CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Optimized cell growth and poly(3-hydroxybutyrate) synthesis from saponified spent coffee grounds oil.

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C0) and the specific growth rate (µ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C0 = 2.9-23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C0 = 19.1 g/L) saponified SCG oil. Peak µ-values of 0.139 h-1 and 0.145 h-1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates (µ = 0.35 h-1) than on the crude SCG oil (µ = 0.23 h-1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA. KEY POINTS: • The bioavailability of plant oil substrates can be improved via saponification. • Cell growth and inhibition were accurately described by the Han-Levenpsiel model. • Mixing crude and saponified oils enable variation of free fatty acid content.

Characterization of an (R)-specific enoyl-CoA hydratase from Streptomyces sp. strain CFMR 7: A metabolic tool for enhancing the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as a commercial bioplastic due to its biodegradability, thermoplastic and mechanical properties. The properties of this copolymer are greatly affected by the composition of 3HHx monomer. One of the most efficient ways to modulate the composition of 3HHx monomer in P(3HB-co-3HHx) is by manipulating the (R)-3HHx-CoA monomer supply. In this study, a new (R)-specific enoyl-CoA hydratase originating from a non-PHA producer, Streptomyces sp. strain CFMR 7 (PhaJSs), was characterized and found to be effective in supplying 3HHx monomer during in vivo production of P(3HB-co-3HHx) copolymer. The P(3HB-co-3HHx) copolymer produced from the Cupriavidus necator transformant that harbors phaJSs, PHB-4/pBBR1-CBP-M-CPF4JSs, showed enhanced 3HHx incorporation of up to 11 mol% without affecting the P(3HB-co-3HHx) production when palm oil was used as the carbon source. In addition, both kcat and kcat/Km of PhaJSs were higher toward the C6 than the shorter C4 substrates, underscoring the preference for 3-hydroxyhexanoyl-CoA. These results suggest that PhaJSs has a significant ability to supply 3HHx monomers for PHA biosynthesis via ß-oxidation and can be applied for metabolic engineering of robust PHA-producing strains.

Impact of various ß-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active ß-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important ß-ketothiolases for C6 metabolism in ß-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six ß-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

High amounts of medium-chain-length polyhydroxyalkanoates subunits can be accumulated in recombinant Cupriavidus necator with wild-type synthase.

Recombinant Cupriavidus necator H16/pMPJAS03, expressing a P. putida KT2440 enoyl-CoA hydratase (phaJ), was able to synthesize short-chain-length/ medium-chain-length (scl-mcl) PHA copolymers with a high content of mcl subunits using its native poly(3-hydroxyalkanoate) synthase. The cells were cultivated on fructose with canola oil or canola oil/decanoic acid (DA) mixtures in fed-batch fermentations. The recombinant C. necator H16 (without any synthase modification) produced a polymer composed of 3-hydroxybutyrate (3HB) with mcl-subunits, including 3-hydroxyhexanoate (3HHx), and about 300-fold more 3-hydroxyoctanoate (3HO) than the yields reported in previous studies, as well as a significant amount of 3-hydroxydecanoate (3HD). Increasing the DA content in the feed from 0% to 15% v/v increased the molar content of the 3HD subunits from 1.2 to 2.1 mol%. The presence of larger monomers, such as 3HO and 3HD, decreased the crystallinity and melting temperature and modified the mechanical properties of the polymers. Thus, replacing either of the two gene products (phaJ or phaC1) required to produce PHA from CoA-3-hydroxy fatty acids with broader spectrum enzymes, is suitable for the production of commercially useful scl-mcl-PHA.

Innovative co-production of polyhydroxyalkanoates and methane from broken rice.

