ES2315176A1 - Aerobic bacterial electrode for a fuel cell anode, which does not include redox mediators or a proton exchange membrane - Google Patents
Aerobic bacterial electrode for a fuel cell anode, which does not include redox mediators or a proton exchange membrane Download PDFInfo
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- ES2315176A1 ES2315176A1 ES200701534A ES200701534A ES2315176A1 ES 2315176 A1 ES2315176 A1 ES 2315176A1 ES 200701534 A ES200701534 A ES 200701534A ES 200701534 A ES200701534 A ES 200701534A ES 2315176 A1 ES2315176 A1 ES 2315176A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M4/00—Electrodes
- H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
- H01M4/8605—Porous electrodes
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M4/00—Electrodes
- H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
- H01M4/88—Processes of manufacture
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M8/00—Fuel cells; Manufacture thereof
- H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
- Y02P70/50—Manufacturing or production processes characterised by the final manufactured product
Abstract
Description
Electrodo bacteriano aeróbico para ánodo de una pila de combustible sin mediadores redox ni membrana intercambiadora de protones.Aerobic bacterial electrode for anode of a fuel cell without redox mediators or membrane proton exchanger
La presente invención se relaciona con electrodos microbianos para pilas de combustible en las cuales el combustible es materia orgánica. Por tanto, se relaciona con la biotecnología y más concretamente con el uso de células bacterianas como biocatalizadores y más en particular como biocatalizadores redox y más en particular con la biotecnología de la producción directa de energía eléctrica a partir de biomasa.The present invention relates to microbial electrodes for fuel cells in which the Fuel is organic matter. Therefore, it relates to the biotechnology and more specifically with the use of bacterial cells as biocatalysts and more particularly as biocatalysts redox and more particularly with production biotechnology Direct electric power from biomass.
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La actual situación energética hace necesaria una apuesta clara por la implantación de las Pilas de Combustible como uno de los medios más seguros, limpios y eficaces de convertir la energía contenida en diversos combustibles en electricidad (M.S. Dreseselhaus and I.L. Thomas, Alternative energy technologies. Nature, 414, 332- 337, 2001). En este contexto cabe resaltar el hecho de que en los últimos años se ha asistido a una intensa investigación en Biopilas de Combustible llamadas así porque en los electrodos de la pila interviene elementos biológicos, enzimas redox o células microbianas en ánodos y en cátodos (G. Tayhas, R. Palmore, and G.M. Whitesides, Microbial and Enzymatic Biofuel Cells in Enzymatic conversion of biomass for fuel production, M.E. Himmel, J.O. Baker and R.P. Overend, eds., American Chemical Society Symposium series, n° 566, ACS, Washington, DC., 271-290, 1994). La utilización de células microbianas como biocatalizadores permite utilizar como combustible de la pila materia orgánica (glucosa, lactato, etanol, sucrosa, celulosa, etc.), por tanto es posible obtener energía eléctrica a partir de biomasa procedente de aguas residuales, deshechos agrícolas o deshechos industriales. De este modo, la pila no solo serviría para generar energía eléctrica sino también para eliminación de residuos (F. Scholz and U. Schroder, Bacterial Batteries. Nat. Biotechnol., 21, 1151-1152, 2003; L. T. Angenent, K. Karim, M. H. Al-Dahhan, B. A. Wrenn and R. Domínguez-Espinosa, Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends Biotechnol., 22, 477-485, 2004). Generalmente, el uso de biocatalizadores microbianos en pilas de combustible requiere la presencia de mediadores redox en disolución para conseguir la comunicación electrónica entre el microorganismo y el electrodo (A. K. Shukla, P. Suresh, S. Berchmans and A. Rajendran, Biological fuel cells and their applications. Curr. Sci., 87, 455-468, 2004), pero en los últimos años se ha descubierto que algunas bacterias reductoras de metales son capaces de ceder electrones directamente al electrodo (H. J. Kim, H. S. Park, M. S. Jun, I. S. Chang, M. Kim, B. H. Kim, A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enzyme Microbiol. Technol., 30, 145-152, 2002; D. R. Bond, D. R. Lovley, Electricity production by Geobacter sulfurreducens attached to electrodes. Appl. Environ. Microbiol., 69, 1548-1555, 2003; S. Chaudhuri, D. R. Lovley, Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat. Biotechnol., 21, 1229-1232, 2003). En todos los casos descritos en la literatura estos microorganismos solo funcionan en condiciones estrictamente anaeróbicas, por lo tanto su utilización en ánodos de biopilas de combustible obliga a emplear una membrana intercambiadora de protones que separe ánodo y cátodo (Patentes WO0104061, WO2005001981, DE10315792, US2004241528 y JP10233226). Esto tiene el inconveniente de que estas membranas intercambiadoras de protones son caras y pueden limitar cinéticamente el funcionamiento de la pila de combustible (B. Min, S. Cheng, B. E. Logan, Electricity generation using membrane and salt bridge microbial fuel cells, Water Res. 39, 1675-1686, 2005; J. L. Cohen, D. A. Westly, A. Pechenik, H. D. Abruña, Fabrication and preliminary testing of a planar membraneless microchannel fuel cell, J. Power Sources 139, 96-105, 2005). Muy recientemente, se ha publicado una patente de una pila de combustible microbiana sin mediadores redox y sin membrana intercambiadora de protones pero sigue requiriendo condiciones anaeróbicas para el funcionamiento de las bacterias que actúan como biocatalizadores, por lo cual las aguas residuales que proporcionan el combustible deben tratarse anaeróbicamente y el compartimiento catódico debe estar separado del compartimiento anódico por lana de vidrio y cuentas de vidrio para evitar la difusión de O_{2} desde el compartimiento catódico (patente US2005208343).The current energy situation requires a clear commitment to the implementation of the Fuel Cells as one of the safest, cleanest and most efficient means of converting the energy contained in various fuels into electricity (MS Dreseselhaus and IL Thomas, Alternative energy technologies . Nature , 414, 332- 337, 2001). In this context, it is worth highlighting the fact that in recent years there has been an intense investigation in Fuel Biopiles, so called because biological elements, redox enzymes or microbial cells are involved in anodes and cathodes (G. Tayhas, R. Palmore, and GM Whitesides, Microbial and Enzymatic Biofuel Cells in Enzymatic conversion of biomass for fuel production, ME Himmel, JO Baker and RP Overend, eds., American Chemical Society Symposium series, No. 566, ACS, Washington, DC., 271-290, 1994). The use of microbial cells as biocatalysts allows organic matter (glucose, lactate, ethanol, sucrose, cellulose, etc.) to be used as fuel in the cell, therefore it is possible to obtain electrical energy from biomass from wastewater, agricultural waste or industrial waste In this way, the battery would not only serve to generate electricity but also for waste disposal (F. Scholz and U. Schroder, Bacterial Batteries . Nat. Biotechnol., 21, 1151-1152, 2003; LT Angenent, K. Karim , MH Al-Dahhan, BA Wrenn and R. Dominguez-Espinosa, Production of bioenergy and biochemicals from industrial and agricultural wastewater . Trends Biotechnol., 22, 477-485, 2004). Generally, the use of microbial biocatalysts in fuel cells requires the presence of redox mediators in solution to achieve electronic communication between the microorganism and the electrode (AK Shukla, P. Suresh, S. Berchmans and A. Rajendran, Biological fuel cells and their applications Curr. Sci., 87, 455-468, 2004), but in recent years it has been discovered that some metal reducing bacteria are capable of transferring electrons directly to the electrode (HJ Kim, HS Park, MS Jun, IS Chang, M. Kim, BH Kim, A mediator-less microbial fuel cell using a metal reducing bacterium , Shewanella putrefaciens . Enzyme Microbiol. Technol., 30, 145-152, 2002; DR Bond, DR Lovley, Electricity production by Geobacter sulfurreducens attached to electrodes Appl. Environ. Microbiol., 69, 1548-1555, 2003; S. Chaudhuri, DR Lovley, Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells . Nat. Biotechnol., 21, 1229-1232, 2003). In all cases described in the literature these microorganisms only work under strictly anaerobic conditions, therefore their use in anodes of fuel biopiles requires the use of a proton exchange membrane that separates anode and cathode (Patents WO0104061, WO2005001981, DE10315792, US2004241528 and JP10233226). This has the disadvantage that these proton exchange membranes are expensive and can kinetically limit the operation of the fuel cell (B. Min, S. Cheng, BE Logan, Electricity generation using membrane and salt bridge microbial fuel cells , Water Res. 39, 1675-1686, 2005; JL Cohen, DA Westly, A. Pechenik, HD Abruña, Fabrication and preliminary testing of a planar membraneless microchannel fuel cell , J. Power Sources 139, 96-105, 2005). Very recently, a patent for a microbial fuel cell without redox mediators and without a proton exchange membrane has been published but still requires anaerobic conditions for the functioning of the bacteria that act as biocatalysts, so the wastewater that provides the fuel must be treated anaerobically and the cathodic compartment must be separated from the anodic compartment by glass wool and glass beads to prevent the diffusion of O2 from the cathodic compartment (US2005208343).
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La presente invención proporciona nuevos electrodos biológicos para pilas de combustible microbianas que no requieran membrana transportadora de protones para separar el cátodo del ánodo, ni el uso de mediadores rédox en disolución para asegurar la comunicación electrónica eficaz entre células bacterianas y el ánodo. En la presente invención se describe por primera vez la adsorción de células bacterianas del género Acidiphilium spp. sobre un electrodo de un material conductor de electricidad. Las células adsorbidas son capaces de oxidar compuestos orgánicos cediendo directamente los electrones de la reacción al electrodo sin necesidad del uso de mediadores redox en disolución y no viéndose afectado el proceso por la presencia de oxígeno molecular en el medio.The present invention provides new biological electrodes for microbial fuel cells that do not require proton transport membrane to separate the cathode from the anode, nor the use of redox mediators in solution to ensure effective electronic communication between bacterial cells and the anode. In the present invention, the adsorption of bacterial cells of the genus Acidiphilium spp. on an electrode of an electrically conductive material. Adsorbed cells are capable of oxidizing organic compounds by directly yielding the electrons of the reaction to the electrode without the need for the use of redox mediators in solution and the process not being affected by the presence of molecular oxygen in the medium.
Por lo tanto, un primer aspecto de la presente invención lo constituye un electrodo biológico, en adelante electrodo biológico de la presente invención, basado en la utilización de células del género bacteriano Acidiphilium spp. adsorbidas sobre un electrodo conductor para producción de electricidad a partir de materia orgánica de diferentes orígenes, al menos en condiciones aeróbicas, donde las células del género Acidiphilium se caracterizan porque presentan una región homologa al polinucleótido que se muestra en la figura 8 (SEQ ID N° 1), correspondiente a la región 16S de su RNA ribosómico nuclear, de al menos un 80% de identidad, preferentemente de al menos un 90% de identidad más preferentemente de al menos un 95% de identidad y, en una realización aún más preferente de la invención, de al menos un 99% de identidad con el polinucleótido mostrado en dicha figura (SEQ ID N° 1)Therefore, a first aspect of the present invention is a biological electrode, hereinafter the biological electrode of the present invention, based on the use of cells of the bacterial genus Acidiphilium spp. adsorbed on a conducting electrode for the production of electricity from organic matter of different origins, at least in aerobic conditions, where cells of the Acidiphilium genus are characterized in that they have a homologous region to the polynucleotide shown in Figure 8 (SEQ ID N 1), corresponding to the 16S region of its nuclear ribosomal RNA, of at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity and, in an even more embodiment of the invention, of at least 99% identity with the polynucleotide shown in said figure (SEQ ID No. 1)
La información que se proporciona en esta memoria, es suficiente para permitir a un taxónomo molecular identificar a otras cepas que estén dentro de este género.The information provided in this memory is enough to allow a molecular taxonomist Identify other strains that are within this genus.
