Production of polyhydroxybutyrate by coupled saccharification-fermentation of inulin.

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/ginulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of Mw, and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization.

Polyhydroxybutyrate production by Cupriavidus necator in a corn biorefinery concept.

The high costs of bioplastics' production may hinder their commercialization. Development of new processes with high yields and in biorefineries can enhance diffusion of these materials. This work evaluated the production of polyhydroxybutyrate (PHB) from the combination of milled corn starchy fraction hydrolysate and crude glycerol as substrates by the strain Cupriavidus necator LPB 1421. After optimization steps, maximum accumulation of 62 % of PHB was obtained, which represents 11.64 g.L-1 and productivity of 0.162 g.Lh-1. In a stirred tank bioreactor system with 8 L of operational volume, 70 % of PHB accumulation was reported, representing 14.17 g.L-1 of the biopolymer with 0.197 g.Lh-1 productivity. PHB recovery was conducted using a chemical digestion method, reaching >99 % purity. Therefore, the potential application of milled corn as substrate for PHB production was confirmed. The developed bioplastic process could be coupled to a bioethanol producing unit creating the opportunity of a sustainable and economic biorefinery.

Application of the solid-state fermentation process and its variations in PHA production: a review.

Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

Genomics and transcriptomics insights into luteolin effects on the beta-rhizobial strain Cupriavidus necator UYPR2.512.

Cupriavidus necator UYPR2.512 is a rhizobial strain that belongs to the Beta-subclass of proteobacteria, able to establish successful symbiosis with Mimosoid legumes. The initial steps of rhizobium-legumes symbioses involve the reciprocal recognition by chemical signals, being luteolin one of the molecules involved. However, there is a lack of information on the effect of luteolin in beta-rhizobia. In this work, we used long-read sequencing to complete the genome of UYPR2.512 providing evidence for the existence of four closed circular replicons. We used an RNA-Seq approach to analyse the response of UYPR2.512 to luteolin. One hundred and forty-five genes were differentially expressed, with similar numbers of downregulated and upregulated genes. Most repressed genes were mapped to the main chromosome, while the upregulated genes were overrepresented among pCne512e, containing the symbiotic genes. Induced genes included the nod operon and genes implicated in exopolysaccharides and flagellar biosynthesis. We identified many genes involved in iron, copper and other heavy metals metabolism. Among repressed genes, we identified genes involved in basal carbon and nitrogen metabolism. Our results suggest that in response to luteolin, C. necator strain UYPR2.512 reshapes its metabolism in order to be prepared for the forthcoming symbiotic interaction.

PHA granule formation and degradation by Cupriavidus necator under different nutritional conditions.

Polyhydroxyalkanoates (PHA) are polymers produced by microorganisms with increasing commercialization potential; Cupriavidus necator has been the model microorganism to research PHA production. Despite many contributions concerning the formation and degradation of PHA granules, as well as the morphological changes in cells, these phenomena have not been univocally explained yet. Thus, this study aims to integrate the microscopic and analytical analysis to characterize changes in bacterial cell/PHA granules morphology, PHA content, and yield coefficients under different cultivation strategies of C. necator ATCC 17697. The cell size and morphology, granule size and amount, residual biomass, and PHA concentration along the fermentation and degradation depend greatly on nutritional conditions and cultivation time of C. necator. It was proposed to calculate a yield coefficient for the residual biomass production in the PHA utilization stage, related to the bacteria's ability to survive without a carbon source in the culture medium by utilizing the accumulated PHA previously. Maximum granule length reached 1.07 µm after 72 h of PHA accumulation stage under optimum nutritional conditions. This value is twice the values previously reported for C. necator. It is important since the larger PHA granules facilitate the recovery of PHA and different application development.

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.

Comparison of acid and enzymatic hydrolysis of pectin, as inexpensive source to cell growth of Cupriavidus necator.

