s12934 023 02276 y

El‑Zawawy et al.
Microbial Cell Factories (2024) 23:23 Microbial Cell Factories

https://doi.org/10.1186/s12934-023-02276-y
RESEARCH Open Access
Bioproduction and optimization

of newly characterized melanin pigment
from Streptomyces djakartensis NSS‑3 with its
anticancer, antimicrobial, and radioprotective
properties
Nessma A. El‑Zawawy1*, El‑Refaie Kenawy2, Sara Ahmed1 and Shimaa El‑Sapagh1
Abstract
Background Melanin is a natural pigment that is considered a promising biomaterial for numerous biotechnologi‑
cal applications across several industries. Melanin has biomedical applications as antimicrobial, anticancer, and anti‑
oxidant properties. Additionally, in the pharmaceutical and cosmetic industries, it is used in drug delivery and as a
radioprotective agent. Also, melanin has environmental uses in the fields of bioremediation and the food industry. The
biosynthesis of melanin pigment is an area of interest for researchers due to its multifunctionality, high compatibility,
and biodegradability. Therefore, our present work is the first attempt to characterize and optimize the productivity
of melanin pigment from Streptomyces djakartensis NSS-3 concerning its radioprotection and biological properties.
Results Forty isolates of soil actinobacteria were isolated from the Wadi Allaqui Biosphere Reserve, Egypt. Only
one isolate, ACT3, produced a dark brown melanin pigment extracellularly. This isolate was identified according
to phenotypic properties and molecular phylogenetic analysis as Streptomyces djakartensis NSS-3 with accession
number OP912881. Plackett–Burman experimental design (PBD) and response surface methodology (RSM) using
a Box-Behnken design (BBD) were performed for optimum medium and culturing conditions for maximum pigment
production, resulting in a 4.19-fold improvement in melanin production (118.73 mg/10 mL). The extracted mela‑
nin pigment was purified and characterized as belonging to nitrogen-free pyomelanin based on ultraviolet–visible
spectrophotometry (UV–VIS), Fourier transform infrared (FT-IR), Raman spectroscopy, scanning electron microscopy
(SEM), energy dispersive X-ray spectroscopy (EDX), and NMR studies. Purified melanin demonstrated potent scaveng‑
ing activity with IC50 values of 18.03 µg/mL and revealed high potency as sunscreens (in vitro SPF = 18.5). Moreover,
it showed a nontoxic effect on a normal cell line (WI38), while it had a concentration-dependent anticancer effect
on HCT116, HEPG, and MCF7 cell lines with IC50 = 108.9, 43.83, and 81.99 µg/mL, respectively. Also, purified melanin
had a detrimental effect on the tested MDR bacterial strains, of which PA-09 and SA-04 were clearly more susceptible
to melanin compared with other strains with MICs of 6.25 and 25 µg/mL, respectively.
Conclusion Our results demonstrated that the newly characterized pyomelanin from Streptomyces djakartensis NSS-3
has valuable biological properties due to its potential photoprotective, antioxidant, anticancer, antimicrobial, and lack
*Correspondence:
Nessma A. El‑Zawawy
[email protected]
Full list of author information is available at the end of the article
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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El‑Zawawy et al. Microbial Cell Factories (2024) 23:23 Page 2 of 26
of cytotoxic activities, which open up new prospects for using this natural melanin pigment in various biotechnologi‑
cal applications and avoiding chemical-based drugs.
Keywords Melanin bioproduction, Actinobacteria, Response surface optimization, Radioprotection, Antimicrobial
Introduction structure and a negative charge. In many organisms, it is

