Introduction
Hydroxy fatty acids (HFAs)
Hydroxy fatty acids (HFAs) are long-chain fatty acids characterized by the presence of one or more hydroxyl (-OH) groups and a terminal carbonyl (-COOH) group These compounds are naturally occurring in waxes, the cerebellum, and the lipids of various organisms, including animals, plants, and microorganisms HFAs exhibit enhanced stability, viscosity, reactivity, and solubility in solvents compared to standard fatty acids.
Despite their remarkable properties, HFAs are not commercially viable due to high production costs and environmentally unfriendly synthetic methods The traditional chemical synthesis of HFAs faces significant challenges, including multiple reaction steps, low selectivity, harsh conditions, toxic reagents, and limited product diversity These issues hinder the commercialization of HFAs However, the discovery of microorganisms that can synthesize HFAs marks a promising advancement in enhancing the value of these fatty acids These microbial strains can convert fatty acids or vegetable oils into HFAs using various enzymes, primarily involving four key enzymes: hydratase and cytochrome P450.
Various enzymes, including monooxygenase, lipoxygenase, and hydroxylase, play crucial roles in metabolizing different hydroxy fatty acids (HFAs) such as 13-hydroxyoctadecadienoic acid (HODE), 10-hydroxystearic acid, and ω-hydroxy fatty acids These compounds are produced through distinct enzymatic pathways involving lipoxygenase, hydratase, and cytochrome P450 monooxygenase Microbial processes for HFA production offer significant advantages over chemical synthesis, including environmental sustainability and reduced production costs.
1.1.2.1 α-HFA and β-HFA α-HFA and β-HFA are fatty acids that possess a hydroxyl group near carboxyl group These two HFAs are important for bacterial endotoxins, yeast and fungi [6] In addition, these fatty acids also possess antimicrobial activity and work as part of the ring structure in some peptide antibiotics [7] α-HFA and β-HFA are synthesized through hydroxylation of fatty acids with the support of the enzyme α-hydroxylases The product of this process is usually 2-hydroxy and (or) 3-hydroxy fatty acids Matsunaga et al conducted the conversion of myristic acid to hydroxymyristic acids with this enzyme in
Bacillus subtilis produces a combination of α- and β-hydroxymyristic acids, with a predominant presence of the β-hydroxylated variant In contrast, α-hydroxylases derived from Sphingomonas paucimobilis exclusively hydroxylate fatty acids at the α-position.
1.1.2.2 ω-HFAs ω-Hydroxy fatty acids (ω-HFAs) are long chain fatty acids containing one -OH group at the position near the end of the circuit ω-HFAs are often used as monomer for the synthesis of polymer materials because they possess the longest chain of carbon chains [10] These fatty acids are also used to prepare α,ω-dicarboxylic acids and ω-amino carboxylic acids which are used as initial materials for the synthesis of polyamides and polyesters [11] In addition, ω-HFAs are used as intermediates in food, chemical and pharmaceutical [12] Fatty acids are converted into ω-HFAs under the catalytic action of cytochrome P450 monooxygenases P450 is a large and diverse enzyme family with a multitude of enzymes whose activity targets fatty acid objects These enzymes are found in many microorganisms and enzymes used for ω-hydroxylation of fatty acids present in Bacillus species such as Bacillus subtilis, Bacillus megaterium [13], Bacillus cereus and Bacillus anthracis [14]
7,10-dihydroxy-8(E)-octadecenoic acid (DOD) is a hydroxy fatty acid (HFA) characterized by two hydroxyl groups in its carbon chain, produced from oleic acid by Pseudomonas sp PR3 in 1991 Subsequent studies have utilized triolein, olive oil, and safflower oil as substrates for DOD production DOD exhibits properties such as reducing surface tension and demonstrating antibacterial activity, making it a valuable compound in various applications.
DOD, featuring two hydroxyl groups, shows significant promise in the synthesis of polymers, particularly polyurethane Hydroxylation takes place at the C7 and C10 positions of oleic acid, facilitated by enzymes from the P450 family, as highlighted in the study by Kim et al.
Oleic acid undergoes hydroxylation primarily at the C10 position, leading to a relocation of the double bond from C9-C10 to C8-C9 This process is followed by hydroxylation at C7, facilitated by the enzyme P450, resulting in the complete conversion of oleic acid into DOD Therefore, cytochrome P450 monooxygenases are crucial for the production of DOD.
Tri-HFAs, characterized by their three hydroxyl groups in the molecular structure, are produced by various microorganisms from different fatty acids For example, Pseudomonas aeruginosa PR3 effectively converts fatty acids into 7,10,12-trihydroxy fatty acids.
8(E)-octadecenoic acid is derived from ricinoleic acid, while 9,10,13-THOD and 9,12,13-THOD are produced from linoleic acid Furthermore, B megaterium ALA2 can convert linoleic acid into 12,13,17-trihydroxy-9(Z)-octadecenoic acid The enzymes responsible for the hydroxylation that leads to the formation of tri-hydroxy fatty acids (tri-HFAs) are believed to be P450 oxygenases.
Figure 1.1 Biosynthetic pathway of oleic acid to 7,10-dihydroxy-8(E)- octadecenoic acid
Hydroxy fatty acids (HFAs) serve as essential raw materials for the synthesis of various biopolymers, lubricants, polyurethanes, bio-plastics, soaps, resins, and nylons Additionally, they are utilized in the production of stabilizers, paints, and plasticizers as additives HFAs also act as precursors for flavor production through bacterial and yeast fermentation The polymers derived from HFAs exhibit superior properties compared to traditional polymers, including non-toxicity, enhanced thermal and chemical resistance, high biological compatibility, and increased pliability These advanced polymers find extensive applications in everyday products such as clothing, footwear, cosmetics, medical equipment, and containers for food, medicine, and detergents.
Numerous studies indicate that hydroxy fatty acids (HFAs) possess significant medicinal properties, making them valuable in medical applications For instance, 15-hydroxyeicosatetraenoic acid derived from arachidonic acid exhibits antifungal, anticancer, and anti-inflammatory effects Additionally, 10-hydroxyoctadecadienoic acid, which is converted from oleic acid, demonstrates α-glucosidase inhibitory activity Furthermore, 14,21-dihydroxydocosahexanoic acid is effective in treating wounds in diabetic patients, while 9,12,13-trihydroxyoctadecenoic acid serves as a vaccine adjuvant Collectively, HFAs show immense potential in both industrial and medical fields, presenting themselves as a sustainable and eco-friendly energy source.
7 to the environment and in the future can be used as source material to replace petroleum resources, which are depleting day by day.
Pseudomonas aeruginosa PR3 and Pseudomonas aeruginosa KNU-2B
Pseudomonas aeruginosa PR3 is a microorganism known for its ability to convert fatty acids into hydroxy fatty acids (HFAs), specifically producing mono-, di-, and tri-HFAs from various fatty acid sources.
In 1991, the PR3 strain was isolated from a pig farm water sample in Morton, Illinois, and subsequently cultured by Hou's research team They discovered a compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), achieving a production yield exceeding 60% when PR3 was grown with oleic acid PR3 showcases its versatility by producing various hydroxy fatty acids (HFAs), including mono-HFA, 10(S)-hydroxy-8(E)-octadecenoic acid from oleic acid, and tri-HFAs such as 7,10,12-trihydroxy-8(E)-octadecenoic acid from ricinoleic acid and 9,12,13-trihydroxy-10(E)-octadecenoic acid from linoleic acid Beyond converting fatty acids into HFAs, PR3 can also generate HFAs from vegetable oils Given the high cost of fatty acids, exploring alternative raw materials like vegetable oil is essential for ensuring a cost-effective supply of essential fatty acids for HFA production.
