<p>U okviru ove doktorske disertacije prikazan je postupak izolacije i karakterizacije denitrifikatora uz optimizaciju biotehnolo&scaron;kih uslova proizvodnje biomase i ispitivanje efikasnosti proizvedene biomase u pogledu bioči&scaron;ćenja različitih građevinskih materijala.<br />U okviru istraživanja koja su obuhvaćena ovom doktorskom disertacijom, izolovan je nov soj denitrifikatora koji je pomoću metode MALDI TOF identifikovan kao Pseudomonas stutzeri. U svim eksperimentima karakteristike i performanse novoizolovanog soja P. stutzeri, interno označenog kao D1, su poređene sa referentnom kulturom P. stutzeri ATCC 17588, koji je model mikroorganizam na kome se izučava proces denitrifikacije. Primenom PCR metode utvrđeno je postojanje četiri ključna denitrifikujuća gena (napA, nirS, norB i nosZ) u genomu P. stutzeri D1 i referentnog soja, čime je pokazano da ispitivani sojevi imaju genetsku predispoziciju za izvođenje potpune aerobne denitrifikacije. Takođe, uz primenu Boks-Benkenovog eksperimentalnog dizajna i metode odzivne povr&scaron;ine, definisan je sastav hranljive podloge za dobijanje maksimalnog sadržaja biomase P. stutzeri D1 (1 g/L glukoze, 3 g/l KNO₃ i 4 g/L peptone) i P. stutzeri ATCC 17588 (2 g/L KNO₃ i 4 g/L peptona). Osim toga, optimizovani su i odabrani bioprocesni parametri za proizvodnju biomase oba denitrifikatora &ndash; temperatura 30&deg;C, pH vrednost kultivacionog medijuma 7 jedinica i veličina inokuluma 5% (v/v). Validacija optimizovanih parametara kultivacije izvr&scaron;ena je izvođenjem nove serije ogleda pri definisanim uslovima u laboratorijskim bioreaktorima ukupnih zapremina 3 L i 7 L, čime je pokazana stabilnost bioprocesa prilikom uvećanja zapremine kultivacione tečnosti uz ostvarenje maksimalnog sadržaja biomase od 9 log CFU/ml za oba ispitivana soja uz minimalno opterećenje otpadnog toka. Rezultati primene denitrifikatora u vidu bioaktivnog sistema (pulpa sa suspenzijom denitrifikatora) koji je nano&scaron;en na povr&scaron;inu ve&scaron;tački kontaminiranih model supstrata opeke ukazuju na visoku efikasnost uklanjanja nitrata i to 95 % u slučaju P. stutzeri D1 i 72% u slučaju referentnog soja. Ispitivanjem bioaktivnih sistema primenjenih na kontaminirane uzorke stenskog materijala konstatovano je da se najveća efikasnost postiže tokom prvih 24 h, kada je efikasnost bioaktivnog sistema sa P. stutzeri ATCC 17588 47,7%, a bioaktivnog sistema sa P. stutzeri D1 čak 89,2%.<br />U okviru doktorske disertacije izolovanjem i karakterizacijom visoko efikasnog denitrifikatora P. stutzeri D1 i detaljnim prikazom postupka optimizacije bioprocesnih parametara i validacije optimizovanih bioprocesnih parametara u laboratorijskim bioreaktorima načinjeni su prvi koraci ka definisanju idejnog re&scaron;enja i uvećanju razmera predloženog bioprocesa. Takođe, visoka efikasnost odabranih denitrifikatora u postupcima bioči&scaron;ćenja različitih građevinskih materijala, koja je postignuta u laboratorijskim uslovima predstavlja dobar osnov za nastavak istraživanja u realnim uslovima, kako na građevinskim materijalima, tako i u tretmanima zemlji&scaron;ta i otpadnih voda.</p> / <p>The main goal of this PhD thesis is to establish the procedure of isolation and characterization of denitrifiers along with the optimization of biotechnological conditions for the biomass production and testing the efficiency of the produced biomass in terms of biocleaning of various building materials.<br />Within the research involved in this PhD thesis, novel denitrifier strain was isolated and identified as Pseudomonas stutzeri by using MALDI TOF. In all experiments, the characteristics and performances of the newly isolated strain of P. stutzeri (internally denoted as D1) were compared with the reference strain P. stutzeri ATCC 17588, which is a model microorganism for studying the denitrification process. By using PCR method the presence of four key denitrification genes (napA, nirS, norB and nosZ) in genome of the both strains was established indicating their genetic predisposition to perform complete aerobic denitrification. Also, the composition of the cultivation medium for obtaining the maximum content of biomass of P. stutzeri D1 (1 g/L glucose, 3 g/l KNO₃ and 4 g/L peptone) and P. stutzeri ATCC 17588 (2 g/L KNO₃ and 4 g/L peptone) was detemined by employing Box-Bhenken experimental design and RSM. In addition, the selected bioprocess parameters were optimized for the production of biomass of the both denitrifiers &ndash; temperature 30 &deg;C, pH value of the cultivation medium of 7 units and inoculum size of 5% (v/v). Validation of the optimized bioprocess parameters was carried out by performing new series of experiments under previously define conditions in laboratory bioreactors with total volumes of 3 L and 7 L. The results of validation showed the stability of bioprocess while increasing the volume of the cultivation broth with the achievment of a maximum biomass content og 9 log CFU/ml for the both strains with minimum load of waste stream. The results of the application of the denitrifiers as a part of bioactive system (poultice with a suspension of denitrifiers) used onto the artificially contaminated brick model supstrates indicate a high efficiency of nitrate removal, namely 95% in the case of P. stutzeri D1 and 72% in the case of reference strain. By examing bioactive systems applied to contaminated stone material it was found that the highest efficiency in nitrate removal was achieved during the first 24 h (47.7% for the reference strian and 89.2% for the P. stutzeri D1).<br />The results obtained in this PhD thesis including the established procedure for isolation and characterization of denitrifiers and detailed presentation of the optimization and validation process of bioprocess parameters represent the first steps made towards defining the conceptual solution and scale up of the proposed bioprocess. Moreover, the high efficiency of selected denitrificators in the biological treatment of various building materials, achieved in laboratory conditions is a good basis for continuing research in real conditions on construction materials, as well as in soil and wastewater treatment.</p>

Selenium (Se) in the aquatic environment is predominantly found as the soluble selenium oxyanions; selenate (SeO42-), and selenite (SeO32-). These oxyanions are toxic, and they readily bio-accumulate in the food chain. However, numerous studies have proven the viability of microbial remediation in reducing them to elemental selenium (Se0) which is considered to be biologically inert and relatively less toxic. Of the various microorganisms that have been employed in Se bioremediation, Pseudomonas stutzeri NT-I has shown great potential in removing high concentrations of SeO42- and SeO32-. Of these two selenium oxyanions, SeO32- is more toxic, is the most reactive and is usually found in mildly oxidising acidic environments. Therefore, the focus of this study is on selenite. In this study Pseudomonas stutzeri NT-I was used in aerobic batch reduction of various SeO32- concentrations (0.5 to 10 mM) to Se0 under already known optimum conditions of temperature 35±2 °C, pH 7 – 8 and salinity 5 g.L-1 NaCl. The selenium (Se) reduction efficiency of strain NT-I was assessed under varying conditions, such as the presence and absence added nitrogen and glucose substrates, inhibited metabolic activity and bacterial cells. Moreover, the variation in the different parameters such the oxidation reduction potential (ORP), metabolic activity (MA) and the concentrations of SeO32-, glucose and total organic carbon (TOC) were also monitored. Key results indicated that the rate and amount of SeO32 reduction was influenced by the concentration to be reduced. For an initial SeO32- concentration of 0.5 mM, reduction was gradual and after 36 h, approximately 75 % had been reduced to Se0 which was equivalent to 0.375 mM. In the reduction of 10 mM SeO32- however, the reduction rate was rapid and even though the overall percentage reduction averaged around 18 %, this was equivalent to 1.8 mM This indicated that the increased initial reduction rate was a result of increased biomass activity in response to increased selenite concentration. This response is likely a defence mechanism employed by strain NT-I to detoxify its surrounding in elevated SeO32- concentrations. The results of these biological experiments were modelled, and the non-growth kinetics were found to fit the adapted Monod equation with k_s and k_Se values of 4.723 mM and 2.869 mmol.(h.g)-1 respectively. Pseudomonas stutzeri NT-I’s capability of being able to survive in very high selenium concentrations make it an attractive and versatile microbial species suitable for the bioremediation of selenium laden industrial wastewater. / Dissertation (MSc (Applied Science: Environmental Technology))--University of Pretoria, 2020. / Chemical Engineering / MSc (Applied Science: Environmental Technology) / Restricted

O presente estudo teve como objetivo avaliar a comunidade envolvida na desnitrificação autotrófica pela oxidação de sulfetos, aplicada ao pós-tratamento de efluentes anaeróbios. O enriquecimento da comunidade bacteriana e da comunidade desnitrificante autotrófica foi realizado a partir de amostras da biomassa coletadas de três reatores verticais de leito fixo operados em condições distintas, sendo, redução autotrófica de nitrato, redução autotrófica de nitrito e redução autotrófica de nitrato com excesso de sulfeto. Após a determinação da melhor condição de enriquecimento, a cultura foi purificada, identificada por meio de ferramentas da biologia molecular e caracterizada quanto às melhores condições de crescimento. O enriquecimento foi bem sucedido com a biomassa dos três reatores. No entanto, a condição de redução de nitrato com relação \'N\'/\'S\' igual a 0,8 foi a que apresentou maior concentração de microrganismos desnitrificantes autotróficos. A bactéria isolada foi identificada como Pseudomonas stutzeri. A velocidade específica máxima de crescimento da cultura (\'mü\'máx) foi de 0,037/h, com tempo de duplicação de 18,7 horas. O rendimento celular (Y) do composto nitrogenado foi de 0,15 gSSV.g/\'N\' e a velocidade de desnitrificação foi de aproximadamente 0,24 g\'N\'/gSSV.h. Os dados obtidos indicaram a viabilidade da aplicação da espécie isolada no processo de desnitrificação autotrófica utilizando sulfeto como doador de elétrons. / The aim of this study was to evaluate the community involved on autotrophic denitrification by sulfide oxidation applied to the post-treatment of anaerobic effluents. The enrichment of the bacterial community and autotrophic denitrifier community was accessed in three immobilized bed reactors operated at the conditions of autotrophic reduction of nitrate, autotrophic reduction of nitrite and autotrophic reduction of nitrate under excess of sulfide. Following the determination of the best enrichment conditions, the culture was purified, identified by molecular biology tools and the best growth conditions were characterized. Enriched cultures were obtained for the three operational conditions, but the best condition for the growth of autotrophic denitrifiers microorganisms was at \'N\'/\'S\' ratio of 0,8. The isolated microorganism was identified as Pseudomonas stutzeri. The maximum specific growth rate (\'mü\'máx) was 0.037/h, with a doubling time of 18.7 hours. The growth yield (Y) of nitrogen compound was 0.15 gSSV/g\'N\' and the specific rate of nitrogen utilization was approximately 0.24 g\'N\'/gSSV.h. The results indicated the viability of application of this microorganism for autotrophic denitrification using sulfide as electron donor.

O presente estudo teve como objetivo avaliar a comunidade envolvida na desnitrificação autotrófica pela oxidação de sulfetos, aplicada ao pós-tratamento de efluentes anaeróbios. O enriquecimento da comunidade bacteriana e da comunidade desnitrificante autotrófica foi realizado a partir de amostras da biomassa coletadas de três reatores verticais de leito fixo operados em condições distintas, sendo, redução autotrófica de nitrato, redução autotrófica de nitrito e redução autotrófica de nitrato com excesso de sulfeto. Após a determinação da melhor condição de enriquecimento, a cultura foi purificada, identificada por meio de ferramentas da biologia molecular e caracterizada quanto às melhores condições de crescimento. O enriquecimento foi bem sucedido com a biomassa dos três reatores. No entanto, a condição de redução de nitrato com relação \'N\'/\'S\' igual a 0,8 foi a que apresentou maior concentração de microrganismos desnitrificantes autotróficos. A bactéria isolada foi identificada como Pseudomonas stutzeri. A velocidade específica máxima de crescimento da cultura (\'mü\'máx) foi de 0,037/h, com tempo de duplicação de 18,7 horas. O rendimento celular (Y) do composto nitrogenado foi de 0,15 gSSV.g/\'N\' e a velocidade de desnitrificação foi de aproximadamente 0,24 g\'N\'/gSSV.h. Os dados obtidos indicaram a viabilidade da aplicação da espécie isolada no processo de desnitrificação autotrófica utilizando sulfeto como doador de elétrons. / The aim of this study was to evaluate the community involved on autotrophic denitrification by sulfide oxidation applied to the post-treatment of anaerobic effluents. The enrichment of the bacterial community and autotrophic denitrifier community was accessed in three immobilized bed reactors operated at the conditions of autotrophic reduction of nitrate, autotrophic reduction of nitrite and autotrophic reduction of nitrate under excess of sulfide. Following the determination of the best enrichment conditions, the culture was purified, identified by molecular biology tools and the best growth conditions were characterized. Enriched cultures were obtained for the three operational conditions, but the best condition for the growth of autotrophic denitrifiers microorganisms was at \'N\'/\'S\' ratio of 0,8. The isolated microorganism was identified as Pseudomonas stutzeri. The maximum specific growth rate (\'mü\'máx) was 0.037/h, with a doubling time of 18.7 hours. The growth yield (Y) of nitrogen compound was 0.15 gSSV/g\'N\' and the specific rate of nitrogen utilization was approximately 0.24 g\'N\'/gSSV.h. The results indicated the viability of application of this microorganism for autotrophic denitrification using sulfide as electron donor.