Bacterial Nanocellulose Production Research Articles

Effect of addition of γ-poly glutamic acid on bacterial nanocellulose production under agitated culture conditions.

Bacterial nanocellulose (BNC), a natural polymer material, gained significant popularity among researchers and industry. It has great potential in areas, such as textile manufacturing, fiber-based paper, and packaging products, food industry, biomedical materials, and advanced functional bionanocomposites. The main current fermentation methods for BNC involved static culture, as the agitated culture methods had lower raw material conversion rates and resulted in non-uniform product formation. Currently, studies have shown that the production of BNC can be enhanced by incorporating specific additives into the culture medium. These additives included organic acids or polysaccharides. γ-Polyglutamic acid (γ-PGA), known for its high polymerization, excellent biodegradability, and environmental friendliness, has found extensive application in various industries including daily chemicals, medicine, food, and agriculture. In this particular study, 0.15g/L of γ-PGA was incorporated as a medium additive to cultivate BNC under agitated culture conditions of 120rpm and 30℃. The BNC production increased remarkably by 209% in the medium with 0.15g/L γ-PGA and initial pH of 5.0 compared to that in the standard medium, and BNC production increased by 7.3% in the medium with 0.06g/L γ-PGA. The addition of γ-PGA as a medium additive resulted in significant improvements in BNC production. Similarly, at initial pH levels of 4.0 and 6.0, the BNC production also increased by 39.3% and 102.3%, respectively. To assess the characteristics of the BNC products, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis were used. The average diameter of BNC fibers, which was prepared from the medium adding 0.15g/L γ-PGA, was twice thicker than that of BNC fibers prepared from the control culture medium. That might be because that polyglutamic acid relieved the BNC synthesis from the shear stress from the agitation. This experiment held great significance as it explored the use of a novel medium additive, γ-PGA, to improve the production and the glucose conversion rate in BNC fermentation. And the BNC fibers became thicker, with better thermal stability, higher crystallinity, and higher degree of polymerization (DPv). These findings lay a solid foundation for future large-scale fermentation production of BNC using bioreactors.

Citrate-buffered Yamanaka medium allows to produce high-yield bacterial nanocellulose in static culture using Komagataeibacter strains isolated from apple cider vinegar

Bacterial nanocellulose (BNC) is a sustainable, renewable, and eco-friendly nanomaterial, which has gained great attentions in both academic and industrial fields. Two bacterial nanocellulose-producing strains (CVV and CVN) were isolated from apple vinegar sources, presenting high 16S rRNA gene sequence similarities (96%–98%) with Komagataeibacter species. The biofilm was characterized by scanning electron microscopy (SEM), revealing the presence of rod-shaped bacteria intricately embedded in the polymeric matrix composed of nanofibers of bacterial nanocellulose. FTIR spectrum and XRD pattern additionally confirmed the characteristic chemical structure associated with this material. The yields and productivities achieved during 10 days of fermentation were compared with Komagataeibacter xylinus ATCC 53524, resulting in low levels of BNC production. However, a remarkable increase in the BNC yield was achieved for CVV (690% increase) and CVN (750% increase) strains at day 6 of the fermentation upon adding 22 mM citrate buffer into the medium. This effect is mainly attributed to the buffering capacity of the modified Yakamana medium, which allowed to maintain pH close to 4.0 until day 6, though in combination with additional factors including stimulation of the gluconeogenesis pathway and citrate assimilation as a carbon source. In addition, the productivities determined for both isolated strains (0.850 and 0.917 g L−1 d−1) compare favorably to previous works, supporting current efforts to improve fermentation performance in static cultures and the feasibility of scaling-up BNC production in these systems.

Advances in the Production of Sustainable Bacterial Nanocellulose from Banana Leaves.

