This dissertation examines the microbial and functional diversity in Kartchner Caverns, a limestone cave in Arizona, USA. Kartchner is highly oligotrophic due to the lack of photosynthesis and the limited inputs of organic material from the surface. This characteristic poses a challenge for microbial life in the cave. The first objective of this work was to evaluate the bacterial richness, diversity and taxonomic composition of speleothems surfaces within Kartchner Caverns in order to gain insight into the distribution patterns associated with these communities. Secondly, the metabolic strategies used by cave communities to survive harsh cave conditions were investigated based on phylogenetic associations and metagenomics. Both objectives were directed toward answering the questions "who are there?" and "what are they doing?". The 454-pyrotag analysis of the V6 region of the 16S rRNA gene revealed an unexpectedly high bacterial diversity with each speleothem supporting a unique bacterial community profile. A focused study on one room of the cave revealed three community types: Type 1 was dominated by the phylum Proteobacteria; Type 2 by Actinobacteria; and Type 3 by Acidobacteria. Phylogenetic associations of the sequences generated by the 454 sequencing and by a Sanger clone library suggested cave microbial communities are supported by chemoautotrophic activities such as nitrite and iron oxidation. Results from the phylogenetic associations guided the metagenomic analysis which supports the presence of chemoautotrophic activities in the cave. Genes for two complete CO2 fixation mechanisms, the Calvin-Benson-Bashan and the rTCA cycles were identified in the cave metagenome, as well as genes for ammonia and nitrite oxidation. These genes are associated with both Bacteria and Archaea suggesting members of both domains are acting as primary producers in the cave ecosystem. Comparative analysis of cave samples to other environments suggests an overabundance of DNA repair mechanisms which could be potentially used by cave communities to overcome the toxicity due to high concentrations of calcium on the speleothem surfaces. This work provides the first comprehensive analysis of the microbial diversity and potential strategies used by microbial communities to survive under the extreme conditions found in a semi-arid limestone cave environment.

This thesis describes the use of different DNA sequencing technologies to investigate the structure and function of microbial communities in two extreme environments, boreal acid sulfate soil and the terrestrial deep biosphere. The first of the two investigated environments was soils containing un-oxidized metal sulfides that are termed ‘potential acid sulfate soil’ (PASS) materials. If these materials are exposed to atmospheric oxygen by either natural phenomena (e.g., land uplift) or human activities (e.g., drainage) then the metal sulfides become oxidized and the PASS becomes acidic and is defined as an ‘acid sulfate soil’ (ASS). The resulting acid and metal release from metal sulfide oxidation can lead to severe environmental damage. Although acidophilic microorganisms capable of catalyzing acid and metal release have been identified from many sulfide mineral containing environments, the microbial community of boreal PASSs/ASSs remains unclear. This study investigated the physicochemical and microbial characteristics of PASSs and ASSs from the Risöfladan experimental field in Vasa, Finland. Sanger sequencing of 16S rRNA gene sequences of microorganisms present in the PASSs and ASSs were mostly assigned to acidophilic species and environmental clones previously identified from acid- and metal-contaminated environments. Enrichment cultures inoculated from the ASS demonstrated that the acidophilic microorganisms were responsible for catalyzing acid and metal release from PASSs/ASSs. Lastly, the study investigated how to mitigate metal sulfide oxidation and the concomitant formation of sulfuric acid by treating ASSs in situ with CaCO3 or Ca(OH)2 suspensions. The DNA sequencing still identified acidophilic microorganisms after the chemical treatments. However, the increased pH during and after treatment suggested that the activity of the acidophiles might be inhibited. This study was the first to identify the microbial community present in boreal PASSs/ASSs and suggested that treatment with basic compounds may inhibit microbial catalysis of metal sulfide dissolution. The second studied environment was the deep, dark terrestrial subsurface that is suggested to be both extremely stable and highly oligotrophic. Despite the scarcity of carbon and energy sources, the deep biosphere is estimated to constitute up to 20% of the total biomass on earth and thus, represents the largest microbial ecosystem. However, due to the difficulties of accessing this environment and our inability to cultivate the indigenous microbial populations, details of the diversity and metabolism of these communities remain largely unexplored. This