Dissertations / Theses on the topic 'Pollutants and Bacteria'

The luxCDABE genes (i.e. full cassette) were inserted into the bacterial strain used in this study, E. coli HB101, using a multi copy plasmid, (pUCD607). A number of experiments were carried out in this thesis to study the potential of using the biosensor, E. coli HB101 (pUCD607), for ecotoxicity testing. Growth and bioluminescence of E. coli HB101 (pUCD607) were characterised and optimised, as well as the stability of this biosensor to a range of environmental parameters. The biosensors were found to be sensitive to a range of pollutants and provided a highly consistent bioluminescence response under conditions likely to be encountered in environmental toxicity testing. Assessment of the potential of the lux-based bioassay for revealing the combination of toxicities of metals was carried out. This is necessary because samples in ecotoxicity testing may contain a number of types of pollutants. The biosensors showed high sensitivity of response to mixed metals and identified the combined toxicity of the mixture. Biosensor bioluminescence could also be used to diagnose the distinction between toxicity after 15 and 30 minutes exposure. Results suggested that exposure time is an important factor affecting on the toxicity of metals in mixtures. The results from challenging lux-based biosensors with heavy metals with various concentration of Cl- demonstrated the effect of Cl- ion complex formation for metals on the bioluminescence of E. coli HB101 (pUCD607). The bioluminescence response of the biosensor also related to the results from computer modelling (GEOCHEM) and was used to diagnose the effect of Cl- on metal toxicity. The results showed that E. coli HB101 (pUCD607) is sensitive to complexes of metals such as are formed by Cl- ion. Immobilised cells of E. coli HB101 (pUCD607) were used to investigate the toxicity of metals, both singly and in mixture. This work aimed to assess the potential of immobilised cells, as an alternative form of biosensor, for use in ecotoxicity testing. Bioluminescence response of immobilised cells varied with the test solution pH and showed that it has great potential for use in low pH environments. Immobilised cell biosensors demonstrated sufficient sensitivity to identify the toxicity of individual and mixed metals. High levels of bioluminescence of immobilised cells lasted up to 5 hours after bead production, suggesting that biosensor immobilisation increases the flexibility of the toxicity assay.

L'activitat humana representa una de les majors causes d'entrada d'una gran varietat de substàncies en els ecosistemes fluvials. L'objectiu principal d'aquest treball es investigar els efectes que els tòxics orgànics poden exercir en els biofilms fluvials. El riu Llobregat ha estat sotmès a fortes pressions, fet que l'ha portat a uns nivells molt elevats de contaminació. En aquest estudi s'ha observat una influència dels plaguicides presents al riu en la distribució de la comunitat de diatomees, així com efectes en el biofilm a nivell funcional i estructural. Experiments amb canals experimentals han mostrat que l'herbicida diuron i el bactericida triclosan poden ocasionar una cadena d'efectes en els biofilms, incloent efectes directes i també efectes indirectes en les relacions entre els components del biofilm. Experiments amb cultius algals han mostrat que aquests tòxics, aplicats en barreja, poden tenir una major toxicitat de la prevista pels models, resultant en efectes sinèrgics.
Human activity is responsible for the entrance of many substances to the aquatic environment. The main objective of this study is to investigate the effects of organic toxicants on fluvial biofilms. The river Llobregat has been submitted to urban, industrial and agricultural pressures, and as such is a highly contaminated river. The diatom community was influenced by the presence of pesticides. Effects on both biofilm function and structure were attributed to the pesticides. Laboratory experiments with experimental channels have shown that the herbicide diuron and the bactericide triclosan may cause a chain of effects on biofilms, which include direct effects and indirect effects on the relationship between biofilm components. Experiments with algal cultures have shown that these toxicants, applied in mixtures, can have higher toxicity than the toxicity predicted by models, resulting in synergistic effects.

The aim of the PhD project was to make possible systematic studies of the bio-aerosols behavior in different atmospheric conditions, with the final goal to assess the link between pollution levels and bio-aerosol dispersion and impact. The research has been carried out at ChAMBRe (Chamber for Aerosol Modelling and Bio-aerosol Research), a 2.3 m3 stainless steel atmospheric simulation chamber. Experiments conducted inside confined artificial environments, such as the Atmospheric Simulations Chambers (ASCs), where atmospheric conditions and composition are controlled, can provide valuable information on bio-aerosols viability and their interaction with other atmospheric constituents. The first phase of the PhD project was dedicated to the characterization of the chamber, the related instrumentation and the design and development of the experimental set-up. An experimental protocol for chamber studies on bio-aerosols was developed and thoroughly tested with two bacteria model strains (B. subtilis and E. coli). An intense effort has been dedicated to fully characterize the performance of three different nebulization systems specifically designed for bioaerosol applications to assess their application in experiments at ASCs. A WIBS-NEO provides the size-segregated, real-time monitoring of the total bio-aerosol concentration inside the chamber. With a clean atmosphere maintained inside ChAMBRe, the ratio between injected and extracted viable bacteria turned out to be reproducible at 11 % and 13% level with E. coli and B. subtilis respectively. After assessing this way the reproducibility and sensitivity of the whole experimental procedure, the first tests to explore the possible correlation between bacteria viability and air quality were carried out. The two bacteria models, B. subtilis and E. coli, were subjected to high concentrations of nitrogen oxides and soot particles, two of the most common pollutants emitted by anthropogenic sources.

A constitutive lux-marked biosensor, E. coli HB101 pUCD607, was developed as a novel, acute, 'fingerprinting' technique by which individual pollutants, and whole effluents, could be identified and quantified. In this way, the advantages of both chemical analysis and toxicology were encompassed in one technique. An assay technique for obtaining temporal response curves was developed by injecting the biosensor into toxic samples, and then measuring luminescence continuously for 5 minutes. An algorithm was formulated, and implemented as a simple computer program, by which 'unknown' temporal response-curves could be compared against a database of 'reference' pollutant response-curves. Two different techniques were derived for analysing the output data; a binomial (yes or no) 'best-fit' technique, and a continuous 'comparative-fit' technique. These techniques were validated by 94% identification success for inter-comparisons of 7 pollutants, each at 5 concentrations, and 100% identification success of four pollutant spikes in three environmental samples. The technique was tested on three complex environmental effluents. Cu was identified as the main pollutant in a distillery effluent, and Zn the main pollutant in a metal processing effluent. Identification was successful despite the presence of complexing substrates and co-pollutants. The technique could also differentiate between three complex effluents and four artificial complex effluents without the need for actual pollutant identification. In conclusion, this technology can be used to 'fingerprint' any form of pollution that is bioavailable to the biosensor, without the need for prior knowledge of sample chemistry. This represents a potentially valuable tool for toxicity assessment and screening.

De nos jours, l'évolution du climat, les rejets anthropiques et l’augmentation de la population mondiale concourent à un accroissement du nombre de bactéries préoccupantes, des rejets médicamenteux et des toxines dans l’environnement. L’ochratoxine A, l’estradiol, et certaines bactéries font partie des contaminants polluant la nature et menaçant la santé des êtres vivants. Dans une optique de détection de ces éléments potentiellement dangereux, nous avons travaillé sur le développement d’un marquage original d'oligonucléotides. Ce marquage est basé sur l’utilisation de complexes métalliques électroactifs comme des sondes redox. Ces complexes sont basés sur les ligands macrocycles DOTA et NOTA, d’ordinaire majoritairement utilisés dans l’imagerie médicale, fonctionnalisés avec du fer (III). L’étude de leurs propriétés électrochimiques, réalisée par voltampérométrie cyclique, a démontré qu’ils présentaient de nombreux atouts faisant concurrence aux composés redox les plus répandus. Nous avons notamment cherché à appliquer ce marquage d’oligonucléotide à la construction de biocapteurs, avec en premier essai un génocapteur pour la détection d’ADN de bactéries Vibrio. La conception du capteur et la détection de cible a été suivie par spectroscopie d’impédance. Toutefois, l’analyse par impédance n’a pas permis d’obtenir les résultats espérés, et, afin d’étendre le champ de notre étude, une autre méthode a été testée.C’est pourquoi, nous avons cherché à coupler directement les complexes métalliques à des aptamères via une réaction entre une fonction thiol et un groupement maléimide. Dans l’étape suivante, les biocapteurs ont été construits en immobilisant les aptamères modifiés sur des électrodes. En parallèle, les interactions aptamères-cible ont été quantifiées par des analyses par thermophorèse ou MST pour confirmer certains résultats et valider les caractéristiques de liaison des aptamères
Nowadays, climate change, anthropogenic releases and the increase in the world's population are contributing to an increase in the number of bacteria of concern, drug releases and toxins into the environment. Ochratoxin A, estradiol, and some bacteria are among the contaminants polluting nature and threatening the health of living beings. In order to detect these potentially harmful elements, we worked on the development of an original oligonucleotide labeling. This marking is based on the use of electroactive metal complexes such as redox probes.These complexes are based on the macrocycle ligands DOTA and NOTA, usually mainly used in medical imaging, functionalized with iron (III). The study of their electrochemical properties, carried out by cyclic voltammetry, has shown that they have many advantages competing with the most common redox compounds. In particular, we sought to apply this oligonucleotide labeling to the construction of biosensors, with the first test of a genosensor for the detection of DNA from Vibrio bacteria. Sensor design and target detection were followed by impedance spectroscopy. However, impedance analysis did not achieve the expected results, and in order to extend the scope of our study, another method was tested. Therefore, we sought to couple metal complexes directly to aptamers via a reaction between a thiol function and a maleimide. In the next step, the biosensors were built by immobilizing the modified aptamers on electrodes. In parallel, aptamer-target interactions were quantified by thermophoresis or MST analyses to confirm certain results and validate the binding characteristics of aptamers

This study aimed to investigate the RBT outcome as a function of the bacterial inoculum during adaptation to degrade the HAs dichloroacetic acid (DCA), trichloroacetic acid (TCA) and 2-monochloropropionic acid (2MCPA). The HAs investigated were ranked, in order of greatest recalcitrance; TCA > 2MCPA > DCA.  DCA degradation was associated with enrichment of a Ralstonia like phylotype and dehII expression.  TCA degradation in replicate RBTs was consistently associated with a Bradyrhizobium like phylotype and dehI gene expression, regardless of the inoculum concentration used, suggesting that TCA imposes a highly selective pressure on the community. TCA degradation was affected by the inoculum concentration, as the rate of degradation was slower at the lowest inoculum concentration used, implying that TCA degradation was accomplished by cometabolism. 2MCPA was associated with various Alpha-, Beta- and Gammaproteobacteria, and the presence and expression of both dehI and dehII genes. Biodegradation of 2MCPA was strongly influenced by inoculum concentration. At lower inoculum concentrations, 2MCPA was commonly associated with a biphasic dechlorination curve, which has not been reported previously.  Biphasic dechlorination curves were attributed to the enrichment of an initial degrading organism, always associated with dehII expression, which was succeeded following 50% dechlorination by a second organism, in association with dehI expression, which were supposed to act sequentially on the L- and D-2MCPA isomers, respectively. The delayed enrichment of a dehI containing organism was attributed to their low environmental abundance, relative to dehII containing organisms. This study highlights the utility of cultivation-independent methods to link more precisely community structure and function during adaptation to degrade xenobiotic compounds.

