Bioplastic production by harnessing cyanobacteria-rich microbiomes for long-term synthesis.

Departing from the conventional axenic and heterotrophic cultures, our research ventures into unexplored territory by investigating the potential of photosynthetic microbiomes for polyhydroxybutyrate (PHB) synthesis, a biodegradable polyester that presents a sustainable alternative to conventional plastics. Our investigation focused on a cyanobacteria-enriched microbiome, dominated by Synechocystis sp. and Synechococcus sp., cultivated in a 3 L photobioreactor under non-sterile conditions, achieving significant PHB production-up to 28 % dry cell weight (dcw) over a span of 108 days through alternating cycles of biomass growth and PHB accumulation. Nile Blue staining and Transmission Electron Microscope visualization allowed to successfully confirm the presence of PHB granules within cyanobacteria cells. Furthermore, the overexpression of PHA synthase during the accumulation phase directly correlated with the increased PHB production. Also, gene expression changes revealed glycogen as the primary storage compound, but under prolonged macronutrient stress, there was a shift of the carbon flux towards favoring PHB synthesis. Finally, analysis through Raman, Fourier- transform infrared spectroscopy and proton Nuclear Magnetic Resonance further validated the extracted polymer as PHB. Overall, it was demonstrated for the first time the feasibility of using phototrophic microbiomes to continuous production of PHB in a non-sterile system. This study also offers valuable insights into the metabolic pathways involved.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

PHB+aPHA Blends: From Polymer Bacterial Synthesis through Blend Preparation to Final Processing by Extrusion for Sustainable Materials Design.

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 ß 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.

Biodegradation of PHB/PBAT films and isolation of novel PBAT biodegraders from soil microbiomes.

Polyhydroxyalkanoates (PHAs) are important candidates for replacing petroleum-based plastics. This transition is urgent for the development of a biobased economy and to protect human health and natural ecosystems. PHAs are biobased and biodegradable polyesters that when blended with other polymers, such as poly(butylene adipate-co-terephthalate) (PBAT), acquire remarkable improvements in their properties, which allow them to comply with the requirements of packaging applications. However, the biodegradation of such blends should be tested to evaluate the impact of those polymers in the environment. For instance, PBAT is a compostable aliphatic-aromatic copolyester, and its biodegradation in natural environments, such as soil, is poorly studied. In this work, we evaluated the biodegradation of a bilayer film composed of PHB and PBAT, by a soil microbiome. The bilayer film reached 47 ± 1 % mineralization in 180 days and PHB was no longer detected after this period. The increased crystallinity of the PBAT residue was a clear sign of biodegradation, indicating that the amorphous regions were preferentially biodegraded. Seven microorganisms were isolated, from which 4 were closely related to microorganisms already known as PHB degraders, but the other 3 species, closely related to Streptomyces coelicoflavus, Clonostachys rosea and Aspergillus insuetus, were found for the first time as PHB degraders. Most remarkably, two fungi closely related to Purpureocillium lilacinum and Aspergillus pseudodeflectus (99.83 % and 100 % identity by ITS sequencing) were isolated and identified as PBAT degraders. This is very interesting due to the rarity of isolating PBAT-degrading microorganisms. These results show that the bilayer film can be biodegraded in soil, at mesophilic temperatures, showing its potential to replace synthetic plastics in food packaging.

Methylosinus trichosporium OB3b bioaugmentation unleashes polyhydroxybutyrate-accumulating potential in waste-activated sludge.

BACKGROUND: Wastewater treatment plants contribute approximately 6% of anthropogenic methane emissions. Methanotrophs, capable of converting methane into polyhydroxybutyrate (PHB), offer a promising solution for utilizing methane as a carbon source, using activated sludge as a seed culture for PHB production. However, maintaining and enriching PHB-accumulating methanotrophic communities poses challenges. RESULTS: This study investigated the potential of Methylosinus trichosporium OB3b to bioaugment PHB-accumulating methanotrophic consortium within activated sludge to enhance PHB production. Waste-activated sludges with varying ratios of M. trichosporium OB3b (1:0, 1:1, 1:4, and 0:1) were cultivated. The results revealed substantial growth and methane consumption in waste-activated sludge with M. trichosporium OB3b-amended cultures, particularly in a 1:1 ratio. Enhanced PHB accumulation, reaching 37.1% in the same ratio culture, indicates the dominance of Type II methanotrophs. Quantification of methanotrophs by digital polymerase chain reaction showed gradual increases in Type II methanotrophs, correlating with increased PHB production. However, while initial bioaugmentation of M. trichosporium OB3b was observed, its presence decreased in subsequent cycles, indicating the dominance of other Type II methanotrophs. Microbial community analysis highlighted the successful enrichment of Type II methanotrophs-dominated cultures due to the addition of M. trichosporium OB3b, outcompeting Type I methanotrophs. Methylocystis and Methylophilus spp. were the most abundant in M. trichosporium OB3b-amended cultures. CONCLUSIONS: Bioaugmentation strategies, leveraging M. trichosporium OB3b could significantly enhance PHB production and foster the enrichment of PHB-accumulating methanotrophs in activated sludge. These findings contribute to integrating PHB production in wastewater treatment plants, providing a sustainable solution for resource recovery.

Genetic characterization of a novel Salinicola salarius isolate applied for the bioconversion of agro-industrial wastes into polyhydroxybutyrate.

BACKGROUND: Polyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production. RESULTS: Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. CONCLUSIONS: Herein, Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.

Cellulose/Polyhydroxybutyrate (PHB) Composites as a Sustainable Bio-Based Feedstock to 3D-Printing Applications.

In this work, we have studied the potential application for 3D-printing of a polymer made from combining a biodegradable and biocompatible polymer (i.e., polyhydroxybutyrate, PHB) with natural bio-based fiber (i.e., cellulose). To this end, a masterbatch at 15 wt.% in filler content was prepared by melt-blending, and then this system was "diluted" with pure PHB in a second extrusion phase in order to produce filaments at 1.5 and 3 wt.% of cellulose. For comparison, a filament made of 100% virgin PHB pellets was prepared under the same conditions. All the systems were then processed in the 3D-printer apparatus, and specimens were mainly characterized by static (tensile and flexural testing) and dynamic mechanical analysis. Thermogravimetric analysis, differential scanning calorimetry, spectroscopic measurements, and morphological aspects of PHB polymer and composites were also discussed. The results showed a significant negative impact of the process on the mechanical properties of the basic PHB with a reduction in both tensile and flexural mechanical properties. The PHB-cellulose composites showed a good dispersion filler in the matrix but a poor interfacial adhesion between the two phases. Furthermore, the cellulose had no effect on the melting behavior and the crystallinity of the polymer. The addition of cellulose improved the thermal stability of the polymer and minimized the negative impact of extrusion. The mechanical performance of the composites was found to be higher compared to the corresponding (processed) polymer.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Nonsterilized Fermentation of Crude Glycerol for Polyhydroxybutyrate Production by Metabolically Engineered Vibrio natriegens.

Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

Evaluation of the effects of zein incorporation on physical, mechanical, and biological properties of polyhydroxybutyrate electrospun scaffold for bone tissue engineering applications.

