Likelihood of polyhydroxyalkanoates production using canola oil cake and specific bacterial isolates for eco-friendly bioplastics

Abstract

Polyhydroxyalkanoates (PHA) are a suitable replacement for conventional petroleum plastics. The global push for environmentally friendly plastics is driving research into innovative, sustainable solutions. This study emphasizes enhancing the production and properties of PHA by using consortia comprising known bacterial strains grown in a glucose-rich, peptone-deficient (GRPD) medium supplemented with 1% canola oil cake as a renewable carbon source. The addition of canola oil cake as an additive accelerated the growth by increasing biomass by 74.22% and simultaneously boosted the PHA yield by 68.67%. Employing confocal microscopy, the PHA granules within the bacterial cells were visualised. Chemical fingerprinting of the PHA film using Fourier transform infrared spectroscopy (FTIR) displayed unique functional groups – carbonyl (–CO–) and ester (–COO–) – at an explicit peak of 1718 cm⁻¹, demonstrating that the polymer was polyhydroxybutyrate (PHB), for Bacillus cereus strain ST001 and Enterobacter cloacae strain AP001; however Rhodococcus ruber strain TESIII revealed a peak at 1653 cm⁻¹ implying the existence of methylene (–CH–). The resultant PHA was identified as poly(3-hydroxybutyrate-co-hydroxyvalerate) [PHBV]. Significant results were documented. A tensile strength of 0.13 MPa and a thermal degradation temperature of 240–260 °C were demonstrated by the resulting PHA. It was further recorded that there was an increase in the intensity of 76.92% of tensile strength in PHA samples when bacteria were cultured in GRPD supplemented with the renewable carbon source. These findings explain the potential of canola oil cake as a cost-effective, renewable feedstock additive for enhancing PHA production and material quality, thus paving the way for future advancements in sustainable bioplastic development.

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1. Introduction

Plastic production reached approximately 390.7 million tonnes worldwide in 2021, with a staggering 90.2% of this total being derived from fossil fuels. Aligning with the sustainable development goals for clean energy, a viable alternative to fossil fuels is bio-based and bio-attributed plastics that account for almost 1.5%.¹ Conventional plastics cause serious environmental pollution as they can remain in the environment for more than a hundred years.² Polyhydroxyalkanoates (PHAs) belong to the class of bio-based polyesters that provide a sustainable and renewable alternative to fossil fuels. These polymers display outstanding features, including biodegradability, biocompatibility, and non-toxicity.³ PHAs are produced by a diverse group of bacteria, typically in response to stress caused by an excess of carbon.⁴ The discovery of PHA inclusion bodies dates back to 1888, when Beijerinck first observed them. Later, between 1923–1951, Maurice Lemoigne conducted pioneering research on PHA production and characterization using Bacillus megaterium under anaerobic conditions. His groundbreaking work earned him the esteemed title of ‘Father of PHAs’. In 1959, Doudoroff and Stanier explored PHB assimilation,⁵ and Hayward et al. anticipated the work on the synthesis and breakdown of PHB by bacteria.⁶ Despite the dominance of petroleum plastics, W. R. Grace & Co. submitted a patent for the production and extraction of PHB and later developed a moulded plastic-like product comprising PHB in 1963.^7,8 The following two decades were imperative for findings associated with the direction of PHAs, as there were noteworthy studies linked to countless developments on the production and extraction of PHAs, as well as their evolution as biomaterials. In the 1980s, countless novel approaches to improvement were revealed, comprising the utilization of innumerable carbon and nitrogen sources, enumerating the pathways of PHA production, and using different bacteria and substrates for PHA production.⁹ Braunegg et al. conducted a study emphasizing the ability of PHA to replace conventional plastics.⁹ In the early 2000s, the combinatorial use of a microbial consortium comprising extremophilic bacteria and the use of renewable substrates for PHA production drew attention due to their ability to survive in extreme environmental conditions.^10,11 The last few years have seen a profound literature related to research work on PHAs, encompassing the testing of various substrates for the sustainable production of PHA biomaterials^12,13 (Fig. 1A).

Fig. 1 [A] A century of PHA advancements: from early beginnings to modern applications (late 1800s–2020). [B] Derivatives of PHA.

Recent studies have shown that the chirality of the α-carbon influences the thermal and mechanical properties of PHA. The stereoisomeric configuration plays an crucial role, as both configurations (S and R) regulate the physical properties of PHA. As discussed by Miyahara et al.^13,14 3-hydroxy-2-methylbutyrate (3H2MB3) and 3-hydroxy-2-methylvalerate (3H2MV), forms of PHA which contain α-methylated monomers, serve as a novel class of bio-based materials with significant properties. Different derivatives of PHA, such as PHB, PHV, and the copolymer of PHBV, are shown in (Fig. 1B). PHA, akin to petroleum plastics, has immense potential in the biomedical, environmental, nanotechnology, and agricultural sectors.¹⁵ Industrial applications of PHA encompass the synthesis of bioplastic films suitable for food packaging,¹⁶ 3D printing,¹⁷ wood plastic composites,¹⁸etc., whereas agricultural applications involve the development of mulch films,¹⁹ agricultural nets,²⁰ grow bags,²¹etc. PHA can be a suitable biomaterial for applications in the biomedical sector for bone tissue engineering,²² drug delivery,²³ and the development of heart valves and artificial blood vessels,²⁴etc. With these versatile applications, there are some drawbacks to the mass utilization of the PHA molecule, like high production cost, and differences in mechanical properties, including lower tensile strength and flexibility, brittleness, lower thermal stability, and reduced production efficiency.²⁵

Waste oil cakes obtained from the oil milling industry can be divided into edible and non-edible oil cakes. According to the literature, oil cakes are a good source of carbon and nitrogen, as well as potassium and phosphorus when used in minimum quantities. Edible oil cakes can be used as cattle feed as they are generated from agro-waste and are rich in nitrogen, amino acids, proteins, antioxidants, fibre, etc., which makes them a good source of nutrition, whereas non-edible oil cakes are used for the production of biogas, biofertilizer, antibiotics, enzymes, bioelectricity, and bioactive compounds, as well as biopolymers such as PHA.²⁶

PHA market growth was anticipated to reach $93.5 million by 2021, up from $73.6 million in 2016. Primarily, glucose and sucrose are frequently used as carbon sources for microbial growth, which increases production costs and makes the process expensive. Almost 40–50% of the production expenses depend on carbon sources, and researchers have observed a reduction in the cost by using cheaper carbon sources obtained from utilizing various wastes.²⁷ The CAPEX (total capital cost) and annual operating cost (OPEX) can be calculated based on different parameters such as direct fixed capital, working capital, and instrumentation. Around 40% of feedstock expenses can be reduced by using renewable sources such as canola oil cake. Equipment purchase costs constitute about 30–40%, whereas instrumentation accounts for approximately 20–25%. Raw material costs depend on substrate type and can be estimated as 30–50% of OPEX. Waste canola oil cake significantly reduces production costs compared with refined carbon sources like glucose or sucrose.²⁸ According to a recent study, the manufacturing cost of PHA also varies depending on the feedstocks used. First-generation feedstocks increase the production cost of PHA to $4 to $6 per kg as compared with second-generation feedstocks ($2–$3 per kg).²⁹ Utilization of waste feedstocks, such as whey or waste lipids, affects the overall production expenses. The WHEYPOL and ANIMPOL projects achieved production costs below €3 and €2 per kg of PHA using surplus whey and animal fats with crude glycerol.³⁰ The climate change impact of PHA production varies widely, ranging from −2.3 to 6.9 kg CO₂-eq per kg PHA, based on the selected feedstocks and life cycle processes. Negative values indicate carbon sequestration. The amount of carbon dioxide emissions for PHA production after using sugarcane and corn starch feedstocks is estimated to be 2.72 kg CO₂-eq per kg PHA and 4.26 kg CO₂-eq per kg PHA, respectively. Studies reporting values below 0.49 kg CO₂-eq per kg PHA account for carbon storage, where atmospheric CO₂ temporarily captured in the polymer is treated as sequestration. However, fermentation remains the most energy-intensive phase, contributing significantly to the overall climate change potential of PHA production. These findings highlight the importance of feedstock selection, carbon accounting, and energy optimization in minimizing environmental impacts and also support the idea of using suitable renewable carbon sources as substrates for PHA synthesis.³¹ The life cycle assessment studies of PHA focus on various ecological impacts and considerable variations in greenhouse gas emissions, along with energy use due to different feedstocks, production methods, and applications. A meta-analysis recorded previously showed that PHA contributes to greenhouse gas emissions, with emissions ranging from −2 to +2 kg CO₂-eq per kg PHA, which are comparatively less than the emissions of fossil plastics (2–8 kg CO₂-eq per kg for fossil plastics). PHA has a higher environmental impact in areas like acidification. The use of renewable resources significantly reduces their footprint and makes it comparatively more eco-friendly than fossil-based plastics.³² According to the life cycle assessment study conducted by Martin-Gamboa and coworkers, the mean impact of climate change for PHA production was 1.88 t CO₂-eq per t PHA (which was typically lower than the reported range of 1–6 t CO₂-eq per t PHA), which contributes to a reduction in the net impact of 4 CO₂-eq per t PHA. Also, when compared with polyethylene terephthalate (PET) and polyurethane (PU), PHA production offers 30–60% lower climate impacts.³²

Renewable carbon sources are an excellent, inexpensive alternative to petroleum-based carbon sources and non-petroleum-based carbon sources, and hence are suitable for PHA production. Utilizing renewable sources can potentially contribute to the reduction of PHA production costs, a significant drawback of the process. Studies have reported that the successful utilization of agro-industrial wastes is a promising way to reduce the accumulation while developing sustainable plastics.³³ Bacteria use various carbon sources obtained from industrial and agricultural waste, food waste, and fatty acids. Utilization of waste from biogas treatment facilities as a renewable nutrient source for PHA production required additional costs, approximately 58% for biogas air compression and 15% for raw materials. The use of biogas waste significantly reduced the production cost by 4.2 € per kg. On comparative assessment, it is relatively high compared with petroleum plastics, but the cost is reasonable in the biopolymer market.³⁴ The final market price of PHA depends on the productivity, yield per substrate, cost of raw material, and downstream processing,³¹ which can be reduced by utilizing different renewable resources. Agro-industrial wastes such as non-recyclable fibre waste and acid whey as substrate serve as significant sources for PHA production as they contain sufficient quantities of carbohydrate, fats, proteins, and mineral salts. An excess of carbon sources and a nutrient-starved environment promote the growth of PHA-producing bacteria. These carbon sources, including glucose and sucrose as well as expensive non-renewable nutrient sources, present as a substrate during the fermentation process, contribute to approximately half of the production of PHA.³⁵ Synthesizing PHA from renewable carbon sources from industrial and agricultural wastes enables a cost-effective and environmentally friendly production process, contributing to the valorisation of abundant, inexpensive, and sustainable production of PHA.³⁶

Hence, instead of using fossil fuels for the production of petroleum plastics, lignocellulosic biomass, dairy wastes, animal fats, sludge waste, and plant oils are explored as inexpensive carbon sources for the development of PHA-based bioplastics.³⁷Loktanella sp. SM43 grown in lignocellulosic biomass containing two sugars, glucose and xylose,³⁸ and purple photosynthetic bacteria grown in hydrolysate of lignocellulosic waste³⁹ were used for PHA production. Dairy industry process waste (whey) was used as a renewable substrate for growing Bacillus megaterium (MTCC 428) and Ralstonia eutropha (MTCC 8320) as PHA producers.⁴⁰ The extremophilic bacterium Paracoccus homiensis (DSM 17862) was examined to check its ability to produce PHA by utilizing cheese whey as a waste carbon source for the production of PHA.⁴¹ Activated sludge can also be used.^42,43 Another innovative approach for PHA production using renewable sources is with the help of microalgae. Microalgae have a natural ability to produce various lipids, carbohydrates, and proteins, including PHAs. They can grow on waste sources and utilize them for PHA production.⁴⁴ Microalgae such as Neochloris oleoabundans and Scenedesmus almeriensis were used for starch extraction, while Calothrix scytonemicola, rich in PHA, was used for PHA extraction. The extracted PHA can be combined with starch and other natural polymers to produce bioplastic films.⁴⁵ PHA extracted from Microcystis sp., sourced from algal ponds, demonstrated excellent plasticizing properties, leading to improved mechanical strength.⁴⁶Table 1 summarizes additional studies related to the use of wastes from various industries and agriculture for PHA production and improvement in mechanical properties. This underscores the importance of leveraging waste and renewable resources to optimize PHA production, improving both yield and material properties.

Table 1 Utilization of agro-industrial wastes and improvement in mechanical properties

Sr. no.	Substrate for PHA production	PHA yield	Elongation at break (%)	Tensile strength (MPa)	Young's modulus	Ref.
1	Municipal biowaste (MBW)	—	—	13.6 ± 3.3 MPa	E = 1583 ± 249 MPa	47
2	Waste activated sludge supplemented with acetic acid and propionic acids (HAc and HPr)	0.02 g of PHA per gVSS	—	—	779 to 2893 MPa	48
3	Surplus activated sludge from potato starch manufacturing factory wastewater	15 mol% of 4HB 50% wt% wood flour	—	7.5 MPa	—	49
4	Various substrates of oils and fatty acids	—	162%	4.3 MPa for octanoic acid	—	50
5	Waste glycerol	34.2 ± 2.4 g and 40.6 ± 3.2 g PHAs per 100 g CDW (for substrate S5 and S6 respectively)	1.07 ± 0.35% and 2.15 ± 0.53%	10.30 ± 1.50 MPa and 6.04 ± 1.07 MPa	2.35 ± 0.18 GPa and 0.89 ± 0.97 GPa	51

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Plant oils, as well as residual oils obtained in the form of oil cakes from the oil milling industry, can serve as valuable renewable carbon sources, because they contain a high amount of carbon and nitrogen, which can be beneficial for enhancing PHA production. As reported by Munir et al.⁵¹ ST2 Pseudomonas sp. and CS8 Bacillus sp. co-culture grown in the presence of volatile fatty acids and glucose resulted in a PHA yield of 35% of cell dry weight (CDW). Meanwhile, Cupriavidus necator DSM428 could increase the yield substantially by using renewable sources singly and in combination.⁵² Two different strains of Pseudomonas putida were studied for PHA production using waste cooking oil and crude palm oil sludge.⁵³ It was observed that Pseudomonas putida KT2440 produced 36.4% CDW and PHA,⁵⁴ whereas Pseudomonas putida S12 produced 41% CDW and medium-chain-length-PHA (mcl-PHA).⁵⁵Enterobacter aerogenes was grown in the presence of Calophyllum inophyllum oil cake as an excess source of carbon for PHA production, and it was noted that it was capable of producing 4.2 g L⁻¹ of PHA.³⁶ These studies indicate the ability of various bacteria to utilize waste oils, oilcakes, and also molasses as excess carbon sources for PHA production.

India ranks 3rd globally in canola/rapeseed production, with Rajasthan leading at 48% of the country's total output. A byproduct of the oil milling industry, waste canola oil cake contains 45–50% carbon (by dry weight), mainly stored in the form of proteins, carbohydrates, and fats present.⁵⁶ Fatty acid-rich substrates like canola oil cake help in improving the mechanical or structural properties due to a reduction in crystallinity and the copolymer formation.³⁷ According to the study performed by Guleria et al.⁵⁶ It was seen that the properties of PHA are significantly affected by the composition of the substrate, resulting in a reduction of brittleness and an improvement in the quality of PHA film produced. Impurities, such as proteins, residual oils, lignin, etc., are present in canola oil cake. The presence of these renewable resources, even in small amounts,⁵⁷ may act as structural modifiers affecting the properties of PHA. This makes the canola oil cakes a suitable substrate for microbial fermentation processes. The potential limitations in processing canola oil cake as a substrate for PHA synthesis include various aspects, such as understanding the composition of canola oil cake, the pre-treatment required before using it as a substrate, and the optimization of pre-fermentation and fermentation conditions suitable for the growth of microbes as well as for the release of nutrients present in canola oil during the process. In downstream processing as well, it is necessary to segregate the nutrients effectively for better growth and ultimately the maximum yield of the final product.⁵⁸ This could be a challenge to resolve before utilizing waste oil-based substrates, such as canola oil cake, for PHA production.⁵⁹ According to the literature available, different bacteria were able to use canola/rapeseed oil and many similar leftover cooking oils from canola fish when used as carbon sources for PHA production. Pseudomonas chlororaphis PA23-63 was able to utilize canola oil to produce PHA of 30% cell dry weight. Pseudomonas sp. Gl01, Cupriavidus necator H16, and Bacillus thermoamylovorans, in the presence of glucose and waste rapeseed oil, waste cooking and waste fish oil, and waste canola cooking oil, respectively, were able to produce different types of PHA.⁶⁰

