• 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:
    The invention is particularly related with a method, which use bioaugmentation with anaerobic rumen fungi at varied ratios of inoculums on the performance of anaerobic digesters of microalgae biomass for increasing methane production. In that proposed method the composite obtain that 4 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 into the anaerobic digesters fed with microalgae Haematoccus pluvialis at different inoculums ratios: %1 (F1), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v/v).
    公开号:ES2743548A1
    申请号:ES201990041
    申请日:2016-12-28
    公开日:2020-02-19
    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 improve methane production from microalgae
    [0003] Field of the Invention
    [0004]
    [0005] The present invention refers to the improvement in methane production using anaerobic digesters of microalgae biomass.
    [0006]
    [0007] The invention is particularly related to a process, which uses bioaugmentation with anaerobic rumen fungi with varying proportions of inoculums in the performance of anaerobic digesters of microalgae biomass to increase methane production.
    [0008]
    [0009] Background of the invention
    [0010]
    [0011] Because microalgae have high photosynthetic performance, high growth rates and the characteristic of not requiring an external source of organic carbon, microalgae are believed to be potential sources of bioenergy and biofuel production.
    [0012]
    [0013] The production of biogas comprising hydrogen or methane from anaerobic digestion of algae is currently a somewhat conspicuous technology because it has the capacity to conserve energy and environmentally compatible characteristics. In addition to the environmental conditions that promote microbial activity, substrate degradation is a fairly important parameter through the anaerobic process.
    [0014]
    [0015] There are already some advances present and known in the state of the art that have been provided for bioenergy from biomass.
    [0016]
    [0017] 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.
    [0018]
    [0019] In the international patent document with the number WO2015044721A1 within the known state of the art, the invention relates to the subsequent processing of microalgae biomass to produce different products in a biorefinery process. The invention establishes interconnected stages from the extraction of biomass from microalgae, following several production processes, including alternatives to use the remaining biomass. The invention allows the general process to be improved in a downward direction by adapting each stage of the production process for a complete use of microalgae biomass by producing several value-added products of interest in the commercial field: proteins, biodiesel and biogas or biomethane. In addition, the invention includes wastewater reuse alternatives to be reused in the same processes. The invention has application in the processing of biomass, such as microalgae and other types of biomass, for the production of biofuels and co-products.
    [0020]
    [0021] In the international patent document with the number W09325671A1 known within the state of the art, a method for cloning xylanase clones from an anaerobic rumen fungus that includes the steps of: (I) cultivating a fungus of anaerobic rumen; (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) link the cDNA to a bacteriophage expression vector selected from lambda ZAP, lambda ZAPII or vectors of similar properties; (VI) selecting positive recombinant 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 . patriciarum; (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.
    [0022]
    [0023] Anaerobic digestion can be performed directly on the algae after a new harvest or on microalgae wastes after lipid extraction. With respect to the first, the resistance of the microalgae cell wall can be a significant restrictive factor for cell digestibility. Certain species of microalgae, such as Haematococcus sp., And Chlorella sp., Have recalcitrant cellulose in the cell wall, which protects microalgae against invasion of enzymes and also limits the biodegradability of algae.
    [0024]
    [0025] Bioaugmentation with a combination of fungi in the rumen in an anaerobic process may represent an appropriate alternative to the use of previous chemical treatments of microalgae biomass. Thus, anaerobic rumen fungi are promising to improve the production of biogas from different species of microalgae and macroalgae and also various lignocellulosic substrates.
    [0026]
    [0027] Brief description of the invention and its objectives
    [0028]
    [0029] The objective of this invention is to improve the bioenergetic potential of anaerobic digesters through the use of rumen fungi.
    [0030] Another objective of this invention is to improve the degradation of algae biomass by bioaugmentation using rumen anaerobic fungi.
    [0031]
    [0032] Another objective of the present invention is to obtain an improvement in the production of biomethane from microalgae biomass according to bioaugmentation by anaerobic rumen fungi in varied proportions of inoculums on the performance of anaerobic digesters.
    [0033]
    [0034] In this procedure, anaerobic rumen fungi, Anaeromyces, Neocallimastix, Orpinomyces and Piromyces were used, which have gene clusters that originate from bacteria through horizontal gene transfer. The results imply that rumen fungi improved fermentation and degradation of microalgae biomass because they promoted cell wall degradation, while methane production increased by 41% due to bioaugmentation with rumen fungi during anaerobic processes
    [0035]
    [0036] In general, the findings here indicate that bioaugmentation with a combination of fungi in the rumen in an anaerobic process may represent an appropriate alternative to the use of previous chemical treatments of microalgae biomass. Therefore, anaerobic rumen fungi are promising to improve the production of biogas from different species of microalgae and macroalgae and also various lignocellulosic substrates.
    [0037]
    [0038] Detailed description of the invention
    [0039]
    [0040] In this process of the invention, first, rumen fluid was taken through the rumen fistulas of a cow. The fluid was analyzed by metagenomic analysis to determine the specific fungi of the anaerobic rumen inside. Isolated and cultured rumen fungi were evaluated with strain identification techniques and phylogenetic analysis to characterize anaerobic rumen fungus species. Four species that had a high expression of enzymes that degraded lignocellulose were isolated and selected and mixed in one 30% proportion of Orpinomyces sp., 25% of Piromyces sp. and 25% of Anaeromyces sp., 20% of Neocallimastix frontalis.
    [0041]
    [0042] Subsequently, this mixture was added in anaerobic digesters fed with H. pluvialis microalgae in different proportions of inoculums: 1% (F1), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v / v).
    [0043]
    [0044] To understand the effect of anaerobic rumen fungi on biogas production, a digester did not bio-increase with anaerobic rumen fungi to maintain it as a control digester: 0% (F0). Anaerobic digesters fed with microalgae were prepared semi-continuously with volumes of 2000 ml for 40 days at 41 ° C.
    [0045]
    [0046] The reactors were operated in duplicate under the same conditions. The performance of anaerobic digesters was evaluated through the production of biogas and biomethane. The inhibitory effect of the digesters was controlled with the measurement of volatile fatty acids (AGV or VFA for its acronym in English "volatile fatty acids").
    [0047]
    [0048] Finally, the dynamics of the microbial community during the anaerobic digestion process were distinguished according to the Illumina Miseq and qPCR analyzes. Samples for the entire rumen content comprising liquid and solids were taken through the rumen fistulas of a cow (live weight: 400-450 kg) with confidential techniques performed by veterinarians.
    [0049]
    [0050] The cows were over two years old and fed alfalfa hay, barely grass, legumes, silage and soybean meal during the summer and winter periods. All ruminal fluid samples were washed with nitrogen gas (N2) to provide anaerobic conditions after loading and sealing. Several samples of rumen fluid were stored at -20 ° C to extract the DNA for a subsequent metagenomic study of the rumen fluid.
    [0051]
    [0052] Metagenomics, also known as environmental genomics, was used to determine the abundance and identity of rumen fungi in a sample. The metagenomic study, based on total purified DNA, was thoroughly investigated in Fungal species of the rumen classified by the function of the genes and the analysis of the pathways at the DNA level. Initially, the qualified DNA samples were cut into smaller fragments by nebulization.
    [0053]
    [0054] Next, the T4 DNA polymerase, the Klenow fragment and the T4 polynucleotidokinase converted the fragmentation results into blunt ends. After the addition of an adenine base (A) to the 3 'end of the blunt phosphorylated DNA fragments, the ends of the DNA fragments were joined with adapters. At that time, the short fragments were removed with AMPpure beads. Sample libraries were scored and quantified with an Agilent 2100 Bioanaylzer and an ABI StepOnePlus real-time PCR system.
    [0055]
    [0056] Qualified libraries were sequenced using the Illumina HiSeqTM platform. The ABI StepOnePlus Real-Time PCR System was used to rate and quantify the sample libraries. These libraries were sequenced using the Illumina HiSeqTM platform. The qualified sequencing readings produced by the Illumina platform were preprocessed and then de novo assembled with SOAPdenovo2 and Rabbit.
    [0057]
    [0058] MetaGeneMark was then used to predict genes from assembled [contigous "or" contiguous "] con-partners, building a project-specific gene catalog. Preprocessed readings were also assigned to the IGC database and the mapped genes were retrieved and integrated into the gene catalog. CD-Hit was used to eliminate redundancy.
    [0059]
    [0060] The gene catalog was used to counteract public databases, including nr, Swiss-Prot, COG, KEGG, GO, CAZ and, eggNOG and ARDB to obtain functional and taxonomic annotations. Before the isolation of the anaerobic rumen fungi, in order to grow them, complex media were prepared using the protocols described previously.
    [0061]
    [0062] Two different salt solutions were prepared for use in the media. While saline solution I involved (g / L) KH2P04-3.0; (H) 2SO-3.0; NaCl-6.0; MgSO-0.6 and CaCl-0.6, the salt solution II included K2HP04 (3 g / L). Saline solution I had 150 ml of saline solution II, 150 ml of centrifuged ruminal fluid, 200 ml of bactocasitone (Difco), 10 g of yeast extract (Oxoid), 2.5 g of NaHCO, 6 g of L-cysteine . To 900 ml 1 g of HCl, 2 g of fructose, 2 g of xylose, 2 g of cellobiose, 8 g of trace elements solution, 10 ml of hemin solution, 10 ml of resazurine solution (0.1%, p / v) and 1 ml of deionized water.
