World Journal of Microbiology & Biotechnology 14, 685688

Production, extraction and purication

of violacein: an antibiotic pigment produced
by Chromobacterium violaceum

D. Rettori and N. Duran*

A procedure for the production, extraction, and purication of violacein was developed using Chromobacterium
violaceum (CCT 3496) cultivated on cotton, in modied 1 litre Roux bottles. A surface tray bioreactor was built to
perform these experiments. Violacein was extracted with commercial ethanol, and puried by ltration, Soxhlet
extraction, crystallization and high performance liquid chromatography. The violacein was analysed and identied
by proton and carbon-13 NMR spectroscopies, thermogravimetric analysis, mass spectrometry, UV-VIS spectro-
scopy and infrared spectroscopy. It was concluded that the product was highly puried violacein.

Key words: Antibiotic, bioreactor, Chromobacterium violaceum, pigment, violacein.

Chromobacterium violaceum is a Gram-negative, faculta- In this work we describe, in detail, a simple method-
tively anaerobic, rod-shaped bacterium (Kaufman et al. ology to obtain highly puried violacein.
1986). It is a common saprophyte found in water and soil
from tropical and subtropical regions of the world, and is
generally considered to be non-pathogenic (Ponte & Materials and Methods
Jenkins 1992).
Violacein (Figure 1), the major pigment produced by Microbial Production
this bacterium, is a bactericide (Lichstein & Van de Sand Four ml (but less is possible) of a suspension of C. violaceum CCT
3496 (grown for 12 h at 30 C on an orbital shaker at 120 rev/
1945; Duran et al. 1983), a trypanocide (Caldas et al. 1978;
min) were inoculated into 400 ml of liquid medium (0.5%
Duran et al. 1994), a tumoricide (Duran et al. 1996) and in D-glucose; 0.5% peptone; 0.2% yeast extract) in a 2000 ml Er-
addition it has anti-viral activity (May et al. 1991). lenmeyer ask. Two inoculated asks were incubated at 30 C
for 12 h on an orbital shaker at 120 rev/min. Then, suspensions
of C. violaceum were inoculated onto eight nonsterilized cotton
carpets (90 ml/carpet). Each carpet (approximately 10 20
0.5 cm) was inside a non-sterilized one litre modied Roux
bottle (i.e. one carpet per bottle). The eight bottles were incu-
bated at 30 C for 24 h in a surface tray bioreactor (Rettori &
Duran 1997) without aeration. After this time, the cotton attains
a bright violet color due to intracellular production of violacein
by the bacteria. If production is not attained, aeration of the
system is necessary. The modication of the Roux bottles was
simply a hole of 8 cm in diameter in order to facilitate the
insertion and removal of the cotton carpets.

Figure 1. Molecular structure of violacein (molecular mass Extraction and Purication

343.34). The removed cotton carpets were all squeezed together to
eliminate excess medium and then washed twice with distilled
The authors are with Instituto de Qumica, Laboratorio de Qumica Biologica,
water. The violacein was extracted twice with 500 ml of com-
Universidade Estadual de Campinas, C.P. 6154, Campinas, CEP 13.083-970, ercial ethanol. The ethanolic solution was ltered and then
SP, Brazil; fax: (+55) 19 788 3023. *Corresponding author. evaporated under reduced pressure, yielding 750 mg of crude

1998 Rapid Science Publishers

World Journal of Microbiology & Biotechnology, Vol 14, 1998 685

D. Rettori and N. Duran
extract. This mass underwent Soxhlet purication rstly this diethyl ether extraction step and therefore this ex-
with chloroform (quantitatively), secondly with diethyl ether traction was limited to 34 h.
(for 34 h) and thirdly (extraction of violacein) with ethanol
(almost quantitatively). Evaporation of the ethanol under re-
In Figure 2 (chromatogram corresponding to the
duced pressure gave 40 mg of a semi-puried extract, which HPLC purication step), it can be seen that the fraction
was puried further by crystallization with the solvent pair collected consists of two peaks (30.14 and 32.62 min). In
methanol/water. The crystals were harvested by centrifugation principle each peak could be attributed to different
and dried at 100 C for 24 h. Approximately 10 mg were ob- compounds, but since the NMR spectrum of a sample
tained. Finally, these 10 mg were puried by high performance
liquid chromatography (HPLC) (with a Waters, model PREP. LC
containing both peaks revealed the existence of a highly
4000 System) using the following conditions: ow 7.0 ml/ puried violacein, all the collected fraction corresponds
min; stationary phase DELTA PAK C18 preparative column to violacein. From uorescence spectroscopy (Rettori
(30 mm 30 cm), with pores of 100 A and particles of 15 lm in 1996) it was concluded that both peaks were due to vi-
diameter; column temperature room temperature (23 C); olacein but that each one corresponded to violacein
moving phase 75% methanol: 25% H2O (% in volume); de-
tector wavelength 230 nm; chart speed 0.5 cm/min. Four
molecules in different aggregation states.
injections of a saturated ethanolic solution (previously ltered Comparing our NMR spectra (Figures 3 and 4) with
with 0.5 lm pore diameter size lter) of these 10 mg were made the data in the literature (Hoshino et al. 1987) it can be
(1.5 ml each injection) to the HPLC. The fraction collected for seen that this procedure leads to highly puried viola-
each injection had initial elution time of 26 min and nal elution cein. In Figure 3, the peak at 0 ppm corresponds to
time of 34 min (Figure 2). The solution was evaporated under
reduced pressure until violacein crystals in water remained.
These were harvested by centrifugation and dried at 100 C for
24 h. Approximately 1 mg violacein was obtained.

Characterization and Analysis

Violacein was characterized and analysed by: proton (300 MHz)
and carbon-13 (75 MHz) NMR spectroscopies (with a Varian,
model GEMINI 300) (Figures 3 and 4 respectively); thermo-
gravimetric analysis (TGA) (with a TA Instrument, model HI-
RES TGA 2950) (Figure 5); mass spectrometry (with a Hewlett
Packard, model HP 5988A) (Figure 6); ultravioletvisible (UV
VIS) spectroscopy (with a Hitachi, model U-2000) (Figure 7); and
infrared (IR) spectroscopy (with a Perkin-Elmer FT-IR, model 16
PC) (Figure 8).

Results and Discussion

The Soxhlet purication step with chloroform extracts Figure 3. NMR-1H spectrum of violacein dissolved in DMSO-d6 with
cell wall and membrane residuals (Melo 1996). The an aliquot of CDCl3 containing TMS.
Soxhlet step with diethyl ether solubilizes deoxy-
violacein, the minor pigment produced by C. violaceum
(Duran et al. 1983). A small fraction of violacein is lost in

Figure 2. Chromatogram from a sample (1.5 ml) of saturated etha- Figure 4. Decoupled NMR-13C spectrum of violacein dissolved in
nolic solution of violacein puried by crystallization. DMSO-d6 with an aliquot of CDCl3 containing TMS.

686 World Journal of Microbiology & Biotechnology, Vol 14, 1998

 Production of violacein by C. violaceum

tetramethylsilane (TMS); at 2.5 ppm to the residual

protons of perdeuterated dimethyl sulphoxide (DMSO);
at 3.3 ppm to water; and at 8.3 ppm to chloroform. In
Figure 4, the peak at 0 ppm corresponds to TMS; at
39 ppm to DMSO-d6; and at 79 ppm to chloroform.
However, two unidenitied peaks are still observed at
1.25 ppm (Figure 3) and 98.2 ppm (Figure 4). These two
peaks are of small intensity and they were attributed to
non-signicant impurities. The water peak is very
intense because DMSO is hygroscopic. However, to
determine if this violacein is hydrated or not, a TGA was
carried out.
In Figure 5 the thermogram shows a rst loss of mass Figure 6. Mass spectrum of violacein; electrons energy 70 eV;
at 170 C. This is strong evidence that violacein is free of temperature of probe 250 C.

water, otherwise a loss of mass at a lower temperature

would be expected. The transition at 386 C is due to
decomposition of violacein (Perkampus et al. 1971;
Bycroft 1988).
The mass spectrum (Figure 6) shows the molecular ion
m/z 343; the UVVIS spectrum (Figure 7) shows
strong absorption at the visible region due to resonance of
violacein; and the IR spectrum (Figure 8), according to
the literature (Laatsch et al. 1984; Riveros et al. 1988),
presents stretching bands at 1680 and 1655 cm)1 due to
C@O bonds and a stretching band at 1610 cm)1 due to the
C@C bond.
Figure 7. UVVIS spectrum of violacein in ethanol (51.9 lg/ml); a
quartz 1 ml cuvet with path length of 1 cm was used.

Concluding Remarks
The use of the surface tray bioreactor, the modied Roux
bottles and the cotton carpets greatly facilitates the
handling, by the operator, of all steps related to the
production and extraction of violacein. Production is
inexpensive, achieved in short periods of time (24 h),
and yields highly puried violacein.

Figure 8. IR spectrum of violacein (KBr pellet).

Acknowledgements

The authors wish to thank the Coordenacao de Ape-

rfeicoamento de Pessoal de Nvel Superior (CAPES)
(Braslia, DF, Brazil) for their nancial support, and the
Fundacao Tropical de Pesquisas e Tecnologia `Andre
Tosello' (Campinas, SP, Brazil) for kindly providing the
Figure 5. Thermogram of 0.9410 mg of violacein; heating rate reference strain. We also thank professor Fred Yukio
10 C/min; argon ow 100 ml/min. Fujiwara for a critical reading of the manuscript.

World Journal of Microbiology & Biotechnology, Vol 14, 1998 687

D. Rettori and N. Duran

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