This research introduces an innovative strategy for PHA production, utilizing specialized bacteria from textile effluent-contaminated soil. By harnessing these microorganisms, this approach becomes cost-effective and also fosters waste valorisation, demonstrating a groundbreaking method for sustainable PHA production. Building on our previous discussion, the stress-induced synthesis of PHA within bacterial cells, under conditions of limited availability of nutrients and excess carbon, opens up new avenues for investigating the potential of these microorganisms to produce high-performance PHA. The fact that these bacteria can thrive on diverse waste materials as feedstocks also underscores the viability of scalable and profitable PHA production.⁶¹ Utilizing renewable resources as carbon feedstocks in conjunction with bacterial isolates from extreme environments offers a viable and environmentally friendly approach to PHA production, paving the way for the development of next-generation bioplastics.⁶² The utilization of consortia along with a renewable carbon source for PHA production offers several advantages due to their synergistic effect. A significant advantage of this approach is the reduced requirement for maintaining sterile conditions. Due to the use of consortia, natural growth environments like agro-industrial waste, household waste, wastewater, etc., can be used as substrates, contributing to a reduction of waste generation from industry.⁶³ This can contribute to waste reduction and ultimately lead to a sustainable economy. As per prior reports,^61,64 the characteristics of PHA are formulated as per the media composition and the carbon-to-nitrogen ratio. Interestingly, the use of waste renewable resources can lead to improved thermal stability and melting temperatures of PHA, surpassing those of commercial PHA products. Nevertheless, further optimization is needed to enhance the mechanical strength of PHA.^61,65 A groundbreaking approach can be the use of selected bacteria from extreme environments for PHA production. At the same time, using renewable carbon sources can help mitigate waste generation and enable their utilization to develop a sustainable biopolymer with improved characteristics. Bacterial consortia are more efficient at utilizing the given substrate compared with a single culture. The different nutrient requirements of the bacteria present in the consortia allow them to complement each other's metabolic processes. They are also capable of adapting to versatile and changing environments compared with single bacteria that generally require optimum conditions for growth.⁶⁶ Several studies have been performed using various forms of waste and renewable carbon sources as well as bacterial consortia. Pseudomonas putida NBRC 14164 and Saccharomyces cerevisiae SyBE_Sc01020078 were used in a ratio of 1 : 10 for the production of PHA using MSM containing xylose and octanoate as carbon sources. As a result, 295.7 mg L⁻¹ of mcl-PHAs were produced.⁶⁷ Crude glycerol, which is obtained as a by-product of the biodiesel industry, can be directly converted into PHA by using a microbial consortium. This strategy mainly focuses on enrichment of the media with crude glycerol and facilitates PHA production.⁶⁸ The study conducted by Romero-Frasca and Buitron⁶⁸ revealed that PHA production using activated sludge and freshwater consortia resulted in a yield of 5 mg PHA per g of dried biomass. Along with the bacterial consortia, photoautotrophic and mixotrophic microalgae consortia were also studied for the accumulation of PHA, using nitrogen- and phosphorus-limiting conditions.⁶⁹ It was observed that around 100–130 mg L⁻¹ of PHA was generated.⁷⁰ It is used to utilize renewable waste materials with bacterial consortia for the production of a sustainable bioplastic resource. Biodegradable polymers like PHA, obtained using renewable sources, can contribute to reducing the adverse environmental impacts and carbon footprints, as they are completely degradable and non-toxic.⁷¹Bacillus cereus is one of the most studied bacteria for PHA production, as it is capable of utilizing various carbon sources. A recent study showed the highest production of PHA around 86.4 g L⁻¹ or 62.1% (w/w) using sugarcane bagasse hydrolysate as a carbon source.⁷² Similarly, Bacillus cereus strain S356 also showed a P3HB yield of 5.16 g L⁻¹ and 85.3% of CDW using sugarcane bagasse.⁷³Bacillus sp. 12GS isolated from northern Mexico was able to grow and produce 87% PHA (w/w), and it was observed by biochemical and molecular analysis as a new isolate of Bacillus cereus.⁷⁴ Several studies have been conducted on different strains of Bacillus using various feedstocks for PHA production.^1,75,76 Similarly, cassava chip (CC)-derived hydrolysates were used to cultivate different Enterobacter strains for PHA production, yielding 11.3 ± 2.8 g L⁻¹ of PHB.⁷⁷ Biohydrogen and PHA coproduction was also studied using Enterobacter.⁷⁸ In the case of Rhodococcus strains, studies have reported the production of PHA. Rhodococcus ruber BSRT1-1 showed 43.1 ± 0.5 wt% of dry cell weight (DCW) of PHB after optimisation using RSM.⁷⁹Rhodococcus sp. lz1 was used for PHA production using sodium acetate as a carbon source, yielding 41.87% of PHBV.⁸⁰ Based on this evidence, the potential of the novel bacterial strains isolated from textile effluent-contaminated soil was explored for the production of PHA. The potential of canola oil cake as a renewable carbon source for the production of polyhydroxyalkanoate was investigated in this article. This research leveraged prior bacterial isolates sourced from textile effluent-contaminated soil sampled from multiple locations across India's textile industry hubs for the sustainable production system of PHA.

2. Materials and methods

2.1. Chemicals

All the required chemicals for the preparation of growth media, viz., minimal salt medium (MSM), glucose-rich peptone-deficient medium (GRPD), and nutrient broth (NB), were purchased from SISCO Research Laboratory (SRL), Mumbai, India, as well as HiMedia Laboratories, Mumbai, India. Solvents required for the extraction and purification were procured from Rankem Chemicals, India.

2.2. Methods

2.2.1. Experimental plan and microorganisms. The bacteria previously isolated from textile effluent-contaminated soil were selected for PHA production. The primary isolation of three selected bacterial strains, Bacillus cereus strain ST001 (accession no. MK613453.1), Enterobacter cloacae strain AP001 (accession no. MK613452.1), and Rhodococcus ruber strain TESIII (accession no. MZ683210.1), was performed on a sterile nutrient agar plate. After a period of 24 h of incubation (37 °C), the bacterial isolates were further characterized by primary screening techniques and finally selected for further study based on the results obtained from primary screening. A schematic experimental plan is shown in Fig. 2.

Fig. 2 The experimental plan of the proposed work includes the isolation and screening of PHA from textile effluent-contaminated soil, with extraction and characterisation of the PHA film developed in the presence of canola oil cake and its possibility to function as a bioplastic component.

2.2.2. Culture media. For PHA production, four different types of growth medium, viz. nutrient broth (beef extract, yeast extract, peptone, NaCl, pH = 7.0), minimal salt medium (MSM) [glycerol, Na₂HPO₄, KH₂PO₄, (NH₄)₂SO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, ferrous ammonium citrate, trace elements, pH = 7.0], MSM nitrogen-limiting medium [glycerol, Na₂HPO₄, KH₂PO₄, (NH₄)₂SO₄, MgSO₄·7H₂O, CaCl₂·2H₂O, ferrous ammonium citrate, trace elements, pH = 7.0]⁸¹ and glucose-rich peptone-deficient medium (GRPD) [glucose (10 g L⁻¹), yeast extract, peptone, NaCl, pH = 7.0].⁸² The growth pattern of the isolates was studied.

2.2.3. Morphological characteristics. The bacterial strains B. cereus strain ST001, E. cloacae strain AP001, and R. ruber strain TESIII were streaked on nutrient agar plates. Incubation at 37 °C for 24 h was done. Morphological characteristics were observed after incubation, followed by Gram staining.

2.2.4. Screening of bacteria for PHA production. Primarily, the screening of all the isolates used nutrient agar supplemented with 0.001 g L⁻¹ Nile Blue, which advocated the growth of only the PHA-producing bacteria. After incubation for 24 h at 37 °C, growth was observed under a UV transilluminator. PHA-producing organisms were expected to exhibit fluorescence under UV exposure. 24-hour-old cultures were used for microscopic imaging of PHA producers. Phase contrast microscopy (Olympus IX83, Japan) was employed to visualize the unstained bacterial cultures and observe their presence in the consortia at a magnification of 400× and 1000×. Field emission scanning electron microscopy (FESEM), model JSM-7610F Plus manufactured by JEOL (Tokyo, Japan), was used to confirm the presence and association of the different types of bacterium growing in the consortia when studied based on spatial and temporal conditions. With the increase in magnification (500× to 100 000×), the documentation of the cultures grown for different periods of time was expected to provide a better idea of the type of association of the bacteria in the consortia. For the detection of PHA granules inside the bacterial cells, Nile Red, a selective dye, was used, and fluorescent microscopy (Olympus IX83, Japan) was employed. A fully spectral confocal laser scanning microscope (Carl Zeiss LSM 880, Germany) was used to obtain the superimposed images of the bacterial cells and to understand the intracellular location of PHA granules.

2.2.5. Molecular characterization of PHA-producing bacteria. PHA-producing bacteria were identified based on the primary screening. They were further analysed using in silico methods of molecular characterization by 16S rRNA sequencing to understand the phylogenetic relationship among the related species and also amongst the selected bacterial strains. Three genes are necessary in PHA synthesis, viz., phaA, phaB, and phaC. These genes are responsible for coding three enzymes, PHA synthase, β-ketothiolase, and acetoacetyl-CoA reductase. PHA synthase (phaC gene) facilitates the polymerization of hydroxyalkanoate monomers into PHA.
2.2.5.1. In silico studies for 16S rRNA sequencing. The 16S rRNA sequences of these three bacterial isolates were extracted from the National Center for Biotechnology Information (NCBI) (ncbi.nlm.nih.gov) website in FASTA (Fast Adaptive Shrinkage/Threshold Algorithm) format, and a Basic Local Alignment Search Tool (BLAST) analysis was used to obtain similar sequences. The similar sequences obtained from BLAST, along with query sequences of all the selected bacterial strains mentioned above, were run for multiple sequence alignment using the CLUSTAL-W (https://www.genomenet.jp) program⁸³ for identification of the similarities among all the sequences based on 16S rRNA (Fig. S1).
2.2.5.2. In silico analysis of the PHA synthase (phaC) gene. As stated for 16S rRNA, sequences were extracted in FASTA format from the NCBI search tool, and the CLUSTAL W program was used to perform MSA using the identical FASTA sequences as mentioned above. The presence of the phaC gene confirms the ability of bacteria to produce PHA.⁸⁴ The phaC gene of Bacillus cereus NZ CP017060.1 was used, and phylogenetic trees for both the MSAs, viz. 16S rRNA and phaC (Fig. S2), were constructed using MEGA-XI [(version 11.0.13) (https://www.megasoftware.net)] software and the bootstrap method using bootstrap values out of 100. MEGA-XI software was utilized to create a phylogenetic tree utilizing the maximum likelihood method and bootstrap values out of 100. Maximum likelihood type trees were constructed to study the phylogenetic similarity among all the sequences.⁸⁵

2.2.6. Effect of canola oil cake on the growth of bacteria and PHA production. The selected bacterial isolates were inoculated in all the selected different media, keeping all the growth conditions common for each of them. Furthermore, they were incubated for 72 h at 37 °C and 150 rpm, respectively. After incubation, all these organisms showed maximum growth in GRPD medium. Hence, GRPD medium was selected for PHA production using the newly discovered bacterial species.⁸² All three bacterial cultures were 24 h old when used as consortia in GRPD medium for PHA production and inoculated in equal volume. The compatibility of bacterial isolates to make a consortium was checked using confocal microscopy. It helped to visualize their growth and the appearance of intracellular PHA granules after 72 h of incubation. Therefore, in parallel with the previous set of experiments, an extra set was also prepared with an additional carbon source, canola oil cake, to understand the role of a renewable carbon source.⁸⁶ Autoclaved canola oil cake (10 g L⁻¹) was added to the GRPD media.⁸⁷ Growth pattern and PHA yield were studied in GRPD medium (control: without canola oil cake) and GRPD medium supplemented with 1% canola oil cake.⁸⁸

