Search Results (34)

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are semi-crystalline elastomers with a low melting point and high elongation at break, allowing for a wide range of applications in domestic, agricultural, industrial, and mainly medical fields. Utilizing low-cost cellulose hydrolyzed sugar as a carbon source and metabolic engineering to enhance synthesis in Pseudomonas putida is a promising strategy for commercializing mcl-PHAs, but little has been attempted to improve the utilization of glucose for synthesizing mcl-PHAs. In this study, a multi-pathway modification was performed to improve the utilization of substrate glucose and the synthesis capacity of PHAs. To enhance glucose metabolism to flow to acetyl-CoA, which is an important precursor of mcl-PHA, multiple genes in glucose metabolism were inactive (branch pathway and negative regulatory) and overexpressed (positive regulatory) in this study. The two genes, gcd (encoding glucose dehydrogenase) and gltA (encoding citrate synthase), involved in glucose peripheral pathways and TCA cycles were separately and jointly knocked out in Pseudomonas putida QSRZ6 (ΔphaZΔhsdR), and the mcl-PHA synthesis was improved in the mutants; particularly, the mcl-PHA titer of QSRZ603 (ΔgcdΔgltA) was increased by 33.7%. Based on the glucose branch pathway truncation, mcl-PHA synthesis was further improved with hexR-inactivation (encoding a negative regulator in glucose metabolism). Compared with QSRZ603 and QSRZ6, the mcl-PHA titer of QSRZ607 (ΔgcdΔgltAΔhexR) was increased by 62.8% and 117.5%, respectively. The mutant QSRZ609 was constructed by replacing the endogenous promoter of gltB encoding a transcriptional activator of the two-component regulatory system GltR/GltS with the ribosome subunit promoter P₃₃. The final mcl-PHA content and titers of QSRZ609 reached 57.3 wt% and 2.5 g/L, an increase of and 20.9% and 27.3% over that of the parent strain QSRZ605 and an increase of 110.4% and 159.9% higher as compared to QSRZ6, respectively. The fermentation was optimized with a feeding medium in shaker flacks; then, the mcl-PHA contents and titer of QSRZ609 were 59.1 wt% and 6.8 g/L, respectively. The results suggest that the regulation from glucose to acetyl-CoA by polygenic modification is an effective strategy for enhancing mcl-PHA synthesis, and the mutants obtained in this study can be used as chassis to further increase mcl-PHA production. Full article

Waste cooking oil is a common byproduct in the culinary industry, often posing disposal challenges. This study explores its conversion into the valuable bioplastic material, medium-chain-length polyhydroxyalkanoate (mcl-PHA), through microbial biosynthesis in controlled bioreactor conditions. Twenty-four bacterial isolates were obtained from oil-contaminated soil and waste materials in Mahd Ad-Dahab, Saudi Arabia. The best PHA-producing isolates were identified via 16S rDNA analysis as Neobacillus niacini and Metabacillus niabensis, with the sequences deposited in GenBank (accession numbers: PP346270 and PP346271). This study evaluated the effects of various carbon and nitrogen sources, as well as environmental factors, such as pH, temperature, and shaking speed, on the PHA production titer. Neobacillus niacini favored waste cooking oil and yeast extract, achieving a PHA production titer of 1.13 g/L, while Metabacillus niabensis preferred waste olive oil and urea, with a PHA production titer of 0.85 g/L. Both strains exhibited optimal growth at a neutral pH of 7, under optimal shaking -flask conditions. The bioreactor performance showed improved PHA production under controlled pH conditions, with a final titer of 9.75 g/L for Neobacillus niacini and 4.78 g/L for Metabacillus niabensis. Fourier transform infrared (FT-IR) spectroscopy and gas chromatography–mass spectrometry (GC-MS) confirmed the biosynthesized polymer as mcl-PHA. This research not only offers a sustainable method for transforming waste into valuable materials, but also provides insights into the optimal conditions for microbial PHA production, advancing environmental science and materials engineering. Full article

The inherent brittleness of polyhydroxybutyrate (PHB), a well-studied polyhydroxyalkanoate (PHA), limits its applicability in flexible and impact-resistant applications. This study explores the potential of blending PHB with a different PHA to overcome brittleness. The synthesis of PHA polymers, including PHB and an amorphous medium-chain-length PHA (aPHA) consisting of various monomers, was achieved in previous works through canola oil fermentation. Detailed characterization of aPHA revealed its amorphous nature, as well as good thermal stability and shear thinning behavior. The blending process was carried out at different mass ratios of aPHA and PHB, and the resulting blends were studied by differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The blends exhibited complex DSC curves, indicating the presence of multiple crystalline forms of PHB. SEM images revealed the morphology of the blends, with PHB particles dispersed within the aPHA matrix. TGA showed similar thermal degradation patterns for the blends, with the residue content decreasing as the PHB content increased. The crystallinity of the blends was influenced by the PHB content, with higher PHB ratios resulting in an increased degree of crystallinity. XRD confirmed the presence of both α and β crystals of PHB in the blends. Overall, the results demonstrate the potential of PHB+aPHA blends to enhance the mechanical properties of biopolymer materials, without com-promising the thermal stability, paving the way for sustainable material design and novel application areas. Full article

Polyhydroxyalkanoate (PHA) polymers are environmentally friendly alternatives to conventional plastics. In support of a circular bioeconomy, they can be produced by growing microbial strains in waste materials, including lignocellulosic biomass, such as Canola fines (straw). In this study, PHA and polyhydroxybutyrate (PHB) production by a selection of seven wild-type actinobacterial strains, including three strains of Gordonia species, were assessed. When grown in defined media and hydrolysates of Canola fines, the highest amounts of PHB were produced by Nocardia gamkensis CZH20^T (0.0476 mg/mL) and Gordonia lacunae BS2^T (0.0479 mg/mL), respectively. Six strains exhibited a substrate preference for cellobiose over glucose, xylose, and arabinose in the hydrolysates. Analysis of Fourier transform infrared spectra indicated that the strains produced co-polymers of short- and medium-chain-length PHAs. None of the core phaABC genes were found on defined operons in the genomes of the top PHB-producing strains (all Gordonia strains, N. gamkensis CZH20^T, and Streptomyces sp. strain HMC19). The Gordonia strains all harbored three phaA genes, a single phaB gene, and, with the exception of strain BG1.3 (with two predicted phaC genes), a single phaC gene. Predictive analyses of the proteins likely to be translated from the phaC genes revealed PhaC proteins of 37.7–39.2 kDa from Gordonia sp. strain BG1.3, G. lacunae BS2^T, and N. gamkensis CZH20^T; PhaC proteins of 106.5–107 kDa from Gordonia sp. strain JC51; and the second PhaC from Gordonia sp. strain BG1.3 and N. gamkensis CZH20^T, possibly representing a new class of PHA synthases. Full article

In recent years, polyhydroxyalkanoates (PHAs) have gained notoriety because of their desirable properties that include proven biodegradability, biocompatibility, and thermal stability, which make them suitable alternatives to fossil-based polymers. However, the widespread use of PHAs is still challenging because of their production costs, which are greatly associated with the cultivation medium used for bacterial cultivation. In Portugal, one-quarter of the forest area is covered by Eucalyptus globulus wood, making its residues a cheap, abundant, and sustainable potential carbon source for biotechnological uses. In this work, eucalyptus bark was used as the sole feedstock for PHA production in a circular bioeconomic approach. Eucalyptus bark hydrolysate was obtained after enzymatic saccharification using Cellic^® CTec3, resulting in a sugar-rich solution containing glucose and xylose. Although with differing performances, several bacteria were able to grow and produce PHA with distinct compositions, using the enzymatic hydrolysate as the sole carbon source. Pseudomonas citronellolis NRRL B-2504 achieved a high cellular growth rate in bioreactor assays (24.4 ± 0.15 g/L) but presented a low accumulation of a medium-chain-length PHA (mcl-PHA) comprising the monomers hydroxydecanoate (HD, 65%), hydroxydodecanoate (HDd, 25%), and hydroxytetradecanoate (HTd, 14%). Burkholderia thailandensis E264, on the other hand, reached a lower cellular growth rate (8.87 ± 0.34 g/L) but showed a higher biopolymer accumulation, with a polyhydroxybutyrate (PHB) content in the cells of 12.3 wt.%. The new isolate, Pseudomonas sp., revealed that under nitrogen availability, it was able to reach a higher accumulation of the homopolymer PHB (31 wt.%). These results, although preliminary, demonstrate the suitability of eucalyptus bark’s enzymatic hydrolysate as a feedstock for PHA production, thus offering an exciting avenue for achieving sustainable and environmentally responsible plastic products from an undervalued forestry waste. Full article

Pseudomonas sp. SG4502 screened from biodiesel fuel by-products can synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glycerol as a substrate. It contains a typical PHA class II synthase gene cluster. This study revealed two genetic engineering methods for improving the mcl-PHA accumulation capacity of Pseudomonas sp. SG4502. One way was to knock out the PHA-depolymerase phaZ gene, the other way was to insert a tac enhancer into the upstream of the phaC1/phaC2 genes. Yields of mcl-PHAs produced from 1% sodium octanoate by +(tac-phaC2) and ∆phaZ strains were enhanced by 53.8% and 23.1%, respectively, compared with those produced by the wild-type strain. The increase in mcl-PHA yield from +(tac-phaC2) and ∆phaZ was due to the transcriptional level of the phaC2 and phaZ genes, as determined by RT-qPCR (the carbon source was sodium octanoate). ¹H-NMR results showed that the synthesized products contained 3-hydroxyoctanoic acid (3HO), 3-hydroxydecanoic acid (3HD) and 3-hydroxydodecanoic acid (3HDD) units, which is consistent with those synthesized by the wild-type strain. The size-exclusion chromatography by GPC of mcl-PHAs from the (∆phaZ), +(tac-phaC1) and +(tac-phaC2) strains were 2.67, 2.52 and 2.60, respectively, all of which were lower than that of the wild-type strain (4.56). DSC analysis showed that the melting temperature of mcl-PHAs produced by recombinant strains ranged from 60 °C to 65 °C, which was lower than that of the wild-type strain. Finally, TG analysis showed that the decomposition temperature of mcl-PHAs synthesized by the (∆phaZ), +(tac-phaC1) and +(tac-phaC2) strains was 8.4 °C, 14.7 °C and 10.1 °C higher than that of the wild-type strain, respectively. Full article

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES). Full article

Steadily increasing R&D activities in the field of microbial polyhydroxyalkanoate (PHA) biopolyesters are committed to growing global threats from climate change, aggravating plastic pollution, and the shortage of fossil resources. These prevailing issues paved the way to launch the third Special Issue of Bioengineering dedicated to future-oriented biomaterials, characterized by their versatile plastic-like properties. Fifteen individual contributions to the Special Issue, written by renowned groups of researchers from all over the world, perfectly mirror the current research directions in the PHA sector: inexpensive feedstock like carbon-rich waste from agriculture, mitigation of CO₂ for PHA biosynthesis by cyanobacteria or wild type and engineered “knallgas” bacteria, powerful extremophilic PHA production strains, novel tools for rapid in situ determination of PHA in photobioreactors, modelling of the dynamics of PHA production by mixed microbial cultures from inexpensive raw materials, enhanced bioreactor design for high-throughput PHA production by sophisticated cell retention systems, sustainable and efficient PHA recovery from biomass assisted by supercritical water, enhanced processing of PHA by application of novel antioxidant additives, and the development of compatible biopolymer blends. Moreover, elastomeric medium chain length PHA (mcl-PHA) are covered in-depth, inter alia, by introduction of a novel class of bioactive mcl-PHA-based networks, in addition to the first presentation of the new rubber-like polythioester poly(3-mercapto-2-methylpropionate). Finally, the present Special Issue is concluded by a critical essay on past, ongoing, and announced global endeavors for PHA commercialization. Full article