Vegetable oils, such as olive oil, canola oil, and palm oil, are cost-effective sources of fatty acids, containing high concentrations of oleic acid (71.3%, 61.8%, and 40%, respectively), which are essential for DOD production Additionally, castor oil is rich in ricinoleic acid (85%-95%), while soybean oil contains 51% linoleic acid; both acids serve as substrates for tri-HFAs However, since fatty acids in vegetable oils are primarily in the form of triglycerides, converting them to mono fatty acids is necessary for HFA production The strain PR3, with its induced-lipase activity, facilitates this conversion, enabling the production of DOD from olive oil and safflower oil.
Pseudomonas aeruginosa PR3 is an efficient producer of hydroxy fatty acids (HFAs), achieving production yields of 55% and 38.6% Notably, PR3 excels in producing 1,3-dihydroxy-2-octanone (DOD), with high DOD content generated even when utilizing fatty acids or vegetable oils as substrates.
Pseudomonas aeruginosa KNU-2B is a newly isolated strain derived from PR3, exhibiting distinct morphological characteristics while retaining 99% similarity in the 16S rRNA gene sequence to Pseudomonas aeruginosa Although KNU-2B can produce hydroxy fatty acids (HFAs) from fatty acids and vegetable oils, its efficiency is lower compared to PR3 Consequently, optimizing the production process for HFAs in the KNU-2B strain is essential.
Table 1.1 Production of DOD and TOD from fatty acid and vegetable oil by P aeruginosa KNU-2B
Oleic acid TM 90% Cell culture 3.03 33.7 120 0.025
Oleic acid TM 90% Cell-free 6.41 71.2 36 0.178
Ricinoleic acid TM 80% Cell culture 0.32 4.00 96 0.0033
Ricinoleic acid TM 80% Cell-free 1.15 14.4 72 0.015
Polyurethane
Polyurethanes (PUs) are versatile polymers characterized by urethane groups in their composition Initially discovered by Bayer and colleagues in 1937, PUs gained prominence during World War II as a replacement for rubber.
Since their introduction in the 1950s, polyurethane (PU) coatings have been produced using various formulas and are now widely utilized in adhesives, coatings, elastomers, and hard foams PUs have garnered global scientific interest due to their unique combination of rubber-like elasticity and the durability of metal, allowing them to effectively replace plastic, rubber, and metal in numerous applications Their versatility has led to applications across diverse fields, including composites, coatings, adhesives, construction engineering, foams, sealants, and medicine, owing to their exceptional biological, physical, chemical, and mechanical properties.
Polyurethane is created through the chemical reaction between polyols, which have two or more hydroxyl (OH) groups, and polyisocyanates, which contain two or more isocyanate (NCO) groups The R’ group in polyisocyanates can be either aliphatic or aromatic, typically with a molecular weight under 200 g/mol In contrast, the R group in polyols is generally a polyether or polyester, possessing a higher molecular weight.
Polyurethanes (PUs), with a molecular weight of 2000 g/mol, can be synthesized from a variety of materials, resulting in a diverse range of products and applications The chemical composition of the initial materials significantly influences the properties of PUs Consequently, PUs are categorized into different types, including thermoplastic, rigid, flexible, binders, and waterborne, each suited for specific applications This classification is illustrated in Figure 1.3.
Figure 1.3 The chemical reaction of Polyurethanes (A) and their classification (B)
Rigid polyurethane foams, created from polyether or polyester polyol combined with methylene diphenyl diisocyanate, feature cross-linked and closed-pore structures Renowned for their exceptional insulating properties, these foams are widely utilized in construction materials, providing temperature stability and noise reduction for both household and commercial applications They serve as effective insulation for windows, roofs, and walls, as well as sealants to prevent air leakage Key industries benefiting from rigid PU foams include refrigeration, construction, and pipeline sectors.
Flexible PU (FPU) foams are created through the reaction of diisocyanate and high molecular weight polyols They are categorized into two types: conventional flexible foams and highly resilient flexible foams Conventional flexible foams are produced using polyols with molecular weights ranging from 3000 to 4000 g/mol and TDI as the diisocyanate, allowing for modifications in physical properties by incorporating additional polyols In contrast, highly resilient flexible foams utilize polyols with molecular weights between 4800 and 12000 g/mol, employing either TDI or MDI as the diisocyanate FPU foams are commonly found in various consumer products, including furniture, beds, carpets, and automotive applications.
17 interior, packaging [41-42] These products are highly appreciated by the following factors: the diversity of foams, long term use, and good breathability
Thermoplastic polyurethanes (TPUs) are highly flexible and resilient materials known for their excellent impact, abrasion, and weather resistance They serve dual purposes, functioning as both a coloring agent and a protective coating to enhance product durability Additionally, due to their water-insoluble, non-ionic, and inert properties, TPUs are widely utilized in various industries, including medical equipment, automotive, construction, and footwear.
Cell-free synthetic biology
Cell-free synthetic biology enables the biosynthesis of proteins and other compounds using biological machinery without the need for living cells This innovative approach offers a more flexible and efficient means of producing desired substances compared to traditional cell-based systems While most synthetic biology processes rely on living cells due to their self-replicating capabilities, challenges such as cell membrane barriers and cellular complexity often hinder synthesis To overcome these obstacles, cell-free synthesis methods have been developed, utilizing three main types of systems: extract-based, purified, and synthetic enzyme pathways.
18 based system, media includes extracts provided from microorganisms such as
E coli [47], insect cells [48], wheat germ [49] or S cerevisiae [50] possessing function of translating and transcription The purified system consists of purified translational components of E coli [51] The final system consists of many enzymes to perform complex metabolic reactions [52] Compared to the cell system, cell-free method has many advantages such as fast synthesis speed, high product productivity, adjustable and controllable the working media, easily synthesizing complex proteins as well as tolerance of toxic products [53] Currently, cell-free synthetic biology is applied in the fields of biological engineering, including protein engineering, metabolic engineering, and artificial cell engineering (Figure 1.6)
Figure 1.6 Engineering protein, metabolism and artificial cell in the open cell-free system
Cell-free synthetic biology is a powerful approach for protein synthesis, enabling precise control over transcription, translation, and metabolic processes within an open system Unlike traditional cell-based methods, which often face challenges like insoluble expression, low stability, inaccurate folding, and low yields, cell-free systems allow for direct manipulation of protein synthesis, free from the constraints of cellular growth This innovative technique simplifies the process and enhances the efficiency of protein production.
Direct access to the reaction environment simplifies protein synthesis, but the process can produce toxic proteins that harm cells and inhibit their growth and division, leading to reduced yields of other proteins Utilizing a cell-free method allows for the production of these toxic proteins without negatively impacting cell development, creating optimal conditions for protein synthesis.
Cell-free systems enable the integration of various biological components or artificial devices, enhancing biological production and broadening the applications of proteins.
Microorganisms are gaining significant attention from researchers for their potential in producing chemicals and fuels To function effectively as a production machine, bacteria must efficiently store nutrients necessary for cell growth and metabolism.
