Browsing by Author "Varela, C"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Biomass content governs fermentation rate in nitrogen-deficient wine musts
    (AMER SOC MICROBIOLOGY, 2004) Varela, C; Pizarro, F; Agosin, E
    Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.
  • No Thumbnail Available
    Item
    Engineering bacterial strains through the chromosomal insertion of the chlorocatechol catabolism tfdICDEF gene cluster, to improve degradation of typical bleached Kraft pulp mill effluent pollutants
    (2002) Bobadilla, R; Varela, C; Céspedes, R; González, B
    Chloroaromatic pollutants from bleached Kraft pulp mill effluents (BKME) are difficult to degrade, because bacterial strains present in BKME aerobic treatments, only partially degrade these compounds, accumulating the corresponding chlorocatechol intermediates. To improve the catabolic performance of chlorocatechol-accumulating strains, we introduced, by chromosomal insertion, the tfd(I)CDEF gene cluster from Ralstonia eutropha JMP134 (pJP4). This gene cluster allows dechlorination and channelling of chlorocatechols into the intermediate metabolism. Two bacterial strains, R. eutropha JMP222 and Pseudomonas putida KT2442, able to produce chlorocatechols from 3-chlorobenzoate (3-CB) were used. Acinetobacter lwoffii RB2 isolated from BKME by its ability to grow on guaiacol as sole carbon source and shown to be able to produce the corresponding chlorocatechols from the BKME pollutants 4-, and 5-chloroguaiacol, was also used. The tfd(I)CDEF gene cluster was inserted in the chromosome of these strains using mini Tn5-derived vectors that allow expression of the Tfd enzymes driven by the lacI(q)/P-trc or tfdR/Ptfd-I regulatory systems, and therefore, responding to the inducers isopropyl-beta-D-thiogalactopyranoside (IPTG) or 3-CB, respectively. Crude extracts of cells from strains JMP222, KT2442 or RB2 engineered with the tfd genes, grown on benzoate and induced with IPTG or 3-CB showed Tfd specific activities of about 15% - 80% of that of the strain JMP134. Dechlorination rates for 3-CB or chloroguaiacols correlated with levels of Tfd enzymes. However, none of the strains containing the chromosomal copy of the tfd(I)CDEF cluster grew on monochloroaromatics as sole carbon source. Experiments with BKME aerobic treatment microcosms showed that the catabolic performance of the engineered bacteria was also lower than the wildtype R. eutropha strain JMP134.
  • No Thumbnail Available
    Item
    Metabolic flux redistribution in Corynebacterium glutamicum in response to osmotic stress
    (2003) Varela, C; Agosin, E; Baez, M; Klapa, M; Stephanopoulos, G
    Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes trehalose first and proline at a later stage of the gradient increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-glycolysis and the tricarboxylic acid cycle-while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and aketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutainicum.