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The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.

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.

International audience; Plasmid expression level heterogeneity in Cupriavidus necator was studied in response to stringent culture conditions, supposed to enhance plasmid instability, through plasmid curing strategies. Two plasmid curing strategies were compared based on their efficiency at generating heterogeneity in batch: rifampicin addition and temperature increase. A temperature increase from 30 degrees to 37 degrees C was the most efficient plasmid curing strategy. To generate a heterogeneous population in terms of plasmid expression levels, successive batches at supra optimal culture temperature (i.e. 37 degrees C) were initially conducted. Three distinct fluorescent subpopulations P-0 (not fluorescent), P-1 (low fluorescence intensity, median = 1 10(3)) and P-2 (high fluorescence intensity, median = 6 10(3)) were obtained. From there, the chemostat culture was implemented to study the long-term stress response under well-controlled environment at defined dilution rates. For dilution rates comprised between 0.05 and 0.10 h(-1), the subpopulation P-2 (62% vs 90%) was favored compared to P1 cells (54% vs 1%), especially when growth rate increased. Our biosensor was efficient at discriminating subpopulation presenting different expression levels under stringent culture conditions. Plus, we showed that controlling growth kinetics had a stabilizing impact on plasmid expression levels, even under heterogeneous expression conditions.

In this study, a phaCR gene encoding PHA synthase was identified in Rhodoligotrophos defluvii which was adjacent to β-ketothiolase encoded by phaAR gene and acetoacetyl-CoA reductase encoded by phaBR gene. Amino acid comparison of PhaCR showed the highest homology of 65.98% with PhaC of R. appendicifer, while its homology with typical class I PHA synthase in Cupriavidus necator was only 42.54%. PHA synthesis genes were then transformed into E. coli harboring phaCABR and phaCRABC which were cultured with 15 g/L glucose respectively, and 20.46 wt% and 16.95 wt% of CDW for poly(3-hydroxybutyrate) (PHB) were accumulated respectively. To further explore the effect of substrate specificity for PHA production, the ptsG gene was then deleted and 15 g/L glucose and 1.5 g/L propionate were co-employed as carbon sources, which enabled the synthesis of poly(3HB-co-3HV) copolymer. As a result, poly(3HB-co-3HV) was accumulated up to 24.74 wt% of CDW, and the highest content of 3-hydroxyvalerate (3HV) was 10.86 mol%. The Td5 was 260 °C, which implied that it possessed good thermal stability, and the Mw of GPC in recombinant strains were between 22 and 26 × 104 g/mol, and the highest PDI was 3.771. The structure of poly (3HB-co-3HV) copolymer was determined through 1H NMR analysis.

Marine macroalgae are potential renewable feedstocks for valuable biomaterials. Among them, alginate is a primary component in brown algae that can be nonenzymatically converted and enzymatically degraded by alginate lyases to 4-deoxy- l -erythro-5-hexoseulose uronic acid (DEH). Here, we constructed alginolytic enzyme complexes comprising two different alginate lyases for synergistic alginate degradation. The complexes showed good thermostability with 60% of the residual activity at high temperature (60 °C). Furthermore, they produced 0.85 and 0.18 mg/mL DEH from alginate and natural brown algae as substrates, respectively. The enzyme complex successfully decomposed brown algal biomass, resulting in a 3.15-fold improvement in DEH when compared to free enzymes. The Ralstonia eutropha strain with alginolytic enzyme complexes on the cell surface showed higher Polyhydroxybutyrate (PHB) production and produced 2.58 g/L PHB from alginate. After the use of alginate, remaining biomass such as fucoidan and laminaran can also be used in the future for high value ingredients in nutritional, medical device, skincare and dermatological products. These results demonstrate that it is possible to create more efficient strategies for producing biodegradable PHB and functional polysaccharides from brown algal substrates.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Purpose To synthesize and investigate polyhydroxyalkanoates (PHAs) with different monomer composition and percentages and polymer films prepared from them. Results Various PHAs: homopolymer poly-3-hydroxybutyrate P(3HB) and 2-, 3-, and 4-component copolymers comprising various combinations of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), and 3-hydroxyhexanoate (3HHx) monomers were synthesized under specialized conditions. Relationships were found between the monomer composition of PHAs and their molecular-weight and thermal properties and degree of crystallinity. All copolymers had decreased weight average molecular weights, Mw (to 390–600 kDa), and increased values of polydispersity (3.2–4.6) compared to the P(3HB). PHA copolymers showed different thermal behavior: an insignificant decrease in Tmelt and the presence of the second peak in the melting region and changes in parameters of crystallization and glass transition. At the same time, they retained thermostability, and the difference between Tmelt and Tdegr was at least 100–120 °C. Incorporation of 4HB, 3HV, and 3HHx monomer units into the 3-hydroxybutyrate chain caused changes in the amorphous to crystalline ratio and decreased the degree of crystallinity (Cx) to 20–40%. According to the degree to which the monomers reduced crystallinity, they were ranked as follows: 4HB – 3HHx – 3HV. A unique set of films was produced; their surface properties and physical/mechanical properties were studied as dependent on PHA composition; monomers other than 3-hydroxybutyrate were found to enhance hydrophilicity, surface development, and elasticity of polymer films. Conclusion An innovative set of PHA copolymers was synthesized and solution-cast films were prepared from them; the copolymers and films were investigated as dependent on polymer chemical composition. Results obtained in the present study contribute to the solution of a critical issue of producing degradable polymer materials.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.

