Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates. / Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

Deux légumineuses (Anthyllis vulneraria et Lotus corniculatus) adaptées aux métaux lourds constituent un matériel d’intérêt pour la phytostabilisation de sites miniers. Leur fonction de fixatrices biologiques d’azote grâce à leur symbiose avec des bactéries symbiotiques permet l’établissement efficace d’une couverture végétale durable limitant la dispersion des métaux dans l’environnement. Nos objectifs ont été d’étudier les effets des métaux lourds et de la plante hôte sur les populations symbiotiques naturellement associées à ces légumineuses en analysant (i) les populations symbiotiques associées à A. vulneraria sur 8 sites contaminés ou non (ii) les populations de rhizobia associées à L. corniculatus qui ont été comparées à celles d’Anthyllis. La distribution des souches de Mesorhizobium isolées de nodosités d’A. vulneraria et provenant de plusieurs sites contaminés ou non dépend des fortes teneurs en métaux lourds des sols qui sélectionnent fortement les souches symbiotiques résistantes et influencent leur composition taxonomique. Les souches appartenant à l’espèce M. metallidurans ont été retrouvées seulement dans les sites fortement contaminés. Deux nouvelles espèces potentielles et résistantes aux métaux semblent exister chacune sur un site minier distinct. L’une d’elle est proche de M. ciceri et de M. loti et tous ses membres présentent la particularité de ne pas posséder de gène cadA, un gène impliqué dans la tolérance aux métaux chez M. metallidurans. Par contre, les sites non contaminés révèlent une diversité taxonomique différente avec la présence de nouvelles espèces de Mesorhizobium sensibles aux métaux lourds. Quatre de ces nouvelles espèces ont été définies. A. vulneraria et L. corniculatus partagent la même diversité taxonomique dans les sites contaminés testés. Par contre, les propriétés symbiotiques des souches varient selon la plante hôte utilisée pour le piégeage. Les souches appartiennent soit au symbiovar (sv.) anthyllidis soit au sv. loti selon le site géographique d’origine et ceci indépendamment des teneurs en métaux lourds dans le sol. A. vulneraria s’associe avec les souches possédant les sv. anthyllidis ou sv. loti. En revanche, L. corniculatus ne s’associe qu’avec des souches du sv. loti. Dans tous les sols qu’ils soient contaminés ou non, A. vulneraria nodule préférentiellement avec le sv. anthyllidis. En conclusion, A. vulneraria et L. corniculatus établissent des symbioses avec les mêmes espèces de Mesorhizobium et s’associent préférentiellement avec un sv. Les taxons retrouvés dépendent fortement des sites d’isolement, ce qui pourrait traduire des adaptations particulières aux conditions environnementales. L’utilisation des ressources biologiques locales est une stratégie que nous recommandons pour la végétalisation d’anciens sites miniers. / Two legumes (Anthyllis vulneraria and Lotus corniculatus) adapted to heavy metals form an interesting material for phytostabilisation strategy in mining sites. As biological nitrogen fixators, these legumes associated with compatible symbiotic bacteria provide an efficient establishment of a sustainable cover vegetation limiting metal dispersion in the environment. Our objectives were to study the effects of heavy metals and the host plant on symbiotic populations naturally associated with these legumes by analyzing (i) symbiotic populations associated with A. vulneraria on 8 contaminated and uncontaminated sites (ii) rhizobial populations associated with L. corniculatus that were compared with those of Anthyllis. The distribution of mesorhizobial strains isolated from A. vulneraria root-nodules from several contaminated and uncontaminated sites depends on high levels of heavy metals in soils by selecting highly resistant strains and impacting the taxonomic composition. Strains belonging to M. metallidurans were only found in highly contaminated sites. Two new potential metal-tolerant species were detected in two distinct mines. One of them was closely related to M. ciceri and M. loti and its members had the feature of not -possessing the cadA gene, a gene involved in metal-tolerance among M. metallidurans strains. By contrast, uncontaminated sites revealed a different taxonomic diversity with new species sensitive to heavy metals. Four of these new species were defined. A. vulneraria and L. corniculatus share the same taxonomic diversity in the contaminated sites tested. By contrast, symbiotic properties of the strains vary depending on the host plant used for trapping. Strains belong either to symbiovar (sv.) anthyllidis or to sv. loti according to geographic origins and independently of heavy metal levels in soils. A. vulneraria associated with strains of sv. anthyllidis or sv. loti. In contrast, L. corniculatus only associated with strains of sv. loti. In contaminated or uncontaminated soils, A. vulneraria was preferentially nodulated by sv. anthyllidis. In conclusion, A. vulneraria and L. corniculatus established symbiotic relationships with the same taxonomic groups of Mesorhizobium but associated with different symbiovars. The finding of taxonomic groups strongly depends on geographical sites, suggesting special adaptations to environmental conditions. Use of local biological resources is the strategy we recommend for revegetation of old mines.

Une nouvelle espèce de micro-algue photosynthétique, Coccomyxa actinabiotis, a récemment été découverte au sein d'une installation nucléaire. Cette algue présente une forte résistance aux radiations ionisantes et accumule certains radionucléides. Le développement d'une biotechnologie de décontamination des effluents liquides des réacteurs nucléaires basée sur C. actinabiotis est étudié du fait de ces propriétés. Ce travail vise plus particulièrement à caractériser et comprendre les processus d'accumulation par l'algue de l'argent et du cobalt, qui sont les radionucléides émetteurs gamma majoritaires dans les effluents liquides des réacteurs du parc électronucléaire français. Cette étude a été menée par quatre approches complémentaires : (a) Les cinétiques et les équilibres d'accumulation de ces métaux ont été déterminés par analyses ICP-MS (b) La distribution intracellulaire des métaux a été cartographiée à l'échelle nanométrique par fluorescence X de rayonnement synchrotron et par microscopie électronique en transmission (c) La spéciation de l'argent et du cobalt accumulés par la micro-algue a été déterminée par spectroscopie d'absorption X et par diffraction de rayons X (d) L'impact biologique de ces métaux sur l'algue a été examiné, notamment par l'étude des perturbations physiologiques, métaboliques et protéomiques associées à leur présence.L'ensemble de ces résultats dévoile une partie des processus à l'œuvre lors de l'accumulation d'argent ou de cobalt par C. actinabiotis. / A new green micro-alga species, Coccomyxa actinabiotis, was recently discovered in a nuclear environment. This alga is highly resistant to ionizing radiation and accumulates some radionuclides. Thanks to its properties, the development of a biotechnology based on C. actinabiotis for the clean-up of nuclear liquid effluents is under consideration. Our work aims more specifically at describing and understanding the alga's silver and cobalt accumulation processes; these metals being the main gamma emitting radionuclides present in liquid effluents issuing from French nuclear facilities.This study was carried out using four complementary approaches: (a) Kinetics and equilibriums of silver and cobalt uptake were assessed by ICP-MS analysis; (b) The subcellular distribution of the metals was mapped at a nanometric scale using synchrotron X-ray fluorescence and transmission electron microscopy; (c) The speciation of silver and cobalt taken up by the alga was assessed by synchrotron X-ray absorption spectroscopy and X-ray diffraction; (d) The biological impact of these metals on the alga was investigated, in particular the physiological, metabolic and proteomic perturbations they induce.Altogether, these results unveiled some of the processes involved in silver and cobalt accumulation by C. actinabiotis.

