The area of research for this thesis concerns the role played by microorganisms in the process of organic breakdown of waste effluent. Although considerable research has been done since the early 1950's, little consideration has been given to the role of the microorganisms in this type of waste water purification.

Objetivo: O objetivo do presente estudo foi desenvolver uma medicação intracanal contendo hidrocloreto de polihexametileno guanidina (PHMG) e hidróxido de cálcio Ca(OH)2, avaliando as suas propriedades e eficácia antimicrobiana. Métodos: O material foi formulado a partir da associação de 2% de PHMG ao pó de hidróxido de cálcio associado ao veículo propilenoglicol. O material foi avaliado pelos testes de análise antimicrobiana, escoamento, espessura de película, análise de pH e liberação de cálcio. Resultados: Os resultados foram analisados através do teste ANOVA de uma via, ANOVA de duas vias e teste tukey, com nível de significância de 5%. A atividade antimicrobiana com a associação de hidrocloreto de polihexametileno guanidina (PHMG) mostrou-se eficaz para Enterococcus faecalis. No teste de escoamento, o hidróxido de cálcio associado com o propilenoglicol mostrou um escoamento maior do que quando associado com o hidrocloreto de polihexametileno guanidina (PHMG) (p˂ 0,05). No teste de espessura de película os grupos não apresentaram diferença. Na análise de pH, os grupos variaram a alcalinidade nos tempos avaliados, durante o período de 28 dias (pH entre 7,5 – 11,5). Na liberação de cálcio, o uso do antimicrobiano hidrocloreto de polihexametileno guanidina (PHMG) não interferiu na liberação de cálcio do material. Conclusão: A adição de (PHMG) ao pó de hidróxido cálcio e ao veículo propilenoglicol não interferiu na liberação de cálcio, além de possuir efeito antimicrobiano, manteve o pH alcalino, não alterando a espessura de película, entretanto apresentou um menor escoamento do material. / Objective: The objective of the present study was to develop an intracanal medication containing polyhexamethylene guanidine hydrochloride (PHMG) and calcium hydroxide Ca (OH)2, evaluating its properties and antimicrobial efficacy. Methods: The material was formulated from the association of 2% PHMG with the calcium hydroxide powder associated with the propylene glycol vehicle. The material was evaluated by antimicrobial analysis, flow, film thickness, pH analysis and calcium release. Results: The results were analyzed through the one-way ANOVA, two-way ANOVA and tukey test, with a significance level of 5%. The antimicrobial activity with the combination of polyhexamethylene guanidine hydrochloride (PHMG) was shown to be effective for Enterococcus faecalis. In the flow test, the calcium hydroxide associated with propylene glycol showed a greater flow than when associated with polyhexamethylene guanidine hydrochloride (PHMG) (p<0.05). In the film thickness test, the groups showed no difference. In the pH analysis, the groups varied the alkalinity at the evaluated times, during the period of 28 days (pH between 7.5 - 11.5). In the release of calcium, the use of the antimicrobial polyhexamethylene guanidine hydrochloride (PHMG) did not interfere with the release of calcium from the material. Conclusion: The addition of (PHMG) to the calcium hydroxide powder and to the propylene glycol vehicle did not interfere with the release of calcium, besides having an antimicrobial effect, it maintained the alkaline pH, without altering the film thickness, however presented a lower material flow.

>Magister Scientiae - MSc / Antimicrobials are chemical compounds that destroy or inhibit the growth of microorganisms. The majority of these antimicrobials are actually natural products or natural product derived with key examples being the pioneer antibiotics penicillin and cephalosporin. Antimicrobials are an extremely important class of therapeutic agents; however, the development of drug resistance and slow pace of new antibiotic discovery is one of the major health issues facing the world today. There is therefore a crucial need to discover and develop new antibacterial agents. In this study, the potential of marine algae as a source of new antibiotics was explored. Crude organic extracts and chromatographic fractions obtained from small-scale extraction of 17 different marine algae were used to prepare a pre-fractionated library that would be tested against several disease causing microorganisms. The activity of the pre-fractionated library and purified compounds was determined against a panel of drug resistant microorganisms namely Acinetobacter baumannii ATCCBAA®-1605™, Enterococcus faecalis ATCC® 51299™, Escherichia coli ATCC® 25922™, Staphylococcus aureus subsp. aureus ATCC® 33591™ and Candida albicans ATCC® 24433™. Finally, cytotoxicity tests of 50 selected library extracts and isolated compounds were done against two cell lines namely MCF-7 (breast cancer) and HEK-293 (kidney embryonic). Based on their antimicrobial activity and interesting chemical profiles, the seaweeds Plocamium sp. and Stypopodium multipartitum were selected for further study. Three new and unusual halogenated monoterpenes (4.16, 4.17 and 4.18) were isolated from Plocamium sp., and an unusual meroditerpenoid (5.8) was isolated from Stypopodium multipartitum. The metabolites were purified using preparative (silica gel) chromatography as well as semipreparative normal phase HPLC. The structures of purified compounds were determined from spectroscopic data, including nuclear magnetic resonance (NMR) spectroscopy. A small library of 153 fractions was generated from collections of South African marine algae. Pre-fractionated crude extracts showed excellent antimicrobial activity against all microbes but particularly against Staphylococcus aureus. The compounds were generally active against the Gram positive bacteria and the yeast. In conclusion, three antimicrobial halogenated monoterpenes and an unusual monoterpene were isolated from a Plocamium sp. and Stypopodium multipartitum respectively. Antimicrobial activity of crude fractions was excellent but that of isolated compounds was not as great as anticipated.

Vibrio vulnificus is a marine pathogen of human health concern, capable of causing potentially fatal wound infections in a select group of the population. Previous studies have indicated this species’ strong negative correlation with salinity, not typically found above 30 ppt. This study assessed the ability of V. vulnificus to become Viable But Nonculturable in response to elevated salinity (35 ppt) as well as investigated novel methods for confirming their entrance into this state. Results showed a complete loss of culturability in both Environmental and Clinical strains of this bacterium by 9 days after inoculation. Using a High Content Imager, it was determined that these pathogens were not dying (< 10%) in response to the treatment and were partially becoming cocci (≈35%). This study indicates the importance of understanding the impact environmental parameters have on this human pathogen, and what it means for reliably detecting them. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Culture independent studies have revealed a greater diversity of Archaea than the two kingdoms initially defined by Carl Woese. Culturing species from the newly discovered archaeal lineages, as with the majority of microbes, has been difficult. To overcome the culturing dilemma, metagenomics is being used to reconstruct environmental genomes. Two metagenomic studies are presented in this thesis, with the aim of recovering newly proposed archaeal genomes from the Nanoarchaeota and Korarchaeota. In the first study, a sediment sample, NZ13, was collected from a terrestrial New Zealand hot spring. Along with the sediment sample, two complex enrichments were sequenced on an Illumina MiSeq platform. Assembly and differential binning recovered two nearly complete genomes of a nanoarchaeote and a korarchaeote. The NZ13 nanoarchaeote is similar to other terrestrial nanoarchaeotes, which lack an ATP synthase and encode genes for glycolysis/gluconeogenesis and archaella. One notable difference is the NZ13 nanoarchaeote contains CRISPR genes, which are absent in other terrestrial nanoarchaeotes, although present in a marine nanoarchaeote, Nanoarchaeum equitans. The NZ13 korarchaeote mirrors Candidatus Korarchaeum cryptofilum, lacking genes for de novo synthesis of purines and several cofactors, while containing an abundance of peptide transporters and amino acid fermentation pathways. The second study focused on sulfide samples collected from deep-sea hydrothermal vent fields in southwestern Pacific Ocean along the Eastern Lau Spreading Center. Ten sulfide samples were sequenced on an Illumina HiSeq platform. Small subunit ribosomal RNA genes were extracted from the metagenome reads and aligned against the SILVA Ref NR 99 123 database. The preliminary results identified which samples could be prioritized for genome reconstruction of uncultured bacterial and archaeal lineages. Three uncultured bacteria, candidate division SR1, Gracilibacteria (GN02), and Parcubacteria (OD1) were identified in several samples. Many uncultured deep-sea hydrothermal archaeal lineages were identified in all samples. In particular, korarchaeotal sequences were in high relative archaeal abundances in two samples, ABE 1 and Vai Lili-2, while few nanoarchaeotal reads were classified.

Includes bibliography.

Probiotic bacteria such as Lactobacillus and Bifidobacterium species are normal inhabitants of healthy gastrointestinal (GI) tract, which may promote beneficial effects on host through limiting the growth of undesirable micro-organisms and modulating the immune system. In the present study, Lactobacillus and Bifidobacterium strains were screened for their in vitro acid and bile tolerance, autoaggregation, coaggregation and hydrophobic abilities to identify potential probiotic bacteria. Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 were selected based on their overall tolerance to in vitro acidic conditions to further investigate their influence on various immune functions and gene expression in mice. Immunofluorescent analysis of small intestine in mice fed with L. acidophilus or L. paracasei demonstrated an increase of immunoglobulin (Ig)-A, interleukin (IL)-10 and interferon (IFN)- producing cells compared to control mice. In summary, L. acidophilus and L. paracasei showed tolerance to various gastric conditions and bile salts. Lactobacillus acidophilus and L. paracasei enhanced gut and systemic immune functions, particularly non-specific and specific immune responses in normal and CT mice. Moreover, L. acidophilus regulated the genes involved in various biological functions in small bowel of normal and CT mice, which provided a basis in understanding the pathways through which these bacteria are beneficial to the host. / Doctor of Philosophy (PhD)

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

The entrainment of microbes from agricultural land into overland flow during rainfall events is recognised as an important source of pathogenic microbes to surface water bodies and yet this transport process is poorly understood. In this study, a method has been developed to separate bacteria into the forms in which they have been postulated to exist in overland flow. Then Escherichia coli was used as a model organism to investigate the transported state of bacteria eroded from cowpats and their subsequent transport in overland flow. Simulated rainfall experiments were used to generate runoff direct from cowpats. Concentrations of E. coli in the runoff direct from cowpats were found to be directly proportional to the concentration in the cowpat, regardless of the age of the cowpat. It was also observed that E. coli were predominantly eroded from cowpats as individual cells. The interactions between E. coli and soil particles in overland flow were then examined in a small laboratory scale model system and showed that E. coli attached to large (>45 [mu]m) soil particles were transported significantly less than unattached cells. However, in the runoff from the model system, E. coli were found to be attached mainly to clay particles that were similar in size to the bacterial cells. Furthermore, the transport of E. coli through the model system appeared to follow the transport of a conservative chemical tracer implying that (a) the cells were being transported as a solute with the bulk of the water flow, and (b) that E. coli attached to small clay particles were as mobile in the overland flow as unattached cells. These observations imply that E. coli predominantly interact with small clay particles that are also being carried along in the overland flow. The transport of E. coli at a larger scale was then investigated using 5-metre long, 1-metre wide buffer strips operated under saturation excess conditions. In buffer strips using intact soils and existing pasture cover, E. coli removal was very poor (26 % removal) at the low flow rate of 2 L min⁻� with no removal observed at the higher flow rates of 6 and 20 L min⁻�. E. coli removal rates were increased to 41 % removal at 2 L min⁻� by cultivating the soils, with the removal rate again decreasing with increasing flow rate. E. coli in the overland flow from the buffer strips did not form into large flocs or attach to large soil particles, but were transported in small neutrally buoyant particles that remain entrained in the overland flow. Under saturation excess runoff conditions, E. coli in overland flow were not effectively removed by buffer strips as the small particles are transported either over the soil surface or, through large pores in the soil. This Thesis has shown that E. coli is transported in overland flow in small particle sizes that are difficult to trap or remove from overland flow thereby explaining the high fluxes of faecal bacteria observed in overland flow from agricultural land.

Metal-reducing microorganisms reduce a variety of metals in metabolic processes coupled to the oxidation of organic compounds. These bacteria play an important role in the biogeochemical cycling of metals and organic matter in anaerobic aquatic and sediment ecosystems. It has been proposed recently that metal-reducing microorganisms also are active in deep subsurface environments such as petroleum reservoirs. Only two metal-reducing bacteria have been isolated from petroleum reservoir fluids, Shewanella putrefaciens and Deferribacter thermophilus. This project studied the occurrence and distribution of metal-reducing microorganisms in petroleum reservoirs. The research focused on the isolation, characterisation and identification of anaerobic bacteria from petroleum reservoirs that were capable of reducing metals and the potential roles of these isolates in the microbial ecology and biogeochemical cycling of petroleum reservoirs. Petroleum reservoirs were selected for this study on the basis of physio-chemical conditions such as temperature, salinity, pH and the presence of organic and inorganic compounds, that were likely to provide a suitable environment for anaerobic bacteria capable of reducing metals. Factors such as the stratigraphic features of the sedimentary basin, age of reservoir and past oil field practices also were considered in choosing the reservoir for study. Seven petroleum reservoirs in the USA and Azerbaijan were chosen for extensive investigations. The physico-chemical conditions in these reservoirs varied substantially. A systematic study of the production water from these petroleum reservoirs revealed a consistent presence of iron- and manganese-reducing microorganisms. It was found that salinity and temperature play a significant and defining role in the occurrence and distribution of these metal-reducing microorganisms. Biotic metal reduction was detected from production waters from all but one of the oil wells sampled. It was significant that the water from this well (Neftcala #1074) was the most saline (78 g/l NaCI). Metal-reducing activity was detected at temperatures up to 70°C. Two pure cultures, strains RED1 for Redwash petroleum reservoir (USA) and NEF1 from the Neftcala petroleum reservoir (Azerbaijan) were isolated and characterized. The strains had diverse physiological and metabolic properties including the ability to oxidize a wide range of carbon compounds and reduce a variety of metals. Their temperature, salinity and pH optima varied markedly. Phylogenetic analyses of the 16S rRNA of strain RED1 showed that the strain represented a new species of a new genus in the domain Bacteria. The bacterium most closely related to strain RED1 is the fermentative Fe(III)-reducer, Pelobacter acetylenicus (similarity value, 92.8%). Strain NEF1 possesses a unique combination of phenotypic traits and a low mol % G+C. From preliminary analyses and comparative biochemistry, NEF1 appears to be a novel metal-reducing bacterium of the Flexistipes group. The bacteria isolated in this study were able to grow at temperatures and salinities consistent with the reservoir from which they were isolated. This indicated that petroleum reservoirs are a new source of physiologically diverse, novel, metal-reducing microorganisms. The bacteria isolated also demonstrated a number of characteristics that would enable them to survive and persist in extreme subsurface conditions and develop a selective ecological advantage in petroleum reservoir environments. Significantly, the metal-reducing bacteria isolated were able to utilize an array of metabolic products produced by bacteria indigenous to petroleum reservoirs. This has resulted in a new proposed model for the ecological succession of bacteria in petroleum reservoirs.