• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A method for the production of biogas which uses a mixture of specific rumen fungi is disclosed. The method shall improve biomethane production and biogas potential of anaerobic digestion.Four isolated species of anaerobic rumen fungi of Orpinomyces sp., Piromyces sp. and Anaeromyces sp., Neocallimastix frontalis were selected and these species were mixed. After that, mixture of rumen fungi containing 4 species was added in the anaerobic digesters fed with animal manure at different inoculums ratios: 5% (R1), 15% (R2), 20% (R3) (v/v).
    公开号:ES2743316A1
    申请号:ES201990040
    申请日:2016-12-28
    公开日:2020-02-18
    发明作者:Orhan Ince；Bahar Ince；Sevcan Aydin；Elif Yildirim
    申请人:Univ Istanbul Teknik；ISTANBUL TEKNIK UNIVERSITESI；
    IPC主号:
    专利说明:

    [0001]
    [0002] Procedure to produce biogas in anaerobic digestion with fungi of the rumen
    [0003]
    [0004] Field of the Invention
    [0005]
    [0006] The present invention relates to the production of biogas in anaerobic digesters.
    [0007]
    [0008] The invention is particularly related to a process, which includes a mixture comprising rumen fungi and improves the production of biomethane and biogas in anaerobic digesters.
    [0009]
    [0010] Background of the invention
    [0011]
    [0012] The benefits of producing environmentally friendly and low-cost energy are of great importance in daily use due to overpopulation and the rapid growth of industries worldwide.
    [0013]
    [0014] Since the main objective of renewable energy is to reduce poverty and allow sustainable development, in recent years many countries have begun to use renewable energy. Another reason why renewable energy has become prominent is that it reduces the reserves of non-renewable energy resources known to cause climate change.
    [0015]
    [0016] Thus, alternative sources of energy, such as solar, geothermal, olamotor, biomass and hydraulics, are considered as possible sources of renewable energy. Biomass is one of the most important alternatives in renewable energy sources, which is described as an organic matter that originates from the photosynthetic capture of solar energy and is stored as chemical energy.
    [0017] Therefore, biomass is an efficient biological material that can be used as fuel providing energy in terms of renewable and sustainable energy. While there are many sources of biomass, including wastes and residues from agricultural crops, municipal solid waste, sewage, crops and forest residues, and industrial and animal waste, animal manure is defined as a primary source of biomass, since generally It is deposited on earth.
    [0018]
    [0019] There are already some advances present and known in the state of the art that have been provided to bioenergy from manure biomass and from sewage sludge.
    [0020]
    [0021] For example, in the Russian patent document with the number RU2419594C1 within the known state of the art, the invention refers to agricultural production, in particular, to complete the treatment and recovery of waste from animal structures to produce electrical energy and thermal, for circulating water and fertilizers. The liquid phase of the overfermentation is evaporated to dry the concentrated fertilizer. Keep in mind that the steam is converted into water to be used as needed in the process. A part of the homogeneous mass is burned to clean the biogas obtained by passing through the water to produce biomethane to provide the consumer. Water is saturated with organic substances that will be used as a liquid fertilizer. The air from the production facilities is collected to facilitate the combustion of the homogeneous mass with greater heat emission. The combustion residue is used as a mineral fertilizer. Exhaust gases are cleaned of solid volatile additives by passing them through water and saturating them with mineral substances for use as mineral fertilizers. The purified exhaust gas is used to generate electricity to power greenhouses.
    [0022]
    [0023] In the international patent document with the number W09325671A1 known within the state of the art, a method is described for cloning xylanase clones from an anaerobic rumen fungus that includes the steps of: (I) cultivating a rumen fungus anaerobic; (II) isolate all RNA from the culture in step (III); (III) isolate the poly A <+> mRNA from all the RNA mentioned in step (II); (IV) build a cDNA expression library; (V) bind the cDNA to a bacteriophage expression vector selected from lambda ZAP, lambda ZAPII or vectors of similar properties; (SAW) selecting recombinant positive xylanase clones in a culture medium incorporating xylan by detecting xylan hydrolysis; and (VII) purify positive recombinant xylanase clones. Xylanase positive recombinant clones produced by the aforementioned method are also provided, as well as xylanase positive recombinant clones having the following properties: (I) production of xylan elimination zones in a culture containing N- derived xylanase cDNA . patríciamm; (II) which has activity in the hydrolysis of xylan but which has no activity in relation to the hydrolysis of CMC or crystalline cellulose. Various cDNA molecules are also provided that can be used in the method mentioned above.