Polyhydroxyalkanoates (PHAs) are polyesters of significant interest due to their biodegradability and properties similar to petroleum-derived plastics, as well as the fact that they can be produced from renewable sources such as by-product streams. In this study, brewer’s spent grain (BSG), the main by-product of the brewing industry, was subjected to a set of physicochemical pretreatments and their effect on the release of reducing sugars (RS) was evaluated. The RS obtained were used as a substrate for further PHA production in Burkholderia cepacia, Bacillus cereus, and Cupriavidus necator in liquid cultures. Although some pretreatments proved efficient in releasing RS (acidthermal pretreatment up to 42.1 gRS L-1 and 0.77 gRS g-1 dried BSG), the generation of inhibitors in such scenarios likely affected PHA production compared with the process run without pretreatment (direct enzymatic hydrolysis of BSG). Thus, the maximum PHA accumulation from BSG hydrolysates was found in the reference case with 0.31 ± 0.02 g PHA per g cell dried weight, corresponding to 1.13 ± 0.06 g L-1 and a PHA yield of 23 ± 1 mg g-1 BSG. It was also found that C. necator presented the highest PHA accumulation of the tested strains followed closely by B. cepacia, reaching their maxima at 48 h. Although BSG has been used as a source for other bioproducts, these results show the potential of this by-product as a no-cost raw material for producing PHAs in a waste valorization and circular economy scheme.

Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2.

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.

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.

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.

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H2 and CO2 and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO2 under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20-60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO2. And, we engineered C. necator to convert CO2 into the LCO, a plant growth enhancer, with titers of ~1.4 mg/L-equivalent to yields in its native source, Bradyrhizobium. We applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding of how a gas-utilizing bacteria can promote sustainable production.

The valorization of agricultural waste is a sustainable way of how to utilize renewable resources wisely. Grape pomace is a by-product of the manufacturing of the must. The potential of Cupriavidus necator, Halomonas halophila and Halomonas organivorans to produce poly(3-hydroxybutyrate) (PHB) by using oil and fermentable sugars derived from grape pomace has been studied. C. necator proved to be the best producer of PHB when utilizing grape pomace extract as a carbon source. After 29.5 h of fermentation of C. necator in 2-L bioreactor, PHB titers reached 8.3 g l−1 and 63 % PHB content in bacteria cell dry mass. The average molecular weight and the polydispersity of accumulated polymer was 512.2 kDa and 1.2, respectively. In addition to fermentable sugars, this work indicates that oil, pigments, phenolics, lignin and cellulose can be isolated from seeds and the rest of the grape pomace to support the cascading utilization of agricultural waste and natural valuable resources recovery.

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 β-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.

Enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. Although nitrilases from several sources have been characterized, there exists a scope for identifying broad spectrum nitrilases exhibiting higher substrate tolerance and better thermostability to develop industrially relevant biocatalytic processes. Through genome mining, we have identified nine novel nitrilase sequences from bacteria and evaluated their activity on a broad spectrum of 23 industrially relevant nitrile substrates. Nitrilases from Zobellia galactanivorans, Achromobacter insolitus and Cupriavidus necator were highly active on varying classes of nitriles and applied as whole cell biocatalysts in lab scale processes. Z. galactanivorans nitrilase could convert 4-cyanopyridine to achieve yields of 1.79 M isonicotinic acid within 3 h via fed-batch substrate addition. The nitrilase from A. insolitus could hydrolyze 630 mM iminodiacetonitrile at a fast rate, effecting 86 % conversion to iminodiacetic acid within 1 h. The arylaliphatic nitrilase from C. necator catalysed enantioselective hydrolysis of 740 mM mandelonitrile to (R)-mandelic acid in 4 h. Significantly high product yields suggest that these enzymes would be promising additions to the suite of nitrilases for upscale biocatalytic application.

International audience; A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L-1 level from CO2 for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO2, H-2 and air inputs and the ability to operate at high pressures. The bioreactor configuration and cultivation strategy enabled growth of Cupriavidus necator strains for long periods, to reach over 3 g L-1 dry cell weight. No negative impact of the high pressure was observed on viability of the strains up to more than 4 bar overpressure. The cultivation was then carried out using an engineered isopropanol producing strain; in this case, 3.5 g L-1 isopropanol was obtained from CO2 as the sole carbon source. This is the first reported demonstration of a successful production from engineered bacteria of product in the g L-1 range on CO2 , raising the prospect of future development of CO2-based bioprocesses.

Due to their advantageous properties, polyhydroxyalkanoates are a promising alternative to conventional petroleum-based plastics. Currently, high production costs in upstream and downstream have to be reduced to make the plastic material competitive. As most of the PHA producing organisms metabolize a wide range of substrates upstream cost can be reduced using carbon rich waste material. For large scale application sophisticated control approaches based on mathematical models can help to further increase the productivity. In the present work, a hybrid cybernetic model approach is presented, which is adapted to experiments with fructose and acetate feeding, respectively. Furthermore, the validated model was used to qualitatively predict the effect of co-substrate feeding.

Process stability is a key operational issue when operating dark fermentation with mixed microbial cultures for hydrogen production. This study aimed at mitigating the instability of hydrogen production by separately adding exogenous pure strains suspected to have key roles in fermentative cultures. Among them, Clostridium acetobutylicum, Clostridium pasteurianum and Lactobacillus bulgaris which became predominant within the mixed culture strongly reduced the spectrum of produced metabolites and H2 production variability. Interestingly, Escherichia coli and Cupriavidus necator, which remained in minor abundance, maintained a high and stable H2 production while lowering the metabolic variability. 16S rRNA revealed that this could correlate to a simplification of the microbial diversity and the non-emergence of spore-forming competitors such as Sporolactobacillus sp. These results illustrate the potential beneficial role of minor OTUs as keystone species on H2-producing complex ecosystem and support the possibility of using them to engineer the ecosystem and maintain high and stable performances.

Dependency on plastic commodities has led to a recurrent increase in their global production every year. Conventionally, plastic products are derived from fossil fuels, leading to severe environmental concerns. The recent coronavirus disease 2019 pandemic has triggered an increase in medical waste. Conversely, it has disrupted the supply chain of personal protective equipment (PPE). Valorisation of food waste was performed to cultivate C. necator for fermentative production of biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The increase in biomass, PHBV yield and molar 3-hydroxy valerate (3HV) content was estimated after feeding volatile fatty acids. The fed-batch fermentation strategy reported in this study produced 15.65 ± 0.14 g/L of biomass with 5.32 g/L of PHBV with 50% molar 3HV content. This is a crucial finding, as molar concentration of 3HV can be modulated to suit the specification of biopolymer (film or fabric). The strategy applied in this study addresses the issue of global food waste burden and subsequently generates biopolymer PHBV, turning waste to wealth.

Artificial synthesis of glucose, the monomer of starch, from renewable resources and CO

The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.

Vinasse and sugarcane molasses are abundant residues from the ethanol mills that were used as substrate for polyhydroxybutyrate (PHB) production in this study. The molasses was previously hydrolyzed by acid (HCl or H2SO4) or enzymatic procedures (invertase or baker’s yeast), and C. necator growth kinetics were compared, whereby enzymatic hydrolysis was chosen for subsequent tests based upon maximum specific growth rate (μmax). The addition of vinasse in a medium containing molasses increased μmax from 0.19 to 0.36 h−1 due to the presence of organic acids in this substrate, which were consumed in the first hours of cultivation. In the production stage, two pulses of molasses were added, reaching 11.7 g·L-1 of PHB and 56% of product accumulation. The nitrogen content in molasses (around 1.8 g·L−1) was responsible for an atypical increase in biomass concentration during the production phase, which renders this substrate interesting, depending on the cultivation method.

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

Microbial degradation is a natural means to combat ubiquitous, harmful and non-covalently bound plasticizers, phthalic acid esters (PAEs), due to their slower photo-degradation and hydrolysis rates. In microbial aerobic degradation of phthalates, phthalate dioxygenase system (PDS) belongs to a large family of Rieske non-heme iron oxygenases with type1A electron-transfer system. Two important proteins of this system include: phthalate dioxygenase reductase (PDR), a flavo-iron-sulfur protein having NADH-dependent oxidoreductase activity; and phthalate dioxygenase oxygenase (PDO), a non-heme iron protein with oxygenase activity. In the present study, phthalate dioxygenase reductase (RePDR) from Ralstonia eutropha CH34, has been cloned, expressed and purified. Further, in order to understand the interactions between RePDO and RePDR, protein-protein docking has been carried out. The homology model structures of the individual proteins served as an input for protein-protein interaction studies and helps in the prediction of complex structure. Important interface residues from both the individual counterparts, i.e. RePDO and RePDR have been identified and the evolutionary relationship between these important interface residues has also been established. Moreover, the stability of these interactions involving highly conserved interface residues, highlights their involvement in structurally conserved interactions and play significant role in phthalate degrading system.

Kenaf biomass (KB) was employed as feedstock for the synthesis of polyhydroxybutyrate (PHB) using Ralstonia eutropha to replace conventional petroleum-derived polymers. Various pretreatments followed by enzymatic saccharification were applied to release monomeric sugars from KB for PHB production. The effects of increasing concentration of Na2CO3 + Na2SO3 (NaC + NaS) pretreated KB hydrolysates (20–40 g/L) on PHB production were investigated. NaC + NaS pretreated KB hydrolysates (30 g/L) exhibited maximum 70.0% PHA accumulation, with PHB titers of 10.10 g/L and PHB yields of about 0.488 g/g of reducing sugar produced within 36 h of fermentation. PHA accumulation, PHB yield and R. eutropha growth performance using KB hydrolysates were found to be comparable with those of synthetic sugar mixture. Characterization of the produced PHB in terms of crystalline structure, and thermal properties was done using various analytical techniques and results coincide with standard PHB. Thus, green liquor pretreated KB hydrolysates deliver a promising and economically feasible carbon substrate for PHB production.

The aim of this study was to conduct an economic assessment of the entire polyhydroxybutyrate (PHB) production process by Cupriavidus necator, using citric molasses as carbon source and employing propylene carbonate as solvent coupled with different extraction pretreatment methods. Four extraction alternatives were assayed. The alternatives that uses high pressure and heat to pretreat the biomass showed equally lower production costs (US$ 4.28 per kg) and better economic indicators among evaluated scenarios. It was observed that method’s extraction capacity is not decisive in selecting the best alternative, but there must be the simultaneous analysis of extractability capacity and expenditures in equipment and utilities. Through five times increase in production capacity, the production cost significantly decreased, reaching US$ 2.71 per kg. Furthermore, achieving high cellular concentration can be responsible for a reduction of up to 18% in the production cost.

As a vital byproduct of chemical intermediates for pesticides and cytotoxin inhibitors, 2-bromo-4-nitrophenol (2B4NP) is toxic to aquatic life. However, bacterial cultures mineralizing 2B4NP have not been reported. In this study, Cupriavidus sp. strain NyZ375 was isolated from aromatics-polluted soil and identified by 16S rRNA gene sequencing. Via an oxidative pathway, it grows on 120 μM 2B4NP as the only carbon source, releasing approximately equivalent amount of nitrite. Three genes (bnpABC) in a 4.8-kb DNA fragment from a draft genome was found to be responsible for the initial reactions in 2B4NP degradation. BnpAB are the monooxygenase and flavin reductase components, respectively, catalyzing the monooxygenation of 2B4NP to bromohydroquinone (BHQ, 63%) and 1,2,4-benzenetriol (BT, 37%) with concomitant nitrite release, in the presence of NADH and FAD. BnpC is a hydroxyquinol 1,2-dioxygenase catalyzing the ring-cleavage oxidation of BT to maleylacetate. The by-product BHQ was not further degraded by strain NyZ375 but was not toxic to its growth at a concentration of 100 μM. It can be tentatively concluded that BnpAB is evolutionary close to the 2,4,6-trichlorophenol monooxygenase TcpAX from Cupriavidus necator JMP134. The 2B4NP catabolism by this new isolate illustrates a pathway distinct from those of all identified 2-chloro-4-nitrophenol utilizers.