Broken rice, a low-cost starchy residue of the rice industry, can be an interesting substrate to reduce the polyhydroxyalkanoates (PHAs) production cost. However, since the most common PHAs-producing strains lack amylases, this waste must be firstly hydrolysed by additional commercial enzymes. In this work, the acidogenesis phase of the anaerobic digestion was exploited as efficient hydrolysis step to convert broken rice into volatile fatty acids (VFAs) to be used as PHAs carbon source by Cupriavidus necator DSM 545, one of the most promising PHAs-producing microbes. Broken rice, both non-hydrolysed and enzymatically hydrolysed, was processed in two continuous stirred tank reactors, at hydraulic retention times (HRT) of 5, 4 and, 3 days, to produce VFAs. The highest VFAs levels were obtained from non-hydrolysed broken rice which was efficiently exploited for PHAs accumulation by C. necator DSM 545. PHAs contents were higher after 96 h of incubation and, noteworthy, reached the highest value of 0.95 g/L in the case of 4 days HRT without any chemicals supplementation, except vitamins. Moreover, in view of a biorefinery approach, the residual solid fraction was used for methane production resulting in promising CH4 levels. Methane yields were very promising again for 4 days HRT. As such, this HRT resulted to be the most suitable to obtain effluents with high promise in terms of both PHAs accumulation and CH4 production. In addition, these results demonstrate that broken rice could be efficiently processed into two valuable products without any costly enzymatic pre-treatment and pave the way for future biorefining approaches where this by-product can be converted in a cluster of added-value compounds. Techno-economical estimations are in progress to assess the feasibility of the entire process, in view of supporting the low-cost conversion of organic waste into valuable products.

Properties of Degradable Polyhydroxyalkanoates (PHAs) Synthesized by a New Strain, Cupriavidus necator IBP/SFU-1, from Various Carbon Sources.

The bacterial strain isolated from soil was identified as Cupriavidus necator IBP/SFU-1 and investigated as a PHA producer. The strain was found to be able to grow and synthesize PHAs under autotrophic conditions and showed a broad organotrophic potential towards different carbon sources: sugars, glycerol, fatty acids, and plant oils. The highest cell concentrations (7-8 g/L) and PHA contents were produced from oleic acid (78%), fructose, glucose, and palm oil (over 80%). The type of the carbon source influenced the PHA chemical composition and properties: when grown on oleic acid, the strain synthesized the P(3HB-co-3HV) copolymer; on plant oils, the P(3HB-co-3HV-co-3HHx) terpolymer, and on the other substrates, the P(3HB) homopolymer. The type of the carbon source influenced molecular-weight properties of PHAs: P(3HB) synthesized under autotrophic growth conditions, from CO2, had the highest number-average (290 ± 15 kDa) and weight-average (850 ± 25 kDa) molecular weights and the lowest polydispersity (2.9 ± 0.2); polymers synthesized from organic carbon sources showed increased polydispersity and reduced molecular weight. The carbon source was not found to affect the degree of crystallinity and thermal properties of the PHAs. The type of the carbon source determined not only PHA composition and molecular weight but also surface microstructure and porosity of the polymer films. The new strain can be recommended as a promising P(3HB) producer from palm oil, oleic acid, and sugars (fructose and glucose) and as a producer of P(3HB-co-3HV) from oleic acid and P(3HB-co-3HV-co-3HHx) from palm oil.

Potential of preventive bioremediation to reduce environmental contamination by pesticides in an agricultural context: A case study with the herbicide 2,4-D.

One of the major problems with pesticides is linked to the non-negligible proportion of the sprayed active ingredient that does not reach its intended target and contaminates environmental compartments. Here, we have implemented and provided new insights to the preventive bioremediation process based on the simultaneous application of the pesticide with pesticide-degrading microorganisms to reduce the risk of leaching into the environment. This study pioneers such a practice, in an actual farming context. The 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) and one of its bacterial mineralizing-strains (Cupriavidus necator JMP134) were used as models. The 2,4-D biodegradation was studied in soil microcosms planted with sensitive (mustard) and insensitive (wheat) plants. Simultaneous application of a 2,4-D commercial formulation (DAM®) at agricultural recommended doses with 105 cells.g-1 dw of soil of the JMP134 strain considerably accelerated mineralization of the herbicide since its persistence was reduced threefold for soil supplemented with the mineralizing bacterium without reducing the herbicide efficiency. Furthermore, the inoculation of the Cupriavidus necator strain did not significantly affect the α- and ß-diversity of the bacterial community. By tackling the contamination immediately at source, the preventive bioremediation process proves to be an effective and promising way to reduce environmental contamination by agricultural pesticides.

Anabolism of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator DSM 545 from spent coffee grounds oil.

Oil extracted from spent coffee grounds (SCG) [yield 16.8 % (w/w)] was discovered to be a highly suitable carbon substrate for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3 HV)] copolymers by Cupriavidus necator DSM 545 in the absence of any traditional 3 HV precursors. Cells cultivated in a 3 L bioreactor (batch) reached a total biomass concentration of 8.9 g L-1 with a P(3HB-co-3 HV) (6.8 mol% 3 HV) content of 89.6 % (w/w). In contrast, cells grown on sunflower oil reached a total biomass concentration of 9.4 gL-1 with a P(3HB-co-3 HV) (0.2 mol% 3 HV) content of 88.1 % (w/w). It is proposed that the organism could synthesize 3 HV monomers from succinyl CoA, an intermediate of the tricarboxylic acid (TCA) cycle, via the succinate-propionate metabolic pathway.

Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and phaJ genes in Cupriavidus necator.

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.

Cupriavidus necator B-10646 growth and polyhydroxyalkanoates production on different plant oils.

The study addresses the growth of the wild-type strain Cupriavidus necator B-10646 and synthesis of polyhydroxyalkanoates by this strain on media containing plant oils with different compositions of fatty acids: palm, Siberian oilseed, and refined and unrefined sunflower seed oils. The study showed that the best carbon substrate was palm oil. Comparison of fatty acid compositions of the starting oils and unutilized residual substrates showed that C. necator B-10646 cells consumed the fatty acids from palm oil evenly while in experiments with other oils, they utilized polyenoic fatty acids first. Higher production parameters of the culture were obtained by preparation of emulsified oil medium using Tween 80 and sodium cocoyl glutamate as emulsifiers. All polyhydroxyalkanoate specimens were terpolymers that contained 3-hydroxybutyrate as the major component and minor amounts of 3-hydroxyvalerate (0.9-1.9 mol%) and 3-hydroxyhexanoate (0.5-1.1 mol%). Molecular weight of polyhydroxyalkanoate specimens depended on the type of plant oil and emulsifier.

Evaluation of BP-M-CPF4 polyhydroxyalkanoate (PHA) synthase on the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil using Cupriavidus necator transformants.

Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of Cupriavidusnecator transformants harbouring the phaCBP-M-CPF4 gene (including PHB¯4/pBBR1-CBP-M-CPF4) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa, along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of ß-ketothiolase (PhaACn) and NADPH-dependent acetoacetyl-CoA reductase (PhaBCn) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB-co-3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 × 106 to 1.8 × 106 Da. PhaCBP-M-CPF4 displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study.

Fed-batch cultivation and adding supplements to increase yields of polyhydroxybutyrate production by Cupriavidus necator from corn stover alkaline pretreatment liquor.

The aim of this study was to evaluate polyhydroxybutyrate (PHB) production and productivity with supplements under fed-batch cultivation at bioreactor scale (1.3 L). In this study, multiple supplements including oxidative enzyme, mediators, surfactants and silicon nanoparticles were added to Cupriavidus necator culture growing on alkaline pretreatment liquor (APL). At 1.3 L bioreactor scale, PHB production reached 3.3 g/L. To further enhance PHB production, fed-batch cultivation with two different feeding strategies were applied. Under single pulse feeding of 300 mL medium, PHB production reached 4.0 g/L. Under 4 pulses feeding of 75 mL medium each time, PHB production reached 4.5 g/L. This is the highest PHB production from lignin that the authors are aware of in literature.

In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy.

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs' and the absorption coefficient µa at 638 nm. A correlation of µs' with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L-1·h-1 and a final biomass of 106 g·L-1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.

Discarded Egg Yolk as an Alternate Source of Poly(3-Hydroxybutyrate-co-3-hydroxyhexanoate).