This PhD thesis in Chemical Science started in November 2015 at the School of Advanced Studies of University of Camerino. The research activity described in this PhD thesis was carried out at University of Camerino in the School of Science and Technology (Dr. Rita Giovannetti’s Research Group) and at École Polytechnique Fédérale de Lausanne in the Institut des Sciences et Ingénierie Chimiques (Prof. César Pulgarin’s Research Group). In this PhD thesis, after a “GENERAL INTRODUCTION” about the aim of the PhD project, are reported an INTRODUCTION regarding environmental and water pollution and Advanced Oxidation Processes, in Chapter 1, and two successive Sections. In Section 1, entitled “GRAPHENE BASED TiO2 NANOCOMPOSITES FOR PHOTOCATALYTIC DEGRADATION OF SYNTHETIC DYES”, are present three Chapters. After a general introduction in Chapter 2 on TiO2 and Graphene-TiO2 photocatalysis, in Chapter 3 and Chapter 4 are reported all obtained results. In particular, in Chapter 3 is reported the first work regarding the preparation and optimization of a new heterogeneous photocatalyst constituted by TiO2 and graphene prepared by exfoliation of graphite, with a special attention into preparation and characterization of graphene dispersion. The new graphene-TiO2 photocatalyst was used for the photodegradation of Alizarin Red S solution under visible light irradiation. Meanwhile, in Chapter 4 is reported the second work regarding the preparation, optimization and characterization of a new heterogeneous photocatalyst constituted by TiO2 and reduced graphene oxide prepared by thermal reduction of graphene oxide, with a special attention into preparation and characterization of reduced graphene oxide dispersion. As in the case of Chapter 3, the new reduced graphene oxide-TiO2 photocatalyst was used for the photodegradation of Alizarin Red S solution under visible light irradiation. In Section 2, entitled “SOLAR DISINFECTION AND PHOTO-FENTON PROCESSES FOR BACTERIA INACTIVATION IN WATER”, are present two Chapters. After a general introduction in Chapter 5 on Solar Disinfection and Photo-Fenton treatments for water depuration, in Chapter 6 are reported all obtained results. In particular, the effect of the presence of various inorganic ions, which individually or in combination with each other can act as scavenger of OH radicals or can produce additional OH radicals or other active radical species on inactivation of Escherichia Coli with Solar Disinfection and Photo-Fenton processes is discussed in detail. Results of this PhD thesis are published and/or submitted for publication in scientific international journals and presented as Congress communications.

Civil Engineering
Ph.D.
Polychlorinated biphenyls (PCBs) are toxic and persistent chemicals that have been largely dispersed into the environment. The biological and abiotic transformations of PCBs often generate hydroxylated derivatives, which have been detected in a variety of environmental samples, including animal tissues and feces, water, and sediments. Because of their toxicity and widespread dispersion in the environment, hydroxylated PCBs (OH-PCBs) are today increasingly considered as a new class of environmental contaminants. Although PCBs are known to be susceptible to microbial degradation under both aerobic and anaerobic conditions, bacterial degradation of OH-PCBs has received little attention. The overall objective of this study is therefore to evaluate the transformation of mono-hydroxylated PCBs by the well characterized aerobic PCB-degrading bacterium, Burkholderia xenovorans LB400. In order to achieve our overall objective, a series of model mono-hydroxylated PCBs have been selected and they are used to determine the toxicity of hydroxylated congeners toward the bacterium B. xenovorans LB400. The biodegradation kinetics and metabolic pathways of the selected OH-PCBs by B. xenovorans LB400 are then characterized using GC/MS. To understand further the molecular basis of the metabolism of OH-PCBs by B. xenovorans LB400, gene expression analyses are conducted using reverse-transcription real-time (quantitative) polymerase chain reaction (RT-qPCR) and microarray technology. More formally, the specific aims of the proposed research are stated as follows: (1) To evaluate the toxicity of selected mono-hydroxylated derivatives of lesser-chlorinated PCBs toward the bacterium B. xenovorans LB400. (2) To assess the degradation of the selected OH-PCBs by B. xenovorans LB400. (3) To gain further understanding of the molecular bases of the metabolism of the selected OH-PCBs by B. xenovorans LB400. Three hydroxylated derivatives of 4-chlorobiphenyl and 2,5-dichlorobiphenyl, including 2'-hydroxy-, 3'-hydroxy-, and 4'-hydroxy- congeners, were significantly transformed by Burkholderia xenovorans LB400 when the bacterium was growing on biphenyl (biphenyl pathway-inducing conditions). On the contrary, only 2'-OH-4-chlorobiphenyl and 2'-OH-2,5-dichlorobiphenyl were transformed by the bacterium growing on succinate (conditions non-inductive of the biphenyl pathway). Gene expression analyses showed that only exposure to 2'-OH-4-chlorobiphenyl and 2'-OH-2,5-dichlorobiphneyl resulted in induction of key genes of the biphenyl pathway, when cells grown on succinate. These observations suggest that 2'OH-PCBs were capable of inducing the genes of biphenyl pathway. These results provide the first evidence that bacteria are able to cometabolize PCB derivatives hydroxylated on the non-chlorinated ring. Genome-wide transcriptional analyses using microarrays showed that 134 genes were differentially expressed in cells exposed to biphenyl, 2,5-dichlorobiphenyl, and 2'-OH-2,5-dichlorobiphneyl as compared to non-exposed cells. A significant proportion of differentially expressed genes were simultaneously expressed or down regulated by exposure to the three target compounds i.e., biphenyl, 2,5-DCB, and 2'-OH-2,5-DCB, which suggests that these structurally similar compounds induce similar transcriptional response of B.xenovorans LB400. Results of this study may have important implications for the natural attenuation of PCBs and fate of OH-PCBs in the environment. The recalcitrance to biodegradation and the high toxicity of some OH-PCBs may provide a partial explanation for the persistence of PCBs in the environment.
Temple University--Theses

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2014
Environmental legislation focusing on wastewater disposal in industries that utilise cyanide and/or cyanide-related compounds has become increasingly stringent worldwide, with many companies that utilise cyanide products required to abide by the Cyanide International Code associated with the approval of process certifications and management of industries which utilise cyanide. This code enforces the treatment or recycling of cyanide-contaminated wastewater. Industries such as those involved in mineral processing, photo finishing, metal plating, coal processing, synthetic fibre production, and extraction of precious metals, that is, gold and silver, contribute significantly to cyanide contamination in the environment through wastewater. As fresh water reserves throughout the world are low, cyanide contamination in water reserves threatens not only the economy, but also endangers the lives of living organisms that feed from these sources, including humans. In the mining industry, dilute cyanide solutions are utilised for the recovery of base (e.g. Cu, Zn, Ni, etc.) and precious metals (e.g. Au, Ag, etc.). However, for technical reasons, the water utilised for these processes cannot be recycled upstream of the mineral bioleaching circuit as the microorganisms employed in mineral bioleaching are sensitive to cyanide and its complexes, and thus the presence of such compounds would inhibit microbial activity, resulting in poor mineral oxidation. The inability to recycle the water has negative implications for water conservation and re-use, especially in arid regions. A number of treatment methods have been developed to remediate cyanide containing wastewaters. However, these chemical and physical methods are capital intensive and produce excess sludge which requires additional treatment. Furthermore, the by-products that are produced through these methods are hazardous. Therefore, there is a need for the development of alternative methods that are robust and economically viable for the bioremediation of cyanide-contaminated wastewater. Biological treatment of free cyanide in industrial wastewaters has been proved a viable and robust method for treatment of wastewaters containing cyanide. Several bacterial species, including Bacillus sp., can degrade cyanide to less toxic products, as these microorganisms are able to use the cyanide as a nitrogen source, producing ammonia and carbon dioxide. These bacterial species secrete enzymes that catalyse the degradation of cyanide into several end-products. The end-products of biodegradation can then be utilised by the microorganisms as nutrient sources. This study focused on the isolation and identification of bacterial species in wastewater containing elevated concentrations of cyanide, and the assessment of the cyanide biodegradation ability of the isolates. Thirteen bacterial isolates were isolated from electroplating wastewater by suppressing the growth of fungal organisms and these species were identified as species belonging to the Bacillus genus using the 16S rDNA gene. A mixed culture of the isolates was cultured in nutrient broth for 48 hours at 37°C, to which FCN as KCN was added to evaluate the species‟ ability to tolerate and biodegrade cyanide in batch bioreactors. Subsequently, cultures were supplemented solely with agro-waste extracts, that is, Ananas comosus extract (1% v/v), Beta vulgaris extract (1% v/v), Ipomea batatas extract (1% v/v), spent brewer‟s yeast (1% v/v) and whey (0.5% w/v), as the primary carbon sources. Owing to the formation of high ammonium concentration from the cyanide biodegradation process, the nitrification and aerobic denitrification ability of the isolates, classified as cyanide-degrading bacteria (CDB) was evaluated in a batch and pneumatic bioreactor in comparison with ammonia-oxidising bacteria (AOB). Furthermore, the effects of F-CN on the nitrification and aerobic denitrification was evaluated assess the impact of F-CN presence on nitrification. Additionally, optimisation of culture conditions with reference to temperature, pH and substrate concentration was evaluated using response surface methodology. Using the optimised data, a continuous biodegradation process was carried out in a dual-stage packed- bed reactor combined with a pneumatic bioreactor for the biodegradation of F-CN and subsequent nitrification and aerobic denitrification of the formed ammonium and nitrates. The isolated bacterial species were found to be gram positive and were able to produce endospores that were centrally located; using the 16S rDNA gene, the species were found to belong to the Bacillus genus. The species were able to degrade high cyanide concentration in nutrient broth with degradation efficiencies of 87.6%, 65.4%, 57.0% and 43.6% from 100 mg F-CN/L, 200 mg F-CN/L, 300 mg F-CN/L, 400 mg F-CN/L and 500 mg F-CN/L respectively over a period of 8 days. Additionally, the isolates were able to degrade cyanide in an agro-waste supported medium, especially in a medium that was supplemented with whey which achieved a degradation efficiency of 90% and 60% from 200 mg F-CN/L and 400 mg F-CN/L, respectively over a period of 5 days. The nitrification ability of the isolates was evaluated and the removal of NH4 +/NO3 - by the CDB and AOB in both shake flasks and pneumatic bioreactor was determined to be pH dependent. The maximum NH4 +/NO3 - removal evaluated over a period of 8 days for CDB and 15 days for AOB, observed at pH 7.7 in shake flasks, was 75% and 88%, respectively, in the absence of F-CN. Similarly, the removal of NH4 +/NO3 - in a pneumatic bioreactor was found to be 97.31% for CDB and 92% for AOB, thus demonstrating the importance of aeration in the designed process. The nitrification by CDB was not inhibited by cyanide loading up to a concentration of 8 mg FCN/ L, while the AOB were inhibited at cyanide loading concentration of 1 mg F-CN/L. The CDB removed the NH4 +/NO3 - in PBSs operated in a fed-batch mode, obtaining efficiencies >99% (NH4 +) and 76 to 98% (NO3 -) in repeated cycles (n = 3) under F-CN (≤8 mg F-CN/L). The input variables, that is, pH, temperature and whey-waste concentration, were optimised using a numerical optimisation technique where the optimum conditions were found to be: pH 9.88, temperature 33.60 °C and whey-waste concentration 14.27 g/L, under which 206.53 mg CN-/L in 96 h can be biodegraded by the microbial species from an initial cyanide concentration of 500 mg F-CN/L. Furthermore, using the optimised data, cyanide biodegradation in a continuous mode was evaluated in a dual-stage packed-bed bioreactor connected in series to a pneumatic bioreactor system used for simultaneous nitrification including aerobic denitrification. The whey-supported Bacillus sp. culture was not inhibited by the free cyanide concentration of up to 500 mg F-CN/L, with an overall degradation efficiency of ≥99% with subsequent nitrification and aerobic denitrification of the formed ammoniu and nitrates over a period of 80 days.