The search of alternative substrates for the synthesis of polyhydroxyalkanoates (PHA) has become an important factor in order to decrease the production costs. Therefore, the use of industrial by-products or waste materials as carbon and energy sources for different PHA-producing microorganisms has been evaluated during the last decades. Recombinant strains of Gordonia polyisoprenivorans VH2 harboring plasmid pAK68, which contains phaCAB from Ralstonia eutropha and plasmid pAK71 comprising phaC1 from Pseudomonas aeruginosa were evaluated for PHA production. Cultivations were performed in shake flasks, using different carbon sources under an N-starvation condition. Having in consideration the rubber degrading capability of the actinomycete, poly(cis-1,4-isoprene) was utilized as sole carbon source. After twenty days of cultivation the PHA content was analyzed using GC-MS. In cultures of G. polyisoprenivorans harboring pAK68, the detection of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units indicated the accumulation of the copolyester poly(3HB-co-3HV). This study proposes a recycling method for rubber waste through its biotransformation into bioplastic.

Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C4 to C8. The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.

The high production cost of polyhydroxybutyrate (PHB) particularly substrate cost limits its industrial applications. Reduced substrate cost by utilizing cheaper carbon source enables to improve its economic viability. Cane final molasses are considered potential feedstock due to its abundance, low cost, and high sugar content. However, sucrose in molasses required to convert to its monomers: fructose and glucose by pretreatments for the bacteria consumption during the fermentation process. In this study, the main components of molasses and sugar content in various pretreated molasses were analyzed. The PHB content and cell dry weight of C. necator biomass fermented utilizing glucose, untreated molasses and pretreated molasses as carbon sources are compared. The molasses consist of 34.49 ± 0.13% sucrose, 5.93 ± 0.07% glucose and 5.00 ± 0.04% fructose respectively. The hydrothermal acid pretreated molasses (oil bath) contains the highest fructose and glucose content which are 21.31 ± 0.04% and 20.32 ± 0.06% respectively. Although the hydrothermal acid pretreated molasses (oil bath) contains the highest fructose and glucose content, the cell dry weight and PHB produced by C. necator using hydrothermal acid pretreated molasses (oven) is the highest which are 2.86 ± 0.82 g and 27% respectively.

Marine invertebrates rely mainly on innate immune mechanisms that include both humoral and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity, are important components of the innate immune defense system in marine invertebrates. They provide an immediate and rapid response to invading microorganisms. The impact of amorphous poly-β-hydroxybutyrate (PHB-A) (1 mg PHB-A L−1) on gene expression of the AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue mussel larvae was investigated during “in vivo” challenge tests with Vibrio coralliilyticus (105 CFU mL−1). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-PCR using the 18srRNA gene as a housekeeping gene. Our data demonstrated that AMPs genes had a tendency to be upregulated in challenged mussel larvae, and the strongest expression was observed from 24 h post-exposure onwards. The presence of both PHB-A and the pathogen stimulated the APMs gene expression, however no significant differences were noticed between treatments or between exposure time to the pathogen V. coralliilyticus. Looking at the phenoloxidase activity in the infected mussels, it was observed that the addition of PHB-A significantly increased the activity.