O Rio Tietê e o rio mais importante do Estado de São Paulo e um dois mais poluídos do Brasil. No seu percurso ele recebe altas concentrações de poluentes orgânicos e industriais que podem alterar as comunidades microbianas autóctones. Uma análise dos perfis metagenômicos realizada em pontos do Rio Tietê com diferentes qualidades de água, permitiu observar a influencia dos parâmetros ambientais na composição e funcionamento das comunidades microbianas presentes no rio. Locais eutróficos mostraram prevalência de micro-organismos e funções associadas à heterotrofia. Em contrapartida, nos locais oligotróficos foram detectadas comunidades microbianas e funções relacionadas a metabolismos autótrofos e litótrofos. Foi visto também que as pressões ambientais podem promover a aquisição, por parte dos micro-organimos, de características que lhes permitam sobreviver às condições ambientais adversas, como a tolerância a metais tóxicos. / The Tietê River is the most important river of the State of São Paulo, in the southeastern of Brazil; it is also one of the most contaminated rivers of the country. On its watercourse, the river receives large amounts of anthropogenic and industrial pollutants that can alter the environmental conditions of the water, leading to possible shifts in the microbial diversity. Metagenomic profiles of river locations with different water qualities showed taxonomic and functional differences related to the sampling site and water quality. Eutrophic sites showed prevalence of microorganisms and functions associated with heterotrophy, in contrast, in oligotrophic sites were detected microbial communities and functions related to autotrophic and litotrophic metabolisms. Also was observed, that environmental pressures can promote the acquisition of characteristics that allow the local microorganism to survive to adverse environmental conditions, such as tolerance to toxic metals.

Two plant species including Polygonum hydropiper L. and Hymenachne acutigluma (Steud.) Gilliland were investigated in their resistance to lead (Pb) and cadmium (Cd) pollution in the soil. Lead-contaminated soil samples were collected from the lead recycling village Dong Mai, Chi Dao commune, Van Lam district, Hung Yen province, Vietnam that had Pb level up to 192,185 mg.kg-1, dry weight (DW). Cadmium-contaminated soil samples were due to supplement of CdCl2.21/2H2O to alluvial soil. Results showed that both species were highly resistant to Pb, however P.hydropiper was better. Similarly, the Cd resistance was higher for P.hydropiper than for H.acutigluma. No morpho-abnormalities of P.hydropiper regarding the impact of lead were recorded, whereas for H.acutigluma, the young leaves had white colour after two months of planting in soil containing lead levels of 192,185 mg.kg-1. The response of both species with Cd in soils included yellowing leaves, withering branches and even dying after 5-15 days exposed to Cd. Lead contents accumulated in above-ground parts of both plants were up to 4,650 and 3,161 mg.kg-1, DW, corresponding to P.hydropiper and H.acutigluma. From the research results on lead resistance and accumulation of two plant species studied, it is suggested that the two species are lead hyperaccumulators can be used for phytoremediation technology to clean contaminated soil. / Hai loài thực vật gồm nghể răm Polygonum hydropiper L. và bấc nhọn Hymenachne acutigluma (Steud.) Gilliland được nghiên cứu về khả năng chống chịu ô nhiễm chì (Pb) và cadmi (Cd) trong đất. Mẫu đất ô nhiễm chì được thu từ làng nghề tái chế chì Đông Mai, xã Chỉ Đạo, huyện Văn Lâm, tỉnh Hưng Yên, Việt Nam có hàm lượng chì lên đến 192.185 mg.kg-1, tính theo trọng lượng khô (DW). Mẫu đất ô nhiễm Cd là do được bổ sung CdCl2.21/2H2O vào đất phù sa. Kết quả cho thấy cả hai loài đều có sức chống chịu chì rất cao, tuy nhiên nghể răm có khả năng tốt hơn. Tương tự như vậy, sức chống chịu Cd của nghể răm cũng cao hơn của bấc nhọn. Không có dấu hiệu bất thường nào của nghể răm đối với tác động của chì được ghi nhận, trong khi ở bấc nhọn thì lá non có màu trắng sau hai tháng trồng trên đất có hàm lượng chì 192.185 mg.kg-1. Phản ứng của cả hai loài thực vật với Cd trong đất gồm có hiện tượng vàng lá, héo ngọn và thậm chí chết sau 5-15 ngày phơi nhiễm với Cd. Hàm lượng chì được tích lũy cao trong phần trên mặt đất của cả hai loài thực vật lên tới 4.650 và 3.161 mg.kg-1, DW, tương ứng cho loài P.hydropiper và H.acutigluma. Từ kết quả nghiên cứu về khả năng chống chịu và tích lũy chì của hai loài thực vật nghiên cứu, có thể đề xuất đây là hai loài siêu tích lũy chì có thể sử dụng trong công nghệ làm sạch đất ô nhiễm.

