In Vitro Response of Rumen Microbiota to the Antimethanogenic Red Macroalga Asparagopsis taxiformis

  • Lorenna Machado
  • Nigel Tomkins
  • Marie Magnusson
  • David J. Midgley
  • Rocky de Nys
  • Carly P. Rosewarne
Host Microbe Interactions

Abstract

The red macroalga Asparagopsis taxiformis has been shown to significantly decrease methane production by rumen microbial communities. This has been attributed to the bioaccumulation of halogenated methane analogues produced as algal secondary metabolites. The objective of this study was to evaluate the impact of A. taxiformis supplementation on the relative abundance of methanogens and microbial community structure during in vitro batch fermentation. Addition of A. taxiformis (2% organic matter) or the halogenated methane analogue bromoform (5 μM) reduced methane production by over 99% compared to a basal substrate-only control. Quantitative PCR confirmed that the decrease in methane production was correlated with a decrease in the relative abundance of methanogens. High-throughput 16S ribosomal RNA gene amplicon sequencing showed that both treatments reduced the abundance of the three main orders of methanogens present in ruminants (Methanobacteriales, Methanomassiliicoccales and Methanomicrobiales). Shifts in bacterial community structure due to the addition of A. taxiformis and 5 μM bromoform were similar and concomitant with increases in hydrogen concentration in the headspace of the fermenters. With high potency and broad-spectrum activity against rumen methanogens, A. taxiformis represents a promising natural strategy for reducing enteric methane emissions from ruminant livestock.

Keywords

Methane Rumen Livestock Seaweed Bromoform 

Supplementary material

248_2017_1086_MOESM1_ESM.docx (14 kb)
Table S1Mean gas production parameters after 72 h incubation (n = 3) (DOCX 13 kb)
248_2017_1086_MOESM2_ESM.xlsx (1.1 mb)
Table S2OTU table with FASTA sequences and taxonomic lineages for experimental and technical replicates. Samples are labelled as treatment_time.Nx, where N is 1, 2 or 3 to denote the experimental replicate; and x is a or b to denote the technical replicate. OTU labelling corresponds to sequences deposited in NCBI Genbank under accession numbers KT168398 – KT174433. (XLSX 1135 kb)
248_2017_1086_Fig4_ESM.gif (85 kb)
Fig. S1

Principal co-ordinate analysis plot showing relationships between microbial communities under different experimental conditions, based on unweighted UniFrac metric of β diversity. BF1: 1 μM bromoform; BF5: 5 μM bromoform Asp: 2% A. taxiformis; BCM: 5 μM bromochloromethane. (GIF 85 kb)

248_2017_1086_MOESM3_ESM.tif (198 kb)
High resolution image (TIFF 197 kb)