There is a wide variety of actinomycetes, all of which are synthesized via the oxidative polymerization of phenolic
gram-positive and filamentous bacteria. They are found or indolic compounds [16]. Microorganism-derived pig-
in natural and artificial environments and play an impor- ments are preferred over plant-based pigments because
tant role in the degradation of organic matter in soil [1]. their production is less susceptible to environmental
These saprophytic, free-living bacteria are considered factors [17]. Generally, the majority of microbial mela-
a major source in the development of antibiotics [2, 3]. nins are produced by converting either tyrosine (by the
These microorganisms are the most economically and DOPA-pathway) or malonyl-coenzyme A (via the DHN-
biotechnologically significant because of their ability to pathway), with the help of distinct groups of enzymes.
produce a wide range of different pigments and bioactive The first process involves the conversion of the melanin
secondary metabolites [4, 5]. precursor, tyrosine, into L-Dopa, which is then trans-
The toxic effects of synthetic pigments have made formed into dopaquinone by the action of tyrosinase and
those derived from microorganisms increasingly appeal- laccase enzymes. The second process involves the inter-
ing [6]. This is because microbial pigments are more reli- nal production of the precursor, malonyl-coenzyme A, by
able, readily available, simple to harvest, and productive the action of polyketide synthases. This is achieved by the
[7]. Natural pigments produced by microorganisms such step-by-step decarboxylative condensation of five mole-
as bacteria, fungi, and yeasts have many advantages in cules of malonyl-coenzyme A, resulting in the formation
different fields when compared to their synthetic coun- of 1,3,6,8-tetrahydroxynaphthalene (THN) [18]. Micro-
terparts [8]. There are numerous industrial and medi- organisms that use the DOPA-pathway are preferred for
cal uses for microbial pigments because they are safe, high-yield melanin synthesis [19].
biodegradable, and non-carcinogenic [9]. Different pig- Melanin’s phenol group, or indole structure, gives it a
ments, including bacteriochlorophylls, flavins, melanin, variety of biological actions in addition to its chemical
carotenoids, phenazine, astaxanthin, and ß-carotene, are and physical characteristics. Prior studies have mostly
byproducts of microbial metabolism [10]. examined the ability of melanin to scavenge free radicals,
Natural pigments, including melanin pigments, can be chelate metal ions, and exhibit antioxidant and antibacte-
produced by actinomycetes, which are also capable of rial properties. Additionally, these studies have discussed
producing a wide variety of these pigments [11]. Moreo- the many applications of melanin in industries such as
ver, melanin can be made by a wide variety of bacteria, agriculture and food production [20, 21]. Applied stud-
including Pseudomonas, Aeromonas, Azotobacter, Myco- ies have gradually focused on biomedical aspects, such
bacterium, Bacillus, and Streptomyces [12]. Streptomy- as magnetic resonance imaging (MRI) contrast agents
ces spp. are the most extensively studied actinomycetes [31, 32], melanin’s anti-tumor [22], immunomodulatory
species for melanin production. The classification of [23, 24], antimicrobial [25, 26], radiation protection [27],
melanin pigments is based on their chemical composi- photothermal properties [28, 29], and other activities that
tions, namely eumelanin, pheomelanin, pyomelanin, and have gradually attracted attention in recent years [30].
allomelanin [13]. Auricularia auricula, a marine strain The physiochemical and nutritional parameters, in par-
of Streptomyces sp., and the soil fungus Cladosporium ticular, must be optimized for microbial growth and mel-
cladosporioides were shown to produce extracellular anin production. It is challenging and time-consuming to
melanin mostly composed of pheomelanin. Moreover, implement a "one factor at a time" optimization method
certain bacteria, fungi, and other species may produce [22]. Several different mathematical techniques with
the black-brown pigment known as pyomelanin. Ralsto- multiple independent variables, like the response surface
nia pickettii, Pseudomonas aeruginosa, and Streptomyces methodology (RSM) and the Plackett–Burman design
avermitilis are significant wild species of bacteria that (PBD) [33] were used to improve optimization, in which
have been linked to pyomelanin synthesis [14]. interaction effects between the variables can also be pre-
The use of microorganisms to produce melanin has dicted for relatively large-scale production of microbial
proven to be a cost-effective and ecologically sound sub- melanins, which could replace current commercial mel-
stitute for the chemical synthesis of melanin [15]. Mela- anin [34]. Therefore, the main objective of this study is
nin is a complex hydrophilic polymer with an irregular to produce, optimize, and characterize melanin pigment
from Streptomyces djakartensis NSS-3 for the first time, all observed visually. Scanning electron microscopy
in addition to assessing its radioprotection and biological (SEM; JEOL, JSM-5200 LV, Electron Microscope Unit,
properties. Faculty of Medicine, Tanta University, Egypt) was used
for microscopic identification. Strain growth charac-
Materials and methods teristics were analyzed by growing the strain at varying
Soil sampling and isolation of actinobacterial strains temperatures (25, 35, 45, and 55 °C), pH levels (5.0, 7.0,
Soil samples were collected randomly from Wadi-Allaqui 9.0, and 10.0), and NaCl concentrations (3.0%, 5%, 7%,
Biosphere Reserve, an extremely arid region which is and 10% w/v). Experiments were carried out according
about 180 km south of Aswan on the eastern side of Lake to Bergey’s Manual of Bacteriology to identify the bio-
Nasser, Egypt. It lies between 22° 00 23° 00 N latitudes, chemical and physiological characteristics of the strain
31° 01 and 32° 80 E longitudes (Additional file 1: Fig. S1). [38].
This region is inhabited by specifically adapted microor- The selected isolate ACT3 was molecularly character-
ganisms that produce different secondary metabolites, ized by sequencing its 16S rDNA gene, as described by
enabling them to survive under extreme environmental El-Zawawy, et al. [39]. Cultures were sent to the Molec-
conditions. Samples were collected aseptically from 10 ular Biology Research Unit at Assiut University, where
to 15 cm below the ground surface. Then, directly deliv- DNA was extracted using a patho-gene-spin DNA/RNA
ered to the lab, where they were air dried for 24 h at 45 °C extraction kit manufactured by the Korean firm Intron
at the Faculty of Science, Tanta University, Tanta, Egypt. Biotechnology. With the 27 F (forward) primer (5-AGA
For bacteriological analysis, one gram of each soil sample GTT TGATCMTGG CTCAG-3) and 1492 R (reverse)
was suspended in 9 ml of distilled water, then vortexed, primer (5-TACGGCTACCTTGTTACGACTT-3), bac-
and diluted serially up to 10−5 dilution using the stand- terial DNA samples were sent to SolGent Company in
ard serial dilution plate method [35]. After that, one mL Daejeon, South Korea, for PCR and rRNA gene sequenc-
of each dilution was plated on starch casein agar (SCA) ing. The PCR amplification was carried out in a manner
(Hi-Media, India) in triplicate. Filter-sterilized cyclohex- similar to that described by Ali, et al. [40]. Sequencing
imide (75 g/mL) and nystatin (25 g/mL) were added to was performed on the isolated amplification products.
SCA medium to prevent the growth of molds and yeasts The obtained sequences were analyzed with the help
[36]. The cultured plates were incubated for 7–14 days at of BLAST, a local alignment search tool hosted on the
30 °C. After being cultured, suspected colonies of actino- NCBI database by the National Center for Biotechnology
bacteria were purified for further assays. Information. GenBank (http://www.ncbi.nlm.nih.gov/
genbank) now contains the sequences. In order to deter-
Screening for the most potent melanin‑producing mine whether or not the obtained sequences were similar
actinobacterium and its morphological, physiological to those already in the NCBI database, a BLAST search
and molecular characterization was performed. MEGA 7.0 was used to generate the 16s
The isolated actinobacteria were screened for the pro- rDNA phylogenetic tree via a neighbor-joining method
duction of melanin by cultivating on peptone yeast [41].
extract iron agar (PYIA) (peptic digest 15 g/L, pep-
tone 5 g/L, yeast extract 1 g/L, ferric ammonium citrate
0.5 g/L, dipotassium phosphate 1 g/L, sodium thiosul- Melanin production and quantification
phate 0.08 g/L, and distilled water 1 L; agar 20 g; pH 7.2). In order to produce a dark melanin pigment, the selected
Plates were incubated at 30 °C for 5–7 days. Pigment isolate ACT3 (108 colony forming unit CFU/mL) was
production was observed every 24 h regularly, as obser- inoculated into modified peptone yeast extract iron
vation of brown to the black coloration around the col- broth (PYI) supplemented with 2 g/L of L-tyrosine and
ony indicates melanin production [12]. The most potent incubated at 30 °C for 7 days with shaking at 160 rpm
melanin-producing actinobacterium was coded as ACT3 [42]. After incubation time, selected cells were collected
and subjected to further identification and melanin by centrifugation at 3500 rpm for 15 min and melanin
extraction. pigment production was quantitatively evaluated by
The morphological characteristics of the chosen measuring optical density ( OD280) of the filtrate spectro-
isolate (ACT3) were studied using macroscopic and photometrically at 280 nm [22]. The components of the
microscopic techniques. The isolate was initially iden- medium are essential in melanin production and opti-
tified through macroscopic characterization on Inter- mization. Therefore, Plackett–Burman design (PBD) and
national Streptomyces Project (ISP) media types ISP-2, Box-Behnken design (BBD) were used to find out the
ISP-3, ISP-4, and ISP-6 [37]. Aerial and substrate myce- role that every component played to optimize melanin
lia, color, and production of diffusible pigments were production.
Melanin optimization 0, and + 1 for low, intermediate, and high values, respec-
