(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) ee Oeganaton = UE WIPO! (43) International Publication Date WO 2018/124984 Al 05 July 2018 (05.07.2018) PCT G1) International Patent Classiieat C12P 5702 (2006.01) 21) International Application Number: PCTITR20161050550 (22) International Fling Dat 28 December 2016 (28.12.2016) 25) Filing Language: English (26) Publication Language: English (71) Applicant: ISTANBUL TEKNIK UNIVERSITESI (TR/TRI; ITU Rekioriuk Binasi ITU Ayazaga Kampusu, Maslak, 34469 Istanbul (TR), (72) Inventors: INCE, Orhan; Istanbul Teknik Universite- si Insaat Fakultesi Cevre Muhendisligi Bolumu Ayaza- a Yerleskesi, Maslak, 34469 Istanbul (TR), INCE, Ba- har; Bogazici Universitesi Cevre Bilimleri Enstitusu His- ‘ar Kampus Bebek, 34342 Istanbul (TR). AYDIN, Sevean; CCinar Mah, 5/5 Sokak No:9 Daire:6, Bagilarilstanbul (TR). YILDIRIM, Elif Nispetiye Cad. Belediye Sitesi Youu Sok. Lale 4 Blok Daire:9 Filer, Besikta/Tstanbul (TR) (74) Agents CANKAYA PATENT MARKA VE DANIS- MANLIK LIMITED SIRKETI; Tunus Caddesi 4009, Kavalier, 06680 Ankara (TR), (81) Designated States (unless otherwise indicated, for every Kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA,CH, CL, CN, CO, CR, CU,CZ, DE, DI, DK, DM, DO, DZ, EC, FE, EG, FS, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, 1D, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA,LC,LK,ER,LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY,MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE; SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,ZW (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, $2,TZ, UG, ZM, ZW), Furasian (AM, AZ, BY, KG, KZ, RU, TH, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, FS, Fl, FR, GB, GR. 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Published: — with international search report (Art. 21(3)) ($4) Title: A METHOD FOR IMPROVING THE BIOGAS POTENTIAL OF ANAEROBIC DIGESTIONS WITH RUMEN FUNGI ¢ (57) Abstract: A method for the production of biogas which uses @ mixture of specific rumen fing is disclosed, The method shall improve biomethane production and biogas potential of anaerobic digestion Four isolated species of anaerabie rumen fing of Orpino= ryces sp, Pitomyces sp, and Anaeromyces sp, Neocallimastixfrontalis were selected and these species were mixed, After tha, mixture sits fed with animal manure at different inoculum ratios: 5% ‘of rumen fungi containing 4 species was added in the anaerobic (RI), 15% (RD), 20% (RS) (v9). = z z & z a ze 5 & ° SWO 2018/124984 10 15 20 25 30 [AMETHOD FOR IMPROVING THE BIOGAS POTENTIAL OF ANAEROBIC DIGESTIONS WITH RUMEN FUNG! DESCRIPTION Technical Field The invention is related with improvement of biogas potential of anaerobic digesters, The invention is particularly related with a method, which includes a composite comprises rumen fungi and improves biomethane production and biogas potential of anaerobic digesters Prior Art Environmentally-friendly and low cost energy production gains are of high importance for everyday use due to overpopulation and fast-growing industries all over the world. Because the main purpose of renewable energy is to reduce poverty and allow sustainable development, many countries have begun using renewable energy in recent years, Another reason why renewable energy has become prominent is that it reduces reserves of non-renewable energy resources that are known to cause climate change. Thus, alternative sources of energy, such as solar, geothermal, wave, biomass, and hydraulic, are considered possible renewable energy resources. Biomass is one of the most important altematives in renewable energy sources, which is described as an organic matter originating from the photosynthetic capture of solar energy and is stocked as chemical energy. Thus, biomass is an efficient biological material that can be used as fuel and provides power in terms of renewable and sustainable energy. Although there are many