Production and Potential Applications of Bioconversion of Chitin and Protein-Containing Fishery Byproducts into Prodigiosin: A Review

The technology of microbial conversion provides a potential way to exploit compounds of biotechnological potential. The red pigment prodigiosin (PG) and other PG-like pigments from bacteria, majorly from Serratia marcescens, have been reported as bioactive secondary metabolites that can be used in the broad fields of agriculture, fine chemicals, and pharmacy. Increasing PG productivity by investigating the culture conditions especially the inexpensive carbon and nitrogen (C/N) sources has become an important factor for large-scale production. Investigations into the bioactivities and applications of PG and its related compounds have also been given increased attention. To save production cost, chitin and protein-containing fishery byproducts have recently been investigated as the sole C/N source for the production of PG and chitinolytic/proteolytic enzymes. This strategy provides an environmentally-friendly selection using inexpensive C/N sources to produce a high yield of PG together with chitinolytic and proteolytic enzymes by S. marcescens. The review article will provide effective references for production, bioactivity, and application of S. marcescens PG in various fields such as biocontrol agents and potential pharmaceutical drugs.


Production of PGs
PG produced by S. marcescens has been considered as a promising aim for drug development due to the described antifungal, immunosuppressive, and antiproliferative bioactivities. Hence, the culture conditions for large-scale production to ameliorate PG productivity becomes an essential issue [75][76][77][78]147]. To gain higher PG productivity, important factors for evaluation include the media composition, inorganic phosphate availability, temperature, and pH [199,200]. In the case of C/N source, the most commonly used media for PG production were commercial media, such as nutrient broth and peptone glycerol broth. For example, Giri et al. found that the PG productivity by S. marcescens was 520 mg/L and 569 mg/L in nutrient broth (28 • C) and peptone glycerol broth (30 • C), respectively [201]. Furthermore, the supplement of fatty acid-containing plant oils was found to show a positive effect on PG productivity which increased to 16,680 mg/L in sesame seed broth (28 • C) [201]. Kamble and Hiwarale reported that S. marcescens showed better PG productivity in nutrient broth than in peptone glycerol broth [173]. Regarding the use of agricultural byproducts as C/N sources, ethanol and cassava wastewater were used for PG production by S. marcescens 389 [190], and S. marcescens UCP1549 [174], respectively. Further, chitin and protein-containing fishery byproducts have been utilized for PG production by S. marcescens TKU011 [59][60][61][62].
For the production of exopolysaccharides (EPS), Paenibacillus polymyxa EJS-3 has been reported to produce the highest EPS productivity (35.26 g/L) by using sucrose (188.2 g/L) and yeast extract (25.8 g/L) as the C/N source [56]. Recently, squid pens were utilized successfully to produce the highest yield of EPS from Paenibacillus sp. TKU023 (41.25 g/L) and P. macerans TKU029 (35.75 g/L) [56]. This inspired us to isolate the PG-producing strain, S. marcescens TKU011 using squid pens as the C/N source. The PG productivity of using chitin and casein, squid pens, shrimp shells, and shrimp heads as the sole C/N source by S. marcescens TKU011 was 4620 mg/L [59], 2480 mg/L, 190 mg/mL, and 30 mg/L, respectively [61]. Table 1 summarizes the comparison of the reported PG yield of S. marcescens by using different C/N sources. Chen et al. reported that a C/N ratio of 6/4 of starch/peptone (1.6% starch; 1.067% peptone) achieved a high PG yield (6700 mg/L) by S. marcescens C3. PG yield of 7070 mg/L was achieved after optimizing the concentrations of FeSO 4 ·4H 2 O (0.56 mM) and MnSO 4 ·4H 2 O (3.25 mM) [172]. Kamble and Hiwarale [173] studied S. marcescens PG production in peptone glycerol broth and nutrient broth and observed that the highest PG productivity was 1335 mg/L and 1845 mg/L, respectively after three days of cultivation [173]. Different from most other studies, the enhancing effects of oil supplements on PG production were not observed in this study [173]. The comparison of PG productivity by S. marcescens SR 1 in nutrient broth and glycerol-yeast extract media was evaluated by Parani and Saha [81]. The results showed that a higher PG productivity (765 mg/L) may be achieved by supplementation of 4% vegetable oil mixture (sunflower, coconut, and olive oil) [81]. Medium containing ethanol (1.5%) and cottonseed meal (1.5%) omitted inorganic salts (phosphate and NaCl) and afforded S. marcescens S389 to produce up to 3000 mg/L PG [190]. Medium supplemented with powdered peanut, coconut, sesame, and castor seed was evaluated for PG production by S. marcescens PP1. The maximum yield of PG reached 1595 mg/L in powdered peanut-supplemented medium. Additionally, PG production by supplementing maltose and glucose to sunflower seed media reached 1556 mg/L and 1525 mg/L, respectively [82]. Peptone (1%) and maltose (0.5%) were used as the C/N source by S. marcescenss subsp. lawsoniana HDZK-BYSB107 for the production of PG (656 mg/L), which showed antibacterial and antitumor activities [143]. 3-[N-morpholino]-Ethanesulfonic acid 475 [199] Cassava wastewater supplemented with 2% mannitol was used by S. marcescens UCP1549 to produce PG (49,500 mg/L) [174]. To avoid using individual fatty acid as a substrate for reducing the cost of industrial production, Giri et al. investigated the effect of different fatty acid-containing media for PG production and found that the higher yield was obtained in peanut seed medium (38,750 mg/L) and sesame seed broth (16,680 mg/L) [201]. Crude glycerol (a waste from biodiesel industry) was supplemented with peptone as a C/N source for PG production by S. marcescens MN5 [202]. Ram horn peptone (0.4%) supplemented in mannitol medium showed enhanced PG production (277.74 mg/L) by S. marcescens MO-1 [170]. To decrease the medium cost by using tannery fleshing (TF) as a C/N source, the proteinaceous byproducts of leather industries, Arivizhivendhan et al. studied the PG production in TF (30%, w/w) and wheat bran (70%, w/w)-containing solid media. The highest PG productivity reached was 70,402 mg per kg of TF [93]. The use of rice bran as a C/N source for S. marcescens PG production was reported by Arivizhivendhan et al., who showed that the PG produced showed antioxidant and antimicrobial activities against foodborne pathogens [92]. Natural substrates such as sweet potato, sesame, and mahua flower extract at different concentrations have also been used for PG production by S. marcescens [171]. The highest PG yield was 4800 mg/L at the final optimized composition of sweet potato powder/casein-containing medium. The purified PG showed antimicrobial [171] and nematicidal [111] activities.
Janthinobacterium lividum BR01 is a psychrotrophic strain that produces PG and heptyl prodigiosin when grown at cool temperatures. The gene cluster of the PG pathway was cloned from J. lividium BR01 and expressed in Escherichia coli, which showed differences in the responsible gene cluster of Serratia sp. [179]. Domröse et al. integrated the PG biosynthesis gene cluster of S. marcescens in Pseudomonas putida KT2440 to construct constitutive PG production strains [188]. The PG productivity obtained was 94 mg/L using the Terrific Broth medium [189]. The PGs productivity of Streptomyces fusant NRCF69, when peanut seed broth, sunflower oil broth, or dairy processing wastewater broth alone or supplemented with 0.5% mannitol were used as the C/N source, was 42,030 mg/L, 40,110 mg/L, 36,700 mg/L, and 47,000 mg/L, respectively [91].
Based on the hydrophobic properties of PG, efficient extraction procedures for PG purification from the culture supernatant of P. putida [188], Serratia sp. KH-95 [206], and S. marcescens SMDR [207] have been studied by adsorption to the materials with hydrophobic surfaces of polyurethane [188], internal adsorbent using acidified methanol and phase separation [206], and macroporous polymeric adsorption resin of Diaion HP-20 resins [207]. The transmembrane transport of PG producing S. marcescens ATCC 8100 and the permeability barrier of the cell membrane were studied by using a model membrane platform with a planar lipid bilayer [151]. The results showed that the mass transfer of the intracellular PG was affected by its size and surface electrical properties and therefore could be modulated by physical and chemical methods [151]. The immobilization strategy to increase PG production was investigated by Chen et al. [172]. The PG productivity of S. marcescens C3 enhanced seven-fold to 15,600 mg/L by using immobilized cells in calcium alginate beads. For increasing the recovery yield of PG, adsorption chromatography was studied to separate and purify PG directly. Wang et al. [208] reported that the use of 0.1% Tween 80 (a nonionic surfactant) may improve the release of PG from the cell envelope. The recovery of PG from the culture broth increased from 50% (using the conventional method) to 83% (with a high loading capacity of the adsorbent X-5 resin) [208]. To investigate the extraction methods which may purify PG with high yield and cost-effectivity, Khanam and Chandra tried six different extraction methods including homogenization, ultrasonication, freezing and thawing, heat treatment, organic solvents, and inorganic acids to evaluate the PG yield. The results showed that the highest amount of extraction was achieved by ultrasonication (98.1%) and the lowest by freezing and thawing (31.8%) methods [209].
The use of bioadsorbents for adsorption of PG was compared among Lactobacillus paracasei subsp. paracasei TKU012 cells, cicada casting, and four chitin-containing materials including squid pens, shrimp shells, α-chitin, and β-chitin [62]. The best result was observed in L. paracasei subsp. paracasei TKU012, followed by cicada casting, shrimp shells, squid pens, β-chitin, and α-chitin. The cells of lactic acid bacterium (strain TKU012) and cicada casting may have the potential to recover and purify PG from the PG-containing culture broth of S. marcescens TKU011 [62]. To increase S. marcescens TKU011 PG production in the medium containing squid pens, the effects of phosphate and ferrous ion supplementation, autoclave treatment, and aeration were studied [60]. The results showed that the 40-min autoclaved medium enhanced PG productivity 2.5-fold to 2480 mg/L [60]. Nguyen et al. further investigated PG production by S. marcescens TKU011 using 1% α-chitin and 0.6% casein as the C/N source and obtained the highest yield of 4620 mg/L. α-Chitin and CaSO 4 were found to play an important role in enhancing PG production by S. marcescens [59].
Generally, peanut seed broth received remarkable PG productivity (47,000 mg/L) which was approximately 10-fold greater than those using chitin-containing fishery byproducts fermented by S. marcescens TKU011. Considering the utilization of obtaining culture broth for biological control, chitin and protein-containing byproducts (1-3 USD/kg) as the C/N source provide PG, and chitinolytic and proteolytic enzymes might also be expected to have potential applications.

Antimicrobial Activity
The antifungal activity of PG produced from S. marcescens is investigated usually against fungal pathogens. The PG from S. marcescens SR 1 showed the maximum inhibitory activity against the fungal pathogens Helminthosporium sativum, Fusarium oxysporium, and Rhizoctonia solani, in decreasing order [81]. Picha et al. reported that the chitin-supplemented agar medium used for the growth and production of PG by S. marcescens PP1 showed high inhibition to the tested fungal pathogens including Alternaria alternata, Aspergillus niger, and F. oxysporum. The tested fungal species of A. niger, Mucor sp., and Rhizopus sp. were resistant to the produced pigment with no clear zone of inhibition [82]. PG has been reported to display antagonistic effects on the tested fungal strains by increasing permeability in the fungal membrane. Among the tested fungi on potato dextrose agar (PDA) plates, F. oxysporum was highly inhibited by the PG treatment. PG enables S. marcescens D1 (an endofungal bacterium) to invade fungal hyphae and spread over the culture of F. oxysporum to result in mycelial death. [213].
