Introduction
The marine environment encompasses approximately 71% of the Earth’s surface and harbours an extraordinary diversity of microorganisms that play essential roles in global biogeochemical cycles, nutrient turnover, climate regulation, and ecosystem functioning (Ocean and Climate, Reference OCEAN AND CLIMATE2019). Among marine microorganisms, bacteria have emerged as prolific producers of structurally diverse secondary metabolites with significant ecological, pharmaceutical, and industrial importance. In recent years, microbial pigments have attracted considerable scientific interest owing to their wide range of biological activities, including antimicrobial, antioxidant, anticancer, antiparasitic, and photoprotective properties (Ramesh et al., Reference Ramesh, Vinithkumar and Kirubagaran2019; Jaber et al., Reference Jaber, Majeed and Al-Hashimi2022).
Marine bacteria constitute an important source of naturally occurring pigments such as carotenoids, phenazines, prodigiosin, violacein, and melanin derivatives (Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020). Early investigations demonstrated that a substantial proportion of marine bacterial populations are chromogenic, highlighting the ocean as a rich reservoir of pigment-producing microorganisms (Zobell, Reference Zobell1941). Growing concerns regarding the environmental persistence, toxicity, and potential carcinogenicity of synthetic colourants have accelerated interest in microbial pigments as sustainable and eco-friendly alternatives for applications in the food, pharmaceutical, cosmetic, textile, and nutraceutical sectors (Shatila et al., Reference Shatila, Yusef and Holail2013; Venil et al., Reference Venil, Dufossé and Renuka2020; Kumar et al., Reference Kumar, Kumar, Ambika, Kumar, Darnal, Thakur, Patial and Singh2022). Compared with synthetic dyes, bacterial pigments are generally biodegradable, biocompatible, and environmentally benign, making them attractive candidates for future industrial exploitation (Venil et al., Reference Venil, Dufossé and Renuka2020; Nawaz et al., Reference Nawaz, Chaudhary, Shah, Dufossé, Fouillaud, Mukhtar and Haq2021).
Beyond their colouring properties, bacterial pigments possess diverse biological functions that contribute to microbial adaptation, survival, and ecological competitiveness. Several microbial pigments exhibit pronounced antimicrobial activities against human and animal pathogens, suggesting their potential as novel alternatives to conventional antimicrobial agents (Ramesh et al., Reference Ramesh, Vinithkumar and Kirubagaran2019; Chaturwedi et al., Reference Chaturwedi, Mainali and Chaudhary2024). The increasing prevalence of antimicrobial resistance has intensified the search for new bioactive compounds from natural sources, particularly those derived from marine microorganisms. Consequently, pigment-producing marine bacteria have become promising targets for the discovery of alternative antibacterial agents with potential biomedical applications (Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020; Nawaz et al., Reference Nawaz, Chaudhary, Shah, Dufossé, Fouillaud, Mukhtar and Haq2021).
Among pigment-producing bacteria, species of Pseudomonas are particularly noteworthy because of their ability to synthesize biologically active pigments, including phenazine derivatives such as pyocyanin, which exhibit antimicrobial, redox-active, and signalling functions (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008; Koyun et al., Reference Koyun, Sirin, Erdem and Aslim2022). Several studies have reported the antibacterial potential of pigments produced by Pseudomonas aeruginosa, highlighting their capacity to inhibit a broad spectrum of pathogenic microorganisms (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008; Abdo et al., Reference Abdo, Fouda, Eid, Fahmy, Elsayed, Khalil, Alzahrani, Ahmed and Soliman2021). Furthermore, genomic analyses have identified carotenoid biosynthetic pathways in marine-associated Pseudomonas species, indicating their potential as valuable sources of bioactive pigments (Fukaya et al., Reference Fukaya, Takemura, Koyanagi, Maoka, Shindo and Misawa2018).
Members of the genus Microbacterium are also recognized for their ecological versatility and metabolic diversity. These Gram-positive actinobacteria are widely distributed in terrestrial and aquatic habitats and are capable of producing yellow carotenoid-like pigments that may contribute to protection against oxidative and environmental stress (Kalsi et al., Reference Kalsi, Drautz-Moses, Uchida, Purbojati, Houghton, Chénard, Wong, Kolundžija, Clare and Kushwaha2019). Although pigment production has been reported in several Microbacterium species, their antibacterial potential remains comparatively underexplored, particularly among marine isolates. Therefore, investigating pigment-producing Microbacterium strains may provide new insights into the discovery of biologically active natural products.
Marine-derived carotenoid pigments have received particular attention because of their antioxidant and therapeutic properties. These pigments function as protective molecules against oxidative damage and environmental stressors while also demonstrating antimicrobial activity against diverse pathogens (Köcher et al., Reference Köcher, Breitenbach, Müller and Sandmann2009; Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020). The commercial significance of carotenoids continues to expand globally, with increasing demand for naturally sourced pigments in food, pharmaceutical, and health-related industries (Yaqoob et al., Reference Yaqoob, Riaz, Shabbir, Zia-Ul-Haq, Alwakeel, Bin-Jumah, Zia-Ul-Haq, Dewanjee and Riaz2021). Consequently, marine bacteria have emerged as attractive and sustainable producers of high-value carotenoid pigments.
The Bay of Bengal represents one of the most ecologically diverse marine ecosystems in South Asia and supports complex microbial communities adapted to dynamic environmental conditions (Akter et al., Reference Akter, Rahman, Ali, Minch, Mehzabin, Siddique, Galib, Yesmin, Azmuda and Adnan2023). Recent investigations have revealed considerable microbial diversity and biotechnological potential within this region, including the isolation of carotenoid-producing marine bacteria possessing bioactive properties (Flora et al., Reference Flora, Afrin, Jui, Bhowmik, Bhuiyan, Rashid, Khatun, Islam, Hossain, Bhuiyan and Saha2026). Despite this growing interest, the pigment-producing bacterial diversity of the Cox’s Bazar coastal region remains insufficiently explored, particularly with respect to the antibacterial activities of their pigments. This knowledge gap limits the effective utilization of indigenous marine microbial resources for biomedical and industrial applications.
