Dissertationen zum Thema „Microbial samples“

In Virginia, over one million households rely on private water supplies (e.g. well, spring, cistern). Previous literature acknowledges bacterial contamination in private water supplies as a significant public health concern in the United States. The present study tested private wells and springs in 20 Virginia counties for total coliforms (TC) and E. coli (EC) along with a suite of chemical contaminants. Sample collection was organized by the Virginia Household Water Quality Program (VAHWQP), a Virginia Cooperative Extension effort managed by faculty in the Biological Systems Engineering Department. Microbial and chemical source tracking were used to identify possible sources of contamination. A logistic regression was employed to investigate potential correlations between TC contamination and chemical parameters (e.g. NO3-, turbidity) as well as homeowner provided survey data describing system characteristics and perceived water quality. TC and EC contamination were quantified via the Colilert (www.idexx.com) defined substrate method for most probable number (MPN) of EC and TC per 100 mL of water. Of the 538 samples collected, 41% (n=221) were positive for TC and 10% (n=53) for EC. Chemical parameters were not statistically predictive of microbial contamination. Well depth, water treatment, and farm location proximate to the water supply were factors in a regression model that predicted presence/absence of TC with 74% accuracy. Microbial and chemical source tracking techniques (Polymerase Chain Reaction (PCR) and fluorometry, respectively) identified 4 of 26 samples as likely contaminated with human wastewater. Application of these source-tracking analyses on a larger scale will prove useful in defining remediation strategies.
Master of Science

Salmonellosis or “food poisoning” is a foodborne infection brought on by the pathogen Salmonella from the ingestion of the bacterium on contaminated foods such as vegetables. Infection from Salmonella leads to the highest incidence of hospitalizations and deaths each year, compared to any other bacterial foodborne illness. South Florida is the second largest agricultural winter vegetable producer in the United States, and contamination of vegetables is often observed in preharvest practices. A hardy bacterium, Salmonella, has been shown to live up to 6 weeks in soil and water up to 42°C without a host. The Florida Everglades is a tropical wetland that plays a large role in South Florida’s watershed. It can be divided into agricultural, conservation, and urban areas that connect Lake Okeechobee to Florida Bay by canals, swamps, and rivers. Inland canals tightly regulate water levels in South Florida as a means of flood control for residential and agricultural land. With the influences of anthropomorphic run off from agricultural and urban use, we hypothesized that microbial communities would significantly differ between three select sites in western (Collier county) versus three sites in more urban eastern Florida (Broward county): natural standing water, manmade drainage canal in agricultural areas, and manmade drainage canals in urban areas. We also hypothesized that pathogenic like Salmonella would be present in these habitats. Deep sequencing and ecological genetics analyses of the 16s rRNA V4 region yielded a total of 163,320 unique bacterial OTUs from a total of 139 samples collected monthly for one year in 2015 and part of 2016. Salmonella is not considered an abundant taxon within the microbial population. With the knowledge that Salmonella resides within the microbial population isolates were cultured from soil and water samples that were taken monthly from each site using a modified version of the Food and Drug Administration Bacterial Analytical Methods manual (FDA-BAM). The culturing resulted in 234 isolates obtained and 31 different serovars of Salmonella. Culturing showed that Salmonella favored months with high standing water and high-water temperatures that would lead to the ideal environment for survival. The most commonly occurring isolates within the sample set are those associated with agricultural animals. Though Salmonella may be a rare taxon within the microbial population given the correct environmental conditions such as warm temperatures it is possible to observe Salmonella year round within the South Florida environment.

There is a need for a sensitive, specific, rapid and cost-effective assay that can be used as an early warning signal of contamination of aquatic ecosystems. The purpose of this work was to develop a sensitive stress-specific microbial bioreporter responsive to pro-oxidants. Furthermore, the bioreporter was designed to be applicable in environments possibly affected by metal processing activities. An E.coli bioreporter was developed containing a plasmid with the katG promoter sequence as the sensing sequence and with mCherry as the reporter protein. The bioreporter responded to metal pro-oxidants (Cd, As, Zn, Pb, Ag and Ag nanoparticles). A new assay growth-medium was developed and contributed to improve the sensitivity of our assay that has the best detection limit to inorganic pro-oxidants compared to other oxidative-stress sensitive bioreporters in the literature. The bioreporter detected pro-oxidants in environmental samples. The assay has a reasonable sensitivity, however, it still lacks sensitivity to detect pro-oxidants at concentrations lower than those shown to be toxic to many aquatic species. Within-lab reproducibility and robustness were determined to be acceptable. For stress-specific bioreporters to be incorporated in regulative legislations and industrial monitoring programs there is a need to improve the sensitivity of these assays, they need to be calibrated with other relevant pro-oxidants, inter-lab reproducibility needs to be established and robustness to environmental samples needs to be further tested. To further validate the sensitivity and ecotoxicological relevance of the bioreporter as a relevant predictive tool, stress-specific bioreporter assays need to be performed in parallel with traditional ecotoxicological assays using contaminated environmental samples.

Philosophiae Doctor - PhD
The El Tatio geyser field (ETGF) is the largest known geothermal field in Chile, forming part of a wide spectrum of extremophilic habitats in the country. The ETGF is NaCl rich, with high concentrations of toxic elements such as Li, As and Cs, which are contributed mainly by volcanic activities in the region. Most previous studies in the area have focused on the geology and geochemistry for mining purposes, as well as on the search for geothermal resources for power generation. Very little is currently known about the composition of the microbial communities of the ETGF, which makes the study reported here of particular novelty.A metagenomic approach, involving the amplification of 16S rRNA gene phylogenetic markers from metagenomic DNA was used to investigate seven different sites within the geyser field. The sample sites were characterized by high temperatures (80-85 °C) and a range of pH values (6.3-8). Various molecular methods, including clone library construction and PCR-DGGE analyses were used to target a wide range of microbial populations within the ETGF sites. Multivariate analysis was also applied to assess differences in the microbial diversity from different sites and to correlate microbial diversity with environmental conditions. Culture-dependent screening of novel nanoarchaeal species was also undertaken.These were coupled with PCR and other detection methods such as fluorescent in situ hybridization (FISH) to trace the presence of nanoarchaeal signals from enriched cultures.The results have shown that the ETGF encompasses a limited microbial diversity represented by only 30 dominant phylotypes, and most likely due to the toxic chemical content of the geyser field. The microbial representatives identified were assigned to OTUs from archaeal,nanoarchaeal and bacterial taxonomic groups. The dominant microbial taxa included members of the Proteobacteria, Firmicutes, Aquificae, Actinobacteria, Euryarchaeota(Halobacteriales, Archaeoglobales), Crenarchaeota (Thermoproteales, Desulfurococcales),together with uncultured representatives of the bacteria, archaea and nanoarchaeota. Notably,representatives of mesophilic, thermophilic and hyperthermophilic taxonomic groups were all detected in ETGF samples. This is attributed to various factors such as temperature gradients and dispersal mechanisms (e.g. natural forces such as rain and volcanic activities). Principal component analysis (PCA) showed significant differences (P < 0.05) in the microbial diversity of the ETGF samples, with principal components (based on the sequenced species from both 16S rRNA clone libraries and PCR-DGGE profiles) explaining up to 62.7% of variance. Furthermore, CCA showed that the differences in phylogenetic diversity were most influenced by temperature and salinity. This was also confirmed by the sequencing results,which showed that hyperthermophilic and haloarchaeal taxa were dominant in the ETGF sites. However, conductivity and pH were also found to contribute to variations in the microbial diversity of the experimental samples, with TDS (total dissolved solids) being a less influential factor. Attempts to generate nanoarchaeal-host co-cultures, and to recover sufficient nanoarchaeal genomic DNA for fosmid and/or large insert cloning for comparative genome analysis, were unsuccessful.This study is the first to employ metagenomic approaches to analyse the microbial diversity of sites in the ETGF, and has expanded our knowledge of microbiota present in this geyser field.

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.

The work in this thesis consisted of two studies: 1) analysis of the microbial biodiversity of multiple ground ice types from the Arctic and the High Arctic; 2)examination of the survival of Cryptococcus NP33 under simulated Martian conditions over 41 days. The first study involved culture-dependent and independent evaluations of the microbial communities found in a buried firnified snow bank, a buried glacier, a pingo and ice wedges. Direct and culturable counts in the various ground ice types differed from each other (104 – 108 cellsmL-1 direct counts; 0 - 105 CFUmL-1 culturable counts), and were only weakly correlated to increasing sample age. Culturable counts were consistently highest in ice wedge samples. All sample isolates were dominated by Actinobacteria. Bacterial pyrosequencing analysis for one ice wedge showed a dominance (<50% of sequences) by Gammaproteobacteria. In an Archaeal clone library of the buried glacier, no clones were closely related to sequenced isolates, but were similar (>90%) to uncharacterized clones from marine environments. The pingo Bacterial clone library clones matched closely to environmental isolates as well as clones from cryoenvironments as well as soil environments. For the survivability study, Cryptotoccus strain NP33 was selected as a candidate organism to undergo Martian simulations. After 41 simulation days, it had a half-life of 10.1 days in simulated sunlightand 16.1 days in darkness. The compiled results suggest that the organism traits most crucial to survival under simulated Martian conditions were desiccation, radiation and freeze-thaw resistance.
Cette thèse contient deux études : 1) la biodiversité de différents types de glacesde sol de l'Arctique et du Grand Arctique, de même que la survie de Cryptococcus NP33dans des conditions martiennes simulées pendant 41 jours. La première étude impliquait des analyses dépendantes et indépendantes des conditions de culture pour évaluer les communautés microbiennes dans une congère névée enterrée, un glacier enterré, un pingo et des coins de glace. Les nombres de cellules totales et les nombres de cellules culturées dans les différents types de glaces de sol variaient (104 – 108 cellulesmL-1 nombre total; 0- 105 CFUmL-1 cellules culturées), et étaient que très faiblement dépendants de l'âge du iispécimen. Les nombres de cellules culturées étaient constamment plus élevées dans les coins de glace. Actinobacteria dominait les isolats de chaque spécimen. Un pyroséquençage bactérien d'un coin de glace a révélé une dominance (>50% desséquences) de Gammaproteobacteria. Dans une librairie de clones d'Archées du glacier enterré, les clones avaient peu de similarité à des isolats environnementaux, mais étaient similaires (>90%) à des clones environnementaux non-caractérisés d'environnements marins. Dans une librairie de clones de Bactéries du pingo, les clones étaient très similaires à des isolats et des clones provenant de cryo-environnements et d'environnements de sol. Pour la simulation martienne, Cryptococcus NP33 a été choisicomme organisme candidat suite à des expériments pour sélectionner des organismes résistant à la dessiccation, au froid et aux concentrations élevées de sel. Au cours de 41 jours dans le simulateur, Cryptococcus NP33 avait une demi-vie de 10.1 jours dans le soleil simulé et 16.1 jours dans le noir. Halorubrum avait un taux de survie de 100%(demi-vie estimée de ~70 - ∞ jours), tandis que d'autres organismes avaient une demi-vie beaucoup moins élevée (~2 - ~8 jours). Les résultats combinés suggèrent que les caractéristiques nécessaires à la survie dans des conditions martiennes simulées étaient la résistance à la dessiccation, la radiation et aux cycles de gel-dégel.

