Isolation and characterization of reductive acetogens from rumen fluid samples of Murrah buffaloes

In the present study, attempts have been made to isolate reductive acetogens from the rumen fluid samples of Murrah buffaloes (Bubalus bubalis). Out of 32 rumen samples 51 isolates were isolated, and based on autotrophic growth for production of acetate and presence of formyltetrahydrofolate synthetase gene (FTHFS) 12 isolates were confirmed as reductive acetogens. Microscopic observations showed that ten isolates as Gram-positive rods (ACB28, ACB29, ACB66, ACB73, ACB81, ACB91, ACB133, ACB229, ACB52, ACB95) and two isolates as Gram-positive cocci (ACB19, ACB89). All isolates tested negative for catalase, oxidase, and gelatin liquefaction, whereas the production of H₂S was detected for two (ACB52 and ACB95) of the above isolates. All these isolates showed autotrophic growth from H₂ and CO₂, and heterotrophic growth with different fermentable sugars, viz., d-glucose, D-fructose, and D-trehalose but failed to grow on salicin, raffinose, and l-rhamnose. Out of the isolates, two showed amylase activity (ACB28 and ACB95), five showed CMCase activity (ACB19, ACB28, ACB29, ACB73 and ACB91), three showed pectinase activity (ACB29, ACB52 and ACB89), whereas none of the isolates was found positive for avicellase and xylanase activity. Based on 16S rDNA gene sequence analysis, the isolates showed their phylogenetic relationship with maximum similarity up to 99% to different strains of earlier reported known acetogens of clostridia group including Clostridium sp. (6), Eubacterium limosum (1), Ruminococcus sp. (1) and Acetobacterium woodii (1) except one, i.e., Vagococcus fluvialis. The results indicate that reductive acetogens isolated from the rumen fluid samples of Murrah buffalos are both autotrophic and heterotrophic in nature and further investigations are required to exploit and explore their potential as an alternate hydrogen sink.