Le dioxyde d’azote (NO2), en tant que second polluant atmosphérique le plus meurtrier en Europe est un des plus préoccupants pour la santé humaine selon l’Agence Européenne de l’Environnement. Il est notamment connu pour être responsable de maladies cardiovasculaires, respiratoires ainsi que pour contribuer au vieillissement cutané et au développement de la dermatite atopique. Des facteurs endogènes à l’hôte tels que le microbiote cutané interviennent également dans cette pathologie. En effet, de nombreuses pathologies cutanées sont corrélées à un déséquilibre (dysbiose) du microbiote bactérien, un acteur essentiel du maintien de l’homéostasie de la peau. Or, il est fortement soupçonné que l’effet des polluants sur la peau implique des mécanismes d’action directe mais également un mécanisme d’action indirecte lié à l’altération du microbiote cutané par le polluant. En conséquence, il est pertinent d’aborder l’effet du NO2 gazeux (gNO2) sur le microbiote cutané bactérien. Cette thèse a donc pour objectif d’évaluer l’impact physiologique, morphologique et moléculaire du NO2 sur des souches bactériennes commensales d’espèces représentatives du microbiote cutané (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Pseudomonas fluorescens, Corynebacterium tuberculostearicum). Selon l’espèce, des réponses différentes au stress nitrosant généré par le gNO2 ont ainsi été mises en évidence ainsi qu’une tolérance plus importante au gNO2 pour certaines d’entre elles. Ces travaux suggèrent par conséquent que le NO2 pourrait contribuer à la formation d’un état dysbiotique du microbiote cutané et participer à l’action indirect du polluant sur la peau
Nitrogen dioxide (NO2), as the second most deadly air pollutant in Europe, is one of the most of concern for human health according to the European Environment Agency. It is notably known to be responsible for cardiovascular and respiratory diseases and also contributes to skin aging and atopic dermatitis. Host endogenous factors such as the cutaneous microbiota are also involved in this pathology, which is common in urban and suburban areas. Indeed, many skin pathologies are correlated to an imbalance (dysbiosis) of the bacterial microbiota, an essential player in the preservation of skin homeostasis. However, it is strongly presumed that the effect of pollutants on the skin involves direct mechanisms of action but also an indirect mechanism linked to the alteration of the cutaneous microbiota by the pollutant. Consequently, it is relevant to address the effect of gaseous NO2 (gNO2) on the cutaneous microbiota. This thesis aims to assess the physiological, morphological and molecular impact of gNO2 on commensal bacterial strains of representative species of the cutaneous microbiota (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Pseudomonas fluorescens, Corynebacterium tuberculostearicum). Depending on the species, different responses to gNO2-generated nitrosative stress were thus highlighted as well as a higher tolerance to gNO2 for some of them. This work therefore suggests that gNO2 could contribute to the formation of a dysbiotic state of the cutaneous microbiota and participate in the pollutant indirect action on the skin

Depuis plusieurs décennies la production mondiale de plastiques ne cesse d’augmenter, menant à une contamination des écosystèmes aquatiques à l’échelle de la planète qui a été récemment estimée à plus de cinq mille milliards de débris de plastiques flottants à la surface des océans. Les microplastiques (particules de plastique < 5 mm), introduits dans l’environnement aquatique directement en tant que microparticules (granulés plastiques industriels, cosmétiques, fibres textiles) ou lors de la fragmentation de plus gros débris plastiques, représentent une préoccupation scientifique et sociétale grandissante. Ces travaux de thèse se sont focalisés sur la contamination par les microplastiques de la rade de Brest (Bretagne, France), un système côtier macrotidal où l’activité anthropique est importante. Les objectifs de ces travaux sont (1) d’évaluer la contamination des matrices environnementales (eau de surface, sédiment subtidal et biote) par les microplastiques, et (2) d’identifier leur rôle potentiel en tant que vecteurs de contaminants chimiques et de bactéries dans la rade de Brest.Pour cela, des développements analytiques ont été nécessaires afin d’améliorer leur extraction des matrices environnementales ainsi que leur caractérisation morphologique et chimique via la microspectrométrie Raman. Les observations in situ ont montré que l’ensemble de l’écosystème de la rade de Brest est contaminé par les microplastiques, avec des concentrations de 0,24 ± 0,35, et 0,97 ± 2,08 (moyenne ± écart-type). L’eau de surface et le sédiment sont contaminés par le polyéthylène, le polypropylène et le polystyrène. Leur distribution à la surface de l’eau semble être liée à l’urbanisation très présente au nord de la rade, mais également à l’hydrodynamisme de cette région marine. Les premiers résultats concernant les bivalves marins ont montré un niveau relativement faible de contamination par les microplastiques (0,01 ± 0,04 et 0,08 ± 0,34 pour les moules et les coques, respectivement), cependant cela est probablement dû aux choix méthodologiques appliqués ici (exclusion des fibres). Les observations in situ ont montré que certains polluants organiques (HAP, PCB et pesticides) étaient détectés sur les microplastiques flottants à des valeurs (non détecté – 49763 ng.g-1, moyenne ± écart-type) similaires de celles retrouvées dans les sédiments et les bivalves locaux, ce qui suggère un risque faible dans le transfert des contaminants chimiques vers les organismes marins en cas d’ingestion. Enfin, les résultats concernant la colonisation bactérienne des microplastiques flottants ont montré des communautés distinctes de celles retrouvées dans l’eau de mer environnante, et l’identification du genre Vibrio en tant que biomarqueur discriminant de cette matrice. Dans l’ensemble, ces travaux fournissent une première évaluation approfondie de l’état de contamination de la rade de Brest par les microplastiques ainsi que de solides recommandations méthodologiques pour des travaux futurs
World production of plastics has increased steadily for the past decades leading to a major contamination of the worldwide aquatic ecosystems recently estimated at more than five trillion plastic pieces floating the surface of the oceans. Microplastics (plastic particles < 5 mm) are introduced into aquatic environments directly as industrial raw material (plastic pellets, cosmetics, clothing) or indirectly via the fragmentation of larger plastics. This emerging contaminant represents an increasing ecological concern for science and society. The present study focused on the microplastic contamination of the Bay of Brest (Brittany, France), a macrotidal coastal ecosystem characterized by intense anthropogenic activity. The main objectives were: (1) to evaluate the contamination of environmental matrices (surface water, subtidal sediment and biota) by microplastics, and (2) to identify their potential role as vector of chemicals and bacteria in the bay of Brest.Methodological developments were first conducted to improve microplastic extraction from environmental matrices as well as their rapid morphological and chemical identification by Raman micro-spectrometry. The field investigations showed that the ecosystem of the bay of Brest is contaminated by microplastics with mean concentrations of 0.24 ± 0.35, and 0.97 ± 2.08 (mean ± standard deviation) in surface water and sediment, respectively. Microplastic contamination in surface water and sediment was dominated by polyethylene, polypropylene and polystyrene microparticles.Spatial microplastic distribution appeared to be related to proximity to urbanized areas and to hydrodynamic in the bay. Preliminarily results of microplastic contamination in marine bivalves demonstrated relatively low contamination (0.01 ± 0.04, and 0.08 ± 0.34 for mussels and cockles, respectively) by microplastics (mainly polyethylene and polypropylene fragments), however this could be partly related to the methodological limitation identified here (e.g. exclusion of fibers). Organic pollutant (PAH, PCB and pesticides) were detected on floating microplastics at levels (not detected – 49,763 ng g-1, mean ± SD) similar to those measured in sediment and bivalves suggesting low risks in transferring hazardous chemicals in local marine organisms upon microplastic ingestion. Finally, distinct bacterial community assemblages were demonstrated on microplastics as compared with surrounding surface water; the Vibrio genus was identified as a discriminant biomarker of the plastic matrix. Overall, this work provides a first and thorough assessment of the microplastic contamination in the bay of Brest and solid methodological recommendations for further work

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Environmental Health in the Faculty of Applied Sciences at the Cape Peninsula University of Technology, 2013
Water is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River). The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes. Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique. Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system. The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons. To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.

