WO2018088884A1 - Method for obtaining a microbial consortium in order to produce hydrogen and hydrolysates using complex substrates - Google Patents

Method for obtaining a microbial consortium in order to produce hydrogen and hydrolysates using complex substrates Download PDF

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WO2018088884A1
WO2018088884A1 PCT/MX2017/000108 MX2017000108W WO2018088884A1 WO 2018088884 A1 WO2018088884 A1 WO 2018088884A1 MX 2017000108 W MX2017000108 W MX 2017000108W WO 2018088884 A1 WO2018088884 A1 WO 2018088884A1
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lactobadllus
stage
qostridium
medium
microbial consortium
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French (fr)
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Jacob GÓMEZ ROMERO
Alberto LÓPEZ LÓPEZ
Octavio GARCÍA DEPRAECT
Elizabeth LEÓN BECERRIL
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Centro De Investigación Y Asistencia En Tecnología Y Diseño Del Estado De Jalisco A.C.
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Publication of WO2018088884A1 publication Critical patent/WO2018088884A1/en

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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
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/02—Acetobacter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/04—Actinomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the field of application of this present invention is in biotechnology, particularly in the treatment of agroindustrial waste, industrial wastewater, urban organic solid waste and lignocellulosic materials by means of a microbial consortium for the production of hydrogen; In addition, production of hydrolysates for the generation of methane as a source of energy, hydrogen by photo-fermentation and lipids by heterotrophic algae cultures.
  • inoculum or microbial consortium as one in which two or more microorganisms coexist for a given smelter; and inoculum or enriched consortium such as that where the type and number of microorganisms desired are increased due to an adaptation process under specific conditions of the method.
  • inoculum or enriched consortium such as that where the type and number of microorganisms desired are increased due to an adaptation process under specific conditions of the method.
  • ios technical problems associated with hydrogen production with microbial consortia remain activity, diversity, stability, robustness, availability and reprodutibilidad inoculum and hydrolytic or proteolfb 'ca inoculum capacity when recalcitrant complex substrates are used as agroindustrial waste, and anaerobic conditions that must be provided to hydrogen production systems.
  • the methods developed to obtain inocula or microbial consortiums enriched with hydrogenic bacteria include: heat treatment, acidification, gasification, aeration, freezing, using high dilution rates "cell washing". Particularly, the heat treatment is one of the most used. However, the heat treatment above 60 ° C, from 1 h to 24 h, is aggressive; since it affects the activity and diversity of the hydrogenic bacterial communities; both optional and anaerobic strict, as well as hydrophilic or lactic acid bacteria that play an important role in the process.
  • the spores of these microorganisms have dedmal reduction times (D) at 80 ° C above 40 min (Table 1). However, other hydrogen producing bacteria are eliminated due to the lack of the sporulation mechanism with reduced D values at temperatures between 30 and 65 ° C, for example, Ethanoiigenens, Megasphaera and anaerobic physicians (Table 1). Escherichia coll, Entembacter, Streptococcus, Klebsiella, dtrobacter, Shigdla, Enterococcus are facultative bacteria that produce hydrogen, also create a natural anaerobic environment or maintain a reduced environment for strict anaerobic bacteria.
  • LAB lactic acid bacteria
  • acetic acid bacteria for example, Acetobacter, Gluconobacter and Gluconacetobacter. Therefore, the co-existence of lactic acid bacteria, acetic acid, with both strict and facultative anaerobic hydrogen producing bacteria should be considered for the robustness and flexibility of microbial consortia for the production of hydrogen or hydrolyzed with high amount of lactate and acetate to produce more hydrogen or methane as a renewable energy source.
  • the sensitivity of strict facultative and anaerobic microorganisms to oxygen tension and redox potential is given by the presence or level of expression of the enzyme superoxide dismutase (SOD).
  • Table 2 shows the activity levels of the SOD and catalase enzymes, as well as the oxygen tolerance time for different microorganisms.
  • the levels of expression or activity of the enzymes SOD and catalase are correlated with the sensitivity of the microorganisms to oxygen and their cell viability. It has been shown that the activity of the SOD enzyme has a bacteriostatic effect, when oxygen is present. For example, Qostridium perfringens shows growth at 0.5% dissolved oxygen (OD), but at higher concentrations growth is inhibited due to the low activity of the SOD enzyme (Table 2).
  • the exclusion of molecular oxygen, together with a very low redox potential environment are essential for methanogenic microorganisms.
  • Methanococcus vottae and M. vannielii sox ⁇ highly sensitive to oxygen due to the absence of the SOD enzyme.
  • methanobacterium thermoautotrophlcum, Methanobrevibacter arboriphilus and Methanosardna barkeri have shown cell viability despite being exposed to air for hours. This suggests that in addition to the intrinsic tolerance due to SOD that some methanogenic species possess, other factors may be responsible for the protection of these microbial species against oxygen.
  • dissolved oxygen is an important substrate for the synthesis of lipid, oleic acid and ergosterol prindpales cell membrane components in lactic acid bacteria or other bacteria, which makes them more tolerant to compounds such as lactic acid and stimulates Its reproduction. Therefore, the combination of these characteristics can be used to eliminate methanogenic bacteria or benefit hydrogen-producing and hydrolytic bacteria.
  • a disadvantage in terms of heat treatment as described in US 6,860,996 B2 is the loss of viability or activity of hydrogen producing bacteria.
  • Buitrón etal. [Bioresource Technology 10 (2010) 9071-9077] describe the selection of inoculum by heat treatment at 104 ° C for 24 h, pulverize the inoculum (850 pm) and store at room temperature. The authors perform an activation and acclimatization of the inoculum using glucose at a concentration of 20 g / L of the powdered inoculum for 7 days.
  • an inert gas to generate anaerobic conditions artificially represents an important operating cost and investment costs, together with maintaining the natural source of the microorganisms, as well as a constant selection of the microorganisms by heat treatment or other method These factors are determinants for an industrial level viability, since it increases the production costs of the process in general.
  • the present invention relates to a method for selecting and adapting a robust microbial consortium with potentdal to produce hydrogen and hydrolysed from complex substrates, which has the ability to maintain its viability in formulated liquid medium, and can be quickly reactivated for its massive or continuous production.
  • B inoculum is used to produce hydrogen as a renewable energy source from complex substrates or model.
  • hydrolysates which can be used in a second stage, which can be methanogenesis to produce methane; photo fermentation to produce hydrogen; or the heterotrophic culture of algae to produce biomass or metabolites of interest (lipids, carbohydrates, ketone bodies, etc.). It is important to note that the invention shows advantages over existing conventional technologies or methods.
  • the invention allows to reproduce or produce the inoculum in a massive and continuous way in bioreactors, keeping the hydrogenic-hydrolytic characteristics of the inoculum.
  • the invention allows only one time to perform the heat treatment at the selection stage, which means reducing the stages in the hydrogen production process, the energy consumption in the system, the investment and the overall energy costs of the process.
  • the inoculum technology has flexibility, reproducibility and robustness which makes it suitable for application on an industrial scale.
  • the present invention relates to a method for obtaining a stable and robust microbial or inoculum consortium, hydrogen producer in biological production systems, enriched with strict hydro-hydrolytic, facultative and anaerobic bacteria.
  • the method of the invention induces a stage of selection of facultative bacteria and strict anaerobes producing hydrogen and hydrolytic bacteria from natural ecosystems or anaerobic digesters.
  • an adapted microbial consortium is obtained, which grows under non-artificial anaerobic conditions, with a viable cell density and remains stable for long periods of time.
  • Said adapted microbial consortium is used to produce hydrogen or hydrolysates as a renewable energy source from pre-treated and non-pretreated complex substrates in an anaerobic digestion or co-digestion process. Its application is specifically in agroindustrial wastes, industrial wastewater, urban organic solid wastes and lymphanocellulosic materials, for example, vinasses, whey, nejayote, molasses, fruit, vegetable and food residues, aerobic or anaerobic sludge, sewage from the industrial industry coffee, beverages and meat and sausage processing, hydrolyzate of lignocellulosic materials such as: bagasse, agave and grape, cereal and grass husk.
  • microbial or inoculum consortia with hydrogenic-hydrolytic characteristics, enriched with strict facultative and anaerobic bacteria, and their growth and production is low Anaerobiosis conditions not artificially generated.
  • the microbial consortium can be used in subsequent fermentations to produce hydrogen as a renewable energy source by performing an activation stage with glucose, lactose, maltose or starch.
  • the liquid medium formulation preserves, reproduces and maintains the consortium of viable hydrogen producing bacteria for a period of up to 2 years.
  • Figure 1 shows the scheme of the method for obtaining a stable and robust microbial or inoculum consortium, hydrogen producer and hydrolysed in biological production systems, and its application.
  • Figure 2 shows the result of the step of selecting a microbial consortium with hydrogenic-hydrolytic characteristics from a natural source, and micro-aeradon, anaerobiosis and thermal cycles, according to the first example of the present invention.
  • Figure 3 shows the step of adapting a microbial consortium to a model substrate with hydrogenic-hydrolytic characteristics, using a sequential batch process, a mineral medium with a certain concentration of dissolved oxygen, in accordance with the second example of the present invention.
  • Figure 4 shows the result of the hydrogen production stage from glucose, lactose, vinasse, whey, fruit-vegetable waste, co-digestion of vinesse-nejayote, whey-fruit-vegetable residue, according to the fifth example of the present invention.
  • Figure 5 shows the generation of (adate and acetate from the hydrogenate of a mixture of vinasse and nejayote, according to the sixth example of the present invention.
  • the invention relates to a method for obtaining a microbial consortium, which induces strict and optional anaerobic and viable bacteria capable of producing hydrogen or hydrolysed from complex substrates such as agro-industrial waste, industrial wastewater, urban organic solid waste and lignocellulosic materials.
  • the invention further includes the adapted microbial consortium, which is used to produce hydrogen or hydrolysates as a renewable energy source by anaerobic digestion or co-digestion of complex p-treated and non-pretreated substrates.
  • the method for obtaining a microbial consortium for the production of hydrogen and hydrolysates from complex substrates comprises the following steps:
  • the adaptation medium using as a base a mineral medium containing salts of KH2PO4, K2HPO4, MgSO » CaCfe, FeSO-» 7H2O and NH4CI, according to Table 3, dissolved in distilled water, keeping the mineral medium under stirring constant from 100 to 300 rpm to reach a dissolved oxygen concentration of 30 to 60%.
  • the salts dissolve 48 to 120 h before being used.
  • a source of carbon which can be glucose, lactose, maltose or starch, according to Table 3.
  • the analysis of liquid samples consists in determining the consumption of carbohydrates by the Phenol-Sulfuric Method or another method to quantify carbohydrates, indirect sanctification of biomass by protein quantification by the Bradford Method or another method to quantify protein, the Total carbohydrate intake should be between 50 and 75%; the amount of protein from 150 to 300 pg / mL in the exponential and stationary phase.
  • the fermentations must be carried out in a sequential batch process for about 2 months, always leaving a percentage not exceeding 50% and At least 10% of fermented medium as a foot of Cuba for the next load of fresh medium and fermentation.
  • Temperature conditions of 35 to 38 ° C, pH 5.0 - 6.0, and stirring speed of 100 to 150 rpm should be controlled.
  • b Recover the biomass from the fermented broth by centrifugation, and re-suspend said biomass in a conservation medium (Table 3 and 4) that contains: potassium monobasic phosphate, potassium dibasic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium doruro; a carbon source selected from: lactose, glucose, and maltose, in a concentration of 10 mg / L; and fatty acids selected from: lactate, acetate and butyrate.
  • the preservative medium has a dissolved oxygen concentration of 12 to 20%, and a pH value of 4.8 to 6.0 units, depending on the source of carbon used.
  • Formulate a mineral medium that contains: potassium monobasic phosphate, potassium basic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium doride, with a pH of 6.0 to 6.5, a dissolved oxygen concentration of 30- 60%, and a selected carbon source of glucose, maltose, lactose, and starch, in a concentration of 5 to 20 g / L.
  • the salts should be dissolved in deionized water for at least 2 d, or more preferably for 5 d under constant stirring, and reach a dissolved oxygen concentration of between 30-60% in the mineral medium.
  • step (a) Inoculate 10 to 15% v / v of the preserved inoculum, in the fresh medium of step (a).
  • C. Perform a fermentation cycle, under controlled pH conditions of 5.5 to 6.4 units, temperature of 35 to 38 ° C, with constant stirring of 100 to 150 rpm. Reactivation fermentation can last up to 48 h of culture, but it is desirable to keep it only for a period of 24 h to continue with the hydrogen production stage.
  • the fresh medium can be a Model substrate such as: glucose, lactose, maltose or starch, or a complex substrate such as: industrial waste including: vinasse, whey, nejayote, molasses, fruit, vegetable and food waste; wastewater including: aerobic or anaerobic sludge, wastewater from the coffee, beverage and meat and sausage processing industry; organic solid wastes including: hydrolysed lignocellulosic materials such as cane bagasse, agave and grapes, grains and grains, etc. Any of these residues in digestion and / or co-digestion, and without dilution thereof. Without the need to create anaerobic artificial conditions by injecting a entrained gas.
  • a Model substrate such as: glucose, lactose, maltose or starch
  • a complex substrate such as: industrial waste including: vinasse, whey, nejayote, molasses, fruit, vegetable and food waste
  • wastewater including: aerobic or anaero
  • the fermentation is carried out under pH 5.5 - 6.0, at a temperature of 35-38 ° C and constant stirring speed of 100 to 150 rpm.
  • the fresh medium formulated with potassium monobasic phosphate, potassium dibasic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium chloride pH 6.0 to 6.5, which contains as carbon source either glucose, lactose, maltose or starch (from 20 to 30 g / L), with the microbial consortium adapted according to stage II, or reactivated according to stage IV, in a proportion of 10 to 15% v / v with respect to the volume of fresh medium to inoculate.
  • the medium also contains a dissolved oxygen concentration of 30-60%
  • the inoculum or microbial consortium with hydrocytic activity, hydrogenic activity obtained and adapted according to the steps and stages defined in the method described above, is made up of strict facultative and anaerobic microorganisms, lactic acid, acetic acid and hydrogen producers.
  • the microbial consortium or inoculum includes: to.
  • Lactic acid microorganisms such as: Bif ⁇ dobacterium psychraerophilum, Lactobadllus casei, Lactobadllus coryniformis, Lactobadllus ddbrueckii, Lactobadllus harbinensis,
  • Lactobadllus hilgardli Lactobadllus parabuchneri, Lactobadllus perolens, Lactobadllus rapi, Lactobadllus rhamnosus, Lactobadllus sunki ⁇ , Lactobadllus vaednostercus, Sporolactobadllus terrae, Sporolactobadllus sp, and
  • Acetic microorganisms such as: Acetobacter tibinongensis, Acetobacter lovaniensfs, Acetobacter orientalls, Acetobacter sp, Acetobacter syzygii.
  • the hydrogen-hydrolytic inoculum can be used with different configurations or designs of anaerobic processes, one-stage process, two stages or multiple stages.
  • the one-stage process represents a process where all the metabolic processes of the microorganisms are carried out inside a reactor.
  • a two or multiple stage process involves the use of more than one reactor, where optimal conditions for the metabolic processes of microorganisms must be provided.
  • hydrolysis, acetogenesis or hydrogenesis can occur in a single reactor.
  • the hydrolyzed organic matter of the first stage can be fed to a second stage.
  • the present invention is applicable to produce hydrogen or hydrogenated organic matter in a single stage.
  • the hydrogenate can be fed to a second, which can be methanogenesis to produce methane; photo-fermentation to produce hydrogen; or the heterotrophic cultivation of algae to produce biomass or meta pellets of interest (lipids, carbohydrates, ketone bodies, etc.).
  • the processes can be in batch or continuous, in one, two or multiple stages.
  • the source of inoculum can come from natural or artificial sources, in this example it was an anaerobic digester from a domestic wastewater treatment plant.
  • An amount of 500 mL of inoculum is placed in a 1 L glass bottle adapted with an injection port for air and inert gas and temperature probe.
  • PM is adjusted from 5.9 to 6.0 units.
  • Air is injected until 3 to 5% of dissolved oxygen is reached and left 10 to 15 min under that condition.
  • an inert gas nitrogen, argon, etc.
  • an inert gas nitrogen, argon, etc.
  • the mineral medium shown in Table 3 was used as a culture medium for the adaptation method, the salts of KH2PO4, K2HPO4, MgSCM, Cadjv FeS0 4 7H 2 0 and NH4CI were dissolved in distilled water (48 to 120 h) before being used, they were kept under constant agitation of 100 to 300 rpm to reach a dissolved oxygen concentration of 30-60%.
  • lactose was used at different concentrations (5, 10, 15, 20 and 30 g / L).
  • the analysis of liquid samples consists in determining the consumption of carbohydrates by the Phenol-Sulfuric Method, indirect quantification of biomass by protein quantification by the Bradford Method, the consumption of total carbohydrates it must be between 50 and 75%; the amount of protein from 150 to 300 pg / mL in the exponential and stationary phase.
  • the hydrolytic-hydrogenic inoculum was kept refrigerated between 4 to 8 ° C, under anaerobic or aerobic conditions. Previously, fermentation was carried out for 36 hours using lactose at a constant pH of 5.5 g and a constant stirring of 100 rpm, then the biomass was recovered by centrifugation. Finally, the biomass was re-suspended in the culture medium specified in Table 3 and 4, and kept refrigerated.
  • the activated inoculum is cultured in a 4 L stirred tank bioreactor under anaerobic conditions not artificially induced, that is, generation of natural anaerobiosis by microorganisms.
  • glucose, lactose are used at a concentration of 10, 20 and 30 g / L
  • whey, vinasses, nejayote, fruit-vegetable residues are used raw (not pretreated) and without dilution.
  • Biogas production is monitored by a digital gasometer.
  • a direct method of gas chromatography is used to measure the evolution of hydrogen activity and production. Biogas samples are obtained from the bioreactor head sword and injected into the gas chromatography equipment. The hydrogen peak is identified and the hydrogen composition is determined from a calibration curve constructed with different hydrogen conodon concentrates.
  • vinegars and nejayote are inoculated with 500 mL of activated inoculum for 24 h, previously described.
  • the culture is carried out in a bioreactor of 5 L stirred tank at 37 ° C, pH 5.0 and stirring speed of 150 rpm for 48 hours. No entrainment gas is injected into the system.
  • the initial dissolved oxygen concentration in the mixture is 10 to 20%.
  • the lactate and acetate concentration is determined by means of a high performance liquid chromatograph.
  • the concentration of acetate and lactate after 48 h of culture is 2,445.5 mg / L and 8,073.6 mg / L, respectively.
  • the lactate content in the hydrolyzate provides a high quality effluent for a methanogenic process, its presence has implications from the energy point of view and generation of buffering capacity.
  • the change of free energy of G ⁇ bbs is -68.8 U / mol when methane is produced from lactate, this value is twice as much as that of acetate (-31.0 kJ / mol); also during the degradation of 1 mol of lactate produces 1 mol acetate, 1 mol of bicarbonate and 2 moles of hydrogen (Pipyn and Verstraete [Biotechnology and Bioengineering 23 (1981) 1145-1154]); Wu etai, [Bioresource Technology 211 (2016) 16-23]).
  • Example 7 Process to produce 5 liters of inoculum with lactose
  • the inoculum is reactivated for 24 h, inoculating with a 50 mL of refrigerated inoculum a volume of 450 mL with mineral medium with salts KH2PO4, K2HPO4, MgSCv » CaCfe, FeSCU 7H 2 0 and NhUCI; and lactose at 20 g / L at an initial dissolved oxygen concentration of 30-60%, this is done at a temperature of 35 ° C, pH 5.5 and stirring speed of 100 rpm for 24 hours.
  • the fermentation process is carried out under the operating conditions specified above.
  • concentration of hydrogen and carbon dioxide in the biogas is continuously monitored by gas chromatography.
  • the cell mass concentration is determined indirectly by protein quantification by the Bradford Method. It should be noted that the process can be successively increased to the desired volume of inoculum to be produced, respecting 10% v / v of the inoculum with respect to the subsequent workload and maintaining the conditions specified above.

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Abstract

The present invention relates to a method for obtaining a stable and robust microbial consortium or inoculum, which produces hydrogen and hydrolysates for the generation of methane as an energy source in biological production systems using complex substrates, specifically agroindustrial waste, industrial wastewater, urban solid organic waste and lignocellulosic materials. The method of the invention ensures the production of an inoculum having a microbial structure with the following advantages: reproducible on a large scale; highly robust, predominance over the microflora native to the substrates; high hydrolytic capacity; generates natural anaerobic conditions, without requiring the injection of an inert gas or the addition of a chemical reducing agent; highly stable, viable for up to 2 years when refrigerated; and reactivation time measured in hours.

Description

MÉTODO PARA OBTENER UN CONSORCIO MICROBIANO PARA PRODUCIR HIDRÓGENO E HIDROUZADOS A PARTIR DE SUSTRATOS COMPLEJOS METHOD FOR OBTAINING A MICROBIAL CONSORTIUM TO PRODUCE HYDROGEN AND HYDROUZED FROM COMPLEX SUBSTRATES
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
El campo de aplicación de esta presente invención es en biotecnología, particularmente en el tratamiento de residuos agroindustriales, aguas residuales industriales, desechos sólidos orgánicos urbanos y materiales lignocelulósicos mediante un consorcio microbiano para la producción de hidrógeno; además producción de hidrolizados para la generación de metano como fuente de energía, hidrógeno por vía foto-fermentativas y lípidos por cultivos heterotrófícos de algas. ANTECEDENTES DE LA INVENCIÓN The field of application of this present invention is in biotechnology, particularly in the treatment of agroindustrial waste, industrial wastewater, urban organic solid waste and lignocellulosic materials by means of a microbial consortium for the production of hydrogen; In addition, production of hydrolysates for the generation of methane as a source of energy, hydrogen by photo-fermentation and lipids by heterotrophic algae cultures. BACKGROUND OF THE INVENTION
EEs bien conocido que una gran variedad de microorganismos mesofilicos tanto facultativos como anaerobios estrictos pueden producir hidrógeno como fuente de energía renovable a partir de sustratos simples o bien sustratos complejos por el proceso de digestión anaerobia (proceso que utiliza solo un sustrato al mismo tiempo) o co-digestión anaerobia (proceso que utiliza dos o más sustratos al mismo tiempo). Las bacterias productoras de hidrógeno, consumidoras de hidrógeno, así como bacterias hidrolrticas son microorganismos que coexisten en fuentes o ecosistemas naturales (pantanos, suelo, composta, etc.) o artificiales (digestores anaerobios o biorreactores). Esto ha llevado a desarrollar métodos para obtener inóculos o consorcios enriquecidos con bacterias productoras de hidrógeno. En la presente invención se entiende como inoculo o consorcio microbiano como aquel en donde coexisten dos o más microorganismos para una determinada fundón; e inoculo o consorcio enriquecido como aquel donde el tipo y número de microorganismos deseados son aumentados debido a un proceso de adaptación bajo condiciones específicas del método. No obstante, ios problemas técnicos asociados con la producción de hidrógeno con consorcios microbianos siguen siendo la actividad, diversidad, estabilidad, robustez, disponibilidad y reprodutibilidad del inoculo, así como la capacidad hidrolítica o proteolfb'ca del inoculo cuando se utilizan sustratos complejos recalcitrantes como los residuos agroindustriales, y las condiciones anaerobias que deben proveerse a los sistemas de producción de hidrógeno. It is well known that a wide variety of mesophilic microorganisms, both facultative and strict anaerobes, can produce hydrogen as a renewable energy source from simple substrates or complex substrates by the anaerobic digestion process (process that uses only one substrate at the same time) or anaerobic co-digestion (process that uses two or more substrates at the same time). Hydrogen-producing, hydrogen-consuming bacteria, as well as hydrolytic bacteria are microorganisms that coexist in natural or artificial sources (swamps, soil, compost, etc.) or artificial (anaerobic digesters or bioreactors). This has led to the development of methods to obtain inoculums or consortia enriched with hydrogen-producing bacteria. In the present invention it is understood as an inoculum or microbial consortium as one in which two or more microorganisms coexist for a given smelter; and inoculum or enriched consortium such as that where the type and number of microorganisms desired are increased due to an adaptation process under specific conditions of the method. However, ios technical problems associated with hydrogen production with microbial consortia remain activity, diversity, stability, robustness, availability and reprodutibilidad inoculum and hydrolytic or proteolfb 'ca inoculum capacity when recalcitrant complex substrates are used as agroindustrial waste, and anaerobic conditions that must be provided to hydrogen production systems.
Hasta ahora, los métodos desarrollados para obtener inóculos o consorcios microbianos enriquecidos con bacterias hidrogénicas incluyen: tratamiento térmico, acidificación, gasificación, aeración, congelamiento, utilizando altas tasas de dilución "lavado de células". Particularmente, el tratamiento térmico es uno de los más utilizados. Sin embargo, el tratamiento térmico por arriba de 60°C, de 1 h a 24 h es agresivo; ya que afecta la actividad y diversidad de las comunidades bacterianas hidrogénicas; tanto facultativas como anaerobias estrictas, así como bacterias hidroifücas o áddo lácticas que juegan un papel importante dentro del proceso. Until now, the methods developed to obtain inocula or microbial consortiums enriched with hydrogenic bacteria include: heat treatment, acidification, gasification, aeration, freezing, using high dilution rates "cell washing". Particularly, the heat treatment is one of the most used. However, the heat treatment above 60 ° C, from 1 h to 24 h, is aggressive; since it affects the activity and diversity of the hydrogenic bacterial communities; both optional and anaerobic strict, as well as hydrophilic or lactic acid bacteria that play an important role in the process.
Ahora bien, se sabe que d tratamiento térmico selecciona bacterias productoras de hidrógeno de las consumidoras de hidrógeno, e.g. metanogénicas (Energía de activación, Ea = 59.0 kJ/mol). Principalmente el género Qostridium y Badllus son resistentes al calor debido al mecanismo de esporulación [Ea, de 227.4 a 318.2 kJ/mol). Entre las espedes de Clostridíum ampliamente reportadas como productoras de hidrógeno tenemos a C tyrobutyricum, C butyricum, C. bdjerinckíí y C. sordelli; del género Badllus están; B. subtilís, B. cereus y B. Ilcheniformis. Las esporas de estos microorganismos tienen tiempos de reducdón dedmal (D) a 80°C por arriba de 40 min (Cuadro 1). Sin embargo, otras bacterias productoras de hidrógeno son eliminadas debido a la falta del mecanismo de esporulación con valores D reducidos a temperaturas entre 30 y 65°C, por ejemplo, Ethanoiigenens, Megasphaera y facultativas anaerobias (Cuadro 1). Escherichia coll, Entembacter, Streptococcus, Klebsiella, dtrobacter, Shigdla, Enterococcus son bacterias facultativas que producen hidrógeno, además crean un ambiente anaerobio natural o mantienen un ambiente reduddo para las bacterias anaerobias estrictas. Por otro lado, bacterias áddo lácticas (LAB, por sus siglas en inglés) del género: Bifidobacterium, Lactobadllus, Sporolactobadllus, Ladococcus, presentan una alta actividad hidroiítica, por tanto, su presenda es importante cuando se trata de sustratos complejos. De igual manera consumen o eliminan el oxígeno disuelto del sistema para establecer un entorno adecuado para las bacterias anaerobias estrictas.  However, it is known that d heat treatment selects hydrogen producing bacteria from hydrogen consumers, e.g. methanogenic (activation energy, Ea = 59.0 kJ / mol). Mainly the genus Qostridium and Badllus are resistant to heat due to the sporulation mechanism [Ea, from 227.4 to 318.2 kJ / mol). Among the Clostridíum species widely reported as hydrogen producers we have C tyrobutyricum, C butyricum, C. bdjerinckíí and C. sordelli; of the genus Badllus are; B. subtilís, B. cereus and B. Ilcheniformis. The spores of these microorganisms have dedmal reduction times (D) at 80 ° C above 40 min (Table 1). However, other hydrogen producing bacteria are eliminated due to the lack of the sporulation mechanism with reduced D values at temperatures between 30 and 65 ° C, for example, Ethanoiigenens, Megasphaera and anaerobic physicians (Table 1). Escherichia coll, Entembacter, Streptococcus, Klebsiella, dtrobacter, Shigdla, Enterococcus are facultative bacteria that produce hydrogen, also create a natural anaerobic environment or maintain a reduced environment for strict anaerobic bacteria. On the other hand, lactic acid bacteria (LAB) of the genus: Bifidobacterium, Lactobadllus, Sporolactobadllus, Ladococcus, have a high hydrocytic activity, therefore, their presence is important when it comes to complex substrates. They also consume or eliminate dissolved oxygen from the system to establish a suitable environment for strict anaerobic bacteria.
Figure imgf000004_0001
Figure imgf000004_0001
Figure imgf000005_0001
Figure imgf000005_0001
Rnalmente, otros géneros de bacterias anaerobias estrictas importantes en el proceso de hidrólisis y acidogénesís son las bacterias ácido acéticas (AAB, por sus siglas en inglés), por ejemplo, Acetobacter, Gluconobacter y Gluconacetobacter. Por lo tanto, la co-existencia de bacterias ácido lácticas, ácido acéticas, con bacterias productoras de hidrógeno tanto anaerobias estrictas como facultativas debe ser considerada para la robustez y flexibilidad de consorcios microbianos para la producción de hidrógeno o hidrolizados con alta cantidad de lactato y acetato para produdr más hidrógeno o metano como fuente de energía renovable. Por otro lado, la sensibilidad de los microorganismos facultativos y anaerobios estrictos a la tensión del oxígeno y potencial redox, está dada por la presencia o nivel de expresión de la enzima superóxido dismutasa (SOD, por sus siglas en inglés). En la Cuadro 2, se presentan los niveles de actividad de las enzimas SOD y catalasa, así como el tiempo de tolerancia al oxígeno para diferentes microorganismos. Los niveles de expresión o actividad de las enzimas SOD y catalasa están correlacionados con la sensibilidad de los microorganismos al oxígeno y su viabilidad celular. Se ha demostrado que la actividad de la enzima SOD tiene un efecto bacteriostático, cuando hay presencia de oxígeno. Por ejemplo, Qostrídium perfringens presenta crecimiento a 0.5% de oxígeno disuelto (OD), pero a concentraciones mayores el crecimiento se inhibe debido a la baja actividad de la enzima SOD (Cuadro 2). Por otro lado, la exclusión de oxígeno molecular, junto con un entorno muy bajo de potencial redox son esenciales para los microorganismos metanogénicos. Particularmente, Methanococcus vottae y M. vannielii sox\ altamente sensitivos al oxígeno debido a la ausencia de la enzima SOD. Sin embargo, algunas metanogénícas tolerantes al oxígeno han sido reportadas; Methanobacterium thermoautotrophlcum, Methanobrevibacter arboriphilus y Methanosardna barkeri, han mostrado viabilidad celular a pesar de estar expuestas al aire por horas. Esto sugiere que además de la tolerancia intrínseca debido a la SOD que algunas espedes metanogénícas poseen, otros factores pueden ser responsables de la protección de estas especies microbianas contra ei oxígeno. Rnally, other genera of strict anaerobic bacteria important in the hydrolysis and acidogenesis process are acetic acid bacteria (AAB), for example, Acetobacter, Gluconobacter and Gluconacetobacter. Therefore, the co-existence of lactic acid bacteria, acetic acid, with both strict and facultative anaerobic hydrogen producing bacteria should be considered for the robustness and flexibility of microbial consortia for the production of hydrogen or hydrolyzed with high amount of lactate and acetate to produce more hydrogen or methane as a renewable energy source. On the other hand, the sensitivity of strict facultative and anaerobic microorganisms to oxygen tension and redox potential is given by the presence or level of expression of the enzyme superoxide dismutase (SOD). Table 2 shows the activity levels of the SOD and catalase enzymes, as well as the oxygen tolerance time for different microorganisms. The levels of expression or activity of the enzymes SOD and catalase are correlated with the sensitivity of the microorganisms to oxygen and their cell viability. It has been shown that the activity of the SOD enzyme has a bacteriostatic effect, when oxygen is present. For example, Qostridium perfringens shows growth at 0.5% dissolved oxygen (OD), but at higher concentrations growth is inhibited due to the low activity of the SOD enzyme (Table 2). On the other hand, the exclusion of molecular oxygen, together with a very low redox potential environment are essential for methanogenic microorganisms. Particularly, Methanococcus vottae and M. vannielii sox \ highly sensitive to oxygen due to the absence of the SOD enzyme. Without However, some oxygen-tolerant methanogens have been reported; Methanobacterium thermoautotrophlcum, Methanobrevibacter arboriphilus and Methanosardna barkeri, have shown cell viability despite being exposed to air for hours. This suggests that in addition to the intrinsic tolerance due to SOD that some methanogenic species possess, other factors may be responsible for the protection of these microbial species against oxygen.
Cuadro 2  Table 2
Microorganismo Actividad enzimática Tiempo de tolerancia al (¾  Microorganism Enzymatic activity Tolerance time to (¾
(unidades/mg proteína) (h)  (units / mg protein) (h)
SOD Catalasa  SOD Catalase
Facultativos  Optional
Lactobadf/us plantarum 49.7 32.2 >72  Lactobadf / us plantarum 49.7 32.2> 72
Escherichia coff 15.3 46.0 >72  Escherichia coff 15.3 46.0> 72
Pseudomonas aeruglnosa 10.9 112.9 >72  Pseudomonas aeruglnosa 10.9 112.9> 72
Klebsilla pneumoniae 10.0 n.r. n.r.  Klebsilla pneumoniae 10.0 n.r. n.r.
Enterobacter doacae 18.3 n.r. n.r.  Enterobacter doacae 18.3 n.r. n.r.
Badllus drculans 18.3 n.r. n.r.  Badllus drculans 18.3 n.r. n.r.
Anaerobios estrictos  Strict anaerobes
Clostrfdium perfringens 14.6 0 >72  Clostrfdium perfringens 14.6 0> 72
Qostridium aminovalerícum 0 0 0.75  Qostridium aminovalerich 0 0 0.75
Qostrídium pasteurianum 0.5 0 n.r.  Qostridium pasteurianum 0.5 0 n.r.
Qostridium acetobutyfícum + n.r. n.r.  Qostridium acetobutycum + n.r. n.r.
Qostridium beijerinddí + n.r. n.r.  Qostridium beijerinddí + n.r. n.r.
Qostridium biférmentans + n.r. n.r.  Qostridium biférmentans + n.r. n.r.
Qostridium sporogenes + n.r. n.r.  Qostridium sporogenes + n.r. n.r.
Anaerobios tolerantes  Tolerant Anaerobes
Bacteriodes fragills 6.8 7.1 48  Bacteriodes fragills 6.8 7.1 48
Bifídobacterium 0.3 0 48  Bifidobacterium 0.3 0 48
adoiescentis  adoiescentis
Streptococcus lactís 131 0 n.r.  Streptococcus lactís 131 0 n.r.
Streptococcus faecalls 0.8 0 n.r. Streptococcus faecalls 0.8 0 n.r.
Figure imgf000006_0001
Figure imgf000006_0001
n.r.: no reportado n.r .: not reported
Finalmente, el oxigeno disuelto es un sustrato importante para ia síntesis de lípidos, áddo oleico y ergosterol prindpales componentes en membrana celular en bacterias áddo lácticas u otras bacterias, lo cual las hace más tolerantes a compuestos como áddo láctico y estimula su reproducción. Por tanto, la combinación de estas características puede ser utilizada para eliminar bacterias metanogénicas o bien beneficiar bacterias hidrógeno productoras e hidrolíticas. Finally, dissolved oxygen is an important substrate for the synthesis of lipid, oleic acid and ergosterol prindpales cell membrane components in lactic acid bacteria or other bacteria, which makes them more tolerant to compounds such as lactic acid and stimulates Its reproduction. Therefore, the combination of these characteristics can be used to eliminate methanogenic bacteria or benefit hydrogen-producing and hydrolytic bacteria.
Cabe poner énfasis en el hecho de que la sintrofía de estos microorganismos dentro del consorcio es muy importante, ya que cada microorganismo juega un rol crucial cuando se trata de sustratos complejos o generar condiciones anaerobias. Por otro lado, la actividad microbiana y composición de los inóculos influyen significativamente sobre el desempeño de la producción de hidrógeno. La diversidad y actividad del inoculo juega un rol vital en la manera de degradar la materia orgánica, ya que reduce el periodo de arranque, así como, la cantidad de inoculo requerido en la operación de una planta a gran escala. Mantener la diversidad y actividad de los consorcios microbianos permite que las comunidades microbianas puedan ser controladas por las condiciones de operación. Es por ello que, el tipo de inoculo y mantener su reprodudbilidad son parámetros operadonales importantes para garantizar la estabilidad en los sistemas de producción.  Emphasis should be placed on the fact that the symmetry of these microorganisms within the consortium is very important, since each microorganism plays a crucial role when it comes to complex substrates or generating anaerobic conditions. On the other hand, the microbial activity and composition of the inoculums significantly influence the performance of hydrogen production. The diversity and activity of the inoculum plays a vital role in the way of degrading organic matter, since it reduces the start-up period, as well as the amount of inoculum required in the operation of a large-scale plant. Maintaining the diversity and activity of microbial consortia allows microbial communities to be controlled by operating conditions. That is why, the type of inoculum and maintaining its reproducibility are important operational parameters to guarantee stability in production systems.
En el estado del arte previo, uno de los métodos desarrollados para seleccionar o enriquecer consorcios con bacterias productoras de hidrogeno ha sido el tratamiento térmico, tal como se menciona en el documento de patente Norteamericano US 6,860,996 B2, que menciona realizar tratamiento térmico a la fuente de bacterias bajo condiciones específicas de pH y temperatura. In the prior art, one of the methods developed to select or enrich consortia with hydrogen-producing bacteria has been heat treatment, as mentioned in US 6,860,996 B2, which mentions performing heat treatment at the source of bacteria under specific conditions of pH and temperature.
Una desventaja en cuanto al tratamiento térmico tal como es descrito en el documento US 6,860,996 B2, es la pérdida de viabilidad o actividad de las bacterias productoras de hidrógeno. Buitrón etal., [Bioresource Technology 10(2010) 9071-9077] describen la selección de inoculo por tratamiento térmico a 104°C por 24 h, pulverizan el inoculo (850 pm) y conservan a temperatura ambiente. Los autores realizan una activación y aclimatación del inoculo utilizando glucosa a una concentración de 20 g/L del inoculo pulverizado durante 7 días. Si bien, las condiciones de conservación del inoculo ofrecen una ventaja, este tratamiento térmico produce una fase de activación prolongada debido al proceso de germinación de las esporas, lo que conlleva a una mayor demanda energética celular ligada a un consumo de nutrientes o sustratos. Esta desventaja impacta en la economía del proceso debido a la alta entrada de energía y costos de operación. Es importante subrayar que los métodos antes mencionados, la generación de condiciones anaerobias artificiales está dada por la inyección de un gas inerte o mezcla de gases {e.g. nitrógeno y bióxido de carbono). A disadvantage in terms of heat treatment as described in US 6,860,996 B2, is the loss of viability or activity of hydrogen producing bacteria. Buitrón etal., [Bioresource Technology 10 (2010) 9071-9077] describe the selection of inoculum by heat treatment at 104 ° C for 24 h, pulverize the inoculum (850 pm) and store at room temperature. The authors perform an activation and acclimatization of the inoculum using glucose at a concentration of 20 g / L of the powdered inoculum for 7 days. Although the inoculum conservation conditions offer an advantage, this thermal treatment produces a prolonged activation phase due to the germination process of the spores, which leads to a higher cellular energy demand linked to a consumption of nutrients or substrates. This disadvantage impacts the economy of the process due to the high energy input and operating costs. It is important to underline that the methods mentioned above, the generation of artificial anaerobic conditions is given by the injection of an inert gas or mixture of gases {e.g. nitrogen and carbon dioxide).
Ahora bien, los expertos en la técnica saben que el tratamiento térmico es un proceso complejo y económicamente costoso, debido al consumo de energía. Dado que su implementadón a nivel industrial requiere una etapa de tratamiento térmico adicional dentro del proceso general, ésta debe ser llevada a cabo en un equipo especial diferente al digestor. En este mismo sentido, incrementar la cantidad de inoculo a tratar térmicamente, Implica que el proceso se vuelva menos eficiente y más costoso. Mariakakis et al., [Hydrogen Energy-Challenges and Perspectives, Prof. Dragica Minie (E<±), InTech (2012), Doi: 10.5772/47750]). Esto se corrobora en muchos trabajos científicos en donde reportan la presencia de metano durante la producción de hidrógeno, esto indica que el tratamiento térmico no es eficiente para eliminar baterías consumidoras de hidrógeno. However, those skilled in the art know that heat treatment is a complex and economically expensive process, due to energy consumption. Since its implementation at the industrial level requires an additional heat treatment stage within the general process, This must be carried out in a special equipment other than the digester. In this same sense, increasing the amount of inoculum to be heat treated implies that the process becomes less efficient and more expensive. Mariakakis et al., [Hydrogen Energy-Challenges and Perspectives, Prof. Dragica Minie (E <±), InTech (2012), Doi: 10.5772 / 47750]). This is corroborated in many scientific papers where they report the presence of methane during hydrogen production, this indicates that the heat treatment is not efficient to eliminate hydrogen-consuming batteries.
Por otro lado, la literatura científica frecuentemente reporta, la inyección de un gas de arrastre o acarreador, por ejemplo, nitrógeno, helio, argón, bióxido de carbono o una mezcla gases para generar condiciones anaerobias artificiales dentro de los sistemas de producción de hidrógeno. Para tal caso se puede citar la patente internacional publicada con el número WO2014147558 enfocada a producir hidrógeno en continuo a partir de efluentes de zootecnia, donde se precisa que las condiciones de anaerobiosis son provocadas por la inyección de nitrógeno como gas de arrastre. Si bien, este documento propone un proceso en continuo para producir hidrógeno y metano como fuente de energía renovable. La inyección continua de un gas de arrastre representa un costo constante adicional por insumos.  On the other hand, scientific literature frequently reports the injection of a carrier or carrier gas, for example, nitrogen, helium, argon, carbon dioxide or a gas mixture to generate artificial anaerobic conditions within hydrogen production systems. For this case, it is possible to mention the published international patent with the number WO2014147558 focused on producing hydrogen continuously from zootechnical effluents, where it is specified that anaerobic conditions are caused by the injection of nitrogen as a entrainment gas. Although, this document proposes a continuous process to produce hydrogen and methane as a renewable energy source. Continuous injection of a entrainment gas represents an additional constant cost for inputs.
Con fines de aplicación industrial, la inyección de un gas inerte para generar condiciones anaerobias artificialmente representa un costo de operación y costos de inversión importantes, aunado a mantener la fuente natural de los microorganismos, así como una constante selección de los microorganismos por tratamiento térmico u otro método. Estos factores son determinantes para una viabilidad a nivel industrial, ya que incrementa los costos de producción del proceso en general. For industrial application purposes, the injection of an inert gas to generate anaerobic conditions artificially represents an important operating cost and investment costs, together with maintaining the natural source of the microorganisms, as well as a constant selection of the microorganisms by heat treatment or other method These factors are determinants for an industrial level viability, since it increases the production costs of the process in general.
En este punto, los consorcios microbianos productores de hidrógeno deben tener robustez, es decir no deben ser fácilmente inhibidos por bacterias autóctonas del sustrato o del medio ambiente. Favaro etal., [International Journal of Hydrogen Energy 38(2013) 11774-11779]) confirma que la selección adecuada del inóculo (por ejemplo, el tratamiento térmico) es crudal para alcanzar altos rendimientos y velocidades de producción de hidrógeno a partir de residuos. Los autores mencionan que la disponibilidad de consorcios o inócuios hidrogénicos es el principal requerimiento para una producción de hidrógeno eficiente. Ren et ai, [Environmental Microbiology 9(5) (2007) 1112-1125] indican que utilizar el mismo inóculo o la reproducibilidad del mismo, la comunidad microbiana puede ser controlada por las condiciones de operación. Finalmente, la operación en continuo con consorcios microbianos robustos y reprodudbles maximiza la producción de hidrógeno y ofrece una ventaja en los costos de producción. En relación a los puntos anteriores, los expertos en la técnica saben que hay necesidad en el diseño, desarrollo y producción de consorcios microbianos o inóculos donde coexistan bacterias productoras de hidrógeno, bacterias ácido lácticas u otras especies que beneficien la producción de hidrógeno; que presenten características de reprodudbilidad, producción rápida en sistemas artificiales y estabilidad, son necesidades técnicas que deben ser resueltas para una aplicación a nivel industrial exitosa. At this point, hydrogen-producing microbial consortia must be robust, that is, they must not be easily inhibited by native bacteria in the substrate or the environment. Favaro et al., [International Journal of Hydrogen Energy 38 (2013) 11774-11779]) confirms that the proper selection of the inoculum (for example, heat treatment) is crucial to achieve high yields and speeds of hydrogen production from waste . The authors mention that the availability of consortia or hydrogenic inoculants is the main requirement for efficient hydrogen production. Ren et ai, [Environmental Microbiology 9 (5) (2007) 1112-1125] indicate that using the same inoculum or its reproducibility, the microbial community can be controlled by operating conditions. Finally, continuous operation with robust and reproducible microbial consortia maximizes hydrogen production and offers an advantage in production costs. In relation to the above points, those skilled in the art know that there is a need in the design, development and production of microbial consortiums or inoculums where hydrogen producing bacteria, lactic acid bacteria or other species that benefit hydrogen production coexist; that have reproducibility characteristics, rapid production in artificial systems and stability, are technical needs that must be met for a successful industrial application.
En resumen, se conduye que hay necesidad de mejorar los métodos para selecdonar, adaptar, mantener, reactivar y reproducir de manera rápida consorcios microbianos con capaddad hidrogénica-hidrolítica para produdr hidrógeno o hidrolizados como fuente de energía renovable a partir de sustratos complejos o modelo.  In summary, it is conducted that there is a need to improve the methods for quickly selecting, adapting, maintaining, reactivating and reproducing microbial consortia with hydrogenic-hydrolytic capacity to produce hydrogen or hydrolyzed as a renewable energy source from complex substrates or model.
