Research Article

Polyhydroxyalkanoates Biopolymer Production by Moderately Halophilic Paracoccus onubensis Strain E3: Extraction, Characterization and Synergistic Activity with Sorafenib Drug against Hepatocellular Carcinoma through Molecular Docking Approach

Hend A. Hamedo, Ahmed E.M. Shokr, Omnia T. Abd-Elsalamand Naglaa Elshafey*

Botany and Microbiology Department, Faculty of Science, Arish University, Al-Arish 45511, Egypt.

Received | December 01, 2024; Revised: December 25, 2024; Accepted | January 09, 2025; Published | January 27, 2025

*Correspondence | Naglaa Elshafey, Botany and Microbiology Department, Faculty of Science, Arish University, Al-Arish 45511, Egypt; Email: [email protected]

Citation | Hamedo, H.A., A.E.M. Shokr, O.T. Abd-Elsalam and N. Elshafey. 2025. Polyhydroxyalkanoates biopolymer production by moderately halophilic Paracoccus onubensis strain E3: extraction, characterization and synergistic activity with sorafenib drug against hepatocellular carcinoma through molecular docking approach. Novel Research in Microbiology Journal, 9(1): 13-27.

DOI | https://dx.doi.org/10.17582/journal.NRMJ/2025/9.1.13.27

Keywords | Polyhydroxyalkanoates, Paracoccus onubensis, Solar saltern, Molecular docking, Hepatocellular carcinoma

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Polyhydroxyalkanoates (PHAs) are bio-polyesters mainly synthesized by prokaryotic microorganisms such as bacteria, which are 0.2–0.5 mm water-insoluble particles found in the bacterial cells or their cytoplasm (Aljuraifani et al., 2019). PHAs accumulate intracellularly in microorganisms as energy storage granules with physicochemical characteristics that resemble those of petrochemical polymers (Mukherjee and Koller, 2023). Polyhydroxybutyrate is the most prevalent type of PHAs, noted for its exceptional optical properties and significant UV resistance, which contribute to its strong market presence (Kumar et al., 2023). This biopolymer possesses several properties, including high crystallinity, biodegradability, and resistance to ultraviolet radiation. Due to these characteristics, this material is utilized in the packaging of food and pharmaceuticals (Grgurević et al., 2024). Various bacterial strains, spanning more than 90 taxa, including Bacillus, Burkholderia, Cupriavidus, and Halomonas sp. have been systematically investigated for their capacity to synthesize short-chain-length polyhydroxyalkanoates (scl-PHAs) (Yoo et al., 2024). Among them, the Halomonas mediterranei is a notable bacterial species for its significant production of bioplastics, because of its quick development, adaptability in metabolism, genetic stability, and effective transformation mechanisms (Martínez et al., 2022; Yoo et al., 2024). Furthermore, agricultural food waste serves as a substitute substrate for biopolymers production, utilizing Haloferax mediterranei, a well-studied strain recognized for its capacity to produce polyhydroxybutyrate (PHB). This strain can prosper and produce bioplastics in high-salinity environments without the need for sterilization (Longo et al., 2024). Synthesis of polyhydroxyalkanoates (PHAs) by halophilic bacteria especially those that can tolerate harsh salt conditions, has several distinct benefits. These include the need for high salinity, which inherently lowers the danger of microbial contamination, capacity to use a wide variety of inexpensive substrates, and an increased intracellular osmotic pressure, which facilitates cell lysis for PHAs extraction (Możejko-Ciesielska et al., 2023). Halophiles possess distinctive adaptive characteristics that enable their survival under highly salt conditions; they have attracted considerable attention due to their remarkable adaptability and potential applications in biotechnology such as enzymes and bioplastics production (Gallo and Aulitto, 2024). But for industrial purposes, the microbial strains should satisfy the following requirements: (i) grow rapidly in cheap carbon sources, (ii) accumulate high PHBs content inside their cells with large size to ease of separation, (iii) have high efficiency for substrate transformation, and (iv) be harmless to animals, humans, and the environment (Chaudhary et al., 2024). Therefore, there is still an ongoing need to identify a variety of halophilic microorganisms with unique characteristics that can produce high-yield PHA’s biopolymers with an ease of extraction. These unique and distinctive polymers have been used in several fields, including medical (i.e., medical materials and drug carriers), agriculture, and packaging (Das et al., 2023; Satchanska et al., 2024). Moreover, these polymers exhibit exceptional physicochemical features, making them highly favorable for drug delivery systems. HCC is a major type of primary liver cancer, responsible for approximately 75–85 % of mortality cases (Massarweh and El-Serag, 2017). Traditional cancer treatments such as surgery, chemotherapy, and radiation are effective but frequently result in non-specific side effects (Fahmy et al., 2024). Sorafenib (SFB) serves as a first-line targeted therapy for patients with advanced HCC, expressing both anti-angiogenic and anti-proliferative effects on the tumor cells. The efficacy of SFB is limited by its off-target distribution, rapid metabolism, and multi-drug resistance. Recent studies have shown that tiny molecules composed of various materials can improve the targeting and therapeutic effectiveness of SFB in its treatment of HCC (Fan et al., 2024). The current state of clinically viable therapy options for liver illnesses is suboptimal, highlighting the urgent need to investigate innovative medications and potential therapeutic approaches to address the existing limitations (Zhu et al., 2023). Due to limitations of the single-drug chemotherapy, organizations have focused on developing drug delivery systems that simultaneously administer various combinations of therapeutic agents (Sari et al., 2024). Combination of these considerations renders the halophilic bacteria as a highly attractive cellular facility for the synthesis of PHAs, therefore providing an environmentally sustainable and economically viable approach to PHAs production (Yoo et al., 2024). Here, the objective of the current study was to identify the promising PHAs-producing moderate halophilic bacterial strain isolated from North Sinai solar saltern, achieving the industrial factors for microbial satisfaction. This study reports the highest yield of PHAs-biopolymer produced under optimized conditions, using cheap carbon and nitrogen sources, and was extracted easily from the moderate halophilic bacterial cells. Moreover, the chemical structures of PHAs indicate their potential against HCC, suggesting their utility as a promising drug delivery system capable of targeting specific sites within the body. Furthermore, an in-silico method suggested that PHA components could lessen the sorafenib’s adverse effects while increasing its anticancer effectiveness.

