# Applications of Natural Violet Pigments from HalophilicChromobacterium violaceum PDF23 for Textile Dyeing with Antimicrobial and Antioxidant Potentials

Authors: Antonyraj Matharasi Perianaika Anahas; Subramanian Kumaran; Mahmoud Kandeel; Gangatharan Muralitharan; Jenifer Silviya; Geja Lakshmi Adhimoolam; Mani Panagal; Sampath Renuka Pugazhvendan; Gopal Suresh; A. Wilson Aruni; Senthil Rethinam; Nainangu Prasannabalaji
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Natural colorants have emanated as a significant substitute to highly toxic synthetic dyes. The present study highlights the dyeing efficiency of violet pigment produced fromChromobacterium violaceum PDF23. A halophilic bacterium C. violaceum PDF23 produce violet pigments were isolated from the Great Salt Lake situated in Chennai, India. Based on morphology and 16S rRNA gene sequencing, a halophilic bacterium was identified as C. violaceum PDF23. The violet dye from C. violaceum PDF23 exhibited antimicrobial efficacy against S. aureus, B. subtilis, E. coli, P. aeruginosa, and C. albicans. The violet pigment exhibited radical scavenging potential with IC50 value of 14.40 μg/mL. The dye ability of colorant was evaluated using mordants on two distinct fabrics, i.e., silk satin and cotton, and colorfastness measurements were recorded. The CIE L∗a∗b∗ qualities of the dyed fabrics were assessed, as well as their colorfastness quality. The current research findings suggest that violet pigment has potent antimicrobial and antioxidant activity and it could be used as alternative to synthetic dye in the textile industry.


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## Body

## 1. Introduction

Anthropogenic activity-induced ecosystem toxicity is a complex and significant issue that society, scientists, and regulatory agencies are struggling around the world [1, 2]. Synthetic dyes have been used extensively in the industrial processes of food, fabrics, health supplements, and pharmaceutical products [3]. Synthetic dyes raise carcinogenic levels in the ecosystem, such as sulfur, phosphorus, nitrogen, and other heavy metal ions, which promote the growth of algae, fungi, and cyanobacteria [4]. The negative consequences of synthetic dye manufacture and application, such as mutagenicity and toxicity, have prompted health-conscious consumers to seek out more eco-friendly and greener colorants known as natural dyes [5–7]. Synthetic dyes have been prohibited worldwide by environmental organizations from the USA, Germany, and Italy due to their fatal and carcinogenic impacts, and natural dyes are being promoted to conserve the environment around the world [8].To combat the drawbacks of synthetic dyes, natural dyes have attracted the interest of researchers and industrialists due to their non-hazardous, bio-degradable, sustainable, nonallergic, anticancer, and environment-friendly properties [9, 10]. Natural dyes have also performed a significant role in preventive medicine, including anti-inflammatory, antihemolytic, antipyretic, antifungal, antioxidant, and antibacterial activities [11]. Natural dyes are recognized to be safer for the environment and produce less harmful waste during textile dyeing than synthetic colorants [7, 12].Natural dyes have a variety of advantages, including light stability, heat stability, and pH stability [13]. Similarly, the use of natural colorants as a textile fabric has gained much importance as a result of similar qualities. The production of natural colors in the food coloring industry has been increased to 10-15% per year, and growing perception of food colorants and the detrimental effects of synthetic colorants is the key reason behind the growing demand. There is a global prohibition on the manufacture and import of synthetic dye-based colors and clothing accessories in several nations throughout the world [12].Natural colorants generated from microorganisms are commonly employed as dyeing agents because of their inexpensive production costs, ease of extraction, higher productivity through strain creation, and lack of temporal fluctuations [13]. Natural colorants produced by bacteria, fungi, algae, and cyanobacteria were used in textile industries [14]. Due to the high demand for natural dyes, industrial manufacturing of these products is rapidly rising. As a result, new varieties of natural colorants have been introduced, dramatically expanding the paradigm of hygiene and protection. The annual rate of the dye industry is predicted to increase about 7%, and the demand is expected to increase $7.79 billion by 2020 [15].For dyeing fabrics, microbial dyes have been used extensively.C. violaceum has been widely investigated in the production of violacein, while different bacterial species have been identified in diverse amounts and process parameters [13, 16]. Due to its therapeutic potential and mechanism of action, violacein pigment produced by Chromobacterium has received extensive interest [17, 18]. Violacein has a wide range of therapeutic potential including broad-spectrum antimicrobial properties, potent bactericidal and anticancer properties, and antimalarial, antiulcerogenic, antiviral, antibiotic, antitumor, and antiparasite properties [19–21]. Many bacterial strains synthesize violacein, including Chromobacter [22, 23], Pseudoalteromonas [24, 25], Janthinobacterium [26, 27], and Duganella [28]. In addition, bacterial strains producing violacein have been isolated from various geographical regions.Mordanting takes significant use of the materials, both color and severity, by combining three types of complexation [29]. In natural fibers, the use of mordants offers great color strength. The most often used mordants are alum, nickel, chromium, iron, copper, and tannic acid [30]. Bio-mordants are non-toxic to humans and the environment, are eco-friendly, and are less expensive for dyeing [12, 31].Radiation technique is gaining prominence in the fabric industry, particularly in dyeing process, because of its technical and economical efficiency, as well as its acceptability [31, 32]. The low color intensity derived from natural dyes has led to the use of advanced approaches such as irradiation techniques in the fabric industries to overcome this constraint [12, 33]. These radiations not only improve the colorant extraction efficiency and improve the dyeing technique faster, but it is also significantly more efficient in terms of duration, expense, manpower, and energy [34]. The ultrasonic radiation process, as a green technique in natural dyeing, offers a lot of potential for isolating colorants from natural sources [35, 36].In this study, we identified a new bacterial strain of halophilicC. violaceum PDF23 that produces a compound of violet pigment with a wide array of biomedical applications. The violet pigments showed wide range of antimicrobial properties, antioxidant capacity, and application in textile dyeing of fabrics. In future, researchers should aim to study whether these properties could be imparted to textiles to obtain fabrics for special applications.


## 2. Materials and Methods

### 2.1. Sampling Site

Soil sediments were collected from 12 different locations at Great Salt Lake located in Chennai, India (Lat.12°43′ 53.99″ N Long. 80 °13′ 0.41″ E) (Figure 1). All of these samples were obtained aseptically in sterile plastic containers, which were placed in the icebox and transported for processing to the laboratory.Figure 1
Map of India, Tamilnadu showing Chennai, Great Salt Lake (10° 50′ N 78° 46′E), where the halophilic sediment samples were collected shown in red.


### 2.2. Bacterial Strain Isolation and Purification

Serial dilution and plating methods were used to isolate bacteria, according to the method of Verma et al. [37]. The 10-1 dilution was made by mixing 1 g of soil in 9 mL of 0.85% saline. An aliquot of 0.01 mL was taken from each dilution (10-2 to 10-6) and plated on petri plates containing nutrient agar and incubated at 25°C. A total of 5 distinct morphological colonies are identified for 12 isolates (PDF11 to PDF23) as shown in Table 1. The violet-colored bacterial isolate was identified as PDF23 among the various bacterial isolates. The PDF23 isolate was purified after three consecutive streaks on nutrient agar medium and stored at -80°C.Table 1
Pigment-producing bacteria were isolated from various sites around Great Salt Lake in Chennai, India.
S. No.OrganismPigmentGram stainingPDF12Yellow(–ve) rodPDF13Yellow(+ve) cocciPDF14Red(–ve) rodPDF15Yellow(+ve) cocciPDF16Red(–ve) rodPDF17Orange(–ve) rodPDF18Pale yellow(+ve) cocciPDF19Red(–ve) rodPDF20Yellow(–ve) rodPDF21Pale yellow(+ve) cocciPDF22Red(–ve) rodPDF23Violet(–ve) rod


### 2.3. Biochemical Analysis of Bacterial Isolate PDF23

Gram staining, cell morphology, and pigment synthesis were used to identify the violet-colored bacterial isolate PDF23. Enzyme activity such as oxidase, catalase, urease, triple sugar iron agar test, citrate test, indole, MR-VP test [38, 39], and motility test were carried out according to method of Litchfield and Gillevet [40].


### 2.4. Molecular Analysis

#### 2.4.1. DNA Extraction

The bacterial isolate PDF23 was grown to reach an A600 of 1.0. After growing, the cells were harvested by centrifugation at 10,000 rpm for 5 min at 4°C. As previously mentioned by [41], total genomic DNA was extracted from bacterial cell pellets. Genomic DNA was resolved on a 1% agarose gel with 1X TAE buffer, and DNA was imaged using a gel documentation unit (Bio-Rad, USA) [39].


#### 2.4.2. Analysis of 16S rRNA Gene by PCR Amplification

Total genomic DNA was employed in PCR amplification of the 16S rRNA gene using primers 27 F (5′-AGAGTTGGATCTGGCTCG-3′) and 1492R (5′-ACCTTGTTACGACTT-3′) [42]. The PCR cycling conditions were set as initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C, 2 min, with a final extension of 10 min at 72°C. The PCR product was separated on 1% agarose gel.


#### 2.4.3. Sequencing and Phylogenetic Tree Construction

The 16S rRNA gene PCR product was purified and sequenced on both strands using 27 F and 1492R primers using the PCR cleanup kit (Qiagen, USA). MEGA X was used to generate a phylogenetic tree using the neighbor joining method [43]. The PDF23 bacterial isolate 16S rRNA nucleotide sequences were submitted to the GenBank database, and accession numbers were received (MT176501).


