Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media

A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.     

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Reactions of two aromatic and two aliphatic amines with methyl 6-O-p-toluenesulfonyl-alpha-D-glucopyranoside or methyl 6-O-p-toluenesulfonyl-beta-D-glucopyranoside were performed on a micro-scale. The synthesis and preparative isolation methods have been developed for quaternary N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-alpha- and -beta-D-glucopyranoside-6-yl)ammonium salts derived from three amines: trimethylamine, 2-methylpyridine, and pyridine. The reaction products were examined with 1H, 13C NMR spectroscopy. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-beta-d-glucopyranoside-6-yl)trimethylammonium tosylate was additionally analyzed with X-ray crystallography.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Cellulose acetate membrane electrophoresis and FTIR spectroscopy as methods of identifying a fucoidan in Fucusvesiculosus Linnaeus

A fucoidan from brown algae such as the common bladder wrack (Fucusvesiculosus Linnaeus) is now widely examined in many countries for its interesting biological and therapeutic properties. In this study, the fucoidan was identified during extraction in hydrochloric acid; its presence was confirmed by FTIR spectroscopy. Two slightly different structures were found in real samples of dried bladder wrack supplied by Flos and Witherba, by comparing them with a reference sample of F. vesiculosus L. A simple, repeatable analytical procedure was developed using apparatus for cellulose acetate membrane electrophoresis and this was supplemented by semi-quantitative analysis.     

The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution

We propose that the proper evolving unit in enzyme evolution is not a single “fittest molecule”, but a cluster of related variants denoted a “quasi-species”. A distribution of variants provides genetic variability and thereby reduces the risk of inbreeding and evolutionary dead-ends. Different matrices of substrates or activity modulators will lead to different selection criteria and divergent evolutionary trajectories. We provide examples from our directed evolution of glutathione transferases illustrating the interplay between libraries of enzyme variants and ligand matrices in the identification of quasi-species. The ligand matrix is shown to be crucial to the outcome of the search for novel activities. In this sense the experimental system resembles the biological environment in governing the evolution of enzymes.     

Comparative antibacterial potential of selected aldehyde-based biocides and surfactants against planktonic Pseudomonas fluorescens

The antimicrobial efficacy of two aldehyde-based biocides (glutaraldehyde, GTA, and ortho-phthalaldehyde, OPA) and two surfactants (cetyltrimethyl ammonium bromide, CTAB, and sodium dodecyl sulphate, SDS) was tested against planktonic Pseudomonas fluorescens. The antimicrobial effects were evaluated by respiratory activity as a measure of the oxygen uptake rate, adenosine triphosphate (ATP) release, outer membrane proteins (OMP) expression and cellular colour changes. The results were compared with the bacterial characteristics without chemical treatment. Tests in the presence of bovine serum albumin (BSA), in order to mimic a disinfection process in the real situation under dirty conditions, were performed according to the European Standard EN-1276. P. fluorescens was completely inactivated with OPA (minimum bactericidal concentration, MBC = 0.5 mM) and CTAB (MBC = 5 mM) and was resistant to GTA and SDS. Only CTAB promoted cellular disruption and consequent ATP release. The antimicrobial action of the chemicals tested was significantly reduced when BSA was introduced into the bacterial cultures, increasing markedly the MBC values. Additionally, the presence of BSA acted as a disruption protective agent when CTAB was applied and stimulated the bacterial respiratory activity when lower concentrations of SDS were tested. The OMP of the bacterial cells was affected by the application of both surfactants. OMP expression remained unaltered after biocide treatment. Bacterial colour change was noticed after treatment with biocides and surfactants. In summary, P. fluorescens was extremely resistant to GTA and SDS, with antimicrobial action being quenched markedly by the reaction with BSA.     