Materials and Methods

Sampling and site description

Three saline soil samples were collected from a solar saltern in North Sinai, Egypt, at about 10 cm of surface soil layer following a prior study conducted by Shokr et al. (2023). These samples were placed in sterile plastic pages and stored at -4 oC till further use.

Isolation of Polyhydroxyalkanoates producing bacteria and growth conditions

Fifty bacterial isolates were obtained via an isolation process. Briefly, 10 g of a soil sample were mixed with 100 ml of sterile seawater, followed by streak plating 0.1 ml onto Nutrient agar (NA) medium supplemented with 4 % NaCl and enriched with 1 % glucose as a carbon source (Hawas et al., 2016). The plates were incubated at 37 °C for 48 h. Sudan Black B (0.05 %) was applied for 30 min. to all culture plates before being rinsed with ethanol. The promising results were represented by the development of plates that were black or dark blue in color, in reference to Hawas et al. (2016); Mustafa et al. (2020). For successful confirmation, the isolates that exhibited a positive result with Sudan Black B stain were re-stained with alcoholic Nile Blue A stain (1 %), and then the plates were exposed to 365 wavelengths of ultraviolet light (UV). Plates that displayed blue fluorescence expressed a positive result (Sohail et al., 2020).

Biochemical and physiological characterization of the promising PHAs producing bacterial strain

For preliminary identification, the biochemical and physiological characteristics involving several in vitro assays (Pardamean et al., 2021), including Gram stain, shape, endospore formation, motility, catalase production, oxidase, starch, indole acetic acid production, citrate, urease production, glucose fermentation, lactose fermentation, optimum pH, and DNA (G+C) content of the promising bacterial strain that can accumulate PHA granules, were determined according to Pardamean et al. (2021).

Scanning electron microscopy (SEM)

The moderate halophilic bacterial strain was cultured in nutrient broth at 37 ºC on a rotary shaker set to 180 rpm for 48 h to prepare them for scanning electron microscopy (SEM). The obtained bacterial culture was filtered through nucleopore filters, treated with 2.5 % glutaraldehyde for fixation, and rinsed with phosphate buffer. The bacterial culture was sequentially dehydrated using acetone solutions with various concentrations (30 %, 50 %, 70 %, 90 %, and 100 %) and then dried using critical point drying. Ultimately, the samples were affixed to metal stubs and covered with a 96 nm coating of gold. Photos were captured using a Hitachi S-570 scanning electron microscope (Hitachi Ltd., Tokyo, Japan) (Jeyanthi and Velusamy, 2016).

