Dissertations / Theses on the topic 'Heavy metal resistance bacteria'

Thesis (M.Sc.) -- University of Limpopo, 2005
Six aerobic bacterial strains [GM 10(1), GM 10 (2), GM 14, GM 15, GM 16 and GM 17] were isolated from an antimony mine in South Africa. Heavy-metal resistance and biosorptive capacities of the isolates were studied. Three of the isolates (GM 15, GM 16 and GM 17) showed different degrees of resistance to antimony and arsenic oxyanions in TYG media. The most resistant isolate GM 16 showed 90 % resistance, followed by GM 17 showing 60 % resistance and GM 15 was least resistant showing 58 % resistance to 80 mM arsenate (AsO4 3-). GM 15 also showed 90 % resistance whereas isolates GM 16 and GM 17 showed 80 % and 45 % resistance respectively to 20 mM antimonate (SbO4 3-). Arsenite (AsO2 -) was the most toxic oxyanion to all the isolates. Media composition influenced the degrees of resistance of the isolates to some divalent metal ions (Zn2+, Ni2+, Co2+, Cu2+ and Cd2+). Higher resistances were found in MH than in TYG media. All the isolates could tolerate up to 5 mM of the divalent metal ions in MH media, but in TYG media, they could only survive at concentrations below 1 mM. Also, from the toxicity studies, high MICs were observed in MH media than TRIS-buffered mineral salt media. Zn2+ was the most tolerated metal by all the isolates while Co2+ was toxic to the isolates. The biosorptive capacities of the isolates were studied in MH medium containing different concentrations of the metal ions, and the residual metal ions were determined using atomic absorption spectroscopy. GM 16 was effective in the removal of Cu2+ and Cd2+ from the contaminated medium. It was capable of removing 65 % of Cu2+ and 48 % of Cd2+ when the initial concentrations were 100 mg/l, whereas GM 15 was found to be effective in the biosorption of Ni2+ from the aqueous solutions. It was capable of removing 44 % of Ni2+ when the initial concentration was 50 mg/l. GM 17 could only remove 20 % of Cu2+ or Cd2+. These observations indicated that GM 16 could be used for bioremediation of xvi Cu2+ and Cd2+ ions from Cu2+ and Cd2+-contaminated aqueous environment, whereas GM 15 could be used for bioremediation of Ni2+.
National Research Foundation and the University of the North Research Unit

Heavy-metals exert considerable stress on the environment worldwide. This study assessed the resistance to and bioremediation of heavy-metals by selected protozoan and bacterial species in highly polluted industrial-wastewater. Specific variables (i.e. chemical oxygen demand, pH, dissolved oxygen) and the growth/dieoff- rates of test organisms were measured using standard methods. Heavy-metal removals were determined in biomass and supernatant by the Inductively Couple Plasma Optical Emission Spectrometer. A parallel experiment was performed with dead microbial cells to assess the biosorption ability of test isolates.

Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ;cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it.
Doctorat en Sciences agronomiques et ingénierie biologique
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The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

Cupriavidus metallidurans CH34 est la bactérie Gram négative considérée comme organisme-modèle pour l’étude de la résistance aux métaux lourds. Notre travail a porté sur sa résistance au cuivre, codée par les gènes cop du plasmide pMOL30. Ces gènes, responsables des différentes étapes de la résistance (compartimentation des systèmes d’efflux entre périplasme et cytoplasme, modification de valence, et d’autres fonctions totalement inconnues) ont suscité notre intérêt.

On distingue dans l’îlot cop des gènes codant pour des fonctions de résistance proprement dite (essentiellement par détoxication active du cytoplasme et du périplasme). En effet, les mutants de copSRABCD, copF, et dans une moindre mesure copJ et copE deviennent sensibles. Les phénotypes des mutants divergent toutefois suivant que la mutation soit sur un cosmide qui ne porte que l’îlot (pMOL1024) ou dans son plasmide d’origine (pMOL30). Un second groupe de mutants (copVTMK, copG, copL, copQ) se distingue par un phénotype plus résistant ou identique à la souche parente, sauf autour de la CMI. Ces gènes interviendraient donc à la CMI pour assurer la résistance la plus élevée et le maintien d'un état viable latent.

La présence de l’îlot cop permet de contenir le taux d’oxygène radicalaire qui reste à un taux basal lorsque les cellules sont adaptées au cuivre environnent. Après un choc de Cu (ou stress aigu), l’îlot cop répond de façon « explosive » au stress, en consommant l’énergie du potentiel membranaire et en augmentant fortement l’activité de la chaîne respiratoire.

La résistance au cuivre est inductible, mais de façon différenciée pour la souche sauvage (CH34) et celle qui ne porte qu l’îlot cop (AE1744) :la CMI de CH34 triple après adaptation au cuivre, alors que celle d’AE1744 est inchangée. Après un choc de Cu, la résistance au cuivre est plus fortement induite pour AE1744 que pour CH34. Ces observations suggèrent que l’îlot cop ait été sélectionné pour sa capacité à répondre à un stress aigu puis intégré dans un ensemble de gènes plus vaste qui répond à des impératifs de stress chronique.

L’analyse biochimique de CopI, une petite protéine bleue à cuivre, montre qu’elle porte un site analogue à celui des oxydases multicuivre. Son rôle pourrait dès lors être celui d’une réductase multicuivre. La protéine CopK lie de façon très spécifique le Cu(I) et il semble que la liaison du cuivre modifie sa structure. L’analyse écologique a montré que des homologues de copK pourraient être présents dans l’ADN extrait de la terre de biotopes chargés en cuivre, et dans les souches cuprorésistantes qu’on y trouve.

La contribution majeure de cette thèse est de montrer que l’effet d’un stress métallique ne se résume pas à deux états physiologiques « mort ou vif ». Il y a lieu de considérer des états transitoires (choc de Cu, adaptation au métal, survie autour de la CMI, persistance) où interviennent des gènes spécifiques dans un ou plusieurs états donnés. Les résultats biochimiques et physiologiques ne nous éclairent pas encore assez sur les interconversions Cu(I)/Cu(II) ni sur les flux de cations notamment vers l'espace extracellulaire. Cette thèse ouvre des perspectives sur des mécanismes (protection à la CMI, phénotype persistant) assurant la survie des bactéries ou leur potentiel de recolonisation lors d'une diminution de la pression toxique :les gènes copT, copV, copK, copM, copB, copG, copL et copQ semblent impliqués dans ces fonctions.

Doctorat en Sciences agronomiques et ingénierie biologique
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The genus Gluconobacter is industrially important due to the ability to accomplish unusual and almost complete oxidation reactions (bioconversions) and to contaminate high sugar content products. Following preliminary evidence that some strains of Gluconobacter were resistant to cadmium, and realizing that cadmium resistance among gram-negative organisms is often encoded by an operon which also encodes cobalt and zinc resistance via an efflux mechanism, 10 strains of Gluconobacter were tested for heavy-metal resistance. Three of the 10 representative strains appeared to be resistant to cadmium chloride, and two were also resistant to cobalt- and zinc chloride. These strains, as well as two cadmium-sensitive strains were analyzed using PCR and sequencing to establish gene homology with Ralstonia eutropha, the most frequently studied Gram-negative bacterium exhibiting cadmium resistance. Amplification of two genes from the czc operon, known to encode cadmium, cobalt and zinc resistance in Ralstonia, was attempted in the three resistant and two sensitive strains of Gluconobacter. The gene, czcA, thought to encode the main pump protein of the efflux mechanism, was found in all Gluconobacter strains tested. However, amplification of a regulatory gene czcD, thought to sense the extracellular metal ion concentration, was not possible in the Gluconobacter strains tested. The PCR products were sequenced and analyzed for homology to the czc operon in Ralstonia. From the data gathered, it appears as though some strains of Gluconobacter contain at least a portion of the czc operon , encoding cadmium, cobalt and zinc resistance in Ralstonia eutropha.
Master of Science

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia
Heavy metal resistance in Marinobacter aquaeolei (Ma.aq) 617 in aerobic conditions was studied for three different ions, cadmium, cobalt and copper. The main aim of this work was the study of a putative copper resistance operon, copSRXAB, located in the chromosome of Marinobacter and the biochemical characterization of a unique copper binding protein CopX (proposed designation), associated with the copper resistance system. Growth under heavy metal ion stress was performed for those three heavy metals and the Minimum Inhibitory Concentration (MIC) / Maximum Tolerant Concentration (MTC) was determined using two different approaches, solid artificial sea water (ASW) plates and liquid ASW medium, supplemented with lactate and yeast extract, as carbon sources. The MIC/MTC of cadmium, cobalt and copper ions was found to be 200 μM, 4-6 mM, and 1.6 mM, respectively. These values classify Ma.aq strain 617 as cadmium, cobalt and copper resistant strain. Moreover, during the cobalt resistance studies we observed the production of an unknown protein or compound, which is proposed be a cobalamine containing protein and/or cobalamine itself. Under the scope of copper resistance, preliminary proteomics analysis of the Ma.aq periplasmic fraction was performed. CopX, identified by MALDI TOF-TOF mass spectrometry, was shown to be differentially expressed under copper stress. This demonstrated that the proposed copper operon, copSRXAB, has a role in the Ma.aq copper resistance. CopX was successfully heterologously expressed in Escherichia coli (Es.coli), and purified for the first time using usually two chromatographic steps (anionic exchangeand size exclusion) with a yield of 5.7 mg or 1.8 mg of purified CopX, per L of LB or M9 medium, respectively. Mass spectrometry Electron Spray Ionisation (ESI) and N-terminus analysis revealed that the signal peptide of CopX comprises 21 residues, and is efficiently processed by the Sec system of Es.coli. Biochemical characterization of CopX proved that it is a periplasmic monomeric type 1 copper protein, with a molecular weight of 17253.25 ± 0.30 Da, determined by mass spectrometry (ESI), that binds approximately 1 copper ion per polypeptide chain. The apparent molecular weight of CopX, 20.4 kDa, determined by size-exclusion chromatography does not depend on the ionic strength. Spectroscopic characterization showed that it presents an intense charge transfer (Scys – Cu ion) band at 440 nm and 580 nm and 720 nm. The extinction coefficient at 580 nm was found to be 3.8 mM-1cm-1, according to the copper content. CopX EPR spectrum is axial. The 15N HSQC NMR spectra of CopX confirms that it is folded, with 131 out of 147 backbone amide resonances identified, showing that it is amenable to NMR solution structure determination. CopX presents some unique features, such as, a ratio between A440nm and A580nm of 0.94 and a high hyperfine coupling constant, 170 G. Taking into account the biochemical properties, CopX is proposed to be part of a new class of the type 1 copper proteins, shown preliminarily for the first time to be associated with copper resistance.

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.

The objectives of this project were to determine the bacterial diversity in a heavily contaminated metal region of Portugal. Both traditional and molecular based methods were used to identify tolerant strains and species. The most tolerant species were subsequently identified and utilized for examining the potential for using them to immobilize specific metals from contaminated waste streams by comparing different support materials. Heterotrophic bacterial populations were isolated and characterized from a contaminated industrial area in Northern Portugal. In a first sampling, 278 strains were isolated in different solid media. To assess the diversity of this ecological site and to select representative strains, the isolates were screened by using Random Amplified Polymorphic DNA (RAPD)-PCR profiles. Phenotypic characterization, phylogenetic analysis by sequencing the 16S rRNA genes and metal tolerance tests with zinc (Zn), cadmium (Cd) and arsenic (As) were performed with the selected strains. Recovered gram-positive isolates were related to class Actinobacteria and Bacilli. The majority of the isolates were related to genera Microbacterium and Bacillus. Strains from the genus Arthrobacter were also well represented. 16S rRNA gene sequence similarity of the gram-negative isolates showed that they were related to classes γ-Proteobacteria, ß-Proteobacteria, a-Proteobacteria and Flavobacteria. The most frequently isolated taxa were γ-Proteobacteria, related with the genus Pseudomonas, where a large number of isolates were clustered. These genera are common in metal contaminated environments. Many of the strains (approx. 17) had a high level of tolerance to the heavy metals tested. A total of 13 isolates were not able to grow when metals were present. In a second sampling the soil rhizosphere was screened for bacterial populations, using metal-based selective media for isolation. About 42 strains were recovered when metal supplemented media was used. The gram-positive population were predominantly Bacilli and Actinobacteria members. Bacillus, Microbacterium and Arthrobacter were the most common gram-positive genera. Gram-negative genera were from the same classes as in the first sampling however Sphingobacteria was present. γ-Proteobacteria and ß-Proteobacteria were the most common taxa. The isolates were shown to be very resistant to Zn and As, with about half of the isolates able to grow with Cd present. Interestingly, no strains could grow in the presence of metal mixtures. Despite the number of strains recovered in both samplings the majority of the isolates were clustered within a very small number of genera. During the sampling periods two strains showing low similarity to other bacteria were isolated. These strains were characterized and studied in detail justifying their classification as representing two novel species of the genus Chryseobacterium. The names proposed for these organisms are Chryseobacterium palustre sp. nov. (type strain 3A10[type strain]) and Chryseobacterium humi sp. nov. (type strain ECP37[type strain]). Three isolates 1C2, 1ZP4 and EC30 belonging to genera Cupriavidus, Sphingobacterium and Alcaligenes respectively, showing high tolerance to heavy metals, were selected for further study in immobilised systems for Zn and Cd removal. In most cases, matrices (alginate, pectate and a synthetic cross-linked polymer) with immobilised bacteria showed better metal removal. 1C2, a strain belonging to the Cupriavidus genera, was able to increased the removal of Zn; EC30, a bacteria related to Alcaligenes, was the most promising candidate for Cd removal, especially when combined with the synthetic polymer. Removal of metals as single or in binary mixtures was also assessed. Cd removal was most effective when single metal solutions were tested using immobilised bacteria and examining metal matrixes. Based on the strains used and the matrices tested, best results were obtained for removal of Zn from binary mixtures with Cd. Potential exists for further studies to exploit these bacterial strain to develop effective bioremediation approaches for the removal of heavy metals from waste water streams.

Antibiotic resistance profiles of fecal coliform isolated from unchlorinated and chlorinated secondary effluent were determined. Of 332 fecal coliforms isolated from chlorinated effluent a mean of 48% were multiply antibiotic resistant. In contrast, of 347 fecal coliforms isolated from unchlorinated effluent a mean of 29% were multiply antibiotic resistant. Resistance to ampicillin, cephalothin, and carbenicillin were significantly higher in the former than the latter. Randomly selected isolates survived and/or grew in sterile and unsterile effluent retaining resistance patterns for 40 days. Resistance factors were transferred in laboratory medium at frequencies from 0 to 1.2 x 10⁻² (number of recombinants/number of recipients) and in sterile neutralized tertiary effluent at frequencies from 0 to 1.0 x 10⁻⁴. Resuscitative techniques were necessary for optimal recovery of fecal coliforms from effluent using selective media. Antibiotic resistance patterns of fecal coliforms isolated from unchlorinated and chlorinated effluent was not associated with chlorine or heavy metal resistance. Multiply antibiotic resistant fecal coliforms from chlorinated effluent were significantly less sensitive to lytic phage than multiply antibiotic sensitive fecal coliforms from unchlorinated effluent (p < .05). Using group discriminate analysis of data, phage typing techniques were shown to be a potential tool for tracing fecal contamination of groundwater.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Our research intends to comprehend the configuration of the resistance (Foucault) as the stylization of life in the contemporary world, taking Heavy Metal as the specific object of study. We believe that Heavy Metal is an ethopoietical device which admits practices of freedom withstanding the reified moral habits since the beginning of the socialization. This is reflected, mainly, in the creation of new individual and communal ways to stylize the life. We also suggest an expansion of Foucault s concept of resistance, considering the idea of consumer society described by Zygmunt Bauman. Our hypothesis understands that the contact with the underground of Heavy Metal provides new ethical manners (Foucault), where the individual take the Heavy Metal as a way of life. At this point, the consumption becomes a key-word since the participation in the underground of Heavy Metal is a way of consumption out of the rules of marketing a practice of freedom, a way of particular existence , being different in both mode and duration
Nossa pesquisa visa compreender a configura??o da resist?ncia (Foucault) enquanto estiliza??o da vida na contemporaneidade, tendo o Heavy Metal enquanto objeto de estudo espec?fico. Acreditamos que o Metal se configure em um dispositivo ethopoi?tico possibilitador de pr?ticas de liberdade frente aos h?bitos morais reificados desde os prim?rdios da socializa??o do sujeito. Isso se reflete, principalmente, na cria??o de novas maneiras de estilizar a vida que s?o individuais e grupais, ao mesmo tempo. Sugerimos tamb?m uma amplia??o do pensamento sobre o tema da resist?ncia, em Foucault, tendo em vista a sociedade de consumo descrita por Zygmunt Bauman. Nossa hip?tese ? a de que o contato com o mundo underground do Heavy Metal ? o possibilitador de novas formas ?ticas (Foucault), onde h? a ades?o e o comprometimento do sujeito com o Heavy Metal enquanto um modo de vida. A partir da?, o consumo se torna uma palavra chave, na medida em que, participar do underground do Heavy Metal - enquanto uma pr?tica de liberdade, um modo de exist?ncia particular constitui tamb?m uma forma de consumo que foge ?s regras gerais do mercado, sendo um consumo diferenciado tanto na forma quanto na sua dura??o

Bacterial organisms continuously maintain homeostasis even in changing environments. This ability to maintain homeostasis is especially critical for pathogenic and opportunistic bacteria, which must adapt to both abiotic and biotic host environments. Both types of environments present unique limitations and conditions. Transition metal homeostasis under these varying conditions is important for bacterial survival. Transition metals such as zinc, cobalt, iron and copper are essential for cell survival, but become toxic if in excess. The host organism often takes advantage of this requirement by greatly limiting access to transition metals to limit infections, but in other environments, toxic levels of metal may be present. Bacterial organisms have developed many mechanisms to maintain transition metal homeostasis. This study focuses on two bacterial systems that are utilized to maintain metal balance; the heme-acquiring iron surface determinant (Isd) system of Bacillus anthracis and the copper and silver export Cus system of Escherichia coli. Host organisms use many proteins and systems to limit iron access from pathogenic bacteria, known as nutrient immunity. B. anthracis must acquire iron from the host organism upon infection and so has evolved multiple iron acquisition systems. The Isd system employs two extracellular proteins, IsdX1 and IsdX2, to remove heme from hemoglobin to use as an iron source. Once bound to heme, these hemophores transfer heme to a cell surface attached protein, IsdC, which further relays the molecule to be transferred into the cell for iron use. This study focused on the kinetics of heme transfer to better understand how acquisition occurs. This study determined that the oxidation state of the iron-heme molecule plays a significant role in the kinetics of heme acquisition by IsdX1 and subsequent transfer to IsdC. This work clarifies and further establishes the mechanism of iron acquisition by B. anthracis during infection. Copper and silver are used in many settings as antimicrobial agents, including as an alternative to antibiotic drugs. Pathogenic and opportunistic bacteria, such as E. coli, experience stress upon contact with copper and silver surfaces and materials. Copper is an essential transition metal, while silver is not biologically used, but both become toxic when in excess due to redox properties and disruption of biological molecules. E. coli utilizes several systems to remove excess copper and silver to resist toxicity. The Cus system, consisting of the soluble CusF and tripartite pump CusCBA, specifically exports copper and silver from the periplasm. Several roles of CusF have been suggested from in vitro data. The components CusAB were hypothesized to be the essential proteins of the CusCBA pump, while the outer membrane unit may not contribute specificity or be necessary for export. This study focused on the role and importance of CusF and outer membrane channel CusC during copper stress in vivo. An in vivo interaction between CusF and CusB was identified during copper stress. The data from this work indicate that cusF and cusC directly affect intracellular copper accumulation. Furthermore, this study revealed that SdsP may play in a secondary role to CusC to complement CusC to maintain copper resistance. This works establishes the importance of CusC as the main outer membrane component during copper export in E. coli.

Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

Soils have been contaminated with cadmium (Cd) by the use of fertilizers, calcareous, pesticides and industrial and/or domestic effluents. It can be leached to groundwater, as well as be taken up by plants potentially leading to reduce growth and yield. It causes different damages to the cell, generating oxidative stress which is responsible for its toxicity, affecting all living organism. A balance in the redox state of the cell to maintain cellular integrity and metabolism is essential for organism tolerance. Thus, the antioxidant response of bacteria exposed to Cd was studied to understand the tolerance mechanism, and be able to develop a methodology to bioremediate contaminated soils. MDA and hydrogen peroxide contents and different enzymes activity of antioxidant system (SOD, CAT, GR and GST) of two strains from Burkholderia genus, one from a soil contaminated with Cd in high concentrations (strain SCMS54) and the other from soil without Cd (strain SNMS32) in two exposure time (5 and 12 h), were analyzed. Stress measurement (MDA and hydrogen peroxide content) and antioxidant enzyme activities (SOD, CAT, GR and GST) increased in almost all treatments in the presence of Cd. These results also indicate that strain SCMS54 (isolated from Cd contaminated soil) presents a higher metabolic diversity and plasticity due the expression of more isoforms of the enzymes SOD, CAT and GR. The strain also accumulates 50% more Cd. We also analyzed the response to Ni of these two strain, observing a similar response to Cd, except for GST enzyme expression, which in strain SCMS54 this enzyme was induced in the presence of Ni, indicating that this enzyme can be essential on Ni tolerance. After that, the strain isolated from Cd contaminated soil (SCMS54) was selected to proceed the studies to evaluate the benefits of tolerant microorganism-tomato plant interaction. The use of plants to remove heavy metals from contaminated soilhas less impact and a lower cost. Soil microorganisms can be able to solubilize or mobilize soil metals acting also as bioremediator. Besides the high tolerance to Cd, the strain SCMS54 can produce indole-acetic acid (IAA), solubilize inorganic phosphate and produce siderophore, revealing its potential in plantmicroorganism mutual and beneficial interaction. When interacting with tomato plants exposed to Cd, this bacterium led to decrease in plant peroxide concentration and chlorosis levels, promoted relative plant growth and reduced the root absorption of Cd resulting in an increase in plant tolerance to this highly toxic heavy metal. Indicating that inoculation of tomato plants with Burkholderia sp. SCMS54 promotes better growth when cultivated in the presence of Cd by a mechanism that appears to decrease Cd concentration in roots as a result of a bacterial-plant root beneficial interaction.
O cádmio (Cd) tem contaminado solos pelo uso de fertilizantes, calcário, agrotóxicos e resíduos industriais e/ou domésticos. Podendo ser lixiviado ao lençol freático ou absorvido pelas plantas,resultando na redução do crescimento e da produtividade. Esse metal afeta todos os organismos vivos e causa diferentes danos às células. A tolerância a esse metal se deve principalmente ao balanço do estado redox da célula para manter a integridade celular e metabolismo.Assim, foram isoladas bactérias de solo contaminado e não contaminado com Cd, selecionando isolados tolerantes a altas concentrações de diferentes metais (Cd, Ni e Zn), em seguida, foi observado a resposta do sistema antioxidante da bactéria na presença do Cd, a fim de auxiliar no desenvolvimento de metodologias para biorremediar solos contaminados. Foi quantificado MDA e peróxido de hidrogênio e a atividade de diferentes enzimas do sistema antioxidante (SOD, CAT, GR e GST) de duas estirpes do gênero Burkholderia tolerantes a todos os metais testados, uma isolada do solo contaminado com altas concentrações de Cd (estirpe SCMS54) e a outra do solo sem Cd (estirpe SNMS32) em dois tempos de exposição (5 e 12 h). Na estirpe SCMS54, as medidas de estresse (peroxidação lipídica e peróxido de hidrogênio) e a atividade das enzimas antioxidantes (SOD, CAT, GR e GST) da maioria dos tratamento com cádmio aumentaram, esta estirpe também expressa mais isoformas de SOD, CAT e GR, além de acumular 50% mais Cd. Esses resultados mostram que a estirpe SCMS54 (isolada do solo contaminado com Cd) apresenta uma maior diversidade metabólica e plasticidade. Foram analisadas também a resposta dessas duas estirpes ao Ni, observando uma resposta semelhante ao Cd, exceto na expressão da enzima GST, que no estirpe SCMS54 foi induzida na presença do Ni, indicando que essa enzima pode ser essencial na tolerância ao Ni. Portanto, a estirpe isoladado solo contaminado com Cd (SCMS54) foi selecionada para prosseguir os estudos e avaliar os benefícios da interação entre microrganismos tolerantes-plantas de tomate na fitorremediação. Essa técnica é usada remover para metais pesados do solo com um menor impacto e baixos custos. Os microrganismos do solo podem solubilizar e mobilizar metais do solo, atuando como biorremediador. Além da alta tolerância ao Cd, a estirpe SCMS54 produz ácido indol acético (AIA), solubiliza fosfato inorgânico e produz sideroforo, mostrando seu potencial na interação benéfica planta-microorganismo. Quando interagindo com as plantas de tomate expostas ao Cd, essa bactéria diminui a concentração de peróxido da planta e a clorose ocasionado pelo Cd,e reduz a absorção de Cd pela raiz resultando em um aumento da tolerância da planta ao metal pesado altamente tóxico. Assim, a inoculação de plantas de tomate com Burkholderia sp. SCMS54 promove crescimento da planta na presença de Cd, desencadeando um mecanismo que diminui a concentração de Cd nas raízes devido a essa interação benéfica bactéria-raiz da planta.

La presente Tesis Doctoral consiste en la determinación de las propiedades de transporte de diferentes especies catiónicas a través de membranas de intercambio catiónico. Las membranas de intercambio iónico son un componente clave de los reactores electroquímicos y de los sistemas de electrodiálisis, puesto que determinan el consumo energético y la eficiencia del proceso. La utilización de este tipo de membranas para el tratamiento de efluentes industriales no es muy extendida debido a los requisitos de elevada resistencia química y durabilidad que deben cumplir las membranas. Otro asunto importante radica en la eficiencia en el transporte de los iones que se quieren eliminar a través de la membrana. Normalmente, existe una competencia por el paso a través de las membranas entre diferentes especies debido al carácter multicomponente de los efluentes a tratar. Sin embargo, una mejora en las propiedades de las membranas de intercambio iónico permitiría la implantación del tratamiento mediante reactores electroquímicos de efluentes industriales con un contenido importante en compuestos metálicos, tales como los baños agotados de las industrias de cromado. La utilización de una tecnología limpia como la electrodiálisis conllevaría diferentes ventajas, entre las cuales destacan la recuperación de los efluentes para su reutilización en el proceso industrial, el ahorro en el consumo de agua y la disminución de la descarga de contaminantes al medio ambiente. La determinación de las condiciones de operación óptimas así como la mejora de las propiedades de transporte de las membranas constituye el principal tema de la presente investigación. Para ello, se emplearán diferentes tipos de membrana. En primer lugar, se estudiará el comportamiento de las membranas poliméricas comerciales que poseen unas propiedades de resistencia química elevadas, las cuales se tomarán como referencia. De forma paralela, se producirán membranas conductoras de iones a partir de materiales cerámicos económicos, ya que la resistencia de los materiales cerámicos a sustancias oxidantes y muy ácidas es mayor que la de los materiales poliméricos. Este punto constituye la parte más innovadora de la investigación, puesto que la mayoría de las membranas de intercambio iónico comerciales están basadas en materiales poliméricos que no pueden resistir las condiciones específicas de los efluentes industriales. Una vez determinadas las condiciones de operación óptimas, se realizarán ensayos en plantas piloto con el fin de confirmar los resultados obtenidos mediante las técnicas de caracterización y determinar el grado de recuperación y coste energético asociado a los procesos electrodialíticos de tratamiento de efluentes industriales.
Martí Calatayud, MC. (2014). STUDY OF THE TRANSPORT OF HEAVY METAL IONS THROUGH CATION-EXCHANGE MEMBRANES APPLIED TO THE TREATMENT OF INDUSTRIAL EFFLUENTS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/46004
TESIS
Premiado

M. Tech. (Department of Biotechnology, Faculty of Engineering and Technology) Vaal University of Technology
The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.

碩士
國立中興大學
生命科學院碩士在職專班
97
Heavy metal pollution has posed one of the serious threat to human health, therefore, seeks an economy and effective method to clear the heavy metal pollution is the urgent matter.Bioremediation is the best method can clear heavy metal pollution completely and will not cause the second pollution. Because the bacterial heavy metal binding proteins can absorb the heavy metal ions, therefore we can use the biological characteristics to eliminate the heavy metal pollution in the soil or water. Take the SilE protein as the example, the SilE protein has played the key role in the resistant mechanism of silver resistant bacterium, therefore, the Ag+ adsorptive capacity of SilE protein will be closely linked to its silver resistance. Six strains were included in this project. E.coli strain J53 contains plasmid pMG101 which have silver resistance. There are nine open reading frames of the silver resistance determinant in the plasmid, including SilE protein. Two recombinant plasmids were constructed by cloning silE gene with or without signal sequence into pET21b vector, resulting SilE-SP and SilE-NSP respectively. For MerP protein, another two constructs were made by cloning MerP with signal sequence or B95P without signal sequence into pET21b vector. All recombinant plasmids were transformed to E.coli BL21 (DE3) strain. One control is with backbone pET21b in E.coli BL21 (DE3). Resistance tests in agar plate and in liquid medium were performed to detect the strength of resistance to Ag+. In disc assay, the strength was pMG101＞SilE-SP＞MerP＞pET21b＞SilE-NSP＞B95P and pMG101＞MerP＞SilE-SP＞pET21b＞SilE-NSP＞B95P for resistance test in liquid medium. Because both SilE and MerP protein can absorb heavy metal ions, adsobability experiment was also done to detect the Ag+ residue in the buffer. The results were 1.89 ± 0.026 mg/l for pMG101，1.92 ± 0.031 mg/l for MerP，1.93 ± 0.006 mg/l for pET21b，1.94 ± 0.021 mg/l for SilE-SP，1.94 ± 0.020 mg/l for SilE-NSP，and 2.10 ± 0.026 mg/l for B95P. In brief, the adsorbability to adsorb Ag+ is pMG101＞MerP＞pET21b＞SilE-SP≒SilE-NSP＞B95P. The results above showed that MerP protein can absorb Ag＋ and the efficiency is only less than pMG101（J53）, therefore, the next experiment was to observe the growth and detect the amount of the Ag＋ can be absorbed by the transgenic plant containing merP gene （P5 transgenic plant）. The host of bacterial merP gene is Arabidopsis. Furthermore, because MerP protein can absorb Hg+2, Ag+ and other heavy metals, the character of adsorption to heavy metal ions needs to be further investigated for the P5 transgenic plant. We found the differences for silver adsorption of P5 transgenic plant embedding in MS medium containing between Ag+ only and both Hg+2 and Ag+.The results indicated that the plant can absorb more Ag+ in the condition containing both Hg+2 and Ag+.

Anthropogenic activities generate waste products that pollute the environment with bacteria and heavy metals. This research assessed pollution of the Kahwa River, Bukavu Town, DRC with cadmium and lead (HMs) and bacterial enteropathogens. A survey of businesses, households and healthcare facilities showed general use of the river to remove effluent and waste. Indicator organisms were cultured at over 200 cfu/100 ml showing faecal contamination of the river water. Antibiotic resistance was shown by enteropathogenic Vibrio cholerae and Salmonella typhi to ampicillin and cotrimoxazole with some sensitivity shown to ciprofloxacin. River water contained HMs at around 40 times the World Health Organisation limit for drinking water. The bacteria, particularly from river sediment, tolerated HMs up to a concentration of 1.5 mg/ml. The presence in the Kahwa River of antibiotic-resistant pathogens showing tolerance to HMs has serious public health implications
Environmental Management
M.Sc. (Environmental management)

Industrial effluents with high concentrations of heavy metals are widespread pollutants of great concerns as they are known to be persistent and non-degradable. Continuous monitoring and treatment of the effluents become pertinent because of their impacts on wastewater treatment plants. The aim of this study is to determine the correlation between heavy metal pollution in water and the location of industries in order to ascertain the effectiveness of the municipal waste water treatment plant. Heavy metal identification and physico-chemical analysis were done using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and multi-parameter probe respectively. Correlation coefficients of the measured values were done to investigate the effect of the industrial effluents on the treatment plants. Heavy metal resistant bacteria were identified and characterised by polymerase chain reaction and sequencing. Leeuwkuil wastewater treatment plants were effective in maintaining temperature, pH, and chemical oxygen demand within South Africa green drop and SAGG Standards whereas the purification plant was effective in maintaining the values of Cu, Zn, Al, temperature, BOD, COD, and TDS within the SANS and WHO standard for potable water. This findings indicated the need for the treatment plants to be reviewed.The industrial wastewater were identified as a point source of heavy metal pollution that influenced Leeuwkuil wastewater treatment plants and the purification plants in Vaal, Vereenining South Africa. Pseudomonas aeruginosa, Serratia marcescens, Bacillus sp. strain and Bacillus toyonensis that showed 100% similarity were found to be resistant to Al, Cu, Pb and Zn. These identified bacteria can be considered for further study in bioremediation.
Environmental Sciences
M. Sc. (Environmental Science)

Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters.
Thesis (Ph.D.)-University of Natal, Pietermartizburg, 1995.

博士
國立中興大學
生命科學系所
98
Mercury and its derivates is known toxic to human beings as well as neurotoxicity to damage brain and led to neurondegeneration disease. In aquatic system, mercury is easily transferred to methylmercury by bacteria in the sediment. As some poisoned case in the past, such as Minamata in Japan in 1950s and Iraq in 1970, it is identified that methylmercury were taken in through food chain. Metallothioneins (MTs) are low-molecular-weight, cysteine-rich intracellular proteins with high affinity to toxic metal cations for detoxification, such as Cd2+, Hg2+ and MeHg+ and also possess the ability of scavenging reactive oxygen species. The tilapia, human and mouse MTs were expressed either in cytosol or fusion to outer membrane protein (OmpC) in the recombinant E. coli. Exposure to mercury or free radicals shows that outer-membrane-target ones had 10 to 20 % higher sequestering ability than cytosolically ones. In scavenging 2,2-azinobis(3-ethylbenzothiazoline-6 -sulfonic acid) diammonium salt free radicals, the outer membrane MTs had 10% higher scavenging ability than cytosolic MTs. Among three MTs, tilapia MT presents best activity of adsorbing mercury and scavenging radicals. Moreover, the caboxy-H2DCFDA was used to detect reactive oxygen species. It is observed that the oxidative stress decrease was dependent on cadmium binding ability as higher metal bonding ability led to lower oxidative stress induced by cadmium. Methylmercury is frequently found in aquatic system and could be amplified through food chain. On the other hand, the organomercurial lyase merB3 gene of Bacillus megaterium MB1 is known to have board spectrum organomercurials resistance enzymes. The purified MerB3 was characterized in vitro with the presence of phenylmercuric acetate (PMA) and methylmercury chloride (MMC) and present similar catalytic activity to MerB1 whereas, MerB2 did not possess the ability to degrade the two organomercurials. As introducing merB3 gene to zebrafish, the LC50 of wild type and transgenic zebrafish in MMC were 376nM and 404nM, respectively, while in PMA were 200nM and 237nM. The transgenesis show better resistance ability than wild type. However, the phenotype of teratogenic effects on tail was no obvious difference for both zebrafish.