Therefore, the present study aimed to isolate and characterize pigment-producing marine bacteria from the coastal waters of Cox’s Bazar, Bangladesh, with particular emphasis on P. aeruginosa and Microbacterium hominis. In addition, the antibacterial activity of pigments extracted from these isolates was evaluated against selected bacterial pathogens. By investigating the antibacterial potential of marine bacterial pigments, this study contributes to the growing search for natural antimicrobial agents and provides further insight into the biotechnological value of marine microbial resources from the Bay of Bengal.
Materials and methods
Sample collection and isolation of pigment-producing bacteria
In August 2023, sediment samples were collected from six distinct coastal locations along the Bay of Bengal near Cox’s Bazar, Bangladesh: Kolatoli, Inani, Himchori, Patuartek, Ukhia 1, and Ukhia 2 (Figure 1). These sites represent diverse marine and intertidal habitats extending across the central to southern coastal belt of Cox’s Bazar. The approximate spatial distance among the sampling sites ranged from 5 to 35 km, encompassing both high-tourism zones and relatively undisturbed areas characterized by varying organic matter content, tidal influence, and anthropogenic impact.
Composition of the pigment-producing bacteria isolated from sediments collected from the coastal area of Cox’s Bazar, Bangladesh.
Kolatoli Beach (21.422° N, 91.981° E) is a heavily visited urban coastal zone near the main Cox’s Bazar beach area, influenced by tourism and fishing activities. The sediment here was fine-grained and moderately moist, with noticeable organic residue from nearshore runoff. Himchori Beach (21.376° N, 91.974° E), located approximately 7 km south of Kolatoli, lies adjacent to the Himchori Marine Protected Area and is characterized by rocky cliffs, moderate tidal flow, and limited pollution. Inani Beach (21.253° N, 92.001° E), about 20 km south of central Cox’s Bazar, features coral fragments and coarse sand with elevated sunlight exposure that favours microbial diversity and pigment production. Patuartek Beach (21.167° N, 92.045° E) represents a relatively less disturbed coastal zone near the southern fringe of the Inani–Teknaf marine corridor, dominated by fine sediments and moderate wave action. Ukhia 1 and Ukhia 2 (21.090° N, 92.100° E; 21.075° N, 92.115° E) are situated inland near the tidal inlets of the Naf River estuarine system, where saline and freshwater inputs converge, creating variable physicochemical gradients that influence microbial community structure.
Sediment samples were aseptically collected from a depth of 6–10 inches (15–25 cm) using sterile Ziplock bags. Each sample (approximately 100 g) was immediately labelled, sealed, and stored in an ice box (4°C) for transportation to the laboratory. Samples were processed within 24 h of collection to minimize microbial alteration. The pH of each sediment sample was measured using a digital pH meter (Jenway 3310, UK), while temperature and other basic environmental parameters were recorded in situ using a portable multiparameter probe.
Although salinity is recognized as a key ecological factor influencing microbial community composition and metabolic activity in marine and estuarine sediments, it was not quantitatively determined for the selected sites in this study. All sampling sites were located within the same coastal stretch of the Bay of Bengal, where seasonal tidal mixing and oceanographic studies have consistently reported relatively narrow salinity variation (typically 28–33 PSU) across short spatial scales during the monsoon season. Hence, it was assumed that salinity differences among the sites would be minimal and unlikely to exert a major confounding influence on pigment-producing bacterial diversity within this restricted geographic range.
Nonetheless, salinity remains a critical environmental determinant of microbial adaptation, osmotic regulation, and secondary metabolite synthesis. Future studies will therefore include quantitative salinity profiling alongside physicochemical characterization, such as dissolved oxygen, nutrient levels, and organic carbon content, to better elucidate correlations between environmental gradients and pigment production in coastal bacterial isolates.
Aerobic heterotrophic marine bacteria were isolated using the serial dilution plate technique with 10-fold serial dilutions ranging from 10−1 to 10−⁶ on Luria–Bertani (LB) agar medium following the method of Eklund and Lankford (Reference Eklund and Lankford1967). The pH of the medium was adjusted to correspond to that of the respective water samples. Plates were incubated aerobically at 37°C for 24 h, as this temperature supports the rapid growth and recovery of mesophilic heterotrophic marine bacteria commonly present in the tropical marine environment of the Bay of Bengal. After incubation, colony-forming units (CFUs) were enumerated, and the number of pigment-producing isolates was recorded. Colonies exhibiting distinct pigmentation were selected and purified by repeated streaking on LB agar plates. Pure isolates were subsequently preserved at 4°C for further analysis (Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020).
Pigment production media
To assess pigment production, five different types of media were used: LB medium, Nutrient Agar, Sea Salt Agar, Zobell Marine Agar, and LB medium supplemented with seawater. All media were purchased from Hi-Media, India. Of these, LB medium supplemented with seawater supported the highest growth and pigment production. This modified medium created optimal conditions for bacterial growth and facilitated the synthesis of vibrant pigments (Zobell, Reference Zobell1941).
Morphological characterization
Morphological characterization of the bacterial isolates was performed based on colony characteristics, including form, size, elevation, margin, surface texture, pigmentation, and optical appearance on culture media (Anzum et al., Reference Anzum, Khan and Hossain2022; Jaber et al., Reference Jaber, Majeed and Al-Hashimi2022). Gram staining was conducted following standard microbiological procedures to determine the Gram reaction and cellular morphology of the isolates (Eklund and Lankford, Reference Eklund and Lankford1967). Microscopic observations were carried out using an Olympus BX41 light microscope (Olympus Corporation, Japan). Based on pigment intensity, colony morphology, and growth characteristics, the two most prominent yellow pigment-producing isolates were selected for further molecular identification and characterization (Chakraborty et al., Reference Chakraborty, Redkar, Munjal, Kumar and Rao2015; Kumar et al., Reference Kumar, Kumar, Ambika, Kumar, Darnal, Thakur, Patial and Singh2022).
DNA extraction and molecular identification
DNA from pigment-producing isolates Y-1 and Y-6 was extracted using a modified heat-thaw method (Kalsi et al., Reference Kalsi, Drautz-Moses, Uchida, Purbojati, Houghton, Chénard, Wong, Kolundžija, Clare and Kushwaha2019). Isolates were grown in Luria Broth at 37°C for 24 h. After incubation, the culture was centrifuged at 10,000 rpm for 10 min, and the resulting pellet was resuspended in 100 µL of RNase-free water. The mixture was heated at 100°C for 5 min, then placed on ice for 10 min. Cold ethanol was added to precipitate the DNA, which was further purified by centrifugation. The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and DNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific, USA).
Pigment-producing bacterial isolates were identified by amplification and sequencing of the 16S rRNA gene. Genomic DNA was extracted from pure bacterial cultures, and the nearly full-length 16S rRNA gene (∼1,500 bp) was amplified by polymerase chain reaction (PCR) using the universal bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). PCR amplification was performed under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1.5 min, annealing at 56°C for 1 min, and extension at 72°C for 1.5 min, with a final extension step at 72°C for 10 min. The amplified PCR products were verified by electrophoresis on 1.5% agarose gel, which revealed a distinct band of approximately 1.5 kb.
The obtained sequences were edited and compared with reference sequences available in the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm to determine their closest phylogenetic affiliations. For phylogenetic analysis, representative 16S rRNA gene sequences of closely related type strains were retrieved from the NCBI GenBank database based on BLAST similarity results. These reference species were selected to include phylogenetically related taxa within the same genus and neighbouring genera to accurately determine the taxonomic position of the isolates. Multiple sequence alignment was performed using the ClustalW algorithm implemented in MEGA version 11. Phylogenetic trees were constructed using the maximum likelihood method based on the Kimura two-parameter model, with branch reliability evaluated by bootstrap analysis using 1,000 replicates. The origin and accession numbers of all reference sequences used in the phylogenetic analysis were obtained from the NCBI GenBank database and are provided in the corresponding phylogenetic tree.
Bacterial growth and pigment production conditions
Bacterial growth and pigment production were evaluated across various temperatures (25°C, 37°C, 40°C, 45°C) and pH levels (3, 4, 5, 6, 7, 8) to determine optimal conditions. Salinity tolerance was also assessed by preparing LB medium with seawater concentrations of 100%, 75%, 50%, 25%, 10%, and 0% (Nawaz et al., Reference Nawaz, Chaudhary, Shah, Dufossé, Fouillaud, Mukhtar and Haq2021). Growth was monitored by measuring absorbance at 600 nm at different time intervals to track bacterial proliferation (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008).
Pigment extraction
Pigment extraction was carried out using a solvent-based protocol to isolate both extracellular and intracellular pigments from the selected isolates. The pigment-producing bacterial strains (Y-1 and Y-6) were cultured in LB medium supplemented with seawater for 6 days at 37°C with continuous shaking at 120 rpm. After incubation, the cultures were centrifuged at 6,000 rpm for 15 min at 4°C to separate the extracellular pigment-containing supernatant from the cell pellets.
The extracellular pigments present in the supernatant were directly extracted using an equal volume of 95% methanol (1:1, v/v) and incubated at 30°C for 30 min with gentle shaking. The mixture was then centrifuged, and the pigment-rich methanolic phase was collected.
For intracellular pigment extraction, the resulting cell pellets were resuspended in 95% methanol and subjected to ultrasonic sonication at 30°C for 30 min to ensure complete cell disruption and pigment release. The lysate was centrifuged again at 6,000 rpm for 15 min, and the supernatant containing the intracellular pigments was collected.
Both extracellular and intracellular pigment extracts were concentrated using a rotary evaporator and subsequently freeze-dried to obtain powdered pigment samples for further characterization (Dawoud et al., Reference Dawoud, Alharbi, Theruvinthalakal, Thekkangil, Kadaikunnan, Khaled, Almanaa, Sankar, Innasimuthu, Alanzi and Rajaram2020).
UV-visible and FTIR spectral analysis
The UV-visible spectra of the extracted pigments were measured in the range of 200–800 nm using an Analytik Specord spectrophotometer (Germany). The wavelengths of maximum absorption (λ max) were recorded to confirm pigment identities. For FTIR analysis, approximately 1–2 mg of each pigment was mixed with 100 mg of KBr powder and pressed into a pellet. FTIR spectra were recorded from 4000 to 400 cm−1 using a PerkinElmer FTIR spectrometer (Frontier, PerkinElmer, UK). The characteristic functional groups were identified based on the absorption peaks (Koyun et al., Reference Koyun, Sirin, Erdem and Aslim2022).
Antibacterial activity
The antibacterial activity of the pigment extracts was evaluated using the agar well diffusion assay following standard procedures (CLSI, Reference CLSI2017). The test organisms included Bacillus subtilis ATCC 11774, Staphylococcus aureus ATCC 9144, Escherichia coli ATCC 11303, and Salmonella typhi ATCC 13311. Fresh bacterial suspensions (adjusted to 0.5 McFarland standard, approximately 1 × 10⁸ CFU/mL) were uniformly spread on Mueller–Hinton Agar plates.
Wells measuring 6 mm in diameter were aseptically punched into the agar plates, and 100 µL of lyophilized pigment extract dissolved in 10% dimethyl sulfoxide (DMSO) was carefully dispensed into each well at active principle concentrations of 0.5, 1.0, 2.5, and 5.0 mg/mL. Tetracycline (10 µg/mL) was used as the positive control, whereas 10% DMSO served as the negative control to confirm that the solvent exhibited no inhibitory effect on microbial growth.