Grass is one of the most abundant plant families on Earth, however it has limited applications beyond the use as animal feed due to its low economic and nutritional value. Here we developed a concept for the increased valorization of grass to microbial protein (MP) for human food and biogas. We followed a four-step approach, starting with lactic acid (LA) fermentation of grass juice (GJ) using Bacillus coagulans, followed by LA extraction via membrane electrolysis, aerobic MP production using Cupriavidus necator and digestion of the side-products for biogas production. Temperature (28–55°C) had a strong effect on the performance of batch GJ fermentations under axenic and non-axenic tests performed with B. coagulans, with the highest LA concentration (16–18 gLA/L) and productivity (0.45–0.56 gLA/L/h) achieved at 45°C. Continuous non-axenic thermophilic (45°C) GJ fermentation enabled the highest LA titers (23 gLA/L) at 6h HRT while 1.5h HRT enabled the highest productivity (9.9 gLA/L/h). The microbial community was dominated (77–97%, HRT

Starch-rich by-products could be efficiently exploited for polyhydroxyalkanoates (PHAs) production. Unfortunately, Cupriavidus necator DSM 545, one of the most efficient PHAs producers, is not able to grow on starch. In this study, a recombinant amylolytic strain of C. necator DSM 545 was developed for the one-step PHAs production from starchy residues, such as broken rice and purple sweet potato waste. The glucodextranase G1d from Arthrobacter globiformis I42 and the α-amylase amyZ from Zunongwangia profunda SM-A87 were co-expressed into C. necator DSM 545. The recombinant C. necator DSM 545 #11, selected for its promising hydrolytic activity, produced high biomass levels with noteworthy PHAs titers: 5.78 and 3.65 g/L from broken rice and purple sweet potato waste, respectively. This is the first report on the engineering of C. necator DSM 545 for efficient amylase production and paves the way to the one-step conversion of starchy waste into PHAs.

The recycling of polyhydroxybutyrate (PHB) via depolymerization-polymerization is here proposed and assessed for the first time. Effective depolymerization of PHB to crotonic acid through a thermolytic distillation at mild conditions (170 °C and 150 mbar), and without the need of any catalyst, gives a crotonic acid-enriched condensate (94% yield and 98% selectivity towards trans-crotonic acid) that was used without any further purification as a substrate for producing renewed PHB with a culture of Cupriavidus necator. The yield of PHB (0.6 g PHB per g of crotonic acid), final PHB content (63%), and PHB-molecular weight (1.5 MDa) confirmed the technical feasibility of this tandem approach, with a 55% overall yield. A preliminary evaluation of the energy consumption of various depolymerization approaches indicated that the thermolytic distillation consumes 20–25% less energy than previously reported methods; the energy requirement for producing crotonic acid from PHBwaste with 50% moisture has a still acceptable energy demand (4 MJ/kgcrotonic acid), comparable to the energy required for obtaining fermentable sugars (4.4 MJ/kgsugar) to be used for feeding bacteria that accumulate PHB.

Valorization of CO2 is key to establishing a carbon–neutral society. However, CO2 possesses a strong C = O with bond energy of 750 kJ/mol, which makes it difficult for electrochemical conversion. As a result, the conversion of CO2 to complex molecules remains a grand challenge and product profiles based on inorganic catalysis are only limited to a few simple chemicals, such as formate, acetate, and ethanol. Here, we employed microbial electrosynthesis to diversify the product spectrum. First, an engineered Cupriavidus necator strain was constructed to produce lycopene from H2 and CO2. Then, by coupling de novo microbial catalyzed lycopene synthesis with inorganic catalyzed water electrolysis, a microbial electrosynthesis (MES) system was developed. Lycopene, representing the most complex nonnative molecules in the MES system, was produced with CO2 as the sole carbon source and H2 derived from electrochemical water splitting as the reducing power. Moreover, the produced lycopene provided long-term cytoprotective capacity against reactive oxygen species, thereby addressing the incompatibility between microbial and inorganic catalysis. Finally, 1.73 mg/L lycopene was produced from real exhaust gas from a coal-fired power plant in the MES system. The present study opens a possible route to turn trash to cash for industrial exhaust gas.

Polyhydroxyalkanoates (PHAs) are diverse biopolyesters produced by numerous microorganisms and have attracted much attention as a substitute for petroleum-based polymers. Despite several decades of study, the detailed molecular mechanisms of PHA biosynthesis have remained unknown due to the lack of structural information on the key PHA biosynthetic enzyme PHA synthase. The recently determined crystal structure of PHA synthase, together with the structures of acetyl-coenzyme A (CoA) acetyltransferase and reductase, have changed this situation. Structural and biochemical studies provided important clues for the molecular mechanisms of each enzyme as well as the overall mechanism of PHA biosynthesis from acetyl-CoA. This new information and knowledge is expected to facilitate production of designed novel PHAs and also enhanced production of PHAs.

