/v); inoculum-1% (v/v); Ca[Cl.sub.2]-100 mM; trace salts (Fe[Cl.sub.3], Mn[Cl.sub.2], [Na.sub.2]Mo[O.sub.4] and ZnS[O.sub.4])-0.03 mM; NaCl-200 mM. All the shake flasks experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml of growth media in an incubator shaker under controlled conditions.
Standardization of procedure for extraction of pigment
Extraction of the pigment was done according to Slater et al.  with slight modification. One milliliter of fermentation broth was centrifuged (10, 000 rpm, 10 min, 4[degrees]C) and one milliliter of methanol was added to the colored pellet and incubated at 60[degrees]C for 20 min. After centrifugation (10, 000 rpm, 10 min, 4[degrees]C) the colored supernatant was analyzed by scanning in a UV-visible spectrophotometer (Shimadzu, Japan) for detecting the absorption maximum (^max). The scanning range selected was 400-600 nm. Absorbance at the ^max was measured.
Biomass estimation 
Ten ml aliquot of the sample was withdrawn from the fermentation medium and centrifuged at 10,000 rpm for 10 min. Sedimented cell pellet was washed twice with sterile distilled water and centrifuged again. The cell pellet was then resuspended in 1 ml sterile distilled water and kept for drying in a hot air oven at 100[degrees]C until a constant weight (24 h) of biomass was obtained. Biomass was expressed as g/L.
The colored supernatant was then analyzed by scanning in a UV-Visible spectrophotometer (Shimadzu, Japan). The total pigment was estimated according to the following formula [21, 22].
TP ([micro]g/L) = AD[V.sub.1]/7.07 x [10.sup.4] [V.sub.2]
Where 'TP' denotes the total pigment yield ([mocro]g/L), 'A' the absorbance of the methanol extract at 535 nm, 'D' is the dilution ratio, '[V.sub.1]' the volume of methanol added, 7.07 x [10.sup.4] is extinction coefficient of prodigiosin and '[V.sub.2]' is the volume of the fermentative liquid.
Purification of the pigment
Pigment produced by the bacterium was purified according to Song et al.  with some modification. Equal volume of petroleum ether was added to the methanol extract in a separatory funnel and mixed well for the phase separation. The hypophase with methanol and water soluble impurities was removed; the petroleum ether phase was washed 4 or 5 times with distilled water to remove residual methanol. The pigment collected from the hypophase was treated with 1 N HCl (9:1; v/v) and concentrated by evaporating the solvent at 40[degrees]C. Silica gel column chromatography was used for removing impurities from the concentrated pigment after phase separation. The sample was eluted with a mixture of n-hexane: ethyl acetate (2:1; v/v) at a flow rate of 1 mL/min. The red-colored fraction was collected from the column and analyzed by scanning in UV-visible spectrophotometer (Shimadzu, Japan) in the scan range of 400-600 nm.
Structure identification of the pigment
The chemical structure of the purified pigment was determined by thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform-infra red spectroscopy (FT-IR), and Fourier transform-Raman spectroscopy (FT-Raman) and also by the effect of pH on the absorption spectrum.
Thin layer chromatography (TLC)
The purified pigment was analysed by thin layer chromatography with silica gel G-60 [F.sub.254] (Merck); using chloroform: methanol (95:5; v/v) as mobile phase and the retention factor was calculated according to the following equation:
[R.sub.f] = Distance travelled by the compound/Distance travelled by the solvent front
Liquid chromatography-mass spectroscopy (LC-MS)
Five [micro]l aliquot of purified sample in methanol was injected into LC-MS equipped with turbo-ion spray source. Parameter settings used in the analysis were as follows: Solvent system methanol: acetonitrile (50:50), flow rate 0.2 ml/min., ion spray voltage: 5500 V, curtain gas: 25.0 lb/[in.sup.2] , collision gas: 6.0 lb/[in.sup.2] , ion source gas 1: 20.0 lb/[in.sup.2], ion source gas 2: 30.0 lb/[in.sup.2], polarity: positive.
Fourier transform-infrared (FT-IR) spectroscopy
The purified pigment sample was subjected to FT-IR spectroscopic analysis (Thermo Nicolet, Avatar 370), equipped with KBr beam splitter with DTGS (Deuterated triglycine sulfate) detector (7800-350 [cm.sup.-1]). The parameters used in the FT-IR analysis were: spectral range: 4000-500 [cm.sup.-1], Resolution: 0.9 [cm.sup.-1] .
Fourier transform-Raman (FT-Raman) spectroscopy
The pigment sample was analysed by FT-Raman spectroscopy (Bruker RFS100/S) equipped with standard InGaAs (Indium gallium arsenide) detector. The parameters used in the analysis were: Raman laser power, 150 mV; resolution, 4 [cm.sup.-1] ; and temperature of measurement, 22[degrees]C.
Proton nuclear magnetic resonance ([sup.1]H NMR) spectroscopy
[sup.1]H NMR spectra was generated in deuterated chloroform (CD[Cl.sub.3]) at 500 MHz using a Bruker Advance II instrument. Tetramethyl silane (TMS) was used as an internal standard.
Effect of pH on absorption spectrum
The pH of the purified pigment dissolved in methanol was pH 7.0. The pH was adjusted to 2.0 using 0.01 N HCl, and pH 10.0 using 0.01 N NaOH. Absorption spectrum of the purified pigment at different pH values was determined using a UV-visible spectrophotometer (Shimadzu, Japan). The scanning range selected was 300-800 nm.
Production of pigment by Serratia sp. BTWJ8 under submerged fermentation
Bioprocess variables that influence the production of pigment by Serratia sp. BTWJ8 under submerged fermentation was optimized towards determining the ideal bioprocess. Initially the medium suitable for pigment production was standardized and the medium that supported maximal pigment was used in the subsequent studies.
Selection of media for pigment production
Zobell Marine broth (ZMB), Nutrient broth (NB), Glycerol asparagine broth (GAB), Seawater yeast extract peptone broth (SWYPB) and Mineral salts tryptone sucrose medium (MSTS) with peptone as nitrogen source were evaluated for the impact of media components on pigment production. The composition of media is given in Table 1. The effect of seawater on pigment production by the bacterium was also studied using the MSTS medium.
Inoculum preparation and incubation
A loopfull of the agar slope culture was transferred onto 5 ml of autoclaved MSTS medium taken in a boiling tube and incubated for 18 h at room temperature (28 [+ or -] 2[degrees]C), at 150 rpm in an orbital shaker. Using 1 % (v/v) level of this preculture as inoculum, 50 ml of MSTS medium taken in an Erlenmeyer flask was inoculated and incubated at room temperature (28 [+ or -] 2[degrees]C) in an orbital shaker for 18 h. Later, one ml of this bacterial culture (Optical density 1.00) was used as inoculum for all the experiments unless otherwise mentioned.
Optimization of bioprocess variables for pigment production by statistical method Plackett-Burman Design (PB Design)
Optimization of process variables using statistical approach for maximal pigment was carried out using Plackett-Burman design  with selected eleven factors after studying the effect of different parameters by "one-variable-at-a-time" method. The eleven factors included the following: incubation period, inoculum, yeast extract, dextrose, NaCl, Ca[Cl.sub.2], [K.sub.2]HP[O.sub.4], K[H.sub.2]P[O.sub.4], trace salts, pH and incubation temperature. The parameters were varied over two levels and the minimum and maximum ranges selected for the parameters are given in Table 2.
The statistical software package Design-Expert[R] 6.0 (Stat Ease Inc., Minneapolis, U.S.A) was used to generate a set of 12 experimental designs. Production was set up by inoculating the media with respective inoculum percentages as suggested by the model and incubated for specified incubation period (12-36 h), at specified temperature (20-30[degrees]C), at 150 rpm. For each experiment, the pigment production was calculated in terms of [micro]g/L. The experiments were done in triplicate. Regression analysis of the experimental data was conducted using statistical software.
Based on the results obtained from the Plackett-Burman design, the fitted first- order model is:
Y = [[beta].sub.0] + [k.summation over (i=1)] [[beta].sub.i][X.sub.i]
Where 'Y' is the predicted response, '[[beta].sub.0]' is the model intercept, '[[beta].sub.i]' is the linear coefficient and '[x.sub.i]' is the level of the independent variable. This model does not describe interaction among factors and it is used to screen and evaluate the important factors that influence the response.
Effect of each variable on the production was determined by calculating their respective E-values .
E = (Total of responses at high level)-(Total of responses at low level)/Number of trials
Response Surface Methodology (RSM)
The significant factors affecting pigment production by Serratia sp. BTWJ8 were optimized using a response surface type Box-Behnken  model experimental design. The treatments considered in the design were concentration of inoculum, sodium chloride, calcium chloride, and incubation temperature.
Based on the results of the "one-variable-at-a-time" experiments and PB Design, the effect of five factors viz; concentration of inoculum (A), concentration of sodium chloride (B), concentration of calcium chloride (C) and incubation temperature (D) was studied on pigment production using Response Surface Methodology. Other components of the medium which were kept constant included yeast extract: 1 % (w/v), dextrose: 10 mM, [K.sub.2]HP[O.sub.4]: 0.45 mM, K[H.sub.2]P[O.sub.4]: 0.5 mM, pH: 6.0, incubation time: 22 h.
Box-Behnken design model is a second-order design that allows estimation of quadratic effects, and is based on combining a two-level factorial design with an incomplete block design. This design was used for creating the quadratic response model.
Each factor in the design was studied at three different levels. All the variables were taken at a central coded value, considering as zero. A design model with 30 runs in 3 blocks of 10 cases was used as exhibited in Table 3, and each independent variable was tested at three levels. The levels were coded in standardized units with the values -1, 0 and +1 representing the lower, middle and higher levels respectively.
Design-Expert[R] 6.0 (Stat-Ease, Inc., Minneapolis, U.S.A) was used to analyze the experimental design. The average of maximum pigment production values recorded was taken as the dependent variable or response (Y). Regression analysis was performed on the data obtained. The results of the Box-Behnken design were then used to fit a quadratic equation by multiple regression procedure. This resulted in an empirical model that related the response measured to the independent variables of the experiment.
The following quadratic model was chosen to represent the relationship fitted between the above four variables.
Y= [[beta].sub.0] + [4.summation over (i=1)] [[beta].sub.i][X.sub.i] + [4.summation over (i=1)] [[beta].sub.ii][X.sub.i.sup.2] + [4.summation over (i=1)] [4.summation over (j=1)] [[beta].sub.ij][X.sub.i][X.sub.j]
Or in the expanded form,
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
In this model, Y represents the dependent variable-pigment yield; [X.sub.1], [X.sub.2], [X.sub.3] and [X.sub.4] are the independent variables denoting inoculum concentration, concentration of sodium chloride, concentration of calcium chloride and incubation temperature respectively. [[beta].sub.1], [[beta].sub.2], [[beta].sub.3] and [[beta].sub.4] are linear coefficients, [[beta].sub.12], [[beta].sub.13], [[beta].sub.14], [[beta].sub.23], [[beta].sub.24] and [[beta].sub.34] are second order interaction coefficients or cross product coefficients and [[beta].sub.11], [[beta].sub.22], [[beta].sub.33] and [[beta].sub.44] are the quadratic coefficients. The design of experiments in terms of actual factors is given in Table 4.
The levels tested were inoculum concentration 0.5, 1.25 and 2.0 %; concentration of sodium chloride 100, 150 and 200 mM; concentration of calcium chloride 50, 75 and 100 mM, and incubation temperature 20, 25 and 30[degrees]C. Analysis of Variance (ANOVA) was performed and 3-dimensional response surface curves were plotted by Design Expert[R] software to study the interaction among various physico- chemical factors.
Validation of the model
In order to validate the response surface model, a random set of experiments was set up according to the conditions predicted by the model. The responses obtained from the trials conducted as mentioned above, following the Box-Behnken design model for four variables, was used to estimate the coefficients of the polynomial model using standard regression techniques. The estimate of "Y" was used to generate an optimal combination of factors that can support maximal pigment production using predictive models from response surface methodology. The software Design-Expert[R] 6.0 (Stat Ease, Minneapolis, U.S.A) was used to fit the response surface-Box-Behnken model to the experimental data. All the experiments were carried out independently in triplicates. Time course experiment was conducted with the optimized conditions determined after statistical optimization of various variables.
Time course study under optimized condition
Time course experiment was conducted with the optimized conditions determined after statistical optimization of various variables. The conditions selected are given in Table 5.
Dyeing of Textile materials
In the present study, scope for probable application of the bacterial pigment was evaluated for different grades of textile materials commercially available in the market which included 'Cotton', 'Chiffon', 'Poplene', '2 by 2', 'Pure silk', 'Century cotton', 'Dupoil silk', '2 by 1', 'Organdi', 'Polyester', 'Terrycotton' and 'Nylon.' Each material was cut into equal size of 2 [cm.sup.2] . Bacterial pigment in methanol (40 [micro]g/L) was used as the stock solution. From this stock solution 100 [micro]l (0.004 [micro]g; w/v), 200 [micro]l (0.008 [micro]g; w/v) and 300 [micro]l (0.012 [micro]g; w/v) was applied to the cloth material in a warm surface and was allowed to dry at room temperature for about 1 h. One set of experiment was done with the application of thiourea as a mordant . The dyed cloth materials were dipped in thiourea solution (1%; w/v) for 30 min. at 70[degrees]C. For all the experiments white cloth material were taken as a control.
Wash performance of the textile materials
All the dyed textile materials were washed with soap solution (sunlight 0.7%; w/v) for 30 min. at room temperature as well as at 40[degrees]C. After 30 min. (random selection) the cloths were washed with tap water and allowed to dry at room temperature (28 [+ or -] 2[degrees]C). Absorbance of the soap solution after washing was measured at 535 nm in a UV-Visible spectrophotometer. Appropriate blank was also used for the experiment. The same procedure was repeated for the dyed textile material treated with thiourea.
The selected bacterial isolate BTWJ8 was identified as Serratia sp. BTWJ8 according to the morphological and biochemical characteristics (Table 6).
Standardization of protocol for isolation of pigment
The pigment was assayed at 535 nm which showed maximum absorption [[lambda].sub.max] . It was inferred from the results obtained that methanol is an ideal solvent (Figure 1) for extracting maximum of the water insoluble membrane bound pigment.
[FIGURE 1 OMITTED]
Purification of the pigment
The absorption pattern of the purified pigment dissolved in methanol is shown in Figure 2. The purified pigment was then used for the characterization studies.
[FIGURE 2 OMITTED]
Structural identification of the pigment
The purified pigment was analysed for its structural identification. A single band with an [R.sub.f] value of 0.42 was obtained after thin-layer chromatography with chloroform: methanol (95:5; v/v) (Figure 3). Prodigiosin ([C.sub.20][H.sub.25][N.sub.3]O) was characterized with a molecular mass of m/z 324, [[M + H].sup.+], which indicates an odd number of nitrogen atoms in the molecule . FT-IR absorption in KBr for the red pigment was dominated by strong bands at 2924.78 [cm.sup.-1] and 2853.67 [cm.sup.-1] (aromatic CH), 1736.21 [cm.sup.-1] and 1710.44 [cm.sup.-1] (C=O), 1611.15 [cm.sup.-1] (aromatic C=C), 1548.24 [cm.sup.-1] (N-H), 1459.18 [cm.sup.-1] (C-H), 1264.91 [cm.sup.-1] (C-N). The spectrum obtained after the FT-Raman analysis showed strong bands at 2924.79 [cm.sup.-1] and 2834.96 [cm.sup.-1] very similar to that of FT-IR spectrum which indicates the presence of aromatic CH bonds in the pigment molecule. In the spectrum, a chemical shift of the methoxy group in the molecule exhibited 8 4.012 ppm as a single peak. In addition, the chemical shift (in CD[Cl.sub.3]) of NH protons in pyrrole ring was [delta] 12.72 ppm. A tripyrrylmethene structure was assigned to prodigiosin by Wrede and Rothhaas .
[FIGURE 3 OMITTED]
Prodigiosin in solution can exist in one of two distinct but readily interconvertible forms or as a mixture of these two forms, depending on the hydrogen ion concentration of the medium [29, 30]. At pH 2.0, the pigment was red and showed a maximum absorption at 535 nm, which is identical to that of prodigiosin hydrochloride . Under neutral condition (pH 7.0), its absorption intensity was decreased and it changed to pink. However, in alkaline condition (pH 10.0) the colour was yellowish orange and its absorption spectrum shifted to 470 nm (Figure 4). All the results were very similar to that of prodigiosin pigment. It is therefore concluded that the identity of the pigment isolated from Serratia sp. BTWJ8 is prodigiosin.
[FIGURE 4 OMITTED]
Production of pigment under submerged fermentation
Among the five different media evaluated for pigment production, MSTS medium supported maximal pigment production (OD 0.453) when compared to Zobell Marine broth (OD 0.228), Nutrient broth (OD 0.008), Seawater yeast extract peptone broth (OD 0.348) and Glycerol asparagine broth (OD 0.213) (Figure 5).
[FIGURE 5 OMITTED]
Further studies were conducted to evaluate the impact of seawater concentration and sodium chloride concentration in the medium on pigment production during fermentation. From the results presented in Figure 6 it is inferred that the pigment production was more in media prepared in 50-100 % seawater. During the course of this experiment it was also observed that in spite of considerable level of pigment production in medium prepared with various concentration of seawater there was loss of pigments during recovery from the fermentation broth. It was observed that the pigments got bound to secreted proteins and thus escaped during the solvent extraction. When secreted proteins from the culture supernatant was precipitated with ammonium sulphate the culture supernatant remained clear indicating that there was more secondary proteins in the culture supernatant when bacteria was grown in the presence of seawater (Figure 1). Hence, MSTS medium prepared in distilled water and with 1 % sodium chloride (OD 0.453) was selected for the further optimization studies, since there was appreciable level of pigment production compared to the combination 25 % seawater + 15 % distilled water (OD 0.210) and there was minimal loss of pigment during harvesting of the pigment.
Optimization of bioprocess variables employing statistical approach for pigment production by Serratia sp. BTWJ8
Statistical approach was used for optimizing the medium that could support maximum pigment production. Initially process variables were optimized using Plackett- Burman design and in the second stage Response Surface Methodology was adopted towards selection of optimal variables and understanding the interrelationship among significant variables.
[FIGURE 6 OMITTED]
[FIGURE 7 OMITTED]
Plackett-Burman Design (PB Design)
Data obtained for the studies conducted on optimization of medium for pigment production by Serratia sp. BTWJ8 using Plackett-Burman design was analysed by Design expert software and a first-order model was fitted to the data obtained from the experiment.
The experimental results of pigment production by Plackett-Burman design are shown in Table 7. With the help of the software the results were analysed and it is inferred that out of the eleven variables screened during PB design, four factors viz; inoculum ([X.sub.1]), NaCl ([X.sub.2]), Ca[Cl.sub.2] ([X.sub.3]), and temperature ([X.sub.4]) were found to be the most significant variables.
First-order model equation
Pigment ([micro]g/L) = 9.43 + 6.52 [X.sub.1] + 4.97 [X.sub.2] - 5.84 [X.sub.3] + 5.15 [X.sub.4]
The statistical significance of the model equation was evaluated by the F-test analysis of variance (ANOVA), which revealed that this regression is statistically significant. The Model F-value of 9.15 implied that the model is significant. Values of "Prob > F" less than 0.05 (actual value 0.0065) indicated that the model terms are significant. "Adequate precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Adequate precision ratio of 8.379 indicated an adequate signal. Thus, this model could be used to navigate the design space.
The effect of individual parameters studied in PB design is presented as Pareto chart in Figure 8. The data evidence that inoculum, sodium chloride and temperature had a positive effect in enhancing pigment production along with their increase in concentration. Whereas, calcium chloride had a negative effect on pigment production along with increase in concentration.
[FIGURE 8 OMITTED]
Response surface methodology
Response surface methodology (using Box-Behnken design experiment) was adopted towards selection of optimal level of the significant variable viz; inoculum ([X.sub.1]), NaCl ([X.sub.2]), Ca[Cl.sub.2] ([X.sub.3]), and temperature ([X.sub.4]) which were identified based on the PB design experiment. The design matrix (Box-Behnken) and the corresponding experimental data obtained are shown in Table 8.
