Aiden J. McLoughlin

Effect of Substrate on the Regulation of Exoprotease Production by Pseudomonas aeruginosa ATCC 10145

Exoprotease production by Pseudomonas aeruginosa ATCC 10145 was growth-associated when cultures were grown on complex substrates such as proteins but it occurred during the decelerating growth phase when the organism was grown on amino acids, mixtures of amino acids or simple carbon sources. NH4Cl and simple carbon sources caused repression. Exoprotease was produced in chemostat cultures in response to growth under any of the nutrient limitations studied (carbon, nitrogen or phosphate). Furthermore, by growing at rates less than approximately 0.1 h-1, the repression of enzyme production could be overcome to a large degree. At low growth rates there was an inverse relationship between growth rate and exoprotease production. Thus, exoprotease production was depressed by available energy sources and was increased in response to any nutrient limitation.

Meat fermentations with immobilized lactic acid bacteria

SummaryTwo meat starter cultures, one ofLactobacillus plantarum and the otherPediococcus pentosaceus, were immobilized in calcium alginate beads and then lyophilized. Upon inoculation into meat, the immobilized cultures were found to ferment more rapidly than comparable free cell cultures.

Production of a liquid inoculum/spawn of Agaricus bisporus

The production of a homogeneous liquid culture of mushroom mycelium with a high density of viable inoculum points is a prerequisite for the adaptation of the liquid culture technology to the mushroom spawn production process. Homogenisation proved unsuitable as a technique to produce a morphology of this nature because of the shear sensitive nature of Agaricus bisporus. To overcome this limitation, a homogeneous culture was produced by exposing culture flasks to alternating periods of shear stress (300 rpm on a shaker table for 60 min day−1) and recovery (23 h day−1 under static conditions).

The regulation of pyocyanin production in Pseudomonas aeruginosa

SummaryPyocyanin was produced only after the exponential phase of growth on all media examined. Pyocyanin was also found to be produced in response to some nutrient limitation (for example, carbon or oxygen). Furthermore, by controlling the growth rate at less than approximately 0.1 h−1 the repression of pyocyanin production could be overcome to a large degree. An inverse relationship existed at low growth rates between growth rate and pyocyanin production, with a decrease in growth rate resulting in increased pyocyanin levels.Therefore, pyocyanin production appeared to be regulated by the energy status of the cell which would be lowered under conditions of low nutrient concentration, resulting in a decrease in growth rate and an increase in the level of pyocyanin produced. Under conditions of readily available nutrients the energy generating capacity of the cell was increased resulting in an increased growth rate and repression of pyocyanin.The ability of uncouplers of oxidative phosphorylation (e.g. CCCP and FCCP) to induce pyocyanin production, and of inhibitors of the membrane-bound ATPase (e.g. DCCD and sodium azide) to repress pyocyanin production, confirmed the existance of an energy mediated regulatory mechanism. Indeed, the evidence presented here along with the reported regulatory role of inorganic phosphate in pyocyanin production, suggests that production of this antibiotic may be regulated by intracellular ATP levels.

Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol–water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.

Plasmid stability and ecological competence in recombinant cultures.

The instability of cell cultures containing plasmid vectors is a major problem in the commercial exploitation of molecular cloning techniques. Plasmid stability is influenced by the nature of the host cell, the type of plasmid and/or environmental conditions. Plasmid encoded properties may confer a selective advantage on the host cell but can be an energy drain due to replication and expression. Stability of recombinant cultures ultimately may be determined by the cost to benefit ratio of plasmid carriage. The relative competition between plasmid containing and plasmid-free or indigenous populations can determine the degree of dominance of recombinant cultures. The use of inocula in biotechnological processes in which dynamic environmental conditions dominate may also result in instabilities resulting from the characteristics of the ecosystem. In such dynamic conditions plasmid stability is just one contribution to culture stability. Strategies to enhance plasmid stability, within such environments, based on manipulation of physiological state of host cells, must consider the responsiveness or plasticity of both cells and populations. The robustness of cells or the responses to stresses or transient environmental conditions can influence the levels of instability detected; for example, instability or mutation in the host genome may lead to enhanced plasmid stability. Competition among subpopulations arising from unstable copy number control may determine the levels of recombinant cells in open versus closed fermenter systems. Thus the ecological competence (ability to survive and compete) of recombinant cells in dynamic or transient environments is fundamental to the understanding of the ultimate dominance or survival of such recombinant cultures and may form the basis of a strategy to enhance or control stability either in fermenter systems or dynamic process environments. The creation of microniches in time and/or space can enhance plasmid stability. Transient operation based on defined environmental stresses or perturbations in fermenter systems or in heterogeneous or dynamic environments found in gel immobilized cultures have resulted in enhanced stability. Spatial organization resulting from immobilization has the additional advantage of regulated cell protection within defined microenvironments and controlled release, depending on the nature of the gel, from these microenvironments or microcosms. This regulation of ecological competence allied to the advantages of microbial cell growth in gel microenvironments combined with the spatial organization (or juxtapositioning of cells, selective agents, nutrients, protectants, etc.) possible through immobilization technology offers new strategies to enhance plasmid and culture stability.

Characterization of the physical properties of yeast flocs

SummaryPhysical characteristics, namely floc density function, floc size distribution, and relative floc strength, of a number of flocculent yeast types were measured. A straight-line relationship was found to exist between log values of size and density for the yeasts examined. Each yeast type had coefficients from this relationship which could be used to interpret settling behaviour. Indices of relative floc strength were also obtained and together with the floc density function allowed fuller interpretation of yeast settling than with simpler theories.

The adsorption of bovine serum albumin by activated sludge

Abstract The rapid removal, from suspension, of between 2–% of bovine serum albumin (BSA) by BSA acclimated activated sludge was attributed to adsorption. The extent of adsorption varied with the substrate to biomass (s/b) ratio. The concentration of BSA adsorped was influenced by both the concentration of BSA and the concentration of activated sludge. The experimental data did not conform to the calssical adsorption equations of Langmuir ( J. Am. chem. Soc. 40 , 1361–1403, 918) or Freundlich ( Colloid and Capillary Chemistry , Methuen, London, 1926) but to a newly developed equation, the activated sludge adsorption equation (ASAE). This new equation was tested and proven by experimental data and by data obtained independently by Banerji et al. ( J. Wat. Pollut. Control Fed. 40 , 161–173, 1968) who investigated starch removal by activated sludge. Following the development of the ASAE, it was found possible to express both the concentration of BSA adsorbed per unit weight activated sludge ( m ) and the concentration of BSA in equilibrium per unit weight activated sludge ( C /b) as a function of the concentration of BSA added to the system per unit weight adsorbent ( C t ). Thus adsorption could be expressed as a function of the substrate to biomass (s/b) ratio.

The Protonmotive Force in Pseudomonas aeruginosa and its Relationship to Exoprotease Production

In Pseudomonas aeruginosa ATCC 10145 a negative correlation was observed between the protonmotive force (delta P) and the amount of exoprotease produced, with a decrease in delta P resulting in an increase in exoprotease. The two components of delta P, the transmembrane pH gradient (delta pH) and the membrane potential (delta psi) were examined independently and it was observed that delta psi varied very little under the conditions which influenced the activities of exoprotease. However, a positive correlation existed between pH and exoprotease production although the intracellular pH varied very little with either changes in growth rate or changes in extracellular pH. It was observed that with a decrease in growth rate, delta pH became more alkaline and increased exoprotease activities were recorded. Furthermore, an increase in extracellular pH to give an artificial alteration in delta pH, and, consequently, a decrease in delta P, increased exoprotease production, thus confirming the importance of delta pH in exoprotease production.

Quantitative determination of the spatial distribution of microbial growth kinetics within alginate beads using an image analysis technique

Comprehensive analyses of spatially organized microbial systems require the use of techniques which can investigate microorganisms in defined regions of space. In this work, an in-situ image analysis method was developed which allowed the quantitative description of Lactobacillus plantarum biomass levels at different locations within alginate beads. Using this technique the kinetic properties of the immobilised biomass were determined in 50 μm increments across the radius of the alginate bead.