Changes in rumen microbial ecology during dietary transition in cattle and sheep: a molecular and metabolic approach

Ruminal acidosis is often characterised by decreased ruminal pH below pH 6.0, increased concentrations of ruminal D and L- lactate and volatile fatty acid concentrations in grain fed ruminants, creating an environment for growth of lactic acid producing bacteria such as S. bovis and Lactobacillus spp. and reduction in cellulolytic bacterial populations e.g. F. succinogenes. This thesis undertook genotypic studies of rumen microbial ecology based on five key bacterial species, Prevotella ruminantium, Fibrobacter succinogenes, Selenomonas ruminantium, Streptococcus bovis, and Lactobacillus spp. using quantitative real time PCR (qRT- PCR) of 16S rRNA genes. This methodology enabled true genetic monitoring of ecological changes rather than traditional phenotypic microbial culture studies. These genetic studies of rumen microbial ecology were aligned with changes in rumen metabolism. Application of qRT-PCR methodology was validated for complete and consistent extraction of DNA from mixed rumen samples to ensure reliable enumeration of rumen bacteria, and finally development of primers for use in the qRT-PCR assays. The qRT-PCR methods were then used to monitor changes in rumen microbial ecology in cattle managed under commercial conditions in feedlots rather than experimental conditions. The key species were stabilised in the rumen microbial ecology within 7 days of introduction of cattle to feedlots irrespective of feeding hay and grain separately or via total mixed rations. Moreover, metabolic indicators of high production potential coincided with the stable populations of the key rumen bacterial species F. succinogenes, P. ruminicola and S. ruminantium and no evidence of elevated S. bovis populations. Developmental changes in rumen bacterial ecology of steers born during either autumn or winter/spring showed similar trends in bacterial populations when adapting to feedlot rations irrespective of time of calving. However, the rumen protozoal populations were reflective of the time of calving with cattle born in winter/spring maintaining higher protozoal populations throughout the feedlot period. In commercial dairy herds, rumen metabolic end products were consistently correlated with changes in key bacterial populations. Rumen acidosis was observed in sheep fed lupins at 3 times maintenance. Decreased populations of F. succinogenes and increased populations of S. bovis with no decrease in rumen pH were observed in sheep fed high-fat soyabean diets. Molecular techniques such as qRT-PCR used here as well as newer molecular genetic approaches such as next generation sequencing will allow for more comprehensive interpretation of ecological changes in the rumen leading to improved management and productivity of cattle and sheep especially during dietary transitions

Maximizing efficiency of rumen microbial protein production.

Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen

Determining the culturability of the rumen bacterial microbiome

The goal of the Hungate1000 project is to generate a reference set of rumen microbial genome sequences. Toward this goal we have carried out a meta-analysis using information from culture collections, scientific literature, and the NCBI and RDP databases and linked this with a comparative study of several rumen 16S rRNA gene-based surveys. In this way we have attempted to capture a snapshot of rumen bacterial diversity to examine the culturable fraction of the rumen bacterial microbiome. Our analyses have revealed that for cultured rumen bacteria, there are many genera without a reference genome sequence. Our examination of culture-independent studies highlights that there are few novel but many uncultured taxa within the rumen bacterial microbiome. Taken together these results have allowed us to compile a list of cultured rumen isolates that are representative of abundant, novel and core bacterial species in the rumen. In addition, we have identified taxa, particularly within the phylum Bacteroidetes, where further cultivation efforts are clearly required. This information is being used to guide the isolation efforts and selection of bacteria from the rumen microbiota for sequencing through the Hungate1000

Characterization of the Rumen Bacterial Communities of Bison Heifers Fed a Grass-Based Diet vs a Grain-Based Free-Choice Diet