The results obtained for the Box-Behnken design experiment were analyzed by ANOVA, which yielded the following regression equation for the level of pigment production (Y):
Pigment, Y ([micro]g/L) = 33.14 + 7.38 [X.sub.1] + 7.26 [X.sub.2] + 2.52 [X.sub.3] - 2.91 [X.sub.4] -3.61 [X.sub.1.sup.2] 4.36 [X.sub.2.sup.2] + 0.22 [X.sub.3.sup.2] - 9.39 [X.sub.4.sup.2] - 1.36 [X.sub.1] [X.sub.2] + 0.058 [X.sub.1] [X.sub.3] + 0.85 [X.sub.1][X.sub.4] - 9.06 [X.sub.2][X.sub.3] + 6.99 [X.sub.2][X.sub.4] - 15.00 [X.sub.3][X.sub.4]
The ANOVA analysis of pigment production showed that Prob > F value was less than 0.05 (actual value 0.008), which indicate that the model is significant. Two linear coefficient, inoculum ([X.sub.1]), NaCl ([X.sub.2]); one quadratic term, temperature ([X.sub.4.sup.2]) interaction coefficient and two interaction coefficient, NaCl and Ca[Cl.sub.2] ([X.sub.2][X.sub.3]); Ca[Cl.sub.2] and temperature ([X.sub.3][X.sub.4]) were found to be significant model terms.
The model coefficients estimated by multiple linear regressions and ANOVA showed that the model was significant with coefficient of determination ([R.sup.2]) of 0.8107. This ensured a satisfactory adjustment of the quadratic model to the experimental data and indicated that approximately 81.07 % of the variability in the dependent variable (response) could be explained by the model. The adjusted [R.sup.2], which is more suited for comparing models with different numbers of independent variables, was 0.6069. All the selected parameters were significant and varied levels of interactions were recorded for the variables in their cumulative effect on pigment production. The coefficient of variance was 30.70. A ratio greater than 4 is desirable as it indicates an adequate signal. Thus, this model could be used to navigate the design space.
Analysis of factors influencing pigment production
Three-dimensional response surface curves were plotted to study the interaction among various physicochemical factors, and to determine the optimum concentration of each individual factor for maximum pigment production. The model predicted maximum pigment production up to 40 [micro]g/L that could be achieved using 1.64 % (v/v) inoculum, 75 mM Ca[Cl.sub.2], 196 mM NaCl, incubation temperature 28[degrees]C and incubation period for 22 h.
Interactions between factors
The pair wise interaction among the factors in terms of pigment production in the optimized set was assessed by examining the response surfaces. Three dimensional response surfaces were generated holding two factors constant at a time and plotting the response obtained for varying levels of the other two. There was a parabolic change in pigment production pattern with respect to inoculum and sodium chloride concentration and maximum pigment yields were recorded over 1.75-2.00 %. Sodium chloride concentration showed maximum response over the range of 175-200 mM (Figure 9). The concentrations of calcium chloride and the incubation temperature were held at their optimum levels 75 mM and 28[degrees]C respectively.
[FIGURE 9 OMITTED]
From the results presented in Figure 10, it is inferred that there was no prominent interaction between concentrations of inoculum and calcium chloride in influencing pigment production. The concentrations of sodium chloride and the incubation temperature were held at their optimum levels 196 mM and 28[degrees]C respectively. The maximum pigment yield was obtained at a temperature range between 23- 28[degrees]C and inoculum concentration in the range between 1.5-2.0 %. There was a positive interaction between inoculum concentration and incubation temperature as it is evidenced by the data presented in the Figure 11. The pigment yield was reduced at temperature below 23[degrees]C and above 29[degrees]C. The concentrations of sodium chloride and calcium chloride were at their optimum levels, 196 mM and 75 mM respectively.
[FIGURE 10 OMITTED]
[FIGURE 11 OMITTED]
Results presented in Figure 12 indicate a very clear interaction between concentration of calcium chloride and sodium chloride in their cumulative effect on pigment production. Maximum pigment yield was obtained in the range of 175-200 mM sodium chloride concentration and 50-62 mM calcium chloride concentration. It is also evident from the data that concentrations lower than 175 mM led to reduction in the pigment yield. The concentrations of inoculum and incubation temperature were at their optimum levels, 1.64% (v/v) and 28[degrees]C respectively.
[FIGURE 12 OMITTED]
Pigment production showed a parabolic trend in response to variation in incubation temperature from 25-28[degrees]C and 175-200 mM sodium chloride concentration (Figure 13). Pigment production decreased at sodium chloride concentration below 120 mM and incubation temperature below 23[degrees]C. Concentrations of calcium chloride and inoculum were at their optimum levels, 75 mM and 1.64 % (v/v). Pigment yields were higher at a temperature range between 20-25[degrees]C and calcium chloride concentration from 87.5-100 mM (Figure 14). Incubation temperature above 25[degrees]C and calcium chloride concentration below 87.5 mM led to decreased pigment production. Concentrations of inoculum and sodium chloride were at their optimum levels, 1.64 % (v/v) and 196 mM respectively.
[FIGURE 13 OMITTED]
[FIGURE 14 OMITTED]
Validation of the deduced response surface model was carried out in shake flasks under conditions predicted by the model. The experimental values were found to be very close to the predicted values and hence, the model was successfully validated (Figure 15).
The optimized conditions for pigment production were as follows: Yeast extract- 1% (w/v); dextrose-10 mM; [K.sub.2]HP[O.sub.4]-0.45 mM; K[H.sub.2]P[O.sub.4]-0.5 mM; calcium chloride-75 mM; sodium chloride-196 mM; inoculum-1.64 % (v/v); pH-6.0; incubation temperature-28[degrees]C and incubation period of 22 h. It was observed that optimal levels of some of the critical factors such as NaCl, Ca[Cl.sub.2] and yeast extract were almost very close for effecting enhanced pigment production, despite the fact they were required at a slightly lesser level, except yeast extract. Reduced level of dextrose (10 mM) could have contributed to enhanced pigment production.
Data obtained for the time course experiment conducted over a period of 48 h under optimized condition testify that pigment production commenced after 8 h of incubation and reached a peak at 24 h of incubation (39.95 [micro]g/L). From the result presented in Figure 16 clearly evidence that further incubation period beyond 24 h resulted in a slight decline in pigment production. Maximum biomass was attained within 24 h of incubation and was found to be decreased during the late hours of incubation. An overall two fold increase in pigment production was achieved after statistical optimization compared to the MSTS media before optimization (21.06 [micro]g/L) (Figure 17).