El género Acidiphilium fue descrito por Harrison en 1981 (Harrison 1981, Int. J. Syst. Bacteriol., 1981, 31, 327-332.). empleando Acidiphilium cryptum Harrison 1981 como especie tipo. Posteriormente se han añadido otras especies al género, un total de 9.The genus Acidiphilium was described by Harrison in 1981 (Harrison 1981, Int. J. Syst. Bacteriol ., 1981, 31 , 327-332.). using Acidiphilium cryptum Harrison 1981 as a type species. Subsequently, other species have been added to the genus, a total of 9.
Acidiphilium acidophilum (Harrison 1983) Hiraishi et al. 1998. Acidiphilium acidophilum ( Harrison 1983 ) Hiraishi et al . 1998
Acidiphilium aminilyticum corrig. Kishimoto et al. 1994, actualmente. Acidiphilium aminilyticum corrig . Kishimoto et al . 1994 , currently.
Acidocella aminolytica (Kishimoto et al. 1994) Kishimoto et al. 1996. Acidocella aminolytica ( Kishimoto et al . 1994 ) Kishimoto et al . 1996
Acidiphilium angustum Wichlacz et al. 1986. Acidiphilium angustum Wichlacz et al . 1986
Acidiphilium facile corrig. Wichlacz et al. 1986. Acidiphilium facile corrig. Wichlacz et al . 1986
Acidiphilium multivorum Wakao et al. 1995. Acidiphilium multivorum Wakao et al . 1995
Acidiphilium organovorum Lobos et al. 1986. Acidiphilium organovorum Lobos et al . 1986
Acidiphilium rubrum Wichlacz et al. 1986. Acidiphilium rubrum Wichlacz et al . 1986
Acidiphilium symbioticum Battacharyya et al. 1991. Acidiphilium symbioticum Battacharyya et al . 1991
De estas 9 especies, una ha surgido como una nueva combinación de Thiobacillus acidophilus (ex Guay and Silver 1975) Harrison 1983, y dos de ella han sido transferidas posteriormente a un nuevo género, Acidocella, como Acidocella aminolytica (Kishimoto et al. 1994) Kishimoto et al. 1996 y Acidocella facilis (Wichlacz et al. 1986) Kishimoto et al. 1996.Of these 9 species, one has emerged as a new combination of Thiobacillus acidophilus ( former Guay and Silver 1975) Harrison 1983, and two of them have subsequently been transferred to a new genus, Acidocella , as Acidocella aminolytica (Kishimoto et al . 1994) Kishimoto et al . 1996 and Acidocella facilis (Wichlacz et al . 1986) Kishimoto et al . nineteen ninety six.
Por las especiales características de estos organismos, que aún no se han estudiado en profundidad, y por las dificultades que supone su descripción, esta será más fácil y fiable si su delimitación taxonómica se basa en métodos de biología molecular (filogenia molecular).Because of the special characteristics of these organisms, which have not yet been studied in depth, and by Difficulties of its description, this will be easier and reliable if its taxonomic delimitation is based on biology methods molecular (molecular phylogeny).
Tanto Acidiphilium como Acidocella pertenecen al Phylum Proteobacteria, a la Clase Alphaproteobacterias del orden Rhodospirillales, de la Familia Acetobacteraceae. El género representante de la Familia es Acetobacter Beijerinck 1898, siendo el tipo del género Acetobacter aceti (Pasteur 1864) Beijerinck 1898.Both Acidiphilium and Acidocella belong to the Phylum Proteobacteria , to the Class Alphaproteobacterias of the order Rhodospirillales , of the Family Acetobacteraceae . The representative genus of the Family is Acetobacter Beijerinck 1898, being the type of the genus Acetobacter aceti (Pasteur 1864) Beijerinck 1898.
Los otros géneros de la Familia, y sus
respectivos tipos, de acuerdo a los criterios taxonómicos del
GenBank,
son:The other genera of the Family, and their respective types, according to the taxonomic criteria of GenBank,
They are:
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Con el fin de definir, a efectos de esta memoria, el género Acidiphylium se realizó un análisis filogenético de las especies tipo de los distintos géneros pertenecientes a la Familia Acetobacteraceae. Como outgroup (grupo externo de comparación) se incluyeron otras especies de la clase Alphaproteobacteria:In order to define, for the purpose of this report, the genus Acidiphylium , a phylogenetic analysis of the type species of the different genera belonging to the Acetobacteraceae Family was performed. As outgroup (external comparison group) other species of the Alphaproteobacteria class were included:
De la Familia Rhodobacteraceae From the Rhodobacteraceae Family
Rhodobacter Imhoff et al. 1984 - Tipo Rhodobacter capsulatis (Molisch 1907) Imhoff et al. 1984. Rhodobacter Imhoff et al . 1984 - Rhodobacter capsulatis type ( Molisch 1907 ) Imhoff et al . 1984 .
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De la Familia Rhodospirillaceae:From the Rhodospirillaceae Family:
Rhodospirillum Molisch 1907 -Tipo Rhodospirillum rubrum (Esmarch 1887) Molisch 1907. Rhodospirillum Molisch 1907 -Type Rhodospirillum rubrum ( Esmarch 1887 ) Molisch 1907 .
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De la Familia Hyphomicrobiaceae:From the Hyphomicrobiaceae Family:
Rhodomicrobium Duchow & Douglas 1949- Tipo Rhodomicrobium vannielii Duchow & Douglas 1949. Rhodomicrobium Duchow & Douglas 1949 - Type Rhodomicrobium vannielii Duchow & Douglas 1949 .
Blastochloris Hiraishi 1997 - Tipo Blastocholris viridis (Drews & Giesbrecht 1966) Hiraishi 1997. Blastochloris Hiraishi 1997 - Type Blastocholris viridis ( Drews & Giesbrecht 1966 ) Hiraishi 1997 .
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De la Familia Erythrobacteracceae:From the Erythrobacteracceae Family:
Erythrobacter Shiba and Simidu 1982 - Tipo Erythrobacter longus Shiba and Simidu 1982. Erythrobacter Shiba and Simidu 1982 - Type Erythrobacter longus Shiba and Simidu 1982 .
Para el análisis arriba mencionado se emplearon polinucleótidos homólogos al que se muestra en la Figura 8 (SEQ ID N°1) y que pertenece al microorganismo empleado para realizar el ejemplo 1 de la presente invención. Estos polinucleótidos homólogos se localizan en la región 16S ribosómico nuclear del material genético de estos microorganismos y fueron obtenidos del GenBank.For the above-mentioned analysis, they were used homologous polynucleotides shown in Figure 8 (SEQ ID N ° 1) and which belongs to the microorganism used to perform the Example 1 of the present invention. These homologous polynucleotides they are located in the nuclear ribosomal 16S region of the material genetic of these microorganisms and were obtained from Genbank.
El alineamiento de estos nucleótidos se hizo
mediante el software SAM (Hughey & Krogh, 1996), obteniendo una
matriz de 75 taxones y 923 pares de bases. La matriz resultante fue
analizada usando la metodología Bayesian Markov Chain Monte Carlo
(B/MCMC) tal y como se recoge en Larget y Simon, 1999; y
Huelsenbeck et al., 2000. El análisis Bayesiano se llevó a
cabo usando MrBayes v3.03b (Huelsenbeck & Ronquist, 2001). La
probabilidad posterior (pp) de cada rama se aproximó por muestreo de
los árboles filogenéticos por el método Markov Chain Monte Carlo
(MCMC). Se empleó el programa MrBayes (Huelsenbeck & Ronquist,
2001) para muestrear los árboles, y el análisis se realizó
asumiendo el modelo general time reversible (Rodriguez et
al., 1990) incluyendo la estimación de los sitios invariantes y
asumiendo una distribución discreta gamma y con seis categorías
(GTR+I+G). No se asumió reloj molecular. Se analizaron 2000000 de
generaciones, comenzando con un árbol al azar, y empleando 6 cadenas
simultaneas. Cada 100 generaciones el árbol resultante se guardó.
Se eliminaron los árboles resultantes de las 200000 primeras
generaciones, obteniendo el árbol consenso de los últimos 18000
árboles, que ya habían alcanzado el estado estacionario. Los clados
con probabilidad igual o superior al 95% se consideraron
soportados
significativamente.The alignment of these nucleotides was done using the SAM software (Hughey & Krogh, 1996), obtaining a matrix of 75 taxa and 923 base pairs. The resulting matrix was analyzed using the Bayesian Markov Chain Monte Carlo (B / MCMC) methodology as recorded in Larget and Simon, 1999; and Huelsenbeck et al ., 2000. Bayesian analysis was carried out using MrBayes v3.03b (Huelsenbeck & Ronquist, 2001). The subsequent probability (pp) of each branch was approximated by sampling the phylogenetic trees by the Markov Chain Monte Carlo (MCMC) method. The MrBayes program (Huelsenbeck & Ronquist, 2001) was used to sample the trees, and the analysis was performed assuming the general time reversible model (Rodriguez et al ., 1990) including estimating invariant sites and assuming a discrete gamma distribution and with six categories (GTR + I + G). No molecular clock was assumed. 2000000 generations were analyzed, starting with a random tree, and using 6 simultaneous chains. Every 100 generations the resulting tree was saved. The resulting trees of the first 200,000 generations were eliminated, obtaining the consensus tree of the last 18,000 trees, which had already reached steady state. Clades with a probability equal to or greater than 95% were considered supported
significantly.
La Figura 7 muestra el análisis filogenético que incluye las especies tipo de la Familia Acetobacteracceae, de acuerdo a los criterios taxonómicos del GenBank, así como todas las especies de Acidiphilium y Acidocella cuyo polinucleótido homólogo al de la Figura 8 (SEQ ID N°1) se encuentra depositado en el GenBank. Se consideraría que un experto en la materia a la luz de lo descrito anteriormente podría determinar si las células de un determinado microorganismo pertenecen al género Acidiphilium mediante un análisis filogenético igual o similar.Figure 7 shows the phylogenetic analysis that includes the type species of the Family Acetobacteracceae , according to the taxonomic criteria of the GenBank, as well as all the species of Acidiphilium and Acidocella whose polynucleotide homologous to that of Figure 8 (SEQ ID No. 1) It is deposited in the GenBank. It would be considered that one skilled in the art in the light of what was described above could determine if the cells of a certain microorganism belong to the genus Acidiphilium by the same or similar phylogenetic analysis.
