Recently, the world has tended to use colorants from natural sources owing to the many problems resulting from the use of industrial colorants in many industries, such as food and medicine [22]. Microbial pigments are of interest because they are often more stable and soluble than those from plant or animal sources [23]. Microorganisms can grow rapidly, which could end in high productivity, and will produce a product throughout the year. Thus, the food industry has become increasingly inquisitive about the utilization of microbial technology to supply colors to be utilized in foods. It is going to help to beat the growing public concern over the adverse health effects of the addition of synthetic colors in food products. Aside from the health benefits, natural colorants will also be a boon to biodiversity since toxic chemicals emitted into the environment when creating synthetic colorants may be eliminated. These natural colorants are utilized in baby foods, cheese, fruit drinks, vitamin-enriched milk products, and some energy drinks. In this way, natural colors may serve the twin purposes of appealing to the eye and providing health advantages for probiotic bacteria in food, in addition to being environmentally beneficial [24].
There are some kinds of micro-organisms that have the pliability to supply pigments in high yields, including species Streptomyces [25]. Streptomyces ssp. can produce an array of pigments having antibacterial or antifungal properties which are applied for human pharmaceutical use [26].
This study focused on the isolation of actinomycetes from samples collected from the El-Mahmoudia canal, in Egypt in diverse forms (soil, water, and sediment). A typical isolation protocol for actinomycetes was followed as described by Rahman [27]. Finally, in total, 50 colonies with different morphology types (shapes/color) forming pin-point shapes were obtained; among them, 12 are colored. Among these 12 chromogenic cultures, the isolate coded LS1 was selected, where it produced a visible amount of red/orange pigmentation in both agar and broth media. A well-developed radial mycelium is characteristic of classic actinomycetes; this mycelium may be split into substrate mycelium and aerial mycelium in accordance with shape and performance [28]. One of the most diverse groups of Gram-positive bacteria, Actinobacteria has the highest G+C content and shows the most complex morphological differentiation, based on filamentous levels of the organization, much like a filamentous fungus. A variety of actinobacteria may generate complex structures such as spores, spore chains, sporangia, and sporangiospores, among other characteristics [29]. Till now, most actinobacteria are characterized and classified on their morphology in the first place. The morphological characteristics are still one of all the foremost basic indexes which give in-depth information on a taxon. Thus, morphological properties of studied isolate LS1 were intensively characterized through several microscopies, picked micrographs, colony characteristics, areal hyphae, and spore formation. LS1 strain showed a typical radial mycelium characteristic for actinomycetes under light microscopies and showed round spore formation under light microscopy and SEM. The stage of spore formation was recognized through the TEM micrograph. Additionally, cells forming pigment appeared in a curved rod shape where it was easily recognized the granulated pigmented products inside the cells at different magnification power.
Over the years, GenBank information based on the 16S rRNA gene has been constructed and it had been successfully utilized for the differentiation of bacteria [30]. Genotypic identification emerged as a complement to determine phenotypic methods. Genotypic identification of bacteria often entails using conserved sequences across phylogenetically informative genetic targets, such as the small subunit 16S rRNA gene [31]. Also, the 16S rDNA sequence comparison has been used as a strong tool for establishing phylogenetic and evolutionary relationships among organisms [32]. So, the 16S rDNA of the studied LS1 isolate was amplified, sequenced, and compared to the general public data in GeneBank. It showed 99.57% sequence homology to Streptomyces sp. TD-050, accordingly the studied bacterium, was designated as Streptomyces sp. LS1. Subsequently, 16s rRNA of LS1 was deposited in GenBank with accession number MW5856041.
Pigments are usually categorized as secondary metabolites, produced mostly inside the cells. So, sonication was applied for LS1 cell disruption, followed by repeated ethanolic extraction (v:v ratio 1:1). There are several laboratory-based approaches for cell disruption, including ultrasonic cavitation, which uses 15–20 kHz ultrasound waves to create an acoustic pressure wave that breaks apart the cell membrane [33]. Similarly, many researchers use sonication for cell rupture and liberating bioactive products like the laccase enzyme [34]. Also, the osmotic shock was used for liberating the pigmented products, especially just in the case of halophilic archaea bacterial strains as reported by Hagazy et al. [21]. Additionally, many protocols for pigment extraction are usually followed using different solvents like hexane, benzene, chloroform, methanol, acetone, ethanol, or mixed solvent [35]. However, during this study, successful extraction for LS1 pigment was observed by using the organic solvent ethanol with a purity of 99.9% (at a volume ratio 1:1). Similarly, Muthusaravanan et al. [16] used the organic solvent (ethanol) for pigment extraction at a volume ratio of 1:1 in a successful way. The carotenoid pigments are most ordinarily found in nature; their absorption is mostly localized within the range 400–500 nm [36]. Additionally, the wavelengths 400, 470, and 500 nm were identified as having the greatest pigment absorption, reflecting the absorption maxima for yellow, orange, and red pigments, respectively [37]. The pigment isolated during this work showed a light-weight absorption characteristic at 400–410 nm, it indicating that might be associated with the carotenoids group of pigment [36].