A century ago, the North American grasslands and prairie ecosystems were dominated by bison. At least 30 million bison roamed the Great Plains when the first explorers arrived. By 1900, there were little over a thousand bison remained in the United States and Canada. Recovery efforts has been made since the 20th century to reestablish the herds and increase the bison population. Today, over 500,000 bison are distributed across North America, with more than 90% of the existing bison population under commercial production. Modern conservation strategies are made via the collaborative efforts of conservationist, producers, and researchers, resulting in increased number of proposed research to better understand bison’s biology. Given that the ruminal bacterial communities of North American bison are one of the most understudied areas of bison research, the aim of the current study was to determine and compare the diversity and composition of ruminal bacteria between bison heifers on two different diets at two different ranches. Stomach tubing was used to collect rumen fluid from lifetime grass-fed heifers between 25 and 30 months of age distributed between 2 ranches located in Standing-Butte (SBR; n=17), SD, and Blue-Creek (BCR; n=17), NE, respectively. A second set of samples was collected after the same individuals had been transitioned to a grain-based free-choice diet for 100 days. Bacterial composition was determined by Illumina MiSeq (2×300) sequencing of PCR amplicons generated from the V1-V3 region of the 16S rRNA gene. Next-Generation Sequence data was analyzed using a combination of custom Perl scripts, and publicly available software (Mothur v.1.40, RDP classifier and NCBI Blast). Taxonomic analysis identified Bacteroidetes and Firmicutes as the dominant phyla across all samples analyzed. A total of 57,132 and 59,133 specieslevel Operational Taxonomic Units (OTUs) were identified in SBR and BCR grass-fed heifers, respectively, in contrast to 13,240 and 22,516 OTUs that were found in the same animals on a grain-based diet. A comparative analysis using the most abundant OTUs from each group was conducted using the Kruskal-Wallis sum-rank test. In the Standing Butte heifers, 28 abundant OTUs were found to be different between diets (P \u3c 0.05), including Bb-00031 (grass = 0.04% vs grain = 1.45%) and Bb-00018 (grass = 0.58% vs grain = 0.06%). In the Blue Creek heifers, 17 of the most abundant OTUs were found to be different between diets (P \u3c 0.05), including Bb-00046 (grass = 1.24% vsgrain = 0.45%) and Bb-00058 (grass = 0.03% vsgrain = 1.22%). Together these results indicate that the rumen of the North American bison harbors highly diverse bacterial communities that undergo dramatic changes in response to changes in diet, and they represent a starting point towards a better understanding of their rumen microbiome, leading to prospective practical applications to bison conservation and production

Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure

The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.Das Pansenmikrobiom ist ein komplexes Ökosystem mit einer großen Vielfalt an Organismen, die kurzkettige Fettsäuren (SCFAs) produzieren und verbrauchen. Es ist von großem Interesse, diese Aktivitäten zu analysieren, da sie sowohl für das Mikrobiom als auch für den Wirt von Nutzen sind. Ziel dieser Arbeit ist es, das Proteom und Metabolom des vorherrschenden Pansenvertreters Prevotella bryantii B14 in Gegenwart verschiedener SCFA und unter Einwirkung des Antibiotikums Monensin in Rein- und Mischkultur (in vitro) darzustellen. Aufgrund der großen Anzahl von Prevotellaceae im Pansenmikrobiom wurde der Vertreter P. bryantii B14 (DSM 11371) ausgewählt, um die biochemischen Faktoren für die erfolgreiche Resistenz gegenüber Monensin und die Auswirkungen von kurzkettigen Fettsäuren auf ihr Wachstum zu untersuchen. Die aktuelle Arbeit besteht aus zwei Publikationen [1, 2], die in Kapitel 2 und 3 eingefügt sind, sowie einem eingereichten Manuskript in Kapitel 4. Die Formatierung der Publikationen und des Manuskriptes wurde an die Dissertation angepasst. Die erste Veröffentlichung (Kapitel 2) befasste sich mit der Supplementierung verschiedener SCFAs und zeigt, dass SCFAs wachstumsfördernd, aber nicht essentiell für P. bryantii B14 sind. Reinkulturen von P. bryantii B14 sind in Hungate-Röhrchen unter anaeroben Bedingungen durchgeführt worden. Gaschromatographie-Flugzeit-Massen-spektrometrie (GC-ToF MS) wurde zur Quantifizierung der langkettigen Fettsäuren (LCFA) von P. bryantii B14 verwendet. Die nicht-markierte und nicht-angereicherte Proteine von P. bryantii B14 wurden mit Hilfe von Massenspektrometrie identifiziert und quantifiziert. Es wurde ein unterschiedliches Wachstumsverhalten in Abhängigkeit von der zugeführten SCFA beobachtet. Eine Auswirkung der strukturellen Eigenschaften der SCFAs hat sich auf die LCFAs und ebenso auf die Anwesenheit verschiedener Membranproteine bemerkbar gemacht. Die Supplementierung von P. bryantii B14 mit verzweigtkettigen Fettsäuren (BCFAs), insbesondere Isovaleriansäure, hat einen Anstieg des Stoffwechsels von 3-Isopropylmalat (IPM) gezeigt, welcher Teil des Stoffwechsels von verzweigtkettigen Aminosäuren (BCAAs) ist. Die Ergebnisse deuten darauf hin, dass die Strukturähnlichkeit von Isovaleriansäure und Valin höchstwahrscheinlich die Umwandlung von BCFA in BCAA begünstigt. Der relative Anstieg der erforderlichen Enzyme BCAA-Stoffwechsel unterstützen diese Sichtweise. Das Ionophor Monensin hat antibiotische Eigenschaften, welche in der Rindermast, aber auch zur Behandlung von Ketose und Azidose bei Wiederkäuern eingesetzt wird. Die zweiten Veröffentlichung (Kapitel 3) zeigte den Einfluss verschiedenen Konzentrationen von Monensin (0, 10, 20 und 50 μM) und verschiedenen Expositionszeiten (9, 24, 48 und 72 h) mit und ohne Monensin auf P. bryantii B14. Das Wachstumsverhalten, extrazelluläre Glukose- und intrazelluläre Natriumkonzentration wurde unter den beschriebenen Bedingungen ermittelt. Das Proteom von P. bryantii B14 wurde mittels markierungsfreier Quantifizierung nach der gleichen Methode wie in Kapitel 2 analysiert. Fluoreszenzmikroskopie wurde zur Unter-suchung der extrazellulären Polysaccharide (EPS) von P. bryantii B14 eingesetzt. Mit fortschreitender Monensin-Exposition hat sich gezeigt, dass P. bryantii B14 die Verbindung zur äußeren EPS abbaute, indem es die Anzahl und Menge der kohlenhydrataktiven Enzyme (CAZyme) erhöhte. Der Anstieg an extrazellulärer Glukose bestätigte die Vermutung, dass die Opferschicht abgebaut wurde. Die beobachtete Reduktion des intrazellulären Natriums ist wahrscheinlich mit der Zunahme der Menge an Ionentransportern verbunden. Weiterhin wurde eine erhöhte Aktivität der Na+-transferrierenden NADH:Quinon-Oxidoreduktase (NQR) unter Monensin-Supplementierung dokumentiert. Die Rolle von mit Monensin supplementierten Prevotella in einer Mischkultur des Pansenmikrobioms ist im letzten Manuskript beschrieben worden (Kapitel 4). Pansensaft ist mit Hilfe der Pansensimulationstechnik (Rumen simulation technique = Rusitec) anaerob inkubiert worden. Das Proteom der festen Phase ist mit einem ähnlichen Ansatz wie in Kapitel 2 und 3 durchgeführt worden. Das Metabolom der flüssigen Phase aus dem Rusitec ist anhand der 1H-Kernresonanz (Nuclear magnetic resonance = NMR) spektroskopie ermittelt worden. Weitere Parameter wie pH-Wert, Gas- und Methan-produktion wurden im Zeitverlauf überwacht. Das Experiment bestand aus drei Phasen, beginnend mit einer Anpassungsphase von 7 Tagen. Es folgte eine Behandlungsphase, in der Monensin über das tägliche verabreichen der totalen Mischration (TMR) für weitere 7 Tage durchgeführt wurde. Die Elutionsphase war die letzte Phase, in der die Monensinzugabe eingestellt und die Überwachung für weitere 3 Tage fortgesetzt wurde. Metabolomics und Proteomics haben gezeigt, dass Mitglieder der Gattung Prevotella unter Monensinzugabe am häufigsten vorkommen. Außerdem verlagerte sich der Pansenstoffwechsel auf eine erhöhte Propionatproduktion. Der Stoffwechsel von Prevotella sp. wurde auf eine erhöhte Succinat-produktion umgestellt. Die vorliegende Arbeit zeigt die Auswirkungen von SCFAs auf verschiedene Stoffwechselfunktionen von P. bryantii B14. Es wurde gezeigt, dass verschiedene Abwehrmechanismen von Prevotella sp. und insbesondere von P. bryantii B14 die anti-biotische Wirkung von Monensin verhindern