[FIGURE 16 OMITTED]
[FIGURE 17 OMITTED]
Dyeing of Textile materials
Results presented in Figure 18 clearly evidence that the pigment produced by Serratia sp. BTWJ8 can be effectively used to dye all the textile materials studied. During the wash performance studies with the textile materials treated with pigment, it was found that the pigment is lost from the cloth after washing in soap solution at room temperature; 28 [+ or -] 2[degrees]C (Figure 19) and also at 40[degrees]C (Figure 20). Whereas, the loss of pigment from the same textile materials treated with mordant was found to be less. So it is inferred that thiourea is an effective mordant for treating the dyed textile materials and it can withstand at hot wash conditions.
[FIGURE 18 OMITTED]
[FIGURE 19 OMITTED]
[FIGURE 20 OMITTED]
The marine Serratia sp. BTWJ8 showed considerable amount of red pigment production both on the agar medium and in the liquid medium and hence was selected as the potential strain for pigment production in the present study. The pigment produced by Serratia sp. BTWJ8 is water insoluble and methanol was found to be an ideal solvent for the maximal extraction of the pigment among the different solvents studied. The results obtained for spectrophotometric and chromatographic analysis indicate that the pigment produced by the strain is prodigiosin or a close derivative. It has been reported that certain strains belonging to genus Serratia as well as other genera of marine bacteria produce prodigiosin, red antibiotic pigment , which is insoluble and accumulates in the cells .
Serratia sp. is reported to produce cell associated red colour pigment prodigiosin [34, 35]. Microscopic observation of S. marcescens colonies showed that prodigiosin pigment was localized in vesicles (extracellular and cell associated) or as intracellular granules .
In the present study, the pigment could be completely transferred to the solvent after incubation at 60[degrees]C in a water bath testifying that the pigment produced by Serratia sp. BTWJ8 is membrane bound. Prodigiosin was reported to display a characteristic absorption spectrum in ethanol, with a maximum at 534 nm  and single peak absorbance at 535 nm [5, 23]. Montaner et al.  extracted prodigiosin by shaking the S. marcescens 2170 cells with a mixture of methanol/1N HCl in the ratio 24:1. Pigment produced by Serratia sp. BTWJ8 recorded maximum absorption at 535 nm suggesting that this pigment is prodigiosin.
Pigments from S. marcescens were purified by extraction with acetone followed by transfer to petroleum ether and the petroleum ether extract was evaporated in vacuo at 30 to 40[degrees]C in order to obtain dry pigment. A single red coloured band was obtained after column chromatography using silica gel column and the same was eluted with n-hexane and ethyl acetate (2:1; v/v) and concentrated by evaporation . In the present study, the red pigment from the Serratia sp. BTWJ8 was purified by extraction with methanol followed by transfer to petroleum ether and dry pigment was obtained by evaporation of the solvent at 40[degrees]C. A single band with an [R.sub.f] value of 0.42 was obtained after thin-layer chromatography with chloroform: methanol (95:5; v/v) solvent system. Song et al.  reported single red prodigiosin band with [R.sub.f] value 0.43. The molecular mass of the pigment produced by Serratia sp. BTWJ8 was 324.2 Da, which corresponds to that of the molecular mass of prodigiosin ([C.sub.20][H.sub.25][N.sub.3]O). Similar result was reported earlier for Serratia sp. [5, 23].
In the present study, the data obtained for the spectroscopic analyses of the red pigment with LC-MS, FT-IR, FT-Raman and [sup.1]H-NMR very clearly testify that the pigment produced by Serratia sp. BTWJ8 is prodigiosin.
Absorption spectrum of the pigment produced by Serratia sp. BTWJ8 was dependent on pH value similar to that reported earlier . Thus at pH 2.0, the pigment was red and showed a maximum absorption at 535 nm, which is identical to that of prodigiosin hydrochloride. Under neutral condition (pH 7.0), its absorption intensity decreased and the colour of the pigment changed to pink. However, in alkaline condition (pH 10.0) the colour was orange and its absorption spectrum shifted to 470 nm. It has been suggested that the nitrogen of the three conjugated pyrrole rings are protonated by NaOH . Prodigiosin has long been known to respond to pH change since the addition of acid causes a bright red colour and the addition of alkali produces an orange shade .
Various nutrients influence the rate and amount of pigment production by bacteria. Hence, in order to increase the potentiality of the bacteria to synthesize maximal quantities of the pigment a suitable medium was developed after conducting a comparative study using different media that are known to be used commonly for carotenoid production. Among the different media evaluated Mineral salts tryptone sucrose medium (MSTS) supported maximal pigment production when compared to Zobell Marine broth, Nutrient broth, Seawater yeast extract peptone broth and Glycerol asparagine broth. Since MSTS medium led to enhanced red pigment production by Serratia sp. BTWJ8, this medium was optimized further towards maximizing pigment production.
It was noted that in spite of more pigment production in MSTS medium prepared in 50-100 % seawater, there was large pigment loss from the cells into the culture supernatant during the recovery of pigments when compared to the medium prepared in distilled water. Hence, an attempt was made to precipitate the culture supernatant with ammonium sulphate in order to ascertain whether the pigment was bound to soluble proteins and hence removed along with supernatant. It was observed that after precipitation the supernatant became colourless denoting that the pigments got bound to the secreted proteins and got removed in the culture supernatant. Probably in the presence of various salts that are natural components of seawater, there was forced binding of the pigment to the proteins and hence removed along with proteins. Further studies to this effect may throw more light on this property of pigment. In an earlier study a water soluble pigment composed of prodigiosin, carbohydrate and protein excreted from Serratia marcescens has been purified by precipitation with ammonium sulphate and dialysis .
Medium optimization by statistical methods has proved to be a powerful and useful tool of biotechnology. Plackett-Burman design offers good and fast screening procedure and mathematically computes the significance of large number of factors in one experiment, which is time saving and maintain convincing information on each component. Only the most effective factors with positive significance would be selected for further optimization studies, while those showing high negative effect on the bioprocess may be dropped in all further experiments . This indicates the effectiveness of Plackett-Burman design as a tool for elucidating the most important variables affecting the response . In the present study, the process variables were statistically optimized initially using Plackett-Burman design and Response Surface Methodology in the second stage for the maximum pigment production. Among the eleven factors namely incubation period, inoculum, yeast extract, dextrose, NaCl, Ca[Cl.sub.2], [K.sub.2]HP[O.sub.4], K[H.sub.2]P[O.sub.4], trace salts, pH and incubation temperature evaluated with Plackett-Burman Design for pigment production. Inoculum ([X.sub.1]), sodium chloride ([X.sub.2]), calcium chloride ([X.sub.3]), and Temperature ([X.sub.4]) were alone found to be the most significant variables. The statistical significance of the model equation evaluated by the F-test analysis of variance (ANOVA) revealed that this regression is statistically significant. The Model F-value of 9.15 implied that the model is significant.