Abstract: The present work investigated what the appropriate methods of hydrolysis of pectin for reducing compounds (RCs) production, employed as a substrate for cell growth of Cupriavidus necator. This microorganism has great importance industrial, because besides potential single cell protein (SCP), is the most studied microorganism for production of polyhydroxybutyrate (PHB), and both processes require high cell concentration with inexpensive substrates For this, it was compared to acid and enzymatic hydrolysis procedures, through rotational central composite experimental design, using pectin concentration (1.0%). It was analyzed as a variable response for both experimental design, the RCs' production. The best conditions of each procedure were used in study kinetics of RCs' production and as a substrate for cell growth of C. necator. The results indicated that the enzymatic hydrolysis method was the most efficient, with a 93.0% yield of RCs, while the yield for acid hydrolysis was 60.0%. The optimum conditions for enzymatic hydrolysis were an enzyme concentration of 10.01 UI/g (International Unit of enzyme per gram of pectin) and an agitation speed of 230.3 rpm. C. necator showed satisfactory growth in the media containing pectin hydrolysates, with specific growth rates (µMax) similar to those reported for other substrates.

Orange and Passion Fruit Wastes Characterization, Substrate Hydrolysis and Cell Growth of Cupriavidus necator, as Proposal to Converting of Residues in High Value Added Product.

Brazil is the world's largest producer of orange and passion fruit, which are destined mainly for industrialization, generating grand volumes of wastes. The solid portion of these residues is a rich source of pectin - composed mainly of galacturonic acid and neutral sugars, which through the hydrolysis process can be used in biological conversion processes, as the production of polyhydroxyalkanoates (PHAs). This way, we characterized these wastes, followed by the extraction and hydrolysis of pectin for employ as a substrate for the cell growth of Cupriavidus necator. The results confirmed the large portion of pectin (almost 40 g.100g-1) and soluble sugars, present in these wastes. The hydrolyzed extract showed as a good source of carbon for the cell growth of C. necator with YX/S 0.56 and 0.44, µMax 0.27 and 0.21 for orange and passion fruit wastes respectively, similar to other carbon sources. This way, the extraction and hydrolysis of orange and passion fruit wastes for the cellular growth of C. necator, can be a good alternative to converting of residues in high value added product.

Hydrogen and polyhydroxybutyrate production from wheat straw hydrolysate using Caldicellulosiruptor species and Ralstonia eutropha in a coupled process.

This report presents an integrated biorefinery concept in which wheat straw hydrolysate was treated with co-cultures of osmotolerant thermophilic bacterial strains, Caldicellulosiruptor saccharolyticus and C. owensensis to obtain hydrogen, while the liquid effluent containing acetate and residual glucose was used as feed for polyhydroxybutyrate (PHB) production by Ralstonia eutropha. The Caldicellulosiruptor spp. co-culture consumed 10.8 g/L of pretreated straw sugars, glucose and xylose, producing 134 mmol H2/L. PHB accumulation by R. eutropha was first studied in minimal salts medium using acetate with/without glucose as carbon source. Addition of salts promoted cell growth and PHB production in the effluent. Fed-batch cultivation in a nitrogen limited medium with 40% (v/v) aeration resulted in a cell density of 15.1 g/L with PHB content of 80.1% w/w and PHB concentration of 12.1 g/L, while 20% aeration gave a cell density of 11.3 g/L with 83.4% w/w PHB content and 9.4 g/L PHB concentration.

Cupriavidus necator strains: zinc and cadmium tolerance and bioaccumulation

Among soil microorganisms, the genus Cupriavidus has garnered particular scientific, economic and ecological interest because of its ability to fix nitrogen and tolerate high concentrations of metals. The aim of this study was to analyze four strains of Cupriavidus necator for their ability to tolerate and bioaccumulate cadmium and zinc. The tolerance of these strains to these metals was assessed in liquid culture medium containing different concentrations of Zn + Cd and in soil solutions and soils contaminated with multiple elements including Zn, Cd, Cu and Pb. The four strains showed high tolerance to Zn and Cd, both in culture medium and when inoculated into contaminated soil solutions or multi-element contaminated soil. The UFLA02-71 strain displayed the highest ability to bioaccumulate these metals. It was able to accumulate 93.76 µmol g cell1 of Zn and 16.03 µmol g cell1 of Cd when cultured in liquid medium with a total heavy metal concentration of 9,140 µmol L1 (9,000 Zn + 140 Cd) and was able to accumulate 16.98 µmol g cell1 of Cd in the soil solution. An increase in the pH of the culture medium resulting from the growth of the C. necator strains reduced the Zn2+ and Cd2+ ions in the medium and increased the concentrations of the ZnHPO4 and CdHPO4 species in solution. Thus, we concluded that they show great potential for use in the bioremediation of HM-contaminated areas.(AU)

Genome characteristics dictate poly-R-(3)-hydroxyalkanoate production in Cupriavidus necator H16.

