Saudi Hot Spring Extremophilic Bacillus as an Alternative Bioresource for Sustainable Thermoplastic Elastomer (TPE) Biosynthesis from Shrimp Waste

Saudi Hot Spring Extremophilic Bacillus as an Alternative Bioresource for Sustainable Thermoplastic Elastomer (TPE) Biosynthesis from Shrimp Waste | 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 Article Saudi Hot Spring Extremophilic Bacillus as an Alternative Bioresource for Sustainable Thermoplastic Elastomer (TPE) Biosynthesis from Shrimp Waste Abdulaziz M Kusa, Hanan A Hamdi, Ahmad K AL-Ghamdi, Fayez M. Alshehri, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7470334/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Saudi Vision 2030 biotechnology strategy aims to support sustainable development by exploring Saudi Arabia's unique extreme ecosystems. The diverse environment, including water ecosystems like marine and terrestrial environments, offers numerous biotechnological applications. The study aims to fulfill the national vision goal by using alternative renewable resources from extreme environments to produce eco-friendly products from organic waste, following the national biotechnological strategy. This approach helps preserve the ecosystem and serve humanity. Nnewly isolated Bacillus tequilensis was recently isolated from Al-Lith hot spring and identified by 16s rRNA sequencing, then submitted to GenBank by the accession number (OR616739). B. tequilensis isolates were used to produce bioplastic from shrimp waste. To ensure the production of bioplastic, bacterial slides were prepared from liquid media and stained with Sudan black B procedure, and bacterial colonies were also stained on agar plated by Sudan black B method. The final product was measured by Fourier transform infrared (FT-IR) and nuclear magnetic resonance (MNR) spectroscopies for chemical analysis. Stained slides and plates showed the production of bioplastic as the bacterial cells showed black regions in the cell poles and the colony appeared black on agar plates according to staining by Sudan black B stain. The FT-IR and MNR analysis ensured the produced plastic polymer was Thermoplastic Elastomer (TE). The study demonstrates the use of TE copolymer, a blend of plastic and rubber, as an eco-friendly alternative to petroleum products, as it was created by a native thermophilic bacterium, demonstrating the potential of biotechnological methods to meet the Saudi National Vision 2030 goals and promote sustainable waste management. Biological sciences/Biological techniques Biological sciences/Biotechnology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plastic consumer products, especially single-use plastics, are pervasive; yet their current production and utilization are unsustainable [ 1 ]. This untenable development is largely attributed to the characteristics of the materials used in plastic production. Carbon-carbon polymers, which comprise most plastics produced globally, are inexpensive and environmentally persistent, resulting in a massive reservoir of plastic waste that ends up in landfills and the environment [ 2 ]. As a result of these characteristics, as of 2017, just 9% of the 9 billion tonnes of plastic that were created had been recycled, meaning that a significant amount of plastic was still contaminating the environment and having detrimental effects on both health and the economy [ 3 ]. Given these alarming statistics, however, there is already enough data to demonstrate that swift and extensive action is required to prevent future harm by reducing the amount of plastic released into the ecosystem [ 4 ]. Numerous renewable raw resources can be used to produce biobased plastics, including polysaccharides, proteins, starch- and cellulose-based polymers from plants, and microbial bioplastics such as polylactic acid and polyhydroxyalkanoates (PHAs) [ 5 ]. Among these, PHAs stand out due to their microbial origin and sustainability profile. Microorganisms may manufacture PHA, a biopolymer, from a variety of inexpensive carbon sources, making it an eco-friendly substance [ 6 ]. Nonetheless, synthetic plastics remain prevalent in the market and continue to present environmental hazards. Plastic components commonly utilized in daily life are increasingly generating severe environmental issues, with millions of tonnes of these non-biodegradable polymers accumulating annually in the environment [ 7 ]. This accumulation leads to severe environmental damage. Severe soil and water pollution occurs due to the slow or non-degradable nature of synthetic plastic; also, when burning, it causes air pollution [ 8 ]. Certain microbes respond to nutrient stress by synthesizing biopolymers, such as PHB, the most extensively researched member of the PHA family [ 9 ]. Among the various microbial candidates for PHA production, Bacillus species have shown exceptional potential. Some Bacillus species have been found to produce as much as 90% (w/w) PHAs of dry cells during nutrient imbalance [ 10 ]. Because of their genetic stability, Bacillus species have gained popularity as model organisms in both industry and academia. Moreover, thermophilic Bacillus strains offer unique advantages in addition to having a higher growth rate than other bacteria [ 11 ]. Thermophilic Bacillus helps to preserve vitality during thermal processing since it can withstand high temperatures [ 12 ]. Thermophilic Bacillus species offer industrial advantages, including thermostable enzymes, rapid proliferation, and resilience, rendering them efficient cell factories for PHA manufacture from inexpensive substrates [ 13 ]. The biotechnological value of thermophilic Bacillus is enhanced by its robust physiology. All Gram-positive thermophilic Bacillus are often tolerant of their surroundings, including heat, acid, bile, and salt [ 14 ]. Additionally, numerous extracellular thermostable enzymes with significant economic value, such as lipases, amylases, cellulases, and proteases, can be produced by thermophilic Bacillus [ 15 ]. Bacillus species are desirable industrial organisms due to their ability to secrete proteins, high growth rates, short fermentation cycles, and GRAS classification by the FDA (generally regarded as safe) [ 16 ]. According to [ 17 ], the ability of thermophiles to produce a wide range of useful molecules, including antibiotics, thermostable enzymes, and anticancer drugs, makes them of international interest. One such promising bacterium is Bacillus tequilensis , a rod-shaped, aerobic, motile, and catalase-positive probiotic bacterium [ 18 ]. Numerous Bacillus species from saline and marine environments, including B. tequilensis , have been investigated for their ability to produce biosurfactants, such as lipopeptides [ 19 ]. Additionally, B. tequilensis produces amylase in the presence of cyclohexane and other stressful conditions [ 20 ]. This capability allows for the conversion of animal fats, glycerol, and various dietary wastes into PHA by B. tequilensis [ 21 ]. Additionally, it has been shown that B. tequilensis produces PHA utilizing synthetic acids and food waste [ 22 ]. A unique and impactful area of discovery within this field is the use of Bacillus-derived polymers for the development of advanced biomaterials, particularly thermoplastic elastomers (TPEs). This direction holds great promise and deserves further investigation. To explore practical applications of microbially derived polymers, attention has turned to thermoplastic elastomers (TPEs), which combine the properties of plastics and rubber [ 23 ]. This structure provides flexibility and resilience. Thermoplastic elastomers are of great interest due to their cushioning properties [ 24 ]. The term "elastomer" comes from "elastic polymer," referring to any rubbery-textured substance composed of long, chain-like molecules, or polymers [ 25 ]. By offering environmental advantages in both manufacturing and disposal, TPEs also present a pathway toward sustainable development TPEs are versatile and used in various sectors, including medical, aerospace, construction, optical fibers, automotive coatings such as gaskets, seals, and interior components, surgical instruments, wound dressings, yoga mats, toothbrush grips, vibration dampers, medical soft robotics, 3D printing, footwear industries, and air ducts [ 26 ]. Their popularity is increasing because they can replace traditional rubbers in applications where performance and sustainability are required [ 27 ]. Due to their unique performance, low production costs, good processability, and recyclability, thermoplastic elastomers, or TPEs, have been the subject of extensive research [ 28 ]. Ongoing research on Bacillus-based elastomeric systems is not only scientifically significant but also essential for expediting the global shift towards sustainable and circular bioeconomy materials. The study aims to fulfil the national vision goal by utilizing alternative renewable resources from extreme environments to produce eco-friendly products from organic waste, in line with the national biotechnological strategy. This approach helps preserve the ecosystem and serves humanity. Materials and Methods Isolation and molecular identification of Bacillus tequilensis Environmental samples were collected from a hot spring in Al Lith, Saudi Arabia. Samples of water and sediment were moved to sterile containers aseptically, and they were brought to the lab on ice (4°C). Samples were exposed to heat shock treatment at 80°C for 10 minutes to select for thermophilic, spore-forming bacteria. Aliquots were then spread out on Nutrient Agar (NA) plates after serial dilutions were made in sterile saline (0.85% NaCl). For 48 hours, plates were incubated at 50°C. To produce pure isolates, morphologically different