Molecular identification of anaerobic fungi isolated from buffalo rumen with their growth rate, cellulase enzyme activity, and fermentation products characteristics




Abstract. Agustina S, Wiryawan KG, Suharti S, Meryandini A. 2022. Molecular identification of anaerobic fungi isolated from buffalo rumen with their growth rate, cellulase enzyme activity, and fermentation products characteristics. Biodiversitas 23: 6448-6455. Anaerobic fungi are essential microbes in the degradation process of feed fiber in the rumen with the ability to produce fibrolytic enzymes and rhizoids that can penetrate feed particles. The activity of cellulase enzymes in fungi is influenced by several factors such as the type of feed and livestock used as a source of isolate. Therefore, this research aimed to analyze the types of anaerobic fungi isolated from buffalo based on their DNA nitrogenous bases composition and also to evaluate the growth rate, cellulase enzyme activity, and fermentation product concentration of anaerobic fungi. The growth rate of fungi was examined by measuring the biomass formed, while cellulase enzyme activity was carried out using CMC, Avicel, and Filter Paper as substrates. The fungal fermentation products were analyzed using the HPLC and GC methods. The results showed that the fungi isolated from buffalo rumen were closely related to anaerobic fungi type Piromyces sp., Caecomyces sp., and Neocallimastix frontalis, with different growth rate, cellulase enzyme activity, and the fermentation products concentration in each type of anaerobic fungi. Therefore, it can be concluded that anaerobic fungi isolated from buffalo rumen can degrade cellulose. It was also discovered that fungi Neocallimastix frontalis had a higher growth rate, cellulase enzyme activity (CMCase, avicelase, and FPase), and fermentation products than Piromyces sp. and Caecomyces sp.


Agustina S, Wiryawan KG, Suharti S, Meryandini A. 2022. The enrichment process and morphological identification of anaerobic fungi isolated from buffalo rumen. BIODIVERSITAS 23: 469-477. DOI: 10.13057/biodiv/d230150.
Asih NK, Harmita, Maggadani BP. 2017. Pengembangan metode analisis agen pengatur keasaman pakan ternak menggunakan metode Kromatografi Cair Kinerja Tinggi. Pharmaceutical Sciences and Research (PSR) 3: 155-164. DOI: 10.7454/psr.v4i3.3771.
Boxma B, Voncken F, Jannink S, van Alen, Akhmanova A, van Weelden SWH, van Hellemond JJ, Ricard G, Huynen M, Tielens AGM, Hackstein JHP. 2004. The anaerobic chytriomycete fungus Piromyces sp. E2 produces ethanol via pyruvate: formate lyase and an alcohol dehydrogenase E. Molecular Microbiology 51: 1389-1399. DOI: 10.1046/j.1365-2958.2003.03912.x.
Budi KL, Wijanarka W, Kusdiyantini E. 2018. Aktivitas enzim selulase yang dihasilkan oleh bakteri Serratia marcescens pada substrat jerami. Jurnal Akademika Biologi (JAB) 7: 35-42.
Cheng YF, Jin W, Mao SY, Zhu WY. 2013. Production of citrate by anaerobic fungi in the presence of co-culture methanogens as revealed by 1H NMR spectrometry. AJAS 26: 1416-1423. DOI: 10.5713/ajas.2013.13134.
Dagar SS, Kumar S, Griffith GW, Edwards JE, Callaghan TM, Singh R, Nagpal AK, Puniya AK. 2015. A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius). Fungal Biol. 119:731-737. DOI: 10.1016/j.funbio.2015.04.005.
Dini IR, Munifah I. 2014. Produksi dan karakterisasi enzim selulase ekstrak kasar dari bakteri yang diisolasi dari limbah rumput laut. JTIP Indonesia 6: 69-75. DOI: 10.17969/jtipi.v6i3.2315.
Dollhofer V, Podmirseg SM, Callaghan TM, Griffith GW, Fliegerova K. 2015. Anaerobic fungi and their potential for biogas production. Adv. Biochem. Eng. Biotechnol. 151:41-61. DOI: 10.1007/978-3-319-21993-6_2.