    [0063]
    [0064] Then, the media were autoclaved for 20 min at 115 ° C. After the autoclave, 0.1% (v / v) of two different vitamin solutions were added to the medium. The vitamin I solution contained (g / L): thiamine HCl, 0.10; riboflavin, 0.20; calcium pantothenate D, 0-60; nicotinic acid, 1.00; nicotinamide, 1.00; folic acid, 0.05; cyanocobalamin, 0.20; biotin, 0.20; pyridoxine.HCl, 0.10; and paminobenzoic acid, 0.01. Vitamin II contained in the solution (mg / L): thiamine HCl, 5; riboflavin, 5; calcium pantothenate D, 5; nicotinic acid, 5; folic acid, 2; cyanocobalamin, 1; biotin, 1; pyridoxine HCl, 10; paminobenzoic acid, 5.
    [0065]
    [0066] 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 added to the medium of isolation to suppress bacterial growth. After the preparation of the medium, all cultures were incubated under CO2 at 39 ° C within a week to reproduce rumen fungi.
    [0067]
    [0068] Fungal DNA was sequenced with strain identification and phylogenetic analysis to identify isolated species of ruminal fluid and cow manure.
    [0069]
    [0070] This analysis was performed with the internally complete transcribed spacer (ITS; partial 18S, complete ITS 1, 5.8S, ITS 2 and partial 28S) and D1 / D2 at the 5 'end of the ribosomal DNA of the large subunit (LSU = large sub unit) being amplified using primer pairs, ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3 ') / ITS4 (5'- TCC TCC GCT TAT TGA TAT GC-3') and L1 5'-GCA TAT CAA TAA GCG GAG GAA AAG-3 ') / NL4 (5'-GGT CCG TGT TTC AAG ACG G-3').
    [0071]
    [0072] The consensus sequences, CATTA / CAACTTCAG (end of 18S / start of 5.8S) and GAGTGTCATTA / TTGACCTCAAT (end of 5.8S / start of 28S) were used to demarcate the different regions of the rRNA locus in a consistent manner as suggested by Hibbett (2016).
    [0073]
    [0074] The Geneious v6 Bioinformatics package that MAFFT uses for the alignment of Sequences and Mr Bayes for phylogenetic analysis were applied to carry out phylogenetic reconstruction.
    [0075]
    [0076] Air enriched with 2% CO2 was photoautotrophically used to grow H. pluvialis strain SCCAP 34/7 . Basal medium in bold with triple N2 and vitamins (3N-BBM + V; CCAP 2015) was used at 25 ° C to grow microalgae cells in a 2L bioreactor photo system. The light source was a 9-chamber "x 9" x 9 "with 8000-10000 lux LED lights.
    [0077]
    [0078] After incubation, the microalgae biomass was obtained, which was concentrated by centrifugation at 3600 x g for 15 min. The concentrated algal biomass presented 11% (based on wet weight) of the total solid (ST), 91.6% (based on dry weight) of volatile solid (SV) and 44, 16 and 26 % (based on dry weight) of proteins, lipids and carbohydrates, respectively.
    [0079]
    [0080] For the methanogenic inoculums, granular sludge was used which was grown in a laboratory-scale sequenced anaerobic batch reactor (1.5 L) (ASBR) for anaerobic sequenced batch reactor. The ASBR had a temperature of 41 ° C, and glucose and acetate (80%: 20%, calculated as COD [chemical oxygen demand]) were used as raw material at an organic loading rate of 1 g COD / (L- day). Different initial concentrations of H. pluvialis (2 g of SV / L of algal biomass) and 3 g of SV / L of methanogenic sludge were used for batch experiments performed at 41 ° C.
    [0081]
    [0082] The culture medium consisting of anaerobic fungi was used in different inoculum proportions: 0% (F0), 1% (F1), 5% (F2), 10% (F3), 15% (F4) and 20% ( F5) (v / v). 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. Next, a trace element and a vitamin solution were prepared and then adjusted according to the procedure described in the previous study.
    [0083]
    [0084] Triplicate samples were taken from the sludges of the F0, F1, F2, F3, F4 and F5 reactors on days 5, 10, 20 and 30 and 40 and 40 during the operation of anaerobic digesters for total DNA isolation. Total DNAs were isolated from mud samples from 1 ml with PureLink Genomic DNA extraction kits. A NanoDrop spectrophotometer determined the concentration.