Por tanto, la presente invención está relacionada con un método para selecdonar y adaptar un consorcio microbiano robusto con potendal para produdr hidrógeno e hidrolizados a partir de sustratos complejos, que tiene la capaddad de mantener su viabilidad en medio líquido formulado, y puede ser reactivado rápidamente para su producción masiva o continua. B inoculo es utilizado para produdr hidrógeno como fuente de energía renovable a partir de sustratos complejos o modelo. O bien, para produdr hidrolizados, que pueden ser utilizados en una segunda etapa, la cual puede ser metanogénesis para produdr metano; foto- fermentadón para produdr hidrógeno; o el cultivo heterotrófico de algas para produdr biomasa o metabolitos de interés (lípidos, carbohidratos, cuerpos cetónicos, etc.). Es importante remarcar que la invendón muestra ventajas sobre las tecnologías o métodos convendonales existentes. Primero: no hay necesidad de crear un ambiente anaerobio artificial. El mantenimiento, reactivadón y producdón del inóculo; así como la producdón de hidrógeno se lleva a cabo sin la inyecdón de un gas inerte. Segundo: la invendón permite reproducir o produdr el inóculo de manera masiva y continua en biorreactores, conservándose las características hidrogénicas-hidrolrticas del inóculo. Tercero: la invendón permite realizar sólo una vez el tratamiento térmico en la etapa de selecdón, lo que implica reducir las etapas en el proceso de producdón de hidrógeno, el consumo de energía en el sistema, la inversión y costos energéticos globales del proceso. Finalmente, la tecnología del inóculo presenta flexibilidad, reprodudbilidad y robustez lo cual la hace apta para su aplicadón a escala industrial.  Therefore, the present invention relates to a method for selecting and adapting a robust microbial consortium with potentdal to produce hydrogen and hydrolysed from complex substrates, which has the ability to maintain its viability in formulated liquid medium, and can be quickly reactivated for its massive or continuous production. B inoculum is used to produce hydrogen as a renewable energy source from complex substrates or model. Or, to produce hydrolysates, which can be used in a second stage, which can be methanogenesis to produce methane; photo fermentation to produce hydrogen; or the heterotrophic culture of algae to produce biomass or metabolites of interest (lipids, carbohydrates, ketone bodies, etc.). It is important to note that the invention shows advantages over existing conventional technologies or methods. First: there is no need to create an artificial anaerobic environment. The maintenance, reactivation and production of the inoculum; as well as hydrogen production is carried out without the injection of an inert gas. Second: the invention allows to reproduce or produce the inoculum in a massive and continuous way in bioreactors, keeping the hydrogenic-hydrolytic characteristics of the inoculum. Third: the invention allows only one time to perform the heat treatment at the selection stage, which means reducing the stages in the hydrogen production process, the energy consumption in the system, the investment and the overall energy costs of the process. Finally, the inoculum technology has flexibility, reproducibility and robustness which makes it suitable for application on an industrial scale.
BREVE DESCRIPCIÓN PE LA INVENCIÓN  BRIEF DESCRIPTION PE THE INVENTION
La presente invendón se relaciona con un método para la obtendón de un consorcio microbiano o inóculo estable y robusto, productor de hidrógeno en sistemas de producdón biológicos, enriqueddo con bacterias hidrogénicas-hidrolíticas, facultativas y anaerobias estrictas. El método de la invendón induye una etapa de selección de bacterias facultativas y anaerobias estrictas productoras de hidrógeno y bacterias hidrolíticas a partir de ecosistemas naturales o digestores anaerobios. Y en una subsiguiente etapa, se obtiene un consorcio microbiano adaptado, que crece bajo condiciones anaerobias no artificiales, con una densidad celular viable y se mantiene estable por largos periodos de tiempo. Dicho consorcio microbiano adaptado, es utilizado para producir hidrógeno o hidrolizados como fuente de energía renovable a partir de sustratos complejos pre-tratados y no-pretratados en un proceso de digestión o co-digestión anaerobia. Su aplicación es específicamente en residuos agroindustriales, aguas residuales industríales, desechos sólidos orgánicos urbanos y materiales lígnocelulósicos, por ejemplo, vinazas, lactosuero, nejayote, melazas, residuos de fruta, verdura y comida, lodos aerobios o anaerobios, aguas residuales de la industria del café, de bebidas y del procesamiento de carnes y embutidos, hidrolizado de materiales lígnocelulósicos como: bagazo de caña, agave y uva, cáscara de cereales y pasto. The present invention relates to a method for obtaining a stable and robust microbial or inoculum consortium, hydrogen producer in biological production systems, enriched with strict hydro-hydrolytic, facultative and anaerobic bacteria. The method of the invention induces a stage of selection of facultative bacteria and strict anaerobes producing hydrogen and hydrolytic bacteria from natural ecosystems or anaerobic digesters. And in a subsequent stage, an adapted microbial consortium is obtained, which grows under non-artificial anaerobic conditions, with a viable cell density and remains stable for long periods of time. Said adapted microbial consortium is used to produce hydrogen or hydrolysates as a renewable energy source from pre-treated and non-pretreated complex substrates in an anaerobic digestion or co-digestion process. Its application is specifically in agroindustrial wastes, industrial wastewater, urban organic solid wastes and lymphanocellulosic materials, for example, vinasses, whey, nejayote, molasses, fruit, vegetable and food residues, aerobic or anaerobic sludge, sewage from the industrial industry coffee, beverages and meat and sausage processing, hydrolyzate of lignocellulosic materials such as: bagasse, agave and grape, cereal and grass husk.
A fin de resolver los problemas del estado de la técnica actual sobre la selección, adaptación, conservación, reactivación y producción de consorcios microbianos o inóculos con características hidrogénlca-hidrolíticas, enriquecidos con bacterias facultativas y anaerobias estrictas, y su crecimiento y producción se da bajo condiciones de anaerobiosis no generadas artificialmente. El consorcio microbiano puede ser utilizado en subsecuentes fermentaciones para producir hidrógeno como fuente de energía renovable realizando una etapa de activación con glucosa, lactosa, maltosa o almidón. La formulación del medio líquido conserva, reproduce y mantiene el consorcio de bacterias productoras de hidrógeno viables por un periodo hasta de 2 años. In order to solve the problems of the current state of the art on the selection, adaptation, conservation, reactivation and production of microbial or inoculum consortia with hydrogenic-hydrolytic characteristics, enriched with strict facultative and anaerobic bacteria, and their growth and production is low Anaerobiosis conditions not artificially generated. The microbial consortium can be used in subsequent fermentations to produce hydrogen as a renewable energy source by performing an activation stage with glucose, lactose, maltose or starch. The liquid medium formulation preserves, reproduces and maintains the consortium of viable hydrogen producing bacteria for a period of up to 2 years.
BREVE DESCRIPCIÓN PE LAS FIGURAS BRIEF DESCRIPTION PE THE FIGURES
Los aspectos novedosos que se consideran característicos de la presente invención se establecerán con particularidad en las reivindicaciones anexas. Sin embargo, la invención misma conjuntamente con otros objetos y ventajas de ella, se comprenderá mejor en la siguiente descripción detallada de ciertas modalidades preferidas de la invención, cuando se lea en relación con los dibujos que se acompañan, en los cuales: The novel aspects that are considered characteristic of the present invention will be established with particularity in the appended claims. However, the invention itself, together with other objects and advantages thereof, will be better understood in the following detailed description of certain preferred embodiments of the invention, when read in relation to the accompanying drawings, in which:
La figura 1, muestra el esquema del método para la obtención de un consorcio microbiano o inoculo estable y robusto, productor de hidrógeno e hidrolizados en sistemas de producción biológicos, y de su aplicación. Figure 1 shows the scheme of the method for obtaining a stable and robust microbial or inoculum consortium, hydrogen producer and hydrolysed in biological production systems, and its application.
La figura 2, muestra el resultado de la etapa de selección de un consorcio microbiano con características hidrogénica-hidrolíticas a partir de una fuente natural, y ciclos de micro- aeradón, anaerobiosis y térmicos, de acuerdo con el primer ejemplo de la presente invención. La figura 3, muestra la etapa de adaptación de un consorcio microbiano a un sustrato modelo con características hidrogénica-hldrolíticas, utilizando un proceso lote secuendal, medio mineral con cierta concentración de oxígeno disuelto, de acuerdo con el segundo ejemplo de la presente invención. Figure 2 shows the result of the step of selecting a microbial consortium with hydrogenic-hydrolytic characteristics from a natural source, and micro-aeradon, anaerobiosis and thermal cycles, according to the first example of the present invention. Figure 3, shows the step of adapting a microbial consortium to a model substrate with hydrogenic-hydrolytic characteristics, using a sequential batch process, a mineral medium with a certain concentration of dissolved oxygen, in accordance with the second example of the present invention.
La figura 4, muestra el resultado de la etapa de producción de hidrógeno a partir de glucosa, lactosa, vinazas, lactosuero, residuos de fruta-verdura, co-digestión de vinazas-nejayote, lactosuero-resíduos de fruta-verdura, de acuerdo con el quinto ejemplo de la presente invención. Figure 4 shows the result of the hydrogen production stage from glucose, lactose, vinasse, whey, fruit-vegetable waste, co-digestion of vinesse-nejayote, whey-fruit-vegetable residue, according to the fifth example of the present invention.
La figura 5, muestra la generación de (adato y acetato a partir del hidroiizado de una mezcla de vinaza y nejayote, de acuerdo con el sexto ejemplo de la presente invención.  Figure 5 shows the generation of (adate and acetate from the hydrogenate of a mixture of vinasse and nejayote, according to the sixth example of the present invention.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La invención se refiere a un método para la obtención de un consorcio microbiano, que induye bacterias anaerobias estrictas y facultativas viables o estables con capacidad de producir hidrógeno o bien hidrolizados a partir de sustratos complejos como residuos agroindustriales, aguas residuales industriales, desechos sólidos orgánicos urbanos y materiales lignocelulósicos. La invención incluye además el consorcio microbiano adaptado, el cual es utilizado para producir hidrógeno o hidrolizados como fuente de energía renovable mediante la digestión o co-digestión anaerobia de sustratos complejos p re-tratados y no-pretratados. MÉTODO PARA LA OBTENCIÓN DE UN CONSORCIO MICROBIANO O INOCULO ESTABLE Y ROBUSTO Y SU REPRODUCCIÓN  The invention relates to a method for obtaining a microbial consortium, which induces strict and optional anaerobic and viable bacteria capable of producing hydrogen or hydrolysed from complex substrates such as agro-industrial waste, industrial wastewater, urban organic solid waste and lignocellulosic materials. The invention further includes the adapted microbial consortium, which is used to produce hydrogen or hydrolysates as a renewable energy source by anaerobic digestion or co-digestion of complex p-treated and non-pretreated substrates. METHOD FOR OBTAINING A STABLE AND ROBUST MICROBIAL CONSORTIUM OR INOCULUS AND ITS REPRODUCTION
El método para la obtención de un consorcio microbiano para la producción de hidrógeno e hidrolizados a partir de sustratos complejos comprende las siguientes etapas:  The method for obtaining a microbial consortium for the production of hydrogen and hydrolysates from complex substrates comprises the following steps:
I. ETAPA DE SELECCIÓN: I. SELECTION STAGE:
a. Tomar una muestra procedente de fuentes naturales como: pantanos, rumen, suelos, composta, o fuentes artificíales como: biodigestores, biorreactores para producción de biogás, plantas de tratamiento, que tratan aguas residuales (domésticas, municipales o industriales) o desechos de origen orgánico, que contiene bacterias productoras de hidrógeno con capacidades hidrolfticas, y depositarla en un contenedor de vidrio, adaptado con un puerto de inyección para aire, gas inerte y sonda de temperatura. b. Ajustar el valor de pH de 5.9 a 6.0 unidades.  to. Take a sample from natural sources such as: swamps, rumen, soil, compost, or artificial sources such as: biodigesters, bioreactors for biogas production, treatment plants, which treat wastewater (domestic, municipal or industrial) or organic waste , which contains hydrogen-producing bacteria with hydrolytic capacities, and deposit it in a glass container, adapted with an injection port for air, inert gas and temperature probe. b. Adjust the pH value from 5.9 to 6.0 units.
c. Realizar una micro-aeración hasta alcanzar 3 - 5 % de oxígeno disuelto y dejar de 10 a 15 min bajo esa condición, después se inyecta un gas inerte como nitrógeno, argón, etc. de entre 80 a 100 ml/min y se realiza un tratamiento térmico de 40 - 50°C y se mantiene de 15 a 20 min, después se enfría en baño frío a una temperatura de 20 - 25°C. C. Perform a micro-aeration until reaching 3-5% of dissolved oxygen and leave for 10 to 15 min under that condition, then an inert gas such as nitrogen, argon, etc. is injected. between 80 to 100 ml / min and a heat treatment of 40 - 50 ° C is carried out and kept for 15 to 20 min, then cooled in a cold bath at a temperature of 20-25 ° C.
d. Realizar segunda micro-aeradón de 3 - 5% de oxígeno, se inyecta gas inerte en las mismas condiciones y nuevamente se vuelve a calentar a una temperatura de 60 - 70°C por un tiempo de 20 min.  d. Perform second micro-aeradon of 3-5% oxygen, inert gas is injected under the same conditions and again heated to a temperature of 60-70 ° C for a time of 20 min.
e. EEnfriar rápidamente con agua a temperatura ambiente en baño frío hasta obtener una temperatura de 18 - 25°C.  and. Cool rapidly with water at room temperature in a cold bath until a temperature of 18-25 ° C is obtained.
Π. ETAPA DE ADAPTACIÓN:  Π ADAPTATION STAGE:
a. Preparar el medio de adaptación, utilizando como base un medio mineral que contiene sales de KH2PO4, K2HPO4, MgSO», CaCfe, FeSO-» 7H2O y NH4CI, de acuerdo con el cuadro 3, disueltas en agua destilada, manteniendo el medio mineral en agitación constante de 100 a 300 rpm para alcanzar una concentración de oxígeno disuelto de 30 a 60%. Preferiblemente las sales se disuelven de 48 a 120 h antes de ser utilizadas.  to. Prepare the adaptation medium, using as a base a mineral medium containing salts of KH2PO4, K2HPO4, MgSO », CaCfe, FeSO-» 7H2O and NH4CI, according to Table 3, dissolved in distilled water, keeping the mineral medium under stirring constant from 100 to 300 rpm to reach a dissolved oxygen concentration of 30 to 60%. Preferably the salts dissolve 48 to 120 h before being used.
b. Adicionar una fuente de carbono (sustrato), que puede ser glucosa, lactosa, maltosa o almidón, de acuerdo con el cuadro 3.  b. Add a source of carbon (substrate), which can be glucose, lactose, maltose or starch, according to Table 3.
c. Ajustar el pH del medio de adaptación a un valor de 6.0 a 6.5 unidades.  C. Adjust the pH of the adaptation medium to a value of 6.0 to 6.5 units.
d. Llevar a cabo la fermentación, en un sistema lote secuendal, adicionando de 10 a 20% v/v del inóculo seleccionado en la etapa I, respecto al volumen final del medio mineral, incrementando gradualmente las concentraciones de sustrato desde 5 g/L a 10, 15, 20, y 30 g/L, manteniendo cada concentración del sustrato por 4 a 7 ciclos de fermentación, cada uno de ellos con una duración de 48 a 72 h, reemplazando al final de cada ddo del 50 al 90% del medio agotado por un volumen igual de medio fresco. Durante la fermentación, el pH debe ajustarse a un valor de 5.0 a 6.0 unidades. Y la concentración de oxígeno disuelto inicial deberá ser de 30 a 60%.  d. Carry out the fermentation, in a sequential batch system, adding 10 to 20% v / v of the inoculum selected in stage I, with respect to the final volume of the mineral medium, gradually increasing the substrate concentrations from 5 g / L to 10 , 15, 20, and 30 g / L, maintaining each concentration of the substrate for 4 to 7 fermentation cycles, each with a duration of 48 to 72 h, replacing at the end of each ddo 50 to 90% of the medium sold out by an equal volume of fresh medium. During fermentation, the pH should be adjusted to a value of 5.0 to 6.0 units. And the initial dissolved oxygen concentration should be 30 to 60%.
e. Monltorear la actividad hidrogénica del inóculo, por medio de cromatografía de gases, realizando inyecciones de biogás continuamente. El sistema es estable cuando la concentración de hidrógeno en el biogás está entre 50 y 70%, sin presentía de metano. Por otro lado, el análisis de muestras líquidas consiste en determinar el consumo de carbohidratos por el Método Fenol-Sulfúrico u otro método para cuantiftcar carbohidratos, santificación indirecta de biomasa por cuantificación de proteína por el Método de Bradford u otro método para cuantíficar proteína, el consumo de carbohidratos totales debe ser entre 50 y 75%; la cantidad de proteína de 150 a 300 pg/mL en la fase exponendal y estadonaria.  and. Monitor the hydrogenic activity of the inoculum, by means of gas chromatography, making biogas injections continuously. The system is stable when the concentration of hydrogen in the biogas is between 50 and 70%, without the presence of methane. On the other hand, the analysis of liquid samples consists in determining the consumption of carbohydrates by the Phenol-Sulfuric Method or another method to quantify carbohydrates, indirect sanctification of biomass by protein quantification by the Bradford Method or another method to quantify protein, the Total carbohydrate intake should be between 50 and 75%; the amount of protein from 150 to 300 pg / mL in the exponential and stationary phase.
Las fermentaciones deben ser realizadas en un proceso lote secuendal por alrededor de 2 meses, dejando siempre un porcentaje no mayor al 50% y mínimamente un 10% det medio fermentado como un pie de cuba para la siguiente carga de medio fresco y fermentación. Las condiciones de temperatura de 35 a 38°C, pH de 5.0 - 6.0, y velocidad de agitación de 100 a 150 rpm deben ser controladas. The fermentations must be carried out in a sequential batch process for about 2 months, always leaving a percentage not exceeding 50% and At least 10% of fermented medium as a foot of Cuba for the next load of fresh medium and fermentation. Temperature conditions of 35 to 38 ° C, pH 5.0 - 6.0, and stirring speed of 100 to 150 rpm should be controlled.
ΠΙ. ETAPA DE CONSERVACIÓN DEL CONSORCIO MICROBIANO  ΠΙ CONSERVATION STAGE OF THE MICROBIAN CONSORTIUM
a. Realizar una fermentación utilizando un medio mineral con una cierta concentración de oxígeno disuelto inicial de 30 a 60%, agitación de 100 a 150 rpm, temperatura de 35 a 38°C y concentración de sustrato modelo de 20 a 30 g/L por un tiempo de 24 a 48 horas.  to. Perform a fermentation using a mineral medium with a certain initial dissolved oxygen concentration of 30 to 60%, stirring of 100 to 150 rpm, temperature of 35 to 38 ° C and model substrate concentration of 20 to 30 g / L for a while from 24 to 48 hours.
b. Recuperar la biomasa del caldo fermentado mediante centrifugación, y re-suspender dicha biomasa en un medio de conservación (Cuadro 3 y 4) que contiene: fosfato monobásico de potasio, fosfato dibásico de potasio, sulfato de magnesio, cloruro de calcio, sulfato de hierro y doruro de amonio; una fuente de carbono seleccionada de: lactosa, glucosa, y maltosa, en una concentración de 10 mg/L; y áddos grasos selecdonados de: lactato, acetato y butirato. El medio de conservadón tiene una concentración de oxígeno disuelto de 12 a 20%, y un valor de pH de 4.8 a 6.0 unidades, dependiendo la fuente de carbono utilizada.  b. Recover the biomass from the fermented broth by centrifugation, and re-suspend said biomass in a conservation medium (Table 3 and 4) that contains: potassium monobasic phosphate, potassium dibasic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium doruro; a carbon source selected from: lactose, glucose, and maltose, in a concentration of 10 mg / L; and fatty acids selected from: lactate, acetate and butyrate. The preservative medium has a dissolved oxygen concentration of 12 to 20%, and a pH value of 4.8 to 6.0 units, depending on the source of carbon used.
c. Mantener el consordo microbiano (inoculo hidrolíbco-hidrogénico) en el medio de conservadón, en refrigeradón a una temperatura de 4 a 8°C, en condidones anaerobias o aerobias.  C. Keep the microbial consortium (hydrolytic-hydrogenic inoculum) in the preservative medium, refrigerated at a temperature of 4 to 8 ° C, in anaerobic or aerobic conditions.
d. Realizar el re-cultivo del inoculo preferentemente cada período de 6 meses a un 1 año, llevando a cabo posteriormente los pasos (a)-(c) de la etapa de conservación para mantener en conservación el inoculo.  d. Perform the re-culture of the inoculum preferably every period of 6 months to 1 year, subsequently carrying out steps (a) - (c) of the conservation stage to keep the inoculum in conservation.
Figure imgf000013_0001
Figure imgf000013_0001
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Figure imgf000014_0001
IV.ETAPA DE REACTIVACIÓN DEL CONSORCIO MICROBIANO  IV. MICROBIAN CONSORTIUM REACTIVATION COVER
a. Formular un medio mineral que contiene: fosfato monobásico de potasio, fosfato di básico de potasio, sulfato de magnesio, cloruro de calcio, sulfato de hierro y doruro de amonio, con un pH de 6.0 a 6.5, una concentración de oxigeno disuelto de 30-60%, y una fuente de carbono seleccionada de glucosa, maltosa, lactosa, y almidón, en una concentración de 5 a 20 g/L.  to. Formulate a mineral medium that contains: potassium monobasic phosphate, potassium basic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium doride, with a pH of 6.0 to 6.5, a dissolved oxygen concentration of 30- 60%, and a selected carbon source of glucose, maltose, lactose, and starch, in a concentration of 5 to 20 g / L.
Preferentemente las sales deben ser disueltas en agua desionizada por lo menos 2 d, o más preferentemente por 5 d en agitación constante, y alcanzar una concentración de oxígeno disuelto de entre 30-60% en el medio mineral.  Preferably the salts should be dissolved in deionized water for at least 2 d, or more preferably for 5 d under constant stirring, and reach a dissolved oxygen concentration of between 30-60% in the mineral medium.
b. Inocular de 10 a 15% v/v del inoculo conservado, en el medio fresco del paso (a). c. Realizar un ciclo de fermentación, en condiciones controladas de pH de 5.5 a 6.4 unidades, temperatura de 35 a 38°C, en agitación constante de 100 a 150 rpm. La fermentación de reactivación puede durar hasta 48 h de cultivo, pero es deseable mantenerla solamente por un período de 24 h para proseguir con la etapa de producción de hidrógeno.  b. Inoculate 10 to 15% v / v of the preserved inoculum, in the fresh medium of step (a). C. Perform a fermentation cycle, under controlled pH conditions of 5.5 to 6.4 units, temperature of 35 to 38 ° C, with constant stirring of 100 to 150 rpm. Reactivation fermentation can last up to 48 h of culture, but it is desirable to keep it only for a period of 24 h to continue with the hydrogen production stage.
d. Verificar la concentración de proteína, que debe alcanzar un valor de 150 a 300 pg/mL. Las manipulaciones pueden realizarse bajo condiciones no estériles, pero es preferible limpiar o lavar equipo, utensilios y áreas de trabajo.  d. Verify the protein concentration, which should reach a value of 150 to 300 pg / mL. Manipulations can be performed under non-sterile conditions, but it is preferable to clean or wash equipment, utensils and work areas.
V. ETAPA DE PRODUCCIÓN DE HIDRÓGENO E HIDROUZADOS  V. STAGE OF HYDROGEN AND HYDROUZED PRODUCTION
a. Inocular una cantidad o volumen de medio fresco respecto al volumen del consorcio microbiano adaptado o reactivado (de 10 a 15%); el medio fresco puede ser un sustrato modelo como: glucosa, lactosa, maltosa o almidón, o un sustrato complejo como: residuos industriales incluyendo: vinazas, lactosuero, nejayote, melazas, residuos de fruta, de verdura y de comida; aguas residuales incluyendo: lodos aerobios o anaerobios, aguas residuales de la industria del café, de bebidas y del procesamiento de carnes y embutidos; desechos sólidos orgánicos incluyendo: hidrol izados de materiales lignocelulósicos como bagazo de caña, agave y uva, cásea ra de cereales y pasto, etc. Cualquiera de estos residuos en digestión y/o co-digestión, y sin dilución de los mismos. Sin la necesidad de crear condiciones artificiales anaerobias por la inyección de un gas de arrastre. to. Inoculate an amount or volume of fresh medium with respect to the volume of the adapted or reactivated microbial consortium (from 10 to 15%); the fresh medium can be a Model substrate such as: glucose, lactose, maltose or starch, or a complex substrate such as: industrial waste including: vinasse, whey, nejayote, molasses, fruit, vegetable and food waste; wastewater including: aerobic or anaerobic sludge, wastewater from the coffee, beverage and meat and sausage processing industry; organic solid wastes including: hydrolysed lignocellulosic materials such as cane bagasse, agave and grapes, grains and grains, etc. Any of these residues in digestion and / or co-digestion, and without dilution thereof. Without the need to create anaerobic artificial conditions by injecting a entrained gas.
b. Cultivar el inoculo activado en un biorreactor de tanque agitado bajo condiciones anaerobias no inducidas artificialmente, es decir generación de anaerobiosis natural por los microorganismos  b. Cultivate the activated inoculum in a stirred tank bioreactor under anaerobic conditions not artificially induced, ie generation of natural anaerobiosis by microorganisms
c. La fermentación se realiza bajo pH de 5.5 - 6.0, a una temperatura de 35 - 38°C y velocidad de agitación constante de 100 a 150 rpm.  C. The fermentation is carried out under pH 5.5 - 6.0, at a temperature of 35-38 ° C and constant stirring speed of 100 to 150 rpm.
VLETAPA DE PRODUCCIÓN MASIVA DEL CONSORCIO MICROBIANO HIDROGÉNICO- HIDROLfrlCO  VLETAPA OF MASS PRODUCTION OF THE HYDROGENIC MICROBIAL CONSORTIUM
a. Inocular ei medio fresco formulado con fosfato monobásico de potasio, fosfato dibásico de potasio, sulfato de magnesio, cloruro de calcio, sulfato de hierro y cloruro de amonio pH de 6.0 a 6.5, que contiene como fuente de carbono ya sea glucosa, lactosa, maltosa o almidón (de 20 a 30 g/L), con ei consorcio microbiano adaptado de acuerdo con la etapa II, o reactivado de acuerdo con la etapa IV, en una proporción de 10 a 15% v/v respecto al volumen del medio fresco a inocular. El medio contiene además una concentración de oxígeno disuelto de 30-60%  to. Inoculate the fresh medium formulated with potassium monobasic phosphate, potassium dibasic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium chloride pH 6.0 to 6.5, which contains as carbon source either glucose, lactose, maltose or starch (from 20 to 30 g / L), with the microbial consortium adapted according to stage II, or reactivated according to stage IV, in a proportion of 10 to 15% v / v with respect to the volume of fresh medium to inoculate. The medium also contains a dissolved oxygen concentration of 30-60%
b. Realizar la fermentación con el medio fresco por un periodo de 24 a 36 h, preferentemente 24 h, bajo condiciones de pH de no menos a 5.5 y no mayores a 6.0 unidades, temperatura de 35 a 38°C, y en agitación constante de 100 a 150 rpm. c. Llevar a cabo el escalonamiento de ios cultivos, considerando siempre un volumen del 10 al 15% v/v de inóculo respecto al volumen de trabajo del biorreactor y tiempos de fermentación de 24 a 36 h en cada cultivo escalonado, hasta alcanzar el volumen de producción de inoculo deseado.  b. Perform the fermentation with the fresh medium for a period of 24 to 36 h, preferably 24 h, under pH conditions of not less than 5.5 and not greater than 6.0 units, temperature of 35 to 38 ° C, and with constant stirring of 100 at 150 rpm C. Carry out the staggering of the crops, always considering a volume of 10 to 15% v / v of inoculum with respect to the work volume of the bioreactor and fermentation times of 24 to 36 h in each staggered crop, until reaching the production volume of desired inoculum.
CONSORCIO MICROBIANO O INÓCULO CON ACTIVIDAD HIDROLÍTICA E HIDROGÉNICA  MICROBIAN OR INOCULATE CONSORTIUM WITH HYDROLYTIC AND HYDROGENIC ACTIVITY
El inóculo o consorcio microbiano con actividad hidroittica, actividad hidrogénica obtenido y adaptado de acuerdo con los pasos y etapas definidos en ei método anteriormente descrito, está conformado por microorganismos facultativos y anaerobios estrictos, ácido lácticos, ácido acéticos e hidrógeno productores. El consorcio microbiano o inóculo incluye: a. Microorganismos hidrógeno productores como: Actinomyces sp, Addamínococeus fermentans, Addamínococeus sp, Qtrobacter sp, Gtrobacter freundii, Qostridium beijerínckü, Qostridium bütyricum, Qostridium luticellaril, Qostridium puniceum, Qostridium roseum, Qostridium tyrobutyricum, Qostridium sp, Escherichia coli, Enterobacter sp, Enterobacter doacae, Ethanoligenens sp, Klebsidla sp, Megasphaera elsdenll, Megasphaera sp, Megasphaera micronudformi, Prevotella buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonla sp, Rhodopseudomonas paiustrís, Se/enomonas ruminantíum, Selenomonas sp The inoculum or microbial consortium with hydrocytic activity, hydrogenic activity obtained and adapted according to the steps and stages defined in the method described above, is made up of strict facultative and anaerobic microorganisms, lactic acid, acetic acid and hydrogen producers. The microbial consortium or inoculum includes: to. hydrogen producing microorganisms such as Actinomyces sp fermentans Addamínococeus, Addamínococeus sp Qtrobacter sp Gtrobacter freundii, Qostridium beijerínckü, Qostridium butyricum, luticellaril Qostridium, Qostridium puniceum, Qostridium roseum, Qostridium tyrobutyricum, Qostridium sp Escherichia coli, Enterobacter species, Enterobacter doacae, Ethanoligenens sp, Klebsidla sp, Megasphaera elsdenll, Megasphaera sp, Megasphaera micronudformi, Prevotella buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonla sp, Rhodopseudomonas paiustrís, Se / enomonas ruminantíum, Selenomom spumonasum
b. Microorganismos ácido lácticos como: Bifídobacterium psychraerophilum, Lactobadllus casei, Lactobadllus coryniformis, Lactobadllus ddbrueckii, Lactobadllus harbinensis,b. Lactic acid microorganisms such as: Bifídobacterium psychraerophilum, Lactobadllus casei, Lactobadllus coryniformis, Lactobadllus ddbrueckii, Lactobadllus harbinensis,
Lactobadllus hilgardli, Lactobadllus parabuchneri, Lactobadllus perolens, Lactobadllus rapi, Lactobadllus rhamnosus, Lactobadllus sunkií, Lactobadllus vaednostercus, Sporolactobadllus terrae, Sporolactobadllus sp, y Lactobadllus hilgardli, Lactobadllus parabuchneri, Lactobadllus perolens, Lactobadllus rapi, Lactobadllus rhamnosus, Lactobadllus sunkií, Lactobadllus vaednostercus, Sporolactobadllus terrae, Sporolactobadllus sp, and
c. Microorganismos áddo acéticos como: Acetobacter tibinongensis, Acetobacter lovaniensfs, Acetobacter orientalls, Acetobacter sp, Acetobacter syzygii. C. Acetic microorganisms such as: Acetobacter tibinongensis, Acetobacter lovaniensfs, Acetobacter orientalls, Acetobacter sp, Acetobacter syzygii.
El inoculo hídrogénico-hidrolftlco puede ser utilizado con diferentes configuraciones o diseños de los procesos anaerobios, proceso de una etapa, dos etapas o múltiples etapas. Al respecto, el proceso de una etapa representa a un proceso donde todos los procesos metabólicos de los microorganismos se realizan dentro un reactor. Un proceso de dos o múltiples etapas involucra el uso de más de un reactor, donde se deben proveer condiciones óptimas para los procesos metabólicos de los microorganismos. Por ejemplo, en un proceso de una etapa, la hidrólisis, acetogénesis o hídrogénesis puede ocurrir en un solo reactor. Mientras, en una segunda etapa o múltiple, la materia orgánica hidrolizada de la primera etapa puede ser alimentada a una segunda etapa.  The hydrogen-hydrolytic inoculum can be used with different configurations or designs of anaerobic processes, one-stage process, two stages or multiple stages. In this regard, the one-stage process represents a process where all the metabolic processes of the microorganisms are carried out inside a reactor. A two or multiple stage process involves the use of more than one reactor, where optimal conditions for the metabolic processes of microorganisms must be provided. For example, in a one-stage process, hydrolysis, acetogenesis or hydrogenesis can occur in a single reactor. Meanwhile, in a second or multiple stage, the hydrolyzed organic matter of the first stage can be fed to a second stage.
La presente invención es aplicable para producir hidrógeno o hidroiizado de la materia orgánica en una sola etapa. El hidroiizado puede ser alimentado a una segunda, la cual puede ser metanogénesis para producir metano; foto-fermentadón para produdr hidrógeno; o el cultivo heterotrófico de algas para produdr biomasa o meta bolitas de interés (lípídos, carbohidratos, cuerpos cetónicos, etc.). Los procesos pueden ser en lote o en continuo, en una, dos o múltiples etapas. The present invention is applicable to produce hydrogen or hydrogenated organic matter in a single stage. The hydrogenate can be fed to a second, which can be methanogenesis to produce methane; photo-fermentation to produce hydrogen; or the heterotrophic cultivation of algae to produce biomass or meta pellets of interest (lipids, carbohydrates, ketone bodies, etc.). The processes can be in batch or continuous, in one, two or multiple stages.
De aquí en adelante, la presente invendón se describirá por medio de algunos ejemplos, pero estos no están destinados a limitar la invención de ninguna manera.  Hereinafter, the present invention will be described by means of some examples, but these are not intended to limit the invention in any way.
EJEMPLOS  EXAMPLES
Ejemlpo 1. Etapa de Selecion del inoculo La fuente de inoculo puede provenir de fuentes naturales o artificiales, en este ejemplo fue un digestor anaerobio de una planta de tratamiento de aguas residuales domésticas. Se coloca una cantidad de 500 mL de inoculo en una botella de vidrio de 1 L adaptada con puerto de inyección para aire y gas inerte y sonda de temperatura. Se ajusta pM de 5.9 a 6.0 unidades. Se inyecta aire hasta alcanzar 3 a 5% de oxígeno disuelto y se deja de 10 a 15 mín bajo esa condición. Posteriormente se inyecta un gas inerte (nitrógeno, argón etc.) de entre 80 a 100 mL/min por 20 min y se calienta en una estufa precalentada a 100°C, alcanzada la temperatura de 50°C se mantiene de 15 a 20 min, después se enfría en baño frío a una temperatura de 20 a 25°C. Nuevamente se inyecta aire hasta alcanzar de 3 a 5% de oxígeno disuelto y se deja de 10 a 15 min; terminada esta operación se inyecta el gas inerte bajo las condiciones antes mencionadas y se expone nuevamente a tratamiento térmico a una temperatura de 60 a 65°C por 20 min. Terminado el tratamiento térmico se hace disminuir la temperatura rápidamente de 20 a 25°C. Terminada la etapa de selección se continúa con ia etapa de adaptación. Example 1. Inoculation Selection Stage The source of inoculum can come from natural or artificial sources, in this example it was an anaerobic digester from a domestic wastewater treatment plant. An amount of 500 mL of inoculum is placed in a 1 L glass bottle adapted with an injection port for air and inert gas and temperature probe. PM is adjusted from 5.9 to 6.0 units. Air is injected until 3 to 5% of dissolved oxygen is reached and left 10 to 15 min under that condition. Subsequently, an inert gas (nitrogen, argon, etc.) of between 80 to 100 mL / min is injected for 20 min and is heated in a preheated oven at 100 ° C, reaching the temperature of 50 ° C it is maintained for 15 to 20 min. , then cooled in a cold bath at a temperature of 20 to 25 ° C. Again, air is injected until 3 to 5% of dissolved oxygen is reached and left for 10 to 15 min; after this operation, the inert gas is injected under the aforementioned conditions and again exposed to heat treatment at a temperature of 60 to 65 ° C for 20 min. Once the heat treatment is finished, the temperature is lowered quickly from 20 to 25 ° C. Once the selection stage is finished, the adaptation stage is continued.
Etempio 2. Etana de Adaptación del inoculo Etempio 2. Etana of Adaptation of the inoculum
El medio mineral mostrado en el Cuadro 3, se utilizó como medio de cultivo para el método de adaptación, las sales de KH2PO4, K2HPO4, MgSCM, Cadjv FeS04 7H20 y NH4CI se disolvieron en agua destilada (de 48 a 120 h) antes de ser utilizadas, éstas se mantuvieron en agitación constante de 100 a 300 rpm para alcanzar una concentración de oxígeno disuelto de 30-60%. Como fuente de carbono se utilizó lactosa a diferentes concentraciones (5, 10, 15, 20 y 30 g/L). A partir del inoculo seleccionado, 200 mL del inoculo se adicionaron a 800 mL de medio mineral con lactosa a 5 g/L, se realizaron 7 ddos de fermentación 48 h, terminado cada dclo se remplazó el 50% del volumen de medio agotado por un volumen igual de medio fresco. Después se incrementó ia concentración de lactosa a 10 g/L, se repitieron 7 ddos de fermentación, remplazando el 50% del medio gastado por medio fresco. Se incrementó la concentración de lactosa a 15 g/L, se realizaron 7 ddos de fermentadón y se remplazó un 50% del medio agotado por fresco. Se incrementó a 20 g/L y finalmente a 30 g/L, con un número de ddos de 4.6 de fermentadón de 72 horas para ambas concentraciones, pero con reemplazamiento 75% y del 90% del medio gastado por medio fresco a 20 y 30 g/L, respectivamente. El pH del medio durante la fermentadón se mantuvo entre 5 y 5.5 unidades. El medio de cultivo se utilizó en condidones no estériles. La actividad hidrogénica del inoculo se monitoreó por cromatografía de gases, realizando inyecciones de blogás continuamente. El sistema es estable cuando la concentradón de hidrógeno en el biogás esté entre 50 y 70%, sin presentía de metano. Por otro lado, el análisis de muestras líquidas consiste en determinar el consumo de carbohidratos por el Método Fenol-Sulfúrico, cuantiflcación indirecta de biomasa por cuantiflcadón de proteína por el Método de Bradford, el consumo de carbohidratos totales debe ser entre 50 y 75%; la cantidad de proteína de 150 a 300 pg/mL en la fase exponencial y estacionaría. The mineral medium shown in Table 3 was used as a culture medium for the adaptation method, the salts of KH2PO4, K2HPO4, MgSCM, Cadjv FeS0 4 7H 2 0 and NH4CI were dissolved in distilled water (48 to 120 h) before being used, they were kept under constant agitation of 100 to 300 rpm to reach a dissolved oxygen concentration of 30-60%. As a carbon source, lactose was used at different concentrations (5, 10, 15, 20 and 30 g / L). From the selected inoculum, 200 mL of the inoculum was added to 800 mL of mineral medium with lactose at 5 g / L, 7 fermentation dips were carried out 48 h, after each dclo 50% of the volume of medium exhausted was replaced by a equal volume of fresh medium. After the lactose concentration was increased to 10 g / L, 7 days of fermentation were repeated, replacing 50% of the spent medium with fresh medium. The lactose concentration was increased to 15 g / L, 7 doubles of fermentadon were made and 50% of the fresh-depleted medium was replaced. It was increased to 20 g / L and finally to 30 g / L, with a number of 4.6 hours of fermentadon of 72 hours for both concentrations, but with 75% replacement and 90% of the medium spent by fresh medium at 20 and 30 g / L, respectively. The pH of the medium during fermentation was maintained between 5 and 5.5 units. The culture medium was used in non-sterile conditions. The hydrogenic activity of the inoculum was monitored by gas chromatography, making injections of blogs continuously. The system is stable when the hydrogen concentration in the biogas is between 50 and 70%, without the presence of methane. On the other hand, the analysis of liquid samples consists in determining the consumption of carbohydrates by the Phenol-Sulfuric Method, indirect quantification of biomass by protein quantification by the Bradford Method, the consumption of total carbohydrates it must be between 50 and 75%; the amount of protein from 150 to 300 pg / mL in the exponential and stationary phase.
Ejemplo 3. Etapa da Consemacián del inoculo  Example 3. Stage of Consecration of inoculum
El inoculo hidrolftico-hidrogénico se conservó en refrigeración entre 4 a 8°C, bajo condiciones anaerobias o aerobias. Previamente se realizó una fermentación durante 36 h utilizando lactosa a 20 g/U pH constante de 5.5 y agitación constante de 100 rpm, después se recuperó la biomasa por centrifugación. Finalmente, la biomasa se re-suspendió en el medio de cultivo especificado en el Cuadro 3 y 4, y se mantuvo en refrigeración.  The hydrolytic-hydrogenic inoculum was kept refrigerated between 4 to 8 ° C, under anaerobic or aerobic conditions. Previously, fermentation was carried out for 36 hours using lactose at a constant pH of 5.5 g and a constant stirring of 100 rpm, then the biomass was recovered by centrifugation. Finally, the biomass was re-suspended in the culture medium specified in Table 3 and 4, and kept refrigerated.
Etemolo 4. Etapa da Reactivación da inoculo Etemolo 4. Reaction stage of inoculum
Para la reactivación del inóculo se realizó una fermentación a condiciones controladas de pH, temperatura y velocidad de agitación, 5.5 a 6.4; 35 a 38°C y 100 a 150 rpm, respectivamente. Previamente las sales del medio mineral (Cuadro 3) fueron disueltas en agua desionizada por lo menos 2 d, preferentemente por 5 d bajo agitación constante de 200 rpm, hasta alcanzar una concentración de oxígeno disuelto de entre 30-60% en el medio mineral. La fermentación de reactivación puede durar hasta 48 h de cultivo, pero es deseable 24 h para proseguir con el proceso de producción de hidrógeno. Alcanzar una concentración de proteína de 150 a 300 μς/mL. Las manipulaciones pueden realizarse bajo condiciones no estériles, pero si preferentemente limpiar o lavar equipo, utensilios y áreas de trabajo. For the reactivation of the inoculum, fermentation was carried out under controlled conditions of pH, temperature and stirring speed, 5.5 to 6.4; 35 at 38 ° C and 100 at 150 rpm, respectively. Previously the salts of the mineral medium (Table 3) were dissolved in deionized water for at least 2 d, preferably for 5 d under constant stirring of 200 rpm, until reaching a dissolved oxygen concentration of between 30-60% in the mineral medium. Reactivation fermentation can last up to 48 h of culture, but it is desirable 24 h to continue with the hydrogen production process. Reach a protein concentration of 150 to 300 μς / mL. Manipulations can be done under non-sterile conditions, but preferably clean or wash equipment, utensils and work areas.
Etemolo 5. Etapa da Producción da hidrogeno a partir de -sustratos simula» y comptejos Etemolo 5. Stage of hydrogen production from -simulates substrates »and complexes
El inóculo activado es cultivado en un bíorreactor de tanque agitado de 4 L bajo condiciones anaerobias no Inducidas artificialmente, es decir generación de anaerobiosis natural por los microorganismos. Para este ejemplo, glucosa, lactosa son utilizados a una concentración de 10, 20 y 30 g/L, lactosuero, vinazas, nejayote, residuos de fruta-verdura son utilizados de forma cruda (no-pretratados) y sin diludón. La producción de biogás es monítoreada mediante un gasómetro digital. Un método directo de cromatografía de gases es utilizado para medir la evolución de actividad y producción de hidrógeno. Muestras de biogás son obtenidas a partir del espado de cabeza del biorreactor e inyectadas al equipo de cromatografía de gases. El pico de hidrógeno es identificado y la composidón de hidrógeno es determinada a partir de una curva de calibración construida con diferentes concentradones conoddas de hidrógeno. The activated inoculum is cultured in a 4 L stirred tank bioreactor under anaerobic conditions not artificially induced, that is, generation of natural anaerobiosis by microorganisms. For this example, glucose, lactose are used at a concentration of 10, 20 and 30 g / L, whey, vinasses, nejayote, fruit-vegetable residues are used raw (not pretreated) and without dilution. Biogas production is monitored by a digital gasometer. A direct method of gas chromatography is used to measure the evolution of hydrogen activity and production. Biogas samples are obtained from the bioreactor head sword and injected into the gas chromatography equipment. The hydrogen peak is identified and the hydrogen composition is determined from a calibration curve constructed with different hydrogen conodon concentrates.
Ejemplo 6. Etapa de Produccio de hidrolozados Example 6. Production stage of hydrolozates
Para ejemplificar el uso del inóculo para producir hidrolizados con alta concentradón de lactato y acetato, una mezda de vinaza y nejayote es utilizada en una proporción 80/20 (% p/p), respectivamente. Para un volumen de 4500 mL vinazas y nejayote se inocula con 500 mL de inóculo activado por 24 h, descrito previamente. El cultivo se realiza en un biorreactor de tanque agitado de 5 L, a 37°C, pH de 5.0 y velocidad de agitación de 150 rpm por 48 horas. No se inyecta gas de arrastre al sistema. La concentración del oxígeno disuelto inicial en la mezcla es de 10 a 20%. La concentración de lactato y acetato es determinada por medio de un cromatógrafo de líquidos de alta resolución. La concentración de acetato y lactato después de 48 h de cultivo es de 2,445.5 mg/L y 8,073.6 mg/L, respectivamente. El contenido de lactato en el hidrolizado, provee un efluente de alta calidad para un proceso metanogénlco, su presencia tiene implicaciones desde el punto de vista energético y generación de capacidad de amortiguamiento. Por ejemplo, el cambio de energía libre de Gíbbs es de -68.8 U/mol cuando se produce metano a partir de lactato, este valor es dos veces más respecto al de acetato (-31.0 kJ/mol); además durante la degradación de 1 mol de lactato produce 1 mol acetato, 1 mol de bicarbonato y 2 moles de hidrógeno (Pipyn y Verstraete [Biotechnology and Bioengineering 23(1981) 1145-1154]; Wu etai, [Bioresource Technology 211 (2016) 16-23]). Por otro lado, en la literatura es reportado que la producción de hidrógeno por vía foto- fermentativa con microorganismos del género Rhodobacter o Rhodospeudomonas es preferible con lactato o acetato como sustratos (Ózgür et al., [International Journal of Hydrogen Energy 35 (2010) 511-517]). Por lo tanto, el hidrolizado presenta un alto contenido en lactato y acetato, el cual puede ser utilizado potencialmente para producir metano o hidrógeno como fuente de energía renovable en una segunda etapa metanogéníca o foto- fermentativa, respectivamente. To exemplify the use of the inoculum to produce hydrolysates with a high concentration of lactate and acetate, a mixture of vinegar and nejayote is used in a proportion 80/20 (% w / w), respectively. For a volume of 4500 mL, vinegars and nejayote are inoculated with 500 mL of activated inoculum for 24 h, previously described. The culture is carried out in a bioreactor of 5 L stirred tank at 37 ° C, pH 5.0 and stirring speed of 150 rpm for 48 hours. No entrainment gas is injected into the system. The initial dissolved oxygen concentration in the mixture is 10 to 20%. The lactate and acetate concentration is determined by means of a high performance liquid chromatograph. The concentration of acetate and lactate after 48 h of culture is 2,445.5 mg / L and 8,073.6 mg / L, respectively. The lactate content in the hydrolyzate provides a high quality effluent for a methanogenic process, its presence has implications from the energy point of view and generation of buffering capacity. For example, the change of free energy of Gíbbs is -68.8 U / mol when methane is produced from lactate, this value is twice as much as that of acetate (-31.0 kJ / mol); also during the degradation of 1 mol of lactate produces 1 mol acetate, 1 mol of bicarbonate and 2 moles of hydrogen (Pipyn and Verstraete [Biotechnology and Bioengineering 23 (1981) 1145-1154]); Wu etai, [Bioresource Technology 211 (2016) 16-23]). On the other hand, it is reported in the literature that the production of hydrogen by photo-fermentation with microorganisms of the genus Rhodobacter or Rhodospeudomonas is preferable with lactate or acetate as substrates (Ózgür et al., [International Journal of Hydrogen Energy 35 (2010) 511-517]). Therefore, the hydrolyzate has a high content of lactate and acetate, which can potentially be used to produce methane or hydrogen as a renewable energy source in a second methanogenic or photo-fermentative stage, respectively.
Producción secuendal masiva de inoculo Mass sequential production of inoculum
Ejemplo 7: Proceso para producir 5 litros de inoculo con lactosa  Example 7: Process to produce 5 liters of inoculum with lactose
Se reactiva el inoculo por 24 h, inoculando con 50 mL de inoculo refrigerado un volumen de 450 mL con medio mineral con sales KH2PO4, K2HPO4, MgSCv», CaCfe, FeSCU 7H20 y NhUCI; y lactosa a 20 g/L a una concentración de oxígeno disuelto inicial de 30-60%, esto se realiza a una temperatura de 35°C, pH de 5.5 y velocidad de agitación de 100 rpm por 24 horas. Después, se inocula con 500 mL del inoculo activo, 4,500 mL de medio mineral fresco con lactosa y oxígeno disuelto en un biorreactor de tanque agitado de 5 L de capacidad nominal, el proceso de fermentación se realiza bajo las condiciones de operación antes especificadas. La concentración de hidrógeno y bióxido de carbono en el biogás es monitoreada continuamente por cromatografía de gases. La concentración de masa celular es determinada indirectamente por cuantificación de proteína por el Método de Bradford. Cabe señalar que el proceso puede incrementarse sucesivamente hasta el volumen deseado de inoculo a producir, respetando el 10%v/v del inoculo respecto al volumen de trabajo subsecuente y manteniendo las condiciones antes especificadas. The inoculum is reactivated for 24 h, inoculating with a 50 mL of refrigerated inoculum a volume of 450 mL with mineral medium with salts KH2PO4, K2HPO4, MgSCv », CaCfe, FeSCU 7H 2 0 and NhUCI; and lactose at 20 g / L at an initial dissolved oxygen concentration of 30-60%, this is done at a temperature of 35 ° C, pH 5.5 and stirring speed of 100 rpm for 24 hours. Then, it is inoculated with 500 mL of the active inoculum, 4,500 mL of fresh mineral medium with lactose and dissolved oxygen in a stirred tank bioreactor of 5 L of nominal capacity, the fermentation process is carried out under the operating conditions specified above. The concentration of hydrogen and carbon dioxide in the biogas is continuously monitored by gas chromatography. The cell mass concentration is determined indirectly by protein quantification by the Bradford Method. It should be noted that the process can be successively increased to the desired volume of inoculum to be produced, respecting 10% v / v of the inoculum with respect to the subsequent workload and maintaining the conditions specified above.

Claims

REIVINDICACIONES
1. Un método para la obtención de un consorcio microbiano estable y robusto para la producción de hidrógeno y/o hidroiizados como fuente de energía renovable a partir de sustratos compiejos pre-tratados y no-pretratados en un proceso de digestión o co- digestión anaerobia caracterizado porque comprende las etapas y pasos siguientes:  1. A method for obtaining a stable and robust microbial consortium for the production of hydrogen and / or hydrogenated as a renewable energy source from pre-treated and non-pre-treated complex substrates in an anaerobic digestion or co-digestion process characterized in that it comprises the following steps and steps:
I. Etapa de selección:  I. Selection stage:
a. tomar una muestra procedente de fuentes naturales y/o de fuentes artificiales, en el cual está contenido un consorcio microbiano que incluye bacterias productoras de hidrógeno y bacterias con capacidades hidrolíticas tanto facultativas como estrictas anaerobias;  to. take a sample from natural sources and / or artificial sources, which contains a microbial consortium that includes hydrogen producing bacteria and bacteria with both facultative and strict anaerobic hydrolytic capabilities;
b. ajustar la muestra a un valor de pH de 5.9 a 6.0 unidades;  b. adjust the sample to a pH value of 5.9 to 6.0 units;
c. realizar una micro-aeradón hasta alcanzar 3 - 5 % de oxígeno disuelto durante un período de 10 a 15 min, inyectando posteriormente un gas inerte de entre 80 a 100 ml/min, después incrementar la temperatura de 40 - 50°C por un período de 15 a 20 min, después enfriar en baño frío a una temperatura de 20 - 25°C;  C. Perform a micro-aeradon until reaching 3-5% dissolved oxygen for a period of 10 to 15 min, subsequently injecting an inert gas between 80 to 100 ml / min, then increase the temperature from 40 - 50 ° C for a period from 15 to 20 min, then cool in a cold bath at a temperature of 20-25 ° C;
d. realizar una segunda micro-aeración de 3 - 5% de oxígeno, se inyecta gas inerte en las mismas condiciones del paso (c), y se vuelve a calentar a una temperatura de 60 - 70°C por un tiempo de 20 min;  d. perform a second micro-aeration of 3-5% oxygen, inert gas is injected under the same conditions as in step (c), and reheated to a temperature of 60-70 ° C for a time of 20 min;
e. enfriar rápidamente con agua a temperatura ambiente en baño frío hasta obtener una temperatura de 18 - 25°C;  and. cool rapidly with water at room temperature in a cold bath until a temperature of 18-25 ° C is obtained;
II. Etapa de adaptación:  II. Adaptation stage:
a. preparar un medio de adaptación, basado en un medio mineral, y mantenerlo en agitación constante para alcanzar una concentración 30 a 60% de oxígeno disuelto; b. adicionar un sustrato como fuente de carbono;  to. prepare an adaptation medium, based on a mineral medium, and keep it under constant agitation to reach a concentration of 30 to 60% dissolved oxygen; b. add a substrate as a carbon source;
c. ajustar el pH del medio de adaptación a un valor de 6.0 a 6.5 unidades;  C. adjust the pH of the adaptation medium to a value of 6.0 to 6.5 units;
d. llevar a cabo la fermentación, en un sistema lote secuencial, incorporando de un 10 a 20%v/v del consorcio microbiano seleccionado en la etapa I, respecto al volumen final del medio mineral, incrementando gradualmente la concentración del sustrato cada 4 a 7 ciclos de fermentación, cada ddo con una duradón de 48 a 72 h, y reemplazando al final de cada ddo del 50 al 90% del medio agotado por un volumen igual de medio fresco;  d. carry out the fermentation, in a sequential batch system, incorporating from 10 to 20% v / v of the microbial consortium selected in stage I, with respect to the final volume of the mineral medium, gradually increasing the concentration of the substrate every 4 to 7 cycles of fermentation, each ddo with a duration of 48 to 72 h, and replacing at the end of each ddo 50 to 90% of the spent medium with an equal volume of fresh medium;
e. mon ¡torear la actividad hidrogénica del consorcio microbiano;  and. monitor the hydrogenic activity of the microbial consortium;
m. Etapa de conservación del consorcio microbiano: a. realizar una fermentación en medio mineral con una concentración de oxígeno disuelto inídal de 30 a 60%, agitación de 100 a 150 rpm, temperatura de 35 a 38°C y concentración de sustrato de 20 a 30 g/L por un tiempo de 24 a 48 horas; m. Conservation stage of the microbial consortium: to. perform a fermentation in mineral medium with an initial dissolved oxygen concentration of 30 to 60%, stirring of 100 to 150 rpm, temperature of 35 to 38 ° C and substrate concentration of 20 to 30 g / L for a time of 24 to 48 hours;
b. recuperar la biomasa del caldo fermentado mediante centrifugación, y re-suspender dicha biomasa en un medio de conservación que contiene: fosfato monobásico de potasio, fosfato dibásico de potasio, sulfato de magnesio, cloruro de calcio, sulfato de hierro y cloruro de amonio; una fuente de carbono seleccionada de: lactosa, glucosa, y maltosa; y ácidos grasos seleccionados de: lactato, acetato y butirato; con una concentración de oxígeno disuelto de 12 a 20%, y un valor de pH de 4.8 a 6.0 unidades; c. mantener el consorcio microbiano en el medio de conservación, a una temperatura de 4 a 8°C, en condiciones anaerobias o aerobias.  b. recover the biomass from the fermented broth by centrifugation, and re-suspend said biomass in a preservation medium containing: monobasic potassium phosphate, potassium dibasic phosphate, magnesium sulfate, calcium chloride, iron sulfate and ammonium chloride; a carbon source selected from: lactose, glucose, and maltose; and fatty acids selected from: lactate, acetate and butyrate; with a dissolved oxygen concentration of 12 to 20%, and a pH value of 4.8 to 6.0 units; C. keep the microbial consortium in the preservation medium, at a temperature of 4 to 8 ° C, under anaerobic or aerobic conditions.
d. realizar el re-cultivo del consorcio microbiano cada período de 6 meses a un 1 año, llevando a cabo posteriormente los pasos (a)-(c) de la etapa de conservación;  d. carry out the re-cultivation of the microbial consortium every period of 6 months to 1 year, subsequently carrying out steps (a) - (c) of the conservation stage;
IV. Etapa de reactivación del inoculo:  IV. Reactivation stage of inoculum:
a. formular un medio mineral, con un pH de 6.0 a 6.5, una concentración de oxígeno disuelto de 30-60%, y una fuente de carbono seleccionada de glucosa, maltosa, lactosa, y almidón, en una concentración de 5 a 20 g/L;  to. formulate a mineral medium, with a pH of 6.0 to 6.5, a dissolved oxygen concentration of 30-60%, and a selected carbon source of glucose, maltose, lactose, and starch, in a concentration of 5 to 20 g / L ;
b. inocular de 10 a 15% v/v del consorcio microbiano conservado, en el medio fresco del paso (a};  b. inoculate 10 to 15% v / v of the conserved microbial consortium, in the fresh medium of step (a);
c. realizar un ddo de fermentación de 24 a 48 h, en condidones de pH de 5.5 a 6.4 unidades, temperatura de 35 a 38°C, en agitadón constante de 100 a 150 rpm;  C. carry out a fermentation period of 24 to 48 h, in pH conditions of 5.5 to 6.4 units, temperature of 35 to 38 ° C, in constant agitation of 100 to 150 rpm;
d. verificar la concentración de proteína, que debe alcanzar un valor de 150 a 300 pg/mL d. verify the protein concentration, which should reach a value of 150 to 300 pg / mL
V. Etapa de producdón de hidrógeno e hidrolizados: V. Stage of hydrogen production and hydrolysates:
a. inocular el consorcio microbiano adaptado o reactivado en un volumen de medio fresco, en una proporción de 10 a 15%, en un biorreactor de tanque agitado bajo condidones anaerobias no induddas artificialmente;  to. inoculate the adapted or reactivated microbial consortium in a volume of fresh medium, in a proportion of 10 to 15%, in a stirred tank bioreactor under anaerobic conditions not artificially induced;
b. mantener la ferméntadón bajo condiciones de pH de 5.5 - 6.0, temperatura de 35 - 38°C, y velocidad de agitadón constante de 100 a 150 rpm;  b. keep the ferméntadón under conditions of pH of 5.5 - 6.0, temperature of 35 - 38 ° C, and constant agitation speed of 100 to 150 rpm;
VLEtapa de producdón masiva del consorcio microbiano hidrogénico-hidrolrtico  VL Mass production stage of the hydrobial-hydrolytic microbial consortium
a. inocular un medio mineral, con pH de 6.0 a 6.5, que contiene una fuente de carbono selecdonada de: glucosa, lactosa, maltosa o almidón, en una concentradón de 20 a 30 g/L, y una concentradón de oxígeno disuelto de 30-60%, con el consorcio microbiano adaptado de la etapa II, o reactivado de la etapa IV, en una proporción de 10 a 15% v/v; b. realizar la fermentación por un periodo de 24 a 36 h, bajo condiciones de pH de 5.5 a 6.0 unidades, temperatura de 35 a 38°C, y en agitación constante de 100 a 150 rpm; c. llevar a cabo el escalamiento de los cultivos, considerando siempre un volumen del 10 al 15% v/v de inoculo respecto al volumen de trabajo del biorreactor y tiempos de fermentación de 24 a 36 h en cada cultivo incrementado, hasta alcanzar ei volumen de producción de consorcio microbiano deseado. to. inoculate a mineral medium, with a pH of 6.0 to 6.5, which contains a carbon source selected from: glucose, lactose, maltose or starch, in a concentration of 20 to 30 g / L, and a dissolved oxygen concentration of 30-60 %, with the microbial consortium adapted from stage II, or reactivated from stage IV, in a proportion of 10 to 15% v / v; b. carry out the fermentation for a period of 24 to 36 h, under pH conditions of 5.5 to 6.0 units, temperature of 35 to 38 ° C, and with constant stirring of 100 to 150 rpm; C. carry out the scaling of the crops, always considering a volume of 10 to 15% v / v of inoculum with respect to the work volume of the bioreactor and fermentation times of 24 to 36 h in each increased crop, until reaching the production volume of desired microbial consortium.
2. El método de acuerdo con la reivindicación 1, caracterizado porque en el paso (a) de la etapa I, la muestra procedente de fuentes naturales y/o de fuentes artificiales se coloca en un contenedor de vidrio adaptado con un puerto de inyección para aire, gas inerte y sonda de temperatura.  2. The method according to claim 1, characterized in that in step (a) of step I, the sample from natural sources and / or artificial sources is placed in a glass container adapted with an injection port for air, inert gas and temperature probe.
3. El método de acuerdo con la reivindicación 1 o 2, caracterizado porque la fuente de donde proviene la muestra a tratar o adaptar es una fuente natural incluyendo: pantanos, rumen, suelos, composta; o una fuente artificial que induye: biodigestores, biorreactores para producción de biogás, plantas de tratamiento que tratan aguas residuales de tipo domésticas, municipales o industríales.  3. The method according to claim 1 or 2, characterized in that the source from which the sample to be treated or adapted comes from is a natural source including: swamps, rumen, soil, compost; or an artificial source that induces: biodigesters, bioreactors for biogas production, treatment plants that treat domestic, municipal or industrial wastewater.
4. El método de acuerdo con una de las reivindicaciones 1-3, caracterizado porque el gas Inerte que se inyecta tras la micro-aeración del paso (c), etapa I, se selecciona de nitrógeno o argón.  4. The method according to one of claims 1-3, characterized in that the Inert gas that is injected after the micro-aeration of step (c), step I, is selected from nitrogen or argon.
5. El método de acuerdo con una de las reivindicaciones 1-4, caracterizado porque el medio mineral contiene sales de
Figure imgf000022_0001
disueltas en agua destilada. 5. The method according to one of claims 1-4, characterized in that the mineral medium contains salts of
Figure imgf000022_0001
dissolved in distilled water.
6. El método de acuerdo con la reivindicación 5, caracterizado porque las sales del medio mineral se disuelven en agua destilada preferiblemente de 48 a 120 h antes de ser utilizadas.  6. The method according to claim 5, characterized in that the salts of the mineral medium are dissolved in distilled water preferably from 48 to 120 h before being used.
