Application of Bacterial Pigments as Colorant

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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering andBiotechnology ISSN 0273-2289Volume 167Number 5 Appl Biochem Biotechnol (2012)167:1220-1234DOI 10.1007/s12010-012-9553-7

Production and Characterizationof Violacein by Locally IsolatedChromobacterium violaceum Grown inAgricultural Wastes

Wan Azlina Ahmad, Nur ZulaikhaYusof, Nordiana Nordin, Zainul AkmarZakaria & Mohd Fazlin Rezali

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Production and Characterization of Violacein by LocallyIsolated Chromobacterium violaceum Grownin Agricultural Wastes

Wan Azlina Ahmad & Nur Zulaikha Yusof &Nordiana Nordin & Zainul Akmar Zakaria &

Mohd Fazlin Rezali

Received: 12 October 2011 /Accepted: 6 January 2012 /Published online: 26 January 2012# Springer Science+Business Media, LLC 2012

Abstract The present work highlighted the production of violacein by the locallyisolated Chromobacterium violaceum (GenBank accession no. HM132057) in variousagricultural waste materials (sugarcane bagasse, solid pineapple waste, molasses,brown sugar), as an alternative to the conventional rich medium. The highest yieldfor pigment production (0.82 g L−1) was obtained using free cells when grown in 3 gof sugarcane bagasse supplemented with 10% (v/v) of L-tryptophan. A much loweryield (0.15 g L−1) was obtained when the cells were grown either in rich medium(nutrient broth) or immobilized onto sugarcane bagasse. Violacein showed similarchemical properties as other natural pigments based on the UV–Vis, Fourier transforminfrared spectroscopy, thin-layer chromatography, nuclear magnetic resonance, andmass spectrometry analysis. The pigment is highly soluble in acetone and methanol,insoluble in water or non-polar organic solvents, and showed good stability betweenpH 5–9, 25–100 °C, in the presence of light metal ions and oxidant such as H2O2.However, violacein would be slowly degraded upon exposure to light. This is the firstreport on the use of cheap and easily available agricultural wastes as growth medium forviolacein-producing C. violaceum.

Keywords Chromobacterium . Violacein . Characterization . Stability . Agriculture wastesmaterial . Sugarcane bagasse

Appl Biochem Biotechnol (2012) 167:1220–1234DOI 10.1007/s12010-012-9553-7

W. A. Ahmad (*) :N. Z. Yusof :N. NordinDepartment of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Johor, Malaysiae-mail: [email protected]

Z. A. ZakariaInstitute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor,Malaysia

M. F. RezaliNational Metrology Laboratory, SIRIM Berhad, Lot PT 4803, Bandar Baru Salak Tinggi, 43900 Sepang,Selangor, Malaysia

Author's personal copy

Introduction

Pigments are widely used in food, cloth, painting, cosmetics, pharmaceuticals, and plastics.However, uncontrolled industrial applications of synthetic pigments may result in serioushazard to human health and a potential environmental disaster. Therefore, it is imperative tofind an alternative pigment which can be biodegraded and easily available at a minimalproduction cost [1]. Natural pigments are biodegradable [2] and can be obtained from variousresources such as plants, microorganisms, animals, and minerals. Of these, pigments obtainedfrom animal, plant, and fungal sources may be less susceptible to exploitation due to thestructural complexity of the pigment-bearing tissue or difficulty to harvest the pigment as it isnormally formed during critical points of development within a complex life cycle [3]. Amongpigment-producing microbial species, bacteria offer certain advantages based on its flexibility,short life cycle, and simple propagation technique [4]. Some example of the pigment-producingbacterial species include Flavobacterium sp. which produces the yellow pigment zeaxanthin[5], Agrobacterium auranticum (pink-red pigment, astaxanthin [5]),Micrococcus sp. (various-colored pigments, carotenoids [6]), Pseudomonas aeruginosa (blue-green pigment [7]), Serra-tia marcescens (red pigment [7]), and Chromobacterium sp. (violet pigment [7]).

Chromobacterium violaceum, a Gram-negative bacteria belonging to the Rhizobiaceaefamily, is a saprophyte found in soil and water in tropical and subtropical areas. In most cases, itis a minor component of the total microflora [8]. Its colonies are slightly convex, not gelatinous,regular, and violets, although irregular variants and non-pigmented colonies can also be found[9]. The pigment responsible for the violet colony is violacein [3-(1, 2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydene)-1,3-dihydro-2H-indol-2-one] which have a strongbactericidal, trypanocidal, tumoricidal, mycobactericidal, and antioxidant [10]. As an example,violacein was reported to display antibacterial activities toward Staphylococcus, Streptococcus,Bacillus, Mycobacterium, Neisseria, and Pseudomonas [11].