La bacterie appelee gu5, isolee du sol, identifiee comme un pseudomonas, utilise les acides vanillique, isovanillique ou 4-hydroxybenzoique comme seule source de carbone et d'energie. L'acide protocatechuique a ete identifie comme intermediaire commun du catabolisme de ces substrats. La demethylation des acides vanillique et isovanillique est realisee par deux systemes enzymatiques distincts. Ces reactions de demethylation n'ont pu etre observees dans les lysats bacteriens. La 4-hydroxybenzoate 3-hydroxylase, nadph dependante a ete purifiee et etudiee. C'est une enzyma flavinique contenant du fad non lie de facon covalente a la proteine. Elle presente une structure quaternaire dimerique avec deux sous-unites de 50000daltons. En outre, selon le substrat de croissance, une protocatechuate 3,4-dioxygenase ou une protocatechuate 4,5-dioxygenase a ete observee. Ceci indique la coexistence des voies "ortho" et "meta" chez cette bacterie

Depuis plusieurs décennies la production mondiale de plastiques ne cesse d’augmenter, menant à une contamination des écosystèmes aquatiques à l’échelle de la planète qui a été récemment estimée à plus de cinq mille milliards de débris de plastiques flottants à la surface des océans. Les microplastiques (particules de plastique < 5 mm), introduits dans l’environnement aquatique directement en tant que microparticules (granulés plastiques industriels, cosmétiques, fibres textiles) ou lors de la fragmentation de plus gros débris plastiques, représentent une préoccupation scientifique et sociétale grandissante. Ces travaux de thèse se sont focalisés sur la contamination par les microplastiques de la rade de Brest (Bretagne, France), un système côtier macrotidal où l’activité anthropique est importante. Les objectifs de ces travaux sont (1) d’évaluer la contamination des matrices environnementales (eau de surface, sédiment subtidal et biote) par les microplastiques, et (2) d’identifier leur rôle potentiel en tant que vecteurs de contaminants chimiques et de bactéries dans la rade de Brest.Pour cela, des développements analytiques ont été nécessaires afin d’améliorer leur extraction des matrices environnementales ainsi que leur caractérisation morphologique et chimique via la microspectrométrie Raman. Les observations in situ ont montré que l’ensemble de l’écosystème de la rade de Brest est contaminé par les microplastiques, avec des concentrations de 0,24 ± 0,35, et 0,97 ± 2,08 (moyenne ± écart-type). L’eau de surface et le sédiment sont contaminés par le polyéthylène, le polypropylène et le polystyrène. Leur distribution à la surface de l’eau semble être liée à l’urbanisation très présente au nord de la rade, mais également à l’hydrodynamisme de cette région marine. Les premiers résultats concernant les bivalves marins ont montré un niveau relativement faible de contamination par les microplastiques (0,01 ± 0,04 et 0,08 ± 0,34 pour les moules et les coques, respectivement), cependant cela est probablement dû aux choix méthodologiques appliqués ici (exclusion des fibres). Les observations in situ ont montré que certains polluants organiques (HAP, PCB et pesticides) étaient détectés sur les microplastiques flottants à des valeurs (non détecté – 49763 ng.g-1, moyenne ± écart-type) similaires de celles retrouvées dans les sédiments et les bivalves locaux, ce qui suggère un risque faible dans le transfert des contaminants chimiques vers les organismes marins en cas d’ingestion. Enfin, les résultats concernant la colonisation bactérienne des microplastiques flottants ont montré des communautés distinctes de celles retrouvées dans l’eau de mer environnante, et l’identification du genre Vibrio en tant que biomarqueur discriminant de cette matrice. Dans l’ensemble, ces travaux fournissent une première évaluation approfondie de l’état de contamination de la rade de Brest par les microplastiques ainsi que de solides recommandations méthodologiques pour des travaux futurs
World production of plastics has increased steadily for the past decades leading to a major contamination of the worldwide aquatic ecosystems recently estimated at more than five trillion plastic pieces floating the surface of the oceans. Microplastics (plastic particles < 5 mm) are introduced into aquatic environments directly as industrial raw material (plastic pellets, cosmetics, clothing) or indirectly via the fragmentation of larger plastics. This emerging contaminant represents an increasing ecological concern for science and society. The present study focused on the microplastic contamination of the Bay of Brest (Brittany, France), a macrotidal coastal ecosystem characterized by intense anthropogenic activity. The main objectives were: (1) to evaluate the contamination of environmental matrices (surface water, subtidal sediment and biota) by microplastics, and (2) to identify their potential role as vector of chemicals and bacteria in the bay of Brest.Methodological developments were first conducted to improve microplastic extraction from environmental matrices as well as their rapid morphological and chemical identification by Raman micro-spectrometry. The field investigations showed that the ecosystem of the bay of Brest is contaminated by microplastics with mean concentrations of 0.24 ± 0.35, and 0.97 ± 2.08 (mean ± standard deviation) in surface water and sediment, respectively. Microplastic contamination in surface water and sediment was dominated by polyethylene, polypropylene and polystyrene microparticles.Spatial microplastic distribution appeared to be related to proximity to urbanized areas and to hydrodynamic in the bay. Preliminarily results of microplastic contamination in marine bivalves demonstrated relatively low contamination (0.01 ± 0.04, and 0.08 ± 0.34 for mussels and cockles, respectively) by microplastics (mainly polyethylene and polypropylene fragments), however this could be partly related to the methodological limitation identified here (e.g. exclusion of fibers). Organic pollutant (PAH, PCB and pesticides) were detected on floating microplastics at levels (not detected – 49,763 ng g-1, mean ± SD) similar to those measured in sediment and bivalves suggesting low risks in transferring hazardous chemicals in local marine organisms upon microplastic ingestion. Finally, distinct bacterial community assemblages were demonstrated on microplastics as compared with surrounding surface water; the Vibrio genus was identified as a discriminant biomarker of the plastic matrix. Overall, this work provides a first and thorough assessment of the microplastic contamination in the bay of Brest and solid methodological recommendations for further work

The study of the defense mechanisms in invertebrates started with marine animals. The first report on the cellular defense in invertebrates was the observation of phagocytosis in starfish larvae and Daphnia (Metchnikoff, 1884). It was suggested that phagocytes play a significant role in the defense mechanisms of all evolutionary stages of animals starting from unicellular animals to mammals. The study of hemolysins in the horse-shoe crab by Noguchi (1903) was the first study on the humoral immunity of invertebrates. Since 1960, there was a renewed interest in the study of defense mechanisms in invertebrates, which was suggested to be because of two reasons: (1) to find out those mechanisms in invertebrates that are also discovered in vertebrates and (2) the development of intensive mariculture requires better knowledge of the factors that may lead to mortality of the crustaceans and mollusc due to failure of their defense systems. Marine invertebrates served as experimental materials for phylogenetic study of defense mechanisms in Animal Kingdom from late 191h century to the beginning of20lh century (Mori, 1990). Among invertebrates, the defense systems of molluscs and insects are being studied extensively in order to understand the basics of invertebrate inullune system. During the past three decades, there has evolved a considerable interest in how the molluscs defend themselves against the invaders. Immunity of molluscs is comprised of cell-mediated immunity and humoral mechanisms. The cell-mediated immunity is the function of hemocytes, while humoral immunity is provided by the serum factors. There are many evidences, which suggest that these two are interrelated (Cheng, 1990). Hemolymph in invertebrates is analogous to blood in vertebrates. The blood of vertebrates consists of plasma and cells. The cells are red blood corpuscles and white blood corpuscles.

