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Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by 1H NMR Spectrometry

  • Cheng, Yan Fen (Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University) ;
  • Jin, Wei (Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University) ;
  • Mao, Sheng Yong (Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University) ;
  • Zhu, Wei-Yun (Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University)
  • Received : 2013.03.04
  • Accepted : 2013.05.26
  • Published : 2013.10.01

Abstract

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and ${\alpha}$-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and ${\alpha}$-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

Keywords

References

  1. Akhmanova, A., F. G. J. Voncken, K. M. Hosea, H. Harhangi, J. T. Keltjens, H. J. M. op den Camp, G. D. Vogels, and J. H. P. Hackstein. 1999. A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism. Mol. Microbiol. 32:1103-1114. https://doi.org/10.1046/j.1365-2958.1999.01434.x
  2. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296.
  3. Bauchop, T. and D. O. Mountfort. 1981. Cellulose fermentation by a rumen anaerobic fungus in both the absence and the presence of rumen methanogens. Appl. Environ. Microbiol. 42:1103-1110.
  4. Beckonert, O., H. C. Keun, T. M. Ebbels, J. Bundy, E. Holmes, J. C. Lindon, and J. K. Nicholson. 2007. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat. Protoc. 2:2692-2703. https://doi.org/10.1038/nprot.2007.376
  5. Boxma, B., F. Voncken, S. Jannink, T. van Alen, A. Akhmanova, S. W. van Weelden, J. J. van Hellemond, G. Ricard, M. Huynen, A. G. Tielens, and J. H. Hackstein. 2004. The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate: formate lyase and an alcohol dehydrogenase E. Mol. Microbiol. 51:1389-1399. https://doi.org/10.1046/j.1365-2958.2003.03912.x
  6. Carrieri, D., K. McNeely, A. C. De Roo, N. Nennette, I. Pelczer, and G. C. Dismukes. 2009. Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation. Magn. Reson. Chem. 47:S138-S146. https://doi.org/10.1002/mrc.2420
  7. Cheng, Y. F., J. E. Edwards, G. G. Allison, W. Y. Zhu, and M. K. Theodorou. 2009. Diversity and activity of enriched ruminal cultures of anaerobic fungi and methanogens grown together on lignocellulose in consecutive batch culture. Bioresour. Technol. 100:4821-4828. https://doi.org/10.1016/j.biortech.2009.04.031
  8. Cheng, Y. F., S. Y. Mao, C. X. Pei, J. X. Liu, and W. Y. Zhu. 2006. Detection and diversity analysis of rumen methanogens in co-cultures with anaerobic fungi. Acta Microbiologica Sinica 46: 879-883.
  9. Chikayama, E., M. Suto, T. Nishihara, K. Shinozaki, T. Hirayama, and J. Kikuchi. 2008. Systematic NMR analysis of stable isotope labeled metabolite mixtures in plant and animal systems: coarse grained views of metabolic pathways. PLoS ONE 3: e3805. https://doi.org/10.1371/journal.pone.0003805
  10. Grivet, J. P. 2001. NMR and microorganisms. Curr. Issues Mol. Biol. 3:7-14.
  11. Grivet, J. P. and A. M. Delort. 2009. NMR for microbiology: In vivo and in situ applications. Prog. Nucl. Magn. Reson. Spectrosc. 54:1-53. https://doi.org/10.1016/j.pnmrs.2008.02.001
  12. Hungate, R. E. 1982. Methane formation and cellulose digestion biochemical ecology and microbiology of the rumen ecosystem. Experimenta 38:189-192. https://doi.org/10.1007/BF01945072
  13. Jin, W., Y. F. Cheng, S. Y. Mao, and W. Y. Zhu. 2011. Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane. Bioresour. Technol. 102:7925-7931. https://doi.org/10.1016/j.biortech.2011.06.026
  14. Joblin, K. N., G. E. Naylor, and A. G. Williams. 1990. Effect of Methanobrevibacter smithii on xylanolytic activity of anaerobic ruminal fungi. Appl. Environ. Microbiol. 56:2287-2295.
  15. Kwon, M., J. Song, J. K. Ha, H. S. Park, and J. Chang. 2009. Analysis of functional genes in carbohydrate metabolic pathway of anaerobic rumen fungus Neocallimastix frontalis PMA02. Asian-Aust. J. Anim. Sci. 22:1555-1565. https://doi.org/10.5713/ajas.2009.80371
  16. Liu J. H. 2009. Metabolic profiles of natural co-cultures of anaerobic fungi and methanogens in vitro and the production of lactate. Ma.D. Thesis, Nanjing Agricultural University, Nanjing, China.
  17. Marvin-Sikkema, F. D., A. J. Richardson, C. S. Steward, J. C. Gottschal, and R. A. Prins. 1990. Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi. Appl. Environ. Microbiol. 56:3793-3797.
  18. Mountfort, D. O., R. A. Asher, and T. Bauchop. 1982. Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri. Appl. Environ. Microbiol. 44:128-134.
  19. Nakashimada, Y., K. Srinivasan, M. Murakami, and N. Nishio. 2000. Direct conversion of cellulose to methane by anaerobic fungus Neocallimastix frontalis and defined methanogens. Biotechnol. Lett. 22:223-227. https://doi.org/10.1023/A:1005666428494
  20. Orpin, C. G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J. Gen. Microbiol. 91:249-262. https://doi.org/10.1099/00221287-91-2-249
  21. O'Fallon, J. V., R. W. Wright, and R. E. Calza. 1991. Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188. J. Biochem. 274:595-599.
  22. Pham, L. H., J. Vater, W. Rotard, and C. Mugge. 2005. Identification of secondary metabolites from Streptomyces violaceoruber TU22 by means of on-flow LC-NMR and LC-DAD-MS. Magn. Reson. Chem. 43:710-723. https://doi.org/10.1002/mrc.1633
  23. Teunissen, M. J., E. P. W. Kets, H. J. M. Op den Camp, J. H. J. Huis in't Veld, and G. D. Vogels. 1992. Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities. Arch. Microbiol. 157:176-182.
  24. Theodorou, M. K., D. R. Davies, and C. G. Orpin. 1995. Nutrition and survival of anaerobic fungi. In: Anaerobic fungi: Biology, Ecology and Function (Ed. D. O. Mountfort and C. G. Oprin). Marcel Dekker, New York, USA. pp. 107-128.
  25. Theodorou, M. K., G. Mennim, D. R. Davies, W. Y. Zhu, A. P. Trinci, and J. L. Brookman. 1996. Anaerobic fungi in the digestive tract of mammalian herbivores and their potential for exploitation. Proc. Nutr. Soc. 55:913-926. https://doi.org/10.1079/PNS19960088

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