Many poultry eggs are discarded worldwide because of infection (i.e., avian flu) or presence of high levels of pesticides. The possibility of adopting egg yolk as a source material to produce polyhydroxyalkanoate (PHA) biopolymer was examined in this study. Cupriavidus necator Re2133/pCB81 was used for the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) or poly(3HHx), a polymer that would normally require long-chain fatty acids as carbon feedstocks for the incorporation of 3HHx monomers. The optimal medium contained 5% egg yolk oil and ammonium nitrate as a nitrogen source, with a carbon/nitrogen (C/N) ratio of 20. Time course monitoring using the optimized medium was conducted for 5 days. Biomass production was 13.1 g/l, with 43.7% co-polymer content. Comparison with other studies using plant oils and the current study using egg yolk oil revealed similar polymer yields. Thus, discarded egg yolks could be a potential source of PHA.

O2-tolerant [NiFe]-hydrogenases of Ralstonia eutropha H16: Physiology, molecular biology, purification, and biochemical analysis.

Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H2⇌2H++2e- even in the presence of O2. Catalytic and probably also noncatalytic mechanisms protect their active sites from being inactivated by reactive oxygen species, which makes them attractive subjects of investigation from both fundamental and applied perspectives. Prominent representatives of the O2-tolerant [NiFe]-hydrogenases have been isolated from the chemolithoautotrophic model organism Ralstonia eutropha H16, which can thrive in a simple mineral medium supplemented with the gases H2, O2, and CO2. In this chapter, we describe methods for cultivation and genetic manipulation of R. eutropha, both of which are prerequisites for the reproducible manufacturing of high-quality hydrogenase preparations. The purification procedures for two different O2-tolerant [NiFe]-hydrogenases from R. eutropha are described in detail, as well as the corresponding biochemical procedures used for the determination of the catalytic properties of these sophisticated enzymes.

Production and optimization of polyhydroxyalkanoates from non-edible Calophyllum inophyllum oil using Cupriavidus necator.

Polyhydroxyalkanoates (PHA) are biodegradable polymers found in the cellular masses of a wide range of bacterial species and the demand for PHA is steadily growing. In this work we have produced PHA from a low-cost substrate, Calophyllum inophyllum oil, using Cupriavidus necator. Effects of various process parameters such as Oil concentration, Nitrogen source and inoculum size on the production of PHA were studied using Response Surface Methodology. A quadratic equation was used in the model to fit the experimental data. It was found that the model could satisfactorily predict the PHA yield (R2=99.17%). Linear, quadratic and interaction terms used in the model were found to be statistically significant. Maximum PHA yield of 10.6gL-1 was obtained under the optimized conditions of oil concentration - 17.5%, inoculum concentration - 50mL/L and nitrogen content - 1.125gL-1, respectively. The product obtained was characterized using FTIR and NMR to confirm that it was PHA. The results demonstrate that C. inophyllum oil, a non-edible oil, can be potentially used as a low-cost substrate for the production of PHA.

An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm.

Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.

Polyhydroxyalkanoate production from sucrose by Cupriavidus necator strains harboring csc genes from Escherichia coli W.

Cupriavidus necator H16 is the most promising bacterium for industrial production of polyhydroxyalkanoates (PHAs) because of their remarkable ability to accumulate them in the cells. With genetic modifications, this bacterium can produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), which has better physical properties, as well as poly(3-hydroxybutyrate) (PHB) using plant oils and sugars as a carbon source. Considering production cost, sucrose is a very attractive raw material because it is inexpensive; however, this bacterium cannot assimilate sucrose. Here, we used the sucrose utilization (csc) genes of Escherichia coli W to generate C. necator strains that can assimilate sucrose. Especially, glucose-utilizing recombinant C. necator strains harboring the sucrose hydrolase gene (cscA) and sucrose permease gene (cscB) of E. coli W grew well on sucrose as a sole carbon source and accumulated PHB. In addition, strains introduced with a crotonyl-CoA reductase gene (ccr), ethylmalonyl-CoA decarboxylase gene (emd), and some other genetic modifications besides the csc genes and the glucose-utilizing mutations produced PHBHHx with a 3-hydroxyhexanoate (3HHx) content of maximum approximately 27 mol% from sucrose. Furthermore, when one of the PHBHHx-producing strains was cultured with sucrose solution in a fed-batch fermentation, PHBHHx with a 3HHx content of approximately 4 mol% was produced and reached 113 g/L for 65 h, which is approximately 1.5-fold higher than that produced using glucose solution.