    [0024]
    [0025] In the patent document of the United States of America, number US6458580B1 within the known state of the art, a method for promoting the growth of at least one anaerobic fungus in the rumen of a ruminant animal is described, the method comprises the step of administer to the rumen an effective amount of a sulfur source resistant to degradation.
    [0026]
    [0027] Despite the fact that it is advantageous to purify animal manure as a fertilizer for soils and to obtain nutrients for food crops, recent studies show that the limitation of land for the disposal of large amounts of waste [landfills] and Limited feeding processes have become a problem in recent times. In addition, public health and the environment are threatened because animal manure is the main source of bad smell, harmful pathogens and harmful gases, which are toxic and harmful to living organisms. Therefore, the use of animal manure as a source of biofuel has become crucial to avoid the accumulation of waste and environmental damage.
    [0028]
    [0029] That is why bioaugmentation is used, which is a procedure for the enrichment of specific microorganisms that is used in anaerobic digesters to improve the yields of hydrolysis, nutrient recovery and biogas production, although some studies have evaluated the Bioaugmentation of anaerobic digestion processes with ruminal fluid and anaerobic ruminal bacteria.
    [0030]
    [0031] As can be understood from the documents mentioned above, Different methods are already used to improve the composition and increase the production of biogas in anaerobic digesters.
    [0032]
    [0033] Brief description of the invention and its objectives
    [0034]
    [0035] The objective of this invention is the production of biogas in anaerobic digesters through the use of rumen fungi.
    [0036]
    [0037] Another objective of this invention is to obtain an improvement in the production of biomethane from animal manure by bioaugmentation using anaerobic fungi in the rumen.
    [0038]
    [0039] In this procedure, it can be seen that the effects of bioaugmentation of anaerobic rumen fungi in various proportions of inoculums on the production of biogas from anaerobic digesters fed with animal manure. The highest biogas production was observed in the R2 digester (15%) with a rate of 5500 mL / d, almost 60% of the total biogas, due to the addition of anaerobic rumen fungi.
    [0040]
    [0041] In terms of increased biogas production, rumen anaerobic fungi appear to be a promising alternative to improve biogas production from different types of lignocellulosic compounds due to their non-specific extracellular ligninolytic enzyme system.
    [0042]
    [0043] Detailed description of the invention
    [0044]
    [0045] In the present invention, all rumen samples comprising liquids and solids were taken by means of dairy animal rumen fistulas (live weight 400-450 kg) using secret procedures performed by veterinarians. A bovine was over two years old and was fed with animal fodder, barely grass, vegetables, silage and soy protein during the summer and winter periods.
    [0046] The rumen samples were collected at 6 h after feeding from the focal part of the rumen. All ruminal fluid samples were washed with nitrogen gas (N2) to provide anaerobic conditions as a result of stacking and fixation.
    [0047]
    [0048] A part of the rumen fluid samples was stored at -20 ° C to extract its DNA for metagenomic examination of the rumen fluid. From that rumen fluid, isolated and cultured rumen fungi were analyzed by using strain analysis techniques and phylogenetic analysis to identify rumen anaerobic fungal species.
    [0049]
    [0050] Four isolated species of rumen anaerobic fungi ( Orpinomyces sp., Piromyces sp. And Anaeromyces sp., Neocallimastix frontalis) were selected and these species were mixed in a proportion of 30% of Orpinomyces sp., 25% of Piromyces sp., And 25% of Anaeromyces sp., And 20% of Neocallimastix frontalis. Next, a mixture of rumen fungi containing four species in different inoculum ratios was added: 5% (R1), 15% (R2), 20% (R3) (v / v).
    [0051]
    [0052] To understand the effect of anaerobic rumen fungi on biogas production, one of the digesters did not bio-increase with anaerobic rumen fungi to have it as a control digester: 0% (R0).
    [0053]
    [0054] Anaerobic digesters fed with animal manure were prepared and operated with volumes of 900 ml for 40 days at 40 ° C to understand the effect of rumen anaerobic fungi on biogas production. Biogas and biomethane production was measured to assess the performance of anaerobic digesters. The effect of digester inhibition was controlled with the measurement of volatile fatty acids (AGV or VFA for its acronym in English "volatile fatty acids").
