bs_bs_banner Determining the culturability of the rumen bacterial microbiome Christopher J. Creevey,1,2 William J. Kelly,3,4* list of cultured rumen isolates that are representative Gemma Henderson3,4 and Sinead C. Leahy3,4 of abundant, novel and core bacterial species in the 1Animal and Bioscience Research Department, Animal rumen. In addition, we have identified taxa, particu- and Grassland Research and Innovation Centre, larly within the phylum Bacteroidetes, where further Teagasc, Grange, Dunsany, Co. Meath, Ireland. cultivation efforts are clearly required. 2Institute of Biological, Environmental and Rural This information is being used to guide the isola- Sciences, Aberystwyth University, Aberystwyth, tion efforts and selection of bacteria from the rumen Ceredigion, UK. microbiota for sequencing through the Hungate1000. 3Grasslands Research Centre, AgResearch Limited, Palmerston North, New Zealand. Introduction 4New Zealand Agricultural Greenhouse Gas Research Ruminants have evolved a symbiotic relationship with a Centre, Palmerston North, New Zealand. complex microbiome consisting of bacteria, archaea, fungi, protozoa, and viruses located in their fore-stomach Summary (reticulorumen) that allows these animals to utilize the lignocellulose component of plant material as their main The goal of the Hungate1000 project is to generate a energy source. The microbial degradation of lignocellu- reference set of rumen microbial genome sequences. lose, and fermentation of the released soluble sugars, Toward this goal we have carried out a meta-analysis produces short-chain fatty acids that are absorbed across using information from culture collections, scientific the rumen epithelium and utilized by the ruminant for literature, and the NCBI and RDP databases and growth, while the microbial cells pass from the rumen to linked this with a comparative study of several rumen the digestive tract where they become the main source of 16S rRNA gene-based surveys. In this way we have amino acids and protein for the animal. Other fermenta- attempted to capture a snapshot of rumen bacterial tion end-products, including hydrogen, carbon dioxide, diversity to examine the culturable fraction of the formate and methyl-containing compounds, are important rumen bacterial microbiome. Our analyses have substrates for methanogenesis. Consequently, rumen revealed that for cultured rumen bacteria, there are microbes are central to the optimal digestive functioning many genera without a reference genome sequence. of the animal, and the lack of sufficient understanding of Our examination of culture-independent studies high- the rumen microbiome is one of the major knowledge lights that there are few novel but many uncultured gaps hindering effective enhancement and modification of taxa within the rumen bacterial microbiome. Taken rumen function (Kingston-Smith et al., 2013). together these results have allowed us to compile a Bacteria are the most numerous organisms in the rumen microbiome, being present at 1010 to 1011 g−1 of Received 3 February, 2014; revised 14 May, 2014; accepted 2 June, content and making up more than 50% of the cell mass. 2014. *For correspondence. E-mail [email protected]; Tel. Studies of rumen bacteria began with the development of (+64) 6 351 8118; Fax (+64) 6 351 8032. techniques for cultivating strictly anaerobic organisms and Microbial Biotechnology (2014) 7(5), 467–479 doi:10.1111/1751-7915.12141 have attempted to answer three questions: ‘Who is Funding Information This work was made possible through a Global there?’, ‘How many?’ and ‘What are they doing? As a Research Alliance Senior Scientist Award from the Livestock Emis- consequence, most of the knowledge we have of rumen sions & Abatement Research Network awarded to CJC. CJC is cur- rently part of the BBSRC (Biotechnology and Biological Sciences bacteria has arisen from traditional microbiological Research Council) Institute Strategic Programme Grant, Rumen methods involving the isolation and cultivation of pure Systems Biology, (Grant Number BB/J004413/1) and was also funded strains and species. Microscopic examination of rumen from the Science Foundation Ireland (SFI) Stokes Lectureship Pro- gramme (Reference Number: 07/SK/B1236A; http://www.sfi.ie) during contents shows that the rumen contains a rich diversity of this work. SCL was awarded a New Zealand Agricultural Greenhouse microbes, and many different bacteria have been isolated Gas Research Centre (NZAGRC) Travel Award to progress part of this from this environment. Some, such as Lampropedia and work. The Hungate1000 project is funded by the New Zealand Gov- ernment