DEVELOPMENT OF A 2-DIMENSIONAL FINITE VOLUME MODEL TO ASSESS HYDRODYNAMIC AND MICROBIAL CONTROLS ON DNAPL DISSOLUTION AND DETOXIFICATION

Tetrachloroethene (PCE) and trichloroethene (TCE) form dense non-aqueous phase liquids (DNAPLs), which are persistent groundwater contaminants. DNAPL dissolution can be "bioenhanced" via dissolved contaminant biodegradation at the DNAPL-water interface. This research hypothesized that: (1) competitive interactions between different dehalorespiring strains can significantly impact the bioenhancement effect, and extent of PCE dechlorination; and (2) hydrodynamics will affect the outcome of competition and the potential for bioenhancement and detoxification. A two-dimensional coupled flowtransport model was developed, with a DNAPL pool source and multiple microbial species. In the scenario presented, Dehalococcoides mccartyi 195 competes with Desulfuromonas michiganensis for the electron acceptors PCE and TCE. Simulations under biostimulation and low velocity (vx) conditions suggest that the bioenhancement with Dsm. michiganensis alone was modestly increased by Dhc. mccartyi 195. However, the presence of Dhc. mccartyi 195 enhanced the extent of PCE transformation. Hydrodynamic conditions impacted the results by changing the dominant population under low and high vx conditions.

Measuring the Impact of Melaleuca quinquenervia Biochar Application on Soil Quality, Plant Growth, and Microbial Gas Flux

Biochar has been heralded a mechanism for carbon sequestration and an ideal amendment for improving soil quality. Melaleuca quinquenervia is an aggressive and wide-spread invasive species in Florida. The purpose of this research was to convert M. quinquenervia biomass into biochar and measure how application at two rates (2% or 5% wt/wt) impacts soil quality, plant growth, and microbial gas flux in a greenhouse experiment using Phaseolus vulgaris L. and local soil. Plant growth was measured using height, biomass weight, specific leaf area, and root-shoot ratio. Soil quality was evaluated according to nutrient content and water holding capacity. Microbial respiration, as carbon dioxide (CO2), was measured using gas chromatography. Biochar addition at 5% significantly reduced available soil nutrients, while 2% biochar application increased almost all nutrients. Plant biomass was highest in the control group, p2 flux decreased significantly in both biochar groups, but reductions were not long term.

Quorum Sensing and Microbial Interactions in Coral Black Band Disease and Coral-Associated Bacteria

The black band disease (BBD) microbial consortium often causes mortality of reef-building corals. Microbial chemical interactions (i.e., quorum sensing (QS) and antimicrobial production) may be involved in the BBD disease process. Culture filtrates (CFs) from over 150 bacterial isolates from BBD and the surface mucopolysaccharide layer (SML) of healthy and diseased corals were screened for acyl homoserine lactone (AHL) and Autoinducer-2 (AI-2) QS signals using bacterial reporter strains. AHLs were detected in all BBD mat samples and nine CFs. More than half of the CFs (~55%) tested positive for AI-2. Approximately 27% of growth challenges conducted among 19 isolates showed significant growth inhibition. These findings demonstrate that QS is actively occurring within the BBD microbial mat and that culturable bacteria from BBD and the coral SML are able to produce QS signals and antimicrobial compounds. This is the first study to identify AHL production in association with active coral disease.

Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Irrigation with Treated Wastewater: Potential Impacts on Microbial Function and Diversity in Agricultural Soils

The reuse of treated wastewater could be a promising measure to attenuate the water scarcity burden. In agriculture, irrigation with wastewater may contribute to improve production yields, reduce the ecological footprint and promote socioeconomic benefits. However, it cannot be considered exempt of adverse consequences in environmental and human health. Apart from the introduction of some biological and chemical hazardous agents, the disturbance of the indigenous soil microbial communities and, thus, of vital soil functions impacting soil fertility may occur. The consequences of these disturbances are still poorly understood. This chapter summarises the physicochemical and microbiological alterations in soil resultant from irrigation with treated wastewater that are described in scientific literature. These alterations, which involve a high complexity of variables (soil, wastewater, climate, vegetal cover), may have impacts on soil quality and productivity. In addition, possible health risks may arise, in particular through the direct or indirect contamination of the food chain with micropollutants, pathogens or antibiotic resistance determinants. The current state of the art suggests that irrigation with treated wastewater may have a multitude of long-term implications on soil productivity and public health. Although further research is needed, it seems evident that the analysis of risks associated with irrigation with treated wastewater must take into account not only the quality of water, but other aspects as diverse as soil microbiota, soil type or the cultivated plant species.

