Constant fed-batch cultivation with glucose and propionate as co-substrate: A strategy to fine-tune polyhydroxyalkanoates monomeric composition in Pseudomonas spp.

Pseudomonas sp. LFM693 is a 2-methylisocitrate lyase (prpB) disrupted mutant. This enzyme catalyzes a step in the 2-methylcitrate cycle, the only known and described pathway for propionate oxidation in this organism. The affected mutants can efficiently produce PHA containing even and odd-chain length hydroxyalkanoates (HAeven/odd) in the presence of propionate and glucose. In this study, a constant fed-batch configuration was utilized to control the composition of PHA and decrease the toxicity of propionate. The incorporation of HAodd into the copolymer was linear, ranging from 7 to approximately 30 %, and correlated directly with the propionate/glucose molar ratio in the feeding solution. This allowed for the molecular composition of the mclPHA to be fine-tuned with minimum process monitoring and control. The average PHA content was 52 % cell dry weight with a molar composition that favored 3-hydroxyalkanoates containing C8, C9, and C10. The conversion factor of propionate to HAodd varied between 0.36 and 0.53 mol·mol-1 (YHAodd/prop.), which are significantly lower than the theoretical maximum efficiency (1.0 mol·mol-1). These results along with the lack of 2-methylisocitrate as a byproduct provides further support for the evidence that the mutant prpB- is still capable of oxidizing propionate.

A potential hybrid nanocomposite of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and fullerene for bone tissue regeneration and sustained drug release against bone infections.

Developing a multifunctional biomaterial for bone filling and local antibiotic therapy is a complex challenge for bone tissue engineering. Hybrid nanocomposites of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with nanohydroxyapatite (nHA), fullerene (C60), and vancomycin (VC) were produced by injection. Fullerene was successfully impregnated with VC, as seen in FTIR. The crystallinity degree of PHBHV was slightly reduced in the presence of C60 and VC (64.3 versus 60.8 %), due to the plasticizing effect of these particles. It also resulted in a decrease in the glass transition temperature (Tg), observed by differential scanning calorimetry (DSC). Dense PHBHV/nHA/C60/VC had a flexural elastic modulus 29 % higher than PHBHV, as a result of the good interface between PHBHV, C60, and nHA - particles of high elastic modulus. Dense disks released 25.03 ± 4.27 % of VC for 14 days, which demonstrated its potential to be an alternative treatment to bone infections. Porous scaffolds of PHBHV/nHA/C60/VC were 3D printed with a porosity of 50 % and porous size of 467 ± 70 µm, and had compression elastic modulus of 0.022 GPa, being a promising material to trabecular bone replacement. The plasticizing effect of C60 improved the printability of the material. The hybrid nanocomposite was non-cytotoxic and showed good ability in adhering macrophage cells.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and amino-functionalized nanodiamond bionanocomposites for bone tissue defect repair.

Injection-molded nanocomposites of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) with 6 % of 3-hydroxyvalerate (HV) and amino-nanodiamonds (nD-A) were produced and characterized to investigate the effect of functionalized nanodiamonds on mechanical and biological behavior to bone replacement application. To prepare mixtures of PHBHV and nD-A in different concentrations, nD-A was dispersed in chloroform by sonication with 40 % of amplitude. Three specimens were characterized by infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (DRX), differential scanning calorimetry (DSC), 3-point flexural tests, dynamic mechanical analysis (DMA), and scanning electron microscopy (SEM). FTIR and TGA evidenced the existence of interactions between the nD-A and PHBHV. The crystallinity degree of PHBHV slightly reduced (~9 %) in nanocomposites and the morphology of the crystals changed. Nanocomposites achieved satisfactory dispersion and distribution of nD-A for low concentrations. Elastic modulus (E) increased from 1.96 ± 0.20 (PHBHV) to 2.59 ± 0.19 GPa (PHBHV/1.0%nD-A) (30 %). Despite the relatively limited dispersion, PHBHV/2.0 % nD-A had the best combination of E, strength, and maximum deformation. It had the highest glass transition temperature (43.1 vs 40.3 °C of PHBHV) and the best adhesion coefficient and reinforcement effectiveness. PHBHV-nD-A did not induce toxicity in 7 days and allowed cell fixation and expansion. These bionanocomposites should be considered for supplementary studies for bone tissue engineering.

Glucose metabolism in Pseudomonas aeruginosa is cyclic when producing Polyhydroxyalkanoates and Rhamnolipids.

Pseudomonas aeruginosa is an important chassis for production of polyhydroxyalkanoates (PHA) and rhamnolipids (RHL). Advances in the understanding of the biosynthesis metabolism of these biocompounds are crucial for increasing yield. 13C-Metabolic Flux Ratio Analysis (13C-MFA) is a technique to estimate in vivo metabolic fluxes ratios. PHA and RHL are essentially non-growth associated products of biotechnological interest and both contain hydroxyalkanoates (HAs), whose labeling patterns could be accessed by GC-MS. In this study, to reveal the relative contributions of the Entner-Doudoroff (ED) pathway and the non-oxidative Pentose Phosphate (PP) pathway to PHA and RHL production, 13C-MFA was performed in Pseudomonas aeruginosa LFM634 when supplied with labeled glucose. This bacterial strain lacks both functional EMP and the oxidative PP branch. Labeling patterns in HAs were measured. Experiments with [U-13C] glucose indicated a low flux though PP pathway. An optimal design of labeling experiment showed that [6-13C] glucose would be the best substrate to enable an estimation of the ED flux with high accuracy. Results of experiments performed with this isotope indicated that about two-thirds of glyceraldehyde 3-phosphate is recycled through a cyclic ED architecture, suggesting that P. aeruginosa utilizes that cycle to regulate the NADPH/Acetyl-CoA ratio for PHA and RHL biosynthesis.

Burkholderia sacchari (synonym Paraburkholderia sacchari): An industrial and versatile bacterial chassis for sustainable biosynthesis of polyhydroxyalkanoates and other bioproducts.

This is the first review presenting and discussing Burkholderia sacchari as a bacterial chassis. B. sacchari is a distinguished polyhydroxyalkanoates producer strain, with low biological risk, reaching high biopolymer yields from sucrose (0.29 g/g), and xylose (0.38 g/g). It has great potential for integration into a biorefinery using residues from biomass, achieving 146 g/L cell dry weight containing 72% polyhydroxyalkanoates. Xylitol (about 70 g/L) and xylonic acid [about 390 g/L, productivity 7.7 g/(L.h)] are produced by the wild-type B. sacchari. Recombinants were constructed to allow the production and monomer composition control of diverse tailor-made polyhydroxyalkanoates, and some applications have been tested. 3-hydroxyvalerate and 3-hydroxyhexanoate yields from substrate reached 80% and 50%, respectively. The genome-scale reconstruction of its metabolic network, associated with the improvement of tools for genetic modification, and metabolic fluxes understanding by future research, will consolidate its potential as a bioproduction chassis.

Antibiofilm effect of mono-rhamnolipids and di-rhamnolipids on carbon steel submitted to oil produced water.

The objective of this study was to compare the potential of mono-rhamnolipids (mono-RML) and di-rhamnolipids (di-RML) against biofilm formation on carbon steel coupons submitted to oil produced water for 14 days. The antibiofilm effect of the RML on the coupons was analyzed by scanning electron microscopy (SEM) and by epifluorescence microscopy, and the contact angle was measured using a goniometer. SEM analysis results showed that all RML congeners had effective antibiofilm action, as well as preliminary anticorrosion evaluation confirmed that all RML congeners prevented the metal deterioration. In more detail, epifluorescence microscopy showed that mono-RML were the most efficient congeners in preventing microorganism's adherence on the carbon steel metal. Image analyses indicate the presence of 15.9%, 3.4%, and <0.1% of viable particles in di-RML, mono/di-RML and mono-RML pretreatments, respectively, in comparison to control samples. Contact angle results showed that the crude carbon steel coupon presented hydrophobic character favoring hydrophobic molecules adhesion. We calculated the theoretical polarity of the RML congeners and verified that mono-RML (log P = 3.63) presented the most hydrophobic character. This had perfect correspondence to contact angle results, since mono-RML conditioning (58.2°) more significantly changed the contact angle compared with the conditioning with one of the most common surfactants used on oil industry (29.4°). Based on the results, it was concluded that rhamnolipids are efficient molecules to be used to avoid biofilm on carbon steel metal when submitted to oil produced water and that a higher proportion of mono-rhamnolipids is more indicated for this application.