    [0055]
    [0056] Finally, Illumina Miseq was used to identify the dynamics of the microbial community and qPCR was used to determine the number of active cells of the anaerobic fungi of the rumen. Subsequently, the metagenomic data obtained from all purified DNA were analyzed in detail to determine the rumen fungus classification and gene function.
    [0057] First, the qualified DNA samples were nebulized into smaller fragments. Then, T4 DNA polymerase, T4 polynucleotide kinase and Klenow fragment were used to convert the protrusions into blunt ends. The adapters were ligated to each DNA fragment after the addition of adenine to the 3 'end of the phosphorylated blunt ends. Ampure beads were used to get rid of the short fragments.
    [0058]
    [0059] Subsequently, the qualification and quantification of the sample libraries with Agilent2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System was evaluated. The libraries were sequenced using the Illumina HiSeq ™ platform. The initially qualified sequencing readings produced by the Illumina platform were refined and subjected to de novo assembly via SOAPdenovo2 and Rabbit.
    [0060]
    [0061] Assets [from English "contig" or "contiguous"] assembled to predict genes through MetaGeneMark were used to construct a project-specific gene catalog.
    [0062]
    [0063] After mapping the preprocessed readings in the IGC database, the genes were obtained and added to the gene catalog. The redundancy was eliminated using CD-Hit. Finally, the BLAST analysis of the gene catalog with some databases was performed for the purpose of functional and taxonomic annotation.
    [0064]
    [0065] Anaerobic rumen fungi were grown in complex media using the protocols described previously. Salt solution contained (g / L) KH2P04, 3.0; (H) 2S0, 3.0; NaCl, 6.0; MgSO, 0.6; CaCl, 0.6 were prepared for use in media. Saline solution, 150 ml; centrifuged with rumen liquid, 200 ml; Bactocasitone (Difco), 10 g; yeast extract (Oxoid), 2.5 g; NaHCO, 6 g; L-cysteine, HC1, 1 g; fructose, 2 g; xylose, 2 g; cellobiose, 2 g; resazurin solution (0-1%, w / v), 8 g; trace element solution, 10 ml; a solution of haemin, 10 ml and deionized water up to 900 ml was added to the media.
    [0066]
    [0067] Then, the media were autoclaved for 20 min at 115 ° C. After treating the media in an autoclave, a 0.1% (v / v) vitamin solution was added. 0.1% (v / v) antibiotic solution containing penicillin (5 g / L), streptomycin (5 g / L), neomycin (5 g / L) and chloramphenicol (5 g / L) was also added to the isolation medium to suppress bacterial growth.
    [0068]
    [0069] After preparing the media, all cultures were incubated under CO2 at 39 ° C for one week to reproduce rumen fungi. When the anaerobic fungi of the rumen reached the optical density, they were anaerobically transferred to discontinuous reactors to increase anaerobic digestion of animal manure.
    [0070]
    [0071] Fungal species isolated from ruminal fluid and cow manure were identified by sequencing the fungal DNA through strain identification and phylogenetic analysis. A complete internal transcribed spacer (ITS: partial 18S, complete ITS 1, 5.8S, ITS 2 and partial 28 S) was used to perform strain identification and phylogenetic analysis of isolated anaerobic fungi.
    [0072]
    [0073] Primer pairs ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3 ') / ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') and L15'-GCA TAT CAA TAA GCG GAG GAA AAG were used -3 ') / NL4 (5'-GGT CCG TGT TTC AAG ACG G-3') to amplify the D1 and D2 domain at the 5 'end of the large subunit (LSU = large sub unit) of ribosomal DNA, respectively.
    [0074]
    [0075] As suggested by Hibbett, different regions of the rRNA locus were demarcated using the consensus sequences CATTA / CAACTTCAG (end of 18S / start of 5.8S) and GAGTGTCATTA / TTGACCTCAAT (end of 5.8S / start of 28S) in a consistent manner. Sequence alignment using MAFFT and phylogenetic analysis using Mr Bayes of the Geneious v6 bioinformatics package were performed to reconstruct the phylogenetic analysis.
    [0076]
    [0077] The impact of rumen anaerobic fungi on the production of biogas and biomethane was determined using lots of anaerobic reactors. Granular sludge cultured in a laboratory-scale serial discontinuous anaerobic reactor (900 mL) (ASBR ) was used as anaerobic batch reactor reactor as methanogenic inoculums.