7. El método de acuerdo con una de las reivindicaciones 5-6, caracterizado porque el medio mineral se mantiene en agitación constante de 100 a 300 rpm para alcanzar una concentración de oxigeno disuelto de 30 a 60%. 7. The method according to one of claims 5-6, characterized in that the mineral medium is kept under constant stirring at 100 to 300 rpm to reach a dissolved oxygen concentration of 30 to 60%.
8. El método de acuerdo con la reivindicación 1, caracterizado porque el sustrato adicionado como fuente de carbono en el paso (b) de la etapa Π, se selecciona de glucosa, lactosa, maltosa y almidón.  8. The method according to claim 1, characterized in that the substrate added as a carbon source in step (b) of step Π, is selected from glucose, lactose, maltose and starch.
9. El método de acuerdo con la reivindicación 8, caracterizado porque ei sustrato se adiciona en una concentración inicial de 5 g/L, y se incrementa gradualmente a 10, 15, 20, y 30 g/L cada 4 a 7 ddos de fermentación. 9. The method according to claim 8, characterized in that the substrate is added at an initial concentration of 5 g / L, and is gradually increased to 10, 15, 20, and 30 g / L every 4 to 7 fermentation days .
10. El método de acuerdo con la reivindicación 1, caracterizado porque en el paso (d), etapa II, la fermentación se lleva a cabo bajo condiciones de pH de 5.0 a 6.0 unidades; y con una concentración de oxígeno disuelto de 30 a 60%. 10. The method according to claim 1, characterized in that in step (d), stage II, the fermentation is carried out under pH conditions of 5.0 to 6.0 units; and with a dissolved oxygen concentration of 30 to 60%.
11. El método de acuerdo con la reivindicación 10, caracterizado porque la fermentación se realiza en un proceso lote secuendal por alrededor de 2 meses, manteniendo un porcentaje de 10 a 50% del medio fermentado como un pie de cuba para la siguiente carga de medio fresco y fermentación.  11. The method according to claim 10, characterized in that the fermentation is carried out in a sequential batch process for about 2 months, maintaining a percentage of 10 to 50% of the fermented medium as a bowl foot for the next medium load Fresh and fermentation.
12. El método de acuerdo con una de las reivindicaciones 10-11, caracterizado porque la fermentación se realiza bajo condiciones de temperatura de 35 a 38°C, pH de 5.0 - 6.0, y velocidad de agitación de 100 a 150 rpm.  12. The method according to one of claims 10-11, characterized in that the fermentation is carried out under conditions of temperature of 35 to 38 ° C, pH of 5.0 - 6.0, and stirring speed of 100 to 150 rpm.
13. El método de acuerdo con la reivindicación 1, caracterizado porque el monitoreo del paso (e), etapa II, se realiza por medio de cromatografía de gases, aplicando inyecciones de biogás continuamente; y, las muestras líquidas se analizan mediante determinación del consumo de carbohidratos por el método Fenol-Sulfúrico u otro método para cuantificar carbohidratos; determinación indirecta de biomasa por cuantificadón de proteína mediante el Método de Bradford u otro método para cuantificar proteína.  13. The method according to claim 1, characterized in that the monitoring of step (e), stage II, is carried out by means of gas chromatography, applying biogas injections continuously; and, the liquid samples are analyzed by determination of carbohydrate consumption by the Phenol-Sulfuric method or another method to quantify carbohydrates; Indirect determination of biomass by protein quantification using the Bradford Method or another method to quantify protein.
14. El método de acuerdo con una de las reivindicaciones 1 o 13, caracterizado porque la etapa Π se considera terminada al tener en el monitoreo del paso (e), resultados de consumo de carbohidratos totales entre 50 y 75%; y cantidad de proteína de 150 a 300 ug/mL en la fase exponendal y estacionaria.  14. The method according to one of claims 1 or 13, characterized in that step Π is considered finished by having in the monitoring of step (e), results of total carbohydrate consumption between 50 and 75%; and protein amount of 150 to 300 ug / mL in the exponential and stationary phase.
15. El método de acuerdo con la reivindicadón 1, caracterizado porque el sustrato que se adidona como fuente de carbono al medio mineral en el paso (a), etapa III, se selecciona de: glucosa, lactosa, maltosa y almidón.  15. The method according to claim 1, characterized in that the substrate that is added as a carbon source to the mineral medium in step (a), step III, is selected from: glucose, lactose, maltose and starch.
16. El método de acuerdo con la reivindicadón 15, caracterizado porque el sustrato se adidona en una concentración de 10 mg/L  16. The method according to claim 15, characterized in that the substrate is added at a concentration of 10 mg / L
17. El método de acuerdo con la reivindicadón 1, caracterizado porque en el paso (a), etapa 17. The method according to claim 1, characterized in that in step (a), step
IV, las sales se disuelven en agua desinonizada y en agitación constante para alcanzar una concentradón de oxígeno disuelto de entre 30-60% en el medio mineral, preferiblemente 2 días antes de ser utilizadas; y más preferiblemente 5 días antes de ser utilizadas. IV, the salts are dissolved in de-ionized water and under constant agitation to reach a dissolved oxygen concentration of between 30-60% in the mineral medium, preferably 2 days before being used; and more preferably 5 days before being used.
18. S método de acuerdo con la reivindicadón 1, caracterizado porque la fermentación del paso (c), etapa IV, tiene una duradón de hasta 48 h; y preferiblemente, la duradón es de 24 h para proseguir con la etapa de producdón de hidrógeno. 18. S method according to claim 1, characterized in that the fermentation of step (c), stage IV, has a duration of up to 48 h; and preferably, the duration is 24 hours to continue with the hydrogen production step.
19. El método de acuerdo con la reivindicadón 1, caracterizado porque en el paso (a), etapa19. The method according to claim 1, characterized in that in step (a), step
V, el medio fresco se selecdona de: un sustrato modelo como glucosa, lactosa, maltosa o almidón; un sustrato complejo como residuos industríales induyendo: vinazas, lactosuero, nejayote, melazas, residuos de fruta, de verdura y de comida; aguas residuales incluyendo: lodos aerobios o anaerobios, aguas residuales de la industria del café, de bebidas y del procesamiento de carnes y embutidos; desechos sólidos orgánicos incluyendo: hldrol izados de materiales lignocelulósicos como bagazo de caña, agave y uva, cascara de cereales y pasto; cualquiera de estos residuos en digestión y/o co-digestión, y sin dilución de los mismos. V, the fresh medium is selected from: a model substrate such as glucose, lactose, maltose or starch; a complex substrate as industrial waste inducing: vinasses, whey, Nejayote, molasses, fruit, vegetable and food waste; wastewater including: aerobic or anaerobic sludge, wastewater from the coffee, beverage and meat and sausage processing industry; organic solid wastes including: hldrol hoisted from lignocellulosic materials such as cane bagasse, agave and grapes, cereal husks and grass; any of these residues in digestion and / or co-digestion, and without dilution thereof.
20. Un consorcio microbiano obtenido y adaptado por el método que se describe en las reivindicaciones 1-19, caracterizado porque induye:  20. A microbial consortium obtained and adapted by the method described in claims 1-19, characterized in that it induces:
a. Microorganismos hidrógeno productores como: Actínomyces sp, Addaminococcus fermentans, Addaminococcus sp, Qtrobacter sp, Qtrobactsr freundii, Qostridium beijerinckii, Qostridium butyricum, Qostridium iutice/iarii, Qostridium puniceum, Qostridium roseum, Qostridium tyrobutyricum, Qostridium sp, Escherichia co/i, Enterobacter sp, Enterobacter doacae, Ethano/igenens sp, Klebsidla sp, Megasphaera e/sden/i, Megasphaera sp, Megasphaera mlcronudtorml, Prevotella buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonia sp, Rhodopseudomonas palustris, to. Producing hydrogen micro-organisms such as: Actínomyces sp, Addaminococcus fermentans, Addaminococcus sp, Qtrobacter sp, Qtrobactsr freundii, Qostridium beijerinckii, Qostridium butyricum, Qostridium iutice / iarii, Qostridium puniceum, Qostridium sputumium, Qostridium Enteroium sputumium, Qostridium intertriumiumumumumumum, Qostridium spumumium, Qostridium intertriumiumumumumumum, Qostridium spumiumiumumumium , Enterobacter doacae, Ethano / igenens sp, Klebsidla sp, Megasphaera e / sden / i, Megasphaera sp, Megasphaera mlcronudtorml, Prevotella buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonia sp, Rhodopseudomonas palustris,
Se/enomonas ruminantíum, Se/enomonas sp, Se / enomonas ruminantíum, Se / enomonas sp,
b. Microorganismos ácido lácticos como: Bifídobacterium psychraeropNlum, Lactobacfllus case/, Lactobacfí/us coryniformis, Lactobacillus delbrueckil, Lactobadllus harbinensis, Lactobadllus hilgardii, Lactobadllus parabuchneri, Lactobadllus perolens, Lactobadllus rapi, Lactobadllus rhamnosus, Lactobadllus sun/di, Lactobadllus vacdnostercus, b. Lactic acid microorganisms such as: Bifídobacterium psychraeropNlum, Lactobacfllus case /, Lactobacfí / us coryniformis, Lactobacillus delbrueckil, Lactobadllus harbinensis, Lactobadllus hilgardii, Lactobadllus parabuchneri, Lactobadllus dusts
Spofoiactobadllus terree, Sporolactobadllus sp, y Spofoiactobadllus terree, Sporolactobadllus sp, and
c. Microorganismos ácido acéticos como: Acetobacter dbinongensis, Acetobacter lovaniensis, Acetobacter orientalis, Acetobacter sp, Acetobacter syzygií.  C. Acetic acid microorganisms such as: Acetobacter dbinongensis, Acetobacter lovaniensis, Acetobacter orientalis, Acetobacter sp, Acetobacter syzygií.
21. Consorcio microbiano de acuerdo a la reivindicación 20, caracterizado porque tiene actividad hidrolrtica; actividad hidrogénica; y porque está conformado por microorganismos facultativos y anaerobios estrictos.  21. Microbial consortium according to claim 20, characterized in that it has hydrolytic activity; hydrogenic activity; and because it is made up of facultative microorganisms and strict anaerobes.
22. Uso del consorcio microbiano de las reivindicaciones 20-21, para llevar a cabo un proceso de digestión o co-digestión anaerobia, bajo condiciones anaerobias artificiales no generadas "no inyección de gases inertes".  22. Use of the microbial consortium of claims 20-21, to carry out an anaerobic digestion or co-digestion process, under artificial anaerobic conditions not generated "no injection of inert gases".
23. El uso de acuerdo con la reivindicación 22, en donde el proceso de digestión o co-digestión anaerobia es para producir hidrógeno bajo condiciones anaerobias artificiales no generadas "no inyección de gases inertes", a partir de sustratos modelo o complejos/recalcitrantes como vinazas, lactosuero, nejayote, residuos de fruta y verdura; directamente a partir de los carbohidratos presentes en el medio o residuos; o bien de ácidos grasos como lactato y acetato producidos por la hidrólisis. 23. The use according to claim 22, wherein the anaerobic digestion or co-digestion process is to produce hydrogen under artificial anaerobic conditions not generated "no injection of inert gases", from model or complex / recalcitrant substrates such as vinasse, whey, nejayote, fruit and vegetable waste; directly from the carbohydrates present in the medium or waste; or of fatty acids such as lactate and acetate produced by hydrolysis.
24. El uso de acuerdo con una de las reivindicaciones 22-23, en el que los procesos de digestión o co-digestión anaerobia presentan una fase hidrolftíca o hidrogénica dependiendo de las condiciones de operación y tipo de sustrato utilizado. 24. The use according to one of claims 22-23, wherein the anaerobic digestion or co-digestion processes have a hydrolytic or hydrogenic phase depending on the operating conditions and type of substrate used.
25. El uso de acuerdo con la reivindicación 24, en donde los microorganismos predominantes en la fase hidrogénica son: Actinomyces sp, Addaminococcus fermentans, 25. The use according to claim 24, wherein the predominant microorganisms in the hydrogenic phase are: Actinomyces sp, Addaminococcus fermentans,
Addaminococcus sp, Gtrobacter sp, Gtrobacter freundíl, Qostridium beijerinckii, Qostridium butyrícum, Qostridium lutice/laríi, Qostridium puniceum, Qostridium roseum, Qostridium tyrobutyricum, Qostridium sp, Escherichia coli, Enterobacter sp, Enterobacter doacae, Ethanoiigenens sp, Klebsieila sp, Megasphaera eisdenii, Megasphaera sp, Megasphaera micronutiformi, Prevoteila buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonia sp, Rhodopseudomonas palustris, Selenomonas ruminantium y Selenomonas sp Addaminococcus sp, Gtrobacter sp, freundíl Gtrobacter, Qostridium beijerinckii, Qostridium butyricum, Qostridium Lutice / Larii, Qostridium puniceum, Qostridium roseum, Qostridium tyrobutyricum, Qostridium sp, Escherichia coli, Enterobacter species, Enterobacter doacae, Ethanoiigenens sp, Klebsieila sp, Megasphaera eisdenii, Megasphaera sp, Megasphaera micronutiformi, Prevoteila buccae, Prevotella sp, Pseudomonas sp, Pseudomonas putida, Ralstonia sp, Rhodopseudomonas palustris, Selenomonas ruminantium and Selenomonas sp
26. El uso de acuerdo con la reivindicación 24, en donde los microorganismos predominantes en la fase hidrolftíca son: Bifídobacterium psychraerophilum, Lactobadllus casef, Lactobadllus corynifórmis, Lactobadllus ddbrueddi, Lactobadllus harbinensfs, Lactobadllus hilgardii, Lactobadllus parabuchneri, Lactobadllus pero/ens, Lactobadllus rapó Lactobadllus rhamnosus, Lactobadllus sunkii, Lactobadllus vacdnostercus, Sporolactobadllus terrae, Sporoiactobatillus sp, Acetobacter dbinongensis, Acetobacter lovaniensfs, Acetobacter orientalis, Acetobacter sp, y Acetobacter syzygii.  26. The use according to claim 24, wherein the predominant microorganisms in the hydrolytic phase are: Bifidobacterium psychraerophilum, Lactobadllus casef, Lactobadllus corynifórmis, Lactobadllus ddbrueddi, Lactobadllus harbinensfs, Lactobadllus hilgardii, Lactobadllus lactobadllus budobadustus, Lactobadllus budobadus, Lactobadllus budobadus, Lactobadllus budobadus, Lactobadllus budobadus, Lactobadllus labubadus, Lactobadllus budobadus, Lactobadllus budobadus, Lactobadllus budobadus, Lactobadllus budobatob, Lactobadllus labatobusti Lactobadllus rhamnosus, Lactobadllus sunkii, Lactobadllus vacdnostercus, Sporolactobadllus terrae, Sporoiactobatillus sp, Acetobacter dbinongensis, Acetobacter lovaniensfs, Acetobacter orientalis, Acetobacter sp, and Acetobacter syzygii.
27. El uso de acuerdo con una de las reivindicaciones 22-26, en donede el consorcio microbiano se utiliza para la realización de procesos anaerobios de una sola etapa que ocurren un solo reactor. 27. The use according to one of claims 22-26, wherein the microbial consortium is used for the performance of single-stage anaerobic processes that occur in a single reactor.
28. B uso de acuerdo con una de las reivindicaciones 22-27, en donde el consorcio microbiano se utiliza para la realización de procesos anaerobios de dos o múltiples etapas involucrando el uso de más de un reactor.  28. B use according to one of claims 22-27, wherein the microbial consortium is used for the performance of two or multiple stage anaerobic processes involving the use of more than one reactor.
29. El uso de acuerdo con la reivindicación 28, en donde la materia orgánica hidrolizada de una primera etapa puede ser alimentada a una segunda etapa metanogénica o foto- fermentativa.  29. The use according to claim 28, wherein the hydrolyzed organic matter of a first stage can be fed to a second methanogenic or photo-fermentative stage.
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