Interest in bacterial nanocellulose (BNC) has grown due to its purity, mechanical properties, and biological compatibility. To address the need for alternative carbon sources in the industrial production of BNC, this study focuses on banana leaves, discarded during harvesting, as a valuable source. Banana midrib juice, rich in nutrients and reducing sugars, is identified as a potential carbon source. An optimal culture medium was designed using a simplex-centroid mixing design and evaluated in a 10 L bioreactor. Techniques such as Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) were used to characterize the structural, thermal, and morphological properties of BNC. Banana midrib juice exhibited specific properties, such as pH (5.64), reducing sugars (15.97 g/L), Trolox (45.07 µM), °Brix (4.00), and antioxidant activity (71% DPPH). The model achieved a 99.97% R-adjusted yield of 6.82 g BNC/L. Physicochemical analyses revealed distinctive attributes associated with BNC. This approach optimizes BNC production and emphasizes the banana midrib as a circular solution for BNC production, promoting sustainability in banana farming and contributing to the sustainable development goals.

Production of bacterial nanocellulose as green adsorbent matrix using distillery wastes for dye removal: a combined approach for waste management and pollution mitigation

Production of bacterial nanocellulose as green adsorbent matrix using distillery wastes for dye removal: a combined approach for waste management and pollution mitigation

Multiphase Under-Liquid Biofabrication With Living Soft Matter: A Route to Customize Functional Architectures With Microbial Nanocellulose.

The growth of aerobic microbes at air-water interfaces typically leads to biofilm formation. Herein, a fermentative alternative that relies on oil-water interfaces to support bacterial activity and aerotaxis is introduced. The process uses under-liquid biofabrication by structuring bacterial nanocellulose (BNC) to achieve tailorable architectures. Cellulose productivity in static conditions is first evaluated using sets of oil homologues, classified in order of polarity. The oils are shown for their ability to sustain bacterial growth and BNC production according to air transfer and solubilization, both of which impact the physiochemical properties of the produced biofilms. The latter are investigated in terms of their morphological (fibril size and network density), structural (crystallinity) and physical-mechanical (surface area and strength) features. The introduced under-liquid biofabrication is demonstrated for the generation of BNC-based macroscale architectures and compartmentalized soft matter. This can be accomplished following three different routes, namely, 3D under-liquid networking (multi-layer hydrogels/composites), emulsion templating (capsules, emulgels, porous materials), and anisotropic layering (Janus membranes). Overall, the proposed platform combines living matter and multi-phase systems as a robust option for material development with relevance in biomedicine, soft robotics, and bioremediation, among others.

Bacterial nanocellulose: A versatile biopolymer production using a cost-effective wooden disc based rotary reactor.

Bacterial nanocellulose (BNC) has various unique qualities, including high mechanical strength, crystallinity, and high water-holding capacity, which makes it appropriate for a wide range of industrial applications. But its lower yield coupled with its high production cost creates a barrier to its usage. In this study, we have demonstrated the better yield of BNC from an indigenous strain Komagataeibacter rhaeticus MCC-0157 using a rotary disc bioreactor (RDB) having a wooden disc. The RDB was optimized based on the type of disc material, distance between the disc, and rotation speed to get the highest yield of 13.0 g/L dry material using Hestrin-Schramm (H-S) medium. Further, the bioreactor was compared for the BNC production using reported medium, which is used for static condition; the RDB showed up to fivefold increase in comparison with the static condition reported. Komagataeibacter rhaeticus MCC-0157 was previously reported to be one of the highest BNC producing stains, with 8.37 g/L of dry yield in static condition in 15 days incubation. The designed RDB demonstrated 13.0 g/L dry yield of BNC in just 5 days. Other characteristics of BNC remain same as compared with static BNC production, although the difference in the crystallinity index was observed in RDB (84.44%) in comparison with static (89.74%). For the first time, wooden disc was used for rotary bioreactor approach, which demonstrated higher yield of BNC in lesser time and can be further used for sustainable production of BNC at the industrial level.

Targeting Bacterial Nanocellulose Properties through Tailored Downstream Techniques.

Bacterial nanocellulose (BNC) is a biodegradable polysaccharide with unique properties that make it an attractive material for various industrial applications. This study focuses on the strain Komagataeibacter medellinensis ID13488, a strain with the ability to produce high yields of BNC under acidic growth conditions and a promising candidate to use for industrial production of BNC. We conducted a comprehensive investigation into the effects of downstream treatments on the structural and mechanical characteristics of BNC. When compared to alkaline-treated BNC, autoclave-treated BNC exhibited around 78% superior flexibility in average, while it displayed nearly 40% lower stiffness on average. An SEM analysis revealed distinct surface characteristics, indicating differences in cellulose chain compaction. FTIR spectra demonstrated increased hydrogen bonding with prolonged interaction time with alkaline solutions. A thermal analysis showed enhanced thermal stability in alkaline-treated BNC, withstanding temperatures of nearly 300 °C before commencing degradation, compared to autoclaved BNC which starts degradation around 200 °C. These findings provide valuable insights for tailoring BNC properties for specific applications, particularly in industries requiring high purity and specific mechanical characteristics.

Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

Isolation of cellulose-producing bacteria (Komagataeibacter Saccharivorans) from rotten sapodilla fruit

Bacterial nanocellulose (BNC) is a nanocellulose produced by bacteria with high purity, crystallinity level, water binding ability, a high degree of polymerization, and excellent mechanical characteristics. The selection of BNC-producing bacteria is one of the critical stages in the production of BNC. This study collected samples from fruit sources and was selected to determine isolates that could produce BNC. Based on the 16s rRNA strain analysis, sapodilla isolate has an identity percentage above 98%, so it can be concluded that it has similarities with the bacteria Komagataeibacter saccharivorans. From eleven sources of fruit, two isolates that have the potential to produce cellulose, namely isolate sapodilla, were obtained. The confirmed sapodilla isolate is an acetic acid bacteria, Komagataeibacter saccharivorans. The yield of BNC-made isolate sapodilla Komagataeibacter saccharivorans (0.432 g/L). Confirmed sapodilla isolates that produce cellulose were mainly determined as cellulose I (adsorption at around 3345, 1430, 1160, and 900 cm−1). Few celluloses II (adsorption at about 1335, 1315, and 1280 cm−1 and a blue-shift of the number of waves from 1430 to around 1425 cm−1) and has a crystallinity index of 52.387 % on HS (Hestrin Scrahm) media with diameter nanofibril about 86.46 nm.

Boosting bacterial nanocellulose production from chemically recycled post-consumer polyethylene terephthalate

The circular economy is emerging with new sustainable solutions to the ever-growing plastic waste challenge, garnering increasing attention. In this study, the possibility to modify expensive Hestrin–Schramm medium (HS) for bacterial nanocellulose (BNC) production and replace significant amounts of glucose with terephthalic acid (TPA) derived after reactive extrusion processing of mixed plastic waste yielding post consumer TPA (pcTPA), was evaluated from laboratory scale to fermentation at pilot scale. Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Thermogravimetric Analysis (TGA) were used to assess the structural, thermal, and morphological properties of BNC and its generated derivatives. The study's findings highlight the positive impact of pcTPA on BNC yield, surpassing the performance of conventional TPA. The presence of pcTPA in the medium resulted in a BNC yield of 4.01 g/L in a scale-up step of 100 mL cultivation, while the positive control using glucose resulted in a yield of 3.57 g/L. The efficiency of glucose substitution with pcTPA increased with each scale-up step, ultimately reaching a 320% yield increase in comparison to the positive control. Additionaly, the procedure that enhanced the materials' thermoplasticity in the form of derivatives has been established resulting in the production of BNC laurate and BNC octanoate derivatives with melting temperatures of 270 °C and 280 °C, respectively. Overall, this study investigates the potential of this approach as an important circular economic solution, enabling an increased sustainable perspective for polyethylene terephthalate (PET) circularity and significantly a much needed cost reduction for BNC production with enhanced thermoplasticity.