Chan Pui-Ling.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 169-182).
Text in English; abstracts in English and Chinese.
Chan Pui-Ling.
Acknowledgements --- p.i
Abstracts --- p.ii
Table of Contents --- p.v
List of Plates --- p.ix
List of Figures --- p.xii
List of Tables --- p.xiv
Abbreviations --- p.xviii
Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Indoor Air Quality (IAQ): An overview --- p.1
Chapter 1.1.1 --- Importance of indoor air quality --- p.2
Chapter 1.1.2 --- Common indoor air pollutants --- p.2
Chapter 1.1.3 --- Airborne bacteria --- p.4
Chapter 1.1.3.1 --- Possible sources of airborne bacteria --- p.4
Chapter 1.1.3.2 --- Health effects of the airborne bacteria --- p.5
Chapter a. --- Sick building syndromes --- p.5
Chapter b. --- Building-related illness --- p.7
Chapter 1.1.4 --- Importance of studying airborne bacteria --- p.12
Chapter 1.2 --- Situation in Hong Kong --- p.13
Chapter 1.2.1 --- Outdoor air quality --- p.14
Chapter 1.2.2 --- Indoor air quality --- p.14
Chapter 1.2.2.1 --- Hong Kong studies --- p.16
Chapter 1.2.3 --- Air quality objectives in Hong Kong --- p.18
Chapter 1.3 --- Different sampling methods --- p.18
Chapter 1.4 --- Identification of bacteria --- p.24
Chapter 1.5 --- Site selection --- p.26
Chapter 2 --- Objectives --- p.28
Chapter 3 --- Materials and methods --- p.29
Chapter 3.1 --- Samples collection --- p.29
Chapter 3.1.1 --- Sampling site --- p.29
Chapter 3.1.2 --- Complete Biosampler System --- p.29
Chapter 3.1.3 --- Sampling preparation --- p.33
Chapter 3.1.4 --- Sampling procedures --- p.33
Chapter 3.2 --- Recovery of the airborne bacteria --- p.36
Chapter 3.2.1 --- Cultural medium --- p.36
Chapter 3.2.2 --- Recovery procedures --- p.36
Chapter 3.2.3 --- Frozen stocks --- p.37
Chapter 3.3 --- Indentification of bacterial strains --- p.37
Chapter 3.3.1 --- Gram stain --- p.37
Chapter 3.3.1.1 --- Chemical reagents --- p.37
Chapter 3.3.1.2 --- Gram stain procedures --- p.38
Chapter 3.3.2 --- Oxidase test --- p.38
Chapter 3.3.2.1 --- Chemical reagents --- p.38
Chapter 3.3.2.2 --- Oxidase test procedures --- p.41
Chapter 3.3.3 --- Midi Sherlock® Microbial Identification System (MIDI) --- p.41
Chapter 3.3.3.1 --- Culture medium --- p.41
Chapter 3.3.3.2 --- Chemical reagents --- p.41
Chapter 3.3.3.3 --- MIDI procedures --- p.41
Chapter 3.3.4 --- Biolog MicroLogTM system (Biolog) --- p.41
Chapter 3.3.4.1 --- Culture medium --- p.41
Chapter 3.3.4.2 --- Chemical reagents --- p.44
Chapter 3.3.4.3 --- Biolog procedures --- p.44
Chapter 3.3.5 --- DuPont Qualicon RiboPrinter® Microbial Characterization System (RiboPrinter) --- p.46
Chapter 3.3.5.1 --- Culture medium --- p.46
Chapter 3.3.5.2 --- Chemical reagents --- p.46
Chapter 3.3.5.3 --- RiboPrinter procedures --- p.46
Chapter 4 --- Results --- p.50
Chapter 4.1 --- Sample naming system --- p.50
Chapter 4.2 --- Interpretation of results --- p.50
Chapter 4.2.1 --- Midi Sherlock® Microbial Identification System (MIDI) --- p.51
Chapter 4.2.2 --- Biolog MicroLog´ёØ System (Biolog) --- p.51
Chapter 4.2.3 --- DuPont Qualicon RiboPrinter® Microbial Characterization System (RiboPrinter) --- p.52
Chapter 4.3 --- Sample results --- p.53
Chapter 4.3.1 --- Sample 1 (Spring) --- p.53
Chapter 4.3.2 --- Sample 2 (Summer-holiday) --- p.62
Chapter 4.3.3 --- Sample 3 (Summer-school time) --- p.71
Chapter 4.3.4 --- Sample 4 (Autumn) --- p.81
Chapter 4.3.5 --- Sample 5 (Winter) --- p.90
Chapter 4.4 --- Bacterial profile of the student canteen --- p.100
Chapter 4.5 --- The cell and colony morphology of the dominant bacteria --- p.100
Chapter 4.6 --- Comparison between samples --- p.121
Chapter 4.6.1 --- Spatial variation --- p.121
Chapter 4.6.1.1 --- Spatial effect on bacterial abundance --- p.121
Chapter 4.6.1.2 --- Spatial effect on species diversity --- p.121
Chapter 4.6.2 --- Daily variation --- p.126
Chapter 4.6.2.1 --- Daily effect on bacterial abundance --- p.126
Chapter 4.6.2.2 --- Daily effect on species diversity --- p.126
Chapter 4.6.3 --- Seasonal variation --- p.126
Chapter 4.6.3.1 --- Seasonal effect on bacterial abundance --- p.126
Chapter 4.6.3.2 --- Seasonal effect on species diversity --- p.130
Chapter 4.7 --- Temperature effect on individual airborne bacterial population --- p.130
Chapter 4.7.1 --- Gram positive bacteria --- p.130
Chapter 4.7.2 --- Gram negative bacteria --- p.130
Chapter 4.8 --- Effect of relative humidity on individual airborne bacterial population --- p.137
Chapter 4.8.1 --- Gram positive bacteria --- p.137
Chapter 4.8.2 --- Gram negative bacteria --- p.137
Chapter 5 --- Discussion --- p.143
Chapter 5.1 --- Bacterial profile --- p.143
Chapter 5.1.1 --- Bacterial diversity --- p.143
Chapter 5.1.2 --- Information of the identified bacteria from the student canteen --- p.144
Chapter 5.1.3 --- Pathogenicity --- p.153
Chapter 5.1.4 --- Summary on the bacterial profile --- p.153
Chapter 5.2 --- Comparison between samples --- p.160
Chapter 5.2.1 --- Spatial variation (Sampling point 1 against Sampling point 2) --- p.160
Chapter 5.2.2 --- Daily variation (Morning against Afternoon) --- p.161
Chapter 5.2.3 --- Seasonal variation --- p.162
Chapter 5.2.4 --- Summer holiday against Summer school time --- p.163
Chapter 5.2.5 --- Summary on the factors affecting the bacterial content --- p.164
Chapter 5.3 --- Summary on indoor air quality of the student canteen in terms of bacterial level. --- p.166
Chapter 6 --- Conclusions --- p.168
Chapter 7 --- References --- p.169
Appendix 1 --- p.183
Appendix 2 --- p.187

Dissertação de mestrado, Inovação Química e Regulamentação (Erasmus Mundus), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016
Pharmaceutical active compounds are an important group of emerging pollutants that have raised an increasing interest in the scientific community due to their ubiquitous presence in the environment and their difficult degradation. Some of these drugs are extensively used as non-prescription drugs and after their intake, are excreted with urine and faeces either as active substance or metabolites. These substances come into wastewater treatment plants (WWTP) where some compounds are not efficiently removed, being able to reach surface, groundwater and subsequently, drinking water. Microorganisms (single isolates) have the potential to degrade a wide range of xenobiotics and recalcitrant contaminants. The tendency is more reinforced in their communities due to a great synergistic interaction between members of the consortium. In this study, the overall objectives aimed at investigating bacterial biodegrading communities from two different wastewater treatment plants (a passive lagoon system and an activated mud treatment system with aeration) for their abilities in effectively biodegrading and mineralizing paracetamol (APAP) and determining the optimum conditions required to achieve the outcome. The study examined the aerobic biodegradation of paracetamol by microbial communities from WWTPs in Faro using residual water and minimum salt medium (MSM) as growth media. From an obscure 25 ºC incubated aerobic aerified (110mL/min) bioreactor with paracetamol as the only carbon and energy sources, the biodegradability of paracetamol was evaluated by direct sample analysis after a 24, 48, 72 and 120hrs growth period. An elution gradient HPLC analysis for paracetamol biodegradations and the identification of its associated metabolite respectively showed a 99.9% elimination within 72hrs and a complete degradation after 120hrs for aerified samples with residual water and 97% elimination within 120hrs for aerified samples with MSM. Tentative identified peaks corresponded to the following metabolites: 4-aminophenol, hydroquinone and p-benzoquinone. The Hach-spectrophotometry analysis of the chemical oxygen demand (COD mg/L) showed a progressive decrease in the values within most batch samples hence suggesting a possible usage of the paracetamol during the process. IC50 measurements by UV-vis spectrometer produced concentration values far to be toxic for the organisms. In a nutshell, the sludge contains aerobic microorganisms capable of totally degrading APAP and the resulting metabolites to obtain energy without any other source of carbon and energy. Degradation is faster with aeration but slower without.

碩士
國立臺灣大學
生物環境系統工程學研究所
105
In recent years, highly industrial development causes many environmental pollutants releasing into the environment, such as heavy metals, agricultural pesticides and emerging contaminants, thereby threaten people’s health around the world. Despite chemical analysis has been proven highly sensitive and accuracy, it still has limitation for large scale screening due to the cost and time consuming. The goal of this study is to use a sulfur-oxidizing bacterium as a biosensor to detect environmental pollutants.in order to current complement chemical analysis. In this study, an acidophilic sulfur-oxidizing bacterium was isolated from the soil of the Sulfur Valley near Yangmingshan. We developed the biosensor based on the bacterium’s sulfur-oxidizing ability to H2SO4 is inhibited by the environmental pollutants toxicity in the presence of O2 and S0, thereby preventing the decrease in pH and the increase in EC. Based on the 16S rDNA sequence analysis, the bacterium SV5 has 99% sequence similarity to Acidicaldus organivorans strain Y008. The optimal growth medium for SV5 is ferrous iron/yeast extract liquid medium (FYM), and the optimal temperature and pH is 37 ℃ and pH 2.5, respectively. The results showed that although SV5 could not effectively detect DEHP and triadimenol, it was able to detect 10-fold EPA effluent standard concentration of arsenic, 100-fold EPA effluent standard concentration of cadmium, and 1-fold EPA effluent standard concentration of chromium in 4 days. Finally, this study demonstrated that chromium inhibits sulfur-oxidizing protein activity of SV5. In conclusion, the sulfur-oxidizing biosensor in this study is easy to operate and cost-effective. It can not only compromise current chemical analysis but also increase the efficiency of large scale screening of environmental pollutants.