In a 0.2% colloidal chitin supplemented Luria broth, S. marcescens B2 produced PG and four extracellular chitinolytic enzymes which showed antifungal activities against the phytopathogens R. solani (caused cyclamen damping-off and rice sheath blight), F. oxysporum (caused fusarium wilt), and Botrytis cinerea (caused gray mold) [85][86][87]. These four chitinolytic enzymes were detected among the extracellular proteins of S. marcescens B2. Among the four chitinolytic enzymes produced by S. marcescens B2, two enzymes showed an inhibitory effect against the spore germination of B. cinerea. The PG extracted and purified from the bacterial cells of S. marcescens B2 also showed inhibition against the spore germination of B. cinerea. A synergistic effect of the S. marcescens B2 produced PG and chitinolytic enzymes was also observed against the tested fungal pathogens [85][86][87]. Gutiérrez-Román et al. [88] also reported that the combination of S. marcescens CFFSUR-B2 produced PG and chitinolytic enzymes showed synergistic inhibitory effect on the germination and germ tube growth of Mycosphaerella fijiensis ascospores. Based on the results of toxic effects similar to that of benzimidazole on ascospore germination, the authors suggested that the combination of PG and chitinolytic enzymes may have potential use in the biocontrol of black Sigatoka disease caused by Mycosphaerella fijiensis [88]. S. marcescens ETR17 produced PG, several hydrolytic enzymes (chitinase, protease, cellulase, and lipase), and plant growth-enhancing compounds (iodoacetic acid and siderophore). This strain was isolated as a biocontrol bacterium which showed a remarkable level of inhibitory activities against several foliar and root pathogens of tea [89]. Due to no hemolysin production, the authors concluded that S. marcescens ETR17 can be applied to minimize the use of chemical fungicides for disease control in tea gardens [89]. Woodhams et al. [95] reported that S. marcescens PG caused remarkable growth inhibition of chytrid fungi Batrachochytrium dendrobatidis and B. salamandrivorans at minimal inhibitory concentrations of 10 and 50 µM, respectively [95]. Streptomyces fusant NRCF69 PGs have also exhibited antimycotic activity against clinical dermatophyte isolates (Trichophyton, Microsporum, and Epidermophyton) [91].
The PG produced by S. marcescens PP1 showed higher inhibitory activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) than Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) [82]. Arivizhivendhan et al. reported the inhibitory activity of S. marcescens produced against P. aeruginosa [93]. Similar results of antibacterial activities by S. marcescens 2170 PG have also been reported by Herráez et al. [94]. PG produced by S. marcescens UFPEDA 398 showed inhibitory activity against 20 strains of oxacillin-resistant S. aureus. The minimum inhibitory concentrations and minimum bactericidal concentrations ranged from 1-4 µg/mL and 2-16 µg/mL, respectively [83].
Arivizhivendhan et al. also reported that the PG from S. marcescens showed effective antioxidant and antibacterial activities [92]. The extracts of aloe leaf and cucumber fruit are known to have photoprotective activity. The application of PG as antibacterial and antioxidant additives to the extracts of aloe leaf and cucumber fruit has been evaluated for the potential of developing commercial sunscreens for human skin protection [96]. Nakashima et al. reported that a bacterial strain (MS-02-063) that produces large amounts of PGs showed equivalent antibacterial activity to those of tetracycline against some pathogenic Gram-positive bacteria including S. aureus [99]. Pseudoalteromonas rubra synthesizes PG, cycloprodigiosin, and four PG derivatives that differ in the length of the alkyl chain. The antimicrobial activities of the produced PG, cycloprodigiosin, and 2-methyl-3-hexyl-prodiginine have been examined against E. coli, Staphylococcus aureus, Salmonella typhi, and Candida albicans and it was found that cycloprodigiosin potently inhibited S. aureus at a concentration of 20 µg/mL [100].
The antimicrobial effect of PG against microorganisms including antibiotic-resistant pathogens and phytofungal pathogens may provide a potential platform for their use as microbial disinfectants in the fields of pharmaceutical and agricultural biocontrol, respectively.

