Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml⁻¹ optical density at 550 nm [OD₅₅₀] unit⁻¹), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml⁻¹ OD₅₅₀ unit⁻¹), trehalose (9.9 nmol ml⁻¹ OD₅₅₀ unit⁻¹), and betaine (19.8 nmol ml⁻¹ OD₅₅₀ unit⁻¹). Two independent genetic modifications of E. coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.     

A manipulative experiment to evaluate predicted changes in intertidal, macro-faunal assemblages after contamination by heavy metals

Concentrations of heavy metals (zinc, copper and lead) were manipulated experimentally to test the hypotheses about effects on intertidal, soft-sediment assemblages of animals in two sand-flats in Port Hacking, Australia. Hypotheses about changes in the structure and composition of whole assemblages and changes in mean abundance and variability of individual taxa were tested. Specific hypotheses were derived a priori from repeated observations of assemblages in urban and non-urban areas of Port Hacking.After manipulation, concentrations of metals were similar to those in sediments near urban areas. Nevertheless, responses of assemblages to the increased concentrations of metals were weak. Polychaetes, spionids and amphipods responded to experimental treatments. Changes were, however, not consistent among times and places and generally not in agreement with what had been predicted. Significant spatial and temporal variability were detected for all variables investigated. Increased concentrations of metals did not affect variability or overall structure of assemblages. Thus, there was little evidence that increased concentrations of metals caused benthic assemblages in pristine areas to become more similar to those in areas contaminated by human activities.Several potential explanations for the discrepancy between previously observed correlative patterns and the results presented here are discussed. These include a critical assessment of different aspects of the experimental study, such as lack of statistical power, insufficient basis for prediction, artefacts due to experimental procedures and issues to do with the difference between evidence based on correlation and manipulation. Explicit comparisons showed that there were significant effects of physical disturbance due to repeated sampling and that assemblages of animals at the start of the experiment were different from those previously observed in uncontaminated areas. These observations were surprising and made interpretation more difficult. Nevertheless, it is possible that the metals investigated really contribute only marginally to previously observed differences between urban and non-urban areas. Repeated comparisons between observations of patterns and manipulative experiments like these can only improve the basis for prediction and the power of current mechanistic models.     

PINUS BUCHANANII,A NEW SPECIES BASED ON A PETRIFIED CONE FROM THE OLIGOCENE OF WASHINGTON

A silicified seed cone of Oligocene age from the Olympic Peninsula of Washington represents a new species of Pinus. The cone was about 8 cm long and 3 cm at its widest diameter in the living condition. Its scale apices are thickened and each has a dorsal umbo. Internal cone construction confirms the assignment of the new species to Pinus and suggests affinity with the subsections Australes and Ponderosae of the section Pinus, subgenus Pinus. The cone is peculiar in having a stout bract trace that is slightly concave on its adaxial side and in having resin canals that diverge from the axial secretory system toward the bract but constrict markedly and terminate before entering the bract.     

The needle necropsy.

The technique of limited necropsy by histological examination of needle tissue cores obtained percutaneously is indicated when a full necropsy is not justified owing to the risk of infection or when tissue for special investigations is needed soon after death without recourse to full necropsy facilities. The method is ideal for detecting conditions producing diffuse changes in an organ. Because the cadaver is preserved essentially intact relatives who refuse permission for a standard necropsy might consent to a needle necropsy if this option is offered to them.