study was carried out at Äspö Hard Rock Laboratory, Sweden and utilized second-generation sequencing to identify the taxonomic composition and genetic potential of planktonic and biofilm populations. Community DNA sequencing of planktonic cells from three water types at varied age and depth (‘modern marine’, ‘undefined mixed’, and ‘old saline’) showed the existence of ultra-small cells capable of passing through a 0.22 μm filter that were phylogenetically distinct communities from the >0.22 μm fraction. The reduced cell size and/or genome size suggested a potential adaptation to the oligotrophic environment in the terrestrial deep biosphere. The identified planktonic communities were dominated by Proteobacteria, Candidate divisions, unclassified archaea, and unclassified bacteria. Functional analysis of the assembled genomes showed that the planktonic population from the shallow modern marine water demonstrated a predominantly anaerobic and heterotrophic lifestyle. In contrast, the deeper, old saline water was more closely aligned with the hypothesis of a hydrogen-driven deep biosphere. Metagenomic analysis of subsurface biofilms from ‘modern marine’ and ‘old saline’ water types suggested only a subset of populations were involved in initial biofilm formation. The identified biofilm populations from both water types were distinct from the planktonic community and were suggested to be dominated by hydrogen fed, chemolithoautotrophic and diazotrophic populations.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-07-21T17:04:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE ASPSP.pdf: 2001369 bytes, checksum: f6de58de3e0c281db510010c6c904ea9 (MD5) / Made available in DSpace on 2016-07-21T17:04:37Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE ASPSP.pdf: 2001369 bytes, checksum: f6de58de3e0c281db510010c6c904ea9 (MD5) Previous issue date: 2015-11-27 / capes / A área no entorno do arquipélago de São Pedro e São Paulo (ASPSP) (0°55 '10"N e 29º20'33" W) foi investigada, com o objetivo de caracterizar a estrutura da comunidade fitoplanctônica, seus padrões de distribuição horizontal e vertical, especialmente na termoclina que coincide com a profundidade máxima de clorofila (PMC) e identificar os valores de biomassa em carbono orgânico através do biovolume celular. Duas expedições foram realizadas a bordo do navio Hidro-Oceanográfico Cruzeiro do Sul, entre os dias 21 a 23 de julho de 2010 e 29 de setembro a 01 de outubro de 2011, ambas na estação seca da região. Foram estabelecidos dois transectos perpendiculares e opostos no entorno do arquipélago e determinados três pontos de coleta para cada um, em diversas profundidades: superfície, 25 metros, 50 metros, 10 metros acima da PMC, na PMC e 10 metros abaixo da PMC. Foram coletadas amostras no período diurno e noturno. Utilizou-se uma rede cilindro-cônica com malha de 20 μm de abertura para arrastos oblíquos e garrafas de Niskin para as coletas de amostras quali-quantitativas e físico-químicas. Duas massas de água foram identificadas (Água Tropical e Água Central do Atlântico Sul), uma termoclina ficou evidente entre 40 e 100 metros de profundidade. Um total de 128 espécies de organismos fitoplanctônicos foi identificado, pertencentes a quatro filos, sendo 22 espécies consideradas novos registros para a região. A densidade fitoplanctônica total variou de 1 a 183 x103 células.L-1. Trichodesmium thiebautii Gomont ex Gomont, Oxytoxum longiceps Schiller e Protoperidinium minimum (Pavillard) Schiller tiveram correlações significativas com os parâmetros físicos e químicos. A análise de agrupamento evidenciou a formação de cinco grupos em função da profundidade. Trichodesmium thiebautii foi à única considerada constante e dominante no entorno do arquipélago nas duas expedições. Os dados morfológicos encontrados neste estudo estão coerentes com as medidas encontradas por diversos autores na literatura citada. No filo cianobactérias, Trichodesmium hildebrandtii Gomont apresentou os maiores valores de biovolume celular e biomassa de carbono (1.603 μm3 e 254 pgC.μm-3, respectivamente). Em relação ao filo Dinophyta, Pyrocystis fusiformis Thomson apresentou os maiores valores de biovolume celular e biomassa em carbono (1.660,269 μm3 e 149.693 pgC.μm-3, respectivamente) e Prorocentrum balticum Loeblich apresentou os menores valores (1.145 μm3 e 161 pgC.μm-3, respectivamente). Para o filo Ochrophyta, Planktoniella sol (C.G.Wallich) Schutt, foi a única espécie analisada do grupo, com 63.107 μm3 de biovolume celular e 2.250 pgC.