Os manguezais são ecossistemas complexos tipicamente encontrados na interface entre a terra e o mar. Apesar de sua grande importância ecológica estes ambientes estão em risco, devido à proximidade de áreas com elevada exposição a poluentes, como hidrocarbonetos policíclicos aromáticos (HPAs) liberados em derramantos de petróleo. Em nosso trabalho nós exploramos a diversidade e abundância taxonômica e funcional de bactérias em três manguezais localizados sob diferentes estágios de preservação no Estado de São Paulo. Os resultados mostraram que a concentração total de HPAs nos sedimentos foram diferentes entre todos os pontos de amostragem, com o sedimento de manguezal apresentando as concentrações mais elevadas. Genes <font face=\"Symbol\">a-ARHDs foram encontrados em todos os locais de amostragem, revelando a presença de enzimas envolvidas no metabolismo do bifenilo, naftaleno, dibenzofurano, 3-fenilpropanoato e benzeno. A PCR em tempo real demonstrou um maior número de cópias do gene <font face=\"Symbol\">a-ARHD e 16S rRNA nas áreas contaminadas. A análise das seqüências obtidas pelo sequenciamento do gene 16S rRNA mostrou estruturas de comunidades distintas para todas as amostras. O filo mais frequente foi Proteobacteria e o número de Unidades Taxonômicas Operacionais (OTUs) detectadas nas amostras da área preservada foi superior às daárea contaminada. A análise das seqüências obtidas para o pirosequenciamento do gene bph indicou um maior número de Famílias Proteicas Operacionais (FPOs) no ambiente com atividade antrópica. Em conclusão, os resultados permitiram acessar e identificar uma extensa diversidade de bactérias, incluindo os genes ARHD, 16S rRNA e bph. Tais dados podem oferecer novas abordagens para melhorar a recuperação de tais ambientes. / Mangroves are complex ecosystems typically found at the interface between land and sea. Despite its great ecological importance, these environments are at risk due to the proximity of areas with high exposure to pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) released by oil spill. In our work we explore the diversity and taxonomic and functional abundance of bacteria in three mangroves under different stages of preservation located in the state of São Paulo. The results showed that the total concentration of PAHs in sediments were different between all the sampling sites, with mangrove sediment having the higher concentrations. <font face=\"Symbol\">a-ARHDs genes were found in all the sampling sites, revealing the presence of enzymes involved in the metabolism of biphenyl, naphthalene, dibenzofuran, 3-phenylpropanoate and benzene. The real-time PCR demonstrated an increased number of copies of the <font face=\"Symbol\">a-ARHD and 16S rRNA genes in the contaminated areas. The analysis of the sequences obtained by pyrosequencing of 16S rRNA gene showed distinct communities structures for all samples. The phylum Proteobacteria was more frequent and the number of Operational Taxonomic Units (OTUs) detected in pristine samples area was higher than contaminated area. The analysis of the sequences obtained for pyrosequencing of bph gene indicated a greater number of Operational Protein Families (OPFs) in the environment with human activity. In conclusion, the results allow to access and identify a wide variety of bacteria, including ARHD genes, 16S rRNA and bph. Such data may provide new approaches for improving the recovery of such environments.