It was stated that both Raman spectroscopy and infrared spectroscopy are effective in analyzing the total fingerprint of carotenoids. IR spectroscopy provides information about the outer structures, whereas Raman proved with a signature of the inner structure with minimal information about the outer structure. The combined use of Raman and infrared spectroscopy could be a good way to see a full spectral fingerprint of carotenoids. Accordingly, further characterization of the studied LS1 pigment was applied using these advanced and fast analytical tools (Raman and FT-IR-spectroscopy), and so GC-MS was used for elucidation of the chemical structure. It was noticed through Raman spectroscopy, the studied red LS1 pigment features a band at 1300 cm−1 which corresponded to CH3 umbrella mode, and a band at 1400 cm−1 which corresponded to CH3 and CH2 deformations. This finding was virtually identical to that reported by Kushwaha et al. [36], who hypothesized that strong Raman bands in the region of 1511–1530 cm−1, 1153–1159 cm−1, and 1003–1010 cm−1 indicate bacterial carotenoids. Furthermore, Hegazy et al. [21] discovered the presence of three regions with two strong and weak signal intensities for orange carotenoids in the pigment produced by Natrialba sp., with a robust band at 1380 cm−1, which corresponds to the CH3 umbrella mode, and a weak band at 1883 cm−1, which appreciates C=C bonds. Additionally, four Raman bands of LS1 pigment with relatively equal signal intensities at area 1700–2100 cm−1 are corresponded to C=C and C=O bonds; these confirm the relatedness of the studied LS1 pigment to the carotenoids group.
FTIR, on the opposite side, could be a technique that is employed to get a spectrum, emission, and photoconductivity of a solid, liquid, or gas. FTIR that operates within the mid-infrared region (4000–400 cm−1) may be a powerful tool for qualitative analysis of fats, oils, and palm carotene [38]. Hence, the studied pigment was subjected to FT-IR spectroscopy analysis, it was found several peaks at 541.05 cm−1 correspond to haloalkane stretching frequency, at 1077.28 cm−1 corresponds to C-OH, and at 1237.38 cm−1 is assigned to C-O-C. Also, peaks at 1407.12 and 1455.34 cm−1 correspond to CH3 and CH2 stretching frequency, respectively. The strong peak at 1639 cm−1 is assigned to the C=C alkene stretching which suggests that some aliphatic compounds existed in LS1 pigment extract; this was verified through GC-MS as well. The presence of such spectral bands confirmed that the studied LS1 pigment resembles a high extent to the carotenoids, especially peaks at 1407 cm−1 which appear that related to the bending vibration of methylene –CH2 as recorded by Hosseini and Jafari [39] when using beta-carotene standard. The height that appears at 2932 cm−1 of the studied pigment may well be attributed to the β-ionone ring of beta-carotene because of the C-H, (–CH3) symmetrical bending [36].
Optimization of media components, culture parameters, and strain improvement are essential tools to improve the performance of the bacterial system which helps to extend the yield of its products economically. This system has been applied for the optimization of various process parameters and medium composition [40]. Submerged pigment production is riddled with several biotechnological processes and environmental parameters such as temperature, pH, salt, nitrogen, and carbon sources [41]. It is vital to control them in industrial bioprocesses. Metabolically, the implications of them are associated with changes within the activities of proteins; therefore, the culture conditions can control some activities like cellular growth, production of primary and secondary metabolites, fermentation, and so the oxidation processes of the cell. Within a few investigations, the ideal conditions for Streptomyces ssp.’s red pigment production have been explored [42].
Microorganisms’ growth and development are greatly influenced by environmental parameters such as temperature, which also has an impact on a variety of biosynthetic processes such as pigment production. Also, the biosynthesis of a pigment is significantly tormented by the physiological parameter and temperature [43]. To seek out the optimal temperature for pigment production by the investigated LS1 strain of Streptomyces sp., it had been cultivated under various temperatures (25–30–37°C). As a result, it was discovered that the ideal temperature for pigment production is 30°C. Streptomyces sp. strain LS1 is thought to have a favorable physiological characteristic that allows it to maintain this ideal temperature. This observation was in agreement with studies with Monascus cultures, red pigment production was highest at 30°C, and decreased at temperatures beyond 40°C in the midst of a rise within the production of yellow pigments [44]. In other studies the growth conditions for red pigment production by a novel strain of Bacillus sp. located at 34°C, which is the optimum for pigment production by this novel isolate, while Pseudomonas aeruginosa strain was reported to supply maximum pigment production optimally at 37° [45].
Pigment production by an organism is affected largely by the pH of the medium within which the microorganism is grown. Slight changes in pH can even alter the shade of color produced [46]. The influence of pH on the assembly of red pigment by LS1 was studied at different pH values starting from 5 to 9 pH. The results showed that maximum production of red pigment by investigated LS1 strain occurred at pH 7. However, the acidic (pH 5) showed along with the all-time low synthesis of the pigment, and no pigment at all was found at basic/alkaline pH (8 and 9). This agreed with studies that found a maximum melanin activity at neutral pH 7, while further increase in pH reduced the melanin by the actinomycetes isolate in starch nitrate medium [47]. Inversely, Mortazavian et al. [48] found that the most effective results for the assembly of yellow pigments were obtained at initial pH values from 3.0 to 3.5, while the finest results for the assembly of red pigments were reached at pH levels between 7.0 and 7.5.
The effect of salt on pigment production was evaluated through the individual addition of the subsequent salts to the medium: NaCl, CaCl2, CaCO3, and MgSO4.7H2O at concentrations 3 g L−1 against unsalted medium (control). The significantly highest level of pigment formation by studied LS1 strain was detected in presence of NaCl, followed by MgSO4, while no pigment production in the presence of CaCl2 or CaCO3. This observation was in agreement with studies of melanin production from Actinobacterium Nocardiopsis alba MSA10, the best melanin production (3.4 mg mL−1) has been obtained at 2.5% of salinity [49]. Another study found CaCl2 (10 mM) slightly enhanced the pigment production by Paecilomyces sinclairii [50]. According to Chaskes and Tyndall [51], there is a correlation between nitrogen sources and pigment production. This study tested the effect of various nitrogen sources on pigment production by LS1 strain in the presence of 1% starch (carbon source). Different formulations for nitrogen sources (organic/inorganic) were tested individually or in combinations. The organic YE alone has shown the maximal production of both pigment and biomass (growth) (13.38 mg% and 0.272 g%), among all tested formulations, followed by a mixture of YE and peptone, while low production values upon lonely using peptone or tryptone. Limited growth and pigmentation were found when using mixed organic and inorganic nitrogen source. However, there were no growth and no pigment just in case of using either beef extract or malt extract alone. This observation was in agreement with studies done by Budihal et al. [52]; they screened nine nutrient parameters and found that YE is the finest for maximum carotenoid production. Another study has shown that organic nitrogen sources promote greater mycelial growth as compared to inorganic nitrogen sources. Pigment production was stimulated by meat peptone, casein peptone, the peptone–YE combination, and corn steep powder, but red pigment synthesis was severely hindered by soy peptone and malt extract [53].
A stimulatory effect of carbon source on LS1 pigment production was estimated through replacing starch (10 g L−1) with other various carbon sources at the identical concentration in presence of a preferred nitrogen source from the previous experiment. Among the tested carbon sources, the highest level of pigment formation by studied strain LS1 was detected with fructose, dextrin, and starch followed by lactose, galactose, and mannose, and a low level of pigment formation was detected by sorbose, ribose, gluconic acid, and sucrose, and no pigment production in glucose, glycerol, xylose, and citric acid. This observation was in agreement with El-Batal and Al Tamie [54]; they found starch is the best source for the highest production of melanin by Aspergillus oryzae. Similarly, the same results were found by Venkatachalam et al. [55], who reported that starch was the foremost effective carbon source for the assembly of melanin, followed by glycerol and fructose. However, Hewedy and Ashour [49] reported that Kluyveromyces marxianus and Streptomyces chibaensis produced brown pigment in presence of xylose as a carbon source.