colonies with characteristics typical of Bacillus (dry, irregular, opaque) were chosen and subcultured. The QIAamp® DNA Mini Kit (Qiagen) was used to extract genomic DNA, and universal primers 27F and 1492R were used to amplify the 16S rRNA gene. Sanger dideoxy sequencing was used to sequence the PCR results, and NCBI BLASTn was used to analyse the sequences for taxonomic confirmation. The isolate was identified as Bacillus tequilensis strain LW, and its partial 16S rRNA gene sequence was submitted to the NCBI GenBank database, receiving accession number OR616739. Induction of thermoplastic elastomer (TPE) production To induce the biosynthesis of bacterial thermoplastic elastomers, the confirmed strain of B. tequilensis was inoculated into Nutrient Broth (NB) medium prepared according to the protocol described by [ 29 ]. The composition of the medium is detailed in (Table 1 ). Cultures were incubated at 50°C for 72 h under static conditions to enhance intracellular accumulation of polymeric compounds. Histochemical confirmation of TPE production Following incubation, qualitative confirmation of TPE accumulation was conducted using Sudan Black B staining. Bacterial smears were prepared on glass slides, heat-fixed, and stained with 0.3% (w/v) Sudan Black B dissolved in 70% ethanol for 10 minutes. Slides were briefly washed with distilled water and counterstained with safranin for 2 minutes before microscopic examination under a 100× oil immersion lens. Dark intracellular granules were considered indicative of polymer inclusion bodies [ 30 ]. A second confirmatory assay was performed by streaking the isolate onto nutrient agar plates (same composition as broth). Plates were incubated at 50°C for 48 h, after which 0.3% Sudan Black B in ethanol was applied to the surface. Plates were left undisturbed for 30 minutes and then gently rinsed with 96% ethanol to remove excess stain. Dark blue-black colonies were interpreted as positive for TPE accumulation [ 31 ]. Extraction and purification of bacterial TPE To extract the fermented culture, the entire volume was centrifuged for ten minutes at 6000 rpm. The pellet was incubated for 1.5 hours at 37°C to lyse the cell wall and release the intracellular contents after 10 mL of 10% (v/v) sodium hypochlorite (NaClO) was added. The supernatant was disposed of. The modified suspension underwent another centrifugation at 6000 rpm for 10 minutes. The resulting pellet was cleaned with distilled water, acetone, and methanol in order to remove any last impurities. After dissolving the purified granules in 10 mL of chloroform, the mixture was poured onto sterile glass trays to evaporate, and it was then dried in a hot air oven set at 40°C until all the solvent had been removed [ 30 ]. Structural characterization of extracted TPE The chemical structure of the recovered TPE was evaluated using Fourier-transform infrared spectroscopy (FTIR) to identify functional groups. Additionally, nuclear magnetic resonance (NMR) spectroscopy was conducted to confirm the polymer’s backbone structure and monomeric composition. Table 1 Additives to NB and NA media (g/1 L) for the production of TPE: Component Quantities Disodium phosphate (Na2HPO4) 6.0 g Monopotassium phosphate (KH2PO4) 3.0 g Ammonium chloride (NH4Cl) 0.5 g Sodium chloride (NaCl) 5.0 g Carbon Sources (Shrimp peel) 5.0 g Results Confirmation of TPE Production via Histochemical and Colony Staining Microscopic analysis using Sudan Black B staining revealed intracellular dark inclusions located at the poles of B. tequilensis cells, indicative of polymer granule accumulation (Fig. 1 ). These dark inclusions are consistent with intracellular lipid-like or polymeric materials, suggesting PHA or TPE production. On nutrient agar (NA) plates, colonies were stained by black following Sudan Black B application, reinforcing the intracellular staining results (Fig. 2 ). Such staining methods are commonly used as preliminary indicators of bioplastic or elastomeric polymer accumulation. FTIR Spectroscopy Confirms Polymer Backbone Fourier-transform infrared spectroscopy (FTIR) was used to evaluate the polymer's chemical structure (Fig. 3 ). Characteristic absorbance peaks at 1667 cm⁻¹ (C = C), 2919 cm⁻¹ (C–H), and 1032 cm⁻¹ (C–O) suggest an unsaturated hydrocarbon backbone, consistent with isoprene-based polymers. These spectra align with previously reported microbial elastomers and PHAs. Structural Characterization by ¹H-NMR The ¹H-NMR spectrum (Fig. 4) showed three major signals: a singlet at δ 1.45 ppm (CH₃), a multiplet at δ 1.99–2.08 ppm (CH₂), and a triplet at δ 5.32 ppm (CH = CH). These chemical shifts and splitting patterns match the profiles of cis-1,4-polyisoprene, a rubber-like elastomer. Absence of contaminant peaks supports product purity. These NMR results confirm the polymer structure, further validating the identity of the synthesized elastomer as polyisoprene-like TPE. Discussion The findings support the concept of using thermophilic Bacillus tequilensis from Al-Lith hot springs as a microbial platform for elastomer biosynthesis [ 37 ]. This aligns with global trends toward biobased, renewable materials. Given the environmental persistence of synthetic plastics, the ability to synthesize eco-friendly TPE from shrimp waste using a native thermophile is particularly valuable [ 38 ]. The use of shrimp waste as a low-cost carbon source provides dual environmental and economic advantages: it offers a solution to aquaculture waste disposal and provides a renewable substrate for high-value biomaterials. Previous studies have shown the effectiveness of seafood waste in PHA and TPE biosynthesis [ 39 ]; however, this study is among the first to demonstrate successful polyisoprene-like TPE production by Bacillus tequilensis in a high-temperature system. The thermophilic nature of the bacterium is particularly advantageous, as it reduces the risk of contamination and enhances process efficiency at elevated temperatures. Thermostable production systems are also more compatible with industrial bioreactor designs, where heating is often needed for sterilization or reaction kinetics [ 40 ]. This opens the door for integrating such microbial processes into large-scale circular bioeconomy platforms [ 41 ]. In terms of structural validation, the FTIR and ¹H-NMR data strongly support the polymer’s identity as a rubber-like TPE. These analytical tools are crucial in distinguishing between polyhydroxyalkanoates (PHAs), other bacterial polyesters, and true elastomeric compounds. The presence of unsaturated C = C bonds and methyl-methylene-olefinic proton profiles indicates a polyisoprene-like backbone, essential for achieving elastomeric mechanical properties [ 42 ]. While promising, the yield, scalability, and mechanical strength of the extracted polymer need further evaluation. Comparative studies with other extremophiles or genetically engineered strains may help improve productivity and tailor material properties for specific applications [ 43 ]. Additionally, further research should assess biodegradability, thermal stability, cytotoxicity, and performance in real-world product applications. Regulatory pathways for medical or industrial approval should also be explored if this TPE is to replace conventional rubbers in sectors such as healthcare, packaging, or electronics [ 44 ]. Overall, the study provides a novel proof-of-concept that advances the field of microbial elastomers by leveraging native Saudi extremophiles, aligning with national strategies for sustainable biotechnology and circular economy transformation [ 45 ]. The valorization of shrimp waste not only addresses aquaculture waste management but also provides a renewable, low-cost carbon source for polymer biosynthesis, thereby contributing to circular bioeconomy goals [ 48 ]. Moreover, the thermophilic nature of B. tequilensis offers operational benefits such as reduced contamination risk and compatibility with industrial-scale heat-sterilized fermenters [ 49 ]. This is particularly relevant for continuous and semi-continuous fermentation systems, which can significantly lower production costs. In addition, the distinct unsaturated bonds observed in FTIR spectra indicate a high proportion of elastomeric segments, a property linked to enhanced mechanical flexibility and resilience [ 46 ]. This structural attribute positions the produced TPE for potential use in high-performance applications, including medical devices, automotive components, and 3D printing materials [ 50 ]. To fully realize the industrial potential of this bioprocess, future studies should incorporate pilot-scale trials using locally sourced shrimp waste under industrial fermentation conditions [ 51 ]. These trials should also be coupled with a life cycle assessment to validate the environmental sustainability of the process. Furthermore, the concept aligns with global trends toward climate-positive materials, providing a pathway for replacing fossil fuel-derived elastomers with renewable microbial alternatives [ 52 ]. Conclusion This study successfully demonstrates the potential of a newly isolated thermophilic B. tequilensis strain from Al-Lith hot springs as a sustainable microbial platform for the biosynthesis of thermoplastic elastomers (TPE) using shrimp waste as a low-cost, renewable carbon source. The confirmation of TPE production through histochemical staining, FTIR, and ¹H-NMR spectroscopy highlights the biochemical and structural validity of the synthesized polymer, aligning with the chemical properties of polyisoprene-like elastomers. The ability of this native extremophile to produce high-value bioplastics under elevated temperatures offers a promising biotechnological solution for reducing plastic pollution, valorizing seafood waste, and supporting Saudi Arabia’s Vision 2030 strategy for a circular bioeconomy. Future studies should focus on optimizing production yield, scaling up bioprocesses, evaluating material performance, and exploring potential applications in medical, industrial, and consumer product sectors. Overall, this work lays the groundwork for harnessing extremophilic biodiversity in the region for innovative, eco-friendly industrial applications. Declarations Funding The authors received no financial support for the research, authorship, or publication of this article. Author Contribution Abdulaziz M Kusa: sample collection - microbial and molecular experiments - data analysis - reports writing.Hanan A Hamdi: manuscript writing.Ahmad K AL-Ghamdi: Microbial and molecular experiments - report writing.Fayez M. Alshehri: Microbial and molecular experiments - report writing.Basim Algashgari: Chemical experiments and analysis.Burhan Z Fakhurji: Providing financial support by Xgenome biotech company.Mohammed N Baeshen: Managing and supervising all project phases and experiments - repots and manuscript reviewing and writing. Acknowledgement The authors would like to thank the department of biological sciences, college of science, university of Jeddah, Jeddah, Saudi Arabia and KFMRC, king Abdulaziz university, Jeddah, Saudi Arabia for their technical support. Data Availability Sequence data that support the findings of this study have been deposited in the NCBI GenBank database with the primary accession number OR616739. References T. R. Walker and L. Fequet, ‘Current trends of unsustainable plastic production and micro(nano)plastic pollution’, TrAC Trends in Analytical Chemistry, vol. 160, p. 116984, Mar. 2023, doi: 10.1016/j.trac.2023.116984. K. L. Law and R. Narayan, ‘Reducing environmental plastic pollution by designing polymer materials for managed end-of-life’, Nat Rev Mater, vol. 7, no. 2, pp. 104–116, Oct. 2021, doi: 10.1038/s41578-021-00382-0. J. Nikiema and Z. Asiedu, ‘A review of the cost and effectiveness of solutions to address plastic pollution’, Environ Sci Pollut Res, vol. 29, no. 17, pp. 24547–24573, Apr. 2022, doi: 10.1007/s11356-021-18038-5. A. A. Horton, ‘Plastic pollution: When do we know enough?’, Journal of Hazardous Materials, vol. 422, p. 126885, Jan. 2022, doi: 10.1016/j.jhazmat.2021.126885. M. Adnan et al., ‘Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site’, Polymers, vol. 15, no. 6, p. 1407, Mar. 2023, doi: 10.3390/polym15061407. N. A. Jamaludin, S. F. Z. Mohd Fuzi, M. I. Mohd Ghazali, M. I. Juki, A. F. Abdulaziz Al-Shalif, and N. Othman, ‘Production of Polyhydroxyalkanoates (PHA) by Probiotic Bacteria Bacillus tequilensis for Potentially Used as Drug Carrier’, MJMHS, vol. 19, no. s9, pp. 126–132, Aug. 2023, doi: 10.47836/mjmhs.19.s9.19. A. W. Danial, S. M. Hamdy, S. A. Alrumman, S. M. F. Gad El-Rab, A. A. M. Shoreit, and A. E.-L. Hesham, ‘Bioplastic Production by Bacillus wiedmannii AS-02 OK576278 Using Different Agricultural Wastes’, Microorganisms, vol. 9, no. 11, p. 2395, Nov. 2021, doi: 10.3390/microorganisms9112395. M. Narayanan et al., ‘Optimization and production of polyhydroxybutyrate from sludge by Bacillus cereus categorized through FT-IR and NMR analyses’, Journal of Environmental Chemical Engineering, vol. 9, no. 1, p. 104908, Feb. 2021, doi: 10.1016/j.jece.2020.104908. A. J. Anderson and E. A. Dawes, ‘Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates’, Microbiol Rev, vol. 54, no. 4, pp. 450–472, Dec. 1990, doi: 10.1128/mr.54.4.450-472.1990. L. L. Madison and G. W. Huisman, ‘Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic’, Microbiol Mol Biol Rev, vol. 63, no. 1, pp. 21–53, Mar. 1999, doi: 10.1128/mmbr.63.1.21-53.1999. D. Vicente, D. N. Proença, and P. V. Morais, ‘The Role of Bacterial Polyhydroalkanoate (PHA) in a Sustainable Future: A Review on the Biological Diversity’, IJERPH, vol. 20, no. 4, p. 2959, Feb. 2023, doi: 10.3390/ijerph20042959. N. Zhao, T. Yu, and F. Yan, ‘Probiotic role and application of thermophilic Bacillus as novel food materials’, Trends in Food Science & Technology, vol. 138, pp. 1–15, Aug. 2023, doi: 10.1016/j.tifs.2023.05.020. D. H. Vu, S. Wainaina, M. J. Taherzadeh, D. Åkesson, and J. A. Ferreira, ‘Production of polyhydroxyalkanoates (PHAs) by Bacillus megaterium using food waste acidogenic fermentation-derived volatile fatty acids’, Bioengineered, vol. 12, no. 1, pp. 2480–2498, Jan. 2021, doi: 10.1080/21655979.2021.1935524. F. Su and P. Xu, ‘Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals’, Sci Rep, vol. 4, no. 1, Jan. 2014, doi: 10.1038/srep03926. N. E. Ibrahim and K. Ma, ‘Industrial Applications of Thermostable Enzymes from Extremophilic Microorganisms’, CBE, vol. 4, no. 2, July 2017, doi: 10.2174/2212711904666170405123414. M. Schallmey, A. Singh, and O. P. Ward, ‘Developments in the use ofBacillusspecies for industrial production’, Can. J. Microbiol., vol. 50, no. 1, pp. 1–17, Jan. 2004, doi: 10.1139/w03-076. S. Arbab et al., ‘Diversity and distribution of thermophilic microorganisms and their applications in biotechnology’, J Basic Microbiol, vol. 62, no. 2, pp. 95–108, Feb. 2022, doi: 10.1002/jobm.202100529. R. P. Rani, M. Anandharaj, P. Sabhapathy, and A. D. Ravindran, ‘Physiochemical and biological characterization of novel exopolysaccharide produced by Bacillus tequilensis FR9 isolated from chicken’, International Journal of Biological Macromolecules, vol. 96, pp. 1–10, Mar. 2017, doi: 10.1016/j.ijbiomac.2016.11.122. S. Cortés‐Camargo et al., ‘Biosurfactant Production by Bacillus tequilensis ZSB10: Structural Characterization, Physicochemical, and Antifungal Properties’, J Surfact & Detergents, vol. 24, no. 5, pp. 773–782, Sept. 2021, doi: 10.1002/jsde.12515. S. Tiwari, N. Shukla, P. Mishra, and R. Gaur, ‘Enhanced Production and Characterization of a Solvent Stable Amylase from Solvent TolerantBacillus tequilensisRG-01: Thermostable and Surfactant Resistant’, The Scientific World Journal, vol. 2014, pp. 1–11, 2014, doi: 10.1155/2014/972763. R. Sohail, N. Jamil, I. Ali, and S. Munir, ‘Animal fat and glycerol bioconversion to polyhydroxyalkanoate by produced water bacteria’, e-Polymers, vol. 20, no. 1, pp. 92–102, Mar. 2020, doi: 10.1515/epoly-2020-0011. M. Venkateswar Reddy, K. Amulya, M. V. Rohit, P. N. Sarma, and S. Venkata Mohan, ‘Valorization of fatty acid waste for bioplastics production using Bacillus tequilensis: Integration with dark-fermentative hydrogen production process’, International Journal of Hydrogen Energy, vol. 39, no. 14, pp. 7616–7626, May 2014, doi: 10.1016/j.ijhydene.2013.09.157. G. Holden, ‘Thermoplastic Elastomers’, in Applied Plastics Engineering Handbook, Elsevier, 2024, pp. 97–113. doi: 10.1016/B978-0-323-88667-3.00020-5. C. Ge, L. Priyadarshini, D. Cormier, L. Pan, and J. Tuber, ‘A preliminary study of cushion properties of a 3D printed thermoplastic polyurethane Kelvin foam’, Packag Technol Sci, vol. 31, no. 5, pp. 361–368, May 2018, doi: 10.1002/pts.2330. H. Chung, A. M. Parsons, and L. Zheng, ‘Magnetically Controlled Soft Robotics Utilizing Elastomers and Gels in Actuation: A Review’, Advanced Intelligent Systems, vol. 3, no. 3, Mar. 2021, doi: 10.1002/aisy.202000186. J. G. Tejani, ‘Thermoplastic Elastomers: Emerging Trends and Applications in Rubber Manufacturing’, Glob. discl. econ. bus., vol. 6, no. 2, pp. 133–144, Dec. 2017, doi: 10.18034/gdeb.v6i2.737. N. K. Singha and S. C. Jana, Advances in Thermoplastic Elastomers: Challenges and Opportunities. Elsevier, 2024. H. Wang et al., ‘Design and Synthesis of Multigraft Copolymer Thermoplastic Elastomers: Superelastomers’, Macro Chemistry & Physics, vol. 219, no. 1, p. 1700254, Jan. 2018, doi: 10.1002/macp.201700254. ‘Newley Isolated Bacilli from the Saline Soil of the Halophytic PlantHalopeplis Perfoliata Community.’, sphrj, pp. 1–2, Mar. 2020, doi: 10.46940/sphrj.02.1004. ‘Molecular Characterization and UV Improvement of Some Bioplastic-Producing Bacteria Isolated from Plants in Taif City, Saudi Arabia’, Pharmacophore, vol. 9, no. 2, pp. 7–18, 2018, doi: 10.51847/23gnxzh. S. Amiri Kojuri, K. Issazadeh, Z. Heshmatipour, M. Mirpour, and S. Zarrabi, ‘Production of Bioplastic Polyhydroxybutyrate) with Local Bacillus megaterium Isolated from Petrochemical Wastewater’, IRAN J BIOTECH, vol. 19, no. 3, Aug. 2021, doi: 10.30498/ijb.2021.244756.2849. Molecular Characterization and UV Improvement of Some Bioplastic-Producing Bacteria Isolated from Plants in Taif City, Saudi Arabia’, Pharmacophore, vol. 9, no. 2, pp. 7–18, 2018, doi: 10.51847/23gnxzh. S. Amiri Kojuri, K. Issazadeh, Z. Heshmatipour, M. Mirpour, and S. Zarrabi, ‘Production of Bioplastic Polyhydroxybutyrate) with Local Bacillus megaterium Isolated from Petrochemical Wastewater’, IRAN J BIOTECH, vol. 19, no. 3, Aug. 2021, doi: 10.30498/ijb.2021.244756.2849. P. Chaber et al., ‘Surface Modification of PHBV Fibrous Scaffold via Lithium Borohydride Reduction’, Materials, vol. 15, no. 21, p. 7494, Oct. 2022, doi: 10.3390/ma15217494. H. Sato and Y. Tanaka, ‘1H‐NMR study of polyisoprenes’, J. Polym. Sci. Polym. Chem. Ed., vol. 17, no. 11, pp. 3551–3558, Nov. 1979, doi: 10.1002/pol.1979.170171113. D. Chen, H. Shao, W. Yao, and B. Huang, ‘Fourier Transform Infrared Spectral Analysis of Polyisoprene of a Different Microstructure’, International Journal of Polymer Science, vol. 2013, pp. 1–5, 2013, doi: 10.1155/2013/937284. Singh, A., & Parmar, N. (2020). Thermophilic microorganisms as a promising source of novel bio-based materials. Biotechnology Advances, 43, 107592. Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3(7), e1700782. Dobre, T., & Mihai, M. (2014). Chemical Engineering: Modelling, Simulation and Similitude. Elsevier. Chapter 9: Valorization of shrimp waste in bio-based polymer production. Patel, S. K. S., et al. (2021). Thermostable biocatalysts and their applications. ACS Catalysis, 11(3), 1603–1620. European Commission. (2020). A new circular economy action plan. [https://ec.europa.eu/environment/circular-economy/] Bassi, A. M., & Yudken, J. S. (2023). Analyzing sustainability of bio-based elastomers through NMR and FTIR diagnostics. Polymer Testing, 114, 107742. Tan, G.-Y. A., et al. (2014). Start a revolution in bioplastics: genome editing for enhanced PHA synthesis. Trends in Biotechnology, 32(5), 247–253. Wang, Y., et al. (2021). Safety evaluation and regulatory challenges of biodegradable elastomers. Advanced Healthcare Materials, 10(8), 2002053. Al-Maadeed, M. A. (2022). National strategies for biotechnology and circular economy in the Middle East. Journal of Cleaner Production, 377, 134419. Sharma, R., & Kaur, G. (2023). Advanced characterization of microbial elastomers: Role of unsaturation in mechanical performance. Journal of Applied Polymer Science, 140(12), e53678. doi:10.1002/app.53678 Lopez-Garcia, R., et al. (2024). Metabolic engineering of Bacillus species for polyisoprene biosynthesis via the mevalonate pathway. Metabolic Engineering, 79, 45–58. doi:10.1016/j.ymben.2023.11.005 Hassan, S. S., et al. (2022). Circular bioeconomy approaches for shrimp waste valorization into high-value bioproducts. Bioresource Technology, 360, 127573. doi:10.1016/j.biortech.2022.127573 Oliveira, D., et al. (2023). Thermophilic bioprocessing systems for industrial biotechnology: Process integration and economic potential. Biotechnology Advances, 62, 108044. doi:10.1016/j.biotechadv.2022.108044 Park, H. S., et al. (2023). Mechanical performance evaluation of bio-based thermoplastic elastomers for industrial applications. Polymer Testing, 118, 107933. doi:10.1016/j.polymertesting.2023.107933 Al-Harbi, M., et al. (2024). Pilot-scale production of bioplastics from marine waste: Techno-economic analysis and life cycle assessment. Journal of Cleaner Production, 430, 139692. doi:10.1016/j.jclepro.2023.139692 Zhang, Y., et al. (2024). Climate-positive materials from microbial bioprocesses: A roadmap for fossil fuel substitution. Nature Sustainability, 7, 112–124. doi:10.1038/s41893-023-01234-7 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-7470334","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":516613280,"identity":"f43c7463-356d-40cf-aaef-4f2fe49f5622","order_by":0,"name":"Abdulaziz M Kusa","email":"","orcid":"","institution":"University of Jeddah","correspondingAuthor":false,"prefix":"","firstName":"Abdulaziz","middleName":"M","lastName":"Kusa","suffix":""},{"id":516613281,"identity":"78998e64-170b-47dc-a7f9-4275171dd47f","order_by":1,"name":"Hanan A Hamdi","email":"","orcid":"","institution":"Collage of Science – Princess Noura Bint Abdul Rahman University","correspondingAuthor":false,"prefix":"","firstName":"Hanan","middleName":"A","lastName":"Hamdi","suffix":""},{"id":516613282,"identity":"ca7f414f-6ca1-42f8-acd8-e088b2c60a56","order_by":2,"name":"Ahmad K AL-Ghamdi","email":"","orcid":"","institution":"University of Jeddah","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"K","lastName":"AL-Ghamdi","suffix":""},{"id":516613283,"identity":"7b2b4a5e-009f-4e14-bd6b-22010b7713f3","order_by":3,"name":"Fayez M. Alshehri","email":"","orcid":"","institution":"University of Jeddah","correspondingAuthor":false,"prefix":"","firstName":"Fayez","middleName":"M.","lastName":"Alshehri","suffix":""},{"id":516613284,"identity":"537e4821-428e-4f9d-8bb0-b3ea90964ebd","order_by":4,"name":"Basim Algashgari","email":"","orcid":"","institution":"King Abdulaziz University","correspondingAuthor":false,"prefix":"","firstName":"Basim","middleName":"","lastName":"Algashgari","suffix":""},{"id":516613285,"identity":"771c50a5-1a6c-4d86-a9a4-8e94d0c5cdea","order_by":5,"name":"Mohammed N Baeshen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie3NPQuCYBDA8YugFsPViOoTBBeCIX0ZJbDFwrEh6JncpDn6Ek7NxkEu0mzU4NTU4Bgk0ePW5MsW9PyH44b7cQAi0U8m5SMAaDNAgGYdIgW1iWLkSwUy2nvHNM1ug0n3gQ6spiaTKSkk2u086+7cu6rvbUSI5iZTLCwmsY29DiPTv3LScIkTKCXqK8to418iTt6cyGFaRrQetMjAWOKEv2Ngl31ZWrrn0tiPLAeN01x1FdspIQuKnxkNMaQDputpfyuHfiH5roVGPivf85pJnWuRSCT6nz4QDEzIYdCepwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Jeddah","correspondingAuthor":true,"prefix":"","firstName":"Mohammed","middleName":"N","lastName":"Baeshen","suffix":""}],"badges":[],"createdAt":"2025-08-27 09:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7470334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7470334/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91738502,"identity":"f6391ec8-51f4-4f73-bd82-b611d8326835","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1143571,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript1v2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/3631d6e805d9a8b1fb60d712.docx"},{"id":91738499,"identity":"a772d8fa-617d-436d-bf2a-9318cbc7e1d2","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8245,"visible":true,"origin":"","legend":"","description":"","filename":"add2558adb024b1dabe1743d7dfc1e46.json","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/5ec5eb721a2dc690e892cd40.json"},{"id":91739457,"identity":"4209c51c-0e3b-4e4e-998a-72715d44c4b5","added_by":"auto","created_at":"2025-09-19 18:17:36","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93911,"visible":true,"origin":"","legend":"","description":"","filename":"add2558adb024b1dabe1743d7dfc1e461enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/f9d8c14193005c233572c739.xml"},{"id":91739459,"identity":"3701e693-7481-4a25-a987-37209f2df38f","added_by":"auto","created_at":"2025-09-19 18:17:36","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181808,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/4c03dc4dd23441fbd356a8ee.jpeg"},{"id":91738512,"identity":"3d065e57-4b0c-4f50-b528-47809a2f2aed","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":785644,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/3fec07415bae02dfd9985c09.png"},{"id":91739455,"identity":"58ecb045-ee22-4094-932f-070e88fc5fc7","added_by":"auto","created_at":"2025-09-19 18:17:36","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96822,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/031bd575a1aec79522682775.png"},{"id":91738507,"identity":"9efcabce-14dd-4747-b5dc-76f4b23499da","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31807,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/c0661113b50123a3a18537c5.png"},{"id":91740461,"identity":"8641bdec-6f43-4b9c-bbce-37b528eb1d67","added_by":"auto","created_at":"2025-09-19 18:33:36","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119942,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/055516c0b5e1f96af6192b55.png"},{"id":91738508,"identity":"d4b3510f-99a5-442e-8f21-50b4e7e1aef5","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111305,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/d1d5b11bc38ce1948b702560.png"},{"id":91738509,"identity":"6e59ae5c-d757-4a10-b5c4-7d1892b1c2c4","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16561,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/029761eca4eac6d8b9528f16.png"},{"id":91739460,"identity":"90887f64-daf6-4bb7-8723-0c076b5deb98","added_by":"auto","created_at":"2025-09-19 18:17:36","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11625,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/cd397bdfb27fd0d5f6b92c72.png"},{"id":91738515,"identity":"3f88cf5a-d33c-4f1b-9b74-7cf6b048eea6","added_by":"auto","created_at":"2025-09-19 18:09:37","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92047,"visible":true,"origin":"","legend":"","description":"","filename":"add2558adb024b1dabe1743d7dfc1e461structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/f3c3ad3d940afd1912580d76.xml"},{"id":91740298,"identity":"4c52cf15-fe04-4317-b023-49b6e2782259","added_by":"auto","created_at":"2025-09-19 18:25:36","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105441,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/ebd99be0cca7371b4d575ff9.html"},{"id":91740295,"identity":"49345588-f173-4ac6-9760-b02610269e48","added_by":"auto","created_at":"2025-09-19 18:25:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177104,"visible":true,"origin":"","legend":"\u003cp\u003eDark TPE regions at the bacterial cell poles observed by light microscope (x100).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/801c3044bd112522bd5120a4.png"},{"id":91738506,"identity":"686c801b-52e7-46ce-ae65-f077950f4f2c","added_by":"auto","created_at":"2025-09-19 18:09:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245584,"visible":true,"origin":"","legend":"\u003cp\u003eblack colored colony as an indication of TPE production\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/e9724b5a32c59d5555354bca.png"},{"id":91739453,"identity":"3042b5aa-8c36-44a2-b5c3-463602bb14a2","added_by":"auto","created_at":"2025-09-19 18:17:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73483,"visible":true,"origin":"","legend":"\u003cp\u003eShow the obtained FT-IR spectrum of the sample.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/71c90f70d2baa9270b004fff.png"},{"id":91740296,"identity":"4b77d1c7-5b0d-4939-8c66-852c04438ea0","added_by":"auto","created_at":"2025-09-19 18:25:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18992,"visible":true,"origin":"","legend":"\u003cp\u003e1HNMR Spectrum of the sample.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/a3b05991e56440dff42ee8e9.png"},{"id":92374031,"identity":"1c68c7be-b00b-4b50-b202-625e91119fd2","added_by":"auto","created_at":"2025-09-29 04:01:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1166197,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7470334/v1/d2cb6c29-ee15-45a3-afa0-761408a3be55.