No description available.

Universiẗat, Diss., 2005--Bremen. / ERscheinungsjahr an der Haupttitelstelle: 2004.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

Many strains of Gram-negative bacteria, such as Pseudomonas, are able to utilize a variety of unusual chemicals, including a wide range or aromatic hydrocarbons and their derivatives for growth. Bacteria with the potential to degrade sodium benzoate were isolated, identified and grown as biofilms on sodium benzoate in a laboratory-scale fluidized bed biofilm bioreactor. Four Pseudomonas strains identified as P. aeruginosa (BDS2) P. putida (BDS1 and GR1) and Burkholderia cepecia (GR3FAR) were used in a laboratory-scale FBBR together with two Bacillus strains - Bacillus macroides (SBSY4) and Bacillus simplex (MAR). Sodium benzoate biodegradation capacities of these species were compared under batch and continuous operations. Biofilm and planktonic bacterial growth dynamics were monitored by plate counts, and optical density measurements (230nm) determined benzoate biodegradation. Overall, higher attached and planktonic bacterial counts were determined under batch compared to continuous mode. In addition to this, the ability of attached cells to use sodium benzoate as their sole carbon source was compared to their suspended counterparts in a batch system. There were more attached counts compared to suspended cells and attached cells apparently degraded sodium benzoate better than planktonic cells. Similarly, higher rates of benzoate depletion were found to occur under batch compared to the continuous system. It thus appeared that more cell growth implied more substrate consumption. SEM showed attached cells and microcolonies of all the isolates on GAC, indicating their biofilmforming abilities.

Thesis (M. Sc.)--University of Hong Kong, 1994. / Includes bibliographical references.

L’objectif de ce travail était de développer des films antimicrobiens à base de poly(alcool vinylique)(PVOH) réticulés par de l’acide citrique (CTR). La démarche choisie a consisté à incorporer dans le polymère du benzoate de sodium (NaBz), seul ou sous forme de complexe avec l’hydroxypropyl-β-cyclodextrine (HPβCD). La combinaison des fonctions résiduelles du CTR greffé sur le polymère et du NaBz libre permet d’envisager des effets antimicrobiens par contact et par diffusion. Les films ont été réalisés par casting à partir de solutions aqueuses et réticulés par un traitement thermique. La réaction de réticulation a été mise en évidence et suivie à travers l’étude des composés résiduels, l’évolution de la Tg et des fonctions COOH résiduelles en fonction du temps de réticulation. L’étude de différentes compositions a permis de sélectionner les concentrations de 20 et 30 wt% en CTR pour un compromis entre réticulation suffisante et ductilité du film préservée. Le temps nécessaire pour lier l’ensemble des réactifs diminue avec l’augmentation de la température ou de la quantité de CTR. La réaction de réticulation n’est pas modifiée par la présence d’HPβCD. Avec l’ajout de CTR ou d’HPβCD et la réticulation, les films restent faiblement perméables à O2 et fortement perméables à la vapeur d’eau. Concernant la libération du NaBz dans l’eau, la cinétique est ralentie à partir de 360 min de réticulation et de 20 wt% de CTR. L’HPβCD n’a pas d’influence sur la cinétique de libération mais a permis de doubler la quantité libérée. L’activité antimicrobienne par contact et par diffusion a été mise en évidence pour la majorité des films contre l’ensemble des microorganismes étudiés. / The aim of this work was to develop antimicrobial films based on polyvinyl alcohol (PVOH) crosslinked by citric acid (CTR). In our approach, sodium benzoate (NaBz) was introduced in polymer, alone or in complex form with hydroxypropyl-β-cyclodextrin (HPβCD). Both CTR residual functions grafted on polymer and free NaBz are expected to provide antimicrobial effects by contact and diffusion. Films were elaborated by casting from aqueous solutions and crosslinked by heat treatment. Crosslinking reaction has been highlighted and followed through the study of residual reagents, the evolution of Tg and residual COOH functions according to crosslinking time. From this investigation, a compromise between sufficient crosslinking and preserved film ductility has been found for a CTR concentration between 20 and 30 wt%. The time required to bind all of the reagents decreases with the increase of temperature or amount of CTR. The crosslinking reaction was not modified by the presence of HPβCD. With the addition of CTR or HPβCD and the crosslinking step, the films remain slightly permeable to O2 and highly permeable to water vapor. Considering the NaBz release in water, kinetics is slowed from 360 min of crosslinking and 20 wt% CTR. HPβCD has no major influence on the kinetics of release but allow to double released amount. Antimicrobial activity by contact and diffusion has been demonstrated for the majority of films against all studied microorganisms.

Demands are placed on cattle producers to provide a steady supply of beef at a competitive price. Producers must maximize beef output while minimizing input expenses without compromising product quality. The use of growth implants has become a common practice among cattle producers. The objective of this study was to evaluate the effects of two implant strategies on the average daily gain of suckling calves rotationally grazed on Kentucky – 31 endophyte-infected tall fescue and Kentucky – 31 non-endophyte-infected tall fescue. Eighteen cows with spring calves (N = 18) were used in this study. Calves were grouped by birth date into four blocks. Within each block, calves were stratified by sex and 45d of age body weight into three implant treatment groups for a total of six calves per treatment (n = 6, control 90.3 ± 9.7 kg, zeranol 102.9 ± 10.9 kg, and progesterone (100 mg) and estradiol benzoate (10 mg) 92.4 ± 10.3 kg). Calves were weighed and re-implanted at 129 d of age (84 d after initial implant). Zeranol treated calves were re-implanted using the same implant as the initial implant. Progesterone and estradiol benzoate treated calves were re-implanted after reaching a minimum body weight of 181 kg with either 200 mg progesterone and 20 mg estradiol benzoate or 200 mg testosterone propionate and 20 mg estradiol benzoate depending on sex. Data were analyzed using the REPEATED function in the MIXED procedure of SAS. No interactions were found among sex and treatments for 84d weight gains and 140d weights. There were also no main effects found for 84d weight gains and 140d weight gains. Forage analysis suggested that low crude protein and energy content may have contributed to the low ADG. Low endophyte concentrations may also have played a role.