The data obtained evidence that inoculum, sodium chloride, and temperature had a positive effect in enhancing pigment production along with their increase in concentration. Whereas, calcium chloride had a negative effect on pigment production along with their increase in concentration
Applying Box-Behnken design is an efficient method to optimize the selected factors for maximal production that tests the effect of factors interaction. Besides, it converts the bioprocess factor correlations into a mathematical model that predicts where the optimum is likely to be located. It is worthwhile to advise the microbial industry sponsors to apply these experimental designs to maintain high efficiency and profit bioprocesses . The results obtained for the Box-Behnken design experiment were analyzed by ANOVA and the result showed that Prob > F value was less than 0.05, which indicate that the model is significant. Two linear coefficient, inoculum ([X.sub.1]), sodium chloride ([X.sub.2]); one quadratic term, temperature ([X.sub.4.sup.2]) interaction coefficient and two interaction coefficient, sodium chloride and calcium chloride ([X.sub.2][X.sub.3]); calcium chloride and temperature ([X.sub.3][X.sub.4]) were found to be significant model terms.
The model coefficients estimated by multiple linear regressions and ANOVA showed that the model was significant with coefficient of determination ([R.sup.2]) of 0.8107. This ensured a satisfactory adjustment of the quadratic model to the experimental data and indicated that approximately 81.07 % of the variability in the dependent variable (response) could be explained by the model. The adjusted [R.sup.2], which is more suited for comparing models with different numbers of independent variables, was 0.6069. All the selected parameters were significant and varied levels of interactions were recorded for the variables in their cumulative effect on pigment production.
Three-dimensional response surface curves were plotted to study the interaction among various physicochemical factors, and to determine the optimum concentration of each individual factor for maximum pigment production. The model predicted maximum pigment production up to 40 [micro]g/L that could be achieved using 1.64 % (v/v) inoculum, 75 mM calcium chloride, and 196 mM sodium chloride, at incubation temperature 28[degrees]C and incubation period for 22 h.
Validation of the deduced response surface model based on the previous experiments was carried out in shake flasks under conditions predicted by the model. The experimental values were found to be very close to the predicted values and hence, the model was successfully validated. The optimized conditions for pigment production were as follows: Yeast extract-1 % (w/v); Dextrose-10 mM; [K.sub.2]HP[O.sub.4]-0.45 mM; K[H.sub.2]P[O.sub.4]-0.5 mM; Ca[Cl.sub.2]-75 mM; NaCl-196 mM; Inoculum-1.64% (v/v); pH-6.0; Incubation temperature-28[degrees]C and incubation period of 22 h in shake flasks.
Data obtained for the time course experiment conducted over a period of 48 h under optimized conditions testify that pigment production commenced after 6 h of incubation and reached a peak after 24 h of incubation. Further incubation period beyond 24 h resulted in a slight decline in pigment production. The results further suggest that pigment production by Serratia sp. BTWJ8 is growth associated. Maximum biomass was attained within 24 h of incubation and was found to be decreased during the late hours of incubation. An overall 2 fold increase in pigment production was achieved after statistical optimization compared to the MSTS media before optimization at the time of the experiment. So the medium can be successfully optimized for supporting the enhanced growth and simultaneously a high yield of prodigiosin by statistical approach.
Medium composition was optimized for high level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs . Statistical experimental design was employed to enhance carotenoid production from sugar cane molasses in the yeast Rhodotorula glutinis . The effect of the major media constituents of Porphyridium sp. was studied using response surface methodology on the production of phycoerythrin . Based on a three level Box Behnken design involving the variables pH ([X.sub.1]), incubation temperature ([X.sub.2]), and fermentation time ([X.sub.3]), a response surface methodology for the production of carotenoid by a mutant Aspergillus carbonarius CFTRI-UV10046 was standardized .
Prodigiosin, a multifaceted secondary metabolite, is produced by Serratia marcescens, Pseudomonas magneslorubra, Vibrio psychroerythrous and other bacteria [46, 41]. The prodigiosin groups of natural products are a family of tripyrrole red pigments that contain a common 4-methoxy, 2-2 bipyrrole ring system. The biosynthesis of the pigment is a bifurcated process in which mono and bipyrrole precursors are synthesized separately and then assembled to form prodigiosin . Pigmentation by S. marcescens was recorded during the exponential phase, and maximal production occurred in the stationary phase . These data testify the fact that prodigiosin can be regarded as a secondary metabolite .
Stavri and Marx,  argued that if the pigment were of no use to the bacterium the ability to synthesize prodigiosin would have been lost. Synthesis of secondary metabolites such as prodigiosin that have no demonstrable function in the bacteria also offers a paradox in which useful cellular macromolecules (genes and proteins) are involved in synthesis of a useless product. The paradox can be explained by Woodruff's hypothesis  that as cells enter the late phases of growth they face death by accumulation of toxic precursors. Biosynthesis of secondary metabolites converts there toxic substances to an end product that has no specific function for the cell but does prolong life by removal of the lethal substances. Thus, secondary metabolism is of value because removal of the primary metabolites may prolong survival of the microorganisms. During senescence of the bacteria, synthesis of prodigiosin and its intermediates may function indirectly in S. marcescens by removing toxic accumulations of metabolites such as aminoacids . Investigations of prodigiosin biosynthesis by nonproliferating cells, in which cellular growth and multiplication are separated from formation of pigment and its precursors, may provide clues as to the function of secondary metabolites in senescent cells. An understanding of the induction and regulation of the biosynthesis may provide information pertinent to the process of aging in living cells.
After conventional "one-variable-at-a-time" approach the optimized conditions for maximal pigment (21.06 [micro]g/L) included the following: Incubation time-30 h; incubation temperature: 25[degrees]C; initial pH of the media-6.0; agitation-150 rpm; dextrose-30 mM; yeast extract-1% (w/v); inoculum-1 % (v/v); Ca[Cl.sub.2]-100 mM; trace salts (Fe[Cl.sub.3], Mn[Cl.sub.2], [Na.sub.2]Mo[O.sub.4] and ZnS[O.sub.4])-0.03 mM; NaCl-200 mM. Whereas, almost two fold increase in pigment production (36.95 [micro]g/L) was recorded after statistical optimization of the variables which included: Yeast extract-1 % (w/v); dextrose-10 mM; [K.sub.2]HP[O.sub.4]-0.45 mM; K[H.sub.2]P[O.sub.4]-0.5 mM; Ca[Cl.sub.2]-75 mM; NaCl-196 mM; inoculum-1.64 % (v/v); initial pH of the media-6.0; incubation temperature 28[degrees]C and incubation period of 22 h. It was observed that optimal levels of some of the critical factors such as NaCl, Ca[Cl.sub.2] and yeast extract were almost very close for effecting enhanced pigment production, despite the fact they were required at a slightly lesser level, except yeast extract. Reduced level of dextrose (10 mM) could have contributed to enhanced pigment production. Statistical optimization demonstrated almost an accurate picture of the actual requirements of the strain for maximal pigment production. So this study strongly supports the statistical method of media optimization for enhancing pigment production by Serratia sp. BTWJ8.