sources of biomass, including agricultural crop wastes and residue,WO 2018/124984 PCT/TR2016/050550 10 15 20 25 30 municipal solid waste, sewage, forestry crops and residue, and industrial and animal residues, animal manure is defined as a primary source of biomass as it is usually disposed into the land. There are some developments present in the known state of the art that have been provide to bioenergy from the biomass of manure and so waiter For example in the Russian Patent document numbered RU2419594C1 within the known state of the art, the invention relates to agricultural production, particularly, to complete treatment and reclamation of animal framing wastes to produce electric and thermal power, circulation water and fertilisers. Liquid phase of over fermented dropping is evaporated to dry concentrated fertiliser. Note here that steam is converted to water to be used for process needs. Portion of homogeneous mass is combusted to clean obtained biogas by passing its through water to produce biomethane to be fed to consumer. Water is saturated with organic substances to be used as liquid fertiliser. Air from production premises is collected to facilitate combusti n of said homogeneous mass with increased heat emission. Residue of combustion is used as a mineral fer ser. Off-gases are cleaned from solid volatile admixtures by passing them through water and saturating with mineral substances for use as mineral fertilisers. Purified off sis used to generate electric power to be fed to green houses. In the Global Patent document numbered WO9325671AI wit the known state of the art, a method of cloning of xylanase clones from an anaerobic rumen fungus including the steps of: (1) cultivation of an anaerobic rumen fungus; (11) isolating total RNA from the culture in step (III), (III) isolating poly A<+> mRNA. from the total RNA referred to in step (Il); (IV) constructing a cDNA expression library; (V) ligating cDNA to a bacteriophage expression vector selected from lambda ZAP, lambda ZAPIL or vectors of similar properties; (VI) screening of xylanase positive recombinant clones in a culture medium incorporating xylan by detection of xylan hydrolysis; and (VII) puri ing xylanase positive recombinantWO 2018/124984 PCT/TR2016/050550 10 15 20 25 clones. There is also provided xylanase positive recombinant clones produced by the above-mentioned method as well as xylanase positive recombinant clones having the following properties: (I) production of xylan clearing zones in a culture containing xylanase cDNA derived from N, patriciarum; (II) having activity in hydrolysis of xylan but having no activity in relation to hydrolysis of CMC or crystalline cellulose, There is also provided various cDNA molecules which may be utilised in the above-mentioned method. In the American Patent document numbered 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, the method comprising the step of administering to the rumen an effective amount of a degradation resistant sulphur source, Despite the fact that disposal of animal manure is advantageous as a soil fertilizer and for harvesting nutrients in feed crops, recent studies show that limited land for disposal of large amount of wastes and lit ed 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 foul odour, harmful pathogens, and noxious gases, which are toxic and harmful to living organisms. Therefore, use of animal manure as a bio-fuel source has become crucial in order to prevent accumulation of wastes and environmental damages. That is why, Bioagumentation - which is a method for the enrichment of specific microorganisms that is used in anaerobic digesters to enhance the yields of hydrolysis, nutrient recovery, and biogas production - used although, some studies have evaluated the bioaugmentation of anaerobic digestion processes with ruminal fluid and anaerobic rumen bacteria.WO 2018/124984 PCT/TR2016/050550 10 15 20 25 30 As it can be understood from the similar mentioned documents above different ‘methods are being used in order to improve a composition to increase the biogas potential for anaerobic digesters. Brief Description of the Invention and its Aims The aim of this invention is improving of biogas potential of anaerobic digesters with using of rumen fungi Another aim of this invention is to gain improvement of biomethane production from animal manure via bioaugmentation using rumen anaerobic fungi In this method, it can be observed that the effects of bioaygmentation of anaerobic rumen fungi in various ratios of inoculums on biogas production of anaerobic digesters fed with animal manure. The highest biogas production was observed in the R2 (15%) digester with a rate of 5500 mL/d, almost 60% of total biogas, due to addition of anaerobic rumen fungi. Anaerobic rumen fungi are more effective on Lentisphaerae, Clostridium, and Methanolinea sp. in terms of the highest biogas production and anaerobic rumen fungi appear to be a promising alternative for improving biogas production from different types of lignocellulosic compounds due to their non-specific extracellular ligninolytic enzymatic system. Detailed Description of the Invention In this invention, all rumen samples comprising of liquid and solids were taken by means of rumen fistulae from a dairy animals (live weight 400-450 kg) with utilizing secret methods by veterinaries. A bovine was older than two years of age and nourished with horse feed roughage, scarcely grass, vegetables, silage and soybean supper amid throughout the summer and winter periods. Rumen samples were gathered at the 6 h in the wake of feeding from the focal part of the rumen. All samples of ruminal liquid were flushed with nitrogen gas (N2) so as to giveWO 2018/124984 PCT/TR2016/050550 10 15 20 25 30 anaerobic conditions in the wake of stacking and fixing. A portion of the samples of rumen liquid were put away at - 20 °C so as to extricate DNA for examination of metagenomic survey of rumen liquid, From that rumen fluid, isolated and cultivated rumen fungi were analysed by using Strain Identification and Phylogenetic Analysis techniques in order to identify species of anaerobic rumen fungi, Isolated 4 species of anaerobic rumen fungi (Orpinomyces sp., Piromyces sp. and Anaeromyces sp., Neocallimastix frontalis) were selected and these species were mixed, After that, ture 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). In order to understand the effect of anaerobic rumen fungi on biogas production, a digester was not biougmentated with anaerobic rumen fungi as a control digester: 0% (RO). Anaerobic digesters fed with animal manure were set up and performed with 900 ml volumes during 40 days at 40°C in order to understand the effect of anaerobic rumen fungi on biogas production. Biogas and biomethane production were measured to evaluate performance of anaerobic digesters. Inhibition effect of the digesters was controlled measurement of VFAs. Finally, Illumina Miseq was used to identify microbial community dynamics and qPCR was used to determine the number of active cells of anaerobic rumen fungi Later, metagenomic data obtained from total purified DNA was elaborately analysed for rumen fungi classification, gene function, First of all, qualified DNA samples were nebulized and into smaller fragments. Then, T4 DNA polymerase, T4 polynucleotide kina Klenow fragment were used to convert overhangs into blunt ends. Adapters were ligated to each DNA fragments upon addition of adenine to 3° end of phosphorylated blunt ends. Ampure beads were used to get rid of short fragments. Later, qualification and quantification of sample libraries were assessed by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System. The libraries were sequence using Illumina HiSeqTM platform. Qualified sequencing reads initially produced by Illumina platform were refined and subjected to de novo assembly via SOAPdenovo2 and Rabbit. Assembled contigs were used to predict genes via MetaGeneMark in order to build project-specific gene catalogue.WO 2018/124984 10 15 20 25 30 After mapping pre-processed reads into IGC database, genes were obtained and added into the gene catalogue. Redundancy was eliminated by using CD-Hit. Finally, BLAST analysis of the gene catalogue with some databases was performed for the purpose of functional and taxonomic annotation. Anaerobic rumen fungi were cultured in complex media by using previously described protocols. Salt solution contained (g/L) KH2P04, 3.0; (NH)2S0, 3.0; NaCl, 6.0; MgSO, 0.6; CaCl, 0.6 were prepared to use in media. Salt solution, 150 ml; centrifuged with rumen fluid. 200 ml; Bactocasitone (Difco), 10 g: yeast extract (Oxoid), 2.5 g; NaHCO, 6 g; L-cysteine, HCI, 1 g: fructose, 2 g; xylose, 2 g: cellobiose, 2 g: resazurin solution (0-1 %, w/v), 8 g; trace elements solution, 10 ml; haemin solution, 10 ml and deionized water to 900 ml were added into media The media was then autoclaved for 20 min at 115 °C. After autoclaving the media, vitamin solution 0.1 % (v/v) was added. Antibiotics solution 0.1 % (viv) containing penicillin (5 g/L), streptomycin (5 g/L), neomycin (5 g/L) and chloramphenicol (5 g/L) was also added to the isolation media to suppress bacterial growth. After preparing the media, all cultures were incubated under CO2 at 3! > during a week in order to reproduce rumen fungi. When the anaerobic rumen fungi reached optical density, they were transferred anaerobically into batch reactors to bioaugment the anaerobic digestion of animal manure. Fungi species isolated from ruminal fluid and cow manure were identified by fungal DNA sequencing through strain identification and phylogenetic analysis. ‘Complete internal transcribed spacer (ITS: partial 18S, complete ITS 1, 5.88, ITS 2, and partial 28S) was utilized to perform strain identification and phylogenetic analysis of isolated anaerobic fungi. Primer pairs ITSI (5’- TCC GTA GGT GAA CCT GCG G-3’/ITS4 (S’- TCC TCC GCT TAT TGA TAT GC-3’) and NLI 5°- GCA TAT CAA TAA GCG GAG GAA AAG-3')/NL4 (5’-GGT CCG TGT TTC AAG ACG G-3°) were used to amplify D1 and D2 domain at 5° end of large- subunit (LSU) ribosomal DNA, respectively. As suggested by Hibbett, differentWO 2018/124984 10 15 20 25 30 regions of the rRNA locus were demarcated using the consensus sequences CATTAICAACTTCAG (end of 18S/start of 5.88) and GAGTGTCATTA/ TIGACCTCAAT (end of 5.8S/start of 28S) in a consistent manner. Sequence alignment using MAFFT and phylogenetic analysis using Mr Bayes from Geneious v6 bioinformatics package were perform to reconstruct phylogenetic analysis. Impact of anaerobic rumen fungi on biogas and biomethane production was determined using anaerobie bacth reactors. Granular sludge cultivated in a laboratory-scale (900 mL) anaerobic sequenced batch reactor (ASBR) was utilized as the methanogenic inoculums. The ASBR was performed at 40°C, and glucose and acetate (80%:20%, calculated as Chemical Oxygen Demand) were utilized as the feedstock at an organic loading rate of | g Chemical Oxygen Demand (COD) W(L-day), Different initial concentration of anaerobic rumen fungi, and 3 g VS/L of methanogenic sludge were used in batch experiments performed at 40°C. Four types of anaerobic rumen fungi were blended at equivalent rate. The culture medium comprising of anaerobic rumen fungi (Orpinomyces sp., Piromyces sp., Anaeromyces sp. and Neocallimastix frontalis) were utilized at various inoculum ratios: 0 % (RO-Control), RI (5%), R2 (15%), R3 (20%) (v/v). Control reactor ‘was not enriched by anaerobic rumen fungi, Fungal inoculum was added just once at the beginning of the experiment. In addition to manure, granular sludge and water were added to get the ideal conditions. After stacking and fixing, anaerobic environment was provided by nitrogen ga: all reactors. Miligas Counter (Ritter Digital Counter, U.S.A.) was used to measure the gas outputs. pH was adjusted to 7-7.4 and alkalinity was added to maintain pH. All reactors were carried out for 40 days. The buffer contained (per L): 1.0 g of NHACI, 0.4 g of K2HPO4.3H20, 0.2 g of MgCl2. 6H20, 0.08 g of CaCI2.2H20, 10 ml of trace element solution, and 10 ml of stock vitamin solution, A stock trace element and vitamin solution were prepared. After preparing, they were adjusted in accordance with the procedure described by our previous studyWO 2018/124984 PCT/TR2016/050550 10 All chemical analysis for alkalinity, total solids (T'S), and volatile solids (VS) were performed in accordance with standard methods. The biogas production was 1 Counter, U.S.A.) in both monitored by using Milligas counters (Ritter Di SBRs, Gas composition and VFA concentration were measured using gas chromatography with a flame ionization detector (Perichrom, France and Agilent Technologies 6890N, USA, respectively) and Elite-FFAP column (30 m X 0,32 mm). The set point of the oven was 100 °C and the maximum temperature of the inlet was 240 °C. In addition, helium gas was utilized as a carrier gas at a rate of 0.8 mL/minWO 2018/124984 PCT/TR2016/050550 CLAIMS 1.) A method for improvement biogas potential of anaerobic digesters with using composite including rumen fungi characterized in that the said 5 method comprises the following steps, - selecting of 4 isolated species of anaerobic rumen fungi with ratio of 30% Orpinomyces sp., 25% Piromyces sp. and 25 % Anaeromyces sp., 20% Neocallimastix frontalis to mixture 10 = adding these 4 species added in the anaerobic digesters fed with animal manure at different inoculums ratios: %5 (R1), 15% (R2), 20% (R3) i)INTERNATIONAL SEARCH REPORT Teterational aplication No PCT/TR2016/050550 Fae ae ADD. atonal Patent Cassiioaton 10) or tobothnatonaelaetioaon and IPC clap cl2N Cl2M ‘Urimum documentation seared (lssfcaonajstem flowed by danaioaion a7 Bam) Dosurrenaton searched Sher an ranrum docurentaion othe Hert Wal auahGasumnlsSre waded ithe hails Seana Teron data bass sonahed Garg he Ep0-Internal, WPI Data oda base ard, where prastoaile search Tne vse) {DOCUMENTS CONSIDERED TO BE RELEVANT ‘aepo07" ‘Caton of dooument, wth Indeaton, where appropri, ote relevant pata x JINDRICH PROCHAZKA ET AL: by rumen fungi", pages 343-351, xP055392528, DE ISSN: 1618-0240, DOI: 10. 1002/e1sc.201100076 the whole document ENGINEERING IN LIFE SCIENCES, vol. 12, no. 3, 27 June 2012 (2012-06-27), "Enhanced 1 biogas yield from energy crops with rumen anaerobic fungi : Increase of biogas yield X] Ferherdoourents ar inte inthe sontnuation of Box ©. ‘See patent amily annex. document dering the general state fhe art which not consires ‘be of parol elvance aor appaton or patent but published on or ater th international “ng date “L eequmort wish may tom dts on pry, info) o wich i peo reason (or speed) “P dgeumentpubahed pr to the stem! ng date bt ater han ‘he peony date Samed lator document published ater he intmatonal ling date or pry ‘ton not sonst wth he sppleation bt ote to understand {hepincipl or feory underngte lventon 7% oon of paris reac ep when te cosurent “Y dcument of paola relevance! the otimed invention cannot be Being obvious toa parson "2° document member ofthe eame patent ay 23 August 2017 ‘Date of maling of he wieranal seek Topo 01/09/2017 European Patent Oe, PB. S818 Ptentinan2 NL“ 2200 HV Riewhe Tel ato) 1483000, Fax (281-70) 340-5016 Boeker, Ruth Fa POTTSAaIO exons hon) Roa DH page 1 of 2INTERNATIONAL SEARCH REPORT Teterational aplication No microorganisms for anaerobic bioconversion of lignocellulosic biomass", BIORESOURCE TECHNOLOGY. , vol. 128, 2013, pages 738-744, P055400736, 6B ISSN: 0960-8524, DOI: 10. 1016/j.biortech.2012.11.073 the whole document PCT/TR2016/050550 ‘Ganon DOCUMENTS CONSIDERED TOE RELEVANT ‘category | Otten of donunent wth heaton wher eppopiate, fe relevent pasa Reever caimno A ZHENG-BO YUE ET AL: "Application of rumen 1 Fam POTTSH210 foninaan Sacer Seah TB page 2 of 2