Bacillus cereus PHB CMST1: A Potential Halophilic Bacterium for Cost-Effective and Sustainable Production of Polyhydroxyalkanoates

Bacillus cereus PHB CMST1: A Potential Halophilic Bacterium for Cost-Effective and Sustainable Production of Polyhydroxyalkanoates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bacillus cereus PHB CMST1: A Potential Halophilic Bacterium for Cost-Effective and Sustainable Production of Polyhydroxyalkanoates Gharishma Roy Sakthivel Latha, Uma Ganapathi, Rubyga Nagarajan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9349740/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Bacteria can spontaneously produce polyhydroxyalkanoate (PHA), a thermoplastic and biodegradable substance. PHA is the best polymer substitute for plastic made from petrochemicals. In this investigation, we isolated sixteen bacterial strains from brine and sediment samples collected from a solar salt works facility. Among the sixteen bacterial strains screened for polyhydroxyalkanoate (PHA) production, two strains, Z6 and Z9, exhibited promising results based on Sudan Black B and Nile Blue A staining. Subsequent morphological, biochemical, and molecular characterization identified strain Z9 as Bacillus cereus PHB CMST1 through 16S rRNA sequencing, which demonstrated superior PHA production compared to the other strain. To optimize PHA production, we employed a one-factor-at-a-time (OFAT) methodology, revealing optimal conditions of 35°C, pH 7, 2% salinity, and a 3-day incubation period, utilizing wheat bran as the carbon source and urea as the nitrogen source. Further optimization using Response Surface Methodology-Central Composite Design (RSM-CCD) indicated that B. cereus strain PHB CMST1 requires 5% wheat bran and 2% urea for enhanced PHA synthesis. The yellowish-green dots in the thin layer chromatography (TLC) plate indicated the presence of PHA. FT-IR analysis confirmed that, 𝛽-glycosidic linkages between the sugar monomers were found. The derivatives of polyhydroxybutyric acid confirmed the monomeric polymer by GC-MS analysis. PHA's XRD study showed wide peaks at 20° and 43°, indicating that the PHA had a semi-crystalline structure. Bacillus cereus shows considerable promise for cost-effective and large-scale production of PHA bioplastics, utilizing wheat bran as a non-expensive carbon source. Antibacterial activity Bacillus cereus Polyhydroxyalkanoate (PHA) Optimization Response Surface Methodology (RSM) Solar Salt works Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction The increasing reliance on conventional plastics in our daily lives has prompted a search for more sustainable alternatives. Microorganisms, including bacteria, fungi, actinomycetes, and yeasts, play a crucial role in the natural production and degradation of biodegradable plastics [ 1 ]. Biopolymers such as polyhydroxyalkanoates (PHAs), including poly(3-hydroxybutyrate) (PHB), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), polylactic acid (PLA), and polycaprolactone, present significant potential to replace traditional plastics [ 2 ]. PHAs are not only biodegradable and biocompatible but also naturally accumulate within bacterial cells as amorphous granules. Since the 1970s, research into PHAs has expanded across scientific and industrial sectors [ 3 ]. Different bacterial species produce PHAs with varying structures, with notable PHA producers found in marine environments, including Alteromonas , Bacillus , Halomonas , Pseudomonas , and Vibrio [ 4 ]. The applications of PHAs span industrial and medical fields, including drug delivery, packaging, agriculture, and printing. Despite the promising potential of PHA-accumulating bacteria for commercial bioplastic production, challenges remain due to the high cost of raw materials. Utilizing affordable and renewable agro-industrial by-products as carbon sources could significantly reduce PHA production costs [ 1 ]. Various organic wastes from the food industry, agricultural residues, and wastewater have been explored as potential substrates. Selecting bacterial strains with high PHA productivity and optimizing growth conditions are essential for facilitating economically viable biosynthesis. The high production costs associated with PHA stem from the use of expensive raw materials. Strategies such as employing diverse carbon sources—ranging from natural products to industrial and agro-industrial wastes—have been implemented to mitigate these costs. Efficient management of bioprocesses is critical for reducing the expenses of commercial biotechnology products. Traditional optimization methods, which often focus on one variable at a time, can lead to inaccurate results and extended timeframes. Moreover, these methods may overlook the interactions between different factors affecting production. In contrast, statistical experimental designs like factorial analysis and response surface methodology (RSM) provide a more reliable framework for optimization. RSM considers both individual and interactive effects of various factors, facilitating a comprehensive approach to optimizing production conditions [ 5 ]. This study aimed to isolate and identify bacteria capable of producing PHA from brine and sediment samples collected from the Puthalam salt pan. Additionally, we sought to optimize the physical and chemical factors influencing PHA production by integrating a one-factor-at-a-time (OFAT) approach with response surface methodology-central composite design (RSM-CCD). Low-cost agro-industrial wastes were utilized as carbon sources, and the resulting PHAs were characterized through techniques such as thin-layer chromatography (TLC), Fourier-transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Materials and Methods Collection of sample and isolation of bacteria Brine and sediment samples were collected from the Puthalam salt pan in the Kanyakumari district (Latitude 8.100361˚ and Longitude 77.479256˚). Serial dilution was carried out using 1 ml of the brine and sediment sample (10 − 1 to 10 − 10 ) to isolate the bacteria. After spreading out the aliquots on sterile Zobell Marine Agar medium plates, the plates were incubated for 24 h at 28°C. The colonies with various distinguishing characteristics were selected; pure cultures were maintained and kept for later usage at 4°C [ 6 ]. Screening of PHAs producing bacteria from the isolated bacteria Sudan Black B (SBB) was used for the primary screening for isolating the PHA producing bacterial colonies. The Zobell Marine Agar plates with the bacterial isolates were flooded with 0.05% SSB for 20 min. The isolates which produce PHAs appeared as bluish black in colour [ 7 ]. Nile Blue A (NBA) staining was carried out as the secondary screening of PHA producing bacterial colonies. The plates with bacterial isolates were strained with 1% NBA for 24 h. The isolates which showed bright orange fluorescence under UV light were selected as PHA accumulators [ 8 ]. Extraction of PHA in the selected PHA producing bacterial isolates The PHA was directly extracted using dispersion methods of sodium hypochlorite and chloroform with slight modification [ 1 , 9 ]. 100 ml of bacterial culture was centrifuged at 8500 x g for 15 min. The pellet was washed with phosphate buffer saline. The cell pellets were air dried and weighed (DCW). The cells were treated with 4% sodium hypochlorite at 37° C for 2 h. The mixture was again centrifuged at 5000 X g for 20 min, and the cell was washed with acetone and methanol in a 1:1 ratio. The cell was dissolved in 5 ml of chloroform and kept at room temperature for evaporation. The PHA obtained was weighed and the weight was noted (determined as the PHA extract weight). Residual biomass (g/l) = Dry Cell Weight (g/l) − Dry weight of extracted PHAs (g/l) PHA accumulation (%) = Dry weight of extracted PHAs (g/l) ×100% / Dry Cell Weight (g/l) Identification of the selected bacteria Morphological and Biochemical based identification The morphology of bacterial colonies in terms of shape, colour, arrangement, size, motility, and Gram-staining were observed using the microscope [ 10 ]. The bacterial isolates were tested for biochemical characterization such as indole production, methyl red (MR), Voges Proskauer (VP), citrate utilization, catalase test, oxidase test, urease production, Triple Sugar Iron (TSI) test, nitrate reduction test, and blood hemolysis [ 11 ] and enzymatic activities such as amylase, gelatinase, protease [ 12 ] and lipase [ 13 ] were also performed. Molecular identification of the selected isolate The genomic DNA of the selected bacterial strain was extracted from culture following the phenol-chloroform method [ 14 ]. PHA-producing bacteria was identified using the 16S rRNA sequence [ 15 ] and using universal primers p27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TAC GGC ACC TTG TTA CGA CTT-3′). Based on the phylogenetic determination, the gene sequence of the strain was submitted to the National Centre for Biotechnological Information (NCBI) to get the accession number. Optimization of media composition to produce PHA One factor at a time (OFAT) The physical, and chemical, culture conditions for PHA production were optimized using one factor at a time (OFAT). The physical factors such as temperature (25°C, 30°C, 35°C, and 40°C); salinity (2%, 3%, 4%, and 5%); pH (5, 6, 7, 8, and 9); and incubation days (1 to 4 days) were optimized for PHA production using minimal salt media. To optimize nutritional culture, the different renewable agro-industrial wastes such as rice bran, wheat bran, tamarind tunnel, and fruit pulp from fruits as carbon sources (2%) and 0.15% nitrogen sources such as beef extract, ammonium chloride, ammonium sulphate, and urea were selected by changing one factor at a time in the minimal salt media [ 16 , 17 ]. Response Surface Methodology-Central Composite Design (RSM-CCD) Based on pre-optimization by OFAT, the renewable agro-industrial substrate wheat bran as carbon source, and urea as nitrogen source, was selected. Using MINITAB 19 software with two factors (carbon and nitrogen sources) and five levels, the combinations of 13 experiments were generated and each factor was studied at five experimental levels: -α (very low), -1 (low), 0 (central point), + 1 (high), +α (very high) [ 18 ]. PHA biofilm preparation 50 mg of PHA extracted from bacterial cells was dissolved in 1 ml of chloroform in a falcon centrifuge tube using a slightly modified version [ 19 ]. The resultant mixture was maintained at 50°C in the oven until all the chloroform had evaporated. PHA coatings developed in the falcon centrifuge tube because of the chloroform evaporating. Characterization of crude PHA UV- Visible Spectrophotometer The extracted PHA was dissolved in chloroform; sulfuric acid was added and heated in a boiling water bath for 20 min at 100 o C. The absorbance of the sample was measured at the range of 200–800 nm using UV-Visible spectrophotometer [ 20 ]. Thin Layer Chromatography After dissolving the PHA biofilm in chloroform, a drop of the mixture was placed on a silica-coated TLC plate. TLC was performed using methanol and chloroform (1:1) for 40 min, and the plates were incubated in an iodine chamber in a water bath for 5 to 10 min [ 18 ]. Fourier-Transform Infrared Spectroscopy (FT-IR) The PHA biofilm extracted from the culture was analyzed using Fourier-transform infrared spectroscopy (FT-IR) with a Shimadzu instrument (Japan). The analysis was conducted at a resolution of 4 cm⁻¹, with 40 scans performed per sample. The spectral frequency range was set between 4000 and 400 cm⁻¹, and the resulting vibration spectrum was recorded as a graphical output [ 21 ]. Gas Chromatography–Mass Spectrometry (GC-MS) For the GC-MS analysis of the PHA biofilm, methanolysis was performed. The analysis was conducted using the Agilent Technologies 7000E and 7010C GC-MS systems (Agilent Technologies, CA, US). Samples were injected in split mode, with the column oven temperature maintained at 50°C and the injection temperature set to 250°C. The flow control was managed in pressure mode [ 22 ]. Thermogravimetric Analysis (TGA) The extracted PHA was subjected to thermogravimetric analysis (TGA) using the EXSTAR SIINT 6300 thermal system. Approximately 20 mg of the dried sample was used for the TGA experiment. Thermograms were generated under an airflow rate of 50 ml/min and a heating rate of 10°C/min, covering a temperature range from 30°C to 800°C. The heat flow and weight loss, measured in milligrams, were plotted against temperature [ 23 ]. X-ray Diffraction (XRD) X-ray diffraction analysis was performed using a D2 Phaser (BRUKER) with CuKα irradiation to determine the crystallinity of the PHA polymer. The sample was analyzed by the X-ray diffractometer over a scanning range of 10° to 80° in 2θ, with a step size of 0.02° [ 24 ]. Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDAX) The surface topology and chemical composition of the PHA films were examined using scanning electron microscopy (SEM). The films were affixed to sample holders coated with carbon tape and subsequently analyzed using the Carl Zeiss EVO18 system [ 25 ]. Antimicrobial activity of PHA with essential oils According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the halo test was performed against pathogens ( Staphylococcus aureus , Streptococcus pyogenes , Listeria sp., Bacillus subtilis , Enterococcus faecalis , Salmonella sp., Shigella sp., Yersinia sp., Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli ) by disc diffusion method. The PHA films loaded with essential oils (20µl), such as lemon grass, eucalyptus, and clove oil, were evaluated for their antibacterial activity. Antibiotic discs containing oxacillin (1 µg/disc) and gentamicin (300 µg/disc) were employed as positive controls against pathogens, while films devoid of essential oils were utilized as negative controls [ 26 ]. Data Analysis Triplicates ( n = 3) of each experiment were carried out. Microsoft Excel’s standard software package was used to undertake a statistical analysis of the results. The findings were shown as mean + standard deviation, or mean ± SD. Results Isolation and Screening for PHA producing bacterial isolates Using serial dilution, sixteen bacterial isolates (seven from the sediment sample and nine from the brine) with distinct morphologies and colours were selected for PHA screening (Fig. 1 ). The sixteen bacterial isolates were tested by Sudan Black B, two isolates were positively stained for primary screening (Fig. 2 a). Following the initial screening, Nile Blue A stain was used for a secondary screening. Two isolates out of sixteen had positive staining (Fig. 2 b) in secondary screening. Following primary and secondary screening, two isolates from the brine sample, Z6 and Z9, were selected for further examination. Extraction for PHA producing bacteria The sodium hypochlorite-chloroform method was employed on the two isolates (Z6, Z9) for PHA extraction. The result showed that the isolates Z6 and Z9 generated 13% and 25% of PHA, respectively (Table 1 ). Z9, which generated the higher quantity of PHA, was selected to undergo further optimization and characterization studies. Table 1 PHA production from the bacterial isolates Strain Dry cell weight (g L − 1 ) PHA extract weight (g L − 1 ) Residual biomass (g L − 1 ) PHA accumulation (%) Z6 6 ± 1 0.8 ± 0.01 5.2 ± 0.2 13 ± 1 Z9 0.8 ± 0.01 0.2 ± 0.01 0.6 ± 0.01 25 ± 1 Identification of selected PHA producing isolates Z9 The chosen isolate, Z9, was rod-shaped, Gram-positive, and motile. Under aerobic conditions, it grew into an off-white-coloured spore form on an agar plate. The isolate was tested positive for Voges-Proskauer, citrate utilization, catalase, urease production, triple sugar iron, nitrate reduction, and hemolytic activity, enzymatic activities such as amylase, gelatinase, protease, and lipase. Based on the results of the morphological and biochemical tests, chosen isolate Z9 was identified as Bacillus sp. (Table 2 ). Phylogenetic analysis of the 16S rRNA sequences revealed that Z9 was confirmed as Bacillus cereus strain PHB CMST1 (Fig. 3 ). The sequence was submitted to NCBI, and accession number OQ804397.1 was obtained. Table 2 Biochemical identification and enzyme assay for the selected isolate Z9 S. No Test Result 1 Indole test Negative (-) 2 Methyl red test Negative (-) 3 Voges-Proskauer test Positive (+) 4 Citrate utilization test Positive (+) 5 Catalase test Positive (+) 6 Oxidase test Negative (-) 7 Urease production Positive (+) 8 Triple sugar iron (TSI) Positive (+) 9 Nitrate reduction Positive (+) 10 Hemolytic Positive (+) Enzymatic assay: 11 Amylase Positive (+) 12 Gelatinase Positive (+) 13 Protease Positive (+) 14 lipase Positive (+) Optimization of media components for PHA production Effect of physical and chemical factors on PHA production by OFAT The findings demonstrated that the greatest amount of PHA was produced at 35°C (4.1 g/L) (Fig. 4 a). At pH 7 (89.6 g/L), PHA synthesis reached its peak (Fig. 4 b). The highest PHA output was reported at 2% (5.2 g/L) (Fig. 4 c) NaCl. After three days of incubation (2 g/L), the highest amount of PHA was produced (Fig. 4 d). The carbon source, wheat bran (62.8 g/L), produced a considerable amount of PHA (Fig. 4 e). The maximum amount of PHA (8.4 g/L) was obtained in the nitrogen source, urea (Fig. 4 f). Response Surface Methodology – Central Composite Design (RSM-CCD) on media components for PHA production The results of experiments with a central composite design and second-order polynomial multiple regression is shown in Table 3 . The experimental and predicted values of the experimental design did not significantly differ. The maximum PHA production was 367.645 g/L at the concentrations of urea 2.14121% (nitrogen source) and wheat bran 4.41421% (carbon source). The model predicted an R 2 value of 99.97% with 0.03% of the variance for PHA production, confirming that it was highly significant. The P values for the linear term, the quadratic coefficient, and the interactive coefficient were 0.000, which is less than 0.05, indicating that they played a significant role in PHA production. The current study found the lack of fit test values for these responses to be insignificant, leading to the acceptance of the model. The Pareto graph (Fig. 5 a) verified the stronger effects in the upper portion and their progression down to the bottom section, with p values less than 0.05, indicating their significant contribution to PHA production compared to other components. From the Fig. 5 b, the Bacillus cereus strain PHB CMST1, the predicted optimal values of wheat bran and urea for PHA production were calculated to be 5% and 2.5%, respectively. Table 3 Response Surface Methodology – Central Composite Design (RSM-CCD) on media components for PHA production and it’s observed and predicted values Run Order Wheat Bran (%) Urea (%) Dry cell weight (g L − 1 ) PHA (g L − 1 ) Experimental Predicted Experimental Predicted 1 1.6 2.1 181.4 180.0 174.8 173.2 2 4.4 0.41 251.6 255.0 235.0 237.2 3 1.6 0.41 149.8 151.9 132.8 132.6 4 3.0 1.3 215.4 215.4 190.2 190.2 5 3.0 2.5 294.8 297.9 288.6 290.5 6 1.0 1.3 135.0 135.6 117.6 119.5 7 3.0 0.05 189.0 185.2 171.0 169.5 8 3.0 1.3 215.4 215.4 190.2 190.2 9 3.0 1.3 215.4 215.4 190.2 190.2 10 4.4 2.1 387.6 386.2 367.8 367.6 11 3.0 1.3 215.4 215.4 190.2 190.2 12 5.0 1.3 355.8 354.5 332.6 331.1 13 1.6 2.1 181.4 180.0 174.2 173.2 PHA biofilm preparation by PHA producing bacteria The prepared biofilm was easily brittle (Fig. 6 ). The isolated Bacillus cereus strain PHB CMST1 produced PHA that fully dissolved in chloroform, a distinctive characteristic of PHA. Characterization of PHA extracted from Bacillus cereus strain PHB CMST1 UV-Visible spectral analysis The UV Visible spectroscopy revealed the absorbance maximum at a range between 235 to 440 nm indicating the presence of PHA (Fig. 7 ). Thin Layer Chromatography The TLC analysis revealed yellowish-green spots on the TLC plate (Fig. 8 ) with R f value of 0.783 indicated the presence of PHAs. Fourier- transform infrared (FT-IR) The FT-IR spectroscopic analysis gave further insights into the chemical structure of the polymer and reflected the monomeric units (Fig. 9 ). There absorption bands were observed at 697 cm − 1 and 825 cm − 1 , which may suggest the presence of 𝛽-glycosidic linkages between the sugar monomers. The band at 1056 cm −1 corresponds to the valence vibration of the carboxylic group (COOH). This demonstrated that plastic had formed, most likely as a polymer with a carboxylic acid side chain. It is crucial to consider the other bands in C-O-C stretching, which occur at 1102 cm −1 , 1132 cm −1 , and 1185 cm −1 , as they play a significant role in identifying and characterizing PHA monomers. There was a peak at 1737 cm −1 indicating the presence of an aliphatic carbonyl group (C = O valence) in the PHA polymer. Another peak at 1660 cm −1 suggested the presence of an alkene C = O valence, while a peak at 1531 cm −1 indicated the presence of an amide C = O valence. The vibrational frequencies at 2875 cm −1 and 2972 cm −1 indicated the presence of influential stretching groups in alkanes, specifically –CH 2 and –CH 3 , these frequencies also reflect the methylene's intensity. At 3069cm −1 and 3272 cm −1 , the band showcased O–H bending, suggesting the presence of hydrogen bonds, the broad nature and low frequency value further support this observation. The fact that the PHA molecule has a strong carbonyl absorption peak at 1737 cm −1 (absorbance of carbonyl band) in the infrared spectrum. Gas chromatography and mass spectroscopy GC-MS analysis determined the monomeric content of the PHA polymer. The main peak's resemblance the monomer composition 3-hydroxybutyrate (3HB) of mcl-PHA biopolymer. The PHA extract revealed five peaks with retention times (RT) of 4.885, 8.165, 10.595, 12.340, and 17.570 min. These include hexanoic acid; hexanedioic acid; ethyl-3-hydroxybutyrate; hexanoic acid, 4-methyl-, methyl ester; and eicosanoic acid. Table 4 , Fig. 10 , displayed the major compounds along with their respective RT, peak area, molecular formula, and molecular weight. Table 4 Derivatives of PHA confirmed through GC-MS analysis Derivatives RT Area % Molecular formula Molecular weight A) Hexanoic acid 4.885 0.74 C 6 H 12 O 2 116 B) Hexanedioic acid 8.165 0.05 C 6 H 10 O 4 146 C) Ethyl 3 hydroxybutyrate 10.595 0.07 C 6 H 12 O 3 132 D) Hexanoic acid, 4-methyl-, methyl ester 12.340 0.02 C 8 H 16 O 2 144 E) Eicosanoic acid 17.570 0.02 C 20 H 40 O 2 312 Thermogravimetric and Differential thermal analysis The thermogravimetric and differential thermal analysis showed a total weight loss of 92% occured at 800°C, with 10% weight loss up to 250°C, 60% up to 550°C, and 20% up to 800°C. Additionally, there are sharp exothermic peaks at 520°C and 600°C. The TG/DTA results provide insights into the thermal degradation processes of the PHA. We can attribute the stepwise weight loss to the following events: 10% weight loss up to 250°C, most likely because bound water molecules and/or volatile components are released; 60% weight loss up to 550°C, because the of the breakdown of PHA polymer chains and releasing of gaseous products; and 20% weight loss up to 800°C was due to the last stages of combustion of the remaining organic material. The sharp exothermic peaks at 520°C and 600°C indicated significant heat release events, suggesting combustion or decomposition processes occurring in the material. The peak at 520°C could be associated with the initial decomposition and combustion of the PHA material, while the peak at 600°C may correspond to the further breakdown and combustion of the remaining polymer chains (Fig. 11 ). X-ray diffraction The X-ray diffraction (XRD) pattern of the polyhydroxyalkanoate (PHA) sample is shown in Fig. 12 . The pattern reveals a sharp peak at 25.62° and a broad hump centered around 19–20°, with another broad region extending towards 40–45°. These observations provide insight into the crystalline and amorphous nature of the material. The sharp peak at 25.62° suggests the presence of some crystalline regions within the polymer, while the broad peaks at 19–20° and around 40° are indicative of significant amorphous content. Based on quantitative analysis by BRUKER Diffrac. EVA software, the degree of crystallinity was calculated to be 5.7%, with the remaining 94.3% contributed to amorphous regions. SEM-EDAX mapping Figure 13 displays the microstructure of PHA derived from Bacillus cereus strain PHB CMST1. The microstructure revealed a porous material with finely linked grains and a strong propensity to form multigrain agglomerates. The chemical composition (wt.%) result revealed 49.8 carbon, 33.9 oxygen, 4.9 sodium, 4.1 phosphorus, 1.4 sulfur, 2.0 chlorine, 1.5 potassium, and 2.4 iron in the sample. These results align with their elemental signals, showcasing the colour-coded mapping of various elements such as carbon, oxygen, sodium, phosphorus, sulfur, chlorine, potassium, and iron (Table 5 and Fig. 14 ). Table 5 Chemical composition in PHA from Bacillus cereus strain PHB CMST1 Element Weight % Atomic % Error % K ratio C K 49.8 61.0 8.7 0.1606 O K 33.9 31.2 10.4 0.0591 NaK 4.9 3.2 9.6 0.0177 P K 4.1 2.0 3.8 0.0322 S K 1.4 0.6 8.9 0.0111 ClK 2.0 0.8 6.1 0.0161 K K 1.5 0.6 8.6 0.00129 FeK 2.4 0.6 9.1 0.0206 Antimicrobial activity using essential oils As shown in Fig. 15 and Table 6 , Gentamicin, the positive control, demonstrated high activity against all eleven tested pathogens. The product PHA did not show any zone of inhibition against the pathogens tested, indicating that it had no antimicrobial activity. Lemongrass oil, along with PHA, had the highest activity among the three essential oils tested with PHA. Eucalyptus oil had the least activity among the three essential oils, along with PHA. The essential clove oil, along with PHA, showed moderate activity against the tested pathogens. Table 6 Zone formation (cm) of PHA films and essential oils in halo test Pathogen Positive control Negative control Lemongrass oil Eucalyptus oil Clove oil Shigella sp. 3.7 ± 0.01 - 3.0 ± 0.1 1.4 ± 0.2 2.1 ± 0.2 Bacillus subtilis 3.5 ± 0.1 - 2.1 ± 0.1 1.0 ± 0.1 2.2 ± 0.1 Staphylococcus aureus 3.4 ± 0.1 - 2.8 ± 0.01 1.0 ± 0.1 2.1 ± 0.2 Pseudomonas aeruginosa 3.8 ± 0.1 - 2.2 ± 0.2 0.9 ± 0.01 2.3 ± 0.1 Salmonella sp. 3.0 ± 0.2 - 3.0 ± 0.1 1.6 ± 0.1 2.4 ± 0.01 S. pyogenes 3.3 ± 0.01 - 2.5 ± 0.1 1.0 ± 0.1 2.0 ± 0.1 Klebsiella pneumonia 4.0 ± 0.1 - 1.5 ± 0.01 1.8 ± 0.01 2.0 ± 0.1 Escherichia coli 3.9 ± 0.02 - 1.7 ± 0.2 1.3 ± 0.1 2.9 ± 0.2 Yersinia sp. 3.4 ± 0.1 - 1.7 ± 0.1 1.0 ± 0.1 2.6 ± 0.1 Listeria sp. 3.4 ± 0.2 - 1.9 ± 0.1 1.4 ± 0.1 2.1 ± 0.1 Enterococcus faecalis 3.5 ± 0.1 - 1.8 ± 0.2 1.0 ± 0.01 1.9 ± 0.1 Discussion The environmental impact of petroleum-based plastics has intensified the global shift towards biodegradable alternatives, such as polyhydroxyalkanoates (PHAs), which offer unique thermoplastic properties. However, the high production costs associated with bioplastics pose significant barriers to widespread adoption. Identifying bacterial strains with enhanced productivity and optimizing their growth conditions are essential for reducing PHA production expenses. Over 300 bacterial species have been linked to PHA accumulation across diverse environments [ 27 ]. In this study, we successfully identified a PHA-producing Bacillus strain from brine samples collected from a salt pan. Previous research, such as that by Martinez-Gutierrez et al. [ 28 ], identified multiple PHA-producing bacteria from hypersaline microbial mats, while Muigano et al. [ 29 ] documented numerous isolates from Kenya’s haloalkaline lakes. Our findings, which yielded 16 isolates from sediment and water samples in the Puthalam salt pan, were screened using Sudan Black B and Nile Blue A staining to assess PHA production. Notably, two strains, Z6 and Z9, exhibited positive results during both primary and secondary screenings [ 30 , 31 ]. Through morphological, biochemical, and molecular characterization, we identified isolate Z9 as Bacillus cereus. Previous studies have highlighted Bacillus species as effective PHA producers due to their simpler extraction processes and ability to secrete hydrolytic enzymes [ 15 , 27 ]. Our results align with existing literature, confirming the significance of Bacillus strains in PHA production [ 32 , 33 ]. Temperature is a critical factor influencing PHA accumulation. Our study indicated minimal PHA production outside the optimal range of 15°C to 50°C, likely due to reduced enzymatic activity at extreme temperatures [ 34 ]. This aligns with findings from Hamdy et al. [ 5 ] and Yasin and Al-Mayaly [ 22 ], who reported peak PHA production at 35°C for various Bacillus cereus strains. pH also plays a vital role in PHA biosynthesis. We maintained a neutral starting pH of 7.0, which facilitated maximum PHA production by minimizing energy costs associated with substrate uptake [ 35 ]. This finding is consistent with other studies that have reported similar optimal pH levels for PHA-producing bacteria [ 16 , 36 ]. The concentration of NaCl can enhance PHA production as it affects osmotic pressure, influencing cell growth and metabolism. Our study found that a 2% NaCl concentration yielded the highest PHA production, supporting the idea that osmotic conditions can promote PHB accumulation by creating a favourable environment for metabolic efficiency [ 37 ]. Extended incubation periods encourage microbial growth, leading to increased PHA accumulation. However, after reaching the logarithmic growth phase, microbial growth declines, which subsequently reduces PHA production. Our findings suggest that the optimal incubation period for maximizing PHA yield is around 72 hours, corroborating observations from previous studies [ 32 , 38 – 40 ]. Microorganisms have the remarkable ability to utilize a variety of carbon sources for the synthesis of polyhydroxyalkanoates (PHAs). These carbon sources serve as substrates for the production of precursors that can be further polymerized into PHAs, depending on the metabolic pathways employed by different bacterial strains. In our study, we explored the feasibility of using agricultural waste as an alternative to glucose for PHA production by Bacillus cereus strain PHB CMST1. We evaluated several carbon sources, including rice bran, wheat bran, tamarind pulp, and various fruit pulps, and measured both the dry cell weight and the resultant PHA accumulation. Our results indicated that wheat bran was the most effective substrate for PHA production. This finding is significant, considering that the cereal industry generates substantial quantities of wheat bran as a byproduct. Wheat bran is composed of approximately 19% starch, 18% protein, 6% lignin, and 38% non-starch polysaccharides, which include cellulose and arabinoxylans [ 41 ]. Previous studies support our findings; for instance, Rezk et al. [ 42 ] reported that Streptomyces incanus effectively utilized wheat bran, which is rich in hemicellulose and cellulose, to produce significant amounts of PHB. Similarly, Adnan et al. [ 43 ] found that the Bacillus flexus HSA3 strain achieved optimal PHB production when grown on wheat bran. Nitrogen is a critical nutrient for the growth of all microorganisms, and it plays a vital role in the synthesis of polyhydroxyalkanoates (PHAs). Different PHA-producing strains exhibit varying preferences for nitrogen sources, including urea, nitrate, and ammonia. The choice of nitrogen source and its concentration significantly impact both microbial growth and PHA production. Limiting nitrogen availability can actually promote PHA accumulation, as studies suggest that under nutrient-limited conditions, the production of PHA may redirect acetyl-CoA from the Krebs cycle toward polymer synthesis [ 1 ]. In our investigation, we assessed the effects of various nitrogen sources—namely beef extract, ammonium chloride, ammonium sulfate, and urea—at a concentration of 0.15% on PHA production by Bacillus cereus strain PHB CMST1. Our results demonstrated that urea was the most effective nitrogen source for enhancing PHA synthesis. Its smaller molecular size and higher polarity facilitate efficient cellular uptake, making it a promising candidate for PHA production [ 44 ]. Supporting our findings, Wang et al. [ 37 ] explored the utilization of various nitrogen sources, including urea, for PHA production in Burkholderia cepacia . Our study further optimized the conditions for PHA production from Bacillus cereus strain PHB CMST1, establishing that the ideal parameters were 35°C, pH 7, 2% salinity, 3 days of incubation, with 2% wheat bran as the carbon source and urea as the nitrogen source . Our study identified the optimal conditions for polyhydroxyalkanoate (PHA) production as 35°C, pH 7, 2% salinity, a 3-day incubation period, 2% wheat bran as the carbon source, and 0.15% urea as the nitrogen source. These findings highlight the significance of customized growth parameters in maximizing PHA yield. Previous research has similarly explored the optimization of media components for PHA production. For instance, Penkhrue et al. [ 45 ] optimized the conditions for producing PHB from Bacillus drentensis BP17 using pineapple peel through response surface methodology (RSM) and central composite design (CCD). Additionally, Mohapatra et al. [ 15 ] demonstrated effective parameter optimization for increasing PHA content in R. eutropha from whey hydrolysate, achieving statistically significant results. Hamdy et al. [ 5 ] also employed RSM-CCD to enhance PHA production in Bacillus cereus strain SH-02, reinforcing the utility of these statistical methods in bioprocess optimization. Patil et al. [ 46 ] conducted a detailed optimization study using a central composite rotatable design, focusing on two process variables, tryptophan and Tween 80 concentrations. Their results showed a close alignment between predicted and experimental PHA production, underscoring the accuracy of their optimization approach. In our investigation, the RSM-optimized medium yielded the highest PHA content and concentration, demonstrating a robust correlation with experimental outcomes. Notably, an optimal method for maximizing PHB synthesis was identified, which involved using 5% wheat