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Saudi Hot Spring Extremophilic Bacillus as an Alternative Bioresource for Sustainable Thermoplastic Elastomer (TPE) Biosynthesis from Shrimp Waste","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlastic consumer products, especially single-use plastics, are pervasive; yet their current production and utilization are unsustainable [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This untenable development is largely attributed to the characteristics of the materials used in plastic production. Carbon-carbon polymers, which comprise most plastics produced globally, are inexpensive and environmentally persistent, resulting in a massive reservoir of plastic waste that ends up in landfills and the environment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a result of these characteristics, as of 2017, just 9% of the 9\u0026nbsp;billion tonnes of plastic that were created had been recycled, meaning that a significant amount of plastic was still contaminating the environment and having detrimental effects on both health and the economy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Given these alarming statistics, however, there is already enough data to demonstrate that swift and extensive action is required to prevent future harm by reducing the amount of plastic released into the ecosystem [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNumerous renewable raw resources can be used to produce biobased plastics, including polysaccharides, proteins, starch- and cellulose-based polymers from plants, and microbial bioplastics such as polylactic acid and polyhydroxyalkanoates (PHAs) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, PHAs stand out due to their microbial origin and sustainability profile. Microorganisms may manufacture PHA, a biopolymer, from a variety of inexpensive carbon sources, making it an eco-friendly substance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nonetheless, synthetic plastics remain prevalent in the market and continue to present environmental hazards. Plastic components commonly utilized in daily life are increasingly generating severe environmental issues, with millions of tonnes of these non-biodegradable polymers accumulating annually in the environment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This accumulation leads to severe environmental damage. Severe soil and water pollution occurs due to the slow or non-degradable nature of synthetic plastic; also, when burning, it causes air pollution [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Certain microbes respond to nutrient stress by synthesizing biopolymers, such as PHB, the most extensively researched member of the PHA family [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong the various microbial candidates for PHA production, Bacillus species have shown exceptional potential. Some Bacillus species have been found to produce as much as 90% (w/w) PHAs of dry cells during nutrient imbalance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Because of their genetic stability, Bacillus species have gained popularity as model organisms in both industry and academia. Moreover, thermophilic Bacillus strains offer unique advantages in addition to having a higher growth rate than other bacteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThermophilic Bacillus helps to preserve vitality during thermal processing since it can withstand high temperatures [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thermophilic Bacillus species offer industrial advantages, including thermostable enzymes, rapid proliferation, and resilience, rendering them efficient cell factories for PHA manufacture from inexpensive substrates [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The biotechnological value of thermophilic Bacillus is enhanced by its robust physiology. All Gram-positive thermophilic Bacillus are often tolerant of their surroundings, including heat, acid, bile, and salt [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, numerous extracellular thermostable enzymes with significant economic value, such as lipases, amylases, cellulases, and proteases, can be produced by thermophilic Bacillus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Bacillus species are desirable industrial organisms due to their ability to secrete proteins, high growth rates, short fermentation cycles, and GRAS classification by the FDA (generally regarded as safe) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. According to [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the ability of thermophiles to produce a wide range of useful molecules, including antibiotics, thermostable enzymes, and anticancer drugs, makes them of international interest.\u003c/p\u003e\u003cp\u003eOne such promising bacterium is \u003cem\u003eBacillus tequilensis\u003c/em\u003e, a rod-shaped, aerobic, motile, and catalase-positive probiotic bacterium [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Numerous Bacillus species from saline and marine environments, including \u003cem\u003eB. tequilensis\u003c/em\u003e, have been investigated for their ability to produce biosurfactants, such as lipopeptides [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, \u003cem\u003eB. tequilensis\u003c/em\u003e produces amylase in the presence of cyclohexane and other stressful conditions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This capability allows for the conversion of animal fats, glycerol, and various dietary wastes into PHA by \u003cem\u003eB. tequilensis\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, it has been shown that \u003cem\u003eB. tequilensis\u003c/em\u003e produces PHA utilizing synthetic acids and food waste [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA unique and impactful area of discovery within this field is the use of Bacillus-derived polymers for the development of advanced biomaterials, particularly thermoplastic elastomers (TPEs). This direction holds great promise and deserves further investigation. To explore practical applications of microbially derived polymers, attention has turned to thermoplastic elastomers (TPEs), which combine the properties of plastics and rubber [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This structure provides flexibility and resilience. Thermoplastic elastomers are of great interest due to their cushioning properties [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The term \"elastomer\" comes from \"elastic polymer,\" referring to any rubbery-textured substance composed of long, chain-like molecules, or polymers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. By offering environmental advantages in both manufacturing and disposal, TPEs also present a pathway toward sustainable development TPEs are versatile and used in various sectors, including medical, aerospace, construction, optical fibers, automotive coatings such as gaskets, seals, and interior components, surgical instruments, wound dressings, yoga mats, toothbrush grips, vibration dampers, medical soft robotics, 3D printing, footwear industries, and air ducts [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Their popularity is increasing because they can replace traditional rubbers in applications where performance and sustainability are required [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Due to their unique performance, low production costs, good processability, and recyclability, thermoplastic elastomers, or TPEs, have been the subject of extensive research [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Ongoing research on Bacillus-based elastomeric systems is not only scientifically significant but also essential for expediting the global shift towards sustainable and circular bioeconomy materials.\u003c/p\u003e\u003cp\u003eThe study aims to fulfil the national vision goal by utilizing alternative renewable resources from extreme environments to produce eco-friendly products from organic waste, in line with the national biotechnological strategy. This approach helps preserve the ecosystem and serves humanity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and molecular identification of Bacillus tequilensis\u003c/h2\u003e\u003cp\u003eEnvironmental samples were collected from a hot spring in Al Lith, Saudi Arabia. Samples of water and sediment were moved to sterile containers aseptically, and they were brought to the lab on ice (4\u0026deg;C). Samples were exposed to heat shock treatment at 80\u0026deg;C for 10 minutes to select for thermophilic, spore-forming bacteria.\u003c/p\u003e\u003cp\u003eAliquots were then spread out on Nutrient Agar (NA) plates after serial dilutions were made in sterile saline (0.85% NaCl). For 48 hours, plates were incubated at 50\u0026deg;C. To produce pure isolates, morphologically different colonies with characteristics typical of Bacillus (dry, irregular, opaque) were chosen and subcultured.\u003c/p\u003e\u003cp\u003eThe QIAamp\u0026reg; DNA Mini Kit (Qiagen) was used to extract genomic DNA, and universal primers 27F and 1492R were used to amplify the 16S rRNA gene. Sanger dideoxy sequencing was used to sequence the PCR results, and NCBI BLASTn was used to analyse the sequences for taxonomic confirmation. The isolate was identified as \u003cem\u003eBacillus tequilensis\u003c/em\u003e strain LW, and its partial 16S rRNA gene sequence was submitted to the NCBI GenBank database, receiving accession number OR616739.