World Journal of Environmental Biosciences
World Journal of Environmental Biosciences
2021 Volume 10 Issue 1

Antimicrobial and Antioxidant potentials  of Carotenoid Pigment Produced by Indigenous Novel Soil Isolate Rhodococcus kroppenstedtii


Chaudhari Varsha Madhukar 1*


1 Department of Microbiology, Faculty of Science, PSGVPMandal’s  SIP Arts, GBPatel Science and STKVS Commerce, College, Shahada, Dist-Nandurbar(MH.), INDIA 425 409.


The present research study evaluated the antioxidant and antimicrobial potentials of intense red carotenoid pigment produced by novel soil isolate.  A novel isolate from the rhizospheric soil of the soya-bean field was found to produce prominent red carotenoid pigment. Based on  phenotype, biochemical and molecular features (16S rRNA sequencing), the isolate was identified as Rhodococcus kroppenstedtii.  Maximum intense red pigment production appeared in yeast extract mannitol broth. Among the various solvents, 80% methanol was the best solvent for the extraction of pigment.  The methanol extracted pigment exhibited efficient antioxidant and antimicrobial activity. Phosphomolybdenum and ferric  analysed the total antioxidant potential of pigment, hence, reducing antioxidant power assay  and the results represented in  ascorbic acid equivalent. The total antioxidant activity of pigment was investigated to be 67.0 ± 0.2 mg AA/g of sample and ferric reducing antioxidant activity was observed to be 80.2 ± 0.31  μg of ascorbic acid equivalent per milligram of the sample. Likewise, another striking applicability of the pigment was investigated and found to exhibit strong antibacterial activity against four target bacterial pathogens of health significance by in vitro techniques as well as antifungal activity against Aspergillus niger, Penicillin chrysogenum, Fusarium oxysporum species.  In conclusion, methanolic extract of the pigment has excellent antioxidant and strong antagonistic activity; this implies the application of pigment as an antioxidant agent in food, pharmaceutical, and cosmetics.

Keywords: Rhodococcus kroppenstedtii, carotenoid pigment,  Antioxidant  and antibacterial activity, Novel soil isolate



Pigments are coloring compounds produced either naturally or synthetically (Koyyati, et al., 2019). Human life is covered with different colors having vital significance in all aspects of life. The pigments have many applications in the coloration of food, textile, paper, cosmetic, plastic, paint industries,  and in leather processing (Mansour and Kairouan, 2018), agriculture, biology (antitumor agents (Numan, et al., 2018; Vishnu and Palaniswamy, 2016), anticancer agents (Afra, et al., 2017), and antimicrobials (Ravikumar, et al., 2016), etc.  Additives usage in food has a vital role in enhancing its quality, improve nutritional value, consistency and provide color to make food more attractive (Chinaza, et al., 2020). Hence, biocolorants can be one of the alternatives to artificial color for addition into any food material (Heer and Sharma, 2017).  Natural pigments produced by bacteria, fungi, plants have better bio-degradability and environment acceptability than synthetic pigments (Mansy and Rathod, 2020; Choudhary and Mallya, 2019). Now a day’s pigment produced from living organisms gained more importance because synthetic colorants are toxic and show harmful  side effects like  hyper-activity in children,  allergenicity, mutagenecity,  carcinogenicity  (Sen, et al., 2019; Oplatowska-Stachowiak and Elliott Christopher, 2015),  neurotoxicity (Akintunde, et al., 2020), genotoxicity (Khana, et al., 2020)  and hurts the  ecosystem. Considering the demands of bio-pigments in various applications, better quality natural colorant with higher stability is the need of the hour. Using  microorganisms for the production of pigment has several advantages, which includes  availability of the cultivation technology, stability of the pigment produced, easy downstream processing, and more cost-effectiveness (Azman, et al., 2018).  Thus,  there is growing intrest in microbial pigment due to their natural character and safety to use (Parmar and Phutela, 2015). Microorganisms provide a readily available alternative source of naturally derived pigments (Rao,  et al., 2017). Various types of pigment such as violacein, carotenoids, quinones,  indigo and melanin are reported from microbes (Caro, et al.,  2017). A carotenoid group of pigments is more widely studied with respect to their applications and verified to show different beneficial effects on human health through serving as precursors of vitamin A, anti-inflammatory effect, antimicrobial and antioxidant activity (Yolmeh, et al., 2016), etc. Pigment-producing bacteria can be isolated from many different sources but soil source being the most common one. Rhodococcus rhodochrous is one of the antioxidative, antimicrobial carotenoid-producing species as previously reported (Haddad, et al., 2017). The present research work revealed  the isolation of novel pigment-producing strains   from  the rhizosphere of soyabean and the investigation of antioxidant and antibacterial activity of carotenoid pigment produced by isolate.  It was found that pigment exhibited excellent antioxidant and antimicrobial activity having potential applications in therapeutics.




Bacterial strains

The bacterial isolate capable of producing an intense red carotenoid pigment, identified as R. kroppenstedtii was isolated from  rhizosphere soil of   soyabean fields  of the  Shahada region . Morphological and biochemical identification of isolate was determined from  MTCC, Chandigarh. The isolate was found to be producing prominent red color pigment on yeast extract mannitol agar plate (YEMA) within 48 hours.  The stock culture of the isolate was maintained on  a YEMA slant at 40C.

Bacterial cultures like Staphylococcus aureus NCIM 2079, Bacilus subtilis  NCIM 2063, E. coli NCIM 2065, Proteus vulgaris NCIM 4175 were procured from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune. Fungal strains like Aspergillus niger  NCIM 1025, Penicilium chrysogenum NCIM 709, Fusarium oxysporum NCIM 1281  were also obtained from NCIM, Pune.


Analysis of isolate by 16S rRNA gene sequencing

16S r RNA nucleotide gene sequencing  was performed for taxonomic characterization of the isolate from the National Centre for Cell Science (NCCS), Pune.


 Sequence assembly and phylogenetic characterization

The 16S rRNA gene sequence data obtained were searched as nucleotide queries  in the nucleotide database of the  NCBI server ( Blast) to find the related sequences. Using the neighbor-joining method in MEGA V 5.04 software  (Saitou and Nei, 1987; Tamura, et al., 2007) and according to the  Kimura two-parameter model (Kimura, 1980),   the phylogenetic tree was constructed after aligning all acquired and related sequences


Biomass and  pigment production

For the biomass and pigment production, the isolate was  inoculated in a 500 mL Erlenmeyer flask   containing 100 mL of sterile yeast extract mannitol broth (YEMB) with 1ml of 24-48 h inoculum. Flask was incubated in a rotary shaker at 120 rpm at 300C for 48 to 72 h until the dark red color of the medium was obtained. After incubation cells were harvested and washed twice with distilled water by repeated centrifugation and allowed to dry to remove complete moisture till constant weight was obtained (Govindaswamy, et al., 1999).


Extraction of pigment

The pigment was extracted from dry cell mass by 10,000 rpm centrifugation for 10 min by the use of 80% HPLC grade methanol (Chaudhari, 2013). The pigment was completely extracted from biomass till the cell pellet appeared colorless.


Ultraviolet-visible spectroscopic studies

UV-VIS spectrophotometer is an important tool for the identification of pigments and dyes. UV-Visible Spectrophotometric analysis (UV Mini 1240 Shimadzu, Japan).  was carried out for the red colored pigment sample extracted in methanol. The scanning range  selected was 200-800 nm and  maximum absorption 𝛌max was measured. Methanol was used as a blank and β-carotene as a Standard.


Antioxidant activity of pigment extract   

Reducing power assay

Reducing power assay is simple and more effective in the analysis of the antioxidant nature of any unknown compounds. This method is based on the reduction of ferric (Fe3+) to ferrous (Fe2+), in the presence of antioxidants (Dontha, 2016). The reducing power of methanol extract of the pigment sample was determined by the slight modification of the method described by Xiao, et al., (2020). Various concentrations ((20 -100 mg/mL) ) of the  test pigment sample were  mixed with 2.5 mL of  50 μM phosphate buffer (pH 6.6). Followed by addition of 2.5 mL 0.1% (w/v) potassium ferricyanide, all the tubes of reactants were placed in a water bath at 50°C for 20 min. 2.5mL  of 1% (w/v) trichloroacetic acid solution was added to the   mixture  with  centrifugation   at 3000 rpm for 10 minutes. 2.5mL of the upper layer was  removed properly  and combined with 0.5 mL of 5 mM ferric chloride solution and the absorbance was measured at 700 nm. Similarly, the blank was run in parallel with all mixture without the pigment sample. Ascorbic acid at various concentrations (20-100μg/mL) was used as an antioxidant standard. The  ferric reducing antioxidant activity was expressed  as a microgram of ascorbic acid equivalent per mg dry weight of the test sample (Al-Laith, et al., 2019). Gradual increased in the absorbance values of reactants indicates an increase in the reducing activity of the pigment sample. (Yao and Qi, 2016). The experimental procedure was  conducted in triplicates and the results were expressed as the mean of triplicates.

Total antioxidant activity by Phophomolybdenum assay

The phosphomolybdenum assay was  used for the quantitative measurement  of the total antioxidation capacity of the pigment sample. It is based on the reduction of Mo (VI) - Mo (V) by the antioxidant and conversion into  green phosphate/Mo (V) complex at acid pH according to the previously reported method of Prieto, et al., 1999; and Mukherjee, et al., (2017).   0.1 ml-test pigment sample (100 μg) was added with a 3 ml of reagent (solution containing 0.6 m/L-1 sulphuric acid, 28mM/L sodium phosphate, and 4 mM/L ammonium molybdate) (Murugan, et al., 2016) and the tubes were kept in boiling water bath at 950C for 90 min. After cooling the absorbance was measured at 695 nm in a UV spectrophotometer  with methanol as a blank. The experimental  reaction was conducted  in triplicate and the mean was  expressed in results. Ascorbic acid in a varying concentration of 100mg/ml was utilized as the reference standard. (Sowndhararajan, et al., 2010). The total antioxidant activity of the test pigment sample was expressed as (mean values) number of mg equivalent of ascorbic acid per gram of the dry weight of the extracted pigment.


Antibacterial  activity of  pigment

Determination of the antimicrobial activity of extracted pigment was carried out by agar disk diffusion technique (Rashid, et al., 2014) against four targets pathogenic bacteria reported affecting health significantly. The pathogenic Gram-positive bacteria like Staphylococcus aureus NCIM 2079, Bacillus subtilis  NCIM 2063, and Gram-negative pathogens like E. coli NCIM 2065, Proteus vulgaris NCIM 4175 were used for the antibacterial activity (0.1mL) by in vitro techniques using nutrient agar plates at 370C for 24-48 h. Antibiotic streptomycin (100 µg/mL) was utilized  as positive control and methanol (100 µL) was utilized as a negative control for comparing activity (Dawoud, et al., 2020)  The paper disk of size 6 mm soaked into pigment (50 and 100 μg/mL) concentrations was used for antibacterial activity.  A  zone of  growth inhibition around the disc was indicative of antimicrobial activity. The  zone diameter  was measured in millimeters. The experiment was repeated  thrice for each pathogenic bacterium and  results were expressed as mean ± Std. deviation.

Antifungal  activity of pigment

Similarly antifungal activity of pigment against some common phytopathogenic fungi namely Aspergillus niger NCIM 1025, Penicillium chrysogenum NCIM 709  and Fusarium oxysporum NCIM 1281 was tested.  Two different concentrations of  50 µL and 100 µL pigment sample soaked in filter paper discs were applied to the PDA agar plate which was inoculated separately with the fungal spore suspension 6 x 106 mL-1 spore.  Commercially  available antibiotic griseofulvin (100 µg/mL) was utilized  as positive control and methanol (100µL) as a negative control. Results were expressed as mean ± Std. deviation.


The intense red carotenoid pigment-producing organism was isolated from the rhizosphere  of a soybean field and identified based on the phenotypic and biochemical characteristics  from MTCC, Chandigarh  as a novel strain Rhodococcus kroppenstedtii.