in support of the Livestock Research Group of the Global Quinella, are distinctive enough to be identified by mor- Research Alliance on Agricultural Greenhouse Gases. phology alone (Clarke, 1979), but most have been © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 468 C. J. Creevey, W. J. Kelly, G. Henderson and S. C. Leahy described on the basis of their metabolism and probable data. This was done not only to assess the diversity functional role in the rumen. Many of the best character- present and to identify cultured taxa that remain unrepre- ized rumen bacteria were described in the pioneering sented in reference genome collections, but also to iden- studies of rumen microbiology which isolated representa- tify taxa that require more cultivation effort. The resulting tives of the functionally significant bacterial groups list of taxa is currently being used by the Hungate1000 for (Bryant, 1959; Hungate, 1966). Knowledge of rumen bac- sequencing of previously unsequenced organisms found terial diversity has increased in subsequent years as addi- in association with ruminants. It is hoped this list of taxa tional genera of rumen bacteria have been isolated and will serve as a resource for the rumen microbiology com- characterized. These culture-based studies of bacterial munity interested in the isolation of novel members of the morphology, physiology and metabolism have revealed a microbiome. Sequencing of these organisms will bring us bacterial community that includes both generalists and closer to completing a comprehensive reference genome specialists able to convert large plant polymers into a collection for the rumen microbiome. variety of smaller organic compounds. However, in recent years, emphasis on bacterial cultur- Results and discussion ing has been reduced and our knowledge of the rumen Rumen bacteria in culture bacteria across different diets, ruminant species and geo- graphical locations has quickly expanded through the Using information from culture collections, scientific litera- insights of culture-independent methods, such as 16S ture, the National Center for Biotechnology Information rRNA gene surveys aimed at classifying and quantifying (NCBI) and Ribosomal Database Project (RDP, Cole et the microbes present (Brulc et al., 2009; Kong et al., al., 2014) databases we identified cultivated rumen bac- 2010; Pitta et al., 2010; De Menezes et al., 2011; Hess teria, which include members of 88 different bacterial et al., 2011; Kim et al., 2011; Li et al., 2011; Fouts et al., genera belonging to nine phyla (Table 1 and Supporting 2012; Jami and Mizrahi, 2012; Kav et al., 2012; Pope Information Table S1). A survey of five international et al., 2012; Wu et al., 2012). These studies are providing culture collections (American Type Culture Collection new answers to the questions ‘Who is there?’ and ‘How (ATCC), Culture Collection University of Göteborg many?’, and it is clear that the number of rumen cultured (CCUG), Leibniz Institute DSMZ-German Collection of representatives has not kept pace with this expansion in Microorganisms and Cell Culture, Japan Collection of knowledge. While 16S rRNA gene sequences are useful Microorganisms (JCM), The Belgian Co-ordinated Collec- to take a census of bacteria present in the community, tions of Micro-organisms (BCCM/LMG) gave 146 bacterial they provide only very limited functional genetic informa- cultures of rumen origin (Supporting Information tion and thus do not address the question of ‘What are Table S2), but members of only seven phyla. These col- they doing?’ As a result, genome sequencing has now lections are dominated by members of the phyla become a starting point for analyses that reveal a Firmicutes, Proteobacteria and Actinobacteria, which microbe’s metabolic potential. However, cultivation and together make up 90% of the cultures listed. Members of isolation of strains remain a challenge, and there is a need the Firmicutes and the family Lachnospiraceae in particu- for physiological studies to verify predictions based on lar, appear more amenable to laboratory culture, as there genome sequence data. are 45 genera of Firmicutes that have been cultured from The Hungate1000 project (www.Hungate1000.org.nz) the rumen. In contrast, it is notable that the phylum seeks to produce a reference set of rumen microbial Bacteriodetes is represented by just five isolates belong- genome sequences from cultivated rumen bacteria and ing to two genera. While these culture collection isolates archaea, together with representative cultures of rumen cover all the major taxonomic groups and include several anaerobic fungi and ciliate