Materials and fabrication methods significantly influence the scaffold's final features in tissue engineering. This study aimed to blend zein with polyhydroxybutyrate (PHB) at 5, 10, and 15 wt%, fabricate scaffolds using electrospinning, and then characterize them. SEM and mechanical analyses identified the scaffold with 10 wt% zein (PHB-10Z) as the optimal sample. Incorporating 10 wt% zein reduced fiber diameter from 894 ± 122 to 531 ± 42 nm while increasing ultimate tensile strength and elongation at break by approximately 53 % and 70 %, respectively. FTIR proved zein's presence in the scaffolds and possible hydrogen bonding with PHB. TGA confirmed the miscibility of polymers. DSC and XRD analyses indicated lower crystallinity for the PHB-10Z than for PHB. AFM evaluation indicated a rougher surface for the PHB-10Z in comparison to PHB. The PHB-10Z demonstrated a more hydrophobic surface and less weight loss after 100 days of degradation in PBS than PHB. The free radical scavenging assay exhibited antioxidant activity for the zein-containing scaffold. Eventually, enhanced cell attachment, viability, and differentiation in the PHB-10Z scaffold drawn from SEM, MTT, ALP activity, and Alizarin red staining of MG-63 cells confirmed that PHB-zein electrospun scaffold is a potent candidate for bone tissue engineering applications.

Osteogenic potential of PHB-lignin/cellulose nanofiber electrospun scaffold as a novel bone regeneration construct.

The electrospun scaffolds could mimic the highly hierarchical structure of extracellular matrix (ECM). Modern tissue engineering focuses on the properties of these microstructures, influencing the biological responses. This research investigates the variation of morphology, crystallinity, bioactivity, mechanical properties, contact angle, mass loss rate, roughness, cell behavior, biomineralization, and the efficacy of polyhydroxybutyrate (PHB)-based nanocomposite. Hence, 6 wt% lignin and 3 wt% cellulose nanofiber were added to the 9 wt% of PHB to prepare a novel electrospun nanocomposite structure (PLC). The outputs indicated more symmetrical circular fibers for PLC mat, higher surface roughness (326 to 389 nm), better hydrophilicity (120 to 60°), smaller crystal size (24 to 16 nm), and more reasonable biodegradability compared to PHB. These changes lead to the improvement of mechanical properties (toughness factor from 300 to 1100), cell behavior (viability from 60 to 100 %), bioactivity (from Ca/P ratio of 0.77 and 1.67), and higher level of alizarin red, and ALP enzyme secretion. Eventually, the osteopontin and alkaline phosphatase expression was also enhanced from ≃2.35 ± 0.15 and 2.1 ± 0.1 folds on the 1st day to ≃12.05 ± 0.35 and 7.95 ± 0.35 folds on 2nd week in PLCs. Accordingly, this newly developed structure could enhance biological responses and promote osteogenesis compared to PHB.

Structure analysis and thermal stability of PHB recovered from sugar industry waste.

The current article emphasized on cultural conditions for Alcaligenes sp. NCIM 5085 to synthesize Polyhydroxybutyrate (PHB) from sugar industry waste during batch fermentation. Alcaligenes sp. NCIM 5085 was found to grow best in conditions that included 40 g l-1 of cane molasses, 1 g l-1 of ammonium sulphate, 10% inoculum with neutral pH and 48 h of incubation time. Sudan Black B staining was employed to verify the PHB synthesis initially, and further TEM analysis was performed to confirm it. The structural analysis of recovered PHB was carried out by using GC-MS, FTIR, 1H NMR and 13C NMR analysis. The absorption peak at 1724.56 cm-1 revealed the presence of C=O (carbonyl) group by FTIR, which is an indicator of PHB presence. Furthermore, results of NMR and GC-MS analysis confirmed the recovered polymer was PHB. The thermal properties of recovered polymer were analyzed by TGA, DTG and DSC and showed thermal stability of PHB. The observed glass transient temperature (Tg) -2.8°C was within the normal PHB range of Tg. However, melting temperature of recovered PHB was 161.7°C, where the degree of crystallinity was lower than standard PHB that widens the application possibilities.

Simulated Environmental Conditioning of PHB Composites Reinforced with Barley Fibres to Determine the Viability of Their Use as Plastics for the Agriculture Sector.

Nowadays, the search for new materials with a sustainable character to reduce the production of residues is under continuous research. In this sense, fully biodegradable composites based on polyhydroxybutyrate and different pretreated fibres coming from barley straw have been fabricated, and their resistance to environmental controlled conditions have been characterized. The materials were already compounded in a kinetic mixer and injection-moulded as specimens for tensile assay to be aged in a Xenotest chamber so as to simulate environmental conditioning. The samples, after accelerated aging, were characterized thus: mechanical characterization (tensile assay), water uptake (immersion and contact angle), and surface observation (optical and SEM microscopy). The incorporation of the fibres helps the composite to keep its structure for a longer time. On the other hand, the presence of the fibres increases the water uptake capacity to allow water permeation in the composite, which allows final degradation, characterised by a significant drop in properties after one month of exposure to simulated environmental conditions.

Plasma surface modification of electrospun polyhydroxybutyrate (PHB) nanofibers to investigate their performance in bone tissue engineering.

Polyhydroxybutyrate (PHB) is a natural-source biopolymer of the polyhydroxyalkanoate (PHA) family. Nanofibrous scaffolds prepared from this biological macromolecule have piqued the interest of researchers in recent years due to their unique properties. Nonetheless, these nanofibers continue to have problems such as low surface roughness and high hydrophobicity. In this research, PHB nanofibers were produced by the electrospinning method. Following that, the surface of nanofibers was modified by atmospheric plasma. Scanning electron microscopy (SEM), water contact angle (WCA), atomic force microscopy (AFM), tensile test, and cell behavior analyses were performed on mats to investigate the performance of treated and untreated samples. The achieved results showed a lower water contact angle (from ≃120° to 43°), appropriate degradation rate (up to ≃20 % weight loss in four months), and outstanding biomineralization (Ca/P ratio of ≃1.86) for the modified sample compared to the neat PHB. Finally, not only the MTT test show better viability of MG63 osteoblast cells, but also Alizarin staining, ALP, and SEM results likewise showed better cell proliferation in the presence of modified mats. These findings back up the claim that plasma surface modification is a quick, environmentally friendly, and low-cost way to improve the performance of nanofibers in bone tissue engineering.

Characterization of polyhydroxybutyrate (PHB) synthesized by newly isolated rare actinomycetes Aquabacterium sp. A7-Y.

Polyhydroxyalkanoates (PHAs) as biodegradable plastics have attracted increasing attention due to its biodegradable, biocompatible and renewable advantages. Exploitation some unique microbes for PHAs production is one of the most competitive approaches to meet complex industrial demand, and further develop next-generation industrial biotechnology. In this study, a rare actinomycetes strain A7-Y was isolated and identified from soil as the first PHAs producer of Aquabacterium genus. Produced PHAs by strain A7-Y was identified as poly(3-hydroxybutyrate) (PHB) based on its structure characteristics, which is also similar with commercial PHB. After optimization of fermentation conditions, strain A7-Y can produce 10.2 g/L of PHB in 5 L fed-batch fermenter, corresponding with 54 % PHB content of dry cell weight, which is superior to the reported actinomycetes species. Furthermore, the phaCAB operon in stain A7-Y was excavated to be responsible for the efficient PHB production and verified in recombinant Escherichia coli. Our results indicate that strain A7-Y and its biosynthetic gene cluster are potential candidates for developing a microbial formulation for the PHB production.

Production of phycobiliproteins, bioplastics and lipids by the cyanobacteria Synechocystis sp. treating secondary effluent in a biorefinery approach.