The effluent released from the dyeing of the synthetic dyes are toxic and cause environmental pollution and harmful to health. The discharge of these waste residues into the environment eventually poison, damage or affect one or more species in the environment, with resultant changes in the ecological balance. There are several attempts being made to evolve ideal processes for safe and effective disposal of dye effluent from industries that use dyestuff. The harmful effects of synthetic dye and chemicals used at the time of dyeing have forced us to concern about the alternative preparation of dye using natural sources. With concern for environment protection becoming so important, there is a challenge to evolve environment friendly technologies and yet be competitive on a global level. So in this study an attempt was made to explore the probable use of natural pigment produced by Serratia sp. BTWJ8 for dyeing purpose in textiles. The textile industry is one amongst the rapidly growing industries worldwide, which utilizes enormous amounts of synthetic dyes. Consequently, the effluent from these textile industries poses serious threat to the environment, which is often very difficult to treat and dispose. This has become a very grave problem in environment conservation and hence natural pigments have drawn the attention of industry as safe alternative.
Results obtained in the present study strongly evidence that the pigment produced by Serratia sp. BTWJ8 has the dyeing property and could be used to dye different grades of textile materials. Further, the wash performance studies with the textile materials treated with pigment and thiourea, which is generally considered as a safe and effective mordant, suggest that there is ample scope for using this pigment as a dye in textile industry. In an earlier study the blue pigment from Janthinobactreium lividum was used to dye natural fibers and the colour shade depending on the material. Dyeing was performed by a simple procedure consisting of either dipping in the pigment extract or boiling with the bacterial cells. It was found that when the dyed material was subjected to post-treatment with thiourea solution, the fading of the bluish-purple colour to light was considerably reduced [26, 34].
To the best of our knowledge this is the first report on the evaluation of prodigiosin as a dyeing agent for use in textile industry and could record satisfactory performance from marine Serratia sp. BTWJ8.
The red pigment produced by Serratia sp. BTWJ8 was isolated and characterized. Various bioprocess parameters affecting pigment production by Serratia sp. BTWJ8 under submerged fermentation were optimized towards maximal pigment production using Mineral salts tryptone sucrose (MSTS) medium. An overall two-fold increase in pigment production was achieved after statistical optimization. The pigment was evaluated for its application as a dye in textile industry. The pigment was taken up by all the textile samples evaluated indicating the dyeing property of the pigment. During the wash performance studies with the textile materials treated with pigment it was found that the pigment is lost from the cloth after wash in soap solution at room temperature (28 [+ or -] 2[degrees]C) and at 40[degrees]C. Whereas, the loss of pigment from the same textile materials treated with thiourea as mordant was found to be less at both the incubation temperatures. So it is inferred that thiourea is an effective mordant for treating the dyed textile materials.
First author is grateful to Kerala State Council for Science Technology and Environment (KSCSTE), Kerala, India for the research fellowship. Authors greatly acknowledge Dr. K. R. K. Menon and Mr. Xavier, Spices Board, Cochin, Kerala, India for their extensive help in the LC-MS analysis and Smt. Lakskmi Varma, Smt. Sathy Chandrasekar, National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala, India for the NMR analysis.
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Jissa G. Krishna * (1), Soorej M. Basheer (1), K.K. Elyas (1,2) and M. Chandrasekaran (1,3)
(1) Microbial Technology Laboratory, Department of Biotechnology, Cochin University of Science and Technology, Cochin 682 022, Kerala, India
* Corresponding Author E-mail: firstname.lastname@example.org
(2) Department of Biotechnology, Calicut University, Kerala, India
(3) Department of Botany & Microbiology, College of Science, King Saud University, Riyadh-11451, Kingdom of Saudi Arabia
Table 1: Media composition. A. Zobell Marine broth (ZMB) Zobell Marine Broth 40.25 g pH 7.0 [+ or -] 2.0 Distilled water 1000 ml B. Nutrient broth (NB) Peptic digest of animal tissue 5.0 g Yeast extract 1.5 g Beef extract 1.5 g NaCl 5.0 g pH 7.4 Distilled water 1000 ml C. Glycerol asparagine broth (GAB) Glycerol 10.0 g Asparagine 1.0 g K2HPO4 1.0 g NaCl 10.0 g pH 7.0 Distilled water 1000 ml D. Sea water yeast extract peptone broth (SWYPB) Peptone 20.0 g Yeast extract 12.0 g Aged sea water 750 ml pH 7.8 Distilled waster 250 ml E. Mineral salts tryptone sucrose medium (MSTS) Tryptone 10.0 g Sucrose 20.0 g MgS[O.sub.4] 4.0 g Ca[Cl.sub.2] 7.0 g [K.sub.2]HP[O.sub.4] 0.08 g K[H.sub.2]P[O.sub.4] 0.07 g NaCl 10.0 g Fe[Cl.sub.3] 5.0 mg Mn[Cl.sub.2] 5.0 mg [Na.sub.2]Mo[O.sub.4] 5.0 mg ZnS[O.sub.4] 5.0 mg pH 6.0 Distilled water 1 L Table 2: Minimum and maximum ranges for the parameters selected in Plackett-Burman Design for optimization of pigment production