### 2.5. Growth and Violacein Production

The growth and synthesis of violacein from PDF23 strain were evaluated in a modified nutrient broth with sodium chloride deprivation. An overnight culture dissolved in saline solution was used as the inoculums at a 5% (v/v) concentration. The experiment was carried out in a 250-mL Erlenmeyer flask and incubated at 25°C under agitation (180 rpm). To determine the increase in biomass and violacein, 1 mL of distilled ethanol was added to the pellet-pigment extract and homogenized to attain the ethanol-phase pigment and 1 mL of culture broth was extracted and separated by centrifugation at 10,000 rpm for 10 min. The filtrate was agitated again, and the pellet and supernatant were extracted. The absorbance of ethanolic supernatant was measured at 575 nm to assess the presence of violacein. For biomass quantification, the pellet was dissolved in 1 mL saline, and the absorbance was measured at 600 nm [44].


### 2.6. Extraction of Violacein Dye

The violet pigment was extracted from a 24-hr-oldC. violaceum cell suspension using the following procedure. Bacteria cells were harvested after an incubation time of 24 hrs, and the cells were collected by centrifugation simultaneously at a speed of 7,000 rpm and 4°C. Pellets were obtained by the extraction methods, and three different solvents were applied to each separate pellet, methanol, acetone, and ethyl acetate, where the solution was also separated by centrifugation at 7,000 rpm for 4°C. The separation technique was replicated till the pellet turned colorless. Violet and blue dyes were extracted from C. violaceum using this extraction process. To remove methanol, acetone, and ethyl acetate from the pigments, the rotary evaporator was used to obtain the pigments in crude form. The time required for condensation at 45°C was 1 h per 250 mL. For 1 L of solution, crude extraction yielded around 0.2 g of pigments [45].


### 2.7. Characterization of Natural Dye

#### 2.7.1. UV-Vis Spectral Analysis

The methanol extract of crude pigment was evaluated using the UV-vis spectrophotometer (Shimadzu UV-1601PC) between 800 and 200 nm for maximum wavelength [46].


#### 2.7.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

FT-IR spectrophotometer (Shimadzu FTIR-8400S) was used to determine violet pigment derived fromC. violaceum PDF23 in the wave number range from 400 to 4000 cm-1. On the diamond glass of the spectrophotometer, approximately 2 μg of dyes were mounted [47]. Various functional groups found in the pigments were identified using the FTIR spectrum.


### 2.8. Applications of Violet Pigment in Textile Dyeing

#### 2.8.1. Fabrics

Two types of fabrics, silk satin and cotton, were used to evaluate the dye-ability of pigments.


#### 2.8.2. Dyeing of Fabric Samples

For all fabric samples of liquidC. violaceum PDF23 dye for both silk and cotton, staining was performed at boiling temperature (100°C). Silk fabric dyeing was performed at a dye concentration of 2%, with a fabric: liquid proportion (1 : 50) and varying mordant concentrations at 60°C and an interval of 60 min. After staining, the colored fabric was rinsed at 60°C for 15 min with 3 g/L of nonionic detergent, then washed with cold water, and allowed to dry at 37°C. For staining of silk satin and cotton fabrics, four parameters affecting color efficiency and intensity were evaluated: mordants, mordant concentration, fabric pretreatment, and natural pigment strength characteristics [48].


#### 2.8.3. Mordant

Ferrous sulfate, sodium silicate, alum, copper sulfate, and calcium hydroxide were used in the forms of mordants [49].


#### 2.8.4. Pretreatment of Fabric

Before proceeding to the pretreatment process, about 1 g of fabric was soaked in alum and Na₂CO₃. The solution was heated for 2 hrs along with the fabrics to ensure that the fabrics were processed efficiently. The fabric was rinsed and dried at 37°C. Silk was pretreated in a similar manner, except sodium carbonate. Pretreatment of the fabric was done to remove pectic compounds and cotton wax found in cotton weave fabrics, as well as particles on the surface of silk fabrics. The sample, which includes alum and Na₂CO3, was prepared for pretreatment of cotton fabrics by dissolving 0.2 g alum and 0.06 g Na₂CO₃ in 30 mL of distilled water, heating and thoroughly mixing by homogenization [50].


### 2.9. Assessment of Dyed Fabric

#### 2.9.1. Color Strength and Colorfastness Standard Tests

The colored fabrics were analyzed in compliance with ISO standard techniques. The transmittance of the colored materials was measured using a UV-vis spectrophotometer (Premier Color Scan, Model: SS 5145A, India). The Kubelka-Munk equation was used to calculate absolute color characteristics (K/S values) [47].
(1)KS=1−R22R−1−R22R0where R is the decimal fraction of dyed fabric reflectance, R0 is the decimal fraction of undyed fabric reflectance, K is the absorption coefficient, and S is the scattering coefficient.


### 2.10. Antimicrobial Activity by Minimum Inhibitory Concentration (MIC)

The antimicrobial efficacy of violet pigments was used to evaluate the percentage of inhibition against human pathogens such asS. aureus, B. subtilis, E. coli, P. aeruginosa, and C. albicans [51]. The bacterial strains were seeded in Mueller Hinton broth incubated at 37°C for 24 hrs, while the yeast grown in Sabouraud dextrose broth incubated at 30°C for 24 hrs. All the test strains were adjusted to 106 CFU/mL (McFarland standard) and used for assay. Different concentrations of violet pigments (0.25, 0.50, 1.0, and 2.0 mg/mL) were loaded into the wells to obtain final volume of 200 μL in microtiter plate. For bacterial strains, microtiter plates were incubated at 37°C and for yeast strains at 30°C. After incubation, the plates were recorded for bacterial and fungal growth inhibition using microtiter plate reader.


### 2.11. Antioxidant Efficacy of Violacein Pigments

The DPPH analysis was performed based on the decolorization efficiency of this molecule in the presence of antioxidants and robust technique for measuring the scavenging ability of free radicals [52]. Ascorbic acid was used as the standard control. In 1 mL of 3 mM DPPH solution, different concentrations of violet dye (5–100 μg/mL) were added and incubated in the dark for 30 min at 30°C. The color transition from violet to yellow was measured at 517 nm. The IC50 value denotes the dye concentration required to scavenge 50% of the free radicals generated by DPPH.Using the following equation, the percent inhibition was quantified:(2)%Inhibition=Ac−As/Ac×100,where Ac denotes control absorbance and As denotes pigment absorbance.


## 2.1. Sampling Site

Soil sediments were collected from 12 different locations at Great Salt Lake located in Chennai, India (Lat.12°43′ 53.99″ N Long. 80 °13′ 0.41″ E) (Figure 1). All of these samples were obtained aseptically in sterile plastic containers, which were placed in the icebox and transported for processing to the laboratory.Figure 1
Map of India, Tamilnadu showing Chennai, Great Salt Lake (10° 50′ N 78° 46′E), where the halophilic sediment samples were collected shown in red.


## 2.2. Bacterial Strain Isolation and Purification

Serial dilution and plating methods were used to isolate bacteria, according to the method of Verma et al. [37]. The 10-1 dilution was made by mixing 1 g of soil in 9 mL of 0.85% saline. An aliquot of 0.01 mL was taken from each dilution (10-2 to 10-6) and plated on petri plates containing nutrient agar and incubated at 25°C. A total of 5 distinct morphological colonies are identified for 12 isolates (PDF11 to PDF23) as shown in Table 1. The violet-colored bacterial isolate was identified as PDF23 among the various bacterial isolates. The PDF23 isolate was purified after three consecutive streaks on nutrient agar medium and stored at -80°C.Table 1
Pigment-producing bacteria were isolated from various sites around Great Salt Lake in Chennai, India.
S. No.OrganismPigmentGram stainingPDF12Yellow(–ve) rodPDF13Yellow(+ve) cocciPDF14Red(–ve) rodPDF15Yellow(+ve) cocciPDF16Red(–ve) rodPDF17Orange(–ve) rodPDF18Pale yellow(+ve) cocciPDF19Red(–ve) rodPDF20Yellow(–ve) rodPDF21Pale yellow(+ve) cocciPDF22Red(–ve) rodPDF23Violet(–ve) rod


## 2.3. Biochemical Analysis of Bacterial Isolate PDF23

Gram staining, cell morphology, and pigment synthesis were used to identify the violet-colored bacterial isolate PDF23. Enzyme activity such as oxidase, catalase, urease, triple sugar iron agar test, citrate test, indole, MR-VP test [38, 39], and motility test were carried out according to method of Litchfield and Gillevet [40].


## 2.4. Molecular Analysis

### 2.4.1. DNA Extraction

The bacterial isolate PDF23 was grown to reach an A600 of 1.0. After growing, the cells were harvested by centrifugation at 10,000 rpm for 5 min at 4°C. As previously mentioned by [41], total genomic DNA was extracted from bacterial cell pellets. Genomic DNA was resolved on a 1% agarose gel with 1X TAE buffer, and DNA was imaged using a gel documentation unit (Bio-Rad, USA) [39].


### 2.4.2. Analysis of 16S rRNA Gene by PCR Amplification

Total genomic DNA was employed in PCR amplification of the 16S rRNA gene using primers 27 F (5′-AGAGTTGGATCTGGCTCG-3′) and 1492R (5′-ACCTTGTTACGACTT-3′) [42]. The PCR cycling conditions were set as initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C, 2 min, with a final extension of 10 min at 72°C. The PCR product was separated on 1% agarose gel.