Molecular studies

DNA extraction, amplification, and 16S rRNA gene sequencing of the promising polyhydroxyalkanoates-producing bacterial strain: During the exponential growth phase of the moderate halophilic bacterial isolate, genomic DNA was obtained, according to the manufacturer’s guidelines for Applied Biotechnology Co. Ltd., Egypt. Using the ABT DNA micro-extraction kit, a 16S rRNA gene was extracted utilizing a pair of bacterial universal primers (Invitrogen, USA), mainly forward: 27F (5-AGA GTT TGA TCC TGG CTC AG-3), reverse: 1492R (5-GGT TAC CTT GTT ACG ACT T-3) (Shokr et al., 2023). The polymerase chain reaction (PCR) was performed under specified conditions utilizing a 50 µl reaction volume, including 30 cycles consisting of a 5 min. pre-denaturation at 95 °C, 1 min. denaturation at 94 °C, 1 min. annealing at 60 °C, 1 min. and 30 sec of extension at 72 °C, a final extension at 72 °C for 10 min., and a final hold at 4 °C for 10 min. The samples were prepared following the instructions provided by Macro Gen Company (South Korea) with a concentration of 50 ng/ l for each PCR product.

To conduct the phylogenetic analysis, similar 16S rRNA gene sequences were retrieved from the NCBI database. The multiple sequence alignment tool CLUSTAL-W was utilized to align a partial sequence. The MEGA X version 11 software package was then used to build a neighbor-joining tree (Tamura et al., 2021).

Optimization of the growth conditions for the polyhydroxyalkanoates-producing bacterial strain

Approximately 100 µl of bacterial culture in the exponential growth phase were added to 100 ml of mineral salt medium (MSM) broth, composed of the following components per liter of dist. water: K2HPO4 1.73 g, KH2PO4 0.68 g, MgSO4·7H2O 0.1 g, FeSO4·7H2O 0.03 g, NH4NO3 1.0 g, CaCl2·2H2O 0.02 g, and NaCl 4.0 g, with a final pH of 7.0 (Schlegel et al., 1961). The broth was supplemented with 20 g/ l glucose as a carbon source, as suggested by Aramvash et al. (2015). Afterward, the broth culture was incubated on a rotary shaker (37 °C/ 150 rpm). The effect of various growth factors on bacterial PHA production was tested. Accordingly, different pH ranges (6.5–7.5) in constant glucose and peptone as carbon and nitrogen sources, respectively, for varying periods (48 h - 96 h), were investigated to choose the optimum conditions for bacterial growth and production of PHAs. Cellular growth was measured using a Spectrophotometer (JENWAY 6300, United Kingdom) set to 600 nm. All assays were performed in triplicate.

Extraction of polyhydroxyalkanoates biopolymer from moderately halophilic Paracoccus onubensis strain E3

To extract the polyhydroxyalkanoates polymer, 10 ml of a bacterial incubated broth culture (at the optimum growth condition) were centrifuged at 10,000 rpm for 15 min. The supernatant was disposed of and the pellet was dissolved in 10 ml sodium hypochlorite, followed by incubation at 30 °C. After 2 h of incubation, the produced mixture was centrifuged at 5000 rpm for 15 min., and then washed with equal volumes of dist. water, acetone, and methanol, respectively. To obtain dry weight of the extracted polymer, the pellets were dissolved in 5 ml of hot chloroform, followed by evaporation through transferring the fluid onto a sterilized glass pan at 4 °C and then weighed. For dry-cell bio-mass measurement, 10 ml of the culture were centrifuged at 10,000 rpm for 15 min., and the obtained pellets were dried in an oven at 55 °C to a constant weight. Residual biomass (g/ l) was calculated as the difference between dry cell weight and dry weight of extracted PHBs (Hawas et al., 2016).

Characterization of polyhydroxyalkanoates biopolymer

Fourier transform infrared spectroscopy (FTIR) analysis: The PHAs product was characterized by using the FTIR spectrometer (Thermo Scientific Nicolet iS50, US) to identify its functional groups. Briefly, 1 mg of PHAs was finely mixed with 10 mg of spectral pure anhydrous potassium bromide crystal, and the powder was turned into a pellet. The transmitted light intensity was measured using the FT-IR spectrophotometer in relation to the absorption wavelength of 4000-400 cm-1 (Getachew and Woldesenbet, 2016).