Nitrogen cycling processes can be tracked using quantitative Polymerase Chain Reaction (qPCR) to determine the presence and qReverse Transcriptase-PCR (qRT-PCR) to determine expression of key genes, or ‘biological markers’, for nitrogen metabolism. Nitrification is catalyzed in part, by two enzymes: ammonia monooxygenase (AMO; NH3 NH2OH) and nitrite oxidoreductase (NXR; NO2- NO3-). For denitrification, four enzymes act sequentially: nitrate reductase (NAR/NAP; NO3- NO2-), nitrite reductase (NIR; NO2- NO), nitric oxide reductase (NOR; NO  N2O), and nitrous oxide reductase (NOS; N2O  N2). A principle of wastewater treatment (WWT) is to remove excess nitrogen by taking advantage of natural nitrogen cycling or biological nitrogen removal (BNR). This process involves using microorganisms to bring influent ammonia through nitrification and denitrification to release nitrogen gas, which does not contribute to eutrophication. A novel shortcut nitrogen removal configuration could increase nitrogen removal efficiency by promoting nitritation/denitritation, reducing the classic nitrogen cycle by removing the redundant oxidation/reduction step to nitrate (NO3-). Here, three nitrogen transformations were used to track the three main phases in the nitrogen cycle; ammonia monooxygenase for nitrification, nitrite oxidoreductase for shortcut, and nitrous oxide reductase for denitrification. Primers for qPCR and qRT-PCR were designed to capture as much sequence diversity as possible for each step. Genes from bacteria known to perform the nitrogen transformations of interest (amoA, nxrB, nosZ) were used to BLAST-query the Integrated Microbial Genomes & Microbiomes database (img.jgi.doe.gov) to find homologs from organisms commonly found in WWT. These sequences were then aligned to find regions sufficiently conserved for primer design. These PCR primers were tested against standards for each gene and used to track nitrogen transformation potential and expression in a novel lab-scale algal photo-sequencing batch reactor which promotes shortcut nitrogen removal from wastewater across three solids retention times (SRT, or mean cell residence time); 5, 10 and 15 days. SRT 15 had the greatest total nitrogen removal with nitritation and denitritation observed. Nitrate was not detected in the first cycle and shortcut nitrogen removal was supported by low levels of nxrB genes and transcripts. Simultaneous nitrification/denitrification was supported by elevated concentrations of nosZ during the light period and less nitrite produced than ammonium consumed. Nitritation was predominantly performed by Betaproteobacteria amoA and nitrous oxide reduction was predominantly from nosZ group I (Proteobacteria-type).

Raman spectroscopy, together with multivariate statistical analyses, has proven to be a near real-time analytical technique capable of phenotyping cells, tissues and organs. This dissertation will show exclusively the application of the Raman spectroscopy phenotypic profiling method to; (i) microbial toxicity, (ii) ex-vivo organ perfusion, and (iii) subcellular location targeting. Real-time analytical methods for monitoring living biological systems will enable study of the physiological changes associated with growth, genetic manipulations, and adverse environmental conditions. Most existing analytical methods (NMR exempt), though highly accurate, must be performed off-line and most require destruction of the studied sample. These attributes make these methodologies less desirable to the study of physiological changes of cells, tissues, and organs. In this work, Raman spectroscopy has been identified and shown to be a good candidate for real-time analysis mainly because it can be performed: (i) in near real-time, (ii) non-destructively and with minimal sample preparation, (iii) through a glass barrier (i.e., can be performed in situ), and (iv) with minimal spectral interference from water. Here, Raman spectroscopy was used in combination with multivariate statistics to analyze the differing toxic effects of 4-C chain alcohols on E. coli. Good correlations were established between Raman spectra and off-line analytical techniques used to measure: (i) saturated, unsaturated, and cyclopropane fatty acids; (ii) amino acid composition of total protein; and (iii) cell membrane fluidity. Also, Raman 'fingerprint' analysis was used to discriminate among different phenotypic responses of cells. In addition, this methodology was applied to analyze perfusates of organs maintained by the VasoWave® organ perfusion system. Raman fingerprints can be used to assess organ health, and it is believed this data can be used to inform decisions such as whether or not to transplant an organ. Finally, molecular biology techniques were used to design and produce specific protein targets harboring a silver binding domain fusion, which upon release migrate to specific subcellular locations. By employing the related technique of surface-enhanced Raman scattering (SERS), which produces a highly amplified Raman signal in the presence of metallic nanoparticle substrates (e.g., silver nanoparticles), different regions of the E. coli cell structure were studied. The target regions studied by the technique included: (i) outer cell membrane, (ii) periplasm, and the (iii) cytoplasm.
Ph. D.