O efeito de alterações climáticas sobre os micro-organismos ainda é incerto, pois pouco se conhece sobre as espécies que habitam regiões extremas como o gelo, solo antártico, e o solo permanentemente congelado (permafrost). O permafrost tem como característica a preservação de material biológico por milhões de anos, servindo como fonte para estudos de evolução e biogeografia de micro-organismos. O objetivo deste trabalho foi estudar a diversidade microbiana em amostras de gelo, solo exposto por recuo de geleira e permafrost polares, e a diversidade funcional do gene alcano monoxigenase (alk). Métodos independentes de cultivo baseados no gene 16S rRNA foram utilizados, como DGGE, clonagem e pirossequenciamento. As geleiras da Ilha Rei George (Península Antártica) e do Pólo Sul Geográfico possuem cerca de 3.104 cél./mL e são compostas por micro-organismos diferentes, com predominância dos Filos Proteobacteria, Actinobacteria, Firmicutes e Cyanobacteria, muitos dos quais já descritos em outros ambientes criogênicos. O solo em frente à geleira Baranowski apresenta uma estrutura de comunidade diferente do gelo. O solo exposto por recuo de geleira apresenta uma sucessão ecológica, com predominância de heterotróficas durante todo o processo. Fixadores de nitrogênio no solo foram compostos por cianobactérias no início, e por Rhodopseudomonas e Rhodobacter no final da sucessão. Estes resultados foram melhor observados com o pirossequenciamento. As mudanças observadas podem estar relacionadas ao aumento de K, Mg+, NH4+, NO3- e/ou CO2 detectados após 15-20 anos de exposição do solo. A comunidade de permafrosts varia com o local e a idade de congelamento (de 5.000 a 8 milhões de anos). O gene alkM foi detectado em permafrosts do Ártico com 3 milhões de anos, e o gene alkB em amostras do Ártico com 15.000 e 120.000 anos, e em solos modernos da Antártica. Alguns clones indicam que podem representar novos genes para alcano monoxigenases. As contribuições deste projeto abrangem os objetivos do Ano Polar Internacional (IPY 2007-2009), sobretudo na avaliação da ecologia microbiana da Antártica. / The effect of climate changes on microorganisms is still unclear, because little is known about the species that inhabit the extreme regions as the glacial ice, antarctic soils and the permanently frozen soil (permafrost). The permafrost is able to preserve the sedimented biological materials by thousands or even millions of years, being an important source for microbiological studies. The objective was to study the microbial diversity in cryogenic samples: glacial ice, soil exposed by glacial retreat and polar permafrosts, as well as to study the functional diversity of alkane monooxygenase genes (alk) in the permafrost. Cultivationindependent methods based on the 16S rRNA gene were used, as DGGE, clone library and 454 Pyrosequencing. Analysis of the King George Island (Antarctic Peninsula) glaciers and the South Pole ice revealed about 3x104 cells/mL each, and different micro-organisms were detected, predominantly members from Proteobacteria, Actinobacteria, Firmicutes and Cyanobacteria, many of which already described in other cryogenic environments. The soil in front of the Baranowski Glacier has a different community structure compared with the ice. Soils exposed by glacier retreat revealed an ecological succession, and heterotrophic bacteria occurred all through the process. Nitrogen-fixing populations were composed by cyanobacteria at the early stages, and shifted to Rhodopseudomonas and Rhodobacter in the older soils. The observed changes may be related to an increase of K, Mg+, NH4 +, NO3- and/or CO2, detected after 15-20 years of soil exposure. The community of permafrosts varies by location and age (5,000 - 8 millions of years). The alkM gene was detected in old Arctic permafrosts (3 millions of years), while alkB genes were found on Arctic samples from 15,000 to 120,000 years, and in Antarctic modern soils. Some of these clones may represent new alk genes. The contributions of this project covers the goals of the International Polar Year (IPY 2007-2009), particularly in assessing the microbial ecology of Antarctica.

Este projeto teve como objetivo a construção de biosensores para a detecção de compostos monoaromáticos do grupo BTEX a partir dos componentes da via de degradação de compostos monoaromáticos codificada no plasmídeo TOL de Pseudomonas putida, mais precisamente: o promotor Pu e a proteína reguladora XylR, que ativa o promotor Pu após a ligação ao efetor monoaromático. Um objetivo secundário foi a verificação da existência de sequências reguladoras desconhecidas a montante do promotor Pu, construindo três variantes com fragmentos de Pu que se estendem por diferentes comprimentos a montante do promotor (Pu202 pb, Pu396 pb e Pu802 pb), cuja existência foi sugerida em trabalho anterior do laboratório. O promotor Pu foi ligado ao gene indicador para fosfatase alcalina isolado de E. coli. Todos os componentes das três variantes de biossensores foram clonados com sucesso. A construção de um dos plasmídeos de biossensoramento com a variante mais curta de Pu (Pu202) foi concluída. / Monoaromatic compounds are mainly responsible for the contamination of areas, this is because they are components of gas and the fuel stations most of accidents sites. Such compounds are highly toxic and, in between non-polar compounds, present high solubility and vapor pressure which assists them in dispersion at groundwaters and soil. This work aim to develop a biosensor based on alkaline phosphatase indicator gene under the regulation of the Pu promoter and its regulatory protein, XylR, that is activated by monoaromatic. Moreover, this work will continue a previous project of the research group that indicated a possible regulatory region not described for Pu, that hypothesis will be tested by producing different plasmids biosensors with varying sizes of Pu (202 bp, 396 bp and 802 bp). All biosensor fragments were purified and cloned on pGem T Easy and biosensor with Pu 202 pb was produced. Next goals are finishing others biosensors assemble and perform induction tests.

Amostras de biofilme de biomassa suspensa de tanque de aeração de lodo ativado, de córrego contaminado com esgoto e de vários pontos de planta de tratamento de água foram analisadas com relação à presença de bactérias indicadoras e patogênicas. Nos testes presuntivos foram detectados todos os microrganismos-alvo (Coliformes, Salmonella spp, Klebsiella spp., Staphylococcus spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., Clostridium spp., Shigella spp., Aeromonas spp., Campylobacter spp. e Legionella spp.), porém nos testes confirmatórios por PCR e teste bioquímico somente as espécies E. coli, Salmonella enterica subsp. Enterica serovar Typhi, K. pneumoniae, V. cholerae, A. hydrophila, e L. pneumophila foram confirmadas para algumas cepas selecionadas dos testes presuntivos. S. flexneri foi somente confirmada por teste bioquímico e S. aureus somente por PCR. Nenhuma amostra de C. jejuni foi confirmada por nenhum dos testes. Estes resultados demonstram que os meios seletivos para testes presumptivos não se mostraram confiáveis uma vez que muitas amostras presuntivas não foram confirmadas por PCR ou teste bioquímico específico. Estes resultados demonstram o potencial de biofilmes como reservatório de patógenos. / Biofilm samples from activated sludge reactors, surfaces from water treatment plants and water samples from a creek contaminated with raw sewage were analyzed for the presence of microbial indicator bacteria and pathogens. All target organisms (Coliforms, Salmonella spp, Klebsiella spp., Staphylococcus spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., Clostridium spp., Shigella spp., Aeromonas spp., Campylobacter spp. and Legionella spp.) were detected by using presumptive testing media. Only a small proportion of positive colonies from presumptive tests were confirmed as pathogenic strains of E. coli, Salmonella enterica subsp. Enterica serovar Typhi, K. pneumoniae, V. cholerae, A. hydrophila, and L. pneumophila in confirmatory testing by selective PCR and biochemical tests. S. flexneri was only confirmed in biochemical tests, S. aureus only by PCR and no colony of C. jejuni was confirmed positive by either PCR or biochemical testing. Presumptive media are therefore not safe means for assessing pathogen load in biofilm samples. These results demonstrate the importance of microbial biofilms as reservoirs for microbial pathogens.

Bacterio-phytoplanktonic coupling and bacterial growth efficiency (BGE) measurements were used to analyze microbial trophic dynamics and the influence of environmental factors in Florida Bay, Florida. Phytoplankton gross primary productivity (GPP) was measured using 24-hour in situ oxygen incubations; bacterial productivity (BP) was measured using 3H- thymidine incorporation. Weak bacterio-phytoplanktonic coupling was observed over the sampling period. BP was more influenced by local total nitrogen concentrations while GPP was more evenly distributed. BGE rates were low but consistent with marine and estuarine ecosystems worldwide. Results suggest that bacterioplankton growth in Florida Bay is relatively uncoupled from phytoplankton production, which may be due in part to the low levels of phytoplankton biomass in the water column, the large amount of seagrass-derived DOM production in this shallow lagoon, the loading of nitrogen and organic matter associated with terrestrial runoff, and/or their combination.

246 samples of bulk and packaged spices from retail stores in the western, southeastern, southern, midwestern, and northeastern areas of the U.S. were examined for the presence of Clostridium -perfringens. Isolates were checked for the presence of the lecithinase gene (cpa) and enterotoxin genes (cpe) by PCR. Enterotoxin formation during sporulation was investigated using the Oxoid Toxin Detection Kit. Forty-three confirmed isolates (from 17% of total samples) were cpa-positive. Of those, 27 were cpe-positive. Together, levels of C. perfringens spores ranged from 3.6-2400/gm. The amount of enterotoxin in cell extracts ranged from 2-16 ng/ml. Some of the SEM images of isolated spore (# 78) and one plasmid-borne ent control (FD-153) showed an organized surface structure termed “candy-wrapper”. This extracellular structure remained after treatment with 0.1 % SDS for 1 hr, suggesting it was not composed of membrane debris from the mother cell. The D values of spores ranged from 1.19- 3.31 min. The addition of lysozyme in the plating medium elevated the recovery rate of heat-treated spores. The growth rate of a cocktail of spores from spices (# 31, # 32, # 45) between 4 to 5 hr after inoculation was determined with a doubling time of 6.82 min in hamburger. A cocktail of spores of plasmid-borne ent control showed an optimum growth rate between 5 to 8 hr after inoculation with doubling time of 15.98 min. However; spice isolate cocktail, plasmid-borne ent control cocktail (FD-5603 and FD-153), and a chromosome-borne ent control (NTCT 8239) were unable to germinate and outgrowth at 20oC. Inoculation in laboratory medium FTG indicated the same result as hamburger at 20oC. The ability of C. perfringens spores in spices to potentially survive cooking procedures can be followed by germination and growth of vegetative cells during improper cooling to levels associated with foodborne illness caused by this organism. Our results suggest that retail spices are potential vehicles of transmission of enterotoxin-positive C. perfringens.

In a large agricultural operation, such as the one at Cal Poly San Luis Obispo, disposal of deceased animals is an immense issue. The cost of transporting and rendering every dead animal is inhibitory to the general function of the agricultural operations and their thin budget. Therefore, we propose that composting mortalities could be an economical alternative. Composting is a recognized method for taking animal waste products along with carbon waste and turning it into a pathogen-free, nutrient-rich topsoil. Carcass composting is in fact performed in other countries and states to varying degrees of success. However, the California EPA limits carcass composing to only private land. Therefore, the purpose of this work was to determine the efficacy of killing pathogens by composting using bench top composting models. Ultimately, our goal is to provide “proof of concept” data in order to gain permission for a full-scale carcass compost pile to be set up at Cal Poly San Luis Obispo. Using thermo tolerant Salmonella senftenberg as an indicator organism, we performed bench top trials of traditional and carcass compost in the lab. Samples were inoculated with S. senftenberg and kept at 55°C for 15 days in accordance with the California EPA and Test Method for the Examination of Composting and Compost (TMECC). Samples were then plated and processed for multiple tube analysis and most probable number. Samples were also partitioned for a viability qPCR with propidium monoazide (PMA) to compare to the classic techniques. Using these methods we were then able to track and produce thermal death time data for S. senftenberg in both traditional and carcass compost. By comparing the types of compost, we were able to determine that the composting method presented by the California EPA and the TMECC produces safe, pathogen free compost, even when inoculated carcasses were introduced. However, even with removal of dead cells by PMA, qPCR did not outperform the classical microbiological methods for as tracking pathogen killing.

South Florida contains one of the largest subtropical wetlands in the world, and yet not much is known about the microbes that live in these surface waters. These microbes play an important role in chemical cycling and maintaining good water quality for both human and ecosystem health. The hydrology of Florida’s surface waters is tightly regulated with the use of canal and levee systems run by the US Army Corps of Engineers and The South Florida Water Management District. These canals run through the Everglades, agriculture, and urban environments to control water levels in Lake Okeechobee, the Water Conservation Areas, and the surrounding farm lands. I hypothesized that there would be noticeable shifts in the microbial communities (also known as “microbiomes”) at the agriculture and urban sites due to anthropogenic influences such as agricultural and sewage runoff. It is also hypothesized that the diversity and stability of these sites will differ from the natural environment Grassy Waters Preserve (GWP), which we studied as a control. The northern section of GWP is a rain-fed Everglades ecosystem with little influence from manmade canal systems, so GWP can represent wetlands before human influences. High-throughput 16s rRNA sequencing was conducted on 112 GWP, canal, and agricultural water samples taken over a one-year period from September 2016 to November 2017. Data were processed in Qiime2 using DADA2 and resulted in 67732 unique taxa. Nineteen metadata factors were measured for 87 of the sampling points to investigate environmental effects. These factors explained 25% (r2=0.25, p=0.002) of the variation between sample locations. Conductivity was found to have the highest effect on microbial diversity (r2=0.078, p=0.002) while latitude and month also significantly influenced the microbial makeup. Urban and agricultural sites were found to have higher stability with lower variation in microbiomes over the course of study. The GWP site was found to have a high seasonality, probably due to its dependence on rain. The most abundant taxa for all sites (urban, agriculture, and control) were; family Spirochaetaceae, phylum Actinobacteria, and family Burkholderiaceae, respectively. Contamination of GWP and canal sites was also investigated using SourceTracker code. Intracoastal waters that receive canal water were found to be heavily influenced in the peak wet season when there is high flow through from the canals. GWP had little influence from farm lands compared to a high influence of agriculture on the urban sites.

Nanoparticles are particles with sizes between 1 and 100 nanometers (nm). Owing to their unique chemical, electrical, mechanical, optical, and piezoelectric properties, zinc oxide nanoparticles (ZnO-NPs) are finding widespread use in numerous applications with yearly production over 550 tons per year. Increasing use of ZnO NPs, and NPs in wastewater discharges from domestic and industrial sources will have significant potential for adverse impacts on aquatic phototrophic organisms. Comparative studies on microalgae species response to ZnO NPs and variation in tolerance among species is still mostly unexplored. The proposed research aims to evaluate interspecies’ variation in tolerance to ZnO NPs among marine and freshwater microalgae. Multi-well culture plate and flask culture screening methods were utilized for assessing microalgae species’ tolerance to various levels of ZnO NPs. Microalgae cell morphology changes in response to nano ZnO exposure were explored using both the Optical Coherence Microscope (OCM) and SEM. Availability of Nano ZnO tolerant microalgae species may provide an impetus for future studies to understand the mechanism of tolerance and potential applications in NPs bioremediation in aquatic systems.

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight.