Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus using non conventional low-cost carbon sources, such as glycerol remaining from biodiesel production and grape bagasse, a residue of wine production. The carbon sources assayed showed their suitability for microbial cellulose production, with relatively high production values such as 10.0 g/l for the culture medium with glycerol from biodiesel as carbon source and corn steep liquor as nitrogen source; and 8.0 g/l for the culture medium containing grape bagasse and corn steep liquor. Glucose, commercial glycerol and cane molasses were also assayed as carbon souces for comparison. The bacterial celluloses produced were characterized by means of scanning electron microscopy, Xray diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis. Morphological analysis showed that bacterial cellulose microfibrils produced from the non-conventional media used were several micrometers long and had rectangular cross-sections with widths and thicknesses in the range of 35-70 nm and 13-24 nm, respectively. X-ray patterns showed crystallinity levels in the range of 74-79% (area method), whereas both X-ray patterns and infrared spectroscopy evidenced the presence of bands characteristic of Cellulose I polymorph. Thermal properties were similar to those found for the pellicle obtained from glucose. The study performed showed the suitability of using wine residues or glycerol remaining from increasing biodiesel production as cheap carbon sources for production of bacterial cellulose microfibrils, with similar characteristics as those obtained by use of more expensive carbon sources such as glucose or commercial glycerol. On the other hand, the low cost nitrogen sources used (corn steep liquor or diammonium phosphate) also contributed to the economy of the bioprocess.Fil: Vazquez, Analia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Tecnologías y Ciencias de la Ingeniería; ArgentinaFil: Foresti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Tecnologías y Ciencias de la Ingeniería; ArgentinaFil: Cerrutti, Patricia. Universidad de Buenos Aires. Facultad de Ingeniería; ArgentinaFil: Galvagno, Miguel Angel. Universidad de Buenos Aires. Facultad de Ingeniería; Argentina. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentin

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% ؎ 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% ؎ 3%. This strain grew in a simple medium at a specific growth rate of 0.69 ؎ 0.07 h ؊1 , whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 ؎ 0.06 h ؊1 . The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.

A recombinant E. coli strain (K24K) was constructed and evaluated for poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20°C higher than those of PHBs from the natural producer strains.Polyhydroxyalkanoates (PHAs) are a group of polyesters produced by a large number of bacteria, which accumulate them in intracellular granules as a response to environmental stress and nutrient imbalance (7, 10). These thermoplastics have properties that vary according to their monomer compositions. A copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate was commercialized in the 1980s, but high production costs have hindered the use of PHAs as commodity plastics, since their final price is considerably higher than that of petrochemical-based synthetic plastic materials (16). Growing concern about environmental pollution has renewed interest in the development of PHAs, which are totally biodegraded by microorganisms present in most environments (11). Also, these polymers can be produced from different renewable carbon sources (11).Poly(3-hydroxybutyrate) (PHB) production costs can be reduced by several means, including the use of cheap substrates, such as whey, favored in countries with important dairy industries, or the enhancement of product yield, e.g., by using recombinant Escherichia coli (11,18). E. coli is a suitable host as a heterologous expression background for foreign genes that can be easily manipulated and improved by means of recombinant DNA methodologies. Also, high-cell-density cultivation strategies for numerous E. coli strains are well established (15,32). E. coli cells that accumulate large amounts of PHB become fragile, facilitating the isolation and purification of the biopolymer, and the bacterium does not express PHA-degrading enzymes (2).PHB is the best known PHA and has been studied most often as a model product in the development of fermentation strategies. In the majority of PHB-accumulating species, it is synthesized in three sequential enzymatic steps: a 3-ketothiolase condenses two acetyl-coenzyme A (CoA) moieties to form acetoacetyl-CoA; a NADPH-dependent a...

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.

The effects of vacuum-drying and freeze-drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10-20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the Tg values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 degrees C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems.

No abstract

ArcA is a global regulator that switches on the expression of fermentation genes and represses the aerobic pathways when Escherichia coli enters low oxygen growth conditions. The metabolic profile of E. coli CT1062 (ΔarcA)and CT1061 (arcA2) grown in microaerobiosis with glycerol as carbon source were determined and compared with E. coli K1060, the arcA⁺ parent strain. Both arcA mutants achieved higher biomass yields than the wild-type strain. The production of acetate, formate, lactate, pyruvate, succinate and ethanol were determined in the supernatants of cultures grown on glycerol under microaerobic conditions for 48 h. The yield of extracellular metabolites on glycerol showed lower acid and higher ethanol values for the mutants. The ethanol/acetate ratio was 0.87 for the parent strain, 2.01 for CT1062, and 12.51 for CT1061. Accordingly, the NADH/NAD⁺ ratios were 0.18, 0.63, and 0.97, respectively. The extracellular succinate yield followed a different pattern, with yield values of 0.164 for K1060, 0.442 for CT1062 and 0.214 for CT1061. The dissimilarities observed can be attributed to the different effects exerted by the deletion and point mutations in a global regulator.

Aims:  Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results:  Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l−1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l−1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l−1 calcium d‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l−1 with a volumetric productivity of 0·34 ± 0·02 g l−1 h−1. Conclusions:  Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study:  Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.