by Serratia sp. BTWJ8. S1. No. Factors Level Minimum Maximum 1. Incubation (h) 12 36 2. Inoculum (%) 0.50 2 3. Yeast extract (%) 0.50 1 4. Dextrose (mM) 10 100 5. NaCl (mM) 100 200 6. Ca[Cl.sub.2] (mM) 50 100 7. [K.sub.2]HP[O.sub.4] (mM) 0 0.45 8. KH2PO4 (mM) 0 0.5 9. Trace salts (mM) 0 0.03 10. pH 5 7 11. Temperature ([degrees]C) 20 30 Table 3: Box-Behnken design for 4 variables at 3 levels-3 blocks and 30 runs for optimization of pigment production by Serratia sp. BTWJ8. BLOCK RUN Inoculum Sodium Calcium Incubation (A) chloride (B) chloride[C] temperature (D) [X.sub.1] [X.sub.2] [X.sub.3] [X.sub.4] (%) (mM) (mM) ([degrees]C) 1 1 0 0 -1 +1 1 2 0 0 -1 -1 1 3 0 0 +1 +1 1 4 +1 -1 0 0 1 5 0 -1 0 0 1 6 +1 +1 0 0 1 7 0 0 0 0 1 8 0 0 +1 0 1 9 -1 +1 0 -1 1 10 -1 -1 0 0 2 11 +1 0 0 0 2 12 0 -1 -1 -1 2 13 0 +1 +1 0 2 14 +1 0 0 +1 2 15 0 0 0 0 2 16 0 +1 -1 0 2 17 -1 0 0 +1 2 18 0 -1 +1 0 2 19 -1 0 0 -1 2 20 0 0 0 0 3 21 0 0 0 0 3 22 0 +1 0 +1 3 23 -1 0 +1 0 3 24 0 -1 0 +1 3 25 +1 0 -1 0 3 26 +1 0 +1 0 3 27 -1 0 -1 0 3 28 0 -1 0 -1 3 29 0 +1 0 -1 3 30 0 0 0 0 Table 4: Box-Behnken design for pigment production by Serratia sp. BTWJ8. BLOCK RUN Inoculum Sodium (A) chloride (B) [X.sub.1] (%) [X.sub.2] (mM) 1 1 1.25 150 1 2 1.25 150 1 3 1.25 150 1 4 2.00 100 1 5 1.25 150 1 6 2.00 200 1 7 1.25 150 1 8 1.25 150 1 9 0.50 200 1 10 0.50 100 2 11 2.00 150 2 12 1.25 100 2 13 1.25 200 2 14 2.00 150 2 15 1.25 150 2 16 1.25 200 2 17 0.50 150 2 18 1.25 100 2 19 0.50 150 2 20 1.25 150 3 21 1.25 150 3 22 1.25 200 3 23 0.50 150 3 24 1.25 100 3 25 2.00 150 3 26 2.00 150 3 27 0.50 150 3 28 1.25 100 3 29 1.25 200 3 30 1.25 150 BLOCK Calcium Incubation chloride (C) temperature (D) [X.sub.3] (mM) [X.sub.4] ([degrees]C) 1 50 30 1 50 20 1 100 30 1 75 25 1 75 25 1 75 25 1 75 25 1 100 20 1 75 25 1 75 25 2 75 20 2 50 25 2 100 25 2 75 30 2 75 25 2 50 25 2 75 30 2 100 25 2 75 20 2 75 25 3 75 25 3 75 30 3 100 25 3 75 30 3 50 25 3 100 25 3 50 25 3 75 20 3 75 20 3 75 25 Table 5: Optimized media composition. Yeast extract 1 % (w/v) Dextrose 10 mM [K.sub.2]HP[O.sub.4] 0.45 mM K[H.sub.2]P[O.sub.4] 0.5 mM pH 6.0 Incubation time 22 h Inoculum concentration 1.64 % (v/v) Incubation temperature 28[degrees]C Ca[Cl.sub.2] 75 mM NaCl 196 mM Table 6: Morphological and biochemical characteristics of Serratia sp. BTWJ8. Variable Characteristics Colony and cell Colony shape Round morphology Colony size Medium Edge Smooth Surface Smooth Opacity Opaque Elevation Convex Colour Red Motility Motile Cell shape Rod Cell size Small Biochemical Gram staining Negative characteristics MOF Fermentation with gas production H2S production Negative Indole Negative Methyl Red Negative Voges-Proskauer Positive Citrate ulilization test Positive Catalase Positive Oxidase Negative Glucose Positive Lactose Negative Sucrose Positive Mannitol Positive Urease Negative Enzyme profile Protease Positive Lipase Positive Alpha amylase Positive Table 7: The matrix of the Plackett-Burman design experiment, together with the observed experimental data for pigment production by Serratia sp. BTWJ8. Run Incubation Inoculum Yeast Dextrose NaCl period (h) (%) extract (mM) (mM) [X.sub.1] [X.sub.2] (%) [X.sub.4] [X.sub.5] [X.sub.3] 1 12 0.50 0.50 10 100 2 36 0.50 1.00 100 100 3 12 0.50 1.00 100 200 4 36 2.00 0.50 100 200 5 36 2.00 1.00 10 200 6 12 2.00 0.50 10 100 7 12 2.00 1.00 10 200 8 12 2.00 1.00 100 100 9 36 0.50 1.00 10 100 10 12 0.50 0.50 100 200 11 36 2.00 0.50 100 100 12 36 0.50 0.50 10 200 Run Ca[Cl.sub.2] [K.sub.2]HP K[H.sub.2] Trace (mM) [O.sub.4] (mM) P[O.sub.4] (mM) salts [X.sub.6] [X.sub.7] [X.sub.8] (mM) [X.sub.9] 1 50 0.00 0.0 0.00 2 100 0.00 0.0 0.00 3 50 0.45 0.5 0.00 4 50 0.45 0.0 0.00 5 100 0.00 0.5 0.00 6 100 0.45 0.5 0.00 7 50 0.00 0.0 0.03 8 100 0.45 0.0 0.03 9 50 0.45 0.5 0.03 10 100 0.00 0.5 0.03 11 50 0.00 0.5 0.03 12 100 0.45 0.0 0.03 Run pH Temperature Pigment [X.sub.10] ([degrees]C) ([micro]g/L) [X.sub.11] 1 5 20 0.014 2 7 30 0.89 3 7 20 0.084 4 5 30 36.48 5 5 20 14.99 6 7 30 2.927 7 7 30 32.48 8 5 20 0.367 9 5 30 14.13 10 5 30 0.565 11 5 20 8.458 12 5 20 1.78 Table 8: Effect of individual variable on pigment production by Serratia sp. BTWJ8 studied using Box-Behnken design experiment. BLOCK RUN Inoculum Sodium Calcium Temperature *Pigment (%) chloride chloride ([degrees]C) ([micro] [X.sub.1] (mM) (mM) [X.sub.4] g/L) Y [X.sub.2] [X.sub.3] 1 1 1.25 150 50 30 36.71 1 2 1.25 150 50 20 7.84 1 3 1.25 150 100 30 0.865 1 4 2.00 100 75 25 26.83 1 5 1.25 150 75 25 34.23 1 6 2.00 200 75 25 38.32 1 7 1.25 150 75 25 22.57 1 8 1.25 150 100 20 31.98 1 9 0.50 200 75 25 17.0 1 10 0.50 100 75 25 0.084 2 11 2.00 150 75 20 36.73 2 12 1.25 100 50 25 2.842 2 13 1.25 200 100 25 36.49 2 14 2.00 150 75 30 20.71 2 15 1.25 150 75 25 28.18 2 16 1.25 200 50 25 37.62 2 17 0.50 150 75 30 1.29 2 18 1.25 100 100 25 37.95 2 19 0.50 150 75 20 20.71 2 20 1.25 150 75 25 37.95 3 21 1.25 150 75 25 37.95 3 22 1.25 200 75 30 38.32 3 23 0.50 150 100 25 35.33 3 24 1.25 100 75 30 11.62 3 25 2.00 150 50 25 33.85 3 26 2.00 150 100 25 37.95 3 27 0.50 150 50 25 31.46 3 28 1.25 100 75 20 24.24 3 29 1.25 200 75 20 22.97 3 30 1.25 150 75 25 37.95 * Pigment production (Experimental value) is considered as the Response (Y)
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|Author:||Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Chandrasekaran, M.|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Feb 1, 2011|
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