Antiparasitic Activity
The isolation of antiparasitic compounds from microbial extracts has been widely investigated. The antiparasitic activities of S. marcescens produced PG have been evaluated against Plasmodium falciparum and Trypanosoma brucei gambiense. Combinations of PG with phytosynthesized silver and gold nanoparticles showed a remarkable decrease in the IC 50 values on both parasites (2.7-to 3.6-fold) without an increase in cytotoxicity to the mammalian cells [102]. The antagonist effect of S. marcescens 2170 produced PG has been tested against parasite Trypanosoma cruzi cells. The results showed that the concentration of PG required to suppress T. cruzi growth was significantly lower than that required for benznidazole (0.25 mg/L and 4.9 mg/L, respectively) [94]. Isaka et al. reported three compounds (metacycloprodigiosin, bafilomycin A 1 , and spectinabilin) extracted from Streptomyces spectabilis BCC 4785 possessed potent antimalarial activity against P. falciparum K1. Among them, metacycloprodigiosin exhibited potent inhibitory activity (IC 50 of 0.0050 µg/mL) against P. falciparum K1 with much lower cytotoxicity [105]. Marchal et al. observed that the presence of a nitrogen atom in the A-ring of PGs is needed for antimalarial activity [107]. Later, the antimalarial activities of four natural and three synthetic PGs were estimated against P. falciparum [108]. From a culture of α-proteobacteria, Lazaro et al. purified heptyl prodigiosin which showed antimalarial activity similar to that of quinine against the chloroquine-sensitive strain P. falciparum 3D7 [109].
The lack of useful nematicides and the serious damage caused by plant-parasitic nematodes have led to an urgent requirement to isolate some natural remedy for their control [102]. The antinematicidal activity of S. marcescens produced PG has been studied against juvenile stages of Radopholus similis and Meloidogyne javanica. The results showed better activity of this PG (LC 50 values, 83 and 79 µg/mL, respectively) than those of the positive control of copper sulfate (LC 50 values, 380 and 280 µg/mL, respectively). The PG also showed antagonist effect on nematode egg-hatching ability [102].

Insecticidal Activity
Bioagents produced from microbes offer alternatives to chemical pesticides as they can be more selective and safer than chemical insecticides. The insecticidal potency of S. marcescens TKU011 PG was investigated and compared with those of food colorants Tartrazine (Y4) and Allura Red AC (R40) against Drosophila larvae [60,61]. The LC 50 of PG (0.23 µg/µL), Y4 (0.449 µg/µL), and R40 (30 µg/µL) using a five-day exposure period showed potential toxicity of the biopigment PG and the food colorant Y4 against Drosophila larvae [60]. The order of survival rates of Drosophila larvae after five-day feeding with 30 µL of cell broth, broken cell, cell pellet, or cell-free culture supernatant of S. marcescens TKU011 were 0%, 6.25%, 12.5%, and 37.5%, respectively, indicating a direct relationship with their PG content. The fractions with a high PG concentration also exhibited a high insecticidal activity [61]. Patil [110]. Pure PG may be an important molecule for the control of Aedes aegypti and Anopheles stephensi mosquitoes at their larval and pupal stages [111]. Suryawanshi et al. [111] analyzed the effect of the pure PG isolated from S. marcescens NMCC 75 against larval and pupal stages of Aedes aegypti and Anopheles stephensi mosquitoes. The pure PG showed mosquito larvicidal activities with LC 50 values of 14, 15.6, 18, and 21 µg/mL against the early second, third, fourth instars, and pupal stages of Aedes aegypti, respectively, and with LC 50 values of 19.7, 24.7, 26.6, and 32.2 µg/mL, respectively, against the early second, third, and fourth instars and pupae of Anopheles stephensi, respectively. The results of variation of enzymes (esterases, acetylcholine esterases, phosphatases, and proteases) and total proteins in the fourth instar larvae of Aedes aegypti indicated the intrinsic difference in biochemical features as a result of PG treatment [111]. Zhou et al. observed PG produced by S. marcescens SCQ1 (an entomopathogenic strain isolated from silkworm) causes acute septicemia in silkworms [112]. Asano et al. investigated the synergistic effects of PG, chitinases, and chitin-binding protein from S. marcescens ATCC274 on the insecticidal activity of δ-endotoxin (Cry 1C of Bacillus thuringiensis) against the common cutworm, Spodoptera litura [113]. The results showed that only PG exhibited a remarkable synergistic inhibitory activity with Cry 1C which was lethal and growth inhibitory. The supernatants from S. marcescens containing PG and partially purified PG exhibited a similar synergistic activity on the insecticidal activity of Cry1C [113].

Anti-Cancer Activity
Several studies have demonstrated the anti-cancer activity of PG in various types of cancer. Zhou et al. reported the PG produced by S. marcescens SCQ1 (an entomopathogenic strain) showed potent anticancer activity on human lung adenocarcinoma A549 cells and concluded that S. marcescens SCQ1 may have potential to be used as an anti-cancer compound [112]. To study the role of mitochondria in PG-induced apoptosis, Llagostera et al. evaluated the apoptotic action of PG in GLC4 small cell lung cancer cell line by Hoechst 33342 staining and found that PG induces apoptosis in both caspase-dependent and caspase-independent pathways [119]. The high cytotoxic sensitivity of the human small cell lung doxorubicin-resistant carcinoma (GLC4/ADR) cell line to PG through apoptosis activation was further studied and concluded that the results support PG as a potential drug for the treatment of lung cancer because of its ability to overcome the multidrug resistance phenotype produced by MRP-1 overexpression [120]. The synergistic effects between PG (produced by Vibrio sp. C1-TDSG02-1) and doxorubicin (a chemotherapy drug) against oral squamous cell carcinoma (OSCC) cells was reported by Lin and Wen [118]. Based on the results of PG-priming, autophagy could sensitize OSCC cells by promoting doxorubicin influx without regulation of doxorubicin transporter. Lin and Wen also concluded that the PG (produced by Vibrio sp. C1-TDSG02-1)-priming might be a promising adjuvant approach for the chemotherapy of OSCC [118]. Chiu et al. reported the attenuation of tumors accumulated in the mice trachea by PG (produced by Vibrio sp. C1-TDSG02-1) treatment and concluded the potential of PG as a potential chemotherapeutic agent for lung cancer regimens regardless of doxorubicin resistance [116].
Montaner and Perez-Tomas evaluated the apoptotic action of PG in colon cancer cells (DLD-1 and SW-620 human colon adenocarcinoma cells, NRK, and Swiss-3T3 nonmalignant cells) and found that metastatic SW-620 cells were more sensitive to PG than DLD-1. According to the results in both cancer cell lines, the authors suggested that PG induces apoptosis in colon cancer cells [130]. The effect of PG on proliferation and expression of apoptotic-related genes in HT-29 cells was evaluated by Hassankhani et al., who also suggested that PG-induced apoptosis may ascribe to the inhibition of Bcl-2 and survivin in HT-29 cells and these genes may be promising molecular targets of PG [128]. Dalili et al. [129] evaluated the antiproliferative activities of S. marcescens PTCC 1111 produced PG in HT-29 and T47D cancer cell lines. The results showed that HT-29 cells were more sensitive than T47D cells to PG [129]. PG showed higher apoptotic effect than doxorubicin in HT-29 cells. The authors, therefore, suggested the use of PG as a promising antineoplastic agent that triggers apoptosis in different cancer cell lines [129]. Kavitha et al. indicated strong anti-cancer and apoptotic activity of S. marcescens PG against human cervical carcinoma cancer, according to the results of dose-dependent inhibition of human cervical carcinoma cell (Hela-229 cell line) proliferation [137]. Díaz-Ruiz et al. found that treatment of human gastric carcinoma cells (HGT-1 cell line) with PG showed a constant decrease in viability due to apoptosis and suggested that PG induces apoptosis in HGT-1 cells [133].
Soto-Cerrato et al. observed potently cytotoxic activity of PG in both estrogen receptor-positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines and suggested PG as an interesting and potent new pro-apoptotic agent for the treatment of breast cancer despite the presence of multidrug resistance transporter molecules [122,123]. Lu et al. reported that PG could downregulate RAD51 (an attractive target for anticancer drugs) in multiple human breast carcinoma cell lines irrespective of p53 status [126].
To evaluate the immunosuppressive and apoptotic mechanisms of PG, Monge et al. examined the variation of protein expression on exposure to apoptotic concentrations of PG in mitoxantrone (MCF-7-MR) resistant MCF-7 cancer cell line and found that the identified proteins were involved in various cellular functions, including cell defense, DNA repair, and cellular organization [121]. Sam and Pourpak reported that as molecular targets of PG, P53 and survivin contribute to caspase-3-dependent apoptosis in acute lymphoblastic leukemia cells where PG represents an attractive p53-and survivin-modulating agent [134]. Campàs et al. demonstrated that PG induces apoptosis of B-cell chronic lymphocytic leukemia (B-CLL) cells through caspase activation [114]. Liu et al. evaluated the undecylprodigiosin (UPG) extracted from the fermentation broth of Saccharopolyspora sp. (a sponge Mycale plumose-derived actinomycete) and found a significant cytotoxic activity of UPG against five cancer cell lines, especially on murine leukemia P388 [115]. Liu et al. further indicated that UP inhibits proliferation of P388 by inducing G2/M phase arrest and apoptosis, which was related to the activation of P38, JNK rather than ERK1/2 signaling [214]. Based on the observation that PG significantly increased the rate of growth inhibition and decreased metabolic activity of HepG2 cells in a dose-and time-dependent manner, Yenkejeh et al. suggested PG as an attractive compound that may provide a novel approach to the hepatocellular carcinoma-targeted therapy [135].
Based on the results of dose-dependent inhibition of human cervix carcinoma cell (Hela-229 cell line) proliferation by S. marcescens, Kavitha et al. suggested strong anticancer and apoptosis activity of PG against human cervical carcinoma cancer [137]. Montaner et al. [142] studied the effects of S. marcescens 2170 culture supernatant on the viability of different hematopoietic cancer cell lines (Jurkat, NSO, HL-60, and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) and found that the cytotoxic effect was due to apoptosis [142]. The results of using mutants of S. marcescens (strains OF, WF, and 933) that do not synthesize PG suggested the involvement of PG in this apoptosis [142,143]. Li et al. [143] investigated the anticancer activities and mechanism of activity of S. marcescens HDZK-BYSB107 PG by using human choriocarcinoma (JEG3) and prostate cancer cell lines (PC3) in vitro and JEG3 and PC3 tumor-bearing nude mice in vivo [143]. The bacterial PG was observed to induce apoptosis in JEG3 cells, and PG significantly inhibited the growth of JEG3 and PC3 cells, in a dose and time-dependent manner [143]. Nguyen et al. [59] found that the PG purified from fermentation of chitin-containing medium by S. marcescens TKU011 showed potent anticancer activities against A549, Hep G2, MCF-7, and WiDr with IC 50 values of 0.06, 0.04, 0.04, and 0.2 µg/mL, respectively. For comparison, mitomycin C, a commercial anti-cancer compound was also tested, and it showed weaker activity with IC 50 values of 0.11, 0.1, 0.14, and 0.15 µg/mL, respectively [59]. Muthukumar et al. [145] studied the antioxidant, anti-inflammatory, and cytotoxic properties of PG produced by S. marcescens VITAPI and observed potent radical scavenging effect of the extracted PG at 86%, which was significant in comparison to ascorbic acid as a standard [145]. The in vitro anti-inflammatory effect of PG in controlled experimental conditions revealed its protection at 88% and inhibition in a concentration-dependent manner. The cytotoxic bioassay of PG showed the IC 50 value as 50 µg/mL at 63% cytotoxicity [145]. El-Bondkly et al. [91] reported the cytotoxic activities of PG pigments produced by Streptomyces fusant NRCF69 against three human cancer cell lines, including colon cancer cell line (HCT-116), liver cancer cell line (HEPG-2), and breast cancer cell line (MCF-7) [91]. The synthesis and evaluation of a series of ten PGs that bear ester and amide substitution about the C-ring of the PG skeleton were reported by Lund et al. [136]. The PG bearing C-ring esters and amides obtained by chemical synthesis displayed anticancer activity, particularly when featuring a hexyl chain [136].

