Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

COMPETITION BETWEEN STAURASTRUM LUETKEMUELLERII (CHLOROPHYCEAE) AND MICROCYSTIS AERUGINOSA(CYANOPHYCEAE) UNDER VARYING MODES OF PHOSPHATE SUPPLY1

The desmid Staurastrum luetkemuellerii Donat et Ruttner and the cyanobacterium Microcystis aeruginosa Kütz. were grown in mixed cultures with various phosphate (P_i) additions. One pulse of P_i each day (semi-continuous cultures) favored M. aeruginosa whereas S. luetkemuellerii was favored when the same quantity of P_i was supplied continuously (chemostats). Both species coexisted under P limitation provided that the nutrient was supplied in an appropriate mode. The ability of each species to compete for P depended on their P_i uptake characteristics and their capability to retain the accumulated P_i. High affinity in uptake at low P_i concentrations contributed considerably to the growth eficiency of S. luetkemuellerii under continuous supply of P_iM. aeruginosa was, however, consistently superior to S. luetkemuellerii in accuniulatiug the newly added P, but had a high rate of P_i release. In both -types of cultures, a net high of P went from M. aeruginosa to S. luetkemuellerii. The kinetic characteristics of the two species were used to simulate the outcome of competition experiments. Simulations agreed with the experimental data f both uptake and P_i release were considered in the model. The zlariable P*(the concentration of P_i at which the net uptake is equal to μ·Q_P is a function of uptake and release of P_i but could not explain the chemostat results. S. luetkemuellerii was the winner in many experiments even if its P*was higher thou that of M. aeruginosa. Thus, in the present case P_c (the concentration at which the net uptake is zero) was a better predictor of the ability to compete for P_i under steady state as well as transient conditions in the P_i supply.     

Aggressiveness and kinship in brown trout (Salmo trutta) parr

In a series of experiments, kin-biased behavior of young browntrout (Salmo trutta) was observed. The aggressiveness shownby groups of familiar siblings (siblings reared together sincefertilization) and groups of unfamiliar siblings (siblings rearedapart since fertilization) was significantly lower comparedto that of mixed groups of two unrelated sibling groups (offspringof two different pairs of parents). The evolution of kin-biasedbehavior, as shown by a reduction in aggressiveness, is assumedto have evolved through a kin-selective mechanism.[Behav Ecol7: 445-450 (1996)]     

Use of swimming speed and egg ratio as predictors of the status of rotifer cultures in aquaculture

This study evaluated the use of egg ratio (eggs rotifer^–1) and swimming speed (mm min^–1) as prediction criteria for production and culture quality in mass cultures of the rotifer Brachionus plicatilis. Egg ratio was determined to be a suitable predictor of rotifer growth and production in the cultures. Low egg ratios (i.e., 0–0.17 eggs rotifer^–1) indicate reduced rotifer population over time (i.e., negative net population growth rates). However, at this time egg ratio dynamics are not suitably understood to predict in advance a sudden population collapse.Swimming speed of reproductive, egg-carrying females in the exponential growth phase was 40–45 mm min^–1. During exponential growth swimming speed was independent of the food used. Lower swimming speeds were obtained in late stationary phase (10–25 mm min^–1) when yeast was used as a food source. Both environmental factors (e.g., accumulating metabolites) and changes in nutritional state of the rotifers may have affected the swimming speed, but environmental factors appear to be the most important. We believe that swimming speed has the potential of becoming an accurate predictor of culture quality in mass cultures of rotifers.     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

Change in social status and risk of low birth weight in Denmark: population based cohort study.

OBJECTIVE: To estimate the risk of having a low birthweight infant associated with changes in social, environmental, and genetic factors. DESIGN: Population based, historical cohort study using the Danish medical birth registry and Statistic Denmark''s fertility database. SUBJECTS: All women who had a low birthweight infant (< 2500 g) (index birth) and a subsequent liveborn infant (outcome birth) in Denmark between 1980 and 1992 (exposed cohort, n = 11,069) and a random sample of the population who gave birth to an infant weighing > or = 2500 g and to a subsequent liveborn infant (unexposed cohort, n = 10,211). MAIN OUTCOME MEASURES: Risk of having a low birthweight infant in the outcome birth as a function of changes in male partner, area of residence, type of job, and social status between the two births. RESULTS: Women in the exposed cohort showed a high risk (18.5%) of having a subsequent low birthweight infant while women in the unexposed cohort had a risk of 2.8%. After adjustment for initial social status, a decline in social status increased the absolute risk of having a low birthweight infant by about 5% in both cohorts, though this was significant only in the unexposed cohort. Change of male partner did not modify the risk of low birth weight in either cohort. CONCLUSION: Having had a low birthweight infant and a decline in social status are strong risk factors for having a low birthweight infant subsequently.