| Essential oils can reduce ruminal methane emission in short time.It’s crucial to reveal the response and adaptation machanism by rumen microorganisms to essential oils to develop the long-term effect by them.So,this study was developed by citrus essential oil(CEO),using ruminal liquid which contained microorganisms collected by different period of CEO-inserting(non-inserted period,responded period,adapted period and intermitted period).And then taking CEO as the only carbon sourse,select the rumen microorganism which can adapt to CEO.Basing the differentially expressed genes of selected microbes cultured by medium adding CEO or not,reveal the adaptation machnisms of rumen microorganism.Experiment 1:the response of rumen microorganisms.Experiments were performed over an 8-week period,and the first 2 weeks served as a preliminary feeding period.Five Hu sheep(EO sheep)were assigned to be fed the basal diet with no CEO supplementation as a control on week 3.CEO were inserted into the rumen through the cannula at a dose of 0.8 mL/L rumen volume just before morning feeding from weeks 4-6.Afterwards,sheep were raised for 2 additional weeks without CEO to examine whether the effect was lasting or not.Collect the rumen liquids just before morning feeding in the last day of week 3(W0_NE),week 4(W1_EO),week 6(W3_EO)and week 8(W5_NE)to get the rumen microorganism sample.The in vitro batch experiment was developed by 4 peroids,2 substrats(corn meal/CM and Chinese wild hay/WH)and 2 dose(additional CEO dose as 0 and 0.8 ml/L).Get the rumen liquid sample after 48 h fermentation.The qPCR results indicated that,the change pattern of methanogens was different with CM or WH.For the CM substrate,the methanogens copies weren’t affect by the effect of periods and additional CEO dose.For the WH substrate,additional CEO dose increased the methanogens copies.16s amplicon sequencing results showed that,(1)For the CM substrate,the relative abundance of the Methanobrevibacter gottschalkii clade,Treponema 2 and Succinivibrio decreased,and the relative abundance of the Methanobrevibacter ruminantium clade,Olsenella,Oribacterium and Streptococcus increased in the response period(W1_EO),in the same time the CH4 yield decreased.While the relative abundance of the Mbb.gottschalkii clade,Treponema 2 and Succinivibrio increased in the adapted period(W3_EO)with CH4 yield increased comparing by W0_NE.For the WH substrate,the relative abundance of the Mbb gottschalkii clade,Succinivibrio,Ruminococcus 2 and RuMinococcaceae decreased,and the relative abundance of the Mbb.ruminantiuM clade,RuMinococcus 1,Prevotella 1 and Treponema 2 increased with the CH4 yield decreased in the response period(W1_EO).While the relative abundance of the Mbb gottschalkii clade decreased and Succinivibrio,Ruminococcus 2 increased in the adapted period(W3_EO)with CH4 yield increased comparing by W0_NE.During intermitted period(W5_NE),relative abundance of RuMinococcus 2 was increased compared to adapted period and non-inserted period.While that of Streptococcus and Prevotella 1 was increased compared to adapted period,but it was not changed compared to non-inserted period.Relative abundance of Ruminococcus 1 was not changed during adapted period but it was significantly increased compared to non-inserted period.In W5_NE,relative abundance of Succinivibrio and Treponema 2 was not changed during W3_EO.Relative abundance of Succinivibrio was decreased while that of TreponeMa 2 was increased compared to W0_NE.Relative abundance of Stretococcus and Olsenella was not changed in W5_NE compared to both W3_EO and W0_NE.