Screening for the main factors affecting melanin production tively) (Additional file 1: Table S2) was used to fit a poly-
by Plackett–Burman experimental design (PBD) nomial model [45]. The whole design was composed of
Optimization of the medium for maximal melanin seventeen experimental trials with five central points to
pigment production using the selected isolate ACT3 estimate the repeatability of the method. All trials were
through statistical analysis was done using Plackett–Bur- performed in triplicate, and the average melanin yield
man design (PBD) as a first optimization step to select was used as a response. The data were analyzed by multi-
the most critical factors that have positive and significant ple regression analysis using Design Expert software, and
effects on melanin production. The statistical software then the polynomial equation to represent melanin yield
used was Design-Expert 7.0 (Stat Ease Inc., Minneapolis, as a function of the independent variables tested was
U.S.A). Plackett–Burman design is a sort of two level frac- derived.
tional factorial design, which identifies the most critical
environmental conditions and media components from a Y = β0 − βi Xi + βij Xi Xj + βii X2i
group of candidates by applying the few numbers of tests.
PB design used to detect which elements required for ele- where y is the predicted response, β0 is the intercept
vated melanin production by selected isolate [43]. In this term, βi is the linear coefficient, βij is the quadratic coef-
study, a total of 13 process parameters affecting melanin ficient, βii is the interaction coefficient, and XiXj repre-
yield and six dummy variables were analyzed in 20 trials sent the independent variables [46]. Three-dimensional
to calculate the standard error. These variables includ- surface plots were used to express the fitted polynomial
ing incubation period, pH, temperature, inoculum size, equation. To maximize the response, the level of each
agitation speed, yeast extract, peptone, peptic digest, variable was optimized using the design expert numerical
L-tyrosine, copper sulphate, ferric ammonium citrate, optimization method.
dipotassium phosphate, and sodium thiosulphate; were
added at two levels: low (−1) and high (+ 1) as in Addi- Experimental validation
tional file 1: Table S1. The experiment was carried out in To detect the validity of the model and find out the accu-
20 runs to investigate the effect of the specified variables racy and stability of the model, one predicted trial, esti-
on the formation of melanin. All trials were done in trip- mated by the BBD numerical optimization, was selected
licate, and the average of melanin production was used as as a check point and tested experimentally to calculate
the response. The statistical significance of the first-order the percent of deviation. For further validation of the
model was determined using Fisher’s test for analysis of results obtained using BBD, a comparative analysis was
variance (ANOVA) [44]. Plackett–Burman experimental carried out on melanin production before and after opti-
design is based on the first order model: mization (under the optimal conditions predicted by the
model).
Y = β0 + βi Xi
Extraction and purification of melanin
where, Y is the response or dependent variable (melanin After optimization of melanin production from selected
production); it will always be the variable we aim to pre- isolate ACT3, melanin was extracted as in Fig. 1 by cen-
dict, β0 is the model intercept and βi is the linear coef- trifuging the darkly pigmented medium at 3500 rpm for
ficient, and Xi is the level of the independent variable 15 min (using a refrigerated Eppendorf 5810R) and sepa-
which will help us explain melanin production. rating the bacterial cells (pelleted) from the supernatant
[22]. The crude pigment granules (melanin) were sepa-
rated by acidifying the supernatant with 3 mol/L HCl to
Response surface methodology using Box Behnken design pH 3, at room temperature for 24 h, and then centrifug-
(BBD) ing it at 3500 rpm for 15 min. To obtain the pure pig-
According to the Plackett–Burman design results, ment, melanin pellets were washed with distilled water
response surface methodology (RSM) with Box–Behnken four times before being centrifuged once more. Lyophi-
experimental design (BBD) was conducted to gain opti- lization and long-term storage at 20 °C allowed the puri-
mal levels of the most three significant factors positively fied pigment to be preserved for later physiochemical
enhancing the production of melanin. The optimal levels and spectroscopic studies [47]. Thin-layer chromatog-
of these three variables for maximum melanin produc- raphy (TLC) was used to confirm the purified melanin
tion were determined by generating response surface [48]. The purified pigment was compared with standard
graphs to visualize the effect of each variable alone and synthetic melanin (Sigma-Aldrich R) using a silica gel
in combination. Each variable at three levels (coded − 1, chromatography plate (Merck TLC Silica Gel 60 F254) as
Fig. 1 Schematic diagram of melanin extraction from S. djakartensis NSS-3
the stationary phase and the solvent system (Petroleum and incubating for 2, 4, and 6 h at the respective tempera-
ether: Ethyl acetate: 95% Ethanol: Ammonia, 4: 4: 6: 1, tures. All of these tests were done compared to standard
v/v) as the mobile phase. The purified pigment and stand- synthetic melanin.
ard melanin were spotted on TLC. The developed plates Ultraviolet (UV) analysis with a Perkin Elmer Lambda
were dried in an oven, the spots were visualized under a 4B UV–vis spectrophotometer was used to verify the
UV chamber, and the retention factor (Rf) value for puri- melanin purification process [50]. All of the chemical
fied melanin was calculated and compared to the stand- functional groups in purified melanin were identified by
ard one. After scratching the melanin band from TLC, performing an FT-IR analysis with an IR spectropho-
physiochemical and spectroscopic analysis were carried tometer (Perkin-Elmer 1430) [51]. Using a Bruker RFS 27
out to confirm the purification of melanin. spectrometer, FT-Raman spectroscopy (SENTERRA II
RAMAN microscopy, Bruker) has reported and assigned
Physiochemical and spectroscopic analysis of melanin a wide variety of vibrational modes in purified melanin
Physiochemical analyses of the purified melanin pig- sample [52]. Melanin’s 13 C and 1H NMR spectra were
ment were carried out using a modified approach men- recorded using a JEOL GSX 400 instrument at 30 °C in
tioned by Muthuraj, et al. [49]. The solubility pattern order to confirm the presence of the functional groups
of the purified melanin pigment was tested by dissolv- [39].
ing melanin (5mg/ml) in various inorganic and organic Energy Dispersive X-ray Spectroscopy (EDX) was used
solvents including distilled water, 1N NaOH, 1N HCl, to permit qualitative and quantitative analysis of elemen-
dimethyl sulfoxide as well as methanol, absolute etha- tal composition of purified melanin sample [53]. After
nol, chloroform, benzene, acetone, and ethyl acetate. The fixing the melanin samples for 2 h in 2% glutaraldehyde
dissolution was observed after standing for 30 min. For in 0.1 M sodium cacodylate buffer, the samples were
the analysis of bleaching, KMnO4 were utilized. Also, in washed in the same solution and allowed to dry. Dried
order to determine if the pigment precipitated, 3N HCl sample was metalized with gold (Denton Vacuum Desk
and a 1% FeCl3 solution were utilized. Moreover, the heat V) and then mounted in aluminum sample holders. Scan-
stability of purified melanin was scanned by subjection ning electron microscopy (JEOL, JSM-5200 LV) was then
of melanin to various temperatures at 60, 80, and 100 °C used to examine the purified melanin sample.
Radioprotection measurement co., St. Louis, USA) supplemented with 10% fetal bovine
In order to determine the sun protection factor (SPF), the serum (GIBCO, UK). Antibiotics added were 100 U/
absorbance in the 290–320 nm spectrum was measured mL penicillin and 100 μg/mL streptomycin at 37 °C in a
in an ethanol solution containing 100 mg of purified mel- 5% CO2 incubator. The cells were seeded at a density of
anin pigment from selected isolate ACT3. The SPF value 1 × 104 cells/well in a 96-well plate at 37 °C for 48h under
of the purified melanin has been determined using the 5% CO2. After incubation, the cells were treated with dif-
Mansur equation [54]. ferent concentrations (0.05, 0.5, 5, 50 and 500 μg/mL) of
purified melanin. Following 24 h of incubation, 0.02 mL
SPF = CF x A [] x I []x EE []
of MTT solution (5 mg/mL) was added to each well and
where CF = 10 is the correction factor, A is the melanin incubated for 4 h in a humidified atmosphere of 5% CO2.
absorbance, λ is the wavelength, I is the solar intensity Purple formazan crystals formed due to MTT reduc-
spectrum, and EE is the erythema effect. tion by viable cells in each well was dissolved in 100 μL
of dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA).
Antioxidant capacity Cells that had not been treated with purified melanin pig-
The antioxidant activity of purified melanin pigment ment were used as a control. The absorbance at 570 nm
from selected isolate ACT3 was assessed using DPPH (2, was measured using a plate reader (EXL 800, USA). Cell
2-diphenyl-1-picrylhydrazyl) assay according to Singh viability percentage (CV%) was calculated using the
[55]. In brief, four milligrams of 0.02 mM DPPH was dis- formula:
solved in 100 ml of methanol and stored at 4 °C to serve CV% = (Absorbance of treated samples
as a standard stock solution. Two milliliters of freshly
/Absorbance of untreated sample) × 100
made stock solution were combined with one milliliter of
purified melanin pigment at various concentrations (1.25, Doxorubicin was used as a standard anticancer drug
2.5, 5, 10, 25, 50, and 100 g/mL). The experiment’s nega- for comparison.
tive control was methanol, and the positive control was
ascorbic acid (Vitamin C). The absorbance of the mix- Antibacterial activity.
tures was measured at 517 nm after 30 min of incubation Four multidrug-resistant bacterial strains obtained from
at room temperature in the dark. The free-radical scav- our prior studies were used in the current study. Strains