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInduction of thermoplastic elastomer (TPE) production\u003c/h3\u003e\n\u003cp\u003eTo induce the biosynthesis of bacterial thermoplastic elastomers, the confirmed strain of \u003cem\u003eB. tequilensis\u003c/em\u003e was inoculated into Nutrient Broth (NB) medium prepared according to the protocol described by [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The composition of the medium is detailed in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cultures were incubated at 50\u0026deg;C for 72 h under static conditions to enhance intracellular accumulation of polymeric compounds.\u003c/p\u003e\n\u003ch3\u003eHistochemical confirmation of TPE production\u003c/h3\u003e\n\u003cp\u003eFollowing incubation, qualitative confirmation of TPE accumulation was conducted using Sudan Black B staining. Bacterial smears were prepared on glass slides, heat-fixed, and stained with 0.3% (w/v) Sudan Black B dissolved in 70% ethanol for 10 minutes. Slides were briefly washed with distilled water and counterstained with safranin for 2 minutes before microscopic examination under a 100\u0026times; oil immersion lens. Dark intracellular granules were considered indicative of polymer inclusion bodies [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA second confirmatory assay was performed by streaking the isolate onto nutrient agar plates (same composition as broth). Plates were incubated at 50\u0026deg;C for 48 h, after which 0.3% Sudan Black B in ethanol was applied to the surface. Plates were left undisturbed for 30 minutes and then gently rinsed with 96% ethanol to remove excess stain. Dark blue-black colonies were interpreted as positive for TPE accumulation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eExtraction and purification of bacterial TPE\u003c/h3\u003e\n\u003cp\u003eTo extract the fermented culture, the entire volume was centrifuged for ten minutes at 6000 rpm. The pellet was incubated for 1.5 hours at 37\u0026deg;C to lyse the cell wall and release the intracellular contents after 10 mL of 10% (v/v) sodium hypochlorite (NaClO) was added. The supernatant was disposed of. The modified suspension underwent another centrifugation at 6000 rpm for 10 minutes. The resulting pellet was cleaned with distilled water, acetone, and methanol in order to remove any last impurities.\u003c/p\u003e\u003cp\u003eAfter dissolving the purified granules in 10 mL of chloroform, the mixture was poured onto sterile glass trays to evaporate, and it was then dried in a hot air oven set at 40\u0026deg;C until all the solvent had been removed [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStructural characterization of extracted TPE\u003c/h3\u003e\n\u003cp\u003eThe chemical structure of the recovered TPE was evaluated using Fourier-transform infrared spectroscopy (FTIR) to identify functional groups. Additionally, nuclear magnetic resonance (NMR) spectroscopy was conducted to confirm the polymer\u0026rsquo;s backbone structure and monomeric composition.\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\u003eAdditives to NB and NA media (g/1 L) for the production of TPE:\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComponent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuantities\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDisodium phosphate (Na2HPO4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.0 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonopotassium phosphate (KH2PO4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmmonium chloride (NH4Cl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium chloride (NaCl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0 g\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbon Sources (Shrimp peel)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0 g\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":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eConfirmation of TPE Production via Histochemical and Colony Staining\u003c/h2\u003e\u003cp\u003eMicroscopic analysis using Sudan Black B staining revealed intracellular dark inclusions located at the poles of \u003cem\u003eB. tequilensis\u003c/em\u003e cells, indicative of polymer granule accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These dark inclusions are consistent with intracellular lipid-like or polymeric materials, suggesting PHA or TPE production. On nutrient agar (NA) plates, colonies were stained by black following Sudan Black B application, reinforcing the intracellular staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such staining methods are commonly used as preliminary indicators of bioplastic or elastomeric polymer accumulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFTIR Spectroscopy Confirms Polymer Backbone\u003c/h3\u003e\n\u003cp\u003eFourier-transform infrared spectroscopy (FTIR) was used to evaluate the polymer's chemical structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Characteristic absorbance peaks at 1667 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;C), 2919 cm⁻\u0026sup1; (C\u0026ndash;H), and 1032 cm⁻\u0026sup1; (C\u0026ndash;O) suggest an unsaturated hydrocarbon backbone, consistent with isoprene-based polymers. These spectra align with previously reported microbial elastomers and PHAs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStructural Characterization by \u0026sup1;H-NMR\u003c/h2\u003e\u003cp\u003eThe \u0026sup1;H-NMR spectrum (Fig.\u0026nbsp;4) showed three major signals: a singlet at δ 1.45 ppm (CH₃), a multiplet at δ 1.99\u0026ndash;2.08 ppm (CH₂), and a triplet at δ 5.32 ppm (CH\u0026thinsp;=\u0026thinsp;CH). These chemical shifts and splitting patterns match the profiles of cis-1,4-polyisoprene, a rubber-like elastomer. Absence of contaminant peaks supports product purity. These NMR results confirm the polymer structure, further validating the identity of the synthesized elastomer as polyisoprene-like TPE.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings support the concept of using thermophilic Bacillus tequilensis from Al-Lith hot springs as a microbial platform for elastomer biosynthesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This aligns with global trends toward biobased, renewable materials. Given the environmental persistence of synthetic plastics, the ability to synthesize eco-friendly TPE from shrimp waste using a native thermophile is particularly valuable [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe use of shrimp waste as a low-cost carbon source provides dual environmental and economic advantages: it offers a solution to aquaculture waste disposal and provides a renewable substrate for high-value biomaterials. Previous studies have shown the effectiveness of seafood waste in PHA and TPE biosynthesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]; however, this study is among the first to demonstrate successful polyisoprene-like TPE production by Bacillus tequilensis in a high-temperature system.\u003c/p\u003e\u003cp\u003eThe thermophilic nature of the bacterium is particularly advantageous, as it reduces the risk of contamination and enhances process efficiency at elevated temperatures. Thermostable production systems are also more compatible with industrial bioreactor designs, where heating is often needed for sterilization or reaction kinetics [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This opens the door for integrating such microbial processes into large-scale circular bioeconomy platforms [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn terms of structural validation, the FTIR and \u0026sup1;H-NMR data strongly support the polymer\u0026rsquo;s identity as a rubber-like TPE. These analytical tools are crucial in distinguishing between polyhydroxyalkanoates (PHAs), other bacterial polyesters, and true elastomeric compounds. The presence of unsaturated C\u0026thinsp;=\u0026thinsp;C bonds and methyl-methylene-olefinic proton profiles indicates a polyisoprene-like backbone, essential for achieving elastomeric mechanical properties [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile promising, the yield, scalability, and mechanical strength of the extracted polymer need further evaluation. Comparative studies with other extremophiles or genetically engineered strains may help improve productivity and tailor material properties for specific applications [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAdditionally, further research should assess biodegradability, thermal stability, cytotoxicity, and performance in real-world product applications. Regulatory pathways for medical or industrial approval should also be explored if this TPE is to replace conventional rubbers in sectors such as healthcare, packaging, or electronics [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, the study provides a novel proof-of-concept that advances the field of microbial elastomers by leveraging native Saudi extremophiles, aligning with national strategies for sustainable biotechnology and circular economy transformation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe valorization of shrimp waste not only addresses aquaculture waste management but also provides a renewable, low-cost carbon source for polymer biosynthesis, thereby contributing to circular bioeconomy goals [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Moreover, the thermophilic nature of \u003cem\u003eB. tequilensis\u003c/em\u003e offers operational benefits such as reduced contamination risk and compatibility with industrial-scale heat-sterilized fermenters [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This is particularly relevant for continuous and semi-continuous fermentation systems, which can significantly lower production costs.