Adicionalmente, en la identificación de un
microorganismo como perteneciente al género Acidiphilium son
aplicables los parámetros siguientes, sea aisladamente o en
combinación con los anteriores. Dado que las cepas de
Acidiphilium son afines en cuanto a su evolución, puede
esperarse que la homología global de los genomas al nivel de los
nucleótidos, y más concretamente a nivel de la región 16 S del RNA
ribosómico nuclear, y más concretamente al polinucleótido de la
región 16S del RNA ribosómico nuclear que se recoge en la Figura 8
(SEQ ID N°1), sea de un 80% o mayor, y más preferiblemente de un
90% o mayor. La correspondencia entre la secuencia genómica de
la(s) cepa(s) de Acidiphilium
putativa(s) y la secuencia de otro microorganismo se puede
determinar por métodos conocidos en la técnica. Por ejemplo,
aquéllas se pueden determinar por una comparación directa de la
información de secuencia del polinucleótido procedente de
Acidiphilium putativo, y la secuencia del polinucleótido que
se muestra en la Figura 8 de esta memoria. Por ejemplo, también,
aquéllas se pueden determinar por hibridación de los
polinucleótidos en condiciones que forman dúplex estables entre
regiones homólogas, seguido por digestión con nucleasa(s)
específica(s)
monocatenaria(s), seguido por
determinación del tamaño de los fragmentos digeridos.Additionally, in the identification of a microorganism as belonging to the genus Acidiphilium the following parameters are applicable, either in isolation or in combination with the above. Since Acidiphilium strains are related in their evolution, the global homology of genomes can be expected at the level of nucleotides, and more specifically at the level of the 16 S region of nuclear ribosomal RNA, and more specifically the polynucleotide of the 16S region of the nuclear ribosomal RNA shown in Figure 8 (SEQ ID No. 1), is 80% or greater, and more preferably 90% or greater. The correspondence between the genomic sequence of the putative Acidiphilium strain (s) and the sequence of another microorganism can be determined by methods known in the art. For example, they can be determined by a direct comparison of the polynucleotide sequence information from putative Acidiphilium , and the polynucleotide sequence shown in Figure 8 of this specification. For example, also, those can be determined by hybridization of the polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with specific nuclease (s)
single chain (s), followed by determination of the size of the digested fragments.
El porcentaje de homología se ha determinado
midiendo la identidad entre el polinucleótido que se muestra en la
Figura 8 (SEQ ID N°1) (perteneciente a la cepa del género
acidiphillium utilizada para llevar a cabo el ejemplo 1 de la
presente invención) y todos los polinucleótidos homólogos del
género Acidiphylium que se encontraban recogidos en el
momento de escritura de esta memoria, en el GenBank. Para ello, se
realizó un alineamiento de los polinucleótidos con el programa
Clustal X (Thompson et al. 1997). Se obtuvo una matriz de 34
taxones y 1045 pares de bases. Se calcularon las distancias
genéticas entre el polinucleótidos de la cepa de
acidiphillium utilizada en el ejemplo 1 con la secuencia
consenso SEQ ID N° 1 y los restantes polinucleótidos, y se calculó
la identidad entre los polinucleótidos que presentaron la mayor
distancia genética con él. La identidad que presentaron fue de un
89%. La identidad entre los polinucleótidos que presentaron una
mayor distancia genética fue de un 86%. Por tanto, cabe pensar que
un organismo que pertenezca al género Acidiphilium tendrá un
polinucleótido homólogo al de la Figura 8 (SEQ ID N°1),
perteneciente a la región 16S de su RNA ribosómico nuclear, que
presente, al menos, una identidad del 80% con
éste.The homology percentage has been determined by measuring the identity between the polynucleotide shown in Figure 8 (SEQ ID No. 1) (belonging to the strain of the genus acidiphillium used to carry out example 1 of the present invention) and all the homologous polynucleotides of the genus Acidiphylium that were collected at the time of writing of this report, in the GenBank. For this, an alignment of the polynucleotides with the Clustal X program was performed (Thompson et al . 1997). A matrix of 34 taxa and 1045 base pairs was obtained. The genetic distances between the polynucleotides of the acidiphillium strain used in example 1 with the consensus sequence SEQ ID No. 1 and the remaining polynucleotides were calculated, and the identity between the polynucleotides that had the greatest genetic distance with it was calculated. The identity they presented was 89%. The identity among the polynucleotides that presented a greater genetic distance was 86%. Therefore, it can be thought that an organism belonging to the genus Acidiphilium will have a polynucleotide homologous to that of Figure 8 (SEQ ID No. 1), belonging to the 16S region of its nuclear ribosomal RNA, which has at least one identity of the 80% with
East.
El término "Género", tal y como se utiliza en esta memoria, hace referencia a la categoría de la clasificación biológica (categoría taxonómica) que comprende una o más especies relacionadas filogenéticamente y morfológicamente similares. También se espera que compartan características químicas y metabólicas similares.The term "Gender", as used in this report, refers to the category of the classification biological (taxonomic category) comprising one or more species related phylogenetically and morphologically similar. They are also expected to share chemical characteristics and similar metabolic
En el contexto de esta memoria, la expresión "árbol filogenético" o "árbol evolutivo" hace referencia a la estructura matemática usada para modelizar la historia evolutiva real de un conjunto de unidades taxonómicas (secuencias, individuos, especies, etcétera).In the context of this memory, the expression "phylogenetic tree" or "evolutionary tree" refers to to the mathematical structure used to model the story real evolutionary of a set of taxonomic units (sequences, individuals, species, etc.)
Por "categoría taxonómica" se entiende el nivel de jerarquía utilizado para la clasificación de los organismos."Taxonomic category" means hierarchy level used to classify organisms.
Tal y como se utiliza en esta memoria, un "ciado" hace referencia a un grupo monofilético, siendo un "grupo monofilético" un grupo que incluye todos los taxones descendientes de un taxón ancestral.As used herein, a "ciado" refers to a monophyletic group, being a "monophyletic group" a group that includes all taxa descendants of an ancestral taxon.
El término "polinucleotido", tal como se utiliza en esta memoria, se refiere a una forma polímera de nucleótidos de cualquier longitud, sean ribonucleótidos o desoxirribonucleótidos. Este término se refiere exclusivamente a la estructura primaria de la molécula.The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers exclusively to the primary structure of the molecule.
El término "filogenia" como aquí se usa se refiere a la relación histórica verdadera entre un conjunto de taxones.The term "phylogeny" as used herein is refers to the true historical relationship between a set of taxa
El término "homología", tal y como se utiliza en esta memoria, hace referencia a la semejanza entre do estructuras debida a una ascendencia evolutiva común, y más concretamente, a la semejanza entre los nucleótidos de dos o más polinucleótidos.The term "homology", as it is used in this report, refers to the similarity between do structures due to a common evolutionary ancestry, and more specifically, similarity between nucleotides of two or more polynucleotides
El término "identidad", tal y como se utiliza en esta memoria, hace referencia a la proporción de nucleótidos idénticos entre dos polinucleótidos que se comparan.The term "identity" as it is used in this report, refers to the proportion of identical nucleotides between two polynucleotides that are compare.
Por "norma de reacción" se entiende el conjunto de fenotipos a que da origen un mismo genotipo cuando se desarrolla en distintos ambientes."Reaction rule" means the set of phenotypes that give rise to the same genotype when It develops in different environments.
El término "genotipo", tal como se utiliza en esta memoria, hace referencia a la constitución hereditaria o genética de un individuo; todo el material genético contenido en una célula, al que, por lo general, se denomina material nuclear.The term "genotype", as used in this report, refers to the hereditary constitution or genetics of an individual; all genetic material contained in a cell, which is usually called material nuclear.
El término "fenotipo", tal como se utiliza en esta memoria, se refiere a la suma total de las propiedades estructurales y funcionales observables de un organismo; producto de la interacción entre el genotipo y el medio ambiente.The term "phenotype", as used in this report, it refers to the total sum of the properties structural and functional observables of an organism; product of the interaction between genotype and the environment.
El término "especie tipo" hace referencia a la especie designada como el tipo de un género o un subgénero, siendo el "tipo" bajo el punto de vista taxonómico, el elemento simple de un taxón al cual se le asigna permanentemente el nombre y sobre el que están basadas las características descriptivas que satisfacen las condiciones de disponibilidad o de publicación válidas.The term "type species" refers to the species designated as the type of a genus or a subgenus, being the "type" from the taxonomic point of view, the simple element of a taxon which is permanently assigned the name and on which the characteristics are based descriptive that satisfy the conditions of availability or of valid publication.
Las bacterias del género acidophilium se caracterizan porque pueden reducir ión férrico utilizando compuestos orgánicos como donadores de electrones. Una propiedad sobresaliente de este tipo de bacterias es su capacidad de utilizar el hierro férrico como aceptor final de electrones en condiciones anaerobias, microaerobias y en elevadas concentraciones de oxígeno. Para llevar a cabo la presente invención se aislaron cepas de Acidiphilium spp. de diferentes zonas del río Tinto caracterizadas por sus elevadas concentraciones de hierro, tanto ión ferroso como férrico. El aislamiento se hizo a partir de medio sólido con glucosa como fuente de energía y luego se procedió a comprobar la pureza de las cepas de Acidiphilium durante su crecimiento en medio liquido a pH 2,5 mediante hibridación in situ con sondas especificas. Acidophilium bacteria genus are characterized by ferric ion can be reduced using organic compounds as electron donors. An outstanding property of this type of bacteria is its ability to use ferric iron as the final electron acceptor in anaerobic, microaerobic and high oxygen concentrations. In order to carry out the present invention, strains of Acidiphilium spp. from different areas of the Rio Tinto characterized by its high concentrations of iron, both ferrous and ferric ion. The isolation was made from solid medium with glucose as an energy source and then the purity of the Acidiphilium strains was checked during their growth in liquid medium at pH 2.5 by in situ hybridization with specific probes.
Por tanto, una realización preferida de la invención lo constituye el electrodo biológico de la invención en el que las células del género bacteriano Acidiphilium spp. se aíslan del Río Tinto (Huelva).Therefore, a preferred embodiment of the invention is the biological electrode of the invention in which the cells of the bacterial genus Acidiphilium spp. they are isolated from the Rio Tinto (Huelva).
Estas células bacterianas pueden ser manipuladas genéticamente por las técnicas convencionales actuales de biología molecular para cambiar sus propiedades.These bacterial cells can be manipulated genetically by current conventional biology techniques molecular to change its properties.
Por lo tanto, otra realización preferida de la invención lo constituye el electrodo biológico de la invención en el que las células del género bacteriano Acidiphilium spp. se han modificado mediante ingeniería genética para mejorar sus propiedades.Therefore, another preferred embodiment of the invention is the biological electrode of the invention in which the cells of the bacterial genus Acidiphilium spp. They have been modified by genetic engineering to improve their properties.
Las células bacterianas de Acidiphilium spp. se pueden modificar genéticamente para promover la secreción de hexopolisacáridos dopados con iones de Fe para favorecer su adherencia a la superficie del electrodo y su conectividad eléctrica.Bacterial cells of Acidiphilium spp. they can be genetically modified to promote the secretion of hexopolysaccharides doped with Fe ions to favor their adhesion to the electrode surface and their electrical connectivity.
