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.

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.

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.

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%.

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%.

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.

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.

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.

High-cell-density poly (3-hydroxybutyrate) production from sucrose using Burkholderia sacchari culture in airlift bioreactor.

Burkholderia sacchari IPT 189 poly (3-hydroxybutyrate) (P3HB) production in airlift bioreactor were investigated in batch and fed-batch culture using sucrose as carbon source. In batch experiments it was observed that during the growth phase B. sacchari IPT 189 might display exponential growth even at very low carbohydrate concentration, as long as NH(4)(+) concentration was above 190 mg l(-1). The onset of accumulation phase took place when NH(4)(+) concentration dropped below this value and continued as long as carbohydrate was in excess, even with dissolved oxygen concentration at 0.0% of air saturation. In the fed-batch experiments, nitrogen limitation was used to induce P3HB biosynthesis in a two-phase process. In the first phase, an initial batch followed by a limited sucrose fed regime led to a growth with low-P3HB-content (less than 13%) and up to 60 g l(-1) of biomass concentration in c.a. 25 h. In the second phase, nitrogen concentration limitation induced P3HB accumulation up to 42%, raising the biomass concentration to c.a. 150 g l(-1). Calculated parameters for the experiments were P3HB productivity=1.7 gl(-1) h(-1) and P3HB yield factor from sucrose=0.22 g g(-1).

Cultivation of Bacillus thuringiensis subsp. Kurstaki S93 in a fed-Batch process: effect of the ratio glucose to yeast extract concentrations in the feeding culture media

The feeding media was studied in a fed-batch process for production of the complex spore-d-endotoxin by B. thuringiensis S93. The microorganism was first cultvated in a initial batch followed by an exponential feeding (m=0,25h(-1)) with concentrated culture media (160 g glucose/L) containing different ratios of glucose and autolysed yeast: 8/7, 8/4 and 8/2 (g/g). The batch culture medium was composed of glucose (8 g/L), autolysed yeast (7g/L) and mineral salts. Sporulation and d-endotoxin production were observed only after the end of feeding. To compare the experiments, batch cultivations were also performed with an initial concentration of 8 g/L of glucose and the same ratio of glucose and autolized yeast. Batch cultivations reached lower concentrations of total biomass and spores than the fed-batch ones and percentagens of sporulation higher than 80 per cente. The 8/7 ratio fed-batch cultivations reached the highest biomass concentration, producing however a very low level of sporulation (27per cent) and virtually no d-endotoxin. Cultivations with 8/4 and 8/2 ratios reached the highest concentrations of spores. In those assays, the maximum spores concentration and the maximum sporulation percentage were 8,3x10(9) spores/mL and 90per cent for the 8/4 ratio and 5,6x10(9) spores/mL and 89per cent for the 8/2 ratio.