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2011
Heavy metal resistant microorganisms are often associated with acidic environments, since metals are easily solubilized in acidic milieus. A unique yeast species from the Cryptococcus genus was isolated from two sulfur-rich acidic environments: acid mine drainage in the south of Portugal and a volcanic river in Argentina. The uniqueness of this species lies on the fact that it is the first acidophilic basidiomycetous yeast known to date. Additionally, the two strains chosen for this work (one from each environment) are resistant to high levels of heavy metals (arsenic, cadmium, copper and zinc). Metal resistance mechanisms are only described for neutrophilic yeasts, and mainly involve thiolated peptides and efflux transporters. To unveil the mechanisms that allow this Cryptococcus species to resist high levels of heavy metals, physiological, cytological and molecular approaches were conducted. Since both isolation sites are sulfur-rich, the possibility that this element influences metal resistance was assessed with microscale growth assays by determining minimal inhibitory concentrations of the four metals in differential sulfate availability conditions. Assessment of thiolmediated resistance mechanisms was achieved by incubating cells (grown with and without metal) with the thiol-specific fluorescent probe 5-chloromethylfluorescein-diacetate. Dot blot hybridization was applied to detect gene homologues involved in arsenic extrusion, and vacuolar metal-thiol accumulation in response to arsenic and cadmium. Also, suppression subtractive hybridization was conducted to investigate resistance to cadmium by analyzing transcripts induced upon exposure to this metal. Our results indicate that thiolated peptides are involved in resistance to arsenic and zinc in the Portuguese strain, cadmium in the Argentinean strain, and copper in both strains. Also, both strains presented evidence of an arsenic extrusion mechanism, and of a Cd-induced demand in protein synthesis and folding. Thus, the present work allowed unveiling of heavy metal resistance mechanisms in two strains of this unique novel yeast species, Cryptococcus sp.
Existem diferentes critérios para definir ‘metal pesado’ que consideram diferentes características dos elementos metálicos. No entanto, o critério mais aceite consiste na comparação da gravidade específica do metal em relação à da água: metal pesado é aquele cuja gravidade específica é pelo menos cinco vezes superior à da água. Esta qualidade é atribuída a elementos essenciais como o cobre e o zinco, e a elementos não essenciais como o arsénio e o cádmio (Lide, 2009). Metais pesados, essenciais e não essenciais, podem ser tóxicos para todos os organismos, pelo que ao longo da evolução foram desenvolvidas diversas estratégias de destoxificação em resposta à presença destes elementos em excesso. Em microorganismos, este tipo de estratégias encontra-se principalmente caracterizado em organismos modelo com aplicações clínicas ou na indústria, como Escherichia coli, Candida albicans, Saccharomyces cerevisiae e Schizosaccharomyces pombe. No entanto, os níveis de resistência a metais pesados que estes microorganismos exibem é baixo em comparação com os níveis observados em microorganismos isolados de locais extremos, como por exemplo ambientes aquáticos ácidos ricos em metais pesados (Dopson et al., 2003). Em leveduras, as principais estratégias descritas em leveduras consistem em mecanismos que levam à destoxificação do citoplasma (Perego & Howell, 1997; Tsai et al., 2009). Estes podem ser exercidos por transporte dos metais para o exterior da célula, mediado por transportadores específicos, ou por acumulação dos metais, normalmente complexados com péptidos tiolados, em organitos como o vacúolo (revisto em Tamás & Wysocki, 2010). Microorganismos resistentes a metais pesados estão frequentemente associados a ambientes acídicos, visto que a solubilização destes metais é facilitada em meios com pH baixo. Um exemplo deste tipo de ambiente existe na Faixa Piritosa Ibérica cuja extensão inclui o Rio Tinto, em Espanha, e as minas de São Domigos, no sul de Portugal. Dada a exploração de minérios associada a estes locais, os depósitos estáveis de minerais associados a diferentes metais foram expostos ao ar e água, o que conduziu à lenta reacção espontânea de oxidação desses mesmos minerais (Johnson & Hallberg, 2003). Comunidades de microorganismos litotróficos, presentes nas águas associadas a estes locais, aceleraram o processo de oxidação de minerais, visto que permitiram a regeneração de compostos necessários à reacção de oxidação. Um destes compostos, o ião férrico, em conjunto com o pH baixo permitiu uma manutenção da oxidação e lixiviação de metais que, dada a sua solubilidade mais facilitada em águas ácidas, ficam mais concentrados (López-Archilla et al., 2001; Johnson & Hallberg, 2003). Este tipo de contaminação da água por oxidação de minerais expostos é denominado drenagem ácida de minas. A geologia local das minas de São Domingos é dominada por depósitos sulfúricos polimetálicos e inclui um lago particularmente extremo na Achada do Gamo, com o pH mais baixo de todos os lagos envolventes (pH 1.8) e com concentrações elevadas de enxofre e vários metais (Gadanho et al., 2006). Por outro lado, ambientes aquáticos extremos com pH baixo e elevada concentração de metais podem ter uma origem relacionada com actividade vulcânica, como é o caso do Rio Agrio, localizado na Patagónia Argentina (Pedrozo et al., 2001). Neste caso, a acidez da água do rio é uma consequência directa da produção de ácido sulfúrico no interior do vulcão, devido à actividade geotérmica e consequente emissão de gases do vulcão. Devido a interacções químicas, a acidez das águas leva a um desgaste da geologia vulcânica local e resulta em elevadas concentrações de enxofre e metais na água (Pedrozo et al., 2001; Russo et al., 2008). Na parte superior deste rio (mais próxima do vulcão), o pH é de 2.2 e foram observadas concentrações elevadas de enxofre e metais pesados como ferro, zinco e arsénio (Russo et al., 2008). Uma nova espécie pertencente ao género Cryptococcus foi isolada em dois locais extremos distintos: Achada do Gamo, nas minas abandonadas de São Domingos (Gadanho et al., 2006); e Rio Agrio, na Patagónia Argentina (Russo et al., 2008). Para realizar o presente trabalho foram escolhidas duas estirpes desta espécie – uma de cada local. As estirpes desta espécie requerem pH baixo para crescer eficientemente, o que torna Cryptococcus sp. a primeira levedura basidiomicete acidófila conhecida até à data (Russo et al., 2008). Adicionalmente, apresentam níveis de resistência a metais pesados mais elevadas que as toleradas por microorganismos modelo (Gadanho et al., 2006). No presente estudo foram pesquisados mecanismos pelos quais estas duas estirpes conseguem resistir a níveis elevados dos metais pesados arsénio, cádmio, cobre e zinco, tendo em consideração um eventual papel do enxofre nos mecanismos de resistência aos metais. Para tal, foram usadas abordagens experimentais a nível fisiológico, citológico e molecular. As metodologias aplicadas incluíram ensaios de crescimento em microescala para determinar concentrações mínimas inibitórias para cada metal em diferentes condições de disponibilidade de sulfato. Por análise das diferenças nos níveis de resistência a cada metal pesado em condições diferenciais de sulfato, foi possível compreender a influência que a disponibilidade de sulfato exerce sobre os níveis de resistência a cada metal pesado. Foi também avaliado o eventual papel da forma química do composto metálico adicionado ao meio de cultura (arsenato vs. arsenito no caso do arsénio, e sulfato de metal vs. cloreto de metal para os outros metais). A destoxificação de metais pesados por acumulação de péptidos tiolados está largamente descrita na literatura como sendo uma estratégia generalizada de resistência a metais pesados em leveduras. Por esta razão, a possibilidade da existência desta estratégia na espécie acidófila foi averiguada. Para tal, técnicas citológicas para observação em microscopia de fluorescência foram optimizadas e aplicadas nas duas estirpes, para os quatro metais pesados em estudo. As duas estirpes foram crescidas em meio com e sem metais pesados, e foram incubadas com uma sonda fluorescente específica para grupos tiolados, a diacetato-5-clorometilfluoresceína (CMFDA). A observação destas células em microscopia de fluorescência permitiu avaliar variações na intensidade de fluorescência provocadas pela presença dos metais. A observação de diferenças nas intensidades de fluorescência em condições diferenciais de crescimento (com metal vs. sem metal) foi interpretada como indicação de participação de péptidos tiolados nos mecanismos de destoxificação. Foram também optimizadas e aplicadas metodologias de biologia molecular para averiguar a presença de homólogos de genes envolvidos no efluxo de arsénio, e de homólogos de um gene envolvido na acumulação de arsénio e cádmio no vacúolo da célula. A metodologia aplicada para este fim foi hibridação de DNA por ‘dot blot’ que consiste na utilização de uma sonda, que corresponde ao gene que se pretende detectar, com o DNA genómico das estirpes em análise. Os genes pesquisados incluíram dois homólogos de genes envolvidos no transporte de arsénio para o exterior da célula (arsA e arsB), e um homólogo de um gene envolvido na acumulação vacuolar de arsénio e cádmio (ycf1). As sondas para esta hibridação foram obtidas por PCR a partir do DNA genómico da espécie-tipo do género Cryptococcus, C. neoformans. Para tal, foram desenhados primers ‘forward’ e ‘reverse’ para cada um dos três genes, e foram conduzidas reacções de PCR com um nucleótido marcado com digoxigenina (DIG-dUTPs). Os produtos de PCR obtidos foram então usados como sondas na procura de homologia dos genes indicados nas duas estirpes em estudo. Neste trabalho foi também incluída uma abordagem transcriptómica para análise e identificação de genes diferencialmente expressos em células crescidas na presença e na ausência de cádmio. Para tal, foi extraído RNA de células obtidas em condições diferenciais (com e sem cádmio) em fase exponencial de crescimento e o mRNA foi purificado a partir do RNA total. Posteriormente, foi realizado um processo de hibridação subtractiva supressiva nas duas estirpes, no qual se obteve uma biblioteca de cDNA correspondente a transcritos cuja expressão foi aumentada na presença de cádmio. Após sequenciação, estes transcritos foram identificados comparação com bases de dados de sequências presentes no ‘National Center for Biotechnology Information’ (NCBI). As metodologias aplicadas e consequente análise de resultados permitiram inferir sobre alguns aspectos das estratégias que as duas estirpes de Cryptococcus sp. analisadas utilizam na destoxificação de metais pesados. Nomeadamente, na estirpe Portuguesa predomina a evidência de que se trata de um mecanismo de resistência principalmente mediado por tióis, com possível subsequente acumulação no vacúolo da célula. Por outro lado, no caso da resistência ao arsénio na estirpe Argentina, o principal mecanismo de destoxificação parece ser mediado por efluxo do metal, possivelmente por um homólogo da bomba de efluxo ArsB, associada a uma ATPase homóloga de ArsA. Os resultados obtidos na resistência ao cádmio também indicam variabilidade intra-específica, uma vez que há indicação de um mecanismo de resistência mediado por tióis para a estirpe Argentina. No entanto, com base nos dados obtidos não foi possível sugerir um mecanismo específico para a estirpe Portuguesa. No caso da resistência ao cobre, os resultados sugerem a existência de um mecanismo mediado por tióis em ambas as estirpes, com a possibilidade de um mecanismo adicional que pode operar predominantemente na estirpe Portuguesa. Finalmente, a análise dos resultados obtidos sob exposição das leveduras a elevadas concentrações de zinco sugerem a possibilidade de acumulação deste metal em compartimentos celulares como o vacúolo. Os resultados obtidos neste trabalho demonstram variabilidade intra-específica nos mecanismos de destoxificação de metais pesados em Cryptococcus sp., visto que nem sempre a mesma estratégia de resistência a metais pesados foi observada nas duas estirpes em estudo. Foi também verificada a possibilidade de existência de mais de um mecanismo de resistência a operar em simultâneo para apenas um metal, o que pode justificar a elevada resistência a metais pesados observada nas duas estirpes estudadas. Os mecanismos de destoxificação nesta levedura única deverão, então, ser alvo de análise em maior detalhe em investigação futura.

碩士
元智大學
生物科技與工程研究所
98
Heavy metals are common contaminants found in polluted area. We have identified a gene (heavy metal translocating P-type ATPase; hmtp) that might be involved in heavy metal resistant traits of Enterobacter sp. CD01(a heavy metal resistant strain isolated from heavy metal contaminated soil) in a previous study via fosmid library and in vitro transposon mutagenesis. The complete gene was then identified, cloned and expressed in a suitable Escherichia coli using pGEM-T as the vector. E. coli W3110, RW3110 (zntA::Km), GG48 (DzitB::Cm zntA::Km) and GG51 (DzitB::Cm] were used to study the possible effects of this gene to heavy metal resistance, cadmium and zinc in particular. Among these E. coli strains, RW3110 and GG48 showed more sensitive to cadmium and zinc compared to the wildtype E. coli W3110 and strain GG5, therefore were chosen for the reference hosts for further evaluation of the gene’s effect. The results showed that this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms. In addition to study on the genes involved in heavy metal resistance, we also examined the cadmium adsorption by Enterobacter sp. CD01 and another isolate (Stenotrophomonas sp. CD02). Moreover, the exopolysaccharide production and Transmission electron microscope of Stenotrophomonas sp. CD02 grown in medium containing cadmium were also examined in this study.

碩士
國立中興大學
生命科學系所
94
Cadmium(Cd) and lead(Pb) are widely used in industrialized countries, and both are non-essential heavy metals for the organisms. When they are accumulated, they will become extremely toxic to living organisms. For instance, the Minimata disease caused by mercury contamination, and the Itai-itai disease caused by cadmium contamination had made serious injury to the biological system for its bioaccumulation in the food chain. These heavy metals could be a threat at the low levels. Therefore, mechanisms responsible for minimizing the concentraction of non-essential heavy metals is required for all organisms. This study was initiated to clone and characterise of cadA gene originated from cadCA operon in a gram-positive bacteial plasmid, Staphylococcus aureus plasmid pI258, into Arabidopsis thaliana.The expression of cadA gene in A. thaliana may increase its resistance to cadmium, lead and zinc(Zn) and decrease the heavy metal content in the transgenic plants. The isolated transgenic lines were conferred both in DNA and RNA levels. Analysis of transgenic A. thaliana plants expressing cadA showed that CadA is functionally active and that the plants have enhanced resistance of Cd(II), Pb(II) and Zn(II), while accumulated a greater amounts of Cd(II) or Pb(II). These results suggest that transgenic plants expressing cadA may be an useful tool for phytoremediation.