Gas chromatography-mass spectrometry (GC-MS) analysis: Gas chromatography-mass spectrometry (GC-MS) analysis was performed on the extracted PHAs polymer following the technique provided by Ojha and Das (2017). The concentration of PHAs in each sample was quantified using a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) that was fitted with a direct capillary column TG-5MS (30 mm × 0.25 mm × 0.25 µm film thickness). The column was placed in an oven originally established at a temperature of 50 °C and subsequently elevated at a rate of 5 °C/ min. until it attained a target temperature of 230 °C. The column was maintained at this temperature for 2 min. and then increased to a final temperature of 290 °C at a speed of 30 °C/ min. The final temperature was maintained for 2 min. The injector and MS transfer lines were kept at 250 oC and 260 oC, respectively. The carrier gas used was helium, which was maintained at a constant flow rate of 1 ml/ min. Approximately 3 min. solvent delay was implemented, and 1 µl diluted sample was automatically injected using an usingan AS1300 auto sampler that was connected to the gas chromatography (GC) in a split mode. The mass spectra via electron ionization (EI) were obtained using an ionization voltage of 70 eV. The spectra were collected in the range of 40–1000 m/z in a full-scanned mode, and the ion source temperature was maintained at 200 °C. The components were identified by comparing retention time and mass spectrum with WILEY 09 and NIST 11 mass spectrum databases.

Forward reaction prediction of the synergistic combination between polyhydroxyalkanoates and sorafenib drug against hepatocellular carcinoma (HCC)

A method for predicting the forward reactions was employed to investigate the various combined reactions between the extracted PHA compounds and the sorafenib drug. The ASKCOS MIT system (Sankaranarayanan and Jensen, 2023) was used to predict all potential subsequent reactions at https://askcos.mit.edu/ accessed on 27 November 2024, and the procedure utilized all the previously generated files in SDF format for the reaction.

Molecular docking of polyhydroxyalkanoates ingredients and predicted compounds against hepatocellular carcinoma (HCC)

The crystalline structures of Ornithine Am receptors (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (BCF 3) were obtained from the protein data bank (PDB) with ID 7JX9 (Butrin et al., 2020). Protein receptors were prepared into the auto dock tool 1.5.7 for prediction of the missing atoms, removal of water molecules, protonation and charge addition, and active site and pocket prediction. Subsequently, ten PHA ligand ingredients were obtained from the PubChem database and the predicted compound resulting from the synergistic combination between PHAs and sorafenib drug was prepared for docking analysis using Avogadro 1.2.0 software. The 2D structure of ligand complexes was modeled using BIOVIA Discovery Studio 2021. Molecular docking was conducted using AutoDockTools_1.5.7_vina docking approach. The drug-likeness of PHAs compounds was then assessed using Lipinski’s rule of five (Elshafey et al., 2025).

Statistical analysis

Statistical analysis of the obtained data was performed using the SPSS software (IBM Corporation, New York, USA). A One-Way Analysis of Variance (ANOVA) test was applied to analyze the data statistically, following the mathematical principles described by Snedecor and Cochran (1967), followed by post hoc comparisons using Duncan’s multiple-range tests (Duncan, 1955).

Results

Screening and selection of the potent polyhydroxyalkanoates producing bacterial strain

Fifty bacterial isolates were obtained from a culture-based method of salty soil on NA with glucose as a carbon source. Sudan black-B and Nile blue-A dyeing approaches were used to screen for bacteria that produce PHAs. Results showed that Paracoccus onubensis strain E3 could accumulate PHAs by using Sudan Black-B dye, followed by a microscopic examination that showed an accumulation of black granules within the bacterial cells indicating positive synthesis of PHAs biopolymer. In addition, the plates stained with Nile blue-A were examined under UV light (365 wavelengths) to confirm the accumulation of PHAs.

Biochemical characterization and molecular identification of Paracoccus onubensis stain E3

Paracoccus onubensis stain E3 was Gram-negative, rod-shaped, non-motile, and had the ability to hydrolysis starch and glucose as examined in our previous study conducted by Shoker et al. (2023). Strain E3 exhibited 99.24 % similarity with Paracoccus onubensis and its gene sequence was deposited in GenBank under the accession number OL891886. A phylogenetic tree was generated demonstrating a correlation between moderate halophilic P. onubensis strain E and other bacterial species (Figure 1).

