The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.

Presented work is focused on study of polyhydroxybutyrate production in bacteria. In theoretical part short characterization of PHB was given and the most common representative of wide group of polyhydroxyalkanoates (PHA) were described. Then, production of PHB and copolymer P(HB-co-HV) in selected bacterial strains was experimentally proven. First, PHB production in Bacillus megaterium using synthetic medium was studied. The PHB content in cells was increased during cultivation under limiting conditions, despite low growth. Addition of ethanol into production media resulted in increased PHB synthesis as well as biomass production (21 % PHB of 1,8 g/l biomass). Further, BM medium containing 8 g/l glucose was tested. PHB production was more than 1 g/l at significant growth increase when compared with synthetic medium. The bacteria B.megaterium showed, except glucose, ability to utilize maltose and xylose. Another cultivations were tested with bacterial strain Azotobacter vinelandii, which is capable of copolymer P(HB-co-HV) synthesis. Maximal growth and copolymer content was reached on Burk's medium with 30 g/l of glucose. Addition of peroxide to growth medium influenced P(HB-co-HV) synthesis to 46 % of 2,6 g/l biomass. Bacteria A.vinelandii showed the best growth on maltose, even compared with glucose (54 % copolymer of biomass content). Finally, PHB production on industrial waste product – whey was monitored. Using Plackett-Burman design for statistical media optimization, the whey content was modified. B.megaterium grown on adjusted whey reached 0,5 g/l PHB, 32 % of cell's content.

Chromosomal DNA of A. vinelandii thought to carry multiple gene copies was examined in efforts to visualize its chromosomal structure using electron microscopy. The chromosomal DNA of A. vinelandii may have multiple circular genomic units carrying multiple copies of genes. Three possible branch construction schemes and their replication modes are postulated in this study.

This research describes the changes in cell morphology of Azotobacter vinelandii cells cultured in dialyzed soil medium. This particular culture medium was assumed to provide the bacteria with an environment similar to their natural habitat, the soil.

The objectives of this research were to identify the form of Azotobacter as it exists in situ in the soil; to compare its resistance to that of laboratory grown cysts typical of those described in the literature; and to compare its resistance to that of cells grown on dialyzed soil agar. In addition, the morphology of the cells grown on dialyzed soil agar was examined by light and electron microscopy and then compared to the cysts grown on n-butanol Burk's medium. Dipicolinic acid and oxygen uptake rate were measured in cysts and on cells grown on dialyzed soil agar in order to determine whether the cells grown on dialyzed soil agar were endospores or other dormant form and also to measure the respiratory quotient in these cells.

Iron-sulfur [Fe-S] clusters are protein cofactors that facilitate various life-sustaining biological processes. Their in vivo assembly is accomplished by three different systems known to date. These are: the NIF system which provides [Fe-S] clusters for nitrogenase and other nitrogen-fixing proteins, the SUF system which is induced during conditions of oxidative stress and iron starvation in E. coli, and the ISC system which serves as the housekeeping assembly apparatus. The latter is the focus of this dissertation and includes the proteins IscR, IscS, IscU, IscA, HscB, HscA, Fdx, and IscX. IscU is purified in its cluster-less (apo) form, but can serve as a scaffold to assemble [Fe-S] clusters in vitro in the presence of excess iron and sulfide. To test the scaffold hypothesis and gain insight into the events that occur during [Fe-S] cluster assembly and delivery, we developed two methods that allow the isolation of IscU and other ISC proteins in vivo. In the first method, Azotobacter vinelandii IscU is isolated from its native host, whereas in the second, it is isolated recombinantly from E. coli using a vector that allows expression of the entire isc operon. We found that IscU exists in vivo in two forms: apo-IscU and [2Fe-2S]2+ cluster-loaded IscU which are believed to be conformationally distinct. Both transient and stable IscU-IscS complexes were identified, indicating that the two proteins interact in vivo in a manner that involves their association and dissociation. The [2Fe-2S]2+-IscU species was present as a single entity, whereas significant amounts of apo-IscU were found associated with IscS, suggesting that IscU-IscS dissociation is triggered by the completion of [2Fe-2S] clusters. Both apo and [2Fe-2S]2+-IscU were predominantly monomeric whereas IscU-IscS complexes were determined to have an α2β2 composition. IscU was purified in the absence of the chaperones HscA and HscB and was also shown to accommodate a [2Fe-2S]2+ cluster similar to the one bound to IscU isolated from wild type cells. The findings suggest that [2Fe-2S]2+-IscU exists in one conformation in vivo and that any conformational changes on IscU are exerted after [2Fe-2S] cluster formation. In silico studies showed that a flexible loop containing the conserved LPPVK motif, which is responsible for interactions with HscA, may facilitate cluster exposure to either mediate its delivery to acceptor proteins or participation in the construction of [4Fe-4S] clusters. Experiments with NfuA, a protein similar to the C-terminal domain of NifU, demonstrated that NfuA and similar proteins might serve as [Fe-S] cluster carriers to accomplish the efficient delivery of nascent cofactors to the various recipient proteins. / Ph. D.

Iron-sulfur clusters are simple inorganic cofactors that are ubiquitous in living systems. The assembly of iron sulfur clusters is an essential process and must be carefully controlled in order to limit the release of toxic free iron or sulfide. Thus far there are three known protein systems for iron sulfur cluster assembly including the <i>nif, suf,</i> and <i>isc</i> systems. The <i>nif</i> system makes iron-sulfur clusters for nitrogenase production, while both the <i>suf</i> and <i>isc</i> systems provide iron-sulfur clusters for general cellular use. In <i>Azotobacter vinelandii</i> the isc operon contains eight genes which are transcribed together as a single operon: <i>iscR iscS iscU iscA hscB hscA fdx iscX</i>. The two central <i>isc</i> players include IscS, a cysteine desulfurase, and IscU the proposed site of iron-sulfur cluster assembly. Using <i>A. vinelandii</i> as a model organism, we have sought to better understand the mechanism of <i>in vivo isc</i> cluster assembly. In order test the scaffold hypothesis, we constructed strains that allowed for quick and rapid isolation of IscU. The purification of IscU with a bound [2Fe-2S] cluster strongly supports the model that IscU serves as the site of cluster synthesis <i>in vivo</i>. Additionally, using this same genetic system we isolated an IscU39DA variant with an oxygen stable bound [2Fe-2S] cluster. The IscU39^DA scaffold came in tight α₂β₂ complex with IscS and was not separated by high salt, size exclusion, or reducing conditions. On the other hand, wild-type IscU also associated with IscS in a α₂β₂ complex, but readily dissociated upon increased salt concentration. The tight association of IscU39^DA and IscS was found to occur regardless of the presence of a bound [Fe-S] cluster. We conclude that the IscU Asp-39 residue is essential for mediating the dissociation of IscU and IscS. In addition to studying IscS and IscU, we were interested to further understand how the isc system is regulated in response to external factors. Previous work has demonstrated that IscR controls expression of the isc operon in <i>Escherichia coli</i>. When IscR is holo this protein represses <i>isc</i> expression, while in its apo-form it allows <i>isc</i> expression. In <i>A. vinelandii</i> we found that ∆<i>iscR</i> strains exhibit in a 5 – 7 fold elevation of isc expression. Additionally, ∆<i>iscR</> strains reveal a small growth phenotype on plates, and a tendency to form spontaneous suppressor mutations allowing reversion to wild-type growth. Loss of apo-IscR function was found to cause a more severe effect on growth than the loss of holo-IscR function, suggesting IscR has cellular roles in addition to the regulation of the <i>isc</i> operon. / Ph. D.

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T19:50:10Z : No. of bitstreams: 1 silva_an_me_sjrp.pdf: 5773853 bytes, checksum: 8d32b3256460ece4e4901a20da094ab8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O alginato é um polissacarídeo normalmente extraído de paredes celulares de algas marrons utilizado nas indústrias de alimentos, farmacêuticas e biotecnológicas. A produção é concentrada no cultivo de algas marinhas marrons, mas várias bactérias do gênero Pseudomonas e Azotobacter produzem alginato. A estrutura química dos alginatos produzidos por algas é similar aos sintetizados pela A. vinelandii. Esta bactéria também produz polímeros intracelulares como o poli-hidroxibutirato (PHB), conhecido como bioplástico. Neste trabalho estudou-se a produção simultânea do alginato e PHB pela A. vinelandii utilizando sacarose, glicose e melaço de cana-de-açúcar como fontes de carbono, além de diferentes parâmetros de fermentação em agitador orbital rotatório. Os valores ótimos para a produção destes compostos foram determinados pela metodologia de superfície de resposta (MSR). O 1º experimento realizado para as três fontes de carbono foi um planejamento fatorial fracionado 26-2 (variáveis independentes: concentração da fonte de carbono; concentração de acetato de amônio; concentração de citrato de amônio e ferro (III); pH; temperatura de incubação e tempo de incubação). O 2º experimento baseou-se nos valores ótimos de produção de PHB para cada fonte de carbono e resultou em um planejamento fatorial completo 33-0 (variáveis independentes: concentração da fonte de carbono; temperatura de incubação e tempo de incubação). Verificou-se que a maior produtividade de PHB (100 mg/g de célula/h) utilizando o melaço de cana-de-açúcar ocorreu no tempo de incubação de aproximadamente 10 h, a 60,0ºC e nas concentrações de sólidos solúveis entre 14,0 - 25,0%. A glicose apresentou uma maior produtividade de PHB (60 mg/g de célula/h) no tempo de incubação de aproximadamente 10 h, entre 23,0-26,0ºC e concentração de glicose... / The alginate is a polysaccharide extracted from cell walls of brown seaweed used in the industries of food, pharmaceutical and biotechnology. The production is concentrated in the brown seaweed cultivation, but several bacteria, Pseudomonas and Azotobacter genus, produce alginate. The chemical structure of alginate produced by algae is similar to those synthesized by A. vinelandii. This bacterium also produces intracellular polymers such as polyhydroxybutyrate (PHB), known as bioplastic. In this work the simultaneous alginate and PHB production by A. vinelandii using sucrose, glucose and sugar cane molasses as carbon source, and different fermentation parameters in orbital shaker was studied. The optimum values for the production of these compounds were determined by the response surface methodology (RSM). The 1st experiment conducted for the three carbon sources was a fractionated factorial design 26-2 (independent variables: the carbon source concentration; ammonium acetate concentration; ammonium citrate and iron (III) concentration; pH; temperature and incubation time). The 2nd experiment was based on optimum values for the production of PHB for each carbon source and resulted in a full factorial design 33-0 (independent variables: the carbon source concentration; temperature and incubation time). The highest PHB yield (100 mg/g cell/h) using sugar cane molasses as a carbon source was found in 10 h at 60.0 ºC and solids soluble concentrations between 14.0 and 25.0%. The glucose showed the highest PHB yield (60 mg/g cell/h) in approximately 10 h, at temperature between 23.0 – 26.0 ºC and glucose concentration between 48.0 and 62.0 g/L. The sucrose, showed the highest PHB yield (45 mg/g cell/h) in approximately 18 h, 60.0 ºC and sucrose concentration of 10.0 g/L. For the alginate productivity using the glucose was observed that the yield was more... (Complete abstract click electronic access below)

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University, Diss., 1998--Braunschweig.