Hydrogen has an extensive potential as a clean and renewable energy source. Photosynthetic, non-sulphur, purple bacteria, Rhodobacter sphaeroides O.U.001 produces molecular hydrogen by nitrogenase enzyme. Nitrogenase enzyme is encoded by nifHDK genes and expression of the structural genes, nifHDK, is controlled by NifA which is encoded by nifA gene. The transcription of nifA is under the control of Ntr system and product of prrA gene. Relationship between the genes that have roles in nitrogenase synthesis should be understood well to increase biological hydrogen production. In this work, expression levels of nitrogenase encoding nifH and control genes nifA, prrA were examined at different physiological conditions. In addition to modifications in expression levels, changes in hydrogen production and growth capacity were also investigated in response to different concentrations of ammonium source, oxygen and different light intensities. In this study, it was found that increasing concentrations of ammonium chloride caused decrease in hydrogen production. Glutamate containing medium had the capacity for higher hydrogen production. The expression levels of nifH and nifA genes decreased with the increase in concentrations of ammonium chloride. There was a negative correlation between the expression levels of prrA gene and its target, nifA gene. Hydrogen production was observed even in aerobic conditions of the same media compositions. It was observed that different culture media had changing growth and hydrogen production capabilities at different light intensities. There was no direct proportion between the expression levels of nifH gene and amount of hydrogen at different light intensities.

Rhodobacter sphaeroides O.U.001 is a purple non-sulphur bacterium producing hydrogen under photoheterotrophic, nitrogen limited conditions. Hydrogen is produced by Mo-nitrogenase but substantial amount of H2 is reoxidized by a membrane bound uptake hydrogenase. In this study, hydrogen production and the expression of structural nitrogenase genes were investigated by varying molybdenum and iron ion concentrations. These two elements are found in the structure of Mo-nitrogenase and they are important for functioning of the enzyme. The results showed that hydrogen production and nifD gene expression increased upon increase in molybdenum concentration. Increasing iron concentration had also positive effect on hydrogen production and nifK gene expression. To improve the hydrogen producing capacity of R. sphaeroides O.U.001, hupSL genes encoding uptake hydrogenase were disrupted in two different methods. In the first method, hup genes were disrupted by gentamicin resistance gene insertion. In the second method, part of the hup gene was deleted without using antibiotic resistance gene. The wild type and the hup- mutant cells showed similar growth patterns but substantially more hydrogen was produced by the mutant cells. The genes coding for hox1 hydrogenase of Thiocapsa roseopersicina was aimed to be expressed in R. sphaeroides O.U.001 to produce H2 under nitrogenase repressed and mixotrophic conditions. The hox1 hydrogenase genes of T. roseopersicina were cloned and transferred to R. sphaeroides. Although the cloning was successful, the expression of hydrogenase was not achieved by using either the native promoter of hox1 hydrogenase or the crtD promoter of T. roseopersicina.

Rhodobacter capsulatus is a photosynthetic, purple non-sulfur (PNS) bacterium that produces biohydrogen via photofermentation. Nitrogenase enzyme is responsible for hydrogen production / during fixation of molecular nitrogen into ammonium, hydrogen is produced. Since this process is an energetically expensive process for the cell, hydrogen production is strictly controlled at different levels. When ammonium is present in the environment, hydrogen production completely ceases. The key proteins in the regulation of nitrogenase by ammonium are two PII proteins / GlnB and GlnK. &lsquo / Hyvolution&rsquo / , 6th framework EU project, aims to achieve maximum hydrogen production by combining two hydrogen production processes / dark fermentation and photofermentation. In the first stage of the overall process, biomass is used for hydrogen production in dark fermentation process. Then, the effluent of dark fermentation is further utilized by photosynthetic bacteria to produce more hydrogen. However, the effluent of dark fermentation contains high amount of ammonium, which inhibits photofermentative hydrogen production. In order to achieve maximum hydrogen production, ammonium regulation of nitrogenase enzyme in R.capsulatus has to be released. For this purpose, all PII signal transduction proteins of R.capsulatus (GlnB and GlnK) were targeted to be inactivated by site-directed mutagenesis. The internal parts of glnB and glnK genes were deleted individually without using antibiotic cassette insertion. The successful glnB mutant was obtained at the end of mutagenesis studies. In the case of glnK mutation, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained. The effect of ammonium on glnB mutant R.capsulatus was investigated and compared with wild type. Biomass of the bacterial cultures, pH of the medium and amount of produced hydrogen were periodically determined. Moreover, the concentrations of acetic, lactic, formic and propionic acids in the medium were periodically measured. Both wild type and glnB mutant grew on acetate and effectively utilized acetate. Ammonium negatively affected hydrogen production of glnB mutant and wild type. The ammonium inhibition of hydrogen production did not release in glnB mutant due to the presence of active GlnK protein in the cell / hence, inactivation of one of PII proteins was not enough to disrupt ammonium regulation of the cell. Moreover, kinetic analysis of bacterial growth and hydrogen production were done. Growth data fitted to the Logistic Model and hydrogen production data fitted to the Modified Gompertz Model.

Rhodobacter capsulatus DSM1710 is a purple non-sulfur bacterium capable of hydrogen production via photofermentation. Biohydrogen is a clean and renewable way of hydrogen production, which can be achieved by PNS bacteria in outdoor large scale photobioreactors using sun light. In outdoor conditions bacteria can be exposed to heat and cold stress. In this study in order to understand the effects of heat and cold stress on photofermentative hydrogen production and gene expression profile of R.capsulatus on acetate as the carbon source, microarray analysis was carried out. Since there is no commercially available microarray chip for R.capsulatus, an Affymetrix GeneChip&reg / was designed and it was manufactured by Affymetrix.The experiments were conducted at 30

Hydrogen, which is a clean energy source, is one of the alternatives for fossil fuels. Biological hydrogen production is one of the hydrogen production methods. Rhodobacter capsulatus is a photosynthetic bacterium that produces hydrogen via photofermentation. R. capsulatus can also synthesize some valuable by-products such as Poly-beta- hydroxy butyric acid (PHB), which is a biodegradable bioplastic. In a two stage biohydrogen production system, which is combination of dark fermentation and photofermentation, dark fermentor effluents are used for photofermentation by R.capsulatus. Dark fermentor effluents usually contain high amount of acetate. High amount of acetate may decrease the efficiency of hydrogen production by causing high amount of PHB production. Therefore, it is significant to determine optimum acetate concentration for photofermentation. In this study, the effects of acetate concentration on hydrogen and PHB production by R.capsulatus were investigated by growing bacteria at various acetate concentrations (10 mM-65 mM). In addition, gene expression analysis was performed to investigate the effects of acetate at transcriptional level. For this purpose, expression levels of the genes that encode nitrogenase which is the enzyme that catalyzes hydrogen production and PHB synthase, which is the key enzyme of the PHB synthesis pathway, are examined. Optimum acetate concentration for photofermentation with high hydrogen yield and low PHB amount was determined to be in the range 25 mM-50 mM. nifD expression was found to be high at optimum acetate concentrations and phaC expression was found to be the highest at 65 mM.

No description available.

Vinasse is the liquid waste removed from the base of sugarcane ethanol distillation columns at a ratio of 12-15 liters per liter of alcohol, resulting in an estimated production of approx. 350 billion liters in 2012/2013 in Brazil. Vinasse has a low pH and high chemical oxygen demand, which can cause land desertification when indiscriminately used as fertilizer. Also, underground water contamination is being observed in some regions. We evaluated the potential of vinasse as nutrient source for biohydrogen and volatile fatty acids production by means of anaerobic consortia. Two different vinasse-based media were proposed, using sugarcane juice or molasses as carbon source, and were compared to fermentation in a sucrosesupplemented medium. Pure cultures (4) and consortia (7) were cultured in the propose media and evaluated for volatile fatty acids (VFAs) and biohydrogen production. The consortium LPBAH1, originated from faeces of fruit bat, was selected for fermentation of vinasse supplemented with sugarcane juice and resulted in a higher H2 yield of 7.14 molH2/molsucrose and hydrogen content in biogas of approx. 31% after process optimization. Similarly, the optimized process using the consortium LPBAH2, originated from a lake of a dairy farm, resulted in 3.66 molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process is of great importance for giving a more rational destination to vinasse and expanding Brazilian energy matrix, reducing the dependence of fossil fuels.

TRANSCRIPTIONAL ANALYSIS OF HYDROGENASE GENES IN RHODOBACTER SPHAEROIDES O.U.001 In photosynthetic non-sulphur bacteria, hydrogen production is catalyzed by nitrogenases and hydrogenases. Hydrogenases are metalloenzymes that are basically classified into: the Fe hydrogenases, the Ni-Fe hydrogenases and metal-free hydrogenases. Two distinct Ni-Fe hydrogenases are described as uptake hydrogenases and bidirectional hydrogenases. The uptake hydrogenases are membrane bound dimeric enzymes consisting of small (hupS) and large (hupL) subunits, and are involved in uptake and the recycling of hydrogen, providing energy for nitrogen fixation and other metabolic processes. In this study the presence of the uptake hydrogenase genes was shown in Rhodobacter sphaeroides O.U.001 strain for the first time and hupS gene sequence was determined. The sequence shows 93% of homology with the uptake hydrogenase hupS of R.sphaeroides R.V. There was no significant change in growth of the bacteria at different concentrations of metal ions (nickel, molybdenum and iron in growth media). The effect of metal ions on hydrogen production of the organism was also studied. The maximum hydrogen gas production was achieved in 8.4&micro / M of nickel and 0.1 mM of iron containing media. The expression of uptake hydrogenase genes were examined by RT-PCR. Increasing the concentration of Ni++ up to 8.4&micro / M increased the expression of uptake hydrogenase genes (hupS). At varied concentrations of Fe-citrate (0.01 mM-0.1 mM) expression of hupS was not detected until hydrogen production stopped. These results will be significant for the improvement strategies of Rhodobacter sphaeroides O.U.001 to increase hydrogen production efficiency. In order to examine the presence of hupL genes, different primers were designed. However, the products could not be observed by PCR.

Submitted by Lilian Danielle de Moura Torquato null (litorquato.ibilce@gmail.com) on 2016-12-19T19:48:44Z No. of bitstreams: 1 Tese_ L.D.M. Torquato (12-12-2016)_DEFINITIVA.pdf: 6366289 bytes, checksum: b0113724fd68a4c4328ce9913a55d99b (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-21T11:15:51Z (GMT) No. of bitstreams: 1 torquato_ldm_dr_araiq.pdf: 6366289 bytes, checksum: b0113724fd68a4c4328ce9913a55d99b (MD5) / Made available in DSpace on 2016-12-21T11:15:51Z (GMT). No. of bitstreams: 1 torquato_ldm_dr_araiq.pdf: 6366289 bytes, checksum: b0113724fd68a4c4328ce9913a55d99b (MD5) Previous issue date: 2016-12-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste trabalho foi avaliar a viabilidade do emprego de lodo anaeróbio, originados pelo processo de tratamento de efluentes sanitários, como biomassa para a geração de energia, o qual foi realizada por diferentes processos de conversão: por combustão, sendo este resíduo aplicado isoladamente ou co-processados com bagaço de cana-de-açúcar; por digestão anaeróbia, aplicando-o como inóculo para a produção biológica de hidrogênio. No processo termoquímico, avaliou-se o desempenho térmico e cinético das biomassas citadas, bem como de suas misturas (“blendas”) em diferentes proporções. Os estudos demonstraram que as blendas, apesar da influência das biomassas precursoras, apresentaram propriedades térmicas distintas destas durante o processo de combustão e dependentes da razão de aquecimento empregadas. Em termos de conteúdo energético, inerentemente, o poder calorífico das blendas cresce com a quantidade de bagaço adicionado, que contribui para o aumento da reatividade destes materiais durante a devolatilização. Entretanto, para a combustão em grande escala, a adição de 75% de bagaço ao lodo não se mostrou viável, por levar ao aumento da energia de ativação (Ea) durante o processo. Por outro lado, apesar de necessitar de maior Ea inicialmente, o lodo demonstrou maior estabilidade ao longo do processo, sugerindo sua capacidade de manter o mesmo rendimento por maior tempo. Os estudos preliminares de emissões gasosas demonstraram um perfil inesperado para a blenda com maior proporção de bagaço, o qual pode ser um reflexo dos resultados obtidos durante a caracterização térmica das biomassas avaliadas. Quanto à abordagem bioquímica, foram realizados ensaios voltados à geração de H2 por meio da digestão anaeróbia, empregando o lodo como inóculo e, como substrato, meio de cultura sintético e efluentes da indústria de processamento de frutas cítricas (a água residuária e a vinhaça citrícola). Este estudo demonstrou a potencialidade de ambos os efluentes citrícolas como substratos, bem como reiteraram a viabilidade de aplicação prática do lodo de esgoto LG para a produção biológica de H2, a qual já havia sido comprovada em meio sintético, inclusive em concentrações crescente de sacarose. Este inóculo apresentou maior potencial biológico quando comparado a amostras de lodo de diferentes sistemas de tratamento de efluentes da região de Araraquara. Paralelamente, foi desenvolvido um método termogravimétrico para a análise imediata de biomassas (umidade, materiais voláteis, carbono fixo e cinzas), no qual foi possível a avaliação de todas as referidas propriedades em uma única medida, realizada em um tempo consideravelmente mais curto em relação a métodos padrão. O método desenvolvido foi validado e aplicado a diferentes tipos de biomassas, demonstrando ser preciso mesmo quando aplicado a uma matriz complexa, como o lodo de esgoto. Em todos os estudos, ficou clara a importância da análise térmica para a caracterização destes resíduos quando se propõe sua aplicação energética, haja visto que fornece parâmetros que servem de base à compreensão dos fatores preponderantes para a futura implantação destes em processos em larga escala. As informações fornecidas por este trabalho certamente podem contribuir para agregar valor a materiais outrora tidos como rejeitos, promovendo-os a fontes potenciais de energia (biomassa) renovável e até mesmo limpa (H2), com alta disponibilidade e custo reduzido. / The aim of this study was to evaluate the feasibility of using the anaerobic sludge generated by the treatment of sanitary effluents, as biomass for power generation, which was carried out by different conversion processes: combustion, by applying this residue single or co-processed with sugar cane bagasse; anaerobic digestion, by applying sludge as inoculum for the biological production of hydrogen. In the thermochemical process, the thermal and kinetic performance of the related biomasses, as well as of their mixtures ("blends") were evaluated in different proportions. The studies showed that the blends had different thermal properties during combustion process despite the influence of the precursor biomass, which dependes on the heating rate employed. In terms of energy content, inherently, the heating value of the blends increases as increases the amount of bagasse, which contributes to increase the reactivity of these materials during devolatilization. However, for large-scale combustion, the addition of 75% of bagasse to the sludge was not feasible, since it results in a increase of the activation energy (Ea) during the process. On the other hand, despite the need for a greater input of Ea, the sludge showed greater stability throughout the process, suggesting its capacity to maintain the same yield for a longer time. Preliminary studies of gaseous emissions showed an unexpected profile for the blend with the highest bagasse ratio, which may be a reflection of the results obtained during the thermal characterization of the evaluated biomasses. Regarding the biochemical approach, tests were performed to generate H2 by anaerobic digestion, using sludge as inoculum and, as substrate, synthetic medium and effluents from the citrus processing industry (the wastewater and the citrus vinasse). This study demonstrated the potential of both citrus effluents as substrates, as well as reaffirm the feasibility of practical application of LG sewage sludge to the biological production of H2, which have already been proven in synthetic medium, even in increasing concentrations of sucrose. This inoculum presented higher biological potential when compared to sludge samples from different effluent treatment systems of the region of Araraquara. In parallel, a thermogravimetric method was developed for the proximate analysis of biomasses (moisture, volatile matter, fixed carbon and ash), which enables the direct evaluation of all these properties in a single measurement, carried out in a considerably shorter time in relation to standard methods. The developed method was validated and applied to different types of biomass, showing to be accurate even when applied to a complex sample as sewage sludge. In all studies, it became clear the importance of thermal analysis for the characterization of these residues when it is proposed their energetic application, given that it provides parameters that serve as a basis for the understanding of the prevailing factors for the future application of these materials in large scale processes. The information provided by this work can certainly contribute to add value to materials once considered as tailings, turning them into potential sources of renewable and even clean energy (H2), with high availability and low cost.

O bagaço de cana de açúcar é um resíduo lignocelulósico que, após a sua hidrólise, pode ser utilizado como substrato para a produção de hidrogênio (H2) por fermentação. O objetivo deste trabalho foi realizar pré-tratamentos do bagaço de cana com os ácidos clorídrico (HCl) e fosfórico (H3PO4) para a solubilização de carboidratos, produzindo o mínimo de inibidores, bem como para tornar a sua estrutura mais suscetível à hidrólise enzimática. Além disso, foi verificada a possibilidade de utilização dos hidrolisados na produção biológica de H2 por uma cultura mista de micro-organismos. A otimização das condições de pré-tratamento com os ácidos foi feita por meio de um planejamento experimental, variando-se a concentração entre 0,64 e 7,36 % (m/v), a temperatura de 63,20 a96,80°C e o tempo de 38,40 a 441,60 min. Nos hidrolisados obtidos foram determinadas as concentrações de açúcares redutores totais (ART) e de monossacarídeos, tais como a glicose, a xilose e a arabinose, além de potenciais inibidores de fermentação, o furfural, o hidroximetilfurfural (HMF) e o ácido acético. As condições de pré-tratamento do bagaço, nas quais foram obtidas as maiores concentrações de ART (13,88 g/L) foi utilizando 6,0 % (m/v) de HCl, em 360,00 min., a 90°C. Entretanto, sob estas condições, também foram detectadas as concentrações mais elevadas dos inibidores. A condição ótima para a hidrólise com o HCl, obtida através da análise estatística, na qual a concentração de inibidores foi minimizada e a de ART maximizada foi de 96,80ºC, 441,6 min e 7,36 % (m/v) de ácido. Para o pré-tratamento com o H3PO4, as condições ótimas foram as mesmas encontradas para o HCl, obtendo-se 4,98 g/L de ART. Os bagaços pré-tratados foram submetidos à hidrólise enzimática com a enzima Celluclast® e um extrato enzimático bruto com atividade de xilanases. A maior concentração de ART (20,98 g/L) obtida pelas duas hidrólises (ácido/enzimática) foi no bagaço pré-tratado com HCl no tempo de 360,00 min., 6,0 % (m/v) de ácido a 90°C, o qual também apresentou a maior concentração de inibidores (total de 1,23 g/L). O hidrolisado obtido com HCl que apresentou maior concentração de ART foi utilizado em ensaios de fermentação para a produção de H2 por cultura mista. / Sugarcane bagasse is a lignocellulosic residue that can be used as substrate to produce hydrogen (H2) by fermentation after hydrolysis. This study aimed to optimize the pretreatment of sugarcane bagasse with hydrochloric acid (HCl) and phosphoric acid (H3PO4), to solubilize carbohydrates and produce the minimal amount of inhibitors as well as make its structure more susceptible to enzymatic hydrolysis. In addition, we verified the possibility of using hydrolysates in the biological production of H2 by a mixed culture of microorganisms. We optimized the conditions for bagasse pretreatment with acids using an experimental designwe varied the concentration between 0.64 and 7.36% (w/v), the temperature from 63.20 to 96.80 °C, and the time from 38.40 to 441.60 min. In the hydrolysates, we determined the concentrations of total reducing sugars (TRS); monosaccharides such as glucose, xylose, and arabinose; and potential inhibitors of fermentation like furfural, hydroxymethylfurfural (HMF), and acetic acid. The conditions of bagasse pretreatment 6.0% (w/v) HCl, 360 min. and 90 °C led to the highest TRS concentrations (13.88 g L-1), but also to the highest concentrations of inhibitors. Statistical analysis revealed that the optimum conditions for the hydrolysis of sugar cane bagasse with HCl that minimized the concentration of inhibitors while maximizing the TRS concentration were: 96.80 °C, 441.6 min, and 7.36% (w/v) of acid. The optimum conditions for pre-treatment with H3PO4 were the same as those found for HCl; which yielded 4.98 g L-1 TRS. We subjected the pretreated bagasse to enzymatic hydrolysis with Celluclast® enzyme and to a crude enzyme extract with xylanase activity. For both hydrolyses (acid and enzymatic), the highest TRS concentration (20.98 g L-1) was achieved with the bagasse pretreated with HCl 6.0% (w/v) at 90 °C for 360.00 min., which also furnished the highest concentration of inhibitors (total 1.23 g L-1). The hydrolysate obtained with HCl contained higher TRS concentration was used as substrate in fermentation assays for the production of H2 by mixed culture.