 

Scanning electron microscopy (SEM)

Scanning electron microscopy analysis confirmed the rod-shaped cells of P. onubensis that occurred singly or in pairs. The scale bars indicated the length of bacterial cell as 1µm (Figure 2).

 

Factors affecting Paracoccus onubensis strain E3 growth rate and polyhydroxyalkanoates production

Constantly, carbon sources such as glucose, nitrogen sources such as peptone, and varying pH and time factors were studied to evaluate the best growth rate of P.s onubensis strain E3 and the accumulation of PHAs. The results indicated a positive correlation between bacterial biomass and PHAs biosynthesis. After 72 h of bacterial incubation, the maximum growth rate and PHA production (54.77 mg/l) was attained (Figure 3a). At various tested pH levels, the highest PHAs accumulation (45.52 mg/ l) was obtained at pH 7, which was dramatically reduced after and before this pH value (Figure 3b).

 

Chemical characterization of polyhydroxyalkanoate polymer

Fourier transform infrared spectroscopy (FTIR): The PHAs extracted from moderate halophilic P. onubensis strain E3 (Figure 4a) was analyzed via FTIR to determine the main functional categories present in it. The spectrum of the PHAs showed a strong band at 1649.72 cm−1 for the carbon (C=O) stretching of the ester group, which is a common feature in PHA’s structures. The peak at 3434.05 cm−1 indicated a strong stretching H bond created by the terminal group (-OH). A band at 2923.55 cm−1 was assigned to (C–H) methylene and methyl groups, respectively. Peaks at 1494.58 and 1434.79 cm−1 were related to (-CH2). The FTIR spectrum also peaked at 1230 cm−1 corresponding to the (C–O) group (Figure 4b).

 

Gas chromatography-mass spectrometry (GC–MS): The PHAs polymer produced from moderate halophilic P. onubensisstrain E3 was analyzed using GC-MS to determine its composition. The PHAs was transformed into volatile hydroxycarboxylic acid methyl esters using acidic methanolysis. The distinct peaks identified in the mass spectra aided in determining the carbonyl and hydroxyl terminations of the corresponding PHAs (Table 1).

Molecular docking analysis of polyhydroxyalkanoates against hepatocellular carcinoma (HCC)

Polyhydroxyalkanoates compound extracted from moderate halophilic P. onubensis strain E showed that all its ten ingredients could adhere to Lipinski’s rule, although the six compounds chosen for molecular docking research against the HCC protein were the most effective according to their binding affinity with protein (Figure 5). The six compounds binding affinity scores to the HCC protein ranged from -4.0 to -6.5 kcal/ mol (Table 2). Several types of chemical ligand-receptor interactions can be studied in Discovery Studio binding to receptors. By analyzing these interactions, researchers can gain insight into the mechanisms of drugs action and design new drugs with improved potency and specificity (Figure 5).

 

Table 1: Chemical composition of the biodegradable polyhydroxyalkanoates biopolymer produced by moderate halophilic Paracoccus onubensis strain E3 using GC-MS analysis.

RT	Compound	Area %	Chemical formula	Molecular weight
13.17	Triacetin	1.93	C9H14O6	218
17.31	2,4-di-tert-butylphenol	11.19	C14H22O	206
18.82	Spathulenol	2.32	C15H24O	220
24.24	2-pentyl6-(4-pentylphenyl)2,6-naphthalendicapoxylate	0.93	C28H32O4	432
26.27	Hexadecenoic acid, methylester	4.38	C17H34O2	270
29.41	9,12-Octadecadienoic acid-methyl ester	2.72	C19H34O2	294
30.06	Heptadecanoic acid, 16-methyl-, methyl ester	3.43	C19H38O2	298
32.21	Isopropyl linoleate	6.84	C21H38O2	322
35.01	9,12-Octadecadienoic acid,2-hydroxy-1-(hydroxymethyl)ethyl ester	0.94	C21H38O4	354
36.73	Isopropyl linoleate	1.46	C21H38O2	322

Where; RT: Retention time of the compounds based on GC–MS peaks. The compounds are listed in order of their elution from a TG5MS column.

 

Table 2: Molecular docking analysis of polyhydroxyalkanoates biopolymer ingredients from Paracoccus onubensis strain E3 against hepatocellular carcinoma (HCC) protein.

