BIODIVERSITAS ISSN: 1412-033X Volume 20, Number 4, April 2019 E-ISSN: 2085-4722 Pages: 1055-1062 DOI: 10.13057/biodiv/d200417 Bacteria and methanogen community in the rumen fed different levels of grass-legume silages RONI RIDWAN 1, , IMAN RUSMANA 2 , YANTYATI WIDYASTUTI 1 , KOMANG G. WIRYAWAN 3 , BAMBANG PRASETYA 1 , MITSUO SAKAMOTO 4 , MORIYA OHKUMA 4 1 Research Center for Biotechnology, Indonesian Institute of Sciences. Jl. Raya Jakarta Bogor Km. 46, Cibinong, Bogor 16911, West Java, Indonesia Tel:+62-21-8754587, Fax: +62-21-8754588,  email: roni001@lipi.go.id, rony_biotech@yahoo.com 2 Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor. Jl. Raya Dramaga, Bogor 16680, West Java, Indonesia 3 Department of Animal Nutrition, Faculty of Animal Sciences, Institut Pertanian Bogor. Jl. Raya Dramaga, Bogor 16680, West Java, Indonesia 4 Microbe Division/Japan Collection of Microorganisms RIKEN Bioresource Center, Tsukuba, Ibaraki, Japan Manuscript received: 7 January 2019. Revision accepted: 22 March 2019. Abstract. Ridwan R, Rusmana I, Widyastuti Y, Wiryawan KG, Prasetya B, Sakamoto M, Ohkuma M. 2019. Bacteria and methanogen community in the rumen fed different levels of grass-legume silages. Biodiversitas 20: 1055-1062. This study aimed to investigate the effects of dietary grass-legume silages on the microbial community by using a culture-independent approach. Treatments consisted of R0: 50% Pennisetum purpureum and 50 % concentrate; R1: 20% P. purpureum, 50 % concentrate, and 30% grass-legumes silage; R2: 20% P. purpureum, 35 % concentrate, and 45% grass-legumes silage; and R3; 20% P. purpureum, 20 % concentrate, and 60% grass- legumes silage. The rumen fluid obtained from fistulated cattle was used for T-RFLP, 16S rDNA clone library, and qPCR analyses. The results indicated that bacterial diversity was dominated by Bacteroidetes, Firmicutes, and methanogen by Methanobacteriales, based on partial 16S rDNA sequences. The microbial communities were dominated by Prevotella brevis, P. ruminicola, Succiniclasticum ruminis, and Methanobrevibacter ruminantium, M. smithi, M. thueri, and M. millerae. The increasing silage diet in a rumen suppressed methanogenesis by reducing population distribution of Methanobacteriales, directly or indirectly, by reducing the diversity of bacterial populations. Generally, the increase silage in the diet changed the bacterial and methanogen community. Grass-legume silage diets of less than 45% are potential for ruminant diet to reduce methane production by a decrease of 4% in the relative distribution of methanogens in the rumen. Keywords: 16S rDNA sequence, culture-independent, grass-legumes silage, microbial community INTRODUCTION The rumen a complex microbiome of bacteria and methanogen plays an important role in feed metabolisms. Naturally, methane (CH4) is produced during feed fermentation by methanogens in the rumen, which constitutes an energy loss and reduces the productivity of the ruminant. Ruminant is one contributor of enteric CH4 emissions into the environment from the livestock sector (Patra et al. 2012; Ji and Park 2012), and potent greenhouse gas that contributes to global warming and climate change (IPCC 2014; Bodas et al. 2012). One of the most limiting factors in feeding cattle with forage is nutrient quality and sustainability. Calliandra calothyrsus contains high crude protein (21-30%) and a total tannin 8-14%. Since crude protein supplies total N for microbes to synthesize protein, polyphenolics are a useful nutritional strategy to reduces CH4 emissions (Lopez et al. 2010). The increased level of silage diets in a rumen in vitro fermentation system suppressed both methane production and protozoa population (Ridwan et al. 2014). The combination of grasses and legumes (1:1) is an alternative solution for improving the crude protein content of feed for sustainable ruminant production (Ridwan et al. 2015). Up until recently, information regarding the microbiome community in the rumen of Ongole cattle has been limited. Examination of the microbial community in the rumen, based on cultural methods, has produced limited results that include less than 1-2 % from total microbes and is highly misleading. Molecular analyses, based on culture- independent methods using the 16S rDNA sequence, should be used for additional information of microbial diversity from unculturable rumen microbes. Many useful methods may be used for metagenomic assays based on 16S rDNA, such as terminal-restriction fragment length polymorphism (T-RFLP) (Khafipour et al. 2009; Cadillo et al. 2008), 16S rDNA clone library (Danielsson et al. 2012; Fernando et al. 2010), and qPCR (Tajima et al. 2001; Bustin et al. 2009). Moreover, additional data on microbial diversity and clustering will be advantageous. The objective of this study was to investigate the bacteria and methanogen communities in the rumen of Ongole cattle, fed different levels of silage containing C. calothyrsus.