Optimizing Polyhydroxyalkanoate production using a novel Bacillus paranthracis isolate: A response surface methodology approach.

Microorganisms have emerged as promising resources for producing economical and sustainable bioproducts like Polyhydroxyalkanoate (PHA), a biodegradable polymer that can replace synthetic plastics. In this study, we screened a novel isolate, Bacillus paranthracis RSKS-3 strain, to produce PHA from sewage water, identifying it using Whole Genome Sequence. This study represents the first report on optimizing PHA production using B. paranthracis RSKS-3, employing Design Expert 12.0 software. Our findings reveal that four factors (temperature, inoculum size, potassium dihydrogen phosphate, and magnesium sulfate) significantly affect PHA production in the Plackett-Burman design experiment. Through Response Surface Methodology, we optimized PHA production to 0.647 g/L with specific values for potassium dihydrogen phosphate (0.55 %), inoculum size (3 %), magnesium sulfate (0.055 %), and a temperature of 35 °C, in agreement with the predicted value of 0.630 g/L. This optimization resulted in a substantial 13.29-fold increase in PHA production from 0.34 g/L to 4.52 g/L, underscoring the promising role of B. paranthracis RSKS-3 in eco-friendly PHA production and advancing sustainable bioproduct development.

Effect of mutation of phaC on carbon supply, extracellular polysaccharide production, and pathogenicity of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice. Polyhydroxyalkanoates (PHAs) consitute a diverse group of biopolyesters synthesized by bacteria under nutrient-limited conditions. The phaC gene is important for PHA polymerization. We investigated the effects of phaC gene mutagensis in Xoo strain PXO99A. The phaC gene knock-out mutant exhibited reduced swarming ability relative to that of the wild-type. Under conditions where glucose was the sole sugar source, extracellular polysaccharide (EPS) production by ΔphaC declined by 44.8%. ΔphaC showed weak hypersensitive response (HR) induction in the leaves of non-host Nicotiana tabacum, concomitant with downregulation of hpa1 gene expression. When inoculated in rice leaves by the leaf-clipping method, ΔphaC displayed reduced virulence in terms of lesion length compared with the wild-type strain. The complemented strain showed no significant difference from the wild-type strain, suggesting that the deletion of phaC in Xoo induces significant alterations in various physiological and biological processes. These include bacterial swarming ability, EPS production, transcription of hrp genes, and glucose metabolism. These changes are intricately linked to the energy utilization and virulence of Xoo during plant infection. These findings revealed involvement of phaC in Xoo is in the maintaining carbon metabolism by functioning in the PHA metabolic pathway.

Overexpression of RuBisCO form I and II genes in Rhodopseudomonas palustris TIE-1 augments polyhydroxyalkanoate production heterotrophically and autotrophically.

With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1's genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH4Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH4Cl, and two times under photoelectrotrophic growth with N2 . In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganism Rhodopseudomonas palustris TIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO2, and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of the phaR or phaZ genes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic modifications in the environmental microbe TIE-1 can improve PHA production. When combined with other approaches (such as reactor design, use of microbial consortia, and different feedstocks), genetic and metabolic manipulations of purple nonsulfur bacteria like TIE-1 are essential for replacing petroleum-derived plastics with biodegradable plastics like PHA.

Drying strategies for maximizing polyhydroxybutyrate recovery from microalgae cultivated in a raceway pond: A comparative study.

Polyhydroxybutyrate (PHB) derived from microalgae are considered a promising alternative bioplastic material to replace synthetic plastics. This study evaluated the effects of various drying techniques (sun, freeze, oven and air drying) on PHB recovery from microalgae. Freeze drying recovered the maximum PHBs (6.2%) followed by sun drying (5.2%), air drying (2.3%), oven drying (2%), and the lowest in wet biomass (1.2%). The most energy-intensive drying method was freeze drying (26.83 kW) followed by oven drying (3 kW) while the other methods did not require energy. The minimum time requirement for drying was oven drying (6 h), followed by freeze drying (24 h), sun drying (48-72 h), and air drying (96-120 h) while wet biomass did not require time. In terms of PHB yield per unit time, oven (0.33%/h) is a more effective drying technique than freeze drying (0.25%/h) which produces 24.24% higher PHB yield per unit time. Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) confirmed PHB structure and thermal stability up to 300 °C from dried biomasses compared to wet biomass at 200 °C. This study indicated that drying techniques significantly influence the PHB recovery from microalgae biomass. Findings also revealed that the oven dried technique can be efficiently scaled up for PHB recovery.

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.

Polyhydroxyalkanoates (PHAs) and its copolymer nanocarrier application in cancer treatment: An overview and challenges.

In the modern era, nanomedicine has developed novel drug-delivery strategies to improve chemotherapy. Nanotechnological-based treatment approaches for cancer through targeted tumour drug delivery and stimulus-responsive tumour microenvironment have gained tremendous success in oncology. The application of building block materials of these nanomedicines plays a vital role in cancer remediation. Despite successful application in various medical treatments, nanocarriers' lack of biodegradability and biocompatibility makes their use in a clinical context difficult. In addition, the preparation of current drug delivery systems is a major constraint. The current cancer treatment methods aim to destroy diseased tissue, frequently with the use of radiation and chemotherapy. These treatment options are accompanied by a significant level of toxicity, which has excellent potential to further medical issues in the afflicted patient. Polyhydroxyalkanoate (PHA) polymers are biodegradable and biocompatible polyesters that can potentially be used as nanoparticular delivery systems for cancer treatment. Previously, PHA has shown tremendous application as a packaging material in the food and pharma industry. PHA-based nanocarriers are an effective drug delivery system because of their non-immunogenicity, regulated drug release, high drug loading capacity, and targeted drug delivery. This review focuses on creating and using PHA-based nanocarriers in cancer treatment. Despite its many benefits, PHA-based nanocarriers have yet to progress to clinical trials for drug delivery applications due to several issues, including the polymers' hydrophobic nature and high production costs. This review examines these challenges along with existing alternatives.

Valorization of Sugarcane Bagasse for Co-Production of Poly(3-hydroxybutyrate) and Bacteriocin Using Bacillus cereus Strain S356.

Poly(3-hydroxybutyrate) (P(3HB)) is an attractive biodegradable plastic alternative to petroleum-based plastic. However, the cost of microbial-based bioplastic production mainly lies in the cultivation medium. In this study, we screened the isolates capable of synthesizing P(3HB) using sugarcane bagasse (SCB) waste as a carbon source from 79 Bacillus isolates that had previously shown P(3HB) production using a commercial medium. The results revealed that isolate S356, identified as Bacillus cereus using 16S rDNA and gyrB gene analysis, had the highest P(3HB) accumulation. The highest P(3HB) yield (5.16 g/L, 85.3% of dry cell weight) was achieved by cultivating B. cereus S356 in an optimal medium with 1.5% total reducing sugar with SCB hydrolysate as the carbon source and 0.25% yeast extract as the nitrogen source. Transmission electron microscopy analysis showed the accumulation of approximately 3-5 P(3HB) granules in each B. cereus S356 cell. Proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy analyses confirmed that the polymer extracted from B. cereus S356 was P(3HB). Notably, during cultivation for P(3HB) plastic production, B. cereus S356 also secreted bacteriocin, which had high antibacterial activity against the same species (Bacillus cereus). Overall, this work demonstrated the possibility of co-producing eco-friendly biodegradable plastic P(3HB) and bacteriocin from renewable resources using the potential of B. cereus S356.

Investigating chemical pre-treatment methods: Valorization of wheat straw to enhance polyhydroxyalkanoate (PHA) production with novel isolate Bacillus paranthracis RSKS-3.

Wheat is a crucial food crop worldwide, generating straw upon post-harvest. The straw is often burned to enhance soil fertility, leading to massive air pollution. In this study, wheat straw was investigated for the production of Polyhydroxyalkanoate (PHA) using the novel isolate Bacillus paranthracis RSKS-3. The wheat straw was pulverized and valorized with different acids (2 % and 4 % H2SO4, acetic acid, and hydrochloric acid) and alkalis (2 % and 4 % NaOH, calcium carbonate, and potassium hydroxide). The validation of carbohydrates was done using the Molisch test by analyzing purple-ring production and the DNS test which concluded 4 % H2SO4 as an effective treatment with a maximal sugar yield of 5.04 mg/mL at P < 0.05. The bioconversion efficiency of the extract to PHA resulted in 0.87 g/L by Bacillus paranthracis RSKS-3, later characterized by Ultraviolet (UV)-spectroscopy and FT-IR assessment. The findings of the research offer a potential strategy to mitigate airborne pollutants that result from smouldering wheat straw, thereby contributing significant improvements to sustainable development.

Monodisperse polyhydroxyalkanoate nanoparticles as self-sticky and bio-resorbable tissue adhesives.

Monodisperse nanoparticles of biodegradable polyhydroxyalkanoates (PHAs) polymers, copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB), are synthesized using a membrane-assisted emulsion encapsulation and evaporation process for biomedical resorbable adhesives. The precise control over the diameter of these PHA particles, ranging from 100 nm to 8 µm, is achieved by adjusting the diameter of emulsion or the PHA concentration. Mechanical properties of the particles can be tailored based on the 3HB to 4HB ratio and molecular weight, primarily influenced by the level of crystallinity. These monodisperse PHA particles in solution serve as adhesives for hydrogel systems, specifically those based on poly(N, N-dimethylacrylamide) (PDMA). Semi-crystalline PHA nanoparticles exhibit stronger adhesion energy than their amorphous counterparts. Due to their self-adhesiveness, adhesion energy increases even when those PHA nanoparticles form multilayers between hydrogels. Furthermore, as they degrade and are resorbed into the body, the PHA nanoparticles demonstrate efficacy in in vivo wound closure, underscoring their considerable impact on biomedical applications.

Complete sequence randomness of lactate-based copolymers (LAHBs) with varied lactate monomer fractions employing a series of propionyl-CoA transferases.

Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Current trends in medium-chain-length polyhydroxyalkanoates: Microbial production, purification, and characterization.

Polyhydroxyalkanoates (PHAs) have gained interest recently due to their biodegradability and versatility. In particular, the chemical compositions of medium-chain-length (mcl)-PHAs are highly diverse, comprising different monomers containing 6-14 carbon atoms. This review summarizes different feedstocks and fermentation strategies to enhance mcl-PHA production and briefly discusses the downstream processing. This review also provides comprehensive details on analytical tools for determining the composition and properties of mcl-PHA. Moreover, this study provides novel information by statistically analyzing the data collected from several reports on mcl-PHA to determine the optimal fermentation parameters (specific growth rate, PHA productivity, and PHA yield from various structurally related and unrelated substrates), mcl-PHA composition, molecular weight (MW), and thermal and mechanical properties, in addition to other relevant statistical values. The analysis revealed that the median PHA productivity observed in the fed-batch feeding strategy was 0.4 g L-1 h-1, which is eight times higher than that obtained from batch feeding (0.05 g L-1 h-1). Furthermore, 3-hydroxyoctanoate and -decanoate were the primary monomers incorporated into mcl-PHA. The investigation also determined the median glass transition temperature (-43°C) and melting temperature (47°C), which indicated that mcl-PHA is a flexible amorphous polymer at room temperature with a median MW of 104 kDa. However, information on the monomer composition or heterogeneity and the associated physical and mechanical data of mcl-PHAs is inadequate. Based on their mechanical values, the mcl-PHAs can be classified as semi-crystalline polymers (median crystallinity 23%) with rubber-like properties and a median elongation at break of 385%. However, due to the limited mechanical data available for mcl-PHAs with known monomer composition, identifying suitable processing tools and applications to develop mcl-PHAs further is challenging.