Orientador: Valéria Maia Merzel
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O processo de biodegradação do petróleo em reservatórios pode resultar em mudanças na composição e propriedades físico-químicas de óleos brutos e gases naturais, as quais levam à diminuição do teor de hidrocarbonetos saturados, produzindo óleos mais pesados e com baixo valor econômico. O uso combinado de técnicas dependentes e independentes de cultivo pode nos permitir um melhor entendimento acerca da comunidade de micro-organismos que habita os reservatórios de petróleo, incluindo aqueles responsáveis por esta biodegradação. O conhecimento sobre a composição microbiana, suas funções e interações com outros micro-organismos e com o ambiente pode levar à definição de estratégias de monitoramento e/ou controle da biodegradação em reservatórios. Este estudo teve como finalidade avaliar a diversidade de micro-organismos e genes envolvidos na degradação de hidrocarbonetos presentes em amostras de petróleo provenientes de dois poços terrestres da Bacia Potiguar (RN), identificados como GMR75 (poço biodegradado) e PTS1 (poço não-biodegradado), através da construção de bibliotecas de genes catabólicos (alcano monooxigenases - alk, dioxigenases que hidroxilam anéis aromáticos ¿ ARHDs e 6-oxocyclohex-1-ene-1-carbonyl-CoA hidroxilase - bamA) e sequenciamento em larga escala de metagenoma e metatranscriptoma de enriquecimentos microbianos aeróbios. Os resultados obervados mostraram uma distribuição diferencial dos genes catabólicos entre os reservatórios, sendo o óleo biodegradado mais diverso para os genes alk e bamA. As sequências foram semelhantes aos genes alkB dos gêneros Geobacillus, Acinetobacter e Streptomyces, aos genes ARHD dos gêneros Pseudomonas e Burkholderia, e aos genes bamA do gênero Syntrophus. A análise quantitativa dos genes catabólicos de degradação de hidrocarbonetos presentes e expressos nos enriquecimentos microbianos em diferentes etapas da biodegradação do óleo, através de PCR Tempo Real, demonstrou maior atividade do gene que codifica a enzima dioxigenase nas comunidades microbianas enriquecidas, e os resultados obtidos pela técnica de microarray sugeriram a existência de novas sequências dos genes alk e ARHD provindas do reservatório de petróleo. As análises das sequências obtidas a partir do metagenoma e metatranscriptoma mostraram que a comunidade bacteriana recuperada no enriquecimento aeróbio é bastante diversa, com predominância do Filo Actinobacteria, seguido de Proteobacteria. As sequências com maior abundância e níveis de expressão foram relacionadas aos genes que codificam as proteínas ligase CoA de ácido graxo de cadeia longa, envolvida na degradação de compostos aromáticos; descarboxilase, envolvida com o ciclo do glioxilato, e o fator sigma da RNA polimerase, envolvida com a regulação da resposta ao estresse oxidativo, sugerindo uma adaptação da comunidade microbiana às condições do enriquecimento e um processo inicial de biodegradação dos hidrocarbonetos. Os resultados obtidos neste trabalho fornecem dados inéditos sobre a diversidade de genes catabólicos e de membros da comunidade microbiana potencialmente envolvidos com a degradação do óleo em reservatórios de petróleo
Abstract: The process of oil biodegradation in reservoirs may result in changes in the composition and physico-chemical properties of crude oils and natural gases, which lead to the decrease of the content of saturated hydrocarbons, producing heavy oils and with low economic value. The combined use of both dependent and independet cultivation techniques may allow us to better understand the microbial community inhabiting oil reservoirs, including those microorganisms responsible for oil degradation. The knowledge about the microorganisms, ther functions and interactions with other microorganisms and the environment may lead to the definition of monitoring and/or control strategies of biodegradation in oil reservoirs. This study aimed at evaluating the diversity of microorganisms and genes involved in the degradation of hydrocarbons present in oil samples from two onshore reservoirs at Potiguar Basin (RN), identified as GMR75 (biodegraded) and PTS1 (non- biodegraded), through the construction of catabolic gene libraries (alkane monooxygenases - alk, aromatic ring hydroxylating dioxygenases ¿ ARHD and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydroxylase - bamA) and highthroughput sequencing of metagenome and metatranscriptome from aerobic microbial enrichments. Results observed showed a differential distribution of catabolic genes between the reservoirs, being the biodegraded oil more diverse for the alk and bamA genes. The sequences were similar to alkB genes from Geobacillus, Acinetobacter and Streptomyces genera, to the ARHD genes from Pseudomonas and Burkholderia genera, and to the bamA genes from Syntrophus genus. Quantitative analysis of the hydrocarbon degradation genes present and expressed in the microbial enrichments during the different phases of oil biodegradation by Real-Time PCR showed that there was a higher activity of dioxygenase enzymes in the enriched microbial communities and results from microarray assays suggested the existence of new alk and ARHD gene sequences originated from the oil reservoir. Metagenomic and metatranscriptomic analyses showed a highly diverse bacterial community, dominated by the Phylum Actinobacteria, followed by Proteobacteria. The most abundant and active sequences were affiliated to the Long-chain-fatty-acid-CoA ligase protein, involved in the degradation of aromatic compounds; decarboxylase, which is involved with the glyoxylate cycle, and RNA polymerase sigma factor, which is involved in regulating the oxidative stress response, suggesting an adaptation of the microbial community to the enrichment conditions and an initial process of biodegradation of hydrocarbon compounds. The results obtained in this work bring innovative data on the diversity of catabolic genes and microbial community members potentially involved with oil degradation in petroleum reservoirs
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

A cultivation-independent approach, sequence analysis of 18S rRNA genes PCR-amplified from environmental DNA, was used to explore the diversity and distribution of eukaryotic microbes inhabiting algal mats in two acidic geothermal streams in Yellowstone National Park. The objectives were to: (1) clarify the identity of mat forming algae in Nymph Creek (2) survey microbial species in the Nymph Creek mat over seasonal intervals along a thermal gradient (3) compare microbial species in the Nymph Creek mat with those in Alluvium Creek mats (4) evaluate microbial species in algal mats formed on different substrates in Alluvium Creek. The results show that a novel red alga dominates high temperature regions (~50ºC) of Nymph Creek and two "Chlorella-like" algae predominate the cooler regions (<38ºC). The predominant algae in Alluvium Creek were distinctly different from those in Nymph Creek. Several stramenophiles and fungi were detected in each algal mat.

Neste trabalho caraterizou-se a presença de comunidades microbianas, envolvidas em processos fermentativos, usando amostras arqueológicas. Foram selecionados dois tipos de materiais: fragmentos de ânforas, amplamente utilizadas para o transporte, transformação e armazenamento de produtos alimentares no Império Romano e resíduos de garum, um dos condimentos mais utilizado e amplamente produzido no Império Romano. Através de métodos de cultura, foi possível isolar diversos microrganismos. Os métodos moleculares permitiram detetar a presença de microrganismos. A análise de DNA metagenómico acoplado a SNG permitiu identificar a presença de comunidades microbianas, possibilitando a atribuição de alguns processos fermentativos a estes artefactos arqueológicos. Os resultados obtidos demonstraram a presença de microrganismos compatíveis com fermentações láticas e fermentações alcoólicas. As ânforas de onde são provenientes as peças cerâmicas estudadas possivelmente armazenaram, produziram ou transportaram vinho. Relativamente aos resíduos de garum os resultados revelaram que este é produzido com peixe, muito provavelmente fermentado por bactérias láticas; Abstract: Title: Study of microbial communities present in archaeological samples. In this study it was characterized the presence of microbial communities involved in fermentative processes in archaeological samples. Two types of materials were selected: fragments of amphorae, which were widely used for transporting, processing and storage of food products in the Roman Empire and garum residues, one of the most used and produced condiments in the Roman Empire. It was possible to isolate various microorganisms using culture-based methods. The application of molecular approaches allowed to detect the presence of microorganisms. The analysis of the metagenomic DNA coupled to NGS allowed to identify the presence of microbial communities that made possible to associate these archaeological artefacts to fermentative processes. The results showed the presence of microorganisms compatible with lactic and alcoholic fermentations. The amphorae from which the studied ceramic pieces were collected were probably used for storage, production or transport of wine. The results obtained from the garum analysis revealed that this condiment was produced from fish, probably fermented by lactic bacteria.

Due to massive expansion of the mass spectrometry and constant price growth of the human labour the optimalisation of the microbial samples preparation comes into question. This master thesis deals with design and implementation of a machine learning algorithm for segmentation of images of microbial colonies cultivated on Petri dishes. This algorithm is going to be a part of a controlling software of a MBT Pathfinder device developed by the company Bruker s. r. o. that automates the process of smearing microbial colonies onto a MALDI target plates. In terms of this thesis a several models of neural networks based on the UNet, UNet++ and ENet architecture were implemented. Based on a number of experiments investigating various configurations of the networks and pre-processing of the training datatset there was chosen an ENet model with quadruplet filter count and additional convolutional block of the encoder trained on a dataset pre-processed with round mask.

This study used petrographic thin sections, scanning electron microscopy, and confocal laser microscopy to document microbially mediated dissolution of carbonate reservoir rocks. The samples studied came from three carbonate units that are hydrocarbon reservoirs; the Salem, Sligo, and Smackover formations. These samples were inoculated with bacteria, and then treated with nutrient solutions followed by ethanol to promote generation of acetic acid by bacteria. Dissolution occurred in calcite-dominated rocks and in dolomitized rocks. Noticeable changes first occurred after nine weeks of ethanol treatment and significant change only occurred after twelve weeks of ethanol treatment. The size of the vuggy pores created increased from 1 µm or less to over 5 µm, and rarely over 10 µm, in length.