Edwards JE, Forster RJ, Callaghan TM, Dollhofer V, Dagar SS, Cheng Y, Chang J, Kittelmann S, Fliegerova K, Puniya AK, Henske JK, Gilmore SP, O'Malley MA, Griffith GW, Smidt H. 2017. PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: insights, challenges and opportunities. Frontiers in Microbiology 8:1657. DOI: 10.3389/fmicb.2017.01657.
Edwards JE, Hermes GDA, Kittelman S, Nijsse B, Smidt H. 2019. Assessment of the accuracy of high-throughut sequencing of the ITS1 region of Neocallimastigomycota for community composition analysis. Frontiers in Microbiology 10: 1-11. DOI: 10.3389/fmicb.2019.02370.
Eida MF, Matter IA, El Zaher. 2016. Isolation and characterization of cellulolytic and hemicellulolytic fungi from salt affected soils and compost. JPBS 3: 164-170.
Gruninger RJ, Puniya AK, Callaghan TM, Edwards JE, Youssef N, Dagar SS, Fliegerova K, Griffith GW, Forster R, Tsang A, McAllister T, Elshahed MS. 2014. Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential. FEMS Microbiol. Ecology 90: 1-17. DOI: 10.1111/1574-6941.12383.
Hakim L, Kurniatuhadi R, Rahmawati. 2020. Karakteristik fisiologis jamur halofilik berdasarkan faktor lingkungan dari sumur air asin di Desa Suak, Sintang, Kalimantan Barat. BIOMA: Jurnal Biologi Makassar 5: 227-232. DOI: 10.20956/bioma.v5i2.11299.
Hanafy RA, Elshahed MS, Liggenstoffer AS, Griffith GW, Youssef NH. 2017. Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic fungus from the feces of cattle and sheep. Mycologia 109: 231-243. DOI: 10.1080/00275514.2017.1317190.
Hanafy RA, Elshahed MS, Youssef NH. 2018. Feramyces austinii, gen. nov., sp. nov., an anaerobic gut fungus from rumen and fecal samples of wild Barbary sheep and fallow deer. Mycologia 110: 513-525. DOI: 10.1080/00275514.2018.1466610.
Hanafy RA, Lanjekar VB, Dhakephalkar PK, Callaghan TM, Dagar SS, Griffith GW. 2020. Seven new Neocallimastigomycota genera from wild, zoo-housed, and domesticated herbivores greatly expand the taxonomic diversity of the phylum. Mycologia 112: 1212-1239. DOI: 10.1080/00275514.2019.1696619.
Hanafy RA, Youssef NH, Elshahed MS. 2021. Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the faeces of a wild blackbuck antelope. International Journal of Systematic and Evolutionary Microbiology 71:004832. DOI: 10.1099/ijsem.0.004832.
Hassan R, El-Kadi S, Sand M. 2015. Effect some organic acid on some fungal growth and their toxins production. International Journal of Advances in Biology 2: 1-11.
Hess M, Paul SS, Puniya AK, Giezen MVD, Shaw C, Edwards JE, Fliegerova K. 2020. Anaerobic fungi: past, present, and future. Frotiers in Microbiology 11: 1-18. DOI: 10.3389/fmicb.2020.584893.
Ho YW, Barr DJS. 1995. Clasification of anaerobic gut fungi from herbivores with emphasis on rumen fungi from Malaysia. Mycologia 87: 655-677. DOI: 10.2307/3760810.
Ho YW, Wong MVL, Abdullah N, Kudo H, Jalaludin S. 1996. Fermentation activities of some new species of anaerobic rumen fungi from Malaysia. J. Gen. Appl. Microbiol. 42: 51-59. DOI: 10.2323/jgam.42.51.
Idiawati N, Harfinda EM, Arianie L. 2014. Produksi enzim selulase oleh Aspergillus niger pada ampas sagu. Jurnal Natur Indonesia 16: 1-9. DOI: 10.31258/jnat.16.1.1-9.
Jimenez HR, Edwards JE, Sanderson R, Kingston-Smith AH, McEwan NR, Theodorou MK. 2020. Cut-lenghts of perennial ryegrass leaf-blades influences in vitro fermentation by anaerobic fungus Neocallimastix frontalis. Microorganisms 8: 1774-1788. DOI: 10.3390/microorganisms8111774.
Jin W, Cheng YF, Mao SY, Zhu WY. 2011. Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane. Bioresource Technology 102: 7925-7931. DOI: 10.1016/j.biortech.2011.06.026.
Joblin KN, Naylor GE. 1993. Inhibition of the rumen anaerobic fungus Neocallimastix frontalis by fermentationn products. Applied Microbiology 16: 254-256. DOI: 10.1111/j.1472-765X.1993.tb01412.x.
Joshi A, Lanjekar VB, Dhakephalkar PK, Callaghan TM, Griffith GW, Dagar SS. 2018. Liebetanzomyces polymorphus gen. et sp. nov., a new anarobic fungus (Neocallimastigomycota) isolated from the rumen of a goat. MycoKeys 40: 89-110. DOI: 10.3897/mycokeys.40.28337.
Kirn F, Siddiqa A, Noreen S, Khalid AM, Ishad M. 2018. Optimized production of cellulase (CMCase). International Journal of Applied Biology and Forensics 2: 194-202.
Kittelmann S, Naylor GE, Koolaard JP, Janssen PH. 2012. A proposed taxonomy of anaerobic fungi (Class Neocallimastigomycetes) suitable for Large-Scale Sequence-Based community structure analysis. PloS One 7: e368666. DOI: 10.1371/journal.pone.0036866.
Lange L, Barret K, Pilgaard B, Gleason F, Tsang A. 2019. Enzymes of early-diverging, zoosporic fungi. Appl. Microbiol. and Biotechnol. 103: 6885-6902. DOI: 10.1007/s00253-019-09983-w.
Leger MM, Gawryluk RMR, Gray MW, Roger AJ. 2013. Evidence for hydrogenosomal-type anaerobic ATP generation pathway in Acanthamoeba castellanii. PLOS ONE 8: e69532. DOI: 10.1371/journal.pone.0069532.
Li J, Gu S, Zhao Z, Chen B, Liu Q, Sun T, Sun W, Tian C. 2019. Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila. Fungal Biology and Biotechnology 6: 21-32. DOI: 10.1186/s40694-019-0083-8.
Li Y, Jin W, Cheng Y, Zhu W. 2016. Effect of the associated methanogen Methanobrevibacter thaueri on the dynamic profile of end and intermediate metabolites of anaerobic fungus Piromyces sp. F1. Current Microbiology 73: 434-441. DOI: 10.1007/s00284-016-1078-9.
Li Y, Jin W, Mu C, Cheng Y, Zhu W. 2017. Indigenously associated methanogens intensified the metabolism in hydrogenosomes of anaerobic fungi with xylose as substrate. Journal of Basic Microbiology 57: 933-940. DOI: 10.1002/jobm.201700132.
Li Y, Meng Z, Xu Y, Shi Q, Ma Y, Aung M, Cheng Y, Zhu W. 2021. Interactions between anaerobic fungi and methanogen in the rumen and their biotechnological potential in biogas production from lignocellulosic materials. Microorganisms 9: 190-206. DOI: 10.3390/microorganisms9010190.
Lillington SP, Chrisler W, Haitjema CH, Gilmore SP, Smallwood CR, Shutthanandan V, Evans JE, O’Malley MA. 2021. Cellulosome localization patterns vary across life stages of anaerobic fungi. mBio 12: e0083221. DOI: 10.1128/mbio.00832-21.
Lowe SE, Theodorou MK, Trinci APJ. 1987. Growth and fermentation of an anaerobic rumen fungus on various carbon sourcs and effect of temperature on development. Applied and Environmental Microbiologi 53: 1210-1215. DOI: 10.1128/aem.53.6.1210-1215.1987.
Ma J, Zhong P, Li Y, Sun Z, Sun X, Aung M, Hao L, Cheng Y, Zhu W. 2022. Hydrogenosome, pairing anaerobic fungi and H2-utilizing microorganisms based on metabolic ties to facilitate biomass utilization. Journal of Fungi 8: 338-358. DOI: 10.3390/jof8040338.
Melati I, Mulyasari, Sunarno MTD, Bintang M, Kurniasih T. 2014. Produk enzim selulase dari bakteri TS2b yang diisolasi dari rumput laut dan pemanfaatannya dalam menghidrolisis kulit ubi kayu dan daun ubi kayu sebagai bahan baku pakan ikan. Jurnal Riset Akuakultur 9: 263-270. DOI: 10.15578/jra.9.2.2014.263-270.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31:426-428. DOI: 10.1021/ac60147a030.
Nababan M, Gunam IBW, Wijaya IMM. 2019. Produksi enzim selulase kasar dari bakteri selulolitik. Jurnal Rekayasa dan Manajemen Agroindustri 7: 190-199. DOI: 10.24843/JRMA.2019.v07.i02.p03.
Nagpal R, Puniya AK, Sehgal JP, Singh K. 2011. In vitro fibrolytic potential of anaerobic rumen fungi fron ruminants and non-ruminant herbivores. Mycoscience 52:31-38. DOI: 10.1007/S10267-010-0071-6.
Nagpal R, Puniya AK, Singh K. 2009. In vitro fibrolytic activity of the anaerobic fungus Caecomyces sp., immobilized in alginate beads. Journal of Animal and Feed Sciences 18: 758-768. DOI: 10.22358/jafs/66451/2009.
Nielsen BB, Zhu WY, Dhanoa MS, Trinci APJ, Theodorou MK. 2002. Comparison of the growth kinetics of anaerobic gut fungi on wheat straw in batch culture. Anaerobe 8: 216-222. DOI: 10.1006/anae.2002.0432.
Olukunle OF, Ayodeji AO, Akinloye PO. 2021. Carboxymethyl cellulase (CMCase) from UV-irradiation mutated Bacillus cereus FOA-2 cultivated on plantain (Musa parasidiaca) stalk-based medium: production, purification and characterization. Scientific African 11: e00691. DOI: 10.1016/j.sciaf.2020.e00691.
Panahi HKS, Dehhaghi M, Guillemin GJ, Gupta VK, Lam SS, Aghbashlo M, Tabatabaei M. 2022. A comprehensive review on anaerobic fungi applications in biofuels porduction. Science of The Total Environment 829: 154521. DOI: 10.1016/j.scitotenv.2022.154521.
Paul SS, Bu D, Xu J, Hyde KD, Yu Z. 2018. A phylogenetic census of global diversity of gut anaerobic fungi and a new taxonomy framework. Fungal Diversity 89: 253-266. DOI: 10.1007/s13225-018-0396-6.
Purkan, Purnama HD, Sumarsih S. 2015. Produksi enzim selulase dari aspergillus niger menggunakan sekam padi dan ampas tebu sebagai induser. JURNAL ILMU DASAR 16: 95-102. DOI: 10.19184/jid.v16i2.2768.
Rabee AE, Forster RJ, Elekwachi CO, Kewan KZ, Sabra EA, Shawket SM, Mahrous HA, Khamiss OA. 2019. Community structure and fibrolytic activities of anaerobic rumen fungi in dromedary camels. J. Basic. Microbiol. 59: 101-110. DOI: 10.1002/jobm.201800323.
Rahman A. 1992. Teknologi Fermentasi. Jakarta (ID): Arcan.
Rakhmana S, Saryono, Nugroho TT. 2015. Ekstrakdi DNA dan amplifikasi ITS rDNA isolat fungi endofit LBKURCC67 umbi tanaman dahlia (Dahlia variabilis). JOM FMIPA 2: 145-151.
Reyes AA, Diaz-Hernandez A, Gracida J, Garcia-Almendarez BE, Escamilla-Garcia M, Arredondo-Ochoa T, Regalado C. 2019. Enhanced performace of immobilized xylanase/filter paper-ase on a magnetic chitosan support. Catalysts 9: 966-975. DOI: 10.3390/catal9110966.