bran as the carbon source and 2.5% urea as the nitrogen source [ 47 ]. The coefficient of determination, R 2 , had a value of 99.97%. The R 2 is a metric that quantifies the accuracy of a model's fit, taking on values between 0 and 1. The higher the model's strength and predictive accuracy, the closer the R 2 value approaches unity. Alternatively, lower R 2 values indicate that the response variables are inadequate in accounting for the observed variation. In this experiment, the R 2 values demonstrated that it has the ability to explain over 99% of the variability in PHA content. The adjusted R 2 value was 99.95%, which accounts for the sample size and number of words required to correct the R 2 value [ 40 , 48 ]. In this investigation, the absorption spectrum of Bacillus cereus strain PHB CMST1 displayed peaks between 235 and 440 nm, confirming the presence of polyhydroxyalkanoates (PHAs). This observation aligns with previous research by Mandagutti and Sudhakar [ 31 ], who reported similar findings in PHB extracts from Bacillus paraconglomeratum . Additionally, our study identified yellowish-green spots on TLC plates, corroborating results previously reported by Rao et al [ 18 ]. The infrared spectroscopic analysis of the produced PHAs revealed a significant absorption peak at 1718.50 cm⁻¹, which corresponds to the carbonyl ester (C = O) functional group typical of PHBs [ 49 ]. In our findings, we observed high absorption peaks at 1737 cm⁻¹, 2972 cm⁻¹, and 2875 cm⁻¹, indicating the presence of both C = O and O-H functional groups. Supporting this, Samrot et al. [ 50 ] identified 3-hydroxybutyrate and fatty acid methyl esters as markers for PHB production. The synthesis of fatty acids through a de novo pathway involves the conversion of simple sugars into 3-hydroxyacyl (3HA) precursors. These precursors are subsequently transformed into malonyl-CoA via acetyl-CoA. A specific CoA transferase, known as PhaG, then catalyzes the production of (R)-3-hydroxyfatty acids [ 50 ]. Our analysis using gas chromatography-mass spectrometry (GC-MS) identified derivative products such as butanoic acid, 4-methyl-hexanoic acid methyl ester, and monomethyl hexanedioate, which helped confirm the structure of PHB from Pseudodonghicola xiamenensis . Furthermore, GC-MS results from PHB isolated from Bacillus licheniformis MSBN1 and B. megaterium indicated the presence of 3-hydroxybutyrate, reinforcing the structural integrity of the polymer [ 7 ]. In our study, we also detected these esters and 3-hydroxybutyrate in Bacillus cereus strain PHB CMST1, providing compelling evidence for the existence of PHB in this strain. Bacteria within the Bacillus genus are well-known for accumulating short-chain polyhydroxyalkanoates (PHAs), particularly polyhydroxybutyrate (PHB). Previous studies, such as that by Pillai et al. [ 27 ], have characterized the thermal properties of standard PHB, noting degradation temperatures at 212°C and 266°C, with thermal degradation beginning at 247°C and peaking at 287°C. In our current investigation, thermogravimetric analysis (TGA) revealed distinct exothermic peaks at 520°C and 600°C, indicating the thermal stability of the produced polymer. X-ray diffraction (XRD) analysis further elucidated the structural characteristics of the material. Strong and intense peaks in the diffraction data suggest a crystalline nature, while our findings indicated a semi-crystalline structure with broad peaks at approximately 20 and 43 degrees [ 50 ]. This relatively low crystallinity is typical of many PHA polymers, which often display a mixture of amorphous and semi-crystalline phases [ 51 , 52 ]. The absence of distinct PHB peaks may reflect the conditions under which the polymer was prepared, potentially inhibiting its crystallization [ 53 ]. These diffractogram results are consistent with prior study [ 45 ], reinforcing the understanding of PHA crystallinity. Additionally, Nwinyi and Owolabi [ 54 ] demonstrated that the microstructure and surface morphology of PHA samples, analyzed using scanning electron microscopy coupled with energy dispersive spectroscopy, exhibited porous and interconnected microstructures. These characteristics enhance the extraction efficiency of PHA from bacterial isolates ( Rhodococcus sp., Corynebacterium sp., Lactobacillus sp., and Arthrobacter sp.), a feature commonly shared among various plastic materials. The antimicrobial properties of various essential oils, such as tea tree, rosemary, eucalyptus, and lavender, have been shown to differ significantly when tested against pathogenic bacteria, as noted by Puvaca et al. [ 55 ]. In general, natural polymers tend to lack intrinsic antibacterial activity, with chitosan being a notable exception due to its positively charged amino groups that confer antimicrobial properties [ 56 ]. Consequently, there has been a growing interest in exploring the potential of polyhydroxyalkanoates (PHAs) as matrices for the incorporation of antimicrobial agents. Research has extensively examined the incorporation of various antimicrobial fillers into biopolymers, including metals, chemicals, natural extracts, essential oils, and nanoparticles [ 57 ]. These antimicrobial agents can interact synergistically with polymers to form composites that enhance functional performance. Our findings support this notion, as we observed that while PHA alone did not demonstrate antibacterial activity, it exhibited significant antibacterial effects when combined with essential oils, particularly lemongrass oil. B. cereus has emerged as a compelling model organism for the production of polyhydroxyalkanoates (PHAs) due to its remarkable biological diversity and adaptability. This species thrives in various environments, equipping it with a range of versatile traits that enhance its productivity. Notably, B. cereus can utilize waste substrates as carbon sources for PHA synthesis, which not only supports sustainable production practices but also improves the economic viability of the process. The PHAs produced by B. cereus are characterized by desirable properties such as low brittleness, non-toxicity, and excellent biocompatibility, making them suitable for a wide array of applications. Our findings further indicate that both PHA and essential oils exhibit antibacterial properties. This suggested that researchers are finding B. cereus increasingly significant in this field of study [ 58 ]. Conclusion Polyhydroxyalkanoates (PHAs) demonstrate efficient biodegradation by natural organisms in challenging environments, positioning them as a superior alternative to conventional plastics. The utilization of agricultural waste offers a sustainable and economically viable substrate for PHA production at a commercial scale. This study successfully identified and optimized critical factors affecting PHA synthesis. By integrating traditional and statistical optimization techniques, optimal conditions were established to enhance both biomass accumulation and PHA yield. The results highlight Bacillus cereus as a promising strain for PHA production using wheat bran. In industrial applications, leveraging cost-effective raw materials like wheat bran can significantly improve PHA production rates, thereby enhancing its potential as an environmentally friendly bioplastic. Declarations Acknowledgments This work was supported by the Researchers Supporting Project Number (RSPD2025R991), King Saud University, Riyadh, Saudi Arabia. Author contributions SLGR: Investigation, Visualization, Methodology, Formal analysis, Writing – original draft. GU and TC: Conceptualization, Investigation, Supervision, Funding acquisition, Writing – review & editing. JRA, NR, SJN, and ENS: Writing – review & overall editing. Funding King Saud University, Riyadh, Saudi Arabia (Project Number RSPD2025R991), Data availability The data related to this research are available with the corresponding author and may be made available upon prior request. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District - 629 502, India 2 Department of Botany and Microbiology, College of Science, King Saud University, P.O. 2455, Riyadh 11451, Saudi Arabia. References Shah S, Kumar A. Production and characterization of polyhydroxyalkanoates from industrial waste using soil bacterial isolates. Brazilian J Microbiolo. 2021;52:715–26. Jost V. Packaging related properties of commercially available biopolymers–An overview of the status quo. Express Polym Lett. 2018;12(5):429–35. Guzik MW. Polyhydroxyalkanoates, bacterially synthesized polymers, as a source of chemical compounds for the synthesis of advanced materials and bioactive molecules. Appl Microbiolo Biotechnolo. 2021;105(20):7555–66. Suzuki M, Tachibana Y, Kasuya K-i. Biodegradability of poly (3-hydroxyalkanoate) and poly (ε-caprolactone) via biological carbon cycles in marine environments. Polym J. 2021;53(1):47–66. Hamdy SM, Danial AW, Gad El-Rab SM, Shoreit AA, Hesham AE-L. Production and optimization of bioplastic (Polyhydroxybutyrate) from Bacillus cereus strain SH-02 using response surface methodology. BMC Microbiol. 2022;22(1):183. Nwosu I, Abu G, Agwa K. Isolation, screening and characterization of exopolysaccharide producing bacteria. Microbiol Res J Int. 2019;29:1–9. Mostafa YS, Alrumman SA, Alamri SA, Otaif KA, Mostafa MS, Alfaify AM. Bioplastic (poly-3-hydroxybutyrate) production by the marine bacterium Pseudodonghicola xiamenensis through date syrup valorization and structural assessment of the biopolymer. Sci Rep. 2020;10(1):8815. Mascarenhas J, Aruna K. Screening of polyhydroxyalkonates (PHA) accumulating bacteria from diverse habitats. J Global Biosci. 2017;6(3):4835–48. Thapa C, Shakya P, Shrestha R, Pal S, Manandhar P. Isolation of polyhydroxybutyrate (PHB) producing bacteria, optimization of culture conditions for PHB production, extraction and characterization of PHB. Nepal J Biotech. 2018;6(1):62–8. Kumar K, Rajput R, Sharma R. Antagonistic activity of bacterial species isolated from soil against fungi. World J Pharm Res. 2018;7:760–8. Masi C, Gemechu G, Tafesse M. Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent. Ann Microbiol. 2021;71:1–11. Luang-In V, Yotchaisarn M, Saengha W, Udomwong P, Deeseenthum S, Maneewan K. Protease-producing bacteria from soil in nasinuan community forest, mahasarakham province, Thailand. Biomed Pharmacolo J. 2019;12(2):587–95. Joshi R, Bisht TS. Isolation and screening of lipase enzyme from bacterial strains isolated from Sambhar lake. Int J Pharm Biol Sci. 2019;9(5):2321–3272. Onajobi IB, Idowu EO, Adeyemi JO, Samson OJ, Ogunyinka PI, Fagade OE. In vitro antibacterial activities and molecular characterization of bacterial species isolated from farmlands against selected pathogens. Biotechnolo Rep. 2020;27:e00513. Mohapatra S, Pattnaik S, Maity S, Sharma S, Akhtar J, Pati S, Samantaray DP, VARMA A. Comparative analysis of PHAs production by Bacillus megaterium OUAT 016 under submerged and solid-state fermentation. Saudi J Biol Sci. 2020;27(5):1242–50. Sriyapai T, Chuarung T, Kimbara K, Samosorn S, Sriyapai P. Production and optimization of polyhydroxyalkanoates (PHAs) from Paraburkholderia sp. PFN 29 under submerged fermentation. Electron J Biotechnol. 2022;56:1–11. Dienye B, Agwa O, Abu G. Molecular Characterization, optimization and production of PHA by indigenous bacteria using alternative nutrient sources as substrate. Microbiol Res J Int. 2022;32(11–12):12–26. Rao A, Haque S, El-Enshasy HA, Singh V, Mishra BN. RSM–GA based optimization of bacterial PHA production and in silico modulation of citrate synthase for enhancing PHA production. Biomolecules. 2019;9(12):872. Mohammed S, Ray L. Polyhydroxyalkanoate recovery from newly screened Bacillus sp. LPPI-18 using various methods of extraction from Loktak Lake sediment sample. J Genetic Eng Biotechnolo. 2022;20(1):115. Rodge SP, Dhanavade MJ, Kajale SC, Patil NP. A polyhydroxyalkanoate synthesised by halophilic archaeon, Natrialba swarupiae . Environ Sci: Adv. 2023;2(7):990–1000. Stanley A, Murthy PK, Vijayendra S. Characterization of polyhydroxyalkanoate produced by Halomonas venusta KT832796. J Polym Environ. 2020;28(3):973–83. Yasin AR, Al-Mayaly IK. Study of the fermentation conditions of the Bacillus cereus Strain ARY73 to produce polyhydroxyalkanoate (PHA) from glucose. J Ecol Eng. 2021; 22(8). Lorini L, Martinelli A, Capuani G, Frison N, Reis M, Sommer Ferreira B, Villano M, Majone M, Valentino F. Characterization of polyhydroxyalkanoates produced at pilot scale from different organic wastes. Front Bioeng Biotechnol. 2021;9:628719. Tian J, Zhang R, Wu Y, Xue P. Additive manufacturing of wood flour/polyhydroxyalkanoates (PHA) fully bio-based composites based on micro-screw extrusion system. Mat Des. 2021;199:109418. Thu NTT, Hoang LH, Cuong PK, Viet-Linh N, Nga TTH, Kim DD, Leong YK. Nhi-Cong LT Evaluation of polyhydroxyalkanoate (PHA) synthesis by Pichia sp. TSLS24 yeast isolated in Vietnam. Sci Rep. 2023;13(1):3137. Basnett P, Marcello E, Lukasiewicz B, Nigmatullin R, Paxinou A, Ahmad MH, Gurumayum B, Roy I. Antimicrobial materials with lime oil and a poly (3-hydroxyalkanoate) produced via valorisation of sugar cane molasses. J Funct Biomat. 2020;11(2):24. Pillai AB, Kumar AJ, Thulasi K, Kumarapillai H. Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai. Brazilian J Microbiol. 2017;48:451–60. Martínez-Gutiérrez CA, Latisnere-Barragán H, García-Maldonado JQ, López-Cortés A. Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico. Peer J. 2018;6:e4780. Muigano MN, Anami SE, Onguso JM, Omare GM. The isolation, screening, and characterization of polyhydroxyalkanoate-producing bacteria from hypersaline lakes in Kenya. Bacteria. 2023;2(2):81–97. Aragosa A, Specchia V, Frigione M. Isolation of two bacterial species from argan soil in morocco associated with polyhydroxybutyrate (PHB) accumulation: Current potential and future prospects for the bio-based polymer production. Polymers. 2021;13(11):1870. Mandragutti T, Sudhakar G. Selective isolation and genomic characterization of biopolymer producer—a novel feature of halophile Brachybacterium paraconglomeratum MTCC 13074. J Genetic Eng Biotechnol. 2023;21(1):24. Alshehrei F. Production of polyhydroxybutyrate (PHB) by bacteria isolated from soil of Saudi Arabia. J Pure Appl Microbiol. 2019; 13(2). Quintero-Silva MJ, Suárez-Rodríguez SJ, Gamboa-Suárez MA, Blanco-Tirado C, Combariza MY. Polyhydroxyalkanoates production from cacao fruit liquid residues using a native Bacillus megaterium strain: Preliminary study. J Polym Environ. 2024;2(3):1289–303. Mahato RP, Kumar S, Singh P. Optimization of growth conditions to produce sustainable polyhydroxyalkanoate bioplastic by Pseudomonas aeruginosa EO1. Front Microbiol. 2021;12:711588. Zihayat B, Shakibaie M, Sabouri-Shahrbabak S, Doostmohammadi M, Ameri A, Adeli-Sardou M, Forootanfar H. Medium optimization for polyhydroxyalkanoate production by Pseudomonas pseudoalcaligenes strain Te using D-optimal design. Biocatal Agricul Biotechnol. 2019;18:101001. Khamkong T, Penkhrue W, Lumyong S. Optimization of production of polyhydroxyalkanoates (PHAs) from newly isolated Ensifer sp. strain HD34 by response surface methodology. Processes. 2022;10(8):1632. Wang J, Huang J, Guo H, Jiang S, Qiao J, Chen X, Qu Z, Cui W, Liu S. Effects of different sodium salts and nitrogen sources on the production of 3-hydroxybutyrate and polyhydroxybutyrate by Burkholderia cepacia . Biores Bioprocess. 2021;8:1–14. Desouky S, El-Shiekh H, Elabd M, Shehab A. Screening, optimization and extraction of polyhydroxyalkanoates (PHAs) from Bacillus thuringienesis . J Adv Biol Biotechnol. 2014;1(1):40–54. Ahmady-Asbchin S, Rezaee H, Safari M, Zamanifar P, Siyamiyan D. Production and optimization of polyhydroxybutyrate (PHB) from Bacillus megaterium as biodegradable plastic. Eur J Biol Rese. 2020;10(1):26–34. Geethu M, Chandrashekar HR, Divyashree M. Statistical optimisation of polyhydroxyalkanoate production in Bacillus endophyticus using sucrose as sole source of carbon. Archives Microbiol. 2021;203:5993–6005. Annamalai N, Sivakumar N. Production of polyhydroxybutyrate from wheat bran hydrolysate using Ralstonia eutropha through microbial fermentation. J Biotechnol. 2016;237:13–7. Rezk SA, Emam DAE-M, Swailam HM, Swelim MA. Production of Polyhydroxybutyrate (PHB) from Streptomyces Incanus and the Effect of Gamma Irradiation on its Production. Arab J Nuclear Sci Appl. 2020;53(2):111–7. Adnan M, Siddiqui AJ, Ashraf SA, Snoussi M, Badraoui R, Ibrahim AM, Alreshidi M, Sachidanandan M, Patel M. Characterization and process optimization for enhanced production of polyhydroxybutyrate (PHB)-based biodegradable polymer from Bacillus flexus isolated from municipal solid waste landfill site. Polymers. 