Two plant species including Polygonum hydropiper L. and Hymenachne acutigluma (Steud.) Gilliland were investigated in their resistance to lead (Pb) and cadmium (Cd) pollution in the soil. Lead-contaminated soil samples were collected from the lead recycling village Dong Mai, Chi Dao commune, Van Lam district, Hung Yen province, Vietnam that had Pb level up to 192,185 mg.kg-1, dry weight (DW). Cadmium-contaminated soil samples were due to supplement of CdCl2.21/2H2O to alluvial soil. Results showed that both species were highly resistant to Pb, however P.hydropiper was better. Similarly, the Cd resistance was higher for P.hydropiper than for H.acutigluma. No morpho-abnormalities of P.hydropiper regarding the impact of lead were recorded, whereas for H.acutigluma, the young leaves had white colour after two months of planting in soil containing lead levels of 192,185 mg.kg-1. The response of both species with Cd in soils included yellowing leaves, withering branches and even dying after 5-15 days exposed to Cd. Lead contents accumulated in above-ground parts of both plants were up to 4,650 and 3,161 mg.kg-1, DW, corresponding to P.hydropiper and H.acutigluma. From the research results on lead resistance and accumulation of two plant species studied, it is suggested that the two species are lead hyperaccumulators can be used for phytoremediation technology to clean contaminated soil. / Hai loài thực vật gồm nghể răm Polygonum hydropiper L. và bấc nhọn Hymenachne acutigluma (Steud.) Gilliland được nghiên cứu về khả năng chống chịu ô nhiễm chì (Pb) và cadmi (Cd) trong đất. Mẫu đất ô nhiễm chì được thu từ làng nghề tái chế chì Đông Mai, xã Chỉ Đạo, huyện Văn Lâm, tỉnh Hưng Yên, Việt Nam có hàm lượng chì lên đến 192.185 mg.kg-1, tính theo trọng lượng khô (DW). Mẫu đất ô nhiễm Cd là do được bổ sung CdCl2.21/2H2O vào đất phù sa. Kết quả cho thấy cả hai loài đều có sức chống chịu chì rất cao, tuy nhiên nghể răm có khả năng tốt hơn. Tương tự như vậy, sức chống chịu Cd của nghể răm cũng cao hơn của bấc nhọn. Không có dấu hiệu bất thường nào của nghể răm đối với tác động của chì được ghi nhận, trong khi ở bấc nhọn thì lá non có màu trắng sau hai tháng trồng trên đất có hàm lượng chì 192.185 mg.kg-1. Phản ứng của cả hai loài thực vật với Cd trong đất gồm có hiện tượng vàng lá, héo ngọn và thậm chí chết sau 5-15 ngày phơi nhiễm với Cd. Hàm lượng chì được tích lũy cao trong phần trên mặt đất của cả hai loài thực vật lên tới 4.650 và 3.161 mg.kg-1, DW, tương ứng cho loài P.hydropiper và H.acutigluma. Từ kết quả nghiên cứu về khả năng chống chịu và tích lũy chì của hai loài thực vật nghiên cứu, có thể đề xuất đây là hai loài siêu tích lũy chì có thể sử dụng trong công nghệ làm sạch đất ô nhiễm.

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