### 2.4.3. Sequencing and Phylogenetic Tree Construction

The 16S rRNA gene PCR product was purified and sequenced on both strands using 27 F and 1492R primers using the PCR cleanup kit (Qiagen, USA). MEGA X was used to generate a phylogenetic tree using the neighbor joining method [43]. The PDF23 bacterial isolate 16S rRNA nucleotide sequences were submitted to the GenBank database, and accession numbers were received (MT176501).


## 2.4.1. DNA Extraction

The bacterial isolate PDF23 was grown to reach an A600 of 1.0. After growing, the cells were harvested by centrifugation at 10,000 rpm for 5 min at 4°C. As previously mentioned by [41], total genomic DNA was extracted from bacterial cell pellets. Genomic DNA was resolved on a 1% agarose gel with 1X TAE buffer, and DNA was imaged using a gel documentation unit (Bio-Rad, USA) [39].


## 2.4.2. Analysis of 16S rRNA Gene by PCR Amplification

Total genomic DNA was employed in PCR amplification of the 16S rRNA gene using primers 27 F (5′-AGAGTTGGATCTGGCTCG-3′) and 1492R (5′-ACCTTGTTACGACTT-3′) [42]. The PCR cycling conditions were set as initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C, 2 min, with a final extension of 10 min at 72°C. The PCR product was separated on 1% agarose gel.


## 2.4.3. Sequencing and Phylogenetic Tree Construction

The 16S rRNA gene PCR product was purified and sequenced on both strands using 27 F and 1492R primers using the PCR cleanup kit (Qiagen, USA). MEGA X was used to generate a phylogenetic tree using the neighbor joining method [43]. The PDF23 bacterial isolate 16S rRNA nucleotide sequences were submitted to the GenBank database, and accession numbers were received (MT176501).


## 2.5. Growth and Violacein Production

The growth and synthesis of violacein from PDF23 strain were evaluated in a modified nutrient broth with sodium chloride deprivation. An overnight culture dissolved in saline solution was used as the inoculums at a 5% (v/v) concentration. The experiment was carried out in a 250-mL Erlenmeyer flask and incubated at 25°C under agitation (180 rpm). To determine the increase in biomass and violacein, 1 mL of distilled ethanol was added to the pellet-pigment extract and homogenized to attain the ethanol-phase pigment and 1 mL of culture broth was extracted and separated by centrifugation at 10,000 rpm for 10 min. The filtrate was agitated again, and the pellet and supernatant were extracted. The absorbance of ethanolic supernatant was measured at 575 nm to assess the presence of violacein. For biomass quantification, the pellet was dissolved in 1 mL saline, and the absorbance was measured at 600 nm [44].


## 2.6. Extraction of Violacein Dye

The violet pigment was extracted from a 24-hr-oldC. violaceum cell suspension using the following procedure. Bacteria cells were harvested after an incubation time of 24 hrs, and the cells were collected by centrifugation simultaneously at a speed of 7,000 rpm and 4°C. Pellets were obtained by the extraction methods, and three different solvents were applied to each separate pellet, methanol, acetone, and ethyl acetate, where the solution was also separated by centrifugation at 7,000 rpm for 4°C. The separation technique was replicated till the pellet turned colorless. Violet and blue dyes were extracted from C. violaceum using this extraction process. To remove methanol, acetone, and ethyl acetate from the pigments, the rotary evaporator was used to obtain the pigments in crude form. The time required for condensation at 45°C was 1 h per 250 mL. For 1 L of solution, crude extraction yielded around 0.2 g of pigments [45].


## 2.7. Characterization of Natural Dye

### 2.7.1. UV-Vis Spectral Analysis

The methanol extract of crude pigment was evaluated using the UV-vis spectrophotometer (Shimadzu UV-1601PC) between 800 and 200 nm for maximum wavelength [46].


### 2.7.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

FT-IR spectrophotometer (Shimadzu FTIR-8400S) was used to determine violet pigment derived fromC. violaceum PDF23 in the wave number range from 400 to 4000 cm-1. On the diamond glass of the spectrophotometer, approximately 2 μg of dyes were mounted [47]. Various functional groups found in the pigments were identified using the FTIR spectrum.


## 2.7.1. UV-Vis Spectral Analysis

The methanol extract of crude pigment was evaluated using the UV-vis spectrophotometer (Shimadzu UV-1601PC) between 800 and 200 nm for maximum wavelength [46].


## 2.7.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

FT-IR spectrophotometer (Shimadzu FTIR-8400S) was used to determine violet pigment derived fromC. violaceum PDF23 in the wave number range from 400 to 4000 cm-1. On the diamond glass of the spectrophotometer, approximately 2 μg of dyes were mounted [47]. Various functional groups found in the pigments were identified using the FTIR spectrum.


## 2.8. Applications of Violet Pigment in Textile Dyeing

### 2.8.1. Fabrics

Two types of fabrics, silk satin and cotton, were used to evaluate the dye-ability of pigments.


### 2.8.2. Dyeing of Fabric Samples

For all fabric samples of liquidC. violaceum PDF23 dye for both silk and cotton, staining was performed at boiling temperature (100°C). Silk fabric dyeing was performed at a dye concentration of 2%, with a fabric: liquid proportion (1 : 50) and varying mordant concentrations at 60°C and an interval of 60 min. After staining, the colored fabric was rinsed at 60°C for 15 min with 3 g/L of nonionic detergent, then washed with cold water, and allowed to dry at 37°C. For staining of silk satin and cotton fabrics, four parameters affecting color efficiency and intensity were evaluated: mordants, mordant concentration, fabric pretreatment, and natural pigment strength characteristics [48].


### 2.8.3. Mordant

Ferrous sulfate, sodium silicate, alum, copper sulfate, and calcium hydroxide were used in the forms of mordants [49].


### 2.8.4. Pretreatment of Fabric

Before proceeding to the pretreatment process, about 1 g of fabric was soaked in alum and Na₂CO₃. The solution was heated for 2 hrs along with the fabrics to ensure that the fabrics were processed efficiently. The fabric was rinsed and dried at 37°C. Silk was pretreated in a similar manner, except sodium carbonate. Pretreatment of the fabric was done to remove pectic compounds and cotton wax found in cotton weave fabrics, as well as particles on the surface of silk fabrics. The sample, which includes alum and Na₂CO3, was prepared for pretreatment of cotton fabrics by dissolving 0.2 g alum and 0.06 g Na₂CO₃ in 30 mL of distilled water, heating and thoroughly mixing by homogenization [50].


## 2.8.1. Fabrics

Two types of fabrics, silk satin and cotton, were used to evaluate the dye-ability of pigments.


## 2.8.2. Dyeing of Fabric Samples

For all fabric samples of liquidC. violaceum PDF23 dye for both silk and cotton, staining was performed at boiling temperature (100°C). Silk fabric dyeing was performed at a dye concentration of 2%, with a fabric: liquid proportion (1 : 50) and varying mordant concentrations at 60°C and an interval of 60 min. After staining, the colored fabric was rinsed at 60°C for 15 min with 3 g/L of nonionic detergent, then washed with cold water, and allowed to dry at 37°C. For staining of silk satin and cotton fabrics, four parameters affecting color efficiency and intensity were evaluated: mordants, mordant concentration, fabric pretreatment, and natural pigment strength characteristics [48].


## 2.8.3. Mordant

Ferrous sulfate, sodium silicate, alum, copper sulfate, and calcium hydroxide were used in the forms of mordants [49].


## 2.8.4. Pretreatment of Fabric

Before proceeding to the pretreatment process, about 1 g of fabric was soaked in alum and Na₂CO₃. The solution was heated for 2 hrs along with the fabrics to ensure that the fabrics were processed efficiently. The fabric was rinsed and dried at 37°C. Silk was pretreated in a similar manner, except sodium carbonate. Pretreatment of the fabric was done to remove pectic compounds and cotton wax found in cotton weave fabrics, as well as particles on the surface of silk fabrics. The sample, which includes alum and Na₂CO3, was prepared for pretreatment of cotton fabrics by dissolving 0.2 g alum and 0.06 g Na₂CO₃ in 30 mL of distilled water, heating and thoroughly mixing by homogenization [50].


## 2.9. Assessment of Dyed Fabric

### 2.9.1. Color Strength and Colorfastness Standard Tests

The colored fabrics were analyzed in compliance with ISO standard techniques. The transmittance of the colored materials was measured using a UV-vis spectrophotometer (Premier Color Scan, Model: SS 5145A, India). The Kubelka-Munk equation was used to calculate absolute color characteristics (K/S values) [47].
(1)KS=1−R22R−1−R22R0where R is the decimal fraction of dyed fabric reflectance, R0 is the decimal fraction of undyed fabric reflectance, K is the absorption coefficient, and S is the scattering coefficient.


## 2.9.1. Color Strength and Colorfastness Standard Tests

The colored fabrics were analyzed in compliance with ISO standard techniques. The transmittance of the colored materials was measured using a UV-vis spectrophotometer (Premier Color Scan, Model: SS 5145A, India). The Kubelka-Munk equation was used to calculate absolute color characteristics (K/S values) [47].
(1)KS=1−R22R−1−R22R0where R is the decimal fraction of dyed fabric reflectance, R0 is the decimal fraction of undyed fabric reflectance, K is the absorption coefficient, and S is the scattering coefficient.