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that can be used to replace petroleum-based plastic. In addition, as a medium-chain-length PHA (mcl-PHA), it can be used to provide elastomeric properties in specific applications. Because of these characteristics, recently, there has been much research on mcl-PHA production using inexpensive biomass materials as substrates. In this study, mcl-PHA producers were screened using alkanes (n-octane, n-decane, and n-dodecane) as sources of carbon. The amount of PHA produced by Pseudomonas resinovorans using sole n-octane, n-decane, or n-dodecane was 0.48 g/L, 0.27 g/L, or 0.07 g/L, respectively, while that produced using mixed alkane was 0.74 g/L. As a larger amount of PHA was produced using mixed alkane compared with sole alkane, a statistical mixture analysis was used to determine the optimal ratio of alkanes in the mixture. The optimal ratio predicted by the analysis was a medium with 9.15% n-octane, 6.44% n-decane, and 4.29% n-dodecane. In addition, through several concentration-specific experiments, the optimum concentrations of nitrogen and phosphorus for cell growth and maximum PHA production were determined as 0.05% and 1.0%, respectively. Finally, under the determined optimal conditions, 2.1 g/L of mcl-PHA and 60% PHA content were obtained using P. resinovorans in a 7 L fermenter. Full article

Synthetic plastics derived from fossil fuels—such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene—are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered increasing interest as replaceable materials to conventional plastics due to their broad applicability in various purposes such as food packaging, agriculture, tissue-engineering scaffolds, and drug delivery. Based on the chain length of 3-hydroxyalkanoate repeat units, there are three types PHAs, i.e., short-chain-length (scl-PHAs, 4 to 5 carbon atoms), medium-chain-length (mcl-PHAs, 6 to 14 carbon atoms), and long-chain-length (lcl-PHAs, more than 14 carbon atoms). Previous reviews discussed the recent developments in scl-PHAs, but there are limited reviews specifically focused on the developments of mcl-PHAs. Hence, this review focused on the mcl-PHA production, using various carbon (organic/inorganic) sources and at different operation modes (continuous, batch, fed-batch, and high-cell density). This review also focused on recent developments on extraction methods of mcl-PHAs (solvent, non-solvent, enzymatic, ultrasound); physical/thermal properties (Mw, Mn, PDI, Tm, Tg, and crystallinity); applications in various fields; and their production at pilot and industrial scales in Asia, Europe, North America, and South America. Full article

Immobilizing antifungal polyenes such as nystatin (Nys) and amphotericin B (AmB) into biodegradable formulations is advantageous compared to free drug administration providing sustained release, reduced dosing due to localized targeting and overall reduced systemic drug toxicity. In this study, we encapsulated Nys and AmB in medium chain length polyhydroxyalkanoates (mcl-PHA) microspheres (7–8 µm in diameter). The obtained formulations have been validated for antifungal activity in vitro against a panel of pathogenic fungi including species of Candida, Aspergillus, Microsporum and Trichophyton genera and toxicity and efficacy in vivo using the zebrafish model of disseminated candidiasis. While free polyenes, especially AmB, were highly toxic to zebrafish embryos at the effective (MIC) doses, after their loading into mcl-PHA microspheres, inner organ toxicity and teratogenicity associated with both drugs were not observed, even at 100 × MIC doses. The obtained mcl-PHA/polyene formulations have successfully eradicated C. albicans infection and showed an improved therapeutic profile in zebrafish by enhancing infected embryos survival. This approach is contributing to the antifungal arsenal as polyenes, although the first broad-spectrum antifungals on the market are still the gold standard for treatment of fungal infections. Full article

One of the major impediments to the commercialization of biodegradable plastic is the high cost of substrate. Consequently, there is a continuous search for effective microorganisms and cheaper carbon substrates to reduce the high production cost. In this study, waste transformer oil-degrading bacteria were isolated from soil, wastewater, and sediment samples, using a mineral salt medium (MSM) supplemented with 1% waste transformer oil as the sole carbon source. The isolates were screened for polyhydroxyalkanoates (PHA) production using Nile red staining and fluorescence microscopy. PHA granules accumulation was confirmed using transmission electron microscopy. Oil degradation analysis was accomplished using solvent extraction and gravimetric methods whereas, the bacteria were identified using 16S DNA sequence homology. A total of 62 transformer oil-degrading bacteria were isolated, out of which 16 (26%) showed positive results for Nile red fluorescence microscopy. The identified organisms belong to four different taxonomic genera of Acinetobacter, Bacillus, Proteus, and Serratia. The percentage of oil degradation observed among the different isolates ranged between 19.58% and 57.51%. Analysis of the PHA extracted from the selected isolate revealed the presence of medium chain length polyhydroxyalkanoates (mcl-PHA). The findings of this work have further highlighted the diversity of the bacteria capable of utilizing waste streams such as waste transformer oil. Consequently, the isolates can be explored as agents of converting waste transformer oil into bioplastics. Full article

Polyhydroxyalkanoates (PHAs) represent a promising alternative to commercially used petroleum-based plastics. Pseudomonas oleovorans is a natural producer of medium-chain-length PHA (mcl-PHA) under cultivation conditions with nitrogen limitation and carbon excess. Two-step cultivation appears to be an efficient but more expensive method of PHA production. Therefore, the aim of this work was to prepare a minimal synthetic medium for maximum biomass yield and to optimize selected independent variables by response surface methodology (RSM). The highest biomass yield (1.71 ± 0.04 g/L) was achieved in the optimized medium containing 8.4 g/L glucose, 5.7 g/L sodium ammonium phosphate and 35.4 mM phosphate buffer. Under these conditions, both carbon and nitrogen sources were completely consumed after 48 h of the cultivation and the biomass yield was 1.7-fold higher than in the conventional medium recommended by the literature. This approach demonstrates the possibility of using two-stage PHA cultivation to obtain the maximum amount of biomass and PHA. Full article

This paper focuses on the design of soft sensors for on-line monitoring of the biotechnological process of biopolymer production, in which biopolymers are accumulated in bacteria as an intracellular energy storage material. The proposed soft sensors for on-line estimation of the biopolymer concentration represent an interesting alternative to the traditional off-line analytical techniques of limited applicability for real-time process control. Due to the complexity of biochemical reactions, which make it difficult to create reasonably complex first-principle mathematical models, a data-driven approach to the design of soft sensors has been chosen in the presented study. Thus, regression methods were used in this design, including multivariate statistical methods (PLS, PCR). This approach enabled the creation of soft sensors using historical process data from fed-batch cultivations of the Pseudomonas putida KT2442 strain used for the production of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Specifically, data from on-line measurements of off-gas composition analysis and culture medium capacitance were used as input to the soft sensors. The resulting soft sensors allow not only on-line estimation of the biopolymer concentration, but also the concentration of the cell biomass of the production bacterial culture. For most of these soft sensors, the estimation error did not exceed 5% of the measurement range. In addition, soft sensors based on capacitance measurement were able to accurately detect the end of the production phase. This study thus offers an innovative and practically relevant contribution to the field of monitoring of bioprocesses used for the production of medium-chain-length biopolymers. Full article

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO₂ as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H₂/O₂/CO₂ = 8:1:1). The results of GC and ¹H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO₂ with high cellular content. When 1.0 g/L (NH₄)₂SO₄ was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccr_MeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccr_MeJ_Ac-emd harboring short-chain-length-specific PhaJ. Full article