No.	Compound name	Chemical formula	Binding-energy (Kcal/ mol)
1	9,12-Octadecadienoic acid (z,z) methyl ester	C19H34O2	-4.9
2	Heptadecanoic acid, 16-methyl-, methyl ester	C19H38O2	-4.0
3	Hexadecenoic, methyl ester	C17H34O2	-4.7
4	Isopropyl linoleate	C21H38O2	-4.7
5	Spathulenol	C15H24O	-6.5
6	Triacetin	C9H14O6	-5.5
Drug	Sorafenib	C21H16ClF3N4O3	-8.9

 

Table 3: Molecular docking analysis of the predicted compounds resulting from the synergistic combination between extracted polyhydroxyalkanoates (PHAs) biopolymer and sorafenib drug.

Forward reaction prediction of compounds	Target protien Hepatocellular carcinoma (HCC)	Binding energy (Kcal/ mol)
Predicted compound from 9,12-Octadecadienoic acid (z,z) methyl ester and sorafenib drug	Ornithine Am receptors (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic Acid (BCF3)	20.2
Predicted compound from Isopropyl linoleate and sorafenib drug		21.9
Predicted compound from-Spathulenol and sorafenib drug		23.2
Predicted compound between Triacetin and sorafenib drug interaction		-4.9

 

Forward reaction prediction of the synergistic combination between polyhydroxyalkanoates and sorafenib drug against hepatocellular carcinoma (HCC): In this investigation, in silico analysis of the predicted compounds derived from the synergistic combination between PHAs and sorafenib drug displayed the potential to inhibit HCC. The binding energies between predicted compounds and amino acids of receptor proteins linked to HCC are shown in Table 3, while predicted compounds resulting from combination between 9,12-Octadecadienoic acid (z,z) methyl ester and sorafenib drug showed high binding energy with receptor protein 20.2 Kcal/ mol. This study identified conventional hydrogen bonds with GLY A:325, CYS A:330, and ASN A:326 residues, indicating that unfavorable interactions may play a significant role in HCC treatment, involving various amino acids such as META A:139, TYR A:323, THR A:109, TYR A:299, and ASN A:140 (Figure 6a). 

Additionally, alkyl and pi-alkyl bonds were observed with PRO A:138, ALA A:333, TYR A:123, VAL A:301, LEU A:110, and LEU A:328 (Figure 6a). 

 

Moreover, a predicted compound resulting from the combination between Isopropyl linoleate and sorafenib demonstrated a high binding affinity of 21.9 Kcal/ mol, exhibiting carbon-hydrogen and conventional hydrogen bonds with amino acids GLY A:329 and CYS A:330. Nevertheless, unfavorable interactions were noted with PRO A:300, ASN A:140, MET A:139, ASN A:326, TYR A:323, and TYR A:299, which may contribute to the treatment of the HCC (Figure 6b). Furthermore, the predicted compounds arising from combination between Spathulenol and sorafenib drug displayed higher binding affinity of 23.2 Kcal/ mol with HCC protein, due to the presence of unfavorable pump in amino acids MET A:139, VAL A:301, ASN A:326, and CYS A:330 (Figure 6c). Meanwhile, the predicted compounds arising from combination between Triacetin and sorafenib drug showed binding affinity of -4.9 Kcal/ mol with HCC protein, due to the presence of unfavorable pump in amino acids MET A:139 and conventional hydrogen bond with amino acids GLY:325, ASN A:326, and CYS A:330 (Figure 6d).

 