Por lo tanto, una realización más preferida de la invención lo constituye el electrodo biológico de la invención en el que las células del género bacteriano Acidiphilium spp. se han modificado mediante ingeniería genética para mejorar su adherencia a la superficie del electrodo y su conectividad eléctrica.Therefore, a more preferred embodiment of the invention is the biological electrode of the invention in which the cells of the bacterial genus Acidiphilium spp. they have been modified by genetic engineering to improve their adherence to the electrode surface and their electrical connectivity.
Por ingeniería genética puede también modificarse las propiedades metabólicas de las células de Acidiphilium spp. para aumentar el tipo de compuestos orgánicos que son capaces de utilizar para generar electricidad.Genetic engineering can also modify the metabolic properties of Acidiphilium spp. to increase the type of organic compounds that they are able to use to generate electricity.
Por lo tanto, otra realización aún más preferida de la invención lo constituye el electrodo biológico de la invención en el que las células del género Acidiphilium sp se han modificado mediante ingeniería genética para ampliar sus propiedades metabólicas con compuestos orgánicos diversos.Therefore, another even more preferred embodiment of the invention is the biological electrode of the invention in which cells of the genus Acidiphilium sp have been genetically engineered to extend their metabolic properties with various organic compounds.
El electrodo conductor que forma parte del electrodo biológico de la invención puede ser, a modo de ejemplo y sin que limite el alcance de la invención, de un material de tipo carbonáceo, el cual tenga gran área específica que permita un alto recubrimiento del electrodo con células bacterianas, y por tanto de lugar a altas densidades de intensidad de corriente en el electrodo biológico de la invención.The conductive electrode that is part of the Biological electrode of the invention can be, by way of example and without limiting the scope of the invention, of a type material carbonaceous, which has a large specific area that allows a high electrode coating with bacterial cells, and therefore of place at high densities of current in the electrode Biological of the invention.
Así, otro aspecto de la presente invención lo constituye el electrodo biológico de la invención en el que el electrodo conductor es de un material carbonáceo.Thus, another aspect of the present invention is it constitutes the biological electrode of the invention in which the Conductive electrode is a carbonaceous material.
Una realización preferida de la presente invención lo constituye el electrodo biológico de la invención en el que el material carbonáceo del electrodo conductor es grafito, hilo de carbón o tela de carbón.A preferred embodiment of the present invention constitutes the biological electrode of the invention in that the carbonaceous material of the conducting electrode is graphite, coal thread or carbon cloth.
El material carbonáceo que constituye el electrodo puede ser dopado con iones metálicos para favorecer la conectividad eléctrica entre las células bacterianas adsorbidas y el electrodo (D. H. Park, J. G. Zeikus, Improved fuel cell and electrode designs for producing electricity from microbial degradation, Biotechnol. Bioeng. 81, 348-355, 2003). Asimismo, con el fin de favorecer la adhesión de las células bacterianas a la superficie del electrodo por interacciones electroestáticas la superficie del electrodo puede ser modificada con diferentes grupos funcionales por los métodos descritos por Saveant y colaboradores mediante la reducción electroquímica de sales de aril diazonio adecuadas (M. Delamar, R. Hitmi, J. Pinson, J. M. Saveant, Covalent modification of carbon surfaces by grafting of functionalized aryl radicals produced from electrochemical reduction of diazonium salts, J. Am. Chem. Soc. 114, 5853-5854, 1992; P. Allongue, M. Delamar, B. Desbat, O. Fagebaume, R. Hitmi, J. Pinson, J. M. Saveant, Covalent modification of carbon surfaces by aryl radicals generated from the electrochemical reduction of diazonium salts, J. Am. Chem. Soc. 1997, 119, 201-207).The carbonaceous material constituting the electrode can be doped with metal ions to promote electrical connectivity between adsorbed bacterial cells and the electrode (DH Park, JG Zeikus, Improved fuel cell and electrode designs for producing electricity from microbial degradation , Biotechnol. Bioeng. 81, 348-355, 2003). Likewise, in order to favor the adhesion of the bacterial cells to the electrode surface by electrostatic interactions the electrode surface can be modified with different functional groups by the methods described by Saveant et al. By electrochemical reduction of suitable aryl diazonium salts. (M. Delamar, R. Hitmi, J. Pinson, JM Saveant, Covalent modification of carbon surfaces by grafting of functionalized aryl radicals produced from electrochemical reduction of diazonium salts , J. Am. Chem. Soc. 114, 5853-5854, 1992 ; P. Allongue, M. Delamar, B. Desbat, O. Fagebaume, R. Hitmi, J. Pinson, JM Saveant, Covalent modification of carbon surfaces by aryl radicals generated from the electrochemical reduction of diazonium salts , J. Am. Chem Soc. 1997, 119, 201-207).
Así, una realización más preferida de la presente invención lo constituye el electrodo biológico de la invención en el que el material del electrodo conductor está modificado con iones metálicos o con grupos funcionales en su superficie.Thus, a more preferred embodiment of the the present invention is constituted by the biological electrode of the invention in which the conductive electrode material is modified with metal ions or with functional groups in their surface.
En un aspecto preferente de la invención y con el fin de lograr la adsorción de las células bacterianas sobre el electrodo conductor se inocula un cultivo de bacteria Acidiphilium spp. en una celda estéril electroquímica que contiene una disolución acuosa aeróbica con pH ácido, un compuesto orgánico como nutriente, y en presencia de un soporte de material conductor que actúa como electrodo de trabajo. Además se colocan un electrodo de referencia y un contraelectrodo en la celda. La comunicación eléctrica entre células bacterianas y el electrodo de trabajo se evalúa por la técnica de voltametría cíclica. Tras un periodo de tiempo de incubación (por ejemplo 6 días) se comprueba que ha aumentado el número de células de la bacteria en el medio, que hay células adsorbidas sobre la tela de carbón y que las bacterias adsorbidas son capaces de ceder los electrones procedentes de la oxidación del compuesto orgánico al electrodo en condiciones aeróbicas sin que requiera la adición de mediadores redox en disolución (ver Ejemplo 1).In a preferred aspect of the invention and in order to achieve adsorption of the bacterial cells on the conducting electrode a culture of Acidiphilium spp. Bacteria is inoculated. in a sterile electrochemical cell containing an aerobic aqueous solution with acidic pH, an organic compound as a nutrient, and in the presence of a conductive material support that acts as a working electrode. In addition, a reference electrode and a counter electrode are placed in the cell. The electrical communication between bacterial cells and the working electrode is evaluated by the cyclic voltammetry technique. After a period of incubation time (for example 6 days) it is verified that the number of bacteria cells in the medium has increased, that there are cells adsorbed on the carbon cloth and that the adsorbed bacteria are able to yield the electrons coming of the oxidation of the organic compound to the electrode in aerobic conditions without requiring the addition of redox mediators in solution (see Example 1).
Por tanto, otro aspecto de la presente invención lo constituye un procedimiento de obtención del electrodo biológico de la invención, en adelante el procedimiento de obtención del electrodo biológico de la invención, basado en el crecimiento de células bacterianas del género Acidiphilium spp. en disolución aeróbica y de pH ácido en presencia de un electrodo conductor y un compuesto orgánico que actúa como nutriente.Therefore, another aspect of the present invention is a method of obtaining the biological electrode of the invention, hereinafter the method of obtaining the biological electrode of the invention, based on the growth of bacterial cells of the genus Acidiphilium spp. in aerobic and acidic pH solution in the presence of a conductive electrode and an organic compound that acts as a nutrient.
Para un crecimiento adecuado de las células bacterianas de Acidiphilium spp es aconsejable que el pH de tampón sea entre 2,0 y 4,0. Así mismo, la glucosa es un nutriente eficaz para este fin. La utilización de un material carbonáceo de gran área específica como electrodo conductor favorece la adhesión de un gran número de células bacterianas a esta superficie (ver Figura 2).For an adequate growth of the bacterial cells of Acidiphilium spp it is advisable that the buffer pH be between 2.0 and 4.0. Likewise, glucose is an effective nutrient for this purpose. The use of a large area specific carbonaceous material as a conductive electrode favors the adhesion of a large number of bacterial cells to this surface (see Figure 2).
Así, una realización preferida de la presente invención lo constituye el procedimiento de obtención del electrodo biológico de la invención en el que el pH de la disolución aeróbica es entre 2,0 y 4,0.Thus, a preferred embodiment of the present invention is the method of obtaining the electrode Biological of the invention in which the pH of the aerobic solution It is between 2.0 and 4.0.
Otra realización preferida de la presente invención lo constituye el procedimiento de obtención del electrodo biológico de la invención en el que el nutriente es glucosa.Another preferred embodiment of the present invention is the method of obtaining the electrode Biological of the invention in which the nutrient is glucose.
Aún otra realización particular de la presente invención lo constituye el procedimiento de obtención del electrodo biológico de la invención en el que el material del electrodo conductor es un material carbonáceo.Still another particular embodiment of the present invention is the method of obtaining the electrode biological of the invention in which the electrode material Conductor is a carbonaceous material.
El crecimiento de las células bacterianas de Acidiphilium spp. mejora cuando se añade en el medio de cultivo otros compuestos aparte del nutriente. A modo de ejemplo y sin que limite el alcance de la invención: 2 g/l de (NH_{4})_{2}SO_{4}, 0,1 g/l de KCl, 0,25 g/l de K_{2}HPO_{4}, 0,25 g/l de MgSO_{4}\cdot7H_{2}O, 0,01 g/l Ca(NO_{3})_{2}. El pH óptimo de crecimiento de éstas células es 2,5. Además, el soporte de material carbonáceo sobre el que se adsorben las células bacterianas y que va actuar como electrodo conductor del electrodo biológico de la invención puede ser un disco de tela de carbono.The growth of bacterial cells of Acidiphilium spp. improves when other compounds than the nutrient are added to the culture medium. By way of example and without limiting the scope of the invention: 2 g / l of (NH 4) 2 SO 4, 0.1 g / l of KCl, 0.25 g / l of K 2 HPO 4, 0.25 g / l of MgSO 4 • 7 H 2 O, 0.01 g / l Ca (NO 3) 2. The optimum growth pH of these cells is 2.5. In addition, the support of carbonaceous material on which the bacterial cells are adsorbed and which will act as the conductive electrode of the biological electrode of the invention can be a carbon cloth disk.
Por lo tanto, otra realización preferida de la presente invención lo constituye un procedimiento de obtención del electrodo biológico en el que el pH de la disolución es 2,5 y contiene 2 g/l de (NH_{4})_{2}SO_{4}, 0,1 g/l de KCl, 0,25 g/l de K_{2}HPO_{4}, 0,25 g/l de MgSO_{4}\cdot7H_{2}O, 0,01 g/l Ca(NO_{3})_{2}, y el material del electrodo conductor es tela de carbón.Therefore, another preferred embodiment of the This invention constitutes a method of obtaining the biological electrode in which the pH of the solution is 2.5 and contains 2 g / l of (NH 4) 2 SO 4, 0.1 g / l of KCl, 0.25 g / l of K 2 HPO 4, 0.25 g / l of MgSO 4 • 7H 2 O, 0.01 g / l Ca (NO 3) 2, and the electrode material conductor is carbon cloth.