## 2.10. Antimicrobial Activity by Minimum Inhibitory Concentration (MIC)

The antimicrobial efficacy of violet pigments was used to evaluate the percentage of inhibition against human pathogens such asS. aureus, B. subtilis, E. coli, P. aeruginosa, and C. albicans [51]. The bacterial strains were seeded in Mueller Hinton broth incubated at 37°C for 24 hrs, while the yeast grown in Sabouraud dextrose broth incubated at 30°C for 24 hrs. All the test strains were adjusted to 106 CFU/mL (McFarland standard) and used for assay. Different concentrations of violet pigments (0.25, 0.50, 1.0, and 2.0 mg/mL) were loaded into the wells to obtain final volume of 200 μL in microtiter plate. For bacterial strains, microtiter plates were incubated at 37°C and for yeast strains at 30°C. After incubation, the plates were recorded for bacterial and fungal growth inhibition using microtiter plate reader.


## 2.11. Antioxidant Efficacy of Violacein Pigments

The DPPH analysis was performed based on the decolorization efficiency of this molecule in the presence of antioxidants and robust technique for measuring the scavenging ability of free radicals [52]. Ascorbic acid was used as the standard control. In 1 mL of 3 mM DPPH solution, different concentrations of violet dye (5–100 μg/mL) were added and incubated in the dark for 30 min at 30°C. The color transition from violet to yellow was measured at 517 nm. The IC50 value denotes the dye concentration required to scavenge 50% of the free radicals generated by DPPH.Using the following equation, the percent inhibition was quantified:(2)%Inhibition=Ac−As/Ac×100,where Ac denotes control absorbance and As denotes pigment absorbance.


## 3. Result and Discussion

### 3.1. Isolation of Bacterial Isolates

Samples of soil sediment were obtained from 12 distinct locations at Great Salt Lake located in Chennai, India (Table1). Different bacterial isolates were obtained from soil samples, including C. violaceum, which was rapidly identified due to its distinctive violet colonies (Figure 2). Bacterial strains were isolated using both enrichment and filtration methods, and they thrived at temperatures between 22°C and 37°C. On nutrient agar plates, the colonies were violet in color, oval, slightly elevated, with a whole margin, shiny, clear, and smooth surface. C. violaceum is a nonsporing, motile, gram-negative coccobacillus.Figure 2
Isolated violet pigment producing bacteriumC. violaceum PDF23.


### 3.2. Biochemical and Physiological Characterization of Bacterial Isolate PDF23

The bacterial strainC. violaceum showed positive results for oxidase, methyl red test, and citrate utilization test whereas negative for catalase, urease, indole production, and Voges-Proskauer test. The TSI test was positive for glucose and no production of sulfide. Casein and fat were hydrolyzed by our isolate of C. violaceum, but not starch. It has ability to oxidize and ferment glucose, but it has not capable to oxidize or ferment sucrose. It did not produce hydrogen sulfide or indole on SIM agar, but it did show motility. C. violaceum exhibited an alkaline slant and an acidic butt on TSI medium, with no hydrogen sulfide or gas production. Lactose and sucrose were not used, but proteolysis and glucose fermentation were identified. Table 2 summarizes the results of biochemical tests performed on C. violaceum PDF23 used in this study.Table 2
Biochemical characterization of selected violet pigment producing isolate PDF23.
CharacteristicsPDF23Catalase+Oxidase+Methyl red—Voges-Proskeur—Citrate+Urease—Gelatinase+Hydrogen sulfide production—Triple sugar iron agar testA/AGlucose+Mannitol—Sorbitol—Rhamnose—Sucrose—Arabinose—Lysine—Arginine++ denotes presence and – denotes absence; A/A indicates alkaline slant/acidic butt.The bacterial species identification was influenced by characterization of the isolate through various physiological and biochemical studies. Further, molecular phylogenetic study and the comparison of different biochemical test results with those published from other countries have authenticated its identification [53–55]. Chromobacterium sp. has capability to metabolize various organic substrates and provides the bacterium the advantage of living in natural environments with minimal amounts of carbon or nutrients. Chromobacterium sp. showed uniform phenotypic and biochemical characteristics and were documented from different natural habitats, while diverse physiological characteristics are adapted to adverse environment [53, 54, 56].


### 3.3. Molecular Phylogenetic Analysis

The 16S rRNA gene was amplified by PCR, yielding an amplicon of around 1500 bp, which was used to identify the bacterial isolate PDF23. Following amplification, sequencing of the 16S rRNA gene produced a 602 bp amplicon, which was then submitted to Blast analysis (Table3). The 16S rRNA gene sequence of PDF23 showed 99.67% identical to 16S rRNA gene sequence of C. violaceum strain RG1 (MF144240). C. violaceum PDF23 nucleotide sequence has been submitted to GenBank with the accession number MT176501.1. The 16S rRNA gene sequence analysis of our bacterial isolate showed 99% similarity with C. violaceum.Table 3
Partial 16S rRNA gene sequence ofC. violaceum PDF23 submitted in NCBI database.
GTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTGTGCAAGTCTGATGTGAAAGCCCCGGGCTTAACCTGGGAACGGCATTGGAGACTGCACAGCTAGAGTGCGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGATGACACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGGTTTGAATCCTTGGTAGCGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTGCTCTTGACATGTACGGAACTTGCCAGAGATGGCTTGGTGCCCGAAAGGGAGCCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTThe first 20 16S rRNA gene sequences ofC. violaceum PDF23 showed >99.67% similarity in BLASTN, indicating that the C. violaceum PDF23 isolate grouped together with other C. violaceum strains in a separate clade assisted by 99% bootstrap values. It belongs to a separate clade and also phylogenetically distinct from the three Chromobacterium species previously identified (C. vaccinii strain CV5, C. piscinae WBJR0303, and Chromobacterium sp. LRSZR53) (Figure 3).Figure 3
C. violaceum PDF23 (shown in red line) is closely linked to other violacein-producing C. violaceum species, according to a phylogenetic tree based on 16S rRNA gene sequences received from the NCBI. 0.003 substitutions per nucleotide position are indicated by the scale bar.C. violaceum, a gram-negative bacteria, present a wide range of tropical and subtropical environments in the atmosphere as a saprophyte, especially in water and soil [57]. It is a β-proteobacterium that has reignited biotechnological interest due to its wide economic, therapeutic, and ecological potential [58]. This free-living bacterium has a high degree of adaptability, allowing it to survive in extreme environmental conditions [59]. Similar to our study, a violet pigment producing bacteria C. violaceum UTM5 (HM132057) was isolated and characterized from soil samples taken from wastewater treatment system at oil refinery in Negeri Sembilan, Malaysia [49]. This is the first evidence of the violet pigment-producing halophilic C. violaceum PDF23 being isolated from Great Salt Lake located in Chennai, India.


### 3.4. Production of Natural Dye fromC. violaceum

Three different solvents include methanol, acetone, ethyl acetate, and water and were used to extract natural pigment fromC. violaceum, whereas ethyl acetate extract from the broth medium was used effectively as a dye. Compared to ethyl acetate and acetone extract, methanol solvents have been shown to contain high concentrations of colorants. While extracted using methanol solvent, C. violaceum developed blue colorants, whereas acetone and ethyl extracts generated violet colorants (Table 4).Table 4
Extraction and production of natural pigment fromC. violaceum.
S.no.Solvent extractionProduction of natural dye1.Methanol+++ (blue)2.Acetone++ (violet)3.Ethyl acetate++ (violet)4.Water+ (colorless)Dye strength graded as follows: + – low, ++ – moderate, and +++ – extremely high.The natural dyes have been synthesized in the media intracellularly inside the cells.C. violaceum could therefore produce various types of pigments from various extraction techniques. Earlier studies have shown that dyes derived from different microbial sources, such as fungi, plants, and other bacterial species primarily extracted from intracellular cells. Similarly, Hiroshi [60] stated that a bluish violet dye was extracted from Janthinobacteriun lividum using various solvents at different concentrations.


### 3.5. Characterization of Natural Dye

#### 3.5.1. UV-Vis Spectral Analysis

The strong absorption spectra were observed between 500 to 700 nm. ForC. violaceum, the maximum wavelength at 575 nm is shown in Figure 4. Study by Lu [61] observed that the C. violaceum dye in methanol developed a greater absorption at 576 nm. Therefore, both the maximum absorbance wavelengths agree with existing studies to suggest that bacterial colorants are of the same genus.Figure 4
UV-visible spectrum of violacein pigment produced byC. violaceum PDF23.The violet dye in methanol showed a sharp peak with absorption maximum at 575 nm and confirms the formation of chromophore groups including alkene and carbonyl, which are responsible for electronic absorption. While the carbon atom absorbs positively at a narrower UV-Vis wavelength, due to the presence of n-electrons, the intensity significantly reduces at longer wavelengths. Therefore, the significant characteristic of the alkene groups in relating to the cross-linking effect would be that the higher the influence of double conjugated bonds, the greater the wavelength of absorption (bathochromic shift) [62].


#### 3.5.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

The structural detection of violacein was done using FT-IR spectroscopy (Figure5). The crude methanolic extract results from the FTIR study are as follows: 3700-3000 cm-1 (OH), 3447 cm-1 (N-H), 3061 cm-1 amide, 2878 cm-1 (C = O amide), 1641 cm-1 (C = C), 1544 cm-1 (C-O phenol), and 1422 cm-1 and 1362 cm-1 (C-N). The low absorption wavelength for the carbonyl group could be formed due to the aromatic ring cross-linking effect, resulting in the π electrons of both unsaturated groups being delocalized. This decreases the double bonds formed between the carbon atoms, resulting in a decrease in the wavelength from 1176 cm-1, i.e., typical carbonyl group absorption wavelength at 1039 cm-1, and alkene stretching frequency from 783 cm-1, i.e., typical alkene absorption wavelength at 689 cm-1. The vibration function of the carbonyl group in the double bond structure was formed due to these conditions [62].Figure 5
FT-IR spectra of violacein pigment produced byC. violaceum PDF23.