Discussion

Biodegradable bio-based polymers sourced from various biomasses, including plant, animal, marine bacteria, or forestry materials demonstrate potential in substituting the traditional petrochemical polymers. Extensive research and development efforts have been undertaken for decades regarding potential biodegradable bio based polylactic acid (PLA), polyhydroxyalkanoates (PHAs), and succinate polymers (Jha et al., 2024). In the current study, the promising bacterial strain was identified as P. onubensis strain E by analyzing the 16S RNA gene, which displayed the formation of PHA’s granules. This species is a moderately halophilic bacterium within the family Rhodobacteraceae that exists in marine habitats, encouraging the use of a variety of microorganisms that produce biopolymers and can synthesize specific genes and enzymes (Gutierrez-Patricio et al., 2021).The obtained results of this study aligns with earlier researches conducted by Bordel et al. (2021), which studied the production of PHB granules by P. denitrificans strain Pd1222 using laser scanning confocal microscopy and gas chromatography analysis. Optimal microbial growth conditions may improve and sustain the balanced metabolic response within bacterial cells; hence, microbial products and biomass might be augmented, as supported by Sasidharan et al. (2015); Mohandas et al. (2017). The current results showed that maximum production of PHAs by P. onubensis strain E3 was achieved after 72 h of incubation; however, extending the fermentation period reduced PHAs production. This may be attributed to consumption of the carbon source, resulting in exhaustion of PHAs or decaying of the enzyme activity responsible for PHAs biosynthesis in line with the results reported by Thapa et al. (2018). In addition, the obtained results revealed that the optimum pH for PHAs biosynthesis at natural conditions was pH 7 at 37 oc. in agreement with Sohail et al. (2020), who reported that the highest PHBs production and biomass of Pseudodonghicola xiamenensis was obtained at 35 °C, while the synthesis of PHB by Streptomyces thermophilus using molasses was (0.701 g/ l) at 50 °C (Sasidharan et al., 2015). In this investigation, P. onubensis strain E3 displayed considerable growth adaptability in seawater (NaCl: 4 %) Similarly, Kawata and Aiba (2010) proved that Halomonas sp. accumulated PHA at NaCl requirements (3–15 %) for its optimal growth. Furthermore, Bordel et al. (2021) found that increasing the salt concentration to a higher degree than that of the optimal value decreased P. denitrificans growth and PHB accumulation due to high osmotic stress. Additionally, Yoo et al. (2023) showed that Halomonas getboli accumulates ions and solutes inside the cell to adapt to high salinity environments. Similarly, changes in Halomonas strains brought on by UV irradiation have been shown to promote intracellular solute accumulation (Wang et al., 2023).

In the present study, P. onubensis strain E3 utilized cheap and available carbon source such as glucose, although the carbon source is a significant cost component for the production of PHAs in consistency with Akkoyunlu et al. (2024), who reported that different carbon sources from economic materials such as fructose, formic acid, and carbon dioxide (CO2) were used for industrial scale PHAs production process. On the other hand, Halomonas sp. KM-1 was reported to produce PHAs using glycerol as the sole carbon source (Hagagy et al., 2021). The previous study conducted by Ben-Abdallah et al. (2024) reported the generation of PHBs by several types of halophilic bacteria, mainly Haloarcula marismortui, H. tradensis, and H. quadrata utilizing starch as a carbon source. The primary factor limiting PHAs production is the nitrogen source. According to Sharma et al. (2017), the bacterial strains need a high concentration of nitrogen, as the nitrogen supply decreases, the bacteria begin to producing the polymer as a survival strategy. Current results demonstrated that P. onubensis strain E3 had the maximum growth rate on using peptone as a nitrogen source. These results agree with the findings previously reported by Muigano et al. (2024), who reported that an alkaliphilic and moderately halophilic bacterium H. alkalicola has the ability to accumulate PHAs on using peptone, which was the best nitrogen source that enhanced the growth rate and the accumulation of PHAs within the bacterial cells.