Para mejorar la estabilidad de las células bacterianas adsorbidas sobre el electrodo y evitar su desorción del electrodo hacia la disolución, estás se pueden recubrir con polímeros con grupos funcionales adecuados, ya sea para realizar reacciones de entrecruzamiento entre células bacterianas próximas o para establecer interacciones electroestáticas entre el polímero y las células bacterianas, formándose un aglomerado de las células bacterianas sobre el electrodo retenidas por el polímero.To improve cell stability bacterial adsorbed on the electrode and prevent its desorption of the electrode towards dissolution, these can be coated with polymers with suitable functional groups, whether to perform crosslinking reactions between nearby bacterial cells or to establish electrostatic interactions between the polymer and the bacterial cells, forming an agglomerate of the cells Bacterial on the electrode retained by the polymer.
Por tanto, otra realización aún más preferida de la presente invención lo constituye el procedimiento de obtención del electrodo biológico de la invención caracterizado porque se estabilizan las células bacterianas adsorbidas sobre el electrodo conductor por entrecruzamiento o encapsulación con polímeros funcionales.Therefore, another even more preferred embodiment of The present invention is constituted by the method of obtaining of the biological electrode of the invention characterized in that stabilize adsorbed bacterial cells on the electrode cross-linking or polymer encapsulation conductor functional.
El electrodo biológico de la invención utilizado como ánodo permite obtener energía eléctrica de compuestos orgánicos diversos formando parte de pilas de combustible. Estos compuestos orgánicos pueden provenir de aguas residuales de distinto origen y por tanto puede servir también para descontaminación.The biological electrode of the invention used as an anode it allows to obtain electrical energy of compounds various organic forming part of fuel cells. These organic compounds can come from wastewater from different origin and therefore can also serve to decontamination.
Así, otra realización de la presente invención lo constituye el uso del electrodo biológico de la invención como ánodo de una pila de combustible que utilice materia orgánica de diferentes orígenes para producir electricidad.Thus, another embodiment of the present invention it is constituted by the use of the biological electrode of the invention as anode of a fuel cell that uses organic matter from Different origins to produce electricity.
Como la comunicación electrónica entre las células bacterianas y el electrodo conductor es directa no se requiere la adición de mediadores redox en disolución. Esta es una gran ventaja ya que estos mediadores redox suelen ser muy tóxicos y además su uso implica un mayor coste de la pila. La otra gran ventaja del electrodo biológico de la invención es que funciona en condiciones aeróbicas tan bien como en condiciones anaeróbicas (ver Ejemplo 1), por lo tanto no requiere la eliminación del oxígeno de la disolución acuosa que actúa como combustible ni la separación del compartimiento anódico del catódico (donde se reduce O_{2}) mediante una membrana intercambiadora de protones. Esto último es posible también por que no se utiliza un mediador rédox en disolución que pudiera reaccionar en el cátodo. La eliminación de la membrana intercambiadora de protones y de la necesidad de borbotear con N_{2} la disolución del combustible para hacerla anaerobia, así como la posible ausencia de mediadores redox, suponen una clara simplificación del diseño de la pila de combustible y en consecuencia una considerable reducción de costes.As electronic communication between bacterial cells and the conductive electrode is direct not requires the addition of redox mediators in solution. This is one great advantage since these redox mediators are usually very toxic and in addition its use implies a greater cost of the battery. The other great advantage of the biological electrode of the invention is that it works in aerobic conditions as well as in anaerobic conditions (see Example 1), therefore does not require the removal of oxygen from the aqueous solution that acts as fuel or separation of the cathodic anodic compartment (where O2 is reduced) by means of a proton exchange membrane. The latter is also possible because a redox mediator is not used in solution that could react in the cathode. The elimination of the proton exchange membrane and the need to bubble up with N2 the dissolution of the fuel to make it anaerobic, as well as the possible absence of redox mediators, they represent a clear simplification of the fuel cell design and in consequently a considerable reduction of costs.
Por lo tanto, otro aspecto de la presente invención lo constituye el uso del electrodo biológico de la invención como ánodo de una pila de combustible que no requiere condiciones anaeróbicas ni mediadores rédox en la disolución del compartimiento anódico, evitando la necesidad de una membrana intercambiadora de protones que la separe del compartimiento catódico.Therefore, another aspect of the present invention is the use of the biological electrode of the invention as the anode of a fuel cell that does not require anaerobic conditions or redox mediators in the dissolution of anodic compartment, avoiding the need for a membrane proton exchanger that separates it from the compartment cathodic
El uso del electrodo biológico de la invención como ánodo en una pila de combustible es compatible con un cátodo de reducción de oxígeno convencional o con un cátodo basado en un catalizador biológico.The use of the biological electrode of the invention as anode in a fuel cell is compatible with a cathode of conventional oxygen reduction or with a cathode based on a biological catalyst
Por tanto, una realización preferida de la presente invención lo constituye el uso de una pila de combustible caracterizada porque comprende el electrodo biológico de la invención.Therefore, a preferred embodiment of the The present invention is the use of a fuel cell characterized in that it comprises the biological electrode of the invention.
Figura 1. Voltamogramas cíclicos que muestran las corrientes de oxidación de glucosa obtenidos con un electrodo microbiano de Acidiphilium spp. La curva continua corresponde al experimento control con glucosa 10 mM antes de añadir bacteria, en condiciones aeróbicas y a 30°C; la curva de trazos equivale a las mismas condiciones pero después de seis días de inoculación de bacteria; la curva de puntos corresponde al experimento anterior después de eliminar el oxígeno borboteando N_{2} durante 40 minutos. Velocidad de barrido 50 mV/s. Los potenciales redox son respecto a un electrodo de calomelanos.Figure 1. Cyclic voltamograms showing glucose oxidation currents obtained with a microbial electrode of Acidiphilium spp . The continuous curve corresponds to the control experiment with 10 mM glucose before adding bacteria, in aerobic conditions and at 30 ° C; the stroke curve is equivalent to the same conditions but after six days of bacterial inoculation; the dot curve corresponds to the previous experiment after removing the oxygen by bubbling N2 for 40 minutes. Scanning speed 50 mV / s. The redox potentials are with respect to an electrode of calomelanos.
Figura 2.- Imagen de microscopio de barrido de la superficie del electrodo microbiano. La imagen corresponde al electrodo de la Figura 1 después de seis días de crecer la bacteria Acidiphilium 3.2 sup 5.Figure 2.- Scanning microscope image of the surface of the microbial electrode . The image corresponds to the electrode of Figure 1 after six days of growing the Acidiphilium 3.2 sup 5 bacteria.
Figura 3.- Efecto de un inhibidor de la bacteria en los voltamogramas cíclicos. La curva de línea continua corresponde al experimento control antes de añadir bacteria Acidiphilium 3.2 sup 5; la curva de trazos corresponde al electrodo bacteriano; la curva de puntos se obtiene 23 horas después de añadir fenol al 5%.Figure 3.- Effect of a bacterial inhibitor on cyclic voltamograms . The continuous line curve corresponds to the control experiment before adding Acidiphilium 3.2 sup 5 bacteria; the dashed curve corresponds to the bacterial electrode; the point curve is obtained 23 hours after adding 5% phenol.
Figura 4. Voltamograma cíclico que muestran las corrientes de oxidación de glucosa obtenido con Acidiphilium Sup . La curva continua corresponde al experimento con glucosa justo al añadir la bacteria y la curva de trazos corresponde a las mismas condiciones después de 11 días.Figure 4. Cyclic voltamogram showing glucose oxidation currents obtained with Acidiphilium Sup . The continuous curve corresponds to the experiment with glucose just by adding the bacteria and the dashed curve corresponds to the same conditions after 11 days.
Figura 5. Voltamograma cíclico que muestran las corrientes de oxidación de glucosa obtenido con Acidiphilium Musta . La curva continua corresponde al experimento con glucosa justo al añadir la bacteria y la curva de trazos corresponde a las mismas condiciones después de 15 días.Figure 5. Cyclic voltamogram showing glucose oxidation currents obtained with Acidiphilium Musta . The continuous curve corresponds to the experiment with glucose just by adding the bacteria and the dashed curve corresponds to the same conditions after 15 days.
Figura 6. Voltamograma cíclico que muestran las corrientes de oxidación de glucosa obtenido con Acidiphilium AiRi. La curva continua corresponde al experimento con glucosa justo al añadir la bacteria y la curva de trazos corresponde a las mismas condiciones después de 15 días.Figure 6. Cyclic voltamogram showing glucose oxidation currents obtained with Acidiphilium AiRi . The continuous curve corresponds to the experiment with glucose just by adding the bacteria and the dashed curve corresponds to the same conditions after 15 days.
Figura 7.- Árbol Filogenético que delimita el género Acidiphilium . Se ha obtenido del análisis bayesiano que incluye los poolinucleótidos homólogos al representado en la Figura 8 (SEQ ID N°1) de las especies tipo de los Géneros que integran la Familia Acetobaccteraceae, según el criterio taxonómico del GenBank, y que se encuentran depositados en el GenBank. En el análisis se han incluido todos los polinucleótidos homólogos al de la Figura 8 (SEQ ID N°1) que se han clasificado en el GenBank como pertenecientes al género Acidiphylium y Acidocella. Los clados que presentan líneas engrosadas presentan una probabilidad posterior (p/p) superior al 95% (p/p > 0.95).Figure 7.- Phylogenetic Tree that delimits the genus Acidiphilium . It has been obtained from the Bayesian analysis that includes the homologous poolinucleotides shown in Figure 8 (SEQ ID No. 1) of the type species of the genera that make up the Acetobaccteraceae family, according to the taxonomic criteria of GenBank, and that are deposited in The GenBank The analysis included all polynucleotides homologous to that of Figure 8 (SEQ ID No. 1) that have been classified in the GenBank as belonging to the genus Acidiphylium and Acidocella . Clades with thickened lines have a posterior probability (w / w) greater than 95% (w / w> 0.95).
Figura 7a.- ampliación de la zona superior (Género Acidocella).Figure 7a.- enlargement of the upper area (Genus Acidocella ).
Figura 7b.- ampliación de la zona inferior (Género Acidiphylium).Figure 7b.- enlargement of the lower zone (Genus Acidiphylium ).
Figura 8.- Polinucleótido de la región 16 S del RNA ribosómico nuclear. Obtenido de la cepa de Acidiphilium sp. utilizada para llevar a cabo el ejemplo 1 de la presente invención y que corresponde con la SEQ ID N° 1.Figure 8.- Polynucleotide of the 16 S region of the nuclear ribosomal RNA . Obtained from the strain of Acidiphilium sp. used to carry out example 1 of the present invention and which corresponds to SEQ ID No. 1.
