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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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4.1.4 Effect of Time on Growth of C. violaceum in NB and BS Figure 4.7 shows growth profile of C. violaceum in NB and BS. To get the required data for plotting the graph, samples were shaken for 24 hours at 25⁰C.

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Growth of bacteria in different growth media shows a similar growth phases (lag, log and stationary phase). Death phase was not observed for BS growth medium within 24 hours. It can be seen that growth of C. violacein gives more yield in NB medium due to highest O.D 600 which is around two.

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4.1.5 Bacterial Preservation Long term maintenance of C. violaceum was successfully obtained using paraffin oil. Paraffin oil was reported to increase the lag phase of growth therefore binary fission will be slowed down so it will take longer for the bacteria to reach to the dead phase which occurs because of either loss of limiting nutrients or build-up of toxins they release during log-phase growth (Hartsell, 1952).

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Figure 4.9: Vitality test of preservation method for C.

violaceum There are some advantages of using paraffin oil as preservative factor such as: it greatly reduces the frequency of contamination, especially with molds, thus permitting cultures to be maintained with greater success in surroundings which are not conducive to precise bacteriological work. No preliminary treatment of the cultures and no seals, such as rubber caps, waxes is necessary (Harstel, 1952).

47

4.2 Characterization of Violet Pigment 4.2.1 Characterization of Violet Pigment from SPW The UV-VIS spectrum of the violet pigment obtained from section 3.2.1.2 (Figure 4.10) is shown in Figure 4.11.

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The obtained λmax was 571-773 nm that is indicating the presence of chromophoric groups, hence indicated existing of violacein in the sample. A similar λmax was obtained by Renee and Kendal (2001).

4.2.2 Characterization of Violet Pigment from BS As explained in 3.2.3.2, extraction of purple pigment from BS was carried out using two different solvents which were methanol and ethyl acetate. TLC test showed presence of impurities in pigments extracted from BS with methanol, where sample collected using ethyl acetate seemed to be pure (Figure 4.12). This fact can be explained by assuming that present impurities in the sample extracted from BS using methanol are highly polar; it is also known that ethyl acetate is a semi polar solvent. Therefore, by using ethyl acetate in extraction stage just poorly-water soluble violacein will migrate to the organic phase of solvent and impurities will remain inside the supernatant.

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4.2.2.1 Column Chromatography Present of impurities in samples collected from BS extracted with methanol was the reason of running the column. The first impurity separated from the sample (Figure 4.13) was the brownish material, probably brown pigment of BS.

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Figure 4.13: Brownish fraction of melanoidin, pigment from BS.

As mentioned in 3.2.6, different fractions consisting different colors were collected from the silica column. Unfortunately, TLC result of collected fractions did not show any purification since there was more than one single spot observed on the TLC paper. Although each vial seemed to be different from the other but the same set of spots appeared on TLC sheet after each and every test (Figure 4.14). The test was repeated again and the same results were observed. From the results, it can be concluded that pure violacein was not obtained through column chromatography.

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4.2.2.2 UV-VIS Spectrophotometer Analysis of violacein Results of the UV-VIS analysis by other researchers (Table 4.1) show absorbance in the range of 550-580 nm of full range of UV light indicated from large conjugation C=C existing in violacein.

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Spectrum resulting from running UV-VIS of the sample collected from BS (Figure 4.15) gave a similar peak at 567.50 nm that shows presence of violacein in the solution. High absorption in the spectrum refers to successful production of pigment in new medium.

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4.2.2.3 FTIR Spectroscopic Analysis The IR spectrum (Figure 4.16) of violacein from BS displayed broad absorption band at 3430.1 cm-1 corresponding to O-H stretching. The N-H stretch 3350 cm-1 might be overlapped with band of O-H. A band appeared at 1640.6 cm-1 was assigned for carbonyl of amide groups. The presence of C=C of an intermediate alkene was observed at 1615.9 cm-1. The out of plane band for =C-H was observed at 757.25-727 cm-1. As for pigment extracted from NB, the IR spectrum (APPENDIX

4) proved the existence of O-H (3445.45 cm-1), a carbonyl of an amide (1662.8 cm-1), C=C of an olefinic band (1615.1 cm-1) and out of plane =C-H (760.4 cm-1). The carbonyl of the lactam ring has high absorption value because the one pair on N is delocalized away from the C=O, hence it behave as ketone C=O (1723.54 cm-1). The above data were almost similar with the IR data of violacein spectrum reported by other researchers (Table 4.2).

Table 4.2: Data of FTIR (Gregor and Wolfgang, 2001; Lara et al, 2005).





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Figure 4.16: FTIR spectrum obtained from KBr pellet of violacein from BS 4.

2.2.4 NMR analysis of violacein 13 C-NMR was employed to identify 20 carbons present in structure of violacein. The first sample analyzed by 13C-NMR was the one obtained from 3.2.3.2.

Result showed presence of noises due to the impurities in the sample which covered the peaks assigned to carbon atoms.