μm-3 de biomassa em carbono. Os dados de clorofila a e nutrientes foram considerados baixos em ambas as expedições, confirmando a oligotrofia da área. Estes dados indicam que ASPSP pode ser associado a um efeito ilha na circulação oceânica e que a distribuição horizontal e vertical dos dados hidrológicos, demonstra tratar-se de um ambiente estável em relação à temperatura, salinidade e oxigênio dissolvido. Em relação aos nutrientes, entretanto, ficou comprovado que estes interferem diretamente na composição das cianobactérias, mesmo com a dominância de T. thiebautii. / The area surrounding the São Pedro and São Paulo Archipelago (ASPSP) (0°55 '10"N and 29º20'33" W) was investigated to characterize the structure of the phytoplankton community and its patterns of horizontal and vertical distribution in the water column, especially in the thermocline that coincides with the maximum depth of chlorophyll (DCM). This area was also investigated to identify organic carbon biomass values according to cell biovolume. Samples were collected during two expeditions aboard the hydro-oceanographic ship Cruzeiro do Sul, between July 21 and 23, 2010 and between September 29 and October 1, 2011. This expedition occurred during the dry season. We established two opposite, perpendicular transects and selected three collection points in each transect at varying depths, as follows: surface, 25 meters, 50 meters, 10 meters above the DCM, DCM and 10 meters below the DCM. Samples were collected during the day and night. We used a cylindrical-conical net and Niskin bottles to collect the samples for qualitative, quantitative and physio-chemical analysis. Two water masses were identified (Tropical Water and Central Water of the South Atlantic), and a thermocline was detected between depths of 40 and 100 meters. A total of 128 species of phytoplankton organisms were identified, belonging to four phyla. Of these species, 22 were considered new records for the region. Total phytoplankton density ranged from 1 to 183 x103 cells. L-1. Trichodesmium thiebautii Gomont ex Gomont, Oxytoxum longiceps Schiller and Protoperidinium minimum (Pavillard) Schiller showed a significant correlation with the physical and chemical parameters. Cluster analysis revealed the formation of five groups by depths. Trichodesmium thiebautii was the only species considered constant and dominant in the area surrounding the archipelago for both expeditions. The morphological data found in this study are consistent with the measurements found by various authors in the cited literature. For the phylum cyanobacteria, Trichodesmium hildebrandtii Gomont presented the highest values of cell biovolume and biomass in carbon (1603 μm3 and 254 pgC.μm-3, respectively). In relation to the phylum Dinophyta, Pyrocystis fusiformis Thomson presented the highest values of cell biovolume and biomass in carbon (1.660,269 μm3 and 149,693 pgC.μm-3, respectively) and Prorocentrum balticum (Lohmann) Loeblich presented the lowest values (1145 μm3 and 161 pgC.μm-3, respectively). For the phylum Ochrophyta, Planktoniella sol (C.G.Wallich) Schütt was the only species of the group that was examined, with 63,107 μm3 of cell biovolume and 2250 pgC.μm-3 of biomass in carbon. The data of chlorophyll a and nutrients were considered low in both expeditions, which confirm the oligotrophy of the area. These data indicate that ASPSP can be associated to the island mass effect in the oceanic circulation. Moreover, the horizontal and vertical distribution of the hydrological data in the ASPS shows that this environment is stable in relation to temperature, salinity and dissolved oxygen. However, the results also show that the nutrients interfere directly in the composition of cyanobacteria, even with the dominance of T. thiebautii.

Focalisé sur le groupe phytoplanctonique des diatomées, ce travail de thèse a permis d'étudier l'impact du réchauffement climatique et de l'acidification des océans afin d'appréhender la réponse de ce taxon au changement climatique actuel. L'augmentation de la pCO2 atmosphérique depuis la première révolution industrielle est à l'origine de l'augmentation de la concentration en carbone inorganique dissous (DIC) dans l'océan de surface, et donc d'une acidification des océans à laquelle s'ajoute un réchauffement de l'océan de surface conduisant finalement à l'extension des zones oligotrophes très stratifiées et pauvres en sels nutritifs. Différentes approches in situ et au laboratoire ont donc été utilisées lors de ce travail de thèse afin de mieux comprendre la place des diatomées dans une province oligotrophe et d'apprécier ensuite leur réponse à un réchauffement et une acidification dans un «chémostat oligotrophe», limité en phosphate. / Specifically orientated on the diatoms' phytoplanktonic group, this work tryed to understand the impact of warming and ocean acidification on diatoms and aimed at understand the taxon's response to the ongoing climate change. Amospheric pCO2 increase since the first industrial revolution lead to the augmentation of dissoveld inorganic carbon (DIC) concentration in the surface ocean, and thus to the ocean acidification, accompanied by an ocean surface warming leading finally to the extension of oligotrophic areas well stratified and nutrient depleted. Different in situ and lab techniques were used in order to better understand the diatom role in oligotrophic system and their response to warming and acidification in an oligotrophic chemostat, limited by phosphate.