Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate.

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

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.

PHA granules help bacterial cells to preserve cell integrity when exposed to sudden osmotic imbalances.

Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator, the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.

Model of acetic acid-affected growth and poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545.

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates.

The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic environments.

Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions.

Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator.

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.

Acid pretreatment and enzymatic saccharification of brown seaweed for polyhydroxybutyrate (PHB) production using Cupriavidus necator.

The brown seaweed Sargassum sp. was used as a feedstock to produce polyhydroxybutyarte (PHB) using Cupriavidus necator PTCC 1615. In order to release monomeric sugars, dilute acid hydrolysis of Sargassum sp. biomass was followed by enzymatic saccharification. In addition, the effect of different nitrogen sources was evaluated for PHB production. The fermentation of hydrolysate with the ammonium sulfate as selected nitrogen source resulted PHB yield of 0.54±0.01g/g reducing sugar. Then, NaCl was used as external stress factor which was added to the media. Addition of 8g/L NaCl had a positive impact on high PHB yield of 0.74±0.01g/g reducing sugar. Increasing trend of NaCl concentration to 16g/L was found to inhibit the production of PHB. Based on obtained results using 20g/L of reducing sugar, at desired condition the highest cell dry weight and PHB concentrations were 5.36±0.22 and 3.93±0.24g/L, respectively. The findings of this study reveal that Sargassum sp. is a promising feedstock for biopolymer production. The characteristics of produced PHB were analyzed by FTIR, differential scanning calorimetry and 1H NMR.

Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry.

Remains of a medieval foundry were excavated by archaeologists in 2013 in Verdun (France). Ancient workshops specialized in brass and copper alloys were found with an activity between 13th to 16th c. Levels of Cu, Zn and Pb reached 20000, 7000 and 6000 mg kg-1 (dw), respectively, in several soil horizons. The objective of the present work was to examine the microbial community in this contaminated site. A total of 8-22 106 reads were obtained by shotgun metagenomics in four soil horizons. Bioinformatic analyses suggest the presence of complex bacterial communities dominated by Proteobacteria. The structure of the community was not affected by metals, contrary to the set of metal-resistance genes. Using selective media, a novel strain of Cupriavidus necator (eutrophus), strain B9, was isolated. Its genome was sequenced and a novel metal resistance gene cluster with Hg resistance genes (merRTPCA) followed by 24 copper-resistance genes (actP, cusCBAF, silP, copK1, copH4QLOFGJH3IDCBARS, copH2H1, copK2) was found. This cluster is partly homologous to the cop genes of Cupriavidus gilardii CR3 and C. metallidurans CH34. Proteomics indicated that the four copH genes were differentially expressed: CopH1 and CopH2 were mostly induced by Cd while CopH4 was highly expressed by Cu.

Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha.

Isobutanol (IBT) has attracted much attention from researchers as a next generation drop-in biofuel. Ralstonia eutropha is a gram-negative bacterium which naturally produces polyhydroxybutyrate (PHB), and has been reported to produce IBT after metabolic engineering. Similar to other microbes, R. eutropha experiences toxicity from branched-chain alcohols and is unable to grow in the presence of IBT concentrations higher than 0.5% (v v(-1)). Such low tolerance greatly limits the ability of R. eutropha to grow and produce IBT. In order to study toxicity to the cells, IBT-tolerant strains were developed by experimental evolution, revealing that two genes, previously described as being related to IBT tolerance in Escherichia coli (acrA and acrA6), also presented mutations in R. eutropha evolved strains. The effect on the physiology of the cells of in-frame deletions of each of these genes was assessed in wild type and engineered IBT-producing strains in an attempt to reproduce a tolerant phenotype. The mutant strains' ability to tolerate, consume, and produce IBT were also analyzed. Although deletions of acrA6 and acrA did not significantly improve R. eutropha growth in the presence of IBT, these deletions improved cell survival in the presence of high concentrations of IBT in the extracellular milieu. Moreover, an in-frame acrA deletion in an engineered IBT-producing R. eutropha enhanced the strain's ability to produce IBT, which could potentially be associated with enhanced survival at high IBT concentrations.

Improvement of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator.

In the context of increasing volatility of oil prices, replacement of petroleum based plastics by bioplastics is a topic of increasing interest. Poly(hydroxyalkanoate)s (PHAs) are among the most promising families in this field. Controlling composition of the polymer on the monomeric level remains a pivotal issue. This control is even more difficult to achieve when the polymer is not synthesized by chemists, but produced by nature, in this case, bacteria. In this study mechanism and role of two 3-hydroxyvalerate (3-HV) inducing substrates on the production of PHBV with high, 80%, 3-HV content were evaluated. It was found that levulinic acid contributes to biomass and bio-polymer content enhancement, whereas sodium propionate mainly contributes to 3-HV enhancement. Optimized proportions of feeding substrates at 1 g/L and 2.5 g/L, respectively for levulinic acid and sodium propionate allowed a 100% productivity enhancement, at 3.9 mg/L/hour, for the production of PHBV with 80% 3-HV.

New Insight into the Role of the Calvin Cycle: Reutilization of CO2 Emitted through Sugar Degradation.

Ralstonia eutropha is a facultative chemolithoautotrophic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle for CO2 fixation. This study showed that R. eutropha strain H16G incorporated (13)CO2, emitted by the oxidative decarboxylation of [1-(13)C1]-glucose, into key metabolites of the CBB cycle and finally into poly(3-hydroxybutyrate) [P(3HB)] with up to 5.6% (13)C abundance. The carbon yield of P(3HB) produced from glucose by the strain H16G was 1.2 times higher than that by the CBB cycle-inactivated mutants, in agreement with the possible fixation of CO2 estimated from the balance of energy and reducing equivalents through sugar degradation integrated with the CBB cycle. The results proved that the 'gratuitously' functional CBB cycle in R. eutropha under aerobic heterotrophic conditions participated in the reutilization of CO2 emitted during sugar degradation, leading to an advantage expressed as increased carbon yield of the storage compound. This is a new insight into the role of the CBB cycle, and may be applicable for more efficient utilization of biomass resources.

Characterization of poly-3-hydroxybutyrate (PHB) produced from Ralstonia eutropha using an alkali-pretreated biomass feedstock.

Alkaline pretreatment using NaOH, KOH, or NaOCl has been applied to various types of waste biomass to enhance enzymatic digestibility. Pretreatment (2% NaOH, 121 °C, 30 min) of rice paddy straw (PS) resulted in a maximum yield of 703 mg of reducing sugar per gram of PS with 84.19% hydrolysis yield after a two-step enzymatic hydrolysis process. Ralstonia eutropha ATCC 17699 was tested for its ability to synthesize poly-3-hydroxybutyrate (PHB) using PS hydrolysates as its sole carbon source. It is noteworthy that dry cell weight, polyhydroxyalkanoate (PHA) accumulation and PHB yield with the use of laboratory-grade sugars were similar to those achieved with PS-derived sugars. Under optimized conditions, we observed maximal PHA accumulation (75.45%) and PHB production (11.42 g/L) within 48 h of fermentation. After PHB recovery, the physicochemical properties of PHB were determined by various analytical techniques, showed the results were consistent with the characteristics of a standard polymer of PHB. Thus, the PS hydrolysate proved to be an excellent cheap carbon substrate for PHB production.

Green synthesis and structural characterization of selenium nanoparticles and assessment of their antimicrobial property.

In the present study, selenium nanoparticles were biologically synthesized by non-pathogenic, economic and easy to handle bacterium Ralstonia eutropha. The selenium oxo anion was reduced to selenium nanoparticles in the presence of the bacterium. The bacterium was grown aerobically in the reaction mixture. An extracellular, stable, uniform, spherical selenium nanoparticle was biosynthesized. The TEM analysis revealed that the biosynthesized selenium nanoparticles were spherical in shape with size range of 40-120 nm. XRD and SAED analysis showed that nanocrystalline selenium of pure hexagonal phase was synthesized. The formation of actinomorphic trigonal selenium nanorods was also observed. A mechanism of biosynthesis of selenium nanoparticles by R. eutropha was proposed. The biosynthesized selenium nanoparticles were investigated for their antimicrobial activity against potential pathogens. Selenium nanoparticles showed excellent antimicrobial activity. The 100, 100, 250 and 100 µg/ml selenium nanoparticles were found to inhibit 99 % growth of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Streptococcus pyogenes, respectively. Similarly, the 500 µg/ml of selenium nanoparticles was found to inhibit the growth of pathogenic fungi Aspergillus clavatus. The antimicrobial efficacy of selenium nanoparticle was comparable with commercially available antibiotic drug Ampicillin.

Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system.

Photovoltaic cells have considerable potential to satisfy future renewable-energy needs, but efficient and scalable methods of storing the intermittent electricity they produce are required for the large-scale implementation of solar energy. Current solar-to-fuels storage cycles based on water splitting produce hydrogen and oxygen, which are attractive fuels in principle but confront practical limitations from the current energy infrastructure that is based on liquid fuels. In this work, we report the development of a scalable, integrated bioelectrochemical system in which the bacterium Ralstonia eutropha is used to efficiently convert CO2, along with H2 and O2 produced from water splitting, into biomass and fusel alcohols. Water-splitting catalysis was performed using catalysts that are made of earth-abundant metals and enable low overpotential water splitting. In this integrated setup, equivalent solar-to-biomass yields of up to 3.2% of the thermodynamic maximum exceed that of most terrestrial plants. Moreover, engineering of R. eutropha enabled production of the fusel alcohol isopropanol at up to 216 mg/L, the highest bioelectrochemical fuel yield yet reported by >300%. This work demonstrates that catalysts of biotic and abiotic origin can be interfaced to achieve challenging chemical energy-to-fuels transformations.