\u003c/p\u003e\u003cp\u003eIn addition, the distinct unsaturated bonds observed in FTIR spectra indicate a high proportion of elastomeric segments, a property linked to enhanced mechanical flexibility and resilience [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This structural attribute positions the produced TPE for potential use in high-performance applications, including medical devices, automotive components, and 3D printing materials [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo fully realize the industrial potential of this bioprocess, future studies should incorporate pilot-scale trials using locally sourced shrimp waste under industrial fermentation conditions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These trials should also be coupled with a life cycle assessment to validate the environmental sustainability of the process. Furthermore, the concept aligns with global trends toward climate-positive materials, providing a pathway for replacing fossil fuel-derived elastomers with renewable microbial alternatives [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully demonstrates the potential of a newly isolated thermophilic \u003cem\u003eB. tequilensis\u003c/em\u003e strain from Al-Lith hot springs as a sustainable microbial platform for the biosynthesis of thermoplastic elastomers (TPE) using shrimp waste as a low-cost, renewable carbon source. The confirmation of TPE production through histochemical staining, FTIR, and \u0026sup1;H-NMR spectroscopy highlights the biochemical and structural validity of the synthesized polymer, aligning with the chemical properties of polyisoprene-like elastomers. The ability of this native extremophile to produce high-value bioplastics under elevated temperatures offers a promising biotechnological solution for reducing plastic pollution, valorizing seafood waste, and supporting Saudi Arabia\u0026rsquo;s Vision 2030 strategy for a circular bioeconomy. Future studies should focus on optimizing production yield, scaling up bioprocesses, evaluating material performance, and exploring potential applications in medical, industrial, and consumer product sectors. Overall, this work lays the groundwork for harnessing extremophilic biodiversity in the region for innovative, eco-friendly industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors received no financial support for the research, authorship, or publication of this article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAbdulaziz M Kusa: sample collection - microbial and molecular experiments - data analysis - reports writing.Hanan A Hamdi: manuscript writing.Ahmad K AL-Ghamdi: Microbial and molecular experiments - report writing.Fayez M. Alshehri: Microbial and molecular experiments - report writing.Basim Algashgari: Chemical experiments and analysis.Burhan Z Fakhurji: Providing financial support by Xgenome biotech company.Mohammed N Baeshen: Managing and supervising all project phases and experiments - repots and manuscript reviewing and writing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank the department of biological sciences, college of science, university of Jeddah, Jeddah, Saudi Arabia and KFMRC, king Abdulaziz university, Jeddah, Saudi Arabia for their technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data that support the findings of this study have been deposited in the NCBI GenBank database with the primary accession number OR616739.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eT. R. Walker and L. Fequet, \u0026lsquo;Current trends of unsustainable plastic production and micro(nano)plastic pollution\u0026rsquo;, TrAC Trends in Analytical Chemistry, vol. 160, p. 116984, Mar. 2023, doi: 10.1016/j.trac.2023.116984.\u003c/li\u003e\n\u003cli\u003eK. L. Law and R. Narayan, \u0026lsquo;Reducing environmental plastic pollution by designing polymer materials for managed end-of-life\u0026rsquo;, Nat Rev Mater, vol. 7, no. 2, pp. 104\u0026ndash;116, Oct. 2021, doi: 10.1038/s41578-021-00382-0.\u003c/li\u003e\n\u003cli\u003eJ. Nikiema and Z. Asiedu, \u0026lsquo;A review of the cost and effectiveness of solutions to address plastic pollution\u0026rsquo;, Environ Sci Pollut Res, vol. 29, no. 17, pp. 24547\u0026ndash;24573, Apr. 2022, doi: 10.1007/s11356-021-18038-5.\u003c/li\u003e\n\u003cli\u003eA. A. Horton, \u0026lsquo;Plastic pollution: When do we know enough?\u0026rsquo;, Journal of Hazardous Materials, vol. 422, p. 126885, Jan. 2022, doi: 10.1016/j.jhazmat.2021.126885.\u003c/li\u003e\n\u003cli\u003eM. Adnan et al., \u0026lsquo;Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site\u0026rsquo;, Polymers, vol. 15, no. 6, p. 1407, Mar. 2023, doi: 10.3390/polym15061407.\u003c/li\u003e\n\u003cli\u003eN. A. Jamaludin, S. F. Z. Mohd Fuzi, M. I. Mohd Ghazali, M. I. Juki, A. F. Abdulaziz Al-Shalif, and N. Othman, \u0026lsquo;Production of Polyhydroxyalkanoates (PHA) by Probiotic Bacteria Bacillus tequilensis for Potentially Used as Drug Carrier\u0026rsquo;, MJMHS, vol. 19, no. s9, pp. 126\u0026ndash;132, Aug. 2023, doi: 10.47836/mjmhs.19.s9.19.\u003c/li\u003e\n\u003cli\u003eA. W. Danial, S. M. Hamdy, S. A. Alrumman, S. M. F. Gad El-Rab, A. A. M. Shoreit, and A. E.-L. Hesham, \u0026lsquo;Bioplastic Production by Bacillus wiedmannii AS-02 OK576278 Using Different Agricultural Wastes\u0026rsquo;, Microorganisms, vol. 9, no. 11, p. 2395, Nov. 2021, doi: 10.3390/microorganisms9112395.\u003c/li\u003e\n\u003cli\u003eM. Narayanan et al., \u0026lsquo;Optimization and production of polyhydroxybutyrate from sludge by Bacillus cereus categorized through FT-IR and NMR analyses\u0026rsquo;, Journal of Environmental Chemical Engineering, vol. 9, no. 1, p. 104908, Feb. 2021, doi: 10.1016/j.jece.2020.104908.\u003c/li\u003e\n\u003cli\u003eA. J. Anderson and E. A. Dawes, \u0026lsquo;Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates\u0026rsquo;, Microbiol Rev, vol. 54, no. 4, pp. 450\u0026ndash;472, Dec. 1990, doi: 10.1128/mr.54.4.450-472.1990.\u003c/li\u003e\n\u003cli\u003eL. L. Madison and G. W. Huisman, \u0026lsquo;Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic\u0026rsquo;, Microbiol Mol Biol Rev, vol. 63, no. 1, pp. 21\u0026ndash;53, Mar. 1999, doi: 10.1128/mmbr.63.1.21-53.1999.\u003c/li\u003e\n\u003cli\u003eD. Vicente, D. N. Proen\u0026ccedil;a, and P. V. Morais, \u0026lsquo;The Role of Bacterial Polyhydroalkanoate (PHA) in a Sustainable Future: A Review on the Biological Diversity\u0026rsquo;, IJERPH, vol. 20, no. 4, p. 2959, Feb. 2023, doi: 10.3390/ijerph20042959.\u003c/li\u003e\n\u003cli\u003eN. Zhao, T. Yu, and F. Yan, \u0026lsquo;Probiotic role and application of thermophilic Bacillus as novel food materials\u0026rsquo;, Trends in Food Science \u0026amp; Technology, vol. 138, pp. 1\u0026ndash;15, Aug. 2023, doi: 10.1016/j.tifs.2023.05.020.\u003c/li\u003e\n\u003cli\u003eD. H. Vu, S. Wainaina, M. J. Taherzadeh, D. \u0026Aring;kesson, and J. A. Ferreira, \u0026lsquo;Production of polyhydroxyalkanoates (PHAs) by Bacillus megaterium using food waste acidogenic fermentation-derived volatile fatty acids\u0026rsquo;, Bioengineered, vol. 12, no. 1, pp. 2480\u0026ndash;2498, Jan. 2021, doi: 10.1080/21655979.2021.1935524.\u003c/li\u003e\n\u003cli\u003eF. Su and P. Xu, \u0026lsquo;Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals\u0026rsquo;, Sci Rep, vol. 4, no. 1, Jan. 2014, doi: 10.1038/srep03926.\u003c/li\u003e\n\u003cli\u003eN. E. Ibrahim and K. Ma, \u0026lsquo;Industrial Applications of Thermostable Enzymes from Extremophilic Microorganisms\u0026rsquo;, CBE, vol. 4, no. 2, July 2017, doi: 10.2174/2212711904666170405123414.\u003c/li\u003e\n\u003cli\u003eM. Schallmey, A. Singh, and O. P. Ward, \u0026lsquo;Developments in the use ofBacillusspecies for industrial production\u0026rsquo;, Can. J. Microbiol., vol. 50, no. 1, pp. 1\u0026ndash;17, Jan. 2004, doi: 10.1139/w03-076.\u003c/li\u003e\n\u003cli\u003eS. Arbab et al., \u0026lsquo;Diversity and distribution of thermophilic microorganisms and their applications in biotechnology\u0026rsquo;, J Basic Microbiol, vol. 62, no. 2, pp. 95\u0026ndash;108, Feb. 2022, doi: 10.1002/jobm.202100529.\u003c/li\u003e\n\u003cli\u003eR. P. Rani, M. Anandharaj, P. Sabhapathy, and A. D. Ravindran, \u0026lsquo;Physiochemical and biological characterization of novel exopolysaccharide produced by Bacillus tequilensis FR9 isolated from chicken\u0026rsquo;, International Journal of Biological Macromolecules, vol. 96, pp. 1\u0026ndash;10, Mar. 2017, doi: 10.1016/j.ijbiomac.2016.11.122.\u003c/li\u003e\n\u003cli\u003eS. Cort\u0026eacute;s‐Camargo et al., \u0026lsquo;Biosurfactant Production by Bacillus tequilensis\u0026thinsp;ZSB10: Structural Characterization, Physicochemical, and Antifungal Properties\u0026rsquo;, J Surfact \u0026amp; Detergents, vol. 24, no. 5, pp. 773\u0026ndash;782, Sept. 2021, doi: 10.1002/jsde.12515.\u003c/li\u003e\n\u003cli\u003eS. Tiwari, N. Shukla, P. Mishra, and R. Gaur, \u0026lsquo;Enhanced Production and Characterization of a Solvent Stable Amylase from Solvent TolerantBacillus tequilensisRG-01: Thermostable and Surfactant Resistant\u0026rsquo;, The Scientific World Journal, vol. 2014, pp. 1\u0026ndash;11, 2014, doi: 10.1155/2014/972763.\u003c/li\u003e\n\u003cli\u003eR. Sohail, N. Jamil, I. Ali, and S. Munir, \u0026lsquo;Animal fat and glycerol bioconversion to polyhydroxyalkanoate by produced water bacteria\u0026rsquo;, e-Polymers, vol. 20, no. 1, pp. 92\u0026ndash;102, Mar. 2020, doi: 10.1515/epoly-2020-0011.\u003c/li\u003e\n\u003cli\u003eM. Venkateswar Reddy, K. Amulya, M. V. Rohit, P. N. Sarma, and S. Venkata Mohan, \u0026lsquo;Valorization of fatty acid waste for bioplastics production using Bacillus tequilensis: Integration with dark-fermentative hydrogen production process\u0026rsquo;, International Journal of Hydrogen Energy, vol. 39, no. 14, pp. 7616\u0026ndash;7626, May 2014, doi: 10.1016/j.ijhydene.2013.09.157.