References

  1. 1.
    Kamra D (2005) Rumen microbial ecosystem. Curr Sci 89:124–135Google Scholar
  2. 2.
    Sirohi SK, Singh N, Dagar SS, Puniya AK (2012) Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem. Appl Microbiol Biotechnol 95:1135–1154CrossRefPubMedGoogle Scholar
  3. 3.
    Wright A-DG, Klieve AV (2011) Does the complexity of the rumen microbial ecology preclude methane mitigation? Anim Feed Sci Technol 166:248–253CrossRefGoogle Scholar
  4. 4.
    Janssen PH, Kirs M (2008) Structure of the archaeal community of the rumen. Appl Environ Microbiol 74:3619–3625CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Costa KC, Leigh JA (2014) Metabolic versatility in methanogens. Curr Opin Biotechnol 29:70–75CrossRefPubMedGoogle Scholar
  6. 6.
    Liu Y, Whitman WB (2008) Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci 1125:171–189CrossRefPubMedGoogle Scholar
  7. 7.
    IPCC (2007) Climate change 2007: The physical science basis. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL (eds) Contribution of working group I to the fourth assessment report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, p 1009Google Scholar
  8. 8.
    Ripple WJ, Smith P, Haberl H, Montzka SA, McAlpine C, Boucher DH (2014) Ruminants, climate change and climate policy. Nat Clim Chang 4:2–5CrossRefGoogle Scholar
  9. 9.
    Borrel G, O’Toole PW, Harris HM, Peyret P, Brugère J-F, Gribaldo S (2013) Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis. Genome Biol Evol 5:1769–1780CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hedderich R, Whitman WB (2006) Physiology and biochemistry of the methane-producing Archaea. The prokaryotes 2:1050–1079CrossRefGoogle Scholar
  11. 11.
    Ellermann J, Hedderich R, Böcher R, Thauer RK (1988) The final step in methane formation. Eur J Biochem 172:669–677CrossRefPubMedGoogle Scholar
  12. 12.
    Ermler U, Grabarse W, Shima S, Goubeaud M, Thauer RK (1997) Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation. Science 278:1457–1462CrossRefPubMedGoogle Scholar
  13. 13.
    Denman SE, Tomkins NW, McSweeney CS (2007) Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiol Ecol 62:313–322CrossRefPubMedGoogle Scholar
  14. 14.
    Goel G, Makkar HP, Becker K (2009) Inhibition of methanogens by bromochloromethane: effects on microbial communities and rumen fermentation using batch and continuous fermentations. Br J Nutr 101:1484–1492CrossRefPubMedGoogle Scholar
  15. 15.
    Tomkins N, Colegate S, Hunter R (2009) A bromochloromethane formulation reduces enteric methanogenesis in cattle fed grain-based diets. Anim Prod Sci 49:1053–1058CrossRefGoogle Scholar
  16. 16.
    Abecia L, Toral PG, Martín-García AI, Martínez G, Tomkins NW, Molina-Alcaide E, Newbold CJ, Yáñez-Ruiz DR (2012) Effect of bromochloromethane on methane emission, rumen fermentation pattern, milk yield, and fatty acid profile in lactating dairy goats. J Dairy Sci 95:2027–2036. https://doi.org/10.3168/jds.2011-4831 CrossRefPubMedGoogle Scholar
  17. 17.
    Wood J, Kennedy FS, Wolfe R (1968) Reaction of multihalogenated hydrocarbons with free and bound reduced vitamin B12. Biochemistry 7:1707–1713CrossRefPubMedGoogle Scholar
  18. 18.
    Martinez-Fernandez G, Denman SE, Yang C, Cheung J, Mitsumori M, McSweeney CS (2016) Methane inhibition alters the microbial community, hydrogen flow, and fermentation response in the rumen of cattle. Front Microbiol 7:1122CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Bauchop T (1967) Inhibition of rumen methanogenesis by methane analogues. J Bacteriol 94:171–175PubMedPubMedCentralGoogle Scholar
  20. 20.
    Thauer RK, Kaster A-K, Seedorf H, Buckel W, Hedderich R (2008) Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Micro 6:579–591CrossRefGoogle Scholar
  21. 21.
    Mitsumori M, Shinkai T, Takenaka A, Enishi O, Higuchi K, Kobayashi Y, Nonaka I, Asanuma N, Denman SE, McSweeney CS (2012) Responses in digestion, rumen fermentation and microbial populations to inhibition of methane formation by a halogenated methane analogue. Br J Nutr 108:482–491CrossRefPubMedGoogle Scholar
  22. 22.
    Hristov A, Oh J, Meinen R, Montes F, Ott T, Firkins J, Rotz A, Dell C, Adesogan A, Yang W, Tricarico J, Kebreab E, Waghorn G, Dijkstra J, Oosting S (2013) Mitigation of greenhouse gas emissions in livestock production—a review of technical options for non-CO2 emissions. In: Gerber P, Henderson B, Makkar H (eds) FAO Animal Production and Health Paper No 177, Rome, Italy, p 231Google Scholar
  23. 23.
    Paul NA, Cole L, De Nys R, Steinberg PD (2006) Ultrastructure of the gland cells of the red alga Asparagopsis Armata (bonnemaisoniaceae). J Phycol 42:637–645CrossRefGoogle Scholar
  24. 24.
    Machado L, Magnusson M, Paul NA, Kinley R, de Nys R, Tomkins N (2016) Identification of bioactives from the red seaweed Asparagopsis taxiformis that promote antimethanogenic activity in vitro. J Appl Phycol 28:3117–3126. https://doi.org/10.1007/s10811-016-0830-7 CrossRefGoogle Scholar
  25. 25.
    Paul N, de Nys R, Steinberg P (2006) Chemical defence against bacteria in the red alga Asparagopsis armata: linking structure with function. Mar Ecol Prog Ser 306:87–101CrossRefGoogle Scholar
  26. 26.
