José Gregório Cabrera Gomez

Evaluation of soil gram-negative bacteria yielding polyhydroxyalkanoic acids from carbohydrates and propionic acid

Abstract A screening programme was developed leading to the isolation of 75 strains of soil gram-negative bacteria which are able to produce polyhydroxyalkanoic acids (PHA) from sugar-cane derivatives. The evaluation of these strains was performed with regard to their efficiency in converting carbohydrates or propionic acid into PHA constituents. Several strains were able to use sucrose as well as glucose and fructose to grow and afterwards to accumulate poly-(3-hydroxybutyric acid) (PHB) with promising yields. Seven strains were found to have more than 80% of the theoretical value when converting carbohydrates into PHB and accumulated at least 50% of the cell dry weight as PHB. Ten strains incorporated 3-hydroxyvaleric acid units into the polymer from propionic acid of which 3 gave yields comparable to those of Alcaligenes eutrophus.

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 0.65h−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·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.

Identification of the 2-Methylcitrate Pathway Involved in the Catabolism of Propionate in the Polyhydroxyalkanoate-Producing Strain Burkholderia sacchari IPT101T and Analysis of a Mutant Accumulating a Copolyester with Higher 3-Hydroxyvalerate Content

ABSTRACT Burkholderia sacchari IPT101T induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The prp locus of B. sacchari IPT101T is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes (prpR, prpB, prpC, acnM, and ORF5) exhibited identity to genes located in the prp loci of other gram-negative bacteria. prpC encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da. prpB encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101T and were heterologously expressed in Escherichia coli. The acnM gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in Ralstonia eutropha HF39, are also located in the prp locus. The translational product of ORF1 (prpR) had a calculated molecular mass of 70,598 Da and is a putative regulator of the prp cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the prp locus of B. sacchari, and these open reading frames have not been found in any other prp operon yet. In summary, the organization of the prp genes of B. sacchari is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a B. sacchari mutant since it was revealed in this study that the mutant is defective in prpC.

Biosynthesis of poly (3-hydroxybutyric acidco-3-hydroxy-4-pentenoic acid) from unrelated substrates byBurkholderia sp.

From soil, two strains ofBurkholderia sp. were isolated that synthesized and accumulated a copolyester of 3-hydroxybutyric acid and 3-hydroxy-4-pentenoic acid from single, unrelated carbon sources such as surcose or gluconate. Strain IPT77B, for instance, accumulated from gluconate poly(3-hydroxybutyric acidco-3-hydroxy-4-pentenoic acid) up to 70% (w/w) of the cellular dry matter, and 3-hydroxy-4-pentenoic acid contributed up to 6.9 mol 100/mol constituents. The occurence of the constituent with the vinyl pendant group in the polyester was confirmed by gaschromatographic and nuclear magnetic resonance analysis.

Produção biotecnológica de poli-hidroxialcanoatos para a geração de polímeros biodegradáveis no Brasil

In recent years, several studies have been developed in Brazil to produce biodegradable materials. A particular family of bacterial polymers, the polyhydroxyalkanoates (PHA), has received special attention. PHAs are thermoplastic, biodegradable, biocompatible, are synthesised from renewable resources and can substitute petrochemical plastics in some applications. Different aspects have been focused to increase productivity and to reduce the cost of PHA production: bacterial improvement, use of industrial by-products as raw material, bioreactor design, process operation strategies, downstream process, mathematical modelling, polymer characterisation, application and biodegradability of blends. A production process was transferred to industry and studies to produce new PHA by controlling monomer composition are in progress. All these aspects are presented in this review.

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.

Making Green Polymers Even Greener:Towards Sustainable Production of Polyhydroxyalkanoates from Agroindustrial By-Products

Jose G. C. Gomez1, Beatriz S. Mendez2, Pablo I. Nikel2,3, M. Julia Pettinari2, Maria A. Prieto4 and Luiziana F. Silva1 1Institute of Biomedical Sciences, University of Sao Paulo 2Department of Biological Chemistry, Faculty of Sciences, University of Buenos Aires and National Council for Research (CONICET), 3Institute for Research in Biotechnology, University of San Martin, 4Department of Environmental Biology, Centro de Investigaciones Biologicas, 1Brazil 2,3Argentina 4Spain

Exploring the potential of Burkholderia sacchari to produce polyhydroxyalkanoates

Evaluation of the capability of Burkholderia sacchari to incorporate different monomers into polyhydroxyalkanoates (PHA).

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 CO2 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.

A suitable procedure to choose antimicrobials as controlling agents in fermentations performed by bacteria

This work evaluated the influence of nitrofurantoin, erythromycin and streptomycin at 50, 25 and 12,5% of the minimal inhibitory concentration (MIC) on maximum specific growth rate (µmax) and specific polymer accumulation rate (µPHB) of Alcaligenes eutrophus, considered resistant to those antimicrobials. Nitrofurantoin strongly affected µmax even at 50% MIC. Streptomycin moderately affected µmax only at 50%MIC. Nitrofurantoin showed the most harmful effect on µPHB when 50% MIC was applied and erythromycin was not harmful.