The Effect of Methane Mitigation by Condensed Tannins on Ruminal Homoacetogens in Cattle

Keong, Li-ann (2016) The Effect of Methane Mitigation by Condensed Tannins on Ruminal Homoacetogens in Cattle. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Ruminants are capable of emitting methane gas via the methanogenesis step that occurs during their ruminal fermentation. The use of methane inhibitor has revealed that the Wood-Ljundahl pathway of reductive acetogenesis is a potential alternative hydrogen sink to replace the methanogenesis. Therefore, this functional gene-based study aimed to investigate the effects of methane mitigation by condensed tannins on the ruminal homoacetogens in the cattle. Condensed tannins were supplemented to the DNA of stomach fluid sample in vitro. Primers targeting the fhs and acsB genes from the respective formyltetrahydrofolate synthetase (FTHFS) and acetyl-CoA synthase (ACS) enzymes of Wood-Ljundahl pathway were used. The results provided by the latter gene could be used to compensate the low specificity limitation of FTHFS due to its involvement in other biochemical reactions. Thus, the early amplification result for fhs and acsB genes supported the presence of homoacetogenic bacteria that were proficient of utilising the proposed pathway. The amplicons recovered from the fhs gene were cloned and four clones were selected randomly to determine their nucleotide and deduced amino acid sequences. The resultant sequences were then used to construct bootstrapped phylogenetic trees based on the neighboring joining and maximum likelihood methods with using the reference sequences from the NCBI gene bank. Homoacetogen similarity (HSi) score was also calculated from the most promising translated amino acid sequence to evaluate its FTHFS activity. Novel sequences closely linked to the uncultured rumen bacterium were retrieved from this study and they were found to be strongly affiliated to the known homoacetogens cluster in the phylogenetic tree. Conserved regions were further identified from the deduced amino acid sequence and an intermediate HSi score of 69.6% was obtained. Therefore, these sequences hold the possibility to be originated from the novel homoacetogens.