Cianobactérias são micro-organismos procarióticos fotoautotróficos que apresentam ampla variedade morfológica e metabólica, o que favorece sua colonização nos mais diversos tipos de ambientes. Essa versatilidade favorece a sua utilização em inúmeras aplicações biotecnológicas em áreas como agricultura, energia, nutrição e tratamento de efluentes. Com o aumento da demanda energética e de alimentos, as cianobactérias são consideradas alternativas mais sustentáveis para a biossíntese de produtos de interesse industrial. Nesse contexto, o objetivo deste estudo foi avaliar o crescimento de cianobactérias em meios suplementados com vinhaça e seus efeitos na produção de lipídios e na composição de ácidos graxos, visando a produção de biodiesel. Para tanto, onze linhagens de cianobactérias isoladas de amostras de água coletadas em rios da Amazônia foram identificadas morfologicamente e geneticamente como pertencentes à quatro ordens - Nostocales, Chroococciodipsidales, Synecoccocales e Oscillatoriales. Essas linhagens tiveram o seu crescimento avaliado em meios de cultivo suplementados com vinhaça (1, 2,5, 5 e 10% v/v) e em vinhaça diluída em água ultrapura (2,5, 5 e 10% v/v). Todas as linhagens foram capazes de crescer nas concentrações testadas, porém, nas concentrações mais elevadas de vinhaça, observou-se a inibição do crescimento e/ou o aumento da proliferação de contaminantes heterotróficos. Nas diluições de vinhaça em água ultrapura, nenhuma linhagem apresentou crescimento. Duas linhagens foram selecionadas para análises subsequentes devido ao rápido crescimento (Planktothrix mougeotii CMAA1564) e ao perfil de ácidos graxos favoráveis à produção de biodiesel (Amazoninema brasiliense CMAA1602, gênero inédito). Essas linhagens tiveram seu crescimento investigado nos meios BG11, BG11+1% e BG11+2,5% de vinhaça utilizando os métodos gravimétrico (biomassa seca) e espectrofométrico (densidade ótica - DO680nm e DO750nm), para a identificação das fases de crescimento. As maiores taxas de produtividade lipídica e de biomassa foram observadas nas biomassas coletadas na fase log em meio suplementado com vinhaça a 1%, com destaque para P. mougeotii CMAA1564 (3,18 e 35,87 mg L-1 dia-1, respectivamente). A análise do perfil de ácidos graxos das frações lipídicas mostrou a predominância dos ácidos graxos saturados e monoinsaturados nos óleos das biomassas cultivadas em meio com vinhaça, sendo majoritários os ácidos palmítico, palmitoleico, oleico e alfa-linolênico. A partir da composição de ácidos graxos das frações lipídicas foi possível estimar 11 parâmetros de qualidade do biodiesel derivado dessa matéria-prima lipídica. A maioria das propriedades avaliadas estavam de acordo com as especificações de qualidade do biodiesel estabelecidas na legislação nacional e internacional, com exceção para a viscosidade cinemática que apresentou valores inferiores. Esses resultados são promissores visto a oportunidade de exploração da biodiversidade de cianobactérias ainda pouco estudada para fins biotecnológicos. Essa pesquisa demonstra a viabilidade de utilização do conteúdo lipídico desses organismos como matéria-prima lipídica alternativa para a produção de biodiesel / Cyanobacteria are photoautotrophic prokaryotes that show a wide morphological and metabolic variety, which favors the colonization of the most diverse types of environments. This versatility reflects in its numerous biotechnological applications in areas such as agriculture, energy, nutrition and effluent treatment. With the increasing in energy and food demand, cyanobacteria are considered a more sustainable alternative for the biosynthesis of products of industrial interest. In this context, the aim of this study was to evaluate the cyanobacteria growth in media supplemented with vinasse and its effect on lipid production and fatty acid composition, with focus on biodiesel production. So, eleven cyanobacterial strains from water samples from Amazonian rivers were morphologically and genetically identified as belonging to four orders - Nostocales, Chroococciodipsidales, Synecoccocales e Oscillatoriales. These strains had their growth evaluated in culture media supplemented with vinasse (1, 2,5, 5 and 10% v/v) and in vinasse diluted in ultrapure water (2,5, 5 and 10% v/v). All strains were able to grow at the concentrations tested, however, at higher vinasse concentrations, growth inhibition and/or higher heterotrophic contaminants proliferation was observed. In vinasse diluted in ultrapure water, none of them were able to grow. Two cyanobacterial strains were selected for subsequent analysis due to rapid growth (Planktothrix mougeotii CMAA1564) and favorable fatty acid profile for biodiesel production (Amazoninema brasiliense CMAA1602, a new genus). Then, these strains had their gowth investigated in the media BG11, BG11+1% and BG11+2,5% of vinasse using the gravimetric (dry biomass) and spectrophotometric (optical density - OD680nm e OD750nm) methods, for growth phases identification. The highest lipid and biomass productivities were observed in the biomass collected in the log phase in medium supplemented with 1% of vinasse, wtith the highest values observed for the P. mougeotii CMAA1564 (3,18 e 35,87 mg L-1 dia-1, respectively). The fatty acid profile of the lipid fraction showed the predominance of saturated and monounsaturated fatty acids in the oils of biomass grown in vinasse medium, with palmitic, palmitoleic, oleic and alpha-linolenic acids being the majority. From the fatty acid composition of the oils, it was possible to estimate eleven quality parameters of the biodiesel derived from these lipid feedstocks. Most of the properties evaluated werein accordance with the biodiesel quality specification estabilished in the national and international legislation, except for the kinematic viscosity that presented lower values. These results are promising given the opportunity for exploiting the underestimated cyanobacteria diversity for biotechnological purposes. This research shows the feasibility of using the lipid content of these organisms as an alternative lipid feedstock for biodiesel production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems. / Singapore-MIT Alliance (SMA)

As the demand for farmed shrimp continues to grow worldwide, the use of probiotics to address the sustainability of aquaculture fisheries has gained much attention. Emerging diseases in shrimp aquaculture, such as acute hepatopancreatic necrosis disease (AHPND), have devastating economic impacts in countries that largely depend on this activity. The relevance of this research lies on the fact that it explores the potential of using probiotics to mitigate the negative effects of AHPND in shrimp aquaculture. The scope of these studies includes survival of probiotic microbes in typical aquaculture water conditions, the effectiveness of probiotics in vitro and in vivo against the pathogenic strain of Vibrio parahaemolyticus that causes AHPND, and the effects of probiotics on the bacterial community composition in aquaculture water and gastrointestinal tract of shrimp after an induced AHPND infection. The microorganisms chosen as probiotics for this research include a lactic acid bacterium, a yeast and a photosynthetic bacterium. Informal feedback from shrimp farmers in Thailand and Vietnam revealed positive results against AHPND when using a commercially available probiotic containing multiple species of microorganisms from these probiotic groups. This research was divided into four studies. The first study (Chapter 2) evaluated the growth of the three different probiotic microbes in two different salinity conditions commonly found in intensive shrimp production systems to determine whether they could be further considered as potential candidates. The hypothesis was that the NaCl concentrations of the media may not have an effect on acid production, growth and cell morphology of the microorganisms being evaluated due to their metabolic mechanisms of adaptation to differences in osmotic pressure. The probiotic microbes were cultured in nutrient media enriched with 1 and 2% NaCl. Microbial survival, acidity and cell morphology between treatments were compared using enumeration by serial dilutions and plating, pH measurements and scanning electron microscopy imaging, respectively. The results showed that salinity levels up to 2% NaCl did not affect the growth of lactic acid bacteria and yeast. Photosynthetic bacteria grown in media with 1% NaCl showed a 24-hour delay in comparison to the control and a prolonged lag phase that lasted 48 hours when the media contained 2% NaCl. Therefore, the hypothesis was partially supported. Based on these results, all three probiotic microbes demonstrated to be suitable for application in aquaculture ponds with up to 2% salinity. The second study (chapter 3) aimed at determining the inhibitory effects of eight different formulations of probiotic solutions against the pathogenic strain of V. parahaemolyticus in vitro. The hypothesis of this study was that probiotic solutions containing whole microbial cultures of multiple microbial types including lactic acid bacteria may have a greater inactivation of the pathogen. The probiotic formulations consisted of individual cultures, combinations of the three probiotic microbes, and a commercially available probiotic formulation. The inhibitory effects were evaluated following a disk diffusion test on solid media by comparing diameters of zones of inhibition, and a challenge test in liquid media by comparing pathogen survival after exposure to probiotic solutions. Findings revealed inhibition zones with greater diameters in disks treated with whole microbial cultures (min: 7.83 mm, max: 11.33 mm) versus disks treated with only supernatants (min: 7.00 mm, max: 8.50 mm). Results from the challenge in liquid media tests showed greater inactivation of the pathogen after 48 h (6.56±0.07 to 5.43±0.03 log10 reduction) when treated with lactic acid bacteria alone and in combination with other microbial types. From these results, the hypothesis was supported and it was concluded that probiotic solutions including a lactic acid bacterium, the combination of lactic acid bacterium and photosynthetic bacterium and the combination of lactic acid bacterium, yeast and photosynthetic bacterium may be used to effectively inhibit AHPND in shrimp aquaculture. The third study (chapter 4) explored the effects of probiotic solutions on live shrimp (Litopenaeus vannamei) pretreated with probiotics for 7 days prior to challenging them with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that higher shrimp survival and weight gains would be observed when shrimps are exposed to probiotics solutions with multiple microbial types in the water and feed. Water quality parameters (dissolved oxygen, temperature, acidity, salinity and total ammonia nitrogen), difference in shrimp weight increase and shrimp survival were compared between probiotic treatments and controls. Treatments included: (1) a lactic acid bacterium alone (Pro.Sol1), (2) a lactic acid bacterium and a photosynthetic bacterium (Pro.Sol2), (3) the combination of a lactic acid bacterium, a yeast, and a photosynthetic bacterium (Pro.Sol3), and (4) a commercial probiotic (Com.Pro) and the results showed shrimp survival of 11.7, 26.7, 36.7 and 73.3%, respectively. Also, treatments Pro.Sol3 and Com.Pro resulted in higher weight gains (19.7 and 31.2%, respectively) versus the negative control (11.2%). Moreover, onset of the disease was delayed in all treatments as follows: 12 h with Pro.Sol1, 20 h with Pro.Sol2, 22 h with Pro.Sol3, and 26 h with Com.Pro. From these results, the hypothesis was supported and it was concluded that probiotics have the potential to effectively mitigate the effects of AHPND in the shrimp aquaculture. Finally, the fourth study (chapter 5) evaluated the effects of probiotics on the bacterial diversity of the gastrointestinal tract of shrimp as well as variation of bacterial and fungal diversity in the water before and after challenging shrimp with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that probiotic solutions with multiple microbial types may be able to maintain the microbial composition of the shrimp GI tract and aquaculture water preventing an increase in relative abundance of the family Vibrionaceae. Next generation sequencing was conducted using an Illumina MiSeq™ and primers specific for bacterial V4 hypervariable region of the 16S rRNA gene. The results obtained from the GI tract of shrimp revealed that the relative abundance of the family Vibrionaceae significantly increased in treatments with high mortalities, whereas treatments with higher survivals showed no significant difference in relative abundance of Vibrionaceae family members (P>0.05) in comparison to the negative control. The Shannon diversity index values (abundance and evenness) of the bacterial communities revealed that the treatment with the highest survival had the highest Shannon index value (4.69±0.133) whereas the treatment with lowest survival had the lowest Shannon index value (0.17±0.004). The results obtained from water samples did not show a higher abundance of the family Vibrionaceae, and diversity was maintained after infection (Shannon index 4.64±0.58). Regarding fungal diversity in water samples, Shannon index values revealed no significant changes before (3.627±0.37) and after infection (3.664±0.18) except for Pro.Sol3 (2.859±0.56) and Com.Pro (1.795±0.50), which included yeast in their formulation. Thus, the hypothesis of this study was partially supported since the results revealed that while all probiotics maintained the diversity of microbial composition in the water, only those probiotic solutions with various microbial types in the formulation maintained the diversity of the microbial composition in the GI tract of shrimp providing protection against AHPND.

The marine cyanobacterium Synechococcus WH7803 adapts to changes in light intensity by changing its photosynthetic physiology. This work is a study o f the rate o f photoadaptation of Synechococcus WH7803 in laboratory cultures. Cultures were shifted from constant 8 µEm-2s-1 light to constant 80 µEm-2s-1 light, from constant 80 µEm-2s-1 light to 8 µEm-2s-1 light and from different light regimes to a single fluctuating light regime. The response of high iron cultures grown in modified aquil culture medium at 5*10-6M Fe was compared with the response of low iron cultures grown in modified aquil medium at 1*10-6M Fe. Cultures were assayed every 2 days for at least 10 days, assaying cell population density by fluorescence microscopy, chlorophyll a by spectrophotometry of acetone extracts, total protein by the bicinchoninic acid method, and photosynthesis vs. irradiance (P vs. I) response by H14CO3 uptake at 10 light intensities. Iron nutrition did not strongly affect the rate of photoadaptation as determined from the slopes of P vs. I curves as light approaches zero (the parameter called alpha). The rate of chlorophyll a synthesis was the parameter most strongly affected by iron nutrition, slowing in restricted iron medium. Based on alpha per cell, the adaptation time to the shift from constant 8 µEm-2s -1 light to constant 80 µEm-2s -1 light was less than 2 days. Based on other culture parameters the time to complete adaptation may be closer to 8 days. Based on alpha per cell, the adaptation time to the shift from constant 80 µEm-2s -1 light to constant 8 µEm-2s -1 light was on the order of 2 days. Based on other culture parameters the time to complete adaptation may exceed 10 days.

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

La recherche de sources d’énergie fiables ayant un faible coût environnemental est en plein essor. L’hydrogène, étant un transporteur d’énergie propre et simple, pourrait servir comme moyen de transport de l’énergie de l’avenir. Une solution idéale pour les besoins énergétiques implique une production renouvelable de l’hydrogène. Parmi les possibilités pour un tel processus, la production biologique de l’hydrogène, aussi appelée biohydrogène, est une excellente alternative. L’hydrogène est le produit de plusieurs voies métaboliques bactériennes mais le rendement de la conversion de substrat en hydrogène est généralement faible, empêchant ainsi le développement d’un processus pratique de production d’hydrogène. Par exemple, lorsque l’hydrogène est produit par la nitrogénase sous des conditions de photofermentation, chaque molécule d’hydrogène constituée requiert 4 ATP, ce qui rend le processus inefficace. Les bactéries photosynthétiques non sulfureuses ont la capacité de croître sous différentes conditions. Selon des études génomiques, Rhodospirillum rubrum et Rhodopseudomonas palustris possèdent une hydrogénase FeFe qui leur permettrait de produire de l’hydrogène par fermentation anaérobie de manière très efficace. Il existe cependant très peu d’information sur la régulation de la synthèse de cette hydrogénase ainsi que sur les voies de fermentation dont elle fait partie. Une surexpression de cette enzyme permettrait potentiellement d’améliorer le rendement de production d’hydrogène. Cette étude vise à en apprendre davantage sur cette enzyme en tentant la surexpression de cette dernière dans les conditions favorisant la production d’hydrogène. L’utilisation de résidus organiques comme substrat pour la production d’hydrogène sera aussi étudiée. / The search for alternative energy sources with low environmental impact is in great expansion. Hydrogen, an elegant and simple energy transporter, could serve as means of transporting energy in the future. An ideal solution to the increasing energy needs would imply a renewable production of hydrogen. Out of all the existing possibilities for such a process, the biological production of hydrogen, also called biohydrogen, is an excellent alternative. Hydrogen is the end result or co-product of many pathways in bacterial metabolism. However, such pathways usually show low yields of substrate to hydrogen conversion, which prevents the development of efficient production processes. For example, when hydrogen is produced via nitrogenase under photofermentation conditions, each hydrogen molecule produced requires 4 molecules of ATP, rendering the process very energetically inefficient. Purple non-sulfur bacteria are highly adaptive organisms that can grow under various conditions. According to recent genomic analyses, Rhodospirillum rubrum and Rhodopseudomonas palustris possess, within their genome, an FeFe hydrogenase that would allow them to produce hydrogen via dark fermentation quite efficiently. Unfortunately, very little information is known on the regulation of the synthesis of this enzyme or the various pathways that require it. An overexpression of this hydrogenase could potentially increase the yields of substrate to hydrogen conversion. This study aims to increase our knowledge about this FeFe hydrogenase by overexpressing it in conditions that facilitate the production of hydrogen. The use of organic waste as substrate for hydrogen production will also be studied.

La recherche de sources d’énergie fiables ayant un faible coût environnemental est en plein essor. L’hydrogène, étant un transporteur d’énergie propre et simple, pourrait servir comme moyen de transport de l’énergie de l’avenir. Une solution idéale pour les besoins énergétiques implique une production renouvelable de l’hydrogène. Parmi les possibilités pour un tel processus, la production biologique de l’hydrogène, aussi appelée biohydrogène, est une excellente alternative. L’hydrogène est le produit de plusieurs voies métaboliques bactériennes mais le rendement de la conversion de substrat en hydrogène est généralement faible, empêchant ainsi le développement d’un processus pratique de production d’hydrogène. Par exemple, lorsque l’hydrogène est produit par la nitrogénase sous des conditions de photofermentation, chaque molécule d’hydrogène constituée requiert 4 ATP, ce qui rend le processus inefficace. Les bactéries photosynthétiques non sulfureuses ont la capacité de croître sous différentes conditions. Selon des études génomiques, Rhodospirillum rubrum et Rhodopseudomonas palustris possèdent une hydrogénase FeFe qui leur permettrait de produire de l’hydrogène par fermentation anaérobie de manière très efficace. Il existe cependant très peu d’information sur la régulation de la synthèse de cette hydrogénase ainsi que sur les voies de fermentation dont elle fait partie. Une surexpression de cette enzyme permettrait potentiellement d’améliorer le rendement de production d’hydrogène. Cette étude vise à en apprendre davantage sur cette enzyme en tentant la surexpression de cette dernière dans les conditions favorisant la production d’hydrogène. L’utilisation de résidus organiques comme substrat pour la production d’hydrogène sera aussi étudiée. / The search for alternative energy sources with low environmental impact is in great expansion. Hydrogen, an elegant and simple energy transporter, could serve as means of transporting energy in the future. An ideal solution to the increasing energy needs would imply a renewable production of hydrogen. Out of all the existing possibilities for such a process, the biological production of hydrogen, also called biohydrogen, is an excellent alternative. Hydrogen is the end result or co-product of many pathways in bacterial metabolism. However, such pathways usually show low yields of substrate to hydrogen conversion, which prevents the development of efficient production processes. For example, when hydrogen is produced via nitrogenase under photofermentation conditions, each hydrogen molecule produced requires 4 molecules of ATP, rendering the process very energetically inefficient. Purple non-sulfur bacteria are highly adaptive organisms that can grow under various conditions. According to recent genomic analyses, Rhodospirillum rubrum and Rhodopseudomonas palustris possess, within their genome, an FeFe hydrogenase that would allow them to produce hydrogen via dark fermentation quite efficiently. Unfortunately, very little information is known on the regulation of the synthesis of this enzyme or the various pathways that require it. An overexpression of this hydrogenase could potentially increase the yields of substrate to hydrogen conversion. This study aims to increase our knowledge about this FeFe hydrogenase by overexpressing it in conditions that facilitate the production of hydrogen. The use of organic waste as substrate for hydrogen production will also be studied.

Mercury (Hg) is a global pollutant and potent neurotoxin that bioaccumulates in aquatic and terrestrial food webs as monomethylmercury (MeHg). Anaerobic microbes are largely responsible for MeHg production, which depends on the bioavailability of inorganic Hg substrates to methylators. Hg redox cycling pathways such as Hg reduction play a key role in determining Hg’s availability in the environment. Although abiotic photochemical Hg reduction typically dominates in oxic surface environments, Hg reduction pathways mediated by photosynthetic and anaerobic microbes are thought to play an important role in anoxic habitats where light is limited and MeHg production occurs. Currently, the physiological mechanisms driving phototrophic and anaerobic Hg reduction remain poorly understood. The main objective of my thesis is to provide mechanistic details on novel anaerobic and phototrophic Hg reduction pathways. I used a combination of physiological, biochemical and trace Hg analytical techniques to study Hg reduction pathways in a variety of anaerobic and photosynthetic bacteria. I demonstrated that Hg redox cycling was directly coupled to anoxygenic photosynthesis in aquatic purple non-sulphur bacteria that reduced HgII when cells incurred a redox imbalance. I discovered that terrestrial fermentative bacteria reduced Hg through pathways that relied on the generation of reduced redox cofactors. I also showed that sulphur assimilation controlled Hg reduction in an anoxygenic phototroph isolated from a rice paddy. In addition, I developed methods to explore cryptic anaerobic Hg redox cycling pathways using Hg stable isotope fractionation. At its core, my thesis underscores the intimate relationship between cell redox state and microbial Hg reduction and suggests a wide diversity of microbes can participate in anaerobic Hg redox cycling.

Title: Artificial light-harvesting antenna based on an aggregation of bacteriochlorophyll c with selected pigments Author: Tomáš Malina Department: Department of Chemical Physics and Optics Supervisor of the master thesis: doc. RNDr. Jakub Pšenčík, Ph.D., KCHFO MFF UK Abstract: Solar energy is one of the most important energy sources for all living organisms. The light harvesting takes place in specialised photosynthetic complexes called antennas; they typically contain pigments held by a protein scaffold. Antennas of green bacteria, chlorosomes, are unique in this respect, for they do not need proteins to organise the pigments. The pigments contained in chlorosomes, bacteriochlorophyll (BChl) c, d or e, aggregate spontaneously. This self-aggregation can be used to form an artificial light-harvesting antenna the absorption spectrum of which can be extended by addition of other pigments. Antennas based on aggregation of BChl c with β-carotene and BChl a were prepared by a fast and slow method. The excitation energy transfer efficiency between these pigments was studied. The efficiency of energy transfer from BChl c to BChl a reached up to 95 %, the efficiency of energy transfer from β-carotene to BChl c was lower. An important role of β- carotene in artificial aggregates as well as in chlorosomes is its...