Saputra KA, Pontoh JS, Momuat LI. 2015. Analisis kandungan asam organik pada beberapa sampel gula aren. JURNAL MIPA UNSRAT ONLINE 4: 69-74. DOI: 10.35799/jm.4.1.2015.6908.
Saye LMG, Navaratna TA, Chong JPJ, O’Malley MA, Theodorou MK, Reilly M. 2021. The anaerobic fungi: challenges and oppurtunities for industrial lignocellulosic biofuel production. 9: 694-722. DOI: 10.3390/microorganisms9040694.
Schulz D, Psenkova-Profausova I, Cervena B, Procter M, Neba TF, Modry D, Petrzelkova KI, Qablan MA. 2021. Occurrence and diversity of anaerobic gut fungi in wild forest elephants and buffaloes inhabiting two separated forest ecosystems in Central West Africa. J. of Vertebrate Biology 71: 1-14. DOI: 10.25225/jvb.21033.
Sembiring A. 2019. Isolasi dan uji aktivitas bakteri penghasil selulase asal tanah kandang sapi. BIOSEL 8: 21-28. DOI: 10.33477/bs.v8i1.843.
Sirohi SK, Choudhury PK, Dagar SS, Puniya AK, Singh D. 2013. Isolation, characterization and fibre degradation potential of anaerobic rumen fungi from cattle. Annals of Microbiology 63: 1187-1194. DOI: 10.1007/s13213-012-0577-6.
Solomon KV, Haitjema CH, Henske JK, Gilmore SP, Borges-Rivera D, Lipzen A, Brewer HM, Purvine SO, Wright AT, Theodorou MK, Grigoriev IV, Regev A, Thompson DA, O’Malley MA. 2016. Early-branching gut fungi posses a large, comprehensive array of biomass-degrading enzymes. Science 351: 1192-1195. DOI: 10.1126/science.aad1431.
Stabel M, Hanafy RA, Schweitzer T, Greif M, Aliyu H, Flad V, Young D, Lebuhn M, Elshahed MS, Ochsenreither K, Youssef NH. 2020. Aestipascuomyces dupliciliberans gen. nov, so. Nov., the first cultured representative of the uncultured SK4 Clade from Aoudad sheep and alpaca. Microorganism 8: 1734-1749. DOI: 10.3390/microorganisms8111734.
Taurisia PP, Proborini MW, Nuhantoro I. 2015. Pengaruh media terhadap pertumbuhan dan biomassa cendawan Alternaria alternata (Fries Keissler. JURNAL BIOLOGI UDAYANA 19: 30-33.
Teunissen MJ, Op den Camp HJM, Orpin CG, Huis in t Veld JHJ, Vogels GD. 1991 Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium. Journal of General Microbiology 137: 1401-14908. DOI: 10.1099/00221287-137-6-1401.
Ulfa M, Faridah E, Sumardi, Lee SS, Mansor P, Ie Roux C, Galiana A. 2019. Identifikasi molekuler jenis – jenis jamur mikoriza ekto yang berasosiasi dengan dipterokarpa di hutan hujan tropika sekunder. Jurnal Wana Tropika 9: 1-14.
Vrabl P, Schinagl CW, Artmann DJ, Heiss B, Burgstaller W. 2019. Fungal growth in batch culture - what we could benefit if we start looking closer. Frontiers in Microbiology 10: 2391.DOI: 10.3389/fmicb.2019.02391.
Wang X, Liu X, Groenewld JZ. 2017. Phylogeny of anaerobic fungi (phylum Neocallimastigomycota), with contribution from yak in China. Antonie Van Leeuwunhoek 110: 87-103. DOI: 10.1007/s10482-016-0779-1.
Yanti A, Mursiti S, Widiarti N, Nurcahyo B, Alauhdin M. 2019. Optimalisasi metode penentuan kadar etanol dan metanol pada minuman keras oplosan menggunakan Komatografi Gas (KG). Indonesian Journal of Chemical Science 8: 53-59. DOI: 10.15294/ijcs.v9i2.33173.

Most read articles by the same author(s)

1 2 > >>