enging activity (%) was calculated using the formula: of Pseudomonas aeruginosa (PA-09), Escherichia coli
(EC-03), Klebsiella pneumoniae (KP-01), and Staphylo-
[(Absorbance at blank) − (Absorbance at test)/
coccus aureus (SA-04) were isolated and identified [51,
(Absorbance at blank)] x100 57, 58]. The agar-well diffusion method, as described
The concentration of melanin required to scavenge by Ali, et al. [59], was used to estimate the antibacterial
50% of the radicals (IC50) was determined using a linear activity of purified melanin pigment against the tested
regression curve. strains of bacteria. Fresh overnight cultures of tested
strains, approximately 1 × 108 CFU/ml; were used. One
In vitro cytotoxicity and anticancer activity hundred microliters of cultural suspension of each tested
Both safety and anticancer activity of the purified mela- strain were swabbed unevenly onto individual sterile
nin of selected isolate ACT3 were measured in vitro on Mueller–Hinton agar (MHA) (Hi-Media, India) plates
both non-cancerous cells (Human lung fibroblast (WI38) by sterile cotton swabs. The suspension of melanin at a
and cancerous cells (Colorectal carcinoma Colon cancer concentration of 1 mg/mL was prepared by suspending
(HCT116), Mammary gland Breast cancer (MCF-7) and 10 mg of purified melanin in 10 mL of 1% pure dimethyl
Hepatocellular carcinoma (HEPG-2) which obtained sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri,
from American Collection of Cell Culture (ATCC) via USA) and using it as a stock solution for further stud-
a holding company for biological products and vac- ies. Then, five wells of 6 mm diameter were made using
cines (VACSERA), Cairo, Egypt. Different cell lines a sterilized steel well borer. About 50 μL of melanin solu-
were used to determine the inhibitory effects of purified tions with various concentrations (10, 20, 30, 40, and
melanin pigment on cell growth using standard 3-(4, 5 50 μg/mL) were pipetted into the corresponding wells.
dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide The plates were incubated at 30 °C for 24 h, and an inhibi-
(MTT) assay [56]. This colorimetric assay is based on tion zone appeared around the well, indicating the bioac-
the reduction of the yellow tetrazolium bromide (MTT) tivity of purified melanin pigment. The diameters of the
(Sigma Aldrich, USA), to a purple formazan product. inhibition zones around the respective wells were meas-
The cells were cultured in RPMI-1640 medium (Sigma ured using a metric ruler, expressed as the mean value (in
mm), and compared with the antibiotics streptomycin
and tetracycline as positive controls, and DMSO (1%) as vegetative and aerial mycelium, ACT3 was isolated on
a negative control. Triplicates were maintained, and aver- various ISP media. Different colors were observed in the
age values were calculated. mycelia of this strain, indicating that it can produce pig-
ments (Additional file 1: Table S3). ACT3 was found to
Minimum inhibitory concentration (MIC) and minimum be a gram-positive bacterium with typical growth and
bactericidal concentration (MBC) determination sporulation by physiological and biochemical analysis.
Following an overnight incubation of selected strains at The strain survived successfully at 25 °C, 5% NaCl, and
37 °C in MHB, turbidity was set to about 106 CFU/mL. pH = 7, according to the results. In addition, starch, gela-
The broth microdilution assay was used to determine tin, casein, and urea were all found to be degradable by
the MIC and MBC of purified melanin, as described by the strain (Additional file 1: Table S4). Further, it was
El-Zawawy, et al. [51]. Purified melanin was prepared by observed that the strain had the ability to degrade the fol-
making two-fold serial dilutions in sterile Luria Bertani lowing substrates: starch, gelatin, casein, and urea (Addi-
broth medium, with concentrations ranging from 1.56 g/ tional file 1: Table S4).
mL to 200 g/mL. Placed 100 µL of the melanin dilutions The morphological characteristics of the ACT3 strain,
in each well of a 96-well microtiter plate, inoculated each as shown in Fig. 2A, B, appear as dusty gray aerial myce-
well with 100 µL of the test organism in LB at a final con- lium with brown substrate mycelium colonies growing
centration of 1 05 CFU/mL, and incubated at 37 °C with on ISP-6 medium. Moreover, scanning electron micro-
120 rpm shaking for 24 h. The minimal inhibitory con- graphs revealed that the selected isolate formed a straight
centration (MIC) of purified melanin was determined as to flexuous (rectiflexible) chain of globose spores with
the concentration required for complete inhibition of the a smooth surface (Fig. 2C, D, and E). All these proper-
growth of pathogenic bacteria after 24 h of incubation at ties clearly suggest that the strain ACT3 belongs to the
37 °C. Ten microliters of each set were streaked onto an genus Streptomyces. Similar findings were done by Dast-
MHA plate, and the plates were incubated at 37 °C for ager, et al. [61], who showed that the ability to produce
24 h to determine the MBC. Since the minimal inhibi- melanin was also found in 5–10% of Streptomyces isolates
tory concentration (MIC) of purified melanin defined screened from soil samples in the Gulbarga region. Also,
the 99.5% mortality of selected isolates, we examined the only two strains out of 25 isolates from Melia dubia fields
plates to find the MBC that completely inhibited bacte- in two different locations in Tamil Nadu were capable of
rial growth [57]. producing melanin [62].
Then, molecular identification by 16S rRNA confirmed
Results and discussion ACT3 as Streptomyces djakartensis NSS-3 with accession
Soil actinomycetes are able to produce a wide variety of number OP912881. Streptomyces djakartensis NSS-3
products, including enzymes, bioactive secondary metab- showed 99.87% similarity with Streptomyces djakartensis
olites, melanin, and antibiotics [1] that cannot be pro- strain NBRC 15409 accession No. NR 041178.1 (Fig. 3).
duced by other types of bacteria because of their unique According to our knowledge, this is the first report show-
environment. Actinobacteria are highly desirable due to ing S. djakartensis as a melanin-producing strain from
their potent production capacity of bioactive compounds soil.
[60]. Melanin is a promising biomaterial with numer-
ous biotechnological applications in the pharmaceutical, Melanin quantification and optimization
medical, and environmental sectors [22]. Therefore, the Because of the multiple and diverse factors that affect
first step in the present study was to screen for the most melanin biosynthesis, there is no universally applicable
potent melanin producers from soil actinobacteria. culture media or cultivation condition for growing mela-
nogenic microorganisms. In our study, S. djakartensis
Actinobacterial isolation and identification NSS-3 produced melanin (28.38 mg/10 mL) after 7 days
of melanin‑producing isolates in PYI broth medium supplemented with tyrosine, simi-
A total of forty different actinobacteria were selected lar to melanin produced by Streptomyces glaucescens
from soil samples collected from the Wadi Allaqui Bio- strain NEAE-H (350 mg/L) on the same media [22].
sphere Reserve. Out of forty strains tested in a pre- While yeast melanin by Yarrowia lipolytica was 160 mg/L
liminary screening for melanin production, only isolate in M8 medium [63]. Media constituents and ratios of
ACT3 showed melanin production. The most promising each component vary in microbial melanin production
isolate, ACT3, was selected as a potential isolate for the depending on the producing isolate [64]. Bacteria use
production of melanin, identified on the basis of mor- melanogenesis via the DOPA pathway as a mean to com-
phological, cultural, physiological, and biochemical prop- bat the harmful effects of phenolic chemicals present in
erties. By observing sporulation and the development of the environment, including those produced by bacteria
Fig. 2 Morphological characteristics of ACT3 strain. Dusty aerial mycelium (A); Brown substrate mycelium (B); Scanning electron micrographs
showing spore chain shape and spore surface ornamentation of ACT3 strain at different magnifications (1.000, 7.500, 7.500X) (C, D, E)
during host defense [65]. Therefore, several microor- growth and pigment biosynthesis, and should be care-
ganisms rely on external sources of tyrosine or tyrosine fully considered. Thus, applying statistical optimization
derivatives to carry out the process of melanin produc- methods can aid significantly in increasing the melanin
tion. Scientists that research microbial melanization find yield by detecting the key parameters affecting the pro-
this particularly interesting since melanin is produced cess and the effects of the interaction between those
extracellularly, which eliminates the need for aggressive valuable parameters [67]. Optimization of the melanin
extraction methods. Moreover, these attributes provide production from S. djakartensis NSS-3 has been carried
significant manipulation over the quantity and quality out using Plackett–Burman and Box-Behnken experi-
of the resultant melanin. While tyrosine is recognized as mental designs.
the primary substrate for melanin, other catechol amines, In order to assess the variables that significantly affect
including dopamine and norepinephrine, may also serve melanin production, a PBD design was employed. Thir-
as substrates. It is important to note that melanin pro- teen variables were examined, and their effects on melanin
duced from different sources might have varying struc- production were summarized in Table 1. PBD experimen-
tures because of distinct breakdown processes involving tal results showed a wide variation in melanin production
specific enzymes. This allows for the adjustment of the from 8.67 to 50.22 mg/10 mL of medium. The Pareto chart
physicochemical characteristics and the enhancement of (Fig. 4A) indicated that L-tyrosine, incubation period, fer-
microbial melanin synthesis [66]. Similarly, environmen- ric ammonium citrate, temperature, yeast extract, pH, agi-
tal factors, i.e., temperature, pH, the presence of oxygen tation speed, copper sulphate, and sodium thiosulphate
and aeration during cultivation, can greatly affect the cell exerted positive effects on melanin production (orange

