WO2014033345A1

WO2014033345A1 - Microbial consortium for the production of hydrogen

Info

Publication number: WO2014033345A1
Application number: PCT/ES2013/070617
Authority: WO
Inventors: Emiliano Enrique DÍAZ PORTUONDO; José Luís SANZ MARTÍN; Hayfa RAJHI
Original assignee: Universidad Autónoma de Madrid; Mygen Laboratorio, S. L.
Priority date: 2012-09-03
Filing date: 2013-09-03
Publication date: 2014-03-06
Also published as: ES2456776B1; ES2456776A1

Abstract

The invention relates to a microbial consortium comprising a Clostridium roseum strain with Spanish Type Culture Collection access number CECT8187 and a Streptomyces sp. strain with Spanish Type Culture Collection access number CECT8185. The invention also relates to the use of said consortium for the production of hydrogen, organic acids, solvents or biofilms, and to a method for producing said consortium.

Description

DESCRIPTION

MICROBIAL CONSORTIUM FOR HYDROGEN PRODUCTION

The present invention falls within the field of biofuel production from microorganisms. Specifically, it refers to a microbial consortium which has utility for the production of hydrogen, organic acids and solvents, as well as a procedure for obtaining the consortium.

STATE OF THE TECHNIQUE

The need to reduce the use of fossil fuels is promoting the search and use of new renewable sources of energy. Among them, hydrogen (H2) is presented as one of the most promising energies, since it is an environmentally sustainable fuel (because its combustion produces only water), its generation can be done in a renewable way and can be efficiently converted into electricity through of the fuel cell or used directly.

Currently, annual hydrogen production worldwide exceeds 50 million tons, however 96% is produced through the conversion of non-renewable fossil fuels (Natural Gas, Petroleum and Coal), while only 4 % corresponds to hydrogen produced from renewable sources, mainly through the electrolysis of water.

One of the ways to produce hydrogen in a renewable and sustainable way, would be through biological production from organic waste and using microorganisms (biohydrogen).

This form is currently being studied by various scientific groups, as it has a number of advantages, including its low energy consumption (natural gas reforming to obtain hydrogen must be done at temperatures above 850 ° C), it releases less greenhouse gases in its production (in natural gas reforming to produce 1 Kg of Hydrogen they are generated as waste 1 1 Kg of CO2), or being able to use solid waste or wastewater from different origins as raw material, instead of other non-renewable energy sources (such as methane or coal), also contributing to the purification of this type of Water and waste

In theory, the biological production of hydrogen through microbial activity can only be carried out in two ways:

Photoreduction where light energy is necessary for microorganisms to produce hydrogen. This route is carried out by cyanobacteria, red and green photosynthetic bacteria, and by some unicellular green algae;

Dark fermentation or acidic fermentation: Where organic matter is fermented to Hydrogen, Carbon Dioxide and organic acids (mainly volatile fatty acids). This route is carried out by anaerobic heterotrophic bacteria (strict or facultative).

There are several factors that influence the process of hydrogen production by fermentation and that should be considered to ensure that it is efficiently applied on an industrial scale. Among the most studied are the selection of the appropriate inoculum and some operational parameters such as pH, temperature and partial pressure of H ₂ .

From the microbiological point of view in the fermentation path there are two fundamental problems: the relationship between the producing and consuming bacteria of hydrogen, and the influence of the partial pressure of hydrogen on the hydrogen producing bacteria. H _2- consuming bacteria decrease the yield of H _2- producing bacteria, which means that in the anaerobic reactors currently operating the percentage of hydrogen does not exceed 5%. To avoid this, different strategies that limit the development of consuming bacteria have been used, such as through the use of heat, since an initial thermal shock promotes the production of H _{2 by} eliminating non-sporulant H ₂ consuming microorganisms and selecting producing bacteria of H ₂ sporulants (Sung et al., Proc. 2002 US DOE Hydrogen Program Review. Other alternative methods have also been tested such as acid treatment, alkaline treatment, partial aeration or inhibition by the addition of 2-bromoethanesulfonic acid, chloroform or Iodine-propane Such treatments may in turn go to a greater or lesser extent to the detriment of the producers of H ₂ or not eliminate some acetogens consumers of H ₂ , so their use is currently under discussion and their results questioned ( Zhu, H., Beland, M., 2006, Int J Hydrogen Energy 31, 1980-1988); Hu and Chen, Int J Hydrogen Energ. 2007, 32: 3266-3273).

Starting from complex microbial inoculums that carry out anaerobic digestion there are two fundamental ways of producing H ₂ : favoring the accumulation of H ₂ (usually scarce) in anaerobic consortia degrading organic matter or by isolating H ₂ producing bacteria from of anaerobic ecosystems, which in turn could be mixed with other microorganisms to form artificial consortia.

