<p>U radu je ispitana zastupljenost azotofiksirajućih cijanobakterija, ukupnog broja algi i ukupnog broja bakterija u različitim tipovima zemlji&scaron;ta na jedanaest lokaliteta u Vojvodini, od čega se sedam nalaze u za&scaron;tićenim prirodnim dobrima. Ispitana je brojnost u zavisnosti od dubine pedolo&scaron;kog profila, kao i od godi&scaron;njeg doba. Izvr&scaron;ena je izolacija sojeva azotofiksirajućih cijanobakterija, određena njihova taksonomska pripadnost i osnovne citolo&scaron;ke karakteristike. Ispitana je ultrastruktura vegetativnih ćelija, heterocista i spoljnih struktura na ćelijama fimbrije i pili, transmisionim elektronskim mikroskopom. Izvr&scaron;ena je genetička karakterizacija izolovanih sojeva azotofiksirajućih cijanobakterija PCR metodom analizom STRR fragmenata DNA.<br />Brojnost azotofiksirajućih cijanobakterija i ukupna brojnost algi je bila znatno veća kod hidromorfnih i halomorfnih zemlji&scaron;ta, nego kod automorfnih zemlji&scaron;ta. Najveća prosečna godi&scaron;nja brojnost azotofiksirajućih cijanobakterija, u povr&scaron;inskom sloju 0-5cm, je utvrđena kod zemlji&scaron;ta fluvisol u SRP &ldquo;Koviljsko petrovaradinskom ritu&rdquo;, 150864 jedinki po gramu apsolutno suvog zemlji&scaron;ta. Zemlji&scaron;te sa najnižom brojno&scaron;ću azotofiksirajućih cijanobakterija, je gajnjača u NP Fru&scaron;ka gora, 1582 jed./gr zemlji&scaron;ta u povr&scaron;inskom sloju.<br />Kod svih ispitivanih zemlji&scaron;ta brojnost azotofiksirajućih cijanobakterija je bila najveća u povr&scaron;inskom sloju zemlji&scaron;ta, 0-5 cm dubine, opadala je sa dubinom zemlji&scaron;ta i bila najmanja u najdubljem sloju, 30 - 60 cm. Kod najvećeg broja ispitivanih zemlji&scaron;ta brojnost azotofiksirajućih cijanobakterija je bila najveća tokom zimskog perioda. Azotofiksirajuće cijanobakterije su dominantne u na&scaron;im zemlji&scaron;tima i zastupljene sa 56.27% u odnosu na druge grupe algi.Izolovano je 30 sojeva azotofiksirajućih cijanobakterija: 19 sojeva Nostoc-a, 4 soja Anabaena, 4 Cylindrospermum, i po jedan soj Calothrix, Tolypothrix i Phormidium. Prosečna zastupljenost heterocista, ćelija koje vr&scaron;e azotofiksaciju, kod roda Nostoc je iznosila 8.28%, Anabaena 4.25%, Cylindrospermum-a 2.93%, Calotrix elenkinii 6.19% i Tolypothrix 7.76%.</p><p>Ultrastrukturnim ispitivanjem, TEM mikroskopom, vegetativnih ćelija azotofiksirajućih cijanobakterija uočili smo inkluzije redovnog pojavljivanja: karboksizome (Cs), cijanoficinkse granule (CG), polifosfatne granule (PG), ribozome (R), lipidne granule (&szlig; &ndash;granule) i tilakoide (T), kao i inkluzije neredovnog pojavljivanja: membranom ograničene kristalne inkluzije.<br />Koristeći TEM i tehniku bojenja ćelija sa RR i ultratankih preseka utvrdili smo prisustvo omotača od fimbrija kod tri soja (A.314, A.azollae i N.302) i tipične fimbrije kod dva soja (N.311 i N.9229). Metodom negativnog bojenja NS PTA uočili smo takođe tipične fimbrije, igličastog-dlakastog izgleda, jasnih granica niti kod tri soja (N.302, N.7901 i N.9229), međutim uočili smo i atipične sluzne fimbrije, koje nemaju jasno izražene granice, ali su veoma moćno ra&scaron;irene oko vegetativnih ćelija, kod tri soja (A.314, A.azollae, N.311).<br />Kod simbiotskih-infektivnih sojeva N.7901 i N.9229 javljaju se samo tipične fimbrije iz prve klase, a kod diazotofnih sojeva i simbiotskog - neinfektivnog soja A.azollae javljaju se atipične-sluzne fimbrije iz druge klase.<br />Za ispitivanje sličnosti cijanobakterija metodom PCR-a pomoću STRR konzervativnih sekvenci DNA genoma kori&scaron;ćeno je 39 sojeva azotofiksirajućih cijanobakterija i kod 38 je utvrđeno njihovo prisustvo. Svi sojevi se mogu podeliti u tri grupe, klastera. Prvi klaster je najveći i obuhvata 24 soja i deli se na dva podklastera: Ia koji obuhvata 12 sojeva gde dominiraju sojevi Nostoc-a (8), i podklaster Ib koji obuhvata takođe 12 sojeva, od čega 6 sojeva pripada rodu Anabaena. Podklaster Ia i podklaster Ib pokazuju različitost od 90%. Sva tri simbiozna, infektivna, soja Nostoc-a se nalaze u klasteru I: N.7901, N.9229 i N. 8001. Svaki simbiozni soj Nostoc-a ima genetske sličnosti sa po jednim diazotrofnim sojem Nostoc-a izolovanim iz zemlji&scaron;ta Vojvodine.<br />Klaster II obuhvata sedam (7) sojeva među kojima dominiraju sojevi Cylindrospermum-a, dok klaster III obuhvata 7 sojeva od čega 6 pripadaju rodu Nostoc, a jedan rodu Rivularia.Detaljno poznavanje svojstava izolovanih azotofiksirajućih cijanobakterija doprineće njihovoj budućoj primeni kako u proizvodnji ratarskih i povrtarskih kultura, tako i u biotehnolo&scaron;koj proizvodnji</p> / <p>In this study examined is the frequency of nitrogen-fixing cyanobacteria, total number of algae and total number of bacteria in different soil types on eleven localities in the Vojvodina Province. Seven out of those eleven localities are found in protected nature reserves. Actually, studied was the number of the cyanobacteria and algae depending on the depth of pedological characterization as well as on season. First, isolated were the types of nitrogen-fixing cyanobacteria, determined was their taxonomic origin and basic cytological characteristics. Also examined was the ultrastructure of vegetative cells, heterocysts and other outer structures on fimbriae and pili cells using TEM, transmission electron microscope. Finally, performed was the genetic characterization of isolated types of nitrogen-fixing cyanobacteria using the PCR method and analyzing STRR fragments of DNA.<br />The presence of nitrogen-fixing cyanobacteria and total number of algae was significantly higher with hydromorphic and halomorphic soils than with authomorphic ones. Highest annual average number of nitrogen-fixing cyanobacteria in the topsoil (0-5 cm) was reported with fluvisol soil in Special Nature Reserve &bdquo;Koviljsko petrovaradinski rit&rdquo; (Swamp) and there were 150864 units of bacteria per gram of absolutely dry soil. The soil with the lowest presence of nitrogen-fixing cyanobacteria recorded was cambisol in National Park &ldquo;Fruska gora&rdquo; with 1582 units per gram of soil in the topsoil.<br />With all the researched types of soils the number of nitrogen-fixing cyanobacteria was in the topsoil, 0-5 cm of depth and decreased in line with the depth<br />of soil and lowest was at the deepest layer, 30-60cm. The highest frequency of nitrogen-fixing cyanobacteria was found during the winter season with most of the examined soils. Nitrogen-fixing cyanobacteria are the dominant type of bacteria in our soils and are presented with 56, 27% compared to other types of algae.<br />30 strains of nitrogen-fixing cyanobacteria were isolated: 19 types of Nostoc sp., 4 of Anabaena sp. and one in each genus of Calothrix, Tolypothrix and Phormidium.<br />Using ultrastructural examination and TEM microscope when studying vegetative cells of nitrogen-fixing cyanobacteria observed were the inclusions of regular frequency: carboxysomes (Cs), cyanophycin granules (CG) , polyphosphate granules (PG), ribosomes (R), lipid granules (SS -granule ) and thylakoids ( T ) as well as the inclusion of irregular occurrence: a membrane-bound crystal inclusions.<br />Using TEM technique and staining the cells with the RR and ultra thin cross section, we determined the presence of depletion of the fimbriae with three strains (A.314, A.azollae and N.302) and typical fimbriae with the two strains (N.311 and N.9229). Applying the method of negative staining NS PTA also noticed were a typical fimbriae, needle-hairy like looks with clear boundaries with the three strains (N.302, N.7901, N.9229). However, also observed were atypical mucous fimbriae, which do not have clearly expressed borders, but they are very strongly spread around the vegetative cells, with the three strains (A.314, A.azollae, N.311).<br />With symbiotic - infective strains N.7901 and N.9229 only typical fimbriae of first class occurred, and in diazotroph strains and symbiotic &ndash; non infectious strain A. azollae found were atypical mucous fimbriae of second class.<br />To test the similarity of cyanobacteria by PCR method and using a STRR - conservative DNA sequence of the genome used were 39 strains fixing cyanobacteria and with 38 established was their presence. All strains can be divided into three groups of clusters. The first cluster is the largest and covers 24 strains, and is divided into two subclusters: Ia which includes 12 strains,where predominant are Nostoc strains ( 8 ) , and subcluster Ib , which also implies 12 strains , out of which 6 strains belong to the genus Anabaena. Subcluster Ia and Ib show a difference of 90 %. All three symbiotic , infectious Nostoc strains are classified in a cluster I: N.7901, N.9229 and N. 8001. Each symbiotic Nostoc strain has a genetic similarity with one di-nitrogen Nostoc strain isolated from a lot of different soils in Vojvodina.<br />Cluster II includes seven (7) strains , including strains among which the predominant are Cylindrospermum ones , while cluster III includes 7 strains of which 6 belong to the genus Nostoc and one to genus Rivularia.<br />Detailed knowledge of the properties of isolated fixing cyanobacteria could contribute to their future application both in the production of field crops and vegetables, as well as in biotechnological production.</p>

Made available in DSpace on 2016-06-02T19:29:50Z (GMT). No. of bitstreams: 1 TeseSPG.pdf: 896184 bytes, checksum: 0237c1553b20f09b9264cf38e782d113 (MD5) Previous issue date: 2004-06-30 / The influence of phytoplankton and their excreted materials in natural waters have been considered important issues among metal speciation studies. This work focused on the potential influence of the high molecular weight materials excreted by dominant phytoplankton species of Barra Bonita reservoir (Anabaena spiroides, Microcystis aeruginosa and Aulacoseira granulata) in the speciation and complexation of Cu and Cd. Therefore, this study comprised field and laboratory studies. Seasonal samplings were made during a period of two years in the reservoir. The total, ionic and organic concentrations of Cu and Cd were determined, as well as the complexation parameters with ligands present in the reservoir water. Also, the dissolved organic carbon and physico-chemical parameters of the water column were obtained. A speciation model was applied to metal complexation parameters and total metal concentrations obtained in field investigations. Laboratory cultures of the selected microalgae were made to obtain high molecular weight excreted materials. In a similar manner, the high molecular weight materials were isolated from the reservoir water. These materials were analysed for metal complexation with Cu and Cd, monosaccharide composition and elemental analysis (C, H, N and S). In the reservoir water, Cd speciation was dominated by ionic species, while Cu was mostly associated to the dissolved organic matter. Seasonal samplings presented Cu ligands with similar complexation parameters for the entire period studied. Cd complexation was not detected in the reservoir water. The speciation model resulted in similar Cu2+ concentration to the mean value obtained in field analyses, and indicated higher Cu association to the ligands of the reservoir water. Cu was complexed by all high molecular weight materials studied, while Cd was complexed solely by the cyanophyte materials. Higher proportions of acid monosaccharides in the high molecular weight materials corresponded to higher Cu and Cd complexation parameters, whereas no relation between C, H, N and S proportions and metal complexation properties of the high molecular weight materials was observed. The excreted materials of dominant microalgae from Barra Bonita reservoir had Cu ligands with intermediate to weak binding strength, as those present in the reservoir water. These results indicate the potential influence of such organic materials in metal speciation, bioavailability and mobility of metals with similar behaviour to that of Cu in the reservoir water. / A influência do fitoplâncton e de seus produtos excretados resultantes da fotossíntese em corpos d'água têm sido o enfoque de vários trabalhos que investigam a especiação e o destino de metais. Este estudo teve como hipótese a potencial influência dos materiais de alta massa molecular excretados por três espécies fitoplanctônicas (Anabaena spiroides, Microcystis aeruginosa e Aulacoseira granulata), dominantes no reservatório de Barra Bonita, em relação à especiação e complexação dos metais Cu e Cd na água do reservatório. Para tanto o estudo foi dividido em investigações de campo e experimentos de laboratório. Coletas sazonais por um período de dois anos no reservatório foram avaliadas quanto às espécies dissolvidas totais, orgânicas e iônicas de Cu e Cd, a determinação dos parâmetros de complexação destes metais com ligantes presentes na água, assim como o conteúdo de carbono orgânico dissolvido e os parâmetros físico-químicos da coluna d água. Uma modelagem da especiação química dos metais estudados foi feita com o uso das concentrações totais e os parâmetros de complexação adquiridos nas coletas sazonais. No laboratório foram obtidos os materiais de alta massa molecular excretados em cultivos das microalgas selecionadas, e o material de alta massa molecular dissolvido na água do reservatório, isolado seguindo-se o mesmo procedimento usado para os excretados algais. Estes materiais foram analisados quanto aos parâmetros de complexação com Cu e Cd, e composição monossacarídica e elementar (C, H, N e S). A especiação do Cd na água do reservatório foi dominada por formas iônicas, enquanto o Cu estava em grande maioria associado à matéria orgânica dissolvida. A água do reservatório apresentou, nas coletas sazonais, ligantes para Cu com parâmetros de complexação semelhantes em todo o período estudado. Nas condições experimentais empregadas, não foi detectada a complexação de Cd com ligantes oriundos da água do reservatório. A modelagem da especiação de Cu resultou em uma concentração semelhante de Cu2+ àquela medida em amostras de campo, e maior associação do Cu aos ligantes caracterizados neste estudo. O Cu foi complexado tanto pelos materiais de alta massa molecular das microalgas como também pela água do reservatório, enquanto o Cd foi complexado somente pelo material excretado pelas cianofíceas. Maiores proporções de monossacarídeos ácidos nos materiais corresponderam a parâmetros de complexação de Cu e Cd com valores mais elevados. No entanto, não foi observada uma relação entre as proporções de C, H, N e S e a complexação dos metais. Os excretados das microalgas dominantes no reservatório apresentaram ligantes para Cu com força de associação de intermediária a fraca, assim como os ligantes presentes no reservatório. Estes resultados confirmam o potencial de influência dos excretados na especiação, mobilidade e destino de metais com comportamento semelhante ao Cu, no reservatório de Barra Bonita.

Algae and cyanobacteria occur naturally in source waters and are known to cause extensive problems in the drinking water treatment industry. Cyanobacteria (especially Anabaena sp. and Microcystis sp.) are responsible for many water treatment problems in drinking water treatment works (DWTW) all over the world because of their ability to produce organic compounds like cyanotoxins (e.g. microcystin) and taste and odour compounds (e.g. geosmin) that can have an adverse effect on consumer health and consumer confidence in tap water. Therefore, the monitoring of cyanobacteria in source waters entering DWTW has become an essential part of drinking water treatment management. Managers of DWTW, rely heavily on results of physical, chemical and biological water quality analyses, for their management decisions. But results of water quality analyses can be delayed from 3 hours to a few days depending on a magnitude of factors such as: sampling, distance and accessibility to laboratory, laboratory sample turn-around times, specific methods used in analyses etc. Therefore the use of on-line (in situ) instruments that can supply real-time results by the click of a button has become very popular in the past few years. On-line instruments were developed for analyses like pH, conductivity, nitrate, chlorophyll-a and cyanobacteria concentrations. Although, this real-time (on-line) data has given drinking water treatment managers a better opportunity to make sound management decisions around drinking water treatment options based on the latest possible results, it may still be “too little, too late” once a sudden cyanobacterial bloom of especially Anabaena sp. or Microcystis sp. enters the plant. Therefore the benefit for drinking water treatment management, of changing the focus from real-time results to future predictions of water quality has become apparent. The aims of this study were 1) to review the environmental variables associated with cyanobacterial blooms in the Vaal Dam, as to get background on the input variables that can be used in cyanobacterial-related forecasting models; 2) to apply rule-based Hybrid Evolutionary Algorithms (HEAs) to develop models using a) all applicable laboratory-generated data and b) on-line measureable data only, as input variables in prediction models for harmful algal blooms in the Vaal Dam; 3) to test these models with data that was not used to develop the models (so-called “unseen data”), including on-line (in situ) generated data; and 4) to incorporate selected models into two cyanobacterial incident management protocols which link to the Water Safety Plan (WSP) of a large DWTW (case study : Rand Water). During the current study physical, chemical and biological water quality data from 2000 to 2009, measured in the Vaal Dam and the 20km long canal supplying the Zuikerbosch DWTW of Rand Water, has been used to develop models for the prediction of Anabaena sp., Microcystis sp., the cyanotoxin microcystin and the taste and odour compound geosmin for different prediction or forecasting times in the source water. For the development and first stage of testing the models, 75% of the dataset was used to train the models and the remaining 25% of the dataset was used to test the models. Boot-strapping was used to determine which 75% of the dataset was to be used as the training dataset and which 25% as the testing dataset. Models were also tested with 2 to 3 years of so called “unseen data” (Vaal Dam 2010 – 2012) i.e. data not used at any stage during the model development. Fifty different models were developed for each set of “x input variables = 1 output variable” chosen beforehand. From the 50 models, the best model between the measured data and the predicted data was chosen. Sensitivity analyses were also performed on all input variables to determine the variables that have the largest impact on the result of the output. This study have shown that hybrid evolutionary algorithms can successfully be used to develop relatively accurate forecasting models, which can predict cyanobacterial cell concentrations (particularly Anabaena sp. and Microcystis sp.), as well as the cyanotoxin microcystin concentration in the Vaal Dam, for up to 21 days in advance (depending on the output variable and the model applied). The forecasting models that performed the best were those forecasting 7 days in advance (R2 = 0.86, 0.91 and 0.75 for Anabaena[7], Microcystis[7] and microcystin[7] respectively). Although no optimisation strategies were performed, the models developed during this study were generally more accurate than most models developed by other authors utilising the same concepts and even models optimised by hill climbing and/or differential evolution. It is speculated that including “initial cyanobacteria inoculum” as input variable (which is unique to this study), is most probably the reason for the better performing models. The results show that models developed from on-line (in situ) measureable data only, are almost as good as the models developed by using all possible input variables. The reason is most probably because “initial cyanobacteria inoculum” – the variable towards which the output result showed the greatest sensitivity – is included in these models. Generally models predicting Microcystis sp. in the Vaal Dam were more accurate than models predicting Anabaena sp. concentrations and models with a shorter prediction time (e.g. 7 days in advance) were statistically more accurate than models with longer prediction times (e.g. 14 or 21 days in advance). The multi-barrier approach in risk reduction, as promoted by the concept of water safety plans under the banner of the Blue Drop Certification Program, lends itself to the application of future predictions of water quality variables. In this study, prediction models of Anabaena sp., Microcystis sp. and microcystin concentrations 7 days in advance from the Vaal Dam, as well as geosmin concentration 7 days in advance from the canal were incorporated into the proposed incident management protocols. This was managed by adding an additional “Prediction Monitoring Level” to Rand Waters’ microcystin and taste and odour incident management protocols, to also include future predictions of cyanobacteria (Anabaena sp. and Microcystis sp.), microcystin and geosmin. The novelty of this study was the incorporation of future predictions into the water safety plan of a DWTW which has never been done before. This adds another barrier in the potential exposure of drinking water consumers to harmful and aesthetically unacceptable organic compounds produced by cyanobacteria. / PhD (Botany), North-West University, Potchefstroom Campus, 2015

Algae and cyanobacteria occur naturally in source waters and are known to cause extensive problems in the drinking water treatment industry. Cyanobacteria (especially Anabaena sp. and Microcystis sp.) are responsible for many water treatment problems in drinking water treatment works (DWTW) all over the world because of their ability to produce organic compounds like cyanotoxins (e.g. microcystin) and taste and odour compounds (e.g. geosmin) that can have an adverse effect on consumer health and consumer confidence in tap water. Therefore, the monitoring of cyanobacteria in source waters entering DWTW has become an essential part of drinking water treatment management. Managers of DWTW, rely heavily on results of physical, chemical and biological water quality analyses, for their management decisions. But results of water quality analyses can be delayed from 3 hours to a few days depending on a magnitude of factors such as: sampling, distance and accessibility to laboratory, laboratory sample turn-around times, specific methods used in analyses etc. Therefore the use of on-line (in situ) instruments that can supply real-time results by the click of a button has become very popular in the past few years. On-line instruments were developed for analyses like pH, conductivity, nitrate, chlorophyll-a and cyanobacteria concentrations. Although, this real-time (on-line) data has given drinking water treatment managers a better opportunity to make sound management decisions around drinking water treatment options based on the latest possible results, it may still be “too little, too late” once a sudden cyanobacterial bloom of especially Anabaena sp. or Microcystis sp. enters the plant. Therefore the benefit for drinking water treatment management, of changing the focus from real-time results to future predictions of water quality has become apparent. The aims of this study were 1) to review the environmental variables associated with cyanobacterial blooms in the Vaal Dam, as to get background on the input variables that can be used in cyanobacterial-related forecasting models; 2) to apply rule-based Hybrid Evolutionary Algorithms (HEAs) to develop models using a) all applicable laboratory-generated data and b) on-line measureable data only, as input variables in prediction models for harmful algal blooms in the Vaal Dam; 3) to test these models with data that was not used to develop the models (so-called “unseen data”), including on-line (in situ) generated data; and 4) to incorporate selected models into two cyanobacterial incident management protocols which link to the Water Safety Plan (WSP) of a large DWTW (case study : Rand Water). During the current study physical, chemical and biological water quality data from 2000 to 2009, measured in the Vaal Dam and the 20km long canal supplying the Zuikerbosch DWTW of Rand Water, has been used to develop models for the prediction of Anabaena sp., Microcystis sp., the cyanotoxin microcystin and the taste and odour compound geosmin for different prediction or forecasting times in the source water. For the development and first stage of testing the models, 75% of the dataset was used to train the models and the remaining 25% of the dataset was used to test the models. Boot-strapping was used to determine which 75% of the dataset was to be used as the training dataset and which 25% as the testing dataset. Models were also tested with 2 to 3 years of so called “unseen data” (Vaal Dam 2010 – 2012) i.e. data not used at any stage during the model development. Fifty different models were developed for each set of “x input variables = 1 output variable” chosen beforehand. From the 50 models, the best model between the measured data and the predicted data was chosen. Sensitivity analyses were also performed on all input variables to determine the variables that have the largest impact on the result of the output. This study have shown that hybrid evolutionary algorithms can successfully be used to develop relatively accurate forecasting models, which can predict cyanobacterial cell concentrations (particularly Anabaena sp. and Microcystis sp.), as well as the cyanotoxin microcystin concentration in the Vaal Dam, for up to 21 days in advance (depending on the output variable and the model applied). The forecasting models that performed the best were those forecasting 7 days in advance (R2 = 0.86, 0.91 and 0.75 for Anabaena[7], Microcystis[7] and microcystin[7] respectively). Although no optimisation strategies were performed, the models developed during this study were generally more accurate than most models developed by other authors utilising the same concepts and even models optimised by hill climbing and/or differential evolution. It is speculated that including “initial cyanobacteria inoculum” as input variable (which is unique to this study), is most probably the reason for the better performing models. The results show that models developed from on-line (in situ) measureable data only, are almost as good as the models developed by using all possible input variables. The reason is most probably because “initial cyanobacteria inoculum” – the variable towards which the output result showed the greatest sensitivity – is included in these models. Generally models predicting Microcystis sp. in the Vaal Dam were more accurate than models predicting Anabaena sp. concentrations and models with a shorter prediction time (e.g. 7 days in advance) were statistically more accurate than models with longer prediction times (e.g. 14 or 21 days in advance). The multi-barrier approach in risk reduction, as promoted by the concept of water safety plans under the banner of the Blue Drop Certification Program, lends itself to the application of future predictions of water quality variables. In this study, prediction models of Anabaena sp., Microcystis sp. and microcystin concentrations 7 days in advance from the Vaal Dam, as well as geosmin concentration 7 days in advance from the canal were incorporated into the proposed incident management protocols. This was managed by adding an additional “Prediction Monitoring Level” to Rand Waters’ microcystin and taste and odour incident management protocols, to also include future predictions of cyanobacteria (Anabaena sp. and Microcystis sp.), microcystin and geosmin. The novelty of this study was the incorporation of future predictions into the water safety plan of a DWTW which has never been done before. This adds another barrier in the potential exposure of drinking water consumers to harmful and aesthetically unacceptable organic compounds produced by cyanobacteria. / PhD (Botany), North-West University, Potchefstroom Campus, 2015

<p>Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N₂), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. </p><p>This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium <i>Anabaena</i> PCC 7120. Apart from performing oxygenic photosynthesis, <i>Anabaena</i> PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. </p><p>NtcA from <i>Anabaena</i> PCC 7120 was heterologously expressed in <i>E. coli</i> and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA <i>in vitro </i>using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N₈ – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. </p><p>Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. </p><p>Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif.</p><p>Furthermore, an NtcA binding site was found in the promoter region of the<i> hupSL</i> gene, encoding an uptake hydrogenase in <i>Nostoc punctiforme</i> (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.</p>

Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N2), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium Anabaena PCC 7120. Apart from performing oxygenic photosynthesis, Anabaena PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. NtcA from Anabaena PCC 7120 was heterologously expressed in E. coli and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA in vitro using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N8 – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif. Furthermore, an NtcA binding site was found in the promoter region of the hupSL gene, encoding an uptake hydrogenase in Nostoc punctiforme (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.

Made available in DSpace on 2016-06-02T19:31:40Z (GMT). No. of bitstreams: 1 1888.pdf: 3636737 bytes, checksum: bdcef6a4d1d6ec63fc8a9a2db7c492bc (MD5) Previous issue date: 2008-05-30 / Financiadora de Estudos e Projetos / The cyanobacterium Anabaena spiroides, a cosmopolitan species occurring in eutrophic environments as in Barra Bonita reservoir, is covered by a thick polysaccharide capsule that provides a microenvironment for association of bacterial communities. The aims of this study were: to identify bacteria attached to A. spiroides capsule to evaluate interspecific relationships among bacteria communities and A. spiroides, considering bacteria selectivity and succession dynamics of attached bacteria; as well as the effect of bacterial inoculum (1.2 µm filtered water from Barra Bonita reservoir) on cyanobacterial growth. For this purpose, density, production, biomass and diversity of bacteria attached to cyanobacteria capsules and free-living bacteria were determined in two replicate cultures of A. spiroides inoculated with bacteria from Barra Bonita reservoir. The diversity was verified by the number of bands obtained through separation of PCR amplification products of 16S rDNA from free-living and attached bacterial communities using Denaturing Gradient Gel Electrophoresis (DGGE). Bacteria attached to the capsule were identified by sequencing the fragment of 16S rDNA. A set of cultures were performed to evaluate cyanobacterial growth as affected by Barra Bonita filtered water. A. spiroides cultures without Barra Bonita inoculum were used as control. The results showed that bacterial density, biomass and total production were higher for free-living bacteria, but no significant difference was obtained between attached and free-living bacteria regarding production per cell. The diversity was lower for the attached bacteria than free-living ones. Three strains of attached bacteria present in A. spiroides inoculum, identified as one Acidobacteria and two Alphaproteobacteria, remained up to the beginning of exponential growth phase. At the senescence phase these bacteria were replaced by four strains identified as one Deltaproteobacteria, one Betaproteobacteria, one Bacilli (Firmicutes) and one unidentified strain. This research demonstrated that there were selectivity and succession in the bacterial community attached to A. spiroides, and that the addition of the filtered water from Barra Bonita inoculum accelerates the death of cyanobacterium cultures / A cianobactéria Anabaena spiroides, cosmopolita em ambientes eutrofizados como o reservatório de Barra Bonita, é recoberta por uma espessa cápsula de polissacarídeo que fornece um microambiente para o crescimento de uma comunidade bacteriana particular. Os objetivos deste trabalho foram: identificar as bactérias associadas à cápsula de A. spiroides para detectar possíveis relações interespecíficas entre estas e a cianobactéria, considerando a seletividade e a dinâmica de sucessão das bactérias associadas; verificar o efeito da adição do inóculo bacteriano (água do reservatório de Barra Bonita filtrada em 1,2 µm) no crescimento da cianobactéria. Para tanto, a densidade, produção, biomassa e a diversidade das bactérias livres e aderidas à cianobactéria, assim como a identificação das bactérias aderidas foram determinadas em duas culturas de A. spiroides inoculadas com bactérias do reservatório de Barra Bonita. A diversidade foi verificada pelo número de bandas obtidas em Denaturing Gradient Gel Electrophoresis (DGGE) após a amplificação do 16S rDNA das comunidades bacterianas das frações livre e aderida e as bactérias aderidas à cápsula foram identificadas pelo seqüenciamento do fragmento do 16S rDNA. Em outro experimento, o crescimento da cianobactéria foi verificado pela concentração de clorofila a e carbono orgânico total após a adição da água de Barra Bonita (filtrada em 1,2µm) em quatro culturas experimentais de A. spiroides. Os controles consistiram em culturas de A. spiroides sem o inóculo de Barra Bonita. Os resultados mostraram que a densidade, biomassa e produção total das bactérias foram sempre maiores para as bactérias livres, no entanto, com relação à produção por célula, não houve diferença significativa entre aderidas e livres. Este estudo também mostrou que a diversidade das bactérias aderidas foi menor do que das livres e que três linhagens de bactérias aderidas que estavam presentes no inóculo de A. spiroides, permaneceram até o início da fase de crescimento exponencial. Essas bactérias foram identificadas como uma Acidobacteria e duas Alphaproteobacteria. Na fase de senescência essas bactérias foram substituídas por outras quatro linhagens: uma Deltaproteobacteria, uma Betaproteobacteria e uma Bacilli (Firmicutes) e uma linhagem não identificada. No segundo experimento as concentrações de clorofila e carbono foram menores nas culturas adicionadas do inóculo bacteriano do que nos controles. O presente estudo demonstrou que houve seleção e sucessão das bactérias aderidas a A. spiroides e que a adição da água de Barra Bonita acelera a morte das culturas da cianobactéria

Microalgae cultivation has received much research attention in recent decades due to its high photosynthetic productivity and ability to produce biofuel feedstocks as well as high value compounds for the health food, cosmetics, and agriculture markets. Microalgae are conventionally grown in open pond raceways or closed photobioreactors. Due to the high water contents of these cultivation systems, they require large energy inputs for pumping and mixing the dilute culture, as well as concentrating and dewatering the resultant biomass. The energy required to operate these systems is generally greater than the energy contained in the resultant biomass, which precludes their use in sustainable biofuel production. To address this challenge, we designed a novel photobioreactor inspired by higher plants. In this synthetic leaf system, a modified transpiration mechanism is used which delivers water and nutrients to photosynthetic cells that grow as a biofilm on a porous, wicking substrate. Nutrient medium flow through the reactor is driven by evaporation, thereby eliminating the need for a pump. This dissertation outlines the design, construction, operation, and modeling of such a synthetic leaf system for energy positive biofuel production. First, a scaled down synthetic leaf reactor was operated alongside a conventional stirred tank photobioreactor. It was demonstrated that the synthetic leaf system required only 4% the working water volume as the conventional reactor, and showed growth rates as high as four times that of the conventional reactor. However, inefficiencies in the synthetic leaf system were identified and attributed to light and nutrient limitation of growth in the biofilm. To address these issues, a modeling study was performed with the aim of balancing the fluxes of photons and nutrients in the synthetic leaf environment. The vascular nutrient medium transport system was also modeled, enabling calculation of nutrient delivery rates as a function of environmental parameters and material properties of the porous membrane. These models were validated using an experimental setup in which the nutrient delivery rate, growth rate, and photosynthetic yield were measured for single synthetic leaves. The synthetic leaf system was shown to be competitive with existing technologies in terms of biomass productivity, while requiring zero energy for nutrient and gas delivery to the microorganisms. Future studies should focus on utilizing the synthetic leaf system for passive harvesting of secreted products in addition to passive nutrient delivery. / text

No description available.

No description available.