Proteolytic Bacterial Community Stucture In Rumen

Dietary protein breankdown in the rumen is a complex process. Although the degradation of protein could provide the peptides and amino acids as nitrogen soueces for growth by microorganisms, however, ruminal microorganisms often produce an excess of ruminal ammonia which can freely diffuse across the rumen wall and be converted to urea and excreted leading to a loss of nitrogen, and even up to50%of bacteria protein formed in the rumen also may be broken down subsequently to ammonia. The purposes of this study are to using anearobe cultivation in vitro and through PCR amplification method to construct the16S rDNA gene clone library and PCR-DGGE to build the16S rDNA V3gene clone library, and sequence the target fragments. We analysed the effect of protein and amino acids as different nitrogen source on the rate of amonia produntion, and analysed the structure and biodiversity of monensin-sensitve proteolytic bacteria community, which can provide an insight into the proteolytic bacterial ecosystem of the Holtein cow.In the first study, ammonia productions at different time under different nitrogen sources using anearobe cultivation method in vitro were analyzed. In this expreiment, casein and casein acid hydrolysate were used as different nitogen sources, and these treatment groups contained five different liquid media:NN medium, complete basal medium (control group); medium P basal medium plus casein substrate; medium P-M; medium P with monensin; AA, basal medium plus casein acid hydrolysate substrate; medium AA-M, medium AA with monensin. Simples were collected at0,7,14,21h. The rate of ammonia production was determined.The rate of ammonia produntion of casein acid hydrolysate as nitrogen source was439.04nmol mg-1h-1, which was significantly higher than casein medium (39.34nmol mg-1h-1)(P<0.05). Monensin could significantly inhibite the ammonia procuntion producing by casein medium at7h, and and casein acid hydrolysate medium was inhibited at14h. Monensin lended to inhibite casein acid hydrolysate as the nitrogen and energy sources comparing with casein. In the second experiment, microbial diversity and shifts in bacterial communities among the different treatments was analyzed by DGGE fingerprinting of PCR-ampolified the variable V3region of16S rDNA gene fragments using primers common primer. The Orientation Checker software was used to align the sequences, Muther was used to remove any chimeric rDNA clone, and BioEdit software was used to detect accuracy of bases. All sequences were classified according to the online program GenBank database.The result showed that the DGGE fingerprinting bands of five treatments were different, and the bands media adding moennsin subatrate were less than those without adding it. The diversity of casein as subatrate was the highest, the follwing is casein acid hydrolysate substrate, casein acid hydrolysate substrate with monensin, and casein with monensin. The bacteria displayed casein proteolytic activity were Prevotella sp., Anaerovibrio, Pseudobutyrivibrio and Oribacterium etc, and displayed casein acid hydrolysate degradation activity were Anaerovibrio, Clostridium, Pseudobutyrivibrio, Megasphaera and Prevotella. The monensin-sensitive bacteria growing casein medium were Pseudobutyrivibrio, Acidovorax, Butyrivibrio, Eubacterium and Limnobacter etc, and growing in medium were Clostridium and Pseudobutyrivibrio. The non monensin-sensitive preotolytic bacteria of casein medium were Oribacterium and Selenomonas, and of casein acid hydrolysate were Bacteroides, Enterobacteriaceae, Porphyromonadaceae and Selenomonas.In experiment3, the bacterial communities in the composite samples were further examined and compared using the16S rDNA clone libraries. After removing the sequences of low quality and suspected chimeric sequences,230high-quality sesquences were obtained. The softare Pregap4was used to do quality controlling. Mothur was used to assign sequences to operational taxnomic units (OTUs) based on97%identity criterion and to population composition betweern five clone libraries by using Shann-non etc indices. All sequences were classified from phylum to genus according to the online programs RDP and GenBank.Result showed that phylum Gammaproteobacteria was only to the AA-M (OTU22) and NN (OTU35). Group P had the most abundant OTU and had5more OTUs than P-M, but AA had1less OTU than AA-M. However Shannon, Simpson and Chao1indices indicated group P and AA had more diversity of proteolytic bacteria than that with added monensin groups P-M and AA-M respectively, especially P had the most abundant diversity than other groups. The PCA of casein and casein acid hydrate subatrates were different, and medium which had the same sbstrate had the same PCA after adding monensin. The bacteria displayed casein proteolytic activity were Anaerovibrio, Pseudobutyrivibrio, Oribacterium and Clostridium etc, and displayed casein acid hydrolysate degradation activity were Anaerovibrio, Clostridium, Prevotella and Selenomonas. The monensin-sensitive bacteria growing casein medium were Clostridium, Lachnospiracea, Pseudobutyrivibrio and Prevotella etc, and growinh in medium were Clostridium, Prevotella and Schwartzia. The bacteria resistant monensin of casein medium were Butyrivibrio and Oribacterium, and of casein acid hydrolysate wereMegasphaera and Succinivibrios. Both Prevotella sp. and Anaerovibrio were played a most impotrant role in the protein and amino acids degradation process.