2023;15(6):1407. Dañez JCA, Requiso PJ, Alfafara CG, Nayve FRP, Ventura J-RS. Optimization of fermentation factors for polyhydroxybutyrate (PHB) production using Bacillus megaterium PNCM 1890 in simulated glucose-xylose hydrolysates from agricultural residues. Philipp J Sci. 2020;149(1):163–75. Penkhrue W, Jendrossek D, Khanongnuch C, Pathom-Aree W, Aizawa T, Behrens RL, Lumyong S. Response surface method for polyhydroxybutyrate (PHB) bioplastic accumulation in Bacillus drentensis BP17 using pineapple peel. PLoS ONE. 2020;15(3):e0230443. Patil TD, Ghosh S, Agarwal A, Patel SKS, Tripathi AD, Mahato DK, Kumar P, Slama P, Pavlik A, Haque S. Production, optimization, scale up and characterization of polyhydoxyalkanoates copolymers utilizing dairy processing waste. Sci Rep. 2024;14(1):1620. Mohd Zain NF, Paramasivam M, Tan JS, Lim V, Lee CK. Response surface methodology optimization of polyhydroxyalkanoate production by Burkholderia cepacia BPT1213 using waste glycerol from palm oil-based biodiesel production. Biotechnol Progress. 2021;37(1):e3077. Das R, Pal A, Paul AK. Optimization of process parameters for production of poly (3-hydroxybutyrate) by Bacillus pumilus AHSD 04, a seed borne endophyte of oleaginous plant Arachis hypogaea L. Biointerf Res Appl Chem. 2022;12(4):5280. Samal S, Pati S, Mohapatra S, Maity S, Tanaya K, Devadarshini D, Samantaray D. PHAs production by facultative anaerobic bacteria, Bacillus cereus FM5 through submerged and solid-state fermentation under anoxic condition. Antonie Van Leeuwenhoek. 2023;116(6):521–9. Samrot AV, Samanvitha SK, Shobana N, Renitta ER, Senthilkumar P, Kumar SS, Abirami S, Dhiva S, Bavanilatha M, Prakash P, Saigeetha S, Shree KS, Thirumurugan R. The synthesis, characterization and applications of polyhydroxyalkanoates (PHAs) and PHA-based nanoparticles. Polymers. 2021;13(19):3302. McAdam B, Brennan Fournet M, McDonald P, Mojicevic M. Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polymers. 2020;12(12):2908. Mai J, Kockler K, Parisi E, Chan CM, Pratt S, Laycock B. Synthesis and physical properties of polyhydroxyalkanoate (PHA)-based block copolymers: A review. Int J Biol Macromol. 2024;263:130204. Mottin AC, Ayres E, Oréfice RL, Câmara JJD. What changes in poly (3-hydroxybutyrate)(PHB) when processed as electrospun nanofibers or thermo-compression molded film? Mater Resea. 2016;19(1):57–66. Nwinyi OC, Owolabi TA. Scanning electron microscopy and Fourier transmission analysis of polyhydroxyalkanoates isolated from bacteria species from abattoir in Ota, Nigeria. J King Saud Uni-Sci. 2019;31(3):285–98. Puvača N, Milenković J, Galonja Coghill T, Bursić V, Petrović A, Tanasković S, Pelić M, Ljubojević Pelić D, Miljković T. Antimicrobial activity of selected essential oils against selected pathogenic bacteria: In vitro study. Antibiotics. 2021;10(5):546. Ignatova L, Brazhnikova Y, Omirbekova A, Usmanova A. Polyhydroxyalkanoates (PHAs) from endophytic bacterial strains as potential biocontrol agents against postharvest diseases of apples. Polymers. 2023;15(9):2184. Bustamante-Torres M, Romero-Fierro D, Estrella-Nuñez J, Arcentales-Vera B, Chichande-Proaño E, Bucio E. Polymeric composite of magnetite iron oxide nanoparticles and their application in biomedicine: a review. Polymers. 2022;14(4):752. Martínez-Herrera RE, Alemán-Huerta ME, Rutiaga-Quiñones OM, de Luna-Santillana EJ, Elufisan TO. A comprehensive view of Bacillus cereus as a polyhydroxyalkanoate (PHA) producer: A promising alternative to Petroplastics. Process Biochem. 2023;129:281–92. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 May, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 10 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 07 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9349740","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627765471,"identity":"b17b7943-8f01-4c1e-bbba-76bb01d545e9","order_by":0,"name":"Gharishma Roy Sakthivel Latha","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Gharishma","middleName":"Roy Sakthivel","lastName":"Latha","suffix":""},{"id":627765473,"identity":"3be417a8-d57c-40ee-936e-c4c347077bb2","order_by":1,"name":"Uma Ganapathi","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Uma","middleName":"","lastName":"Ganapathi","suffix":""},{"id":627765475,"identity":"eed269e4-3929-4a62-9f60-039408b6859c","order_by":2,"name":"Rubyga Nagarajan","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Rubyga","middleName":"","lastName":"Nagarajan","suffix":""},{"id":627765480,"identity":"109a02de-e6fa-4fff-8959-b723dcdcdcca","order_by":3,"name":"Anusha John Radha","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Anusha","middleName":"John","lastName":"Radha","suffix":""},{"id":627765481,"identity":"381f9fa9-4422-43a7-9c33-4514ea9d3c07","order_by":4,"name":"Jeraldin Nisha Selvaraj","email":"","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Jeraldin","middleName":"Nisha","lastName":"Selvaraj","suffix":""},{"id":627765482,"identity":"f2ca6b1e-1396-4cc4-9e1b-eafb385db958","order_by":5,"name":"Sholkamy Essam Nageh","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Sholkamy","middleName":"Essam","lastName":"Nageh","suffix":""},{"id":627765483,"identity":"06247c61-0997-478f-a8cc-1d7502811d32","order_by":6,"name":"Citarasu Thavasimuthu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYDACZijNDyISCojSAtUj2QDSYkCKNQYHwCQRGvjb+Q9+rqipS9x8fnXihwcGDPL8Ygfwa5E4zMwseebY4cRtN95ulgA6zHDm7AT8WgyAfpFsYDsA1HJ2A0hLgsFtwlqYfzb8AzpsxtnNP4jVwibZ2MacuIG/dxtxtgD9YmbZ2HfYeMYN3m0WCQYShP3C33/w8c2Gb3Wy/f1nN9/8UWEjzy9NQAuSfWCVEsQqB9t3gBTVo2AUjIJRMJIAAOPeQhX3CrUdAAAAAElFTkSuQmCC","orcid":"","institution":"Manonmaniam Sundaranar University","correspondingAuthor":true,"prefix":"","firstName":"Citarasu","middleName":"","lastName":"Thavasimuthu","suffix":""}],"badges":[],"createdAt":"2026-04-07 23:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9349740/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9349740/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107700820,"identity":"f7961422-ebe3-4321-88ef-da001fdb6212","added_by":"auto","created_at":"2026-04-24 08:01:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":490998,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial strains isolated from solar salt works for PHA screening\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/ab527f49de406e0d2a530b74.png"},{"id":107707919,"identity":"a9342238-2241-43b6-983e-85361b1a85ea","added_by":"auto","created_at":"2026-04-24 09:21:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":404680,"visible":true,"origin":"","legend":"\u003cp\u003ePrimary screening using Sudan Black B (\u003cstrong\u003ea\u003c/strong\u003e) and Secondary screening using Nile Blue A (\u003cstrong\u003eb\u003c/strong\u003e). Red arrow shows the positive result\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/83f811adc5769576a1cab727.png"},{"id":107707905,"identity":"0aa4d72b-9513-4858-937f-a64c4d6829b7","added_by":"auto","created_at":"2026-04-24 09:21:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107636,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationship based on 16S rRNA of the isolate \u003cem\u003eBacillus cereus\u003c/em\u003ePHB CMST1\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/27c1bc4598e43936db5cd6be.png"},{"id":107707431,"identity":"d7108180-c4fc-427c-932e-26f3790e7662","added_by":"auto","created_at":"2026-04-24 09:20:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214081,"visible":true,"origin":"","legend":"\u003cp\u003eOFAT studies \u003cstrong\u003e(a)\u003c/strong\u003e temperature \u003cstrong\u003e(b)\u003c/strong\u003e pH \u003cstrong\u003e(c)\u003c/strong\u003e NaCl \u003cstrong\u003e(d)\u003c/strong\u003e Incubation period \u003cstrong\u003e(e)\u003c/strong\u003edifferent carbon source, and \u003cstrong\u003e(f)\u003c/strong\u003edifferent nitrogen source\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/c4779fe9c101b3c23c2620a2.png"},{"id":107707740,"identity":"059d0638-ed37-4985-8204-4f11f40bc9d2","added_by":"auto","created_at":"2026-04-24 09:21:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003ePareto chart for PHA production\u003cstrong\u003e (b) \u003c/strong\u003eResponse optimizer for PHA production\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/d0790c39d1df03a8d6f09497.png"},{"id":107707230,"identity":"3314cdab-2a98-4248-9290-c5c9c6a7f55d","added_by":"auto","created_at":"2026-04-24 09:19:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":324648,"visible":true,"origin":"","legend":"\u003cp\u003ePHA film prepared from the \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/5cfe8c3499982cd3f00d595d.png"},{"id":107707687,"identity":"55b6bc92-c21d-4b05-af48-c3d30246dab9","added_by":"auto","created_at":"2026-04-24 09:20:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176964,"visible":true,"origin":"","legend":"\u003cp\u003eUV-vis spectral analysis of PHA from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/7050a1971ea6567ec7db26c4.png"},{"id":107700826,"identity":"e1338e3f-ec21-4308-bfdf-bb55af7f43c4","added_by":"auto","created_at":"2026-04-24 08:01:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":95418,"visible":true,"origin":"","legend":"\u003cp\u003eTLC plate indicating the presence of PHA\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/beed7d944b0d3722e4a05cc8.png"},{"id":107707878,"identity":"7c20d03c-5d37-4252-a108-d27450c36772","added_by":"auto","created_at":"2026-04-24 09:21:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":123457,"visible":true,"origin":"","legend":"\u003cp\u003eFourier- transform infrared (FT-IR) spectra of PHA produced from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/e47359852ae8e7264f56f149.png"},{"id":109203711,"identity":"d1b8dd05-5f00-42ad-b563-892b346ae246","added_by":"auto","created_at":"2026-05-13 14:44:03","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":155312,"visible":true,"origin":"","legend":"\u003cp\u003eGC-MS confirmation for derivatives of PHA from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1 (\u003cstrong\u003ea\u003c/strong\u003e) hexanoic acid; (\u003cstrong\u003eb\u003c/strong\u003e) hexanedioic acid; (\u003cstrong\u003ec\u003c/strong\u003e) ethyl-3-hydroxy butyrate; (\u003cstrong\u003ed\u003c/strong\u003e) hexanoic acid, 4-methyl-, methyl ester; and (\u003cstrong\u003ee\u003c/strong\u003e) eicosanoic acid\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/3e5f68df56e2905830461f8b.png"},{"id":107700835,"identity":"8cf12014-a49d-4f1a-ae97-be07a86c4a41","added_by":"auto","created_at":"2026-04-24 08:01:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":60712,"visible":true,"origin":"","legend":"\u003cp\u003eThermogravimetric analysis of PHA produced from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/a2d3769c9c4ff0e8a3be58f9.png"},{"id":107707107,"identity":"64aee826-dc5b-475a-8d13-b561e347ae6f","added_by":"auto","created_at":"2026-04-24 09:19:32","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":90227,"visible":true,"origin":"","legend":"\u003cp\u003eXRD analysis of PHA produced from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/ee92149452725912ae59c8d3.png"},{"id":107868808,"identity":"e4e11ae8-5fd7-44bf-b601-4f7d065d5491","added_by":"auto","created_at":"2026-04-27 07:34:10","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":540892,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographof PHA sheets at different magnification derived from \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003ecereus\u003c/em\u003e strain PHB CMST1 (Scale bar A: 10µM; B: 20µM and C: 2µM)\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/5466d0115b9ecb075a64e974.png"},{"id":107707326,"identity":"4d2319b0-3c5a-47bd-8750-6c5c61367a51","added_by":"auto","created_at":"2026-04-24 09:20:05","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":269364,"visible":true,"origin":"","legend":"\u003cp\u003eMicrostructure of surface morphology, and mapping of elements in PHA from \u003cem\u003eBacillus cereus \u003c/em\u003estrain PHB CMST1\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/74031b36e0707eed7462a0e3.png"},{"id":107707877,"identity":"0d5aecfb-11e3-4f07-87ef-bfbe879162b6","added_by":"auto","created_at":"2026-04-24 09:21:19","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":1192823,"visible":true,"origin":"","legend":"\u003cp\u003eHalo test for PHA biofilm with essential oils (Lemongrass (Le), Eucalyptus (Eu) and Clove oil (Clov), Positive control (+ve), Negative control (-ve)) against eleven pathogens (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e,\u003cem\u003e Streptococcus pyogenes\u003c/em\u003e,\u003cem\u003eListeria \u003c/em\u003esp., \u003cem\u003eBacillus subtilis\u003c/em\u003e,\u003cem\u003e Enterococcus faecalis\u003c/em\u003e,\u003cem\u003eSalmonella \u003c/em\u003esp.,\u003cem\u003e Shigella \u003c/em\u003esp.,\u003cem\u003e Yersinia \u003c/em\u003esp.,\u003cem\u003e Pseudomonas aeruginosa\u003c/em\u003e,\u003cem\u003e Klebsiella pneumoniae\u003c/em\u003e, and\u003cem\u003e Escherichia coli\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/f4af6f409193277bbfabe4b9.png"},{"id":109205920,"identity":"014e89b2-1b98-4147-a3ae-64d3a3ab5167","added_by":"auto","created_at":"2026-05-13 15:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5235422,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9349740/v1/9db4eacf-5615-4cc3-b29b-52102daa8619.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacillus cereus PHB CMST1: A Potential Halophilic Bacterium for Cost-Effective and Sustainable Production of Polyhydroxyalkanoates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing reliance on conventional plastics in our daily lives has prompted a search for more sustainable alternatives. Microorganisms, including bacteria, fungi, actinomycetes, and yeasts, play a crucial role in the natural production and degradation of biodegradable plastics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Biopolymers such as polyhydroxyalkanoates (PHAs), including poly(3-hydroxybutyrate) (PHB), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), polylactic acid (PLA), and polycaprolactone, present significant potential to replace traditional plastics [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PHAs are not only biodegradable and biocompatible but also naturally accumulate within bacterial cells as amorphous granules.\u003c/p\u003e \u003cp\u003eSince the 1970s, research into PHAs has expanded across scientific and industrial sectors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Different bacterial species produce PHAs with varying structures, with notable PHA producers found in marine environments, including \u003cem\u003eAlteromonas\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eVibrio\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The applications of PHAs span industrial and medical fields, including drug delivery, packaging, agriculture, and printing.\u003c/p\u003e \u003cp\u003eDespite the promising potential of PHA-accumulating bacteria for commercial bioplastic production, challenges remain due to the high cost of raw materials. Utilizing affordable and renewable agro-industrial by-products as carbon sources could significantly reduce PHA production costs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Various organic wastes from the food industry, agricultural residues, and wastewater have been explored as potential substrates. Selecting bacterial strains with high PHA productivity and optimizing growth conditions are essential for facilitating economically viable biosynthesis.\u003c/p\u003e \u003cp\u003eThe high production costs associated with PHA stem from the use of expensive raw materials. Strategies such as employing diverse carbon sources\u0026mdash;ranging from natural products to industrial and agro-industrial wastes\u0026mdash;have been implemented to mitigate these costs. Efficient management of bioprocesses is critical for reducing the expenses of commercial biotechnology products. Traditional optimization methods, which often focus on one variable at a time, can lead to inaccurate results and extended timeframes. Moreover, these methods may overlook the interactions between different factors affecting production. In contrast, statistical experimental designs like factorial analysis and response surface methodology (RSM) provide a more reliable framework for optimization. RSM considers both individual and interactive effects of various factors, facilitating a comprehensive approach to optimizing production conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to isolate and identify bacteria capable of producing PHA from brine and sediment samples collected from the Puthalam salt pan. Additionally, we sought to optimize the physical and chemical factors influencing PHA production by integrating a one-factor-at-a-time (OFAT) approach with response surface methodology-central composite design (RSM-CCD). Low-cost agro-industrial wastes were utilized as carbon sources, and the resulting PHAs were characterized through techniques such as thin-layer chromatography (TLC), Fourier-transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCollection of sample and isolation of bacteria\u003c/p\u003e \u003cp\u003eBrine and sediment samples were collected from the Puthalam salt pan in the Kanyakumari district (Latitude 8.100361˚ and Longitude 77.479256˚). Serial dilution was carried out using 1 ml of the brine and sediment sample (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e) to isolate the bacteria. After spreading out the aliquots on sterile Zobell Marine Agar medium plates, the plates were incubated for 24 h at 28\u0026deg;C. The colonies with various distinguishing characteristics were selected; pure cultures were maintained and kept for later usage at 4\u0026deg;C [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScreening of PHAs producing bacteria from the isolated bacteria\u003c/p\u003e \u003cp\u003eSudan Black B (SBB) was used for the primary screening for isolating the PHA producing bacterial colonies. The Zobell Marine Agar plates with the bacterial isolates were flooded with 0.05% SSB for 20 min. The isolates which produce PHAs appeared as bluish black in colour [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNile Blue A (NBA) staining was carried out as the secondary screening of PHA producing bacterial colonies. The plates with bacterial isolates were strained with 1% NBA for 24 h. The isolates which showed bright orange fluorescence under UV light were selected as PHA accumulators [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtraction of PHA in the selected PHA producing bacterial isolates\u003c/p\u003e \u003cp\u003eThe PHA was directly extracted using dispersion methods of sodium hypochlorite and chloroform with slight modification [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 100 ml of bacterial culture was centrifuged at 8500 x g for 15 min. The pellet was washed with phosphate buffer saline. The cell pellets were air dried and weighed (DCW). The cells were treated with 4% sodium hypochlorite at 37\u0026deg; C for 2 h. The mixture was again centrifuged at 5000 X g for 20 min, and the cell was washed with acetone and methanol in a 1:1 ratio. The cell was dissolved in 5 ml of chloroform and kept at room temperature for evaporation. The PHA obtained was weighed and the weight was noted (determined as the PHA extract weight).