### 3.6. Dyeing of Fabric Samples

#### 3.6.1. Color Measurement

The measurements of CIEL∗a∗b∗ for silk satin and cotton fabrics stained with C. violaceum dye are shown in Table 5. The properties of L∗ reflect the lightness or darkness interpreted. The number 0 denotes black, while the number 100 denotes white. The scores of (+a∗) are red and (−a∗) are orange, whereas yellow are (+b∗) and blue are (−b∗). Cotton fabrics have lower L∗ scores than silk satin in terms of dyes. The L∗ score for methanol extract of both silk satin and cotton fabrics has the maximum values of 81.23 and 72.14, respectively, indicating that the dye is similar to the white colors. Ethyl acetate extract yielded the darkest colors as previously described; moreover, they showed higher L∗ scores that were 63.89 and 58.35 for silk satin and cotton fabrics, respectively. The a∗ and b∗ scores of colored fabrics are provided in Table 5 and indicated that all the textile fabrics were stained with C. violaceum extract and were found in the red–yellow region.Table 5
L∗a∗b∗ value for C. violaceum violet pigment dyed with silk fabric.
S. No.FabricsSolventL∗a∗b∗1.Silk satinMethanol81.2312.8230.94Acetone60.475.6118.21Ethyl acetate63.898.9322.85Water42.571.4712.372.CottonMethanol72.1410.5324.71Acetone51.104.658.49Ethyl acetate58.356.4716.12Water31.850.987.95In contrast, Yusoff et al. [28] stated that the dye extracted from actinomycetes provided a low a∗ score in the red region range (+a) as well as a lower L∗ score indicating the closest to white color. Similar findings were reported from the extract of C. violaceum indicating the values (+a) and (-b) as colored on microfiber cloths and polyester fabrics suggesting the red-blue region even when these dyes were processed at different temperatures, although the value of a∗ remains unchanged (+a) as in the red zone. Moreover, the L∗ score for these dyes was more than 50, reflecting white color shades [49].


#### 3.6.2. Colorfastness Properties

Natural dyes also need a metallic mordant to improve the adsorption capacity between the textile fabrics and the dye, and various mordants often significantly reduce the dye from fading or washing out after exposure to light. Colored cotton fabrics and silk statin were mordanted with alum, ferrous sulfate, copper sulfate, and slake lime in this analysis, while the control fabrics were not mordanted. The stained fabrics were tested for rubbing, washing, transpiration, and light colorfastness properties. The assessments were done based on MS ISO specifications with a rating scale of 1 to 5, where 1 indicates worst and 5 denotes the best. The data for the evaluation of transpiration, washing, rubbing, and light fastness are provided in Table6. Wet and dry rubbing durability received a grade of 4 to 5, indicating fair to excellent performance. The fabrics colorfastness to light was assessed by comparing them to blue wool standards on a scale of 1 to 5, with 1 being the least and 5 being the highest.Table 6
Colorfastness qualities of natural dyed cotton fabric and silk satin in the presence of different mordants.
FabricsMordantColorfastnessLightWashingRubbing/crockingPerspirationWaterDCSDryWetCCDCCCDCSilk satinNo12/33/454/54/54/54/54/5Alum1/24/55554/554/55Fe2(SO4)313/44/54/54/54/554/55CuSO414/54/5553/44/54/54/5Ca(OH)224/44/5554/54/554/5CottonNo11/224/544/54/555Alum14/5544/54/554/55Fe2(SO4)324/544/54/53/44/555CuSO42/344/544/54/54/54/54/5Ca(OH)214/53/444/554/54/55S: staining on white fabric; DC: determining color; CC: color change; 1 denotes very poor; 2 denotes poor; 3 denotes fair; 4 denotes good; and 5 denotes excellence.In both cotton and silk satin, the use of slake lime mordant appears to result in darker color than the control fabrics. A golden colored fabric appeared in Fe2(SO4)3, whereas CuSO4 changed the colored fabric into shades of grey. Conversely, alum produced no substantial variation was observed between the color produced on the dyed fabrics and the control samples.As shown in Table6, Fe2(SO4)3 and CuSO4 improve the light fastness of cotton from 1 (control) to 2 and 2/3, respectively, while alum and slaked lime improve the light fastness of silk satin from 1 (control) to 2 and 2/3, respectively. Similarly, Ahmad et al. [49] also stated that the light fastness ranking of silk satin stained with natural violacein dye along with mordant alum and slaked lime was observed to enhance from 1 (control) to 2. It could be attributed to interaction with transition metal ions, which decreases the chromophore valence electrons, resulting in increased resistance to photocatalytic degradation [63]. The findings indicated that in particular, the colored silk and cotton showed low light fastness, giving grades between 1 and 2/3. In general, it is well documented that the characteristics of light fastness were weak among natural colorants that generally offered low grades. For example, the grade of bluish-violet dye derived from Janthinobacterium lividum was as low as 1 [64], whereas dye extracted from the barks of Ixonanthes icosandra Jack offered a score of 2 for the characteristics of light fastness [65].In this work, when alum is being used as the mordant, good colorfastness of the fabric was observed for washing. This is although Al3+ could act as an effective electron acceptor to form link bonds with the dye molecule, preceded by the development of an insoluble complex, thus improving the pigments binding interactions to the fibers [66]. Some colorfastness studies have proven that in the absence of mordant, cotton and silk satin have fair (4) to excellent (5) colorfastness characteristics for transpiration, rubbing/crocking, and water. The strong affinity of the dye to cellulose and protein could be attributed to this effect [49].It has also been demonstrated that dyes would function as both direct pigment and mordant stain, because without the aid of certain other additives, the dyes would bind to the fabric molecules. It could also function to bind it to the fabric in the presence of a synthetic agent known as a mordant. Compared to un-mordanted fabric, the colorfastness result of dyed fabric mordanted with alum is good. A potential perspective for this finding is that the auxochrome groups present in the dye (-OH and -NH2) resulted in the effective production of metal compounds with the fabrics and the ability to form very strong bonds with the dye and fabrics.The evaluation of colorfastness to perspiration is shown in Table6. The result for colorfastness provided a good to excellent grade of 3/4-5 for color change, while the rating for color staining was fair to excellent ranged from 4 to 5. In contrast, the bluish-violet dye derived from Janthino bacteriumlividum provided a fair score of colorfastness characteristics [64]. The effect of colorfastness to washing provided a color change score between 1/2 and 5. It clearly demonstrates that the ranking is comparatively low to excellence.For staining, the grade was high, from 4/5 to 5, for both silk and cotton. As a consequence, this could be attributed that the dye in soap and water faded quickly, and did not affect silk and cotton stains. However, a good ranking of 4 for red and violet dyes derived fromSerratia marcescens and C. violaceum was demonstrated by Zulkifli et al. [67]. While the scores for colorfastness to light and washing were poor for both colorfastness measurements, the ranking towards perspiration and rubbing was excellent providing grades between 4 and 5. Conversely, more advancement would make to enhance the quality of the pigments, particularly for light and washing to colorfastness.


### 3.7. Applications of Violet Pigment in Textile Dyeing Fabrics

#### 3.7.1. Antimicrobial Efficacy of Violet Dye

In the initial screening, crude methanolic extract ofC. violaceum culture was evaluated for the antimicrobial efficacy against two gram-positive (S. aureus and B. subtilis), two gram-negative (E. coli and P. aeruginosa), and one fungal strains (C. albicans). Violacein-containing culture extract was capable of inhibiting growth of all tested strains between 12 and 54% significantly at a high concentration of 2 mg L-1 compared with control. The growth of S. aureus was inhibited for 39% even at concentration of 0.5 mg L-1, whereas the least affected strain was P. aeruginosa. C. albicans was the most susceptible fungal strain with 50 and 15% growth inhibition at 2 and 0.25 mgL-1 of the extract, respectively (Figure 6).Figure 6
Antimicrobial activity of violacein pigment fromC. violaceum PDF23 showing inhibition against selected human pathogens.


#### 3.7.2. Antioxidant Activity of Violacein Pigments ofC. violaceum PDF23

The radical scavenging assay evaluated the antioxidant activity of violet pigments. The IC50 value was 14.40 μg/mL and 28.66 μg/mL for violet pigments and ascorbic acid, respectively (Figure 7).Figure 7
DPPH was used to assess the antioxidant activity ofC. violaceum PDF23 violet pigments. Results are based on an average of three separate experiments and standard deviation is represented by the error bars.These findings suggest that the violet pigments ofC. violaceum PDF23 is more potent in scavenging free radicals than ascorbic acid. The plausible reason for the absence of catalase activity in C. violaceum PDF23 could be the significant antioxidant potential of violet pigments. A crucial role in the advancement of a wide range of pathological chronic diseases such as diabetes and cancer has been proposed for the generation of free radicals [68]. Consequently, treatment with free radical scavenging antioxidants such as microbial dyes C. violaceum PDF23 has the ability to prevent, prolong, or alter many of these diseases.


## 3.1. Isolation of Bacterial Isolates

Samples of soil sediment were obtained from 12 distinct locations at Great Salt Lake located in Chennai, India (Table1). Different bacterial isolates were obtained from soil samples, including C. violaceum, which was rapidly identified due to its distinctive violet colonies (Figure 2). Bacterial strains were isolated using both enrichment and filtration methods, and they thrived at temperatures between 22°C and 37°C. On nutrient agar plates, the colonies were violet in color, oval, slightly elevated, with a whole margin, shiny, clear, and smooth surface. C. violaceum is a nonsporing, motile, gram-negative coccobacillus.Figure 2
Isolated violet pigment producing bacteriumC. violaceum PDF23.