L’écologie microbienne concerne l’étude des microorganismes et de leurs interactions biotiques et abiotiques dans un écosystème donné. Ces vingt dernières années, l’avancement des techniques moléculaires pour analyser la diversité microbienne et, notamment, les nouvelles technologies de séquençages (NGS) ont permis de surmonter les limitations associées aux approches traditionnelles basées sur la culture et la microscopie. Ces approches moléculaires ont conduit à une accumulation des données de diversité microbienne et de potentiel métabolique dans des communautés microbiennes des écosystèmes variés.Cependant, ces efforts ont été principalement appliqués sur des environnements facilement accessibles ou liés à l’humain, comme le plancton (marin principalement) et la flore intestinale. Néanmoins, ceci a conduit à une très forte augmentation de données environnementales et au développement de la bioinformatique par le biais de nombreux outils. Parmi les environnements délaissés des études, les environnements faibles en oxygène sont probablement également porteurs de nouveautés phylogénique ou métaboliques.Afin de palier à cela, nous avons choisi d’explorer deux environnements suboxiques relativement peu étudiés : la cave Movile (Roumanie) et les sédiments du lac Baikal (Sibérie, Russie). Notre but étant de montrer les diversités phylogénétiques et fonctionnelles des microbes de ces biotopes.Pour cela, j’ai d'abord développé un pipeline d’analyse de données métabarcoding (petite sous-unités ribosomique). Ensuite, j’ai appliqué cet outil sur des données de métabarcoding de protistes provenant d’échantillons d’eau et de tapis microbiens de la cave de Movile, un écosystème chemosynthétique pratiquement fermé. Nous avons montré que la diversité des protistes de la cave s’étendait à quasiment tous les grands groupes eucaryotes et provenait à la fois d’origine d’eaux douces et marines. De plus, la plupart ont été affiliées à des groupes d’organismes typiquement anaérobies, ce qui est concordant avec les paramètres abiotiques de la cave. Écologiquement, ces protistes sont des prédateurs mais aussi vraisemblablement des partenaires symbiotiques avec des espèces procaryotes de la cave.Dans une deuxième étude, j’ai eu l’opportunité d’appliquer ce pipeline de métabarcoding sur des données procaryotes et eucaryotes provenant des couches superficielles des sédiments du lac d’eau douce Baikal. Comme attendu, les communautés microbiennes dans ces sédiments sont particulièrement diverses et relativement enrichis en archées. Nous avons aussi pu mettre en évidence des lignées que l’on pensait exclusivement marines dans ces sédiments. Ces lignées sont probablement planctoniques mais s’accumulent au fond par sédimentation. Enfin, les échantillons ont été prélevés dans le but de tester les influences de la profondeur, du bassin et de la latitude sur les communautés. Aucune d’elles ne s’est révélée significative.Dans une troisième étude, j'ai utilisé une approche métagénomique afin de révéler les acteurs écologiquement majeurs dans les sédiments, leurs rôles et de reconstruire leurs génomes. Cela nous a permis notamment de mettre en évidence le rôle primordial des Thaumarchaeota dans le cycle de l’azote et la production primaire de molécules de carbone. Les chloroflexi et les protéobacteries ont aussi un rôle important dans la surface des sédiments du lac Baikal. Ce travail de thèse participe à la connaissance globale de la diversité microbienne sur la planète en mettant en lumière des environnements peu étudiés. De plus, l’étude de la surface des sédiments du lac Baikal apporte de nouvelles données sur le sujet de la transition eau douces/eau marines des microbes. Enfin, la métagénomique a permis de révéler le cycle des nutriments et les microorganismes y participant dans ces échantillons de sédiment. En résumé, ce travail vient mettre en lumière l’écologie microbienne d’écosystèmes suboxiques, notamment la surface des sédiments du lac Baikal
Microbial ecology is the science of micro-organisms and their biotic and abiotic interactions in a given ecosystem. As technology has advanced, molecular techniques have been widely used to overcome the limitations of classical approaches such as culturing and microscopy. Indeed, the development of Next Generation Sequencing (NGS) technologies in the past twenty years has largely helped to unravel the phylogenetic diversity and functional potential of microbial communities across ecosystems.Nonetheless, most of the environments studied through these techniques concentrated on relatively easily accessible, tractable and host-related ecosystems such as plankton (especially in marine ecosystems), soils and gut microbiomes. This has contributed to the rapid accumulation of a wealth of environmental diversity and metagenomic data along with advances in bioinformatics leading to the development of myriads of tools. Oxygen-depleted environments and especially their microbial eukaryote components are less studied and may lead to future phylogenetic and metabolic discoveries.In order to address this, we conducted analyses on two poorly studied suboxic ecosystems: Movile Cave (Romania) and lake Baikal sediments (Siberia, Russia). In this task, we aimed at unveiling the taxonomic and functional diversity of microorganims in these environments.To do so, I first evaluated the available bioinformatics tools and implemented a bioinformatics pipeline for 16S/18S rRNA gene-based metabarcoding analysis, making reasoned methodological choices. Then, as a case study, I carried out metabarcoding analyses of the water and floating microbial mats found in Movile Cave in order to investigate its protist diversity. Our study showed that Movile Cave, a sealed off chemosynthetic ecosystem, harbored a substantial protist diversity with species spanning most of the major eukaryotic super groups. The majority if these protists were related to species of freshwater and marine origins. Most of them were putatively anaerobic, in line with the cave environment, and suggesting that in addition to their predatory role, they might participate in prokaryote-protist symbioses.In a second study, I applied my metabarcoding pipeline to explore unique and relatively unexplored environment of Lake Baikal sediments. I first applied a metabarcoding approach using 16S and 18S rRNA genes to describe prokaryotic as well as protist diversity. Overall, the communities within these ecosystems were very diverse and enriched in ammonia-oxidizing Thaumarchaeota. We also identified several typical marine taxa which are likely planktonic but accumulate in sediments. Finally, our sampling plan allowed us to test whether differences across depth, basin or latitude affected microbial community structure. Our results showed that the composition of sediment microbial communities remained relatively stable across the samples regardless of depth or latitude.In a third study, we applied metagenomics to study the metabolic potential of communities associated to Baikal sediments and to reconstruct metagenome-assembled genomes (MAGs) of dominant organisms. This revealed the considerable ecological importance of Thaumarchaeota lineages in lake Baikal sediments, which were found to be the major autotrophic phyla and also very implicated in the nitrogen cycle. Chloroflexi and Proteobacteria-related species also appeared ecologically important.This PhD thesis reveals the taxonomic diversity of poorly studied suboxic ecosystems and therefore contributes to our knowledge of microbial diversity on Earth. Additionally, the analyses of surface sediment samples in lake Baikal adds new light on freshwater-marine transitions. The metagenomic analyses reported here allowed us to postulate a model of nutrient cycle carried out by microorganismsin these sediments. Overall, this work sheds light on the microbial ecology of oxygen-depleted environments, and most notably lake Baikal surface sediments

Identification of ignitable liquids in fire debris analysis using pattern recognition is an important step in determining the nature of a suspicious fire. Complex mixtures that make up ignitable liquids are susceptible to microbial degradation when fire debris evidence is presented in the form of soil. Microbial degradation results in a selective metabolism of certain classes of compounds required for identification of an ignitable liquid. Various ignitable liquids that may be used to initiate or propagate a fire contain different classes of organic compounds. These include normal alkanes, branched alkanes, cycloalkanes, aromatics, terpenes, and others. In this work, microbial degradation of nine ignitable liquids in soil was evaluated over a period of twenty-six days. The degradation of aromatic compounds in gasoline was faster with toluene and C2-alkylbenzenes than in C3-alkylbenzenes. However, the overall loss of aromatics made gasoline chromatographically unidentifiable. The complete loss of n-alkanes in medium and petroleum distillates resulted in patterns that resembled naphthenic-paraffinic products. Normal alkanes were more susceptible to microbial degradation than isoalkanes, which was specifically demonstrated in medium and heavy petroleum distillates. In diesel, pristane and phytane remained prominent in comparison to the normally prevalent n-alkanes, which could no longer be detected post-degradation. The degradation of isoalkanes and cycloalkanes was evaluated in a naphthenic-paraffinic product. Isoalkanes were degraded significantly faster than cycloalkanes. The remaining peaks in the naphthenic-paraffinic pattern consisted solely of cycloalkane compounds, and could no longer be classified as a naphthenic-paraffinic product. The terpene compounds in turpentine were also observed to be susceptible to degradation by microorganisms. The loss of !-pinene, limonene, and camphene was significantly noticeable in comparison to other terpene compounds, such as 1,4-cineole. Microbial biodegradation in different soil types was investigated. The difference in soil texture can affect the rate of metabolism of ignitable liquids due to the variance of available oxygen, nutrients and mobility of the microbial population. The degradation of isoalkanes, cycloalkanes, aromatics and heavier normal alkanes was faster in clay, whereas normal alkanes of lower molecular weight were degraded more readily in sand. There has been no explanation of this occurrence within the scientific literature, however it could be hypothesized that the difference in microbial flora and water saturation levels could affect the selective degradation between the two soil types. Fire debris evidence is often stored for long periods of time before analysis due to case backlogs. The storage condition of arson-related soil samples is a sensitive subject. If evidence, containing soil, is stored at room temperature, petroleum compounds in any ignitable liquid residues that are present will be degraded within a week. Therefore, it is important to freeze or refrigerate soil samples. The storage of both refrigerated and frozen soil samples containing gasoline were evaluated over six months. Less than 6% of the aromatic compounds distinctive of gasoline remained when stored at 5 °C, while minimal change was observed in the same compounds when stored at -15 °C. Microbial degradation of petroleum-based ignitable liquids is advantageous from the environmental perspective. However, within the forensic community the effect of microbial action could lead to misclassification or inability to identify the presence of an ignitable liquid in fire debris evidence.

博士
國立臺灣師範大學
生命科學系
105
Sex steroid hormones (SHs), a major group of endocrine disrupting agents, are often detected in aquatic environments. The most concerned SHs include estrogens (e.g., 17β-estradiol and estrone) and androgens (e.g., testosterone). Among the proposed remediation strategies, bacterial degradation has been considered an efficient and eco-friendly strategy for removing the SHs from the contaminated ecosystems. In this dissertation, I aimed to investigate the metabolic and phylogenetic diversity related to bacterial degradation of SHs from model organisms to the environemnt. By using culturable bacterial strains as model organisms, I demonstrated that strictly aerobic Sphingomonas sp. strain KC8 degrade estrogens through the 4,5-seco pathway; the essential meta-cleavage dioxygenase was isolated and characterized. Furthermore, through the genomic and transcriptomic analyses, I identified the catabolic gene clusters in the 4,5-seco pathway of strain KC8, and in the 2,3-seco pathway for androgen biodegradation of Steroidobacter denitrificans DSM 18526. The omics studies on the model organisms enabled the environmental investigations of steroid biodegradation, for which I used the following approaches: (i) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identification of signature metabolites, (ii) identification of main catabolic players through next-generation sequencing techniques, and (iii) PCR-based identification of functional genes. This study is the first integrated ‘omics’ investigation on the biochemical mechanisms and phylogenetic diversity of steroid biodegradation in the environment. In brief, Introduction provides the background information of SHs, current knowledge on their biodegradation, and my research objectives. The studies of androgen degradation: the genome of the androgen anaerobic decomposer, Steroidobacter denitrificans was completely sequenced and annotated. Transcriptomic data revealed the gene clusters that were distinctly expressed during anaerobic growth on testosterone; besides, I identified the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, suggested as biomarkers to investigate androgen biodegradation. Based on the available biomarkers of androgen degradation, I investigated the biochemical mechanisms and corresponding microorganisms of androgen degradation in the anaerobic and aerobic sewage. Sewage samples collected from the Dihua Sewage Treatment Plant (Taipei, Taiwan) were incubated with testosterone (1 mM) anaerobically or aerobically. Androgen metabolite analysis indicated that denitrifying bacteria in anaerobic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assay showed androgen degradation in anaerobic sewage by Thauera spp. (mainly T. terpenica) through the action of 1-testosterone hydratase/dehydrogenase. Moreover, the 2.3-seco pathway utilized by T. terpenica 58Eu (DSMZ 12139) was also confirmed. By contrast, bacteria in aerobic sewage degraded androgens via the oxygenase-dependent 9,10-seco pathway, and the metagenomic analysis indicated the apparent enrichment of Comamonas spp. (mainly C. testosteroni) and Pseudomonas spp. in sewage incubated with testosterone. I used the degenerate primers derived from the meta-cleavage dioxygenase gene (tesB) of various proteobacteria to track this essential catabolic gene in the sewage. The amplified sequences showed the highest similarity (87–96%) to tesB of C. testosteroni. Using quantitative PCR, I detected a remarkable increase of the 16S rRNA and catabolic genes of C. testosteroni in the testosterone-treated sewage. The studies of estrogen degradation: Using a tiered functional genomics approach, I deciphered the catabolic enzymes and genes involved in estrogen biodegradation by a wastewater isolate, Sphingomonas sp. strain KC8. I identified the initial intermediates of this catabolic pathway, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. Further genomic and transcriptomic analyses revealed that two gene clusters are specifically expressed in strain KC8 cells grown on 17β-estradiol. I also characterized 17β-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A-ring, respectively. The 4-hydroxyestrone 4,5-dioxygenase gene and the characteristic pyridinestrone acid were detected in two wastewater treatment plants and two suburban rivers in Taiwan. In conclusion, the catabolic genes and characteristic metabolites can be used as the biomarkers to investigate fate and biodegradation potential of estrogens in the environment.

abstract: Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.
Dissertation/Thesis
Additional Paper
M.S. Civil and Environmental Engineering 2011

碩士
明志科技大學
環境與安全衛生工程系環境工程碩士班
103
Previous study has shown that bagasse compost sample could serve as microbial and electron source for anaerobic reduction of tetrachloroethene (PCE). In this study, different types and amendment methods were applied to add additional electron donors into the compost microcosms, to study their effects on the reduction dechlorination efficiency. During 180 days of incubation period, the experimental results showed that adding full amount (200 electron equivalent; EQ) of lactase in the microcosms on Day 0 would show the best PCE removal efficiency among different treatments; while adding full amount of acetate would exhibit the worst removal efficiency. If adding half amount (100 EQ) of lactase or acetate on Day 0 and Day 120 respectively, the PCE removal efficiency would be in the middle. Comparing with acetate, lactase could serve as slower electron release agents, in addition that lactase could provide greater alkalinity. Therefore, the microcosms amended with lactase would have more suitable pH environment for reductive dechlorination of PCE. If adding total 200 EQ lactase or acetate in the microcosms on Day 120, the enhancement of microbial reduction efficient was not as signification when compared with the electron donors were amended on Day 0, probably because the microbial activity decreased during the incubation period. Overall, the results suggested that the additional amendment of lactase would have better reduction efficiency when compared with acetate. The lactase amendment could be done at full amount at the beginning of the experiment, while the acetate amendment should be half amount.

This diploma thesis has been carried out as a part of the project Utilization of long term (passive) sampling methods combined with in situ microcosms for assessment of (bio)degradation potential (PASSES). In the frame of the project groundwater remediation took place in the premises of Farmak a.s. in Olomouc using a pilot photooxidation unit and efficiency of the remediation was monitored through passive and active sampling methods. Pilot photooxidation unit is a technology based on the H2O2/UV-C photochemical oxidation of organic pollutants. In this work optimization tests of the pilot photooxidation unit were performed. The residence time of the groundwater in the photoreactors, required for its sufficient decontamination from pharmaceuticals and aromatic hydrocarbons, was 2.5 hours. 91% degradation of the pharmaceuticals and 80% degradation of aromatic hydrocarbons were reached during this interval. Although the removal efficiency of the pharmaceuticals by the photooxidation unit was high, the pilot photooxidation unit was not able to effectively remove the pharmaceuticals at the studied locality. By comparing the results of the pharmaceuticals from active and passive groundwater sampling during the remediation attempt, passive Polar Organic Chemical Integrative Sampler (POCIS) was found to be...