2.2.7. PHA extraction and purification. The extraction of PHA was initially done via centrifugation. Subsequently, after centrifugation, the fresh weight of biomass was noted. After drying, the pellet's weight was measured repeatedly until a constant weight was obtained, indicating complete desiccation. As it is an intracellular product, cell lysis was done by suspending the pellet in 4% sodium hypochlorite for 2 h. Cells were then centrifuged (10 000 rpm for 10 min) to obtain crude PHA.⁸⁰ The crude PHA was purified using organic solvents like acetone and methanol.⁸⁹ The pellet was washed with an equimolar solution of acetone and methanol.⁹⁰ The crude PHA was dissolved in chloroform, and as the chloroform evaporates, powdered PHA is left. This can be formulated into a flexible PHA film.⁹¹

2.2.8. Quantification of PHA. After extraction, the purity of PHA was checked using the crotonic acid assay. The polymer PHA was dissolved in chloroform, and the concoction was heated in a water bath after the addition of concentrated sulfuric acid to yield crotonic acid. Sulfuric acid was used as a blank, and at 235 nm, the resultant PHA in the form of crotonic acid was measured using a UV-visible spectrophotometer.^92,93 Standard stock of crotonic acid was prepared using a concentration of 1 mg ml⁻¹. Constant intervals were taken between 10 μg ml⁻¹ and 100 μg ml⁻¹, and absorbance was measured at 235 nm. The absorbance of the PHA sample was plotted against the standard graph (Fig. S3). The comparative analysis was done concerning cell dry weight and the concentration of PHA was calculated.

2.2.9. UV-Vis spectroscopy for opacity and transparency analysis of PHA film. The barrier properties of PHA film were analyzed by performing UV-Vis spectroscopy. The film was scanned at a wavelength of 200–700 nm and scanned to analyse the absorbance. The calculation of the opacity of the film was done by taking the ratio of optical density at 350 nm, and the film's thickness was measured (mm). Photometric analysis was done at absorption maxima (λ_max) obtained from the wavelength scan.⁹⁴

2.3. Functional characterization

2.3.1 Fourier transform infrared spectroscopy (FTIR). FTIR was used to examine the functional groups present in PHA film obtained from consortia grown in canola oil cake after extraction and purification.⁷⁹ The FTIR analysis was carried out using a spectral range between 4000–500 cm⁻¹ using a Bruker ALPHA FTIR spectrophotometer (Massachusetts, U.S.) with a resolution of 0.8 cm⁻¹. Automated baseline correction of the raw signal was carried out to minimize errors.⁹⁵

2.3.2 Thermogravimetric analysis (TGA). TGA (Shimadzu DTG-60, Kyoto, Japan) was explored to verify the thermal stability of the PHA film by examining the weight alteration that takes place as it is heated at a constant rate.⁸⁷ The analysis was performed under a nitrogen atmosphere with a ramping rate of 10 °C min⁻¹, ranging from 25 °C to 900 °C.

2.3.3 Field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray (EDX) spectroscopy analysis of PHA film. FESEM was performed with 100 000× magnification to understand the surface morphology of the PHA. The JSM-7610F chamber is equipped with an energy-dispersive X-ray (EDX) detector for elemental investigation of the film. Sharp peaks indicate the elements present in the sample, and the percentage of weight can be calculated accordingly.¹⁵

Scanning electron microscopy (SEM) provides topographical and elemental information at magnification 1000× to 100 000×. The guns improve the spatial resolution by concentrating low- and high-energy electrons at low electrical potential (above 0.02 to 5 kV).¹⁵ FESEM was performed with 100 000× magnification using a JSM-7610F Plus, manufactured by JEOL (Tokyo, Japan), to understand the surface morphology of the PHA film obtained from consortia grown in the presence of canola oil cake. EDX was used to detect the elemental composition of the film.

2.3.4 Nuclear magnetic resonance (NMR) analysis of PHA. An NMR spectrometer (ECZ 400 MHz; JEOL, Tokyo, Japan) was used for the non-destructive quantification of total PHA content and identification of PHA copolymer composition using the ¹H NMR method. Deuterated chloroform (CDCl₃) was used to dissolve the extracted PHA, and then subjected to analysis of chemical shifts.⁹⁶ The NMR data processing and integration of peaks were performed using the software MestReNova-14.0.0-23239.msi (Mestrelab Research, Santiago de Compostela, NW Spain).

2.3.5 Mechanical properties of PHA film. Mechanical properties like tensile strength and elongation at break of the PHA film obtained from consortia using canola oil cake as a renewable carbon source were analyzed using Instron-5967 Universal testing machines (manufactured by Instron, Norwood, MA, US) with a maximum capacity of 30 KN load. Strips of PHA film (60 mm × 10 mm) were prepared and subjected to a minimum load with an increasing load rate of 5 mm min⁻¹.⁹⁷ The tensile strength was calculated based on the ratio of the maximum load taken by the PHA film to the cross-sectional area of the film.⁹⁸

2.3.6 Gas chromatography and mass spectrometry analysis of canola oil cake. 1 g of canola oil cake was immersed in methanol and incubated at 120 RPM overnight. For GC-MS analysis, the extracts were filtered using filter paper and a microsyringe filter. GC-MS was performed using the Shimadzu GCMS-QP2020. The capillary column had an internal diameter of 0.25 mm. The injector temperature and detector temperature were set at 250 °C and 275 °C respectively, and the oven temperature was raised from 50 to 250 °C at an increasing rate of 6 °C per minute. Helium was used as a carrier gas at a rate of 1 ml min⁻¹ linear velocity. Peak identification of individual components was performed by comparing their retention time with their reference standard and reconfirmed using the mass spectral library of standard compounds (NIST-MS Search 2.0 software). The quantification of individual components was done by peak area integration, using a built-in data handling software system.⁹⁹

2.4. Statistical analysis

‘One-way ANOVA’ was carried out to verify the statistical significance at p ≤ 0.05. It was employed by using different types of medium for bacterial growth and ultimately for PHA yield (g L⁻¹). The variability in experimental data was assessed by identifying statistically significant effects, thus emphasising the use of canola oil cake for the production of PHA film.

3. Results and discussion

3.1. Morphological characteristics

The selection of bacteria for the present study was based on their high yield of PHA. A literature survey justified the selection of Bacillus cereus SS105,¹⁰⁰Enterobacter cloacae¹⁰¹ and Rhodococcus.⁷⁹ The textile industry is a substantial contributor to environmental pollution, producing large quantities of waste that can contaminate soil and water resources.¹⁰² These bacteria were found to produce a high amount of PHA. As mentioned earlier, the selected bacteria were isolated from a niche dye-containing environment. This adaptation makes them suitable for withstanding harsh conditions and producing the energy reserve material “PHA” under stress conditions.¹⁰³

Bacteria isolated from textile effluent-contaminated soil were initially compared with the original cultural morphology after being revived from the glycerol stocks. Following 24 hours of incubation, the growth of the colonies showed consistency with the original cultures. B. cereus strain ST001 was observed as sticky, white-coloured colonies. On the other hand, small, translucent colonies of E. cloacae strain AP001 and bright orange-colored small colonies of R. ruber strain TESIII were observed on nutrient agar plates (Fig. 3A–C). Microscopic analysis after Gram staining revealed B. cereus strain ST001 and R. ruber strain TESIII as Gram-positive rods, with E. cloacae strain AP001 as Gram-negative rods (Fig. 3D–F).

Fig. 3 Microorganisms showing characteristic patterns of growth of the bacteria: (A) Bacillus cereus strain ST001, (B) Enterobacter cloacae strain AP001 and (C) Rhodococcus ruber strain TESIII. (A–C) Isolated bacteria from textile effluent-contaminated soil retrieved from glycerol stocks showing similar patterns of growth on nutrient agar media. (D–F) Gram staining of the isolated bacteria. (G–I) Primary screening with Nile blue dye in selective media showing fluorescence.

3.1.1. Screening of PHA producers using selective media. Mascarenhas and Aruna¹⁰⁴ reported that PHA producers showed fluorescence under a UV transilluminator when grown in a suitable medium containing Nile Blue. All the isolates produced fluorescence, indicating their ability to produce PHA (Fig. 3G–I). Similarly, Thirumala et al. demonstrated that the Gram-positive, PHA-producing bacteria exhibited a positive response to Nile Blue staining.¹⁰⁵ Researchers also studied the ability of bacterial isolates from hot springs using Nile Blue and confirmed that out of 144 isolates, 12 showed strong fluorescence under a UV transilluminator.³⁸

3.1.2. Screening of PHA producers using microscopic techniques. Different microscopic techniques were used to visualize the bacteria after incubation for 72 h. Visualization using a phase-contrast microscope helped to identify the bacteria, Fig. 4(A–C), and also detect their presence in the consortia [Fig. 4D]. Thus, the presence of the growing bacterial strains in the consortia indicated their synergistic growth pattern.

Fig. 4 Phase-contrast microscopy of bacterial strains [A] Bacillus cereus strain ST001, (B) Enterobacter cloacae strain AP001 and (C) Rhodococcus ruber strain TESIII (A) consortia grown for 72 h. It indicates the presence of all three bacteria growing individually as well as simultaneously in consortia.

Furthermore, on FESEM analysis, similar morphological characteristics akin to our previous microscopic images were noted (Fig. 5). The bacterial cultures were clearly identified and their presence was verified in the bacterial cultures grown for 24 h [Fig. 5(I)] and 72 h [Fig. 5(II)]. On careful examination, it was notable that almost all the cells had a similar increase in number, justifying their synergistic growth rate in the same culture medium.

Fig. 5 Field emission scanning electron microscopy analysis of Bacillus cereus strain ST001, Enterobacter cloacae strain AP001 and Rhodococcus ruber strain TESIII and consortia grown for [I] 24 h and [II] 72 h, indicating the synergistic growth of all the bacteria in the consortia.

Further studies using different microscopy and staining techniques verified that the PHA was present inside the bacterial cells. Fluorescence imaging performed using Nile Red selective dye (excitation wavelength of 553 nm) showed the red-coloured fluorescence as bright red dots, showcasing the presence of PHA granules in bacteria and consortia, respectively.⁸⁶

Superimposed images of bacteria and consortia confirmed the intracellular location of the PHA granules [Fig. 6B(I–IV)]. It was clear that the same cells as seen through both the imaging techniques contained PHA granules inside their cells. According to Choi et al.,¹⁰⁶ the uniformly stained PHB granules were observed inside the cells of C. necator and recombinant E. coli.¹⁰⁶ Confocal microscopy was employed to observe PHA granules within the cells of Massilia sp.⁸⁵ The presence of fluorescent PHA granules depends on the amount of biomass produced and the concentration of dye used.¹⁰⁷ The bright red spots indicate the considerable amount of intracellular PHA granules.

Fig. 6 Microscopic imaging studies for the detection of PHA inside the cells. A (I, II, III, IV) shows the fluorescence images of Bacillus cereus strain ST001, Rhodococcus ruber strain TESIII, Enterobacter cloacae strain AP001 and consortia; B (I, II, III, IV) show the cells of the respective bacteria and their consortia, with superimposed images of fluorescence and phase contrast microscopy of bacterial strains and consortia indicating the bright red PHA granules inside the bacterial cells.

3.2. Molecular characterization of PHA-producing bacteria

Multiple sequence alignment was performed for 16S rRNA sequences (Fig. S1) using the CLUSTAL-W program followed by phylogenetic analysis using MEGA-XI (version 11.0.13) software using bootstrap analysis out of 100 replicates. A maximum likelihood type of phylogenetic tree was constructed. These bootstrap values, out of 100, provide an idea about the similarity percentage.¹⁰⁸ This method of phylogenetic analysis shows how closely the bacterial species are related to each other. It gives clarity regarding the frequency of a particular branch appearing in repeated phylogenetic tree formation. The values equal to 100 confirm the relatedness among the bacteria, and a conclusion can be drawn. If the values are between 80–95, then it can be inferred that the node is well-supported. Bootstrap values below 50 cannot be considered relevant.¹⁰⁹ The distance bar below indicates the base changes per 100 nucleotides.¹¹⁰

The molecular characterization of these bacteria depicts their relatedness to each other. Fig. 7A shows the phylogenetic tree constructed by using the 16S rRNA sequences extracted from NCBI in FASTA format. It indicates the evolutionary relationship among the three bacteria under study. Mainly three groups are formed by Bacillus strains, Enterobacter strains and Rhodococcus strains with bootstrap values equal to 100 showing complete relatedness to each other. Also, all these three groups of bacteria show a close relationship with each other with bootstrap values of 71 for Bacillus and 92 as well as 56 for Rhodococcus strains. Enterobacter strains show a bootstrap value 100, indicating complete relatedness among them. The distance bar, with a value 0.02, suggests the change in 2 bases per 100 nucleotides.

Fig. 7 Phylogenetic analysis of MK613453.1 B. cereus strain ST001, MK613452.1 E. cloacae strain AP001, MZ683210.1 R. ruber strain TESIII and similar sequences obtained from BLAST analysis. [A] 16S rRNA sequences; [B] detection of the phaC gene by using the bootstrap method and MEGA XI software to check their ability to produce PHA.

The phaC gene was also observed in the nucleotide sequences of B. cereus strain ST001, E. cloacae strain AP001 and R. ruber strain TESIII, justifying their capability to produce PHA inside the cells. Previous studies support the fact that the presence of the phaC gene influences the PHA production in bacterial cells. PHA production by Halomonas sp. SF2003 showed that the PHA synthase genes phaC1 and phaC2 play a vital role in PHA production.¹¹¹Cobetia sp. MC34 and Cobetia marina DSM 4741T were also studied for investigating the role of PHA synthase genes in PHA production, and it was observed that only a single phaC2 gene was present in Cobetia sp. MC34, whereas two variants, viz. phaC1 and phaC2, were noted in C. marina DSM 4741.¹¹² Extremophilic isolates of Pseudomonas and Janthinobacterium (UMAB-08, UMAB-40 and UMAB-56, UMAB-60 respectively) isolated from the Antarctic region showed the presence of PHA synthase genes.¹¹³

Multiple sequence alignment was performed for phaC gene sequences (Fig. S2) using the CLUSTAL-W program, followed by phylogenetic analysis using MEGA-XI (version 11.0.13) software using the bootstrap concept.¹¹⁴ Similarly, phylogenetic analysis was performed using our bacteria of interest, viz. strain ST001, AP001, and TESIII with bootstrap values equal to 98 out of 100, showing the close resemblance between the sequences of bacteria and phaC gene extracted from NZ_CP017060.1 B. cereus. This authenticated NZ_CP017060.1 strain as a standard related to PHA production (Fig. 7B).

3.3. Effect of renewable carbon source canola oil cake on growth of bacteria and PHA production

The growth of PHA producers depends on the nutritional composition of the medium, operational and growth conditions, and most importantly, the additional carbon sources present in the medium. When bacteria were allowed to grow in different types of medium like nutrient broth, minimal salt medium (MSM), and MSM with nitrogen-limiting conditions, it was clearly visible that all the bacterial strains showed minimal growth in MSM with nitrogen-limiting conditions and the highest growth in nutrient broth. MSM contains only basic salts and trace elements, which restrain the bacteria from relying solely on the carbon source provided and result in slow growth of the bacteria (Fig. 8A).¹¹⁵ Although nutrient broth supported the bacterial growth, it produced scant PHA. This may be due to the complex form of the available nitrogen source in the form of peptone and yeast extract.¹¹⁶

Fig. 8 [A] Five different types of growth medium were tested for studying the growth pattern of Bacillus cereus strain ST001, Enterobacter cloacae strain AP001 and Rhodococcus ruber strain TESIII. MSM with limiting N₂ concentration showed the least growth of all the three bacteria whereas GRPD medium supplemented with 1% canola oil cake showed the highest growth in terms of cell dry weight. [B] Comparative analysis of cell dry weight obtained after the growth of bacteria in GRPD medium without canola cake and [C] supplemented with 1% canola oil cake, and PHA extracted from the respective bacteria and their consortia. [D] Quantification of PHA by crotonic acid assay for consortia grown with and without canola oil cake supplementation in GRPD media. All results are represented as mean ± standard deviation (n = 3). Error bars represent standard deviation.

GRPD medium contains excess glucose as a carbon source and nitrogen limitation, which helped in the facilitation of the growth of PHA producers, resulting in a larger amount of PHA accumulation inside the cells.⁸² This medium showed maximum growth for all three bacteria compared with nutrient broth. Canola oil cake was incorporated into the GRPD medium as a renewable and excess carbon source to improve the PHA yield. Addition of 10 g L⁻¹ canola oil cake⁸⁷ into the GRPD medium showed the maximum amount of biomass obtained (Fig. 8A). The p-value obtained by a one-way ANOVA analysis performed to study the growth of biomass was recorded as 2.48 × 10⁻⁷, significantly less than 0.05 (p ≤ 0.05) (Table S1). This indicates that there was a difference in the growth of the bacteria in different media, and GRPD (with canola oil cake) was most suitable for the development of all three bacteria.¹¹⁷

GRPD medium supplemented with canola oil cake was used to produce PHA using all three bacteria individually as well as in consortia. As reported previously by Israni and Shivakumar,⁸⁷ different types of industrial waste, including 10 g L⁻¹ sesame oil cake, utilized as a waste carbon source for PHA production, gave significant results. Canola oil cake was also used at a similar concentration (1% or 10 g L⁻¹) and added to the GRPD medium for the bacterial growth. It was observed that the consortia were able to give a better yield of biomass and PHA as compared with individual bacteria. The growth parameters such as initial pH at 7.0, 37 °C temperature, incubation for 72 h at 150 RPM, etc. were kept constant (Fig. 8B and C). The polymer composition was highly influenced by the carbon source used as a substrate. Waste canola oil cake, consisting of residual oil, carbon, nitrogen and trace elements, was able to alter the elemental composition, molecular structure, surface morphology and mechanical strength of the PHA film produced to some extent. The p-value of obtained by a one-way ANOVA analysis was recorded as 1.93 × 10⁻⁵, which is significantly less than 0.05 (p ≤ 0.05) (Table S2). This indicates that there was a statistically significant difference in the PHA yield relative to cell dry weight among the three bacterial strains tested.¹¹⁷ Further studies will mainly focus on the optimisation of the concentration of canola and its influence on the properties of PHA. This is the preliminary study and underscores the potential of canola oil cake to be utilized as an excess carbon source that could be studied in greater detail in future.