Cell-free synthetic biology offers significant advantages in biological production by decoupling biosynthesis from cell growth, thus eliminating the challenges posed by bacterial survival and by-product formation This innovative approach allows for rapid synthesis times, enhanced control over responses, and increased tolerance to hazardous substances By functioning as an open system, cell-free methods minimize incompatibility and optimize metabolic efficiency Additionally, they enable the design of tailored metabolic processes to maximize the production of desired substances, making them suitable for industrial scaling and improving bio-industrial production capabilities.
An artificial cell is typically composed of bioactive materials encased in a capsule-like membrane, designed to carry out specific functions There are two primary approaches to creating artificial cells: the top-down method and the bottom-up method The top-down approach starts with a living organism and relies on a viable genomic cell, but it is often costly and time-consuming.
The bottom-up method for creating artificial cells is more efficient and less complex than the traditional top-down approach, as it begins from the ground up, facilitating easier cell formation Utilizing a cell-free method within this framework has proven effective in engineering artificial cells that exhibit functionalities akin to living cells These innovative cells have diverse applications, including protein synthesis, guiding protein evolution, and exploring biological networks.
Aims and objectives of this study
Polyurethane is recognized as a highly effective polymer with exceptional properties and diverse applications However, its traditional synthesis relies on toxic polyisocyanates and petroleum-derived polyols, raising environmental concerns To address these issues, bio-based polyurethane has emerged as a sustainable alternative, offering benefits such as cost-effectiveness, reduced environmental impact, and biodegradability The primary raw materials for producing bio-based polyurethanes are largely sourced from vegetable oils.
Hydroxy fatty acids (HFAs), derived from vegetable oils rich in hydroxyl groups, hold potential for synthesizing polyurethanes (PUs) as polyols Despite their promising characteristics, there is currently a lack of research on the use of HFAs in PU synthesis This dissertation aims to explore the feasibility of using HFAs to produce bio-based PUs and to analyze the properties of these innovative materials.
The newly identified strain P aeruginosa KNU-2B can produce hydroxy fatty acids (HFAs) from fatty acids and vegetable oils, although it does so with low yields Given the benefits of cell-free synthesis methods, exploring this approach for HFAs production using the KNU-2B strain presents a valuable opportunity The study aims to investigate these possibilities further.
• To evaluate the possibility of olive oil and castor oil as substrate for the bio-conversion to HFAs
• To investigate the potential of HFAs as an initial material for polyurethane synthesis
• To investigate the properties of bio-based polyurethane synthesized from HFAs
• To investigate the use of cell-free system to enhance the production of HFAs.
Microbial Conversion of Vegetable Oil to 7,10-dihydroxy- 8(E)-octadecenoic acid and Its Application to Bio-Based Polyurethane
Introduction
Polyurethanes (PUs) are versatile polymeric materials characterized by their urethane group, making them essential in various applications due to their unique mechanical, physical, biological, and chemical properties Their cost-effective synthesis has garnered significant attention from researchers, leading to widespread use in fields such as medicine, construction, coatings, sealants, adhesives, foams, and composites PUs can be molded into various forms, including fibers, films, castables, thermoplastics, and foams The typical synthesis of PUs involves the reaction of polyols with multiple hydroxyl groups and isocyanates, although the use of isocyanates is limited due to their toxicity.
Many polyols are derived from petroleum-based materials, leading to environmental concerns Consequently, there is growing interest among scientists globally in producing isocyanates and polyols from renewable resources.
In recent years, renewable resources, particularly vegetable oils, have emerged as vital contributors to sustainable development, addressing environmental issues, waste management, and the depletion of non-renewable resources These oils, derived from sources like palm trees, soybeans, and sunflowers, offer advantages such as biodegradability, cost-effectiveness, and low eco-toxicity, making them increasingly valuable in the chemical and polymer industries Bio-oil derived polymers and composites are now widely used in applications like paints, coatings, adhesives, and biomedicine Vegetable oils, which are triglycerides containing long-chain fatty acids such as oleic, linoleic, and linolenic acids, can be converted into hydroxy fatty acids (HFAs) known for their higher viscosity and reactivity Recent studies have shifted focus to microbial conversion processes, particularly using Pseudomonas aeruginosa PR3, which effectively transforms unsaturated fatty acids like oleic acid into mono-, di-, and tri-hydroxy fatty acids, showcasing significant potential for industrial applications in plastics, lubricants, cosmetics, and paint additives.
The PR3 strain effectively produces 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), making it an ideal choice for utilizing olive oil as a substrate due to its high oleic acid content.
Pseudomonas aeruginosa KNU-2B, isolated from P aeruginosa PR3, can produce 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid and olive oil DOD is a unique compound featuring two hydroxyl groups, allowing it to fully react with isocyanates to create polyurethanes (PUs).
This study introduces the first proof-of-concept for the synthesis of polyurethane (PU) using DOD produced by Pseudomonas aeruginosa KNU-2B with olive oil as a substrate The PU preparation involved a reaction mediated by hexamethylene diisocyanate (HMDI) in various ratios DOD was combined with different weight ratios of polyethylene glycol (PEG) and polycaprolactone diol (PCLDO) and reacted with HMDI to create new PUs The structure of DOD was verified using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry (GC/MS) The synthesized PUs were characterized through FTIR, differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA), and their tensile properties were measured and compared using a universal testing machine (UTM).
Materials and Methods
Olive oil, rich in oleic acid (73%), was sourced from Samchun in Seoul, Korea Key materials including PCLDO (Mn: 2000), PEG (Mn: 200), and dibutyltin dilaurate (DBTDL) were obtained from Sigma-Aldrich in St Louis, Missouri, USA, while PEG (Mn: 20,000) was acquired from Fluka in Buchs, Switzerland Additionally, HMDI was purchased from Daejung in Siheung, Korea, with all other reagents meeting analytical grade standards.
In this study, Pseudomonas aeruginosa KNU-2B was cultured using a standard growth medium formulated with 4 g/L glucose, 1 g/L yeast extract, 4 g/L K2HPO4, 1 g/L (NH4)2HPO4, 0.1 g/L MgSO4, 0.056 g/L FeSO4, and 0.01 g/L MnSO4 The pH of the medium was carefully adjusted to 8.0 using diluted phosphoric acid.
[17] The strain was grown aerobically in a 500-mL flask containing 100 mL of the standard medium at 27°C with shaking at 200 rpm
2.2.3 Production of DOD from Olive Oil
After 24 h of cultivation on glucose, 1.1 mL (1.0% v/v) olive oil was added to the medium for DOD production and the mixture was incubated for an additional 72 h After the cultivation period (total 96 h), the medium was acidified to pH 2 with 6N HCl, then extracted twice with an equivolume
A mixture of ethyl acetate and diethyl ether was used, followed by the removal of solvents via a rotary evaporator The product was then washed with n-hexane and recrystallized in ethyl acetate DOD was filtered and dried using a solvent dryer All experiments were conducted independently and repeated three times, with data presented as mean ± standard deviation.
2.2.4 Preparation of PUs Based on DOD
To prepare the polymer films, one gram of DOD was combined with 1% w/v DBTDL and 3 mL of chloroform in a beaker, mixed for 5 minutes at room temperature, and then HMDI was added for an additional 10 minutes of mixing The molar ratio of NCO to OH was adjusted to 1.0, 1.2, 1.4, and 1.6 mol/mol by varying the amounts of DOD and HMDI The resulting mixture was poured onto a glass plate and post-cured in a vacuum oven at 80°C for 2 hours After the solvent evaporated, the polymer films were peeled off the plate and sliced into specific dimensions for thermo-mechanical testing.
2.2.5 Preparation of PUs Based on DOD and PEG or PCLDO
In a beaker, one gram of polyol was combined with 1% w/v DBTDL and 3 mL of chloroform, stirring for 5 minutes at room temperature The polyols used included DOD and PEG (with molecular weights of 200 and 20,000) as well as PCLDO (with a molecular weight of 2000), which were mixed in different weight ratios HMDI was subsequently added to achieve a final molar NCO/OH ratio.
29 of 1.4 mol/mol and mixed further for 10 min The mixture was casted on a glass plate and heated to 80°C in a vacuum oven for 2h
For GC analysis, the sample was esterified first by diazomethane for
The samples were allowed to stand at room temperature for 10 minutes before being derivatized with a mixture of trimethylsilyl imidazole (TMSI) and pyridine (1:4, v/v) for 45 minutes Analysis was conducted using a GC (Agilent model 6890N) with an HP-5 column (30 m × 320 μm × 0.25 μm), where 1 μL was injected The temperature program started at 70°C, increasing to 200°C at 20°C/min, held for 1 minute, then raised to 240°C at 0.7°C/min and maintained for 15 minutes The detector and injector temperatures were set at 280°C and 230°C, respectively, with heptadecanoic acid methyl ester serving as the internal standard for quantification Product structures were confirmed via GC/MS analysis (Agilent 6890/5973i) using a temperature gradient from 70°C to 170°C at 20°C/min, holding for 1 minute, then from 170°C to 250°C at 5°C/min, and holding at 250°C for 15 minutes, with a helium flow rate of 0.67 mL/min and ionization energy at 70 eV Each sample was injected at a volume of 1 μL.
30 were identified by comparing their fragmentation patterns with those of reference compounds
FTIR analysis was conducted using a Nicolet Magna-IR 200 FTIR spectrometer from Thermo Fisher Scientific Korea Thin films of DOD and various PU samples were prepared with potassium bromide (KBr) pellets, maintaining a sample/KBr ratio of 1:100 Absorbance spectra were recorded over a wave number range of 4000–400 cm−1, following established procedures.
DSC thermograms were acquired using the DSC Q2000 from TA Instruments, with each 10-mg sample undergoing a three-step dynamic curing process The samples were initially heated from 30°C to 200°C at a rate of 20°C/min and held at 200°C for 5 minutes, followed by cooling to −40°C at −20°C/min and maintaining that temperature for another 5 minutes Finally, the samples were reheated to 200°C at 10°C/min Additionally, TGA experiments were performed using the Discovery TGA from TA Instruments to analyze the thermal decomposition behavior of polyurethanes (PUs), where all samples were heated from room temperature to 800°C at a rate of 10°C/min under a nitrogen gas flow of 60 mL/min.
Tensile properties of the polymer samples, measuring 50 mm × 10 mm, were evaluated using a Universal Testing Machine (UTM LR-30 K, Lloyd Instruments, Hampshire, UK) with a 1 kN load cell and a crosshead speed of 5 mm/min at room temperature, in accordance with ASTM D638 standards Each test was conducted with a minimum of five repetitions to ensure reliability of the results.
Results and Discussion
Vegetable oils, containing at least one unsaturated fatty acid in their triacylglyceride structure, offer a promising alternative to petroleum-based polyols The bacterium P aeruginosa KNU-2B effectively converts these unsaturated fatty acids into hydroxy fatty acids (HFAs) via triolein-induced lipase action The resulting bio-based polyols can perform comparably to synthetic polyols in producing polyurethanes (PUs) through isocyanate-catalyzed reactions This study aims to demonstrate the potential of using DOD for the synthesis of new bio-based PUs.
2.3.1 Production of DOD from Olive Oil
Olive oil was used in this work as a renewable and inexpensive model substrate for HFA production P aeruginosa KNU-2B grown on glucose for
24 h could effectively convert 1% olive oil to DOD after an additional 72 h of cultivation The cultures were treated with organic solvents to extract the
The analysis of the product using FTIR and GC/MS revealed key functional groups, with the FTIR spectrum showing hydroxyl groups at a transmittance of 3337 cm−1 and a carboxyl group at 1696 cm−1.
961 cm −1 confirmed that the product was composed of trans-unsaturation (Figure 2.1) The peaks identified by FTIR corroborate with those obtained previously on DOD [85]
The extract was methylated using diazomethane and derivatized with a TMSI and pyridine mixture before GC/MS analysis The GC profile revealed a dominant peak at a retention time of 15.503 minutes, accounting for 94% of the total area, excluding the internal standard The electron impact MS analysis of the methylated TMS derivatives displayed key fragments: m/z 215 for a fragment with one TMS group, m/z 343 for a fragment with two TMS groups and a double bond, and additional peaks at m/z 231 and m/z 359 indicating fragments of the methylated carboxyl group A peak at m/z 472 corresponds to the molecular mass of the TMS derivative of the methylated product The fragmentation patterns, which suggest a double bond at C8-9 and hydroxyl groups at C7 and C10, align with previous findings for DOD identification.
[15,17] The results obtained by FTIR and GC/MS confirm the bioconversion of olive oil to DOD by strain KNU-2B
This study utilized olive oil as a model substrate, which can be substituted with various cost-effective vegetable oils high in oleic acid, such as sunflower oil (82% oleic acid), canola oil (61%), and palm oil (36.6%) Recent findings also indicate that Philippine nut oil and palm oil can be effectively used for DOD production, highlighting the potential of affordable oil substrates for this application.
2.3.2 Synthesis of PUs from DOD (PU-DOD)
The potential for producing polyurethane (PU) from the bioconversion of olive oil-derived diol (DOD), which contains two hydroxyl groups, was investigated by reacting DOD with hexamethylene diisocyanate (HMDI) at varying NCO/OH ratios of 1.0, 1.2, 1.4, and 1.6 mol/mol Initial experiments with low NCO/OH ratios below 1.0 indicated that DOD could not effectively bond with HMDI, resulting in a liquid sample Conversely, at ratios exceeding 1.6 mol/mol, the resulting polymer exhibited increased rigidity and adherence to glass surfaces The Fourier-transform infrared (FTIR) spectrum of the PU-DOD synthesized from DOD and HMDI is illustrated in Figure 2.3.
Figure 2.1 FTIR spectra of the product produced from olive oil by P aeruginosa KNU-2B
Figure 2.2 EI mass spectra of the major product produced from olive oil by
The bands observed at 3410 cm −1 and 1537 cm −1 indicate the presence of the N–H group, corresponding to N–H stretching and bending vibrations Additionally, the amide carbonyl stretch around 1699 cm −1, along with the N–H bending vibration coupled with the C–N stretch at 1130 cm −1, suggests a reaction between the carboxyl group and –N=C=O The detection of a band at 1254 cm −1 for the C–O–C group further confirms the formation of polyurethane (PU), as neither DOD nor HMDI contains C–O–C bonds Furthermore, the lack of a band at 2270 cm −1 for the N=C=O group indicates that all HMDI has reacted with hydroxyl groups during the polymerization process.
The glass transition temperature (Tg) of PU–DOD was also measured
The glass transition temperature (Tg) was identified as the midpoint of the steepest slope in the inflection, defined by the onset and outset temperatures, as illustrated in Figure 2.4 and summarized in Table 2.1 Notably, the Tg of PU-DOD increased with a rising NCO/OH ratio, reaching 11.3°C at a 1.0 mol/mol ratio and escalating to 13.6°C at a 1.4 mol/mol ratio This increase in Tg correlates with the enhanced reaction between the NCO and OH groups as the NCO/OH ratio increases Importantly, no melting temperature peak was observed in the data.
The thermal gravimetric analysis (TGA) and differential thermal gravimetric (DTG) weight loss curves of PU-DOD with varying NCO/OH ratios illustrate that decomposition for all PU-DOD samples initiates at 200°C This process occurs in three distinct steps, with the first step taking place between 200°C and 320°C, primarily associated with the breakdown of unstable urethane bonds, including the dissociation of NCO and OH groups.
The thermal decomposition of primary and secondary amines involves three distinct steps The second step occurs between 320°C and 470°C, indicating the chain scission of polyols, while the third step begins at temperatures exceeding 470°C, attributed to the decomposition of fragments produced in the earlier phase Additionally, the temperature for 10% weight loss (T10%) rises with an increasing NCO/OH ratio, suggesting the potential formation of diisocyanate trimer compounds Moreover, the temperatures at which the maximum DTG peaks (Tmax) are observed for PU-DOD are slightly elevated at higher NCO/OH ratios.
Figure 2.3 FTIR spectra of polyurethane‐7,10‐dihydroxy‐8(E)‐octadecenoic acid (PU-DOD) with different NCO/OH ratios
NCO/OH 1.0 mol/mol NCO/OH 1.2 mol/mol NCO/OH 1.4 mol/mol NCO/OH 1.6 mol/mol
Figure 2.4 Differential scanning calorimetry (DSC) analysis of PU‐DOD with different NCO/OH ratios
Figure 2.5 (a) TGA and (b) derivative thermogravimetry (DTG) thermograms of PU-DOD with different NCO/OH ratios
NCO/OH 1.0 mol/mol NCO/OH 1.2 mol/mol NCO/OH 1.4 mol/mol NCO/OH 1.6 mol/mol
NCO/OH 1.2 mol/mol NCO/OH 1.4 mol/mol NCO/OH 1.6 mol/mol d(TGA weight)/dT (%/o C)
Table 2.1 Thermal and mechanical properties of PU‐DOD
TGA in Nitrogen (°C) Elongation at
T g : glass transition temperature, T 10% : temperature for 10% weight loss, T 50% : temperature for 50% weight loss,
T Max : temperature for maximum decomposition rates
The tensile properties of PU-DOD polymers, detailed in Table 2.1, reveal that increasing the NCO/OH ratio results in greater rigidity, which correlates with a decrease in elongation at break—from 85.6% at a 1.0 mol/mol ratio to 31.2% at a 1.6 mol/mol ratio This shift indicates a transition of PU-DOD from ductile to brittle behavior Notably, the maximum tensile strength of 37.9 MPa was achieved at an NCO/OH ratio of 1.4 mol/mol, highlighting the significance of optimizing this ratio in PU synthesis.
Increasing the NCO/OH ratio to 1.6 mol/mol resulted in a decrease in tensile strength due to trimer formation, leading to increased brittleness in the polymer As the trimer content rose, tensile strength improved while elongation at break diminished This brittleness may also stem from amide formation between the carboxyl group of DOD and the NCO group of HMDI, a reaction that can occur at room temperature and involves mixed carbamic-carboxylic anhydride intermediates Additionally, the CO2 produced during this reaction is derived from isocyanate, although the precise mechanism remains unreported.
Figure 2.6 Stress-strain curves of PU‐DOD with different NCO/OH ratios
2.3.3 Synthesis of PUs from DOD and PEG or PCLDO (PU‐DOD/PEG or PU-DOD/PCLDO)
Attempts were made to enhance the diversity of the PU‐DOD polymers
To achieve this, three kinds of PEG (Mn: 200 and 20,000) or PCLDO (Mn:
In the synthesis of new polyurethanes (PUs) labeled as PU-DOD/PEG200, PU-DOD/PEG20K, and PU-DOD/PCLDO, DOD was combined with HMDI The chemical structures of DOD, PEG, and PCLDO, along with the reaction schemes for the formation of these PUs, are illustrated in Figure 2.7.
The weight ratios of DOD to PEG or PCLDO were set at 2/1, 1/1, and 1/2 FTIR analysis of the samples confirmed the formation of polyurethanes (PUs), evidenced by the presence of N–H group peaks at 3400 cm −1 and 1550 cm −1, a C=O group peak around 1700 cm −1, and a C–O–C group peak at 1250 cm −1 Notably, the absence of a stretching vibration band at 2270 cm −1 indicated the lack of the N=C=O group, consistent with typical FTIR spectra for PUs.
The TGA curves of all polymeric samples exhibited an initial slow degradation followed by a rapid decomposition process This degradation occurred in three distinct steps, similar to PU-DOD: the first step, occurring between 230°C and 350°C, involved the breakdown of unstable urethane bonds; the second step, from 350°C to 470°C, was associated with the degradation of polyol segments; and the final step, above 470°C, marked the complete decomposition of the sample.
Figure 2.7 Reaction schemes for the synthesis of PU‐DOD/ polycaprolactone diol (PCLDO) and PU‐DOD/ polyethylene glycol (PEG)
Figure 2.8 FTIR spectra of (a) PU-DOD/PEG200, (b) PU-DOD/PEG20K, and
(c) PU-DOD/PCLDO 2/1, 1/1, and 1/2 are the weight ratios of DOD to PEG or PCLDO The NCO/OH 1.4 mol/mol was used for synthesizing of these PUs
DOD/PEG200 1/1 DOD/PEG200 1/2 DOD/PEG200 2/1
DOD/PCLDO 1/1 DOD/PCLDO 1/2 DOD/PCLDO 2/1
Figure 2.9 illustrates the weight loss of various polyurethanes (PUs) with three hard segment contents as temperature increases The initial degradation step for PU-DOD/PCLDO and PU-DOD/PEG occurs within a range of 5–15°C, highlighting the influence of hard segment structure on degradation Additionally, alternative chain extenders such as 1,3-propanediol and 1,4-butanediol show promise as potential bio-based PU candidates Notably, the T10% of PU-DOD/PEG and PU-DOD/PCLDO rises with a higher fraction of PEG or PCLDO, with PU-DOD/PEG20K exhibiting the highest T10%.
(290°C) and PU-DOD/PCLDO had the lowest T10% (273°C) Thus, it can be concluded that PU‐DOD/PEG20K possesses higher thermal stability than the others [91]
Conclusions
A bio-based polyol, DOD, was synthesized from olive oil using the bacterium P aeruginosa KNU-2B Polyurethanes (PUs) created from DOD, with an NCO/OH ratio of 1.4 mol/mol, exhibited amorphous characteristics, achieving an elongation at break of 59.3% and a tensile strength of 37.9 MPa The incorporation of DOD with PCLDO or PEG in varying weight ratios further diversified the properties of PU-DOD This research highlights the effective bio-conversion of natural oils into high-functioning additives (HFAs), which serve as essential building blocks for the synthesis of bio-based polyurethanes.