The accumulation of H ₂ can be achieved by inhibiting H ₂ consuming microorganisms from anaerobic digestion. The establishment of a type of H ₂ consumer depends mainly on the type of inoculum, the concentration of H ₂ , the carbon source, the solubility of the electron acceptor, etc. but in most anaerobic environments, hydrogenotrophic methanogenic bacteria are the most common group of H ₂ consuming microorganisms Valdez-Vazquez I, 2009 .. International Journal of Hydrogen Energy 34 (9): 3639-3646). Therefore, for the accumulation of H ₂ , Try to inhibit methanogenesis. In practice it has been seen, in effect, that a pH between 5.5 and 6.5, short hydraulic retention times (<6h), heat shock treatments (80-100 ° C 2-3h) and inhibitors as acetylene are efficient minimizing the loss of H ₂ by methanogenic microorganisms in non-sterile systems Valdez-Vazquez I, 2009 .. International Journal of Hydrogen Energy 34 (9): 3639-3646). By inhibiting H ₂ consuming microorganisms, therefore, stable H ₂ producing microbial communities are achieved. Numerous authors study the structure, diversity and dynamics of these communities since deepening the functioning of the H ₂ producing consortiums is essential to improve the efficiency of this process. In recent studies it has been seen that the metabolites most commonly formed during dark fermentation by consortia, are acetate, propionate, butyrate, ethanol and butanol, suggesting that species of the genus Clostridium are predominant in these H ₂ producing systems (Lay , 2000, Bioechnol Bioeng 68, 269-78).

In studies of inoculum enrichment by thermal shock and acidification, it was observed by scanning electron microscopy that the granules producing H ₂ were typically composed of bacillary bacteria forming spores and fusiform bacilli, suggesting that the dominant species was Clostridium sp . together with Bacillus Fang HHP, Liu H, Zhang T. 2002, Biotechnol Bioeng 78, 44-52).

The alternative approach to the manipulation of organic matter degrading consortiums for biohydrogen production is the isolation of bacteria that release H ₂ as a byproduct of their fermentative metabolism, that is, the production of H ₂ by pure isolated cultures. Several authors have studied the production of H ₂ by isolated strains. (Wang et al. J Appl Microbiol. 2007, 103: 1415-1423) and Chen Chen et al. 2006 Int. J. Hydrogen Ener. 31, 2170-2178., Isolated different strains of Clostridium very efficient in the production of H ₂ . Several strains of Enterobacter and Thermoanaerobacterium) have also been isolated. In addition to the isolation and characterization of H ₂ producing strains, some authors have worked with pure cultures in continuous studies with reactors. (Zhang et al. Process Biochem 2006, 41, 21 18-2123), for example, studied the production of H ₂ by a pure culture of Clostridium acetobutylicum in a percolator filter reactor.

In the process of producing biohydrogen from pure cultures, it should be borne in mind that the strain used may be specialized in the degradation of one type of compound, so it could not use complex substrates. With pure cultures it will therefore be necessary to use simple substrates, which makes it difficult to work with waste. Total degradation of organic matter will not occur since there will be intermediary accumulation in the absence of the last stages of degradation.

Both studies of structure, diversity and dynamics of H ₂ producing communities, as well as isolates of H ₂ producing bacteria show that the Clostridium bacteria are fundamental bacteria for the biological production of H ₂ . Therefore, it is essential to study the biochemical routes that Clostridium follows for the conversion of carbohydrates to H ₂ , C02, organic acids and solvents in the processes of production of H ₂ , both by mixed consortia and pure cultures.

The ability of species of the genus Clostridium to form endospores under unfavorable conditions has been widely used to select them, both from natural environments and from sewage sludge, subjecting the inoculum to thermal shock. Isolates of this genus, however, have a disadvantage when operating in continuous cultures: if the partial pressure of H _{2 is} not maintained at low levels, its metabolism can change and stop producing H ₂ , to produce ethanol or lactate, which reduces or cancels the production of H ₂ . Even in continuous cultures maintained in non-sterile conditions for a long time, a change in populations can take place: from producers of H ₂ to consumers or non-producers, such as acetogens, producers of propionate, lactate or methanogens (Kim et al, Process Biochem. 2006, 41: 199-207). Again the H ₂ partial pressure (PH ₂ ) plays a key role in the evolution of the degradative process.

An alternative to this genus are facultative anaerobic bacteria, especially those belonging to the Enterobacter genus and other members of the Enterobacteraceae family such as Citrobacter or Klebsiella (Ito et al, J Biosc Bioeng. 2005, 100: 260-265). Unlike Clostridium, the production of H ₂ in the enterobacteria is not inhibited at high partial pressures of H ₂ , but its yield is less than half of the production yield of H ₂ of Clostridium sp. or Rhodobacter sp. An extensive literature review on the subject, although focused on research conducted in Japan, one of the leading countries in the bioproduction of H ₂ , has been carried out by Nishio and Nakashimada (Nishio N et al. 2005. J Biosc Bioeng 100 ( 3), 260-265).

On the other hand, among the most important operational parameters to take into account during fermentation, pH and temperature seem to play a key role in the production of H ₂ . Both factors have been studied in multiple works. In most of the reported cases, the optimal pH is 5.5 (Jun et al. J microbiol Biotechnol. 2008; 18: 1 130-1 135; Tang et al. J Biosci Bioeng. 2008, 106: 80-87 ) so this pH is used routinely in multiple jobs. With respect to temperature, it has a considerable effect on the production of H ₂ . Thus, using sewage sludge as inoculum for the generation of H ₂ from slurry, (Tang et al. J Biosci Bioeng. 2008, 106: 80-87) determined, for the mesophilic range, the temperature at which a Optimum performance is 45 ° C. The alternative, working in thermophilic conditions (55-60 ° C), has also been investigated, both with pure cultures of C. uzonii and T. acidotolerans (Koskinen et ai, Biotechnol Bioeng. 2008, 101: 679-690), as mixed (O-Thong et al. Biores Technol. 2009, 100: 909-918) in which Thermoanaerobacterium spp. and Clostridium spp. Although the production of H ₂ is higher at 55 ° C than at 37 ° C (Karlsson et al. Int J Hydrogen Energy 2008, 33: 953-962) and contamination problems by consuming bacteria decrease of H ₂ , the process only has an industrial interest for those wastes (wastewater, mainly) that are generated at high temperature, such as certain canning or paper mill industries.

In spite of everything described above, it is still necessary to search for alternatives and / or improvements for the production of hydrogen by microorganisms so as to improve current production yields.

DESCRIPTION OF THE INVENTION

The present invention relates to a microbial consortium formed by a strain of Clostridium roseum and a strain of Streptomyces sp. which allows a high production of hydrogen (H ₂ which makes this consortium an element of high interest in the biofuel industry.

The present invention therefore provides a mixture of strains, one belonging to the species Clostridium roseum with access number to the Spanish Type Culture Collection (CECT) 8187 which has been isolated by an isolation method under conditions of under vacuum (which allows a better selection of highly producing strains by eliminating the limitation caused by the partial pressure of H ₂ in its production) and another belonging to the species Streptomyces sp. with access number to the Spanish Type Crop Collection (CECT) 8185, isolated from the granular sludge in aerobic conditions. The consortium of the invention, as shown in the examples, has the characteristic that by joining the strain of H5 Clostridium roseum with access number to the Spanish Crop Collection Type CECT8187 (producer of H ₂ ) with the strain EJ1 of Streptomyces sp. With access number to the Spanish Culture Collection Type CECT8185, a greater amount of H _{2 is produced} than using the H5 strain Clostridium roseum or other strains independently, despite the Streptomyces sp. It is not a producer of H ₂ . On the other hand, the selected strains have advantages such as (i) that the specific strain of Streptomyces sp. It does not produce antibiotics contrary to what occurs with most organisms of the same genus, and (ii) also allows the formation of spherical biofilms which agglutinate the microorganism and allow better use in bioreactors. In addition, the strain selected from Streptomyces sp, being an oxygen consumer, allows limiting its presence, which in the consortium causes a better development of the other strains selected for the formation of the consortium. The selected strain of Clostridium roseum, meanwhile, has a higher production of H ₂ separately than some organisms of other species of the genus Clostridium. The consortium formed by at least the 2 strains has the additional advantage that when biofilms are formed, the use in bioreactors minimizes the loss of the strain both by greater protection of the strain and by lower elimination by changing the medium in the bioreactors and therefore They make the hydrogen production process easier and more profitable.

Therefore, a first aspect of the invention relates to a microbial consortium from now on, the microbial consortium of the invention, which comprises the Clostridium roseum strain of access number to the Spanish Type CECT8187 Crop Collection and the Streptomyces strain sp. of access number to the Spanish Crop Collection Type CECT8185.

The scientific classification of strain CECT8187 or strain H5 of the present invention is: Domain Bacteria I Edge Firmicutes I Class Clostridia I Order Clostridiales I Family Clostridiaceae I Genus Clostridium I Species Clostridium roseum. This strain was deposited on July 19, 2012 in the Spanish Type Culture Collection (CECT), Building 3 CUE, Pare Científic Universitat de Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia, Spain).

The scientific classification of strain CECT8185 or strain EJ1 of the present invention is: Domain Bacteria I Edge Actinobacteria I Order Actinomycetales I Suborder Streptomycineae / Family Streptomycetaceae I Genus Streptomyces I Species Streptomyces sp. This strain was deposited on July 19, 2012 in the Spanish Type Culture Collection (CECT), Building 3 CUE, Pare Científic Universitat de Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia, Spain).

"Microbial consortium" in the present invention is understood as a mixture of at least 2 strains of different microorganisms. The consortium of the invention comprises the strain of Streptomyces sp. EJ1 CECT8185 and the strain of Clostridium roseum H5 CECT8187, and additionally other strains of different organisms. The consortium of the invention works more adequately when the Streptomyces sp. It is in an adequate proportion for the formation of granules that integrate the producing strains. In this way, the appropriate ratio can be, for example, between 1: 1 and 1: 1000 with the rest of the strains that form the microbial consortium, although it may vary depending on the experimental conditions. Therefore, in a preferred embodiment of this aspect of the invention, the ratio between CECT8185 and CECT8187 is between 1: 1 and 1: 1000.

The consortium of the invention may additionally include other organisms that, for example, but not limited to, allow to degrade other different substrates and therefore allow better use of the starting material used by increasing the yield of the H ₂ production process. In this case, as in the initial consortium, H ₂ , organic acids or solvents may be produced. These organisms can be for example organisms belonging to the genus Clostridium, which can be used to produce H ₂ . Among the organisms that can be added to the consortium are, for example, strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. Therefore, in a preferred embodiment of the first aspect of the invention, the microbial consortium of the invention further comprises at least one other strain of Clostridium. In a more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In a more preferred embodiment of this aspect of the invention, the ratio between CECT8185 and strains CECT8187, CECT8186, CECT8188 and / or CECT8189 is between 1: 1 and 1: 1000.

Clostridium strain is understood as a strain belonging to the Bacteria Domain; Firmicutes Division; Clostridia class; Clostridial Order; Clostridiaceae family; Clostridium genus.

The scientific classification of strain CECT8186 or strain H1 of the present invention is: Domain Bacteria I Edge Firmicutes I Class Clostridia I Order Clostridiales I Family Clostridiaceae I Genus Clostridium I Species Clostridium saccharobutylicum. This strain was deposited on July 19, 2012 in the Spanish Type Culture Collection (CECT), Building 3 CUE, Pare Científic Universitat de Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia, Spain).

The scientific classification of strain CECT8188 or strain R6 of the present invention is: Domain Bacteria I Edge Firmicutes I Class Clostridia I Order Clostridiales I Family Clostridiaceae I Genus Clostridium I Species Clostridium butyricum. This strain was deposited on July 19, 2012 in the Spanish Type Culture Collection (CECT), Building 3 CUE, Pare Científic Universitat de Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia, Spain). The scientific classification of strain CECT8189 or strain RT2 of the present invention is: Domain Bacteria I Edge Firmicutes I Class Clostridia I Order Clostridiales I Family Clostridiaceae I Genus Clostridium I Species Clostridium diolis. This strain was deposited on July 20, 2012 in the Spanish Type Culture Collection (CECT), Building 3 CUE, Pare Científic Universitat de Valencia, Professor Agustín Escardino, 9, 46980 Paterna (Valencia, Spain).

The microbial consortium of the invention can be used independently or in conjunction with other elements known to the person skilled in the art to, for example, but not limited to, make its application easier, maintain its characteristics longer, confer protection against to external conditions, or complement your activity. These compositions comprising other elements additional to the consortium of the invention would have the same utility as the consortium of the invention. Therefore, another aspect of the invention relates to a composition, hereinafter the composition of the invention, comprising the microbial consortium of the invention.

As shown in the examples, the microbial consortium of the invention has utility for the production of H ₂ , since it has a high production rate. Therefore, another aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of access number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of H ₂ . In a preferred embodiment of this aspect of the invention, the consortium or composition further comprises at least one other strain of Clostridium. In a more preferred embodiment the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection CECT8189 of Clostridium diolis.

The consortium of the invention is especially useful in dark or acidic fermentation processes in which degradation of organic matter is carried out under anaerobic conditions. Therefore, a preferred embodiment of this aspect of the invention refers to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of accession number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of H ₂ , where the production of H ₂ is carried out by fermentation. In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis.

"Dark or acidic fermentation" in the present invention is understood as the process that leads to the production of H ₂ from the degradation by microorganisms of organic matter.

The production of H ₂ can be produced from different starting materials, for example, but not limited to, from pure substrates such as proteins or sugars, from complex substrates or even from mixtures such as residues. The use of the latter is particularly interesting since it allows the use of materials that would otherwise be discarded. In the present invention it is shown that the microbial consortium can be used for the production of H ₂ in complex substrate samples, thus demonstrating its usefulness for use of different wastes as starting material for the production of H ₂ . Therefore, another preferred embodiment of this aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of access number to the Spanish Type CECT8187 Crop Collection and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of H ₂ , preferably by fermentation where the initial substrate is urban waste, industrial waste and / or agroindustrial waste. In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis.

"Urban waste" is understood in the present invention as waste from urban areas only.

"Industrial waste" in the present invention means waste from industrial areas exclusively: Example: Waste water from the industrial process of a brewery.

"Agroindustrial waste" is understood in the present invention as waste from agricultural and / or industrial holdings.

In addition to the production of H ₂ , as demonstrated in the examples, the microbial consortium has a higher production of various organic acids than for example the H5 strain independently.

Therefore, another aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium strain Roseum of access number to the Spanish Crop Collection Type CECT8187 and the strain of Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of organic acids. In a preferred embodiment of this aspect of the invention, the consortium or composition further comprises at least one other strain of Clostridium. In a more preferred embodiment the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In an even more preferred embodiment, the organic acids are selected from the list comprising acetic acid, butyric acid, propionic acid, lactic acid, formic acid or succinic acid.

In the same way that in the case of H ₂ these organic acids can be produced preferably in dark or acidic fermentation processes. Therefore, a preferred embodiment of this aspect of the invention refers to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of accession number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of organic acids, where the production of organic acids is carried out by fermentation. In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In an even more preferred embodiment, the organic acids are selected from the list comprising acetic acid, butyric acid, propionic acid, lactic acid, formic acid or succinic acid. In the case of the formation of organic acids, one can also start from different pure substrates or complex substrates. Therefore, another preferred embodiment of this aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of access number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of organic acids, preferably by fermentation, where the initial substrate is urban waste, industrial waste and / or agroindustrial waste. In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In an even more preferred embodiment, the organic acids are selected from the list comprising acetic acid, butyric acid, propionic acid, lactic acid, formic acid or succinic acid.

By "organic acid" in the present invention is meant a compound that contains at least one carboxyl group. In the present invention it is restricted to compounds formed by C, H and O of low molecular weight which contain at least one carboxyl group, such as, but not limited to, acetic acid, butyric acid, propionic acid, lactic acid, acid formic or succinic acid.

In addition to organic acids and H ₂ , the consortium of the invention, as shown in the examples, also presents the ability to produce solvents such as, but not limited to, ethanol, methanol, acetone and butanol. Therefore, another aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain number of access to the Spanish Culture Collection Type CECT8187 and the strain of Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the production of solvents, preferably ethanol, methanol, acetone and / or butanol. In a preferred embodiment of this aspect of the invention, the consortium or composition further comprises at least one other strain of Clostridium. In a more preferred embodiment the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In an even more preferred embodiment the solvents are selected from the list comprising ethanol, methanol, acetone and butanol.

The production of solvents, in the case of the consortium of the invention, can preferably occur in dark or acidic fermentation processes. Therefore, a preferred embodiment of this aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of accession number to the Spanish Type CECT8187 Crop Collection and the Streptomyces sp strain . of access number to the Spanish Crop Collection Type CECT8185 for the production of solvents, preferably ethanol, methanol, acetone and / or butanol, where the production of solvents is carried out by fermentation. In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis. In addition, as in the previous cases, it is also possible to start from different pure substrates or complex substrates. Therefore, another preferred embodiment of this aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of access number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Collection of Crops Type CECT8185 for the production of solvents, preferably ethanol, methanol, acetone and / or butanol, and more preferably by fermentation, where the initial substrate is urban waste, industrial waste and / or agroindustrial waste . In a more preferred embodiment the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis.

By "solvent" in the present invention, organic solvents are understood as volatile organic compounds that are used to dissolve other compounds without solvent or solute undergoing chemical change.

The examples of the present invention also show that the consortium has the ability to form biofilms. These biofilms allow the agglutination of microorganisms so that they are better protected against the stress to which they may be subjected, for example, in a bioreactor and therefore allow a better use of productive capacity and greater use of resources. Therefore, another aspect of the invention relates to the use of a microbial consortium or a composition comprising the Clostridium roseum strain of accession number to the Spanish Crop Collection Type CECT8187 and the Streptomyces sp. of access number to the Spanish Crop Collection Type CECT8185 for the biofilm formation In a preferred embodiment, the consortium or composition further comprises at least one other strain of Clostridium. In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis.

By "biofilm" or "biofilm" in the present invention is meant a community of microorganisms that grow embedded in a matrix of exopolysaccharides. These biofilms can be found adhered to different surfaces or, as in this invention, form granules that allow the aggregation of microorganisms.

In the present invention, a method is also described by which highly H ₂ producing strains are obtained. This procedure is performed in low vacuum conditions, which prevents the inhibition of growth caused by the accumulation of H ₂ . This allows a better selection of highly producing organisms of H ₂ . Therefore, another aspect of the invention relates to a method for obtaining highly producing strains of H ₂ , which comprises:

a) cultivate a mud sample in a bioreactor under aerobic conditions,

b) isolate colonies present in the mud of (a), and

c) cultivate the colonies of (b) and select the most producing strains, characterized in that it is carried out under low vacuum conditions.

In a preferred embodiment of this aspect of the invention the strain selected in step (c) is selected from the list comprising strain H5 CECT8187, strain H1 CECT8186, strain R6 CECT8188 and strain RT2 CECT8189. In an even more preferred embodiment the strain the strain selected in step (c) is strain H5.

"Under vacuum" in the present invention is understood as a pressure in the range between 100 kPa and 100 Pa, equivalent to 1-0.00 atmospheres.

"Mud" in the present invention is understood to mean a microbial conglomerate or consortium that can have a definite shape or not.

"Granular sludge" in the present invention means anaerobic sludge in the form of a granule that can be formed, for example, but not limited to, in anaerobic UASB reactors (Anaerobic Sludge Blanket Upflow)

"Highly producing H ₂ strains" in the present invention are understood as those strains capable of producing an amount of H ₂ greater than two moles of H ₂ per mole of glucose consumed.

To obtain the consortium of the invention, it is necessary to mix a highly producing strain of H ₂ with strain EJ1 of Streptomyces sp. Therefore, another aspect of the invention relates to a process for obtaining a microbial consortium, comprising:

a) cultivate a mud sample in a bioreactor under aerobic conditions,

b) isolate colonies present in the mud of (a), and

c) cultivate the colonies of (b) and select the most producing strains, d) independently cultivate crushed granular sludge under aerobic conditions

e) select the floc formed

f) insulate on the plaque-forming microorganism, which corresponds to Streptomyces sp.

g) mix the strains of steps (c) and (f) characterized in that steps a, b and c are carried out under low vacuum conditions. In a preferred embodiment of this aspect of the invention the strain selected in step (c) is strain H5 strain CECT8187. In a more preferred embodiment of this aspect of the invention, at least one other Clostridium strain is additionally mixed in step (g). In an even more preferred embodiment, the strain of the genus Clostridium is strain H1 of access number to the Spanish Crop Collection Type CECT8186 of Clostridium saccharobutylicum, strain R6 of access number to the Spanish Crop Collection Type CECT8188 of Clostridium butyricum and / or strain RT2 of access number to the Spanish Crop Collection Type CECT8189 of Clostridium diolis.