    Kinley RD, de Nys R, Vucko MJ, Machado L, Tomkins NW (2016) The red macroalgae Asparagopsis taxiformis is a potent natural antimethanogenic that reduces methane production during in vitro fermentation with rumen fluid. Anim Prod Sci 56:282–289. https://doi.org/10.1071/AN15576 CrossRefGoogle Scholar
  27. 27.
    Machado L, Magnusson M, Paul NA, de Nys R, Tomkins N (2014) Effects of marine and freshwater macroalgae on in vitro total gas and methane production. PLoS One 9:e85289. https://doi.org/10.1371/journal.pone.0085289 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Machado L, Magnusson M, Paul NA, Kinley R, de Nys R, Tomkins N (2016) Dose-response effects of Asparagopsis taxiformis and Oedogonium sp. on in vitro fermentation and methane production. J Appl Phycol 28:1443–1452. https://doi.org/10.1007/s10811-015-0639-9 CrossRefGoogle Scholar
  29. 29.
    Kinley RD, Vucko MJ, Machado L, Tomkins NW (2016) In vitro evaluation of the antimethanogenic potency and effects on fermentation of individual and combinations of marine macroalgae. Am J Plant Sci 7:2038CrossRefGoogle Scholar
  30. 30.
    NHMRC (2013) Australian code for the care and use of animals for scientific purposes. National Health and Medical Research Council, CanberraGoogle Scholar
  31. 31.
    Yu Z, Morrison M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques 36:808–813PubMedGoogle Scholar
  32. 32.
    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998CrossRefPubMedGoogle Scholar
  34. 34.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2013) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42(D1):D633–D642CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Rosewarne CP, Pettigrove V, Stokes HW, Parsons YM (2010) Class 1 integrons in benthic bacterial communities: abundance, association with Tn402-like transposition modules and evidence for coselection with heavy-metal resistance. FEMS Microbiol Ecol 72:35–46CrossRefPubMedGoogle Scholar
  37. 37.
    Anderson M, Gorley R, Clarke K (2008) PERMANOVA + for PRIMER: guide to software and statistical methods. PRIMER-EGoogle Scholar
  38. 38.
    Clarke KR, Gorley RN (2006) PRIMER v6: user manual/tutorial. PRIMER-E Ltd, PlymouthGoogle Scholar
  39. 39.
    Lozupone CA, Hamady M, Kelley ST, Knight R (2007) Quantitative and qualitative β diversity measures lead to different insights into factors that structure microbial communities. Appl Environ Microbiol 73:1576–1585CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kladi M, Vagias C, Roussis V (2004) Volatile halogenated metabolites from marine red algae. Phytochem Rev 3:337–366CrossRefGoogle Scholar
  41. 41.
    Burreson B, Moore RE, Roller P (1975) Haloforms in the essential oil of the alga Asparagopsis taxiformis (Rhodophyta). Tetrahedron Lett 16:473–476CrossRefGoogle Scholar
  42. 42.
    Burreson BJ, Moore RE, Roller PP (1976) Volatile halogen compounds in the alga Asparagopsis taxiformis (Rhodophyta). J Agric Food Chem 24:856–861CrossRefGoogle Scholar
  43. 43.
    Woolard FX, Moore RE, Roller PP (1979) Halogenated acetic and acrylic acids from the red alga Asparagopsis taxiformis. Phytochemistry 18:617–620CrossRefGoogle Scholar
  44. 44.
    Lanigan G (1972) Metabolism of pyrrolizidine alkaloids in the ovine rumen. IV. Effects of chloral hydrate and halogenated methanes on rumen methanogenesis and alkaloid metabolism in fistulated sheep. Crop Pasture Sci 23:1085–1091CrossRefGoogle Scholar
  45. 45.
    Ungerfeld E, Rust S, Boone D, Liu Y (2004) Effects of several inhibitors on pure cultures of ruminal methanogens. J Appl Microbiol 97:520–526CrossRefPubMedGoogle Scholar
  46. 46.
    Seedorf H, Kittelmann S, Henderson G, Janssen PH (2014) RIM-DB: a taxonomic framework for community structure analysis of methanogenic archaea from the rumen and other intestinal environments. Peer J 2:e494CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kittelmann S, Seedorf H, Walters WA, Clemente JC, Knight R, Gordon JI, Janssen PH (2013) Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities. PLoS One 8:e47879CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Denman SE, Fernandez GM, Shinkai T, Mitsumori M, McSweeney CS (2015) Metagenomic analysis of the rumen microbial community following inhibition of methane formation by a halogenated methane analog. Front Microbiol 6:1087. https://doi.org/10.3389/fmicb.2015.01087 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Li X, Norman HC, Kinley RD, Laurence M, Wilmot M, Bender H, de Nys R, Tomkins N (2016) Asparagopsis taxiformis decreases enteric methane production from sheep. Anim Prod Sci AN15883. doi: https://doi.org/10.1071/AN15883

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.MACRO – Centre for Macroalgal Resources and Biotechnology, College of Science and EngineeringJames Cook UniversityTownsvilleAustralia
  2. 2.Centre for Macroalgal Resources and Biotechnology, College of Marine and Environmental SciencesJames Cook UniversityTownsvilleAustralia
  3. 3.CSIRO, Australian Tropical Science and Innovation PrecinctTownsvilleAustralia
  4. 4.Meat & Livestock AustraliaBrisbaneAustralia
  5. 5.CSIRO, Riverside Life Sciences CentreSydneyAustralia
  6. 6.CSIROAdelaideAustralia

Personalised recommendations