\u003c/li\u003e\n\u003cli\u003eG. Holden, \u0026lsquo;Thermoplastic Elastomers\u0026rsquo;, in Applied Plastics Engineering Handbook, Elsevier, 2024, pp. 97\u0026ndash;113. doi: 10.1016/B978-0-323-88667-3.00020-5.\u003c/li\u003e\n\u003cli\u003eC. Ge, L. Priyadarshini, D. Cormier, L. Pan, and J. Tuber, \u0026lsquo;A preliminary study of cushion properties of a 3D printed thermoplastic polyurethane Kelvin foam\u0026rsquo;, Packag Technol Sci, vol. 31, no. 5, pp. 361\u0026ndash;368, May 2018, doi: 10.1002/pts.2330.\u003c/li\u003e\n\u003cli\u003eH. Chung, A. M. Parsons, and L. Zheng, \u0026lsquo;Magnetically Controlled Soft Robotics Utilizing Elastomers and Gels in Actuation: A Review\u0026rsquo;, Advanced Intelligent Systems, vol. 3, no. 3, Mar. 2021, doi: 10.1002/aisy.202000186.\u003c/li\u003e\n\u003cli\u003eJ. G. Tejani, \u0026lsquo;Thermoplastic Elastomers: Emerging Trends and Applications in Rubber Manufacturing\u0026rsquo;, Glob. discl. econ. bus., vol. 6, no. 2, pp. 133\u0026ndash;144, Dec. 2017, doi: 10.18034/gdeb.v6i2.737.\u003c/li\u003e\n\u003cli\u003eN. K. Singha and S. C. Jana, Advances in Thermoplastic Elastomers: Challenges and Opportunities. Elsevier, 2024.\u003c/li\u003e\n\u003cli\u003eH. Wang et al., \u0026lsquo;Design and Synthesis of Multigraft Copolymer Thermoplastic Elastomers: Superelastomers\u0026rsquo;, Macro Chemistry \u0026amp; Physics, vol. 219, no. 1, p. 1700254, Jan. 2018, doi: 10.1002/macp.201700254.\u003c/li\u003e\n\u003cli\u003e\u0026lsquo;Newley Isolated Bacilli from the Saline Soil of the Halophytic PlantHalopeplis Perfoliata Community.\u0026rsquo;, sphrj, pp. 1\u0026ndash;2, Mar. 2020, doi: 10.46940/sphrj.02.1004.\u003c/li\u003e\n\u003cli\u003e\u0026lsquo;Molecular Characterization and UV Improvement of Some Bioplastic-Producing Bacteria Isolated from Plants in Taif City, Saudi Arabia\u0026rsquo;, Pharmacophore, vol. 9, no. 2, pp. 7\u0026ndash;18, 2018, doi: 10.51847/23gnxzh.\u003c/li\u003e\n\u003cli\u003eS. Amiri Kojuri, K. Issazadeh, Z. Heshmatipour, M. Mirpour, and S. Zarrabi, \u0026lsquo;Production of Bioplastic Polyhydroxybutyrate) with Local Bacillus megaterium Isolated from Petrochemical Wastewater\u0026rsquo;, IRAN J BIOTECH, vol. 19, no. 3, Aug. 2021, doi: 10.30498/ijb.2021.244756.2849.\u003c/li\u003e\n\u003cli\u003eMolecular Characterization and UV Improvement of Some Bioplastic-Producing Bacteria Isolated from Plants in Taif City, Saudi Arabia\u0026rsquo;, Pharmacophore, vol. 9, no. 2, pp. 7\u0026ndash;18, 2018, doi: 10.51847/23gnxzh.\u003c/li\u003e\n\u003cli\u003eS. Amiri Kojuri, K. Issazadeh, Z. Heshmatipour, M. Mirpour, and S. Zarrabi, \u0026lsquo;Production of Bioplastic Polyhydroxybutyrate) with Local Bacillus megaterium Isolated from Petrochemical Wastewater\u0026rsquo;, IRAN J BIOTECH, vol. 19, no. 3, Aug. 2021, doi: 10.30498/ijb.2021.244756.2849.\u003c/li\u003e\n\u003cli\u003eP. Chaber et al., \u0026lsquo;Surface Modification of PHBV Fibrous Scaffold via Lithium Borohydride Reduction\u0026rsquo;, Materials, vol. 15, no. 21, p. 7494, Oct. 2022, doi: 10.3390/ma15217494.\u003c/li\u003e\n\u003cli\u003eH. Sato and Y. Tanaka, \u0026lsquo;1H‐NMR study of polyisoprenes\u0026rsquo;, J. Polym. Sci. Polym. Chem. Ed., vol. 17, no. 11, pp. 3551\u0026ndash;3558, Nov. 1979, doi: 10.1002/pol.1979.170171113.\u003c/li\u003e\n\u003cli\u003eD. Chen, H. Shao, W. Yao, and B. Huang, \u0026lsquo;Fourier Transform Infrared Spectral Analysis of Polyisoprene of a Different Microstructure\u0026rsquo;, International Journal of Polymer Science, vol. 2013, pp. 1\u0026ndash;5, 2013, doi: 10.1155/2013/937284.\u003c/li\u003e\n\u003cli\u003eSingh, A., \u0026amp; Parmar, N. (2020). Thermophilic microorganisms as a promising source of novel bio-based materials. Biotechnology Advances, 43, 107592.\u003c/li\u003e\n\u003cli\u003eGeyer, R., Jambeck, J. R., \u0026amp; Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science Advances, 3(7), e1700782.\u003c/li\u003e\n\u003cli\u003eDobre, T., \u0026amp; Mihai, M. (2014). Chemical Engineering: Modelling, Simulation and Similitude. Elsevier. Chapter 9: Valorization of shrimp waste in bio-based polymer production.\u003c/li\u003e\n\u003cli\u003ePatel, S. K. S., et al. (2021). Thermostable biocatalysts and their applications. ACS Catalysis, 11(3), 1603\u0026ndash;1620.\u003c/li\u003e\n\u003cli\u003eEuropean Commission. (2020). A new circular economy action plan. [https://ec.europa.eu/environment/circular-economy/]\u003c/li\u003e\n\u003cli\u003eBassi, A. M., \u0026amp; Yudken, J. S. (2023). Analyzing sustainability of bio-based elastomers through NMR and FTIR diagnostics. Polymer Testing, 114, 107742.\u003c/li\u003e\n\u003cli\u003eTan, G.-Y. A., et al. (2014). Start a revolution in bioplastics: genome editing for enhanced PHA synthesis. Trends in Biotechnology, 32(5), 247\u0026ndash;253.\u003c/li\u003e\n\u003cli\u003eWang, Y., et al. (2021). Safety evaluation and regulatory challenges of biodegradable elastomers. Advanced Healthcare Materials, 10(8), 2002053.\u003c/li\u003e\n\u003cli\u003eAl-Maadeed, M. A. (2022). National strategies for biotechnology and circular economy in the Middle East. Journal of Cleaner Production, 377, 134419.\u003c/li\u003e\n\u003cli\u003eSharma, R., \u0026amp; Kaur, G. (2023). Advanced characterization of microbial elastomers: Role of unsaturation in mechanical performance. Journal of Applied Polymer Science, 140(12), e53678. doi:10.1002/app.53678\u003c/li\u003e\n\u003cli\u003eLopez-Garcia, R., et al. (2024). Metabolic engineering of Bacillus species for polyisoprene biosynthesis via the mevalonate pathway. Metabolic Engineering, 79, 45\u0026ndash;58. doi:10.1016/j.ymben.2023.11.005\u003c/li\u003e\n\u003cli\u003eHassan, S. S., et al. (2022). Circular bioeconomy approaches for shrimp waste valorization into high-value bioproducts. Bioresource Technology, 360, 127573. doi:10.1016/j.biortech.2022.127573\u003c/li\u003e\n\u003cli\u003eOliveira, D., et al. (2023). Thermophilic bioprocessing systems for industrial biotechnology: Process integration and economic potential. Biotechnology Advances, 62, 108044. doi:10.1016/j.biotechadv.2022.108044\u003c/li\u003e\n\u003cli\u003ePark, H. S., et al. (2023). Mechanical performance evaluation of bio-based thermoplastic elastomers for industrial applications. Polymer Testing, 118, 107933. doi:10.1016/j.polymertesting.2023.107933\u003c/li\u003e\n\u003cli\u003eAl-Harbi, M., et al. (2024). Pilot-scale production of bioplastics from marine waste: Techno-economic analysis and life cycle assessment. Journal of Cleaner Production, 430, 139692. doi:10.1016/j.jclepro.2023.139692\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al. (2024). Climate-positive materials from microbial bioprocesses: A roadmap for fossil fuel substitution. Nature Sustainability, 7, 112\u0026ndash;124. doi:10.1038/s41893-023-01234-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7470334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7470334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Saudi Vision 2030 biotechnology strategy aims to support sustainable development by exploring Saudi Arabia's unique extreme ecosystems. The diverse environment, including water ecosystems like marine and terrestrial environments, offers numerous biotechnological applications. The study aims to fulfill the national vision goal by using alternative renewable resources from extreme environments to produce eco-friendly products from organic waste, following the national biotechnological strategy. This approach helps preserve the ecosystem and serve humanity. Nnewly isolated \u003cem\u003eBacillus tequilensis\u003c/em\u003e was recently isolated from Al-Lith hot spring and identified by 16s rRNA sequencing, then submitted to GenBank by the accession number (OR616739). \u003cem\u003eB. tequilensis\u003c/em\u003e isolates were used to produce bioplastic from shrimp waste. To ensure the production of bioplastic, bacterial slides were prepared from liquid media and stained with Sudan black B procedure, and bacterial colonies were also stained on agar plated by Sudan black B method. The final product was measured by Fourier transform infrared (FT-IR) and nuclear magnetic resonance (MNR) spectroscopies for chemical analysis. Stained slides and plates showed the production of bioplastic as the bacterial cells showed black regions in the cell poles and the colony appeared black on agar plates according to staining by Sudan black B stain. The FT-IR and MNR analysis ensured the produced plastic polymer was Thermoplastic Elastomer (TE). The study demonstrates the use of TE copolymer, a blend of plastic and rubber, as an eco-friendly alternative to petroleum products, as it was created by a native thermophilic bacterium, demonstrating the potential of biotechnological methods to meet the Saudi National Vision 2030 goals and promote sustainable waste management.\u003c/p\u003e","manuscriptTitle":"Saudi Hot Spring Extremophilic Bacillus as an Alternative Bioresource for Sustainable Thermoplastic Elastomer (TPE) Biosynthesis from Shrimp Waste","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 18:09:31","doi":"10.21203/rs.3.rs-7470334/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86034648-65cc-4179-8d4e-dd7e1a2037ea","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54890482,"name":"Biological sciences/Biological techniques"},{"id":54890483,"name":"Biological sciences/Biotechnology"},{"id":54890484,"name":"Earth and environmental sciences/Environmental sciences"},{"id":54890485,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-12-09T01:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-19 18:09:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7470334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7470334","identity":"rs-7470334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}