(2)With either CM or WH as the substrate,relative abundance of Mbb.gottschalkii and production of CH4 both decreased after addtional CEO supplementation in all periods.With CM as the substrate,relative abundance of Streptococcus and Oribacterium increased while that of Succinivibrio and Treponema 2 decreased during W1_EO,W3_EO and W5_NE after addtional CEO supplementation.Relative abundance of Ruminococcus 1 increased during adapted period after addtional CEO supplement.With WH as the substrate,relative abundance of Treponema 2 decreased in all periods,while relative abundance of Prevotella 1 decreased during W1_EO,W3_EO and W5_NE after addtional CEO supplementation.Succinivibrio was increased during responsed period and Ruminococcus 1 was increased during W3_EO after addtional CEO supplementation.Relative abundance of Olsenella and Ruminococcus 2 was in creased during W1_EO and W5_NE after addtional CEO supplementation.These results indicated that the microorganisms adapted to CEO were different with CM or WH substrate.For CM substrate,the genera of Mbb.gottschalkii clade,Mbb.ruminantium clade,Treponema 2,Butyrivibrio 2,Olsenella,Oribacterium and Streptococcus responded quickly and decreased the CH4 yield.While supplied CEO steadly,the relative abundance of Mbb.gottschalkii clade,Treponema 2 and Succinivibrio changed differently and decreased the effect of CEO.But for the WH substrate,the Mbb gottschalkii clade,Mbb.ruminantium clade,Succinivibrio,Ruminococcus 2,Ruminococcaceae,Ruminococcus 1 and Treponema 2 responded quickly and decreased the CH4 yield.While supplied CEO steadly,the relative abundance of the Mbb.gottschalkii clade decreased and Ruminococcus 2 and Succinivibrio changed differently with the drop off of CEO effect.Stop inserting CEO recovered the bacteria community structure to W0_NE period and weakened the adaptation of them.As the recovery of the relative abundance of Ruminococcus 2,Prevotella 1 and other bacteria,they likely responded to CEO again.Experiment 2:Rumen microbes were cultured in medium using CEO as sole carbon source.The survived microbe was cultured in two different media with or without CEO for 18h.To identify the different expressed genes(DEGs)which might play an important role in CEO adaptation,the RNA of two groups were extracted and applied for RNA-sequencing.Our results indicate:(1)Klebsiella oxytoca was identified after selection of microbes from CEO as sole carbon source.(2)Total 507 DEGs were identified in comparison of two groups,and 307 genes were up-regulated in CEO groups.Within these,psp shock protein(pspABCDE),envelope stress response protein(pspG),protease(htpX),rod shape-determining protein(rodA),rod shape-determining protein(mreC),and repressor(cpxP)were significantly up-regulated.(3)Total 26 enriched GO terms were enriched by DEGs,while organonitrogen compound metabolic process,organonitrogen compound biosynthetic process,cellular amide metabolic process,Iron binding,metal ion binding,cytoplasm and intracellular were enriched in CEO group.(4)KEGG Pathways such as ribosome,propanoate metabolism,and biosynthesis of antibiotics were enriched by up-regulated DEGs,while pathways such as ABC transporters was enriched by down-regulated DEGs.(5)Expression of phage shock protein operon was significantly higher in CEO group compared to control.The results suggested that CEO may damage microbes by damaging cell membranes.To tolerate the CEO pressure,K oxytoca probably up-regulate genes such as pspABCDEG,htpX,rodA,mreC and cpxP to maintain the cell membrane integrity,promote synthetic pathway of cell components to repaire cell membrane damage and degrade the limonene by propanol metabolism pathway.Overall,composition of bacterial and archaeal community was altered after addition of CEO,and different response of microbes to CEO was identified with different substrates.The Mbb.gottschalkii clade,Mbb.ruminantium clade,Treponema 2,Butyrivibrio 2,Ruminococcus 2,Ruminococcaceae,and Streptococcus were significant changed after CEO addition,and production of CH4 was reduced.Treponema 2、Succinivibrio and Ruminococcus 2 could adapted to CEO and weaken the capability of CEO to mitigate CH4 yield.CEO could inhibit the growth of microbes via damaging the cell wall and membrane and reducing the active transportation.Repairing the cell damages through activation of the pathways involved in cell ingredients synthesis,and by degradation of component of CEO through propanol metabolism may be the potential CEO adapatation machanisms of K.oxytoca. |
PDF Full Text Request