3.4. PHA extraction and purification

PHA polymer, as obtained after centrifugation and solvent processing,^82,91 was cast into film and a comparative analysis was executed with respect to cell dry weight and PHA yield. The highest PHA was obtained by using consortia of the selected bacteria grown in GRPD medium supplemented with 1% canola oil cake.

3.5. Quantification of PHA

Crotonic acid assay was carried out to confirm and determine the concentration of PHA produced. It was observed that the concentration of PHA polymer obtained from B. cereus strain ST001, E. cloacae strain AP001 and R. ruber strain TESIII when grown in GRPD medium supplemented with canola oil cake showed an negligible increase by 7.9%, 10.1% and 25.89% respectively (Fig. 8D). But in the case of the consortium, under similar circumstances, there was a significant increase of approximately 29% in PHA production. As reported by Montiel-Jarillo et al.,¹¹⁸ there was a similar improvement in the yield of PHA and PHB using oil cake. It was also noted that this hike was due to volatile fatty acids extracted from the winterization of the oil cake. In a similar study using Camelina sativa oil seed cake, the PHA films demonstrated improved mechanical and thermal properties.¹¹⁹ Similarly, sesame oil cake and valorized finger millet (Ragi) husk showed an approximately 3.6 to 3.8 fold increase in PHA production using B. megatarium.⁸⁷ Comparable oil-based substrates, such as biodiesel liquid waste used for the production of PHA by Pseudomonas mendocina PSU, showed approximately 77% DCW of PHA content.¹²⁰ It was also documented that, with the use of waste frying oil and propionic acid feeding, the amount of PHBV increased.¹²¹ A halotolerant strain of Bacillus cereus NT-3 showed potential for metabolising volatile fatty acids present in food waste, as a carbon source for PHA synthesis.¹²² Canola oil cake, when used as an additional carbon source, demonstrated better results in terms of PHA yield as mentioned above. This underlined the potential of canola oil cake and consortia of selected bacteria to serve as a suitable renewable carbon source to enhance PHA production using GRPD medium.

3.6. Analysis of the barrier properties of PHA film by UV-Vis spectroscopy

Using UV-Vis spectroscopy, the opacity and transparency of the PHA film were calculated. Both films showed an absorption maximum (λ_max) at 350 nm. A comparison of PHA films revealed that the sample produced using canola oil cake had an opacity value of 25.24. In contrast, the control sample (without canola oil cake) had an opacity value of 25.08. Analysis of the opacity values yielded a transmittance of approximately 0.3%, confirming the film's opaque characteristics and its ability to block UV light transmission effectively. This underscores its robust UV-shielding capabilities. Additionally, a similar investigation was conducted on the UV barrier properties of multilayer PHA films reinforced with cellulose nanocrystals. Interestingly, the results showed that PHB-blend films exhibited greater transparency than PHBV films.¹²³

3.7. Characterization of PHA film obtained from consortium grown using canola oil cake

3.7.1. FTIR for functional group description. The PHA films produced from the consortia under different conditions were quite dissimilar. The two conditions of growth in GRPD medium were without (set I) and with canola oil cake (set II). The film developed using the canola oil cake had a sturdy and robust appearance. FTIR was done to identify and detect the functional groups present in crude PHA obtained from all individual bacteria and their consortium. From the FTIR spectra obtained by analysing the PHA film of set I samples (Fig. 9A), pronounced signals were observed in the regions 3015–2960 cm⁻¹, 2945–2925 cm⁻¹, and 2855–2875 cm⁻¹, corresponding to the stretching of CH₃ asymmetrical, CH₂ anti-symmetrical, and CH₃ symmetrical, respectively.

Fig. 9 PHA film developed from consortia when it was grown in [A] GRPD media and [B] GRPD media supplemented with canola oil cake; FTIR spectra of biopolymer obtained from B. cereus ST001, E. cloacae AP001, R. ruber TESIII and their consortia by using [C] GRPD media and [D] GRPD media supplemented with canola oil cake.

A sharp peak at 1702 cm⁻¹ corresponding to the C=O (carbonyl) and C=O–O (ester) groups, depicted the crystalline nature of the PHA polymer. Similarly, at 2840 cm⁻¹, the –CH₃– symmetrical group is also related to the crystallinity of the PHA polymer (Fig. 9C).¹⁰³ The presence of the –CH₂– anti-symmetrical group at the peak obtained at 2922 cm⁻¹ resembles the lateral chains of monomeric units. Recent reports have indicated the presence of CH₃ asymmetric stretching close to the 3000 cm⁻¹ regions, suggesting the potential combination of C–H–O bonds or hydrogen bridges. The peak at 1449 cm⁻¹ characterizes the –CH₂ bond. Some notable PHA peaks were observed in the stretch of bands between 1032 and 1263 cm⁻¹, which can be correlated to the C–O bonding and the C–O–C group. Moreover, the bending vibrations of the C–H group are represented by the peak at 967 cm⁻¹.¹²⁴

The FTIR spectra of PHA film (Fig. 9B) extracted from bacterial consortia grown in GRPD with canola oil cake (set II), and PHA polymer extracted by using canola oil cake as a carbon source (Fig. 9D), depicted the presence of similar peaks in the spectra of 3015–2960 cm⁻¹, 2945–2925 cm⁻¹, and 2855–2875 cm⁻¹. A variation observed due to the occurrence of another peak at 3116 cm⁻¹ and 2070 cm⁻¹ indicate the presence of –CH– stretching and C=O stretching.¹⁹ A characteristic sharp peak at 1717 cm⁻¹ corresponding to the C=O (carbonyl) and C=O–O (ester) groups was observed, indicating the distinctive feature of PHB polymer.¹⁰³ The anti-symmetrical –CH₂– group and symmetrical –CH₃– group were confirmed by observing the peaks at 2930 cm⁻¹ and 2848 cm⁻¹ respectively.¹⁰³ Peaks belonging to the 3000 cm⁻¹ spectra depict the crystalline structure of the polymer. –CH₂, C–O, and C–O–C bonding was confirmed by observing the peaks obtained at 1464, 1047, and 1270 cm⁻¹, respectively. The peak at 802 cm⁻¹ indicates characteristic C–C stretching and 973 cm⁻¹ represents the alkyl halide group.¹²⁴

All these peaks seen in the polymer in set I (Fig. 9C), and set II (Fig. 9D) did not show many differences. There is substantial evidence to show that B. cereus ST001 and E. cloacae AP001 showed similarity with PHB polymers and R. ruber strain TESIII showed similarity with PHBV. Prior research has indicated that the ester bond and the C–O–C group in the FTIR spectra are indicative of poly-3-hydroxybutyrate (P3HB) monomers¹²⁴ and the spectra obtained between 2956–2872 cm⁻¹ correspond to the spectra of PHBV polymer.¹²⁵ However, both consortia had evidence of PHB, poly(3-hydroxyvalerate) [PHV] and PHBV, yet there were no distinct differences among them. There were only differences in the intensity of the peaks. Hence, the NMR analysis was pursued to find out the functional groups associated with the polymer, while also understanding the type of PHA polymer.

3.7.2. Nuclear magnetic resonance (NMR) of PHA film. The PHA extracted from the consortium of bacteria growing in set I [without the renewable resource canola oil cake in GRPD medium] and set II [with canola oil cake in GRPD medium] was analysed using an NMR spectrometer, and their respective chemical shifts helped in understanding their chemical nature and bonding among different functional groups. The NMR scan of the extracted PHA grown in set I (Fig. 10A) and set II (Fig. 10B) showed distinct peaks showcasing the monomeric and polymeric form of PHA.

Fig. 10 [A] ¹H NMR analysis of PHA film produced by the consortia using GRPD medium for growth of the consortium and [B] PHA film produced by the consortia using GRPD medium along with canola oil cake as an additional renewable carbon source. Negligible differences were observed among the two.

The representative chemical shifts seen in the NMR scans were identified as the PHB peaks at δ = 5.23–5.28 ppm (1H, m) and 2.45–2.64 ppm (1H, m),¹²⁶ with an integration value of 1, which corresponded to methine protons –CH– (multiplet) and methylene –CH₂– (multiplet) respectively (Fig. 10A and B).

Similarly, distinct peaks of PHB were also identified at δ = 1.27–1.28 ppm (3H, d), with an integration value of 3 corresponding to the methyl (–CH₃) group. Hence, the integration value of PHB/PHV was 5. There was apparently no difference between both the sets.

All these three peaks were clearly visible in the spectra. Variations in the NMR spectrum were attributed to differences in signal intensity and multiplicity, with no changes observed in the chemical shift. Both consortia showcased the coexistence of PHB and PHV, suggesting that the copolymer, specifically poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), was present. The methine proton signals at δ = 2.45–2.50, 2.58–2.64 ppm and δ = 5.23–5.28 ppm, at an integration value of 1, are indicative of the presence of both PHB and PHV. Analysing the integration values, the ratios of the PHV to PHB were determined to be 5 : 2. Consequently, the composition of the PHBV copolymer was calculated to be 71% PHB and 28% PHV, with a terminal methyl group supported by methylene and methine groups respectively.¹²⁷

3.7.3. Thermogravimetric analysis (TGA) for thermal stability assessment. TGA was performed to analyse the thermal degradation point of the polymer. As per Israni and Shivkumar,⁸⁷ TGA can aid in understanding the polymer's thermal degradation profile through a single step and a limited range of temperature in the thermal stability analysis. As reported by Akdoğan and colleagues,¹²⁸ the thermal degradation of PHA ranges between 250–261 °C. Similar results were obtained after performing TGA analysis of PHA extracted from consortia when grown in GRPD containing canola oil cake as a carbon source. The maximum decomposition temperature (T_max) was 266.99 °C, and the degradation started at 254.5 °C. At a temperature of 275 °C, the polymer was completely degraded. According to an earlier report, PHA extracted from Enterobacter sp. SEL2 showed a thermal degradation point at 245 °C.¹²⁹ PHB extracted from Rhodococcus ruber BSRT1-1 and Rhodococcus equi showed the thermal degradation point at 288 °C and 276 °C, respectively.¹³⁰ The TGA analysis of PHBV produced by Bacillus cereus MCCB 281 revealed that the degradation temperature was 271 °C, which was higher than the reported degradation of PHB (250 °C).¹³¹ Based on these observations, it can be concluded that the resultant PHA obtained from consortia in set I and set II demonstrated a thermal degradation temperature of 240–270 °C. According to the literature, the thermal stability of PHA can be highly affected by the carbon source and the impurities present after the extraction.

The increase in the thermal degradation temperature of the PHA film obtained from consortia grown in the presence of canola oil cake signifies the positive effect of the renewable carbon source used during the fermentation process.¹³² The thermal degradation point of the polymer obtained from these bacteria was stable above 200 °C and was completely degraded at 244 °C and 259 °C. The weight loss of set I (control) was 94.37% and set II (with using canola oil cake) was 98.11% respectively [Fig. 11A and B]. Similar results were obtained from the study conducted using a mixed microbial culture for PHA production by utilizing sludge as the carbon source, indicating that the thermal degradation temperature was between 250–270 °C for PHBV copolymer. The utilization of valorized Ragi rusk and sesame oil cake as renewable carbon sources for PHA production also showed that the thermal degradation temperature was 266 °C.⁸⁷ Furthermore, a separate study on PHBV copolymer and its composites with polylactic acid (PLA) reported thermal degradation temperatures in the range of 280–350 °C, as determined by TGA, thereby validating the thermal stability of PHBV-based materials.

Fig. 11 TGA profiling of polymer obtained from (A) consortia grown in GRPD media, and (B) consortia grown in GRPD media and canola oil cake showing thermal degradation points at 240 °C and 259 °C.

The comparative TGA analysis revealed that the thermal stability of the PHA film produced by consortia using canola oil cake (set II) has increased from 244 °C to 259 °C, aligning with the literature review.⁸⁷ Furthermore, a separate study on PHBV copolymer and its composites with polylactic acid (PLA) reported the thermal breakdown temperature of in the range of 280–350 °C. As determined by TGA, the results validated the thermal stability of PHBV based materials.^133,134 The blends of poly(3-hydroxybutyrate-co-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) i.e. P(3HB-co-3HV-co-3HHx) showed thermal stability over 240 °C when produced from biomass extracted from a wastewater treatment plant.¹³⁵ This evidence indicates that the use of various substrates for PHA production can impact on its thermal properties and contribute to increasing its degradation temperature. Hence, there is a possibility that, with the use of canola oil cake in GRPD media, the growth of the consortium and the enhanced yield could also augment the thermal features of the PHA biomaterial. Hence, it was expected that the resulting PHA film produced from the consortia would possess high thermal stability.

3.7.4. FESEM for surface topology and EDX for elemental characterisation. FESEM analysis of PHA gives an idea about the smooth surface morphology of the polymer film. The cracks in the film occur because of the composites or trapped air. Also, agglomerates can cause the formation of cracks in the films.¹³⁶ Mechanical properties of PHA films are influenced by crazing, which is the phenomenon of the formation of cracks on the surface of the film.¹³⁷

These cracks generally form due to the addition of composites or due to the presence of impurities. Uneven distribution of polymer also results in the formation of cracks, which results in lower tensile strength.¹³⁸ The roughness index of the PHA film provided the quantitative metrics to justify the formation of cracks. This can be calculated using atomic mass microscopy. It has been reported that pure monomeric films of PHA exhibit a lower roughness index compared with copolymers.¹³⁹ According to FTIR and NMR analysis, discussed in sections 3.7.1 and 3.7.2, the PHA film formed by the bacterial consortium grown in the presence of canola oil cake showed a copolymeric structure. Another study suggested the effect of olive and rapeseed/canola oil on nonwoven electrospun mats by decreasing the degree of crystallinity and roughness leading to the formation of comparatively smooth films.

This can be correlated with our FESEM results, which showed less crack formation on the PHA film produced in the presence of canola oil cake as compared with PHA film produced without using canola oil cake.¹⁴⁰ FESEM was performed using the films obtained from the PHA polymer produced by consortia grown in set I [absence of canola oil cake] (Fig. 12A) and set II [presence of canola oil cake] as an excess carbon source for the growth of bacteria (Fig. 12B). Magnification ranging from 500× to 100 000× was used to visualise the surface morphology of the film. Fig. 12B showed the comparatively smoother surface which indicates the positive effect of canola oil cake on the formation of PHA film and better mechanical strength as compared with PHA film obtained from consortia without using canola oil cake as a carbon source.

Fig. 12 FESEM and EDX analysis of PHA film obtained from consortia of Bacillus cereus strain ST001, Enterobacter cloacae strain AP001 and Rhodococcus ruber strain TESIII [A(a–g)] without using canola oil in GRPD medium (set I); [B] (a–g) with using canola oil in GRPD medium as an excess carbon source (set I); the EDAX pattern indicates the elemental composition of the PHA obtained from the consortia, [A(h)] without using waste canola oilcake as an excess carbon source and [B(h)] when waste canola oil cake was used as excess carbon source.

The chemical composition of the film produced by the consortia was analysed by EDX. Fig. 12A(h) portrays the presence of carbon (53.2 wt%) and oxygen (47.8 wt%) as the principal components in the film obtained from the consortia in set I (control). In contrast, the film from set II [Fig. 12B(h)] exhibits a distinct composition of 65 wt% carbon and 30.6 wt% oxygen. The analysis shows considerably low levels of impurities like silica, sodium and chlorine, which comes from the extraction and casting procedures of PHA film. As recorded previously,¹⁴¹ the carbon source used in the production of PHA affects the elemental composition and molecular structure as well as mechanical strength. All these factors influence the overall composition of PHA film and as per Fig. 12B(h). The spectra obtained from EDX also show the increased carbon content in the PHA film due to the effect of excess carbohydrate content as waste canola oil cake.¹⁴²

The film produced by set I (consortia without canola oil cake supplementation) exhibited an elongation break point at 0.03 mm mm⁻¹ extension, with a minimum force of 0.6 N (Fig. 13A). The tensile strength of this film was approximately 0.1 MPa.

Fig. 13 Tensile strength was calculated based on the maximum load taken by the films produced by the consortia at different break points [A] without using an excess carbon source and [B] in the presence of canola oil cake as a renewable and excess carbon source for PHA production. [C] Comparative analysis of all the mechanical properties, viz. capacity to take a maximum load, tensile strength at maximum load and elongation at break point. All results are represented as mean ± standard deviation (n = 3). Error bars represent standard deviation.

3.7.5. Mechanical properties of PHA film. The mechanical characteristics of PHA films were analysed with the films obtained from two sets of experiments. It was seen that the consortia were capable of producing films of PHA polymer. PHA has variable tensile strength depending on its polymer composition, ranging from a minimum load of 1 MPa to 18–20 MPa.^97,143 The film produced by set I (consortia without using waste canola oil cake) exhibited an elongation break point at 0.03 mm mm⁻¹ extension, with a minimum force of 0.6 N (Fig. 13A). The tensile strength of this film was approximately 0.1 MPa (Fig. 13B). Due to the presence of waste canola oil cake as an excess carbon source, a tensile strength of 0.13 MPa was demonstrated by the resulting PHA film produced by using consortia (Fig. 13C), with the maximum load of 1.19 N and elongation at break point at 0.04 mm mm⁻¹ (Fig. 13B). It clearly shows the positive effect of the utilization of waste canola oil cake as a renewable carbon source for enhancing the mechanical properties of PHA copolymer. Desirable changes in the mechanical properties of PHA film, including enhanced tensile strength and improvement in elongation at the break point, indicate better bioplastic activity. This indicates the positive effect of waste canola oil cake on the production of PHA. The p-value obtained through one-way ANOVA analysis was 0.065, which was slightly greater than 0.05. This indicates that there was no significant change in the mechanical characteristics of the PHA films produced with and without canola oil cake.

