Competition between two aquatic microorganisms for oscillating concentrations of phosphorus

Thesis (Ph.D.) University of Alaska Fairbanks, 1989The availability of limiting nutrients is a critical factor regulating growth of aquatic microorganisms. In at least some aquatic systems the frequency of addition rather than the absolute concentration of nutrients controls community structure. Gnotobiotic continuous cultures were used to examine the growth characteristics of a green alga (Selenastrum capricornutum) and a heterotrophic yeast (Rhodotorula rubra) when phosphorus-limited steady-state populations were subjected to varying concentrations of pulsed phosphorus. The responses of these organisms to phosphorus additions were measured both in single and dual species continuous cultures. Both organisms exceeded the maximum transport rates for phosphorus predicted from batch and steady-state continuous cultures. Carbon limitation did not cause a decline in phosphorus accumulation in R. rubra. Carbon-limited yeast cultures perturbed with phosphorus attained the highest phosphorus per cell values seen in these studies. The phosphorus pool was not significantly diminished in these cultures only because the total yeast biomass was limited by carbon. These results suggest that carbon-limitation of heterotrophic populations may be essential to the existence of phytoplankton in low-nutrient aquatic environments

The theoretical and experimental exploration of the use of predatory bacteria to control biofilms

Membrane based technologies are widely used for treating drinking water in sparsely populated areas, but their effectiveness is significantly diminished by the growth of biofilms and biofouling. Preventing or removing biofilms can increase the life span of membranes and, thus, improve their economic viability. Most cleaning methods involve physical disruption or antimicrobial chemicals and, therefore, require an interruption in the membrane operation. Bdellovibrio, a group of predatory bacteria, are a potential alternative to antimicrobials or physical disruption because of its ability to kill a large range of gram-negative bacterial prey and the inability of their prey to develop genetic resistance. However, the use of Bdellovibrio in industrial application has not been widespread in part due to the lack of understanding of the dynamics between Bdellovibrio and their prey. To compound this, many of the previous investigations into Bdellovibrio and biofilm ecology are limited by inaccurate, uninformative, and labour-intensive methods to quantify the population dynamics, which makes it difficult to build comprehensive models to exploit Bdellovibrio as a control to biofilms in systems like drinking water membranes. This thesis aims to develop a set of novel methods and technologies to accurately investigate Bdellovibrio and the effect they have on dynamics of their prey; Pseudomonas sp, a key gram negative biofilm forming species. This research develops the first protocol to use flow cytometry to accurately and rapidly quantify Bdellovibrio and Pseudomonas sp growth, which makes recording high resolution population dynamics feasible. The protocol was used for the development and experimental validation of mathematical models which aimed to predict Bdellovibrio dynamics in batch and chemostat systems. We show the first experimental observation of Bdellovibrio-prey oscillations, a key component of predation dynamics and a desired phenomenon for the use of Bdellovibrio as a self-sustaining biocontrol. To extend the models for application to systems where biofilms prevail, we demonstrated a new method of deploying flow cytometry and fluorescent assays to quantify and characterise the effect of nutrients on biofilm growth and predation. The findings suggest that extracellular polymeric substances (EPS) play a vital role in the attachment and persistence of biofilm when under Bdellovibrio predation. Thus, in biofilm research, the simple density dependent predator-prey interactions need to be augmented by representing the spatial heterogeneities in biofilm processes and properties such as its detachment, EPS and presence of metabolically damaged cells. For a more nuanced analysis of predator-prey interactions, at the resolution of individual organisms, this research develops a novel microfluidic device to observe Bdellovibrio predation on a 1-D biofilm. This thesis describes both the rationale and novel protocols for combining electron-beam lithography with, the more commonly used, photolithography to create an array of high-resolution channels to constrain biofilms and challenge them with predators. The research demonstrates the opportunities and the technical challenges in using microfluidics. Ultimately, if we are to develop mathematical models that can be parameterised and used effectively in designing strategies for controlling biofilms using predatory bacteria, then observations at the individual scale in microfluidic devices will be invaluable

Metabolic shifts in microorganisms: the case of Lactococcus lactis

A commonly observed organismal response to changing growth rate is a metabolic shift from one mode of metabolism to another. This phenomenon is potentially interesting from a fundamental and industrial perspective because it can influence cellular choices and can limit the capacity of industrial microorganisms to channel nutrients to desired products. The mechanistic cause of the metabolic shift may vary between species, but the presence of such shifts from bacteria to man suggests functional relevance, which may be understood through an evolutionary perspective. One of the many existing hypotheses (reviewed in Chapter 2) states that protein investment costs affect the metabolic strategy employed, and that the implemented strategy is the result of a cost-benefit analysis. To test this experimentally, we performed a global multi-level analysis using the model lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363, which shows a distinct, anaerobic version of the bacterial Crabtree/Warburg effect: at low growth rates it produces “mixed-acids” (acetate, formate and ethanol) and at high growth rates it produces predominantly lactate from glucose. We first standardized growth conditions and established an in vivo–like enzyme assay medium mimicking the intracellular environment for enzyme activity measurements of growing cells of L. lactis (Chapter 3). With standardized experimental procedures we characterized at multiple cellular levels, glucose-limited chemostat cultures of L. lactis at various growth rates. More than a threefold change in growth rate was accompanied by metabolic rerouting with, surprisingly, hardly any change in transcription, protein ratios, and enzyme activities (Chapter 4). Even ribosomal proteins, constituting a major investment of cellular machinery, scarcely changed. Thus, contrary to the original hypothesis, L. lactis displays a strategy where its central metabolism appears always prepared for high growth rate and it primarily employs the regulation of enzyme activity rather than alteration of gene expression. Only at the highest growth rate and during batch growth – conditions associated with glucose excess – we observed down-regulated stress protein levels and up-regulated glycolytic protein levels. We conclude from this that for glucose, transcription and protein expression largely follow a binary feast / famine logic in L. lactis. To delve deeper into the mechanism of regulation of the shift in L. lactis, we tested a mixed-acid fermentative lactose-utilizing L. lactis MG1363 derivative and showed that there is a strong positive correlation between glycolytic flux and the extent of homolactic fermentation: a correlation caused by metabolic regulation (Chapter 5). We subsequently provided new evidence for a causal relationship between the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate (FBP) and the metabolic shift. We showed that 2,5-anhydromannitol, which converts to a non-metabolizable FBP analogue in vivo, almost doubles the flux towards lactate when taken up by the cells. In vitro the activating effect of the analogue on lactate dehydrogenase is similar to native FBP, whereas it had no effect on the enzyme phosphotransacetylase (part of the mixed-acid pathway). The activation concentration of the analogue, however, is much lower than normal intracellular FBP concentrations. This may imply that the activation of lactate dehydrogenase in vivo requires a much higher concentration of FBP, but this remains to be resolved. We subsequently put the regulatory relationships of glycolytic flux, FBP, the redox potential and allosteric effectors on enzymes of the glycolytic and downstream pathways together in a mathematical model to test and investigate whether these interactions can explain the metabolic shift (Chapter 6). Although the model was not able to consistently fit combined data from the chemostats at various dilution rates, and in vivo–NMR data of glucose pulsed non-growing cells, we found for the best fitted model that the parameters most influencing the metabolic shift were those involved in regulation by FBP and inorganic phosphate. In conclusion, L. lactis seems to be always prepared for high growth rate as it carries a high overcapacity of enzymes, a property retained even after evolving for 800 generations under constant environmental conditions. Moreover, its growth rate-related metabolic shift does not appear to be an outcome of growth-rate optimization with protein cost as a major driver. At the mechanistic level, the choice of the strategy is regulated via alterations in metabolite levels, with FBP (and probably phosphate) exerting a central role.</p

Morphological studies on Kluyveromyces Marxianus var; Marxianus NRRLy2415 in suspension culture: a study incorporating computer aided image analysis

The morphology of the lactose fermenting organism, Kluyveromyces marxianus var. marxianus NRRLy2415 was studied in batch and continuous culture. The morphology of the organism was observed to vary significantly from a budding yeastlike form to a branched pseudohyphal form depending on the operating conditions. The following morphological classes were deemed important for the complete description of culture morphology: yeast, elongated yeast, filament, double yeast, double elongated yeast, double filament and pseudohyphae. Image analysis was used to implement the classification system, due to its objectivity in measurement of visual phenomena. The protocol developed was also capable of measuring geometric properties of the cell population including volume, length and width for all cells and hyphal growth unit length for pseudohyphae. The predominant morphology observed for the organism in fully aerobic batch culture was yeast-like. When the agitation rate was decreased, an increased heterogeneity in the morphology was observed, w ith the generation o f more elongated yeast and filamentous forms. This was attributed to partial oxygen limitation in the fermentations. In chemostat culture, the morphology observed was predominantly pseudohyphal. This morphological form was found to dominate over a large range of dilution rates (0 .1-0 .45 h ']) and substrate feed concentrations (5 to 20 g /L lactose). At both extrema of dilution rates a reversion to a yeastlike morphology was observed. The mycelial morphology was attributed to substrate limitation and was demonstrated to be of ecological advantage under substrate limited conditions. Kinetic modelling of substrate metabolism was undertaken for continuous and batch culture results. It was demonstrated that, the stoichiometry of the metabolic pathways studied was identical under all operating conditions. This is significant, as the morphology of the organism varies significantly from batch to continuous culture. A population model was developed which was capable of describing the distributions of cell geometric parameters based on summed lognormal distributions of selected morphological classes. The ultrastructure of pseudohyphae was examined. This study demonstrated significant differences in methods of formation between pseudohyphae and true hyphae and highlighted key issues that need to be addressed if successful modeling of such grow this to be undertaken

DYNAMIC COMPLEXITIES OF A CHEMOSTAT MODEL WITH PULSED INPUT AND WASHOUT AT DIFFERENT TIMES

In this paper, we consider a predator–prey chemostat model with ratio-dependent Monod type functional response and periodic input and washout at different fixed times. We obtain an exact periodic solution with substrate and prey. The stability analysis for this periodic solutions yields an invasion threshold for the period of pulses of the predator. When the impulsive period is more than the threshold, there are periodic oscillations in the substrate, prey, and predator. If the impulsive period further increases, the system undergoes a complex dynamic process. By analyzing bifurcation diagrams, we can see that the impulsive system shows two kinds of bifurcation, which are period-doubling and period-halving.Predator–prey system, impulsive invasion and washout, chemostat, complexity

Book of abstracts of the 10th International Chemical and Biological Engineering Conference: CHEMPOR 2008

This book contains the extended abstracts presented at the 10th International Chemical and Biological Engineering Conference - CHEMPOR 2008, held in Braga, Portugal, over 3 days, from the 4th to the 6th of September, 2008. Previous editions took place in Lisboa (1975, 1889, 1998), Braga (1978), Póvoa de Varzim (1981), Coimbra (1985, 2005), Porto (1993), and Aveiro (2001). The conference was jointly organized by the University of Minho, “Ordem dos Engenheiros”, and the IBB - Institute for Biotechnology and Bioengineering with the usual support of the “Sociedade Portuguesa de Química” and, by the first time, of the “Sociedade Portuguesa de Biotecnologia”. Thirty years elapsed since CHEMPOR was held at the University of Minho, organized by T.R. Bott, D. Allen, A. Bridgwater, J.J.B. Romero, L.J.S. Soares and J.D.R.S. Pinheiro. We are fortunate to have Profs. Bott, Soares and Pinheiro in the Honor Committee of this 10th edition, under the high Patronage of his Excellency the President of the Portuguese Republic, Prof. Aníbal Cavaco Silva. The opening ceremony will confer Prof. Bott with a “Long Term Achievement” award acknowledging the important contribution Prof. Bott brought along more than 30 years to the development of the Chemical Engineering science, to the launch of CHEMPOR series and specially to the University of Minho. Prof. Bott’s inaugural lecture will address the importance of effective energy management in processing operations, particularly in the effectiveness of heat recovery and the associated reduction in greenhouse gas emission from combustion processes. The CHEMPOR series traditionally brings together both young and established researchers and end users to discuss recent developments in different areas of Chemical Engineering. The scope of this edition is broadening out by including the Biological Engineering research. One of the major core areas of the conference program is life quality, due to the importance that Chemical and Biological Engineering plays in this area. “Integration of Life Sciences & Engineering” and “Sustainable Process-Product Development through Green Chemistry” are two of the leading themes with papers addressing such important issues. This is complemented with additional leading themes including “Advancing the Chemical and Biological Engineering Fundamentals”, “Multi-Scale and/or Multi-Disciplinary Approach to Process-Product Innovation”, “Systematic Methods and Tools for Managing the Complexity”, and “Educating Chemical and Biological Engineers for Coming Challenges” which define the extended abstracts arrangements along this book. A total of 516 extended abstracts are included in the book, consisting of 7 invited lecturers, 15 keynote, 105 short oral presentations given in 5 parallel sessions, along with 6 slots for viewing 389 poster presentations. Full papers are jointly included in the companion Proceedings in CD-ROM. All papers have been reviewed and we are grateful to the members of scientific and organizing committees for their evaluations. It was an intensive task since 610 submitted abstracts from 45 countries were received. It has been an honor for us to contribute to setting up CHEMPOR 2008 during almost two years. We wish to thank the authors who have contributed to yield a high scientific standard to the program. We are thankful to the sponsors who have contributed decisively to this event. We also extend our gratefulness to all those who, through their dedicated efforts, have assisted us in this task. On behalf of the Scientific and Organizing Committees we wish you that together with an interesting reading, the scientific program and the social moments organized will be memorable for all.Fundação para a Ciência e a Tecnologia (FCT