碩士
長榮大學
職業安全與衛生學系碩士在職專班
102
Recent researches have indicated that the antibiotic-resistant gene and pollutant-resistant gene are co-exist on plasmid and subjected to conjugative transfer from cell to cell. Under the selective pressure of anthropogenic pollutants, antibiotic-resistant pathogens might present in a higher population than in a related pristine environment. Considering the situations of dense human population and pig farms with unsatisfied wastewater treatment practice, research on how pollution affects the distribution of antibiotic resistant pathogens is deemed necessary. To explicit the relationship between the occurrence of resisting strains and the anthropogenic stress, this research conducted investigation on metal and antibiotic susceptibility of Vibrio spp. in the estuary of Erhjen River, characterized by its long history of metal pollution by the past scrap metal factories and recent industrial activities, in southern Taiwan. Vibrio spp. isolated from Chiku Lagoon in Tainan, a related pristine water body in Southern Taiwan, as well as strains from Beibin in Hwalian, seashore located at Eastern Taiwan, were compared to extend the spatial variability. The study assessed three metals (Cu, Zn, and Ni) and three antibiotics (gentamicin, ciprofloxacin, and tetracycline). The study suggested, however, metal susceptibility of Vibrio spp. isolated from Erhjen River was unrelated to environmental stress with non-significant correlation coefficients (P>0.05) between minimum inhibitory concentrations (MIC) for Cu, Zn, Ni and sediment concentrations for the same metals. There was also no relationship found between metal resistance and antibiotic resistance for the Vibrio spp. isolated from the three sampling area. Correlation coefficients between MIC for heavy metals and MIC for antibiotics were non-significance (P>0.05). Comparing with Chiku and Beibin strains, Vibrio spp. isolated in Erhjen had significantly higher MICs for gentamicin and ciprofloxacin (Kruskal-Wallis ANOVA, P=0.008 and 0.007, respectively). All isolated Vibrio were resistance to tetracycline, suggesting ineffective curability of the antibiotic. The preliminary study explored the ecology of Vibrio in a highly polluted environment. Attempts were made to understand the survival strategy of Vibrio in sediment with potential of heavy metal and antibiotic contaminations. The findings are expected not only benefit to the scientific communities but also to the policy makers who may use the information to protect the seafood consumption safety as well as to regulate the uses of antibiotics and heavy metals in the watershed.

碩士
元智大學
生物科技與工程研究所
98
The aim of this study was isolation and characterization of heavy metal resistant microorganisms from sediments of Er-Jen River in south Taiwan, a river that is well known to be polluted by various contaminants. We have isolated several microorganisms that are highly resistant to heavy metals including cadmium by enrichment culture. One of the isolates was identified as a Pseudomonas sp. according to its 16S rRNA gene sequence analysis and was designed as strain EJ01. Pseudomonas sp. EJ01 was used for further characterization in this study. The minimum inhibitory concentrations of cadmium for strains EJ01 was 7 mM when cells grown in LB medium containing cadmium. At stationary phase, cells of Strain EJ01 aggregated and formed biofilm when concentrations of Cd was high (2 mM and above). This phenomenon might be related to the bacterium’s ability to produce extracellular polysaccharides (EPS). Therefore, EPS produced by strain EJ01 was extracted from stationary phase grown cells to investigate its relationship to the bacterium’s resistance to Cd and the removal of cadmium. Cadmium concentration was reduced 75% in the culture medium when Pseudomonas sp. EJ01 grown in the medium containing 2 mM. The extracted EPS also showed significant ability for adsorption of cadmium in the solution. Transposon mutagenesis was also used to study genes involved in biofilm formation in strain EJ01 and their possible roles in heavy metal resistance. A mutant (EJ01m-3055) was selected as reduced biofilm formation mutant. The mutant also showed decreased ability for cadmium resistance and suggested that biofilm formation and cadmium resistance in Pseudomonas sp. EJ01 is realted.

碩士
國立中興大學
生命科學系所
94
Cadmium(Cd) is a non-essential metal for the organisms, widely used in industrialized countries. The Itai-itai disease caused by cadmium contamination has made a serious impact on human health for its bioaccumulation through the food chain. Cadmium represents a heavy metal pollutant in Taiwan. Under the Cd2+ exposure, fresh water fish experiences growth inhibition and even death. Homeostatic mechanisms are required for all organisms to minimize the toxicity of cadmium. The motivation of this study is to clone and to transfer the cadA gene originated from bacterial plasmid, Staphylococcus aureus plasmid pI258, into Danio rerio. Experiments were conducted to examine whether it would confer resistance to cadmium, lead(Pb), zinc(Zn) as well as decrease the heavy metal content of the transgenic zebrafish. The stable transgenic germ-lines were confirmed both in DNA, and RNA level. Furthermore, fish embryos during developing or larvae in general showed the highest susceptibility in the fish life cycle. Posthatch larvae were continuously exposed to 5 μM Cd, 76.5 μM Zn, or 12.0 μM Pb for 96 hours beginning at the 72nd hour after fertilization, and resulting in a larval survival of near 50％.On the other hand, transient zebrafish expressing cadA showed that CadA was functionally active and that the zebrafish had enhanced resistance to Cd(II), and Zn(II). These results suggest that transgenic zebrafish expressing cadA can enhance metal resistance probably due to the ability to reduce the heavy metal content in fish.

碩士
國立中山大學
生物科學系研究所
91
In the present work, in order to evaluate the seriousness of environmental pollution caused by antibiotics abuse, and by industrial and agricultural pollutants, different strains of Pseudomonas aeruginosa were collected from industrial area, abandoned metal hardware factory, fishery pool, vegetable garden and fruit farm, river mud, and different origins of water bodies in southern Taiwan. The organisms were analyzed for their drug resistance against a variety of antibiotics and agricultural pesticides. They were also analyzed for endurance toward heavy metal ions including mercury, cadmium, arsenic and chromium ions. As the data indicated that, in terms of their resistance to clinical frequently used antibiotics, about 40% of the Pseudomonas aeruginosa isolates of the environment have developed resistance against Cefoperazone, about 20% showed resistance against Tobramycin, and only about 4% revealed resistance against Imipenem and Ceftazidime. As to heavy metal ion resistance, about 27% of the environmental Pseudomonas aeruginosa strains demonstrated resistance against mercury ion, and about 10% exhibited resistance against arsenic ions, whereas no resistance was observed toward chromium ions. In terms of resistance to agricultural pesticides, about 36% environmental isolates demonstrated resistance against Paraquat, but none of the tested Pseudomonas aeruginosa show resistance toward Cartap and Methomyl. To sum up the findings, so far only minor portion of Pseudomonas aeruginosa acquired drug resistance, therefore, immediate measure is required to prevent the spreading of drug-resistant Pseudomonas aeruginosa . It is suggested that all the physicians and pharmacists to prescribe antibiotics should be more careful and responsible manner. Meanwhile, it would also call on the restrained usage of pesticides and antibiotics in the livestock and aquatic product industry, and strengthening pollution control in the industrial sector.

碩士
元智大學
生物科技與工程研究所
99
In this study, microorganisms isolated from contaminated soil (Pseudomonas aeruginosa strain T1 and Ochrobactrum sp. strain T2), heavy metal (Cd) resistant microorganism Enterbacter sp. strain CD01 and BTEX degradative microorganism Enterbacter sp. strain NKNU02 (E02) were investigated for their ability of toluene degradation and tolerance against benzene, toluene or xylenes (BTX) and cadmium. The results indicated that when LB medium containing 500 ppm of benzene, toluene or xylenes, and 1 mM Cd, all strains showed to have a prolonged lag growth phase before cell growth. In a minimal salta (MSB) medium containing 500 ppm toluene, the growth of Ochrobactrum sp. strain T2 could reach 0.3 as measured by optical density at 600 nm (OD600). In MSB medium containing 500 ppm benzene, toluene or xylenes, Pseudomonas aeruginosa strain T1 could grow to OD600 0.1. As for the degradation of toluene, the degradation efficiency is 8.06 ppm per hour for strain T1 when incubated in the medium containing 500 ppm of toluene. On the other hand, Enterbacter sp. strain NKNU02 ( E02 ) was better than other strains in terms of cadmium resistance, which could tolerate up to 500 ppm of BTX in addition of 1mM of cadmium in LB medium. However, the degradation efficiency of strain E02 was still lower than that of strain T1.