The plates were incubated at 37°C for 24 h, after which the zones of inhibition (mm) were measured around each well to determine antibacterial efficacy. The inhibitory activity was expressed as mean ± standard deviation (SD) from three independent experiments, reflecting the concentration-dependent effect of the active pigment principles.
Cytotoxicity assessment using MTT assay
The MTT assay was used to assess the cytotoxicity of pyocyanin (Y-1) and pyoverdine (Y-6) against Vero cells (African green monkey kidney cells). Cells were cultured in DMEM supplemented with 1% penicillin-streptomycin, 0.2% gentamycin, and 10% fetal bovine serum and incubated in a 5% CO₂ atmosphere at 37°C. After reaching confluence, cells were seeded in a 96-well plate at 1.5 × 10⁴ cells per 100 µL and incubated for 24 h. Cells were then exposed to varying concentrations of pyocyanin (5.625, 11.25, 22.5, 45 µg/mL) and pyoverdine (5.625, 11.25, 22.5, 45 µg/mL) for 24 h. MTT solution (25 µL of each well) was added, and the plates were incubated for 48 h. The formazan crystals were solubilized in DMSO, and absorbance was measured at 570 nm. Cell viability was calculated by comparing treated samples with untreated controls, using the following formula:
${\text{Cell viability }}\left( {\text{\% }} \right){\text{ = }}\left( {\frac{{{\text{Atreated}}}}{{{\text{Auntreated}}}}} \right) \times 100$
Duplicate wells were used for each sample, and both were read to ensure reliability.
Statistical analysis
All experiments were conducted in triplicate, and the results are presented as mean ± SD. Prior to statistical analysis, the homogeneity of variance among the datasets was evaluated using Levene’s test. Statistical comparisons between groups were performed using one-way analysis of variance, followed by Tukey’s post hoc test to determine significant differences among treatment means. A probability value of p < 0.05 was considered statistically significant. Data analysis was carried out using Microsoft Excel 2019 and SPSS software (version XX, IBM Corp., USA).
Results
Microbial load of the collected samples
A total of six sediment samples were collected from different quadrants of the Cox’s Bazar area in Bangladesh, a region known for its unique marine ecosystem. The pH of these sediment samples ranged from 7.10 to 7.80, with an average pH of 7.40, indicating a slightly basic environment. This slightly basic pH is conducive to microbial growth in marine ecosystems (Akter et al., Reference Akter, Rahman, Ali, Minch, Mehzabin, Siddique, Galib, Yesmin, Azmuda and Adnan2023).
The aerobic heterotrophic bacterial load in the collected samples varied between 1.08 × 10⁶ and 2.60 × 10⁶ CFU/mL in LB medium and between 1.5 × 10⁶ and 2.84 × 10⁶ CFU/mL in LB medium supplemented with seawater (Table 1). The highest bacterial count (2.84 × 10⁶ CFU/mL) was observed in the LB medium supplemented with seawater at the Ukhia-1 site, while the lowest bacterial count (1.08 × 10⁶ CFU/mL) was found in LB medium at the Patuartek site. These variations likely reflect differences in local environmental conditions, including salinity, temperature, nutrient availability, organic matter content, and microbial community composition, which are known to influence the abundance and distribution of coastal bacterial populations (Akter et al., Reference Akter, Rahman, Ali, Minch, Mehzabin, Siddique, Galib, Yesmin, Azmuda and Adnan2023).
pH and bacterial load (cfu/g) in sediment samples of Cox’s Bazar area
Table 1 Long description
The table reports sediment pH and bacterial load for six Cox’s Bazar sampling sites, measured as total bacterial count and pigmented colony count in two culture conditions: standard LB medium and LB medium prepared with seawater. pH ranges from 7.10 at Ukhia 2 to 7.80 at Inani, with an overall average of 7.40. Total bacterial counts are consistently higher in the seawater condition than in standard LB across every site. The highest total counts occur at Ukhia 1, while the lowest total counts occur at Patuartek in both media. Pigmented colony counts also increase with seawater at every site, with the highest pigmented counts at Ukhia 1 and the lowest at Kolatoli. On average, seawater medium yields higher totals and higher pigmented colonies than standard LB. Values reflect culture-based results and may not represent all bacteria present in the sediment.
Among the media tested, LB medium supplemented with seawater supported more robust growth of pigment-producing bacteria, providing optimal conditions for both bacterial growth and pigment synthesis (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008). On average, 39 pigmented bacterial colonies were observed in LB medium, while 49 pigmented colonies were observed in LB medium supplemented with seawater. These colonies exhibited four distinct colours: yellow (59.11%), orange (26.67%), brown (5.56%), and off-white (8.66%). Yellow pigment-producing bacteria were the most prevalent, and hence, the study focused on the two most prominent yellow pigment-producing isolates, designated Y-1 and Y-6, for further investigation (Figure 1).
Morphological and molecular identification of pigment-producing bacteria
The bacterial isolates Y-1 and Y-6 were both observed under a microscope and found to be rod-shaped and non-sporulating. Isolate Y-1 was identified as Gram-positive, while isolate Y-6 was identified as Gram-negative (Figure 2). Molecular identification was performed through 16S rRNA gene sequencing, with partial gene sequences obtained for both isolates. The sequence of Y-1 exhibited 98.17% similarity to P. aeruginosa, and the sequence of Y-6 exhibited 99.65% similarity to M. hominis (Table 2). The sequences were deposited in the NCBI GenBank with accession numbers PP773550 for Y-1 and PP783876 for Y-6. Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated clear clustering of the isolates within their respective species (Figure 3), confirming their identities as P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012.
Observations of yellow-pigmented bacterial isolates cultured in Petri dishes: (A) P. aeruginosa strain BDIFST240011 and (B) M. hominis strain BDIFST240012. Microscopic observations of bacterial isolates: (C) P. aeruginosa strain BDIFST240011 and (D) M. hominis strain BDIFST240012.
Phylogenetic tree of the pigment-producing bacteria obtained from sediments of the coastal sites of Cox's Bazar, Bangladesh.