## 3.2. Biochemical and Physiological Characterization of Bacterial Isolate PDF23

The bacterial strainC. violaceum showed positive results for oxidase, methyl red test, and citrate utilization test whereas negative for catalase, urease, indole production, and Voges-Proskauer test. The TSI test was positive for glucose and no production of sulfide. Casein and fat were hydrolyzed by our isolate of C. violaceum, but not starch. It has ability to oxidize and ferment glucose, but it has not capable to oxidize or ferment sucrose. It did not produce hydrogen sulfide or indole on SIM agar, but it did show motility. C. violaceum exhibited an alkaline slant and an acidic butt on TSI medium, with no hydrogen sulfide or gas production. Lactose and sucrose were not used, but proteolysis and glucose fermentation were identified. Table 2 summarizes the results of biochemical tests performed on C. violaceum PDF23 used in this study.Table 2
Biochemical characterization of selected violet pigment producing isolate PDF23.
CharacteristicsPDF23Catalase+Oxidase+Methyl red—Voges-Proskeur—Citrate+Urease—Gelatinase+Hydrogen sulfide production—Triple sugar iron agar testA/AGlucose+Mannitol—Sorbitol—Rhamnose—Sucrose—Arabinose—Lysine—Arginine++ denotes presence and – denotes absence; A/A indicates alkaline slant/acidic butt.The bacterial species identification was influenced by characterization of the isolate through various physiological and biochemical studies. Further, molecular phylogenetic study and the comparison of different biochemical test results with those published from other countries have authenticated its identification [53–55]. Chromobacterium sp. has capability to metabolize various organic substrates and provides the bacterium the advantage of living in natural environments with minimal amounts of carbon or nutrients. Chromobacterium sp. showed uniform phenotypic and biochemical characteristics and were documented from different natural habitats, while diverse physiological characteristics are adapted to adverse environment [53, 54, 56].


## 3.3. Molecular Phylogenetic Analysis

The 16S rRNA gene was amplified by PCR, yielding an amplicon of around 1500 bp, which was used to identify the bacterial isolate PDF23. Following amplification, sequencing of the 16S rRNA gene produced a 602 bp amplicon, which was then submitted to Blast analysis (Table3). The 16S rRNA gene sequence of PDF23 showed 99.67% identical to 16S rRNA gene sequence of C. violaceum strain RG1 (MF144240). C. violaceum PDF23 nucleotide sequence has been submitted to GenBank with the accession number MT176501.1. The 16S rRNA gene sequence analysis of our bacterial isolate showed 99% similarity with C. violaceum.Table 3
Partial 16S rRNA gene sequence ofC. violaceum PDF23 submitted in NCBI database.
GTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTGTGCAAGTCTGATGTGAAAGCCCCGGGCTTAACCTGGGAACGGCATTGGAGACTGCACAGCTAGAGTGCGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGATGACACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGGTTTGAATCCTTGGTAGCGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTGCTCTTGACATGTACGGAACTTGCCAGAGATGGCTTGGTGCCCGAAAGGGAGCCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTThe first 20 16S rRNA gene sequences ofC. violaceum PDF23 showed >99.67% similarity in BLASTN, indicating that the C. violaceum PDF23 isolate grouped together with other C. violaceum strains in a separate clade assisted by 99% bootstrap values. It belongs to a separate clade and also phylogenetically distinct from the three Chromobacterium species previously identified (C. vaccinii strain CV5, C. piscinae WBJR0303, and Chromobacterium sp. LRSZR53) (Figure 3).Figure 3
C. violaceum PDF23 (shown in red line) is closely linked to other violacein-producing C. violaceum species, according to a phylogenetic tree based on 16S rRNA gene sequences received from the NCBI. 0.003 substitutions per nucleotide position are indicated by the scale bar.C. violaceum, a gram-negative bacteria, present a wide range of tropical and subtropical environments in the atmosphere as a saprophyte, especially in water and soil [57]. It is a β-proteobacterium that has reignited biotechnological interest due to its wide economic, therapeutic, and ecological potential [58]. This free-living bacterium has a high degree of adaptability, allowing it to survive in extreme environmental conditions [59]. Similar to our study, a violet pigment producing bacteria C. violaceum UTM5 (HM132057) was isolated and characterized from soil samples taken from wastewater treatment system at oil refinery in Negeri Sembilan, Malaysia [49]. This is the first evidence of the violet pigment-producing halophilic C. violaceum PDF23 being isolated from Great Salt Lake located in Chennai, India.


## 3.4. Production of Natural Dye fromC. violaceum

Three different solvents include methanol, acetone, ethyl acetate, and water and were used to extract natural pigment fromC. violaceum, whereas ethyl acetate extract from the broth medium was used effectively as a dye. Compared to ethyl acetate and acetone extract, methanol solvents have been shown to contain high concentrations of colorants. While extracted using methanol solvent, C. violaceum developed blue colorants, whereas acetone and ethyl extracts generated violet colorants (Table 4).Table 4
Extraction and production of natural pigment fromC. violaceum.
S.no.Solvent extractionProduction of natural dye1.Methanol+++ (blue)2.Acetone++ (violet)3.Ethyl acetate++ (violet)4.Water+ (colorless)Dye strength graded as follows: + – low, ++ – moderate, and +++ – extremely high.The natural dyes have been synthesized in the media intracellularly inside the cells.C. violaceum could therefore produce various types of pigments from various extraction techniques. Earlier studies have shown that dyes derived from different microbial sources, such as fungi, plants, and other bacterial species primarily extracted from intracellular cells. Similarly, Hiroshi [60] stated that a bluish violet dye was extracted from Janthinobacteriun lividum using various solvents at different concentrations.


## 3.5. Characterization of Natural Dye

### 3.5.1. UV-Vis Spectral Analysis

The strong absorption spectra were observed between 500 to 700 nm. ForC. violaceum, the maximum wavelength at 575 nm is shown in Figure 4. Study by Lu [61] observed that the C. violaceum dye in methanol developed a greater absorption at 576 nm. Therefore, both the maximum absorbance wavelengths agree with existing studies to suggest that bacterial colorants are of the same genus.Figure 4
UV-visible spectrum of violacein pigment produced byC. violaceum PDF23.The violet dye in methanol showed a sharp peak with absorption maximum at 575 nm and confirms the formation of chromophore groups including alkene and carbonyl, which are responsible for electronic absorption. While the carbon atom absorbs positively at a narrower UV-Vis wavelength, due to the presence of n-electrons, the intensity significantly reduces at longer wavelengths. Therefore, the significant characteristic of the alkene groups in relating to the cross-linking effect would be that the higher the influence of double conjugated bonds, the greater the wavelength of absorption (bathochromic shift) [62].


### 3.5.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

The structural detection of violacein was done using FT-IR spectroscopy (Figure5). The crude methanolic extract results from the FTIR study are as follows: 3700-3000 cm-1 (OH), 3447 cm-1 (N-H), 3061 cm-1 amide, 2878 cm-1 (C = O amide), 1641 cm-1 (C = C), 1544 cm-1 (C-O phenol), and 1422 cm-1 and 1362 cm-1 (C-N). The low absorption wavelength for the carbonyl group could be formed due to the aromatic ring cross-linking effect, resulting in the π electrons of both unsaturated groups being delocalized. This decreases the double bonds formed between the carbon atoms, resulting in a decrease in the wavelength from 1176 cm-1, i.e., typical carbonyl group absorption wavelength at 1039 cm-1, and alkene stretching frequency from 783 cm-1, i.e., typical alkene absorption wavelength at 689 cm-1. The vibration function of the carbonyl group in the double bond structure was formed due to these conditions [62].Figure 5
FT-IR spectra of violacein pigment produced byC. violaceum PDF23.


## 3.5.1. UV-Vis Spectral Analysis

The strong absorption spectra were observed between 500 to 700 nm. ForC. violaceum, the maximum wavelength at 575 nm is shown in Figure 4. Study by Lu [61] observed that the C. violaceum dye in methanol developed a greater absorption at 576 nm. Therefore, both the maximum absorbance wavelengths agree with existing studies to suggest that bacterial colorants are of the same genus.Figure 4
UV-visible spectrum of violacein pigment produced byC. violaceum PDF23.The violet dye in methanol showed a sharp peak with absorption maximum at 575 nm and confirms the formation of chromophore groups including alkene and carbonyl, which are responsible for electronic absorption. While the carbon atom absorbs positively at a narrower UV-Vis wavelength, due to the presence of n-electrons, the intensity significantly reduces at longer wavelengths. Therefore, the significant characteristic of the alkene groups in relating to the cross-linking effect would be that the higher the influence of double conjugated bonds, the greater the wavelength of absorption (bathochromic shift) [62].


