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      Synchrony Degree of Dietary Energy and Nitrogen Release Influences Microbial Community, Fermentation, and Protein Synthesis in a Rumen Simulation System

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          Abstract

          Synchrony of energy and nitrogen release in rumen has been proposed to maximize ruminal microbial fermentation. However, the information regarding bacterial community composition and its metabolism under a higher or lower degree of synchronization is limited. In our study, a 0 to 6 h post-feeding infusion (first half infusion, FHI), 6 to 12 h post-feeding infusion (second half infusion, SHI), and 0 to 12 h post-feeding infusion (continuous infusion, CI) of maltodextrin were used to simulate varying degrees of synchronization of energy and nitrogen release in a rumen simulation system. In addition, the bacterial community, metabolite, enzyme activity, and microbial protein synthesis (MPS) were evaluated. Compared with the FHI and CI, the relative abundance of Fibrobacter, Ruminobacter, BF311, and CF231 decreased in the SHI, but that of Klebsiella and Succinivibrio increased in the SHI. The NH 3-N and branched-chain volatile fatty acids were significantly higher, but propionate content and activities of glutamate dehydrogenase (GDH) and alanine dehydrogenase were significantly lower in the SHI than those in the FHI and CI. The SHI had lower MPS and less efficiency of MPS than the FHI and CI, which indicated that the SHI had a lower degree of synchronization. Correlation analysis showed that MPS was positively related to GDH activity and relative abundance of Fibrobacter but negatively related to NH 3-N and relative abundance of Klebsiella. Therefore, a higher degree of synchronization of energy and nitrogen release increased MPS partly via influencing the bacterial community, metabolism, and enzyme activities of ammonia assimilation in the in vitro fermenters.

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          Analysis, Optimization and Verification of Illumina-Generated 16S rRNA Gene Amplicon Surveys

          The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.
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            Effect of Dietary Forage to Concentrate Ratios on Dynamic Profile Changes and Interactions of Ruminal Microbiota and Metabolites in Holstein Heifers

            A better understanding of global ruminal microbiota and metabolites under extensive feeding conditions is a prerequisite for optimizing rumen function and improving ruminant feed efficiency. Furthermore, the gap between the information on the ruminal microbiota and metabolites needs to be bridged. The aim of this study was to investigate the effects of a wide range of forage to concentrate ratios (F:C) on changes and interactions of ruminal microbiota and metabolites. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and Illumina MiSeq sequencing and gas chromatography time-of-flight/mass spectrometry were used to investigate the profile changes of the ruminal microbes and metabolites, and the interaction between them. The predominant bacterial phyla in the rumen were Bacteroidetes (57.2 ± 2.6%) and Firmicutes (26.8 ± 1.6%), and the predominant anaerobic fungi were Neocallimastigomycota (64.3 ± 3.8%) and Ascomycota (22.6 ± 2.4%). In total, 44, 9, 25, and 2 genera, respectively, were identified as the core rumen bacteria, ciliate protozoa, anaerobic fungi, and archaea communities across all samples. An increased concentrate level linearly decreased the relative abundance of cellulolytic bacteria and ciliates, namely Fibrobacter, Succinimonas, Polyplastron, and Ostracodinium (q < 0.05), and linearly increased the relative abundance of Entodinium (q = 0.04), which is a non-fibrous carbohydrate degrader. Dietary F:C had no effect on the communities of anaerobic fungi and archaea. Rumen metabolomics analysis revealed that ruminal amino acids, lipids, organic acids, and carbohydrates were altered significantly by altering the dietary F:C. With increasing dietary concentrate levels, the proportions of propionate and butyrate linearly increased in the rumen (P ≤ 0.01). Correlation analysis revealed that there was some utilization relationship or productive association between candidate metabolites and affected microbe groups. This study provides a better understanding of ruminal microbiota and metabolites under a wide range of dietary F:C, which could further reveal integrative information of rumen function and lead to an improvement in ruminant production.
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              The Complete Genome Sequence of Fibrobacter succinogenes S85 Reveals a Cellulolytic and Metabolic Specialist

              Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                09 February 2020
                February 2020
                : 8
                : 2
                : 231
                Affiliations
                [1 ]State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; June_zh16@ 123456cau.edu.cn (J.Z.);
                [2 ]College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
                Author notes
                Author information
                https://orcid.org/0000-0001-7474-6057
                https://orcid.org/0000-0001-5194-8780
                Article
                microorganisms-08-00231
                10.3390/microorganisms8020231
                7074744
                32050406
                e8d8cd81-56c4-4fbc-9766-9a8e9f98f1d8
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 26 November 2019
                : 06 February 2020
                Categories
                Article

                synchrony,microbial protein synthesis,rumen,bacterial community,energy,nitrogen

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