The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively. BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety. The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target. In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann’s reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann’s reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above. We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB. The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme. Our structural analysis revealed that despite its low sequence identity of 28%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD. / Die vorliegende Arbeit befasst sich mit der strukturellen und biochemischen Charakterisierung der beiden unterschiedlichen bakteriellen Enzyme BadI von Rhodopseudomonas palustris und MenD von Staphylococcus aureus. Die 2-Ketocyclohexancarboxyl-CoA-Hydrolase BadI ist eines der Schlüsselenzyme des anaeroben Abbaus aromatischer Verbindungen. Der Abbau aromatischer Verbindungen ist essentiell für die Aufrechterhaltung des biogeochemischen Kohlenstoffkreislaufs und der biologischen Beseitigung von Xenobiotika, welche in höheren Konzentrationen eine Gefahr für den menschlichen Organismus darstellen können. Wegen des inerten Charakters aromatischer Verbindungen sind Enzyme, welche deren Abbau katalysieren, von besonderem Interesse für industrielle Anwendungen. BadI ist eines der Schlüsselenzyme für den anaeroben Abbau aromatischer Verbindungen zu aliphatischen Gruppen. Das Hauptaugenmerk dieses Projekts lag auf der Aufklärung des Reaktionsmechanismus, welcher von BadI katalysiert wird. BadI gehört zur Überfamilie der Crotonasen und zeigt hohe Sequenzhomologie mit der zugehörigen Dihydroxynaphthoat-Synthase MenB. Durch die Hydrolyse einer C-C Bindung katalysiert BadI den Schnitt des zyklischen Rings von 2-Ketocyclohexancarboxyl-CoA, welcher zur Bildung der aliphatischen Verbindung Pimelyl-CoA führt. MenB, andererseits, katalysiert die Kondensationsreaktion von O-Succinylbenzyl-CoA zu Dihydronaphthoyl-CoA. Ein umfassender Aminosäuresequenzvergleich zwischen BadI und MenB zeigt, dass die Reste des aktiven Zentrums von MenB aus Mycobacterium tuberculosis (mtMenB) in BadI von R. palustris konserviert sind. MenB ist Teil des Menaquinon Biosynthesewegs und ein potentielles Wirkstoffziel gegen M. tuberculosis, da kein humanes Homolog existiert. Wegen der ausgeprägten Homologie zwischen MenB und BadI und der Tatsache, dass bisher keine MenB-Inhibitor Komplex Strukturen gelöst werden konnten, erweiterten wir unser Interesse auf BadI, da es als Model für mtMenB als Wirkstoffziel dienen könnte. Darüber hinaus besitzt BadI einige einzigartige mechanistische Charakteristika. Wie zuvor erwähnt, hydrolysiert es das Substrate durch eine reverse Dieckmanns Reaktion in Gegensatz zu seinem ähnlichsten Homolog MenB, das einen Ringschluss durch eine Dieckmanns Reaktion katalysiert. Dennoch scheinen die Reste des aktiven Zentrums streng konserviert zu sein. Daher entschieden wir die strukturelle Charakterisierung von BadI anzugehen um Gemeinsamkeiten und Unterschiede zwischen BadI und MenB aufzuzeigen und einen Einblick zu erhalten, wie sie die gegenläufigen Reaktionen durchführen. Wir lösten die ersten Strukturen von BadI in seiner Apo-Form und einer Substrat-Mimik gebundenen Form. Die Kristallstrukturen von BadI zeigten die gleiche Gesamtfaltung wie andere Mitglieder der Crotonase Familie. Allerdings gibt es in BadI kein Anzeichen für Domain-Swapping, wie es in MenB beobachtet wurde. Das Fehlen des Domain-Swappings ist bemerkenswert, da die vertauschte C-terminale helikale Domäne in MenB ein Tyrosin enthält, welches essentiell für die Katalyse ist und auch in BadI konserviert vorliegt. Der Vergleich des aktiven Zentrums zeigt, dass der C-Terminus von BadI so auf seinen Kern/Hauptteil faltet, dass das konservierte Tyrosin an der gleichen Stelle positioniert ist wie in MenB und mit dem Liganden interagieren kann. Die Struktur von BadI bestätigt auch die Rolle eines Serin- und eines Aspartatrests für die Ligandenbindung und bekräftigt damit, dass das konservierte aktive Zentrum an der enzymatischen Reaktion teilnimmt. Die Strukturen zeigen auch eine bemerkenswerte Verschiebung des aktiven Aspartats, welches zwei Hauptkonformationen einnimmt. Strukturelle Analysen zeigten auch die Nähe des Serinrests zu einem Wasser- und Chlormolekül, sowie einem Kohlenstoffrest, an dessen Stelle der Carbonylrest des eigentlichen Substrats läge. Die biochemische Charakterisierung von BadI in enzymkinetischen Untersuchungen bestätigte dass die vorgeschlagenen Reste des aktiven Zentrums an der Substratbindung beteiligt sind. Jedoch ist die Rolle der verschiedenen Reste sehr verschieden, wobei dem Serin eine herausragende Rolle zugedacht wird. Die hier dargestellte Arbeit bestätigt die Mitwirkung des mutmaßlichen aktiven Zentrums und zeigt, dass die Reste des Aktiven Zentrums von BadI eine unterschiedliche Rolle, im Vergleich zu ihrem ähnlichsten Homolog MenB, spielen. MenD, eine SEPHCHC (2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carbonsäure) Synthase, ist an der Menaquinonbiosynthese von S. aureus beteiligt. Obwohl S. aureus gewöhnlich als Kommensale betrachtet wird, kann es als bemerkenswertes Pathogen auftreten, wenn es die Epithelwand durchbricht und eine Vielzahl an Erkrankungen, von einfachen Hautinfektionen bis zu lebensbedrohlichen Zustanden, verursachen. Sogenannte „Small colony variants“ (SCVs), eine langsam wachsende, kleinzellige Subpopulation der Bakterien wurde mit persistenten, rezidivierenden und antibiotika-resistenten Infektionen assoziiert. Diese Varianten weisen einen Mangel von Thiamin, Menaquinon und Hämin auf. Menaquinon ist ein essentieller Bestandteil der Elektronentransport-Kette in grampositiven Organismen. Daher sind Enzyme dieses Stoffwechselwegs attraktive Wirkstoffziele gegen Krankheitserreger wie M. tuberculosis oder Bacillus subtilis. MenD, das Enzym, welches den ersten irreversiblen Schritt des Menaquinon-Biosynthesewegs katalysiert, wurde mit dem SCV Phänotyp von S. aureus in Verbindung gebracht. In dieser Arbeit werden die biochemischen und strukturellen Eigenschaften dieses wichtigen Enzyms untersucht. Unsere strukturelle Untersuchung zeigte, dass trotz einer niedrigen Sequenzidentität von 28%, die Gesamtfaltung von S. aureus MenD (saMenD) mit derjenigen von Escherichia coli MenD (ecMenD), trotz einiger signifikanter Abweichungen, übereinstimmt. Größere strukturelle Unterschiede können nahe des aktives Zentrums des Proteins beobachtet werden, vor allem in der C-terminalen Helix und einer Schleife nahe dem aktiven Zentrum. Die Schleife enthält kritische Reste für die Kofaktorbindung und liegt nur in der ecMenD-ThDP Komplexstruktur definiert vor, während die in der Apo-Form und der Substrat-gebundenen Struktur von ecMenD ungeordnet ist. In unserer saMenD Struktur zeigt sich die Schleife erstmals komplett geordnet in der Apo-Form und stellt eine neue Konformation der Kofaktor-Bindeschleife dar. Die Schleife nimmt eine ungewöhnlich offene Konformation an und die konservierten Reste, welche für die Kofaktorbindung verantwortlich sind, sind zu weit entfernt, um in dieser Position einen produktiven Komplex mit dem Kofaktor zu bilden. Zudem haben biochemische Studien in Verbindung mit den strukturellen Daten zur Identifizierung der Substratbindetasche und der an der Bindung und Katalyse beteiligten Aminosäuren beigetragen. In der vorliegenden Arbeit wurden die biochemischen und strukturellen Charakteristika von saMenD erfolgreich aufgeklärt.

British Columbia salmon aquaculture operations use the chemotherapeutant emamectin benzoate (EMB trade name SLICE®), a synthesized avermectin compound, delivered through feed to decrease sea lice (Lepeophtheirus salmonis) parasite abundance on production fish. Avermectins bind to ion channels in crustaceans and disrupts nerve impulse transmission. Detectable amounts of EMB can accumulate in the depositional area around farms during SLICE® treatment periods, thus presenting potential for exposure to populations of proximate non-target species. The distribution of spot prawn (Pandalus platyceros), an economically important crustacean, overlaps with areas of intensive salmon farm activity. The primary objective of this research was to determine if EMB exposure had a measurable biological effect on spot prawns in the field and in the laboratory. The field component was conducted in the Broughton Archipelago, BC, to determine if emamectin benzoate residues could be detected near actively treating salmon farms, and whether farm proximity affected spot prawn size distribution. Three laboratory experiments tested the mortality, molting and behavioural response of spot prawns to SLICE® feed pellet exposure and acute exposure to EMB through sediment over ten, 30 and 45-day durations. Measurable amounts of EMB was detected in the marine sediment near five farm sites during the field survey and was found to persist between treatment periods. Male and transitional stage spot prawns captured near farm sites attained a greater size and had better body condition compared to reference sites, indicating prawns may benefit from direct or indirect farm food subsidies. However, at several farm sites the size distribution of prawns changed over the sampling period, a trend not observed at reference sites, demonstrating that farm activity may alter prawn population dynamics. Laboratory results indicated that only prawns that had been starved prior to exposure would initially consume SLICE® pellets, but feeding rates declined with subsequent exposures. Depressed consumption rates was not a residual effect of EMB, but rather an aversion to the SLICE® pellet diet as prawns resumed feeding when offered a preferred diet. Sediment EMB exposures to doses 808 µg kg-1 and greater increased prawn mortality, largely due to the inability of molting individuals to successfully complete ecdysis. Exposed individuals accumulated EMB in their abdomen tissue with levels increasing with exposure dose. Prawns exposed to EMB through sediment at concentrations 1419 and 3330 µg kg-1 displayed a significant reduction in olfactory detection and orientation behaviours to food stimuli. This research highlights that spot prawns may avoid SLICE® pellets for preferential food sources, and that only short term EMB exposure 50 to 200 magnitude greater than levels present in the marine environment elicited a measurable response in spot prawn mortality rates, molting success and behaviour. However, preliminary trends in the field survey data indicate that there may be population differences occurring in spot prawns inhabiting areas near treating salmon farms that are not observed in reference populations. These results signify the inherent pitfalls in current management policy that base decisions on short-term acute toxicity laboratory exposure results that may not be indicative of the response of marine populations near active salmon farms to long-term chronic EMB exposure. / Graduate / 0792 / 0329 / 0768

Thesis (M. Phil.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 58-64).