## 3.5.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

The structural detection of violacein was done using FT-IR spectroscopy (Figure5). The crude methanolic extract results from the FTIR study are as follows: 3700-3000 cm-1 (OH), 3447 cm-1 (N-H), 3061 cm-1 amide, 2878 cm-1 (C = O amide), 1641 cm-1 (C = C), 1544 cm-1 (C-O phenol), and 1422 cm-1 and 1362 cm-1 (C-N). The low absorption wavelength for the carbonyl group could be formed due to the aromatic ring cross-linking effect, resulting in the π electrons of both unsaturated groups being delocalized. This decreases the double bonds formed between the carbon atoms, resulting in a decrease in the wavelength from 1176 cm-1, i.e., typical carbonyl group absorption wavelength at 1039 cm-1, and alkene stretching frequency from 783 cm-1, i.e., typical alkene absorption wavelength at 689 cm-1. The vibration function of the carbonyl group in the double bond structure was formed due to these conditions [62].Figure 5
FT-IR spectra of violacein pigment produced byC. violaceum PDF23.


## 3.6. Dyeing of Fabric Samples

### 3.6.1. Color Measurement

The measurements of CIEL∗a∗b∗ for silk satin and cotton fabrics stained with C. violaceum dye are shown in Table 5. The properties of L∗ reflect the lightness or darkness interpreted. The number 0 denotes black, while the number 100 denotes white. The scores of (+a∗) are red and (−a∗) are orange, whereas yellow are (+b∗) and blue are (−b∗). Cotton fabrics have lower L∗ scores than silk satin in terms of dyes. The L∗ score for methanol extract of both silk satin and cotton fabrics has the maximum values of 81.23 and 72.14, respectively, indicating that the dye is similar to the white colors. Ethyl acetate extract yielded the darkest colors as previously described; moreover, they showed higher L∗ scores that were 63.89 and 58.35 for silk satin and cotton fabrics, respectively. The a∗ and b∗ scores of colored fabrics are provided in Table 5 and indicated that all the textile fabrics were stained with C. violaceum extract and were found in the red–yellow region.Table 5
L∗a∗b∗ value for C. violaceum violet pigment dyed with silk fabric.
S. No.FabricsSolventL∗a∗b∗1.Silk satinMethanol81.2312.8230.94Acetone60.475.6118.21Ethyl acetate63.898.9322.85Water42.571.4712.372.CottonMethanol72.1410.5324.71Acetone51.104.658.49Ethyl acetate58.356.4716.12Water31.850.987.95In contrast, Yusoff et al. [28] stated that the dye extracted from actinomycetes provided a low a∗ score in the red region range (+a) as well as a lower L∗ score indicating the closest to white color. Similar findings were reported from the extract of C. violaceum indicating the values (+a) and (-b) as colored on microfiber cloths and polyester fabrics suggesting the red-blue region even when these dyes were processed at different temperatures, although the value of a∗ remains unchanged (+a) as in the red zone. Moreover, the L∗ score for these dyes was more than 50, reflecting white color shades [49].


### 3.6.2. Colorfastness Properties

Natural dyes also need a metallic mordant to improve the adsorption capacity between the textile fabrics and the dye, and various mordants often significantly reduce the dye from fading or washing out after exposure to light. Colored cotton fabrics and silk statin were mordanted with alum, ferrous sulfate, copper sulfate, and slake lime in this analysis, while the control fabrics were not mordanted. The stained fabrics were tested for rubbing, washing, transpiration, and light colorfastness properties. The assessments were done based on MS ISO specifications with a rating scale of 1 to 5, where 1 indicates worst and 5 denotes the best. The data for the evaluation of transpiration, washing, rubbing, and light fastness are provided in Table6. Wet and dry rubbing durability received a grade of 4 to 5, indicating fair to excellent performance. The fabrics colorfastness to light was assessed by comparing them to blue wool standards on a scale of 1 to 5, with 1 being the least and 5 being the highest.Table 6
Colorfastness qualities of natural dyed cotton fabric and silk satin in the presence of different mordants.
FabricsMordantColorfastnessLightWashingRubbing/crockingPerspirationWaterDCSDryWetCCDCCCDCSilk satinNo12/33/454/54/54/54/54/5Alum1/24/55554/554/55Fe2(SO4)313/44/54/54/54/554/55CuSO414/54/5553/44/54/54/5Ca(OH)224/44/5554/54/554/5CottonNo11/224/544/54/555Alum14/5544/54/554/55Fe2(SO4)324/544/54/53/44/555CuSO42/344/544/54/54/54/54/5Ca(OH)214/53/444/554/54/55S: staining on white fabric; DC: determining color; CC: color change; 1 denotes very poor; 2 denotes poor; 3 denotes fair; 4 denotes good; and 5 denotes excellence.In both cotton and silk satin, the use of slake lime mordant appears to result in darker color than the control fabrics. A golden colored fabric appeared in Fe2(SO4)3, whereas CuSO4 changed the colored fabric into shades of grey. Conversely, alum produced no substantial variation was observed between the color produced on the dyed fabrics and the control samples.As shown in Table6, Fe2(SO4)3 and CuSO4 improve the light fastness of cotton from 1 (control) to 2 and 2/3, respectively, while alum and slaked lime improve the light fastness of silk satin from 1 (control) to 2 and 2/3, respectively. Similarly, Ahmad et al. [49] also stated that the light fastness ranking of silk satin stained with natural violacein dye along with mordant alum and slaked lime was observed to enhance from 1 (control) to 2. It could be attributed to interaction with transition metal ions, which decreases the chromophore valence electrons, resulting in increased resistance to photocatalytic degradation [63]. The findings indicated that in particular, the colored silk and cotton showed low light fastness, giving grades between 1 and 2/3. In general, it is well documented that the characteristics of light fastness were weak among natural colorants that generally offered low grades. For example, the grade of bluish-violet dye derived from Janthinobacterium lividum was as low as 1 [64], whereas dye extracted from the barks of Ixonanthes icosandra Jack offered a score of 2 for the characteristics of light fastness [65].In this work, when alum is being used as the mordant, good colorfastness of the fabric was observed for washing. This is although Al3+ could act as an effective electron acceptor to form link bonds with the dye molecule, preceded by the development of an insoluble complex, thus improving the pigments binding interactions to the fibers [66]. Some colorfastness studies have proven that in the absence of mordant, cotton and silk satin have fair (4) to excellent (5) colorfastness characteristics for transpiration, rubbing/crocking, and water. The strong affinity of the dye to cellulose and protein could be attributed to this effect [49].It has also been demonstrated that dyes would function as both direct pigment and mordant stain, because without the aid of certain other additives, the dyes would bind to the fabric molecules. It could also function to bind it to the fabric in the presence of a synthetic agent known as a mordant. Compared to un-mordanted fabric, the colorfastness result of dyed fabric mordanted with alum is good. A potential perspective for this finding is that the auxochrome groups present in the dye (-OH and -NH2) resulted in the effective production of metal compounds with the fabrics and the ability to form very strong bonds with the dye and fabrics.The evaluation of colorfastness to perspiration is shown in Table6. The result for colorfastness provided a good to excellent grade of 3/4-5 for color change, while the rating for color staining was fair to excellent ranged from 4 to 5. In contrast, the bluish-violet dye derived from Janthino bacteriumlividum provided a fair score of colorfastness characteristics [64]. The effect of colorfastness to washing provided a color change score between 1/2 and 5. It clearly demonstrates that the ranking is comparatively low to excellence.For staining, the grade was high, from 4/5 to 5, for both silk and cotton. As a consequence, this could be attributed that the dye in soap and water faded quickly, and did not affect silk and cotton stains. However, a good ranking of 4 for red and violet dyes derived fromSerratia marcescens and C. violaceum was demonstrated by Zulkifli et al. [67]. While the scores for colorfastness to light and washing were poor for both colorfastness measurements, the ranking towards perspiration and rubbing was excellent providing grades between 4 and 5. Conversely, more advancement would make to enhance the quality of the pigments, particularly for light and washing to colorfastness.


## 3.6.1. Color Measurement

The measurements of CIEL∗a∗b∗ for silk satin and cotton fabrics stained with C. violaceum dye are shown in Table 5. The properties of L∗ reflect the lightness or darkness interpreted. The number 0 denotes black, while the number 100 denotes white. The scores of (+a∗) are red and (−a∗) are orange, whereas yellow are (+b∗) and blue are (−b∗). Cotton fabrics have lower L∗ scores than silk satin in terms of dyes. The L∗ score for methanol extract of both silk satin and cotton fabrics has the maximum values of 81.23 and 72.14, respectively, indicating that the dye is similar to the white colors. Ethyl acetate extract yielded the darkest colors as previously described; moreover, they showed higher L∗ scores that were 63.89 and 58.35 for silk satin and cotton fabrics, respectively. The a∗ and b∗ scores of colored fabrics are provided in Table 5 and indicated that all the textile fabrics were stained with C. violaceum extract and were found in the red–yellow region.Table 5
L∗a∗b∗ value for C. violaceum violet pigment dyed with silk fabric.
S. No.FabricsSolventL∗a∗b∗1.Silk satinMethanol81.2312.8230.94Acetone60.475.6118.21Ethyl acetate63.898.9322.85Water42.571.4712.372.CottonMethanol72.1410.5324.71Acetone51.104.658.49Ethyl acetate58.356.4716.12Water31.850.987.95In contrast, Yusoff et al. [28] stated that the dye extracted from actinomycetes provided a low a∗ score in the red region range (+a) as well as a lower L∗ score indicating the closest to white color. Similar findings were reported from the extract of C. violaceum indicating the values (+a) and (-b) as colored on microfiber cloths and polyester fabrics suggesting the red-blue region even when these dyes were processed at different temperatures, although the value of a∗ remains unchanged (+a) as in the red zone. Moreover, the L∗ score for these dyes was more than 50, reflecting white color shades [49].


## 3.6.2. Colorfastness Properties

Natural dyes also need a metallic mordant to improve the adsorption capacity between the textile fabrics and the dye, and various mordants often significantly reduce the dye from fading or washing out after exposure to light. Colored cotton fabrics and silk statin were mordanted with alum, ferrous sulfate, copper sulfate, and slake lime in this analysis, while the control fabrics were not mordanted. The stained fabrics were tested for rubbing, washing, transpiration, and light colorfastness properties. The assessments were done based on MS ISO specifications with a rating scale of 1 to 5, where 1 indicates worst and 5 denotes the best. The data for the evaluation of transpiration, washing, rubbing, and light fastness are provided in Table6. Wet and dry rubbing durability received a grade of 4 to 5, indicating fair to excellent performance. The fabrics colorfastness to light was assessed by comparing them to blue wool standards on a scale of 1 to 5, with 1 being the least and 5 being the highest.Table 6
Colorfastness qualities of natural dyed cotton fabric and silk satin in the presence of different mordants.
FabricsMordantColorfastnessLightWashingRubbing/crockingPerspirationWaterDCSDryWetCCDCCCDCSilk satinNo12/33/454/54/54/54/54/5Alum1/24/55554/554/55Fe2(SO4)313/44/54/54/54/554/55CuSO414/54/5553/44/54/54/5Ca(OH)224/44/5554/54/554/5CottonNo11/224/544/54/555Alum14/5544/54/554/55Fe2(SO4)324/544/54/53/44/555CuSO42/344/544/54/54/54/54/5Ca(OH)214/53/444/554/54/55S: staining on white fabric; DC: determining color; CC: color change; 1 denotes very poor; 2 denotes poor; 3 denotes fair; 4 denotes good; and 5 denotes excellence.In both cotton and silk satin, the use of slake lime mordant appears to result in darker color than the control fabrics. A golden colored fabric appeared in Fe2(SO4)3, whereas CuSO4 changed the colored fabric into shades of grey. Conversely, alum produced no substantial variation was observed between the color produced on the dyed fabrics and the control samples.As shown in Table6, Fe2(SO4)3 and CuSO4 improve the light fastness of cotton from 1 (control) to 2 and 2/3, respectively, while alum and slaked lime improve the light fastness of silk satin from 1 (control) to 2 and 2/3, respectively. Similarly, Ahmad et al. [49] also stated that the light fastness ranking of silk satin stained with natural violacein dye along with mordant alum and slaked lime was observed to enhance from 1 (control) to 2. It could be attributed to interaction with transition metal ions, which decreases the chromophore valence electrons, resulting in increased resistance to photocatalytic degradation [63]. The findings indicated that in particular, the colored silk and cotton showed low light fastness, giving grades between 1 and 2/3. In general, it is well documented that the characteristics of light fastness were weak among natural colorants that generally offered low grades. For example, the grade of bluish-violet dye derived from Janthinobacterium lividum was as low as 1 [64], whereas dye extracted from the barks of Ixonanthes icosandra Jack offered a score of 2 for the characteristics of light fastness [65].In this work, when alum is being used as the mordant, good colorfastness of the fabric was observed for washing. This is although Al3+ could act as an effective electron acceptor to form link bonds with the dye molecule, preceded by the development of an insoluble complex, thus improving the pigments binding interactions to the fibers [66]. Some colorfastness studies have proven that in the absence of mordant, cotton and silk satin have fair (4) to excellent (5) colorfastness characteristics for transpiration, rubbing/crocking, and water. The strong affinity of the dye to cellulose and protein could be attributed to this effect [49].It has also been demonstrated that dyes would function as both direct pigment and mordant stain, because without the aid of certain other additives, the dyes would bind to the fabric molecules. It could also function to bind it to the fabric in the presence of a synthetic agent known as a mordant. Compared to un-mordanted fabric, the colorfastness result of dyed fabric mordanted with alum is good. A potential perspective for this finding is that the auxochrome groups present in the dye (-OH and -NH2) resulted in the effective production of metal compounds with the fabrics and the ability to form very strong bonds with the dye and fabrics.The evaluation of colorfastness to perspiration is shown in Table6. The result for colorfastness provided a good to excellent grade of 3/4-5 for color change, while the rating for color staining was fair to excellent ranged from 4 to 5. In contrast, the bluish-violet dye derived from Janthino bacteriumlividum provided a fair score of colorfastness characteristics [64]. The effect of colorfastness to washing provided a color change score between 1/2 and 5. It clearly demonstrates that the ranking is comparatively low to excellence.For staining, the grade was high, from 4/5 to 5, for both silk and cotton. As a consequence, this could be attributed that the dye in soap and water faded quickly, and did not affect silk and cotton stains. However, a good ranking of 4 for red and violet dyes derived fromSerratia marcescens and C. violaceum was demonstrated by Zulkifli et al. [67]. While the scores for colorfastness to light and washing were poor for both colorfastness measurements, the ranking towards perspiration and rubbing was excellent providing grades between 4 and 5. Conversely, more advancement would make to enhance the quality of the pigments, particularly for light and washing to colorfastness.


## 3.7. Applications of Violet Pigment in Textile Dyeing Fabrics

### 3.7.1. Antimicrobial Efficacy of Violet Dye

In the initial screening, crude methanolic extract ofC. violaceum culture was evaluated for the antimicrobial efficacy against two gram-positive (S. aureus and B. subtilis), two gram-negative (E. coli and P. aeruginosa), and one fungal strains (C. albicans). Violacein-containing culture extract was capable of inhibiting growth of all tested strains between 12 and 54% significantly at a high concentration of 2 mg L-1 compared with control. The growth of S. aureus was inhibited for 39% even at concentration of 0.5 mg L-1, whereas the least affected strain was P. aeruginosa. C. albicans was the most susceptible fungal strain with 50 and 15% growth inhibition at 2 and 0.25 mgL-1 of the extract, respectively (Figure 6).Figure 6
Antimicrobial activity of violacein pigment fromC. violaceum PDF23 showing inhibition against selected human pathogens.


### 3.7.2. Antioxidant Activity of Violacein Pigments ofC. violaceum PDF23

The radical scavenging assay evaluated the antioxidant activity of violet pigments. The IC50 value was 14.40 μg/mL and 28.66 μg/mL for violet pigments and ascorbic acid, respectively (Figure 7).Figure 7
DPPH was used to assess the antioxidant activity ofC. violaceum PDF23 violet pigments. Results are based on an average of three separate experiments and standard deviation is represented by the error bars.These findings suggest that the violet pigments ofC. violaceum PDF23 is more potent in scavenging free radicals than ascorbic acid. The plausible reason for the absence of catalase activity in C. violaceum PDF23 could be the significant antioxidant potential of violet pigments. A crucial role in the advancement of a wide range of pathological chronic diseases such as diabetes and cancer has been proposed for the generation of free radicals [68]. Consequently, treatment with free radical scavenging antioxidants such as microbial dyes C. violaceum PDF23 has the ability to prevent, prolong, or alter many of these diseases.


## 3.7.1. Antimicrobial Efficacy of Violet Dye

In the initial screening, crude methanolic extract ofC. violaceum culture was evaluated for the antimicrobial efficacy against two gram-positive (S. aureus and B. subtilis), two gram-negative (E. coli and P. aeruginosa), and one fungal strains (C. albicans). Violacein-containing culture extract was capable of inhibiting growth of all tested strains between 12 and 54% significantly at a high concentration of 2 mg L-1 compared with control. The growth of S. aureus was inhibited for 39% even at concentration of 0.5 mg L-1, whereas the least affected strain was P. aeruginosa. C. albicans was the most susceptible fungal strain with 50 and 15% growth inhibition at 2 and 0.25 mgL-1 of the extract, respectively (Figure 6).Figure 6
Antimicrobial activity of violacein pigment fromC. violaceum PDF23 showing inhibition against selected human pathogens.


## 3.7.2. Antioxidant Activity of Violacein Pigments ofC. violaceum PDF23

The radical scavenging assay evaluated the antioxidant activity of violet pigments. The IC50 value was 14.40 μg/mL and 28.66 μg/mL for violet pigments and ascorbic acid, respectively (Figure 7).Figure 7
DPPH was used to assess the antioxidant activity ofC. violaceum PDF23 violet pigments. Results are based on an average of three separate experiments and standard deviation is represented by the error bars.These findings suggest that the violet pigments ofC. violaceum PDF23 is more potent in scavenging free radicals than ascorbic acid. The plausible reason for the absence of catalase activity in C. violaceum PDF23 could be the significant antioxidant potential of violet pigments. A crucial role in the advancement of a wide range of pathological chronic diseases such as diabetes and cancer has been proposed for the generation of free radicals [68]. Consequently, treatment with free radical scavenging antioxidants such as microbial dyes C. violaceum PDF23 has the ability to prevent, prolong, or alter many of these diseases.


## 4. Conclusion

Microbial pigments are a potential source of natural dyes, with a wide range of applications in the textile industry. The study highlights the natural dye production from microbes isolated from Great Salt Lake in Chennai, India. The PDF23 bacterial isolate was identified asC. violaceum, which differed from other members of the genus phylogenetically. Violet pigment was observed to be capable of dyeing both cotton and silk satin. Interestingly, fabrics dyed with violet pigment scored fair (3/4) to excellent (5) in all of the colorfastness properties (except light). Violet pigment was efficiently used in textile dyeing in this study, highlighting that it can be used as a substitute for synthetic dye. This is the first report of violet colored pigments isolated from C. violaceum PDF 23 (Great Salt Lake) with antimicrobial and antioxidant properties and textile dyeing applications.



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*Source: 3885396-2022-07-23.xml*