Several strains of pseudomonads which are normally found in natural water and soil environments, including Pseudomonas fluorescens, Pseudomonas aeruginosa, and strains initially identified as Pseudomonas paucimobilis and Pseudomonas maltophilia, were used in this thesis and their stress responsive proteins were surveyed at the physiological level. Additionally, 16S rRNA gene comparison was employed to classify the pseudomonads and related species at the genetic level. In order to see to what extent the pseudomonads and related species responded to heat shock, cells were initially subjected to temperature upshift from 28 to 37 deegree centegrade then disrupted by sonication or cells lysed by boiling in sample buffer, and the extracted total proteins were resolved on 1-D or 2-D SDS-PAGE. Their growth characteristics in different media (varying from rich to minimal) were determined and the physiological impacts on growth were examined at various concentrations of selected chemicals (metal ions, phenolics), to establish toxic and sub-lethal levels for use in determining stress responses. Production of novel proteins or elevated levels of normal proteins following exposure to different concentrations of toxicants was examined in detail in strain VUN10,077 (formerly Ps. paucimobilis) by sampling throughout the growth cycle and using 35S-methionine incorporation into newly synthesized proteins. Indigenous strain VUN10,077 presented abnormal rRNA pattern in standard RNA gels and 16S-like rRNA was analysed using Northern blot associated with 16S rRNA gene analysis which confirmed the identity of the known pseudomonads. An bands of MW 2.5 kb (faint), 1.38 kb, 1.2 kb and 0.1 kb were detected in RNA gels. The 1.38 kb band corresponded to 16S rRNA gene analysis, as shown by Northern blot analysis. 16S rRNA gene sequencing homology showed that strain VUN10,077 was a Brevundimonas species. The pseudomonads and related species accumulated a GroEL-like protein under heat stress when cells were grown in LB broth and disrupted by sonication, and 2-D SDS-PAGE conditions were established using sonically disrupted cells of Stenotrophomonas maltophilia (formerly Ps. maltophilia). Cells lysed by boiling represented the total protein profile and included particulate or membrane-associated proteins, 70 kDa-like and 44-46 kDa proteins for strain VUN10,077. Selected pollutants impacted on the cell viability and sub-lethal levels of these were determined as follows; 2.0 gL-1 for CdCl2, 2.0 gL-1 for CuCl2.2H2O, 2.0 gL-1 for NiCl2.6H2O, 1.0 gL-1 for CoCl2.6H2O, 0.025 gL-1 for HgCl2, 0.25 gL-1 for SDS, 0.2 gL-1 for NaAsO2 and 0.5 gL-1 for phenol, from A600 readings and viable counts. Major HSPs were present at relatively lower levels or were barely increased compared to controls, whereas low molecular weight proteins were significantly changed. A 20 kDa protein was commonly found in cadmium, copper, cobalt, nickel, arsenite and phenol stresses. However, a DnaK-like protein strongly reacted against commercially available DnaK antibodies on Western blot analysis and a visibly increased signal was seen for heat, cadmium, zinc, cobalt, phenol stresses and control, where represents in that order, whereas changes in the GroEL-like protein were less specific. Several minimal or defined minimal media were developed and used in heat stress and different protein profiles were seen. Medium (2) contained M9 salts medium (supplemented with glucose) plus casamino acids and medium (5) contained M9 salts medium (supplemented with glucose), vitamin solution, trace elements solution and casamino acids. Media (2) and (5) were found to be suitable for studying the physiological changes in strain VUN10,077, but growth rates in medium (2) were much less than seen in medium (5). Strain VUN10,077 presented major HSPs (70, 58 and 18 kDa proteins) in medium (5), similar to seen in Brain Heart Infusion (BHI) media. Pulse-chase 35S-methionine labelling methods were established and known major HSPs were newly synthesised by strain VUN10,077 under heat and cadmium stresses, and these proteins gradually declined while 55 kDa, 50 kDa and 18 kDa proteins were detected under ongoing stress conditions. Presumptive groEL gene analysis was attempted and partial conserved groEL sequences were determined in the pseudomonads and related species, where similarity was 98-100%. In parallel, a water-based lux system was established to examine physiological impacts of selected pollutants at the cellular level. Lux genes cloned from a marine bacterium were introduced into the background of the indigenous strain VUN10,077, providing a potentially sensitive tool for toxicity screening for terrestrial and fresh water samples. To show the use of bioluminescence in the indigenous (naturally non-bioluminescent) organism, research was performed on stable marking and optimisation of light output including marking of the organisms with luxAB or the whole lux cassette, which necessitated establishing gene transfer systems using available transposition vectors, optimisation of conditions for light output and stability. This approach would alleviate the requirement for salt or osmotic stabilizers in the test system to maintain bioluminescence or viability, which currently is a requirement of the Microtox (TM) system which employs Photobacterium phosphoreum. Expression of bioluminescence genes in the indigenous strain employed the luminescence system of Vibrio species which is encoded by a gene cluster with a divergent transcriptional pattern. As strain VUN10,077 was a genetically uncharacterised natural isolate, this work was achieved by establishing the parameters necessary for introducing genes after determining antibiotic sensitivity to determine which marker genes could be used in these backgrounds. The genes for two polypeptides (LuxA, LuxB) which form luciferase were transferred into strain VUN10,077. Introduction of the luxAB bioluminescence genes into a range of bacteria has been facilitated by using plasmid, pUT::Tn5-luxAB (de Lorenzo et al., 1990), which carries a tetracycline resistance gene. The plasmid was introduced by bi- and tri-parental conjugation employing a helper plasmid pRK2013, if necessary, so that antibiotic resistance can occur in the transconjugants if transposition occurs. Alternatively, electroporation was used to introduce bioluminescence lux genes into strain VUN10,077 when cells were harvested at early-log and stationary phase. One of the engineered water-based luxAB-marked strain (VUN3,600) was used to detect the presence of toxic substances (determining selectivity and sensitivity). This bacterial luciferase system produces bioluminescence when supplied with an aliphatic aldehyde substrate, where the luxAB genes were expressed on the chromosome. The substrate delivery system and preparation of cells for bioluminescence toxicity test were achieved by diluting ndecyl aldehyde 10 (3)-fold in Milli-Q water and resuspending cells in tap-water after mild centrifugation, when cells were harvested at A600 1.0-1.2. When a variety of common pollutants were tested in the water-based monitoring system, cells of VUN3,600 responded sensitively, rapidly and differentially to Cd, Pb, Hg, Zn, Co ions and SDS, and with less sensitivity to Ni, and As; responses were also dose-related in a short-term assay. DMF, a solvent used to dissolve or extract several organic compounds in environmental testing, stimulated light output but in a controlled fashion. Selected substituted heterocyclic compounds tested and phenol, d- nitrophenol and strontium failed to cause dose-related inhibition, whereas di-, tri-, tetra- and penta-chlorophenol decreased light output in a long-term assay. DMF-dissolved PAH compounds were tested in the long-term assay and naphthalene, phenanthrene, fluorene, fluoranthene and benzo[a]pyrene presented dose-related responses while benz[a] anthracene and di-benzo[a]anthracence were not suitable for use in this type of assay. Although there is need to investigate extensive toxicity assays to gain broad acceptance, the water-based monitoring system showed repeatable and reliable toxicity assessment and has great possibility of usage in aquatic samples in either water-soluble or DMF-soluble forms. This study allowed explanation of the stress responses of strain VUN10,077 to be examined at a variety of levels. Bioluminescence was the most sensitive approach to determining the lowest concentrations of pollutants which affected cell physiology, as this was obvious at concentrations lower than required to cause changes in viability or induce stress responses.

Several strains of pseudomonads which are normally found in natural water and soil environments, including Pseudomonas fluorescens, Pseudomonas aeruginosa, and strains initially identified as Pseudomonas paucimobilis and Pseudomonas maltophilia, were used in this thesis and their stress responsive proteins were surveyed at the physiological level. Additionally, 16S rRNA gene comparison was employed to classify the pseudomonads and related species at the genetic level. In order to see to what extent the pseudomonads and related species responded to heat shock, cells were initially subjected to temperature upshift from 28 to 37 deegree centegrade then disrupted by sonication or cells lysed by boiling in sample buffer, and the extracted total proteins were resolved on 1-D or 2-D SDS-PAGE. Their growth characteristics in different media (varying from rich to minimal) were determined and the physiological impacts on growth were examined at various concentrations of selected chemicals (metal ions, phenolics), to establish toxic and sub-lethal levels for use in determining stress responses. Production of novel proteins or elevated levels of normal proteins following exposure to different concentrations of toxicants was examined in detail in strain VUN10,077 (formerly Ps. paucimobilis) by sampling throughout the growth cycle and using 35S-methionine incorporation into newly synthesized proteins. Indigenous strain VUN10,077 presented abnormal rRNA pattern in standard RNA gels and 16S-like rRNA was analysed using Northern blot associated with 16S rRNA gene analysis which confirmed the identity of the known pseudomonads. An bands of MW 2.5 kb (faint), 1.38 kb, 1.2 kb and 0.1 kb were detected in RNA gels. The 1.38 kb band corresponded to 16S rRNA gene analysis, as shown by Northern blot analysis. 16S rRNA gene sequencing homology showed that strain VUN10,077 was a Brevundimonas species. The pseudomonads and related species accumulated a GroEL-like protein under heat stress when cells were grown in LB broth and disrupted by sonication, and 2-D SDS-PAGE conditions were established using sonically disrupted cells of Stenotrophomonas maltophilia (formerly Ps. maltophilia). Cells lysed by boiling represented the total protein profile and included particulate or membrane-associated proteins, 70 kDa-like and 44-46 kDa proteins for strain VUN10,077. Selected pollutants impacted on the cell viability and sub-lethal levels of these were determined as follows; 2.0 gL-1 for CdCl2, 2.0 gL-1 for CuCl2.2H2O, 2.0 gL-1 for NiCl2.6H2O, 1.0 gL-1 for CoCl2.6H2O, 0.025 gL-1 for HgCl2, 0.25 gL-1 for SDS, 0.2 gL-1 for NaAsO2 and 0.5 gL-1 for phenol, from A600 readings and viable counts. Major HSPs were present at relatively lower levels or were barely increased compared to controls, whereas low molecular weight proteins were significantly changed. A 20 kDa protein was commonly found in cadmium, copper, cobalt, nickel, arsenite and phenol stresses. However, a DnaK-like protein strongly reacted against commercially available DnaK antibodies on Western blot analysis and a visibly increased signal was seen for heat, cadmium, zinc, cobalt, phenol stresses and control, where represents in that order, whereas changes in the GroEL-like protein were less specific. Several minimal or defined minimal media were developed and used in heat stress and different protein profiles were seen. Medium (2) contained M9 salts medium (supplemented with glucose) plus casamino acids and medium (5) contained M9 salts medium (supplemented with glucose), vitamin solution, trace elements solution and casamino acids. Media (2) and (5) were found to be suitable for studying the physiological changes in strain VUN10,077, but growth rates in medium (2) were much less than seen in medium (5). Strain VUN10,077 presented major HSPs (70, 58 and 18 kDa proteins) in medium (5), similar to seen in Brain Heart Infusion (BHI) media. Pulse-chase 35S-methionine labelling methods were established and known major HSPs were newly synthesised by strain VUN10,077 under heat and cadmium stresses, and these proteins gradually declined while 55 kDa, 50 kDa and 18 kDa proteins were detected under ongoing stress conditions. Presumptive groEL gene analysis was attempted and partial conserved groEL sequences were determined in the pseudomonads and related species, where similarity was 98-100%. In parallel, a water-based lux system was established to examine physiological impacts of selected pollutants at the cellular level. Lux genes cloned from a marine bacterium were introduced into the background of the indigenous strain VUN10,077, providing a potentially sensitive tool for toxicity screening for terrestrial and fresh water samples. To show the use of bioluminescence in the indigenous (naturally non-bioluminescent) organism, research was performed on stable marking and optimisation of light output including marking of the organisms with luxAB or the whole lux cassette, which necessitated establishing gene transfer systems using available transposition vectors, optimisation of conditions for light output and stability. This approach would alleviate the requirement for salt or osmotic stabilizers in the test system to maintain bioluminescence or viability, which currently is a requirement of the Microtox (TM) system which employs Photobacterium phosphoreum. Expression of bioluminescence genes in the indigenous strain employed the luminescence system of Vibrio species which is encoded by a gene cluster with a divergent transcriptional pattern. As strain VUN10,077 was a genetically uncharacterised natural isolate, this work was achieved by establishing the parameters necessary for introducing genes after determining antibiotic sensitivity to determine which marker genes could be used in these backgrounds. The genes for two polypeptides (LuxA, LuxB) which form luciferase were transferred into strain VUN10,077. Introduction of the luxAB bioluminescence genes into a range of bacteria has been facilitated by using plasmid, pUT::Tn5-luxAB (de Lorenzo et al., 1990), which carries a tetracycline resistance gene. The plasmid was introduced by bi- and tri-parental conjugation employing a helper plasmid pRK2013, if necessary, so that antibiotic resistance can occur in the transconjugants if transposition occurs. Alternatively, electroporation was used to introduce bioluminescence lux genes into strain VUN10,077 when cells were harvested at early-log and stationary phase. One of the engineered water-based luxAB-marked strain (VUN3,600) was used to detect the presence of toxic substances (determining selectivity and sensitivity). This bacterial luciferase system produces bioluminescence when supplied with an aliphatic aldehyde substrate, where the luxAB genes were expressed on the chromosome. The substrate delivery system and preparation of cells for bioluminescence toxicity test were achieved by diluting ndecyl aldehyde 10 (3)-fold in Milli-Q water and resuspending cells in tap-water after mild centrifugation, when cells were harvested at A600 1.0-1.2. When a variety of common pollutants were tested in the water-based monitoring system, cells of VUN3,600 responded sensitively, rapidly and differentially to Cd, Pb, Hg, Zn, Co ions and SDS, and with less sensitivity to Ni, and As; responses were also dose-related in a short-term assay. DMF, a solvent used to dissolve or extract several organic compounds in environmental testing, stimulated light output but in a controlled fashion. Selected substituted heterocyclic compounds tested and phenol, d- nitrophenol and strontium failed to cause dose-related inhibition, whereas di-, tri-, tetra- and penta-chlorophenol decreased light output in a long-term assay. DMF-dissolved PAH compounds were tested in the long-term assay and naphthalene, phenanthrene, fluorene, fluoranthene and benzo[a]pyrene presented dose-related responses while benz[a] anthracene and di-benzo[a]anthracence were not suitable for use in this type of assay. Although there is need to investigate extensive toxicity assays to gain broad acceptance, the water-based monitoring system showed repeatable and reliable toxicity assessment and has great possibility of usage in aquatic samples in either water-soluble or DMF-soluble forms. This study allowed explanation of the stress responses of strain VUN10,077 to be examined at a variety of levels. Bioluminescence was the most sensitive approach to determining the lowest concentrations of pollutants which affected cell physiology, as this was obvious at concentrations lower than required to cause changes in viability or induce stress responses.