Figure 3 Long description
The image displays two phylogenetic trees labeled A and B. Tree A shows the evolutionary relationships among various strains of Pseudomonas aeruginosa. The tree is rooted and branches out to show different strains, with bootstrap values indicated at the nodes. The Pseudomonas aeruginosa strain BDIFST240011 is highlighted. Tree B illustrates the phylogenetic relationships among strains of Microbacterium hominis, also with bootstrap values at the nodes. The Microbacterium hominis strain BDIFST240012 is highlighted. Both trees depict the genetic similarities and differences among the strains, with branch lengths representing evolutionary distances.
Molecular identification of potential yellow pigment-producing bacterial isolates
Table 2 Long description
The table reports molecular identification results for two potential yellow pigment-producing bacterial isolates, listing isolate number, scientific name, strain code, query cover, E value, and percent identity match. Isolate Y-1 is identified as Pseudomonas aeruginosa with strain BDIFST240011, full query cover, an E value reported as zero, and 98.17 percent identity. Isolate Y-6 is identified as Microbacterium hominis with strain BDIFST240012, full query cover, an E value reported as zero, and 99.65 percent identity. Both isolates have complete query coverage and the same E value, indicating similarly strong database matches by those measures. Y-6 has the higher identity match than Y-1, suggesting a closer sequence match for Y-6. The table includes only two isolates, so broader trends across more samples cannot be inferred.
Optimization of the medium
Both isolates, P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012, exhibited optimal growth and pigment production when cultured in LB medium supplemented with seawater. The results demonstrated that a temperature of 37°C and a pH range of 5.7–6.0 were optimal for both bacterial growth and pigment synthesis (Figure 4). For P. aeruginosa, the growth rate and pigment yield increased steadily with seawater concentrations up to 50%, after which they remained relatively stable at 75% and 100% salinity levels. In contrast, M. hominis showed the highest biomass accumulation and pigment intensity at 75% seawater, indicating this strain’s preference for moderately saline environments (Ramesh et al., Reference Ramesh, Vinithkumar and Kirubagaran2019). Quantitative pigment estimation revealed that M. hominis produced the highest pigment concentration (1.84 ± 0.06 mg/mL) under 75% seawater, while P. aeruginosa exhibited a comparable yield (1.79 ± 0.04 mg/mL) at 50% seawater, confirming that salinity acts as a key modulator of pigment biosynthesis in both strains.
Growth optimization of Pseudomonas aeruginosa and Microbacterium hominis at the various treatments: temperature (A), pH (B), and seawater (C).
Figure 4 Long description
The image A showing a bar graph with the vertical axis labeled OD at 600 nm, ranging from 0 to 1.5. The horizontal axis is labeled Temperature, with categories 25 degree celsius, 30 degree celsius, 37 degree celsius, 40 degree celsius and 45 degree celsius. Two bar series are shown: Y-1 (Pseudomonas aeroginosa) and Y-6 (Microbacterium hominis). At 25 degree celsius, Y-1 is about 0.7 and Y-6 is about 0.1. At 30 degree celsius, Y-1 is about 0.4 and Y-6 is about 0.2. At 37 degree celsius, Y-1 is about 1.2 and Y-6 is about 0.3. At 40 degree celsius, Y-1 is about 0.1 and Y-6 is about 0.4. At 45 degree celsius, Y-1 is about 0.75 and Y-6 is about 0.55. The image B showing a bar graph with the vertical axis labeled OD at 600 nm, ranging from 0 to 1.5. The horizontal axis is labeled pH, with categories 3, 5, 6, 7, 8 and 9. Two bar series are shown: Y-1 (Pseudomonas aeroginosa) and Y-6 (Microbacterium hominis). At pH 3, Y-1 is about 0.35 and Y-6 is about 0.1. At pH 5, Y-1 is about 0.3 and Y-6 is about 0.4. At pH 6, Y-1 is about 1.1 and Y-6 is about 1.1. At pH 7, Y-1 is about 0.3 and Y-6 is about 0.5. At pH 8, Y-1 is about 0.05 and Y-6 is about 0.25. At pH 9, Y-1 is about 0.35 and Y-6 is about 0.3. The image C showing a bar graph with the vertical axis labeled OD at 600 nm, ranging from 0 to 0.4. The horizontal axis is labeled Seawater Concentration, with categories 100 percent, 75 percent, 50 percent, 25 percent, 10 percent and 0 percent. Two bar series are shown: Y-1 (Pseudomonas aeroginosa) and Y-6 (Microbacterium hominis). At 100 percent, Y-1 is about 0.33 and Y-6 is about 0.23. At 75 percent, Y-1 is about 0.33 and Y-6 is about 0.30. At 50 percent, Y-1 is about 0.33 and Y-6 is about 0.27. At 25 percent, Y-1 is about 0.30 and Y-6 is about 0.27. At 10 percent, Y-1 is about 0.12 and Y-6 is about 0.26. At 0 percent, Y-1 is about 0.10 and Y-6 is about 0.15.
Although comparative differences in growth and pigment intensity were visually and quantitatively evident among the tested conditions, a t-test was not performed between the variables. This decision was based on the experimental design, which focused on preliminary optimization rather than statistical hypothesis testing. The primary objective was to identify qualitative and trend-based optima for pigment synthesis under different physicochemical conditions rather than to evaluate treatment means for statistical significance. Each condition was tested in triplicate, and data were summarized as mean values with corresponding SDs to ensure biological reproducibility.
Furthermore, the study involved multiple independent variables, temperature, pH, and seawater concentration, that are known to interact in complex, non-linear ways. Therefore, a simple pairwise t-test was deemed statistically inappropriate. Instead, descriptive and comparative analyses were applied to interpret the observed growth and pigment production patterns, providing a robust foundation for subsequent quantitative optimization and scale-up studies.
Production and extraction of pigments
Pigment extraction was conducted using a methanol solvent extraction method. The isolates Y-1 and Y-6 were cultured in LB medium supplemented with seawater and incubated at 37°C while shaking at 120 rpm for 6 days. The culture supernatants were collected by centrifugation, and the pellets were resuspended in methanol. Ultrasonication was performed to facilitate pigment extraction, followed by concentration using a rotary evaporator. The final products were freeze-dried, and the yield was measured to ensure high-purity pigment production (Dawoud et al., Reference Dawoud, Alharbi, Theruvinthalakal, Thekkangil, Kadaikunnan, Khaled, Almanaa, Sankar, Innasimuthu, Alanzi and Rajaram2020).
Characterization of pyocyanin and pyoverdine pigments
To confirm the identity of the pigments, UV-visible spectroscopy and FT-IR spectroscopy were employed. UV-visible absorption spectra for pyocyanin (P. aeruginosa Y-1) revealed a peak in the range of 300–400 nm, consistent with the characteristic absorption of pyocyanin (Figure 5a) (Koyun et al., Reference Koyun, Sirin, Erdem and Aslim2022). Pyoverdine (from M. hominis Y-6) exhibited absorption peaks around 360 nm, which corresponds to the absorption pattern of carotenoid pigments (Figure 5b). The FT-IR spectra of both pigments exhibited characteristic peaks. For pyocyanin, peaks at 3325.80 cm−1, 2955.49 cm−1, and 1719.46 cm−1 were identified, which correspond to hydroxyl, alkyl, and carbonyl groups, respectively (Figure 5c). Similarly, the FTIR spectrum of pyoverdine exhibited peaks at 3325.80, 2118.10, and 1685.57 cm−1, indicating the presence of hydroxyl, alkyl, and carbonyl groups, further supporting its identification as a carotenoid pigment (Figure 5d) (Köcher et al., Reference Köcher, Breitenbach, Müller and Sandmann2009).
Characterization of pigments from P. aeruginosa (pyocyanin) and M. hominis (pyoverdine) using spectroscopic analyses. (A) UV-Vis spectrum of pyocyanin; (B) UV-Vis spectrum of pyoverdine; (C) FTIR spectrum of pyocyanin; (D) FTIR spectrum of pyoverdine.
Figure 5 Long description
Insufficient visual information to describe this element accurately.
Antibacterial activity of pigments
The antibacterial activity of pyocyanin and pyoverdine was evaluated using the agar well diffusion method (Table 3). Pyocyanin exhibited the most substantial inhibition against B. subtilis at 5.0 mg/mL, with a zone of inhibition of 19.2 ± 0.25 mm. In contrast, pyoverdine also demonstrated significant inhibition against B. subtilis (18.0 ± 0.50 mm) but showed limited activity against Gram-negative bacteria, with no inhibition observed against E. coli and S. typhi (Figure 6). This finding suggests that pyocyanin has a more potent effect against Gram-positive bacteria, while pyoverdine’s antibacterial activity appears to be more restricted (Koyun et al., Reference Koyun, Sirin, Erdem and Aslim2022).
(A) Antibacterial activity of varying concentrations of yellow pigment from P. aeruginosa strain BDIFST240011 (pyocyanin). (B) Antibacterial activity of varying concentrations of yellow pigment from M. hominis strain BDIFST240012 (pyoverdine). ‘PC’ denotes tetracycline as the positive control, while ‘NC’ represents 10% DMSO as the negative control.
The antibacterial activity of different concentrations of pigments (pyocyanin and pyoverdine) derived from P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012 was assessed using the agar well diffusion assay
Table 3 Long description
Agar well diffusion results report mean inhibition zone diameters in millimeters for two pigments, pyocyanin and pyoverdine, tested at 0.5, 1.0, 2.5, and 5.0 mg/mL against B. subtilis, S. aureus, E. coli, and S. typhi. Pyocyanin shows no inhibition at 0.5 mg/mL for any organism, then begins inhibiting B. subtilis and S. aureus at 1.0 mg/mL with zones around 6 to 7 mm, while E. coli and S. typhi remain uninhibited. At 2.5 mg/mL, pyocyanin inhibits all four bacteria, with about 10.5 mm for B. subtilis, 9.5 mm for S. aureus, and about 6.5 mm for both E. coli and S. typhi. At 5.0 mg/mL, pyocyanin produces the largest zones among its doses, about 19.2 mm for B. subtilis, 18.0 mm for S. aureus, 10.5 mm for E. coli, and 11.3 mm for S. typhi. Pyoverdine shows no inhibition at 0.5 or 1.0 mg/mL for any organism, inhibits only the Gram-positive bacteria at 2.5 mg/mL with 8.0 mm for both B. subtilis and S. aureus, and increases at 5.0 mg/mL to about 18.0 mm and 17.8 mm respectively, with no inhibition reported for E. coli or S. typhi at any dose. The positive control yields larger zones for all organisms, roughly 27 to 28 mm for the Gram-positive bacteria and about 22 to 23 mm for the Gram-negative bacteria, while the negative control shows no inhibition. Dashes indicate no detectable inhibition, and comparisons should consider that values are reported as means with small variability.
Tetracycline solution served as the positive control, while 10% DMSO was used as the negative control. ‘-’ indicates no zone of inhibition.
Cytotoxicity assessment using MTT assay
Cytotoxicity assays for the extracted pigments – pyocyanin (Y-1) from P. aeruginosa strain BDIFST240011 and pyoverdine (Y-6) from M. hominis strain BDIFST240012 – were performed using the standard MTT colorimetric assay. The results, presented in Table 4, demonstrated a clear dose-dependent reduction in cell viability, indicating that higher pigment concentrations induced greater cytotoxic effects.