Discovery of periplasmic solute binding proteins with specificity for ketone bodies: ß-hydroxybutyrate binding proteins.

Polyhydroxyalkanoates are a class of biodegradable, thermoplastic polymers which represent a major carbon source for various bacteria. Proteins which mediate the translocation of polyhydroxyalkanoate breakdown products, such as ß-hydroxybutyrate (BHB)-a ketone body which in humans serves as an important biomarker, have not been well characterized. In our investigation to screen a solute-binding protein (SBP) which can act as a suitable recognition element for BHB, we uncovered insights at the intersection of bacterial metabolism and diagnostics. Herein, we identify SBPs associated with putative ATP-binding cassette transporters that specifically recognize BHB, with the potential to serve as recognition elements for continuous quantification of this analyte. Through bioinformatic analysis, we identified candidate SBPs from known metabolizers of polyhydroxybutyrate-including proteins from Cupriavidus necator, Ensifer meliloti, Paucimonas lemoignei, and Thermus thermophilus. After recombinant expression in Escherichia coli, we demonstrated with intrinsic tryptophan fluorescence spectroscopy that four candidate proteins interacted with BHB, ranging from nanomolar to micromolar affinity. Tt.2, an intrinsically thermostable protein from Thermus thermophilus, was observed to have the tightest binding and specificity for BHB, which was confirmed by isothermal calorimetry. Structural analyses facilitated by AlphaFold2, along with molecular docking and dynamics simulations, were used to hypothesize key residues in the binding pocket and to model the conformational dynamics of substrate unbinding. Overall, this study provides strong evidence identifying the cognate ligands of SBPs which we hypothesize to be involved in prokaryotic cellular translocation of polyhydroxyalkanoate breakdown products, while highlighting these proteins' promising biotechnological application.

The biological and therapeutic assessment of a P(3HB-co-4HB)-bioactive glass-graphene composite biomaterial for tissue regeneration.

An ideal wound dressing should create a healing environment that relieves pain, protects against infections, maintains moisture, removes debris, and speeds up wound closure and repair. However, conventional options like gauze often fall short in fulfilling these requirements, especially for chronic or nonhealing wounds. Hence there is a critical need for inventive formulations that offer efficient, cost-effective, and eco-friendly alternatives. This study focuses on assessing the innovative formulation based on a microbial-derived copolymer known as poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB) bioactive glass and graphene particles, and exploring their biological response in vitro and in vivo-to find the best combination that promotes cell adhesion and enhances wound healing. The formulation optimized at concentration of bioactive glass (1 w/w%) and graphene (0.01 w/w%) showed accelerated degradation and enhanced blood vessel formation. Meanwhile biocompatibility was evaluated using murine osteoblasts, human dermal fibroblasts, and standard cell culture assays, demonstrating no adverse effects after 7 days of culture and well-regulated inflammatory kinetics. Whole thickness skin defect using mice indicated the feasibility of the biocomposites for a faster wound closure and reduced inflammation. Overall, this biocomposite appears promising as an ideal wound dressing material and positively influencing wound healing rates.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Polyhydroxyalkanoate Copolymer Production by Recombinant Ralstonia eutropha Strain 1F2 from Fructose or Carbon Dioxide as Sole Carbon Source.

Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB-4, cannot produce PHA. Strain 1F2, derived from strain PHB-4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO2. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 106 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA.

Performance of Recycled Polylactic Acid/Amorphous Polyhydroxyalkanoate Blends.

Blends of polylactic acid (PLA) with amorphous polyhydroxyalkanoate (aPHA) are less brittle than neat PLA, thus enabling their use as biodegradable packaging. This work investigated the impact of recycling on the properties of neat PLA and PLA/aPHA blends with 90 and 75 wt. % PLA. After the materials were subjected to five heat histories in a single-screw extruder, the mechanical, rheological, and thermal properties were measured. All recycled compounds with 100% PLA and 75% PLA had similar decomposition behavior, whereas the decomposition temperatures for the blends with 90% PLA decreased with each additional heat cycle. The glass transition and melting temperatures were not impacted by reprocessing, but the crystallinity increased with more heat cycles. The complex viscosity of the reprocessed PLA and PLA/aPHA blends was much lower than for the neat PLA and increasing the number of heat cycles produced smaller reductions in the complex viscosity of 100% PLA and the blend with 90% PLA; no change in complex viscosity was observed for blends with 75% PLA exposed to 2 to 5 heat cycles. The tensile properties were not affected by reprocessing, whereas the impact strength for the 75% PLA blend decreased with reprocessing. These properties suggest that users will be able to incorporate scrap into the neat resin for thermoformed packaging.

Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Impact of the environmental parameters on single cell protein production and composition by Cupriavidus necator.

Due to the rapid increase in the world's population, many developing countries are facing malnutrition problems, including famine and food insecurity. Particularly, the deficiency of protein sources becomes a serious problem for human and animal nutrition. In this context, Single Cell Proteins, could be exploited as an alternative source of unconventional proteins. The aim of the study was to investigate SCP production and composition by Cupriavidus necator under various environmental conditions, temperature and pH values. A mono-factorial approach was implemented using batch bioreactor cultures under well-controlled conditions. Results were compared in terms of bacterial growth and SCP composition (proteins, nucleic acids, amino acids and elemental formula). Complementary analyses were performed by flow cytometry to study cell morphology, membrane permeability and the presence of Poly(3-hydroxybutyrate) (PHB) production. Our data confirmed the ability of C. necator to produce high amount of proteins (69 %DW at 30 °C and pH7). The results showed that temperature and pH independently impact SCP production and composition. This impact was particularly observed at the highest temperature (40 °C) and also the lowest pH value (pH5) providing lower growth rates, cell elongation, changes in granularity and lower amounts of proteins (down to 44 %DW at pH5) and nucleic acids. These low percentages were related to the production of PHB production (up to 44 %DW at 40 °C) which is the first report of a PHB accumulation in C. necator under nutrient unlimited conditions.

Unlocking growth potential in Halomonas bluephagenesis for enhanced PHA production with sulfate ions.

The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.

Ammonia-dependent reducing power redistribution for purple phototrophic bacteria culture-based biohydrogen production.

The global energy crisis has intensified the search for sustainable and clean alternatives, with biohydrogen emerging as a promising solution to address environmental challenges. Leveraging photo fermentation (PF) process, purple phototrophic bacteria (PPB) can harness reducing power derived from organic substrates to facilitate hydrogen production. However, existing studies report much lower H2 yields than theoretical value when using acetate as carbon source and ammonia as nitrogen source, primarily attributed to the widely employed pulse-feeding mode which suffers from ammonia inhibition effect on nitrogenase. To address this issue, a continuous feeding mode was applied to avoid ammonia accumulation in this study. On the other hand, other pathways like carbon fixation and polyhydroxyalkanoate (PHA) formation could compete reducing power with H2 production. However, the reducing power allocation under continuous feeding mode is not yet clear. In this study, the reducing power allocation and hydrogen production performance were evaluated under various ammonia loading, using acetate as carbon source and infrared LED at around 50 W·m-2 as light source. The results show that (a) The absence of ammonia resulted in the best performance for hydrogen production, with 44 % of the reducing power distributed to H2 and the highest H2 volumetric productivity, while the allocation of reducing power to hydrogen production stopped when ammonia loading was above 7.6 mg NH4-N·L-1·d-1; (b) when PPB required to eliminate reducing power under ammonia limited conditions, PHA production was the preferred pathway followed by the hydrogen production pathway, but once PHA accumulation reached saturation, hydrogen generation pathway dominated; (c) under ammonia limited conditions, the TCA cycle was more activated rendering higher NADH (i.e. reducing power) production compared with that under ammonia sufficient conditions which was verified by metagenomics analysis, and all the hydrogen production, PHA accumulation and carbon fixation pathways were highly active to dissipate reducing power. This work provides the insight of reducing power distribution and PPB biohydrogen production variated by ammonia loading under continuous feeding mode.