M. Tech.
Background: Waterborne disease contributes significantly to the total global disease burden. Populations in rural areas of South Africa depend on untreated waters for consumption and sanitation. Contamination of public water supplies by harmful bacteria such as pathogenic E. coli poses a major risk for public health. Ingestion of these pathogenic microorganisms present in the contaminated and untreated waters could cause infection, leading to systemic inflammatory responses manifested by the production of various proinflammatory cytokines. To date, there is no human system test available to detect whether water, following ingestion, would cause disease (i.e. whether the water is infectious). The current water testing methods only test for the presence of indicator organisms, such as faecal coliforms, total coliforms, and Escherichia coli. A reliable in-vitro bioassay that could assess whether the water would cause an inflammatory response was investigated in this study. Objectives: Pro-inflammatory cytokines and whole-blood have been used in similar studies to detect the inflammatory responses following exposure to specific stimulants such as dust, lipopolysaccharide (LPS), E. coli and various others. It has been reported that larger numbers of these contaminants induced higher levels of pro-inflammatory cytokine expression. This implies that the pro-inflammatory cytokine expression could be used as a marker of infection since, inflammation occurs in response to infection. Successful infection is thus necessary for inflammation to occur, and high levels of pro-inflammatory cytokine expression confirm that infection has occurred. Thus if pro-inflammatory cytokines could serve as indicators for infection, these cytokines could be used as indicators for bacterial pathogenicity of water.

Successful bioremediation of polluted soils is often limited by the bioavailability of organic contaminants to degrading bacteria. Recent studies revealed that fungal hyphae have the potential to promote bacterial dispersal, and thus raised the idea of specifically stimulating the establishment of fungal networks in soils to increase contaminant bioavailability. Can such bacterial dispersal networks improve biodegradation performance considerably? If so, how are the improvements affected by abiotic conditions and by the spatial structure of dispersal networks? This doctoral thesis aims at answering these research questions. To this end, laboratory experiments are performed and a bacterial simulation model is developed, incorporating both microbiological and ecological theory. Manifold simulations and analyses of the microbial ecosystems’ spatiotemporal dynamics under different environmental scenarios reveal key factors and processes controlling biodegradation performance and determining benefits from bacterial dispersal networks.

Bacteria were previously isolated from two separate sites, one contaminated with lead and the other with Polycyclic Aromatic Hydrocarbons (PAHs). Alcaligenes sp. AO22 and Arthrobacter sp. E9 were identified from the lead contaminated site, while Stenotrophomonas maltophilia was identified from the PAH contaminated site. Minimum Inhibitory Concentration assays (MICs) were previously performed on Alcaligenes sp. AO22 and Arthrobacter sp. E9 and they were found to be resistant to varying levels of heavy metals and polymerase chain reactions suggesting the presence of mercury (mer), copper (pco) and cadmium, zinc and cobalt (czc) resistance genes. S. maltophilia VUN 10010, was previously investigated for its ability to degrade pyrene and other HMW PAHs as a sole carbon and energy source. The purpose of the current project was to further characterise the MICs of these isolates, along with biofilm capabilities. The genetic basis of their heavy metal resistance was also investigated.

The focus of this work was to develop a general methodology for modeling water quality in coastal waterbodies. The methods were developed in the context of modeling bacterial total maximum daily loads (TMDLs), but the general approach is applicable to a wide variety of pollutants. The study area for this dissertation was the Copano Bay watershed, which is located on the Texas Gulf Coast. The developed approach combines simple modeling techniques, of the type recommended by state and national advisory groups, in a GIS (geographic information system) framework, resulting in a methodical, easily transferred approach. This work addresses coastal systems where water quality is a function of operations in non-tidal rivers, tidal rivers, and bays, combined with the effects of watershed contributions. An uncertainty analysis was done to quantify a subset of the variance in the modeled results. Outcomes from this work include modeling tools, a documented workflow for modeling water quality in coastal watersheds, procedures to quantify the uncertainty associated with the developed approach, insight to the factors affecting water quality in the study area, and mean annual bacterial TMDLs for the impaired waterbodies of the Copano Bay watershed.
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碩士
國立雲林科技大學
環境與安全衛生工程系
107
Total petroleum hydrocarbons (TPHs) are widely used in daily life and industrial processes. Based on regulations, users must be responsible for TPHs remediation when TPHs pollute the soil and groundwater. TPHs are mainly composed of alkanes containing different carbon numbers, such as gasoline and kerosene with a carbon number between C6 and C12, and diesel with a carbon number of C12-C20. Gasoline, kerosene, diesel and other fuels belong to light non-aqueous phase liquid (LNAPL) because they have a lower specific gravity than water. When it is exposed to the soil at a high concentration, it is difficult to naturally degrade in the environment, especially alkanes with high carbon number. The physical and chemical methods such as backfilling and chemical oxidation are costly and easily produce by-products causing environmental impact. Biological method is superior to physical and chemical methods because is environmentally friendly and low cost. However, it is important to monitor environmental parametrs, TPHs degrading bacteria and microbial community during bioremediation so that bioremediation strategy can be adjusted to achieve the expected goal. In this research, biopile combined with biostimulation (CleanGreenPH) and bioaugmentation (OleoBact) was applied for TPHs contaminated soil remediation. TPHs , organic carbon, Kjeldahl nitrogen and phosphorus concentrations, bacterial community and functional genes were periodically analyzed during TPH-contaminated soil remediation to understand the bioremediation effectiveness. In addition, laboratory experiments were also carried out for further evaluating the effects of the biologics and nutrients. By controlling the operating parameters, it is more effective to explore the effects of biologics and nutrients on TPHs bioremediation. The results of the biopile monitoring indicated that after increasing the biologics and nutrients adding frequency, the Kjeldahl nitrogen concentration of biopile A increased, and then decreased owing to the use of microorganisms. The amount of viable bacteria stopped decreasing and slightly increased, and TPHs degradation was also improved. DGGE sequencing results showed that many TPHs degrading bacteria mentioned in the reference, such as Serratia, Thermomonas, Alcanivorax, Colwellia, Shewanella, Nitrosomonas, Nitrosococcus, Thauera and Lysobacter were detected. The results of the next-generation sequencing showed that the dominant genus in the bacterial community were Alcanivorax, Lysobacter and Sphingomonas. Laboratory experiments demonstrated that the total organic carbon and total Kjeldahl nitrogen of the experimental groups A, B, C, D and E decreased within 35 day, but the phosphorus concentration of each group had no significant change. The TPH degradation efficiencies of groups A, B, C and D were -2.09%, 65.02%, 91.82% and 41.67%, respectively. Within the fisrt 5 days, the TPH concentration of group E had significantly decreased 2207.5 mg/kg, and the degradation efficiency was about 56.0%. At the end of the experiment, the TPH concentration of group E was only 54.7 mg/kg, and the degradation efficiency was 98.9%. The results of DGGE indicated that TPHs degrading bacteria such as Bacillus sp., Lysobacter sp. and Pseudomonas sp. were present in each group in the beginning. Owing to the addition of the OleoBact, Groups D, E and F additionally contained Lysinibacillus sp. and Bacillus sp.. At the end of the experiment, the bacterial community of all groups changed. Lysinibacillus sp., the bacterium from the OleoBact, disappeared. The native bacterium Pseudomonas sp. also decreased. This indicated that the bacteria from the OleoBact could not become the dominant species in the TPHs contaminated soil. Overall, biostimulation and bioaugmentation could affect the microbial community in the TPH-contaminated soil. High concentration of TPHs was almost completely degraded within a short period.