Para llevar a cabo la presente invención se aislaron las células de Acidiphilium a partir de aguas del Río Tinto (Huelva) utilizando suporte sólidos cuya la composición es:To carry out the present invention, Acidiphilium cells were isolated from waters of the Rio Tinto (Huelva) using solid supports whose composition is:
Se ajusta el pH a 4.5 mediante H_{2}SO_{4}, y se esteriliza en la autoclave a 120° a 0,5 atm durante 30 minutos para evitar la caramelización de glucosa.The pH is adjusted to 4.5 by H 2 SO 4, and sterilized in the autoclave at 120 ° at 0.5 atm for 30 minutes to prevent caramelization of glucose.
Después de crecer en medio solidó se cogen la colonias y se siembran en medio liquido que tiene los mismos nutrientes que el suporte solidó menos el agar y el pH en este caso fue ajustado a 2.5After growing in solid medium, the colonies and are sown in liquid medium that has the same nutrients that support solidified less agar and pH in this case was adjusted to 2.5
Se realizaron experimentos de voltametria cíclica para la caracterización electroquímica de Acidiphilium 3.2 en disolución cuya l a composición es 2 g/l de (NH_{4})_{2}SO_{4}, 0,1 g/l de KCl, 0,25 g/l de K_{2}HPO_{4}, 0,25 g/l de MgSO_{4}\cdot7H_{2}O, 0,01 g/l Ca(NO_{3})_{2}. a pH 2.5.Cyclic voltammetry experiments were performed for electrochemical characterization of Acidiphilium 3.2 in solution whose composition is 2 g / l of (NH 4) 2 SO 4, 0.1 g / l of KCl, 0, 25 g / l of K 2 HPO 4, 0.25 g / l of MgSO 4 • 7 H 2 O, 0.01 g / l Ca (NO 3) 2. at pH 2.5.
Los experimentos se llevaron a cabo a temperatura de 30°C que es la temperatura optima de crecimiento de esta bacteria, se aplicaron velocidades de barrido de 0.05 V/s.The experiments were carried out at temperature of 30 ° C which is the optimum growth temperature of this bacterium, scanning speeds of 0.05 V / s were applied.
Todas las bacterias utilizadas para llevar a cabo los ejemplos 2-4 pertenecen al género acidiphillium spp y tienen al menos un 80% de homología con la secuencia consenso mostrada en la figura 8 (SEQ ID N°1).All bacteria used to carry out examples 2-4 belong to the genus acidiphillium spp and have at least 80% homology with the consensus sequence shown in Figure 8 (SEQ ID No. 1).
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Ejemplo 1Example one
En una celda electroquímica estéril y termoestatizada a 30°C se colocan un electrodo de trabajo hecho de un disco de tela de carbono de 13 mm de diámetro, un electrodo de referencia de calomelanos y un contraelectrodo de platino. La celdilla electroquímica contiene una disolución a pH 2,5 de 2 g/l de (NH_{4})_{2}SO_{4}, 0,1 g/l de KCl, 0,25 g/l de K_{2}HPO_{4}, 0,25 g/l de MgSO_{4}\cdot7H_{2}O, 0,01 g/l Ca(NO_{3})_{2}. A continuación se añade glucosa 10 mM y se inocula un cultivo de la bacteria perteneciente al género Acidiphilium spp (Depósito realizado ante la CECT (Colección Española de Cultivos Tipo) con referencia de identificación: acidiphilium 3.2 sup5, n° de depósito: CECT 7285 y fecha de depósito: 23 de mayo de 2007) con la secuencia consenso mostrada en la figura 8 (SEQ ID N° 1) y obtenida del río Tinto. A los seis días de incubación se comprueba que ha aumentado el número de células de la bacteria en el medio por microscopía y recuento directo de las células teñidas con DAPI (4',6'-diaminofenilindol), alcanzándose 4 x 10^{9} células/ml. Por voltametría cíclica se comprueba el efecto catalítico de oxidación de glucosa con el electrodo biológico de la invención y se compara con el experimento control antes de inocular bacteria (Figura 1). El efecto catalítico de oxidación de glucosa en presencia de oxígeno, no varía en condiciones anaeróbicas (Figura 1). La Figura 2 muestra las imágenes obtenidas por microscopía electrónica de barrido del electrodo biológico de la invención, en las cuales se ven claramente las células bacterianas adsorbidas sobre los hilos de la tela de carbón. En la Figura 3 se muestran las voltametrías cíclicas realizadas con otro electrodo biológico preparado del mismo modo antes y después de añadir fenol en concentración del 5%. El fenol es un inhibidor potente de las células bacterianas y prácticamente suprime las propiedades catalíticas del electrodo biológico de la invención.A working electrode made of a 13 mm diameter carbon cloth disk, a calomelan reference electrode and a platinum counter electrode are placed in a sterile electro-thermostatic cell at 30 ° C. The electrochemical cell contains a solution at pH 2.5 of 2 g / l of (NH4) 2 SO4, 0.1 g / l of KCl, 0.25 g / l of K_ 2} HPO4, 0.25 g / l MgSO4 · 7H2O, 0.01 g / l Ca (NO3) 2. Next, 10 mM glucose is added and a culture of the bacterium belonging to the genus Acidiphilium spp (Deposit made before the CECT (Spanish Type Culture Collection) is inoculated with identification reference: acidiphilium 3.2 sup5, deposit number: CECT 7285 and Deposit date: May 23, 2007 ) with the consensus sequence shown in Figure 8 (SEQ ID No. 1) and obtained from the Tinto river. After six days of incubation, it is verified that the number of bacteria cells in the medium has increased by microscopy and direct count of DAPI stained cells (4 ', 6'-diaminophenylindole), reaching 4 x 10 9 cells / ml. By cyclic voltammetry, the catalytic effect of glucose oxidation is checked with the biological electrode of the invention and compared with the control experiment before inoculating bacteria (Figure 1). The catalytic effect of glucose oxidation in the presence of oxygen does not vary under anaerobic conditions (Figure 1). Figure 2 shows the images obtained by scanning electron microscopy of the biological electrode of the invention, in which the adsorbed bacterial cells are clearly seen on the threads of the carbon cloth. Figure 3 shows the cyclic voltammetries performed with another biological electrode prepared in the same way before and after adding phenol in 5% concentration. Phenol is a potent inhibitor of bacterial cells and virtually suppresses the catalytic properties of the biological electrode of the invention.
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Ejemplo 2Example 2
Se repite el experimento del ejemplo 1, manteniendo las mismas condiciones, pero construyendo el electrodo exclusivamente con células de Acidiphilium sp. cepa Sup. y sin medir el efecto inhibidor del fenol. El voltamograma cíclico se mide en el momento de inocular la bacteria y 11 días después de la inoculación. Los resultados se muestran en la Figura 4.The experiment of Example 1 is repeated, maintaining the same conditions, but constructing the electrode exclusively with Acidiphilium sp. Sup. strain and without measuring the inhibitory effect of phenol. The cyclic voltamogram is measured at the time of inoculation of the bacteria and 11 days after inoculation. The results are shown in Figure 4.
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Ejemplo 3Example 3
Se repite el experimento del ejemplo 1, manteniendo las mismas condiciones, pero construyendo el electrodo exclusivamente con células de Acidiphilium sp. cepa Musta y sin medir el efecto inhibidor del fenol. La curva continua corresponde al experimento con glucosa justo al añadir la bacteria y la curva continua corresponde a las mismas condiciones después de 15 días. Los resultados se muestran en la Figura 5.The experiment of Example 1 is repeated, maintaining the same conditions, but constructing the electrode exclusively with Acidiphilium sp. Musta strain and without measuring the inhibitory effect of phenol. The continuous curve corresponds to the experiment with glucose just by adding the bacteria and the continuous curve corresponds to the same conditions after 15 days. The results are shown in Figure 5.
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Ejemplo 4Example 4
Se repite el experimento del ejemplo 1, manteniendo las mismas condiciones, pero construyendo el electrodo exclusivamente con células de Acidiphilium sp. cepa AiRi y sin medir el efecto inhibidor del fenol. Los resultados se muestran en la Figura 6.The experiment of Example 1 is repeated, maintaining the same conditions, but constructing the electrode exclusively with Acidiphilium sp. AiRi strain and without measuring the inhibitory effect of phenol. The results are shown in Figure 6.
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Alarico S., Rainey F. A., Empadinhas N., Schumann P., Nobre M. F. y M. S. Da Costa. 2002. Rubritepida flocculans gen. nov., sp. nov., a new slightly thermophilic member of the \alpha-1 subclass of the Proteobacteria. Systematic and Applied Microbiology 25:198-206. Alarico S., Rainey FA, Empadinhas N., Schumann P., Nobre MF and MS Da Costa . 2002 Rubritepida flocculans gen. nov., sp. Nov., a new slightly thermophilic member of the α-1 subclass of the Proteobacteria . Systematic and Applied Microbiology 25 : 198-206.
Allongue P., Delamar M., Desbat B., Fagebaume O., Hitmi R., Pinson J. y J. M. Saveant. 1997. Covalent modification of carbon surfaces by aryl radicals generated from the electrochemical reduction of diazonium salts. Journal of American Chemical Society 119:201-207. Allongue P., Delamar M., Desbat B., Fagebaume O., Hitmi R., Pinson J. and JM Saveant . 1997 Covalent modification of carbon surfaces by aryl radicals generated from the electrochemical reduction of diazonium salts. Journal of American Chemical Society 119 : 201-207.
Angenent, L. T., Karim, K., Al-Dahhan, M. H., Wrenn B. A. y R. Domínguez-Espinosa. 2004. Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends in Biotechnology. 22:477-485. Angenent , LT, Karim , K., Al-Dahhan , MH, Wrenn BA and R. Domínguez-Espinosa . 2004 Production of bioenergy and biochemicals from industrial and agricultural wastewater. Trends in Biotechnology . 22 : 477-485.
Asai T. 1935. Taxonomic studies on acetic acid bacteria and allied oxidative bacteria isolated from fruits. A new classification of the oxidative bacteria. Journal of the Agricultural Chemical Society of Japan 11:674-708. Asai T. 1935 . Taxonomic studies on acetic acid bacteria and allied oxidative bacteria isolated from fruits. A new classification of the oxidative bacteria. Journal of the Agricultural Chemical Society of Japan 11 : 674-708.
Beijerinck M. W. 1898. Über die Arten der Essigbakterien. Zentralblatt für Bakteriologie, Parasitenkunde, lnfektionskrankheiten and Hygiene, Abteilung II, 4:209-216. Beijerinck MW 1898 . Über die Arten der Essigbakterien. Zentralblatt für Bakteriologie, Parasitenkunde, lnfektionskrankheiten and Hygiene, Abteilung II , 4 : 209-216.
Bond D. R. y D. R. Lovley. 2003. Electricity production by Geobacter sulfurreducens attached to electrodes. Applied and Enviromental. Microbiology 69:1548-1555. Bond DR and DR Lovley . 2003 Electricity production by Geobacter sulfurreducens attached to electrodes. Applied and Enviromental. Microbiology 69 : 1548-1555.
Chaudhuri S., y D. R. Lovley. 2003. Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nature Biotechnology 21:1229-1232. Chaudhuri S., and DR Lovley . 2003 Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nature Biotechnology 21 : 1229-1232.