Y = 35.35 + 10.41 A + 3.36 B − 4.44 C
+ 0.3818 D − 0.5609 E + 3.57 F
+ 1.51 G − 2.86 H + 8.29 J + 1.59 K
+ 5.45 L − 1.97 M − 0.3603 N + 10.53 AC

− 4.22 AD + 2.54 CF − 9.22 FG
where Y is the predicted melanin yield and A is the incu-

bation period, B is pH, C is temperature, D is inoculum
size, E is agitation speed, F is yeast extract, G is peptone,
H is peptic digest, J is L-tyrosine, K is copper sulfate, L
is ferric ammonium citrate, M is dipotassium phosphate,
and N is sodium thiosulfate.
Based on the results of PBD, the most effective
parameters affecting melanin production are L-tyros-
ine, incubation period, and ferric ammonium citrate,
which were selected for further optimization using
RSM with BBD. Response surface methodology was
employed in this work to detect the levels required for
Fig. 3 Phylogenetic tree based on 16S rDNA gene sequencing the applied parameters to produce the desired value
of Streptomyces djakartensis NSS-3 strain with GenBank accession
of the response in a limited number of tests [68]. The
no. OP912881 (arrowed) aligned with closely related sequences
of bacterial strains accessed from the GenBank interactions between the different parameters have also
been evaluated using this method [69]. Many research-
ers have found that the Box-Behnken design method
columns), whereas peptone, peptic digest, inoculum size, of RSM is a reliable and effective tool for the optimiza-
and dipotassium phosphate had a negative effect (blue tion and formulation of a wide range of processes [70].
columns). The predicted versus actual melanin produc- The BB experiment was designed and conducted using
tion plot (Fig. 4B) confirmed the model’s adequateness by the most effective parameters, as shown in Table 3. The
showing that the experimental results agree closely with results obtained were submitted to ANOVA using the
the theoretical values predicted by the model equation. The Design Expert software. A t-test analysis of the poly-
results of the response’s statistical analysis are shown in nomial model’s statistical significance yielded the data
Table 2. The P-value was used to determine how each com- presented in Table 4. The polynomial model was found
ponent affected the production of melanin. The coefficient to be statistically significant (p = 0.0378) in an analysis
of determination R2 was used to assess the model’s fitness. of variance (ANOVA). The coefficient of determina-
The R2 value was 0.9990, which indicates that the model tion (R2 = 0.8410) suggested that the variation in mela-
explains 99% of the variability in the response. Therefore, nin yield could be attributed to medium components,
in the current investigation, the model is a reliable estima- which was consistent with the data. Furthermore, A
tor of melanin production. The adjusted determination and B have been reported as significant model terms.
coefficient’s value (Adj.R2 = 0.9909) and the predicted R A significant value (0.0014) indicated a lack of fit. Our
squared of 0.8508 are similarly quite high, indicating a high findings indicated that the model’s quality was satisfac-
significance of the model. This indicated that the predicted tory, suggesting that it could accurately describe the
and observed values were very closely aligned. The experi- relationship between the various medium components.
mental design’s analysis of variance (ANOVA) was calcu- Moreover, it has been reported that A and B are signifi-
lated, and the model F-value of 122.51 implies the model cant model terms. The lack of fit value was significant
is significant. The analysis showed that L-tyrosine (J) was (0.0014). Results showed that the quality of the model
determined to be the most significant factor affecting mela- was adequate and might describe the real relationship
nin production by S. djakartensis strain NSS-3 at 99.77% among medium components. The quadratic polynomial
confidence, followed by incubation period (A) at 99.76% equation estimated by the model regression analysis
confidence, and ferric ammonium citrate (L) at 99.58% was as follows:
confidence. The design expert’s regression equation is given
by:
Table 1 The applied Plackett–Burman experimental design for the production of melanin pigment by S. djakartensis NSS-3
Run No Coded levels of independent variables Melanin production (mg/10 mL) Residuals
A B C D E F G H J K L M N O P Q R S T Actual value Predicted value
El‑Zawawy et al. Microbial Cell Factories
1 +1 −1 −1 +1 +1 +1 +1 −1 +1 −1 +1 − 1 −1 −1 −1 1 1 −1 1 42.38 42.57 − 0.1922