Figure 2.9 TGA and derivative thermogravimetry (DTG) thermograms of (a)
The synthesis of polyurethane (PU) composites, specifically PU-DOD/PEG200, PU-DOD/PEG20K, and PU-DOD/PCLDO, was conducted using weight ratios of DOD to PEG or PCLDO at 2:1, 1:1, and 1:2, respectively This process utilized an NCO/OH ratio of 1.4 mol/mol to achieve the desired material properties.
DOD/PEG200 1/1 DOD/PEG200 1/2 DOD/PEG200 2/1
DOD/PEG200 1/1 DOD/PEG200 1/2 DOD/PEG200 2/1
DOD/PEG20K 1/1 DOD/PEG20K 1/2 DOD/PEG20K 2/1
DOD/PEG20K 1/1 DOD/PEG20K 1/2 DOD/PEG20K 2/1
DOD/PCLDO 1/1 DOD/PCLDO 1/2 DOD/PCLDO 2/1
DOD/PCLDO 1/1 DOD/PCLDO 1/2 DOD/PCLDO 2/1
Figure 2.10 Tensile properties of (a) PU-DOD/PEG200, (b) PU-
DOD/PEG20K, and (c) PU-DOD/PCLDO These PUs were synthesized using an NCO/OH ratio of 1.4 mol/mol
Table 2.2 Thermal and mechanical properties of PUs from DOD with PEG or PCLDO
Weight Ratio of DOD to PEG or
(first/second) PU‐DOD/PEG200
T g : glass transition temperature, T 10% : temperature for 10% weight loss, T 50% : temperature for 50% weight loss,
T Max : temperature for maximum decomposition rates
Microbial Conversion of Castor Oil to 7,10,12-trihydroxy- 8(E)-octadecenoic acid and Its Application to Bio-Based Polyurethane
Introduction
Hydroxy fatty acids (HFAs) are unique carboxylic acids characterized by one or more -OH groups, offering enhanced reactivity, stability, and viscosity compared to standard fatty acids Their distinct properties make HFAs valuable in various industries, including lubricants, waxes, cosmetics, and coatings Additionally, HFAs have significant medical applications; for example, 9,12,13-trihydroxy-octadecenoic acid serves as a vaccine adjuvant, while 15-hydroxyeicosatetraenoic acid exhibits anticancer and antifungal properties Furthermore, 14,21-dihydroxy-docosahexanoic acid is effective in treating diabetic wounds Due to their limited natural occurrence in plants, recent research has focused on bioconverting unsaturated carboxylic acids into HFAs, utilizing raw materials like oleic, linoleic, linolenic, and ricinoleic acids for microbial production of hydroxy fatty acids.
Vegetable oils, such as those derived from sunflower, palm, rapeseed, cotton, olives, soybeans, and coconuts, serve as effective substrates for the production of hydroxy fatty acids (HFAs) due to their unique chemical structure, which includes long carbon chains with carboxylic acids like linoleic, oleic, linolenic, and ricinoleic acids in the form of triglycerides These oils are advantageous because they are cost-effective, universally convenient, inherently biodegradable, and exhibit low ecotoxicity towards humans Additionally, they can be utilized in a wide range of applications, including the preparation of polymers and composites for paints, adhesives, coatings, and biomedical uses.
Pseudomonas aeruginosa PR3 is a notable bacterial strain capable of synthesizing mono-, di-, and trihydroxy fatty acids from various unsaturated fatty acids, particularly utilizing ricinoleic acid to produce 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) The presence of three hydroxyl groups in TOD's molecular structure allows it to function as a polyol, making it suitable for reactions with isocyanates in polyurethane (PU) synthesis Castor oil, rich in ricinoleic acid, serves as an effective raw material for TOD production Additionally, a new strain, P aeruginosa KNU-2B, has been isolated from PR3, demonstrating similar capabilities.
55 to convert olive oil into 7,10-dihydroxy-8(E)-octadecenoic acid like PR3 strain Therefore, this strain can be used to produce TOD from castor oil
Polyurethane (PU) is a versatile class of polymeric materials characterized by urethane bonds, which have garnered significant attention for their unique mechanical, chemical, and biological properties Due to these characteristics, PUs are increasingly explored for various commercial applications, including medical scaffolds, coatings, foams, and composites They can be synthesized in several forms, such as fibers, films, castables, thermoplastics, and foams The traditional synthesis of PUs involves the reaction between isocyanates, which contain at least two NCO groups, and polyols with two or more hydroxyl groups However, the use of isocyanates is limited due to their toxicity, while most polyols used in synthesis are primarily derived from petroleum sources.
[67] Therefore, producing polyols and isocyanates by means of renewable materials is expected to draw substantial attention of several researchers all over the world [76–80]
In this study, castor oil was converted into triol (TOD) using Pseudomonas aeruginosa KNU-2B, which was then utilized to synthesize new polyurethanes (PUs) with hexamethylene diisocyanate (HMDI) at varying ratios TOD was also blended with polycaprolactone diol (PCLDO) and polyethylene glycol (PEG) at different proportions before reacting with HMDI to create PUs The structure of TOD was confirmed using Fourier transform-infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), and gas chromatography/mass spectrometry (GC/MS) The characterization of the synthesized PUs was performed through thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC), and FTIR, while their tensile properties were evaluated using a universal testing machine (UTM).
Materials and Methods
Castor oil (having 85-95% ricinoleic acid), pyridine, 1- (trimethylsilyl)imidazole (TMSI), dibutyltin dilaurate (DBTDL), PCLDO (Mn:
In this study, all chemicals utilized were of analytical grade, including PEG (Mn: 200) and PEG (Mn: 20,000) sourced from Sigma-Aldrich and Fluka, respectively, along with HMDI obtained from Daejung.
Pseudomonas aeruginosa KNU-2B was successfully cultivated to produce TOD from castor oil using a standard medium composed of glucose, yeast extract, K2HPO4, (NH4)2HPO4, MgSO4, MnSO4, and FeSO4 The medium was adjusted to a pH of 8 with diluted phosphoric acid (20% v/v) The aerobic culture was maintained in a 500 mL Erlenmeyer baffled flask containing 100 mL of the standard medium.
3.2.3 Production of TOD from castor oil
To produce TOD, 1.0 mL of castor oil (1% v/v) was added to the culture after 24 hours and shaken for four days Following cultivation, the media was acidified to pH 2.0 with 6N HCl, and extraction was performed twice using ethyl acetate The solvent was subsequently removed using a rotary evaporator, and the extract was washed with n-hexane before recrystallization with ethyl acetate The final yield of purified TOD from castor oil was 0.014 g/g of substrate, with a purity of 95% as determined by gas chromatography (GC).
3.2.4 Preparation of PUs from TOD
To prepare the solution, one gram of TOD was dissolved in 3 mL of tetrahydrofuran and combined with 1% w/v DBTDL at room temperature in a glass container, mixing for 5 minutes Following this, HMDI was added to the container and stirred thoroughly for an additional 10 minutes.
The molar ratio of NCO groups to OH groups ranged from 1.4 to 2.0 mol/mol Subsequently, the mixture was placed on a pre-labeled glass plate and heated in a vacuum oven at 80°C for 5 hours.
3.2.5 Preparation of blended PUs: PU-TOD/PEG and PU-TOD/PCLDO
One gram of polyol, either PEG or PCLDO mixed with TOD, was dissolved in 3 mL of tetrahydrofuran and combined with 1% w/v DBTDL at room temperature for 5 minutes Different weight ratios of TOD to PCLDO or PEG were examined HMDI was then added to the mixture at an NCO/OH ratio of 2.0 mol/mol and stirred for an additional 10 minutes The resulting blend was processed according to the previously outlined method.
The samples underwent esterification with diazomethane at room temperature for 10 minutes, followed by a derivatization process using a TMSI and pyridine mixture (1:4, v/v) for a minimum of 45 minutes The derivatized samples were analyzed using a GC (Agilent 6890N) equipped with an HP-5 column (30 m × 320 µm × 0.25 µm) The chemical structure of the resulting product was confirmed through GC/MS analysis conducted on an Agilent 6890/5973i, employing a temperature gradient from 70°C to 170°C at a rate of 20°C.
The experimental procedure involved heating at a rate of 59°C per minute, maintaining a temperature of 170°C for 1 minute, followed by a gradual increase to 250°C at a rate of 5°C per minute, and holding at 250°C for 15 minutes The carrier gas used was helium, maintained at a split ratio of 30:1, with a column flow rate of 33.6 mL per minute.
The 1H-NMR analysis of the sample was conducted using a Bruker Avance III 400MHz spectrometer in Billerica, Massachusetts, with deuterated methanol serving as the solvent Additionally, FTIR analysis was performed utilizing the Nicolet Magna model.
The IR 200 FTIR spectrometer was utilized to analyze thin films of TOD and various polymer samples, which were prepared by mixing with potassium bromide (KBr) at a sample/KBr ratio of 1:100 Absorbance spectra were collected over a wave number range of 4000 to 400 cm⁻¹.
About 10 mg of each sample was dynamically cured in three stages At first, the individual sample was heated from 30°C to 250°C at 20°C per min Subsequently they were kept at 250°C for 5 min, cooled to -50°C with a cooling rate of 20°C per min, and kept at -50°C for 5 min Eventually, they were heated again to 250°C with a heating rate of 10°C per min DSC Q2000 (TA Instruments, New Castle, DE, USA) was used to collect DSC thermograms TGA experiment was performed using Discovery TGA (TA Instruments, New Castle, DE, USA) to investigate the thermal decomposition
60 behavior of the polymers The polymer samples were kept for heating from room temperature to 800°C at a rate of 10°C per min under N2 gas flowing at
All polymer samples were weighed (W1) after drying under vacuum for
24 h Then, the samples were extracted with tetrahydrofuran for 24 h, dried, and weighed (W2) The gel content (%) was determined as W2/W1 × 100
After the synthesis, PU samples were extracted from the glass plate and cut into dimensions of 5 cm × 1 cm for tensile property evaluation The thickness of all samples varied between 100 and 200 μm Tensile testing was conducted using a Universal Testing Machine (UTM, LR-30 K, Lloyd Instruments, Hampshire, UK) in accordance with ASTM D638 standards, employing a 1 kN load cell and a crosshead speed of 5 mm per minute at room temperature Each experiment was repeated a minimum of five times to ensure accuracy.
Results and discussion
Certain hydroxy fatty acids (HFAs) with three hydroxyl groups have been synthesized, including 12,13,17-trihydroxy-9(Z)-octadecenoic acid by B megaterium ALA2 and 9,10,13-trihydroxy-11(E)-octadecenoic acid by P aeruginosa PR3, utilizing linoleic acid as a substrate Additionally, P aeruginosa PR3 has been shown to produce TOD through the direct conversion of ricinoleic acid Furthermore, P aeruginosa KNU-2B, a new strain isolated from PR3, exhibits similar capabilities.
The PR3, KNU-2B strain has demonstrated the ability to convert castor oil into TOD for the first time, highlighting castor oil's potential as a source due to its high ricinoleic acid content (85-95%) The process involves inducing lipase activity to hydrolyze castor oil, releasing ricinoleic acid, which is subsequently transformed into TOD by KNU-2B This resulting TOD, characterized by three hydroxyl groups, serves as a valuable polyol source for the synthesis of polyurethanes (PUs).
Figure 3.1 Bio-conversion of castor oil to TOD by P aeruginosa KNU-2B
3.3.1 Production of TOD from castor oil
GC analysis identified a significant peak at a retention time of 20.5 minutes, accounting for 95% of the total peak area Additionally, FTIR analysis confirmed the presence of -OH groups, as indicated by specific transmittance patterns.
3337 cm -1 , and -COOH groups at 1706 cm -1 The transmittance at 1061 cm -1 established that the material consisted of trans-unsaturation
The MS spectrum of the methylated TMS derivative of the major product reveals key fragments: the peak at m/z 187 indicates a fragment with one TMS group at C12, while m/z 231 corresponds to a fragment with a carbonyl group and one TMS group at C7 The peak at m/z 359 represents a fragment containing both a carbonyl group and TMS groups at C7 and C10 Additionally, the peak at m/z 431 signifies a larger fragment with three TMS groups and a double bond The elimination of one TMS group from m/z 431, 359, and 329 results in fragments at m/z 341, 269, and 239, respectively, suggesting the presence of a double bond between C8-9 and three -OH groups at C7, C10, and C12 Notably, the MS spectrum closely resembles that of TOD derived from ricinoleic acid.
Figure 3.2 FTIR spectra of the major product produced from castor oil by P aeruginosa KNU-2B
Figure 3.3 GC/MS of the major product produced from castor oil by P aeruginosa KNU-2B
The 1 H-NMR data of TOD revealed a peak at 5.65 ppm corresponding to an olefinic proton, while the protons nearest the carbonyl group have a peak at 2.27 ppm (Figure 3.4) Three tertiary protons of carbons that contain hydroxyl groups have peaks at 4.30, 4.01, and 3.78 ppm, with first two being near the double bond All methylene groups appear from 1.31 to 1.64 ppm and the methyl group has a peak at 0.90 ppm The NMR spectrum is identical to the spectrum of TOD reported by Kuo et al [20] In addition, the coupling constant of the olefinic proton was 15.6 Hz, representing the trans- stereochemistry of the double bond
The cultivation of strain KNU-2B from castor oil primarily produces TOD, confirming that castor oil can effectively substitute ricinoleic acid as a raw material for TOD synthesis.
Figure 3.4 1 H-NMR spectra of product produced from castor oil by P aeruginosa KNU-2B
3.3.2 Synthesis of PUs from TOD (PU-TOD)
Because TOD produced from castor oil by the strain KNU-2B has three -OH groups, it could be considered as a polyol unit for the synthesis of
In this study, PU-TOD was synthesized by reacting TOD with HMDI, using NCO to OH ratios ranging from 1.4 to 2.0 mol/mol Ratios below 1.4 mol/mol resulted in ineffective reactions, leaving the product in a liquid state Residual ricinoleic acid, possessing a single OH group, can hinder polymer network propagation by causing chain termination, potentially contributing to the liquid state of the polymer Conversely, ratios exceeding 2.0 mol/mol led to excessive rigidity, making it impossible to remove the polymer from the glass plate FTIR analysis served as an efficient method to assess the formation of PU-TOD, with spectra indicating distinct bands corresponding to various NCO/OH ratios.
The analysis revealed key absorption bands at 3385 cm -1 and 1547 cm -1, which correspond to N-H stretching and bending, respectively, while a band near 1700 cm -1 indicated the presence of the C=O group Additionally, the detection of a band at 1250 cm -1 confirmed the synthesis of polyurethane (PU) since neither TOD nor HMDI contained the C-O-C bond The absence of the 2270 cm -1 band associated with the N=C=O group indicated nearly complete consumption of HMDI Furthermore, the gel content of the PU-TOD samples exceeded 93%, providing further evidence of successful formation.
As the NCO/OH ratio in the cross-linked polymeric network of TOD and HMDI increased to 2.0 mol/mol, the gel content of the polymer sample rose to 99.4%, resulting in a harder polymer.
The glass transition temperature (Tg) of PU-TOD was analyzed using Differential Scanning Calorimetry (DSC), with results summarized in Table 3.2 Tg was determined by the mid-point temperature of the inflection point, defined by the onset and outset temperatures Notably, the Tg of PU-TOD increased as the NCO/OH ratio rose, starting at 11.3°C for a 1.4 mol/mol ratio and reaching 22.5°C at a 2.0 mol/mol ratio The presence of three hydroxyl groups in TOD facilitates the formation of a cross-linked structure with HMDI during polymerization, resulting in higher cross-linking density and consequently elevated Tg with increasing NCO/OH ratios.
T g Compared to PUs based on 7,10-dihydroxy-8(E)-octadecenoic acid (PU-DOD) [93], T g of PU-TOD was higher due to the formation of cross-linking that was absent for PU-DOD
Table 3.1 Gel content of PU-TOD, PU-TOD/PEG and PU-TOD/PCLDO
NCO/OH ratio (mol/mol)
Weight ratio of TOD to PEG or PCLDO
Figure 3.5 FTIR spectra of PU-TOD with different NCO/OH ratios and PU-
CO (synthesized from castor oil and HMDI with an NCO/OH of 2.0 mol/mol)
Figure 3.6 DSC analysis of PU-TOD with different NCO/OH ratios
NCO/OH 1.8 mol/mol NCO/OH 1.6 mol/mol
The thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) results for PU-TOD with varying NCO/OH ratios, as illustrated in Figure 3.7, indicate that decomposition begins at 200°C and occurs in three distinct stages The first stage, occurring between 200°C and 300°C, involves the breakdown of unstable urethane bonds, resulting in the formation of primary and secondary amines The second stage, from 300°C to 470°C, is characterized by the cleavage of polyol chains, while the final degradation stage follows thereafter.
At 470°C, the decomposition of fragments occurs during the second stage, indicating a significant thermal transition An increase in the NCO/OH ratio leads to a higher temperature for 10% weight loss (T 10%), attributed to the formation of trimeric diisocyanate compounds Consequently, the thermal stability of PU-TOD improves in the initial phase with a rising NCO/OH ratio.
The tensile properties of PU-TOD reveal a significant relationship between hardness and the NCO/OH ratio, as detailed in Table 3.2 and Figure 3.8 Specifically, as the NCO/OH ratio increased, the hardness of PU-TOD rose, leading to a decrease in elongation at break from 26.4% at a ratio of 1.4 mol/mol to 8.16% at 2.0 mol/mol, indicating a transition from ductile to brittle behavior Conversely, the tensile strength showed an upward trend with increasing NCO/OH ratio, peaking at 45.4 MPa when the ratio reached 2.0 mol/mol.
Figure 3.7 (a) TGA and (b) derivative thermogravimetry (DTG) thermograms of PU-TOD with different NCO/OH ratios
W ei ght pe rc ent age (%)
NCO/OH 1.4 mol/mol NCO/OH 1.6 mol/mol NCO/OH 1.8 mol/mol NCO/OH 2.0 mol/mol
NCO/OH 1.4 mol/mol NCO/OH 1.6 mol/mol NCO/OH 1.8 mol/mol NCO/OH 2.0 mol/mol
The cross-linking density of polymers increases with the ratio of TOD to HMDI, resulting in harder materials and enhanced tensile strength Additionally, the presence of trimer affects the tensile properties of PU-TOD, as higher NCO/OH ratios lead to increased trimer content, making the polymer more brittle and reducing its elongation at break while enhancing tensile strength In contrast, PU synthesized from castor oil and HMDI (PU-CO), which contains 85 to 95% ricinoleic acid, exhibits significantly lower tensile strength (0.57 MPa) compared to PU-TOD, indicating inferior tensile properties.
The analysis revealed that CO exhibited a band for the N=C=O group at 2302 cm-1, indicating that HMDI did not react with castor oil Consequently, the degree of cross-linking in PU-CO was lower than in PU-TOD, resulting in reduced tensile strength As HMDI is a linear aliphatic diisocyanate, PUs synthesized from it generally exhibit low tensile strength However, PU-TOD demonstrated superior tensile strength compared to other PUs derived from vegetable oil-based polyols with HMDI, even surpassing some PUs made from aromatic diisocyanates, highlighting the distinct advantage of PU-TOD.
Figure 3.8 Tensile properties of PU-TOD with different NCO/OH ratios and
PU-CO (synthesized from castor oil and HMDI with an NCO/OH of 2.0 mol/mol)
E longa ti on a t bre ak (%)
Table 3.2 Thermal and mechanical properties of PU-TOD
T g : glass transition temperature, T 10% : temperature for 10% weight loss, T 50% : temperature for 50% weight loss,
T Max : temperature for maximum decomposition rates
3.3.3 Synthesis of PUs from TOD and its blend (PU-TOD/PEG or PU- TOD/PCLDO)
Conclusions
A green polyol, TOD, was effectively produced from castor oil using Pseudomonas aeruginosa KNU-2B, demonstrating significant potential in the polyurethane industry Polyurethanes (PUs) created from TOD and HMDI, with an NCO/OH ratio of 2.0 mol/mol, exhibited impressive mechanical properties, including an elongation at break of 8.16% and a tensile strength of 45.4 MPa Modifications using PEG or PCLDO at various weight ratios enhanced the elongation at break of PU-TOD, although the tensile strength decreased Thermogravimetric analysis revealed that PU-TOD/PEG200 exhibited superior thermal stability compared to other polymers This research highlights the promising applications of green polyols like TOD in the development of advanced polyurethane materials.
Figure 3.12 Tensile properties of (a) PU-TOD/PEG200, (b) PU- TOD/PEG20K, and (c) PU-TOD/PCLDO These PUs were synthesized using an NCO/OH ratio of 2.0 mol/mol
Weight ratio of TOD to PEG200
T ens ile s tr ength (M Pa)
Weight ratio of TOD to PEG20K
T ens ile s tr ength (M Pa)
Weight ratio of TOD to PCLDO
T ens ile s tr ength (M Pa)
Table 3.3 Thermal and mechanical properties of PU-TOD/PEG and PU-TOD/PCLDO
T 10% : temperature for 10% weight loss, T 50% : temperature for 50% weight loss,
T Max : temperature for maximum decomposition rates These PUs were synthesized using an NCO/OH ratio of 2.0 mol/mol
Weight ratio of TOD to
(first/second) PU-TOD/PEG200