Effects of High Concentrations of Crude Glycerin on Rumen Microbial Populations in Sheep

Crude glycerin, the major byproduct of biodiesel industry, can alter ruminal kinetic parameters. The objective of this trial was to evaluate the effects of high concentrations of crude glycerin on rumen microbial groups. Isonitrogenous (18.4% CP) and isoenergetic (2.7 Mcal ME/kg DM) diets, composed of corn silage, soybean hulls, soybean meal, mineral premix, cracked corn grain and crude glycerin (83% glycerol, 95% DM, 6% salt, and less than 0.01% methanol) included at 0 (G0), 10 (G10), 20 (G20), or 30% (G30) of the total diet (DM basis), in a roughage:concentrate ratio of 40:60, were used as substrates in an in vitro rumen batch culture trial. Three adult male rumen-cannulated sheep were used as rumen fluid donors. The incubation was carried out in 160-mL serum bottles (n=30) containing 0.5 g test feed (placed into F57 Ankom^® filter bags), 50 mL buffer solution, and 25 mL ruminal fluid. The bottles were sealed and placed into a forced-air oven (39^oC). After 24 h of fermentation, bottles were placed into an ice bucket to halt microbial activity. DNA extraction was performed using commercial kits, and used for the quantitative PCR assay, using primers for bacteria (BACT), rumen fungi (FUNG), Ruminococcus flavefaciens (RUMI), Fibrobacter succinogenes (FIBRO), and rumen methanogens (METH). The relative population sizes of microbial groups were expressed as proportion of BACT 16S rDNA. The ∆Ct values were calculated by subtracting the Ct (threshold cycle) value of target gene from the Ct value of reference gene. The relative expression of target groups was calculated from the ∆Ct values as 2^−∆Ct and the relative quantification as 100×(2^∆Ct)⁻¹ (%). Data were analyzed using the MIXED procedure of SAS, and whenever the F-test was significant, contrast analyses (linear, quadratic, 0 × glycerin) were performed. No interaction of treatment × inoculum was observed in this trial (P > 0.10). The Ct of reference gene was not different (P > 0.10) among treatments (average = 14.9). Experimental substrates also did not alter (P > 0.10) relative proportions of FUNG (0.003%), RUMI (0.003%), FIBRO (0.05%), but tended to linearly (P = 0.07) decrease METH (13.9, 10.2, 10.8, 9.2%, respectively for G0, G10, G20 and G30). In conclusion, crude glycerin, even in high concentrations (up to 30% DM) has minimal impact in the amount of rumen BACT and proportions of FUNG, RUMI and FIBRO, but tends to linearly decrease METH.