Cupriavidus necator H16 is a well-recognized enterprise with efficient manufacturing machineries to produce diverse polymers belonging to polyhydroxyalkanoates (PHAs) family. The genome fingerprints, including PHA machinery proteins and fatty acid metabolism, had educated engineering strategies to enhance PHAs production. This outstanding progress has enlightened us to present an exhaustive examination of the ongoing research, addressing the great potential design of genome features towards PHA production and furthermore, we show how those acquired knowledge have been explored in other biotechnological applications. This updated-review concludes that the combination of an optimal strain selection, suitable metabolic engineering and a large-scale fermentation on oil substrates is critical to endow the ability of incorporating mcl-PHAs monomers in this organism.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures.

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

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.

Poly(3-Hydroxybutyrate) Production in Repeated fed-Batch with Cell Recycle Using a Medium with low Carbon Source Concentration.

Among approaches applied to obtain high productivity and low production costs in bioprocesses are high cell density and the use of low cost substrates. Usually low cost substrates, as waste/agroindustrial residues, have low carbon concentration, which leads to a difficulty in operating bioprocesses. Real time control of process for intracellular products is also difficult. The present study proposes a strategy of repeated fed-batch with cell recycle to attain high cell density of Cupriavidus necator and high poly(3-hydroxybutyrate) (P(3HB)) productivity, using a substrate with low carbon source concentration (90 g l(-1)). Also, the use of the oxygen uptake rate data was pointed out as an on line solution for process control, once P(3HB) is an intracellular product. The results showed that total biomass (X), residual biomass (Xr) and P(3HB) values at the end of the culture were 61.6 g l(-1), 19.3 g l(-1) and 42.4 g l(-1) respectively, equivalent to 68.8 % of P(3HB) in the cells, and P(3HB) productivity of 1.0 g l(-1) h(-1). Therefore, the strategy proposed was efficient to achieve high productivity and high polymer content from a medium with low carbon source concentration.

Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha.

Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbR(AF)) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbR(AF) function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbR(AF) was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbR(AF) was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbR(AF) activity.

Influence of pH on the molecular weight of poly-3-hydroxybutyric acid (P3HB) produced by recombinant Escherichia coli.

The production of ultrahigh molecular weight poly-3-hydroxybutyric acid (P3HB) from carbohydrates by recombinant Escherichia coli harboring genes from Ralstonia eutropha was evaluated. In shaken-flask experiments, E. coli XL1 Blue harboring plasmid pSK::phaCAB produced P3HB corresponding to 40 and 27% of cell dry weight from glucose and xylose, respectively. Cultures in bioreactor using glucose as the sole carbon source at variable pH values (6.0, 6.5, or 7.0) allowed the production of P3HB with molecular weight varying between 2.0 and 2.5 MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5-1.0 MDa). Contrary to reports of other authors, no influence of pH was observed on the molecular weight of the polymer produced. Using xylose, P3HB with high molecular weight was also produced, indicating the possibility to produce these polymers from lignocellulosic materials.

Use of enzymes in extraction of polyhydroxyalkanoates produced by Cupriavidus necator.

Poly(3-hydroxybutyrate) (P(3HB)) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV), are biodegradable thermoplastic polymers. They are members of the polyhydroxyalkanoate (PHA) family, synthesized and accumulated as a carbon and energy reserve by a variety of microorganisms. The aim of this study was to evaluate the use of the proteases Corolase® L10, Alcalase® 2.4L, Corolase® 7089 and Protemax® FC and glycosidases Celumax® BC, Rohament® CL and Rohalase® Barley for the recovery of P(3HB) and P(3HB-co-3HV) synthesized by Cupriavidus necator. The enzyme Celumax® BC provided better lysis of the bacterial cell membrane and the results for the optimization of the operating conditions showed that this enzyme is most stable in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). The optimization of the operating conditions showed that the enzyme Celumax® BC provided better lysis of the bacterial cell in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). These conditions resulted in lysis of the membrane of the bacteria with a recovery of 93.2% P(3HB-co-3HV) with 94% purity. The results showed that the use of enzymes for the polymer extraction is an efficient process that assists in the cell disruption of Cupriavidus necator.

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species.

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using Cupriavidus necator.

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l(-1)) was achieved using soybean oil at 40 g l(-1) and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l(-1) h(-1), and the calculated Y(p/s) value was 0.85 g g(-1). Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.

Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4-hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134.

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.