Isolation And Identification Of A Lactate-Utilizing Bacterium From Rumen Of Goats And Its Metabolic Characteristics In Vitro

Rumen is a nature anaerobic habitat,which is inhabited by diverse and strictly manipulated populations of ruminal micro-organisms.Ruminal micro-organisms degrade feedstuffs and discard the soluble end products,and these end products are available to the host.Seeking methods for manipulating ruminal fermentation to enhance the health and productive performance of animal is always a hot topic in the research field of ruminal nutrition.This study was designed to investigate the characteristics of metabolism of a lactate-utilizing bacterium L9 from goat rumen.In addition, experiments were performed to evaluate the effects of strain L9 and its associative effects with yeast on in vitro fermentation of mixed ruminal micro-organisms under different substrate conditions. This thesis was described in the following four sections.In the first section,a lactate-utilizing strain L9 was isolated from rumen of goat feeded with high concentrate by using modified Hungate technique and anaerobic culture technique.Five bacterial strains were isolated and purified by using LH medium supplemented with 90 mmol L^-1 lactate as the sole carbon source.The ensuring selection method was accompanied by the lactate detection that could determine the lactate utilization ability of the strains.Strain L9 had the highest ability to utilize lactate,which could ferment 99.67%of 90 mmol L^-1 lactate in 24 h.In the second section,the study was designed to identify strain L9 by morphology,biochemical and molecular methods.Strain L9 was an Gram negative bacterium,which resembled the traits of crescent.The result of sequencing the 16S rRNA gene revealed that 27 of total 33 sequences with homology higher than 97%with strain L9 belonged to Selenomonas ruminantium,which indicated that strain L9 was closely related to Selenomonas ruminantium.According to the characteristics of morphology,physiology and biochemistry tests and comparison of 16S rRNA gene sequence,this Strain L9 was identified as selenomonas ruminantium and was designated as S.ruminantium L9.In the third section,the study was designed to investigate the metabolic characteristics of strain L9 by means of the fermentation system in vitro.The growth of strain L9 could be supported by the LH media supplemented with lactate and reached logarithmic phase at 12h.98.67%of 90 mmol L^-1 lactate in LH media could be fermented in 12 h.LH media supplemented with glucose,maltose, cellobiose,lactose and lactate supported the growth of strain L9,but LH media supplemented with maltose,cellobiose,lactose and glucose better supported it.When strain L9 was grown in LH medium with glucose,maltose,cellobiose and lactose,these carbon source could be fermented simultaneously with production of lactate,however,most of the lactate could be degraded in 24h. Lactate,glucose,maltose,cellobiose and lactose in LH medium were mainly converted to propionate after 24h fermentation.Strain L9 tolerated to lower pH,and obtained best growth at pH5.5～6.5.In the fourth section,experiment was conducted to investigate the effect of strain L9 and associative effects of strain L9 with yeast on the fermentation in in vitro batch cultures with different substrate(Leymus chinensis,corn and starch).The results showed that strain L9 increased significantly pH value and decreased significantly lactate concentration under all substrate condition from 6 to 18h.Strain L9 associated with yeast could enhance the ability of strain L9 to increase the pH value and decrease the lactate concentration.With the strain L9 added,the TVFA concentration, propionate concentration and propionate percentage was higher than those of control,while the ratio of acetate to propionate was lower than those of control.In the condition of cron substrate,the ratio of acetate to propionate of the treatment of strain L9 associated with yeast was lowest and was 0.43 lower than those of control(P＜0.01) at 18h,while the TVFA concentration,propionate concentration, propionate percentage and acetate concentration were higher than the control and all the treatment. Among all the substrate,the TVFA concentration of starch test was highest.In the condition of starch substrate,the TVFA concentration of the treatment of strain L9 associated with yeast was apparently higher than other treatments and that of control,the propionate percentage was 132.8% (P＜0.01) higher than that of control,and meanwhile,the ratio of acetate to propionate was 0.5 (P＜0.01) lower than that of control.Strain L9 increased propionate concentration of all the substrate.Without strain L9 addition,yeast have no significant effects on the in vitro fermentation of mixed ruminal micro-organisms,however,yeast increased the lactate accumulation under the condition of starch substrate.In conclusion,strain L9 had an effect on all the substrate,which could be optimized when strain L9 was associated with yeast.