–  –  –

The 13C chemical shifts of all the present carbons in violacein structure have been studied and summarized in Table 4.3. Labeled carbons shows carbon number 16 and 11 are shifted downfield because of the influence of the electronegative oxygen atom where carbon number 13 is shielded to up field.

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Simultaneously, 1H-NMR spectrum was recorded in DMSO-d6. The sample showed similar peaks as reported in references (Figure 4.18).

Figure 4.18: 1H-NMR spectrum of preliminary purified violacein from BS.

–  –  –

Violacein pigments were tested for their stability in acidic and basic pH.

From the study, it was found that the pigment was not stable toward varying pH range where in extreme acidic condition the solution turned to blue color and in extreme alkaline condition, the solution was green in color (Figure 4.19). Initial pH of solution was 5.83 which had a dark violet color (Figure 4.20).

–  –  –

Changes of the color due to varying the pH can be explained by changing of the structure in extreme acidic and alkaline pH. For instance, in alkaline condition excess OH- in the solution deprotonates N-H groups which cause formation of anion.

Localization of negative charge of the anion to the ring system due to the long conjugation effect causes violacein to be absorbed at higher wavelength (694.7 nm).

Therefore it can be concluded that changes of the structure is changing the absorbance of the light hence reflected color from the sample deferrers.

CHAPTER 5

–  –  –

5.1 CONCLUSION In this project, the growth of Chromobacterium violaceum on agricultural wastes such as SPW and BS was studied. To achieve that target different growth factors (temperature, pH, concentration of media) were examined.

From the growth profile of C. violaceum, the purple pigment found to be the secondary metabolite products because the pigments were produced after 6 hours of the active stage of growth.

Effect of different temperatures (25°C, 30°C, and 37°C) has been studied on different growth cultures to show the optimum temperature suitable for growth of bacteria and production of pigment. All three media include Solid Pineapple Waste, Brown Sugar and Nutrient Broth were capable of intensive growth and pigment production at 25°C.

–  –  –

The pigment has been tested for stability towards pH. Appearance of different colors after adjusting the pH at extreme acidic and extreme alkaline showed nonstability of the pigment in different range of pH value.

–  –  –

Maintenance of the bacteria through this study was carried out using paraffin oil preservation method.

5.2 Future Study It was mentioned earlier on in the literature review that the most crucial amino acid required for formation of violacein structure is L-tryptophan. In this project it was aimed to produce violacein pigment without the use of chemicals in order to reduce manufacturing costs.

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Bromberg, N., and Duran, N. (2001). Violacein biotransformation by basdiomycetes and bacteria. Letters in Applied Microbiology. 33, 316-319.

Carina, A., Oliver, G., and Maria, C. A. (2001). Effect of different complex carbon sources on growth and bacteriocin synthesis of Enterococcus faecium. International Journal of Food Microbiology. 63, 235-241.

Carmen, V. F., Carina, L. B., Henri, H. V., Giselle, Z. J., Nelson, D., and Maikel, P. P.

(2004). Molecular mechanism of violacein-mediated human leukemia cell death.

Blood Journal. 104, 1459-1464.

Caroline, C., Sonia, A., Michel, F., and Chantal, L. (2008). A versatile preparation of azobenzene-dye functionalized colored polymer nanoparticles by surface 62 modification. Journal of Polymer Science Part A: Polymer Chemistry. 46, 3375 – 3386.

Chau, T. T. and David, A. M. (1995). Pineapple Waste - A Novel Substrate for Citric Acid Production by Solid-State Fermentation. Biotechnology Letters. 17: 1107-1110.

Daniel, M. M. (1986). Handbook of U.S. Colorant for Food, Drugs, and Cosmetics. (2nd ).

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14 DeMoss, R. D., and Evans, N. R. (1959). Incorporation of C-Labaled substrates into violacein. Department of Microbiology, University of Illinois, Urbana, Illenois. 79, 129-133.

DeMoss, R, D., and Happle, N. R.(1958). Nutritional Requirements of Chromobacterium violaceum. Department of Microbiology, University of Illinois, Urbana, Illenois. 77, 137-141.

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Georgeta, M. S., Sergiu, A. C., Nicole, M., Walter, S., and Eugen, S. (2004). Direct Dyes Derived from 4,40-Diaminobenzanilide Synthesis, Characterization and Toxicity Evaluation of a Disazo Symmetric Direct Dye. Turk Journal of Chemistry. 28, 579Goodwin, T. W. (2002). Chemistry and biochemistry of plant pigments. London, New York, Academic Press.

Gregor, W., and Wolfgang, S. (2001). A short synthesis of the bacterial pigment Violacein and Deoxyviolacein. Synthesis. 5, 759-762.

Haisheng, W., Peixia, J., Yuan, L., Zhiyong, R., Ruibo, J., Xin-Hui, X., Kai, L., and Dong, W. (2008). Optimization of culture condition for violacein production by a new strain of Duganella sp. B2. Biochemical Engineering Journal. 44, 119-124.

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Hartmut, L., and Ronald, H. T. (1984). Journal of the Chemical Society Perkin Transactions ΙΙ. Spectroscopic Properties of violacein and Related Compounds: Crystal Structure of teramethylviolacein. 1331-1339.

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