Sunflower-based biorefinery: poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from crude glycerol, sunflower meal and levulinic acid.

Polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production was developed in bioreactor cultures using the strain Cupriavidus necator DSM 7237 cultivated on crude glycerol, sunflower meal (SFM) hydrolysates and levulinic acid as the sole fermentation feedstocks. Bacterial growth and PHB production was influenced significantly by the free amino nitrogen and inorganic phosphorus content of the SFM hydrolysate. Fed-batch bioreactor fermentations led to the production of 27gL(-1) PHB with an intracellular content of 72.9% (w/w). Continuous feeding of levulinic acid led to the production of up to 23.4gL(-1) P(3HB-co-3HV) with an intracellular content of 66.4% (w/w) and a 3HV content of 22.5mol%. A maximum 3HV content of 31mol% was achieved at earlier fermentation time (53h). Thus, levulinic acid could be combined with biodiesel industry by-products for the production of high P(3HB-co-3HV) concentration, intracellular content and industrially useful 3HV content.

Antimicrobial agents act differently on Staphyloccocus aureus and Ralstonia eutropha flavohemoglobins.

Flavohemoglobins (FlavoHb) play a key role in bacterial resistance to nitrosative stress and NO signaling modulation. In this study, we cloned, expressed, and characterized the flavoHb from the opportunistic pathogen, Staphylococcus aureus. The higher amino-acid sequence homology is shared with that from Saccharomyces cerevisiae which was therefore used to build a model structure by homology modeling. Interestingly, the high sequence homology with S. cerevisiae did not correlate with the enzymatic and kinetic properties which are much similar to those of Escherichia coli. In vitro and aerobically, we showed that S. aureus and Ralstonia eutropha flavoHbs accept cytochrome c and oxygen as substrates. Based on this feature, we investigated the preferences for both substrates depending on miconazole or econazole addition and found that the inhibitor chemical composition is determinant.

Optimization of polyhydroxyalkanoates fermentations with on-line capacitance measurement.

The aim of this work was to provide an effective methodology for optimization of the polyhydroxyalkanoates (PHAs) fermentation with Ralstonia eutropha by the on-line capacitance measurement. The present study found the capacitance values could reflect variations of microbial morphology and viability. Furthermore, oxygen uptake rate, specific oxygen uptake rate and specific growth rate were measured in real-time and compared with the capacitance value. In addition, a fed-batch control strategy based on the on-line capacitance measurement was proposed to improve the PHAs production by 22%.

A fluorescence-based microbial sensor for the selective detection of gold.

This study presents a fluorescence-based microbial sensor for the detection of metal ions as a novel analytical tool for environmental applications. Our results demonstrate the effectiveness of whole-cell sensors in the selective detection of gold ions. Two heavy-metal-tolerant proteobacteria, Cupriavidus metallidurans and Ralstonia eutropha, were examined and showed great specificity. This work highlights the potential of employing engineered microbial strains as robust analytical tools.

Phosphorus limitation strategy to increase propionic acid flux towards 3-hydroxyvaleric acid monomers in Cupriavidus necator.

Properties of polyhydroxybutyrate-co-hydroxyvalerate (P(3HB-co-3HV)) depend on their 3HV content. 3HV can be produced by Cupriavidus necator from propionic acid. Few studies explored carbon distribution and dynamics of 3HV and 3HB monomers production, and none of them have been done with phosphorus as limiting nutrient. In this study, fed-batch cultures of C. necator with propionic acid, as sole carbon source or mixed with butyric acid, were performed. Phosphorus deficiency allowed sustaining 3HV production rate and decreasing 3HB production rate, leading to an instant production of up to 100% of 3HV. When a residual growth is sustained by a phosphorus feeding, the maximum 3HV percentage produced from propionic acid is limited to 33% (Mole.Mole(-1)). The association of a second carbon source like butyric acid lead to higher conversion of propionic acid into 3HV. This study showed the importance of the limiting nutrient and of the culture strategy to get the appropriate product.

Impact of sustaining a controlled residual growth on polyhydroxybutyrate yield and production kinetics in Cupriavidus necator.

In this study a complementary modeling and experimental approach was used to explore how growth controls the NADPH generation and availability, and the resulting impact on PHB (polyhydroxybutyrate) yields and kinetics. The results show that the anabolic demand allowed the NADPH production through the Entner-Doudoroff (ED) pathway, leading to a high maximal theoretical PHB production yield of 0.89 C mole C mole(-1); whereas without biomass production, NADPH regeneration is only possible via the isocitrate dehydrogenase leading to a theoretical yield of 0.67 C mole C mole(-1). Furthermore, the maximum specific rate of NADPH produced at maximal growth rate (to fulfil biomass requirement) was found to be the maximum set in every conditions, which by consequence determines the maximal PHB production rate. These results imply that sustaining a controlled residual growth improves the PHB specific production rate without altering production yield.