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«AKRAM NESHATI A Dissertation Submitted To The Faculty Of Science In Partial Fulfillment Of The Requirement For The Award Of The Degree In Masters of ...»

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Centrifugation is another possible way to extract the pigment as Yuan et al (2008) centrifuged the cells first at 8000 g for 5 minutes and the supernatant was discarded. The cell pallets were then rinsed with deionized water, followed by centrifugation to recover the cells by discharging the supernatant again. The recovered cells were fully mixed with ethanol. The mixture of the cells and ethanol was treated by ultra-sonication until the cells were completely bleached. With this protocol, it was confirmed that there was no residual pigment in the cell pellets after the extraction.

2.5 Violacein Characterization

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2.5.1 UV-VIS UV and visible spectrometry are usually the simplest analysis method to perform and require the least amount of sample, often as little as 0.1 mg. Where possible, spectra of the unknown compound should be compared with those of known compound in several solvents. UV-VIS spectrometry is fast, reliable and relatively simple procedures for identifying dyestuffs and should be used whenever possible. Their use required only a modest amount of training whereas the necessary equipment is moderate to expensive in price (Daniel, 1986).

2.5.2 FTIR For pigment analysis, FTIR is considered as a complementary technique that can provide the molecular and structural information of organic and inorganic materials (Douglas et al, 2003).

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2.5.3 NMR Recently proton nuclear magnetic resonance (NMR) has been used to identify both primary and secondary pigments. Good spectra of the certified water-soluble colors have already been obtained and published using a mixed, deuterated solvent (water : dimethylsulfoxide; D2O:DMSO-d6, 2:1v/v) at 100-105°C. NMR is one of the least sensitive, most complicated of the spectral techniques in use today but it is an excellent tool for identification purposes also for studying the structure of organic compounds (Daniel, 1986).

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3.1 Materials All the glassware used during this experiment were washed with distilled water and sealed and sterilized by autoclaving at 121°C for 15 minutes (HVE-50, Hirayama). All reagents and chemicals were of analytical grade.

3.1.1 Bacteria

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3.1.2 Growth Media 3.1.2.1 Nutrient Broth Nutrient broth (NB) was used as a liquid growth medium for preparing active culture. NB powder (8 g) (MERCK, Germany) was dissolved in 1 litter of distilled water and sterilized by autoclaving at 121°C for 15 minutes.

3.1.2.2 Nutrient Agar (NA) Nutrient agar (NA) was prepared by dissolving 20 g of nutrient agar (MERCK, Germany) powder in 1 litter of distilled water. The medium was sterilized by autoclaving at 121°C for 15 minutes. The molten agar was cooled to about 50°C before being poured into sterile Petri dishes. The agar was allowed to harden and then incubated for 24 hours at 30°C to ensure that the medium was free from contamination.

3.1.2.3 Solid Pineapple Waste (SPW)

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3.1.2.4 Brown Sugar (BS) Stock Solution To prepare the Brown Sugar (BS) stock solution, 40 g BS was dissolved in 1 L distilled water. The solution was heated, stirred and filtered using filter paper (Advantec, Japan) 125 mm to remove insoluble particles. pH of solution was set at 8 using 0.1 M NaOH before autoclaving at 105°C for 15 minutes.

3.1.3 Tryptophan Solution Solutions of DL-tryptophan and L-tryptophan were prepared by dissolving

0.1 g of the amino acid in 100 mL of distilled water. The solution was sterilized using 0.45 µm cellulose acetate membrane.

3.1.4 Active Culture

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3.2.1 Growth of Chromobacterium violaceum 3.2.1.1 Cultivation of C. violaceum in Nutrient Broth at Different Temperatures NB (22.5 mL) was poured into a sterile 250 mL conical flask under laminar flow followed by the addition of 2.5 mL of active culture. The mixture was shaken for 24 hours at 200 rpm in an orbital shaker at room temperature. Similar flask were prepared but grown at 30°C and 37°C.

3.2.1.2 Cultivation of C. violaceum on SPW at Different Temperatures SPW (10 g) was added to 22.5 mL of distilled water. Ethanol (1.25 mL) (5% of total volume) was added to the mixture in order to kill other available microorganisms. pH of the mixture was adjusted to 7 with the addition of adequate amount of 1M NaOH.

The mixture was then transferred to sterilized 250 mL flask under laminar flow followed by the addition of 2.5 mL of active culture. The mixture was shaken for 24 hours at 200 rpm in an orbital shaker at room temperature. Similar flasks were prepared but grown at 30°C and 37°C.

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Addition of BS solution, DL-tryptophan, L-tryptophan as a supplement of growth was also studied in this project. Prepared BS solution added to the flask containing 5 g SPW in different ratio as follow: 1mL, 5 mL, and 10 mL. A control solution was prepared with addition of 2.5 mL active culture to 22.5 mL BS. Growth of bacteria was observed only in the control solution.

3.2.1.2.1 Effect of Tryptophan on Growth of C. violaceum

Study of effects of tryptophan initiated by addition of DL-tryptophan solution into seven 250 mL flask containing 10 g SPW, 2.5 mL active culture and 1.25 mL ethanol. Distilled water was added to each flask and the pH adjusted to 7. The amount of DL-tryptophan added is as shown in Table 3.1. (The control flask contains





22.5 mL DL-Tryptophan solution and 2.5 mL active culture).

Table 3.1: Sample preparation with addition of DL-tryptophan

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Growth of C. violaceum was also studied in the presence of L-tryptophan.

Table 3.2 shows sample’s ingredients while that of the control is shown in Table 3.

3.

Table 3.2: Preparation of samples in the presence of L-tryptophan

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3.2.1.3 Effect of Temperature on Growth of C. violaceum The effect of temperature on growth of C. violaceum was also studied samples were prepared by addition of 22.5 mL of BS solution and 2.5 mL of active culture into three flasks. The mixture was shaken at 200 rpm for 24 hours at 3 different temperatures (25°C, 30°C and 37°C).

After 24 hours of incubation, pigments were extracted from the samples and UV-VIS analyses of the samples were taken.

3.2.2 Growth Profile of C. violaceum Grown in NB and BS Growth of C. violaceum was also monitored in NB and BS. Active culture of C. violaceum (30 mL) was inoculated into NB (300 mL) and BS (300 mL) in Erlenmeyer flask. Each of the samples were complemented with a cell-free control set. The culture and control were shaken at 200 rpm and 25°C for 24 hours. At regular intervals, the turbidity of the culture was determined using a spectrophotometer (Spectronic 21D, Milton Roy) at 600 nm. The growth profile was obtained by plotting OD600 versus time.

34 3.2.3 Extraction of Violet Pigment 3.2.3.1 Extraction of Violet Pigment from SPW To extract violacein from SPW, firstly, the mixture was filtered with a filter paper (0.45 µm). After filtration, the filter paper was rinsed with distilled water several times. The filtered pineapple pieces were placed into a clean conical flask and 20 mL of methanol added to it. The mixture was left to shake for 15 minutes at 200 rpm to leach the pigments into solution.

Pigment was extracted using ethyl acetate (Figure 3.1) (MeMoss and Evans, 1959). Supernatant (30 mL) was poured into a separatory funnel and 10 mL ethyl acetate added to it. The mixture was then shaken and after releasing its gas the funnel was placed into the holder in order to separate the organic and aqueous phases. The organic layer which contains the pigments was kept in a sealed container.

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3.2.3.2 Extraction and Preliminary Purification of Violet Pigment from BS Two methods were employed to extract the violet pigment from BS samples.

The first method is as stated in 3.2.3.1. The first step was filtering the sample and then rinsing it with distilled water. Precipitated pigment on the filter paper was then washed with methanol till the filter paper becomes white in color. The pigment was then extracted using ethyl acetate as described in 3.2.3.1.

To check for purity of the sample, the ethyl acetate and methanolic fractions were spotted on a TLC plate and developed using n-hexane : ethyl acetate (4:6) solvent system.

The second method is as follows; sample (30 mL) was poured into a separatory funnel and added with 6 mL of ethyl acetate (5:1). The funnel was shaken to allow separation of the organic and aqueous phase. The organic layer was poured into a plate and left in the fume cupboard to evaporate the solvent.

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3.2.4 Characterization of Violacein 3.2.4.1 Characterization of Violacein using UV-VIS UV-VIS analysis was carried out for samples extracted from NB, SPW, and BS media. UV-VIS samples were prepared from the extracted pigments from NB and BS using the following ratio 2:2:1:1 (violacein from filter paper, violacein from supernatant, methanol ethyl acetate). A similar procedure was applied to the purple pigments extracted from SPW.

3.2.4.2 Characterization of Violacein using FTIR Dried samples obtained from 3.2.3.2 were analyzed using FTIR analysis.

KBr pellets were prepared out of each sample with grinding dry violacein with KBr powder to get a fine powder. The concentration of the sample in KBr was in the range of 0.2% to 1%.

3.2.4.3 Characterization of Violacein using NMR

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3.2.5 Stability Test of Pigment Towards pH Changes The effect of pH on the stability of the pigment extracted from BS was carried out by adjusting the pH at extreme acidic pH (2) and extreme alkaline (13) using and 0.1 M HCl and 0.1 M NaOH respectively.

3.2.6 Column Chromatography Preparation Column chromatography was carried out using a column with the following diameter: 12 cm height and 18 mm width. The column was then packed with silica gel (column chromatography grade) and washed twice with n-hexane. The concentrated sample obtained in methanol was placed on the top of the column and separation initiated with the addition of solvent. Different fractions consisting of different colors ranging from dark violet to bright pink were collected.

3.3 Bacterial Preservation

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Figure 3.2: C.

violaceum preserved on agar slant in the presence of paraffin oil.

C. violaceum was successfully maintained throughout this study by a very simple method of preservation in paraffin oil. Maintenance was carried out by streaking the bacteria on agar slant, followed by 48 hours of incubation at 30°C in order to obtain higher amounts of colonies (Harstel, 1952).

Final step of preservation was pouring sterile paraffin oil into agar slant to a height of one centimeter above the top of the slant’s surface. Prepared slant was kept in the fridge for almost four months.

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4.1 Effect of Growth Parameters on Chromobacterium violaceum 4.1.1 Effect of Temperature on growth of C. violaceum in NB The optimum growth temperature of C. violaceum and production of pigment was monitored in nutrient broth (NB). The results are shown in Figure 4.1.

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At 25°C, the color of the medium was dark purple 6 hours after inoculation (Figure 4.1(a)). However, the color of the culture medium was bright violet at 30°C and majority of pigment precipitated in the medium (Figure 4.1(b)) and at 37°C no violet pigment was observed.

4.1.2 Effect of Temperature on growth of C. violaceum in SPW Figure 4.2 shows the growth of C. violaceum in SPW at different temperatures and intensity of produced pigments.

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Out of three flasks, pigment production was only observed at 25°C (Figure 4.2 (a)) and 30°C (Figure 4.3 (b)). However, when this experiment was repeated, no pigment production was observed. This could be due to a different batch of SPW used to carry out the experiment.

–  –  –

After three days, no colonies of C. violaceum were observed on the plate.

C. violaceum has been replaced by other microorganism present in the SPW and compete for the available nutrients that lead to insufficient carbon source to support bacterial growth (Nordiana Nordin, 2006). Fermentation process also leads to the decrease in the pH (5.89) of the medium and makes it unsuitable for bacterial growth (Chua and David, 1995).

Figure 4.3: Effect of incubation period on growth of C.

violaceum. 1) Streaking right after inoculation; 2) after 1 day; 3) after 3 days.

The effect of supplementation on growth of C. violaceum in SPW was also looked into. Addition of BS did not have a positive effect on growth of C. violacein in the SPW culture (Figure 4.4).

–  –  –

Among all samples prepared with addition of DL-tryptophan and L-tryptophan (section 3.2.1.2.1), only control cultures and samples number 3 and 5 from Table 3.1, showed production of pigment after a day of cultivation.

Reproducibility of obtained result from growth of C. violaceum in SPW studied by repeating the experiment. This time, none of the prepared growth media showed growth of bacteria.

4.1.3 Effect of Temperature on growth of C. violaceum in Brown Sugar Growth of C. violaceum in brown sugar (BS) medium was observed at 25°C and 30°C. However, pigment was only produced at 25°C as can be seen in Figure

4.5. At 37°C, no growth and pigment production was observed.

–  –  –

It can be concluded that production of pigment in BS medium is highly temperature dependent and the optimum growth temperature for C. violaceum and production of violacein reported as 25°C.

Production of pigment is also studied at different concentrations of BS medium. Violacein absorbance of extracted pigments from different concentration of BS medium suggested the highest amount of pigment production belonged to sample number 17 (Figure 4.6).

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