\u003c/p\u003e \u003cp\u003eResidual biomass (g/l)\u0026thinsp;=\u0026thinsp;Dry Cell Weight (g/l)\u0026thinsp;\u0026minus;\u0026thinsp;Dry weight of extracted PHAs (g/l)\u003c/p\u003e \u003cp\u003ePHA accumulation (%)\u0026thinsp;=\u0026thinsp;Dry weight of extracted PHAs (g/l) \u0026times;100%\u003cb\u003e/\u003c/b\u003e Dry Cell Weight (g/l)\u003c/p\u003e \u003cp\u003eIdentification of the selected bacteria\u003c/p\u003e \u003cp\u003eMorphological and Biochemical based identification\u003c/p\u003e \u003cp\u003eThe morphology of bacterial colonies in terms of shape, colour, arrangement, size, motility, and Gram-staining were observed using the microscope [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The bacterial isolates were tested for biochemical characterization such as indole production, methyl red (MR), Voges Proskauer (VP), citrate utilization, catalase test, oxidase test, urease production, Triple Sugar Iron (TSI) test, nitrate reduction test, and blood hemolysis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and enzymatic activities such as amylase, gelatinase, protease [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and lipase [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] were also performed.\u003c/p\u003e \u003cp\u003eMolecular identification of the selected isolate\u003c/p\u003e \u003cp\u003eThe genomic DNA of the selected bacterial strain was extracted from culture following the phenol-chloroform method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. PHA-producing bacteria was identified using the 16S rRNA sequence [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and using universal primers p27F (5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-TAC GGC ACC TTG TTA CGA CTT-3\u0026prime;). Based on the phylogenetic determination, the gene sequence of the strain was submitted to the National Centre for Biotechnological Information (NCBI) to get the accession number.\u003c/p\u003e \u003cp\u003eOptimization of media composition to produce PHA\u003c/p\u003e \u003cp\u003eOne factor at a time (OFAT)\u003c/p\u003e \u003cp\u003eThe physical, and chemical, culture conditions for PHA production were optimized using one factor at a time (OFAT). The physical factors such as temperature (25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C, and 40\u0026deg;C); salinity (2%, 3%, 4%, and 5%); pH (5, 6, 7, 8, and 9); and incubation days (1 to 4 days) were optimized for PHA production using minimal salt media. To optimize nutritional culture, the different renewable agro-industrial wastes such as rice bran, wheat bran, tamarind tunnel, and fruit pulp from fruits as carbon sources (2%) and 0.15% nitrogen sources such as beef extract, ammonium chloride, ammonium sulphate, and urea were selected by changing one factor at a time in the minimal salt media [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResponse Surface Methodology-Central Composite Design (RSM-CCD)\u003c/p\u003e \u003cp\u003eBased on pre-optimization by OFAT, the renewable agro-industrial substrate wheat bran as carbon source, and urea as nitrogen source, was selected. Using MINITAB 19 software with two factors (carbon and nitrogen sources) and five levels, the combinations of 13 experiments were generated and each factor was studied at five experimental levels: -α (very low), -1 (low), 0 (central point), +\u0026thinsp;1 (high), +α (very high) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePHA biofilm preparation\u003c/p\u003e \u003cp\u003e50 mg of PHA extracted from bacterial cells was dissolved in 1 ml of chloroform in a falcon centrifuge tube using a slightly modified version [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The resultant mixture was maintained at 50\u0026deg;C in the oven until all the chloroform had evaporated. PHA coatings developed in the falcon centrifuge tube because of the chloroform evaporating.\u003c/p\u003e \u003cp\u003eCharacterization of crude PHA\u003c/p\u003e \u003cp\u003eUV- Visible Spectrophotometer\u003c/p\u003e \u003cp\u003eThe extracted PHA was dissolved in chloroform; sulfuric acid was added and heated in a boiling water bath for 20 min at 100\u003csup\u003eo\u003c/sup\u003eC. The absorbance of the sample was measured at the range of 200\u0026ndash;800 nm using UV-Visible spectrophotometer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThin Layer Chromatography\u003c/p\u003e \u003cp\u003eAfter dissolving the PHA biofilm in chloroform, a drop of the mixture was placed on a silica-coated TLC plate. TLC was performed using methanol and chloroform (1:1) for 40 min, and the plates were incubated in an iodine chamber in a water bath for 5 to 10 min [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFourier-Transform Infrared Spectroscopy (FT-IR)\u003c/p\u003e \u003cp\u003eThe PHA biofilm extracted from the culture was analyzed using Fourier-transform infrared spectroscopy (FT-IR) with a Shimadzu instrument (Japan). The analysis was conducted at a resolution of 4 cm⁻\u0026sup1;, with 40 scans performed per sample. The spectral frequency range was set between 4000 and 400 cm⁻\u0026sup1;, and the resulting vibration spectrum was recorded as a graphical output [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGas Chromatography\u0026ndash;Mass Spectrometry (GC-MS)\u003c/p\u003e \u003cp\u003eFor the GC-MS analysis of the PHA biofilm, methanolysis was performed. The analysis was conducted using the Agilent Technologies 7000E and 7010C GC-MS systems (Agilent Technologies, CA, US). Samples were injected in split mode, with the column oven temperature maintained at 50\u0026deg;C and the injection temperature set to 250\u0026deg;C. The flow control was managed in pressure mode [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThermogravimetric Analysis (TGA)\u003c/p\u003e \u003cp\u003eThe extracted PHA was subjected to thermogravimetric analysis (TGA) using the EXSTAR SIINT 6300 thermal system. Approximately 20 mg of the dried sample was used for the TGA experiment. Thermograms were generated under an airflow rate of 50 ml/min and a heating rate of 10\u0026deg;C/min, covering a temperature range from 30\u0026deg;C to 800\u0026deg;C. The heat flow and weight loss, measured in milligrams, were plotted against temperature [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eX-ray Diffraction (XRD)\u003c/p\u003e \u003cp\u003eX-ray diffraction analysis was performed using a D2 Phaser (BRUKER) with CuKα irradiation to determine the crystallinity of the PHA polymer. The sample was analyzed by the X-ray diffractometer over a scanning range of 10\u0026deg; to 80\u0026deg; in 2θ, with a step size of 0.02\u0026deg; [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDAX)\u003c/p\u003e \u003cp\u003eThe surface topology and chemical composition of the PHA films were examined using scanning electron microscopy (SEM). The films were affixed to sample holders coated with carbon tape and subsequently analyzed using the Carl Zeiss EVO18 system [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimicrobial activity of PHA with essential oils\u003c/p\u003e \u003cp\u003eAccording to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the halo test was performed against pathogens (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e, \u003cem\u003eListeria\u003c/em\u003e sp., \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e sp., \u003cem\u003eShigella\u003c/em\u003e sp., \u003cem\u003eYersinia\u003c/em\u003e sp., \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e) by disc diffusion method. The PHA films loaded with essential oils (20\u0026micro;l), such as lemon grass, eucalyptus, and clove oil, were evaluated for their antibacterial activity. Antibiotic discs containing oxacillin (1 \u0026micro;g/disc) and gentamicin (300 \u0026micro;g/disc) were employed as positive controls against pathogens, while films devoid of essential oils were utilized as negative controls [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eTriplicates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) of each experiment were carried out. Microsoft Excel\u0026rsquo;s standard software package was used to undertake a statistical analysis of the results. The findings were shown as mean\u0026thinsp;+\u0026thinsp;standard deviation, or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIsolation and Screening for PHA producing bacterial isolates\u003c/p\u003e \u003cp\u003eUsing serial dilution, sixteen bacterial isolates (seven from the sediment sample and nine from the brine) with distinct morphologies and colours were selected for PHA screening (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sixteen bacterial isolates were tested by Sudan Black B, two isolates were positively stained for primary screening (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Following the initial screening, Nile Blue A stain was used for a secondary screening. Two isolates out of sixteen had positive staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) in secondary screening. Following primary and secondary screening, two isolates from the brine sample, Z6 and Z9, were selected for further examination.\u003c/p\u003e \u003cp\u003eExtraction for PHA producing bacteria\u003c/p\u003e \u003cp\u003eThe sodium hypochlorite-chloroform method was employed on the two isolates (Z6, Z9) for PHA extraction. The result showed that the isolates Z6 and Z9 generated 13% and 25% of PHA, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Z9, which generated the higher quantity of PHA, was selected to undergo further optimization and characterization studies.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePHA production from the bacterial isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDry cell weight (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePHA extract weight (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResidual biomass (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePHA accumulation (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIdentification of selected PHA producing isolates Z9\u003c/p\u003e \u003cp\u003eThe chosen isolate, Z9, was rod-shaped, Gram-positive, and motile. Under aerobic conditions, it grew into an off-white-coloured spore form on an agar plate. The isolate was tested positive for Voges-Proskauer, citrate utilization, catalase, urease production, triple sugar iron, nitrate reduction, and hemolytic activity, enzymatic activities such as amylase, gelatinase, protease, and lipase. Based on the results of the morphological and biochemical tests, chosen isolate Z9 was identified as \u003cem\u003eBacillus\u003c/em\u003e sp. (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Phylogenetic analysis of the 16S rRNA sequences revealed that Z9 was confirmed as \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The sequence was submitted to NCBI, and accession number OQ804397.1 was obtained.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiochemical identification and enzyme assay for the selected isolate Z9\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIndole test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative (-)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethyl red test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative (-)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVoges-Proskauer test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitrate utilization test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatalase test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxidase test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative (-)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrease production\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTriple sugar iron (TSI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNitrate reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHemolytic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eEnzymatic assay:\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGelatinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elipase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOptimization of media components for PHA production\u003c/p\u003e \u003cp\u003eEffect of physical and chemical factors on PHA production by OFAT\u003c/p\u003e \u003cp\u003eThe findings demonstrated that the greatest amount of PHA was produced at 35\u0026deg;C (4.1 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). At pH 7 (89.6 g/L), PHA synthesis reached its peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The highest PHA output was reported at 2% (5.2 g/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) NaCl. After three days of incubation (2 g/L), the highest amount of PHA was produced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The carbon source, wheat bran (62.8 g/L), produced a considerable amount of PHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The maximum amount of PHA (8.4 g/L) was obtained in the nitrogen source, urea (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eResponse Surface Methodology \u0026ndash; Central Composite Design (RSM-CCD) on media components for PHA production\u003c/p\u003e \u003cp\u003eThe results of experiments with a central composite design and second-order polynomial multiple regression is shown in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The experimental and predicted values of the experimental design did not significantly differ. The maximum PHA production was 367.645 g/L at the concentrations of urea 2.14121% (nitrogen source) and wheat bran 4.41421% (carbon source). The model predicted an R\u003csup\u003e2\u003c/sup\u003e value of 99.97% with 0.03% of the variance for PHA production, confirming that it was highly significant. The \u003cem\u003eP\u003c/em\u003e values for the linear term, the quadratic coefficient, and the interactive coefficient were 0.000, which is less than 0.05, indicating that they played a significant role in PHA production. The current study found the lack of fit test values for these responses to be insignificant, leading to the acceptance of the model. The Pareto graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) verified the stronger effects in the upper portion and their progression down to the bottom section, with p values less than 0.05, indicating their significant contribution to PHA production compared to other components. From the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, the \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1, the predicted optimal values of wheat bran and urea for PHA production were calculated to be 5% and 2.5%, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResponse Surface Methodology \u0026ndash; Central Composite Design (RSM-CCD) on media components for PHA production and it\u0026rsquo;s observed and predicted values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRun Order\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eWheat Bran (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUrea (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDry cell weight (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePHA (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eExperimental\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePredicted\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eExperimental\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003ePredicted\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e180.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e174.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e173.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e251.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e255.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e235.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e237.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e149.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e151.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e132.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e132.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e294.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e297.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e288.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e290.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e135.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e135.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e117.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e119.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e189.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e185.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e171.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e169.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e387.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e386.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e367.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e367.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e215.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e190.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e355.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e354.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e332.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e331.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e180.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e174.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e173.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePHA biofilm preparation by PHA producing bacteria\u003c/p\u003e \u003cp\u003eThe prepared biofilm was easily brittle (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The isolated \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1 produced PHA that fully dissolved in chloroform, a distinctive characteristic of PHA.\u003c/p\u003e \u003cp\u003eCharacterization of PHA extracted from \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1\u003c/p\u003e \u003cp\u003eUV-Visible spectral analysis\u003c/p\u003e \u003cp\u003eThe UV Visible spectroscopy revealed the absorbance maximum at a range between 235 to 440 nm indicating the presence of PHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThin Layer Chromatography\u003c/p\u003e \u003cp\u003eThe TLC analysis revealed yellowish-green spots on the TLC plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e) with R\u003csub\u003ef\u003c/sub\u003e value of 0.783 indicated the presence of PHAs.\u003c/p\u003e \u003cp\u003eFourier- transform infrared (FT-IR)\u003c/p\u003e \u003cp\u003eThe FT-IR spectroscopic analysis gave further insights into the chemical structure of the polymer and reflected the monomeric units (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e9\u003c/span\u003e). There absorption bands were observed at 697 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 825 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which may suggest the presence of \u0026#120573;-glycosidic linkages between the sugar monomers. The band at 1056 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e corresponds to the valence vibration of the carboxylic group (COOH). This demonstrated that plastic had formed, most likely as a polymer with a carboxylic acid side chain. It is crucial to consider the other bands in C-O-C stretching, which occur at 1102 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, 1132 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, and 1185 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, as they play a significant role in identifying and characterizing PHA monomers. There was a peak at 1737 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e indicating the presence of an aliphatic carbonyl group (C\u0026thinsp;=\u0026thinsp;O valence) in the PHA polymer. Another peak at 1660 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e suggested the presence of an alkene C\u0026thinsp;=\u0026thinsp;O valence, while a peak at 1531 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e indicated the presence of an amide C\u0026thinsp;=\u0026thinsp;O valence. The vibrational frequencies at 2875 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 2972 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e indicated the presence of influential stretching groups in alkanes, specifically \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e and \u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e, these frequencies also reflect the methylene's intensity. At 3069cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 3272 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, the band showcased O\u0026ndash;H bending, suggesting the presence of hydrogen bonds, the broad nature and low frequency value further support this observation. The fact that the PHA molecule has a strong carbonyl absorption peak at 1737 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e (absorbance of carbonyl band) in the infrared spectrum.\u003c/p\u003e \u003cp\u003eGas chromatography and mass spectroscopy\u003c/p\u003e \u003cp\u003eGC-MS analysis determined the monomeric content of the PHA polymer. The main peak's resemblance the monomer composition 3-hydroxybutyrate (3HB) of mcl-PHA biopolymer. The PHA extract revealed five peaks with retention times (RT) of 4.885, 8.165, 10.595, 12.340, and 17.570 min. These include hexanoic acid; hexanedioic acid; ethyl-3-hydroxybutyrate; hexanoic acid, 4-methyl-, methyl ester; and eicosanoic acid. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e, displayed the major compounds along with their respective RT, peak area, molecular formula, and molecular weight.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDerivatives of PHA confirmed through GC-MS analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDerivatives\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArea %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular weight\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA) Hexanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.885\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB) Hexanedioic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC) Ethyl 3 hydroxybutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD) Hexanoic acid, 4-methyl-, methyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE) Eicosanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThermogravimetric and Differential thermal analysis\u003c/p\u003e \u003cp\u003eThe thermogravimetric and differential thermal analysis showed a total weight loss of 92% occured at 800\u0026deg;C, with 10% weight loss up to 250\u0026deg;C, 60% up to 550\u0026deg;C, and 20% up to 800\u0026deg;C. Additionally, there are sharp exothermic peaks at 520\u0026deg;C and 600\u0026deg;C. The TG/DTA results provide insights into the thermal degradation processes of the PHA. We can attribute the stepwise weight loss to the following events: 10% weight loss up to 250\u0026deg;C, most likely because bound water molecules and/or volatile components are released; 60% weight loss up to 550\u0026deg;C, because the of the breakdown of PHA polymer chains and releasing of gaseous products; and 20% weight loss up to 800\u0026deg;C was due to the last stages of combustion of the remaining organic material.\u003c/p\u003e \u003cp\u003eThe sharp exothermic peaks at 520\u0026deg;C and 600\u0026deg;C indicated significant heat release events, suggesting combustion or decomposition processes occurring in the material. The peak at 520\u0026deg;C could be associated with the initial decomposition and combustion of the PHA material, while the peak at 600\u0026deg;C may correspond to the further breakdown and combustion of the remaining polymer chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eX-ray diffraction\u003c/p\u003e \u003cp\u003eThe X-ray diffraction (XRD) pattern of the polyhydroxyalkanoate (PHA) sample is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e. The pattern reveals a sharp peak at 25.62\u0026deg; and a broad hump centered around 19\u0026ndash;20\u0026deg;, with another broad region extending towards 40\u0026ndash;45\u0026deg;. These observations provide insight into the crystalline and amorphous nature of the material. The sharp peak at 25.62\u0026deg; suggests the presence of some crystalline regions within the polymer, while the broad peaks at 19\u0026ndash;20\u0026deg; and around 40\u0026deg; are indicative of significant amorphous content. Based on quantitative analysis by BRUKER Diffrac. EVA software, the degree of crystallinity was calculated to be 5.7%, with the remaining 94.3% contributed to amorphous regions.\u003c/p\u003e \u003cp\u003eSEM-EDAX mapping\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e13\u003c/span\u003e displays the microstructure of PHA derived from \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1. The microstructure revealed a porous material with finely linked grains and a strong propensity to form multigrain agglomerates. The chemical composition (wt.%) result revealed 49.8 carbon, 33.9 oxygen, 4.9 sodium, 4.1 phosphorus, 1.4 sulfur, 2.0 chlorine, 1.5 potassium, and 2.4 iron in the sample. These results align with their elemental signals, showcasing the colour-coded mapping of various elements such as carbon, oxygen, sodium, phosphorus, sulfur, chlorine, potassium, and iron (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition in PHA from \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAtomic %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eError %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003csub\u003eratio\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1606\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0591\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0177\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0322\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0111\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0161\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00129\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0206\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAntimicrobial activity using essential oils\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e15\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Gentamicin, the positive control, demonstrated high activity against all eleven tested pathogens. The product PHA did not show any zone of inhibition against the pathogens tested, indicating that it had no antimicrobial activity. Lemongrass oil, along with PHA, had the highest activity among the three essential oils tested with PHA. Eucalyptus oil had the least activity among the three essential oils, along with PHA. The essential clove oil, along with PHA, showed moderate activity against the tested pathogens.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eZone formation (cm) of PHA films and essential oils in halo test\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathogen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLemongrass oil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEucalyptus oil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eClove oil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eShigella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS. pyogenes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumonia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYersinia\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eListeria\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe environmental impact of petroleum-based plastics has intensified the global shift towards biodegradable alternatives, such as polyhydroxyalkanoates (PHAs), which offer unique thermoplastic properties. However, the high production costs associated with bioplastics pose significant barriers to widespread adoption. Identifying bacterial strains with enhanced productivity and optimizing their growth conditions are essential for reducing PHA production expenses. Over 300 bacterial species have been linked to PHA accumulation across diverse environments [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we successfully identified a PHA-producing \u003cem\u003eBacillus\u003c/em\u003e strain from brine samples collected from a salt pan. Previous research, such as that by Martinez-Gutierrez et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], identified multiple PHA-producing bacteria from hypersaline microbial mats, while Muigano et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] documented numerous isolates from Kenya\u0026rsquo;s haloalkaline lakes. Our findings, which yielded 16 isolates from sediment and water samples in the Puthalam salt pan, were screened using Sudan Black B and Nile Blue A staining to assess PHA production. Notably, two strains, Z6 and Z9, exhibited positive results during both primary and secondary screenings [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThrough morphological, biochemical, and molecular characterization, we identified isolate Z9 as Bacillus cereus. Previous studies have highlighted Bacillus species as effective PHA producers due to their simpler extraction processes and ability to secrete hydrolytic enzymes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our results align with existing literature, confirming the significance of Bacillus strains in PHA production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTemperature is a critical factor influencing PHA accumulation. Our study indicated minimal PHA production outside the optimal range of 15\u0026deg;C to 50\u0026deg;C, likely due to reduced enzymatic activity at extreme temperatures [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This aligns with findings from Hamdy et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and Yasin and Al-Mayaly [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], who reported peak PHA production at 35\u0026deg;C for various \u003cem\u003eBacillus cereus\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003epH also plays a vital role in PHA biosynthesis. We maintained a neutral starting pH of 7.0, which facilitated maximum PHA production by minimizing energy costs associated with substrate uptake [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This finding is consistent with other studies that have reported similar optimal pH levels for PHA-producing bacteria [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe concentration of NaCl can enhance PHA production as it affects osmotic pressure, influencing cell growth and metabolism. Our study found that a 2% NaCl concentration yielded the highest PHA production, supporting the idea that osmotic conditions can promote PHB accumulation by creating a favourable environment for metabolic efficiency [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtended incubation periods encourage microbial growth, leading to increased PHA accumulation. However, after reaching the logarithmic growth phase, microbial growth declines, which subsequently reduces PHA production. Our findings suggest that the optimal incubation period for maximizing PHA yield is around 72 hours, corroborating observations from previous studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroorganisms have the remarkable ability to utilize a variety of carbon sources for the synthesis of polyhydroxyalkanoates (PHAs). These carbon sources serve as substrates for the production of precursors that can be further polymerized into PHAs, depending on the metabolic pathways employed by different bacterial strains. In our study, we explored the feasibility of using agricultural waste as an alternative to glucose for PHA production by \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1. We evaluated several carbon sources, including rice bran, wheat bran, tamarind pulp, and various fruit pulps, and measured both the dry cell weight and the resultant PHA accumulation. Our results indicated that wheat bran was the most effective substrate for PHA production. This finding is significant, considering that the cereal industry generates substantial quantities of wheat bran as a byproduct. Wheat bran is composed of approximately 19% starch, 18% protein, 6% lignin, and 38% non-starch polysaccharides, which include cellulose and arabinoxylans [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Previous studies support our findings; for instance, Rezk et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] reported that \u003cem\u003eStreptomyces incanus\u003c/em\u003e effectively utilized wheat bran, which is rich in hemicellulose and cellulose, to produce significant amounts of PHB. Similarly, Adnan et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] found that the \u003cem\u003eBacillus flexus\u003c/em\u003e HSA3 strain achieved optimal PHB production when grown on wheat bran.