Cupriavidus necator H16 Re2058/pCB113 could effectively utilize oil sources to synthesize P(3HB-co-3HHx). In this study, P(3HB-co-3HHx) biosynthesis procedure with optimally controlled (between 2–28) 3HHx mol% was established for C. necator H16 Re2058/pCB113 by regulating the amounts of dual carbon sources supplied; date molasses and date seed oil. Alone, date molasses supplied at 20 g/L total sugar equivalents to recombinant C. necator resulted in pure P(3HB) biosynthesis, with accumulation up to 28 wt% of cell dry weight (CDW), and effective total biomass of 4 g/L. Whereas, date seed oil supply alone or in combination with date molasses resulted in P(3HB-co-3HHx) biosynthesis. P(3HB-co-3HHx) with as high as 28 mol% 3HHx, up to 80 wt% of CDW and total biomass of 7.6 g/L were produced using 10 g/L date seed oil as the sole carbon source. The results of PHA production in the presence of date seed oil as a sole carbon source was obtained with a modest yield at a range of 0.38–0.62 g-PHA per g-date oil used. Gel permeation chromatography (GPC) analyses of these P(3HB-co-3HHx) copolymers exhibited weight average molecular weights (Mw) between 500 − 690 kDa. Differential scanning calorimetry (DSC) analyses showed Tm values of 157 °C and 85 °C for P(3HB) and P(3HB-co-28 mol% 3HHx), respectively. NMR analyses (1H and 13C) of P(3HB-co-3HHx) copolymers confirmed the incorporation of 3HHx monomers between 2–28 mol% depending on the supplied dual carbon source composition.

We aimed to develop a non-toxic and simple recovery procedure for poly(3-hydroxybutyrate) based on the use of green solvent. In this study, 2-methyltetrahydrofuran, 1,3-propanediol, 1,3-dioxolane and ethyl lactate were investigated and compared with chloroform in the extraction of poly(3-hydroxybutyrate) from Cupriavidus necator strain A-04. A water-miscible system was developed using 1,3-dioxolane and water. PHB was extracted from dried cells at 80 °C for 6 h in 1,3-dioxolane then water was used to cause phase separation of PHB film from 1,3-dioxolane solution resulted in the recovery yield 92.7% ± 1.4% and purity up to 97.9% ± 1.8%. The physical properties and molecular weight distribution of the PHB recovered were not significantly affected by the 1,3-dioxolane extraction. The PHB obtained from wet cells by 1,3-dioxolane extraction at 80 °C for 6 h had a MW of 4.86 × 105, MN of 3.74 × 105, polydispersity index of 1.30. The recovery process is non-toxic and very simple, thus reducing purification costs.

Ralstonia eutropha is a well-known microbe reported for polyhydroxyalkonate (PHA) production, and unable to utilize sucrose as carbon source. Two strains, Ralstonia eutropha H16 and Ralstonia eutropha 5119 were co-cultured with sucrose hydrolyzing microbes (Bacillus subtilis and Bacillus amyloliquefaciens) for PHA production. Co-culture of B. subtilis:R. eutropha 5119 (BS:RE5) resulted in best PHA production (45% w/w dcw). Optimization of the PHA production process components through response surface resulted in sucrose: NH4Cl:B. subtilis: R. eutropha (3.0:0.17:0.10:0.190). Along with the hydrolysis of sucrose, B. subtilis also ferments sugars into organic acid (propionic acid), which acts as a precursor for HV monomer unit. Microbial consortia of BS:RE5 when cultured in optimized media led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV) with 66% w/w of dcw having 16 mol% HV fraction. This co-culture strategy overcomes the need for metabolic engineering of R. eutropha for sucrose utilization, and addition of precursor for copolymer production.

Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.

Highlights • We investigate a kinetic model for the mevalonate pathway which includes inhibition effects and a sink of acetyl-CoA. • Of the enzymes in the pathway, upregulating HMG-CoA reductase has the most significant positive effect on improving pathway efficiency. • Upregulating pyruvate dehydrogenase complex and HMG-CoA synthase can also help, but only in conjunction with the upregulation of HMG-CoA reductase. • We confirm our theoretical predictions by introducing the mevalonate pathway into Cupriavidus necator., The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway.