碩士
逢甲大學
環境工程與科學所
99
A previous epidemiological study of agriculture workers indicated that farmers have the greatest number of work-related health complaints. Most of the complaints are related to respiratory problems such as coughing, shortness of breath and asthma symptoms. Exposure to grain dusts, mould spores, organic and inorganic dusts, insecticides, bacteria, fungi and bacterial endotoxins, farm workers may result in health risk. Studies indicated that the contamination tends to cause cellular responses which result in or are indirectly associated with lung disease. We conduct a study in different farms to estimate farmers’ exposure to bacterial pollutant in work and provided protection by surgical masks and N95 filtering facepiece respirators against airborne bacterial pollutant in agricultural farms. Our results show that the concentrations of bacteria, Gram-negative bacteria and endotoxin concentration respectively range from &lt;LOD to 186768 CFU/m3, from &lt;LOD to 58067 CFU/m3 and from &lt;LOD to 43027 EU/m3 in different farms. The highest concentration of bacteria appears in poultry, and the highest concentration of Gram-negative bacteria and endotoxin appears in the corn. Apart from that, we also notice Gram-negative bacteria and endotoxin are all collected within the particle size over 1.8 μm, the ratio for each are 98%,99.0%,and 97.4%. Through Pearson correlation analysis, we found out, correlation coefficient of bacteria, Gram-negative bacteria and endotoxin each are 0.4236(p=0.0068) and 0.4869(p=0.0011). By statistics, both bacteria and Gram-negative with endotoxin are medium positive correlation. Further more, research also found out that over the particle size of 1.8 μm. Bacteria and Gram-negative bacteria with endotoxin each appear medium correlation (r=0.4432(p=0.0033) and 0.4899(p=0.0010)).Between the particle size 1-1.8 μm and &lt;1.0 μm, no correlation exists. The reason of that might be, due to the huge amount of bacterial fragment corrections under a tiny particle size, the endotoxin still viable and bacteria (Gram-negative bacteria) are not. The geometric means of workplace profection factors of N95 filtering facepiece respirators and surgicall masks against bacteria, Gram-negative bacteria and endotoxin each are 75.5 and 29.9, 19.4 and 11.2, 35.0 and 20.6. The geometric means of workplace profection factors of N95 filtering facepiece respirators against for bacteria, Gram-negative bacteria, and endotoxin each are 2.4, 1.7, and 1.1 higher than surgicall masks. The assigned protection factor (APF) of N95 filtering facepiece respirators against bacteria, Gram-negative bacteria and endotoxin each are 1.9, 2.0, and 2.4, which are far below the OSHA standard specification of 10. In addition, we found that regardless of N95 masks or surgical masks, its WPF values will increase with larger particle size. The particle size range >1.8 μm of the geometric mean WPF of bacterial contaminants are greater than 10, while the particle size range 1 -1.8 μm and &lt;1.0 μm of the geometric mean WPF are less than 10. From the results, we could know that the APF of half facepiece set by the United States OSHA may be overestimated. The particle size should be taken into account when setting the APF value. From statistic, the WPF for endotoxin with the WPF for bacteria and Gram-negative bacteria are significantly medium positive correlation. The correlation coefficients each are 0.600(p=0.0001) and 0.638(p=0.0001). This means that the endotoxin can be used as an indicator to assess the protection provided by masks against bacteria and Gram-negative bacteria in farms. Moreover, the study found the fit factor (FF) and WPFs of bacterial pollutant are no statistically significant correlation (p> 0.05), which means the fit test results can not replace the workplace protection factor. Carbon dioxide, temperature, and relative humidity and other environmental factors together with bacteria, Gram-negative bacteria, endotoxin, and the results of microbial viability found no statistically significant correlation (p> 0.05). The results show carbon dioxide, temperature, and relative humidity and other environmental factors will not affect the distribution of bacterial pollutant in farms.

Les champignons mycorhiziens à arbuscules (CMA) forment un groupe de champignons qui appartient à l'embranchement des Gloméromycètes (Glomeromycota). Les CMA forment des associations symbiotiques, connus sous le nom des mycorhizes à arbuscules avec plus de 80 % des plantes vasculaires terrestres. Une fois que les CMA colonisent les racines de plantes, ils améliorent leurs apports nutritionnels, notamment le phosphore et l'azote, et protègent les plantes contre les différents pathogènes du sol. En contrepartie, les plantes offrent un habitat et les ressources de carbone nécessaires pour le développement et la reproduction des CMA. Des études plus récentes ont démontré que les CMA peuvent aussi jouer des rôles clés dans la phytoremédiation des sols contaminés par les hydrocarbures pétroliers (HP) et les éléments traces métaliques. Toutefois, dans les écosystèmes naturels, les CMA établissent des associations tripartites avec les plantes hôtes et les microorganismes (bactéries et champignons) qui vivent dans la rhizosphère, l'endosphère (à l'intérieur des racines) et la mycosphère (sur la surface des mycéliums des CMA), dont certains d'entre eux jouent un rôle dans la translocation, l’immobilisation et/ou la dégradation des polluants organiques et inorganiques présents dans le sol. Par conséquent, la diversité des CMA et celle des microorganismes qui leur sont associés sont influencées par la concentration et la composition des polluants présents dans le sol, et aussi par les différents exsudats sécrétés par les trois partenaires (CMA, bactéries et les racines de plantes). Cependant, la diversité des CMA et celle des microorganismes qui leur sont associés demeure très peu connue dans les sols contaminés. Les interactions entre les CMA et ces microorganismes sont aussi méconnus aussi bien dans les aires naturelles que contaminées. Dans ce contexte, les objectifs de ma thèse sont: i) étudier la diversité des CMA et les microorganismes qui leur sont associés dans des sols contaminés par les HP, ii) étudier la variation de la diversité des CMA ainsi que celle des microorganismes qui leur sont associés par rapport au niveau de concentration en HP et aux espèces de plantes hôtes, iii) étudier les correlations (covariations) entre les CMA et les microorganismes qui leur sont associés et iv) comparer les communautés microbiennes trouvées dans les racines et sols contaminés par les HP avec celles trouvées en association avec les CMA. Pour ce faire, des spores et/ou des propagules de CMA ont été extraites à partir des racines et des sols de l'environnement racinaire de trois espèces de plantes qui poussaient spontanément dans trois bassins de décantation d'une ancienne raffinerie de pétrole située dans la Rive-Sud du fleuve St-Laurent, près de Montréal. Les spores et les propagules collectées, ainsi que des échantillons du sol et des racines ont été soumis à des techniques de PCR (nous avons ciblés les genes 16S de l'ARNr pour bactéries, les genes 18S de l'ARNr pour CMA et les régions ITS pour les autres champignons), de clonage, de séquençage de Sanger ou de séquençage à haut débit. Ensuite, des analyses bio-informatiques et statistiques ont été réalisées afin d'évaluer les effets des paramètres biotiques et abiotiques sur les communautés des CMA et les microorganismes qui leur sont associés. Mes résultats ont montré une diversité importante de bactéries et de champignons en association avec les spores et les propagules des CMA. De plus, la communauté microbienne associée aux spores des CMA a été significativement affectée par l'affiliation taxonomique des plantes hôtes et les niveaux de concentration en HP. D'autre part, les corrélations positives ou négatives qui ont été observées entre certaines espèces de CMA et microorganismes suggérèrent qu’en plus des effets de la concentration en HP et l'identité des plantes hôtes, les CMA peuvent aussi affecter la structure des communautés microbiennes qui vivent sur leurs spores et mycéliums. La comparaison entre les communautés microbiennes identifiées en association avec les spores et celles identifiées dans les racines montre que les communautés microbiennes recrutées par les CMA sont différentes de celles retrouvées dans les sols et les racines. En conclusion, mon projet de doctorat apporte de nouvelles connaissances importantes sur la diversité des CMA dans un environnement extrêmement pollué par les HP, et démontre que les interactions entre les CMA et les microorganismes qui leur sont associés sont plus compliquées que ce qu’on croyait précédemment. Par conséquent, d'autres travaux de recherche sont recommandés, dans le futur, afin de comprendre les processus de recrutement des microorganismes par les CMA dans les différents environnements.
Arbuscular mycorrhizal fungi (AMF) are an important soil fungal group that belongs to the phylum Glomeromycota. AMF form symbiosic associations known as arbuscular mycorrhiza with more than 80% of vascular plants on earth. Once AMF colonize plant roots, they promote nutrient uptake, in particular phosphorus and nitrogen, and protect plants against soil-borne pathogens. In turn, plants provide AMF with carbon resources and habitat. Furthermore, more recent studies demonstrated that AMF may also play key roles in phytoremediation of soils contaminated with petroleum hydrocarbon pollutants (PHP) and trace elements. Though, in natural ecosystems, AMF undergo tripartite associations with host plants and micoorganisms (Bacteria and Fungi) living in rhizosphere (the narrow region of soil surounding the plant roots), endosphere (inside roots) and mycosphere (on the surface AMF mycelia), which some of them play a key role on translocation, immobilization and/or degradation of organic and inorganic pollutants. Consequently, the diversity and community structures of AMF and their associated microorganisms are influenced by the composition and concentration of pollutants and exudates released by the three partners (AMF, bacteria and plant roots). However, little is known about the diversity of AMF and their associated microorganisms in polluted soils and the interaction between AMF and these microorganisms remains poorly understood both in natural and contaminated areas. In this context, the objectives of my thesis were to: i) study the diversity of AMF and their associated microorganisms in PHP contaminated soils, ii) study the variation in diversity and community structures of AMF and their associated microorganisms across plant species identity and PHP concentrations, iii) study the correlations (covariations) between AMF species and their associated microorganisms and iv) compare microbial community structures of PHP contaminated soils and roots with those associated with AMF spores in order to determine if the microbial communities shaped on the surface of AMF spores and mycelia are different from those identified in soil and roots. To do so, AMF spores and/or their intraradical propagules were harvested from rhizospheric soil and roots of three plant species growing spontaneously in three distinct waste decantation basins of a former petrochemical plant located on the south shore of the St-Lawrence River, near Montreal. The harvested spores and propagules, as well as samples of soils and roots were subjected to PCR (we target 16S rRNA genes for bacteria, 18S rRNA genes for AMF and ITS regions for the other fungi), cloning, Sanger sequencing or 454 high throughput sequencing. Then, bioinformatics and statistics were performed to evaluate the effects of biotic and abiotic driving forces on AMF and their associated microbial communities. My results showed high fungal and bacterial diversity associated with AMF spores and propagules in PHP contaminated soils. I also observed that the microbial community structures associated with AMF spores were significantly affected by plant species identity and PHP concentrations. Furthermore, I observed positive and negative correlations between some AMF species and some AMF-associated microorganisms, suggesting that in addition to PHP concentrations and plant species identity, AMF species may also play a key role in shaping the microbial community surrounding their spores. Comparisons between the AMF spore-associated microbiome and the whole microbiome found in rhizospheric soil and roots showed that AMF spores recruit a microbiome differing from those found in the surrounding soil and roots. Overall, my PhD project brings a new level of knowledge on AMF diversity on extremely polluted environment and demonstrates that interaction of AMF and their associated microbes is much complex that we though previously. Further investigations are needed to better understand how AMF select and reward their associated microbes in different environments.