The results of FTIR and GC-MASS analysis in this study agree with Muigano et al. (2024), who showed that the range of (C=O) as a carbonyl group is a characteristic feature shared by all PHA’s structures. The presence of a strongly stretching (H) bond formed by the terminal (-OH) group resembles the results revealed by Muigano et al. (2024). In addition, GC-MS analysis was conducted in this study to detect the biodegradable components of the extracted PHAs biopolymer, where the molecular ion peak in the mass spectrum and the elemental analysis agreed well with the molecular formula of the PHAs biopolymer. Meanwhile, the PHAs biodegradable components such as hexadecenoic acid and octadecanoic acid were recorded in the GC-MS report, confirming the presence of PHAs. This result is in accordance with this reported by Zahra et al. (2025), revealing the appearance of other ethyl esters, including pentadecanoic acid, hexadecanoic acid, and octadecanoic acid ethyl ester, indicating the presence of medium-chain length PHAs. On the other hand, the human liver constitutes around 2.5 % of the average body mass, rendering it the most substantial organ in the body. It has a critical function in many essential processes such as the breakdown of proteins, lipids, and carbohydrates, and the removal of harmful substances (Sender et al., 2023). The prevention and treatment of HCC continues to be a worldwide concern, due to the lack of reliable diagnostic biomarkers and therapeutic approaches. Additionally, HCC poses diagnostic challenges owing to the absence or ambiguity of its symptoms (Chan et al., 2024). Despite the use of several treatment strategies such as chemotherapy and radiotherapy, the survival rates of liver cancer patients do not experience substantial enhancements. According to Hai et al. (2021), a wide range of sulfur-containing substances with biological activities has been isolated from different marine environments. These substances include secondary metabolites from moderate and halophilic Halomonas sp., which have shown antibacterial and antitumor activity, in addition to antiviral, anti-inflammatory, and antibiotic properties. Thus, these metabolites become important in the fight against cancer and the development of new drugs to treat it (Mahrosh and Mustafa, 2021). On the other hand, finding the precise location of a ligand molecule within the binding or active site of a target or receptor protein is the primary goal of molecular docking techniques. These approaches are also employed to predict the binding patterns between the ligand and the receptor protein (Hassan et al., 2024). Combination therapies have demonstrated greater efficacy compared to monotherapy in the treatment of various cancer diseases. This advantage has historically been attributed to the capacity of combinations to tackle tumor heterogeneity; however, synergistic interaction has emerged as a prevalent explanation and design criterion for the new combinations (Plana et al., 2022). A significant number of studies have concentrated on the development of drug delivery systems that simultaneously deliver various combinations of therapeutic agents to address the limitations associated with the single-drug chemotherapy (Khafaga et al., 2024; Ullah et al., 2025). These findings support our study which indicated that sorafenib-loaded PHAs compound is more effective than either sorafenib or PHAs compound alone. In the current study, combination of sorafenib and PHAs compound as 9,12-Octadecadienoic acid (z,z) methyl ester, Isopropyl linoleate, Spathulenol, and Triacetin demonstrated the potential and was more effective inhibitor of Ornithine Am receptors in treatment of HCC. PHAs molecules have the potential to be used as a drug carrier during treatment of liver cancer, as demonstrated by the drug-like analysis of some compounds recorded in this study. Similarly, several compound features, including maximum number of hydrogen bond acceptors, maximum number of hydrogen bond donors, minimum molecular mass of 500 Daltons, and maximum number of violations, pose significant challenges in the process of medicine development (Hamedo et al., 2023; Sarfraz et al., 2024). In the future, the chosen compounds such as 9,12-Octadecadienoic acid (z,z) methyl ester, Isopropyl linoleate, Spathulenol, and Triacetin may express promising potential as drug carriers for the treatment of hepatocellular carcinoma (HCC).

Conclusions and Recommendations

The promising moderate halophilic bacterial strain P. onubensisstrain E3 (OL891886) was recovered in a pure culture from Solar Saltern, North Sinai, Egypt. This strain showed a high potential for using glucose and peptone as sole carbon and nitrogen sources, respectively, for growth and PHAs production. Maximum production of PHAs by this strain (54.77 mg/ l) was obtained after 72 h of incubation at 37 oC, pH 7, and 4 % NaCl (w/v). FTIR analysis pattern displayed sharp peaks formed around the absorbed bands at 3434.05 cm−1, 2923.55 cm−1, 1649.72 cm−1, 1494.58 cm−1, and 1230 cm−1 corresponding to OH, CH3, C=O, CH2, and C–O groups, respectively, related to PHAs. Future studies will prioritize optimization of PHAs production by utilizing alternative low-cost feed stocks, with the aim of enhancing both material quality and productivity. P. onubensis strain E3 is a promising producer of PHAs biopolymer that can be used as a drug carrier for treatment of liver cancer.

Acknowledgment

None to declare.

Novelty Statement

The novelty is found in the biotechnological capacity of the P. onubensis strain E3 as a sustainable and efficient producer of PHA, especially regarding its prospective use as a drug carrier for liver cancer treatment, distinguishing it from traditional PHA-producing strains. Utilizing alternative low-cost feedstocks to improve material quality and productivity will be the main focus of future research.

Author’s Contribution

Conceptualization: NE, AEMS and HAH.

Methodology: NE and HAH.

Software: NE.

Validation: NE, HAH and AEMS.

Formal analysis: NE.

Investigation: OTA and NE.

Resources: OTA and NE.

Data curation: NE.

Writing-original draft preparation: NE, HAH.

Writing review and editing: HAH and NE.

Visualization: AEMS and NE.

Supervision: NE, HAH.

Funding

No funding was provided for this study.

Ethical approval

None applicable.

Conflict of interests

The authors have declared no conflicts of interest.

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