Foi avaliado o potencial de Halomonas sp. HG01, uma bactéria halófila, isolada de uma lagoa salina no Peru, com relação à produção de polihidroxialcanoatos (PHA). Halomonas sp. HG01 foi capaz de utilizar eficientemente sacarose, glicose e frutose como fontes de carbono. Os melhores desempenhos foram obtidos com glicose ou sacarose. 3-Hidroxibutirato (3HB) foi o único monômero detectado a partir de carboidratos. Precursores de outros monômeros foram avaliados. Monômeros de 3-hidroxivalerato (3HV) foram incorporados ao polímero quando os ácidos propiônico ou valérico foram supridos. 4-Hidroxibutirato (4HB) também foi utilizado como monômero pela PHA sintase de Halomonas sp. HG01. Engenharia evolutiva permitiu aumentar a eficiência de consumo de xilose e glicerol. Cultivos em biorreator permitiram atingir produtividades volumétricas de 0,13; 0,07 e 0,06 g/L.h, quando glicose, xilose e glicerol, respectivamente, foram supridas. Maiores produtividades deverão ser atingidas com aumento na densidade celular dos cultivos. / The potential of Halomonas sp. HG01, a halophilic bacteria isolated from a saline lagoon in Peru, was evaluated regarding the production of polyhydroxyalkanoates (PHA). Halomonas sp. HG01 was able to use efficiently sucrose, glucose and fructose as carbon sources. The best performance was obtained with glucose or sucrose. 3-hydroxybutyrate (3HB) was the only monomer detected from carbohydrates. Precursors of other monomers were evaluated. 3-Hydroxyvalerate (3HV) was inserted in the polymer when propionic or valeric acids were supplied. 4-Hydroxybutirate (4HB) was also used as a monomer for the PHA synthase from Halomonas sp. HG01. Evolutive engineering allowed to increase the uptake efficiency of xylose and glycerol. Cultures in bioreactor allowed achieving volumetric productivities of 0.13; 0.07 and 0.06 g /L.h when glucose, xylose, and glycerol, respectively, were supplied. Higher productivities should be achieved with increased cell density of cultures.

Universiẗat, Diss., 2004--Bonn.

Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T08:43:54Z (GMT). No. of bitstreams: 1 Piubeli_FrancineAmaral_D.pdf: 6673876 bytes, checksum: 678469d11c710488c1e19eda0ee7d179 (MD5) Previous issue date: 2011 / Resumo: A água residuária da extração de petróleo é altamente salina e contém uma mistura complexa de hidrocarbonetos, muitos dos quais são altamente tóxicos. Com o aumento da preocupação em preservar o meio ambiente, as tecnologias voltadas para a recuperação e despoluição de áreas degradadas tem ganhado a atenção. A biorremediação tem sido utilizada como método de biodegradação natural através da otimização de processos biológicos por ser econômica, versátil e a que mais se aproxima de uma despoluição ecologicamente aceitável. No entanto, a biorremediaçao depende de alguns fatores tais como: pH, temperatura, salinidade e pressão. Condições extremas desses fatores podem matar ou inibir espécies que não estejam adaptadas a ambientes extremos, por isso o desenvolvimento e otimização de processos de biorremediação de ambientes extremos contaminados por é de grande relevância. Desta forma, este trabalho teve como objetivos o isolamento e caracterização de bactérias halofílicas presentes na água residuária da extração de petróleo, além do estudo molecular dos genes envolvidos na degradação de compostos aromáticos para que posteriormente utilizássemos essas linhagens na biorremediação desse efluente, outra vertente desse trabalho foi o estudo da diversidade microbiana presente nesta água para auxiliar o direcionamento de esforços na minimização dos impactos causados com a exploração de petróleo. Foram isoladas sete linhagens de bactérias halofílicas da água de produção de petróleo, sendo todas pertencentes a família Halomoneaceae e com potencial para degradação de compostos aromáticos. A linhagem que apresentou maior taxa de crescimento nos compostos testados (ácido benzóico, fenol, ácido p-hidroxibénzoico) foi a denominada df2. Com relação à taxa de degradação dos compostos aromáticos testados, a dp3 degradou cerca 90% do fenol disponível, seguida pela df2 que degradou 80% desse mesmo composto. Quando a fonte de carbono foi o ácido benzóico, a df1 e DP3 degradaram 99% do composto e 70% do ácido p-hidroxibenzóico foi degradado em 12 dias de experimento pela linhagem df2. Para a degradação desses compostos a linhagem Halomonas organivorans seguiu a via de degradação do catecol na rota do ß- cetoadipato compreendendo a codificaçao de diferentes enzimas por catRBCA. A comparação das seqüências da biblioteca de bactérias com as sequências do gene RNA ribossomal 16S presente nos bancos de dados GenBank e RDP revelou a presença de micro-organismos pertencentes aos gêneros Marinobacter e Halomonas e na comparação da técnica clássica de isolamento com a técnica independente de cultivo (biblioteca do gene RNA ribossomal 16S), foi encontrada uma maior diversidade na última. Finalmente de todos os testes realizados na tentativa de biorremediar a água de produção de petróleo, a bioestimulação foi a mais efetiva, sendo conseguida uma diminuição de 77% da carga orgânica com a adição de solução de fosfato e alanina. Estes resultados demonstraram o grande potencial destas linhagens para a degradação de compostos aromáticos, bem como para a biorremediação da água de produção de petróleo, além de descrever a via metabólica utilizada por membros da família Halomoneaceas na degradação de compostos aromáticos, auxiliando assim nos esforços para biorremediação de ambientes salinos contaminados / Abstract: The wastewater from oil extraction (produced water) is highly saline and contains a complex mixture of hydrocarbons, many of which are highly toxic. With increasing concern about preserving the environment, the technologies for recovery and remediation of degraded areas has been gaining attention. Bioremediation has been used as a method of natural biodegradation by optimizing biological processes because it is economical, versatile and it is the closest to a ecologically acceptable decontamination. However, bioremediation depends on factors such as pH, temperature, salinity and pressure. Extremes of these factors can inhibit or kill species that are adapted to extreme environments, so the development and optimization of bioremediation processes for contaminated extreme environments is of great importance. Thus, this work aimed at the isolation and characterization of halophilic bacteria present in the wastewater from oil extraction, and molecular studies of the genes involved with the degradation of aromatic compounds so that later we could use these strains in the bioremediation of this wastewater. Another aspect of this work was to study the microbial diversity present in this wastewater to assist in directing the efforts to minimize the impact of oil drilling. Seven strains of halophilic bacteria were isolated from of the produced water of oil production, all belonging to the family Halomoneaceae having potential for degradation of aromatic compounds. The strain that showed the highest growth rate in the tested compounds (benzoic acid, phenol, p-hydroxybenzoic acid) was strain DF2. Regarding the rate of degradation of the aromatic compounds tested, dp3 degraded about 90% of available phenol, followed by DF2 that degraded 80% of this compound. When the carbon source was benzoic acid, DP3 and df1 and degraded 99% and strain DF2 degraded 70% when the compound was p-hydroxybenzoic acid. For the degradation of these compounds the strain Halomonas organivorans followed the catechol degradation pathway ending in the formation of ß-Ketoadipate and was shown to produce various enzyme activities encoded by catRBCA. Comparison of the 16S rRNA gene sequences of the isolates with the sequences present in the GenBank and RDP databases revealed the isolates belonged to the genera Marinobacter and Halomonas. Comparing these results with those obtained by classical taxonomic techniques demonstrated that the 16S ribosomal RNA gene analysis (culture independent method), found greater diversity. Finally, of all tests performed to bioremediate the produced water, biostimulation by addition of nutrients was the most effective, and achieved a reduction of 77% of the organic load with the addition of phosphate combined with alanine. These results demonstrate the great potential of these strains for the degradation of aromatic compounds, as well as for the bioremediation of produced water from crude oil production, and describe the metabolic pathway used by members of the family Halomoneaceas for the degradation of aromatic compounds, thus assisting in efforts to develop methods for the bioremediation of contaminated saline environments / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Universiẗat, Diss., 2003--Münster (Westfalen).

This diploma thesis deals with the use of thermal analysis in the study on effects of microbial inhibitors. The main aim of this work was to determine the utilization of the method, which is mainly used in different fields of science and research. Three bacterial strains: Cupriavidus necator H16, its mutant strain Cupriavidus necator PHB-4 and Halomonas halophila, were used as model microorganisms. The inhibitory effect of levulinic acid on growth and metabolic activity was monitored by microcalorimetry. It was found that bacteria were able to adapt to levulinic acid to a certain concentration - Cupriavidus necator to 5 g/l and Halomonas halophila to 2 g/l. The thermal analysis results were compared to a conventional method, which is commonly used to study the growth of microorganisms.

Many plants are highly sensitive to salt in the soil, and their growth and yield can be greatly hindered by as little as less than 1% salt concentration in the soil. Additionally, soil salinity is a growing issue globally and affects significant areas in Utah. Halophytes are plants that are adapted to grow in saline soils and have been widely studied for their physiological and molecular characteristics, but little is known about their associated microbiomes. Bacteria were isolated from the rhizosphere and as root endophytes of Salicornia rubra, Sarcocornia utahensis, and Allenrolfea occidentalis, three native Utah halophytes. Several strains of halophilic bacteria have been isolated and screened for the ability to stimulate plant growth in saline conditions despite the high salt concentrations. Halomonas, Bacillus, and Kushneria species were consistently isolated both from the soil and as endophytes from roots of all three plant species at all collection times. Of the isolates tested for the ability to stimulate growth of alfalfa under saline conditions, Halomonas and Kushneria strains stimulated plant growth in the presence of 1% NaCl. The same bacteria used in the inoculation were recovered from surface sterilized alfalfa roots, indicating the ability of the inoculum to become established as an endophyte. This raises the question of whether these plant associated halophilic isolates contain genes that aid in plant growth promotion. We are interested in genomic sequencing of our Halomonas and Kushneria strains and performing genomic analysis to determine if there is a difference in genes between plant associated and non-plant associated halophilic isolates. We explored the hypothesis that certain bacterial properties have been selected for to aid plant growth. This was accomplished by performing whole genome sequencing of 26 Kushneria and Halomonas strains, both plant and non-plant associated. These strains came from freezer stocks of previously collected isolates as well as field trips to collect more samples. Halophilic bacteria were isolated from bulk soil, rhizosphere, and halophyte tissues (root and shoot tissues). The non-plant associated (bulk soil) halophilic Kushneria and Halomonas strains aided in determining if there are specific bacterial genes that are expressed in plant associated strains. Whole genome sequencing of the isolates was performed on the Oxford Nanopore platform. The sequence data was then assembled and annotated. The genomes were then included in a genome wide association study was performed. The results from the GWAS show that there is not a significant difference between plant and non-plant associated isolates, disproving our hypothesis. The results also show that few known genes for phytohormone synthesis were present in the pangenome, highlighting the need for further research to determine how these halophilic isolates aid in plant growth promotion in saline soils.

Polyuronic acids are an important constituent of seaweed and plants, and therefore represent a significant part of global biomass, providing an abundant carbon source for both terrestrial and marine heterotrophic bacteria. Through the action of polysaccharide lyases, polyuronic acids are degraded into unsaturated monouronic acid units, which are fed into the Entner-Doudoroff pathway where they are converted into pyruvate and glyceraldehyde-3-phosphate. The first step of this pathway was thought to occur non- enzymatically. A highly conserved sequence, kdgF was found in alginate and pectin utilization loci in a diverse range of prokaryotes, in proximity to well established enzymes catalyzing steps downstream in the Entner-Doudoroff pathway and I hypothesized that KdgF was involved in the catalysis of the first step of this pathway. The kdgF genes from both Yersinia enterocolitica and a locally acquired Halomonas sp. were expressed in Escherichia coli and their activity was examined using unsaturated galacturonic acid depletion activity assays. To gain perspective on the general structure of KdgF, x-ray crystallography was used to obtain a crystal structure of both HaKdgF and YeKdgF. These crystal structures provided insight into the molecular details of catalysis by the KdgF proteins, including their putative catalytic residues and a coordinated metal binding site for substrate recognition. To elucidate amino acids that may be involved in binding and/or catalysis, mutants were created in HaKdgF, and lack of activity was observed in four mutants (Asp102A, Phe104A, Arg108A, and Gln55A). The research done in this study suggests that KdgF proteins use a metal binding site coordinated by three histidines and several additional residues to cause a change in monouronic acid, thereby, affecting the unsaturated double bond. This suggests that KdgF is involved in the first step in the Entner-Doudoroff pathway, which is the linearization of unsaturated monouronic acids. / Graduate

This master thesis deals with the development of a minibioreactor for microbial biotechnologies. The AlgaTox system – an analytical photobioreactor from BVT Technologies – was the default unit of the new device. The working volume of the new minibioreactor is in the range of 4 to 8 ml. The minibioreactor was composed of a minithermostat, a reaction vessel, an oxygen electrode, a temperature and pH probe, accessories for the supply of air to the liquid and an insertion for the transport of liquid from / to the reactor. The functionality of the assembled device and its characteristics were measured. Furthermore, an operating procedure for the decontamination process using a hydrogen peroxide mist was developed for the new equipment. An operating procedure for culturing microorganisms in a minibioreactor was also set up. Pilot cultivations of Halomonas halophila were demonstrated in the prepared equipment. In these cultivations, three assemblies for air supply to the liquid were tested. However, none of the assemblies was able to provide a sufficient supply of oxygen to cell culture – the dissolved oxygen value always dropped to 0%. For the further development, a new assembly was designed for the supply of air to the liquid - an aeration ring from a membrane tube.

Soil salinity is an increasing problem facing agriculture in many parts of the world. Climate change and irrigation practices have led to decreased yields of large areas of farmland due to increased salt levels in the soil. Irrigation introduces salts to the soil that with time accumulate and threaten crop yield. In arid climates like Utah, the practice of irrigation is especially threatening to salt-sensitive crops including alfalfa (Medicago sativa). Plants that have tolerance to salt are needed to feed livestock and the world's population. One approach to address this problem is to introduce genes encoding salt tolerance into the genomes of salt-sensitive plants through genetic engineering, but this approach has limitations. These limitations include the misinformed public perception of genetically modified organisms (GMOs). Even if the GMO salt-tolerant plants could produce palatable foods, in regions of the world with saline soils incongruous with farming, if consumers refuse to purchase the food then the engineering and upfront costs of production are negated. Another fairly new approach involves the isolation and development of salt-tolerant (halophilic) plant-associated bacteria. Several reports are now available demonstrating how the use of halophilic inoculants enhance plant growth in salty soil. This enhanced plant growth is most likely associated with changes in plant gene expression; however, the mechanisms behind this growth stimulation are not yet clear. Halomonas elongata 1H9, a rhizobacteria native to Goshen UT, has been identified as a plant growth-promoting rhizobacteria (PGPR) when used as an inoculant added to alfalfa seedlings grown in salty soils. Plants grown in the presence of this Halomonas sp. and 1% salt demonstrated an average increase of 2.4x the biomass of alfalfa plants grown without inoculum in salty soils. This suggests that this Halomonas sp. positively influences plant salt tolerance, which raises the question as to how the bacteria stimulate plant growth under these conditions. To identify and characterize plant genes induced by Halomonas elongata, transcriptional analysis was performed using RNA-sequencing (RNA-seq). This analysis identified a variety of differentially expressed genes (DEGs) including transcription factors (e.g. MYB14, GATA transcription factor 9, Ethylene-responsive transcription factors ER017 and ER109) and plant enzymes involved in growth and development (e.g. xyloglucan endotransglucosylase and phosphodiesterase). This was followed by gene validation via real-time quantitative PCR (RT-qPCR), the gold standard for RNA-seq validation, however this process was never successfully completed. Suggestions for next steps are included in the discussion section of chapter 3.

This diploma thesis deals with the study of the utilization of Raman spectroscopy and Raman tweezers for analysis and isolation of polyhydroxyalkanoates (PHA) producing bacteria. Using gas chromatography with FID detection, we determined the polyhydroxybutyrate (P(3HB)) content of the PHA biomass of bacterial strains Burkholderia cepacia, Halomonas halophila, Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 and Lactobacillus delbrueckii, which is not a producer of polyhydroxyalkanoates but this bactrea was selected as representative of Gram-positive bacteria. Subsequently, thanks to Raman microspectroscopy, Raman tweezers and FT-IR spectrometer in combination with Raman FT-module, we were able to confirm or disprove the presence of P(3HB) in bacteria. Furthermore, the thesis describes Cupriavidus necator H16, which is a model organism for the production of P(3HB), and his mutant strain Cupriavidus necator H16/PHB-4. The bacterial strain Cupriavidus necator H16 was cultivated in a production mineral medium of various nitrogen contents, while cultivation was also carried out in liquid Nutrient Broth. By this cultivation we were able to reach various P(3HB) content in bacterial biomass, the spectra were subsequently compared with the spectrum of the bacterial strain Cupriavidus necator H16/PHB-4. Raman spectroscopy is well used to characterize the composition of individual bacterial cells, is a fast, versatile, and virtually non-invasive tool for studying cells.