Polyhydroxyalkanoates bioproduction from bench to industry: Thirty years of development towards sustainability.

The pursuit of carbon neutrality goals has sparked considerable interest in expanding bioplastics production from microbial cell factories. One prominent class of bioplastics, polyhydroxyalkanoates (PHA), is generated by specific microorganisms, serving as carbon and energy storage materials. To begin with, a native PHA producer, Cupriavidus necator (formerly Ralstonia eutropha) is extensively studied, covering essential topics such as carbon source selection, cultivation techniques, and accumulation enhancement strategies. Recently, various hosts including archaea, bacteria, cyanobacteria, yeast, and plants have been explored, stretching the limit of microbial PHA production. This review provides a comprehensive overview of current advancements in PHA bioproduction, spanning from the native to diversified cell factories. Recovery and purification techniques are discussed, and the current status of industrial applications is assessed as a critical milestone for startups. Ultimately, it concludes by addressing contemporary challenges and future prospects, offering insights into the path towards reduced carbon emissions and sustainable development goals.

Fabrication of bio-based polyamide 56 and antibacterial nanofiber membrane from cadaverine.

A green bioprocess for the fabrication of nanofiber membranes from the biomaterial polyamide 56 (PA56) via electrospinning was proposed. Cadaverine, as the precursor of PA56, was first produced from recombinant Escherichia coli using the whole-cell biotransformation of lysine. PA56 was then fabricated by mixing adipic acid with purified cadaverine obtained from solvent extraction and distillation. The thermal properties of the fabricated PA56 are as follows: a melting point of 250 °C, a crystallization point of 220 °C, and a degradation temperature of 410 °C. A PA56 nanofiber membrane (PAM) was further prepared via electrospinning. Dyed membranes (P-Dye) were obtained by the reaction of Reactive Red 141 dye with the amino group of PAM. Poly-(hexamethylene biguanide) (PHMB) was attached to the P-Dye to create P-Dye-PHMB. On the other hand, PAM with alginate, used to facilitate PHMB attachment (P-Alg-PHMB), was compared with P-Dye-PHMB in terms of antibacterial activity against pathogenic strains of E. coli and Pseudomonas putida. P-Alg-PHMB showed excellent antibacterial efficiency for E. coli (97%) and P. putida (100%). The proposed bioprocess can be used to fabricate novel membranes for biomedical applications and functional textiles.

Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration.

The recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) was covalently bonded on novel polyacrylonitrile (PAN) and polyethylene terephthalate (PET) nanofibers (PAN-PET-PAN donated as AEA) that was first fabricated by electrospinning. The resulting composite materials further crosslinked by the glutaraldehyde, which significantly increased thermostability up to 89.8% and 18.0% after heating at 60 °C for 1 h for immobilized crude and pure SyCA, respectively. After four repetitive attempts in the demonstration of CO2 sequestration, immobilized crude and pure SyCA on AEA also effectively improved the total CaCO3 yields to be 5.8 folds and 2.2 folds compared to free enzyme. Furthermore, the endurance of immobilized crude was investigated on flue gasses, which was retained its activity up to 57% on 50 mM NOx and 61% on 50 mM SOx presence. This is the first report of immobilized thermophilic SyCA on a novel nanofiber at the reusability, durability, sequestration of carbon dioxide, tolerant to sulfur oxides (SOx) and nitrogen oxides (NOx) toxic gases and to prevent air pollution.

Antibacterial efficacy of chitosan- and poly(hexamethylene biguanide)-immobilized nanofiber membrane.

In this study, polyacrylonitrile (PAN) nanofiber membrane was prepared by an electrospinning technique. After alkaline hydrolysis, the ion-exchange nanofiber membrane (P-COOH) was grafted with chitosan molecules to form a chitosan-modified nanofiber membrane (P-COOH-CS). Poly(hexamethylene biguanide) (PHMB) was then covalently immobilized on P-COOH and P-COOH-CS to form P-COOH-PHMB and P-COOH-CS-PHMB, respectively. The nanofiber membranes were subjected to various surface analyses as well as to the evaluations of antibacterial activity against Escherichia coli. The optimal modification conditions for P-COOH-CS-PHMB were attained by water-soluble chitosan at 50 kDa of molecular weight, coupling pH at 7, and 0.05% (w/w) of PHMB. Within 10 min of treatment, the antibacterial rate was close to 100%. Under the similar conditions of antibacterial treatment, the P-COOH-CS-PHMB exhibited a better antibacterial efficacy than the P-COOH-PHMB. When the number of bacterial cells was increased by 2000 folds, both types of nanofiber membranes still maintained the antibacterial rate close to 100%. After five cycles of repeated antibacterial treatment, the antibacterial efficacy of P-COOH-PHMB was 96%, which was higher than that of P-COOH-CS-PHMB (83%). The experimental results revealed that the PHMB-modified nanofiber membranes can be suitably applied in water treatment such as water disinfection and biofouling control.

Exploring fermentation strategies for enhanced lactic acid production with polyvinyl alcohol-immobilized Lactobacillus plantarum 23 using microalgae as feedstock.

Lactic acid (LA) fermentation was conducted with suspended and immobilized cells of an isolated Lactobacillus plantarum 23 strain using various fermentation strategies. Glucose and an alternative, relatively inexpensive carbon source - the hydrolysate of microalga Chlorella vulgaris ESP-31, were used as the carbon source. Batch fermentation using immobilized cells of L. plantarum 23 could enhance LA titer and yield by 43% and 39%, respectively, when compared with the suspended culture. Fed-batch culture integrated with in situ LA removal via ion exchange raised LA productivity by 72% by overcoming product inhibition. The highest LA productivity from glucose with PVA immobilized cells was 14.22 g/L/h, achieved under continuous operation at 50% w/v loading of immobilized beads and hydraulic retention time (HRT) of 2 h. PVA immobilized L. plantarum 23 could also use microalgal hydrolysate as the renewable carbon source, and the highest LA productivity was 9.93 g/L/h under continuous fermentation at 4 h HRT.

Effective purification of lysozyme from chicken egg white by tris(hydroxymethyl)aminomethane affinity nanofiber membrane.

Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.

Enhancement of C-phycocyanin purity using negative chromatography with chitosan-modified nanofiber membrane.

In this work, a chitosan-modified nanofiber membrane was fabricated and used to examine the permeation characteristics of C-phycocyanin (CPC) obtained from Spirulina platensis. The effects of NaCl concentration (0.1-1.0 M), chitosan coupling pH (6-8), chitosan coupling concentration (0.1-3.0%), algal solution pH (6-8), algal mass concentration (0.1-1.0% dw/v), and membrane flux (4.08 × 10-2-2.04 × 10-1 mL/min·cm2) on the penetration performance of the membrane for CPC were investigated. The results show that the order of binding selectivity of the membrane for these proteins is contaminating proteins (TP) > allophycocyanin (APC) > CPC. TP and APC molecules were more easily adsorbed by the chitosan-modified membrane, and the CPC molecules most easily penetrated the membrane without being adsorbed, enhancing CPC purity. The purification factor and total mass flux were 3.3 fold and 66%, respectively, in a single step.

Antibacterial activity of quaternized chitosan modified nanofiber membrane.

The electrospinning PAN nanofiber membrane (P-CN) was hydrolysed to convert carboxylic groups as reaction sites and covalently graft chitosan molecule. The chitosan derivatives with quaternary ammonium groups exerted greater efficiency against bacteria as compared to pure chitosan. Hence, the chitosan modified membrane (P-CS), can be functionalized with quaternary amine (i.e., glycidyl trimethyl ammonium chloride, GTMAC) to form quaternized chitosan nanofiber membrane (designated as P-HTCC) under various conditions (acidic, neutral, and alkaline). N-quaternized derivatives of chitosan modified membrane (N-HTCC) showed 72% and 60% degree of quaternization (DQ) under acidic and neutral conditions, respectively. Under alkaline condition, additional quaternization of N, O-HTCC via its amino and hydroxyl groups, has improved up to 90% DQ of the chitosan. The antibacterial activity of the quaternized chitosan modified membrane prepared from acetic acid medium is stronger than that prepared from water and alkaline media. Also, antibacterial activity of quaternized chitosan is stronger than chitosan modified membrane against E. coli. The microbiological assessments showed that the water-stable P-HTCC nanofiber membrane under modification in acidic medium exerted antibacterial activity up to 99.95% against E. coli. Therefore, the P-HTCC membrane exhibited high potential to be integrated into microfiltration membrane to effectively disinfect E. coli.

Purification of lysozyme from chicken egg white using nanofiber membrane immobilized with Reactive Orange 4 dye.

Nanofiber membrane chromatography integrates liquid membrane chromatography and nanofiber filtration into a single-step purification process. Nanofiber membrane can be functionalised with affinity ligands for promoting binding specificity of membrane. Dye molecules are a good affinity ligand for nanofiber membrane due to their low cost and high binding affinity. In this study, a dye-affinity nanofiber membrane (P-Chitosan-Dye membrane) was prepared by using polyacrylonitrile nanofiber membrane modified with chitosan molecules and immobilized with dye molecules. Reactive Orange 4, commercially known as Procion Orange MX2R, was found to be the best dye ligand for membrane chromatography. The binding capacity of P-Chitosan-Dye membrane for lysozyme was investigated under different operating conditions in batch mode. Furthermore, desorption of lysozyme using the P-Chitosan-Dye membrane was evaluated systematically. The recovery percentage of lysozyme was found to be ~100%. The optimal conditions obtained from batch-mode study were adopted to develop a purification process to separate lysozyme from chicken egg white. The process was operated continuously using the membrane chromatography and the characteristic of the breakthrough curve was evaluated. At a lower flow rate (i.e., 0.1 mL/min), the total recovery of lysozyme and purification factor of lysozyme were 98.59% and 56.89 folds, respectively.

High-level production of a thermoacidophilic beta-glucosidase from Penicillium citrinum YS40-5 by solid-state fermentation with rice bran.

A high yield of beta-glucosidase (EC 3.2.1.21) of 159.1 U/g-solid activity on 4-nitrophenyl beta-d-glucopyranoside (pNPG) was achieved by rice bran-based solid-state fermentation (SSF) of the recently characterized fungus Penicillium citrinum YS40-5. The enzyme was both thermophilic and acidophilic at the optimized temperature and pH of 70 degrees C and 5.0, respectively. Over 95% of the original beta-glucosidase activity was maintained after a prolonged storage at ambient temperature for 4 weeks. The kinetic parameters V(max), K(m) and K(I) were 85.93 U/mg, 1.2 mM and 17.59 mM with pNPG, and 72.49 U/mg, 32.17 mM and 8.29 mM with cellobiose, respectively. The protein band with beta-glucosidase activity was characterized by native PAGE followed by MUG-zymogram analysis, and its identity confirmed by nanoLC-MS/MS. A 3.43-fold synergistic effect by combining this beta-glucosidase with Trichoderma reesei cellulases was observed, indicating this enzyme could potentially be used for improving the efficiency of cellulosic bioconversion.