This research explicitly investigates the utilization of Chlorella Vulgaris sp. microalgae as a renewable source for lipid production, focusing on its application in bioplastic manufacturing. This study employed the supercritical fluid extraction technique employing supercritical CO2 (sCO2) as a green technology to selectively extract and produce PHA's precursor utilizing CO2 solvent as a cleaner solvent compared to conventional extraction method. The study assessed the effects of three extraction parameters, namely temperature (40-60 °C), pressure (15-35 MPa), and solvent flow rate (4-8 ml/min). The pressure, flowrate, and temperature were found to be the most significant parameters affecting the sCO2 extraction. Through Taguchi optimization, the optimal parameters were determined as 60 °C, 35 MPa, and 4 ml/min with the highest lipid yield of 46.74 wt%; above-average findings were reported. Furthermore, the pretreatment process involved significant effects such as crumpled and exhaustive structure, facilitating the efficient extraction of total lipids from the microalgae matrix. This study investigated the microstructure of microalgae biomatrix before and after extraction using scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). Fourier-transform infrared spectroscopy (FTIR) was utilized to assess the potential of the extracted material as a precursor for biodegradable plastic production, with a focus on reduced heavy metal content through inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis. The lipid extracted from Chlorella Vulgaris sp. microalgae was analysed using gas chromatography-mass spectrometry (GC-MS), identifying key constituents, including oleic acid (C18H34O2), n-Hexadecanoic acid (C16H32O2), and octadecanoic acid (C18H36O2), essential for polyhydroxyalkanoate (PHA) formation.
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microorganisms, under unbalanced growth conditions, as a carbon storage compound. PHAs are composed of various monomers such as 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Silk fibroin (SF) derived from Bombyx mori cocoons, is a widely studied protein polymer commonly used for biomaterial applications. In this study, non-woven electrospun films comprising a copolymer of 3HB and 3HHx [P(3HB-co-3HHx)], SF and their blends were prepared by electrospinning technique. The growth and osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were studied using different types of fabricated electrospun films. The differentiation study revealed that electrospun P(3HB-co-3HHx)/SF film supports the differentiation of hUC-MSCs into the osteogenic lineage, confirmed by histological analysis using Alizarin Red staining, energy dispersive X-ray (EDX) and quantitative real-time PCR analysis (qPCR). Electrospun P(3HB-co-3HHx)/SF film up-regulated the expression of osteogenic marker genes, alkaline phosphatase (ALP) and osteocalcin (OCN), by 1.6-fold and 2.8-fold respectively, after 21 days of osteogenic induction. In conclusion, proliferation and osteogenic differentiation of hUC-MSCs were enhanced through the blending of P(3HB-co-3HHx) and SF. The results from this study suggest that electrospun P(3HB-co-3HHx)/SF film is a promising biomaterial for bone tissue engineering.
A simple procedure for recovering biodegradable polymer from bacterial cells has been developed using economical and environmentally friendly solvent or chemicals. Recombinant bacterium, Cupriavidus necator harboring pBBR1MCS-C2 plasmid polyhydroxyalkanoate (PHA) synthase gene was used for the production of copolymer P(3HB-co-3HHx) from crude palm kernel oil (CPKO). NaOH was chosen in this study as it could give high purity and recovery yield. Increase of NaOH concentration had resulted in an increase of the PHA purity, but the recovery yield had decreased. The greater improvement of PHA purity and recovery were achieved by incubating the freeze-dried cells (10-30 g/L) in NaOH (0.1 M) for 1-3 h at 30°C and polishing using 20% (v/v) of ethanol. The treatment caused negligible degradation of the molecular weight of PHA recovered from the bacterial cells. The present review also highlights other extraction methods to provide greater insights into economical and sustainable recovery of PHA from bacterial cells.
Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8 g L-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1 g L-1 , and 48 H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2 M Tris-HCl buffer (pH 9, strength = 0.2 M, 30 °C) at 0.21 g L-1 H-1 and 98.6 ± 1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.
In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.
Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.
Chemical recycling of bio-based polymers polyhydroxyalkanoates (PHAs) by thermal degradation was investigated from the viewpoint of biorefinery. The thermal degradation resulted in successful transformation of PHAs into vinyl monomers using alkali earth compound (AEC) catalysts. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) were smoothly and selectively depolymerized into crotonic (CA) and 2-pentenoic (2-PA) acids at lower degradation temperatures in the presence of CaO and Mg(OH)(2) as catalysts. Obtained CA from 3-hydroxybutyrate sequences in PHBV was copolymerized with acrylic acid to produce useful water-soluble copolymers, poly(crotonic acid-co-acrylic acid) that have high glass-transition temperatures. The copolymerization of CA derived from PHA pyrolysis is an example of cascade utilization of PHAs, which meets the idea of sustainable development.
The (R)-3-hydroxyacyl-ACP-CoA transferase catalyses the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serves as the ultimate precursor for polyhydroxyalkanoate (PHA) polymerisation from unrelated substrates in pseudomonads. PhaG was found to be responsible for channelling precursors for polyhydroxyalkanoate (PHA) synthase from a de novo fatty acid biosynthesis pathway when cultured on carbohydrates, such as glucose or gluconate. The phaG gene was cloned from Pseudomonas sp. USM 4-55 using a homologous probe. The gene was located in a 3660 bp Sal I fragment (GenBank accession number EU305558). The open reading frame (ORF) was 885 bp long and encoded a 295 amino acid protein. The predicted molecular weight was 33251 Da, and it showed a 62% identity to the PhaG of Pseudomonas aeruginosa. The function of the cloned phaG of Pseudomonas sp. USM 4-55 was confirmed by complementation studies. Plasmid pBCS39, which harboured the 3660 bp Sal I fragment, was found to complement the PhaG-mutant heterologous host cell, Pseudomonas putida PhaGN-21. P. putida PhaGN-21, which harboured pBCS39, accumulated PHA that accounted for up to 18% of its cellular dry weight (CDW). P. putida PhaGN-21, which harboured the vector alone (PBBR1MCS-2), accumulated only 0.6% CDW of PHA.
Polyhydroxyalkanoates (PHAs) has been investigated for more than eighty years. Ever since then, the scientists are kept on synthesizing and developing new polymers and application to suit human interests nowadays. The resourcefulness of PHAs has made them a good candidates for the study of their potential in a variety of areas from biomedical/medical fields to food, packaging, textile and household material. In medical field (specifically in tissue engineering application), producing a biocompatible 3-D scaffold with adaptable physical properties are essential. However, to the best of our knowledge, scaffolds from PHB and PHBV with thickness greater than 1 mm have not been produced yet. In this work, PHB and PHBV porous 3-D scaffolds with an improved thickness greater than 4 mm was fabricated using conventional method of solvent-casting particulate-leaching (SCPL). A preliminary assessment on the improved thickness 3-D scaffolds was carried out to examine its pore interconnectivity by using non-invasive color staining method. The vertical cross sections image of the stained scaffolds was analyzed by image analyzer software. This technique was considered simple, fast and cost effective method prior to the usage of super accurate analytical instruments (micro-computed tomography). The results showed that over 80% of the pores have been interconnected with the adjacent pores. Moreover, there was a good correlation between the predicted pore interconnectivity and porosity. These results indicated how well a simple technique can do by giving an overview of the internal morphology of a porous 3-D structure material.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
Overconsumption of plastic goods and improper handling of petroleum-derived plastic waste have brought a plethora of negative impacts to the environment, ecosystem and human health due to its recalcitrance to degradation. These drawbacks become the main driving force behind finding biopolymers with the degradable properties. With the advancement in biopolymer research, polyhydroxyalkanoate (PHA) and poly(lacyic acid) (PLA) and its composites have been alluded to as a potential alternative to replace the petrochemical counterpart. This review highlights the current synthesis process and application of PHAs and PLA and its composites for food packaging materials and coatings. These biopolymers can be further ameliorated to enhance their applicability and are discussed by including the current commercially available packaging products. Factors influencing biodegradation are outlined in the latter part of this review. The main aim of this review article is to organize the scattered available information on various aspects of PHAs and PLA, and its composites for packaging application purposes. It is evident from a literature survey of about 140 recently published papers from the past 15 years that PLA and PHA show excellent physical properties as potential food packaging materials.
PHAs (polyhydroxyalkanoates) have emerged as biodegradable plastics more strongly in the 20th century. A wide range of bacterial species along with fungi, plants, oilseed crops and carbon sources have been used extensively to synthesize PHA on large scales. Alteration of PHA monomers in their structures and composition has led to the development of biodegradable and biocompatible polymers with highly specific mechanical properties. This leads to the incorporation of PHA in numerous biomedical applications within the previous decade. PHAs have been fabricated in various forms to perform tissue engineering to repair liver, bone, cartilage, heart tissues, cardiovascular tissues, bone marrow, and to act as drug delivery system and nerve conduits. A large number of animal trials have been carried out to assess the biomedical properties of PHA monomers, which also confirms the high compatibility of PHA family for this field. This review summarizes the synthesis of PHA from different sources, and biosynthetic pathways and biomedical applications of biosynthesized polyhydroxyalkanoates.
A novel method for the assay of polyhydroxyalkanoate (PHA)-degrading ability of triacylglycerol lipases was developed. By applying the natural affinity of lipases towards hydrophobic interfaces, a sensitive and rapid densitometry analysis for the evaluation of hydrolytic activity of lipase droplets towards PHA-coated surface was successfully carried out. We found that 12 out of 14 tested lipases which are of fungal, bacterial and animal origin were able to hydrolyze P(3HB-co-92 mol% 4HB) thin film. The patterns and opacity of the hydrolysis spots of lipases on PHA films allowed easy comparison of PHA-hydrolytic strength of lipases. Lipase from the bacterium Chromobacterium viscosum exhibited the highest PHA-degrading activity. The hydrolytic activity of lipases on water insoluble PHA, emulsified p-nitrophenyl laurate and olive oil were also compared and interestingly some lipases showed better activity when PHA was used as a substrate.
Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.
Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain.
Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by a wide range of bacteria, which serve as a promising candidate in replacing some conventional petrochemical-based plastics. PHA synthase (PhaC) is the key enzyme in the polymerization of PHA, and the crystal structures were successfully determined using the catalytic domain of PhaC from Cupriavidus necator (PhaCCn-CAT) and Chromobacterium sp. USM2 (PhaCCs-CAT). Here, we review the beneficial mutations discovered in PhaCs from a structural perspective. The structural comparison of the residues involved in beneficial mutation reveals that the residues are near to the catalytic triad, but not inside the catalytic pocket. For instance, Ala510 of PhaCCn is near catalytic His508 and may be involved in the open-close regulation, which presumably play an important role in substrate specificity and activity. In the class II PhaC1 from Pseudomonas sp. 61-3 (PhaC1Ps), Ser325 stabilizes the catalytic cysteine through hydrogen bonding. Another residue, Gln508 of PhaC1Ps is located in a conserved hydrophobic pocket which is next to the catalytic Asp and His. A class I, II-conserved Phe420 of PhaCCn is one of the residues involved in dimerization and its mutation to serine greatly reduced the lag phase. The current structural analysis shows that the Phe362 and Phe518 of PhaC from Aeromonas caviae (PhaCAc) are assisting the dimer formation and maintaining the integrity of the core beta-sheet, respectively. The structure-function relationship of PhaCs discussed in this review will serve as valuable reference for future protein engineering works to enhance the performance of PhaCs and to produce novel biopolymers.
Biodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its β-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.
Polyhydroxyalkanoate (PHA) is a type of polyesters produced in the form of accumulated intracellular granules by many microorganisms. It is viewed as an environmentally friendly bioproduct due to its biodegradability and biocompatibility. The production of the PHA using oil substrates such as waste oil and plant oil, has gained considerable attention due to the high product yield and lower substrate cost. Nevertheless, the PHA fermentation using oil substrate is complicated due to the heterogenous fatty acid composition, varied bio-accessibility and possible inhibitory effect on the bacterial culture. This review presents the current state-of-the-art of PHA production from oil-based substrates. This paper firstly discusses the technical details, such as the choice of bacteria strain and fermentation conditions, characteristic of the oil substrate as well as the PHA composition and application. Finally, the paper discusses the challenges and prospects for up-scaling towards a cleaner and effective bioprocess. From the literature review, depending on the cell culture and the type of PHA produced, the oil platform can have a PHA yield of 0.2-0.8 g PHA/g oil substrate, with PHA content mostly from 40 to 90% of the cell dry weight. There is an on-going search for more effective oil-utilising PHA producers and lower cost substrate for effective PHA production. The final application of the PHA polymer influences the treatment needed during downstream processing and its economic performance. PHA with different compositions exhibits varied decomposition behaviour under different conditions, requiring further insight towards its management towards a sustainable circular economy.
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyester which are biosynthesized from the intracellular cells of microalgae through the cultivation of organic food waste medium. Before cultivation process, food waste must undergo several pre-treatment techniques such as chemical, biological, physical or mechanical in order to solubilize complex food waste matter into simpler micro- and macronutrients in which allow bio-valorisation of microalgae and food waste compound during the cultivation process. This work reviews four microalgae genera namely Chlamydomonas, Chlorella, Spirulina, and Botryococcus, are selected as suitable species due to rapid growth rate, minimal nutrient requirement, greater adaptability and flexibility prior to lower the overall production cost and maximized the production of PHAs. This study also focuses on the different mode of cultivation for the accumulation of PHAs followed by cell wall destabilization, extraction, and purification. Nonetheless, this review provides future insights into enhancing the productivity of bioplastic derived from microalgae towards low-cost, large-scale, and higher productivity of PHAs.
High PHA production and storage using palm oil mill effluent (POME) was investigated using a laboratory batch Bio-PORec® system under aerobic-feeding conditions. Results showed that maximum PHA was obtained at a specific rate (q(p)) of 0.343 C-mol/C-molh when air was supplied at 20 ml/min. The PHA yield was found to be 0.80 C-mol/C-mol acetic acid (HAc) at microaerophilic condition and the mass balance calculation showed that PHA production increased up to 15.68±2.15 C-mmol/cycle. The experiments showed that short feeding rate, limited requirements for electron acceptors (e.g. O(2), NO(3)) and nutrients (N and P) showed lower tendency of glycogen accumulation and contributed more to PHA productivity.