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.

Draft Genome Sequence of Halomonas sp. HG01, a Polyhydroxyalkanoate-Accumulating Strain Isolated from Peru.

Halomonas sp. strain HG01, isolated from a salt mine in Peru, is a halophilic aerobic heterotrophic bacterium accumulating poly-3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from different carbon sources. Here, we report the draft genome sequence of this isolate, which was found to be 3,665,487 bp long, with a G+C content of 68%.

Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440.

Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.

Draft Genome Sequence of Pseudomonas sp. Strain LFM046, a Producer of Medium-Chain-Length Polyhydroxyalkanoate.

Pseudomonas sp. LFM046 is a medium-chain-length polyhydroxyalkanoate (PHAMCL) producer capable of using various carbon sources (carbohydrates, organic acids, and vegetable oils) and was first isolated from sugarcane cultivation soil in Brazil. The genome sequence was found to be 5.97 Mb long with a G+C content of 66%.

Draft Genome Sequence of the Polyhydroxyalkanoate-Producing Bacterium Burkholderia sacchari LMG 19450 Isolated from Brazilian Sugarcane Plantation Soil.

Burkholderia sacchari LMG 19450, isolated from the soil of a sugarcane plantation in Brazil, accumulates large amounts of polyhydroxyalkanoates from sucrose, xylose, other carbohydrates, and organic acids. We present the draft genome sequence of this industrially relevant bacterium, which is 7.2 Mb in size and has a G+C content of 64%.

Perspectives on the production of polyhydroxyalkanoates in biorefineries associated with the production of sugar and ethanol.

Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bacterial thermoplastic polymers that can be obtained from renewable resources. The high impact of the carbon source in the final cost of this polymer has been one of the major limiting factors for PHA production and agricultural residues, mainly lignocellulosic materials, have gained attention to overcome this problem. In Brazil, production of 2nd generation ethanol from the glucose fraction, derived from sugarcane bagasse hydrolysate has been studied. The huge amounts of remaining xylose will create an opportunity for the development of other bioprocesses, generating new products to be introduced into a biorefinery model. Although PHA production from sucrose integrated to a 1G ethanol and sugar mill has been proposed in the past, the integration of the process of 2G ethanol in the context of a biorefinery will provide enormous amounts of xylose, which could be applied to produce PHA, establishing a second-generation of PHA production process. Those aspects and perspectives are presented in this article.

Polyhydroxyalkanoate biosynthesis and simultaneous remotion of organic inhibitors from sugarcane bagasse hydrolysate by Burkholderia sp.

Burkholderia sp. F24, originally isolated from soil, was capable of growth on xylose and removed organic inhibitors present in a hemicellulosic hydrolysate and simultaneously produced poly-3-hydroxybutyrate (P3HB). Using non-detoxified hydrolysate, Burkholderia sp. F24 reached a cell dry weight (CDW) of 6.8 g L(-1), containing 48 % of P3HB and exhibited a volumetric productivity (PP3HB) of 0.10 g L(-1) h(-1). Poly-3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (P3HB-co-3HV) were produced using xylose and levulinic acid (LA) as carbon sources. In shake flask cultures, the 3HV content in the copolymer increased from 9 to 43 mol% by adding LA from 1.0 to 5.0 g L(-1). In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04 g L(-1) of CDW containing 49 % of P3HB and PP3HB of 0.28 g L(-1 )h(-1). Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed.

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.

PHB biosynthesis in catabolite repression mutant of Burkholderia sacchari.

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose.

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101. The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl- mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates.

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHAMCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h(-1). Limitation and inhibition owing to NH4+ ions were observed, respectively, at 400 and 3500 mg of NH4+/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHAMCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHAMCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHAMCL content. Using the last strategy, 21% PHAMCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH4+ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHAMCL and a productivity of 0.8 g/(L x h) were obtained in 42 h of fed-batch operation. The PHAMCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.