Bioactivity of extracts from extremophile microalgae Coccomyxa sp. on chloride cells in operculum tissue of Fundulus heteroclitus

Microalgae have been studied because of their great potential as a source of new compounds with important value for biotechnology and to understand their strategies of survival in extreme environments. The microalgae Coccomyxa sp., studied in this thesis, is a poly-extremophile witch was isolated from the acid mine drainage of S. Domingos mine. This environment is characterized by low pH (<3) and high concentration of metals, such as copper and iron. The main purpose of the present work was to evaluate the potential bioactivity in an ex-vivo animal model (Fundulus heteroclitus), and expression on selected genes, of cellular extracts obtained from cultures of Coccomyxa sp. at pH 7 without or with exposure to copper (0.6mM Cu²+). The extracts of Coccomyxa sp. cultured at pH 7 exposed to copper show a great potential to be used as epithelial NKCC inhibitors, revealing their potential use as diuretics, but did not show significant effects on gene expression. Coccomyxa sp. could be a good source of cellular extracts with a great potential to be used in pharmaceutical and biotechnology industries.

Aqueous geochemistry and oxygen isotope compositions of acid mine drainage from the Río Tinto, SW Spain, highlight inconsistencies in current models

The Rio Tinto river in SW Spain is a classic example of acid mine drainage and the focus of an increasing amount of research including environmental geochemistry, extremophile microbiology and Mars-analogue studies. Its 5000-year mining legacy has resulted in a wide range of point inputs including spoil heaps and tunnels draining underground workings. The variety of inputs and importance of the river as a research site make it an ideal location for investigating sulphide oxidation mechanisms at the field scale. Mass balance calculations showed that pyrite oxidation accounts for over 93% of the dissolved sulphate derived from sulphide oxidation in the Rio Tinto point inputs. Oxygen isotopes in water and sulphate were analysed from a variety of drainage sources and displayed delta O-18((SO4-H2O)) values from 3.9 to 13.6 parts per thousand, indicating that different oxidation pathways occurred at different sites within the catchment. The most commonly used approach to interpreting field oxygen isotope data applies water and oxygen fractionation factors derived from laboratory experiments. We demonstrate that this approach cannot explain high delta O-18((SO4-H2O)) values in a manner that is consistent with recent models of pyrite and sulphoxyanion oxidation. In the Rio Tinto, high delta O-18((SO4-H2O)) values (11.2-13.6 parts per thousand) occur in concentrated (Fe = 172-829 mM), low pH (0.88-1.4), ferrous iron (68-91% of total Fe) waters and are most simply explained by a mechanism involving a dissolved sulphite intermediate, sulphite-water oxygen equilibrium exchange and finally sulphite oxidation to sulphate with O-2. In contrast, drainage from large waste blocks of acid volcanic tuff with pyritiferous veins also had low pH (1.7). but had a low delta O-18((SO4-H2O)) value of 4.0 parts per thousand and high concentrations of ferric iron (Fe(III) = 185 mM, total Fe = 186 mM), suggesting a pathway where ferric iron is the primary oxidant, water is the primary source of oxygen in the sulphate and where sulphate is released directly from the pyrite surface. However, problems remain with the sulphite-water oxygen exchange model and recommendations are therefore made for future experiments to refine our understanding of oxygen isotopes in pyrite oxidation. (C) 2009 Elsevier B.V. All rights reserved.

Produzione di bioidrogeno in dark fermentation da scarti dell'industria agroalimentale mediante l'impiego di batteri ipertermofili

La presente tesi di dottorato ha come argomento la produzione d’idrogeno per via fermentativa sfruttando il metabolismo anaerobico di particolari batteri estremofili del genere Thermotoga. In questo lavoro, svolto in seno al progetto Bio-Hydro, sfruttando reattori batch da 116 mL, è stato selezionato il ceppo migliore di Thermotoga fra i quatto ceppi testati: T. neapolitana. Una volta individuato il candidato batterico migliore è stato individuato il valore ottimale di pH (8.5 a t.amb) per la produzione d’idrogeno. Un intenso lavoro è stato svolto sul medium di coltura permettendone la minimizzazione e rendendolo così economicamente sostenibile per il suo utilizzo nel reattore da 19L; in questo caso il glucosio è stato completamente sostituito con due sottoprodotti agroindustriali individuati in precedenza, il melasso di barbabietola e il siero di latte. Sono stati poi eliminati i gravosi micronutrienti e le vitamine. È stata sfruttata la capacità di T. neapolitana di produrre biofilm e sono stati testati 4 diversi supporti in vetro sinterizzato e ceramici, tali test hanno permesso di individuare Biomax come supporto migliore. Sono stati svolti studi sul metabolismo di T. neapolitana volti ad individuare le concentrazioni inibenti di ogni substrato testato, l’inibizione da prodotto (idrogeno) e l’inibizione da ossigeno. Tutte queste prove hanno dato le conoscenze di base per la conduzione di esperienze su reattore da 19L. L’innovativo reattore di tipo SPCSTR è stato interamente studiato, progettato e costruito presso il DICMA. dell’Università di Bologna. La conduzione di esperienze batch su SPCSTR ha dato la possibilità di verificare il funzionamento del nuovo tipo d’impianto. Presso il Wageningen UR (NL), è stata svolta la selezione del miglior ceppo di Caldicellulosisruptor fra 3 testati e del miglior supporto per la produzione d’idrogeno; è stato poi costruito testato e condotto in continuo l’innovativo reattore CMTB.

Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.