    [0078]
    [0079] The ASBR was performed at 40 ° C, and glucose and acetate (80%: 20%, calculated as chemical oxygen demand) were used as raw material at an organic loading rate of 1 g of chemical oxygen demand (COD) ) / (L-day). Different concentrations were used initials of anaerobic rumen fungi and 3 g of VS / L of methanogenic sludge in batch experiments conducted at 40 ° C.
    [0080]
    [0081] Four types of rumen anaerobic fungi were mixed at an equivalent rate. The culture medium comprises anaerobic rumen fungi (Orpinomyces sp., Piromyces sp., Anaeromyces sp. And Neocallimastix frontalis) was used in various inoculum proportions: 0% (RO-Control), R1 (5%), R2 (15 %), R3 (20%) (v / v). The control reactor was not enriched by anaerobic rumen fungi. Fungal inoculum was added only once at the beginning of the experiment. In addition to manure, granular sludge and water were added to obtain the ideal conditions.
    [0082]
    [0083] After stacking and fixation, gaseous nitrogen provided an anaerobic environment in all reactors. The Miligas counter (Ritter Digital Counter, U.S.A.) was used to measure the gas outputs. The pH was adjusted to 7-7.4 and alkalinity was added to maintain the pH. All reactors were operated for 40 days. The buffer contained (per L): 1.0 g of NH4Cl; 0.4 g of K2HP04.3H20; 0.2 g of MgC12.6H20; 0.08 g of CaC12.2H20, 10 ml of trace element solution and 10 ml of mother vitamin solution.
    [0084]
    [0085] A trace element and a vitamin solution were prepared. After preparation, they were adjusted according to the procedure described in our previous study.
    [0086]
    [0087] All chemical analyzes of alkalinity, total solids (TS) and "volatile solids" (VS) were performed according to standard methods. Biogas production was controlled through the use of Milligas meters (Ritter Digital Counter, U.S.A.) in both SBR.
    [0088]
    [0089] Gas composition and AGV concentration were measured by gas chromatography with a flame ionization detector (Perichrom, France and Agilent Technologies 6890N, USA, respectively) and an Elite-FFAP column (30 mx 0.32 mm). The oven setpoint was 100 ° C and the maximum inlet temperature was 240 ° C. In addition, helium gas was used as carrier gas at a rate of 0.8 ml / min.
    权利要求:
    Claims (1)
    [1]
    1. Procedure for producing biogas in anaerobic digesters through the use of mixtures that include rumen fungi, characterized in that the procedure comprises the following steps:
    - select four species of anaerobic fungi isolated from the rumen in a proportion of 30% of Orpinomyces sp., 25% of Piromyces sp., 25% of Anaeromyces sp., and 20% of Neocallimastix fmntalis to the mixture,
    - add these four species mixed in anaerobic digesters fed with animal manure in different proportions of inoculums: 5% (R1), 15% (R2), 20% (R3) (v / v).
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    同族专利:
    公开号 | 公开日
    ES2743316B1|2021-02-10|
    WO2018124984A1|2018-07-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20080187975A1|2006-12-18|2008-08-07|Richard Allen Kohn|Process for rapid anaerobic digestion of biomass using microbes and the production of biofuels therefrom|
    BRPI1001753A2|2010-06-02|2014-04-29|Univ Fed Sergipe|MODEL OF A TECHNOLOGICAL BIOPROCESS FOR BIOGAS GENERATION FROM SUGAR CANE|
    EP2740799A2|2012-12-07|2014-06-11|EADS Deutschland GmbH|Procecss for producing fuel employing algae and ruminal microorganisms|
    NZ252937A|1992-06-17|1996-10-28|Commw Scient Ind Res Org|Cloning recombinant xylanase from anaerobic rumen fungus|
    AUPO579397A0|1997-03-21|1997-04-17|Australian Wool Research & Promotion Organisation|Fungal sulphur source and method of using the same|
    RU2419594C1|2010-04-14|2011-05-27|Государственное образовательное учреждение высшего профессионального образования "Оренбургский государственный университет"|Method of animal farming wastes treatment and reclamation|CN110591922B|2019-07-16|2020-08-04|甘肃省科学院生物研究所|Pear penis and straw fermentation method for producing hydrogen and application thereof|
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    优先权:
    申请号 | 申请日 | 专利标题
    PCT/TR2016/050550|WO2018124984A1|2016-12-28|2016-12-28|A method for improving the biogas potential of anaerobic digestions with rumen fungi|
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