De Ley J. 1961. Comparative carbohydrate metabolism and a proposal for a phylogenetic relationship of the acetic acid bacteria. Journal of General Microbiology 24, 31-50.From Law J. 1961 . Comparative carbohydrate metabolism and a proposal for a phylogenetic relationship of the acetic acid bacteria. Journal of General Microbiology 24 , 31-50.
De Ley J. y Frateur J.
1974. Genus Acetobacter Beijerinck 1898, 215.
In: R.E. Buchanan and N.E. Gibbons (eds): Bergey's Manual of
Determinative Bacteriology, eighth edition, The Williams &
Wilkins Co, Baltimore, pp. 276-
278.From Law J. and Frateur J. 1974 . Genus Acetobacter Beijerinck 1898 , 215. In: RE Buchanan and NE Gibbons (eds): Bergey's Manual of Determinative Bacteriology, eighth edition, The Williams & Wilkins Co, Baltimore, pp. 276-
278.
Delamar M., Hitmi R., Pinson J. y J. M. Saveant. 1992. Covalent modification of carbon surfaces by grafting of functionalized aryl radicals produced from electrochemical reduction of diazonium salts. Journal of American Chemical Society 114:5853-5854. Delamar M., Hitmi R., Pinson J. and JM Saveant . 1992 . Covalent modification of carbon surfaces by grafting of functionalized aryl radicals produced from electrochemical reduction of diazonium salts. Journal of American Chemical Society 114 : 5853-5854.
Dreseselhaus M. S. y I.L. Thomas. 2001. Alternative energy technologies. Nature 414: 332- 337. Dreseselhaus MS and IL Thomas . 2001 Alternative energy technologies. Nature 414 : 332- 337.
\newpage\ newpage
Greenberg D. E., Porcella S. F., Stock F., Wong A., Conville P. S., Murray P. R., Holland S. M. y A. M. Zelazny. 2006. Granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae. International Journal of Systematic and Evolutionary Microbiology 56:2609-2616. Greenberg DE, Porcella SF, Stock F., Wong A., Conville PS, Murray PR, Holland SM and AM Zelazny . 2006 Granulibacter bethesdensis gen. nov., sp. Nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae. International Journal of Systematic and Evolutionary Microbiology 56 : 2609-2616.
Duchow E. y Douglas, H. C. 1949. Rhodomicrobium vannielii, a new photoheterotrophic bacterium. Journal of Bacteriology, 58:409-416.] Duchow E. and Douglas , HC 1949 . Rhodomicrobium vannielii , a new photoheterotrophic bacterium. Journal of Bacteriology , 58 : 409-416.]
Harrison Jr. A.P. 1981. Acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments. International Journal of Systematic Bacteriology 31: 327-332. Harrison Jr. AP 1981 . Acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments. International Journal of Systematic Bacteriology 31 : 327-332.
Henneberg W. 1897. Beiträge zur Kenntnis der Essigbakteriën. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten and Hygiene. Abteilung II. 3:223-231. Henneberg W. 1897. Beiträge zur Kenntnis der Essigbakteriën. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten and Hygiene. Abteilung II 3 : 223-231.
Hiraishi A., Matsuzawa Y., Kanbe T. y N. Wakao. 2000: Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments. International Journal of Systematic and Evolutionary Microbiology 50: 1539-1546. Hiraishi A., Matsuzawa Y., Kanbe T. and N. Wakao . 2000 : Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments. International Journal of Systematic and Evolutionary Microbiology 50 : 1539-1546.
Hiraishi A., Nagashima K. V. P., Matsuura K. Shimada K. Takaichi S., Wakao N. y Y. Katayama 1998. Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria: its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. nov. International Journal of Systematic Bacteriology 48, 1389-1398. Hiraishi A., Nagashima KVP, Matsuura K. Shimada K. Takaichi S., Wakao N. and Y. Katayama 1998 . Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria: its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. Nov. International Journal of Systematic Bacteriology 48 , 1389-1398.
Huelsenbeck J. P. y F. Ronquist. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. Huelsenbeck JP and F. Ronquist . 2001 MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17 : 754-755.
Huelsenbeck J. P., Rannala B. y J. P. Masly. 2000. Accommodating phylogenetic uncertainty in evolutionary studies. Science 288:2349-2350. Huelsenbeck JP, Rannala B. and JP Masly . 2000 Accommodating phylogenetic uncertainty in evolutionary studies. Science 288 : 2349-2350.
Hughey R. y A. Krogh. 1996. Hidden Markov models for sequence analysis: Extension and analysis of the basic method. CABIOS 12:95-107. Hughey R. and A. Krogh . 1996 Hidden Markov models for sequence analysis: Extension and analysis of the basic method. CABIOS 12 : 95-107.
Imhoff J. F., Trüper H. G. y N. Pfennig. 1984. Rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria". International Journal of Systematic Bacteriology 34:340-343. Imhoff JF, Trüper HG and N. Pfennig . 1984 . Rearrangement of the species and generates of the phototrophic "purple nonsulfur bacteria". International Journal of Systematic Bacteriology 34 : 340-343.
Johnson D. B., Stallwood B. Kimura S. y K. B. Hallberg. 2006. Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium. Archives of Microbioogy 185:212-221. Johnson DB, Stallwood B. Kimura S. and KB Hallberg . 2006 Isolation and characterization of Acidicaldus organivorus , gen. nov., sp. Nov .: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium. Archives of Microbioogy 185 : 212-221.
Kämpfer P., Andersson M. A., Jäckel U. & Salkinoja-Salonen M. 2003. Teichococcus ludipueritiae gen. nov. sp. nov., and Muricoccus roseus gen. nov. sp. nov. representing two new genera of the \alpha-1 subclass of the Proteobacteria. Systematic and Applied Microbiology. 26:23-29. Kämpfer P., Andersson MA, Jäckel U. & Salkinoja-Salonen M. 2003 . Teichococcus ludipueritiae gen. Nov. sp. nov., and Muricoccus roseus gen. Nov. sp. Nov. representing two new genera of the α-1 subclass of the Proteobacteria. Systematic and Applied Microbiology . 26 : 23-29.
Kämpfer P., Busse H. J., Rosselló-Mora R., Kjellin E. y E. Falsen. 2004. Rhodovarius lipocyclicus gen. nov. sp. nov., a new genus of the \alpha-1 subclass of the Proteobacteria. Systematic and Applied Microbiology. 27:511-516. Kämpfer P., Busse HJ, Rosselló-Mora R., Kjellin E. and E. Falsen . 2004 Rhodovarius lipocyclicus gen. Nov. sp. Nov., a new genus of the α-1 subclass of the Proteobacteria. Systematic and Applied Microbiology . 27 : 511-516.
Kanamori T., Rashid N., Morikawa M., Atomi H. y Imanaka T. 2002. Oleomonas sagaranensis gen. nov., sp. nov., represents a novel genus in the alpha-Proteobacteria. FEMS Microbiology Letters. 17:217(2):255-61. Kanamori T., Rashid N., Morikawa M., Atomi H. and Imanaka T. 2002 . Oleomonas sagaranensis gene. nov., sp. nov., represents a novel genus in the alpha-Proteobacteria. FEMS Microbiology Letters . 17: 217 (2): 255-61.
Kim H. J., Park H. S., Jun M. S., Chang I. S., Kim M. y B. H. Kim. 2002. A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enzyme and Microbial Technology. 30:145-152. Kim HJ, Park HS, Jun MS, Chang IS, Kim M. and BH Kim . 2002 A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens . Enzyme and Microbial Technology . 30 : 145-152.
Kishimoto N., Kosako Y. y T. Tano 1993. Acidiphilium aminolytica sp. nov.: an acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment. Current Microbiology. 27:131-136. Kishimoto N., Kosako Y. and T. Tano 1993 . Acidiphilium aminolytica sp. nov .: an acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment. Current Microbiology 27 : 131-136.
Kishimoto N., Kosako Y., Wakao N., Tano T. y A. Hiraishi. 1995. Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the genus Acidocella gen. nov., and emendation of the genus Acidiphilium. Systematic and Applied Microbiology. 18:85-91. Kishimoto N., Kosako Y., Wakao N., Tano T. and A. Hiraishi . 1995 Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the genus Acidocella gen. nov., and emendation of the genus Acidiphilium . Systematic and Applied Microbiology . 18 : 85-91.
Larget B. y D. Simon. 1999. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Molecular Biology & Evolution 16:750-759. Larget B. and D. Simon . 1999 Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Molecular Biology & Evolution 16 : 750-759.
Lisdiyanti P., Kawasaki H., Wisyastuti Y., Saono S., Seki T., Yamada Y., Uchimura T. y K. Komagata. 2002. Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the \alpha-Proteobacteria. International Journal of Systematic and Evolutionary Microbiology. 52: 813-818. Lisdiyanti P., Kawasaki H., Wisyastuti Y., Saono S., Seki T., Yamada Y., Uchimura T. and K. Komagata . 2002 Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the \ alpha- Proteobacteria. International Journal of Systematic and Evolutionary Microbiology . 52 : 813-818.
Lobos J. H., Chisolm T. E., Bopp L. H. y D. S. Holmes. 1986. Acidiphilium organovorum sp. nov., an acidophilic heterotroph isolated from a Thiobacillus ferrooxidans culture. International Journal of Systematic Bacteriology. 36:139-144. Lobos JH, Chisolm TE, Bopp LH and DS Holmes . 1986 Acidiphilium organovorum sp. nov., an acidophilic heterotroph isolated from a Thiobacillus ferrooxidans culture. International Journal of Systematic Bacteriology . 36 : 139-144.
Loganathan P. y S. Nair. 2004. Swaminathania salitolerans gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (Porteresia coarctata Tateoka). International Journal of Systematic and Evolutionary Microbiology. 54: 1185-1190. Loganathan P. and S. Nair . 2004 Swaminathania salitolerans gen. nov., sp. Nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice ( Porteresia coarctata Tateoka). International Journal of Systematic and Evolutionary Microbiology . 54 : 1185-1190.
Meyer O., Stackebrandt E. y G. Auling. 1993. Reclassification of ubiquinone Q-10 containing carbooxidotrophic bacteria: transfer of "[Pseudomonas] carboxydovorans" OM5 to Oligotropha, gen. nov., as Oligotropha carboxidovorans, comb. nov., transfer of "[Alcaligenes] carboxydus" DSM 1086 to Carbophilus, gen. nov., as Carbophilus carboxidus, comb. nov., transfer of "[Pseudomonas] compransoris" DSM 1231 to Zavarzinia, gen. nov., as Zavarzinia compransoris, comb. nov., amended description of the new genera. Systematic and Applied Microbiology. 16, 390-395. Meyer O., Stackebrandt E. and G. Auling . 1993 Reclassification of ubiquinone Q-10 containing carbooxidotrophic bacteria: transfer of "[ Pseudomonas ] carboxydovorans " OM5 to Oligotropha , gen. Nov., as Oligotropha carboxidovorans , comb. Nov., transfer of "[ Alcaligenes ] carboxydus " DSM 1086 to Carbophilus , gen. Nov., as Carbophilus carboxidus , comb. Nov., transfer of "[ Pseudomonas ] Compransoris " DSM 1231 to Zavarzinia , gen. Nov., as Zavarzinia Compransoris , comb. Nov., amended description of the new genera. Systematic and Applied Microbiology . 16 , 390-395.
Molisch H. 1907. Die Purpurbakterien nach neuen Untersuchungen. G. Fischer, Jena I-VII, 1, pp. 1-95 Molisch H. 1907 . Die Purpurbakterien nach neuen Untersuchungen. G. Fischer, Jena I-VII, 1, pp. 1-95
Pasteur L. 1864. Mémoire sur la fermentation acétique. Annales Scientifiques de l'École Normale Supérieure Paris, 1:113-158. Pasteur L. 1864 . Mémoire sur la fermentation acétique. Annales Scientifiques de l'École Normale Supérieure Paris , 1 : 113-158.
Reddy G. S. N., Nagy M. y F. Garcia-Pichel. 2006. Belnapia moabensis gen. nov., sp. nov., an aiphaproteobacterium from biological soil crusts in the Colorado Plateau, USA. International Journal of Systematic and Evolutionary Microbiology. 56:51-58. Reddy GSN, Nagy M. and F. Garcia-Pichel . 2006 Belnapia moabensis gen. nov., sp. Nov., an aiphaproteobacterium from biological soil crusts in the Colorado Plateau, USA. International Journal of Systematic and Evolutionary Microbiology . 56 : 51-58.
Rihs J. D., Brenner D. J., Weaver R. E., Steigerwalt A. G., Hollis D. G. y V. L. Yu. 1993. Roseomonas, a new genus associated with bacteremia and other human infections. Journal of Clinical Microbiology. 31:3275-3283. Rihs JD, Brenner DJ, Weaver RE, Steigerwalt AG, Hollis DG and VL Yu . 1993 Roseomonas , a new genus associated with bacteremia and other human infections. Journal of Clinical Microbiology . 31 : 3275-3283.
Rodriguez F., Oliver J.F., Marin A. y J. R. Medina. 1990. The general stochastic model of nucleotide substitutions. Journal of Theoretical Biology 142:485-501. Rodriguez F., Oliver JF, Marin A. and JR Medina . 1990 The general stochastic model of nucleotide substitutions. Journal of Theoretical Biology 142 : 485-501.
Saitoh S., Suzuki T. y Y. Nishimura. 1998. Proposal of Craurococcus roseus gen. nov., sp. nov. and Paracraurococcus ruber gen. nov., sp. nov., novel aerobic bacteriochlorophyll a-containing bacteria from soil. International Journal of Systematic Bacteriology. 48:1043-1047. Saitoh S., Suzuki T. and Y. Nishimura . 1998 Proposal of Craurococcus roseus gen. nov., sp. Nov. and Paracraurococcus ruber gen. nov., sp. nov., novel aerobic bacteriochlorophyll a -containing bacteria from soil. International Journal of Systematic Bacteriology . 48 : 1043-1047.
Shiba T. y Simidu U. 1982. Erythrobacter longus gen. nov., sp. nov., an aerobic bacterium which contains bacteriochlorophyll a. Inernational Journal of Systematic Bacteriology, 32,:211-217. Shiba T. and Simidu U. 1982 . Erythrobacter longus gen. nov., sp. nov., an aerobic bacterium which contains bacteriochlorophyll a . International Journal of Systematic Bacteriology , 32 ,: 211-217.
Scholz F. y U. Schroder. 2003. Bacterial Batteries. Nature Biotechnology. 21:1151-1152. Scholz F. and U. Schroder . 2003 Bacterial Batteries Nature Biotechnology 21 : 1151-1152.
Shukla, A. K., Suresh, P., Berchmans, S. y A. Rajendran, 2004. Biological fuel cells and their applications. Curent Science. 87:455-468. Shukla , AK, Suresh , P., Berchmans , S. and A. Rajendran , 2004 . Biological fuel cells and their applications. Curent Science 87 : 455-468.
Tayhas G., Palmore R., y G. M. Whitesides. 1994. Microbial and Enzymatic Biofuel Cells in Enzymatic conversion of biomass for fuel production, M. E. Himmel, J.O. Baker and R.P. Overend, eds., American Chemical Society Symposium series, n° 566, ACS, Washington, DC., 271-290. Tayhas G., Palmore R., and GM Whitesides . 1994 Microbial and Enzymatic Biofuel Cells in Enzymatic conversion of biomass for fuel production, ME Himmel, JO Baker and RP Overend, eds., American Chemical Society Symposium series, No. 566, ACS, Washington, DC., 271-290.
Thompson J. D., Gibson T. J., Plewniak F, Jeanmougin F, y D. G. Higgins. 1997. The Clustal X windows interface; flexible strategies for multiple sequence alignmen aided by quality analysis tools. Nucleic Acids Research. 25:4876-4882. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and DG Higgins . 1997 The Clustal X windows interface; flexible strategies for multiple sequence alignmen aided by quality analysis tools. Nucleic Acids Research . 25 : 4876-4882.
Urakami T. Tamaoka J., Suzuki K. I. y K. Komagata. 1989. Acidomonas gen. nov., incorporating Acetobacter methanolicus as Acidomonas methanolica comb. nov. International Journal of Systematic Bacteriology. 39: 50-55. Urakami T. Tamaoka J., Suzuki KI and K. Komagata . 1989 Acidomonas gene. nov., incorporating Acetobacter methanolicus as Acidomonas methanolica comb. Nov. International Journal of Systematic Bacteriology . 39 : 50-55.
Vasilyeva L. V. 1985. Stella, a new genus of soil prosthecobacteria, with proposals for Stella humosa sp. nov. and Stella vacuolata sp. nov. International Journal of Systematic Bacteriology. 35, 518-521. Vasilyeva LV 1985 . Stella , a new genus of soil prosthecobacteria, with proposals for Stella humosa sp. Nov. and Stella vacuolata sp. Nov. International Journal of Systematic Bacteriology . 35 , 518-521.
Wakao N., Nagasawa N., Matsuura T., Matsukura H., Matsumoto T., Hiraishi A., Sakurai Y. y H. Shiota. 1994. Acidiphilium multivorum sp. nov., an acidophilic chemoorganotrophic bacterium from pyritic acid mine drainage. Journal of General and Applied Microbiology. 40:143-159. Wakao N., Nagasawa N., Matsuura T., Matsukura H., Matsumoto T., Hiraishi A., Sakurai Y. and H. Shiota . 1994 Acidiphilium multivorum sp. nov., an acidophilic chemoorganotrophic bacterium from pyritic acid mine drainage. Journal of General and Applied Microbiology . 40 : 143-159.
Wichlacz P. L., Unz R. F. y T. A. Langworthy. 1986. Acidiphilium angustum sp. nov., Acidiphilium facilis sp. nov., and Acidiphilium rubrum sp. nov.: acidophilic heterotrophic bacteria isolated from acidic coal mine drainage. International Journal of Systematic Bacteriology. 36, 197-201. Wichlacz PL, Unz RF and TA Langworthy . 1986 Acidiphilium angustum sp. Nov., Acidiphilium facilis sp. Nov., and Acidiphilium rubrum sp. Nov .: Acidophilic heterotrophic bacteria isolated from acidic coal mine drainage. International Journal of Systematic Bacteriology . 36 , 197-201.
Yamada Y., Hoshino K. y T. Ishikawa. 1997. The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to generic level. Bioscience Biotechnology and Biochemistry. 61:1244-1251. Yamada Y., Hoshino K. and T. Ishikawa . 1997 The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to generic level. Bioscience Biotechnology and Biochemistry . 61 : 1244-1251.
Yamada Y., Katsura K., Kawasaki H., Widyastuti Y., Saono S. Seiki T., Uchimura T y K. Komagata 2000. Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the \alpha-Proteobacteria. International Journal of Systematic and Evolutionary Microbiology. 50:823-829. Yamada Y., Katsura K., Kawasaki H., Widyastuti Y., Saono S. Seiki T., Uchimura T and K. Komagata 2000 . Asaia bogorensis gene. nov., sp. nov., an unusual acetic acid bacterium in the α- Proteobacteria . International Journal of Systematic and Evolutionary Microbiology . 50 : 823-829.
Yukphan P., Malimas T., Potacharoen W., Tanasupawat S., Tanticharoen M. y Yamada Y. 2005. Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the \alpha-Proteobacteria. Journal of General Applied Microbiology, 51, 301-311. Yukphan P., Malimas T., Potacharoen W., Tanasupawat S., Tanticharoen M. and Yamada Y. 2005 . Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the \ alpha- Proteobacteria. Journal of General Applied Microbiology , 51 , 301-311.
Yurkov V., Stackebrant E., Holmes A., Fuerst J. A., Hugenholtz P., Golecki J., Gad'on N., Gorlenko V. M., Kompantseva E. I. & Drews G. 1994. Phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of Roseococcus thiosulfatophilus gen. nov., sp. nov., Erythromicrobium ramosum gen. nov., sp. nov., and Erythrobacter litoralis sp. nov. International Journal of Systematic Bacteriology. 44:427-434. Yurkov V., Stackebrant E., Holmes A., Fuerst JA, Hugenholtz P., Golecki J., Gad'on N., Gorlenko VM, Kompantseva EI & Drews G. 1994 . Phylogenetic positions of novel aerobic, bacteriochlorophyll a -containing bacteria and description of Roseococcus thiosulfatophilus gen. nov., sp. Nov., Erythromicrobium ramosum gen. nov., sp. nov., and Erythrobacter litoralis sp. Nov. International Journal of Systematic Bacteriology . 44 : 427-434.
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| PCT/ES2008/070100 WO2008148923A1 (en) | 2007-06-04 | 2008-05-26 | Aerobic bacterial electrode for a fuel cell anode, which does not include redox mediators or a proton exchange membrane |
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Non-Patent Citations (6)
| Title |
|---|
| HALLBERG, K. B., et al. Macroscopic streamer growths in acidic, metal-rich mine waters in North Wales consist of novel and remarkably simple bacterial communities. Appl. Environ. Microbiol., Marzo 2006, vol. 72, nº 3, páginas 2022-2030. Ver todo el documento. * |
| IM, H. J., et al. A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enz. Microb. Technol., 4 febrero 2002, vol. 30, nº 2, páginas 145-152. Ver todo el documento. * |
| JONG, B. C., et al. Enrichment, preformance and microbial diversity of a thermophilic mediatorless microbial fuel cell. Environ. Sci. Technol., 15 octubre 2006, vol. 40, nº 20, páginas 6449-6454. Ver todo el documento. * |
| LOVLEY, D. R. Microbial fuel cells: novel microbial physiologies and engineering approaches. Current Op. Biotechnol., 5 mayo 2006 (on line), vol. 17, páginas 327-332. Ver todo el documento. * |
| PHUNG N. T., et al. Analysis of microbial diversity in oligotrophic mirobial fuel cells using 16S rDNA sequences. FEMS Microbiol. Lett., 10 febrero 2004 (on line), vol. 233, nº 1, páginas 77-82. Ver todo el documento. * |
| RABAEY, K., et al. Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl. Environ. Microbiol., Septiembre 2004, vol. 70, nº 9, páginas 5373-5382. Ver todo el documento. * |
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