Successful commercialization ventures for microbial products are normally hampered bythe high cost of synthetic growth mediums. In view of this, various studies have been carriedout to explore the possibility of using other types of growth medium which is cheaper andeasily available such as agricultural wastes materials [12]. Besides providing a profitablemeans of reducing the substrate cost, the use of these agro-industrial residues in bioprocessescan also reduce its environmental impact [13].

Materials and Methods

Isolation and Identification of Bacterium

In this study, C. violaceum was isolated from soil sample collected from the vicinity of awastewater treatment plant in one oil refinery premise in Negeri Sembilan, Malaysia. C.violaceum was isolated using the culture-enrichment technique where 1 g of soil sample wasaseptically transferred into a 250-mL Erlenmeyer flask containing 25 mL of nutrient broth(NB) (8 g L−1) followed by incubation at 30 °C, 200 rpm for 24 h. Continuous subculturingonto fresh nutrient agar (20 g L−1) was carried out at 30 °C for 24 h until pure colonies ofbacteria were obtained. One small violet punctiform bacterial colony (designated as P1) wasselected and identified using the 16S rRNA gene sequencing analysis (Vivantis TechnologySdn. Bhd., Malaysia), where a 100% similarity with C. violaceum (EU 372837, AJ 871127)was obtained from a nucleotide sequence of 1061 bp (Fig. 1). The strain was deposited intoGenBank and allocated with accession number HM132057.

Appl Biochem Biotechnol (2012) 167:1220–1234 1221


Production of Violacein in Complex Media

The production of crude violacein was carried out using a series of 1-L Erlenmeyer flaskscontaining 200 mL of either Luria–Bertani (LB) [tryptone 10 g/L, yeast extract 5 g/L, NaCl10 g/L], NB [peptone 10 g/L, NaCl 5 g/L, yeast extract 3 g/L], tryptic soy broth (TSB)[peptone from casein 17 g/L, peptone from soymeal 3 g/L; D(+)-glucose 2.5 g/L; NaCl 5 g/L;K2HPO4 2.5 g/L], and peptone glycerol broth (PGB) [meat extract 10 g/L, peptone 10 g/L,glycerol 10% (v/v)]. pH of all the mediums set at 7.0±0.2. Bacterial inoculums (10%, v/v)were then added into the growth mediums followed by incubation at 200 rpm, 30 °C for24 h. Samples were withdrawn at specified time intervals to determine cell concentration(OD600), pH, and pigment concentration.

Production of Violacein in Various Agricultural Wastes

Active cultures of C. violaceum, 10% (v/v) were inoculated into a series of 500-mL Erlenmeyerflasks containing a mixture of sugarcane bagasse (SCB) (1%, 3%, and 5%, w/v) and 50 mL of100 mg/L L-tryptophan (at pH 7.0±0.2) followed by incubation at 200 rpm, 30 °C for 24 h.Following centrifugation (5,000 rpm, 20 min, 4 °C), violacein was extracted from the spentmedium (ethyl acetate; 1:5) as well as from the bacterial cells pellet (absolute methanol). Thebacterial cell pellets were dried at room temperature until constant weight prior to the determi-nation of cell dry weight. Stock solution of L-tryptophan was prepared by dissolving of 1 g of L-tryptophan powder in 1 L of a slightly basic distilled water. The solution was then neutralizedusing 0.1 M HCl followed by filter sterilization using a 0.45-μm Whatman filter paper.

Similar experimental setup was repeated using other types of agricultural waste, namelysolid pineapple waste (SPW) (1%, 3%, and 5%, w/v), brown sugar (BS) (0.4, 2.0, and4.0 g L−1), molasses (MOL) (0.1%, 0.5%, and 1%, v/v), and in 100 mg/L of L-tryptophan only.SPW was obtained from one local pineapple manufacturing facility in Tampoi, Johor; SCBfrom a local wet market; MOL from local supplier while BS were obtained from a local sundryshop. SCB and SPWwere dried, cut into 1.2–1.5 cm in length, and kept at 4 °C until further use.The stock solutions of BS andMOLwere prepared by dissolving either 40 g of BS or 0.5 mL ofmolasses in 1 L of distilled water followed by autoclaving at 105 °C, 101.3 kPa for 15 min.

Production of Violacein by SCB-Immobilized C. violaceum in Flow-Through Column

A glass column with an inner diameter (i.d.) of 1.5 cm, outer diameter (o.d.) 2.0 cm, andheight of 20 cm was used. Inlet and outlet points were set at 2 cm from the bottom and topsections of the column respectively. Silicone tubing (i.d., 2.0 mm; o.d., 4.0 mm) was fitted to

Fig. 1 The inter-relationship between isolate P1 and top 10 Blast hits from NCBI

1222 Appl Biochem Biotechnol (2012) 167:1220–1234


the inlet and outlet points while 3 cm3 of inert stones was packed at the top of the column toensure good flow distribution and retention of the column content. Following this, SCB waspacked inside the column to a volume of 27 cm3, which is considered as the working volumeof the column. Active cultures of C. violaceum (100 mL) were continuously pumped into thecolumn for 24 h using a down-flow mode at a flow rate of 3.5 mL min−1 to allow initialbacterial attachment. Using similar flow rate, 100 mL of 100 mg/L of L-tryptophan was thenpumped into the column for 24 h to promote bacterial growth. Violacein produced was thenrepeatedly extracted using fresh changes of 100 mL absolute methanol until completeelution was achieved.

Extraction of Pigment

Violet pigment was extracted from 24-h grown cell suspension of C. violaceum using thefollowing procedure; after centrifugation (7,500 rpm, 20 min), the bacterial cell pellets werewashed with distilled water followed by extraction using absolute methanol. The methanolicextract was concentrated using rotary evaporator (BÜCHI R-210, Switzerland) at 50 °Cfollowed by drying for 3 days at 60 °C. The dried extract was then characterized using UV–Vis, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR),and electron spray ionization coupled with mass spectrometry (ESI-MS). Supernatantobtained from the centrifugation was extracted for violacein using ethyl acetate (4:1) wherethe organic layer obtained was collected while the aqueous layer discarded. Violaceincontent in the organic layer was concentrated using rotary evaporator, determined for dryweight prior to use in the solubility and stability studies (Table 1).

Characterization of Pigment

The methanolic extract was determined for maximum wavelength, λmax using UV–Visspectrophotometer (Shimadzu UV-1601PC) between 800 and 200 nm. The dried bacterialpellet (1 mg) was finely ground with 200 mg KBr (Scharlau), pressed under 0.414 barfollowed by analysis using the FTIR spectrophotometer (Shimadzu, FTIR 8300) between4000 and 400 cm−1 and thin-layer chromatography (TLC) using a 0.20-mm pre-coated silicagel aluminum sheets (Merck Kieselgel 60F254) and developed using a mixture of benzene/

Table 1 The solubility of violet pigment (100 mg/L) in different types of solvent

Solvent at 25 °C Response Resultant color λmax (nm)

Water − Colorless NA

HCl (0.1 M) − Colorless NA

NaOH (0.1 M) +++ Light yellow 500

Acetone +++ Purple 557

Methanol +++ Blue 571

Ethyl acetate + Purple 555

n-Hexane − Colorless NA

Chloroform − Colorless NA

Paraffin oil − Colorless NA

Cooking oil − Colorless NA

+ slightly soluble, +++ highly soluble, − not soluble, NA not determined



acetone (2:1) [14]. Spots developed were sprayed with vanillin–H2SO4 solution followed byvisual inspection under UV light at 254 and 356 nm. The vanillin–H2SO4 solutionconsists of vanillin (0.5 g), methanol (80 mL), acetic acid (10 mL), and concentratedH2SO4 (5 mL). The powdered form pigment was analyzed using the NMR spectro-scopic analysis for 1H (400 MHz) and 13C (100 MHz) using Bruker Avance 400NMR spectrometer. The chemical shifts were reported in parts per million relative totetramethylsilane in deuterated dimethyl sulfoxide (DMSO) as solvent. The powderedpigment (0.5 g) was dissolved in DMSO prior to analysis using the ESI-MS. Theanalysis was carried out at the National Metrology Laboratory, Standard and IndustrialResearch Institute of Malaysia, Malaysia.

The solubility of the pigment was evaluated using various types of solvents, namelywater, HCl (0.1 M), NaOH (0.1 M), cooking oil, paraffin oil, acetone, methanol, ethylacetate, and n-hexane. Five milliliters of the solvents was added into a series of test tubescontaining 100 mg/L of pigment in methanol (stock pigment 4.2 g/L dry wt.) prior to mixingat 200 rpm for 12 h at room temperature. The λmax and absorbance were recorded using aHACH DR-5000 UV–Vis spectrophotometer. The stability of pigment toward pH and lightwas evaluated by varying the pH of 100 mg/L pigment in methanol (stock pigment4.2 g/L dry wt.) between 2 and 11, using either 1 M of NaOH or 1 M of HCl,followed by irradiating the pigment solutions with a low-pressured Hg dischargePhillips fluorescent light (18 W; 1 m distance). The effect of pH on pigment’sstability was recorded immediately after the pH adjustment while extent of photo-degradation was monitored using spectrophotometer at regular time intervals. Anotherset of experiment kept in the dark acted as control. The stability of pigment towardheat was evaluated using water bath at 20 °C, 40 °C, 60 °C, 80 °C, and 100 °C for15, 30, 60, 90, and 120 min, respectively. To study the effects of various metal species(500 mg/L of either Pb, Al, Mg, Ca, Cu), reducing agent (20–500 mg/L Na2SO3), andoxidizing agent (50–500 mg/L H2O2, 20–100 mg/L KMnO4) on the stability of pigment,100 mg/L of pigment (in methanol) was mixed with these solutions and left at roomtemperature for 2 h.

Results obtained from the pigment characterization studies are as follows; Rf00.43(benzene/acetone02:1); IR (KBr) υmax cm−1—3,700–3,000 (OH), 3,256.53 (−NH),1,657.07, 1,635.13 (C0O amide), 1,613.18 (C0C), 1,223.69 (C–O phenol), 1,543.93,1,223.69 (C–N); UV–λmax (MeOH) nm—585.72; 1H NMR (DMSO, 400 MHz)—δ 6.79(1H,dd, J02.0 Hz and 8.8 Hz, H-7), 6.83 (1H, d, J07.6 Hz, H-22), 6.94 (1H, t, J07.6 Hz, H-20), 7.20 (1H, t, J07.6 Hz, H-21), 7.23 (1H, d, J02.0 Hz, H-5), 7.35 (1H, d, J08.8 Hz, C-8),7.55 (1H, d, J02.0 Hz, H-13), 8.06 (1H, d, J02.4 Hz, H-2), 8.93 (1H, d, J07.6 Hz, H-19),9.32 (s, 6-OH), 10.60 (s, 15-NH), 10.72 (s, 10-NH), 11.87 (s, 1-NH); 13C NMR (DMSO,100 MHZ)—δ 97.5 (C-13), 105.1 (C-5), 106.2 (C-3), 109.5 (C-22), 113.6 (C-7), 113.9 (C-8),119.2 (C-17), 121.3 (C-20), 122.9 (C-18), 126.1 (C-4), 126.8 (C-19), 129.9 (C-2), 130.1 (C-21), 132.1 (C-9), 137.5 (C-12), 142.3 (C-23), 148.1 (C-14), 153.4 (C-6), 170.7 (C-16), 172.1(C-11); ESI-MS m/z (relative intensity)—342.34 [ [M-H]−, C20H13O3N3]; ESI-MS/MS m/z(m/z 342.34)—298.42, 209.07, 157.16.

Statistical Analysis

All data collected were analyzed for variance using ANOVA, and probability tests wereconducted to determine the degree of differences between the means obtained with P<0.05regarded as significant while P<0.01 was considered very significant. All assays werecarried out in duplicate.



Results and Discussion

Production of Violacein in Various Complex Media

The use of different growth media, i.e., NB, LB, TSB, and PGB, directly affects the growthand production of violacein by C. violaceum. The OD600 reached a maximum of 1.83 after24 h in LB followed by 1.81 (TSB), 1.66 (NB), and 1.15 (PGB). However, this conditiondoes not directly correlates with the pigment production with the highest pigment production(0.15 g L−1) achieved when C. violaceum was grown in NB and lowest in TSB (0.05 g L−1).This observation could be due to the higher concentration of peptone and yeast extractpresent in NB compared to other medium. Peptone was reported to impact to greatest effecton cellular mass and crude violacein production (Mendes et. al. 2001) [10]. However, highglucose concentration (2.5 g/L) in TSB medium repressed the violacein production eventhough the cell concentration was higher compared to in NB. This could suggest theexistence of genetic repression mechanisms during the production of violacein by C.violaceum in high concentration of glucose. The cellular mass of some microorganismswas reported to increase during rapid metabolisms of carbon sources (e.g., glucose) whereduring this time the production of secondary metabolite, such as violacein, decreases.However the use of carbon sources of slower consumption (e.g., sucrose, lactose) resultsin a decrease of cellular growth and an increase in secondary metabolite production [15]. Nopigment production was observed when the bacteria was grown in the PGB medium (rich inpeptone) indicating the important role of yeast extract (present in all of the other mediums)in inducing pigment production by C. violaceum. This also indirectly implies that violaceinis not required for growth and survival of C. violaceum [16].

Production of Violacein Using Various Agricultural Wastes

The highest production of violacein (0.82 g L−1) was achieved when C. violaceum wasgrown in the SCB3 medium (3 g of SCB/100 ppm of L-tryptophan) while the lowest(0.19 g L−1) in SCB1 medium (1 g SCB/100 ppm of L-tryptophan) as shown in Fig. 2.The violacein production was observed both on the SCB (solid support) and the culturebroth for the SCB3 medium while violacein was only produced in the culture broth in theSCB1 medium. Other medium formulations showed negligible or poor pigment yields (ingrams per liter); SPW1 0.01, SPW3 0.07, SPW5 0.03, BS1 0.02, BS5 0.02, BS10 0.08, MOL1

0.05, MOL5 0.03, MOL10 0.03, and SCB5 not detected. In view of this, SCB3 was chosen forsubsequent pigment production studies. SCB-immobilized cells of C. violaceum were alsoable to produce 0.15 g L−1 of pigment after 24 h in a flow-through column system.

Agricultural waste materials used in this study is known to consist of high concentrationsof sucrose, glucose, and fructose [17] and substantial amounts of organic acids (citric,maleic, ascorbic), protein, phenolic compounds, and several trace elements which is crucialfor bacterial growth [18]. SCB for example contain cellulose (40% wt.), hemicelluloses(27% wt.), and chitin (10% wt.) [19]. These lignocellulosic components can serve as carbonsource for the bacterial growth and also as immobilizing material for the bacterial cells. C.violaceum has been reported to utilize cellulose (as C source) based on the presence ofcellulases enzymes [20] via the conversion of glucose into various types of aromatic aminoacids (such as tryptophan). Even though the hexose monophosphate pathway is incompletedue to the lack of genes encoding for 6-phosphogluconate dehydrogenase, cellulose degra-dation was not affected which could be due to the presence of other enzymes required in thepathway [21]. This characteristic should enable C. violaceum to convert cellulose from SCB



into smaller sugar units followed by the production of violacein (which consist of 2 U oftryptophan), since glucose has been reported as one of the crucial factors for violaceinproduction [10]. However, glucose also acts as the limiting factor in violacein production asit can inhibit even distribution of dissolved oxygen leading to anoxia which can retardviolacein production by C. violaceum, which occurs aerobically [22].

Characterization of Pigment

The violet pigment (in methanol) showed a maximum peak with strong absorption at585.72 nm which is mainly due to the presence of chromophore groups, such asalkene and carbonyl (responsible for electronic absorption). Even though the carbonylgroup absorbs strongly at shorter UV–Vis wavelength, the intensity decreases athigher wavelength owing to the participation of n electrons. Hence, the significantrole of the alkene groups in contributing toward the conjugation effect where the greater thenumber of conjugated double bonds, the longer the absorption wavelength (bathochromic shift)[23].

Results from the FTIR analysis of the crude methanolic extract are as follows: 3,700–3,000 cm−1 (OH), 3,256.53 cm−1 (N–H), 1,657.07 cm−1 amide, 1,635.13 cm−1 (C 0 Oamide), 1,613.18 cm−1 (C 0 C), 1,223.69 cm−1 (C–O phenol), and 1,543.93, 1,223.69 (C–N). Lower absorption frequency for the carbonyl group can be attributed to the conjugationeffect with an aromatic ring resulting in the delocalization of the π electrons of bothunsaturated groups. This reduces the double bond character of both the bonds causing adecrease in the frequency from 1,718 cm−1, i.e., normal absorption frequency for carbonyl,to 1,657.07 cm−1 and alkene stretching frequency from 1,645 cm−1, i.e., normal absorptionfrequency for alkene, to 1,613.18 cm−1. These conditions are attributable to the resonancestructure of carbonyl containing group in double bond structure [23]. From the TLCanalysis, two single spots with Rf values of 0.43 (indicative of violacein) and 0.5

Fig. 2 Production of pigment by C. violaceum grown in 100 mg/L tryptophan and various concentrations ofsugar cane bagasse (SCB), solid pineapple waste (SPW), brown sugar (BS), and molasses (MOLS). Error barsrepresent standards deviations of the means (n02)



(representative of deoxyviolacein; obtained after the addition of vanillin–H2SO4 reagent)were recorded. This condition is supported with that reported by DeMoss and Evans [22].

The 1H NMR spectrum (Fig. 3a) showed the presence of 13 protons with three singletsignals observed at the down-field region that corresponds to indole NH at δ 11.87 ppm,lactam NH at δ 10.72 ppm, and isatin NH at δ 10.60 ppm. The appearance of the three low-

Fig. 3 1H NMR (a) and 13C NMR (b) spectrum of violet pigment



field signals from indole and pyrrole NH protons was due to the presence of electron-withdrawinggroup substituents attached to the pyrrole ring which can be attributed to the H-bonding with thesolvent. These three distinct and sharp NH signals are the most striking features of violacein asreported by Yada et al. [24]. The three aromatic −H in the indole skeleton which corresponds tothemeta-couple and ortho-couple signals were detected at δ 6.79 ppm (dd, J02.0, 8.8 Hz) that canbe attributed to H-7, δ 7.23 ppm (d, J02.0 Hz) to H-5 and δ 7.35 ppm (d, J08.8 Hz) to H-8.Signals representative of the H-5 and H-7 are a distinct feature for the binding between thephenolic hydroxyl group and the ortho group in the indole skeleton [25]. Four protons wereobserved in the aromatic isatin skeleton at δ 8.93 ppm (d, J07.6 Hz, H-19), δ 6.94 ppm (t, J07.6 Hz, H-20), δ 7.20 ppm (t, J07.6 Hz, H-21), and δ 6.83 ppm (d, J07.6 Hz, H-22). A doubletpeak at δ 7.55 ppm with J02.0 Hz was assigned to H-13 in the lactam skeleton.

The 13C NMR spectrum (Fig. 3b) exhibited 20 signals that correspond to the carbonylcarbons at δ 170.7 ppm (C-16) and δ 172.1 ppm (C-11) as well as 18 other carbons at δ 97.5(C-13), 105.1 (C-5), 106.2 (C-3), 109.5 (C-22), 113.6 (C-7), 113.9 (C-8), 119.2 (C-17),121.3 (C-20), 122.9 (C-18), 126.1 (C-4), 126.8 (C-19), 129.9 (C-2), 130.1 (C-21), 132.1 (C-9), 137.5 (C-12), 142.3 (C-23), 148.1 (C-14), and 153.4 (C-6). The signal at δ 153.4 stronglyindicates that the pigment analyzed was violacein rather than deoxyviolacein whose C signalnormally appears at the higher magnetic field due to the absence of hydroxyl residues [26].

Mass Spectrometry

In the negative ion ESI-MS spectrum, a quasimolecular ion peak was observed at m/z 342.34[M–H]−. The MS/MS spectrum of the ion at m/z 342.34 showed the fragmented ions at m/z298.42, m/z 209.07, and m/z 157.16. All of these peaks correspond to the molecular formulaof C20H13O3N3 as suggested previously for other violacein-producing bacterium such asJanthinobacterium lividium [27].

The Solubility of Violet Pigment

The solubility of violet pigment relies on the polarity and charges carried by the pigmentmolecules and property of the solvent. The pigment was determined not to dissolve in water,alkaline water, oils, and non-polar organic solvent, such as n-hexane and chloroform, but has highaffinity toward polar solvents such as acetone andmethanol. The pigment showed good solubilityin alkaline water (with color change to light yellow) but only slightly soluble in ethyl acetate.

The high solubility of violacein in polar solvents can be attributed to the presence ofcarbonyl, hydroxyl, and amine groups in its structural units namely hydroxyindol, oxoindol,and pyrrolidone, which assists in the formation of H-bonding with the solvent molecules(acetone and methanol). In alkaline water solution, the hydroxyl group will be deprotonatedleading to the formation of enolate ions which is highly water soluble.

The Stability of Pigment

Stability Toward pH, Light, and Temperature

Violacein showed good stability toward light with more than 80% residual pigment deter-mined after 30 days at pH 5 to 9. However, at high pH value (pH 11), violacein turnedcolorless after 24 h, both in the presence and absence of light, with more than 90% degree ofdecomposition. In highly acidic condition (pH 2), violacein showed moderate degree ofdecomposition with around 65% of residual pigment remained in solution after 30 days. In



general, adjusting the solution’s pH to 5–9 resulted in the lowest effect on pigment intensity(based on the amount of residual pigment remaining in solution), hence should be a usefulworking pH for subsequent studies. On another note, the rate of color reduction is fasterupon exposure to light (Fig. 4).

The degradation rate of violacein upon exposure to light can be calculated using the first-order kinetic as described in Eq. 1 where Ri is residual percentage of pigment at specifiedtime interval, Ro is initial residual percentage of pigment, and k is the degradation rateconstant which can be obtained from the slopes of regression lines by plotting ln (Ri /Ro) as afunction of time. The half life (t1/2) of the pigment was calculated using the first orderArrhenius equation (Eq. 2).

lnRi

Ro

� �¼ �kt ð1Þ

t12¼ 0:693

kð2Þ

Table 2 summarizes the degradation constant, k and half-life, t1/2 for violacein at differentpH values and after exposure to light. Most of the residual pigments were degraded after24 h with pH 11 resulted in the highest degradation rate. In the absence of light, the

Fig. 4 Stability of violet pigment at various pH upon exposure to fluorescent light (a) and kept in the dark (b)for 30 days



degradation rate for the pigment was very slow at pH 5–9, hence not possible to beelucidated using the first-order kinetics.

Violacein has been reported to change to darker blue at pH 2 and to green at pH 13 [28].In alkaline condition, excess OH− ions from NaOH deprotonates the phenolic group of thehydroxyindol and amine group of oxoindol and pyrrolidone causing the formation of anionand destruction in the conjugated structure of the pigment. This can be supported from theUV–Vis analysis where at pH 11, the degradation of pigment is clearly demonstrated fromthe total reduction of absorption at λmax with no shifting observed (Fig. 5). Similar resultswere reported by Wang et al. and Oetari et al. regarding the instability of curcumin at neutral

Table 2 Degradation constants, k, and half-life, t1/2 profiles for violacein at different pH values after exposureto light

Condition pH k (h−1) R2 t1/2 (h)

With light 2 6×10−4 0.9819 1,155

5 2×10−4 0.9907 3,465

7 2×10−4 0.9740 3,465

9 2×10−4 0.9904 3,465

Without light 2 2×10−4 0.9344 3,465

0.00

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.60

A

pH 11 pH 2 pH 5 pH 7 pH 9

300.0 350 400 450 500 550 600 650 700 750 800.0

300.0 350 400 450 500 550 600 650 700 750 800.00.00

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.80

nm

A

a

b

pH 11 pH 2 pH 5 pH 7 pH 9

nm

Fig. 5 UV–Vis absorption spectra of violacein at pH 2–11 at a 0 h and b after 24 h



to alkaline pH conditions [29, 30]. At alkaline pH, proton would be reduced from thephenolic group on curcumin, leading to the destruction of pigment structure.

The susceptibility of violacein toward light was due to the absorption of light in the UVandvisible range which leads to the excitation of electrons from chromophore groups to unstableand short-lived excited state. The excess energy trapped in the excited molecules caused higherreactivity of the violacein molecules toward undesirable chemical reactions such as photo-oxidation [31]. Some natural functions of microbial pigments have been reported by Liu andNizet which among others are as protection against ultraviolet radiation and oxidizing agents[7]. Violacein (in methanol) also showed good stability when heated for 2 h at temperaturesranging from 40 °C to 100 °Cwith residual pigments in solution around 97% (Fig. 6). This mayindicate its suitability to be applied in various industrial applications.

Stability Toward Metal Ions

After 1 h mixing of the pigment with 500 mg/L of either the transition or alkaline metal solutions,only the transition metals (Pb, Cu) resulted in color change of violacein while the strong violetremained in the alkaline metal solutions (Al, Mg, Ca). The precipitation of Pb interferes with theabsorbance for pigment–Pb solution while Cu resulted in a 50% reduction in violet absorption(Table 3). Further investigation on the effects of Cu(II) ions on violacein showed a disproportionalrelationship between the intensity of pigment and the concentration of Cu(II) ions (Table 4).

Fig. 6 Stability of pigment at different temperatures. Error bars represent standard deviation of the means(n02); P≤0.05

Table 3 Effect of 500 mg/ L Pb, Cu, Al, Mg, and Ca ions on the stability of violacein

Condition λmax Absorbance Color change

Violet pigment (control) 567 0.707±0.034 Violet

Violet pigment+500 mg/L Pb 574 1.057±0.133 Cloudy violet

Violet pigment+500 mg/L Al 574 0.696±0.089 Violet

Violet pigment+500 mg/L Mg 574 0.765±0.006 Violet

Violet pigment+500 mg/L Ca 574 0.672±0.018 Violet

Violet pigment+500 mg/L Cu 570 0.462±0.055 Gray

Values shown are means of n02; P≤0.05



Greater impact of transition metals to the intensity of violacein compared to light metal ions hasalso been reported by other researcher [32].

Zhang et al. reported the formation of sediment upon the mixing of Pb with blue pigmentextract from Streptomyces coelicolor while the presence of Fe(II) ions ≥0.5 mM woulddenature the pigment [32]. Betalain degradation was also accelerated in the presence ofmetal cations such as Fe(II), Fe(III), Sn(II), Al(III), Cr(III), and Cu(II) due to the formationof metal–pigment complexes accompanied by bathochromic and hypochromic shift [33].

Stability Toward Oxidizing and Reducing Agents

The introduction of 25–100 mg/ L of KMnO4 solution (oxidizing agent) into the violaceinsolution resulted in absorbance decrease from 1.054 to a maximum of 0.526 with shift inλmax from 570 to 537 nm. Color of the solutions also changed from violet to light orange–dark brown. For 50–500 mg/L Na2SO3 (reducing agent), the absorbance fluctuates between0.915 and 1.545 from an initial value of 1.054. Minimal shift in λmax (from 570 to 573 nm)was also recorded with color change from violet to dark blue–pale yellow (Table 5).Violacein also showed good resistance toward H2O2 with negligible results recorded interms of absorbance, color change, and λmax (data not shown).

Table 4 Effect of 50, 100, 250, and 500 mg/L Cu(II) ions on the stability of violacein

Cu (II) concentration (mg/L) λmax Absorbance Color change

0 570 0.900±0.110 Violet

50 570 0.581±0.152 Light purple

100 570 0.294±0.103 Light orange

250 570 0.140±0.003 Yellow

500 570 0.110±0.006 Yellow

Values are mean±SD (n02); P≤0.05

Table 5 Effect of 25–100 mg/L of KMnO4 or Na2SO3 on the stability of violacein

Oxidizing/reducing agent (mg/L) λmax Absorbance Color change

KMnO4

0 570 1.054 Violet

25 537 0.163±0.024 Light orange

50 537 0.262±0.000 Brown

75 537 0.396±0.013 Brown

100 537 0.526±0.005 Dark brown

Na2SO3

0 570 1.054 Violet

50 570 1.234±0.011 Violet

100 570 1.053±0.052 Violet

250 573 1.545±0.050 Dark blue

500 573 0.915±0.000 Pale yellow

Values are means of n02; P≤0.05



Oxidative bleaching effect of KMnO4 on the violacein structure may occur at thehydroxyl group of hydroxyindol which results in the formation of ketone and MnO2 asprecipitates. The oxidation can also take place at the alkene double-bond linking thepyrrolidone and oxoindol structure, leading to the formation of glycol and MnO2 asprecipitates. The purple permanganate color disappear; instead, the MnO2 formed wasobserved as brown precipitates. Violacein has been reported to possess significant antioxi-dant activity and can protect against lipid peroxidation caused by hydroxyl radicals, animportant role of microbial pigments [34].

Conclusion

This study successfully demonstrated the use of cheap and easily available agricultural wastematerials (sugarcane bagasse, solid pineapple waste, molasses, brown sugar) as growthmedium for violacein-producing C. violaceum. Due to the relatively fast propagation timefor bacteria compared to other color-producing microorganisms (yeast, fungi), this finding isimportant as it projects the useful role of the agricultural waste material as nutrient duringcommercial-scale propagation of the bacteria. In view of this factor together with desirablephysicochemical properties and biodegradability feature, it is envisaged that bacterial pig-ments such as violacein would be thrusted into commercial application sooner rather thanlater.

Acknowledgments The authors acknowledge the contribution from the Ministry of Agriculture, Malaysiafor the Technofund grant (TF0310F080), the Ministry of Science, Technology and Innovation, Malaysia forthe National Science Fellowship scholarship, and the Ministry of Higher Education, Malaysia for the MyPhDscholarship to Nur Zulaikha Yusof and Nordiana Nordin at Universiti Teknologi Malaysia.

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Application of Bacterial Pigments as Colorant

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