An assessment of time-dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches

Time-dependent effects of lead (Pb) toxicity were studied in Algerian mice (Mus spretus) treated with Pb acetate via drinking water (1 g Pb acetate/L) for different periods of exposure (15, 45, and 90 d). End points included the determination of hepatic Pb concentration and the assessment of some morphophysiological, biochemical and cytogenetical parameters. A control group receiving distilled water was also monitored for comparative purposes. Hepatic Pb accumulation increased with the time of exposure and was significantly higher in treated mice when compared to controls. In association with significant body mass loss in Pb-exposed mice, for 15 and 45 d, a significant increase in the relative spleen mass was observed after 45 d of intoxication. Pb-exposed mice also showed significant decreases in red blood cells, hematocrit, and mean corpuscular hemoglobin. On the contrary, changes in plasma transferases (aspartate aminotransferase and alanine aminotransferase) and hepatic superoxide dismutase activities did not reach statistical significance. A significant increase in the frequency of micronucleated polychromatic bone marrow erythrocytes was also found in the 90-d-exposed mice, compared to nontreated mice and the other exposed groups. Exposure to Pb acetate resulted also in a slight time-dependent decrease of the polychromatic-normochromatic ratio. These results support the concept that a long-term chronic exposure to Pb induced alterations upon some morphophysiological and genetic paramaters in Algerian mice.     

Redox Fluctuations Frame Microbial Community Impacts on N-cycling Rates in a Humid Tropical Forest Soil

Fluctuating soil redox regimes may facilitate the co-occurrence of microbial nitrogen transformations with significantly different sensitivities to soil oxygen availability. In an upland humid tropical forest, we explored the impact of fluctuating redox regimes on gross nitrogen cycling rates and microbial community composition. Our results suggest that the rapidly fluctuating redox conditions that characterize these upland soils allow anoxic and oxic N processing to co-occur. Gross nitrogen mineralization was insensitive to soil redox fluctuations. In contrast, nitrifiers in this soil were directly affected by low redox periods, yet retained some activity even after 3–6 weeks of anoxia. Dissimilatory nitrate reduction to ammonium (DNRA) was less sensitive to oxygen exposure than expected, indicating that the organisms mediating this reductive process were also tolerant of unfavorable (oxic) conditions. Denitrification was a stronger sink for NO₃⁻ in consistently anoxic soils than in variable redox soils. Microbial biomass and community composition were maintained with redox fluctuation, but biomass decreased and composition changed under static oxic and anoxic soil regimes. Bacterial community structure was significantly correlated with rates of nitrification, denitrification and DNRA, suggesting that redox-control of soil microbial community structure was an important determinant of soil N-cycling rates. Specific nitrogen cycling functional groups in this environment (such as nitrifiers, DNRA organisms, and denitrifiers) appear to have adapted to nutrient resources that are spatially and temporally variable. In soils where oxygen is frequently depleted and re-supplied, characteristics of microbial tolerance and resilience can frame N cycling patterns.     

Colony Structure and Seasonal Differences in Light and Nitrogen Modify the Impact of Sessile Epifauna on the Giant Kelp Macrocystis pyrifera (L.) C Agardh

The presence of the stoloniferous hydroid Obelia geniculata (L.) had no effect on the pigment concentration or nitrogen status of underlying blade tissue of the giant kelp Macrocystis pyrifera (L.) C. Agardh. The sheet-like colonies of the bryozoan Membranipora membranacea (L.) markedly reduced the pigment concentration of colonized blade tissue, but only during winter. Reductions in pigment concentration are most likely a result of damage to underlying tissue due to some factor related to the presence of bryozoan colonies on blade surfaces. Blade tissue colonized by M. membranacea also had higher δ¹⁵N signatures than surrounding bryozoan-free tissue, possibly indicating the provision of nitrogen to M. pyrifera by bryozoan colonies. Results show that seasonal changes in nitrogen and colony size can strongly modify the effect of epifauna on macroalgae they colonize. Unlike bryozoans, hydroid colonies provided no barrier to nitrogen uptake by colonized M. pyrifera tissue and enhanced ammonium uptake was observed for tissue colonized by O. geniculata during nitrogen limitation. Epifauna with stoloniferous growth forms such as hydroids are more likely to have benign or even mutualistic relationships with macroalgae they colonize than the sheet-like colonies of bryozoans.