Bacterial nanocellulose-Robust preparation and application—A literature review

Increasing attention is being paid to bacterial nanocellulose (BNC) because of its environment-friendly properties. Researchers investigated the role of microbial hosts in BNC production due to the benefits of cellulose produced by microbes. Several research groups have developed techniques to make BNC on a large scale with the goal of developing new methods. A 3D network of micro and nanofibrils in BNC synthesized from several bacterial strains makes these BNC useful for reinforcing nanostructured composites that have increased Young’s modulus, tensile strength, purity, crystallinity, and water holding capacity. To overcome the barriers associated with the industrial scale production of BNC, different production techniques will be used, including static culture, cell-free production, agitated/shaking culture, using a variety of receptors for fermentation, and low-cost substrates as carbon sources. By in-situ and ex-situ fermentation processes, metal/metal oxide nanoparticle composites are among the most widely used materials in diagnostic and regenerative medicine. The purpose of the review is to update the researchers regarding the lucid production process and versatile applications of bacterial nanocellulose in biomedical field. We shall mainly discuss about the different methods for bacterial cellulose production and some of its applications in this mini-review.

Bacterial nanocellulose and its application in heavy metals and dyes removal: a review.

This review discusses the application of bacterial nanocellulose (BNC) and modified BNC in treating wastewater containing heavy metals and dye contaminants. It also highlights the challenges and future perspectives of BNC and its composites. Untreated industrial effluents containing toxic heavy metals are systematically discharged into public waters. In particular, lead (Pb), copper (Cu), cadmium (Cd), nickel (Ni), zinc (Zn), and arsenic (As) are very harmful to human health and, in some cases, may lead to death. Several methods such as chemical precipitation, ion exchange, membrane filtration, coagulation, and Fenton oxidation are used to remove these heavy metals from the environment. However, these methods involve the use of numerous chemicals whilst producing high amount of toxic sludge. Meanwhile, the development of the adsorption-based technique has provided an alternative way of treating wastewater using BNC. Bacterial nanocellulose requires less energy for purification and has higher purity than plant cellulose. In general, the optimum growth parameters are crucial in BNC production. Even though native BNC can be used for the removal of heavy metals and dyes, the incorporation of other materials, such as polyethyleneimine, graphene oxide, calcium carbonate and polydopamine can improve sorption efficiencies.

Bacterial nanocellulose production using cost-effective, environmentally friendly, acid whey based approach

This study investigates the production of bacterial nanocellulose (BNC) film using a symbiotic culture of bacteria and yeasts (SCOBY) in acid whey medium. The yields were 59.58 g/L for wet and 2.75 g/L for freeze-dried BNC films in four days. The film contained cellulose nanofibrils with diameters ranging from 20 nm to 100 nm with a 34.3 % crystallinity index as confirmed by X-ray diffraction. Incorporating glycerol as a plasticizer modified the mechanical and thermal properties of the BNC film, reduced brittleness, and enhanced strain from 0.75 % to above 16 %. Fourier Transform Infrared Spectroscopy analysis identified intermolecular hydrogen bonding between the BNC film and glycerol. The glass transition temperature increased from roughly 158 °C to above 166 °C. The study validates the potential of acid whey as a viable alternative substrate for the production of BNC. The production method providing environmental benefits, cost-efficiency, and scalability, it also delivers an effective circular economy solution.

Use of brewer's residual yeast for production of bacterial nanocellulose with Gluconacetobacter hansenii

Bacterial nanocellulose (BNC) has attained elevated interest due to its versatile structure and high resistance characteristics. Accordingly, efforts have been made in order to reduce its production costs, such as the employment of its by-products as a nutrient broth to yield the microorganism. Residual brewer's yeast is an excellent recourse, due to its high nutritional value and availability. Therefore, research which aimed to contribute to the development of a low cost, efficient and biosustainable technology for BNC production with Gluconacetobacter hansenii was carried out. BNC was obtained from residual brewer's yeast hydrolysate at pH 7.0 and five days of incubation at 30 °C in static culture. The hydrolysate was characterized by the amount of sugars, fatty acids, total proteins and ash content. Subsequently, BNC obtained was characterized in terms of yield, carbon conversion ratio, hydrodynamic size, crystallinity, morphology, Fourier-transform infrared spectroscopy, and surface analysis. Residual brewer's yeast hydrolysate proved to be efficient in BNC production via gluconeogenesis with consumption of alanine, threonine and glycerol, obtaining 1.9 times the yield of the chemically defined broth adopted as standard. Additionally, properties observed in the obtained BNC were equal to those obtained from conventional chemical medium. The research contributed to bacterial nanocellulose production using by-products from the brewing industry.

PRODUCTION OF BACTERIAL NANOCELLULOSE IN ALTERNATIVE CULTURE MEDIA UNDER STATIC AND DYNAMIC CONDITIONS

Bacterial nanocellulose (BNC) was produced in three different culture media: the standard HS medium, and the alternative much less costly analytical glycerol/corn steep liquor (AG-CSL) and glycerol remaining from biodiesel production/corn steep liquor (BG-CSL) media. Both static and agitated conditions were assessed, and the effect of the culture medium, agitation speed and cultivation time used was evaluated on BNC production and selected products characteristics (morphology, specific surface area and crystallinity). Results showed that the cheaper alternative culture media assayed led to increased production of BNC, and that agitation speed modulation allowed producing either BNC pellicles or BNC pellets with varying size, shape, crystallinity and cellulose nanofibers packing.

Nata de fique: A cost-effective alternative for the large-scale production of bacterial nanocellulose

Bacterial nanocellulose (BNC) is a biopolymer with an expanding market due to its properties. One important aspect related to BNC production is the culture medium composition, which is rich in carbon and nitrogen, and it represents 30% of the total cost of production. The aim of this work was to evaluate the use of fique juice, a by-product of the fique fiber isolation process, as an alternative source to produce BNC using the Komagataeibacter medellinensis strain, denominated as nata de fique. First, the fique juice was chemically characterized to determine its nutrient content. The nata de fique yield was calculated as gram of dry cellulose per littler of the medium; the physicochemical characterization of the nata de fique was analyzed using Scanning Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FTIR), Thermogravimetric analysis (TGA), X-Ray diffraction and BET (Brunauer-Emmett-Teller) surface area analysis. Finally, the environmental impact and financial analysis were also determined. It was found that using fique juice as a culture medium, the BNC production yield was 2.2 g BNC/L medium compared to BNC-HS, which was 1.14 g BNC/L medium, due to the chemical composition of fique juice, which presented a higher content of nitrogen and additives like Mg2+ and Ca2+. In the morphological analysis, the same nanoribbon structure was observed, and no changes in the crystalline parameters were detected. Nevertheless, there was a slight decrease in the thermal stability temperature, the crystalline index, and the surface area in the BNC produced with fique juice, due to the presence of some remaining impurities after the purification step. In the environmental analysis, nata de fique decreased the water consumption, carbon footprint, and raw material consumption, and the BNC production cost was twice cheaper than the HS culture medium. This research demonstrates that nata de fique is an alternative source of BNC regarding economic and environmental impact.

Review of Bacterial Nanocellulose as Suitable Substrate for Conformable and Flexible Organic Light-Emitting Diodes.

As the development of nanotechnology progresses, organic electronics have gained momentum in recent years, and the production and rapid development of electronic devices based on organic semiconductors, such as organic light-emitting diodes (OLEDs), organic photovoltaic cells (OPVs), and organic field effect transistors (OFETs), among others, have excelled. Their uses extend to the fabrication of intelligent screens for televisions and portable devices, due to their flexibility and versatility. Lately, great efforts have been reported in the literature to use them in the biomedical field, such as in photodynamic therapy. In tandem, there has been considerable interest in the design of advanced materials originating from natural sources. Bacterial nanocellulose (BNC) is a natural polymer synthesized by many microorganisms, notably by non-pathogenic strains of Komagataeibacter (K. xylinus, K. hansenii, and K. rhaeticus). BNC shows distinct physical and mechanical properties, including its insolubility, rapid biodegradability, tensile strength, elasticity, durability, and nontoxic and nonallergenic features, which make BNC ideal for many areas, including active and intelligent food packaging, sensors, water remediation, drug delivery, wound healing, and as conformable/flexible substrates for application in organic electronics. Here, we review BNC production methods, properties, and applications, focusing on electronic devices, especially OLEDs and flexible OLEDs (FOLEDs). Furthermore, we discuss the future progress of BNC-based flexible substrate nanocomposites.

Bacterial nanocellulose production using Cantaloupe juice, statistical optimization and characterization

The bacterial nanocellulose has been used in a wide range of biomedical applications including carriers for drug delivery, blood vessels, artificial skin and wound dressing. The total of ten morphologically different bacterial strains were screened for their potential to produce bacterial nanocellulose (BNC). Among these isolates, Bacillus sp. strain SEE-3 exhibited potent ability to produce the bacterial nanocellulose. The crystallinity, particle size and morphology of the purified biosynthesized nanocellulose were characterized. The cellulose nanofibers possess a negatively charged surface of − 14.7 mV. The SEM images of the bacterial nanocellulose confirms the formation of fiber-shaped particles with diameters of 20.12‒47.36 nm. The TEM images show needle-shaped particles with diameters of 30‒40 nm and lengths of 560‒1400 nm. X-ray diffraction show that the obtained bacterial nanocellulose has crystallinity degree value of 79.58%. FTIR spectra revealed the characteristic bands of the cellulose crystalline structure. The thermogravimetric analysis revealed high thermal stability. Optimization of the bacterial nanocellulose production was achieved using Plackett–Burman and face centered central composite designs. Using the desirability function, the optimum conditions for maximum bacterial nanocellulose production was determined theoretically and verified experimentally. Maximum BNC production (20.31 g/L) by Bacillus sp. strain SEE-3 was obtained using medium volume; 100 mL/250 mL conical flask, inoculum size; 5%, v/v, citric acid; 1.5 g/L, yeast extract; 5 g/L, temperature; 37 °C, Na2HPO4; 3 g/L, an initial pH level of 5, Cantaloupe juice concentration of 81.27 percent and peptone 11.22 g/L.

Enhanced production of fibrous bacterial cellulose in Gluconacetobacter xylinus culture medium containing modified protein of okara waste

In Gluconacetobacter xylinus cultivation for bacterial nanocellulose production, agro-industrial wastes, soybean residual okara, okara extracted protein, and modified okara protein, were used as a protein source. In comparison with homogenized raw okara and protein extracted from raw okara, acetic-acid modified protein provided the higher cellulose yield (2.8g/l at 3 %w/v protein concentration) due to the improved protein solubility in the culture medium (89 %) and smaller particle size (0.2μm) leading to facile uptake by the bacteria. Importantly, pH of the culture medium containing the modified protein measured before and after the cultivation was similar, suggesting the buffering capacity of the protein. Nanocellulose fibers were then produced densely in the network of hydrogels with high crystallinity nearly 90 %. Based on the results, economic constraints around nanocellulose production could be alleviated by valorization of okara waste, which provided enhanced sustainability.

Exploration of a novel and efficient source for production of bacterial nanocellulose, bioprocess optimization and characterization

The demand for bacterial nanocellulose is expected to rise in the coming years due to its wide usability in many applications. Hence, there is a continuing need to screen soil samples from various sources to isolate a strain with a high capacity for bacterial nanocellulose production. Bacillus sp. strain SEE-12, which was isolated from a soil sample collected from Barhiem, Menoufia governorate, Egypt, displayed high BNC production under submerged fermentation. Bacillus sp. strain SEE-12 was identified as Bacillus tequilensis strain SEE-12. In static cultures, BNC was obtained as a layer grown in the air liquid interface of the fermentation medium. The response surface methodology was used to optimise the process parameters. The highest BNC production (22.8 g/L) was obtained using 5 g/L peptone, 5 g/L yeast extract, 50%, v/v Cantaloupe juice, 5 g/L Na2HPO4, 1.5 g/L citric acid, pH 5, medium volume of 100 mL/250 mL conical flask, inoculum size 5%, v/v, temperature 37 °C and incubation time 6 days. The BNC was purified and characterized by scanning electron microscopy (SEM), Energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and transmission electron microscopy (TEM).