Algicidal Activity
Red tides (harmful algal blooms) are caused by some toxic phytoplankton, and lead to massive economic losses and cause marine environmental disturbances. The PG producing strain Hahella chejuensis KCTC 2396 (a marine bacterium) was evaluated by Kim et al. as an effective and environment-friendly strategy to control red tide outbreaks [148]. The lytic activity of this promising molecule against Cochlodinium polykrikoides cells at very low concentrations (1 ppb) was serendipitously detected, making H. chejuensis a strong candidate among the biological agents to control red tides [148]. Nakashima et al. [149] reported that the pigment, PG-L-1 produced by a marine bacterium strain MS-02-063 (γ-proteobacterium) exhibited potent algicidal activity against various red tide phytoplanktons in a concentration-dependent manner [99,149]. The authors suggested that PG-L-1 produced by strain MS-02-063 is controlled by the homoserine lactone quorum sensing. This bacterium and other algicidal bacteria may be effective in regulating the blooms of harmful flagellate algae through the quorum sensing system [149].

Dyes
The environmental pollution concerns raised because of the use of chemically synthetic pigment have led to increasing interest in the natural ones. Microbial pigments have lately attracted increasing attention in textile dyeing because of their sustainability and cleaner production [75][76][77][78]151,152,169,210]. Recently, PG has become a research hotspot for its bright colors and antibacterial function. To promote the application of PG in textile dyeing, a novel idea of preparing dye liquid based on fermentation broth was put forward via increasing the proportion of extracellular pigments [151]. With the improvement in transmembrane transfer efficiency of S. marcescens ATCC8100, PG was produced as the proportion of extracellular pigments, and the complicated biological separation process could be avoided and the application of microbial pigments in textile dyeing can be promoted [151].
In the ethanol solution, PG has good stability under natural indoor light but gets rapidly decomposed under intense sunlight [210]. PG is an eco-friendly colorant to dye fabrics, including synthetic and natural fibers. Synthetic fabrics (such as polyamide and acrylic) dyed with PG have high colorfastness to washing and exhibit antimicrobial activities against E. coli and Staphylococcus aureus. Liu et al. [210] reported the promising prospects of S. marcescens jx1-1, with high PG yield and purity, in food, cosmetic, and textile industries.
A strain of Vibrio sp. KSJ45 produced large quantities of bright red PGs with an elementary composition of C 20 H 25 N 3 O that could be used to dye many fibers including wool, nylon, acrylics, and silk. Fabrics dyed with the PGs produced from Vibrio sp. KSJ45 demonstrated antibacterial activity [101]. Vaidyanathan et al. [211] have studied the application of a novel red biochrome (514 Da in size) produced by solid-state cultivation of Serratia sakuensis subsp. nov. strain KRED in the dyeing of silk, wool, and cotton fabrics. The results showed that silk, wool, and cotton fabrics dyed with this new natural red compound have high color strength and dye uptake values along with good colorfastness as well as antibacterial activity [211]. Mehta and Shah [169] studied the application of PG in the candle industry. The mixture of PG with translucent wax was homogenized and poured into the mold and the candles were left to cool down. After de-molding, the translucent candle showed a more intense coloration, which was similar to the colored candles available in the market, the authors proposed that the synthetic coloring agents can be replaced by natural colorants extracted from microorganisms. The results of the present study on the isolation and application of PG extracted from S. marcescens in the coloration of translucent candles revealed that the pigment PG can be considered as a possible alternate source of colorant in various industries. Table 2 summarizes the bioactivities and applications of PGs reported.

Conclusions and Perspectives
Pigments of various kinds and forms have been used as additives or supplements in the food industry, cosmetics, pharmaceuticals, biocontrol, and other applications. Recently, in response to the problems of the synthetic pigments that cause toxicity and carcinogenicity in the human body, the inclination to use natural pigments as adding natural materials for human health and safety has gradually expanded. This review contains the most recent information on the production of PGs from various bacteria especially S. marcescens. For PG production, the commercial media of peptone and yeast extract supplemented with vegetable oil have been used for enhancing PG productivity. Considering the bioactivities of PG, anti-cancer applications have been most widely studied. Compared to the use of expensive media supplemented with vegetable oils, chitin and protein-containing fishery byproducts have more potential for PG production.
The discovery of inexpensive PG not only solves environmental problems but also promotes the economic value of marine wastes. Furthermore, the chitinolytic and proteolytic enzymes together with PG produced by S. marcescens TKU011 using fishery byproducts can enhance antimicrobial activities and may be a potential source of biological control agents.

Conflicts of Interest:
The authors declare no conflict of interest.