This allows future studies to explore the potential factors affecting the mechanical strength. Also, studies based on the biochemical profiling of canola oil cake can be conducted for a detailed understanding of its effect on the mechanical characteristics of PHA.¹¹⁷ Although the mechanical strength of PHA has been enhanced, further improvement is still necessary to render it suitable for applications like food packaging.^144,145 As reported in the literature, the fatty acid profile of canola oil includes palmitic acid, hexadecenoic acid, eicosenoic acid, dodecanoic acid, and octadecenoic acid along with erucic acid. These have a crucial effect on the durability and crystallinity of films, biodegradation, enhanced yield and thermal properties.⁵⁰ In the present study, we investigated the effect of using canola oil cake on the mechanical properties of PHA. There was a slight increase in tensile strength. Further studies will focus on the detailed analysis of fatty acid profiling of canola oil cake. The biodegradability of PHA film has been previously assessed in different studies subjecting it to various environmental conditions such as soil, anaerobic digestion, marine and fresh water, home and industrial composting, etc. As per the reports, 90% PHA film biodegrades within a maximum of 6 years in marine water, 56 days in fresh water and 2 years in soil.¹⁴⁵ PHA has better stability at higher temperature as compared with poly(lactic acid) and polypropylene. It also has better barrier properties in visible UV regions.¹⁴⁶

3.7.6. Gas chromatography and mass spectrometry (GC-MS) of canola oil cake. It was previously reported that over 50 compounds were found in canola oil cake according to GC-MS spectra.⁹⁹ These extracts were reported to contain a large number of hydrocarbons in varying percentages. A few fatty acids such as dodecanoic acid, octadecenoic acid, eicosenoic acid and hexadecenoic acid¹⁴⁷ along with erucic acid (cis-13-docosenoic acid) or erucic acid methyl ester were observed in the GC-MS spectra. These compounds have their own unique properties. 14-Methyl-5α-cholest-8-en-3β-ol-15-one, cholest-8-ene-3β,15β-diol, 2-(1,1-dimethylethyl)-cholestan-3-one, 3,5-dehydro-6-methoxy-cholest-22-ene-21-ol, etc. were reported as sterol esters along with a considerable amount of hydrocarbon. Cholest-8-ene-3β,15β-diol was reported for the first time in rapeseed meal by GC-MS analysis, which was earlier reported to be present in red algae Acanthophora spicifera.¹⁴⁷ According to earlier reports,¹⁴⁸ β-sitosterol was found to be the most prevalent sterol in rapeseed oil along with traces of cholesterol, brassicasterol, campesterol, stigmasterol and D5-avenasterol. A high amount of oleic acid was discovered from the GC-MS analysis of rapeseed/canola oil cake with other constituents such as 2,3,5-trimethoxy toluene, toluene, 4-ethyl-phenol, phenol, 3-methyl-1H-indole, 9-octadecenoic acid, methyl ester, 2,6-dimethoxy-phenol, p-cresol, hexadecanitrile, palmitic acid, etc.¹⁴⁹ Based on the peak area (%) obtained from GC-MS analysis,¹⁵⁰ 9-octadecenoic acid was found to be most abundant followed by two other compounds, namely 9,12-octadecadienoic acid and n-hexadecanoic acid. According to this study,¹⁵⁰ triglycerides are mainly observed from GC-MS analysis. Based on the evidence mentioned above, it was understood that the composition of canola oil cake mainly involves hydrocarbons, fatty acids, triglycerides, sterols, methyl ester, etc. that are possibly having a positive effect on bacterial growth as there was a significant increase observed in biomass as well as PHA yield. GC-MS analysis of canola oil cake was performed using methanol as a solvent and the presence of a total of 77 compounds was detected. Compounds like propanoic acid derived from volatile fatty acids can significantly influence the copolymerization of PHB and PHV, resulting in the formation of PHBV. As discussed in FTIR and NMR, there were characteristic peaks and chemical shifts which support this observation^19,125 (Fig. 14).

Fig. 14 GC-MS analysis of canola oil cake using methanol as a solvent. [A] Original spectra containing 77 compounds. [B] Magnified view of the GC-MS spectra showing some of the [C] volatile organic compounds relevant to the characteristics of the PHA related to functional and mechanical properties.

4. Mechanism of the influence of the carbon source on PHA synthesis

PHA production involves a complex interplay of biochemical pathways and enzymes, which facilitate the synthesis of hydroxyacyl-CoA and its subsequent polymerization into PHA.¹⁰⁶ Acetoacyl CoA synthesis, β-oxidation and biosynthesis of fatty acids are the three main pathways involved in PHA production. The route followed by the bacteria depends on the carbon source present in the growth medium for PHA production.¹⁵¹ The pha gene cluster is responsible for the regulating PHA production at different levels. Enzymes encoded by the pha cluster play a crucial role in stabilizing PHA biosynthesis and regulating various processes, including polymer stabilization, protein regulation, monomer synthesis, polymerization, and ultimately degradation.¹⁵²

When the available carbon source is sugar, bacteria follow pathway I, in which sugar is metabolised through the TCA cycle and acetoacetyl CoA gets formed by the enzymatic conversion of acetyl CoA by β-ketothiolase encoded by the phaA gene. The phaB gene encodes acetoacetyl CoA reductase which catalyses the formation of 3-hydroxybutyryl-CoA, an intermediate in PHA synthesis. This molecule is polymerised into PHA, due to the action of PHA synthase (phaC) enzyme.¹¹³ The second pathway followed by the bacteria can happen when the provided carbon source is in the form of fatty acids. In this pathway, β-oxidation of fatty acids produces various intermediates, which later get converted to 3-hydroxybutyryl-CoA due to the action of acetoacetyl CoA reductase (phaB) and enoyl-CoA hydratase (phaJ). 3-Hydroxybutyryl-CoA, in the presence of PHA synthase, gets polymerised into PHA.¹⁵² In de novo fatty acid biosynthesis, different precursors for PHA are formed, such as 3-hydroxyacyl-ACP, which accelerates the formation of an intermediate, 3-hydroxybutyryl-CoA, in the presence of acyl-ACP–CoA trans-acylase (phaG).¹⁵³ It later gets converted to PHA polymer¹¹³ (Fig. 15).

Fig. 15 Mechanism involved in PHA production using bacteria isolated from textile effluent-contaminated soil and GRPD growth media containing waste canola oil cake as a renewable carbon source.

In this study, the ability of novel PHA-producing bacteria, isolated from textile effluent-contaminated soil, was investigated to showcase the usage of waste canola oil cake as a renewable carbon source for PHA production. As per the reports, canola oil cakes consist of residual oils, fats and a high carbon content which is suitable for PHA production.¹⁵⁴ A considerable change in the mechanical properties of PHA was observed, such as tensile strength and elongation at break point, and the ability to take a maximum load was increased. It confirms the ability of bacteria to utilize glucose present in GRPD medium and fatty acids as well as residual oils present in waste canola oil cake to increase the quantity and to enhance the quality of PHA produced.^155,156

5. Conclusion

In conclusion, the use of waste canola oil cake as a renewable carbon source demonstrates a feasible approach to enhance the yield as well as quality of PHA. As an agro-industrial waste product, rich in carbon, canola oil cake could significantly enhance the mechanical as well as thermal properties of PHA, escalating its applications in the bioplastic industry. According to industrial standards, the tensile strength of plastics to be applicable in various bioplastic industries should be in between 10–30 MPa. The canola oil cake significantly improved the PHA yield and mechanical properties such as tensile strength under maximum load. The PHA film produced using waste canola oil cake was able to withstand a maximum load up to 1.19 N as compared with 0.6 N by PHA film produced without using canola oil cake. Hence the approach of developing PHA films from the consortia using canola oil cake has the potential to meet industrial standards by incorporating more experimental studies based on optimisation. Additionally, this study focuses on the agenda of cost-reduction of the production process and promotes sustainable development by utilizing waste resources, increasing the quality of PHA and contributing to the circular economy. Future research directions will prioritize process optimisation and improvement and the enhancement of mechanical properties. Additionally, investigations will focus on utilizing agro-industrial wastes to scale up PHA production, exploring blends with other natural polymers, and minimizing waste generation. This could contribute to the enhancement of PHA applications in various sectors promoting sustainable development.

Author contributions

The authors confirm the contribution to the paper as follows: Siddhi Joshi: material development: draft manuscript preparation and interpretation of results; Mousumi Debnath: study conception and design; resources and funding acquisition and supervision; draft manuscript preparation; revision of manuscript and interpretation of results. Both authors reviewed the results and approved the final version of the manuscript.

Conflicts of interest

The authors declare that they have no conflicts of interest to report regarding the present study.

Abbreviations

PHA	Polyhydroxyalkanoate
PHB	Polyhydroxybutyrate
PHBV	Poly(3-hydroxybutyrate-co-hydroxyvalerate)
MSM	Mineral salt medium
NB	Nutrient broth
GRPD	Glucose-rich peptone-deficient medium
FTIR	Fourier transform infrared spectroscopy
TGA	Thermogravimetric analysis
NMR	Nuclear magnetic resonance
FESEM	Field emission scanning electron microscopy
EDX	Energy dispersive X-ray (EDX) spectroscopy analysis
GC-MS	Gas chromatography and mass spectrometry
UTM	Universal Tensile Machine
MEGA-XI	Molecular Evolutionary Genetics Analysis
NCBI	National Centre for Biotechnology Information
BLAST	Basic Local Alignment Search Tool

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5bm00787a.

Acknowledgements

The authors would like to thank the Central Analytical Centre and Sophisticated Analytical Instrumentation Centre at Manipal University Jaipur and Material Research Centre at Malviya National Institute of Technology in Jaipur, the instrumental support. Thanks are also due to Prof. Prabhat Nath (BITS Pilani, Rajasthan, India), Prof. Shankar Manoharan and Tomal Das from IIT Jodhpur, Rajasthan, India for the microscopy facility and imaging of PHA granules. The author, SJ, is thankful for the financial assistance provided to carry out her research work through the Dr. Ramdas Pai Scholarship, established by Manipal University Jaipur. The work was financially supported by DST-FIST project (DST/2022/1012) from Govt. of India sanctioned to Department of Biosciences, Manipal University Jaipur and MRB grant from the Manipal Research Board (DoR/RB|2023/SG-03).

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