Marilda Keico Taciro

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 (83xa0gxa0l−1) was achieved using soybean oil at 40xa0gxa0l−1 and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5xa0gxa0l−1xa0h−1, and the calculated Yp/s value was 0.85xa0gxa0g−1. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.

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 6.8xa0gxa0L−1, containing 48xa0% of P3HB and exhibited a volumetric productivity (PP3HB) of 0.10xa0gxa0L−1xa0h−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 43xa0mol% by adding LA from 1.0 to 5.0xa0gxa0L−1. In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04xa0gxa0L−1 of CDW containing 49xa0% of P3HB and PP3HB of 0.28xa0gxa0L−1xa0h−1. Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed.

Combining molecular and bioprocess techniques to produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with controlled monomer composition by Burkholderia sacchari

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl-1h-1. The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions.

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

ABSTRACT 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%.

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 27xa0% 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 2.5xa0MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5–1.0xa0MDa). 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.

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (YP3HB/Xilxa0=xa00.35xa0g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

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

ABSTRACT 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

ABSTRACT 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%.

Metabolic pathways analysis in PHAs production by Pseudomonas with 13C-labeling experiments

Abstract Metabolic flux analysis is a useful tool for metabolism characterization and verification of genetic modification effects; it is a support for decisions on biotechnological process improvement. Commercial production of biodegradable polymers, specifically polyhydroxyalkanoates PHAs, is restricted by production costs, which may be cut by increasing yield from substrate to product, since carbon source for PHAs production accounts up to 50% of the total production costs; additionally, in Pseudomonas sp . LMF046 the experimental yield is between 60-70% of theoretical maximum yield. This work presents metabolic pathways identification, flux quantification and analysis on this strain, employing position isotopomer balancing and measurements of labeling patterns in the polymer by GC-MS. Initial results –using [1- 13 C]glucose-allow to rule out carbohydrate catabolism by EMP pathway, whereas final ones –using a mixture of [U- 13 C]glucose and natural glucose– allow to estimate fraction of glucose metabolized by ED and PP pathways. Metabolic network includes eight intracellular metabolite and 324 isotopomer balances, and it is solved in 1.3 seconds in a Core i5 PC. Sensitivity analysis shows inclusion of carbon natural labeling improves the prediction. The estimated ratio for sugar metabolism into PP and ED pathways, 1.35:0.55, that corresponds to 77% of the theoretical yield, leads to the conclusion that the glucose metabolism in larger proportion by the Pentoses pathway is the main reason for a low yield. The problem has been solved satisfactorily, and a sensitivity analysis shows that it is necessary to reduce uncertainty on labeling measurements.