Cyanobacteria have been identified as promising organisms to reuse nutrients from waste effluents and produce valuable compounds such as lipids, polyhydroxyalkanoates (PHAs), and pigments. However, almost all studies on cyanobacterial biorefineries have been performed under lab scale and short cultivation periods. The present study evaluates the cultivation of the cyanobacterium Synechocystis sp. in a pilot scale 30 L semi-continuous photobioreactor fed with secondary effluent for a period of 120 days to produce phycobiliproteins, polyhydroxybutyrate (PHB) and lipids. To this end, the harvested biomass from the semi-continuous photobioreactor was transferred into 5 L vertical column batch photobioreactors to perform PHB and lipid accumulation under nutrient starvation. Three hydraulic retention times (HRT) (6, 8 and 10 days) were tested in the semi-continuous photobioreactor to evaluate its influence on biomass growth and microbial community. A maximum biomass concentration of 1.413 g L-1 and maximum productivity of 173 mg L-1 d-1 was reached under HRT of 8 days. Microscopy analysis revealed a shift from Synechocystis sp. to Leptolyngbya sp. and green algae when HRT of 6 days was used. Continuous, stable production of phycobiliproteins in the semi-continuous photobioreactor was obtained, reaching a maximum content of 7.4%dcw in the biomass. In the batch photobioreactors a PHB content of 4.8%dcw was reached under 7 days of nitrogen and phosphorus starvation, while a lipids content of 44.7%dcw was achieved under 30 days of nitrogen starvation. PHB and lipids production was strongly dependent on the amount of nutrients withdrawn from the grow phase. In the case of lipids, their production was stimulated when there was only phosphorus depletion. While Nitrogen and phosphorus limitation was needed to enhance the PHB production. In conclusion, this study demonstrates the feasibility of cultivating cyanobacteria in treated wastewater to produce bio-based valuable compounds within a circular bioeconomy approach.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2.

BACKGROUND: A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization. RESULTS: Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2-fixing metabolism and PHB production were investigated. CONCLUSIONS: The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Selected Simple Natural Antimicrobial Terpenoids as Additives to Control Biodegradation of Polyhydroxy Butyrate.

In this experimental research, different types of essential oils (EOs) were blended with polyhydroxybutyrate (PHB) to study the influence of these additives on PHB degradation. The blends were developed by incorporating three terpenoids at two concentrations (1 and 3%). The mineralization rate obtained from CO2 released from each sample was the factor that defined biodegradation. Furthermore, scanning electron microscope (SEM), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) were used in this research. The biodegradation percentages of PHB blended with 3% of eucalyptol, limonene, and thymol after 226 days were reached 66.4%, 73.3%, and 76.9%, respectively, while the rate for pure PHB was 100% after 198 days, and SEM images proved these results. Mechanical analysis of the samples showed that eucalyptol had the highest resistance level, even before the burial test. The other additives showed excellent mechanical properties although they had less mechanical strength than pure PHB after extrusion. The samples' mechanical properties improved due to their crystallinity and decreased glass transition temperature (Tg). DSC results showed that blending terpenoids caused a reduction in Tg, which is evident in the DMA results, and a negligible reduction in melting point (Tm).

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Potential use of Bacillus paramycoides for the production of the biopolymer polyhydroxybutyrate from leftover carob fruit agro-waste.

This study was designed to investigate, at a laboratory scale, the possibility of valorizing the leftover carob fruits to produce the eco-friendly biopolymer polyhydroxybutyrate (PHB) by using the bacterial strain Bacillus paramycoides, which has been isolated from the botanical garden of Skikda University in Algeria. The PHB production was tested under various conditions: a pH of 3-8, temperature range of 30-44 °C, carob extracted molasses concentration of 2-8% v/v, an incubation time of 24-96 h and an agitation speed of 150-300 rpm. The effects of different nitrogen sources and carob extracted molasses treatment types were also investigated. The PHB concentration was determined quantitatively as crotonic acid by measuring the absorbance at 300 nm. Cell growth was quantified by measuring the density of the culture at 600 nm. The presence of PHB was confirmed by applying high-performance liquid chromatography (HPLC) using an Aminex HPX-87H and implementing gas chromatography analysis. The best yield of PHB synthesis was obtained by using 6% v/v of 5 M H2SO4 treated with carob molasses as a carbon source, with peptone as a nitrogen source; incubation was conducted at 37 °C for 96 h at an agitation speed of 300 rpm (114.95 mg/L). The HPLC analysis confirmed the synthesis of PHB by B. paramycoides to have a chromatogram retention time of 22.5 min. Carob waste was successfully valorized to PHB.