\u003c/p\u003e \u003cp\u003eNitrogen is a critical nutrient for the growth of all microorganisms, and it plays a vital role in the synthesis of polyhydroxyalkanoates (PHAs). Different PHA-producing strains exhibit varying preferences for nitrogen sources, including urea, nitrate, and ammonia. The choice of nitrogen source and its concentration significantly impact both microbial growth and PHA production. Limiting nitrogen availability can actually promote PHA accumulation, as studies suggest that under nutrient-limited conditions, the production of PHA may redirect acetyl-CoA from the Krebs cycle toward polymer synthesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In our investigation, we assessed the effects of various nitrogen sources\u0026mdash;namely beef extract, ammonium chloride, ammonium sulfate, and urea\u0026mdash;at a concentration of 0.15% on PHA production by \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1. Our results demonstrated that urea was the most effective nitrogen source for enhancing PHA synthesis. Its smaller molecular size and higher polarity facilitate efficient cellular uptake, making it a promising candidate for PHA production [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Supporting our findings, Wang et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] explored the utilization of various nitrogen sources, including urea, for PHA production in \u003cem\u003eBurkholderia cepacia\u003c/em\u003e. Our study further optimized the conditions for PHA production from \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1, establishing that the ideal parameters were 35\u0026deg;C, pH 7, 2% salinity, 3 days of incubation, with 2% wheat bran as the carbon source and urea as the nitrogen source .\u003c/p\u003e \u003cp\u003eOur study identified the optimal conditions for polyhydroxyalkanoate (PHA) production as 35\u0026deg;C, pH 7, 2% salinity, a 3-day incubation period, 2% wheat bran as the carbon source, and 0.15% urea as the nitrogen source. These findings highlight the significance of customized growth parameters in maximizing PHA yield. Previous research has similarly explored the optimization of media components for PHA production. For instance, Penkhrue et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] optimized the conditions for producing PHB from \u003cem\u003eBacillus drentensis\u003c/em\u003e BP17 using pineapple peel through response surface methodology (RSM) and central composite design (CCD). Additionally, Mohapatra et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] demonstrated effective parameter optimization for increasing PHA content in R. eutropha from whey hydrolysate, achieving statistically significant results. Hamdy et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] also employed RSM-CCD to enhance PHA production in \u003cem\u003eBacillus cereus\u003c/em\u003e strain SH-02, reinforcing the utility of these statistical methods in bioprocess optimization. Patil et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] conducted a detailed optimization study using a central composite rotatable design, focusing on two process variables, tryptophan and Tween 80 concentrations. Their results showed a close alignment between predicted and experimental PHA production, underscoring the accuracy of their optimization approach. In our investigation, the RSM-optimized medium yielded the highest PHA content and concentration, demonstrating a robust correlation with experimental outcomes. Notably, an optimal method for maximizing PHB synthesis was identified, which involved using 5% wheat bran as the carbon source and 2.5% urea as the nitrogen source [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe coefficient of determination, R\u003csup\u003e2\u003c/sup\u003e, had a value of 99.97%. The R\u003csup\u003e2\u003c/sup\u003e is a metric that quantifies the accuracy of a model's fit, taking on values between 0 and 1. The higher the model's strength and predictive accuracy, the closer the R\u003csup\u003e2\u003c/sup\u003e value approaches unity. Alternatively, lower R\u003csup\u003e2\u003c/sup\u003e values indicate that the response variables are inadequate in accounting for the observed variation. In this experiment, the R\u003csup\u003e2\u003c/sup\u003e values demonstrated that it has the ability to explain over 99% of the variability in PHA content. The adjusted R\u003csup\u003e2\u003c/sup\u003e value was 99.95%, which accounts for the sample size and number of words required to correct the R\u003csup\u003e2\u003c/sup\u003e value [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this investigation, the absorption spectrum of \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1 displayed peaks between 235 and 440 nm, confirming the presence of polyhydroxyalkanoates (PHAs). This observation aligns with previous research by Mandagutti and Sudhakar [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], who reported similar findings in PHB extracts from \u003cem\u003eBacillus paraconglomeratum\u003c/em\u003e. Additionally, our study identified yellowish-green spots on TLC plates, corroborating results previously reported by Rao et al [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The infrared spectroscopic analysis of the produced PHAs revealed a significant absorption peak at 1718.50 cm⁻\u0026sup1;, which corresponds to the carbonyl ester (C\u0026thinsp;=\u0026thinsp;O) functional group typical of PHBs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our findings, we observed high absorption peaks at 1737 cm⁻\u0026sup1;, 2972 cm⁻\u0026sup1;, and 2875 cm⁻\u0026sup1;, indicating the presence of both C\u0026thinsp;=\u0026thinsp;O and O-H functional groups. Supporting this, Samrot et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] identified 3-hydroxybutyrate and fatty acid methyl esters as markers for PHB production.\u003c/p\u003e \u003cp\u003eThe synthesis of fatty acids through a de novo pathway involves the conversion of simple sugars into 3-hydroxyacyl (3HA) precursors. These precursors are subsequently transformed into malonyl-CoA via acetyl-CoA. A specific CoA transferase, known as PhaG, then catalyzes the production of (R)-3-hydroxyfatty acids [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our analysis using gas chromatography-mass spectrometry (GC-MS) identified derivative products such as butanoic acid, 4-methyl-hexanoic acid methyl ester, and monomethyl hexanedioate, which helped confirm the structure of PHB from \u003cem\u003ePseudodonghicola xiamenensis\u003c/em\u003e. Furthermore, GC-MS results from PHB isolated from Bacillus licheniformis MSBN1 and \u003cem\u003eB. megaterium\u003c/em\u003e indicated the presence of 3-hydroxybutyrate, reinforcing the structural integrity of the polymer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In our study, we also detected these esters and 3-hydroxybutyrate in \u003cem\u003eBacillus cereus\u003c/em\u003e strain PHB CMST1, providing compelling evidence for the existence of PHB in this strain.\u003c/p\u003e \u003cp\u003eBacteria within the \u003cem\u003eBacillus\u003c/em\u003e genus are well-known for accumulating short-chain polyhydroxyalkanoates (PHAs), particularly polyhydroxybutyrate (PHB). Previous studies, such as that by Pillai et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], have characterized the thermal properties of standard PHB, noting degradation temperatures at 212\u0026deg;C and 266\u0026deg;C, with thermal degradation beginning at 247\u0026deg;C and peaking at 287\u0026deg;C. In our current investigation, thermogravimetric analysis (TGA) revealed distinct exothermic peaks at 520\u0026deg;C and 600\u0026deg;C, indicating the thermal stability of the produced polymer. X-ray diffraction (XRD) analysis further elucidated the structural characteristics of the material. Strong and intense peaks in the diffraction data suggest a crystalline nature, while our findings indicated a semi-crystalline structure with broad peaks at approximately 20 and 43 degrees [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This relatively low crystallinity is typical of many PHA polymers, which often display a mixture of amorphous and semi-crystalline phases [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The absence of distinct PHB peaks may reflect the conditions under which the polymer was prepared, potentially inhibiting its crystallization [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. These diffractogram results are consistent with prior study [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], reinforcing the understanding of PHA crystallinity. Additionally, Nwinyi and Owolabi [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] demonstrated that the microstructure and surface morphology of PHA samples, analyzed using scanning electron microscopy coupled with energy dispersive spectroscopy, exhibited porous and interconnected microstructures. These characteristics enhance the extraction efficiency of PHA from bacterial isolates (\u003cem\u003eRhodococcus\u003c/em\u003e sp., \u003cem\u003eCorynebacterium\u003c/em\u003e sp., \u003cem\u003eLactobacillus\u003c/em\u003e sp., and \u003cem\u003eArthrobacter\u003c/em\u003e sp.), a feature commonly shared among various plastic materials.\u003c/p\u003e \u003cp\u003eThe antimicrobial properties of various essential oils, such as tea tree, rosemary, eucalyptus, and lavender, have been shown to differ significantly when tested against pathogenic bacteria, as noted by Puvaca et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In general, natural polymers tend to lack intrinsic antibacterial activity, with chitosan being a notable exception due to its positively charged amino groups that confer antimicrobial properties [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Consequently, there has been a growing interest in exploring the potential of polyhydroxyalkanoates (PHAs) as matrices for the incorporation of antimicrobial agents. Research has extensively examined the incorporation of various antimicrobial fillers into biopolymers, including metals, chemicals, natural extracts, essential oils, and nanoparticles [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. These antimicrobial agents can interact synergistically with polymers to form composites that enhance functional performance. Our findings support this notion, as we observed that while PHA alone did not demonstrate antibacterial activity, it exhibited significant antibacterial effects when combined with essential oils, particularly lemongrass oil.\u003c/p\u003e \u003cp\u003e \u003cem\u003eB. cereus\u003c/em\u003e has emerged as a compelling model organism for the production of polyhydroxyalkanoates (PHAs) due to its remarkable biological diversity and adaptability. This species thrives in various environments, equipping it with a range of versatile traits that enhance its productivity. Notably, \u003cem\u003eB. cereus\u003c/em\u003e can utilize waste substrates as carbon sources for PHA synthesis, which not only supports sustainable production practices but also improves the economic viability of the process. The PHAs produced by \u003cem\u003eB. cereus\u003c/em\u003e are characterized by desirable properties such as low brittleness, non-toxicity, and excellent biocompatibility, making them suitable for a wide array of applications. Our findings further indicate that both PHA and essential oils exhibit antibacterial properties. This suggested that researchers are finding \u003cem\u003eB. cereus\u003c/em\u003e increasingly significant in this field of study [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePolyhydroxyalkanoates (PHAs) demonstrate efficient biodegradation by natural organisms in challenging environments, positioning them as a superior alternative to conventional plastics. The utilization of agricultural waste offers a sustainable and economically viable substrate for PHA production at a commercial scale. This study successfully identified and optimized critical factors affecting PHA synthesis. By integrating traditional and statistical optimization techniques, optimal conditions were established to enhance both biomass accumulation and PHA yield. The results highlight \u003cem\u003eBacillus cereus\u003c/em\u003e as a promising strain for PHA production using wheat bran. In industrial applications, leveraging cost-effective raw materials like wheat bran can significantly improve PHA production rates, thereby enhancing its potential as an environmentally friendly bioplastic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Researchers Supporting Project Number (RSPD2025R991), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSLGR: Investigation, Visualization, Methodology, Formal analysis, Writing \u0026ndash; original draft. GU and TC: Conceptualization, Investigation, Supervision, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing. JRA, NR, SJN, and ENS: Writing \u0026ndash; review \u0026amp; overall editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKing Saud University, Riyadh, Saudi Arabia (Project Number RSPD2025R991),\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data related to this research are available with the corresponding author and may be made available upon prior request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eCentre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari District - 629 502, India\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2 \u0026nbsp;\u003c/sup\u003eDepartment of Botany and Microbiology, College of Science, King Saud University, P.O. 2455, Riyadh 11451, Saudi Arabia. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShah S, Kumar A. Production and characterization of polyhydroxyalkanoates from industrial waste using soil bacterial isolates. Brazilian J Microbiolo. 2021;52:715\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJost V. Packaging related properties of commercially available biopolymers\u0026ndash;An overview of the status quo. Express Polym Lett. 2018;12(5):429\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzik MW. Polyhydroxyalkanoates, bacterially synthesized polymers, as a source of chemical compounds for the synthesis of advanced materials and bioactive molecules. Appl Microbiolo Biotechnolo. 2021;105(20):7555\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki M, Tachibana Y, Kasuya K-i. Biodegradability of poly (3-hydroxyalkanoate) and poly (ε-caprolactone) via biological carbon cycles in marine environments. Polym J. 2021;53(1):47\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamdy SM, Danial AW, Gad El-Rab SM, Shoreit AA, Hesham AE-L. Production and optimization of bioplastic (Polyhydroxybutyrate) from \u003cem\u003eBacillus cereus\u003c/em\u003e strain SH-02 using response surface methodology. BMC Microbiol. 2022;22(1):183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwosu I, Abu G, Agwa K. Isolation, screening and characterization of exopolysaccharide producing bacteria. Microbiol Res J Int. 2019;29:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMostafa YS, Alrumman SA, Alamri SA, Otaif KA, Mostafa MS, Alfaify AM. Bioplastic (poly-3-hydroxybutyrate) production by the marine bacterium \u003cem\u003ePseudodonghicola xiamenensis\u003c/em\u003e through date syrup valorization and structural assessment of the biopolymer. Sci Rep. 2020;10(1):8815.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMascarenhas J, Aruna K. Screening of polyhydroxyalkonates (PHA) accumulating bacteria from diverse habitats. J Global Biosci. 2017;6(3):4835\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThapa C, Shakya P, Shrestha R, Pal S, Manandhar P. Isolation of polyhydroxybutyrate (PHB) producing bacteria, optimization of culture conditions for PHB production, extraction and characterization of PHB. Nepal J Biotech. 2018;6(1):62\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar K, Rajput R, Sharma R. Antagonistic activity of bacterial species isolated from soil against fungi. World J Pharm Res. 2018;7:760\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasi C, Gemechu G, Tafesse M. Isolation, screening, characterization, and identification of alkaline protease-producing bacteria from leather industry effluent. Ann Microbiol. 2021;71:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuang-In V, Yotchaisarn M, Saengha W, Udomwong P, Deeseenthum S, Maneewan K. Protease-producing bacteria from soil in nasinuan community forest, mahasarakham province, Thailand. Biomed Pharmacolo J. 2019;12(2):587\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi R, Bisht TS. Isolation and screening of lipase enzyme from bacterial strains isolated from Sambhar lake. Int J Pharm Biol Sci. 2019;9(5):2321\u0026ndash;3272.