Tinjauan bacaan : penghasilan protease melalui kaedah fermentasi mikrob / Nursyuhadah Othman, Darah Ibrahim and Wan Nordini Hasnor Wan Ismail

Enzim adalah suatu protein globul yang dihasilkan oleh sel-sel hidup yang seterusnya akan digunakan untuk memangkinkan tindak balas biokimia yang spesifik. Ciri enzim adalah bersifat spesifik dalam setiap tindak balas yang dimangkinkannya, bertindak pada kadar penukaran yang tinggi dan mempamerkan tindak balasnya pada keadaan fisiologi yang tertentu. Enzim protease merupakan enzim yang memangkinkan penguraian ikatan polipeptida di dalam molekul protein kompleks. Enzim protease, (EC 3.4.21-24), terdapat di dalam mikroorganisma, tumbuhan dan haiwan. Walau bagaimanapun, mikroorganisma adalah sumber yang lebih digemari kerana pertumbuhannya yang cepat, tidak menggunakan ruang yang luas untuk proses pengkulturan dan mudah dimanipulasi secara genetik untuk menghasilkan enzim-enzim baru yang boleh digunakan untuk pelbagai tujuan. Kaedah pemfermentasian mikroorganisma bagi menghasilkan protease lebih digemari kerana ia dapat mengurangkan kos apabila substrat yang digunakan terdiri daripada sisa pertanian atau sisa domestik. Enzim protease mempunyai pelbagai kegunaan dalam pelbagai industri bagi memudahkan kehidupan kita seharian. Selain itu, kesedaran masyarakat tentang betapa pentingnya menggunakan produkproduk bioteknologi dalam urusan harian berbanding penggunaan bahan sintetik yang boleh mencemarkan alam sekitar dan memerlukan tenaga buruh yang ramai, misalnya dalam pengurusan dan pemprosesan sisa, telah meningkatkan lagi permintaan terhadap penghasilan enzim proteas

Control of lactate accumulation in ruminants using Prevotella bryantii

Overfeeding ruminants with readily fermentable carbohydrates alters the normal balance of microbes in the rumen compartment causing changes in the fermentation pattern and rumen function. The resulting increase in the molar concentrations of ruminal lactate and/or volatile fatty acids (VFAs), and a decrease in ruminal pH may lead animals to experience acute or subacute acidosis. The objective of this study was to test the hypothesis that lactic acid accumulation can be inhibited by inoculating the rumen with volatile acid-producing bacteria that would compete with lactic acid-producing bacteria for substrate (starch).;We used strain 25A of Prevotella bryantii that had been selected as a rapid starch fermenter from a mixed rumen population as a rumen inoculum for in vitro or in vivo ruminal fermentations. In studies conducted in vitro; strain 25A reduced the accumulation of lactate by 90% when added to mixed rumen fermentations that contained excess soluble carbohydrates, and cultures had greater amounts of succinate and propionate compared with the controls. Morphological, biochemical and genetic analysis (sequencing of the 16S rRNA gene) indicated that strain 25A is related to Prevotella bryantii. Amylolytic strain 25A showed an ability to reduce ruminal lactate accumulation (85%) in goats in a model of acute acidosis, and was able to drive starch fermentation to VFAs and away from lactate accumulation. When the ability of this bacterium to prevent lactate accumulation was tested in lactating cows, ruminal pH was favourable (above 6) and lactate accumulation was reduced, but significant changes in ruminal VFA profile and animal milk yields were not observed