FROM GENES TO GENOMES: LOCAL ADAPTATION AND ADAPTIVE POTENTIAL IN TWO ARCTIC SEABIRDS

By investigating the mechanisms underlying the evolution and the maintenance of local adaptations we can help predict how species will adapt to future environmental change. In this thesis I investigate local adaptation and adaptive potential in thick-billed and common murres (Uria lomvia and U. aalge), two arctic seabirds of international conservation concern. Thanks to the recent development of new genomic methods, I address three major themes that are relevant for both the development of evolutionary theory and conservation: 1) the role of gene flow in the origin and maintenance of adaptation; 2) levels and distribution of standing genetic variation, and their contribution to adaptive potential; and 3) the genomic mechanisms maintaining an adaptive dimorphism within a single interbreeding population. First, I review the literature on genomics of local adaptation with gene flow and find that adaptation can be maintained despite gene flow, that gene flow itself can promote adaptation, and that genetic architecture is important in the origin and maintenance of local adaptations. Second, I genotype genome-wide markers and toll-like receptor genes (TLRs) to investigate local adaptation and adaptive potential in thick-billed murres. Thick-billed murres do not show signatures of local adaptation to their breeding grounds, but outlier loci group birds according to their non-breeding distributions, suggesting that selection and/or demographic connectivity in the winter may explain patterns of differentiation in this species. Genetic variation at TLRs does not decrease with increasing latitude as predicted, but tests of selection and measures of genetic diversity suggest differences in local selective regimes at most genes. Thick-billed murres show high levels of standing genetic variation and their adaptive potential will mostly depend on rate and magnitude of environmental change. Finally, I improve and annotate the assembly of the highly heterozygous genome of the thick-billed murre. Using this assembly as a reference, I perform whole genome analyses to investigate the genomic basis of an adaptive dimorphism in Atlantic common murres. I show for the first time that a 60 kb complex copy number variant in a non-coding region maintains differences in plumage and cold adaptation despite high gene flow.

Heat shock response in bacteria with large genomes: lessons from rhizobia

Rhizobia are important soil bacteria due to their ability to establish nitrogen-fixing symbioses with legume plants. In this dual lifestyle, as free-living bacteria or as plant symbiont, rhizobia are often exposed to different environmental stresses. The present chapter overviews the current knowledge on the heat shock response of rhizobia, highlighting how these large genome bacteria respond to heat from a transcriptional point of view. Response to heat shock in rhizobia involves genome wide changes in the transcriptome that may affect more than 30% of the genome and involve all replicons. In addition to the expected upregulation of genes already known to be involved in stress response (dnaK, groEL, ibpA, clpB), the reports on the heat shock response in rhizobia also showed particular aspects of stress response in these resourceful bacteria. The transcriptional response to heat in rhizobia includes the overexpression of a large number of genes involved in transcription and carbohydrate transport and metabolism. Additional studies are needed in order to better understand the transcriptional regulation of stress response in bacteria with large genomes.

Physicochemical and microbial analysis of feces from horses fed diets containing citrus pulp.

This study aimed to evaluate the e ect of diets containing increasing levels of citrus pulp on the physic-chemical and microbiological characteristics of horses feces. Five mares, at an average age of 3.5 years old and body weight of 492 ± 44.5 kg were arranged in a 5 x 5 Latin Square. The experimental diet consisted of 60% coast-cross hay and 40 % of concentrate with increasing levels of citrus pulp (0, 7, 14, 21, and 28 %). To determine the fecal pH, samples were collected directly from the oor, immediately after defecation, in the rst feces the day at 07:00 a.m., and color and fecal consistency were evaluated. For microbiological analysis, an aliquot was reserved in plastic bags, frozen, and sent to the microbiological laboratory for further analysis. Lactic acid bacteria were counted for Lactobacillus spp. and Streptococcus spp. from fecal samples under anaerobic conditions. The diet produced di erences (P<0.05) in feces consistency 98% had normal and rm stools, while 2% had loose ruminant-type feces. We observed no di erence (P<0.05) for color, verifying 100% of greenish feces, normal for equines. There was no e ect (P>0.05) on pH and on the number of Lactobacillus spp. and Streptococcus spp. The inclusion of up to 28% citrus pulp concentrates for horses did not promote change in the physio-chemical characteristics and on the population of lactic acid-producing bacteria in feces.

Microbial activities in soils cultivated with transgenic maize expressing Bacillus thuringiensis Cry1 Ab and Cry1 F genes.

A área brasileira plantada com milho geneticamente modificado (GM) expressando genes Cry derivados da bactéria do solo Bacillus thuringiensis (Bt) aumentou de 4,9% (5,0 milhões de hectares) da área total plantada em 2009 para 81,4% (15,83 milhões de hectares) em 2014. No entanto, estudos sobre os efeitos da tecnologia Bt-milho sobre microrganismos não alvo em solos tropicais são incipientes. Dessa forma, foi realizado experimento de campo para avaliar a atividade fisiológica das comunidades bacterianas associadas com genótipos de milho Bt plantados em Latossolo Vermelho Escuro do Cerrado e solos hidromórficos da planície com inundações localizadas. Um híbrido não transgênico (30F35) e seus homólogos transgênicos 30F35Y (Cry1Ab) e 30F35H (Cry1F) foram plantados com delineamento de blocos casualizados com quatro repetições. Solos rizosféricos e não rizosféricos coletados de plantas no estádio de florescimento foram submetidos aos ensaios de diversidade metabólica com Biolog e atividades enzimáticas de urease, arginase, fosfatase ácida e fosfatase alcalina. Solos rizosféricos apresentaram maior atividade microbiana e não foram detectadas diferenças significativas entre os genótipos em todos os parâmetros bioquímicos e de solo avaliados. Os resultados sugerem que o milho Bt não afeta negativamente a comunidade microbiana dos solos tropicais.

Effect of nitrogen fertilization on soil microbial biomass in an Oxisol cultivated with irrigated barley in the Brazilian Cerrado.

Abstract:The aim of this study was to evaluate the effect of different nitrogen doses and five period of sample collection, on soil microbial biomass - nitrogen (SMB-N), total nitrogen (total N) and percentual ratio of the microbial biomass and total N (SMB-N/total N) in a Oxisol cultivated with barley (Hordeum vulgare L.). The experiment was installed in June, 2005, in an area located at Embrapa Cerrados, Federal District. The experimental design was a randomized block, with three replicates. The plots received doses of nitrogen: 20 - 40 - 80 kg ha-1 N and a control without it and the subplots were period of soil sample. Three applications of N were realized: 10 kg ha-1 on the 5th day (06/14) after sowing; the rest of N was parceled in two applications with fertigation, on tillage, on the 27th (07/08) DAP, e no 43rd (07/22) DAP. Soil samples layer (0 - 10 cm deep) were collected for (SMB-N) determination and total N in six periods: 02 days before of the first fertigation; 02 days after of the first fertigation; 04 days before of the last fertigation and 04 days after of the last fertigation; on flowering stage and after harvesting. There was effect of the doses of N and the period of soil collection on the SMB-N, total N and in the ratio SMB-N/total N. The average values of total N revealed steadier in short-term (cycle of the culture) and this was not a good parameter to evaluate the behavior and N transformations in the soil-plant system. Resumen: El objetivo de este estudio fue evaluar el efecto de diferentes dosis de nitrógeno y cinco período de muestreo en la biomasa microbiana del complejo suelo - nitrogeno (BMS-N), nitrógeno total (N total) y la relación porcentual de la biomasa microbiana y N total (BMS-N/N total) en un Oxisol cultivado con cebada (Hordeum vulgare L.). El estudio se inició en junio de 2005 en la estación experimental de la Empresa de Pesquisa Agropecuaria (Embrapa-Cerrados), Distrito Federal, Brazil. El experimento se dispuso en bloques al azar con tres repeticiones. Las parcelas recibieron dosis de nitrógeno: 20 - 40 - 80 kg/ha de N más un control sin N, y las subparcelas fueron el periodo de muestro. Las aplicaciones de N se realizaron de la forma siguiente: cinco días después de la siembra (dds) se aplicaron 10 kg/ha y el resto de la dosis se aplicó con fertirrigación en dos dosis 27 y 43 dds. Las muestras de suelo (0-10 cm de profundidad) para determinar BMS-N y N total fueron tomadas, 2 días antes e igual número de días después de la primera fertirrigación y 4 días antes y después de la última, en floración y después de la cosecha. No se encontró efecto de las dosis de N y el período de muestreo en el BMS-N, N total y en la relación BMS-N/N total. Los valores medios de N total fueron más estable en el corto plazo (ciclo de la cultivo) lo que indica que éste no es un buen parámetro para evaluar la dinámica del N y sus transformaciones en el sistema suelo-planta.

Prediction of G protein coupled receptors from plant genomes.

2016

Microbial diversity and metabolic potential in caves

Caves are dark and oligotrophic habitats where chemotrophic microbial communities interact with the inorganic mineral rocks and cooperate organizing themselves in complex biological formations, which are visible in caves as biofilms, biodeposits or biospeleothems. In these environments, microorganisms contribute to the turnover of the matter and activate peculiar enzymatic reactions leading to the modification of the mineral rocks and to the production of metabolites with possible industrial and pharmaceutical interest. In this PhD thesis, various molecular and geomicrobiological approaches were used to investigate the microbial diversity and potential activities in different cave systems, i.e. the orthoquartzite cave Imawarì Yeuta, the sufidic cave Fetida and the ice cave Cenote Abyss. This is aimed at gathering indications on the possible interactions that support microbial growth and its impact in cave environments. As a result, microbial taxa and functions associated to light-independent chemolithotroph and heterotrophic activities were identified in the three caves, indicating the involvement of microorganisms in i) silica mobilization and amorphization processes and the formation of a novel type of silica-based stromatolite in Imawarì Yeuta Cave, ii) the formation of three types of biofilm/biodeposit involved in sulphur cycle and in the speleogenesis of Fetida Cave, iii) the development of biofilms and their maintenance under psychrophilic conditions in samples collected from ice in Cenote Abyss. Additionally, the metabolic potentials of around one hundred isolates derived from these cave systems were evaluated in terms on anti-microbial activity. The results pointed out that unexplored and oligotrophic caves are promising environments for novel bioactive molecules discovery.

Computational resources for structural and metagenomic characterization of functions in bacteria and bacterial communities. Antimicrobial resistance, pathways for xenobiotic degradation and relationship between gut microbiome and ageing.

Prokaryotic organisms are one of the most successful forms of life, they are present in all known ecosystems. The deluge diversity of bacteria reflects their ability to colonise every environment. Also, human beings host trillions of microorganisms in their body districts, including skin, mucosae, and gut. This symbiosis is active for all other terrestrial and marine animals, as well as plants. With the term holobiont we refer, with a single word, to the systems including both the host and its symbiotic microbial species. The coevolution of bacteria within their ecological niches reflects the adaptation of both host and guest species, and it is shaped by complex interactions that are pivotal for determining the host state. Nowadays, thanks to the current sequencing technologies, Next Generation Sequencing, we have unprecedented tools for investigating the bacterial life by studying the prokaryotic genome sequences. NGS revolution has been sustained by the advancements in computational performance, in terms of speed, storage capacity, algorithm development and hardware costs decreasing following the Moore’s Law. Bioinformaticians and computational biologists design and implement ad hoc tools able to analyse high-throughput data and extract valuable biological information. Metagenomics requires the integration of life and computational sciences and it is uncovering the deluge diversity of the bacterial world. The present thesis work focuses mainly on the analysis of prokaryotic genomes under different aspects. Being supervised by two groups at the University of Bologna, the Biocomputing group and the group of Microbial Ecology of Health, I investigated three different topics: i) antimicrobial resistance, particularly with respect to missense point mutations involved in the resistant phenotype, ii) bacterial mechanisms involved in xenobiotic degradation via the computational analysis of metagenomic samples, and iii) the variation of the human gut microbiota through ageing, in elderly and longevous individuals.