In another preferred embodiment, strain EJ1 of Streptomyces sp. it is mixed in step (g) in the form of spherical biofilms.

In another preferred embodiment the strains of step (g) are mixed in a ratio of between 1: 1 to 1: 1000.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. A and B Shows the formation of spherical particles by strain EJ1 (CECT8185) of Streptomyces sp. EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the usefulness of the microbial consortium for the production of H2, organic acids and solvents. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described

below they illustrate the invention without limiting its scope of application.

Example 1: Isolation of microorganisms producing H ₂ under low vacuum conditions.

For the isolation of H ₂ producing bacteria and obtaining an enriched culture, different types of bioreactors were worked on (Volume: 20 - 300 ml_). Initially for starting, between 1% and up to 20% of a sludge were inoculated, which can come from different sources such as the anaerobic sewage treatment plant of a brewery (strains H5 CECT8187 and H1 CECT8186), sludge from an aerobic sewage treatment plant urban sludge from an anaerobic digester of urban solid waste (strain R6 CECT8188) and river sediment (strain RT2 CECT8189) in culture medium. The process proceeded at 30 ° C and was always applied under vacuum (101325 Pa - 1, 333 Pa) or (760 torr- 0.01 torr). During the enrichment stage, a volume of inoculum, between 0.1% and 5% of the useful volume, was transferred to another bioreactor with Reactor Medium (MR), which was incubated under the same conditions. Similarly, between 3 and up to 15 successive passes were made. Subsequently, from the enriched culture obtained, 30 ml roll-tubes and petri dishes (with the same culture medium) were inoculated using anaerobic jugs and jugs under low vacuum conditions (between 100 kPa and 100 Pa), until obtaining isolated colonies . Finally, the isolated colonies were re-dominated in bioreactors with liquid medium (Volume: 20 ml_ - 120 ml_), they grew at 30 ° C (in the same culture medium) and cultures that produced more H _{2 were selected} (measured with Honeywell MDA Scientific Midas® Gas Detector and with a Bruker 450-GC gas chromatograph). The pure H _2- producing cultures were extracted with DNA (Fast DNA spin kit for soil, MPbiomedical) and an amplification of the 16S RNAr was performed by PCR (primers 27F of sequence SEQ ID NO: 1 (AGAGTTTGATCMTGGCTCAG) and 1492R of sequence SEQ ID NO: 2 (TACGGYTACCTTGTTACGACTT) and was sent to be sequenced (BIGDYE kit) The sequences obtained were analyzed using the Blast computer tool (http://blast.ncbi.nlm.nih.gov/) and with Ribosomal Datábase Project (RDP) (http://rdp.cme.msu.edu/), to determine its phylogeny (table 2). With this isolation procedure, several strains of microorganisms highly producing H ₂ (table 2) were obtained, since the problem caused by the inhibition caused by the partial pressure of H ₂ was eliminated.

Table 1: Means used in the examples:

It was used for the Macronutrient Solution

1 mL growth of Solution

Means, medium

Streptomyces sp and Micronutrients

Extract from

H ₂ Component producers Carbohydrate: 4g / l Meat (EC) ^*

Meat extract

^* 4g COD / L Flush up to 1000 mL with dH ₂ 0

It was used for the Macronutrient Solution

growth and 1 mL of Solution

Micronutrient enrichment

Growing crops Carbohydrate Component: 2g / l

Reactor Medium

of H ₂ and in the Sucrose

(MR) ^*

Formation of Protein Component: 1 g / L

consortia Meat extract; 0.5 g / l Extract

of yeast

^* 4g COD / L Flush up to 1000 mL with dH ₂ 0 Macronutrient Solution

1 ml_ of Solution

He was employed at the

Micronutrients

kinetic study

Carbohydrate component: 4g / l

Average glucose growth of

Glucose

(MG) H ₂ producers

Flush up to 1000 mL with dH ₂ 0 Adjust to pH7

^* 4g COD / L

Sterilize in autoclave (120 ° C; 0.5 atm; 20 min) or by filtration (22μΜ)

Bacto-Tryptone 10 g / L, Extract from

5 g / L and 5 g / L NaCI were used for yeast.

Medium LB (1X) growth of the Adjust to pH7

strain EJ1 Sterilize in autoclave (120 ° C; 0.5 atm; 20 min)

Table 2: List of microorganisms producing H ₂ isolated.

Example 2: Optimization of the physical-chemical parameters of the fermentation for the microorganisms producing H ₂ isolated. All trials were done in triplicate. All bioreactors were inoculated with an initial Optical Density (OD) inoculum of 0.001 to 610 mm. The assays for pH optimization (5; 5.5; 6.5; 7.5) were incubated at 30 ° C. The tests for temperature optimization were performed at an initial pH of 6.5 and incubated at 25 ° C, 30 ° C, 35 ° C and 40 ° C. Samples were taken at 14h, 24h and 48h (and in some cases 72h) of incubation, determining in all cases the following parameters: H ₂ produced, pH of the medium, optical density of the medium (OD), chemical oxygen demand of the medium (COD), and final fermentation products. H ₂ measurements were made using a MDA Scientific Midas® Gas HoneyweII Detector and / or gas chromatography using a Bruker 450-GC chromatograph coupled to a TCD detector, with Varian CP2056 0.6m x1 / 8 "Ultimetal column Cromosorb GHP 100-120, working in bypass. Injector, detector and column temperatures were 150, 200 and 50 ° C respectively. Nitrogen was used as a carrier gas with a flow rate of 25 ml min ^" ¹ . pH were performed with the ThermoScientific-Orion 2STAR pH meter, the Optical Density was determined with a Pharmacia LKB-Novaspec II spectrophotometer and the Chemical Oxygen Demand with a Hatch device. The final fermentation products (volatile organic acids, glucose and their metabolic derivatives) were quantified by high performance liquid chromatography (HPLC) with a Vartar Prostar kit in order to study the fermentation products. The most relevant results are shown in table 3:

Table 3: Parameters of pH Temperat strains

Production

optimal optimum

average of H ₂ Production of others

Strain of

(ml_ H ₂ / g COD elements (mM) growth growth

removed)

to o (° C)

H5 5.5 - 7.5 35 397 In MR medium: 18.8 CECT818 butyric acid; 4.2 acetic acid 7; 0.7 propionic acid

H1 In MR medium: 13 acid

CECT818 5.5-7.5 35 252 butyric; 7.6 acetic acid 6; 2.7 ethanol

In MG medium: 1, 7 acid

R6

butyric acid 7.2 CECT818 6.5 35 264

acetic; 3.3 acid 8

propionic; 4.6 ethanol

RT2

In EC medium: 4.9 CECT818 acid 6.5 35 475

acetic; 18, 2 ethanol 9

H5 CECT

In EC medium: 0.09 acid 8187 +

formic, 4.4 acid EJ1 7.0 30-35 480

acetic; 19.9 CECT818 acid

butyric; 2.6 Ethanol 5

In general, a parallel increase in H ₂ production and optical density (610 nm) was observed in all bioreactors, with a decrease in the pH of the medium caused by the production of organic acids.

Example 3: Formation of a microbial consortium between an H ₂ producing microorganism (strain H5 CECT8187) and the oxygen consuming microorganism {Streptomyces strain EJ1 CECT8185). 100 μΙ_ of strain H5 (CECT8187) was inoculated in a bioreactor (120 ml_) under anaerobic conditions in glucose medium (2 g COD / L) and supplemented with macronutrient solution (g / l) (NH ₄ CI (0.20 -0.4), KH ₂ P0 ₄ (0.1 - 0.55), MgS0 ₄ 7H ₂ 0 (0.1 -0.4)) and micronutrients (mg / l) (2 FeCI ₂ 4H ₂ 0; 0.05 H ₃ B0 ₃ ; 0.05 ZnCI ₂ ; 0.04 CuCI ₂ 2H ₂ 0; 0.5 MnCI ₂ .6H ₂ 0; 0.05 (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4H ₂ 0; 0, 09 AICI ₃ .6H20, 2 CoCI ₂ .6H20; 0.09 NiCI ₂ .6H ₂ 0; 0.16 Na ₂ Se0.5H ₂ 0; 1 EDTA; 0.2 resazurine), until the exponential phase is reached. Anaerobic conditions were obtained by gasifying the head space with N ₂ : C0 ₂ (80:20), and adding L-cysteine (0.5 g / L) to the medium, leaving it until the redox indicator, the resazurine, turns from pink to colorless, a symptom that there is no free oxygen in the middle.

On the other hand, 100 μΙ_ of Strain EJ1 (CECT8185) was inoculated in a bioreactor (250 ml_) under aerobic conditions in MR (4 g-COD / L) or Medium LB 1X, under stirring conditions between 80 - 150 rpm until achieve spherical biofilm formation. Subsequently, several biofilms of the Streptomyces strain EJ1 culture (CECT8185) were crushed and mixed with strain H5 (CECT8187) in OD ratio (610 nm) between EJ1: H5 (Between 1: 1 to 1 - 1000). This mixture of microorganisms is grown in a discontinuous bioreactor in a medium MR 4 g COD / L), supplemented by micronutrient and micronutrient solutions. This MR has an initial concentration of dissolved oxygen between 1 - 10 mg of O ₂ / L of H ₂ O. Consortium growth was done at 30 ° C and with horizontal agitation (50 - 200 rpm) with an average time of 96 hours After the incubation time, a spherical biofilm was obtained where strain H5 has been agglutinated by Streptomyces strain EJ1. This fact was confirmed by the techniques of FISH and DGGE. These biofilms (H5-Streptomyces) were inoculated in a bioreactor (120 mL) under anaerobic conditions in MR medium (4 g-COD / L) and in a short time a production of H ₂ higher than that registered by the pure H5 culture was detected under the same conditions (table 4). Table 4: Production of H ₂ of strain H5 (CECT8187), of the H5 consortium (CECT8187) + EJ1 (CECT8185), and of the H5 consortium (CECT8187) + H1 (CECT8186) + R6 (CECT8188) + RT2 (CECT8189) + EJ1 (CECT8185)

Example 4: Formation of a microbial consortium between four microorganisms producing H ₂ (clostridia strains H5, H1, R6 and RT2) and the oxygen-consuming microorganism {Streptomyces sp. strain EJ1).