    [0085]
    [0086] The V4-V5 hypervariable region of the 16S rRNA gene was reproduced with region-specific primers that were designed to contain an illuminates adapter and 518F-926R barcode sequences for bacteria and 518F-958R for archaea. A double round of PCR and dual indexing in a Peltier Thermal Cycler with PTC-200 DNA Engine [PTC-200 DNA Engine Peltier Thermal Cycler] was used to generate the sample amplicons.
    [0087]
    [0088] A picogreen assay and a fluorometer (96-well SpectraMax GeminiXPS plate reader) were used to adjust amplicon concentrations. After concentration determination, they were placed in the same amounts (-100 ng) in a single tube. The following technique was applied to ward off short undesirable fragments and clean the amplicon group.
    [0089]
    [0090] First, the size of the combination was determined using AMPure beads (from Beckman Coulter), and the product was characterized in a 1% gel that was cut and purified with a Qiagen MiniElute PCR purification kit, after which determined the size of the combination with AMPure pearls again.
    [0091]
    [0092] The PCR containing specific Illumina adapter primers was used to adjust the quality of the amplicon group. Thereafter, the PCR products were passed through a DNA 1000 chip for Agilent 2100 bioanalyzer. The last set of amplicons was accepted on the condition that long fragments would not be identified after PCR and if there were short fragments, the procedure would be repeated again.
    [0093]
    [0094] The KAPA 454 library quantification kit (from KAPA Biosciences) and the StepOnePlus real-time PCR system from Applied Biosystems quantified the amplicon group, which was determined to be clean. Finally, the protocol follows MiSeq illuminates 300 base pairs to obtain the sequences.
    [0095]
    [0096] QPCR was applied to all anaerobic digesters to assess optimal fungal concentrations. The qPCR assay was performed in triplicate using an ABI system 7500 SDS with all amplified digesters using prior primer sets specific for anaerobic fungi (direct primer: 5'- GAGGAAGT AAAAGTCGT A AC AAGGTTTC-3 ', reverse primer: 5'-CAAATTC AC AAAGGGT AGGATGATTT-3').
    [0097]
    [0098] An optimal primer concentration of 350 nm and a final MgC12 concentration of 4 mM were used for the qPCR under the following cycle conditions: denaturation at 94 ° C for 4 min, followed by 35 cycles of 96 ° C for 45 s, 56 ° C for 45 s, and 72 ° C for 1 min with a final extension after 72 ° C for 5 min. Detailed information on the qPCR analysis has already been described
    [0099]
    [0100] The analysis of alkalinity, total solids (ST) and volatile solids (SV) was performed in an appropriate manner with standard methods. Biogas production was measured with Milligas counters in both ASBR. Gas chromatography with a flame ionization detector and an Elite-FFAP column (30 m X 0.32 mm) measured the gas compositions and AGV concentrations. 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.
    [0101]
    [0102] Histograms, q-q graphs and the Shapiro-Wilk 'test were performed to examine the normality of the data. The homogeneity of the variation was also investigated by the Levene test. Variation analysis (ANOVA) or t-tests of independent samples were used to verify variations in biogas production and the dynamics of the microbial community between various anaerobic fungus inoculum ratios and F0. To facilitate multiple comparisons, the Tukey test was also applied.
    [0103]
    [0104] The test values were interpreted as mean and standard deviation. The applicability of the microbial community and inoculum relationships were determined by the Pearson test in terms of correlation. Significant differences were detected at the level p <0.05.
    权利要求:
    Claims (1)
    [1]
    1. Procedure to improve methane production by using anaerobic digesters of microalgae biomass characterized by the fact that the process comprises the following stages:
    - select four species of anaerobic fungi isolated from the rumen, and mix in a proportion of 30% of Orpinomyces sp., 25% of Piromyces sp. and 25% of Anaeromyces sp., 20% of Neocallimastix fmntalis.
    - add these four species to anaerobic digesters fed with Haematococcus pluvialis microalgae in different inoculum proportions: 1% (F1), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) ( v / v).
    类似技术:
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    ES2743548B1|2021-02-19|Procedure to improve methane production from microalgae
    ES2743316B1|2021-02-10|Procedure to produce biogas in anaerobic digestions with rumen fungi
    同族专利:
    公开号 | 公开日
    ES2743548B1|2021-02-19|
    WO2018124988A1|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|
    RU2419594C1|2010-04-14|2011-05-27|Государственное образовательное учреждение высшего профессионального образования "Оренбургский государственный университет"|Method of animal farming wastes treatment and reclamation|
    WO2015044721A1|2013-09-30|2015-04-02|Desert Bioenergy|Microalgae biorefinery for biofuel and valuable products production|
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