A microbial production process was developed to convert CO2 and valeric acid into tailored poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) bioplastics. The aim was to understand microbial PHBV production in mixotrophic conditions and to control the monomer distribution in the polymer. Continuous sparging of CO2 with pulse and pH-stat feeding of valeric acid were evaluated to produce PHBV copolyesters with predefined properties. The desired random monomer distribution was obtained by limiting the valeric acid concentration (below 1 g L - 1 ). 1H-NMR, 13C-NMR and chromatographic analysis of the PHBV copolymer confirmed both the monomer distribution and the 3-hydroxyvalerate (3HV) fraction in the produced PHBV. A physical-based model was developed for mixotrophic PHBV production, which was calibrated and validated with independent experimental datasets. To produce PHBV with a predefined 3HV fraction, an operating diagram was constructed. This tool was able to predict the 3HV fraction with a very good accuracy (2% deviation).

Waste paper, a major fraction of municipal solid waste, has a potential to serve as renewable feedstock for the biorefineries of fuels, chemicals and materials due to rich in cellulose and abundant at low cost. This study evaluates the possibility of waste office paper (WOP) to serve as a potential feedstock for the biorefinery production of poly (3-hydroxybutyrate). In this study, the WOP was pretreated, enzymatically saccharified and the hydrolysate was used for PHB production. The hydrolysate mainly consists of glucose (22.70 g/L) and xylose (1.78 g/L) and the corresponding sugar yield was about 816 mg/g. Ammonium sulphate and C/N ratio 20 were identified as most favorable for high yield of PHB. The batch fermentation of Cupriavidus necator using the pretreated WOP hydrolysate resulted in cell biomass, PHB production and PHB content of 7.74 g/L, 4.45 g/L and 57.52%, respectively. The volumetric productivity and yield achieved were 0.061 g/L/h and 0.210 g/g sugar, respectively. The results suggested that WOP could be a potential alternative feedstock for the biorefinery production of bioplastics.

This study evaluates a biorefinery concept for producing poly(3-hydroxybutyrate) (PHB) with the cyanobacterial strain Synechocystis salina. Due to this reason, pigment extraction and cell disruption were investigated as pre-treatment steps for the harvested cyanobacterial biomass. The results demonstrated that at least pigment removal was necessary to obtain PHB with processable quality (weight average molecular weight: 569-988kgmol-1, melting temperature: 177-182°C), which was comparable to heterotrophically produced PHB. The removed pigments could be utilised as additional by-products (chlorophylls 0.27-1.98mgg-1 TS, carotenoids 0.21-1.51mgg-1 TS, phycocyanin 0-127mgg-1 TS), whose concentration depended on the used nutrient source. Since the residual biomass still contained proteins (242mgg-1 TS), carbohydrates (6.1mgg-1 TS) and lipids (14mgg-1 TS), it could be used as animal feed or converted to biomethane (348 mn3 t-1VS) and fertiliser. The obtained results indicate that the combination of photoautotrophic PHB production with pigment extraction and utilisation of residual biomass offer the highest potential, since it contributes to decrease the environmental footprint of the process and because biomass could be used in a cascading way and the nutrient cycle could be closed.

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.

Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB-4 strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB-4 strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB-4 (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs.

The current study demonstrates the enhanced production capability of strain Cupriavidus sp. ISTL7 for polyhydroxyalkanoates (PHA) using acetate and glucose (4.93 ± 0.4571 g L-1) which was characterised analytically by GC–MS, FTIR and NMR analysis. Whole genome sequencing of strain ISTL7 unveiled an array of PHA metabolism genes which included phaA, phaB and phaC. Life cycle assessment of the protocol established that the production was most sustainable with the carbon source acetate. + Glucose as compared to acetate/glucose alone. It also concluded that solvent extraction of PHA and energy consumption during the process requires optimization to sustain the production on ecological fronts. Additionally, acetoacetyl-CoA reductase (phaB) gene was molecularly cloned, expressed and purified (27 KDa, 2.63 mg/ml). Conclusively, Cupriavidus sp. ISTL7 is a potential strain for PHA production with a scope of improvement on energy fronts which would transform the production environmentally and economically appealing.