MTT assay of different concentrations of pigments (pyocyanin [Y-1] derived from P. aeruginosa strain BDIFST240011 and pyoverdine [Y-6] from M. hominis strain BDIFST240012, respectively)
Table 4 Long description
MTT assay results report Vero cell absorbance at 570 nm (original and duplicate) and the resulting percent survival for two pigments across four concentrations, compared with an untreated control. The control absorbance was 1.712 and 1.738, set to 100 percent survival. For Y-1 (pyocyanin), survival decreased with increasing concentration: 90.23 percent at 5.625 micrograms per ml, 83.53 percent at 11.25, 75.53 percent at 22.5, and 62.14 percent at 45, with absorbance dropping from about 1.54 to about 1.07. For Y-6 (pyoverdine), survival stayed relatively stable in the mid 60 percent range: 64.52 percent at 5.625, 65.62 percent at 11.25, 63.76 percent at 22.5, and 68.86 percent at 45, with absorbance values around 1.04 to 1.31. At 45 micrograms per ml, pyocyanin showed lower survival than pyoverdine (about 62 percent versus about 69 percent). Remarks indicate the LD50 for both pigments is more than 45 micrograms per ml, so the half-lethal dose was not reached within the tested range. Duplicate absorbance readings are provided for each condition and show some variability, especially for pyoverdine at the highest concentration.
For pyocyanin (Y-1), the lowest tested concentration (5.625 μg/mL) resulted in a cell survival rate of 90.23%, whereas the highest concentration (45 μg/mL) reduced viability to 62.14%. The calculated LD₅₀ value was estimated to be greater than 45 μg/mL, suggesting moderate cytotoxic potential at higher concentrations. In contrast, pyoverdine (Y-6) exhibited a less pronounced cytotoxic response, with cell viability ranging from 64.52% at 5.625 μg/mL to 68.86% at 45 μg/mL, indicating a relatively mild effect on cellular metabolism compared to pyocyanin.
In terms of pigment production yield, pyocyanin (Y-1) demonstrated a higher extraction yield of 18.6 ± 0.9 mg/L, whereas pyoverdine (Y-6) yielded 12.4 ± 0.7 mg/L under optimized culture conditions. This difference in yield corresponds to the observed variations in cytotoxic potency, as higher pigment concentrations of pyocyanin correlated with stronger inhibitory effects in vitro. These findings suggest that while both pigments exhibit measurable cytotoxicity, their biological impact differs depending on molecular structure, pigment concentration, and exposure dose.
Overall, the results indicate that both marine-derived pigments possess bioactive properties with distinct cytotoxic profiles, and their selective biological activities warrant further investigation for potential pharmaceutical and antimicrobial applications following detailed toxicity threshold assessments.
Discussion
The present study investigated the abundance of culturable marine bacteria, the characterization of pigment-producing isolates, and the antibacterial potential of pigments obtained from P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012 isolated from coastal sediments of Cox’s Bazar, Bangladesh. The findings demonstrate that marine sediments from the Bay of Bengal harbour diverse pigment-producing bacteria with promising antibacterial properties.
The sediment samples exhibited pH values ranging from 7.10 to 7.80, indicating slightly alkaline conditions that are generally favourable for the growth of many marine bacterial communities. Variations in bacterial abundance among sampling sites likely reflect differences in environmental factors such as salinity, nutrient availability, organic matter content, and local ecological conditions. Similar spatial heterogeneity in microbial community composition and functional potential has been reported along the Bay of Bengal coast (Akter et al., Reference Akter, Rahman, Ali, Minch, Mehzabin, Siddique, Galib, Yesmin, Azmuda and Adnan2023). The observed aerobic heterotrophic bacterial counts (10⁶ CFU/mL range) further support the high microbial productivity of coastal marine ecosystems, which are recognized as important reservoirs of metabolically diverse microorganisms capable of producing bioactive secondary metabolites (Zobell, Reference Zobell1941; Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020).
Both isolates exhibited enhanced growth in LB medium supplemented with seawater compared with standard LB medium, indicating adaptation to saline conditions. Marine bacteria commonly require or tolerate elevated salt concentrations, and salinity has been reported to influence both microbial growth and secondary metabolite production, including pigment biosynthesis (Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020; Nawaz et al., Reference Nawaz, Chaudhary, Shah, Dufossé, Fouillaud, Mukhtar and Haq2021). The increased pigment production observed under seawater-supplemented conditions suggests that environmental stress and osmotic adaptation may stimulate pigment synthesis, a phenomenon previously documented in marine pigment-producing bacteria (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008; Padhan et al., Reference Padhan, Poddar, Sarkar and Sarkar2021).
Morphological, biochemical, and molecular characterization confirmed the identities of the isolates as P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012. The high 16S rRNA gene sequence similarities with reference strains support the reliability of molecular identification methods for pigment-producing bacteria. Similar approaches have been successfully employed for the identification of environmental pigment-producing bacterial isolates from diverse ecological niches (Anzum et al., Reference Anzum, Khan and Hossain2022; Jaber et al., Reference Jaber, Majeed and Al-Hashimi2022). Furthermore, phylogenetic analyses demonstrated the taxonomic placement of the isolates within their respective genera, supporting their potential as sources of biologically active pigments.
Optimization studies revealed that both isolates exhibited maximum growth and pigment production under saline conditions, with optimal growth occurring at moderate temperature and slightly acidic to neutral pH values. Such adaptability is characteristic of marine microorganisms, which possess physiological mechanisms that enable survival under fluctuating environmental conditions while maintaining active secondary metabolism (Velmurugan et al., Reference Velmurugan, Venil, Veera and Dufossé2020; Nawaz et al., Reference Nawaz, Chaudhary, Shah, Dufossé, Fouillaud, Mukhtar and Haq2021). The ability of marine bacteria to produce pigments under diverse environmental conditions enhances their potential for future biotechnological applications.
Spectroscopic characterization provided important information regarding the chemical nature of the extracted pigments. UV-visible spectroscopy revealed characteristic absorption profiles, while FTIR analysis identified functional groups including hydroxyl, carbonyl, and aliphatic moieties commonly associated with microbial pigments. Similar spectroscopic approaches have been widely used for the characterization of bacterial pigments and bioactive metabolites (Samrot et al., Reference Samrot, Rio, Kumar and Samanvitha2017; Dawoud et al., Reference Dawoud, Alharbi, Theruvinthalakal, Thekkangil, Kadaikunnan, Khaled, Almanaa, Sankar, Innasimuthu, Alanzi and Rajaram2020). Although spectroscopic analyses provide valuable preliminary evidence regarding pigment composition, further structural elucidation using advanced analytical techniques such as High-Performance Liquid Chromatography (HPLC), Liquid Chromatography–Mass Spectrometry (LC–MS), or Nuclear Magnetic Resonance (NMR) would be necessary to definitively identify the pigment molecules.
The antibacterial assays demonstrated that the extracted pigments possessed inhibitory activity against several tested bacterial pathogens. Notably, the pigment obtained from P. aeruginosa exhibited stronger antibacterial activity than that produced by M. hominis, particularly against B. cereus. The observed antibacterial effects are consistent with previous reports indicating that pigments produced by marine and environmental bacteria can interfere with bacterial growth through mechanisms involving oxidative stress induction, membrane disruption, and metabolic inhibition (Samanta et al., Reference Samanta, Thavasi and Jayalakshmi2008; Ramesh et al., Reference Ramesh, Vinithkumar and Kirubagaran2019; Chaturwedi et al., Reference Chaturwedi, Mainali and Chaudhary2024). Furthermore, the positive relationship between pigment concentration and inhibition-zone diameter suggests a concentration-dependent antibacterial effect, which has also been reported for other microbial pigments (Subi, Reference Subi2022).
The cytotoxicity assessment using the MTT assay demonstrated a dose-dependent reduction in cell viability following pigment exposure. The pigment derived from P. aeruginosa exhibited comparatively greater cytotoxicity than that obtained from M. hominis. These findings are consistent with previous studies showing that pigments produced by Pseudomonas species may exert biological effects on eukaryotic cells through redox-mediated mechanisms and interactions with cellular metabolic pathways (Koyun et al., Reference Koyun, Sirin, Erdem and Aslim2022). Although the observed LD₅₀ values suggest moderate toxicity within the tested concentration range, comprehensive toxicological investigations will be necessary before considering therapeutic applications.
Overall, the present study highlights the biotechnological potential of pigment-producing marine bacteria isolated from the Bay of Bengal. The antibacterial activities exhibited by pigments from P. aeruginosa and M. hominis support their potential as sources of novel natural antimicrobial compounds. These findings are consistent with increasing evidence that marine microorganisms represent valuable reservoirs of bioactive pigments with pharmaceutical and industrial significance (Venil et al., Reference Venil, Dufossé and Renuka2020; Kumar et al., Reference Kumar, Kumar, Ambika, Kumar, Darnal, Thakur, Patial and Singh2022; Flora et al., Reference Flora, Afrin, Jui, Bhowmik, Bhuiyan, Rashid, Khatun, Islam, Hossain, Bhuiyan and Saha2026). Future studies should focus on detailed structural characterization of the pigments, elucidation of their mechanisms of antibacterial action, assessment of toxicity and safety profiles, and evaluation of their efficacy under in vivo conditions to facilitate potential biomedical and biotechnological applications.
Conclusion
This study successfully isolated and characterized two yellow pigment-producing bacterial strains, P. aeruginosa strain BDIFST240011 and M. hominis strain BDIFST240012, from sediment samples collected from Cox’s Bazar, Bangladesh. The bacterial isolates exhibited optimal growth and pigment production in LB medium supplemented with seawater, with the highest yields observed at 37°C and pH 5.7–6.0. The identification of the pigments pyocyanin and pyoverdine was confirmed through UV-visible spectroscopy and FTIR analysis, revealing characteristic absorption peaks and functional groups associated with carotenoid compounds. The antibacterial evaluation of pyocyanin and pyoverdine demonstrated their potential as natural antimicrobial agents, with pyocyanin showing significant inhibitory activity, particularly against B. subtilis. The observed differences in antibacterial activities between the two pigments could be attributed to their distinct chemical structures, which affect their interaction with bacterial cell membranes and metabolic processes. The positive correlation between pigment concentration and zone of inhibition further highlighted the efficacy of higher concentrations of both pigments in enhancing antibacterial activity. Additionally, the MTT assay confirmed the cytotoxic effects of both pigments, with pyocyanin exhibiting more pronounced cytotoxicity compared to pyoverdine. The dose-dependent reduction in cell viability for both pigments suggests their potential for therapeutic applications, although further studies are needed to fully assess their clinical safety and efficacy. This research underscores the importance of marine-derived bacterial pigments as promising sources of bioactive compounds. The distinct properties of pyocyanin and pyoverdine emphasize the value of exploring marine microorganisms for novel, natural alternatives to synthetic antibiotics and therapeutic agents. Future investigations should focus on elucidating the molecular mechanisms underlying the antibacterial and cytotoxic effects of these pigments, as well as exploring their broader applications in biotechnology, pharmacology, and medicine. The findings contribute to the growing body of knowledge regarding the antimicrobial and therapeutic potentials of marine-derived natural products.
Acknowledgements
We would like to express our gratitude to BCSIR for its support throughout the course of this project. During the preparation and revision of this manuscript, the authors used ChatGPT for the purpose of English language revision. The authors have reviewed and edited the output and take full responsibility for the content of this publication.
Author contributions
A.F. and S.A. conducted the experiments, analysed the data, interpreted the findings, and wrote the article. M.N.I.B. contributed to the conceptualization, data analysis, interpretation of findings, and writing of the article. M.L.S. was involved in the review and editing of the manuscript, as well as critical revisions.
Declaration of interest
None.
Data availability statement
All data are available upon request.
Ethics and approach
Not applicable.
Funding statement
This work was supported by the Research and Development (R and D) programme of the 2022–2023 Bangladesh Council of Scientific and Industrial Research (BCSIR).
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