Since YE was observed to support pigment production, further studies were conducted to optimize the concentration of YE required for maximal production of pigment. Results indicate that 3 g L−1 of YE supported maximum pigment production. The findings of El-Naggar and El-Shweihy [56], who utilized a basal medium consisting of YE, which considerably boosted the biomass and also the pigment content of R. gelatinosus, were in accordance with this finding. In another study, the utmost activity was shown in meat peptone containing medium 2 g L−1 for the red pigment production by P. sinclairii [57].
Under the previous optimal conditions, the significantly highest level of pigment formation by Streptomyces sp. LS1 strain was detected at level 8 g L−1 fructose. Budihal et al. [52] applied 1% starch for melanin production by Streptomyces sp. DSK2 and Gunasekaran and Poorniammal [57] applied 1.5% starch for pigment production by Paecilomyces sinclairii, while a low level of starch (0.2%) is optimum for the growth of melanin pigment producer Streptomyces virginiae as described by Deepthi and Rosamma [58].
To search out the optimal level of NaCl (the best salt), the previous experiment was repeated in presence of various levels of tested salt ranging (1-5 g L−1). The significantly highest level of pigment formation by studied LS1 strain was detected with level 3 g L−1 in presence of fructose (carbon source). At the lowest level of salt (1 g L−1), the smallest amount of pigmentation was shown. This observation was in agreement with some studies, where 2.5% of salinity increased the melanin production by Vibrio cholera [59]. It is known that hyperosmotic stress induces melanin production; this explained why melanin production occurred at a higher concentration of NaCl [59]. Also, Farkas and Monagha [60] reported that the best melanin production (3.4 mg mL−1) has been obtained at 2.5% of salinity. Yet, these differences between their results and ours related to the intraspecific variability and strain dependence
The antimicrobial activity of carotenoid pigment was studied against five species of marine bacterial pathogens of G+ve (B. subtilis ATCC 6633 and S. aureus ATCC 6538) and G−ve (E. coli ATCC 10418, K. pneumoniae ATCC 13883 and P. aeruginosa ATCC 9027) bacterial strains. The results indicated that G−ve (P. aeruginosa ATCC 9027 and K. pneumoniae ATCC 13883) microorganisms were more susceptible to carotenoid pigment extracted from Streptomyces sp. LS1 strain than the G+ve (B. subtilis ATCC 6633). This observation was in agreement with studies done by Manimala and Murugesan [61]. Fucoxanthin’s antibacterial activity on 13 aerobically grown bacterial strains was evaluated in another investigation. It was observed to have a significantly stronger impact on G+ve than G−ve bacterial strains [62]. The antifouling (AF) activity of the pigment was tested; it was noted the pigment has a remarkable valuable effect on biofouling reduction. This finding was in line with other studies with dried and fresh macroalgae Chondrus crispus (Rhodophyceae) crude ethanol extracts. To determine AF effectiveness, these extract was evaluated against five marine bacterial strains, five phytoplankton strains, and two macroalgae. Compared to the fresh source, the dried algae extract had a lower minimum inhibitory concentration (25μg mL−1) against the growth of bacteria and phytoplankton species than the fresh algal extract. The extracts were shown to have anti-germination activity against both Undaria pinnatifida and Ulva intestinalis spores in macroalgae tests, at a concentration of 25–50μg mL−1. The initial efficacy of AF paint with crude extract was found to persist for 6 weeks in a field study. The biocidal ability of photocatalytic TiO2-based nano compounds (also in combination with Ag and Cu nanoparticles) applied on travertine surfaces by spray-coating to limit or inhibit algal fouling. Stone’s aesthetic compatibility with colorimetry has been evaluated [63]. Antimicrobial and antifouling are mainly attributed to the synergistic effects of identified active compounds in the ethanolic extract as shown through GC-Ms starting from alcohol, phenol, fatty acids, and ester. These active compounds such as alcohol-related (2,3-Butanediol), phenolic-related compounds (Phenol, 2-methoxy-3-(2-propenyl)- and phenol, 2-methoxy-4-(2-propenyl)-, acetate), and fatty acid-related (vaccinic, hexadecanoic, octadecanoic) are characterized by several biological activities as potent antibacterial, antifungal, antiviral, and antioxidant.