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008. / Bibliography: p. 257-274. / Literature review -- General materials and methods -- Characterisation of strain collection -- Distribution of integrons and gene cassettes in pseudomonas -- Genomic context of pseudomonas integrons -- Evolutionary analysis of pseudomonas spp. integrons 199 -- Final discussion -- Appendix -- References. / Integrons associated with mobile genetic elements have played a central role in the emergence and spread of multiple antibiotic resistance in many pathogenic bacteria. However, the discovery of integrons in the chromosomes of diverse, non-pathogenic bacteria suggests that integrons have a broader role in bacterial evolution. The Pseudomonas stutzeri species complex is a well studied model for bacterial diversity. Members of the complex are genetically closely related, but sub-taxa are not able to be defined by exclusively shared sets of phenotypic characters. Rather, on the basis of total DNA:DNA similarity, Ps. stutzeri strains have been divided into 17 different groups (termed genomovars). Two Ps. stutzeri strains have been found to contain Chromosomal Integrons (CIs). This thesis involved exploration of the hypothesis that a CI was present in the common ancestor of the Ps. stutzeri species complex and assessed the impact of integrons on diversity across all Pseudomonads. The history and significance of integrons is discussed in Chapter 1 as part of a literature review, and general materials and methods are provided in Chapter 2. Chapters 3 - 6 comprise the sections in which data generated during my PhD project are presented. A comprehensive analysis of the relationships between the strains being analysed is presented in Chapter 3. In Chapter 4, results of PCR and hybridisation screening for integrons across the strain collection are presented. In Chapter 5 the recovery of additional integrons and in depth sequence analysis of the recovered integrons are described. Finally, Chapter 6 contains statistical analyses of integron-associated genes and Chapter 7 contains a final discussion the most significant findings. Twenty-three Pseudomonas spp. strains were screened for the presence of integrons. All but three were found to contain integron-like sequences; however, most integron sequences recovered contained inactivated core integrons. viii Despite having a chromosomal locus, integrons in Pseudomonas were found to have properties indicative of frequent horizontal transfer. Evidence was also obtained which suggests that integrons have been acquired at the same locus on multiple independent occasions. This has not been observed in other families of chromosomal integrons and suggests that the loci at which integrons in Pseudomonas are found are hotspots for recombination. / Mode of access: World Wide Web. / xiii, 274 p. ill

Bioremediation of seleniferous water is gaining more momentum, especially when it comes to bacterial reduction of the selenium oxyanions. More and more bacterial strains that are able to reduce selenium are being isolated. These bacteria need to be studied further to determine whether they are suited for industrial application. In this study, the reduction of Se(VI) to Se(0) by Pseudomonas stutzeri NT-I was examined using batch experiments with the bacteria suspended in MSM. For the determination of the optimum conditions for the growth of the bacteria, the linearized rate during the exponential phase for different conditions were compared. A pH of 7, temperature of 37 ⁰C, salinity of 20 g.L-1 NaCl and initial concentration of 5 mM selenate were found to be the best at promoting growth. To determine the optimum conditions for the reduction of selenium, the amount of Se(0) recovered from the plug after 16 hours of incubation was measured. A pH of 8, temperature of 37 ⁰C and salinity of 5 g.L-1 resulted in the most Se(0) recovered. The kinetics of the reduction of Se(VI) to Se(0) was found to follow the adapted Monod equation. An increase in the initial Se(VI) concentration positively affected the reduction rate indicating that substrate saturation had not yet been reached. One kmax could be fitted to each of the two reactions but not one Ks. It was found that Ks decreased with increasing initial selenate concentration. Visually it can be deduced that inhibition starts playing a role in the reduction of selenate at a concentration of 4 mM. Pseudomonas stutzeri NT-I is an exemplary selenium reducing agent and deserves more attention, not only for industrial application but also in the research world, for further understanding of the complex mechanism behind metal reduction in bacteria. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted