Projects
2019 - Current
Sustainable bio-based 1,3-propanediol production from C5/C3 sources by metabolically engineered clostridia
This joint research proposal focuses on the sustainable and bio-based production of the chemical commodity 1,3-propanediol (PPD) by bacteria of the genus Clostridium. PPD can be manufactured by chemical synthesis or by fermentation. Currently, about 150000 tons of PPD are produced per year, and the value is expected to increase to around 225000 tons by 2022. The research proposal is based on two renewable sources of raw material, that is, the C5 fraction of hydrolysis of lignocellulose and the residual glycerol C3 fraction from biodiesel production. Both are produced in large quantities both in Germany and in the State of São Paulo, resulting from the production of biodiesel and the hydrolysis of lignocellulose in biorefineries. Thus, the project contemplates several topics of this notice, that is, the development of sustainable bioproducts through the use / processing of biomass to obtain bio-based products aiming at the use of residual raw materials. The use of two different bio-based raw materials will be carried out in order to promote the growth of bacteria in pentoses (C5) with the generation of excess reducing equivalents (NADH), which will allow the complete conversion of glycerol into PPD. The current fermentation pathways of Clostridium for the use of glycerol are hampered by the fact that 50% of the glycerol needs to be oxidized to produce NADH, necessary for the reduction of another 50% of glycerol in PPD. Thus, the use of C5 to promote the growth of bacteria with NADH production will lead to a high conversion of glycerol into PPD. This will be achieved by the construction of Clostridium mutants without the ability to use reducing equivalents for the production of ethanol, hydrogen, isopropanol and lactate, obtained through the inactivation of the respective genes. Fermentation experiments will reveal which raw materials are most suitable and whether the simultaneous use of pentose and glycerol will be hampered by catabolic repression. In this case, targeted mutagenesis will also be employed to inactivate the regulator responsible for CcpA repression. The clostridia strain foreseen in this project will employ a glycerol dehydratase independent of vitamin B12. Another objective of the joint proposal is targeted engineering and the expression of a vitamin B12-dependent glycerol dehydratase from Klebsiella pneumoniae, in order to increase its activity. Finally, we also want to address a future use of renewable energy, using bioelectrochemistry. Therefore, we will test if the electricity generated by solar, wind or hydropower can be used as a source of reducing equivalents for the reduction of glycerol in PPD. Strains of wild and recombinant clostridia will be immobilized on electrodes and the biofilms will be tested to capture and use the electrons provided to produce PPD by electrophoresis in a microbial electrolytic cell (MEC). The joint research proposal will also be complemented by exchange programs for graduate students and researchers that will be funded by international programs, independent of this project.
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2018 - Current
Obtaining fuels, products and energy from Agroindustrial waste in biological and bioelectrochemical systems: a contribution to the bioeconomy.
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Description: The bioeconomy is a subject of great worldwide interest, as it comprises the use of renewable biological resources from land and sea to produce bioproducts, such as food, fuel, materials and energy. Obtaining bioproducts and energy becomes even more interesting economically if the raw materials are by-products and / or residues from industrial activities. We been studying sustainable technologies for the recovery of biofuels and energy from wastewater and agro-industrial waste. In previous research projects, the Br21 strain of Clostridium beijerinckii (accession number KT626859) was isolated in our laboratory, and since then its metabolism has been explored for the production of H2 by fermentation. This project aims to increase knowledge about the biotechnological potential of the isolated strain, investigating, in addition to biohydrogen, the formation of other metabolism bioproducts, such as organic acids, ethanol, acetone, butanol and isopropanol. As raw materials will be used pure carbohydrates normally present in renewable raw materials (glucose and xylose), Synthetic lignocellulosic hydrolysates containing carbohydrates and derivatives of biomass hydrolysis (5-hydroxymethylfurfural, syringaldehyde and vanillin), in addition to hydrolyzate of lignocellulosic residues, such as sugar cane straw. Fermentation tests will be carried out to determine the main products of the metabolism of C. beijerinckii Br21 and the kinetic variables of the bioprocesses from these raw materials. In a second stage of the project, an integrated fermentation / bioelectrochemical system will be investigated, aiming at the recovery of H2 produced by C. beijerinckii from carbohydrates and the use of fermentation by-products in a microbial fuel cell (MFC) to generate electrical energy. In this way, this project will contribute to the bioeconomy by maximizing the use of lignocellulosic raw materials for the production of H2, bioproducts and energy.
2015- 2017
Production of third generation biohydrogen from algae biomass and its hydrolysis derivatives (concluded)
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Description: The development of technologies for obtaining renewable and less polluting energy is a matter of worldwide interest. Hydrogen (H2) is a clean fuel; burning it generates only water. H2 can be produced biologically (biohydrogen), especially by fermentation, from renewable raw materials rich in carbohydrates. Recently, algae biomasses with a high concentration of polysaccharides have been studied as substrates for the production of biohydrogen, called third generation. Algae have high CO2 sequestration and productivity, compared to other biomasses, they are aquatic organisms, and therefore their cultivation does not compete with food production. In addition, algae do not have lignin, which facilitates their hydrolysis. This project will use biomass from the macroalgae, Kappaphycus alvarezii, consisting of about 50 to 70% of carbohydrates, especially carrageenan and cellulose, as a substrate for the production of H2. In a first stage of the project, H2-producing microorganisms will be isolated from anaerobic sludges that will be identified by molecular techniques. The isolate should have the ability to produce H2 from glucose and galactose, the main monosaccharides derived from algal hydrolysis. Subsequently, the effect of compounds potentially inhibiting fermentation, derived from algal hydrolysis, such as 5-hydroxymethylfurfural (HMF), levulinic acid and formic acid, on the production of H2 by the isolated microorganism will be investigated. In a second stage of the project, the algae biomass will be used in its integral form and / or as hydrolyzate for the production of H2. The hydrolyzate will be obtained from the optimal hydrolysis conditions with hydrochloric acid, established by an experimental design, having as variables the concentration of the acid, the time and the temperature of the treatment. Therefore, this project will verify the potential of K. alvarezzi algae biomass as a substrate for the production of third generation biohydrogen, in addition to studying the effect of monosaccharides and inhibitors, derived from the hydrolysis of algae biomass, on the production of H2 by fermentation.
2010-2014
Biological Hydrogen Production by Mixed Culture using Sugarcane Bagasse as Substrate (concluded)
Description: This project aims at the production of hydrogen, a clean fuel, alternative to fossil fuels, since its combustion produces mainly H2O. This gas will be produced biologically, through the fermentation of carbohydrates. It is known that the organic production of hydrogen (biohydrogen) only becomes economically competitive, if the sources of carbohydrates are of low cost, as the residues of the agribusiness. Bearing in mind that the State of São Paulo is the largest sugarcane processor in Brazil and that this activity generates a large amount of residues rich in carbohydrates, especially sugarcane bagasse, the use of this residue as a substrate for production will be studied of biohydrogen. However, the carbohydrate present in the bagasse must be made available for its application in fermentation. In this project an optimization of the bagasse hydrolysis will be carried out using an experimental design, having as variables the granulometry, the acid concentration, the time and the temperature of the treatment. As inoculum mixed culture will be used, that is, sludge from an anaerobic effluent treatment system, submitted to different types of pre-treatments for the enrichment of hydrogen-producing bacteria. The conditions suitable for the production of hydrogen by fermentation, such as the initial substrate / microorganism ratio (So / Xo) and temperature (35 and 50 ° C) using sugarcane bagasse as a carbon source, will be investigated in anaerobic batch tests. The operation of bioreactors for long periods, based on the conditions established in the batch tests, will provide information about the possibility of stable production of biohydrogen from this substrate. In addition, the microbiota present in the bioreactors will be monitored using a molecular tool based on DNA, which will allow the evaluation of the genetic variability of the main organisms involved in fermentation.
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Generation of energy in Microbial Fuel Cell from renewable raw materials.
Currently, there is great interest in the development of sustainable technologies for the production of fuels and energy, mainly from effluents and agro-industrial waste. Among the technologies, microbial fuel cells (MFCs) stand out. MFCs, or microbial fuel cell in English, MFC, are systems that use bacteria as biocatalysts for the oxidation of organic and inorganic compounds to generate electric current. Electrons from oxidation are transferred to an electrode at the anode, and from there to the cathode through a conductive material. In this project, energy production in a MFC will be studied using effluents rich in organic acids as substrate. For this purpose, a MFC will be built, and the anode will consist of a carbon tissue electrode and will contain a mixed anaerobic culture of microorganisms (sludge) as a biocatalyst. The anode and cathode compartments will be separated by a Nafion® proton exchanger membrane using a platinum electrode on the cathode side that will be in direct contact with the air. The anode will be fed periodically with an effluent containing organic acids as a carbon source, which must be oxidized to generate energy. The main electrochemical parameters generally evaluated in MFC studies will be determined, such as open circuit potential and power. The concentration of organic acids will be determined by high performance liquid chromatography. As a result, it is expected to quantify the power generated in the system and how it varies depending on the consumption of organic acids. In this way, initial conditions will be established for the generation of energy from effluents rich in organic acids.
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Produção de biocombustíveis e bioprodutos por cepas de Clostridium a partir de derivados da hidrólise de biomassa lignocelulósica.
Atualmente há um grande interesse no desenvolvimento de tecnologias sustentáveis para a produção de combustíveis e energia, principalmente a partir de efluentes e resíduos agroindustriais. Dentre as tecnologias destacam-se as células a combustível microbiológicas ou Microbianas (CCMs). As CCMs, ou microbial fuel cell em inglês, MFC, são sistemas que utilizam bactérias como biocatalizadores da oxidação de compostos orgânicos e inorgânicos para geração de corrente elétrica. Os elétrons oriundos da oxidação são transferidos para um eletrodo no ânodo, e deste para o cátodo através de um material condutor. Neste projeto será estudada a produção de energia em uma CMC utilizando efluentes ricos em ácidos orgânicos como substrato. Para tal será construída uma CMC, sendo que o ânodo será constituído por um eletrodo de tecido de carbono e conterá uma cultura mista anaeróbia de microrganismos (lodo) como biocatalizador. Os compartimentos anôdicos e catódicos serão separados por uma membrana trocadora de prótons Nafion® utilizando um eletrodo de platina no lado catódico que ficará em contato direto com o ar. O ânodo será alimentado periodicamente com um efluente contendo ácidos orgânicos como fonte de carbono, que deverão ser oxidados para gerar energia. Serão determinados os principais parâmetros eletroquímicos geralmente avaliados em estudos de CMC, tais como potencial de circuito aberto e potência. A concentração dos ácidos orgânicos será determinada por cromatografia líquida de alta eficiência. Como resultados espera-se quantificar a potência gerada no sistema e como esta varia em função do consumo dos ácidos orgânicos. Desta forma serão estabelecidas condições iniciais para a geração de energia a partir de efluentes ricos em ácidos orgânicos.
Co-cultivation of Clostridium beijerinckii strains for the biotechnological production of n-butanol
Obtaining bioproducts and energy becomes economically more interesting if the raw materials are by-products and / or residues from industrial activities. We been studying sustainable technologies for the recovery of biofuels and energy from wastewater and agro-industrial waste. In previous research projects, the Br21 strain of Clostridium beijerinckii was isolated in our laboratory, and since then its metabolism has been explored for the production of different products of commercial interest. However, this microorganism alone is not capable of forming the biofuel butanol. This project aims to investigate the formation of butanol by cultivating the strain Br21 with others of Clostridium beijerinckii with the capacity to form n-butanol. Carbohydrates present in renewable raw materials (glucose and xylose) will be used as raw materials. In this way, we want to contribute to the development of a sustainable process for obtaining n-butanol, a potential substitute for gasoline.