Identification of  isolate based on 16S rRNA sequence

Identification of isolate was confirmed by 16S rRNA gene amplification and sequencing from NCCS, Pune.  PCR amplification of 16S rRNA gene sequences using the universal primers yielded an amplicon with a size of approximately 1415 bp. The comparison of BLAST search of 16S rRNA gene sequences of pigment-producing isolate with 16srRNA gene sequences of NCBI GenBank database  revealed the highest identity of the isolate with Rhodococcus species.  16S r RNA sequences were submitted to NCBI GenBank database ( under the name, Rhodococcus kroppenstedtii with accession number JN873342.   The phylogenetic tree  (Figure 1) based on the 16S r RNA  sequences was constructed  showing  the relationship of the pigment-producing organism with sequences of related genera.


Figure 1. Phylogenetic tree of Rhodococcus kroppenstedtii


Ultraviolet–Visible spectroscopy

UV-VIS Spectrophotometer with 200-800 nm range was used for scanning the pigment extracted in methanol. Pigment exhibited the presence of a single major peak with absorption maxima at 472 nm.  The maximum absorption of the pigment between wavelengths of 300–600 nm indicates the presence of carotenoids  (Dawoud, et al., 2020). Moreover, the earlier research of  Ichiyama et al,  1989  reported  about the carotenoid pigment in the genus of Rhodococcus the maximum absorption of pigment production in nanometers was (430-425), (440-435), (470-489).  However, according to the literature (Rodriguez-Amaya, 2001; Finkel’shtein,  2016), most of the carotenoid pigments have a maximum absorption range between 375 -505 nm.                      

Antioxidant activity of pigment extract

Reducing power assay

The reducing capacity of the pigment was observed to be remarkable. The pigment produced by isolate R. kroppenstedtii exhibited stronger antioxidant capacity. The reducing power was expressed in terms of a microgram of ascorbate equivalent per mg dry weight of the test sample.  Reducing power of the pigment sample was found to increase with gradually increasing (Table 1) the concentration of sample from 20 to 100 mg.mL-1 which indicated the higher reductive potential of the pigments. The experimental results revealed that the red color of the test solution changes to various shades of blue color because of the reducing power of the pigment sample. Presence of reducer radical causes the conversion of the ferricyanide complex to the ferrous form with the formation of pearls Prussian blue measured at 700 nm. The maximum antioxidant activity was found to be 80.2 ± 0.31 μg of ascorbic acid equivalent per milligram of the sample. The ferric-reducing capacity  pigment serves as a significant  potential antioxidant  activity indicator of  the pigment (Mogadem, et al., 2021).

Table 1. Reductive capacity of pigment

Pigment concentration mg.mL-1

OD values at 700 nm

Antioxidant activity    (μg of ascorbic acid)



25.2 ± 0.6



39.5 ± 0.12



58.5 ± 0.53



62.4 ±0.71



80.2± 0.31

Values are means ± Std. dev.(n=3)

Total antioxidant activity by phosphomolybdenum assay

Phospomolybdenum assay was used for evaluation of the total antioxidant capacity of the pigment and expressed as the number of equivalents of ascorbic acid (AA) (Soulef,  et al., 2020; Fidrianny and Rika, 2016).   Experimental results revealed that pigment exhibited stronger total antioxidant activity of 67.0 ± 0.2 mg AA/g of the sample.


Antibacterial activity of pigment

Experimental results of the present study revealed that (Table 2) the pigment extracted from a novel strain of R. kroppenstedtii exhibited effective antibacterial activity as compared with standard antibiotic streptomycin. Among the four pathogenic strains used to study antibacterial activity, organisms showed variation in the growth inhibition by test pigment sample.  The highest zone of growth inhibition was observed against the pathogens Staphylococcus aureus (21.0 ± 0.2 mm). This was compared with the activity of streptomycin (19.3 ± 0.5 mm) against S. aureus. Similarly bacterial pathogens Bacillus subtilis (12.0 ± 0.21mm) and E.coli (10.0 ± 0.42mm) were inhibited at higher concentration (100 μg/mL) of pigment while a minor zone of growth inhibition was observed against the Proteus species (3.0 ± 0.2mm). This data obtained revealed that the Staphylococcus and Bacillus, species were found to be highly susceptible to Rhodococcus pigment and more effectively inhibited, followed by E.coli and  Proteus.  The antimicrobial activity of the pathogen may vary due to difference in cell wall composition and susceptibility of each pathogen to pigment extract.



Table 2.  Antibacterial activity of pigment against human pathogens

Bacterial pathogens

Zone Diameter (mm)

Concentration  of  pigment

Streptomycin (PC)

(100 μg/mL)

Methanol (NC)

(100 μL)

100 (μg/mL)

50 (μg/mL)


12.0 ± 0.21

8.0 ± 0.4

10.7 ± 0.1

0.5 ± 0.2


21.0 ± 0.2

11.0 ± 0.12

19.3 ± 0.5

0.4 ± 0.6

E. coli

10.0 ±0.42

7.0 ± 0.3

11.1 ± 0.2

0.4 ± 0.3

P. vulgaris

3.0 ±0.2

2.0 ± 0.5

10.0 ± 0.5

0.3 ± 0.4

PC=Positive control, NC=Negative control    Values are the mean ± Std. deviation (n=3)



The literature survey reported that the carotenoid type of pigment produced by Sporobolomyces spwas found to be highly inhibitory for E. coli with an inhibition zone of 28 mm.

The carotenoid pigment possessed good activity against S. aureus  26 mm zone (Manimala and Murugesan, 2014). The antimicrobial activity of Rhodococcus rhodochrous pigment against Staphylococcus aureus was with a 21 mm inhibition zone (Haddad, et al., 2017) According to the literature, the  pigment  extracted from Micrococcus roseus  has antimicrobial activity higher against Gram-positive bacteria than Gram-negative bacteria (Yolmesh, et al., 2018). Thus, this result of Rhodococcus pigment demonstrates the potential application of pigment in pharmaceutical and cosmetic products.

Antifungal activity of pigment

After incubation, inhibition of mycelial growth in fungus was observed but very minor zones of inhibitions were observed against the fungal pathogens after 3-7 days. The data obtained (Table 3) indicate that pigment extracted from Rhodococcus has negligible antifungal activity as compared to antibacterial activity.



Table 3. Antifungal activity of pigment against fungal pathogens



Zone Diameter (mm)

Pigment Concentratios


(PC) 100 (μg/mL)

Methanol (NC)

(100 μL)

100 (μL)

50   (μL)

A. niger

0.5 ± 0.12

2.7 ± 0.5

2.7 ± 0.5

0.1 ± 0.5


0.2 ± 0.5

2.2 ± 0.3

2.2 ± 0.3

0.15 ± 0.3


0.43 ±0.1

2.8 ± 0.6

2.8 ± 0.6

0.18 ± 0.2

PC=Positive control, NC=Negative control    Values are the mean ± Std. deviation (n=3)



At higher pigment concentration inhibition of mycelial growth and reduction in length of mycelia was observed in A.niger and F.oxysporum followed by inhibition of P.chrysogenum as compared to that in control. According to the literature survey (Ferdes, et al., 2009). Sometimes fungal  strains are less or more inhibited by natural dyes


The bacterial isolate producing prominent red pigment was newly isolated from a rhizospheric soil sample collected from  the soyabean field of the surrounding local area of Shahada. Morphological, biochemical, and molecular identification revealed the unknown isolate as  R. kroppenstedtii with accession number JN873342. The maximum pigment production was observed in yeast extract mannitol broth and agar plates.  Methanol extract of pigment has absorption maxima recorded at 472 nm, which revealed the presence of carotenoids.  The pigment had strong total antioxidant activity determined by phosphomolybdenum assay and reducing power assay. In addition, pigments have excellent antimicrobial activity against pathogenic bacteria and minor antifungal activity. It can be concluded that pigment has the potential use in the food, cosmetic and pharmaceutical industry as a natural antioxidant colorant. Studies in the future will be conducted on the large-scale production of pigment for industrial applications.

ACKNOWLEDGMENTS: The author is thankful to the Department of Microbiology, PSGVPMandal’s ASC College, Shahada for providing the necessary facilities for this work.


FINANCIAL SUPPORT: The author is thankful to UGC (WRO), Pune,  for providing financial support in this  research work.



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