100 μΙ_ of each of the H ₂ producing strains (H5, H1, R6 and RT2) were inoculated separately in bioreactors (120 mL) under anaerobic conditions in glucose medium (4 g-COD / L) and supplemented with solution macronutrients and micronutrients, until reaching the exponential phase. Anaerobic conditions were obtained by gasifying the head space with N ₂ : CO ₂ (80:20) and adding L-cysteine (0.5 g / L) to the medium, leaving it until the redox indicator, the resazurine, turns pink colorless, a symptom that there is no oxygen free in the middle. On the other hand, 100 μΙ_ of strain EJ1 was inoculated in a bioreactor (250 mL) under aerobic conditions in MR medium (4 g-COD / L) or LB medium (1X), under agitation conditions between 80 - 150 rpm until achieve spherical biofilm formation.

Subsequently, several biofilms of the Streptomyces Cepa EJ1 culture were crushed and mixed at the same time with the four strains H5, H1, R6 and RT2 in OD ratio (610 nm) between EJ1: H5: H1: R6: RT2 (1: 10 : 10: 10: 10). This mixture of microorganisms is grown in a discontinuous bioreactor in an MR medium (4 g COD / L), supplemented by micronutrient and micronutrient solutions. This mixed medium has an initial dissolved oxygen concentration of between 1-10 mg of O2 / L of H2O. The consortium is grown at 30 ° C and with horizontal agitation (50-200 rpm) at an average time of 96 hours. After the incubation time, a spherical biofilm was obtained where the four strains H5, H1, R6 and RT2 have been agglutinated by Streptomyces strain EJ1. This fact was confirmed by the techniques of FISH and DGGE. These biofilms (H5-H1 -R6-RT2-Sfreptomyces) were inoculated in a bioreactor (120 mL) under anaerobic conditions in mixed medium (4 gDQO / L) and in a short time a significant H ₂ production was detected superior to the production of strain H5 independently (table 4).

Example 5: Isolation of Streptomyces sp. strain EJ1.

Between 0.5 and 1 gram of crushed anaerobic granular sludge was inoculated in 100 ml of MR medium (4 g COD / L) or LB medium. The organisms were subsequently grown in aerobic conditions at 30 ° C for 24 hours at 120 rpm in horizontal agitation. The floc-forming microorganisms were separated from the liquid medium and successive plate insulations were performed under aerobic conditions at 30 ° C until pure cultures were obtained. These axenic cultures were grown again in aerobic conditions in MR medium at 30 ° C, 120 rpm in horizontal agitation and the strain that forms flocs was selected, in Shape of spherical biofilms (Figure 1). Analysis of its 16S rRNA showed that it was Streptomyces sp. (strain EJ1 CECT 8185).

Claims

one . - Microbial consortium comprising strain H5 of Clostridium roseum of number CECT 8187 and strain EJ1 of Streptomyces sp. of CECT number 8185.

2. - Microbial consortium according to claim 1, further comprising at least one other strain of the genus Clostridium.

3. - Microbial consortium according to claim 2 wherein the strain of the genus Clostridium is strain H1 of number CECT8186 of Clostridium saccharobutylicum, strain R6 of number CECT8188 of Clostridium butyricum and / or strain RT2 of number CECT8189 of Clostridium diolis.

4. - Composition comprising a microbial consortium according to any of claims 1 to 3.

5. - Use of the microbial consortium according to any one of claims 1 to 3 or of a composition according to claim 4 for the production of hydrogen.

6. - Use of the microbial consortium according to any of claims 1 to 3 or of a composition according to claim 4 for the production of organic acids.

7. - Use according to claim 6 wherein the organic acids are acetic acid, butyric acid, propionic acid, lactic acid, formic acid and / or succinic acid.

8. - Use of the microbial consortium according to any one of claims 1 to 3 or of a composition according to claim 4 for the production of solvents.

9. - Used according to claim 8 wherein the solvents are ethanol, methanol, acetone and / or butanol.

10. - Use according to any of claims 5 to 9 wherein the production of hydrogen, organic acids and / or solvents takes place by fermentation.

eleven . - Use of the microbial consortium or the composition according to any of claims 5 to 10 wherein the initial substrate is urban waste, industrial waste and / or agroindustrial waste.

12. - Use of the microbial consortium according to any of claims 1 to 3 or a composition according to claim 4 for biofilm formation.

13. - Procedure for obtaining a highly hydrogen producing strain comprising:

a) cultivate a mud sample in a bioreactor under aerobic conditions,

b) isolate colonies present in the mud of (a), and

14. Procedure according to claim 13 wherein the highly producing strain isolated in step (c) is Clostridium roseum strain H5

CECT8187.

15. - Method of obtaining a microbial consortium comprising obtaining a highly hydrogen producing strain according to claim 14 which further comprises: d) cultivating crushed granular sludge under aerobic conditions

e) select the floc formed f) insulate on the plaque-forming microorganism, which corresponds to Streptomyces sp. CECT8185

g) mix the strains of steps (c) and (f).

16. Method according to claim 15 wherein in step (g) at least one other strain of the genus Clostridium is also mixed.

17. Method according to claim 16 wherein the strain of the genus

Clostridium is strain H1 of number CECT8186 of Clostridium

saccharobutylicum, strain R6 of number CECT8188 of Clostridium butyricum and / or strain RT2 of number CECT8189 of Clostridium diolis.