2 −1 +1 −1 −1 −1 −1 +1 +1 −1 +1 +1 − 1 −1 1 1 1 1 −1 1 46.63 47.16 − 0.5322
3 −1 +1 −1 +1 −1 −1 −1 −1 +1 +1 − 1 +1 +1 −1 −1 1 1 1 1 41.86 41.62 0.2439
4 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 10.70 10.60 0.0961
(2024) 23:23
5 +1 −1 +1 −1 +1 −1 −1 −1 −1 +1 +1 − 1 +1 1 −1 −1 1 1 1 38.66 38.13 0.5322

6 −1 −1 +1 +1 −1 +1 +1 −1 −1 +1 +1 +1 +1 −1 1 −1 1 −1 −1 8.67 9.45 − 0.7761
7 +1 +1 −1 −1 +1 +1 +1 +1 −1 +1 − 1 +1 −1 −1 −1 −1 1 1 −1 23.20 23.01 0.1922
8 −1 −1 −1 +1 +1 −1 +1 +1 −1 −1 +1 +1 +1 1 −1 1 −1 1 −1 41.31 40.68 0.6283
9 −1 +1 +1 −1 −1 +1 +1 +1 +1 −1 +1 − 1 +1 −1 −1 -1 −1 1 1 19.30 18.62 0.6799
10 +1 −1 −1 −1 −1 +1 +1 −1 +1 +1 −1 −1 +1 1 1 1 −1 1 −1 43.02 42.92 0.0961
11 −1 −1 +1 +1 +1 +1 −1 +1 −1 +1 −1 −1 −1 −1 1 1 −1 1 1 11.86 11.80 0.0516
12 +1 +1 −1 +1 +1 −1 −1 +1 +1 +1 +1 − 1 +1 −1 1 −1 −1 −1 −1 40.52 40.96 − 0.4361
13 −1 +1 +1 −1 +1 +1 −1 −1 +1 +1 +1 +1 −1 1 −1 1 −1 −1 −1 38.75 38.56 0.1922
14 +1 −1 +1 −1 −1 −1 −1 +1 +1 −1 +1 +1 −1 −1 1 1 1 1 −1 43.30 43.73 − 0.4361
15 +1 +1 −1 +1 −1 +1 −1 −1 −1 −1 +1 +1 −1 1 1 −1 −1 1 1 45.63 45.29 0.3400
16 +1 +1 +1 −1 +1 −1 +1 −1 −1 −1 ss− 1 + 1 +1 −1 1 1 −1 −1 1 47.90 48.29 − 0.3845
17 +1 +1 +1 +1 −1 +1 −1 +1 −1 −1 −1 −1 +1 1 −1 1 1 −1 −1 49.04 49.19 − 0.1477
18 −1 −1 −1 −1 +1 +1 −1 +1 +1 −1 − 1 +1 +1 1 1 −1 1 −1 1 35.75 36.19 − 0.4361
19 +1 −1 +1 +1 −1 −1 +1 +1 +1 +1 − 1 +1 −1 1 −1 −1 −1 −1 1 50.22 49.78 0.4361
20 −1 +1 +1 +1 +1 −1 +1 −1 +1 −1 − 1 +1 −1 1 1 −1 1 1 −1 28.28 28.43 − 0.1477
A (incubation period), B (pH), C (temperature), D (inoculum size), E (agitation speed), F (yeast extract), G (peptone), H (peptic digest), J (L-tyrosine), K (copper sulfate), L (ferric ammonium citrate), M (dipotassium
phosphate), and N (sodium thiosulphate)
Page 10 of 26
Fig. 4 Pareto chart depicts the degree to which each variable influences melanin production by S. djakartensis NSS-3 (A); Correlation
between the experimentally actual and predicted values for melanin production by S. djakartensis NSS-3 according to the Plackett–Burman
experimental results (B)
Table 2 Analysis of variance (ANOVA) and regression statistics for the experimental results of Plackett Burman design of melanin
production by S. djakartensis NSS-3
Source SS df MS F-value P-value Confidence
level (%)
Model 3420.80 17 201.22 122.51 0.0081* 99.19

Incubation period (A) 674.99 1 674.99 410.94 0.0024* 99.76
pH (B) 129.51 1 129.51 78.85 0.0124* 98.76
Temperature (C) 303.13 1 303.13 184.55 0.0054* 99.46
Inoculum size (D) 1.69 1 1.69 1.03 0.4174* 58.26
Agitation speed (E) 4.12 1 4.12 2.51 0.2541* 74.59
Yeast extract (F) 74.58 1 74.58 45.40 0.0213* 97.87
Peptone (G) 34.02 1 34.02 20.71 0.0450* 95.5
Peptic digest (H) 117.63 1 117.63 71.62 0.0137* 98.63
L-tyrosine (J) 713.46 1 713.46 434.37 0.0023* 99.77
Copper sulphate (K) 36.45 1 36.45 22.19 0.0422* 95.78
Ferric ammonium citrate (L) 389.38 1 389.38 237.06 0.0042* 99.58
Dipotassium phosphate (M) 51.08 1 51.08 31.10 0.0307* 96.93
Sodium thiosulphate (N) 1.49 1 1.49 0.9056 0.4417* 55.83
Residual 3.29 2 1.64
Cor Total 3424.09 19 R-Squared 0.9990
Std. Dev 1.28
Mean 35.35 Adjusted R-Squared 0.9909
C.V. % 3.63 Predicted R-Squared 0.8508
Adeq Precision 33.1686
*Significant values, SS sum of squares, MS mean of square, F Fishers, s function, P: Level of significance, CV %- the coefficient of variation %
R1 = 103.44 + 16.26 ∗ A + 13.80 ∗ B + 1.80 ∗ C

− 2.17 ∗ A ∗ B + 3.00 ∗ A ∗ C − 11.21
Table 3 Seventeen trials of Box-Behnken design representing
melanin production by S. djakartensis NSS-3 ∗ B ∗ C − 24.04 ∗ A2 − 6.44 ∗ B2 + 1.46 ∗ C2
Run No Experimental Melanin yield (mg/10 mL)
parameters
where R1 is the melanin yield, A is the coded value for
L-tyrosine, B is the coded value for the incubation period,
A B C Actual value Predicted value and C is the coded value for ferric ammonium citrate.
1 −1 1 0 42.38 40.73 The main effects of independent variables on melanin
2 0 −1 −1 60.75 77.59 production and their interactions were represented using
3 1 0 −1 89.51 72.67 a three-dimensional response surface and contour plots.
4 0 1 −1 99.20 100.85 The 3D response surface plots and contour plots for sig-
5 0 1 1 60.50 65.80 nificant pair-wise combinations of the three variables
6 0 0 0 105.50 92.32 (AB, AC, and BC) are presented in Fig. 5. The melanin
7 0 −1 1 50.22 63.40 yield was plotted on the z-axis of each three-dimensional
8 1 −1 0 107.22 101.92 response surface plot (Fig. 5A, B, and C) against two
9 0 0 0 75.30 71.65 independent variables that were investigated simultane-
10 0 0 0 110.12 121.66 ously, while the third variable was held constant at zero
11 0 0 0 109.20 97.66 (intermediate value). There was an insignificant inter-
12 1 0 1 99.20 102.85 action between the tested variables. The correlation
13 0 0 0 105.20 103.44 between each of the two variables does not help much
14 −1 −1 0 102.40 103.44 in increasing melanin production [71]. It was observed
15 1 1 0 98.75 103.44 that the increase in L-tyrosine concentration and incu-
16 −1 0 −1 106.33 103.44 bation period value led to an increase in melanin pro-
17 −1 0 1 104.50 103.44
duction. While an increase in ferric ammonium citrate
Table 4 ANOVA of the fitted quadratic polynomial model of melanin production by S. djakartensis NSS-3
Source Sum of Squares df Mean square F value P-value Prob > F
Model 6904.38 8 767.15 4.12 0.0378 Significant

A-L tyrosine 2114.45 1 2114.45 11.34 0.0120
B-Time 1523.52 1 1523.52 8.17 0.0244
C-Ferric ammonium 25.99 1 25.99 0.14 0.7199
citrate
BC 18.84 1 18.84 0.10 0.7599
A2 36.00 1 36.00 0.19 0.6736
B2 502.21 1 502.21 2.69 0.1447
C2 2432.44 1 2432.44 13.05 0.0086
BC2 174.65 1 174.65 0.94 0.3653
Residual 8.97 8 8.97 0.048 0.8326
Lack of Fit 1304.86 4 186.41 47.47 0.0014 Significant
Pure Error 1269.21 4 423.07
Cor Total 35.65 16 R2 = 0.8410
led to a decrease in melanin production.With the aid of production by S. djakartensis NSS-3 as the organism
BBD numerical optimization, the optimal conditions reduces ferric citrate by ferric reductase, converting fer-
for melanin production were determined to be 3.71 g/L ric into ferrous, and these ferrous metal ions are criti-
L-tyrosine, 12.75 days of incubation, and 0.57 g/L ferric cal for melanin production. Wang, et al. [76] reported
ammonium citrate (Fig. 6). After applying the numeri- that iron supplementation promoted the generation of
cal optimization design, the yield of melanin increased melanin by inducing the synthesis of tyrosinase. For
4.19-fold as compared to the yield before the entire model validation, the model was verified by culturing
optimization step (Fig. 7). A similar study used an iden- the strain S. djakartensis NSS-3 in the predicted opti-
tical approach to maximize S. glaucescens NEAE-H pro- mum medium obtained from BBD. The maximum yield
ductivity of melanin [22]. Results showed that 31.65 g for melanin (118.737 mg/10 mL) obtained from model
melanin/0.1 mL of medium could be obtained with an verification experiments was found to be very close to the
incubation time of 6 days, a protease-peptone concentra- predicted response (115.89 mg/10 mL), giving a devia-
tion of 5 g/L, and a ferric ammonium citrate concentration of 2.8%, suggesting the high accuracy of the model.
tion of 0.5 g/L. As a result, BB design was found to be an accurate and
In our study, L-tyrosine had a significant effect on decisive tool for predicting the extraction yield of mela-
melanin production by S. djakartensis NSS-3, as it is nin from S. djakartensis NSS-3. According to the previ-
considered a precursor for melanin synthesis. A similar ously illustrated data, it can be expected that in order to
study showed that Bacillus safensis is capable of synthe- achieve the highest production of melanin pigment by S.
sizing approximately 6.69 g/L of melanin when the cul- djakartensis NSS-3, the formula of the medium should be
ture medium is supplemented with tyrosine [72]. Our as follows (g/L): yeast extract (1.5 g/L); peptone (3 g/L);
findings are similar to those of Quadri and Agsar [73], peptic digest (10 g/L); L-tyrosine (3.71 g/L); copper sul-
who found that thermo-alkaliphilic Streptomyces pro- fate (0.1 g/L); ferric ammonium citrate (0.57 g/L); dipo-
duced the most melanin when given tyrosine as a sim- tassium phosphate (0.5 g/L); sodium thiosulfate (0.1 g/L);
ple nitrogen source. In contrast, marine Pseudomonas inoculum size, 1 mL; pH 7; incubation period of 13 days
stutzeri isolated from seaweed was shown to produce at 40 °C with an agitation speed of 200 rpm.
considerable levels of melanin, with a melanin concen-
tration of 6.7 g/L after 10 h of incubation without tyros- Extraction and physiochemical properties of melanin
ine supplementation [74]. According to our findings, Purified melanin pigment is separated using the TLC
the optimal time for producing melanin was the 13th technique after standardizing the solvent proportions to
day of incubation. This finding corroborated the work of a 4:4:6:1 ratio of petroleum ether: ethyl acetate: 95% etha-
Babitskaya, et al. [75], who found that the optimal time nol: conc. ammonia, respectively. A chromatogram was
to extract melanin pigment from Aspergillus carbonicus generated, and separated spots of pigment were observed
was between the 15th and 25th days of incubation. Fer- in the UV lamp. Retention factor (Rf) value was calcu-
ric ammonium citrate has a positive effect on melanin lated to be 0.8. The separated bands revealed an equal
Fig. 5 The 3D surface response and contour plots showing the effect of L-tyrosine (A, A1); incubation period (B, B1); and ferric ammonium citrate
(C, C1) and their mutual interaction on melanin production
Fig. 6 Predicted solution for maximum melanin pigment production using BBD numerical optimization
found to be soluble in dimethyl sulfoxide (DMSO), potas-

sium hydroxide, and sodium hydroxide (1N) solutions.
The temperature tolerance and stability of purified mel-
anin were tested, and it was extremely resistant to tem-
perature. Visible peaks in the UV spectrophotometer at
255 nm indicated temperature stability. Additionally, the
extracted melanin precipitated when exposed to a solu-
tion of 3N HCl and 1% ferric chloride. Hydrogen perox-
ide (30% v/v) was used to remove the color, and K MnO4
was added to transform the brown pigment into a clear
Fig. 7 Melanin production before and after RSM optimization solution. Physicochemical comparisons of the standard
synthetic melanin and the purified melanin are identical,
as shown in Additional file 1: Table S5, similar to melanin
reported from other microorganisms [78–80] and char-
Rf value with standard synthetic melanin as in Additional
acterized by its dark brown color and is partially soluble
file 1: Fig. S2, similar to the TLC analysis of melanin
in water and the majority of organic and inorganic sol-
done by Diraviyam, et al. [77]. As a first step in identi-
vents. Our results are consistent with those of Kamarud-
fying and characterizing the purified melanin, traditional
heen, et al. [81], who found that the extracellular melanin
physicochemical tests could be used because of melanin’s
pigment produced by marine Nocardiopsis spp. was
rare solubility and reactivity. The chemical characteriza-
insoluble in ethyl acetate and chloroform but soluble in
tion of the dark brown melanin pigment extracted from
dimethyl sulfoxide and alkaline water (pH = 10).
S. djakartensis NSS-3 is presented in Additional file 1:
Table S5. The purified brown powder was found to be
partially soluble in water as well as in absolute ethanol, Characterization of melanin
methanol, chloroform, acetone, benzene, and ethyl ace- As shown in Fig. 8A, the maximum absorption wave-
tate. After vigorous shaking, the purified pigment was length of the UV–vis spectrum of purified melanin
produced by S. djakartensis NSS-3 was 255 nm, and its
Fig. 8 Spectroscopic analysis of the purified melanin produced by S. djakartensis NSS-3. Uv–visible spectrum (A); FTIR spectroscopic spectrum (B);
Raman spectrum (C)
optical density gradually decreased as the wavelength synthesized by S. djakartensis NSS-3 has shown reso-
increased toward the visible wavelength, revealing the nances in both aliphatic and aromatic regions (Fig. 9A).
typical nature of the melanin absorbance. This result was The peak at 2.47 ppm indicated C14-H and C23-H
similar to the previous study of Dadachova, et al. [82], [93, 94], while the peak centered at 3.36 ppm has been
who revealed that the melanin molecule might contain assigned to the presence of methyl or methylene groups
a conjugated double bond system or an aromatic ring attached to oxygen atoms [95], as has been found in other
structure. Furthermore, there was no absorption peak at melanin pigments [93, 94, 96]. The resonances between
260 or 280 nm, indicating that melanin did not contain 6.79 and 9.8 ppm in NMR spectra have been assigned to
nucleic acids or proteins [83]. When compared to stand- protons attached to the substituted aromatic and het-
ard melanin [84], melanin pigment extracted from Cryp- ero-aromatic regions [97]. In the 13C-NMR spectra, the
tococcus rajasthanensis KY627764 showed similar results, peaks between 120 and 140 ppm could be generated by
with absorbance peaks at 244 and 220 nm. In addition, protonated and non-protonated aromatic carbons. The
our spectral data were consistent with those published by resonance peaks of mostly protonated aliphatic carbons
Elsayis, et al. [85], who found that the absorbance max- were detected within 10–40 ppm (Fig. 9B). The results
ima of UV spectral data of extracted melanin from Hor- obtained in the present study corroborated the observa-
taea werneckii AS1 was at 255 nm. tions made earlier by Chatterjee, et al. [98] and Prados-
The FT-IR spectrum can be used to identify melanin Rosales, et al. [99]. Based on these findings, we suspect
because it provides details about the structure’s primary that the resultant pigment may be pyomelanin and not
functional groups [86]. Melanin is a complex macromol- eumelanin, which lacks nitrogen. Similar results have
ecule with a characteristic infrared spectrum featuring been shown by Hocquet et al. [100] and Mahmood et al.
a series of broad, intense absorption peaks. Each broad [101].
peak is generated by a large number of functional groups In the present study, SEM micrographs of purified
[87]. The FTIR spectra of purified melanin pigment melanin isolated from S. djakartensis NSS-3 are shown
derived from S. djakartensis NSS.3 (Fig. 8B) showed in Fig. 10A, B. Purified melanin appeared as aggregates
a number of significant peaks that suggest the follow- with an amorphous (irregular) shape pattern, similar to
ing peak assignments: OH group (3442.31 cm−1), CH2 past reports of purified bacterial melanin [102]. However,
stretching (2372.01 cm−1), C = O or aromatic stretching melanin obtained from Streptomyces glaucescens NEAE-
(1645.95 cm−1), ionized C OO− groups (1397.17 cm−1), H has been found to be small and sphere-shaped [22].
combination of C-O bonds (1105.01 cm−1) and weak Further to knowing the compositional pattern, energy
−1
bands below 700 cm represent the alkene C-H replace- dispersive X-ray (EDX) spectroscopy analysis revealed
ment in melanin. These FTIR features are identical with that the main elements detected on the surface of the
those of pyomelanin produced by different microorgan- purified melanin were carbon and oxygen (Fig. 10F),
isms [88–90]. Moreover, the molecular vibration and which are considered the key elements in melanin struc-
crystal structures of the purified melanin were analyzed ture [67]. Carbon and oxygen are present by atomic per-
using Raman spectroscopy. The Raman spectra of puri- centage (79.11 and 20.89%, respectively) in the absence of
fied melanin in the range of 500–5000 cm−1 are shown nitrogen. The elemental map of purified melanin pigment
in Fig. 8C. The recorded spectra displayed multiple bands produced by S. djakartensis NSS-3 was demonstrated
between 1000 and 1800 cm−1; the 1236.09 cm−1 band in Fig. 10C, D and E. The absence of nitrogen serves as
corresponds to the phenolic C–OH stretching vibration additional support, which reflects that the extracted pig-
and the carboxylic acid C–O stretching vibration, while ment may be pyomelanin [103]. Many strains, including
the 1396.83 cm−1 band corresponds to the aromatic C–C Pseudomonas aeruginosa and Legionella pneumophila
linear stretching vibration. Two bands at 1570.48 cm−1 [104, 105], produce pyomelanin during the catabolism of
and 1609.38 cm−1 can be attributed to the stretching in tyrosine or phenylalanine via the oxidation of Homogen-
C = C [91]. Moreover, these results confirm that melanin tistic acid (HGA), which is consistent with our findings
derived from Streptomyces djakartensis NSS-3 closely that to produce pyomelanin, HGA must undergo auto-
resembles the RAMAN spectra results of pyomela- oxidation and self-polymerization, which requires the
nin from Aspergillus fumigatus, as RAMAN bands at involvement of 4-hydroxyphenylpyruvic acid dioxygenase
1381 cm−1 and 1583 cm−1 served as a spectral signature (4-HPPD) and HGA-oxidase [106, 107].
for pyomelanin [92].
Furthermore, 1H and 13C-NMR spectra of purified Sunscreen protection factor (SPF)
melanin contain a number of chemical shifts that can be Melanin has developed commercial applications as
used to confirm the molecular structure of melanin. 1H a UV protector in cosmetics and food [108]. The
NMR spectral analysis of the purified melanin pigment UV-absorption and photostability of melanin are
Fig. 9 1HNMR (A); and.13CNMR (B) of purified melanin produced by S. djakartensis NSS-3
Fig. 10 SEM images of purified melanin produced by S. djakartensis NSS-3 at different magnifications (A, B); Energy dispersive spectroscopy (EDS)
and elemental mapping analysis showing elemental composition of purified melanin (C, D, E, F)
Table 5 SPF result of extracted melanin from S. djakartensis NSS-

3
Wavelength (nm) Abs. EE(λ) × I(λ)
290 0.0150
310 0.1964
315 0.0939
320 0.0180
Sample SPF value
Extracted melanin 18.5
EE erythema effect spectrum, I solar intensity spectrum, Abs the absorbance of Fig. 11 Antioxidant activity of purified melanin and ascorbic acid
sunscreen product as standard
significantly higher than those of previously reported and it has fewer side effects compared to organic sun-
metabolites of palythine and mycosporine-like amino screens [112, 113].
acids [109]. Melanin can protect the body from the Melanin has phenolic hydroxyl and carboxyl active
sun because of its physiological and photoprotec- groups, enabling it to absorb UV light and function as a
tive properties. Melanin is resistant to breakdown physical barrier that disperses UVR. Additionally, it acts
and may eliminate as much as 90% of the heat gener- as an absorbent filter that diminishes the penetration of
ated by exposure to sunlight [110]. Currently, melanin UV radiation through the epidermis [114]. Studies have
is primarily used as a dye in the lenses of sunglasses. shown that melanin interacts with DNA and functions
In this case, the advantage is particularly highlighted as a photosensitizer, generating reactive oxygen species
because of the pigment’s natural origin and its ability to (ROS) upon exposure to UVA radiation [115]. In addi-
reduce high-energy visible light. In our study, the high tion, melanin also enhances the secretion of histamine,
SPF value of the purified melanin from S. djakartensis which has a role in the development of sun-induced red-
NSS-3 was determined to be 18.5 (Table 5), suggesting ness and swelling in people with fair skin [116].
that it may be effective in protecting the skin from the
harmful effects of UV rays when used as a sunscreen. Antioxidants and cytotoxicity
Sunscreens with an SPF of 15 are recommended by the To determine the possible biomedical applications of
FDA to protect against sunburn, skin cancer, and pre- purified melanin, we examined its potential antioxidant
mature aging of the skin [111]. Therefore, purified mel- and anticancer activities. Antioxidants protect cells from
anin from S. djakartensis NSS-3 is promising for future free radicals and have a protective function for DNA and
formulations of sunscreens and personal care products many other biologically important compounds [117].
Purified melanin pigment from S. djakartensis NSS-3 was
tested for antioxidant activity against DPPH free radi- Furthermore, cytotoxicity evaluation is an important
cals. A deep violet DPPH solution can be used to meas- part of developing safe and effective medicines [124,
ure radical concentration because its color changes from 125]. In this study, we used in vitro models of WI38,
deep violet to pale yellow or even colorless upon adding HCT116, HEPG, and MCF7 cell lines to test the cytotox-
extracted melanin pigment [118]. As the concentration of icity of melanin using the MTT assay. Figure 12 depicted
extracted melanin pigment increased, so increased their the cytotoxic effects of purified melanin against cancer-
DPPH activity, indicating a dose-dependent behavior. It ous and noncancerous cell lines. The results showed that
exhibited scavenging activity of 32.2–94.82% at concen- purified melanin was found to be non-toxic toward the
trations of 1.25–100 µg/mL, with an average IC50 value WI38 cell line, permitting normal cell metabolism and
of 18.03 µg/mL as shown in Fig. 11. Purified melanin pig- growth (IC50 greater than 500 μg/mL). These results con-
ment has comparable antioxidant activity with standard firmed the high safety of purified melanin for normal
ascorbic acid (IC50 = 16.83 µg/mL). The results obtained cells as a biocompatible agent as it has shown negligible
were similar to earlier studies, which also revealed that effect against the normal cell line. On the other hand, the
the antioxidant activity of melanin is dose-dependent. purified melanin pigment was able to reduce the viability
Rao and Rao [119] found free radical scavenging activ- of tumor cells in a dose-dependent manner, as shown in
ity in the range of 56.58–68.91%, while Arun et al. [120] Fig. 12. Longer exposure had additional toxicity for the
found it to be in the range of 87–96%. Melanin is a poly- cells. The cell viability of all tested cell lines was remark-
mer with molecules that contain unpaired electrons and ably inhibited in the presence of melanin at a concentra-
can either donate or accept an electron. Melanin pigment tion of 0.5 μg/mL or higher. The IC50 of purified melanin
has many unpaired valence electrons, making it a great was 108.9, 43.83, and 81.99 μg/mL for HCT116, HEPG,
scavenger for free radicals and other reactive oxygen and MCF7, respectively. Importantly, the comparison of
species. Melanin is an antioxidant that fights free radi- all IC50 results clearly indicated that the purified mela-
cals through a series of one-electron transfer reactions, nin demonstrated higher cytotoxicity against cancer
suggesting its use in cosmetic products that minimize cells in comparison with a normal cell line. Therefore,
toxin-induced tissue damage [121]. Moreover, melanin S. djakartensis NSS-3 melanin pigment can be used as a
has a strong affinity and great capacity for direct binding potential natural antitumor agent. Similar results were
with metal ions like Fe2+ [122]. The presence of aromatic reported on the HEP2 carcinoma cell line by Arun,
components and functional groups in melanin, includ- et al. [120], who reported that inhibition of cell viability
ing hydroxyl, carboxyl, amine, and phenol groups, allows was concentration-dependent and that 60 µg of mela-
melanin to chemically interact with many organic and nin inhibited cell viability by 53%. Without harming or
inorganic compounds [123]. adversely affecting other healthy cells or bodily tissues,
Fig. 12 In vitro cytotoxicity and anticancer activities of various concentrations of the purified melanin pigment of S. djakartensis NSS-3
El‑Zawawy et al. Microbial Cell Factories
Table 6 Mean of diameter of inhibition zones obtained by purified melanin pigment against tested bacterial strains
(2024) 23:23
Microbial strains Mean of diameter of inhibition zone (mm) ± standard deviation
Different concentrations of melanin pigment (µg/mL) MIC (µg/mL) MBC (µg/mL) Antibiotic (Positive control) DMSO (1%)
(Negative
10 20 30 40 50 Streptomycin Tetracycline control)
(10 µg/mL) (10 µg/mL)
Gram-positive bacteria
Staphylococcus aureus (SA-04) 0.0 ± 0.0 9.3 ± 0.47 13.0 ± 0.0 12.0 ± 0.0 19.5 ± 0.02 25 50 30.5 ± 0.05 15.0 ± 0.47 0.0 ± 0.0
Gram-negative bacteria
Escherichia coli )EC-03( 0.0 ± 0.0 0.0 ± 0.0 8.3 ± 0.47 9.6 ± 0.94 12.5 ± 0.05 25 100 25.0 ± 0.00 13.3 ± 0.47 0.0 ± 0.0
Klebsiella pneumonia )KP-01( 0.0 ± 0.0 8.3 ± 0.47 11.0 ± 0.0 14.3 ± 0.47 16.0 ± 0.13 12.5 100 27.3 ± 0.47 15.0 ± 0.02 0.0 ± 0.0
Pseudomonas aeruginosa )PA-09( 11.3 ± 0.0 14.6 ± 0.94 16.0 ± 0.0 21.0 ± 0.0 35.3 ± 0.47 6.25 25 27.3 ± 0.47 15.0 ± 0.00 0.0 ± 0.0
Page 21 of 26
melanin offers anti-tumor properties that include apop- growth of P. aeruginosa, and some Bacillus species. Xu
tosis promotion and angiogenesis inhibition [126]. Mela- [129] investigated the antibacterial efficacy of Lach-
nin has good photothermal conversion efficiency and num YM30 melanin and discovered that it was effec-
can absorb light from the ultraviolet to the near-infrared tive against a wide range of microorganisms, including
range. This means that it can be used as a photother- S. aureus. According to many studies, the antibacterial
mal agent in multimodal imaging-guided photothermal properties of melanin may be attributed to its ability
therapy (PTT) to target tumor cells or tissues specifically to disrupt cell membranes [130]. This disruption can
[127]. impair the functioning of bacteria, increase the leak-
age of cell contents, enhance the uptake of non-protein
Antimicrobial activities nitrogen, reduce the membrane potential, and inhibit
The antibacterial activity of purified melanin was the formation of biofilms regulated by quorum sens-
investigated against four MDR bacterial strains, ing (QS) systems, thereby inhibiting these systems in
namely Staphylococcus aureus (SA-04), Escherichia bacteria [131].
coli (EC-03), Klebsiella pneumoniae (KP-01), and Pseu-
domonas aeruginosa (PA-09). The antibacterial activity Conclusions
of the purified melanin in different concentrations (10, S. djakartensis NSS-3 is a soil-isolated actinobacterium
20, 30, 40, and 50 µg/mL) was quantitatively assessed whose melanin pigment has been studied for the first
on the basis of zones of growth inhibition. The zone of time. In this research, RSM optimization demonstrated
growth inhibition was accurately measured and com- the capability of improving this bacterium to produce
pared with the standard antibiotics given in Table 6. abundant melanin pigment using L-tyrosine. The phe-
Among the Gram-negative bacteria, P. aeruginosa was nolic structure of pyomelanin, isolated from S. dja-
the most inhibited by melanin, with a zone of inhibi- kartensis NSS-3, was found to have multiple biological
tion of 35.3 ± 0.47 mm at the highest concentration properties, including the ability to scavenge free radicals
(50 μg/mL) and 11.3 ± 0.0 at the lowest concentration and provide high radioprotection activity. It was found
(10 μg/mL). The present study clearly indicates that that purified melanin pigment had no cytotoxic effect on
melanin exhibited strong antibacterial activity against healthy cells, demonstrating its safe nature. The results
all isolates, even at low concentrations. Melanin also showed that purified melanin has powerful anti-
showed a considerable amount of microbial growth microbial and antitumor effects against various micro-
inhibition, and the values of MIC and MBC are shown organisms and cancer cell lines. The overall findings of
in Table 6. MIC values of melanin gave the lowest val- this study showed the potential of pyomelanin as a novel
ues of 6.25 µg/mL against P. aeruginosa among Gram- biomaterial with improved biological properties, with
negative bacteria and 25 µg/mL against S. aureus further future studies on its mode of action for use in dif-
among Gram-positive bacteria. ferent medical and biotechnological applications.
The melanin pigment showed considerable anti-
microbial activity against the tested bacteria, with Supplementary Information
inhibitory growth zones varying in diameter depend- The online version contains supplementary material available at https://doi.
ing on the bacterial species. As the amount of mela- org/10.1186/s12934-023-02276-y.
nin increased, the diameter of the inhibition zones
Additional file 1: Table S1. Experimental independent variables at two
increased as well. The highest antimicrobial activity levels used for the production of melanin by ACT3 using Plackett Burman
was observed against P. aeruginosa ATCC 902 (PA- design. Table S2. Experimental variables for Box-Behnken design at dif‑
09), followed by S. aureus (SA-04) and K. pneumoniae ferent levels. Table S3. Cultural characteristics of ACT3 strain on different
culture media. Table S4. Physiological and biochemical characterization
(KP-01). The least antimicrobial activity was noticed of isolated ACT3 strain. Table S5. Physicochemical properties of melanin
against E. coli (EC-03). The MIC and MBC values of produced by S. djakartensis NSS-3 and standard synthetic melanin. Figure
melanin on pathogenic bacteria demonstrated that it S1. Soil samples collected from Wadi-Allaqui Biosphere Reserve on the
eastern side of Lake Nasser, Egypt. Figure S2. TLC analysis of purified
has the potential to suppress bacterial cell develop- melanin pigment (A) compared with standard synthetic melanin (B).
ment even at low bacteriostatic concentrations. These
findings indicated that biosynthesized melanin had
Acknowledgements
antibacterial activity which might potentially be used The authors thank Scientific Research Centre and Measurement, Tanta Uni‑
in development of novel antimicrobials or antibiotics versity, Tanta, Egypt for monitoring the characterization and identification of
melanin from bacterial extract.
adjuvants that enhance activity of existing drugs. Anti-
microbial assessment results are consistent with those Author contributions
discovered by other authors. Laxmi [128] found that NAE: Conceptualization, methodology, writing-original draft, review and edit‑
ing. EK: Conceptualization and supervision. SA: Methodology, writing-original
melanin derived from Providencia rettgeri hindered the
draft. SE: Methodology, formal analysis, data curation, writing, review and 9. Mussagy CU, Winterburn J, Santos-Ebinuma VC, Pereira JFB. Produc‑
editing. All authors read and approved the final manuscript. tion and extraction of carotenoids produced by microorganisms. Appl
Microbiol Biotechnol. 2019;103:1095–114. https://doi.org/10.1007/
Funding s00253-018-9557-5.
Open access funding provided by The Science, Technology & Innovation 10. Huang L, Liu M, Huang H, Wen Y, Zhang X, Wei Y. ” Recent advances and
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank progress on melanin-like materials and their biomedical applications.
(EKB). This research did not receive any specific Grant from funding agencies. Biomacromolecules. 1858;2018(19):1868. https://doi.org/10.1021/acs.
biomac.8b00437.
Availability of data and materials 11. Charousova´ I, Medo J, Hleba L, C´sarova´ M, Javorekova´ S. Antimicro‑
The datasets generated during and/or analyzed during the current study bial activity of actinomycetes and characterization of actinomycin-pro‑
are available from the corresponding author on reasonable request. All data ducing strain KRG-1 isolated from Karoo, South Africa. Brazilian J Pharm
generated or analyzed during this study are included in this published article Sci. 2019;55:1–11.
(and its Additional file 1). 12. Polapally R, Mansani M, Rajkumar K, Burgula S, Hameeda B, Alhazmi
A, et al. Melanin pigment of Streptomyces puniceus RHPR9 exhib‑
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Consent for publication Almansour AI, Perumal K, Nayaka S. Penicillium citrinum NP4 medi‑
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El‑Zawawy et al.

Microbial Cell Factories (2024) 23:23 Microbial Cell Factories

RESEARCH Open Access

Bioproduction and optimization

Introduction structure and a negative charge. In many organisms, it is

Fig. 1 Schematic diagram of melanin extraction from S. djakartensis NSS-3

where Y is the predicted melanin yield and A is the incu-

1 +1 −1 −1 +1 +1 +1 +1 −1 +1 −1 +1 − 1 −1 −1 −1 1 1 −1 1 42.38 42.57 − 0.1922

5 +1 −1 +1 −1 +1 −1 −1 −1 −1 +1 +1 − 1 +1 1 −1 −1 1 1 1 38.66 38.13 0.5322

Model 3420.80 17 201.22 122.51 0.0081* 99.19

R1 = 103.44 + 16.26 ∗ A + 13.80 ∗ B + 1.80 ∗ C

Model 6904.38 8 767.15 4.12 0.0378 Significant

found to be soluble in dimethyl sulfoxide (DMSO), potas-

Table 5 SPF result of extracted melanin from S. djakartensis NSS-

Microbial strains Mean of diameter of inhibition zone (mm) ± standard deviation

Whitman WB. Bergey’s Manual® of systematic bacteriology. New York:

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