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOnajobi IB, Idowu EO, Adeyemi JO, Samson OJ, Ogunyinka PI, Fagade OE. \u003cem\u003eIn vitro\u003c/em\u003e antibacterial activities and molecular characterization of bacterial species isolated from farmlands against selected pathogens. Biotechnolo Rep. 2020;27:e00513.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohapatra S, Pattnaik S, Maity S, Sharma S, Akhtar J, Pati S, Samantaray DP, VARMA A. Comparative analysis of PHAs production by \u003cem\u003eBacillus megaterium\u003c/em\u003e OUAT 016 under submerged and solid-state fermentation. Saudi J Biol Sci. 2020;27(5):1242\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSriyapai T, Chuarung T, Kimbara K, Samosorn S, Sriyapai P. Production and optimization of polyhydroxyalkanoates (PHAs) from Paraburkholderia sp. PFN 29 under submerged fermentation. Electron J Biotechnol. 2022;56:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDienye B, Agwa O, Abu G. Molecular Characterization, optimization and production of PHA by indigenous bacteria using alternative nutrient sources as substrate. Microbiol Res J Int. 2022;32(11\u0026ndash;12):12\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao A, Haque S, El-Enshasy HA, Singh V, Mishra BN. RSM\u0026ndash;GA based optimization of bacterial PHA production and \u003cem\u003ein silico\u003c/em\u003e modulation of citrate synthase for enhancing PHA production. Biomolecules. 2019;9(12):872.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed S, Ray L. Polyhydroxyalkanoate recovery from newly screened \u003cem\u003eBacillus\u003c/em\u003e sp. LPPI-18 using various methods of extraction from Loktak Lake sediment sample. J Genetic Eng Biotechnolo. 2022;20(1):115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodge SP, Dhanavade MJ, Kajale SC, Patil NP. A polyhydroxyalkanoate synthesised by halophilic archaeon, \u003cem\u003eNatrialba swarupiae\u003c/em\u003e. Environ Sci: Adv. 2023;2(7):990\u0026ndash;1000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanley A, Murthy PK, Vijayendra S. Characterization of polyhydroxyalkanoate produced by \u003cem\u003eHalomonas venusta\u003c/em\u003e KT832796. J Polym Environ. 2020;28(3):973\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasin AR, Al-Mayaly IK. Study of the fermentation conditions of the \u003cem\u003eBacillus cereus\u003c/em\u003e Strain ARY73 to produce polyhydroxyalkanoate (PHA) from glucose. J Ecol Eng. 2021; 22(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLorini L, Martinelli A, Capuani G, Frison N, Reis M, Sommer Ferreira B, Villano M, Majone M, Valentino F. Characterization of polyhydroxyalkanoates produced at pilot scale from different organic wastes. Front Bioeng Biotechnol. 2021;9:628719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian J, Zhang R, Wu Y, Xue P. Additive manufacturing of wood flour/polyhydroxyalkanoates (PHA) fully bio-based composites based on micro-screw extrusion system. Mat Des. 2021;199:109418.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThu NTT, Hoang LH, Cuong PK, Viet-Linh N, Nga TTH, Kim DD, Leong YK. Nhi-Cong LT Evaluation of polyhydroxyalkanoate (PHA) synthesis by Pichia sp. TSLS24 yeast isolated in Vietnam. Sci Rep. 2023;13(1):3137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasnett P, Marcello E, Lukasiewicz B, Nigmatullin R, Paxinou A, Ahmad MH, Gurumayum B, Roy I. Antimicrobial materials with lime oil and a poly (3-hydroxyalkanoate) produced via valorisation of sugar cane molasses. J Funct Biomat. 2020;11(2):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePillai AB, Kumar AJ, Thulasi K, Kumarapillai H. Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai. Brazilian J Microbiol. 2017;48:451\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Guti\u0026eacute;rrez CA, Latisnere-Barrag\u0026aacute;n H, Garc\u0026iacute;a-Maldonado JQ, L\u0026oacute;pez-Cort\u0026eacute;s A. Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico. Peer J. 2018;6:e4780.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuigano MN, Anami SE, Onguso JM, Omare GM. The isolation, screening, and characterization of polyhydroxyalkanoate-producing bacteria from hypersaline lakes in Kenya. Bacteria. 2023;2(2):81\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAragosa A, Specchia V, Frigione M. Isolation of two bacterial species from argan soil in morocco associated with polyhydroxybutyrate (PHB) accumulation: Current potential and future prospects for the bio-based polymer production. Polymers. 2021;13(11):1870.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMandragutti T, Sudhakar G. Selective isolation and genomic characterization of biopolymer producer\u0026mdash;a novel feature of halophile \u003cem\u003eBrachybacterium paraconglomeratum\u003c/em\u003e MTCC 13074. J Genetic Eng Biotechnol. 2023;21(1):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlshehrei F. Production of polyhydroxybutyrate (PHB) by bacteria isolated from soil of Saudi Arabia. J Pure Appl Microbiol. 2019; 13(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintero-Silva MJ, Su\u0026aacute;rez-Rodr\u0026iacute;guez SJ, Gamboa-Su\u0026aacute;rez MA, Blanco-Tirado C, Combariza MY. Polyhydroxyalkanoates production from cacao fruit liquid residues using a native Bacillus megaterium strain: Preliminary study. J Polym Environ. 2024;2(3):1289\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahato RP, Kumar S, Singh P. Optimization of growth conditions to produce sustainable polyhydroxyalkanoate bioplastic by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e EO1. Front Microbiol. 2021;12:711588.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZihayat B, Shakibaie M, Sabouri-Shahrbabak S, Doostmohammadi M, Ameri A, Adeli-Sardou M, Forootanfar H. Medium optimization for polyhydroxyalkanoate production by \u003cem\u003ePseudomonas pseudoalcaligenes\u003c/em\u003e strain Te using D-optimal design. Biocatal Agricul Biotechnol. 2019;18:101001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhamkong T, Penkhrue W, Lumyong S. Optimization of production of polyhydroxyalkanoates (PHAs) from newly isolated \u003cem\u003eEnsifer\u003c/em\u003e sp. strain HD34 by response surface methodology. Processes. 2022;10(8):1632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Huang J, Guo H, Jiang S, Qiao J, Chen X, Qu Z, Cui W, Liu S. Effects of different sodium salts and nitrogen sources on the production of 3-hydroxybutyrate and polyhydroxybutyrate by \u003cem\u003eBurkholderia cepacia\u003c/em\u003e. Biores Bioprocess. 2021;8:1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesouky S, El-Shiekh H, Elabd M, Shehab A. Screening, optimization and extraction of polyhydroxyalkanoates (PHAs) from \u003cem\u003eBacillus thuringienesis\u003c/em\u003e. J Adv Biol Biotechnol. 2014;1(1):40\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmady-Asbchin S, Rezaee H, Safari M, Zamanifar P, Siyamiyan D. Production and optimization of polyhydroxybutyrate (PHB) from \u003cem\u003eBacillus megaterium\u003c/em\u003e as biodegradable plastic. Eur J Biol Rese. 2020;10(1):26\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeethu M, Chandrashekar HR, Divyashree M. Statistical optimisation of polyhydroxyalkanoate production in \u003cem\u003eBacillus endophyticus\u003c/em\u003e using sucrose as sole source of carbon. Archives Microbiol. 2021;203:5993\u0026ndash;6005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnamalai N, Sivakumar N. Production of polyhydroxybutyrate from wheat bran hydrolysate using \u003cem\u003eRalstonia eutropha\u003c/em\u003e through microbial fermentation. J Biotechnol. 2016;237:13\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRezk SA, Emam DAE-M, Swailam HM, Swelim MA. Production of Polyhydroxybutyrate (PHB) from \u003cem\u003eStreptomyces Incanus\u003c/em\u003e and the Effect of Gamma Irradiation on its Production. Arab J Nuclear Sci Appl. 2020;53(2):111\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdnan M, Siddiqui AJ, Ashraf SA, Snoussi M, Badraoui R, Ibrahim AM, Alreshidi M, Sachidanandan M, Patel M. Characterization and process optimization for enhanced production of polyhydroxybutyrate (PHB)-based biodegradable polymer from Bacillus flexus isolated from municipal solid waste landfill site. Polymers. 2023;15(6):1407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa\u0026ntilde;ez JCA, Requiso PJ, Alfafara CG, Nayve FRP, Ventura J-RS. Optimization of fermentation factors for polyhydroxybutyrate (PHB) production using \u003cem\u003eBacillus megaterium\u003c/em\u003e PNCM 1890 in simulated glucose-xylose hydrolysates from agricultural residues. Philipp J Sci. 2020;149(1):163\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePenkhrue W, Jendrossek D, Khanongnuch C, Pathom-Aree W, Aizawa T, Behrens RL, Lumyong S. Response surface method for polyhydroxybutyrate (PHB) bioplastic accumulation in \u003cem\u003eBacillus drentensis\u003c/em\u003e BP17 using pineapple peel. PLoS ONE. 2020;15(3):e0230443.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil TD, Ghosh S, Agarwal A, Patel SKS, Tripathi AD, Mahato DK, Kumar P, Slama P, Pavlik A, Haque S. Production, optimization, scale up and characterization of polyhydoxyalkanoates copolymers utilizing dairy processing waste. Sci Rep. 2024;14(1):1620.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohd Zain NF, Paramasivam M, Tan JS, Lim V, Lee CK. Response surface methodology optimization of polyhydroxyalkanoate production by \u003cem\u003eBurkholderia cepacia\u003c/em\u003e BPT1213 using waste glycerol from palm oil-based biodiesel production. Biotechnol Progress. 2021;37(1):e3077.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas R, Pal A, Paul AK. Optimization of process parameters for production of poly (3-hydroxybutyrate) by Bacillus pumilus AHSD 04, a seed borne endophyte of oleaginous plant \u003cem\u003eArachis hypogaea\u003c/em\u003e L. Biointerf Res Appl Chem. 2022;12(4):5280.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamal S, Pati S, Mohapatra S, Maity S, Tanaya K, Devadarshini D, Samantaray D. PHAs production by facultative anaerobic bacteria, \u003cem\u003eBacillus cereus\u003c/em\u003e FM5 through submerged and solid-state fermentation under anoxic condition. Antonie Van Leeuwenhoek. 2023;116(6):521\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamrot AV, Samanvitha SK, Shobana N, Renitta ER, Senthilkumar P, Kumar SS, Abirami S, Dhiva S, Bavanilatha M, Prakash P, Saigeetha S, Shree KS, Thirumurugan R. The synthesis, characterization and applications of polyhydroxyalkanoates (PHAs) and PHA-based nanoparticles. Polymers. 2021;13(19):3302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAdam B, Brennan Fournet M, McDonald P, Mojicevic M. Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polymers. 2020;12(12):2908.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMai J, Kockler K, Parisi E, Chan CM, Pratt S, Laycock B. Synthesis and physical properties of polyhydroxyalkanoate (PHA)-based block copolymers: A review. Int J Biol Macromol. 2024;263:130204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMottin AC, Ayres E, Or\u0026eacute;fice RL, C\u0026acirc;mara JJD. What changes in poly (3-hydroxybutyrate)(PHB) when processed as electrospun nanofibers or thermo-compression molded film? Mater Resea. 2016;19(1):57\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwinyi OC, Owolabi TA. Scanning electron microscopy and Fourier transmission analysis of polyhydroxyalkanoates isolated from bacteria species from abattoir in Ota, Nigeria. J King Saud Uni-Sci. 2019;31(3):285\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuvača N, Milenković J, Galonja Coghill T, Bursić V, Petrović A, Tanasković S, Pelić M, Ljubojević Pelić D, Miljković T. Antimicrobial activity of selected essential oils against selected pathogenic bacteria: \u003cem\u003eIn vitro\u003c/em\u003e study. Antibiotics. 2021;10(5):546.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgnatova L, Brazhnikova Y, Omirbekova A, Usmanova A. Polyhydroxyalkanoates (PHAs) from endophytic bacterial strains as potential biocontrol agents against postharvest diseases of apples. Polymers. 2023;15(9):2184.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBustamante-Torres M, Romero-Fierro D, Estrella-Nu\u0026ntilde;ez J, Arcentales-Vera B, Chichande-Proa\u0026ntilde;o E, Bucio E. Polymeric composite of magnetite iron oxide nanoparticles and their application in biomedicine: a review. Polymers. 2022;14(4):752.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Herrera RE, Alem\u0026aacute;n-Huerta ME, Rutiaga-Qui\u0026ntilde;ones OM, de Luna-Santillana EJ, Elufisan TO. A comprehensive view of \u003cem\u003eBacillus cereus\u003c/em\u003e as a polyhydroxyalkanoate (PHA) producer: A promising alternative to Petroplastics. Process Biochem. 2023;129:281\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"blue-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Blue Biotechnology](https://bluebiotechnology.biomedcentral.com)","snPcode":"44315","submissionUrl":"https://submission.springernature.com/new-submission/44315/3","title":"Blue Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibacterial activity, Bacillus cereus, Polyhydroxyalkanoate (PHA), Optimization, Response Surface Methodology (RSM), Solar Salt works","lastPublishedDoi":"10.21203/rs.3.rs-9349740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9349740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteria can spontaneously produce polyhydroxyalkanoate (PHA), a thermoplastic and biodegradable substance. PHA is the best polymer substitute for plastic made from petrochemicals. In this investigation, we isolated sixteen bacterial strains from brine and sediment samples collected from a solar salt works facility. Among the sixteen bacterial strains screened for polyhydroxyalkanoate (PHA) production, two strains, Z6 and Z9, exhibited promising results based on Sudan Black B and Nile Blue A staining. Subsequent morphological, biochemical, and molecular characterization identified strain Z9 as Bacillus cereus PHB CMST1 through 16S rRNA sequencing, which demonstrated superior PHA production compared to the other strain. To optimize PHA production, we employed a one-factor-at-a-time (OFAT) methodology, revealing optimal conditions of 35\u0026deg;C, pH 7, 2% salinity, and a 3-day incubation period, utilizing wheat bran as the carbon source and urea as the nitrogen source. Further optimization using Response Surface Methodology-Central Composite Design (RSM-CCD) indicated that \u003cem\u003eB. cereus\u003c/em\u003e strain PHB CMST1 requires 5% wheat bran and 2% urea for enhanced PHA synthesis. The yellowish-green dots in the thin layer chromatography (TLC) plate indicated the presence of PHA. FT-IR analysis confirmed that, \u0026#120573;-glycosidic linkages between the sugar monomers were found. The derivatives of polyhydroxybutyric acid confirmed the monomeric polymer by GC-MS analysis. PHA's XRD study showed wide peaks at 20\u0026deg; and 43\u0026deg;, indicating that the PHA had a semi-crystalline structure. \u003cem\u003eBacillus cereus\u003c/em\u003e shows considerable promise for cost-effective and large-scale production of PHA bioplastics, utilizing wheat bran as a non-expensive carbon source.\u003c/p\u003e","manuscriptTitle":"Bacillus cereus PHB CMST1: A Potential Halophilic Bacterium for Cost-Effective and Sustainable Production of Polyhydroxyalkanoates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 08:01:32","doi":"10.21203/rs.3.rs-9349740/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-12T23:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231658019606949931210430267999039555050","date":"2026-04-22T11:21:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100330199185590922268111609889820834406","date":"2026-04-22T08:57:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329155757842971635578051405919334384713","date":"2026-04-19T15:14:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-16T21:45:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-11T01:55:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T06:43:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Blue Biotechnology","date":"2026-04-07T23:11:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"blue-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Blue Biotechnology](https://bluebiotechnology.biomedcentral.com)","snPcode":"44315","submissionUrl":"https://submission.springernature.com/new-submission/44315/3","title":"Blue Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59f0022c-cd83-4bfc-b899-877fa87fcda8","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T23:38:05+00:00","index":21,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T08:01:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 08:01:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9349740","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9349740","identity":"rs-9349740","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}