Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction1

Abstract

The effects of two kinds of Escherichia coli strains, wild-type E. coli W3110 or E. coli nir-Ptac, which has enhanced nitrite reduction activity, on in vitro CH4 production and nitrate and nitrite reduction in cultures of mixed ruminal microorganisms was investigated using continuous incubation systems. Escherichia coli nir-Ptac, a derivative of wild-type E. coli W3110, was constructed by replacing self promoter of nir BD operon encoding subunits of nitrite reductase in E. coli W3110 by tac promoter to make the expression of nir BD higher and constitutive. The nitrite reductase activity of E. coli nir-Ptac was approximately twice as high as E. coli W3110. The culture media consisted of 400 mL of strained ruminal fluid taken from two nonlactating Holstein cows receiving a basal diet of orchardgrass hay at maintenance level (55 g of DM/kg of BW0.75 daily), and 400 mL of autoclaved artificial saliva. Treatments were arranged in two separate 3 x 3 factorials consisting of nitrate (NaNO3; 0, 5, or 10 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac, or nitrite (NaNO2; 0, 1 or 2 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac. The control culture contained no chemical or microbial additives. Escherichia coli cells were inoculated into in vitro mixed ruminal cultures at approximately 2 x 10(8) to 10(9) cells/mL. Methane production by ruminal microorganisms was decreased markedly (P < 0.001) by the addition of nitrate and nitrite, and by the inoculation of cultures with E. coli W3110 or E. coli nir-Ptac (P < 0.01). With mixed nitrite-containing cultures, E. coli nir-Ptac inhibited (P < 0.001) in vitro nitrite accumulation and CH4 production more than E. coli W3110, which may be due to the tac promoter-enhanced nitrite reductase activity of E. coli nir-Ptac accelerating electrons to be consumed for nitrite reduction rather than CH4 biosynthesis. In conclusion, anaerobic cultures of E. coli W3110 or E. coli nir-Ptac may decrease CH4 production in the rumen. The inoculation of E. coli W3110 or, especially, E. coli nir-Ptac to mixed ruminal microorganisms may decrease nitrite toxicity when ruminants consume high-nitrate-containing forages and when nitrite is applied to abate ruminal CH4 production.

Microbial ecosystem and methanogenesis in ruminants

“…Wolinella was shown to reduce methane production in vitro but only if nitrate was added to the incubation media (Iwamoto et al, 2002). These effects were also demonstrated in vivo using strains of Escherichia coli with high nitrate/nitrite-reducing activity (Sar et al, 2005a and2005b).…”

The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

“…The highest decrease was observed with W. succinogenes, with low nitrite accumulated in the culture media compared with the other two nitrate-reducing bacteria (Iwamoto et al, 2002). Nitritereducing Escherichia coli strains decreased nitrite accumulation in vitro as well as in vivo when added with nitrate (Sar et al, 2005b and2005c). Propionibacterium acidipropionici, which has the ability to reduce nitrite into N 2 O (Rehberger and Hibberd, 2000) is commercially available to avoid nitrite toxicity in cows fed high nitrate forages.…”

Otimização da pecuária nacional de forma sustentável

“…Desta forma, quando o nitrato está presente no rúmen, o H 2 é utilizado na redução de nitrato a nitrito a amônia, diminuindo assim a produção de metano (UNGERFELD & KOHN, 2006). Outra hipótese é que a inibição da produção de CH 4 pode ser atribuída à toxicidade direta de nitrito e outros produtos da redução de nitrato a amônia sobre as archeas metanogênicas (BOZIC et al, 2009;SAR et al, 2005), mas, no presente experimento, não foi mensurada a expressão genica das bactérias ruminais. Os resultados da PGT e MOVD não foram alterados (P>0,05) pela adição de nitrato em substituição a proteína da soja.…”